ABSTRACT
Hepatic stellate cells (HSCs) are thought to play key roles in the development of liver fibrosis. Extensive evidence has established the concept that αV integrins are involved in the activation of latent transforming growth factor ß (TGF-ß), a master regulator of the fibrotic signaling cascade. Based on mRNA and protein expression profiling data, we found that αVß1 integrin is the most abundant member of the αV integrin family in either quiescent or TGF-ß1-activated primary human HSCs. Unexpectedly, either a selective αVß1 inhibitor, Compound 8 (C8), or a pan-αV integrin inhibitor, GSK3008348, decreased TGF-ß1-activated procollagen I production in primary human HSCs, in which the role of ß1 integrin was confirmed by ITGB1 siRNA. In contrast with an Activin receptor-like kinase 5 (Alk5) inhibitor, C8 and GSK3008348 failed to inhibit TGF-ß1 induced SMAD3 and SMAD2 phosphorylation, but inhibited TGF-ß-induced phosphorylation of ERK1/2 and STAT3, suggesting that αVß1 integrin is involved in non-canonical TGF-ß signaling pathways. Consistently, ITGB1 siRNA significantly decreased phosphorylation of ERK1/2. Furthermore, a selective inhibitor of MEK1/2 blocked TGF-ß1 induced phosphorylation of ERK1/2 and decreased TGF-ß1 induced procollagen I production, while a specific inhibitor of STAT3 had no effect on TGF-ß1 induced procollagen I production. Taken together, current data indicate that αVß1 integrin can regulate TGF-ß signaling independent of its reported role in activating latent TGF-ß. Our data further support that αVß1 inhibition is a promising therapeutic target for the treatment of liver fibrosis.
Subject(s)
Hepatic Stellate Cells/metabolism , Integrin alpha5beta1/genetics , Procollagen/genetics , Receptor, Transforming Growth Factor-beta Type I/genetics , Smad2 Protein/genetics , Transforming Growth Factor beta1/genetics , Butyrates/pharmacology , Gene Expression Regulation , Hepatic Stellate Cells/cytology , Hepatic Stellate Cells/drug effects , Humans , Integrin alpha5beta1/antagonists & inhibitors , Integrin alpha5beta1/metabolism , MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/genetics , MAP Kinase Kinase 2/metabolism , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Naphthyridines/pharmacology , Phosphorylation/drug effects , Primary Cell Culture , Procollagen/metabolism , Pyrazoles/pharmacology , Pyrrolidines/pharmacology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptor, Transforming Growth Factor-beta Type I/metabolism , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Signal Transduction , Smad2 Protein/metabolism , Smad3 Protein/genetics , Smad3 Protein/metabolism , Transforming Growth Factor beta1/metabolismABSTRACT
In the course of a fragment screening campaign by in silico docking followed by X-ray crystallography, a novel binding site for migration inhibitory factor (MIF) inhibitors was demonstrated. The site is formed by rotation of the side-chain of Tyr-36 to reveal a surface binding site in MIF that is hydrophobic and surrounded by aromatic side-chain residues. The crystal structures of two small inhibitors that bind to this site and of a quinolinone inhibitor, that spans the canonical deep pocket near Pro-1 and the new surface binding site, have been solved. These results suggest new opportunities for structure-based design of MIF inhibitors.
Subject(s)
Enzyme Inhibitors/pharmacology , Macrophage Migration-Inhibitory Factors/antagonists & inhibitors , Binding Sites , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Macrophage Migration-Inhibitory Factors/chemistry , Macrophage Migration-Inhibitory Factors/metabolism , Models, Molecular , Molecular StructureABSTRACT
Biochemical and X-ray crystallographic studies confirmed that hydroxyquinoline derivatives identified by virtual screening were actually covalent inhibitors of the MIF tautomerase. Adducts were formed by N-alkylation of the Pro-1 at the catalytic site with a loss of an amino group of the inhibitor.
Subject(s)
Drug Discovery , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/pharmacology , Hydroxyquinolines/pharmacology , Intramolecular Oxidoreductases/antagonists & inhibitors , Macrophage Migration-Inhibitory Factors/antagonists & inhibitors , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Hydroxyquinolines/chemical synthesis , Hydroxyquinolines/chemistry , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
OBJECTIVE: Previous studies have demonstrated that inhibition of c-Jun N-terminal kinase (JNK) decreases joint destruction in the rat adjuvant arthritis model. The present study was undertaken to investigate whether selective loss of JNK-2 function decreases joint destruction in JNK-2 knockout mice, in order to determine the role of this isoform in inflammatory arthritis. METHODS: Passive collagen-induced arthritis (CIA) was induced in Jnk2(-/-) and wild-type mice by administering anti-type II collagen antibodies. Arthritis was assessed daily using a semiquantitative clinical scoring system. Fibroblast-like synoviocytes (FLS) were prepared from Jnk2(-/-) and wild-type mice, and JNK protein expression was determined by Western blot analysis. Matrix metalloproteinase 13 (MMP-13) expression was determined by Northern blot analysis, and activator protein 1 (AP-1) binding activity by electromobility shift assay (EMSA). RESULTS: The JNK protein level in Jnk2(-/-) mice with CIA was 22% of that in wild-type mice with CIA (P < 0.001), and mainly the 46-kd isoform was expressed in the former group. Surprisingly, clinical arthritis was slightly more severe in the Jnk2(-/-) mice. Histologic scores for synovial inflammation were not significantly different. However, Safranin O-stained sections from the Jnk2(-/-) mice exhibited significantly less joint damage. Although joint destruction was decreased in Jnk2(-/-) mice with CIA, EMSA and Northern blot analysis of total joint extracts revealed similar levels of AP-1 binding and MMP-13 expression in Jnk2(-/-) and wild-type mice. The lack of correlation with AP-1 activity and MMP expression was probably because non-FLS cells in the joint may express more JNK-1 than do FLS. CONCLUSION: JNK-2 is a determinant of matrix degradation, but it has little effect on inflammation in arthritis. Complete inhibition of MMP expression and joint destruction will likely require combined JNK-1 and JNK-2 inhibition.
Subject(s)
Arthritis, Experimental/metabolism , Arthritis, Experimental/pathology , Joints/pathology , Mitogen-Activated Protein Kinases/deficiency , Animals , Collagenases/genetics , Gene Expression , JNK Mitogen-Activated Protein Kinases , Mice , Mice, Knockout/genetics , Mitogen-Activated Protein Kinase 9 , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Protein Isoforms/deficiency , Protein Isoforms/genetics , Transcription Factor AP-1/metabolismABSTRACT
The role of the tumor suppressor p53 as a key regulator of inflammation was examined in murine collagen-induced arthritis (CIA), a model of rheumatoid arthritis. Wild-type DBA/1 mice develop progressive arthritis in this model, in which p53 expression and apoptosis are evident in the synovial cells. In contrast, the joints of p53(-/-) DBA/1 animals with CIA showed increased severity of arthritis using clinical and histological scoring methods with almost no apoptosis. Consistent with this, collagenase-3 expression and cytokine production (interleukin-1 and interleukin-6) in the joints of p53(-/-) mice with CIA were significantly greater than in wild-type mice. Anti-collagen antibody titers, however, were not different. Therefore, p53 expression occurs during inflammation and acts to suppress local inflammatory responses. Because mutations in p53 have been described in the synovial membrane of rheumatoid arthritis patients, the loss of p53 function in synoviocytes or other cells in the joint because of dominant-negative mutations might contribute to invasion and destruction of the joint in this disease.
