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Thyroid hormones (THs) are key regulators of different biological processes. Their action involves genomic and non-genomic mechanisms, which together mediate the final effects of TH in target tissues. However, the proportion of the two processes and their contribution to the TH-mediated effects are still poorly understood. Skeletal muscle is a classical target tissue for TH, which regulates muscle strength and contraction, as well as energetic metabolism of myofibers. Here we address the different contribution of genomic and non-genomic action of TH in skeletal muscle cells by specifically silencing the deiodinase Dio2 or the β3-Integrin expression via CRISPR/Cas9 technology. We found that myoblast proliferation is inversely regulated by integrin signal and the D2-dependent TH activation. Similarly, inhibition of the nuclear receptor action reduced myoblast proliferation, confirming that genomic action of TH attenuates proliferative rates. Contrarily, genomic and non-genomic signals promote muscle differentiation and the regulation of the redox state. Taken together, our data reveal that integration of genomic and non-genomic signal pathways finely regulates skeletal muscle physiology. These findings not only contribute to the understanding of the mechanisms involved in TH modulation of muscle physiology but also add insight into the interplay between different mechanisms of action of TH in muscle cells.

Integrin β3 (ITGB3) is probably related to skeletal metastasis, which is the most frequent complication in breast cancer progression. We aimed to define its role and suitability as target for anti-metastatic therapy. We generated two MDA-MB-231 cell clones with conditional miRNA-mediated ITGB3 knockdown for analyzing the resulting effects in vitro regarding mRNA expression, proliferation and migration, as well the impact on skeletal metastasis in a nude rat model. Furthermore, ITGB3 levels were analyzed in exosomes from plasma of rats with skeletal metastases, and from MDA-MB-231 cells incubated with these vesicles, as well as from exosomes secreted by cells with conditional ITGB3 knockdown. This inhibition of ITGB3 expression decreased cellular proliferation and more distinctly inhibited cellular migration. Reduction and even complete remissions of respective soft tissue and osteolytic lesions were detected after ITGB3 knockdown in vivo. Furthermore, ITGB3 levels were increased in exosomes isolated from plasma of rats harboring MDA-MB-231 lesions as well as in respective cells incubated with these vesicles in vitro. ITGB3 was distinctly decreased in exosomes from cells with ITGB3 knockdown. The observed in vitro and in vivo anti-ITGB3 effects can be explained by downregulation of specific genes, which have roles in angiogenesis (NPTN, RRM2), tumor growth (NPTN), energy metabolism (ISCA1), cytokinesis (SEPT11), migration (RRM2, STX6), cell proliferation, invasiveness, senescence, tumorigenesis (RRM2) and vesicle trafficking (SEPT11, STX6). ITGB3 has a role in breast cancer skeletal metastasis via gene expression modulation, as mirrored for ITGB3 in exosomes, thus it could serve as target for anti-metastatic therapy.

The spike protein of SARS-CoV-2 binds the angiotensin-converting enzyme 2 (ACE2) on the host cell surface and subsequently enters host cells through receptor-mediated endocytosis. Additional cell receptors may be directly or indirectly involved, including integrins. The cytoplasmic tails of ACE2 and integrins contain several predicted short linear motifs (SLiMs) that may facilitate internalization of the virus as well as its subsequent propagation through processes such as autophagy. Here, we measured the binding affinity of predicted interactions between SLiMs in the cytoplasmic tails of ACE2 and integrin β 3 with proteins that mediate endocytic trafficking and autophagy. We validated that a class I PDZ-binding motif mediated binding of ACE2 to the scaffolding proteins SNX27, NHERF3, and SHANK, and that a binding site for the clathrin adaptor AP2 μ2 in ACE2 overlaps with a phospho-dependent binding site for the SH2 domains of Src family tyrosine kinases. Furthermore, we validated that an LC3-interacting region (LIR) in integrin β 3 bound to the ATG8 domains of the autophagy receptors MAP1LC3 and GABARAP in a manner enhanced by LIR-adjacent phosphorylation. Our results provide molecular links between cell receptors and mediators of endocytosis and autophagy that may facilitate viral entry and propagation., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Diabetic nephropathy (dNP), now the leading cause of ESKD, lacks efficient therapies. Coagulation protease-dependent signaling modulates dNP, in part via the G protein-coupled, protease-activated receptors (PARs). Specifically, the cytoprotective protease-activated protein C (aPC) protects from dNP, but the mechanisms are not clear., Methods: A combination of in vitro approaches and mouse models evaluated the role of aPC-integrin interaction and related signaling in dNP., Results: The zymogen protein C and aPC bind to podocyte integrin- β 3 , a subunit of integrin- α v β 3 . Deficiency of this integrin impairs thrombin-mediated generation of aPC on podocytes. The interaction of aPC with integrin- α v β 3 induces transient binding of integrin- β 3 with G α 13 and controls PAR-dependent RhoA signaling in podocytes. Binding of aPC to integrin- β 3 via its RGD sequence is required for the temporal restriction of RhoA signaling in podocytes. In podocytes lacking integrin- β 3 , aPC induces sustained RhoA activation, mimicking the effect of thrombin. In vivo , overexpression of wild-type aPC suppresses pathologic renal RhoA activation and protects against dNP. Disrupting the aPC-integrin- β 3 interaction by specifically deleting podocyte integrin- β 3 or by abolishing aPC's integrin-binding RGD sequence enhances RhoA signaling in mice with high aPC levels and abolishes aPC's nephroprotective effect. Pharmacologic inhibition of PAR1, the pivotal thrombin receptor, restricts RhoA activation and nephroprotects RGE-aPC high and wild-type mice. Conclusions aPC-integrin- α v β 3 acts as a rheostat, controlling PAR1-dependent RhoA activation in podocytes in diabetic nephropathy. These results identify integrin- α v β 3 as an essential coreceptor for aPC that is required for nephroprotective aPC-PAR signaling in dNP., (Copyright © 2020 by the American Society of Nephrology.)

Therapy resistance in leukemia may be due to cancer cell-intrinsic and/or -extrinsic mechanisms. Mutations within BCR-ABL1, the oncogene giving rise to chronic myeloid leukemia (CML), lead to resistance to tyrosine kinase inhibitors (TKI), and some are associated with clinically more aggressive disease and worse outcome. Using the retroviral transduction/transplantation model of CML and human cell lines we faithfully recapitulate accelerated disease course in TKI resistance. We show in various models, that murine and human imatinib-resistant leukemia cells positive for the oncogene BCR-ABL1 T315I differ from BCR-ABL1 native (BCR-ABL1) cells with regards to niche location and specific niche interactions. We implicate a pathway via integrin β3, integrin-linked kinase (ILK) and its role in deposition of the extracellular matrix (ECM) protein fibronectin as causative of these differences. We demonstrate a trend towards a reduced BCR-ABL1 T315I+ tumor burden and significantly prolonged survival of mice with BCR-ABL1 T315I+ CML treated with fibronectin or an ILK inhibitor in xenogeneic and syngeneic murine transplantation models, respectively. These data suggest that interactions with ECM proteins via the integrin β3/ILK-mediated signaling pathway in BCR-ABL1 T315I+ cells differentially and specifically influence leukemia progression. Niche targeting via modulation of the ECM may be a feasible therapeutic approach to consider in this setting.

Introduction: Cancer is the second leading cause of death in the United States, surpassed only by cardiovascular disease. However, cancer has now overtaken cardiovascular disease as the main cause of death in 12 countries in Western Europe. The burden of cancer is posing a major challenge to health care systems worldwide and demanding improvements in methods for cancer prevention, diagnosis, and treatment. Alternative and complementary strategies for orthodox surgery, radiotherapy, and chemotherapy need to be developed., Objective: To determine the oncolytic potential of tumor cell-adapted rotavirus in terms of their ability to infect and lysate murine myeloma Sp2/0-Ag14 cells., Materials and Methods: We inoculated rotaviruses Wt1-5, WWM, TRUYO, ECwt-O, and WTEW in Sp2/0-Ag14 cells and we examined their infectious effects by immunocytochemistry, immunofluorescence, flow cytometry, and DNA fragmentation assays., Results: Rotavirus infection involved the participation of some heat shock proteins, of protein disulfide isomerase (PDI), and integrin β3. We detected the accumulation of viral antigens within the virus-inoculated cells and in the culture medium in all the rotavirus isolates examined. The rotavirus-induced cell death mechanism in Sp2/0-Ag14 cells involved changes in cell membrane permeability, chromatin condensation, and DNA fragmentation, which were compatible with cytotoxicity and apoptosis., Conclusions: The ability of the rotavirus isolates Wt1-5, WWM, TRUYO, ECwt-O, and WTEW to infect and cause cell death of Sp2/0-Ag14 cells through mechanisms that are compatible with virus-induced apoptosis makes them potential candidates as oncolytic agents.

Sepsis is a major challenge in clinical practice and responsible for high mortality. Recent studies indicated that integrins participated in toll-like-receptor (TLR)-mediated innate immunity. In the present study, we investigated the mechanism of integrin β3 and TLR4 signaling using a cecal ligation and puncture (CLP)-induced sepsis and lipopolysaccharide (LPS)-treated macrophage cell model. In a lethal CLP model, the survival rate of integrin β3 mice was higher than that of wild-type mice. The levels of alanine aminotransferase, aspartate transaminase, creatinine, blood urea nitrogen , and lactate dehydrogenase in the serum and cluster of differentiation 14 (CD14) protein expression in the tissues were significantly decreased in integrin β3 mice. A similar effect with regard to CD14 down-regulation was observed in septic TLR4 mice. In wild-type macrophages, the inhibition of integrin β3 by P11 or with a specific antibody, inhibited TNF-α, and IL-6 release at the early time period of LPS stimulation. However, during the late periods of LPS stimulation this effect was not noted. CD14 expression levels had no change in such treatment. In contract, LPS-induced TNF-α and IL-6 release and LPS-induced CD14 expression were significantly decreased in integrin β3macrophages. The inhibition of the TLR4 pathway by TAK-242, or in TLR4 mutant macrophages abolished LPS-induced CD14 expression. Integrin β3 pathway activation by vitronectin exhibited no effect in CD14 expression. Furthermore, recombinant CD14 protein stimulation reversed integrin β3 deficiency and caused lower TNF-α and IL-6 release. Moreover, the molecular interaction of TLR4 and integrin β3 was significantly increased following LPS stimulation. In conclusion, integrin β3 positively regulated TLR4-mediated inflammatory responses via CD14 expression in macrophages in septic condition. Specifically targeting integrin β3/TLR4-CD14 signaling pathway may be a potential treatment strategy for polymicrobial sepsis.

Antiplatelet drugs serve as a first-line antithrombotic therapy for the management of acute ischemic events and the prevention of secondary complications in vascular diseases. Numerous antiplatelet therapies have been developed; however, currently available agents are still associated with inadequate efficacy, risk of bleeding, and variability in individual response. Understanding the mechanisms of platelet involvement in thrombosis and the clinical development process of antiplatelet agents is critical for the discovery of novel agents. The functions of platelets in thrombosis are regulated by two major mechanisms: the interaction between surface receptors and their ligands, and the downstream intracellular signaling pathways. Recently, most of the progress made in antiplatelet drug development has been achieved with P2Y receptor antagonists. Additionally, the usage of GP IIb/IIIa receptor antagonists has decreased, because it is associated with a higher risk of bleeding and thrombocytopenia. Agents targeting other platelet surface receptors such as PARs, TP receptor, EP3 receptor, GPIb-IX-V receptor, P-selectin, as well as intracellular signaling factors, such as PI3Kβ, have been evaluated in an attempt to develop the next generation of antiplatelet drugs, reduce or eliminate interpatient variability of drug efficacy and significantly lower the risk of drug-induced bleeding. The aim of this review is to describe the pathways of platelet activation in thrombosis, and summarize the development process of antiplatelet agents, as well as the preclinical and clinical evaluations performed on these agents., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The infiltration of tumor-associated macrophages (TAMs) is associated with tumor progression and poor prognosis in endometrial cancer (EC). Collagen triple helix repeat containing 1 (CTHRC1), a secreted ECM protein, has been reported to have important roles in promoting cancer invasion and metastasis, but the functional role of CTHRC1 and its association with TAMs in EC remain unclear. Here we report that, in EC patients, CTHRC1 expression was up-regulated in endometrial cancer tissues compared with normal endometrium (P < 0.0001), and is positively correlated with tumor grade and depth of myometrial invasion (P = 0.024 and P = 0.0002, respectively). Meanwhile, CTHRC1 expression was positively correlated with an increased number of infiltrating TAMs, especially M2-like TAMs (P = 0.003, P = 0.001). In the tumor microenvironment of EC, CTHRC1 not only promoted myometrial invasion by interacting with Integrin β3-Akt signaling pathway, but also promoted infiltration of M2-like TAMs by upregulating Fractalkine chemokine receptor (CX3CR1) expression in macrophages. Changing levels of recombinant CTHRC1 protein (rCTHRC1) promoted tumor migration and invasion via enhancing macrophage recruitment in vitro. In summary, our findings eventually provided a novel role for CTHRC1 in remodeling the tumor immune microenvironment to promote tumor metastasis in EC patients.

Elevated serotonin (5-HT) blood levels, the first biomarker identified in autism research, has been consistently found in 20-30% of patients with Autism Spectrum Disorder (ASD). Hyperserotonemia is mainly due to greater 5-HT uptake into platelets, mediated by the 5-HT transporter (SERT) located at the platelet plasma membrane. The protein complex involved in platelet SERT trafficking and externalization includes integrin β3, the beta subunit of the platelet membrane adhesive GP IIb/IIIa. Integrin β3 is encoded by the ITGB3 gene, previously identified as a quantitative trait locus (QTL) for 5-HT blood levels in ASD at single nucleotide polymorphism (SNP) rs2317385. The present study aims to identify the functional ITGB3 gene variants contributing to hyperserotonemia. ITGB3 gene sequencing in 20 individuals selected on the basis of rs2317385 genotypes defined four haplotypes encompassing six SNPs located in the ITGB3 gene promoter region, all in linkage disequilibrium with rs2317385. Luciferase assays in two hematopoietic cell lines, K-562 and HEL 92.1.7, demonstrate that ITGB3 gene promoter activity is enhanced by the presence of the C allele at rs55827077 specifically during differentiation into megakaryocytes (P < 0.01), with modulatory effects by flanking SNPs. This same allele is strongly associated with (a) higher 5-HT blood levels in 176 autistic individuals (P < 0.001), (b) greater platelet integrin β3 protein expression (P < 0.05) and (c) enhanced SERT trafficking from the cytosol toward the platelet plasma membrane (P = 4.05 × 10-11). Our results support rs55827077 as the functional ITGB3 gene promoter variant contributing to elevated 5-HT blood levels in ASD and define a mechanistic chain of events linking ITGB3 to hyperserotonemia., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

It is currently unclear why agonist-stimulated platelets require shear force to efficiently externalize the procoagulant phospholipid phosphatidylserine (PS) and release PS-exposed microvesicles (MVs). We reveal that integrin outside-in signaling is an important mechanism for this requirement. PS exposure and MV release were inhibited in β 3 -/- platelets or by integrin antagonists. The impaired MV release and PS exposure in β 3 -/- platelets were rescued by expression of wild-type β 3 but not a Gα 13 binding-deficient β 3 mutant (E 733 EE to AAA), which blocks outside-in signaling but not ligand binding. Inhibition of Gα 13 or Src also diminished agonist/shear-dependent PS exposure and MV release, further indicating a role for integrin outside-in signaling. PS exposure in activated platelets was induced by application of pulling force via an integrin ligand, which was abolished by inhibiting Gα 13 -integrin interaction, suggesting that Gα 13 -dependent transmission of mechanical signals by integrins induces PS exposure. Inhibition of Gα 13 delayed coagulation in vitro. Furthermore, inhibition or platelet-specific knockout of Gα 13 diminished laser-induced intravascular fibrin formation in arterioles in vivo. Thus, β 3 integrins serve as a shear sensor activating the Gα 13 -dependent outside-in signaling pathway to facilitate platelet procoagulant function. Pharmacological targeting of Gα 13 -integrin interaction prevents occlusive thrombosis in vivo by inhibiting both coagulation and platelet thrombus formation., (© 2018 by The American Society of Hematology.)

Smooth muscle cells (SMCs) play a key role in atherogenesis. However, mechanisms regulating expansion and fate of pre-existing SMCs in atherosclerotic plaques remain poorly defined. Here we show that multiple SMC progenitors mix to form the aorta during development. In contrast, during atherogenesis, a single SMC gives rise to the smooth muscle-derived cells that initially coat the cap of atherosclerotic plaques. Subsequently, highly proliferative cap cells invade the plaque core, comprising the majority of plaque cells. Reduction of integrin β3 (Itgb3) levels in SMCs induces toll-like receptor 4 expression and thereby enhances Cd36 levels and cholesterol-induced transdifferentiation to a macrophage-like phenotype. Global Itgb3 deletion or transplantation of Itgb3 (-/-) bone marrow results in recruitment of multiple pre-existing SMCs into plaques. Conditioned medium from Itgb3-silenced macrophages enhances SMC proliferation and migration. Together, our results suggest SMC contribution to atherogenesis is regulated by integrin β3-mediated pathways in both SMCs and bone marrow-derived cells.

Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, β 3 integrins. Previous studies have implicated a cognate cis interaction between the bent conformation β 5 /β 3 integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y 2 R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant α IIb β 3 integrins and (RGD)P2Y 2 R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y 2 R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y 2 R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing α IIb β 3 integrins, virus engagement at the integrin PSI domain, recapitulates physiologic activation of the integrin as indicated by staining with the activation-specific mAB PAC1. The data also show that blocking of the Gα 13 protein from binding to the cytoplasmic domain of the β 3 integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y 2 R provides allosteric resistance to the membrane-normal motion associated with the switchblade model of integrin activation, where the development of tensile force yields physiological integrin activation., (© 2017 Bondu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

De novo expression in the kidney of periostin, a protein involved in odontogenesis and osteogenesis, has been suggested as a biomarker of renal disease. In this study, we investigated the mechanism(s) of induction and the role of periostin in renal disease. Using a combination of bioinformatics, reporter assay, and chromatin immunoprecipitation analyses, we found that NF κ B and other proinflammatory transcription factors induce periostin expression in vitro and that binding of these factors on the periostin promoter is enriched in glomeruli during experimental GN. Mice lacking expression of periostin displayed preserved renal function and structure during GN. Furthermore, delayed administration of periostin antisense oligonucleotides in wild-type animals with GN reversed already established proteinuria, diminished tissue inflammation, and improved renal structure. Lack of periostin expression also blunted the de novo renal expression of integrin- β 3 and phosphorylation of focal adhesion kinase and AKT, known mediators of integrin- β 3 signaling that affect cell motility and survival, observed during GN in wild-type animals. In vitro , recombinant periostin increased the expression of integrin- β 3 and the concomitant phosphorylation of focal adhesion kinase and AKT in podocytes. Notably, periostin and integrin- β 3 were highly colocalized in biopsy specimens from patients with inflammatory GN. These results demonstrate that interplay between periostin and renal inflammation orchestrates inflammatory and fibrotic responses, driving podocyte damage through downstream activation of integrin- β 3 signaling. Targeting periostin may be a novel therapeutic strategy for treating CKD., (Copyright © 2017 by the American Society of Nephrology.)

Osteopontin (OPN), a phosphorylated glycoprotein, is frequently overexpressed in cancer. Among the three OPN isoforms, OPN-a is the most highly expressed in lung cancer cell lines and lung tumors. Overexpression of OPN-a greatly reduced CL1-5 lung adenocarcinoma cell growth, but had no effect on growth in A549 lung adenocarcinoma cells. Examination of the expression of integrins and CD44, which are possible OPN-a receptors, revealed that differences in integrin β3 levels might explain this discrepancy between CL1-5 and A549 cells. When integrin β3 was ectopically expressed in A549 cells, OPN-a inhibited their growth, whereas OPN-a increased cell growth following integrin β3 knockdown in CL1-5 cells. This OPN-a-induced increase in growth appeared to result from activation of the CD44/NFκB pathway. Our results demonstrated that OPN-a inhibits growth of cells with high integrin β3 levels and increases growth via activation of the CD44/NFκB pathway in cells with low integrin β3 levels. Thus, OPN-a, integrin β3, and CD44 interact to affect lung cancer cell growth, and this study may aid in the development of cancer treatment strategies involving these molecules.

Integrin β3 is critical for tumor invasion, neoangiogenesis, and inflammation, making it a promising cancer target. However, preclinical and clinical data of integrin β3 antagonists have demonstrated no benefit or worse outcomes. We hypothesized that integrin β3 could affect tumor immunity and evaluated tumors in mice with deletion of integrin β3 in macrophage lineage cells (β3KOM). β3KOM mice had increased melanoma and breast cancer growth with increased tumor-promoting M2 macrophages and decreased CD8(+) T cells. Integrin β3 antagonist, cilengitide, also enhanced tumor growth and increased M2 function. We uncovered a negative feedback loop in M2 myeloid cells, wherein integrin β3 signaling favored STAT1 activation, an M1-polarizing signal, and suppressed M2-polarizing STAT6 activation. Finally, disruption of CD8(+) T cells, macrophages, or macrophage integrin β3 signaling blocked the tumor-promoting effects of integrin β3 antagonism. These results suggest that effects of integrin β3 therapies on immune cells should be considered to improve outcomes. Cancer Res; 76(12); 3484-95. ©2016 AACR., (©2016 American Association for Cancer Research.)

Objective: Periarticular and subchondral bone erosion in rheumatoid arthritis caused by osteoclast differentiation and activation is a critical index for diagnosis, therapy and monitoring of the disease. Sorting nexin (SNX) 10, a member of the SNX family which functions in regulation of endosomal sorting, has been implicated to play an important clinical role in malignant osteopetrosis. Here we studied the roles and precise mechanisms of SNX10 in the bone destruction of collagen-induced arthritis (CIA) mice., Methods: The role of SNX10 in bone destruction was evaluated by a CIA mice model which was induced in male SNX10(-/-) mice and wild type littermates. The mechanism was explored in osteoclasts induced by receptor activator of nuclear factor κB ligand from bone marrow mononuclear cells of wild type and SNX10(-/-) mice., Results: SNX10 knockout prevented bone loss and joint destruction in CIA mice with reduced serum levels of TNF-α, interleukin 1β and anticollagen IgG 2α antibody. SNX10 deficiency did not block osteoclastogenesis, but significantly impaired osteoclast maturation and bone-resorption function by disturbing the formation of actin belt. The production of TRAP, CtsK and MMP9 in SNX10(-/-) osteoclasts was significantly inhibited, and partially restored by SNX10 overexpression. We further demonstrated that the degradation of NFATc1 was accelerated in SNX10(-/-) osteoclasts causing an inhibition of integrin β3-Src-PYK2 signalling., Conclusions: Our study discloses a crucial role and novel mechanism for SNX10 in osteoclast function, and provides evidence for SNX10 as a promising novel therapeutic target for suppression of immune inflammation and bone erosion in rheumatoid arthritis., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Cancer patients frequently develop skeletal metastases that significantly impact quality of life. Since bone metastases remain incurable, a clearer understanding of molecular mechanisms regulating skeletal metastases is required to develop new therapeutics that block establishment of tumors in bone. While many studies have suggested that the microenvironment contributes to bone metastases, the factors mediating tumors to progress from a quiescent to a bone-destructive state remain unclear. In this study, we hypothesized that the "soil" of the bone microenvironment, specifically the rigid mineralized extracellular matrix, stimulates the transition of the tumor cells to a bone-destructive phenotype. To test this hypothesis, we synthesized 2D polyurethane (PUR) films with elastic moduli ranging from the basement membrane (70 MPa) to cortical bone (3800 MPa) and measured expression of genes associated with mechanotransduction and bone metastases. We found that expression of Integrin β3 (Iβ3), as well as tumor-produced factors associated with bone destruction (Gli2 and parathyroid hormone related protein (PTHrP)), significantly increased with matrix rigidity, and that blocking Iβ3 reduced Gli2 and PTHrP expression. To identify the mechanism by which Iβ3 regulates Gli2 and PTHrP (both are also known to be regulated by TGF-β), we performed Förster resonance energy transfer (FRET) and immunoprecipitation, which indicated that Iβ3 co-localized with TGF-β Receptor Type II (TGF-β RII) on rigid but not compliant films. Finally, transplantation of tumor cells expressing Iβ3 shRNA into the tibiae of athymic nude mice significantly reduced PTHrP and Gli2 expression, as well as bone destruction, suggesting a crucial role for tumor-produced Iβ3 in disease progression. This study demonstrates that the rigid mineralized bone matrix can alter gene expression and bone destruction in an Iβ3/TGF-β-dependent manner, and suggests that Iβ3 inhibitors are a potential therapeutic approach for blocking tumor transition to a bone destructive phenotype., (Published by Elsevier Ltd.)

As an early responder to an inflammatory stimulus, neutrophils (PMNs) must exit the vasculature and migrate through the extravascular tissue to the site of insult, which is often remote from the point of extravasation. Following a central epithelial corneal abrasion, PMNs recruited from the peripheral limbal vasculature migrate into the avascular corneal stroma. In vitro studies suggest PMN locomotion over 2-D surfaces is dependent on integrin binding while migration within 3-D matrices can be integrin-independent. Electron micrographs of injured mouse corneas show migrating PMNs make extensive surface contact not only with collagen fibrils in the extracellular matrix (ECM), but also keratocytes. Evidence supporting involvement of integrins in corneal inflammation has prompted research and development of integrin blocking agents for use as anti-inflammatory therapies. However, the role of integrin binding (cell-cell; cell-ECM) during stromal migration in the inflamed cornea has previously not been clearly defined. In this study in vivo time lapse imaging sequences provided the means to quantify cell motility while observing PMN interactions with keratocytes and other stromal components in the living eye. The relative contribution of β1, β2 and β3 integrins to PMN locomotion in the inflamed mouse cornea was investigated using blocking antibodies against the respective integrins. Of the 3 integrin families (β1, β2 and β3) investigated for their potential role in PMN migration, only β1 antibody blockade produced a significant, but partial, reduction in PMN motility. The preferential migration of PMNs along the keratocyte network was not affected by integrin blockade. Hence, the dominant mechanism for PMN motility within the corneal stroma appears to be integrin-independent as does the restriction of PMN migration paths to the keratocyte network., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Angiogenesis is mediated by signaling through receptor tyrosine kinases (RTKs), Src family kinases and adhesion receptors such as integrins, yet the mechanism how these signaling pathways regulate one another remains incompletely understood. The RTK modulator, Sprouty4 (Spry4) inhibits endothelial cell functions and angiogenesis, but the mechanisms remain to be fully elucidated. In this study, we demonstrate that Spry4 regulates angiogenesis in part by regulating endothelial cell migration. Overexpression of Spry4 in human endothelial cells inhibited migration and adhesion on vitronectin (VTN), whereas knockdown of Spry4 enhanced these behaviors. These activities were shown to be c-Src-dependent and Ras-independent. Spry4 disrupted the crosstalk between vascular endothelial growth factor-2 and integrin αVβ3, the receptor for VTN. Spry4 overexpression resulted in decreased integrin β3 protein levels in a post-transcriptional manner in part by modulating its tyrosine phosphorylation by c-Src. Conversely, knockdown of Spry4 resulted in increased integrin β3 protein levels and tyrosine phosphorylation. Moreover, in vivo analysis revealed that Spry4 regulated integrin β3 levels in murine embryos and yolk sacs. Our findings identify an unanticipated role for Spry4 in regulating c-Src activity and integrin β3 protein levels, which contributes to the regulation of migration and adhesion of endothelial cells. Thus, targeting Spry4 may be exploited as a target in anti-angiogenesis therapies.

We used an in vivo small hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase Syk. In contrast, loss of Itgb3 in normal hematopoietic stem and progenitor cells did not affect engraftment, reconstitution, or differentiation. Finally, using an Itgb3 knockout mouse model, we confirmed that Itgb3 is dispensable for normal hematopoiesis but is required for leukemogenesis. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Identification of tractable signaling molecules essential for leukemogenesis facilitates the development of effective targeted therapies. In this issue of Cancer Cell, Miller and colleagues report that Integrin Beta 3, which is largely dispensable for normal hematopoiesis, plays an important role and is a potential therapeutic target in mixed lineage leukemia., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Integrins can modulate the infiltration of inflammatory cells and the secretion of various inflammatory mediators, essential players in the pathogenesis of colitis. This study explores the role of beta2 and beta3 integrin signaling and their possible role in experimental colitis. A total of 160 adult male Sprague-Dawly rats were divided into 4 equal groups: methylcellulose, bacteria, iodoacetamide and iodoacetamide plus bacteria. Clinical symptoms and signs of colitis were checked daily and colonic tissues were biopsied on days 3, 14, 28, and 56 post induction. Histological studies along with histochemical analysis and polymerase chain reaction of beta2, beta3 and alphavbeta3 were performed according to standard procedures. The symptoms and signs were consistent with previously reported data on active colitis. The highest expression of beta3 integrin was in the combined treatment mostly on platelets, endothelial and inflammatory cells. In the same group, the expression of alphavbeta3 integrin complex reached the highest score after 56 days in all colonic layers. Beta2 integrin expression showed a 3-4-fold increase in the combined treatment group at all time points and kept increasing till day 56. It was mostly expressed in the mucosa and submucosa. In addition, the expression of both &#945;v&#946;3 and &#945;ii&#946;3 integrins was also elevated 2- to 10-fold, respectively, in the same colitis groups throughout the duration of the experiment. In conclusion, the combined treatment of IA and Enteropathogenic E. coli led to a significant upregulation of all the tested integrins throughout the experimental duration. Such upregulation of integrins could have contributed to the increase and chronicity of inflammation.

Overexpression of integrins in dermal fibroblasts is thought to play a key role in the pathogenesis of systemic sclerosis (SSc), but the mechanism is unknown. We evaluated the possibility that microRNAs (miRNAs) are involved in the regulation of integrin β3 in these cells. The miRNA expression profile was determined by miRNA PCR array and real-time PCR. Protein expression of integrin β3 was determined by immunoblotting. In vivo detection of miRNA in paraffin section was performed by in situ hybridization. miR-150 expression was decreased in SSc fibroblasts both in vivo and in vitro. The transfection of miR-150 inhibitor into normal fibroblasts induced expression of integrin β3, phosphorylated Smad3, and type I collagen, whereas forced overexpression of the miRNA resulted in their down-regulation in SSc fibroblasts. Treatment of SSc fibroblasts with 5-AdC revealed that miR-150 down-regulation in these cells is caused by DNA methylation. In addition, we found that miR-150 is detectable and quantitative in serum. Serum miR-150 levels were decreased in SSc patients, and the SSc patients with lower serum miR-150 levels tended to have more severe clinical manifestations. miR-150 may play an important role in the pathogenesis of SSc via overexpression of integrin β3. Investigation of the regulatory mechanisms of tissue fibrosis by miR-150 could lead to development of new diagnostic tools and new treatments using miRNA., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Rodent incisors exhibit pigmentation on their labial surfaces. Although previous studies have shown that this pigment is composed of iron, the existence of other elements has not been investigated. This study found that the lower incisors of CD61, also known as integrin β3, null mice (CD61(-/-)) lacked pigmentation. Although ameloblasts differentiated and formed enamel normally, no ferric ion accumulation was observed in maturation-stage ameloblasts in CD61(-/-) mice. Surface elements of control and CD61-/- lower incisors were compared by x-ray photoelectron spectroscopy (XPS). XPS analysis detected C, Ca, N, O, and P on the labial surfaces of lower incisors of both mice, whereas Fe was detected only in control samples. No peak of non-ferrous metal or other element was detected in either group. Quantitative RT-PCR analysis of 18 iron-transportation-related genes with mRNA from maturation-stage ameloblasts and ALC, a pre-ameloblastic cell line, was performed. The results suggested that CD61 regulates the expressions of Slc11a2 and Slc40a1, both of which are involved in iron transportation in epithelial tissues. These results suggested that the pigment on the labial surface of mouse incisors is composed of Fe and that both anemia and reduction of iron-transporting proteins may cause the loss of pigmentation in CD61(-/-) mice.

Integrins play key roles in the developing and mature nervous system, from promoting neuronal process outgrowth to facilitating synaptic plasticity. Recently, in hippocampal pyramidal neurons, β3 integrin (ITGβ3) was shown to stabilise synaptic AMPA receptors (AMPARs) and to be required for homeostatic scaling of AMPARs elicited by chronic activity suppression. To probe the physiological function for ITGβ3-dependent processes in the brain, we examined whether the loss of ITGβ3 affected fear-related behaviours in mice. ITGβ3-knockout (KO) mice showed normal conditioned fear responses that were similar to those of control wild-type mice. However, anxiety-like behaviour appeared substantially compromised and could be reversed to control levels by lentivirus-mediated re-expression of ITGβ3 bilaterally in the ventral hippocampus. In hippocampal slices, the loss of ITGβ3 activity did not compromise hebbian forms of plasticity--neither acute pharmacological disruption of ITGβ3 ligand interactions nor genetic deletion of ITGβ3 altered long-term potentiation (LTP) or long-term depression (LTD). Moreover, we did not detect any changes in short-term synaptic plasticity upon loss of ITGβ3 activity. In contrast, acutely disrupting ITGβ1-ligand interactions or genetic deletion of ITGβ1 selectively interfered with LTP stabilisation whereas LTD remained unaltered. These findings indicate a lack of requirement for ITGβ3 in the two robust forms of hippocampal long-term synaptic plasticity, LTP and LTD, and suggest differential roles for ITGβ1 and ITGβ3 in supporting hippocampal circuit functions., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Background: Integrins are involved in the process of embryo-endometrium interaction during implantation. We investigated the localization of integrin β3 in the rat blastocyst and Ishikawa cells using an in vitro co-culture model of implantation., Methods: Zona pellucida-free rat blastocysts were co-cultured with the Ishikawa cells (endometrial adenocarcinoma cell line) to observe the attachment between the embryo and endometrium. Immunofluorescence staining was used to investigate the localization of integrin β3 in rat embryos at different stages of development (each n= 3 embryos) and at the embryo/endometrium interface, observed by confocal microscopy. The Ishikawa cells were transfected with integrin β3 small interfering RNA (siRNA) for 48 h and then co-cultured with Day 5 rat blastocysts to observe the effect on attachment., Results: Integrin β3 staining in the rat embryos increased at the blastocyst stage being highly concentrated in the cytoplasm of trophoblast cells (n= 9 embryos). Integrin β3 was localized on the apical surface of the Ishikawa cells (n= 3 experiments). However, integrin β3 relocated to the apical membrane of trophoblast cells in response to attachment to Ishikawa cells (n= 6 embryos). Moreover, when Ishikawa cells were transfected with integrin β3 siRNA, blastocyst attachment was significantly reduced compared with those transfected with control siRNA (16.7 versus 92.3%, respectively, P< 0.05)., Conclusions: Integrin β3, localized apically in the blastocyst and the Ishikawa cells, is important during initial attachment of the blastocyst to endometrial cells. This study provides further evidence of the importance of integrins during implantation and may aid in elucidating the molecular mechanism of implantation failure and infertility in women.

Angiogenesis alleviates hypoxic stress in ischemic tissues or during tumor progression. In addition to endothelial cell proliferation and migration, the angiogenic process requires bone marrow-derived cell (BMDC) recruitment to sites of neovascularization. However, the mechanism of communication between hypoxic tissues and the BM remains unknown. Using 2 models of hypoxia-induced angiogenesis (ischemic hindlimb surgery and subcutaneous tumor growth), we show that platelet infusion promotes BMDC mobilization into the circulation, BMDC recruitment into growing neovasculature, tumor vascularization, and blood flow restoration in ischemic limbs, whereas platelet depletion inhibits these effects. Thus, platelets are required for BMDC recruitment into ischemia-induced vasculature. Secretion of platelet α-granules, but neither dense granules nor platelet aggregation is crucial for BMDC homing and subsequent angiogenesis, as determined using VAMP-8(-/-), Pearl, and integrin Beta 3(-/-) platelets. Finally, platelets sequester tumor-derived promoters of angiogenesis and BMDC mobilization, which are counterbalanced by the antiangiogenic factor thrombospondin-1. A lack of thrombospondin-1 in platelets leads to an imbalance in proangiogenic and antiangiogenic factors and accelerates tumor growth and vascularization. Our data demonstrate that platelets stimulate BMDC homing in a VAMP-8-dependent manner, revealing a previously unknown role for platelets as key mediators between hypoxic tissues and the bone marrow during angiogenesis.

Polymorphism A1/A2 in the β3 subunit of integrins αIIb/β3 and αV/β3 is implicated in the risk of development of embryonic and fetal recurrent pregnancy loss (RPL). In 191 women with RPL, polymorphism A1/A2 was statistically significantly associated with RPL at <10 weeks of gestation (29.3% versus 16.4% in controls), but it was much more pronounced in 67 women with RPL between 10 and 20 weeks of gestation (41.8%), illustrating its role in recurrent fetal loss., (Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Accumulating evidence indicates that a sub-population of cancer cells with stem-like properties, termed tumor-initiating cells (T-ICs), exist in many different kinds of malignancies, which have a pivotal role in tumorigenesis, tumor progression, metastasis and post-treatment relapse. However, how the stem-like properties of T-ICs are regulated remains obscure. Our previous study showed that reduction of let-7 microRNA (miRNA) in breast tumor-initiating cells (BT-ICs) contributes to the maintenance of their self-renewal capacity and undifferentiated status. In this study we show the effect of mir-30 reduction on the stem-like features of BT-ICs. Similar to let-7, mir-30 is reduced in BT-ICs, and the protein level of Ubc9 (ubiquitin-conjugating enzyme 9) and ITGB3 (integrin beta3), the target genes of mir-30, is markedly upregulated. Enforced constitutive expression of mir-30 in BT-ICs inhibits their self-renewal capacity by reducing Ubc9, and induces apoptosis through silencing ITGB3. On the contrary, blocking the miRNA with a specific antisense oligonucleotide (ASO) in differentiated breast cancer cells revived their self-renewal capacity. Furthermore, ectopic expression of mir-30 in BT-IC xenografts reduces tumorigenesis and lung metastasis in nonobese diabetic/severe combined immunodeficient mice, whereas blocking mir-30 expression enhances tumorigenesis and metastasis. Together, our data suggest mir-30 as one of the important miRNAs in regulating the stem-like features of T-ICs.

Aim: To assess the significance of the ITGB3 polymorphism at residue 33 (ITGB3 L33P) in the development of chronic oxaliplatin-induced peripheral neuropathy (OXLIPN)., Methods: Fifty-five patients with advanced colorectal cancer were genotyped, using allele-specific primers and sybr green in real-time PCR. Patients had received adjuvant oxaliplatin-based chemotherapy. The severity of the OXLIPN was defined by means of the clinical total neuropathy score (TNSc). Following the discontinuation of treatment, 34/55 patients (61.8%) developed OXLIPN. Grade I neurotoxicity was revealed in 13 (38.2%) patients and grade II neurotoxicity in 21 (61.8%) patients., Results: Patients without OXLIPN (n = 21) were 19% homozygous for C, 33.3% were heterozygous, and 47.7% were homozygous for T. The corresponding percentages for patients developing any grade of OXLIPN (n = 34) were similar. About half of patients (46.1%) with grade I OXLIPN were heterozygotes (CT), 23.1% were CC, and 30.8% were TT. The majority of patients with grade II OXLIPN were TT (66.7%) with the remaining 33.3% being CT. The TT genotype was associated with increased severity of OXLIPN compared to the genotypes containing the C allele (P = 0.044)., Conclusion: The ITGB3 L33P seems to be unrelated to the development of OXLIPN, but it appears to be related to its severity.

Integrin receptors are essential in the regulation of vital cardiac functions, and impaired integrin activity has been associated with cardiac remodeling. Oxidative stress is known to be involved in apoptosis and cardiac remodeling and thus may profoundly influence cardiac function via integrin modulation. The aim of this study was to determine the expression pattern and functional role of integrins in HL-1 cardiomyocytes under conditions of oxidative stress. Gene expression was studied by end-point and real-time PCR; surface protein expression was studied by flow cytometry; integrin knockdown was accomplished by siRNA gene silencing; and apoptosis was studied by annexin V staining and active caspase-3/7 using flow cytometry. Among the various subunits under study (alphav, alpha5, alpha6, and beta1, beta3, beta4, and beta5), the expression of beta3 integrin was significantly increased at both the mRNA and protein levels in cardiomyocytes exposed to 100 micromol/L hydrogen peroxide for 3 h. Gene silencing of beta3 integrin by using siRNA resulted in a 2-fold increase in cardiomyocyte apoptosis upon treatment with hydrogen peroxide. This increase in apoptosis, as measured by annexin V staining, correlated with an increase in active caspase-3/7. Integrin beta3 plays a vital role in preventing cardiomyocyte apoptosis under conditions of oxidative stress.

beta3-Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in beta3-integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on beta3-null endothelial cells. Here we transplanted beta3-null bone marrow (BM) into wild-type (WT) mice to dissect the role of BM beta3-integrin deficiency in pathological angiogenesis. Mice transplanted with beta3-null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow beta3-integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM beta3-integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with beta3-null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with beta3-null bone marrow a significant proportion of tumour blood vessels are non-functional when compared with tumour blood vessels in WT-transplanted controls. Furthermore, beta3-null-transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT-transplanted animals. BM beta3-integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF-induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with beta3-null bone marrow when compared with WT-transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in beta3-null-transplanted mice. In conclusion, although BM beta3-integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM beta3-integrin in VEGF-induced mobilization of bone marrow-derived cells to the peripheral circulation and for the functionality of those vessels in which BM-derived cells become incorporated.

Hantaviruses cause two vascular permeability-based diseases and primarily infect endothelial cells which form the primary fluid barrier of the vasculature. Since hantavirus infections are not lytic, the mechanisms by which hantaviruses cause haemorrhagic fever with renal syndrome (HFRS) or Hantavirus Pulmonary Syndrome (HPS) are indeterminate. HPS is associated with acute pulmonary oedema and HFRS with moderate haemorrhage and renal sequelae, perhaps reflecting the location of vast microvascular beds and endothelial cell reservoirs available for hantavirus infection. Endothelial cells regulate capillary integrity, and hantavirus infection provides a primary means for altering vascular permeability that contributes to pathogenesis. The central importance of endothelial cells in regulating oedema, vascular repair, angiogenesis, immune cell recruitment, platelet deposition as well as gas exchange and solute delivery suggest that a multitude of inputs and cellular responses may be influenced by hantavirus infection and contribute to pathogenic changes in vascular permeability. Here we focus on understanding hantavirus interactions with endothelial cells which are linked to vascular permeability, and provide insight into the contribution of endothelial cell responses in hantavirus pathogenesis.

Several cell surface molecules have been implicated in rotavirus cell entry, however, their individual relevance during this process is unknown. In this work, the expression of integrins alpha2, beta2, and alpha v beta 3, the heat shock cognate protein 70, and of ganglioside GM1 in different cell lines of human and simian origin was correlated with the infectivity of four rotavirus strains. We observed that different combinations of receptor expression correlated with the infectivity of rotavirus strains, suggesting that the participation of several receptors is important for rotavirus infection. To characterize the relevance of integrins alpha2 and alpha v beta 3 in more detail, their expression was silenced using RNA interference. About 80% decrease in the cell content of integrins resulted in 15-30% decrease of infectivity of strains RRV and Wa when measured by a focus-forming assay, while there was no decrease of infectivity when measured by flow cytometry in integrin-deficient cells. Altogether these data suggest that integrins alpha2 and alpha v beta 3 do not play a major role in the rotavirus entry process.

alpha(v)beta(3) integrin was investigated in multiple myeloma in relation to the in vitro osteoclast-like activity of malignant plasma cells. Myeloma cells from patients with skeleton involvement overexpressed alpha(v)beta(3) and produced erosion pits on bone substrates, whereas this effect was not observed by cells from patients with no evidence of bone disease. We therefore explored the alpha(v)beta(3) transcriptional pathway in the bone-resorbing cells. Silencing of beta(3) chain abrogated the ability to produce erosion pits and extracellular signal-regulated kinase 1/2 phosphorylation resulting in the defective function of cFos and nuclear factor activator T cell 1, the terminal effectors of osteoclast activation. A similar defect occurred in constitutively beta(3)-deficient cells from patients with no skeleton disease. Microarray gene analysis of beta(3)(+) myeloma cells showed that several osteoclast-related genes were up-regulated. Their functions include the activation of receptor pathways beta(3) and c-fms that regulate several osteoclast functions. These data emphasize the postulated role of myeloma cells in multiple myeloma bone disease and suggest that their osteoclast-like activity is regulated, at least in vitro, by the beta(3) subunit of the integrin.

Purpose: To investigate the role of integrin beta3 in invasive features of ovarian cancer SKOV3 cells, by comparing different metastatic subclones., Methods: In the present study, two cell subclones, termed as S1 and S21, which possessed high and low metastatic potential, respectively, were isolated and established from human ovarian cancer parental cell line SKOV3 by the limited dilution method. The expressions of integrin alphav, integrin alphavbeta3, integrin beta3, E-cadherin, FAK and ILK in the two cell subclones were compared by means of real-time RT-PCR or flow cytometry. Subsequently, S21 was transfected with siRNA for integrin beta3 and the effects of transfection were examined by methyl thiazolyl tetrazolium (MTT) assay, colony formation assay, Matrigel invasion assay and cell migration assay., Results: The expressions of integrin alphavbeta3, integrin beta3 and E-cadherin were markedly down-regulated in S1; however, there were no significant differences in the expressions of integrin alphav, FAK and ILK beta. Of note, more than 70% knockdown of integrin beta3 expression was obtained by siRNA technique. The integrin beta3-siRNA-transfected cells showed significant increases in cell proliferation, cell migration and invasive activity in contrast with the mock-transfected cells. The expressions of integrin alphavbeta3 and E-cadherin were lower in the integrin beta3-siRNA-transfected cells compared to the mock control., Conclusion: Integrin beta3, like E-cadherin, may be also a suppressor gene down-regulating invasive features of ovarian cancer cells in SKOV3 cell subclones.

Platelet integrin alphaIIbbeta3 plays an essential role in thrombus formation through interactions with adhesive ligands. Successful parenteral blockade of these interactions has validated alphaIIbbeta3 as a therapeutic target in cardiovascular medicine. However, oral alphaIIbbeta3 antagonists have not been successful and there is an unmet need for more effective anti-platelet drugs. Growing evidence points to the cytoplasmic tails of alphaIIb and beta3, and the beta3 tail in particular, as scaffolds for intracellular proteins that mediate inside-out signaling and regulate alphaIIbbeta3 affinity for ligands. Intracellular protein interactions with the integrin cytoplasmic tails also regulate outside-in signals to the actin cytoskeleton. Here we focus on recent studies that illustrate the relevance of the beta3 cytoplasmic tail as a regulatory scaffold in vivo. We speculate that this scaffold or its interacting proteins may serve as therapeutic targets for the development of future anti-thrombotic drugs.

The integrin family of cell surface receptors integrates cell-extracellular matrix interactions with the cell cytoskeleton and signalling across the cell membrane, resulting in an important role in cell adhesion, mobility and migration, proliferation, and survival. Changes in the number and identity of integrin receptors are common in cancer cells resulting in alteration of the ability of malignant cells to interact with the extracellular matrix, and promoting migration as well as facilitating survival outside the tumour normal environment. beta(3) integrins are potentially involved in every step of the metastatic process and expression of both alpha(IIb)beta(3) and alpha(n)beta(3) is correlated with metastatic ability of tumour cells. The recognition of the RGD binding motif common to the disintegrins and natural integrin ligands such as fibrinogen allowed the development of small molecule beta(3) integrin antagonists, progressing from linear peptides containing the RGD sequence to cyclic peptides with well-defined conformation, and hence to small molecule peptidomimetics with improved pharmacological properties. In this review, we summarize the role of the beta(3)-subfamily of integrins when expressed in normal and tumour tissue, the development of small-molecule antagonists of beta(3) integrins and their potential anti-cancer applications.

All but the smallest-diameter axons in the central nervous system are myelinated, but the signals that initiate myelination are unknown. Our prior work has shown that integrin signaling forms part of the cell-cell interactions that ensure only those oligodendrocytes contacting axons survive. Here, therefore, we have asked whether integrins regulate the interactions that lead to myelination. Using homologous recombination to insert a single-copy transgene into the hypoxanthine phosphoribosyl transferase (hprt) locus, we find that mice expressing a dominant-negative beta1 integrin in myelinating oligodendrocytes require a larger axon diameter to initiate timely myelination. Mice with a conditional deletion of focal adhesion kinase (a signaling molecule activated by integrins) exhibit a similar phenotype. Conversely, transgenic mice expressing dominant-negative beta3 integrin in oligodendrocytes display no myelination abnormalities. We conclude that beta1 integrin plays a key role in the axoglial interactions that sense axon size and initiate myelination, such that loss of integrin signaling leads to a delay in myelination of small-diameter axons.

The multicellular nature of metazoans means that all cellular processes need to be tuned by adhesive interactions between cells and their local microenvironment. The spatial organization of cells within tissues requires sophisticated networks of extracellular signals to control their survival and proliferation, movements and positioning, and differentiated function. These cellular characteristics are mediated by multiple inputs from adhesion systems in combination with soluble and developmental signals. In the present review we explore how one class of adhesion receptor, the integrins, co-operate with other types of receptor to control diverse aspects of cell fate. In particular we discuss: (i) how beta3 and beta1 integrins work together with growth factors to control angiogenesis; (ii) how alpha6beta4 integrin co-operates with receptor tyrosine kinases in normal epithelial function and cancer; (iii) the interplay between beta1 integrins and EGF (epidermal growth factor) receptor; (iv) signal integration connecting integrins and cytokine receptors for interleukins, prolactin and interferons; and (v) how integrins and syndecans co-operate in cell migration.

Purpose: During light-induced photoreceptor degeneration, large amounts of cellular debris are formed that must be cleared from the subretinal space. The integrins alphavbeta5 and alphavbeta3 are involved in the normal physiological process of phagocytosis in the retina. This study was conducted to investigate the question of whether the lack of beta5 and/or beta3 integrin subunits might influence the course of retinal degeneration and/or clearance of photoreceptor debris induced by acute exposure to light., Methods: Wild-type, beta5(-/-) and beta3(-/-) single-knockout, and beta3(-/-)/beta5(-/-) Ccl2(-/-)/beta5(-/-) double-knockout mice were exposed to 13,000 lux of white light for 2 hours to induce severe photoreceptor degeneration. Real-time PCR and Western blot analysis were used to analyze gene and protein expression, light- and electron microscopy to judge retinal morphology, and immunofluorescence to study retinal distribution of proteins., Results: Individual or combined deletion of beta3 and beta5 integrin subunits did not affect the pattern of photoreceptor cell loss or the clearance of photoreceptor debris in mice compared with that in wild-type mice. Invading macrophages may contribute to efficient phagocytosis. However, ablation of the MCP-1 gene did not prevent macrophage recruitment. Several chemokines in addition to MCP-1 were induced after light-induced damage that may have compensated for the deletion of MCP-1., Conclusions: Acute clearance of a large amount of cellular debris from the subretinal space involves invading macrophages and does not depend on beta3 and beta5 integrins.

The hemostatic process is a host defense mechanism to preserve the integrity of the closed high pressure circulatory system. This process must remain inactive but poised to minimize extravasation of blood from the vasculature following tissue injury. Given the complexity of the hemostatic mechanism, paradigms developed from biochemical and cell biological approaches have been revisited by studying thrombus formation in a live animal by intravital microscopy. Many of these paradigms have proven accurate, but others need to be reconsidered given the results of whole animal experiments.

Purpose: This study evaluates the proapoptotic function of integrin beta(3) in human hepatocellular carcinoma (HCC)., Experimental Design: The expression of integrin beta(3) in 67 HCC specimens paired with corresponding neighboring nontumorous tissue was studied by quantitative real-time PCR and Western blot. The proapoptotic function of integrin beta(3) in SMMC-7721 human hepatoma cells overexpressing ITGB3 (gene coding integrin beta(3)) was determined through colony formation, serum starvation, and anoikis assay., Results: Compared with neighboring pathologically normal liver tissue, approximately 60% of the HCC specimens showed a significant down-regulated level of integrin beta(3) expression. Transient expression of integrin beta(3) in SMMC-7721 resulted in an enhanced level of apoptosis and suppression of colony formation. Cell growth inhibition on serum/ligand deprivation and incidences of anoikis were remarkably increased in SMMC-7721 with stable expression of integrin beta(3) in comparison with vector control transfectants. In addition, expression of fibrinogen and vitronectin, two native ligands for integrin alpha(v)beta(3) in liver, was inhibited, which was correlated with the decreased integrin beta(3) expression. Replenishing these ligands to the starved SMMC-7721 stable transfectants effectively restored the proapoptotic function of integrin beta(3)., Conclusions: Down-regulation of integrin beta(3) and its ligands in liver is related to the aggressive growth of HCC. Thus, reconstitution of integrin beta(3) in HCC may be a potential therapeutic approach to inhibit aggressive growth of liver cancer.

The cells of origin and mechanisms that underpin tumor heterogeneity in breast cancer are poorly understood. Here, we have examined three mouse models of mammary tumorigenesis (MMTV-wnt-1, MMTV-neu, and p53(+/-)) for changes in their epithelial cell hierarchy during the preneoplastic and neoplastic stages of tumor progression. In preneoplastic tissue, only MMTV-wnt-1 mice showed a perturbation in their epithelial subpopulations. In addition to an expanded mammary stem cell pool, repopulating cells capable of yielding extensive mammary outgrowths in vivo were revealed in the committed luminal progenitor population. These findings indicate that wnt-1 activation induces the appearance of aberrant progenitor cells, and suggest that both mammary stem and progenitor cells can serve as the cellular targets of wnt-1-induced tumorigenesis. In tumors arising in MMTV-wnt-1 tumors, the luminal epithelial progenitor marker CD61/beta3 integrin identified a cancer stem cell (CSC) population that was highly enriched for tumorigenic capability relative to the CD61(-) subset. CD61 expression also defined a CSC subset in 50% of p53(+/-)-derived tumors. No CSCs, however, could be identified in the more homogeneous MMTV-neu/erbB2 model, suggesting an alternative model of tumorigenesis. Overall, our findings show the utility of the progenitor marker CD61 in the identification of CSCs that sustain specific mammary tumors.

Recent morphological studies have suggested that osteocyte processes are directly attached at discrete locations along the canalicular wall by beta3 integrins at the apex of infrequent, previously unrecognized, canalicular projections. This discovery has led to a new paradigm for the initiation of intracellular signaling, which provides a possible long sought after molecular mechanism for the initiation of intracellular signaling in bone cells. The quantitative feasibility of this hypothesis is explored with a detailed theoretical model, which predicts that axial strains due to the sliding of actin microfilaments about the fixed integrin attachments are in order of magnitude larger than the radial strains in the previously proposed strain amplification theory and two orders of magnitude greater than whole tissue strains.

At synapses, cell adhesion molecules (CAMs) provide the molecular framework for coordinating signaling events across the synaptic cleft. Among synaptic CAMs, the integrins, receptors for extracellular matrix proteins and counterreceptors on adjacent cells, are implicated in synapse maturation and plasticity and memory formation. However, little is known about the molecular mechanisms of integrin action at central synapses. Here, we report that postsynaptic beta3 integrins control synaptic strength by regulating AMPA receptors (AMPARs) in a subunit-specific manner. Pharmacological perturbation targeting beta3 integrins promotes endocytosis of GluR2-containing AMPARs via Rap1 signaling, and expression of beta3 integrins produces robust changes in the abundance and composition of synaptic AMPARs without affecting dendritic spine structure. Importantly, homeostatic synaptic scaling induced by activity deprivation elevates surface expression of beta3 integrins, and in turn, beta3 integrins are required for synaptic scaling. Our findings demonstrate a key role for integrins in the feedback regulation of excitatory synaptic strength.

Junctional epidermolysis bullosa (JEB) is an inherited mechanobullous disease characterized by reduced adherence of the epidermal keratinocytes to the underlying dermis, and is often caused by the absence of functional laminin 332 due to the lack or dysfunction of its beta3 chain. As there are no specific therapies for JEB, we tested whether a protein replacement strategy could be applicable for the restoration of the laminin 332 assembly and reversion of the JEB phenotype in human keratinocytes that lack beta3 subunit. Here, we developed the protocol for production and purification of the biologically active recombinant beta3 chain. Next, we demonstrated that delivery of recombinant beta3 polypeptide into the endoplasmic reticulum of the immortalized beta3-null keratinocytes led to the restoration of the laminin 332 assembly, secretion, and deposition into the basement membrane zone, as confirmed by Western blot analysis, confocal immunofluorescent microscopy in vitro, and on cultured organotypic human JEB skin reconstructs. Although the amount of laminin 332 produced by protein-treated beta3-null keratinocytes is lower than that in normal human keratinocytes, our results demonstrate the applicability of the recombinant proteins for JEB treatment and open new perspectives for the development of novel therapeutics for this inherited, currently intractable, skin disorder.

A combination of experimental and computational approaches was used to provide a structural context for the role of the beta3 integrin subunit ligand-associated metal binding site (LIMBS) in the binding of physiological ligands to beta3 integrins. Specifically, we have carried out (1) adhesion assays on cells expressing normal alphaIIbeta3, normal alphaVbeta3, or the corresponding beta3 D217A LIMBS mutants; and (2) equilibrium and nonequilibrium (steered) molecular dynamics (MD) simulations of eptifibatide in complex with either a fully hydrated normal alphaIIbeta3 integrin fragment (alphaIIb beta-propeller and the beta3 betaA (I-like), hybrid, and PSI domains) or the equivalent beta3 D217A mutant. Normal alphaIIbeta3 expressing cells adhered to immobilized fibrinogen and echistatin, whereas cells expressing the alphaIIbeta3 D217A LIMBS mutant failed to adhere to either ligand. Similarly, the equivalent alphaVbeta3 mutant was unable to support adhesion to vitronectin or fibrinogen. The alphaIIbeta3 D217A mutation increased the binding of mAb AP5, which recognizes a ligand-induced binding site (LIBS) in the beta3 PSI domain, indicating that this mutation induced allosteric changes in the protein. Steered MD simulating the unbinding of eptifibatide from either normal alphaIIbeta3 or the equivalent beta3 D217A mutant suggested that the reduction in ligand binding caused by the LIMBS mutant required the loss of both the LIMBS and the metal ion-dependent adhesion site (MIDAS) metal ions. Our computational results indicate that the LIMBS plays a crucial role in ligand binding to alphaIIbeta3 by virtue of its effects on the coordination of the MIDAS., ((c) 2008 Wiley-Liss, Inc.)