Smad-dependent stimulation of type I collagen gene expression in human skin fibroblasts by TGF-β involves functional cooperation with p300/CBP transcriptional coactivators

Ghosh, Asish K.; Yuan, Weiping; Mori, Yasuji; Varga, John

doi:10.1038/sj.onc.1203693

Cited by 218 publications

(209 citation statements)

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“…Therefore, one of the possible mechanisms by which abrogation of Smad3 gene expression prevented bleomycin-induced lung fibrosis may be mediated by having a direct negative effect on collagen expression in mouse lungs. However, it is also possible that reduction of collagen expression in bleomycin-treated Smad3 null mutant lungs is mediated by other indirect effects, such as increased expression of the transcriptional coactivator p300/CBP or reduced expression of the TGF-␤ pathway inhibitor Smad7 (9,27). The molecular mechanism of Smad3 action on collagen expression needs to be studied further.…”

Section: Discussionmentioning

confidence: 99%

“…Recent evidences have shown that Smad3 is involved in the transcriptional activation of TGF-␤-mediated stimulation of collagen expression (5,32,33). Smad3 is necessary for the formation of transcriptional activation complex in the stimulation of type I collagen gene expression by TGF-␤ ligands in human skin fibroblasts (6,9). Smad3 also acts synergistically with transcriptional factor TFE3 to activate TGF-␤-induced transcription of the plasminogen activator inhibitor-1 gene (16).…”

Section: Discussionmentioning

confidence: 99%

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Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice

Zhao

Shi

Wang

et al. 2002

American Journal of Physiology-Lung Cellular and Molecular Phys

367

287

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice

Zhao

Shi

Wang

et al. 2002

American Journal of Physiology-Lung Cellular and Molecular Phys

367

287

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“…56 The transcriptional coactivators p300/CBP enhance basal as well as TGF-␤-or Smad3-induced collagen ␣2(I) promoter activity, and stimulate the expression of endogenous type I collagen in human dermal fibroblasts. 57 No comparable element has been identified in the collagen ␣1(I) gene.…”

Section: Discussionmentioning

confidence: 99%

The role of Smad3 in mediating mouse hepatic stellate cell activation

et al. 2001

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Activated hepatic stellate cells (HSCs) have a central role as the main extracellular matrix (ECM) protein-producing cell during hepatic fibrogenesis. 1 Hepatic injury induces the HSCs to undergo a transdifferentiation or activation process, 1 which is characterized by loss of their intracellular vitamin A stores, 2 increase in proliferation, 3 changes in cellular morphology to a more myofibroblast-like cell type with expression of smooth muscle ␣-actin (␣-SMA), 4 and an increase in the production of ECM proteins, including type I collagen. 5 In addition, cultured HSCs express all 3 transforming growth factor ␤ (TGF-␤) isoforms 6,7 and TGF-␤ receptor types I, II, and III on the cell surface. 8 Treatment of HSCs in early culture with TGF-␤1 stimulates collagen type I messenger RNA (mRNA) expression 9,10 and protein synthesis, 10,11 inhibits HSC proliferation, 11-13 decreases the expression of matrix metalloproteinases, and increases the expression of tissue inhibitors of matrix metalloproteinases. 14 No change was observed in the level of ␣-SMA in TGF-␤1-treated HSCs. 15 Excessive TGF-␤ is associated with tissue damage caused by scarring in many diseases. 16 Clinical studies have revealed a close correlation between increased TGF-␤1 gene expression and the high expression of collagen type I mRNA in the liver tissue of patients with cirrhosis 17,18 and in experimental rat models of cirrhosis. 19,20 Furthermore, mRNA expression of TGF-␤1, TGF-␤2, and TGF-␤3 is increased in HSCs during fibrosis induced by bile duct ligation in rats. 21 Studies from transgenic mice support the etiologic and fibrogenic role for TGF-␤ in the development of liver fibrosis. Transgenic mice overexpressing mature TGF-␤1 under control of hepatocyte-specific promoters develop hepatic fibrosis with increased interstitial deposition of type I collagen. 22,23 Liver histology of an inducible transgenic mouse model overexpressing the active form of TGF-␤1 showed up-regulation of hepatic collagen type I and III mRNA and activation of HSCs. 24 TGF-␤ signals through its type I and type II receptors, which have serine/threonine kinase activity. The ligand binds to the constitutively active type II receptor, which then recruits and transphosphorylates type I receptor. 25 The activated type I receptor transiently associates with and phosphorylates Smad2 26 and Smad3, which then form heteromeric complexes with Smad4. [27][28][29] These complexes translocate to the nucleus, where the proteins function as transcriptional activators through their interaction with DNA-binding proteins. 30,31 Two inhibitory Smad proteins, Smad6 and Smad7, block Smad-mediated signaling in cells. [32][33][34] To date, Smad proteins are the only TGF-␤ receptor substrates with a demonstrated ability to propagate signals. 35 Several other signaling molecules and pathways are also activated by TGF-␤, including TGF-␤-activated kinase 1 (TAK1) 36 and the mito-

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“…The SMAD pathway plays a fundamental role in regulation of collagen synthesis, and SMADs are necessary to mediate TGF␤-dependent stimulation (29)(30)(31)(32)(33). In light of the singular importance of TGF␤ in both initiating and sustaining fibroblast activation, and given the pivotal role of SMADs as major intracellular effectors of TGF␤-induced profibrotic responses, we undertook extensive characterization of SMAD signaling in a large panel of scleroderma fibroblasts.…”

mentioning

confidence: 99%

“…To prevent protein dephosphorylation, a Phosphatase Inhibitor Mix (Sigma, St. Louis, MO) was added. Nuclear extracts were prepared as previously described (31). Protein concentrations in the cytosolic, nuclear, or whole cell fractions were determined by Bradford assay (Bio-Rad, Hercules, CA).…”

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confidence: 99%

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

Mori

Chen

Varga

2003

Arthritis & Rheumatism

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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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Smad-dependent stimulation of type I collagen gene expression in human skin fibroblasts by TGF-β involves functional cooperation with p300/CBP transcriptional coactivators

Cited by 218 publications

References 61 publications

Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice

Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice

The role of Smad3 in mediating mouse hepatic stellate cell activation

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

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