Increased phosphorylation of transcription factor Sp1 in scleroderma fibroblasts: Association with increased expression of the type I collagen gene

Ihn, Hironobu; Tamaki, Kunihiko

doi:10.1002/1529-0131(200010)43:10<2240::aid-anr11>3.0.co;2-2

Cited by 53 publications

(37 citation statements)

References 39 publications

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“…Recent reports indicate that optimal induction of COL1A2 transcription by TGF␤ involves an interaction between activated SMAD3 and the ubiquitous DNA binding transcription factor Sp1 (32,33). In this regard, it is of interest that Sp1 has been shown to be constitutively phosphorylated and activated in scleroderma fibroblasts (60). These findings suggest that for maximal transactivation of COL1A2 in scleroderma fibroblasts, SMAD activation and Sp1 phosphorylation may both be required.…”

Section: Discussionmentioning

confidence: 99%

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

Mori

Chen

Varga

2003

Arthritis & Rheumatism

172

115

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Objective. Scleroderma is characterized by excessive synthesis and accumulation of matrix proteins in lesional tissues. Transforming growth factor ␤ (TGF␤) plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of fibroblasts; however, the underlying mechanisms are poorly understood. We undertook this study to examine the expression and function of SMADs, recently characterized intracellular effectors of TGF␤ signaling, in scleroderma fibroblasts.Methods. Primary dermal fibroblasts obtained from 14 patients with scleroderma and from 4 healthy adult volunteers were studied. Northern analysis was used to determine the expression of endogenous SMAD messenger RNA (mRNA), and Western analysis was used to determine SMAD protein expression. Intracellular compartmentalization of cellular SMAD proteins in the presence and absence of TGF␤ was studied by antibody-mediated immunofluorescence confocal microscopy. The effect of TGF␤ blockade on SMAD subcellular distribution was determined using anti-TGF␤ antibodies as well as a dominant-negative TGF␤ receptor type II (TGF␤RII) vector to disrupt TGF␤ responses. SMAD-regulated luciferase reporter expression was examined to investigate the potential functional significance of activation and nuclear accumulation of endogenous SMADs in scleroderma fibroblasts.Results. Protein and mRNA levels of SMAD3, but not of SMAD4 or SMAD7, were variably elevated in scleroderma fibroblasts compared with those from healthy controls. In sharp contrast to control fibroblasts, which displayed predominantly cytoplasmic localization of SMAD3/4 in the absence of exogenous TGF␤, in scleroderma fibroblasts SMAD3 and SMAD4 consistently showed elevated nuclear localization. Furthermore, phosphorylated SMAD2/3 levels were elevated and nuclear localization of phosphorylated SMAD2/3 was increased, suggesting activation of the SMAD pathway in scleroderma fibroblasts. Blockade of autocrine TGF␤ signaling with antibodies or by expression of dominant-negative TGF␤RII failed to normalize SMAD subcellular distribution, suggesting that elevated nuclear SMAD import was due to alterations downstream of the TGF␤ receptors. The activity of a SMADresponsive minimal promoter-reporter construct was enhanced in transiently transfected scleroderma fibroblasts.Conclusion. This study is the first to demonstrate apparently ligand-independent constitutive activation of the intracellular TGF␤/SMAD signaling axis in scleroderma fibroblasts. SMAD signaling may be a mechanism contributing to the characteristic phenotype of scleroderma fibroblasts and playing a role in the pathogenesis of fibrosis.Scleroderma is associated with fibrosis of affected tissues due to excessive synthesis and progressive accumulation of connective tissue macromolecules (1). Scleroderma fibroblasts display features of sustained activation in vitro and in vivo. In culture, these cells show elevated synthesis of collagens, fibronectin, proteoglycans, and tissue inhibitors of metalloproteinases, constitutive secretion of i...

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Section: Discussionmentioning

confidence: 99%

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

Mori

Chen

Varga

2003

Arthritis & Rheumatism

172

115

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“…The remaining cells were washed with cold phosphate-buffered saline twice and lysed in lysis buffer (5 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% sodium dodecyl sulfate, 1 g/ml aprotinin, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, and 100 g/ml phenylmethylsulfonyl fluoride). Aliquots of conditioned media (normalized for cell numbers) or cell lysates (normalized for protein concentrations as measured by the Bio-Rad reagent) were subjected to electrophoresis on 7.5% gradient sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose filters (17)(18)(19)). The nitrocellulose filters were then incubated with antibody against human type I collagen, ␤-actin, or STAT6.…”

Section: Methodsmentioning

confidence: 99%

“…Monolayers were washed, and the cells were transfected by the Lipofectin technique (FuGENE 6 Transfectin Reagent; Roche Applied Science) (12)(13)(14)(15) with Ϫ3500 COL1A2/chloramphenicol acetyltransferase constructs. pSV-␤-galactosidase control vector (Promega) was cotransfected to normalize for transfection efficiency (12)(13)(14)(15)(17)(18)(19)(20)(21)(22)(23). After incubation overnight, the medium was replaced with MEM containing 0.1% bovine serum albumin or with MEM containing various amounts of cytokines, and incubation was continued for 48 h. The cells were harvested in 0.25 M Tris-HCl (pH 8) and fractured by freeze thawing.…”

Section: Methodsmentioning

confidence: 99%

Interleukin-13 Stimulates the Transcription of the Human α2(I) Collagen Gene in Human Dermal Fibroblasts

Jinnin¹,

Ihn²,

Yamane

et al. 2004

Journal of Biological Chemistry

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Interleukin (IL)-13 is a novel lymphokine produced by activated Type 2 helper cells. In this study, we examined the target genes of IL-13 by the cDNA microarray analysis in human dermal fibroblasts. We focused on the human ␣2(I) collagen gene, which was one of the IL-13-induced genes by the microarray analysis. IL-13 induced type I collagen protein as well as mRNA in a dose-dependent manner. Actinomycin D, an RNA synthesis inhibitor, significantly blocked the IL-13-mediated up-regulation of ␣2(I) collagen mRNA expression, whereas cycloheximide, a protein synthesis inhibitor, did not block this up-regulation. In addition, IL-13 treatment induced the promoter activity of ␣2(I) collagen by nuclear run-on transcription assay and chloramphenicol acetyltransferase assay. IL-13-mediated transcriptional activation of ␣2(I) collagen gene or type I collagen protein up-regulation was inhibited by the treatment of fibroblasts with a selective phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or STAT6 antisense oligonucleotide, but not by PD98059, a specific inhibitor of MEK/ERK, or SB202190 or SB203580, specific inhibitors of p38 MAPK; IL-13 induced the phosphorylation of PI3K p85 regulatory subunit and STAT6. These results suggest that IL-13 may play a role in the regulation of extracellular matrix and indicate the possible therapeutic value of the blockade of IL-13 signaling pathways via PI3K and STAT6 in fibrosis.

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“…Intriguingly, Sp1 or its family members may contribute directly to the elevated level of expression of target genes in fibrosis. Intriguingly, increased phosphorylation of Sp1 has been shown to be a feature of scleroderma fibroblasts (32). However, the relevance of the phosphorylated residues to Sp1 binding or the elevation of matrix gene expression in SSc fibroblasts is not clear.…”

Section: Sp1 Is Required For the Elevated Levels Of Ctgf Expression Omentioning

confidence: 99%

Constitutive Connective Tissue Growth Factor Expression in Scleroderma Fibroblasts Is Dependent on Sp1

Holmes

Abraham

Chen

et al. 2003

Journal of Biological Chemistry

111

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Fibrotic diseases such as scleroderma (systemic sclerosis, SSc) are characterized by an excessive production of extracellular matrix and profibrotic proteins such as connective tissue growth factor (CTGF). In normal dermal fibroblasts, CTGF is not expressed unless induced by proteins such as tumor growth factor-␤ (TGF␤). Conversely, in fibroblasts cultured from fibrotic lesions CTGF mRNA and protein are constitutively expressed, even in the absence of exogenously added TGF␤. Thus, studying the mechanism underlying CTGF overexpression in SSc fibroblasts is likely to yield valuable insights into the basis of the fibrotic phenotype of SSc and possibly other scarring disease. CTGF overexpression is mediated primarily by sequences in the CTGF promoter. In this report, we identify the minimal promoter element involved with the overexpression of CTGF in SSc fibroblasts. This element is distinct from the element necessary and sufficient for the induction of CTGF expression by TGF␤ in normal fibroblasts. Within this region is a functional Sp1 binding site. Blocking Sp1 activity reduces the elevated, constitutive levels of CTGF promoter activity and protein expression observed in SSc fibroblasts. Relative to those prepared from normal dermal fibroblasts, nuclear extracts prepared from SSc fibroblasts possess increased Sp1 binding activity. Removal of phosphate groups from nuclear extracts enhanced Sp1 binding activity, suggesting that phosphorylation of Sp1 normally reduces Sp1 binding to DNA. Thus, the constitutive overexpression of CTGF in SSc fibroblasts seems to be independent of TGF␤ signaling but dependent at least in part on Sp1.

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Increased phosphorylation of transcription factor Sp1 in scleroderma fibroblasts: Association with increased expression of the type I collagen gene

Abstract: These results demonstrate the involvement of Sp1 in the up-regulation of expression of the alpha2(I) collagen gene in SSc fibroblasts.

Cited by 53 publications

References 39 publications

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

Interleukin-13 Stimulates the Transcription of the Human α2(I) Collagen Gene in Human Dermal Fibroblasts

Constitutive Connective Tissue Growth Factor Expression in Scleroderma Fibroblasts Is Dependent on Sp1

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