91 results on '"Jyrki Heino"'
Search Results
2. Embigin is a fibronectin receptor that affects sebaceous gland differentiation and metabolism
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Kalle Sipilä, Emanuel Rognoni, Johanna Jokinen, Mukul Tewary, Matteo Vietri Rudan, Salli Talvi, Ville Jokinen, Käthe M. Dahlström, Kif Liakath-Ali, Atefeh Mobasseri, Xinyi Du-Harpur, Jarmo Käpylä, Stephen L. Nutt, Tiina A. Salminen, Jyrki Heino, and Fiona M. Watt
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Integrins ,Mice ,Sebaceous Glands ,Integrin beta1 ,Cell Adhesion ,Animals ,Cell Differentiation ,Cell Biology ,Molecular Biology ,General Biochemistry, Genetics and Molecular Biology ,Fibronectins ,Integrin alpha5beta1 ,Developmental Biology - Abstract
Stem cell renewal and differentiation are regulated by interactions with the niche. Although multiple cell populations have been identified in distinct anatomical compartments, little is known about niche-specific molecular factors. Using skin as a model system and combining single-cell RNA-seq data analysis, immunofluorescence, and transgenic mouse models, we show that the transmembrane protein embigin is specifically expressed in the sebaceous gland and that the number of embigin-expressing cells is negatively regulated by Wnt. The loss of embigin promotes exit from the progenitor compartment and progression toward differentiation, and also compromises lipid metabolism. Embigin modulates sebaceous niche architecture by affecting extracellular matrix organization and basolateral targeting of monocarboxylate transport. We discover through ligand screening that embigin is a direct fibronectin receptor, binding to the N-terminal fibronectin domain without impairing integrin function. Our results solve the long-standing question of how embigin regulates cell adhesion and demonstrate a mechanism that couples adhesion and metabolism.
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- 2022
3. C1r Upregulates Production of Matrix Metalloproteinase-13 and Promotes Invasion of Cutaneous Squamous Cell Carcinoma
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Liisa Nissinen, Marjaana Ojalill, Markku Kallajoki, Pilvi Riihilä, Jyrki Heino, Kristina Viiklepp, Veli-Matti Kähäri, Seppo Meri, HUSLAB, Seppo Meri / Principal Investigator, Department of Bacteriology and Immunology, University of Helsinki, and TRIMM - Translational Immunology Research Program
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EXPRESSION ,Skin Neoplasms ,MMP1 ,MMP10 ,Extracellular matrix component ,PROGRESSION ,Dermatology ,Complement factor I ,Matrix metalloproteinase ,Biochemistry ,COMPLEMENT ,HETEROGENEOUS DISORDER ,03 medical and health sciences ,Classical complement pathway ,0302 clinical medicine ,Cell Movement ,Cell Line, Tumor ,Matrix Metalloproteinase 13 ,Animals ,Humans ,Molecular Biology ,Cell Proliferation ,030304 developmental biology ,0303 health sciences ,Complement C1r ,Chemistry ,PERIODONTAL-DISEASE ,Cell migration ,KERATINOCYTE ,Cell Biology ,3. Good health ,Gene Expression Regulation, Neoplastic ,COLLAGENASE-3 MMP-13 ,3121 General medicine, internal medicine and other clinical medicine ,030220 oncology & carcinogenesis ,Metalloendopeptidase activity ,Carcinoma, Squamous Cell ,Cancer research ,GROWTH ,Heterografts ,TUMOR MICROENVIRONMENT ,EHLERS-DANLOS-SYNDROME - Abstract
Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. Previous studies have shown the role of the complement system in cSCC progression. In this study, we have investigated the mechanistic role of serine proteinase C1r, a component of the classical pathway of the complement system, in cSCC. Knockout of C1r in cSCC cells using CRISPR/Cas9 resulted in a significant decrease in their proliferation, migration, and invasion through collagen type I compared with that of wild-type cSCC cells. Knockout of C1r suppressed the growth and vascularization of cSCC xenograft tumors and promoted apoptosis of tumor cells in vivo. mRNA-sequencing analysis after C1r knockdown revealed significantly regulated Gene Ontology terms cell-matrix adhesion, extracellular matrix component, basement membrane, and metalloendopeptidase activity and Kyoto Encyclopedia of Genes and Genomes pathway extracellular matrix-receptor interaction. Among the significantly regulated genes were invasion-associated matrix metalloproteinases (MMPs) MMP1, MMP13, MMP10, and MMP12. Knockout of C1r resulted in decreased production of MMP-1, MMP-13, MMP-10, and MMP-12 by cSCC cells in culture. Knockout of C1r inhibited the expression of MMP-13 by tumor cells, suppressed invasion, and reduced the amount of degraded collagen in vivo in xenografts. These results provide evidence for the role of C1r in promoting the invasion of cSCC cells by increasing MMP production.
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- 2022
4. The binding capacity of α1β1-, α2β1- and α10β1-integrins depends on non-collagenous surface macromolecules rather than the collagens in cartilage fibrils
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Peter Bruckner, Mikko Huhtala, Uwe Hansen, Johannes A. Eble, Christian Gil Girol, Jarmo Käpylä, Jyrki Heino, Birgit Leitinger, Rita Dreier, Christian Woltersdorf, Stephan Niland, Melanie Bonk, and Biotechnology and Biological Sciences Research Council (BBSRC)
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Cartilage, Articular ,0301 basic medicine ,Biochemistry & Molecular Biology ,Fibrillar Collagens ,Integrin ,Chick Embryo ,Fibril ,Chondrocyte ,Integrin alpha1beta1 ,Extracellular matrix ,03 medical and health sciences ,Chondrocytes ,0302 clinical medicine ,Mechanoreception ,Adaptor proteins ,Cell Adhesion ,medicine ,Animals ,Humans ,Cell adhesion ,Discoidin Domain Receptors ,Molecular Biology ,Cells, Cultured ,Integrin binding ,Suprastructure ,biology ,Cell adhesion molecule ,Chemistry ,Cell-matrix-interactions ,Fibrillogenesis ,06 Biological Sciences ,Cell biology ,Immobilized Proteins ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,biology.protein ,Cattle ,Integrin alpha2beta1 ,Integrin alpha Chains ,Protein Binding - Abstract
Interactions of cells with supramolecular aggregates of the extracellular matrix (ECM) are mediated, in part, by cell surface receptors of the integrin family. These are important molecular components of cell surface-suprastructures regulating cellular activities in general. A subfamily of β1-integrins with von Willebrand-factor A-like domains (I-domains) in their α-chains can bind to collagen molecules and, therefore, are considered as important cellular mechano-receptors. Here we show that chondrocytes strongly bind to cartilage collagens in the form of individual triple helical molecules but very weakly to fibrils formed by the same molecules. We also find that chondrocyte integrins α1β1-, α2β1- and α10β1-integrins and their I-domains have the same characteristics. Nevertheless we find integrin binding to mechanically generated cartilage fibril fragments, which also comprise peripheral non-collagenous material. We conclude that cell adhesion results from binding of integrin-containing adhesion suprastructures to the non-collagenous fibril periphery but not to the collagenous fibril cores. The biological importance of the well-investigated recognition of collagen molecules by integrins is unknown. Possible scenarios may include fibrillogenesis, fibril degradation and/or phagocytosis, recruitment of cells to remodeling sites, or molecular signaling across cytoplasmic membranes. In these circumstances, collagen molecules may lack a fibrillar organization. However, other processes requiring robust biomechanical functions, such as fibril organization in tissues, cell division, adhesion, or migration, do not involve direct integrin-collagen interactions.
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- 2017
5. H-Ras activation and fibroblast-induced TGF-β signaling promote laminin-332 accumulation and invasion in cutaneous squamous cell carcinoma
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Jyrki Heino, Veli-Matti Kähäri, Pekka Rappu, Elina Siljamäki, Pilvi Riihilä, and Liisa Nissinen
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0301 basic medicine ,Adult ,Male ,Skin Neoplasms ,Adolescent ,Proto-Oncogene Proteins p21(ras) ,03 medical and health sciences ,Mice ,Young Adult ,0302 clinical medicine ,Laminin ,Transforming Growth Factor beta ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Neoplasm Invasiveness ,Fibroblast ,Molecular Biology ,biology ,Chemistry ,Spheroid ,Fibroblasts ,Coculture Techniques ,Up-Regulation ,HaCaT ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Cancer cell ,biology.protein ,Cancer research ,Carcinoma, Squamous Cell ,Immunohistochemistry ,Female ,Cell Adhesion Molecules ,Immunostaining ,Neoplasm Transplantation ,Transforming growth factor ,Signal Transduction - Abstract
Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. The molecular basis of cSCC progression to invasive and metastatic disease is still incompletely understood. Here, we show that fibroblasts and transforming growth factor-β (TGF-β) signaling promote laminin-332 synthesis in cancer cells in an activated H-Ras-dependent manner, which in turn promotes cancer cell invasion. Immunohistochemical analysis of sporadic UV-induced invasive human cSCCs (n = 208) revealed prominent cSCC cell specific immunostaining for laminin-332 γ2 chain, located in the majority of cases (90%, n = 173) in the invasive edge of the tumors. To mimic the progression of cSCC we established 3D spheroid cocultures using primary skin fibroblasts and HaCaT/ras-HaCaT human keratinocytes. Our results indicate that in 3D spheroids, unlike in monolayer cultures, TGF-β upregulates laminin-332 production, but only in cells that harbour oncogenic H-Ras. Accumulation of laminin-332 was prevented by both H-Ras knock down and inhibition of TGF-β signaling by SB431542 or RAdKD-ALK5 kinase-defective adenovirus. Furthermore, fibroblasts accelerated the invasion of ras-HaCaT cells through collagen I gels in a Ras/TGF-β signaling dependent manner. In conclusion, we demonstrate the presence of laminin-332 in the invasive front of cSCC tumors and report a new Ras/TGF-β-dependent mechanism that promotes laminin-332 accumulation and cancer cell invasion.
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- 2019
6. Extracellular citrullination inhibits the function of matrix associated TGF-beta
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Hannu Larjava, Mark S. Johnson, Jarmo Käpylä, Laura Pirilä, Pekka Rappu, Vipin Ranga, Kalle Sipilä, Jyrki Heino, and Annamari Torittu
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0301 basic medicine ,Arginine ,Integrin ,Amino Acid Motifs ,CHO Cells ,Biology ,Protein Serine-Threonine Kinases ,ta3111 ,Extracellular matrix ,Arthritis, Rheumatoid ,Transforming Growth Factor beta1 ,03 medical and health sciences ,0302 clinical medicine ,Cricetulus ,Cell Line, Tumor ,Cricetinae ,Extracellular ,Animals ,Humans ,Molecular Biology ,030203 arthritis & rheumatology ,Receptor, Transforming Growth Factor-beta Type II ,ta1182 ,Citrullination ,Cell biology ,Extracellular Matrix ,Fibronectin ,030104 developmental biology ,Ectodomain ,Immunology ,biology.protein ,Protein-Arginine Deiminases ,Protein Processing, Post-Translational ,Receptors, Transforming Growth Factor beta ,Transforming growth factor ,Protein Binding ,Signal Transduction - Abstract
In inflammatory arthritis peptidyl arginine deiminase (PAD) enzymes can citrullinate arginine residues in extracellular matrix (ECM) proteins, such as collagens and fibronectin. This may lead to the generation of anti-citrullinated protein antibodies, important diagnostic markers in rheumatoid arthritis. In addition, the citrullination may directly affect protein function. Based on structural analysis, we found that most ECM-associated growth factors (GFs) have arginine residues in their receptor recognition sites. Thus, they are potential functional targets of extracellular citrullination. To examine this further, we focused on the citrullination of transforming growth factor-βs (TGF-β), well-known ECM-associated GFs. PAD-treatment of CHO-LTBP1 cell derived matrix, rich with TGF-β, decreased the level of TGF-β activity as detected by HaCaT and MLEC-PAI-1/Lu reporter cells. Additional experiments indicated that PAD-treatment inhibits the integrin-mediated TGF-β activation since PAD-treatment decreased the binding of integrin αVβ6 ectodomain as well as integrin-mediated spreading of MG-63 and HaCaT cells to β1-latency associated peptide (TGF-β1 LAP). The citrullination of the RGD site, an important integrin recognition motif, was confirmed by mass spectrometry. Furthermore, the citrullination of active TGF-β1 inhibited its binding to recombinant TGF-β receptor II, and prevented its ability to activate TGF-β signaling. Thus, extracellular PAD activity can affect the function of ECM-associated growth factors by different mechanisms. Importantly, the citrullination of both latent and active TGF-β has the potency to regulate the inflammatory process.
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- 2016
7. Keratinocyte Microvesicles Regulate the Expression of Multiple Genes in Dermal Fibroblasts
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Gethin Owen, Hannu Larjava, Anne Rokka, Jiarui Bi, Lari Häkkinen, Weimin Chen, Jyrki Heino, Leeni Koivisto, and Ping Huang
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Keratinocytes ,MAP Kinase Signaling System ,Dermatology ,Biology ,Biochemistry ,Fibroblast migration ,03 medical and health sciences ,0302 clinical medicine ,Transforming Growth Factor beta ,medicine ,Humans ,Extracellular Signal-Regulated MAP Kinases ,Molecular Biology ,Skin ,030304 developmental biology ,Tube formation ,Wound Healing ,0303 health sciences ,integumentary system ,ta1182 ,Granulation tissue ,Transforming growth factor beta ,Cell Biology ,Fibroblasts ,Microvesicles ,Cell biology ,HaCaT ,medicine.anatomical_structure ,Gene Expression Regulation ,030220 oncology & carcinogenesis ,biology.protein ,Keratinocyte ,Wound healing - Abstract
Extracellular vesicles released from cells regulate many normal and pathological conditions. Little is known about the role of epidermal keratinocyte microvesicles (KC-MVs) in epithelial–stromal interaction that is essential for wound healing. We investigated, therefore, whether MV-like structures are present in human wounds and whether they affect wound healing–associated gene expression in dermal fibroblasts. In human wounds, MV-like vesicles were observed during active epithelial migration and early granulation tissue formation. When KC-MVs derived from keratinocyte-like cells (HaCaT) were added to fibroblast cultures, expression of 21 genes was significantly regulated (P
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- 2015
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8. Proline hydroxylation in collagen supports integrin binding by two distinct mechanisms
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Pekka Rappu, Johanna Myllyharju, Jarmo Käpylä, Kalle Sipilä, Antti M. Salo, Tiina A. Salminen, Johanna Jokinen, Kati Drushinin, and Jyrki Heino
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0301 basic medicine ,collagen ,Proline ,post-translational modification (PTM) ,integrin ,Integrin ,Integrin alpha1 ,Glycobiology and Extracellular Matrices ,Crystallography, X-Ray ,Hydroxylation ,Biochemistry ,Collagen Type I ,Prolyl Hydroxylases ,Collagen receptor ,Extracellular matrix ,03 medical and health sciences ,chemistry.chemical_compound ,Hydroxyproline ,Mice ,0302 clinical medicine ,Cell Adhesion ,Animals ,hydroxyproline ,Molecular Biology ,Integrin binding ,connective tissue ,Binding Sites ,biology ,ta1182 ,cell adhesion ,Cell Biology ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,Biophysics ,Triple helix ,Protein Binding - Abstract
Collagens are the most abundant extracellular matrix proteins in vertebrates and have a characteristic triple-helix structure. Hydroxylation of proline residues is critical for helix stability, and diminished prolyl hydroxylase activity causes wide-spread defects in connective tissues. Still, the role of proline hydroxylation in the binding of collagen receptors such as integrins is unclear. Here, we isolated skin collagen from genetically modified mice having reduced prolyl 4-hydroxylase activity. At room temperature, the reduced proline hydroxylation did not affect interactions with the recombinant integrin α2I domain, but at 37 °C, collagen hydroxylation correlated with the avidity of α2I domain binding. Of note, LC–MS/MS analysis of isolated skin collagens revealed no major changes in the hydroxyproline content of the main integrin-binding sites. Thus, the disrupted α2I domain binding at physiological temperatures was most likely due to structural destabilization of the collagenous helix. Integrin α2I binding to the triple-helical GFPGER motif was slightly weaker than to GFOGER (O = hydroxyproline). This phenomenon was more prominent when α1 integrin was tested. Integrin α1β1 expressed on CHO cells and recombinant α1I domain showed remarkably slower binding velocity and weaker avidity to GFPGER when compared with GFOGER. Structural modeling revealed the critical interaction between Arg-218 in α1I and the hydroxyproline residue in the integrin-binding motif. The role of Arg-218 was further validated by testing a variant R218D α1I domain in solid-phase binding assays. Thus, our results show that the lack of proline hydroxylation in collagen can affect integrin binding by a direct mechanism and via structural destabilization of the triple helix.
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- 2018
9. Platelet response to a small molecule inhibitor of α2β1 integrin is associated with ITGA2 C807T dimorphism
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Jonna Nieminen, Anne Marjamäki, Liisa Nissinen, Pauli Ollikka, Jyrki Heino, and Pekka Rappu
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Adult ,Blood Platelets ,Male ,0301 basic medicine ,Genotype ,Pharmacogenomic Variants ,Integrin ,Plasma protein binding ,Biology ,Polymorphism, Single Nucleotide ,Amino Acid Chloromethyl Ketones ,Collagen receptor ,Mice ,Young Adult ,03 medical and health sciences ,Animals ,Humans ,ta318 ,Platelet ,Allele ,Alleles ,ta1182 ,Hematology ,General Medicine ,Middle Aged ,Molecular biology ,Small molecule ,030104 developmental biology ,biology.protein ,Female ,Collagen ,Integrin alpha2beta1 ,Protein Binding - Abstract
High expression of the collagen receptor, α2β1 integrin, on platelets of ITGA2 807T-allele carriers has been identified as a risk factor for thromboembolic conditions, and α2β1 inhibitors are considered to be potential therapeutic agents. In 59 genotyped individuals, we measured α2 expression levels on platelets and analyzed platelet adhesion to collagen under flow conditions. A sulfonamide-type small-molecule inhibitor of α2β1 integrin decreased average platelet adhesion in individuals with the C/T807T genotype but not in those harboring C807C. Thus, genotype can be used to select a human subpopulation that has the highest probability of showing a positive response to α2β1 inhibitors.
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- 2015
10. 447 Complement component C1r upregulates the expression of matrix metalloproteinase-1, -13 and -10 and promotes invasion of cutaneous squamous carcinoma cells
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Marjaana Ojalill, Liisa Nissinen, Seppo Meri, Veli-Matti Kähäri, Kristina Viiklepp, Pilvi Riihilä, and Jyrki Heino
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COMPLEMENT COMPONENT C1r ,Chemistry ,Cancer research ,Cell Biology ,Dermatology ,Matrix metalloproteinase ,Molecular Biology ,Biochemistry ,Squamous carcinoma - Published
- 2019
11. Leukocyte integrins αLβ2, αMβ2 and αXβ2 as collagen receptors—Receptor activation and recognition of GFOGER motif
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Matti Lahti, Jarmo Käpylä, and Jyrki Heino
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Receptors, Collagen ,Amino Acid Motifs ,Integrin ,Integrin alphaXbeta2 ,Macrophage-1 Antigen ,HL-60 Cells ,Complement receptor ,Biochemistry ,Collagen receptor ,Extracellular matrix ,Leukemia, Promyelocytic, Acute ,Cell surface receptor ,Cell Line, Tumor ,Cell Adhesion ,Humans ,Binding site ,Antibodies, Blocking ,Cell adhesion ,Binding Sites ,biology ,Cell adhesion molecule ,Chemistry ,Cell Biology ,Molecular biology ,Lymphocyte Function-Associated Antigen-1 ,Protein Structure, Tertiary ,biology.protein ,Collagen ,Protein Binding - Abstract
Integrins α L β 2 , α M β 2 and α X β 2 are expressed on leukocytes. Their primary ligands are counter transmembrane receptors or plasma proteins, such as intercellular cell adhesion molecule-1 (ICAM-1) or components of complement system (iC3b, iC4b), respectively. Function blocking antibodies for these integrins may also reduce cell adhesion to collagens. To make the first systematical comparison of human α L β 2 , α M β 2 and α X β 2 as collagen receptors, we produced the corresponding integrin αI domains both in wild-type and activated form and measured their binding to collagens I–VI. In the “closed” (wild-type) conformation, the α L I and α M I domains bound with low avidity to their primary ligands, and the interaction with collagens was also very weak. Gain-of-function mutations α L I306G, α L K287C/K294C and α M I316G are considered to mimic “open”, activated αI domains. The binding of these activated αI domains to the primary ligands was clearly stronger and they also recognized collagens with moderate avidity ( K d L I domain favored collagen I ( K d ≈ 80 nM) when compared to collagen IV. The integrin α X I domain acted in a very different manner since already in native, wild-type form it bound to collagen IV and iC3b ( K d ≈ 200–400 nM). Antibodies against α X β 2 and α M β 2 blocked promyelocytic leukemia cell adhesion to the collagenous GFOGER motif, a binding site for the β 1 integrin containing collagen receptors. In brief, leukocyte β 2 integrins may act as collagen receptors in a heterodimer specific manner.
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- 2013
12. BioImageXD: an open, general-purpose and high-throughput image-processing platform
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Silja Tiitta, Varpu Marjomäki, Daniel J. White, Mikko Karjalainen, Jyrki Heino, Pasi Kankaanpää, Joacim Päivärinne, Lassi Paavolainen, and Jonna Nieminen
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ta113 ,SIMPLE (military communications protocol) ,Computer science ,business.industry ,ta1182 ,Computational Biology ,Image processing ,Cell Biology ,Bioinformatics ,Biochemistry ,Visualization ,High-Throughput Screening Assays ,User-Computer Interface ,Software ,Workflow ,Imaging, Three-Dimensional ,Human–computer interaction ,business ,Cluster analysis ,Molecular Biology ,Throughput (business) ,Algorithms ,Biotechnology ,Graphical user interface - Abstract
BioImageXD puts open-source computer science tools for three-dimensional visualization and analysis into the hands of all researchers, through a user-friendly graphical interface tuned to the needs of biologists. BioImageXD has no restrictive licenses or undisclosed algorithms and enables publication of precise, reproducible and modifiable workflows. It allows simple construction of processing pipelines and should enable biologists to perform challenging analyses of complex processes. We demonstrate its performance in a study of integrin clustering in response to selected inhibitors.
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- 2012
13. Structure of Collagen Receptor Integrin α1I Domain Carrying the Activating Mutation E317A
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Anna-Maria Brandt, Henri Niskanen, Vimal Parkash, Jyrki Heino, Jarmo Käpylä, Tiina A. Salminen, Matti Lahti, Eva Bligt, Pekka Patrikainen, and Johanna Jokinen
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Receptors, Collagen ,Integrin alpha1 ,Integrin ,CHO Cells ,Plasma protein binding ,Crystallography, X-Ray ,Biochemistry ,Collagen Type I ,Protein Structure, Secondary ,Integrin alpha1beta1 ,Collagen receptor ,Protein structure ,Cricetinae ,Cell Adhesion ,Animals ,Humans ,skin and connective tissue diseases ,Cell adhesion ,Molecular Biology ,biology ,Chemistry ,Cell Biology ,Adhesion ,Molecular biology ,Rats ,Mutation ,Protein Structure and Folding ,Helix ,Biophysics ,biology.protein ,Integrin, beta 6 ,sense organs ,Collagen ,Protein Binding - Abstract
We have analyzed the structure and function of the integrin α(1)I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α(1)I C139S/E317A was a higher avidity collagen binder than α(1)I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α(1)I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α(2)I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α(2)I structure, has not changed its position in the activated α(1)I variant. During the integrin activation, Glu(335) on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the β(1) subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu(335). This indicates that the stabilization of helix 7 into its downward position is not required if the α(1) MIDAS is already open. To conclude, the activated α(1)I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg(287)-Glu(317) ion pair has just broken during the integrin activation.
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- 2011
14. Molecular mechanism of α2β1 integrin interaction with human echovirus 1
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Timo Hyypiä, Varpu Marjomäki, Daniel J. White, Jarmo Käpylä, Mikko Huhtala, Jyrki Heino, Kalle Sipilä, Liisa Nissinen, Johanna Jokinen, J. Santeri Puranen, Mark S. Johnson, Pasi Kankaanpää, and Maria Salmela
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Models, Molecular ,Protein Conformation ,media_common.quotation_subject ,Integrin ,CHO Cells ,In Vitro Techniques ,Biology ,p38 Mitogen-Activated Protein Kinases ,CD49c ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Collagen receptor ,Cricetulus ,Cricetinae ,Chlorocebus aethiops ,Animals ,Humans ,Binding site ,Internalization ,Molecular Biology ,media_common ,Binding Sites ,General Immunology and Microbiology ,General Neuroscience ,Recombinant Proteins ,Enterovirus B, Human ,Protein Structure, Tertiary ,Cell biology ,Amino Acid Substitution ,Integrin alpha M ,Biochemistry ,Mutagenesis, Site-Directed ,biology.protein ,Receptors, Virus ,Integrin, beta 6 ,Integrin alpha2beta1 ,Signal transduction ,Signal Transduction - Abstract
Conformational activation increases the affinity of integrins to their ligands. On ligand binding, further changes in integrin conformation elicit cellular signalling. Unlike any of the natural ligands of alpha2beta1 integrin, human echovirus 1 (EV1) seemed to bind more avidly a 'closed' than an activated 'open' form of the alpha2I domain. Furthermore, a mutation E336A in the alpha2 subunit, which inactivated alpha2beta1 as a collagen receptor, enhanced alpha2beta1 binding to EV1. Thus, EV1 seems to recognize an inactive integrin, and not even the virus binding could trigger the conformational activation of alpha2beta1. This was supported by the fact that the integrin clustering by EV1 did not activate the p38 MAP kinase pathway, a signalling pathway that was shown to be dependent on E336-related conformational changes in alpha2beta1. Furthermore, the mutation E336A did neither prevent EV1 induced and alpha2beta1 mediated protein kinase C activation nor EV1 internalization. Thus, in its entry strategy EV1 seems to rely on the activation of signalling pathways that are dependent on alpha2beta1 clustering, but do not require the conformational regulation of the receptor.
- Published
- 2009
15. Effects of conformational activation of integrin α1I and α2I domains on selective recognition of laminin and collagen subtypes
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Anna Domogatskaya, Matti Lahti, Tiina A. Salminen, Mark S. Johnson, Karl Tryggvason, Jyrki Heino, Mira Tulla, Jarmo Käpylä, J. Santeri Puranen, and Anna-Maria Brandt
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Models, Molecular ,biology ,EGF-like domain ,Integrin alpha1 ,Integrin ,Integrin alpha2 ,Protein Data Bank (RCSB PDB) ,Alpha (ethology) ,Cell Biology ,Plasma protein binding ,Arginine ,Molecular biology ,Protein Structure, Tertiary ,Collagen receptor ,Cell biology ,Protein structure ,Laminin ,Mutation ,biology.protein ,Humans ,Protein Isoforms ,Collagen ,Protein Binding - Abstract
Collagen receptor integrins alpha 1 beta 1 and alpha 2 beta 1 can selectively recognize different collagen subtypes. Here we show that their alpha I domains can discriminate between laminin isoforms as well: alpha 1I and alpha 2I recognized laminin-111, -211 and -511, whereas their binding to laminin-411 was negligible. Residue Arg-218 in alpha1 was found to be instrumental in high-avidity binding. The gain-of-function mutation E318W makes the alpha 2I domain to adopt the "open" high-affinity conformation, while the wild-type alpha 2I domain favors the "closed" low-affinity conformation. The E318W mutation markedly increased alpha 2I domain binding to the laminins (-111, -211 and -511), leading us to propose that the activation state of the alpha 2 beta 1 integrin defines its role as a laminin receptor. However, neither wild-type nor alpha 2IE318W domain could bind to laminin-411. alpha 2IE318W also bound tighter to all collagens than alpha 2I wild-type, but it showed reduced ability to discriminate between collagens I, IV and IX. The corresponding mutation, E317A, in the alpha 1I domain transformed the domain into a high-avidity binder of collagens I and IV. Thus, our results indicate that conformational activation of integrin alpha 1I and alpha 2I domains leads to high-avidity binding to otherwise disfavored collagen subtypes.
- Published
- 2008
16. Leukocyte Integrins α L β 2 , α M β 2 and α X β 2 as Collagen Receptors ‐ Receptor Activation and Recognition of GFOGER Motif
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Matti Lahti, Jyrki Heino, and Jarmo Käpylä
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biology ,Chemistry ,Integrin ,Biochemistry ,Collagen receptor ,Complement system ,Cell biology ,Cell surface receptor ,Genetics ,biology.protein ,iC3b ,Avidity ,Cell adhesion ,Molecular Biology ,Intracellular ,Biotechnology - Abstract
Integrins αLβ2, αMβ2 and αXβ2 are expressed on leukocytes. Their primary ligands are counter transmembrane receptors or plasma proteins, such as intercellular cell adhesion molecule-1 (ICAM-1) or components of complement system (iC3b, iC4b), respectively. Function blocking antibodies for these integrins may also reduce cell adhesion to collagens. To make the first systematical comparison of human αLβ2, αMβ2 and αXβ2 as collagen receptors, we produced the corresponding integrin αI domains both in wild-type and activated form and measured their binding to collagens I-VI. In the "closed" (wild-type) conformation, the αLI and αMI domains bound with low avidity to their primary ligands, and the interaction with collagens was also very weak. Gain-of-function mutations αL I306G, αL K287C/K294C and αM I316G are considered to mimic "open", activated αI domains. The binding of these activated αI domains to the primary ligands was clearly stronger and they also recognized collagens with moderate avidity (Kd
- Published
- 2015
17. Histidine-rich glycoprotein blocks collagen-binding integrins and adhesion of endothelial cells through low-affinity interaction with α2 integrin
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Satoshi Honjo, Kalle Sipilä, Jyrki Heino, Sonia Tugues, Staffan Johansson, Francis P. Roche, and Lena Claesson-Welsh
- Subjects
Vascular Endothelial Growth Factor A ,Histidine-rich glycoprotein ,Integrin ,Gene Expression ,Plasma protein binding ,Cell Line ,Extracellular matrix ,Myoblasts ,chemistry.chemical_compound ,Mice ,Cell Adhesion ,Human Umbilical Vein Endothelial Cells ,Animals ,Humans ,Binding site ,Cell adhesion ,Molecular Biology ,biology ,Chemotaxis ,ta1182 ,Proteins ,Heparan sulfate ,Molecular biology ,Recombinant Proteins ,Protein Structure, Tertiary ,Mice, Inbred C57BL ,Protein Subunits ,chemistry ,Ectodomain ,biology.protein ,Female ,Heparitin Sulfate ,Integrin alpha2beta1 ,Protein Binding - Abstract
The plasma protein histidine-rich glycoprotein (HRG) affects the morphology and function of both endothelial cells (ECs) and monocytes/macrophages in cancer. Here, we examined the mechanism of action of HRG's effect on ECs. HRG suppressed adhesion, spreading and migration of ECs specifically on collagen I (COL I) whereas ECs seeded on other extracellular matrix proteins were insensitive to HRG. HRG did not bind specifically to COL I or to the α-integrin binding site on collagen, GFOGER. Furthermore, HRG's inhibition of EC adhesion was not dependent upon heparan sulfate (HS) moieties as heparitinase-treated ECs remained sensitive to HRG. C2C12 cells expressing α2 integrin, the major collagen-binding α-integrin subunit in ECs, showed increased binding of HRG compared with wild type C2C12 cells lacking the α2 subunit. Recombinant α2 I-domain protein bound HRG and to a higher extent when in active conformation. However, the α2 I-domain bound weakly to HRG compared with COL I and the purified α2β1 ectodomain complex failed to retain HRG. We conclude that HRG binds to α2 integrin through low-affinity interactions in a HS-independent manner, thereby blocking EC-adhesion to COL I.
- Published
- 2015
18. An arthritogenic alphavirus uses the α1β1 integrin collagen receptor
- Author
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Andreas Suhrbier, Janet M. Davies, Robert W. Slade, Johannes A. Eble, Jyrki Heino, May La Linn, and Christoph Lübken
- Subjects
α1β1 integrin ,Collagen Type IV ,Integrin alpha1 ,Integrin ,Alphavirus ,Biology ,Virus Replication ,Antibodies ,Virus ,Integrin alpha1beta1 ,Collagen receptor ,Mice ,Ross River virus ,Virology ,Animals ,Humans ,Mice, Knockout ,Collagen IV ,Virus receptor ,Fibroblasts ,biology.organism_classification ,Molecular biology ,Solubility ,Integrin alpha M ,biology.protein ,Receptors, Virus ,Integrin, beta 6 ,Receptors, Adrenergic, beta-1 ,Receptor ,HeLa Cells - Abstract
Ross River (RR) virus is an alphavirus endemic to Australia and New Guinea and is the aetiological agent of epidemic polyarthritis or RR virus disease. Here we provide evidence that RR virus uses the collagen-binding alpha1beta1 integrin as a cellular receptor. Infection could be inhibited by collagen IV and antibodies specific for the beta1 and alpha1 integrin proteins, and fibroblasts from alpha1-integrin-/- mice were less efficiently infected than wild-type fibroblasts. Soluble alpha1beta1 integrin bound immobilized RR virus, and peptides representing the alpha1beta1 integrin binding-site on collagen IV inhibited virus binding to cells. We speculate that two highly conserved regions within the cell-receptor binding domain of E2 mimic collagen and provide access to cellular collagen-binding receptors.
- Published
- 2005
19. The Fibril-associated Collagen IX Provides a Novel Mechanism for Cell Adhesion to Cartilaginous Matrix
- Author
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Juha Jäälinoja, Varpu Marjomäki, David E. Birk, Liisa Nissinen, Jyrki Heino, Mira Tulla, Leena Ala-Kokko, Petri Nykvist, Tiina Viitasalo, Joni Ylostalo, Richard W. Farndale, Jarmo Käpylä, Anna-Marja Säämänen, and Piia Vehviläinen
- Subjects
Integrin alpha1 ,Integrin alpha2 ,Ligands ,Polymerase Chain Reaction ,Biochemistry ,Collagen receptor ,Mice ,Cricetinae ,Receptor ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Chemistry ,Chinese hamster ovary cell ,Recombinant Proteins ,Cell biology ,Collagen ,Integrin alpha Chains ,Protein Binding ,Molecular Sequence Data ,Integrin ,Chondrosarcoma ,CHO Cells ,Fibril ,Collagen Type IX ,Cell Line ,Chondrocytes ,Microscopy, Electron, Transmission ,Cell Line, Tumor ,Cell Adhesion ,Escherichia coli ,Animals ,Humans ,Immunoprecipitation ,Amino Acid Sequence ,RNA, Messenger ,Binding site ,Cell adhesion ,Molecular Biology ,Binding Sites ,Sequence Homology, Amino Acid ,Cell Biology ,Protein Structure, Tertiary ,Rats ,Microscopy, Electron ,Collagen, type I, alpha 1 ,Cartilage ,Mutation ,Mutagenesis, Site-Directed ,biology.protein ,RNA ,Peptides - Abstract
Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express alpha1beta1, alpha2beta1, or alpha10beta1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant alphaI domains in Escherichia coli. For each of the four alphaI domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both alpha1I- and alpha2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by alpha2I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the alpha2I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix.
- Published
- 2004
20. Bacterial heat shock protein 60 may increase epithelial cell migration through activation of MAP kinases and inhibition of α6β4 integrin expression
- Author
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Leeni Koivisto, Veli-Jukka Uitto, Jyrki Heino, and Liangxuan Zhang
- Subjects
Keratinocytes ,MAPK/ERK pathway ,animal structures ,Pyridines ,SB 203580 ,p38 mitogen-activated protein kinases ,Integrin ,Biophysics ,Biology ,Epithelial cell migration ,Biochemistry ,Cell Line ,Collagen receptor ,chemistry.chemical_compound ,Bacterial Proteins ,Cell Movement ,Heat shock protein ,Cell Adhesion ,Animals ,Humans ,Enzyme Inhibitors ,Molecular Biology ,Flavonoids ,Integrin alpha6beta4 ,Dose-Response Relationship, Drug ,fungi ,Imidazoles ,Epithelial Cells ,Chaperonin 60 ,Cell Biology ,Molecular biology ,Cell biology ,Enzyme Activation ,ErbB Receptors ,Protein Subunits ,Matrix Metalloproteinase 9 ,chemistry ,Mitogen-activated protein kinase ,biology.protein ,Matrix Metalloproteinase 2 ,Mitogen-Activated Protein Kinases - Abstract
Exogenous heat shock proteins may modify cell behavior of infected epithelium. The effect of heat shock protein 60 (hsp60) of Actinobacillus actinomycetemcomitans and Escherichia coli, and human recombinant hsp60 on migration of HaCaT skin keratinocytes was studied using the Boyden chamber assay. Hsp60 from different species increased cell migration by two- to fivefold and this effect was inhibited by ERK inhibitor PD 98059, p38 inhibitor SB 203580, and a function-blocking epidermal growth factor receptor (EGFR) antibody. Hsp60 reduced the expression of alpha6-integrin mRNA and its protein levels on the cell surface but had no effect on the expression of beta4, beta1, alpha1, alpha5 or alphav integrin subunits. Hsp60 also significantly inhibited cell adhesion to laminin-5, a ligand of alpha6beta4 integrin. These results suggest that exogenous hsp60 released from bacteria or inflammatory cells may promote epithelial cell migration through activation of EGFR and MAP kinases, and inhibition of alpha6beta4 integrin expression.
- Published
- 2004
21. Jararhagin-derived RKKH Peptides Induce Structural Changes in α1I Domain of Human Integrin α1β1
- Author
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Yvonne Nymalm, Jarmo Käpylä, J. Santeri Puranen, Thomas K.M. Nyholm, Jyrki Heino, Heidi Kidron, Tomi T. Airenne, J. Peter Slotte, Tiina A. Salminen, Mark S. Johnson, and Olli T. Pentikäinen
- Subjects
Models, Molecular ,Protein Conformation ,Stereochemistry ,Integrin ,Alpha (ethology) ,Peptide ,Crystallography, X-Ray ,Binding, Competitive ,Biochemistry ,Collagen Type I ,Protein Structure, Secondary ,Integrin alpha1beta1 ,Protein structure ,Crotalid Venoms ,Humans ,Magnesium ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Peptide sequence ,Fluorescent Dyes ,chemistry.chemical_classification ,Binding Sites ,Calorimetry, Differential Scanning ,Molecular Structure ,biology ,Metalloendopeptidases ,Cell Biology ,Peptide Fragments ,Recombinant Proteins ,Spectrometry, Fluorescence ,chemistry ,Jararhagin ,Helix ,biology.protein ,Crystallization - Abstract
Integrin alpha(1)beta(1) is one of four collagen-binding integrins in humans. Collagens bind to the alphaI domain and in the case of alpha(2)I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In alpha(2)I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound "open" conformation of alpha(2)I differs significantly from the "closed" conformation seen in the structure of apo-alpha(2)I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to alpha(1)I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of alpha(1)I with Mg(2+) and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-alpha(1)I ("closed" conformation) and a model structure of the alpha(1)I ("open" conformation) based on the closely related structure of alpha(2)I reveals that the binding site is partially blocked to ligands by Glu(255) and Tyr(285) in the "closed" structure, whereas in the "open" structure helix C is unwound and these residues are shifted, and the "RKKH" peptides fit well when docked. The "open" conformation of alpha(2)I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of alpha(1)I and shown experimentally for alpha(1)I using a fluorescent hydrophobic probe.
- Published
- 2004
22. The Selective Regulation of αVβ1 Integrin Expression Is Based on the Hierarchical Formation of αV-containing Heterodimers
- Author
-
Pekka Koistinen and Jyrki Heino
- Subjects
Integrins ,Protein subunit ,Cell ,Integrin ,Biology ,Models, Biological ,Biochemistry ,Antigens, CD ,Complementary DNA ,Tumor Cells, Cultured ,medicine ,Humans ,Receptors, Vitronectin ,Melanoma ,Molecular Biology ,Cell Membrane ,Cell Biology ,Transfection ,Integrin alphaV ,Fibronectins ,Cell biology ,Gene Expression Regulation, Neoplastic ,Fibronectin ,medicine.anatomical_structure ,biology.protein ,Vitronectin ,Collagen ,Dimerization ,Intracellular ,Protein Binding - Abstract
The integrin beta1 subunit can form a heterodimer with 12 different alpha subunits. According to the present model, the expression level of any alphabeta complex is regulated by the availability of the specific alpha subunit, whereas beta1 subunit is constantly present in a large excess. The expression of several heterodimers containing the alphaV subunit seems to be regulated by an identical mechanism. The fact that many cells express alphaVbeta1 heterodimer, and that this fibronectin/vitronectin receptor may be selectively regulated, compromises the present model of the regulation of beta1 and alphaV integrins. We have tried to solve this problem by assuming that distinct alphabeta heterodimers are formed with different tendency. To test the hypothesis, we analyzed WM-266-4 melanoma cells transfected with a cDNA construct coding for an intracellular single-chain anti-alphaV integrin antibody. We could see 70-80% reduction in the cell surface expression of alphaV subunit. However, the only one of the alphaV integrins reduced on the cell surface was alphaVbeta1. This suggests that the cell surface expression level of alphaVbeta1 is dependent on the number of alphaV subunits available after the formation of other alphaV-containing heterodimers. Thus, there seems to be a hierarchy in the complex formation between alphaV and its different beta-partners. These observations explain how alphaVbeta1 can be specifically regulated without concomitant changes in the expression of other alphaV or beta1 integrins.
- Published
- 2002
23. Internalization of Echovirus 1 in Caveolae
- Author
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Timo Hyypiä, Hilkka Reunanen, Varpu Marjomäki, Liisa Nissinen, Vilja Pietiäinen, Johanna Ivaska, Heli Matilainen, Jyrki Heino, Paula Upla, and Pasi Huttunen
- Subjects
Integrins ,Receptors, Collagen ,Echovirus ,media_common.quotation_subject ,Caveolin 1 ,Immunology ,Integrin ,Caveolae ,medicine.disease_cause ,Caveolins ,Microbiology ,Clathrin ,03 medical and health sciences ,Capsid ,Virology ,Caveolin ,Enterovirus Infections ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Internalization ,030304 developmental biology ,media_common ,0303 health sciences ,Microscopy, Confocal ,biology ,030302 biochemistry & molecular biology ,Molecular biology ,Enterovirus B, Human ,Virus-Cell Interactions ,Cell biology ,Microscopy, Electron ,Viral replication ,Insect Science ,biology.protein ,Rabbits ,beta 2-Microglobulin - Abstract
Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor α2β1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, α2β1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.
- Published
- 2002
24. Collagen XXII binds to collagen-binding integrins via the novel motifs GLQGER and GFKGER
- Author
-
Markus Meier, Guido Veit, Donald Gullberg, Jörg Stetefeld, Manuel Koch, Florence Ruggiero, Johannes A. Eble, Daniela Zwolanek, and Jyrki Heino
- Subjects
Integrins ,Amino Acid Motifs ,Integrin ,CHO Cells ,Biochemistry ,Protein Structure, Secondary ,Collagen receptor ,Fibril-Associated Collagens ,Cricetulus ,Protein structure ,Cell Line, Tumor ,Animals ,Humans ,Receptor ,Molecular Biology ,biology ,Chemistry ,Chinese hamster ovary cell ,ta1182 ,Cell Biology ,biology.organism_classification ,Protein Structure, Tertiary ,Cell biology ,Mice, Inbred C57BL ,Cell culture ,Immunology ,biology.protein ,Protein Binding ,Triple helix - Abstract
Collagen XXII, a FACIT (fibril-associated collagen with interrupted triple helices), is expressed at the myotendinous junction and the articular surface of joint cartilage. Cellular receptors like collagen-binding integrins are known to bind collagens with distinct binding motifs following the sequence GXOGER. In the present study, we demonstrate the sequences GLQGER and GFKGER as novel binding motifs between collagen XXII and collagen-binding integrins, especially α2β1 integrin. Solid-phase assays and surface plasmon resonance spectroscopy revealed a direct interaction between α2β1 integrin and the motif GFKGER. In addition, immunohistochemical analysis demonstrated partial co-localization of collagen XXII, α2β1 integrin and α11β1 integrin at the myotendinous junction. Furthermore, computational modelling of the motifs GLQGER and GFKGER showed perfect fitting of the sequences into the binding pocket of collagen-binding integrins. Taken together, we demonstrated that collagen XXII interacts with collagen-binding integrins via the new motifs GLQGER and GFKGER.
- Published
- 2014
25. Selective Binding of Collagen Subtypes by Integrin α1I, α2I, and α10I Domains
- Author
-
Liisa Nissinen, Olli T. Pentikäinen, Mira Tulla, Ulla Impola, Mark S. Johnson, Tiina Viitasalo, Jarmo Käpylä, Jyrki Heino, and Petri Nykvist
- Subjects
Type IV collagen ,Integrin alpha2 ,Integrin alpha Chains ,Alpha (ethology) ,Cell Biology ,Biology ,Molecular Biology ,Biochemistry ,Molecular biology ,Type I collagen ,Binding domain ,Collagen receptor ,G alpha subunit - Abstract
Four integrins, namely alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1), form a special subclass of cell adhesion receptors. They are all collagen receptors, and they recognize their ligands with an inserted domain (I domain) in their alpha subunit. We have produced the human integrin alpha(10)I domain as a recombinant protein to reveal its ligand binding specificity. In general, alpha(10)I did recognize collagen types I-VI and laminin-1 in a Mg(2+)-dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When alpha(10)I was tested together with the alpha(1)I and alpha(2)I domains, all three I domains seemed to have their own collagen binding preferences. The integrin alpha(2)I domain bound much better to fibrillar collagens (I-III) than to basement membrane type IV collagen or to beaded filament-forming type VI collagen. Integrin alpha(1)I had the opposite binding pattern. The integrin alpha(10)I domain was similar to the alpha(1)I domain in that it bound very well to collagen types IV and VI. Based on the previously published atomic structures of the alpha(1)I and alpha(2)I domains, we modeled the structure of the alpha(10)I domain. The comparison of the three I domains revealed similarities and differences that could potentially explain their functional differences. Mutations were introduced into the alphaI domains, and their binding to types I, IV, and VI collagen was tested. In the alpha(2)I domain, Asp-219 is one of the amino acids previously suggested to interact directly with type I collagen. The corresponding amino acid in both the alpha(1)I and alpha(10)I domains is oppositely charged (Arg-218). The mutation D219R in the alpha(2)I domain changed the ligand binding pattern to resemble that of the alpha(1)I and alpha(10)I domains and, vice versa, the R218D mutation in the alpha(1)I and alpha(10)I domains created an alpha(2)I domain-like ligand binding pattern. Thus, all three collagen receptors appear to differ in their ability to recognize distinct collagen subtypes. The relatively small structural differences on their collagen binding surfaces may explain the functional specifics.
- Published
- 2001
26. Bacterial Heat Shock Protein-60 Increases Epithelial Cell Proliferation through the ERK1/2 MAP Kinases
- Author
-
Veli-Jukka Uitto, Steven L. Pelech, Lianxuan Zhang, Daniel Grenier, Jyrki Heino, and Denis Mayrand
- Subjects
Keratinocytes ,MAPK/ERK pathway ,Proto-Oncogene Proteins c-jun ,p38 mitogen-activated protein kinases ,Ribosomal Protein S6 Kinases, 90-kDa ,p38 Mitogen-Activated Protein Kinases ,Heat shock protein ,Humans ,Enzyme Inhibitors ,Phosphorylation ,Cyclic AMP Response Element-Binding Protein ,Protein kinase A ,Cell Line, Transformed ,Mitogen-Activated Protein Kinase 1 ,Mitogen-Activated Protein Kinase 3 ,biology ,Cell growth ,Kinase ,Epithelial Cells ,Actinobacillus ,Bacterial Infections ,Chaperonin 60 ,Cell Biology ,MAP Kinase Kinase Kinases ,Molecular biology ,Cell biology ,Mitogen-activated protein kinase ,biology.protein ,Mitogen-Activated Protein Kinases ,Proto-Oncogene Proteins c-fos ,Cell Division - Abstract
Heat shock proteins (hsp) have important roles in the regulation and protection of both prokaryotic and eukaryotic cells, especially during environmental stress. Hsps are also important bacterial virulence factors. We investigated whether bacterial hsp60 can alter epithelial cell mitogen-activated protein kinase (MAPK) signaling and cell proliferation. Human skin keratinocytes (HaCaT cell line) were cultured in the presence of hsp60 purified from Actinobacillus actinomycetemcomitans, an important oral pathogen. Protein kinases in the ERK1/2 and p38 MAPK signaling pathways were probed with kinase-specific and phosphorylation-site-specific antibodies on Western blots. In quiescent cultures, hsp60 increased ERK1/2 phosphorylation in a sustained manner and p38 phosphorylation transiently. Hsp60 also increased epithelial cell proliferation by about 30%. Inhibition of the ERK1/2 pathway by PD 98059 (a MEK1 inhibitor) reversed partially ERK1/2 phosphorylation and totally cell proliferation indicating that the ERK1/2 MAPK pathway is involved in the hsp60-induced cell growth. This was supported by findings that hsp60 stimulated phosphorylation of RSK1/2 and cyclic AMP response element-binding protein and increased expression of transcription factors c-Jun and c-Fos. Recombinant human hsp60 did not alter ERK1/2 or p38 phosphorylation and had no effect on epithelial cell proliferation. Inhibition of p38 MAPK pathway by SB 203580 increased both ERK1/2 phosphorylation and cell proliferation demonstrating that the inhibitor can either directly or indirectly activate the ERK1/2 MAPK pathway. The results show that exogenous bacterial hsp60 is able to activate ERK1/2 phosphorylation and thereby cause increased epithelial proliferation. In case of mucosal infection this effect may either lead to increased wound repair or participate in the pathological mechanism of some bacterial diseases that involve increased epithelial proliferation.
- Published
- 2001
27. Three-Dimensional Collagen Regulates Collagen Gene Expression by a Mechanism That Requires Serine/Threonine Kinases and Is Independent of Mechanical Contraction
- Author
-
L. Nissinen, M. Potila, Jyrki Heino, and A. Broberg
- Subjects
Cell signaling ,Transcription, Genetic ,Integrin ,Integrin alpha2 ,Biophysics ,Bone Neoplasms ,Protein Serine-Threonine Kinases ,Biology ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Feedback ,Collagen receptor ,Antigens, CD ,1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ,Gene expression ,Cell Adhesion ,Tumor Cells, Cultured ,Humans ,Molecular Biology ,Regulation of gene expression ,Osteosarcoma ,Tissue Inhibitor of Metalloproteinase-2 ,Kinase ,Cell Biology ,Cell biology ,Kinetics ,Collagen, type I, alpha 1 ,biology.protein ,Collagen ,Matrix Metalloproteinase 1 ,Signal transduction ,Signal Transduction - Abstract
Integrin alpha1beta1, one of the cellular collagen receptors, can participate in the regulation of collagen accumulation by acting as a negative feedback regulator. The molecular mechanism behind this phenomenon has been unknown. We have plated cells inside three-dimensional collagen and analyzed a set of chemical inhibitors for various signal transduction pathways. Only two wide-spectrum serine/threonine kinase inhibitors, H-7 and iso-H-7 could prevent the down-regulation of alpha1(I) collagen mRNA levels in cells exposed to three-dimensional collagen. In monolayer iso-H-7 slightly down-regulated collagen gene expression, indicating that inside collagen it affected integrin signaling rather than having a direct stimulatory effect on collagen mRNA levels. The effect of iso-H-7 was not dependent on its ability to inhibit protein kinases A, C, or G. H-7 and iso-H-7 could also inhibit collagen gel contraction, but this mechanism was independent of collagen gene regulation. Three-dimensional collagen could also up-regulate the mRNA levels of several matrix metalloproteinases (MMPs) but H-7 and iso-H-7 had no effect on the regulation of MMP genes. Our data indicate that three-dimensional collagenous matrix regulates distinct cellular signaling pathways and that collagen gene regulation is independent of the other effects of the matrix.
- Published
- 2001
28. Distinct Recognition of Collagen Subtypes by α1β1 and α2β1Integrins
- Author
-
Taina Pihlajaniemi, Jyrki Heino, Johanna Ivaska, Petri Nykvist, Jarmo Käpylä, and Hongmin Tu
- Subjects
Chinese hamster ovary cell ,Integrin ,Type II collagen ,Cell Biology ,Transfection ,Biology ,Biochemistry ,Molecular biology ,Collagen receptor ,Collagen, type I, alpha 1 ,biology.protein ,Cell adhesion ,Receptor ,Molecular Biology - Abstract
Two integrin-type collagen receptors, α1β1 and α2β1, are structurally very similar. However, cells can concomitantly express the both receptors and they might have independent functions. Here, Chinese hamster ovary (CHO) cells, which lack endogenous collagen receptors, were transfected with either α1 or α2 integrin cDNA. Cells were allowed to adhere to various collagen types and their integrin function was tested by observing the progression of cell spreading. The cells expressing α1β1 integrin could spread on collagen types I, III, IV, and V but not on type II, while α2β1 integrin could mediate cell spreading on collagen types I-V. Type XIII is a transmembrane collagen and its interaction with the integrins has not been previously studied. CHO-α1β1 cells could spread on human recombinant type XIII collagen, unlike CHO-α2β1 cells. Integrins α1β1 and α2β1recognize collagens with the specific αI domains. The α1I and α2I domains were produced as recombinant proteins, labeled with europium and used in a sensitive solid-phase binding assay based on time-resolved fluorescence. α1I domain, unlike the α2I domain, could attach to type XIII collagen. The results indicate, that α1β1 and α2β1have different ligand binding specificity. Distinct recognition of different collagen subtypes by the αI domains can partially explain the differences seen in cell spreading. However, despite the fact that CHO-α1β1 cells could not spread on type II collagen α1I domain could bind to this collagen type. Thus, the cell spreading on collagens may also be regulated by factors other than the integrins.
- Published
- 2000
29. Integrin and dystrophin associated adhesion protein complexes during regeneration of shearing-type muscle injury
- Author
-
Teppo L. N. Järvinen, Liisa Nissinen, Hannu Kalimo, Markku Järvinen, Jyrki Heino, Janne Kääriäinen, and Minna Kääriäinen
- Subjects
Male ,Integrins ,Duchenne muscular dystrophy ,Muscle Fibers, Skeletal ,Integrin ,Dystrophin ,Rats, Sprague-Dawley ,Extracellular matrix ,Muscular Diseases ,Glycoprotein complex ,Sarcoglycans ,medicine ,Animals ,Regeneration ,Dystroglycans ,Muscle, Skeletal ,Cell adhesion ,Genetics (clinical) ,Membrane Glycoproteins ,biology ,Myogenesis ,Vinculin ,Blotting, Northern ,medicine.disease ,Immunohistochemistry ,Molecular biology ,Rats ,Cytoskeletal Proteins ,Disease Models, Animal ,Neurology ,Pediatrics, Perinatology and Child Health ,biology.protein ,Laminin ,Neurology (clinical) ,Cell Adhesion Molecules - Abstract
In shearing injury both the myofibres and connective tissue framework are breached and the muscle tendon continuity is disrupted. During regeneration the firm myofibre to extracellular matrix (ECM) adhesion must be re-established. We have analysed the expression of selected molecules implementing this adhesion in regenerating myofibres 2-56 days after transection of rat soleus muscle using quantitative immunohistochemistry and Northern blotting. Beta1 integrin mRNA level and alpha7 integrin and vinculin immunoreactivities were transiently increased in both the intact and regenerating parts of the transected myofibres by day 5-7 with normalization by day 10-14. After day 14, alpha7 integrin and vinculin accumulated at the tips of the regenerating myofibres, indicating formation of new mini-myotendinous junctions (mMTJ). Immunoreactivities for dystrophin and associated proteins as well as merosin appeared in regenerating myotubes by day 3-4 reaching control levels by day 56. Our results suggest that integrin and dystrophin associated molecules are complementary in myofibre-ECM adhesion. During regeneration, ruptured myofibres temporarily reinforce their integrin mediated lateral adhesion until mMTJs are formed. Thereby the load on the newly formed scar and the risk of rerupture are reduced. Dystrophin associated molecules appear later and replace integrin on the lateral aspects, while both complexes are abundant at the mMTJs. These molecular events correspond to our previous results on tensile strength.
- Published
- 2000
30. Integrin α2β1 Mediates Isoform-Specific Activation of p38 and Upregulation of Collagen Gene Transcription by a Mechanism Involving the α2 Cytoplasmic Tail
- Author
-
Johanna Ivaska, Veli-Matti Kähäri, Jyrki Heino, Hilkka Reunanen, Leeni Koivisto, and Jukka Westermarck
- Subjects
collagen ,Integrins ,Receptors, Collagen ,Transcription, Genetic ,integrin ,Integrin ,cytoplasmic domain ,CDC42 ,Biology ,p38 MAPK ,Transfection ,CD49c ,p38 Mitogen-Activated Protein Kinases ,Collagen receptor ,Tumor Cells, Cultured ,Humans ,Protein Isoforms ,Cell Biology ,Molecular biology ,Cell biology ,Up-Regulation ,Enzyme Activation ,Integrin alpha M ,biology.protein ,Integrin, beta 6 ,Original Article ,Signal transduction ,Mitogen-Activated Protein Kinases ,ITGA6 ,Signal Transduction - Abstract
Two collagen receptors, integrins alpha1beta1 and alpha2beta1, can regulate distinct functions in cells. Ligation of alpha1beta1, unlike alpha2beta1, has been shown to result in recruitment of Shc and activation of the Ras/ERK pathway. To identify the downstream signaling molecules activated by alpha2beta1 integrin, we have overexpressed wild-type alpha2, or chimeric alpha2 subunit with alpha1 integrin cytoplasmic domain in human osteosarcoma cells (Saos-2) lacking endogenous alpha2beta1. The chimeric alpha2/alpha1 chain formed a functional heterodimer with beta1. In contrast to alpha2/alpha1 chimera, forced expression of alpha2 integrin resulted in upregulation of alpha1 (I) collagen gene transcription in response to three-dimensional collagen, indicating that the cytoplasmic domain of alpha2 integrin was required for signaling. Furthermore, signals mediated by alpha2beta1 integrin specifically activated the p38alpha isoform, and selective p38 inhibitors blocked upregulation of collagen gene transcription. Dominant negative mutants of Cdc42, MKK3, and MKK4 prevented alpha2beta1 integrin-mediated activation of p38alpha. RhoA had also some inhibitory effect, whereas dominant negative Rac was not effective. Our findings show the isoform-specific activation of p38 by alpha2beta1 integrin ligation and identify Cdc42, MKK3, and MKK4 as possible downstream effectors. These observations reveal a novel signaling mechanism of alpha2beta1 integrin that is distinct from ones previously described for other integrins.
- Published
- 1999
31. Echovirus 1 Infection Induces both Stress- and Growth-Activated Mitogen-Activated Protein Kinase Pathways and Regulates the Transcription of Cellular Immediate-Early Genes
- Author
-
Jyrki Heino, Pasi Huttunen, Timo Hyypiä, Pia Vihinen, and Liisa Nissinen
- Subjects
Gene Expression Regulation, Viral ,MAPK/ERK pathway ,Transcription, Genetic ,JUNB ,p38 mitogen-activated protein kinases ,Biology ,Kidney ,Antiviral Agents ,03 medical and health sciences ,Virology ,Proto-Oncogenes ,Gene expression ,Cell Adhesion ,Tumor Cells, Cultured ,Animals ,Humans ,RNA, Messenger ,Enzyme Inhibitors ,Phosphorylation ,Protein kinase A ,Genes, Immediate-Early ,030304 developmental biology ,Regulation of gene expression ,0303 health sciences ,030302 biochemistry & molecular biology ,Haplorhini ,Isoxazoles ,Semliki forest virus ,Molecular biology ,Enterovirus B, Human ,3. Good health ,Cell biology ,Transcription Factor AP-1 ,Poliovirus ,Protein Biosynthesis ,Mitogen-activated protein kinase ,Calcium-Calmodulin-Dependent Protein Kinases ,biology.protein ,Receptors, Virus ,Collagen ,Sarcoma, Experimental - Abstract
We have previously shown that echovirus 1 (EV1) infection increases the mRNA levels of cellular immediate-early (IE) genes in host cells. Here we provide further evidence that the induction of junB, c-jun, and c-fos genes is due to active viral macromolecular synthesis rather than to the interaction of EV1 with its receptor, alpha2beta1 integrin. Nuclear run-on transcription assays indicated that differences in mRNA levels in infected and uninfected cells are brought about by regulation at the transcriptional level. EV1 infection induced the phosphorylation of both the stress-related p38 mitogen-activated protein kinase (MAPK) and the growth signal-related ERK1/2 MAPKs. Studies with selective MAPK inhibitors revealed that p38 was the main inducer of junB expression, whereas both MAPK pathways were involved in the induction of c-fos. Activation of AP-1 genes was also observed to occur during infections with other enteroviruses and with Semliki Forest A7(74) virus, suggesting that the phosphorylation of MAPKs and induction of AP-1 gene expression may be important regulators of host cell behavior during viral infections.
- Published
- 1998
32. Transcription of α2 Integrin Gene in Osteosarcoma Cells Is Enhanced by Tumor Promoters
- Author
-
Jukka Westermarck, Veli-Matti Kähäri, Liisa Nissinen, Leeni Koivisto, and Jyrki Heino
- Subjects
Integrins ,Time Factors ,Transcription, Genetic ,Integrin alpha3 ,Integrin ,Integrin alpha2 ,CD18 ,Integrin alpha5 ,CD49c ,CD49b ,Collagen receptor ,Antigens, CD ,Okadaic Acid ,Cell Adhesion ,Tumor Cells, Cultured ,Humans ,Collagenases ,RNA, Messenger ,Osteosarcoma ,biology ,Activator (genetics) ,Integrin beta1 ,Cell Biology ,Integrin alphaV ,Blotting, Northern ,Flow Cytometry ,Molecular biology ,Up-Regulation ,Integrin alpha M ,Carcinogens ,biology.protein ,Tetradecanoylphorbol Acetate ,Integrin, beta 6 ,Collagen ,Matrix Metalloproteinase 1 - Abstract
Integrin alpha2beta1 is a heterodimeric transmembrane receptor for collagens. In osteogenic cells the expression of alpha2beta1 integrin is induced by both Kirsten sarcoma virus and chemical transformation. The association of alpha2 integrin with transformed cell phenotype was studied further by testing the effects of two tumor promoters, 12-O-tetradecanoylphorbol 13-acetate (TPA) and okadaic acid (OA), on human MG-63 osteosarcoma cells. TPA, an activator of protein kinase C, increased the cell surface expression of alpha2 integrin and the corresponding mRNA levels. Nuclear run-on assays indicated that TPA activated the transcription of alpha2 integrin gene. TPA also slightly increased the expression of alpha3 integrin but had no effect on the transcription of alpha5, alphav, or beta1 integrin subunits. OA, an inhibitor of serine/threonine phosphatases, increased alpha2 integrin gene transcription and mRNA levels, but in contrast to TPA, OA decreased alpha3 integrin expression. The increased expression of alpha2 integrin on TPA-treated MG-63 cells led to faster cell spreading on type I collagen. Our results link the enhanced transcription of alpha2 integrin gene to tumor progression and show the independent regulation of alpha2 integrin compared to other integrin genes.
- Published
- 1998
33. Suprabasal expression of epidermal alpha2beta1 and alpha3beta1 integrins in skin treated with topical retinoic acid
- Author
-
Lari Häkkinen, Nina Johansson, Jyrki Heino, Juha Peltonen, Veli-Matti Kähäri, Jukka Westermarck, and Heikki Aho
- Subjects
Adult ,Keratinocytes ,Male ,Integrins ,Pathology ,medicine.medical_specialty ,Receptors, Collagen ,Integrin ,Cell Culture Techniques ,Fluorescent Antibody Technique ,Alpha (ethology) ,Tretinoin ,Human skin ,Dermatology ,Administration, Cutaneous ,Receptors, Laminin ,Keratolytic Agents ,medicine ,Humans ,Beta (finance) ,integumentary system ,biology ,Integrin alpha3beta1 ,Molecular biology ,Fibronectin ,HaCaT ,medicine.anatomical_structure ,biology.protein ,Epidermis ,Carrier Proteins ,Keratinocyte - Abstract
In normal adult human skin, expression of epidermal integrins is confined to keratinocytes in the basal layer. However, suprabasal expression of alpha 2, alpha 3 and beta 1 integrin subunits is noted in hyperproliferative epidermis in wound repair and psoriasis. In this study, we examined the effect of topical all-trans-retinoic acid (RA), known to induce epidermal hyperplasia, on expression of integrins in human epidermis. Immunostaining of vehicle-treated skin revealed expression of alpha 2, alpha 3 and beta 1, as well as alpha 6 and beta 4 integrin subunits entirely on basal keratinocytes. Topical application of RA (0.1%) for 2 weeks resulted in marked suprabasal expression of alpha 2, alpha 3 and beta 1 integrin subunits, whereas alpha 6 and beta 4 staining remained on basal keratinocytes. Staining for putative ligands of alpha 2 beta 1 and alpha 3 beta 1 integrins, i.e. type IV collagen, laminin-5 and fibronectin, was not detected in the epidermal layer in RA- or vehicle-treated skin. Treatment of HaCaT keratinocytes in culture with RA (1 mumol/L) enhanced alpha 2 and beta 1 mRNA abundance. Furthermore, RA slightly up-regulated the expression of alpha 2, alpha 3 and beta 1 integrin subunits on primary epidermal keratinocytes and HaCaT cells in culture with no effect on cell proliferation. These results provide evidence that RA-elicited epidermal hyperplasia is associated with aberrant suprabasal expression of alpha 2 beta 1 and alpha 3 beta 1 integrins, and that this also involves direct stimulation of keratinocyte integrin expression by RA.
- Published
- 1998
34. Novel α2β1 integrin inhibitors reveal that integrin binding to collagen under shear stress conditions does not require receptor preactivation
- Author
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Kalle Sipilä, Maria Salmela, Liisa Nissinen, Anne Marjamäki, Johanna Jokinen, Olli T. Pentikäinen, Jarkko T. Koivunen, Marjo Pihlavisto, Jonna Nieminen, Jarmo Käpylä, and Jyrki Heino
- Subjects
Blood Platelets ,Integrin ,Platelet Membrane Glycoproteins ,Biochemistry ,CD49c ,Collagen Type I ,Collagen receptor ,Cell Line ,Mice ,Cell Adhesion ,Animals ,Humans ,Binding site ,Receptor ,Molecular Biology ,Integrin binding ,Sulfonamides ,biology ,Molecular Structure ,Chemistry ,ta1182 ,Cell Biology ,Mice, Inbred C57BL ,Integrin alpha M ,biology.protein ,Biophysics ,Integrin, beta 6 ,Stress, Mechanical ,Integrin alpha2beta1 ,Protein Binding - Abstract
The interaction between α2β1 integrin (GPIa/IIa, VLA-2) and vascular collagen is one of the initiating events in thrombus formation. Here, we describe two structurally similar sulfonamide derivatives, BTT-3033 and BTT-3034, and show that, under static conditions, they have an almost identical effect on α2-expressing CHO cell adhesion to collagen I, but only BTT-3033 blocks platelet attachment under flow (90 dynes/cm(2)). Differential scanning fluorimetry showed that both molecules bind to the α2I domain of the recombinant α2 subunit. To further study integrin binding mechanism(s) of the two sulfonamides, we created an α2 Y285F mutant containing a substitution near the metal ion-dependent adhesion site motif in the α2I domain. The action of BTT-3033, unlike that of BTT-3034, was dependent on Tyr-285. In static conditions BTT-3034, but not BTT-3033, inhibited collagen binding by an α2 variant carrying a conformationally activating E318W mutation. Conversely, in under flow conditions (90 dynes/cm(2)) BTT-3033, but not BTT-3034, inhibited collagen binding by an α2 variant expressing E336A loss-of-function mutation. Thus, the binding sites for BTT-3033 and BTT-3034 are differentially available in distinct integrin conformations. Therefore, these sulfonamides can be used to study the biological role of different functional stages of α2β1. Furthermore, only the inhibitor that recognized the non-activated conformation of α2β1 integrin under shear stress conditions effectively blocked platelet adhesion, suggesting that the initial interaction between integrin and collagen takes place prior to receptor activation.
- Published
- 2012
35. Integrin α2β1 Is a Positive Regulator of Collagenase (MMP-1) and Collagen α1(I) Gene Expression
- Author
-
Jukka Westermarck, Veli-Matti Kähäri, Jyrki Heino, Terhi Riikonen, Leeni Koivisto, and Arsi Broberg
- Subjects
biology ,Chemistry ,Integrin ,Cell Biology ,Transfection ,Biochemistry ,Molecular biology ,Collagen receptor ,Collagen, type I, alpha 1 ,Integrin alpha M ,Collagenase ,medicine ,biology.protein ,Integrin, beta 6 ,Molecular Biology ,Type I collagen ,medicine.drug - Abstract
A classical model for studying the effects of extracellular matrix is to culture cells inside a three-dimensional collagen gel. When surrounded by fibrillar collagen, many cell types decrease the production of type I collagen, and the expression of interstitial collagenase (matrix metalloproteinase-1; MMP-1) is simultaneously induced. To study the role of the collagen-binding integrins α1β1 and α2β1 in this process, we used three different osteogenic cell lines with distinct patterns of putative collagen receptors: HOS cells, which express only α1β1 integrin, MG-63 cells, which express only α2β1 integrin, and KHOS-240 cells, which express both. Inside collagen gels, α1(I) collagen mRNA levels were decreased in HOS and KHOS-240 cells but not in MG-63 cells. In contrast, MMP-1 expression was induced in KHOS-240 and MG-63 cells but not in HOS cells. Transfection of MG-63 cells with α2 integrin cDNA produced cell clones overexpressing α2β1 integrin. Transfection of MG-63 cells with α2 integrin cDNA in an antisense orientation reduced the expression level of α2 integrin. These cell clones showed induction and reduction of mRNA levels for MMP-1, respectively. HOS cells normally lacking α2β1 integrin were forced to express it, and this prevented the down-regulation in the levels of α1(I) collagen mRNA when cells were grown inside collagen gels. The data indicate that the level of MMP-1 expression is regulated by the collagen receptor α2β1 integrin. The down-regulation of collagen α1(I) is mediated by another receptor. Integrin α2β1 may compete with it and thus be a positive regulator of collagen synthesis.
- Published
- 1995
36. Melanoma-Associated Cancer-Testis Antigen 16 (CT16) Regulates the Expression of Apoptotic and Antiapoptotic Genes and Promotes Cell Survival
- Author
-
Hannu Larjava, Camilla Nylund, Laura L. Elo, Pia Vihinen, Laura Laato, Aleksi Heino, Pekka Rappu, Jyrki Heino, Markku Kallajoki, Eveliina Pakula, Seppo Pyrhönen, and Gethin Owen
- Subjects
Melanomas ,Small interfering RNA ,Skin Neoplasms ,lcsh:Medicine ,Apoptosis ,Biochemistry ,Malignant transformation ,Metastasis ,Gene expression ,Molecular Cell Biology ,Basic Cancer Research ,Promoter Regions, Genetic ,lcsh:Science ,Melanoma ,Skin Tumors ,Regulation of gene expression ,Multidisciplinary ,Cell Death ,Systems Biology ,Malignant Melanoma ,Immunohistochemistry ,Oncology ,DNA methylation ,Cytochemistry ,Cancer/testis antigens ,Medicine ,Melanoma-Specific Antigens ,Immunocytochemistry ,Research Article ,Cell Survival ,Blotting, Western ,Malignant Skin Neoplasms ,Dermatology ,Biology ,In Vitro Techniques ,Real-Time Polymerase Chain Reaction ,ta3111 ,Cell Line, Tumor ,medicine ,Gene silencing ,Humans ,Cell Proliferation ,lcsh:R ,ta1182 ,Proteins ,Cancers and Neoplasms ,medicine.disease ,ta3122 ,Molecular biology ,Cancer research ,lcsh:Q ,Tumor Suppressor Protein p53 - Abstract
Cancer-testis (CT) antigens are predominantly expressed in testis or placenta, but absent in most adult tissues. During malignant transformation CT genes are often activated. CT antigen 16 (CT16, PAGE5) is frequently expressed in advanced melanoma but its biological function has been unknown. To examine the role of CT16 in cell survival we knocked it down in A2058 melanoma cells using specific siRNAs and exposed the cells to cancer drug cisplatin known to induce apoptosis. As a result, cell survival was markedly decreased. To study the effects of CT16 on cell survival in more detail, the cellular gene expression profiles were investigated after CT16 silencing in CT16 positive A2058 melanoma cells, as well as after CT16 overexpression in CT16 negative WM-266-4 melanoma cells. Among the 11 genes both upregulated by CT16 silencing and downregulated by CT16 overexpression or vice versa, 4 genes were potentially apoptotic or antiapoptotic genes. CT16 was recognized as a positive regulator of antiapoptotic metallothionein 2A and interleukin 8 genes, whereas it inhibited the expression of apoptosis inducing dickkopf 1 (DKK1) gene. In addition CT16 enhanced the expression of fatty acid binding protein 7, a known promoter of melanoma progression. The effect of CT16 on DKK1 expression was p53 independent. Furthermore, CT16 did not regulate apoptotic genes via DNA methylation. In twenty melanoma metastasis tissue samples average DKK1 mRNA level was shown to be significantly (p
- Published
- 2012
37. Collagen XXIII, novel ligand for integrin alpha2beta1 in the epidermis
- Author
-
Johannes A. Eble, Guido Veit, Stephan Niland, Jarmo Käpylä, Manon C. Zweers, Akemi Ishada-Yamamoto, Jyrki Heino, Beate Eckes, Manuel Koch, Daniela Zwolanek, and Thomas Krieg
- Subjects
Keratinocytes ,Integrin ,Glycobiology and Extracellular Matrices ,Ligands ,Biochemistry ,Laminin 111 ,Collagen receptor ,Cell Line ,Focal adhesion ,Extracellular matrix ,medicine ,Cell Adhesion ,Humans ,Cell adhesion ,Molecular Biology ,Basement membrane ,Focal Adhesions ,biology ,Chemistry ,Cell Biology ,Surface Plasmon Resonance ,Immunohistochemistry ,Cell biology ,medicine.anatomical_structure ,Integrin alpha M ,biology.protein ,Collagen ,Epidermis ,Integrin alpha2beta1 - Abstract
Cellular receptors for collagens belong to the family of β(1) integrins. In the epidermis, integrin α(2)β(1) is the only collagen-binding integrin present. Its expression is restricted to basal keratinocytes with uniform distribution on the cell surface of those cells. Although α(2)β(1) receptors localized at the basal surface interact with basement membrane proteins collagen IV and laminin 111 and 332, no interaction partners have been reported for these integrin molecules at the lateral and apical membranes of basal keratinocytes. Solid phase binding and surface plasmon resonance spectroscopy demonstrate that collagen XXIII, a member of the transmembrane collagens, directly interacts with integrin α(2)β(1) in an ion- and conformation-dependent manner. The two proteins co-localize on the surface of basal keratinocytes. Furthermore, collagen XXIII is sufficient to induce adhesion and spreading of keratinocytes, a process that is significantly reduced in the absence of functional integrin α(2)β(1).
- Published
- 2011
38. Lumican inhibits cell migration through α2β1 integrin
- Author
-
Christine Terryn, Corinne Perreau, Hélène Bobichon, Yanusz Wegrowski, Jyrki Heino, Stéphane Brézillon, François-Xavier Maquart, Clemens M. Franz, Jarmo Käpylä, Johannes A. Eble, and Cedric Zeltz
- Subjects
Lumican ,Integrin alpha2 ,CHO Cells ,CD49c ,Focal adhesion ,Cricetulus ,Cell Movement ,Cell Line, Tumor ,Cricetinae ,Animals ,Humans ,Phosphorylation ,Melanoma ,biology ,Chinese hamster ovary cell ,Cell migration ,Cell Biology ,Transfection ,Molecular biology ,Cell biology ,Proteoglycan ,Chondroitin Sulfate Proteoglycans ,Cell culture ,Keratan Sulfate ,Focal Adhesion Protein-Tyrosine Kinases ,biology.protein ,Integrin alpha2beta1 ,Protein Binding - Abstract
Lumican, an extracellular matrix protein of the small leucine-rich proteoglycan family, has been shown to impede melanoma progression by inhibiting cell migration. In the present study, we show that lumican targets α2β1 integrin thereby inhibiting cell migration. A375 melanoma cells were transfected with siRNA directed against the α2 integrin subunit. Compared to A375 control cells, the anti-migratory effect of lumican was abrogated on transfected A375 cells. Moreover, lumican inhibited the chemotactic migration of Chinese hamster ovary (CHO) cells stably transfected with α2 integrin subunit (CHO-A2) but not that of wild-type CHO cells (CHO-WT) lacking this subunit. In contrast to CHO-WT cells, we observed in time-lapse microscopy a decrease of CHO-A2 cell migration speed in presence of lumican. Focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) and total FAK were analysed in CHO-WT and CHO-A2 cells. A significant decrease of the ratio pFAK/FAK was shown in presence of recombinant human lumican. Using solid phase assays, a direct binding between lumican and the α2β1 integrin was demonstrated. This interaction did not involve the glycan moiety of lumican and was cation independent. Lumican was also able to bind the activated I domain of the α2 integrin subunit with a K(d)≥200nM. In conclusion, we demonstrated for the first time that the inhibition of cell migration by lumican depends on a direct binding between the core protein of lumican and the α2β1 integrin.
- Published
- 2010
39. Regulation of integrin-type cell adhesion receptors by cytokines
- Author
-
P Santala and Jyrki Heino
- Subjects
Interleukin 5 receptor alpha subunit ,Gi alpha subunit ,Cell Biology ,Biology ,Biochemistry ,CD49c ,Collagen receptor ,Interleukin 10 receptor, alpha subunit ,Cell biology ,Integrin alpha M ,biology.protein ,Integrin, beta 6 ,Molecular Biology ,G alpha subunit - Abstract
Integrin heterodimers which share a common beta 1 subunit are the major cellular receptors for many extracellular matrix proteins. Here, we show that two inflammatory mediators, interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), can regulate the expression of the alpha 1 beta 1 integrin heterodimer, known to be a laminin and collagen receptor. In human skin fibroblasts 10 units/ml IL-1 beta increase the biosynthesis of the alpha 1 integrin subunit an average of 4.5-fold. Furthermore, IL-1 beta can turn on alpha 1 subunit expression in MG-63 human osteosarcoma cells even in conditions where the untreated MG-63 cells do not express it in detectable amounts. The effect of TNF-alpha on alpha 1 subunit expression is similar. Both IL-1 beta and TNF-alpha increased MG-63 cell adhesion on laminin. The effect of transforming growth factor-beta 1 (TGF-beta 1) on integrin expression in MG-63 cells has been previously described (Heino, J., and Massague, J. (1989) J. Biol. Chem. 264, 21806-21811). TGF-beta 1 decreases the biosynthesis of alpha 3 subunit but increases the production of alpha 2 subunit. IL-1 beta potentiates the effects of TGF-beta 1. Furthermore, in the presence of TGF-beta 1 the increase in the expression of alpha 1 subunit by IL-1 beta is even larger. Thus, IL-1 beta and TGF-beta 1, which usually have antagonistic functions in connective tissue, can regulate integrin expression in a synergistic way.
- Published
- 1991
40. Comparative Effects of Interleukin-1 and Tumor Necrosis Factor-α on Collagen Production and Corresponding Procollagen mRNA Levels in Human Dermal Fibroblasts
- Author
-
G. Loyau, Veli-Matti Kähäri, Eero Vuorio, Jean-Pierre Pujol, Jyrki Heino, Alain Mauviel, and D J Hartmann
- Subjects
Adult ,Male ,medicine.medical_specialty ,medicine.medical_treatment ,Dermatology ,Biology ,Biochemistry ,Extracellular matrix ,Dermal fibroblast ,Internal medicine ,Translational regulation ,medicine ,Humans ,RNA, Messenger ,Prostaglandin E2 ,Fibroblast ,Molecular Biology ,Cells, Cultured ,Skin ,Tumor Necrosis Factor-alpha ,Infant, Newborn ,Cell Biology ,Fibroblasts ,Molecular biology ,Recombinant Proteins ,Fibronectins ,Kinetics ,Procollagen peptidase ,Endocrinology ,medicine.anatomical_structure ,Cytokine ,Tumor necrosis factor alpha ,Collagen ,Procollagen ,Interleukin-1 ,medicine.drug - Abstract
The effects of recombinant human Interleukin-1 alpha (IL-1 alpha), Interleukin-1 beta (IL-1 beta), and Tumor Necrosis Factor-alpha (TNF-alpha) on collagen biosynthesis were studied in vitro using dermal fibroblast cultures. Both forms of IL-1 and TNF-alpha induced a dose-dependent inhibition of both types I and III collagen synthesis, as measured by radioimmunoassay, gel electrophoresis, or collagenase-sensitive material. This effect was accompanied by a significant release of prostaglandin E2 into the culture medium. However, indomethacin, a potent inhibitor of prostaglandin synthesis, could not prevent the inhibitory effect of the three cytokines on collagen synthesis. Measurement of type I and type III procollagen mRNA levels in IL-1 treated cells revealed that both IL-1 alpha and IL-1 beta were potent enhancers of procollagen gene expression at pretranslational level. On the other hand, TNF-alpha was found to reduce the steady-state levels of type I and III procollagen mRNA in a dose-dependent manner. Quantitation of IL-1 beta and TNF-alpha transcripts following TNF-alpha treatment of fibroblasts indicated that this cytokine can induce IL-1 beta gene expression in these cells. By contrast, TNF-alpha mRNA remained at a constant level after TNF-alpha exposure. These data suggest that IL-1 and TNF-alpha, two cytokines that share several biologic activities, modulate collagen deposition in dermal fibroblasts by mechanisms that are clearly different: TNF-alpha appears to act at a transcriptional level to inhibit collagen synthesis, whereas IL-1 inhibitory action involves important translational regulation, still unknown, that counterbalances its stimulatory effect on procollagen mRNA levels. Moreover, our data suggest the existence of local fibroblastic cytokine production that may be involved in the modulation of extracellular matrix deposition.
- Published
- 1991
41. Control of junB and extracellular matrix protein expression by transforming growth factor-beta 1 is independent of simian virus 40 T antigen-sensitive growth-sensitive growth-inhibitory events
- Author
-
Jyrki Heino, Lars Rönnstrand, David M. Livingston, J W Ludlow, Marikki Laiho, James A. DeCaprio, and Joan Massagué
- Subjects
Proto-Oncogene Proteins c-jun ,JUNB ,Platelet Membrane Glycoproteins ,Transfection ,Cell Line ,Transforming Growth Factor beta ,Animals ,RNA, Messenger ,Genes, Retinoblastoma ,Lung ,Molecular Biology ,Regulation of gene expression ,Thrombospondin ,biology ,Cell Cycle ,Cell Biology ,Transforming growth factor beta ,Cell cycle ,Molecular biology ,Extracellular Matrix ,Fibronectins ,DNA-Binding Proteins ,Fibronectin ,Plasminogen Inactivators ,Gene Expression Regulation ,Mink ,Cell culture ,biology.protein ,Thrombospondins ,Research Article ,Transforming growth factor - Abstract
Treatment of Mv1Lu mink lung epithelial cells with transforming growth factor-beta 1 (TGF-beta 1) prevents phosphorylation of the retinoblastoma susceptibility gene product, RB, in late G1 phase of the cell cycle, which is thought to retain RB in a growth-suppressive state. This effect is paralleled by cell cycle arrest in late G1 (M. Laiho, J. A. DeCapric, J. W. Ludlow, D. M. Livingston, and J. Massagué, Cell 62:175-185, 1990). Arrest can be prevented by expression of simian virus 40 T antigen, which binds to underphosphorylated RB, presumably blocking its growth-suppressive activity. The response of cells to TGF-beta 1, however, is complex and includes changes in the levels of expression of genes encoding nuclear transcription factors and extracellular matrix components. To define the relationships among various components of the TGF-beta 1 response, we have investigated the effect of TGF-beta 1 on cells whose growth-inhibitory response to this factor is prevented by T antigen. TGF-beta 1 addition to exponentially growing Mv1Lu cells increased the levels of junB mRNA and of three extracellular matrix proteins: plasminogen activator inhibitor-1, fibronectin, and thrombospondin. Kinetically, the effects on junB and plasminogen activator inhibitor-1 expression occurred faster (half-maximal at 1 to 2 h) than the effects on fibronectin and thrombospondin expression (half-maximal at 6 to 10 h). These effects either preceded or overlapped, respectively, the withdrawal of Mv1Lu cells from the cell cycle. Expression of a transfected T-antigen gene in Mv1Lu cells, however, did not prevent any of these responses to TGF-beta 1. The results indcate that TGF-B1-stimulated expression of junB and extracellular matrix proteins in Mv1Lu cells can occur independently of the T-antigen-sensitive events that lead to growth arrest.
- Published
- 1991
42. Control of JunB and Extracellular Matrix Protein Expression by Transforming Growth Factor-β1 Is Independent of Simian Virus 40 T Antigen-Sensitive Growth-Inhibitory Events
- Author
-
Marikki Laiho, Lars Rönnstrand, Jyrki Heino, James A. Decaprio, John W. Ludlow, David M. Livingston, and Joan Massagué
- Subjects
Cell Biology ,Molecular Biology - Published
- 1991
43. A Raft-derived, Pak1-regulated Entry Participates in α2β1 Integrin-dependent Sorting to Caveosomes
- Author
-
Jyrki Heino, G. Herma Renkema, Varpu Marjomäki, Timo Hyypiä, Elina Kakkonen, Paula Upla, Heli Paloranta, Pasi Kankaanpää, Mikko Karjalainen, and Prisca Liberali
- Subjects
Time Factors ,Endosome ,Antigens, Polyomavirus Transforming ,Integrin ,Caveolae ,Clathrin ,Caveolins ,Models, Biological ,Amiloride ,Membrane Microdomains ,Cell Line, Tumor ,Caveolin ,Humans ,Molecular Biology ,Dynamin ,Microscopy, Confocal ,biology ,Cell Biology ,Articles ,Cell biology ,Enterovirus B, Human ,Integrin alpha M ,p21-Activated Kinases ,Type C Phospholipases ,biology.protein ,Integrin, beta 6 ,Integrin alpha2beta1 - Abstract
We have previously shown that a human picornavirus echovirus 1 (EV1) is transported to caveosomes during 2 h together with its receptor alpha2beta1 integrin. Here, we show that the majority of early uptake does not occur through caveolae. alpha2beta1 integrin, clustered by antibodies or by EV1 binding, is initially internalized from lipid rafts into tubulovesicular structures. These vesicles accumulate fluid-phase markers but do not initially colocalize with caveolin-1 or internalized simian virus 40 (SV40). Furthermore, the internalized endosomes do not contain glycosylphosphatidylinositol (GPI)-anchored proteins or flotillin 1, suggesting that clustered alpha2beta1 integrin does not enter the GPI-anchored protein enriched endosomal compartment or flotillin pathways, respectively. Endosomes mature further into larger multivesicular bodies between 15 min to 2 h and concomitantly recruit caveolin-1 or SV40 inside. Cell entry is regulated by p21-activated kinase (Pak)1, Rac1, phosphatidylinositol 3-kinase, phospholipase C, and actin but not by dynamin 2 in SAOS-alpha2beta1 cells. An amiloride analog, 5-(N-ethyl-N-isopropanyl) amiloride, blocks infection, causes integrin accumulation in early tubulovesicular structures, and prevents their structural maturation into multivesicular structures. Our results together suggest that alpha2beta1 integrin clustering defines its own entry pathway that is Pak1 dependent but clathrin and caveolin independent and that is able to sort cargo to caveosomes.
- Published
- 2008
44. Interleukin-1β prevents the stimulatory effect of transforming growth factor-β on collagen gene expression in human skin fibroblasts
- Author
-
Jyrki Heino and T Heinonen
- Subjects
medicine.medical_specialty ,Gene Expression ,Alpha (ethology) ,Human skin ,Biology ,Biochemistry ,Transforming Growth Factor beta ,Skin Physiological Phenomena ,Internal medicine ,medicine ,Humans ,RNA, Messenger ,Beta (finance) ,Fibroblast ,Molecular Biology ,Cells, Cultured ,Skin ,Cell Biology ,Transforming growth factor beta ,Fibroblasts ,Molecular biology ,Recombinant Proteins ,Stimulation, Chemical ,Endocrinology ,medicine.anatomical_structure ,Cell culture ,Connective tissue metabolism ,biology.protein ,Collagen ,Research Article ,Interleukin-1 ,Transforming growth factor - Abstract
Transforming growth factors beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) are well-characterized strong inducers of collagen gene expression. A 100 pM concentration of TGF-beta 1 or TGF-beta 2 increases pro alpha 1(I) collagen mRNA levels in human skin fibroblasts 6.6-fold and 7.0-fold respectively, and also increases the accumulation of procollagens in the cell culture medium. Interleukin-1 beta (IL-1 beta) is an inflammatory mediator which also regulates connective tissue metabolism. A small concentration of IL-1 beta (0.01-1.0 unit/ml) slightly increases pro alpha 1(I) collagen mRNA levels (2.2-fold). Here we provide evidence that IL-1 beta prevents the stimulatory effect of TGFs-beta on collagen synthesis in human skin fibroblasts. An IL-1 beta concentration of 1 unit/ml is enough to keep pro alpha 1(I) collagen mRNA levels at control values in cells stimulated by 100 pM-TGF-beta 1. Thus the results indicate that IL-1 beta inhibits collagen synthesis in cells activated by TGFs-beta, whereas it does not significantly change or might even stimulate collagen gene expression in non-activated cells.
- Published
- 1990
45. Cell adhesion to collagen and decreased myogenic gene expression implicated in the control of myogenesis by transforming growth factor beta
- Author
-
Jyrki Heino and Joan Massagué
- Subjects
Transcription, Genetic ,Proto-Oncogene Proteins c-jun ,JUNB ,Cellular differentiation ,Muscle Proteins ,Biology ,Biochemistry ,Cell Line ,Extracellular matrix ,Proto-Oncogene Proteins ,Gene expression ,Cell Adhesion ,Animals ,Myocyte ,RNA, Messenger ,Molecular Biology ,Myogenin ,Myogenesis ,Muscles ,Cell Differentiation ,Cell Biology ,Transforming growth factor beta ,Protein-Tyrosine Kinases ,musculoskeletal system ,Molecular biology ,Rats ,DNA-Binding Proteins ,Kinetics ,Gene Expression Regulation ,Transforming Growth Factors ,biology.protein ,Collagen ,tissues ,Transcription Factors - Abstract
Transforming growth factor beta 1 (TGF-beta 1) is an inhibitor of skeletal muscle myoblast differentiation. Myoblast differentiation is dependent on the expression of certain myogenic differentiation genes and is affected by cell interaction with the extracellular matrix. We have searched for events in the differentiation process of L6E9 rat myoblasts that may be involved in the inhibitory action of TGF-beta 1. Elevated expression of the myogenic differentiation gene, myogenin, which is thought to play a central role in the differentiation process, occurs 10 h after the shift of L6E9 myoblasts to differentiation medium. Elevation of myogenin mRNA is blocked by TGF-beta 1 added at the time of the shift. This effect is preceded by, and might be related to, a rapid up-regulation of junB mRNA observed in TGF-beta 1-treated L6E9 myoblasts. However, TGF-beta 1 can also block myogenic differentiation in cells transfected with the myogenin gene under the control of a constitutive SV40 viral promoter. The nature of a mechanism that could mediate the inhibitory action of TGF-beta 1 without blocking myogenin mRNA expression is suggested by the observations that (a) TGF-beta 1 upregulates type I collagen expression and deposition in L6E9 myoblasts, (b) a fibrillar type I collagen layer inhibits L6E9 myoblast differentiation, and (c) inhibition of L6E9 myoblast differentiation by a type I collagen layer occurs without a block in myogenin expression. Thus, the data suggest that inhibition of L6E9 myoblast differentiation by TGF-beta 1 may be accomplished by at least two mechanisms acting in concert. One mechanism leads to a block in the expression of myogenin whereas the other mechanism may involve TGF-beta 1-induced changes in cell adhesion that either block the action of myogenic differentiation gene products or prevent the function of other as yet unknown components of the myogenic differentiation pathway.
- Published
- 1990
46. Calpain 1 and 2 Are Required for RNA Replication of Echovirus 1▿
- Author
-
Camilla Nylund, Matti Waris, Varpu Marjomäki, Timo Hyypiä, Jyrki Heino, Liisa Nissinen, and Paula Upla
- Subjects
Proteases ,Immunoelectron microscopy ,Immunology ,Parechovirus ,Virus Replication ,Microbiology ,Cell Line ,Viral entry ,Virology ,Humans ,Gene Silencing ,Enzyme Inhibitors ,Microscopy, Immunoelectron ,Microscopy, Confocal ,biology ,Calpain ,Cytoplasmic Vesicles ,RNA ,Molecular biology ,Cell biology ,Virus-Cell Interactions ,Enterovirus B, Human ,Viral replication ,Cell culture ,Insect Science ,Calpain-2 ,biology.protein ,RNA, Viral - Abstract
Calpains are calcium-dependent cysteine proteases that degrade cytoskeletal and cytoplasmic proteins. We have studied the role of calpains in the life cycle of human echovirus 1 (EV1). The calpain inhibitors, including calpeptin, calpain inhibitor 1, and calpain inhibitor 2 as well as calpain 1 and calpain 2 short interfering RNAs, completely blocked EV1 infection in the host cells. The effect of the inhibitors was not specific for EV1, because they also inhibited infection by other picornaviruses, namely, human parechovirus 1 and coxsackievirus B3. The importance of the calpains in EV1 infection also was supported by the fact that EV1 increased calpain activity 3 h postinfection. Confocal microscopy and immunoelectron microscopy showed that the EV1/caveolin-1-positive vesicles also contain calpain 1 and 2. Our results indicate that calpains are not required for virus entry but that they are important at a later stage of infection. Calpain inhibitors blocked the production of EV1 particles after microinjection of EV1 RNA into the cells, and they effectively inhibited the synthesis of viral RNA in the host cells. Thus, both calpain 1 and calpain 2 are essential for the replication of EV1 RNA.
- Published
- 2007
47. Analysis of an ascidian integrin provides new insight into early evolution of collagen recognition
- Author
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Jyrki Heino, Mira Tulla, Juha Jäälinoja, Richard W. Farndale, Mark S. Johnson, Leena Ala-Kokko, Mikko Huhtala, and Jarmo Käpylä
- Subjects
Models, Molecular ,Integrins ,animal structures ,Ascidian ,Evolution ,Protein Conformation ,Integrin ,Molecular Sequence Data ,Biophysics ,Sequence alignment ,Biochemistry ,Conserved sequence ,Collagen receptor ,Evolution, Molecular ,Protein structure ,Structural Biology ,Genetics ,Animals ,Humans ,Ciona intestinalis ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Conserved Sequence ,biology ,fungi ,Hydrogen Bonding ,Cell Biology ,biology.organism_classification ,Ciona ,embryonic structures ,biology.protein ,Collagen ,MIDAS ,Oligopeptides ,Sequence Alignment - Abstract
AlphaI domain integrins have been found in the ascidian Ciona intestinalis. We produced Ciona alpha1I domain as a recombinant protein. It did not recognize fibril-forming collagens or bind to GFOGER or other similar motifs in triple-helical peptides. No GFOGER motifs were found in Ciona collagens. As Ciona alpha1I bound to collagen IX, we propose that before the emergence of GFOGER-dependent collagen receptors in vertebrates, alphaI domain integrins might have been able to bind to collagen with alternative mechanisms.
- Published
- 2007
48. Two synergistic activation mechanisms of alpha2beta1 integrin-mediated collagen binding
- Author
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Mark S. Johnson, Wendy Lee Connors, Pasi Kankaanpää, Mira Tulla, Johanna Jokinen, Paula Upla, Jyrki Heino, J. Santeri Puranen, and Daniel J. White
- Subjects
Receptors, Collagen ,Protein Conformation ,Protein subunit ,Integrin ,Bone Neoplasms ,CHO Cells ,Ligands ,Biochemistry ,Collagen Type I ,Collagen receptor ,Cricetulus ,Cricetinae ,Cell Adhesion ,Animals ,Humans ,Avidity ,Cell adhesion ,Receptor ,Molecular Biology ,Protein kinase C ,Protein Kinase C ,Receptor Aggregation ,Osteosarcoma ,biology ,Chemistry ,Cell Biology ,Molecular biology ,Cell biology ,Extracellular Matrix ,Mutation ,cardiovascular system ,biology.protein ,Carcinogens ,Tetradecanoylphorbol Acetate ,Integrin alpha2beta1 ,Protein Binding - Abstract
Activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces ligand-independent aggregation of a cell surface collagen receptor, alpha2beta1 integrin. Concomitantly, TPA increases the avidity of alpha2beta1 for collagen and the number of conformationally activated alpha2beta1 integrins. The structural change was shown using a monoclonal antibody 12F1 that recognizes the "open" (active) conformation of the inserted domain in the alpha2 subunit (alpha2I). Amino acid residue Glu-336 in alpha2 subunit is proposed to mediate the interaction between alpha2I domain and beta1 subunit. Glu-336 seems to regulate a switch between open and "closed" conformations, since the mutation alpha2E336A inhibited the TPA-related increase in the number of 12F1 positive integrins. E336A also reduced cell adhesion to collagen. However, E336A did not prevent the TPA-related increase in adhesion to collagen or alpha2beta1 aggregation. Thus, alpha2beta1 integrin avidity is regulated by two synergistic mechanisms, first an alpha2E336-dependent switch to the open alpha2I conformation, and second an alpha2E336-independent mechanism temporally associated with receptor aggregation.
- Published
- 2007
49. Early Chordate Origin of the Vertebrate Integrin αI Domains
- Author
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Konstantin Denessiouk, Bhanupratap Singh Chouhan, Mark S. Johnson, Jarmo Käpylä, Jyrki Heino, and Alexander I. Denesyuk
- Subjects
Models, Molecular ,Computer and Information Sciences ,Lineage (genetic) ,Sequence analysis ,Integrin alpha1 ,Integrin ,lcsh:Medicine ,Sequence alignment ,Chordate ,Research and Analysis Methods ,Protein Structure, Secondary ,Collagen receptor ,Evolution, Molecular ,biology.animal ,Evolutionary Modeling ,Animals ,Humans ,Evolutionary Systematics ,lcsh:Science ,Molecular Biology Techniques ,Molecular Biology ,Phylogeny ,Taxonomy ,Data Management ,Evolutionary Biology ,Evolutionary Theory ,Molecular Biology Assays and Analysis Techniques ,Multidisciplinary ,biology ,Lamprey ,lcsh:R ,ta1182 ,Biology and Life Sciences ,Computational Biology ,Vertebrate ,Phylogenetic Analysis ,Anatomy ,biology.organism_classification ,Phylogenetics ,Evolutionary biology ,Vertebrates ,biology.protein ,lcsh:Q ,Research Article - Abstract
Half of the 18 human integrins α subunits have an inserted αI domain yet none have been observed in species that have diverged prior to the appearance of the urochordates (ascidians). The urochordate integrin αI domains are not human orthologues but paralogues, but orthologues of human αI domains extend throughout later-diverging vertebrates and are observed in the bony fish with duplicate isoforms. Here, we report evidence for orthologues of human integrins with αI domains in the agnathostomes (jawless vertebrates) and later diverging species. Sequence comparisons, phylogenetic analyses and molecular modeling show that one nearly full-length sequence from lamprey and two additional fragments include the entire integrin αI domain region, have the hallmarks of collagen-binding integrin αI domains, and we show that the corresponding recombinant proteins recognize the collagen GFOGER motifs in a metal dependent manner, unlike the α1I domain of the ascidian C. intestinalis. The presence of a functional collagen receptor integrin αI domain supports the origin of orthologues of the human integrins with αI domains prior to the earliest diverging extant vertebrates, a domain that has been conserved and diversified throughout the vertebrate lineage.
- Published
- 2014
50. Functional display of an alpha2 integrin-specific motif (RKK) on the surface of baculovirus particles
- Author
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Christian Oker-Blom, Reetta Riikonen, Jyrki Heino, Heli Matilainen, Mark S. Johnson, Nina Rajala, and Olli T. Pentikäinen
- Subjects
0301 basic medicine ,Models, Molecular ,Cancer Research ,Insecta ,viruses ,media_common.quotation_subject ,Amino Acid Motifs ,Green Fluorescent Proteins ,Integrin alpha2 ,Peptide ,Enzyme-Linked Immunosorbent Assay ,CHO Cells ,Biology ,Gene delivery ,Green fluorescent protein ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,Cricetinae ,Animals ,Cloning, Molecular ,Internalization ,media_common ,chemistry.chemical_classification ,Microscopy, Confocal ,Phospholipase C ,Wild type ,Gene Transfer Techniques ,biology.organism_classification ,Flow Cytometry ,Molecular biology ,Recombinant Proteins ,Protein Structure, Tertiary ,Autographa californica ,030104 developmental biology ,Enzyme ,Oncology ,chemistry ,Microscopy, Fluorescence ,Mutagenesis ,030220 oncology & carcinogenesis ,Type C Phospholipases ,Electrophoresis, Polyacrylamide Gel ,Peptides ,Baculoviridae ,Viral Fusion Proteins ,Plasmids ,Protein Binding - Abstract
The use of baculovirus vectors shows promise as a tool for gene delivery into mammalian cells. These insect viruses have been shown to transduce a variety of mammalian cell lines, and gene transfer has also been demonstrated in vivo. In this study, we generated two recombinant baculovirus vectors displaying an integrin-specific motif, RKK, as a part of two different loops of the green fluorescent protein (GFP) fused with the major envelope protein gp64 of Autographa californica M nucleopolyhedrovirus. By enzyme linked immunosorbent assays, these viruses were shown to bind a peptide representing the receptor binding site of an α2 integrin, the α2I-domain. However, the interaction was not strong enough to overcome binding of wild type gp64 to the unknown cellular receptor(s) on the surface of α2 integrin-expressing cells (CHO-α2β1) or enhance the viral uptake. After treatment of these cells with phospholipase C, internalization of all viruses was blocked or decreased significantly. However, one of the RKK displaying viruses, AcGFP(K)gp64, was still able to internalize into CHO-α2β1 cells, although at a lower level as compared to non-treated cells. This may indicate the possible utilization of a PLC independent alternative route via, in this case, the α2β1 integrin.
- Published
- 2005
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