13 results on '"Bucala, R."'
Search Results
2. Sustained mitogen-activated protein kinase (MAPK) and cytoplasmic phospholipase A2 activation by macrophage migration inhibitory factor (MIF). Regulatory role in cell proliferation and glucocorticoid action.
- Author
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Mitchell, R A, Metz, C N, Peng, T, and Bucala, R
- Abstract
Macrophage migration inhibitory factor (MIF) is an important pro-inflammatory mediator with the unique ability to counter-regulate the inhibitory effects of glucocorticoids on immune cell activation. MIF is released from cells in response to glucocorticoids, certain pro-inflammatory stimuli, and mitogens and acts to regulate glucocorticoid action on the ensuing inflammatory response. To gain insight into the molecular mechanism of MIF action, we have examined the role of MIF in the proliferation and intracellular signaling events of the well characterized, NIH/3T3 fibroblast cell line. Both endogenously secreted and exogenously added MIFs stimulate the proliferation of NIH/3T3 cells, and this response is associated with the activation of the p44/p42 extracellular signal-regulated (ERK) mitogen-activated protein kinases (MAP). The MIF-induced activation of these kinases was sustained for a period of at least 24 h and was dependent upon protein kinase A activity. We further show that MIF regulates cytosolic phospholipase A2 activity via a protein kinase A and ERK dependent pathway and that the glucocorticoid suppression of cytokine-induced cytoplasmic phospholipase A2 activity and arachidonic acid release can be reversed by the addition of recombinant MIF. These studies indicate that the sustained activation of p44/p42 MAP kinase and subsequent arachidonate release by cytoplasmic phospholipase A2 are important features of the immunoregulatory and intracellular signaling events initiated by MIF and provide the first insight into the mechanisms that underlie the pro-proliferative and inflammatory properties of this mediator.
- Published
- 1999
3. Hydroxyalkenal formation induced by advanced glycosylation of low density lipoprotein.
- Author
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Al-Abed, Y, Liebich, H, Voelter, W, and Bucala, R
- Abstract
Advanced glycosylation end products (AGEs) have been identified to be present on both the apolipoprotein and lipid components of low density lipoprotein (LDL) and to act to prevent its recognition and uptake by high affinity, tissue LDL receptors. Lipid-linked AGEs form readily in vitro by the covalent addition of glucose to the amine-containing head groups of phospholipids. This process is accompanied by oxidation of the unsaturated fatty acid side chains and occurs in the absence of exogenously added transition metals or free radical generating systems, suggesting that AGE formation may contribute significantly to lipid oxidation in vivo. To assess more precisely the chemical basis of AGE-induced oxidative modification, we performed gas chromatography-mass spectrometry analysis of the lipid products which form over time during LDL-advanced glycosylation in vitro. Negative ion chemical ionization mass spectroscopy of two major compounds that were identified were consistent with the structures of the fatty acid oxidation products 4-hydroxyhexenal and 4-hydroxynonenal. These data support the concept that AGE formation in close proximity to unsaturated fatty acyl groups leads to lipid oxidation and provide additional evidence that advanced glycosylation is an important pathogenic modification of the LDL particle in vivo.
- Published
- 1996
4. Detection of an advanced glycosylation product bound to protein in situ.
- Author
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Chang, J C, Ulrich, P C, Bucala, R, and Cerami, A
- Abstract
Protein amino groups can react with glucose without the aid of enzymes to form stable Amadori products containing 1-amino-1-deoxyketose residues. These adducts can undergo subsequent rearrangements and dehydrations to form various brown and fluorescent pigments. Recently, a chromophore, 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI), was isolated from acid hydrolysates of bovine serum albumin (BSA) and poly-L-lysine which had been incubated with glucose. To confirm the presence of FFI in situ, a radioimmunoassay was developed. A derivative of FFI, 4-furanyl-2-furoyl-1H-imidazole-1-hexanoic acid, was coupled to BSA and used to immunize rabbits. A radioactive FFI derivative was synthesized by reaction of 2-furyl-glyoxal with gamma-amino-[2,3-3H]butyric acid to form FFI-[3H]butyric acid. The resultant antiserum showed binding affinity to FFI and cross-reactivity for related compounds. FFI bound to proteins was liberated by acid hydrolysis or digestion by proteinase K prior to measurement. A linear relationship was seen between the amount of FFI equivalent detected and the amount of acid hydrolysate or enzymatic digest assayed. Poly-L-lysine and BSA incubated with glucose showed a time-dependent increase in the amounts of fluorescence and FFI equivalence. The detection of a time-related increase in the amount of FFI or a closely related structure in enzymatically digested proteins implicates it as an in situ product on proteins which have undergone the Maillard reaction with glucose. Of physiological significance is that FFI could also be detected in human globin and serum albumin from normal individuals. Thus, proteins exposed to glucose in vitro and in vivo form FFI as an in situ glycosylation product.
- Published
- 1985
- Full Text
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5. Identification of the major site of apolipoprotein B modification by advanced glycosylation end products blocking uptake by the low density lipoprotein receptor.
- Author
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Bucala, R, Mitchell, R, Arnold, K, Innerarity, T, Vlassara, H, and Cerami, A
- Abstract
Advanced glycosylation end products (AGEs) arise from glucose-derived Amadori products and have been implicated in the pathogenesis of diabetic vascular disease. We recently reported the presence of an AGE-modified form of low density lipoprotein (LDL) that circulates in high amounts in patients with diabetes or renal insufficiency and that exhibits impaired plasma clearance kinetics. We utilized AGE-specific antibodies to identify the major sites of AGE modification within protease-digested preparations of apolipoprotein B that impair the binding of the AGE-modified form of LDL by human fibroblast LDL receptors. The predominant site of AGE immunoreactivity was found to lie within a single, 67-amino acid region located 1791 residues NH2-terminal of the putative LDL receptor binding domain. These data point to the high reactivity and specificity of this site for AGE formation and provide further evidence for important structural interactions between the LDL receptor binding domain and remote regions of the apolipoprotein B polypeptide.
- Published
- 1995
6. A small-molecule allele-selective transcriptional inhibitor of the MIF immune susceptibility locus.
- Author
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Li J, Leng L, Pantouris G, Manjula R, Piecychna M, Abriola L, Hu B, Lolis E, Armstrong ME, Donnelly SC, and Bucala R
- Subjects
- Humans, Alleles, Microsatellite Repeats, Promoter Regions, Genetic, Animals, Macrophages metabolism, Macrophages immunology, Macrophages drug effects, Transcription, Genetic drug effects, Mice, CCAAT-Enhancer-Binding Proteins genetics, CCAAT-Enhancer-Binding Proteins metabolism, Macrophage Migration-Inhibitory Factors genetics, Macrophage Migration-Inhibitory Factors antagonists & inhibitors, Macrophage Migration-Inhibitory Factors metabolism, Macrophage Migration-Inhibitory Factors immunology, Intramolecular Oxidoreductases genetics, Intramolecular Oxidoreductases antagonists & inhibitors, Intramolecular Oxidoreductases metabolism
- Abstract
Functional variants of the gene for the cytokine macrophage migration inhibitory factor (MIF) are defined by a 4-nucleotide promoter microsatellite (-794 CATT
5-8 , rs5844572) and confer risk for autoimmune, infectious, and oncologic diseases. We describe herein the discovery of a prototypic, small molecule inhibitor of MIF transcription with selectivity for high microsatellite repeat number and correspondingly high gene expression. Utilizing a high-throughput luminescent proximity screen, we identify 1-carbomethoxy-5-formyl-4,6,8-trihydroxyphenazine (CMFT) to inhibit the functional interaction between the transcription factor ICBP90 (namely, UHRF1) and the MIF -794 CATT5-8 promoter microsatellite. CMFT inhibits MIF mRNA expression in a -794 CATT5-8 length-dependent manner with an IC50 of 470 nM, and preferentially reduces ICBP90-dependent MIF mRNA and protein expression in high-genotypic versus low-genotypic MIF-expressing macrophages. RNA expression analysis also showed CMFT to downregulate MIF-dependent, inflammatory gene expression with little evidence of off-target metabolic toxicity. These findings provide proof-of-concept for advancing the pharmacogenomic development of precision-based MIF inhibitors for diverse autoimmune and inflammatory conditions., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
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7. A selective small-molecule inhibitor of macrophage migration inhibitory factor-2 (MIF-2), a MIF cytokine superfamily member, inhibits MIF-2 biological activity.
- Author
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Tilstam PV, Pantouris G, Corman M, Andreoli M, Mahboubi K, Davis G, Du X, Leng L, Lolis E, and Bucala R
- Subjects
- Humans, Inflammation enzymology, Inflammation metabolism, Intramolecular Oxidoreductases chemistry, MSH Release-Inhibiting Hormone chemistry, Neoplasms enzymology, Neoplasms metabolism, Protein Structure, Secondary, Signal Transduction, Intramolecular Oxidoreductases metabolism, MSH Release-Inhibiting Hormone metabolism
- Abstract
Cytokine macrophage migration inhibitory factor-2 (MIF-2 or D-dopachrome tautomerase) is a recently characterized second member of the MIF cytokine superfamily in mammalian genomes. MIF-2 shares pro-inflammatory and tumorigenic properties with the clinical target MIF (MIF-1), but the precise contribution of MIF-2 to immune physiology or pathology is unclear. Like MIF-1, MIF-2 has intrinsic keto-enol tautomerase activity and mediates biological functions by engaging the cognate, common MIF family receptor CD74. Evidence that the catalytic site of MIF family cytokines has a structural role in receptor binding has prompted exploration of tautomerase inhibitors as potential biological antagonists and therapeutic agents, although few catalytic inhibitors inhibit receptor activation. Here we describe the discovery and biochemical characterization of a selective small-molecule inhibitor of MIF-2. An in silico screen of 1.6 million compounds targeting the MIF-2 tautomerase site yielded several hits for potential catalytic inhibitors of MIF-2 and identified 4-(3-carboxyphenyl)-2,5-pyridinedicarboxylic acid (4-CPPC) as the most functionally potent compound. We found that 4-CPPC has an enzymatic IC
50 of 27 μm and 17-fold selectivity for MIF-2 versus MIF-1. An in vitro binding assay for MIF-1/MIF-2 to the CD74 ectodomain (sCD74) indicated that 4-CPPC inhibits MIF-2-CD74 binding in a dose-dependent manner (0.01-10 μm) without influencing MIF-1-CD74 binding. Notably, 4-CPPC inhibited MIF-2-mediated activation of CD74 and reduced CD74-dependent signal transduction. These results open opportunities for development of more potent and pharmacologically auspicious MIF-2 inhibitors to investigate the distinct functions of this MIF family member in vivo ., (© 2019 Tilstam et al.)- Published
- 2019
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8. A novel allosteric inhibitor of macrophage migration inhibitory factor (MIF).
- Author
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Bai F, Asojo OA, Cirillo P, Ciustea M, Ledizet M, Aristoff PA, Leng L, Koski RA, Powell TJ, Bucala R, and Anthony KG
- Subjects
- Allosteric Regulation drug effects, Antigens, Differentiation, B-Lymphocyte chemistry, Cells, Cultured, Fibroblasts cytology, Histocompatibility Antigens Class II chemistry, Humans, Protein Binding drug effects, Protein Structure, Quaternary, Antigens, Differentiation, B-Lymphocyte metabolism, Azo Compounds chemistry, Azo Compounds pharmacology, Fibroblasts metabolism, Histocompatibility Antigens Class II metabolism, Intramolecular Oxidoreductases antagonists & inhibitors, Intramolecular Oxidoreductases chemistry, Intramolecular Oxidoreductases metabolism, Macrophage Migration-Inhibitory Factors antagonists & inhibitors, Macrophage Migration-Inhibitory Factors chemistry, Macrophage Migration-Inhibitory Factors metabolism, Trypan Blue chemistry, Trypan Blue pharmacology
- Abstract
Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.
- Published
- 2012
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9. Ribosomal protein S19 interacts with macrophage migration inhibitory factor and attenuates its pro-inflammatory function.
- Author
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Filip AM, Klug J, Cayli S, Fröhlich S, Henke T, Lacher P, Eickhoff R, Bulau P, Linder M, Carlsson-Skwirut C, Leng L, Bucala R, Kraemer S, Bernhagen J, and Meinhardt A
- Subjects
- Animals, Apoptosis genetics, Autoimmune Diseases genetics, Autoimmune Diseases metabolism, Cell Adhesion genetics, Humans, Inflammation genetics, Inflammation metabolism, Macrophage Migration-Inhibitory Factors genetics, Mice, Mutation, NIH 3T3 Cells, Oxidoreductases genetics, Oxidoreductases metabolism, Protein Binding genetics, Rats, Receptors, Interleukin-8B agonists, Receptors, Interleukin-8B genetics, Ribosomal Proteins genetics, Sepsis genetics, Sepsis metabolism, Surface Plasmon Resonance, Endothelial Cells metabolism, Macrophage Migration-Inhibitory Factors metabolism, Monocytes metabolism, Receptors, Interleukin-8B metabolism, Ribosomal Proteins metabolism
- Abstract
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in the pathogenesis of inflammatory disorders such as infection, sepsis, and autoimmune disease. MIF exists preformed in cytoplasmic pools and exhibits an intrinsic tautomerase and oxidoreductase activity. MIF levels are elevated in the serum of animals and patients with infection or different inflammatory disorders. To elucidate how MIF actions are controlled, we searched for endogenous MIF-interacting proteins with the potential to interfere with key MIF functions. Using in vivo biotin-tagging and endogenous co-immunoprecipitation, the ribosomal protein S19 (RPS19) was identified as a novel MIF binding partner. Surface plasmon resonance and pulldown experiments with wild type and mutant MIF revealed a direct physical interaction of the two proteins (K(D) = 1.3 x 10(-6) m). As RPS19 is released in inflammatory lesions by apoptotic cells, we explored whether it affects MIF function and inhibits its binding to receptors CD74 and CXCR2. Low doses of RPS19 were found to strongly inhibit MIF-CD74 interaction. Furthermore, RPS19 significantly compromised CXCR2-dependent MIF-triggered adhesion of monocytes to endothelial cells under flow conditions. We, therefore, propose that RPS19 acts as an extracellular negative regulator of MIF.
- Published
- 2009
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10. Macrophage migration inhibitory factor induces B cell survival by activation of a CD74-CD44 receptor complex.
- Author
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Gore Y, Starlets D, Maharshak N, Becker-Herman S, Kaneyuki U, Leng L, Bucala R, and Shachar I
- Subjects
- Animals, Antigens, Differentiation, B-Lymphocyte chemistry, Antigens, Differentiation, B-Lymphocyte genetics, B-Lymphocytes cytology, B-Lymphocytes immunology, Base Sequence, Cell Survival drug effects, DNA Primers genetics, Histocompatibility Antigens Class II chemistry, Histocompatibility Antigens Class II genetics, Hyaluronan Receptors chemistry, Hyaluronan Receptors genetics, In Vitro Techniques, Intramolecular Oxidoreductases metabolism, Macrophage Migration-Inhibitory Factors metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Multiprotein Complexes, Recombinant Proteins metabolism, Recombinant Proteins pharmacology, Signal Transduction drug effects, Antigens, Differentiation, B-Lymphocyte metabolism, B-Lymphocytes drug effects, Histocompatibility Antigens Class II metabolism, Hyaluronan Receptors metabolism, Intramolecular Oxidoreductases pharmacology, Macrophage Migration-Inhibitory Factors pharmacology
- Abstract
Macrophage migration inhibitory factor (MIF) is an upstream activator of innate immunity that regulates subsequent adaptive responses. It was previously shown that in macrophages, MIF binds to a complex of CD74 and CD44, resulting in initiation of a signaling pathway. In the current study, we investigated the role of MIF in B cell survival. We show that in B lymphocytes, MIF initiates a signaling cascade that involves Syk and Akt, leading to NF-kappaB activation, proliferation, and survival in a CD74- and CD44-dependent manner. Thus, MIF regulates the adaptive immune response by maintaining the mature B cell population.
- Published
- 2008
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11. Structural and functional characterization of a secreted hookworm Macrophage Migration Inhibitory Factor (MIF) that interacts with the human MIF receptor CD74.
- Author
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Cho Y, Jones BF, Vermeire JJ, Leng L, DiFedele L, Harrison LM, Xiong H, Kwong YK, Chen Y, Bucala R, Lolis E, and Cappello M
- Subjects
- Amino Acid Sequence, Ancylostoma, Animals, Cricetinae, Cytokines metabolism, Humans, Intestinal Mucosa metabolism, Mesocricetus, Molecular Sequence Data, Protein Binding, Rabbits, Recombinant Proteins chemistry, Sequence Homology, Amino Acid, Antigens, Differentiation, B-Lymphocyte chemistry, Antigens, Differentiation, B-Lymphocyte physiology, Histocompatibility Antigens Class II chemistry, Histocompatibility Antigens Class II physiology, Macrophage Migration-Inhibitory Factors metabolism
- Abstract
Hookworms, parasitic nematodes that infect nearly one billion people worldwide, are a major cause of anemia and malnutrition. We hypothesize that hookworms actively manipulate the host immune response through the production of specific molecules designed to facilitate infection by larval stages and adult worm survival within the intestine. A full-length cDNA encoding a secreted orthologue of the human cytokine, Macrophage Migration Inhibitory Factor (MIF) has been cloned from the hookworm Ancylostoma ceylanicum. Elucidation of the three-dimensional crystal structure of recombinant AceMIF (rAceMIF) revealed an overall structural homology with significant differences in the tautomerase sites of the human and hookworm proteins. The relative bioactivities of human and hookworm MIF proteins were compared using in vitro assays of tautomerase activity, macrophage migration, and binding to MIF receptor CD74. The activity of rAceMIF was not inhibited by the ligand ISO-1, which was previously determined to be an inhibitor of the catalytic site of human MIF. These data define unique immunological, structural, and functional characteristics of AceMIF, thereby establishing the potential for selectively inhibiting the hookworm cytokine as a means of reducing parasite survival and disease pathogenesis.
- Published
- 2007
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12. Pigment epithelium-derived factor inhibits advanced glycation end product-induced retinal vascular hyperpermeability by blocking reactive oxygen species-mediated vascular endothelial growth factor expression.
- Author
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Yamagishi S, Nakamura K, Matsui T, Inagaki Y, Takenaka K, Jinnouchi Y, Yoshida Y, Matsuura T, Narama I, Motomiya Y, Takeuchi M, Inoue H, Yoshimura A, Bucala R, and Imaizumi T
- Subjects
- Adult, Animals, Diabetic Retinopathy physiopathology, Electrophysiology, Female, Humans, Male, Middle Aged, Rats, Reference Values, Signal Transduction physiology, Capillary Permeability physiology, Eye Proteins physiology, Glycation End Products, Advanced antagonists & inhibitors, Nerve Growth Factors physiology, Reactive Oxygen Species metabolism, Retinal Vessels physiology, Serpins physiology, Vascular Endothelial Growth Factor A genetics
- Abstract
Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis, suggesting that loss of PEDF contributes to proliferative diabetic retinopathy. However, the role of PEDF against retinal vascular hyperpermeability remains to be elucidated. We investigated here whether and how PEDF could inhibit the advanced glycation end product (AGE) signaling to vascular hyperpermeability. Intravenous administration of AGEs to normal rats not only increased retinal vascular permeability by stimulating vascular endothelial growth factor (VEGF) expression but also decreased retinal PEDF levels. Simultaneous treatments with PEDF inhibited the AGE-elicited VEGF-mediated permeability by down-regulating mRNA levels of p22(phox) and gp91(phox), membrane components of NADPH oxidase, and subsequently decreasing retinal levels of an oxidative stress marker, 8-hydroxydeoxyguanosine. PEDF also inhibited the AGE-induced vascular hyperpermeability evaluated by transendothelial electrical resistance by suppressing VEGF expression. Furthermore, PEDF decreased reactive oxygen species (ROS) generation in AGE-exposed endothelial cells by suppressing NADPH oxidase activity via down-regulation of mRNA levels of p22(PHOX) and gp91(PHOX). This led to blockade of the AGE-elicited Ras activation and NF-kappaB-dependent VEGF gene induction in endothelial cells. These results indicate that the central mechanism for PEDF inhibition of the AGE signaling to vascular permeability is by suppression of NADPH oxidase-mediated ROS generation and subsequent VEGF expression. Substitution of PEDF may offer a promising strategy for halting the development of diabetic retinopathy.
- Published
- 2006
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13. Adhesion-dependent signaling by macrophage migration inhibitory factor (MIF).
- Author
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Liao H, Bucala R, and Mitchell RA
- Subjects
- Animals, Autocrine Communication, Cells, Cultured, Cyclin D1 metabolism, Cyclin-Dependent Kinase 4, Cyclin-Dependent Kinases metabolism, E2F Transcription Factors, Enzyme Activation, Extracellular Matrix metabolism, Fibroblasts cytology, Fibronectins metabolism, Genes, Reporter, Integrins metabolism, Macrophage Migration-Inhibitory Factors genetics, Mice, Mice, Knockout, Mitogen-Activated Protein Kinases metabolism, Protein Kinase C antagonists & inhibitors, Protein Kinase C metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Retinoblastoma Protein genetics, Retinoblastoma Protein metabolism, Transcription Factors genetics, Transcription Factors metabolism, Transcription, Genetic, Cell Adhesion physiology, Cell Cycle Proteins, Cell Movement physiology, DNA-Binding Proteins, Fibroblasts metabolism, MAP Kinase Signaling System physiology, Macrophage Migration-Inhibitory Factors metabolism, Proto-Oncogene Proteins
- Abstract
Proper stimulation of cell cycle progression and DNA synthesis requires cooperating signals from integrin and growth factor receptors. We previously found that the proinflammatory peptide, macrophage migration inhibitory factor (MIF), functions as an autocrine mediator of growth factor-dependent ERK MAP kinase activation and cell cycle progression. We now report that MIF secretion is induced by cell adhesion to fibronectin in quiescent mouse fibroblasts. Adhesion-mediated release of MIF subsequently promotes integrin-dependent activation of MAP kinase, cyclin D1 expression, and DNA synthesis. Secretion of MIF requires protein kinase C activity, and recombinant MIF reconstitutes the activation of MAP kinases in the presence of protein kinase C inhibition. Finally, we show that cells deficient in MIF have significantly higher retinoblastoma tumor suppressor and lower E2F transcriptional activities. These results suggest that MIF is an important autocrine mediator of adhesion-dependent signaling events and may provide mechanistic insight into how MIF regulates proliferative and oncogenic processes.
- Published
- 2003
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