Involvement of αvβ5 integrin–mediated activation of latent transforming growth factor β1 in autocrine transforming growth factor β signaling in systemic sclerosis fibroblasts

Abstract

Objective. To confirm the involvement of ␣v␤5 in the self-activation system in systemic sclerosis (SSc) fibroblasts.Methods. Levels of ␣v␤5 expression were analyzed by immunoprecipitation. The promoter activity of the human ␣2(I) collagen gene was determined by transient transfection assay. Phosphorylation levels and DNA binding ability of Smad3 were investigated by immunoprecipitation and DNA affinity precipitation, respectively. The localization of active transforming growth factor ␤ (TGF␤) was determined by coculture assay using TMLC cells (mink lung epithelial reporter cells that stably express a portion of the plasminogen activator inhibitor 1 promoter). The morphologic features of cells were determined by immunofluorescence analysis.Results. Levels of ␣v␤5 expression were significantly elevated in SSc fibroblasts compared with normal fibroblasts. Treatment with anti-␣v␤5 antibody or ␤5 antisense oligonucleotide significantly reduced human ␣2(I) collagen gene promoter activity in SSc fibroblasts. In SSc fibroblasts pretreated with TGF␤1 antisense oligonucleotide, the exogenous latent TGF␤1 stimulation significantly increased human ␣2(I) collagen gene promoter activity; this effect was significantly reduced in the presence of anti-␣v␤5 antibody. Phosphorylation levels and DNA binding ability of Smad3 in SSc fibroblasts were significantly reduced by treatment with ␤5 antisense oligonucleotide. The luciferase activity of TMLC cells cocultured with SSc fibroblasts was significantly elevated compared with that of TMLC cells cocultured with normal fibroblasts and was significantly reduced in the presence of anti-␣v␤5 antibody. Anti␣v␤5 antibody reversed the myofibroblastic features of SSc fibroblasts.Conclusion. Up-regulated expression of ␣v␤5 contributes to the establishment of autocrine TGF␤ signaling in SSc fibroblasts through activation of endogenous latent TGF␤1.Scleroderma (systemic sclerosis [SSc]) is an acquired disorder that typically results in fibrosis of the skin and internal organs (1). Previous findings indicate that the pathogenesis of this disorder includes inflammation, autoimmune attack, and vascular damage, leading to fibroblast activation (2). However, cultured SSc fibroblasts, which are free of such environmental factors, continue to produce excessive amounts of extracellular matrix (ECM) proteins (3,4), suggesting that once activated, these cells establish a constitutive self-activation system. One of the major cytokines involved in this process is transforming growth factor ␤1 (TGF␤1) (5). The principal effect of TGF␤ on mesenchymal cells is its stimulation of ECM deposition, as evidenced by the findings that 1) SSc fibroblasts express elevated levels of TGF␤ receptors, and this correlates with elevated levels of ␣2(I) collagen messenger RNA (mRNA) (6-9) and 2)

Thrombin-mediated Proteoglycan Synthesis Utilizes Both Protein-tyrosine Kinase and Serine/Threonine Kinase Receptor Transactivation in Vascular Smooth Muscle Cells

“…Latent TGF-␤ can be activated by various members of the integrin family of cell surface molecules (27)(28)(29)(30). Inside out integrin signaling has been associated with cytoskeletal rearrangement and ROCK signaling cascades.…”

Integrin-Mediated Transforming Growth Factor-β Activation, a Potential Therapeutic Target in Fibrogenic Disorders

“…In vitro, TGF-␤ conformational activation can result from treatment of latent TGF-␤ with heat, denaturants, reactive oxygen species, 43 acidification, 44 interaction with the extracellular-matrix molecule thrombospondin-1, or interactions with the integrins ␣v␤3, ␣v␤5, and ␣v␤6 [45][46][47][48][49][50] Proteolytic activation occurs through interactions with matrix metalloprotein (MMP)-2 or -9, plasmin or the inte- 50 B: The ␣v␤8 metalloproteolytic (MMP-14) mechanism of activation results in release of the mature TGF-␤ peptide from the SLC, which can diffuse as a paracrine signal to adjacent cell types. 52 This mechanism of activation of TGF-␤ may be facilitated by the MMP-14-dependent cleavage of LTBP, causing release of the SLC from the large latent complex (LLC).…”

“…69 In humans, expression of the ␤3 and ␤5 integrin subunits has been shown to be up-regulated in dermal fibroblasts of skin samples from scleroderma patients. 46,48 Furthermore, increased expression of these integrins has been shown to increase autocrine activation of TGF-␤ and support conversion of dermal fibroblasts to a profibrotic phenotype. 46 -48 Whether ␣v␤3, ␣v␤5, or other integrins that bind with low affinity to TGF-␤ account for a component of TGF-␤ activation in pathological tissues remains to be determined using in vivo models.…”

Induction of an Epithelial Integrin αvβ6 in Human Cytomegalovirus-Infected Endothelial Cells Leads to Activation of Transforming Growth Factor-β1 and Increased Collagen Production

“…28,29 The propeptide of TGF-␤1, latency-associated peptide-␤1, contains an Arg-Gly-Asp (RGD) motif that is recognized by a subset of integrins having in common the integrin ␣v subunit 18,19,40 -42 and ␣5␤1. 43 Furthermore, the integrins ␣v␤6 and ␣v␤8 have been shown to activate TGF-␤1 in vivo. 18,19 To examine whether CMV infection alters the expression level of ␣v integrin ␤ subunit partners and integrin ␣5, we infected HUVECs with VR1814, a pathogenic clinical CMV strain, and quantified the surface expression of integrins ␤1, ␤3, ␤5, ␤6, ␤8, and ␣5 by flow cytometry at 10 days after infection.…”