Fli1 and Ets1 Have Distinct Roles in Connective Tissue Growth Factor/CCN2 Gene Regulation and Induction of the Profibrotic Gene Program

Nakerakanti, Sashidhar; Kapanadze, Bagrat; Yamasaki, Masaomi; Markiewicz, Margaret; Trojanowska, Maria

doi:10.1074/jbc.m600466200

Cited by 91 publications

(123 citation statements)

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“…Chromatin immunoprecipitation (ChIP). ChIP was performed as described previously (20). Adult dermal fibroblasts were treated with either AdGFP or AdTGF␤RI (AdALK-5) for 18 hours.…”

Section: Smad1 Signaling Pathway In Ssc 2531mentioning

confidence: 99%

“…Half of the AdGFP-treated cells were also treated with TGF␤ (1 ng/ml) for 90 minutes. The cells were crosslinked and lysed, and nuclei were isolated as described previously (20). Chromatin was sheared by sonication to yield an average fragment size of 400 bp and microcentrifuged at 14,000 revolutions per minute, and supernatant containing soluble chromatin was collected.…”

Section: Smad1 Signaling Pathway In Ssc 2531mentioning

confidence: 99%

“…The PCR reaction was carried out for either 28 or 33 cycles, and the product was visualized by agarose gel electrophoresis. A 213-bp region (-194 to ϩ19) of the CCN2 promoter encompassing the Sp-1 site was amplified using the primers described previously (20).…”

Section: Smad1 Signaling Pathway In Ssc 2531mentioning

confidence: 99%

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Smad1 pathway is activated in systemic sclerosis fibroblasts and is targeted by imatinib mesylate

Pannu¹,

Asano²,

Nakerakanti³

et al. 2008

Arthritis & Rheumatism

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Objective. Activation of Smad1 signaling has recently been implicated in the development of fibrosis. The goal of the present study was to gain further insights into activation of the Smad1 pathway in fibrosis in systemic sclerosis (SSc) and to determine whether this pathway is targeted by the antifibrotic drug imatinib mesylate.Methods. Levels of phosphorylated Smad1 and total Smad1 were examined in SSc and control skin biopsy samples by immunohistochemistry and in cultured fibroblasts by Western blotting. Activity of the CCN2 promoter was examined by a luciferase reporter gene assay. Interactions of Smad1 with the CCN2 promoter were examined by in vitro and in vivo DNA binding assays. Expression of the nonreceptor tyrosine kinase c-Abl and Smad1 was blocked using respective small interfering RNA.Results. Total and phosphorylated Smad1 levels were significantly elevated in SSc skin biopsy samples and in cultured SSc fibroblasts and correlated with elevated CCN2 protein and CCN2 promoter activity. DNA binding assays demonstrated that Smad1 was a direct activator of the CCN2 gene. Small interfering RNA-mediated depletion of Smad1 in SSc fibroblasts normalized the production of CCN2 and collagen. Imatinib mesylate blocked activation of the Smad1 pathway in transforming growth factor ␤-stimulated control fibroblasts and reversed activation of this pathway in SSc fibroblasts. Likewise, blockade of c-Abl abrogated activation of the Smad1 pathway in SSc fibroblasts.Conclusion. Our findings demonstrate that activation of Smad1 signaling occurs in a subset of SSc patients and contributes to persistent activation of SSc fibroblasts. Demonstration that the Smad1/CCN2 pathway is blocked by imatinib mesylate further clarifies the mechanism of the antifibrotic effects of this compound.

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“…Chromatin immunoprecipitation (ChIP). ChIP was performed as described previously (20). Adult dermal fibroblasts were treated with either AdGFP or AdTGF␤RI (AdALK-5) for 18 hours.…”

Section: Smad1 Signaling Pathway In Ssc 2531mentioning

confidence: 99%

Section: Smad1 Signaling Pathway In Ssc 2531mentioning

confidence: 99%

See 1 more Smart Citation

Smad1 pathway is activated in systemic sclerosis fibroblasts and is targeted by imatinib mesylate

Pannu¹,

Asano²,

Nakerakanti³

et al. 2008

Arthritis & Rheumatism

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“…9,27,28 As such, we cannot rule out the possibility that the PBE may contain more than one binding site. The various PARP-1 -binding sites differ considerably in sequence, suggesting that PARP-1 may bind to these sites as part of an enhancer/promoter binding complex containing other DNA binding factors and co-activators.…”

Section: Discussionmentioning

confidence: 99%

“…7,8 In addition, a Smad-binding element (SBE) and an Ets1-binding site are necessary for TGF-␤ induction of CCN2 in skin fibroblasts. 5,7,9 In addition, we and others also demonstrated that TGF-␤ is an important inducer of CCN2 in epithelial cells. [2][3][4][5]10 Although several consensus gene regulatory motifs, including SBE, BCE-1, Ets1, and Sp1, have been identified in the murine CCN2 promoter, their functional relevance in renal tubular epithelial cells has not yet been demonstrated; therefore, in this study, we investigated the mechanism underlying CCN2 transcription in tubular epithelial cells.…”

mentioning

confidence: 98%

Poly(ADP-Ribose) Polymerase-1 Enhances Transcription of the Profibrotic CCN2 Gene

Okada¹,

Inoue²,

Kikuta³

et al. 2008

Journal of the American Society of Nephrology

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In the fibrotic kidney, tubular epithelial cells express CCN2, formerly known as connective tissue growth factor. Because little is known about the transcriptional regulation of this profibrotic protein, this study investigated the mechanism underlying epithelial cell-selective upregulation of CCN2 in fibrosis. It was found that a previously unidentified cis-regulatory element located in the promoter of the murine CCN2 gene plays an essential role in basal and TGF-␤1-induced gene transcription in tubular epithelial cells; this element acts in conjunction with the Smad-binding element and the basal control element-1. By protein mass fingerprint analysis and de novo sequencing, poly(ADP-ribose) polymerase-1 (PARP-1) was identified as a trans-acting protein factor that binds to this promoter region, which we termed the PARP-1-binding element. In vivo, knockdown of PARP-1 in proximal tubular epithelial cells significantly reduced CCN2 mRNA levels and attenuated interstitial fibrosis in the obstructed kidney. Thus, the PARP-1/PARP-1 binding element complex functions as a nonspecific, fundamental enhancer of both basal and induced CCN2 gene transcription in tubular epithelial cells. This regulatory complex may be a promising target for antifibrotic therapy.

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The c-Abl tyrosine kinase controls protein kinase Cδ-induced Fli-1 phosphorylation in human dermal fibroblasts

Bujor¹,

Asano²,

Haines³

et al. 2011

Arthritis & Rheumatism

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Objective We have previously demonstrated that in response to TGFβ, Fli1 activity is repressed through a series of sequential posttranslational modifications, consisting of PKCδ induced Thr312 phosphorylation, acetylation by PCAF, and detachment from the collagen promoter. In this study, we further investigated the upstream events that lead to Fli1 phosphorylation in response to TGFβ. Methods Fibroblasts were isolated from SSc and matched control patients. Western blot was used to analyze protein levels and quantitative real time RT-PCR was used to measure mRNA expression. Cells were transduced with constitutively active PKCδ adenovirus or transiently transfected with a bcr-abl overexpressing plasmid. Subcellular localization of PKCδ was examined by immunocytochemistry. Results Western blot analysis of cell lysates demonstrated that the levels of P-Fli1 (Thr321) were up-regulated in SSc fibroblasts, correlating with increased collagen type I and c-abl protein. Experiments using a constitutively activated form of c-abl, siRNA against c-abl and the specific tyrosine kinase inhibitor Imatinib, demonstrate that c-abl is required for the TGFβ induced phosphorylation of Fli1. Additionally, we show that c-abl kinase activity is required for PKCδ nuclear localization. Conclusions Our results demonstrate that in SSc fibroblasts c-abl is an upstream regulator of the pro-fibrotic PKCδ/P-Fli1 pathway, via induction of PKCδ nuclear localization. Additionally, the finding that Fli1 is phosphorylated at higher levels in SSc fibroblasts supports the notion that the c-abl/PKCδ/P-Fli1 pathway is constitutively activated in these cells. Thus, blocking the TGFβ/c-abl/PKCδ/P-Fli1 pathway could be an attractive alternative approach for scleroderma therapy.

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Fli1 and Ets1 Have Distinct Roles in Connective Tissue Growth Factor/CCN2 Gene Regulation and Induction of the Profibrotic Gene Program

Cited by 91 publications

References 44 publications

Smad1 pathway is activated in systemic sclerosis fibroblasts and is targeted by imatinib mesylate

Smad1 pathway is activated in systemic sclerosis fibroblasts and is targeted by imatinib mesylate

Poly(ADP-Ribose) Polymerase-1 Enhances Transcription of the Profibrotic CCN2 Gene

The c-Abl tyrosine kinase controls protein kinase Cδ-induced Fli-1 phosphorylation in human dermal fibroblasts

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