Differential Expression of Transforming Growth Factor-β Receptors I and II and Activation of Smad 3 in Keloid Fibroblasts

Chin, Gyu S.; Liu, Wei; Peled, Ziv M.; Lee, Thomas Y.; Steinbrech, Douglas S.; Hsu, Meier; Longaker, Michael T.

doi:10.1097/00006534-200108000-00022

Cited by 176 publications

(142 citation statements)

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“…Similar ligand-independent SMAD activation has been described in hepatic stellate cells derived from fibrotic livers (56) and in dermal fibroblasts derived from keloid lesions (57). Constitutive SMAD activation is therefore likely to contribute to the profibrotic phenotype of scleroderma fibroblasts.…”

Section: Discussionsupporting

confidence: 57%

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: Discussionsupporting

confidence: 57%

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

Mori

Chen

Varga

2003

Arthritis & Rheumatism

172

115

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“…Bettinger et al also found a significant increase in the response rate to TGF-ß in KFs, with an increase in fibroblast proliferation and collagen synthesis (15). Increased rates of proliferation and collagen synthesis suggest an altered response of KFs to this cytokine, which might reflect a modification in the regulatory pathway that occurs at the receptor level or during intracellular transduction (4,9). According to the literature, TGF-ß1 and -ß2 mRNA expression was found to be higher in KFs than in NFs or hypertrophic scar fibroblasts, whereas TGF-ß3 mRNA expression was significantly lower (3,11,25).…”

Section: Discussionmentioning

confidence: 98%

“…They grow invasively into the surrounding healthy skin and are not confined to the border of the initial wound. They seldom show a tendency to regress spontaneously (2)(3)(4). The prevalence between the male and female gender is equally distributed.…”

Section: Introductionmentioning

confidence: 99%

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Effect of the abrogation of TGF-β1 by antisense oligonucleotides on the expression of TGF-β-isoforms and their receptors I and II in isolated fibroblasts from keloid scars

Bran

Goessler²,

Schardt³

et al. 2010

Int J Mol Med

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Abstract. Disequilibrium of dermal wound repair can result in continued accumulation of ECM and excessive scar formation. In susceptible genetically predisposed individuals, keloid formation can be observed. Keloid disease represents a benign dermal fibroproliferative tumor that is unique to humans. TGF-ß is known to play a key role in the pathogenesis of this disease which is still not fully understood. The isoforms TGF-ß1 and TGF-ß2 have profibrotic properties, whereas TGF-ß3 may have antifibrotic functions. TGF-ß exerts its influence by binding to type I and type II TGF-ß receptors, thereby forming a complex and activating specific downstream effector molecules. The aim of this study was to investigate the effect of TGF-ß1 targeting by antisense oligonucleotides on the RNA synthesis and protein expression of TGF-ß isoforms and their receptors in keloid-derived fibroblasts. In tissue samples with normal fibroblasts (NFs) serving as control samples, expression of TGF-ß1 and -ß2 was decreased when compared to keloid fibroblasts (KFs), while expression of TGF-ß3 and of TGF-ßRII was significantly higher in NFs. In the ELISA assay, abrogation of TGF-ß1 led to a significant decrease in TGF-ß1 and -ß2 (p<0.05). Expression of TGF-ß2 mRNA was reduced. Expression of TGF-ß3 mRNA revealed contrary patterns in KFs from different patients while expression of TGF-ßRI was found to be equal during the measurement period. TGF-ßRII mRNA expression was increased after 48 and 72 h respectively. There is growing evidence for a regulatory mechanism between TGF-ß1 and its receptors. Our findings support this theory by suggesting interrelations between the different TGF-ß isoforms and their receptors. Abnormal response of KFs to TGF-ß might reflect a modification in the regulatory pathway that occurs at the receptor level or during intracellular transduction. Improving the understanding of TGF-ß in keloid disease could lead to the development of clinically useful therapeutic modalities for treatment of keloid disease or even allow identification of preventive strategies.

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“…It has been widely accepted that the dysregulation of the TGF-β/Smad pathway plays a key role in hypertrophic or keloid scar formation in human skin tissues. A number of studies have clearly demonstrated that the increased expression of TGF-β receptors (types I and II) and the increased phosphorylation of Smad3 in keloid fibroblasts compared to normal HSFs, as well as the suppression of the TGF-β1/Smad pathway by certain chemicals such as curcumin, may exert chemopreventive effects, thus preventing keloid formation (7)(8)(9).…”

Section: Discussionmentioning

confidence: 99%

Downregulation of type I collagen expression in silibinin-treated human skin fibroblasts by blocking the activation of Smad2/3-dependent signaling pathways: Potential therapeutic use in the chemoprevention of keloids

Cho

Lee

2013

International Journal of Molecular Medicine

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Abstract. The inhibition of the Smad2/3 pathway is a key step involved in the downregulation of type I collagen synthesis, thus preventing keloid formation in tissue. In this study, we investigated the effect of silibinin on the proliferation of human skin fibroblasts (HSFs), as well as its effect on the expression of type I collagen, matrix metalloproteinase (MMP)-1, Smad2 and Smad3. Our results showed that the proliferation rates of the fibroblasts were not markedly decreased in a dose-and time-dependent manner following treatment with silibinin. Even though silibinin did not exert any cytotoxic effects on HSFs, the expression of type I collagen was markedly decreased in a dose-and time-dependent manner in the silibinin-treated HSFs. Consistent with this finding, the decreased promoter activity of type I collagen was observed in the HSFs following treatment with silibinin. The MMP-1 and MMP-2 expression levels were increased in the silibinin-treated HSFs. Moreover, the silibinin-induced downregulation of type I collagen was associated with the inhibition of Smad2/3 activation in the transforming growth factor-β1 (TGF-β1)-treated HSFs. We further demonstrated that silibinin attenuated the translocation of Smad2 and Smad3 to the nucleus in the TGF-β1-treated HSFs. Taken together, our data indicate that silibinin has the potential to prevent fibrotic skin changes by inducing the downregulation of type I collagen expression; this effect was partly mediated by the inhibition of the Smad2/3-dependent signaling pathway in HSFs.

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Differential Expression of Transforming Growth Factor-β Receptors I and II and Activation of Smad 3 in Keloid Fibroblasts

Cited by 176 publications

References 31 publications

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

Effect of the abrogation of TGF-β1 by antisense oligonucleotides on the expression of TGF-β-isoforms and their receptors I and II in isolated fibroblasts from keloid scars

Downregulation of type I collagen expression in silibinin-treated human skin fibroblasts by blocking the activation of Smad2/3-dependent signaling pathways: Potential therapeutic use in the chemoprevention of keloids

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