A Polymorphism in the<i>CTGF</i>Promoter Region Associated with Systemic Sclerosis

Fonseca, Carmen; Lindahl, Gisela; Ponticos, Markella; Sestini, Piersante; Renzoni, Elisabetta A.; Holmes, Alan M.; Spagnolo, Paolo; Pantelidis, Panagiotis; Leoni, Patricio Clark Di; McHugh, Neil; Stock, Carmel; Xu, Shiwen; Denton, Christopher P.; Black, Carol M.; Welsh, Kenneth I.; Bois, Roland M. du; Abraham, David

doi:10.1056/nejmoa067655

Cited by 182 publications

(146 citation statements)

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“…In strong support of this concept in the clinical setting, we recently reported findings from a functional genetic study in which a common sequence variant in the CTGF promoter was genetically associated with SSc, particularly in patients with the antitopoisomerase autoantibody specificity and fibrosing alveolitis (11). The disease-associated allele was demonstrated to have higher transcriptional activity, providing the first direct causative evidence that CTGF is involved in susceptibility to lung fibrosis in SSc (11).…”

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confidence: 85%

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Pivotal role of connective tissue growth factor in lung fibrosis: MAPK‐dependent transcriptional activation of type I collagen

Ponticos

Holmes

et al. 2009

Arthritis & Rheumatism

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Objective. Connective tissue growth factor (CTGF; CCN2) is overexpressed in systemic sclerosis (SSc) and has been hypothesized to be a key mediator of the pulmonary fibrosis frequently observed in this disease. CTGF is induced by transforming growth factor ␤ (TGF␤) and is a mediator of some profibrotic effects of TGF␤ in vitro. This study was undertaken to investigate the role of CTGF in enhanced expression of type I collagen in bleomycin-induced lung fibrosis, and to delineate the mechanisms of action underlying the effects of CTGF on Col1a2 (collagen gene type I ␣2) in this mouse model and in human pulmonary fibroblasts.Methods. Transgenic mice that were carrying luciferase and ␤-galactosidase reporter genes driven by the Col1a2 enhancer/promoter and the CTGF promoter, respectively, were injected with bleomycin to induce lung fibrosis (or saline as control), and the extracted pulmonary fibroblasts were incubated with CTGF blocking agents. In vitro, transient transfection, promoter/ reporter constructs, and electrophoretic mobility shift assays were used to determine the mechanisms of action of CTGF in pulmonary fibroblasts.Results. In the mouse lung tissue, CTGF expression and promoter activity peaked 1 week after bleomycin challenge, whereas type I collagen expression and Col1a2 promoter activity peaked 2 weeks postchallenge. Fibroblasts isolated from the mouse lungs 14 days after bleomycin treatment retained a profibrotic expression pattern, characterized by greatly elevated levels of type I collagen and CTGF protein and increased promoter activity. In vitro, inhibition of CTGF by specific small interfering RNA and neutralizing antibodies reduced the collagen protein expression and Col1a2 promoter activity. Moreover, in vivo, anti-CTGF antibodies applied after bleomycin challenge significantly reduced the Col1a2 promoter activity by ϳ25%. The enhanced Col1a2 promoter activity in fibroblasts from bleomycintreated lungs was partly dependent on Smad signaling, whereas CTGF acted on the Col1a2 promoter by a mechanism that was independent of the Smad binding site, but was, instead, dependent on the ERK-1/2 and JNK MAPK pathways. The CTGF effect was mapped to the proximal promoter region surrounding the inverted CCAAT box, possibly involving CREB and c-Jun. In human lung fibroblasts, the human COL1A2 promoter responded in a similar manner, and the mechanisms of action also involved ERK-1/2 and JNK signaling.Conclusion. Our results clearly define a direct profibrotic effect of CTGF and demonstrate its contribution to lung fibrosis through transcriptional activation

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confidence: 85%

“…The disease-associated allele was demonstrated to have higher transcriptional activity, providing the first direct causative evidence that CTGF is involved in susceptibility to lung fibrosis in SSc (11).…”

mentioning

confidence: 95%

Pivotal role of connective tissue growth factor in lung fibrosis: MAPK‐dependent transcriptional activation of type I collagen

Ponticos

Holmes

et al. 2009

Arthritis & Rheumatism

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213

151

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“…A polymorphism in the CTGF promoter region, increasing CTGF expression levels, has recently been associated with systemic sclerosis. 35 CTGF has also been identified as a marker of fibrosis in chronic renal and heart allograft rejection, disease states that are also associated with sustained endothelial injury. 36,37 Blockade of CTGF expression by systemic injection of siRNAs was recently shown to prevent fibrosis in a model of chronic renal allograft rejection.…”

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confidence: 99%

Caspase-3-mediated secretion of connective tissue growth factor by apoptotic endothelial cells promotes fibrosis

et al. 2009

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Apoptosis of endothelial cells (ECs) is an early pathogenic event in various fibrotic diseases. In this study, we evaluated whether paracrine mediators produced by apoptotic ECs play direct roles in fibrogenesis. C3H mice injected subcutaneously with serumfree medium conditioned by apoptotic ECs (SSC) showed increased skin thickness and heightened protein levels of a-smoothmuscle actin (aSMA), vimentin and collagen I as compared with mice injected with medium conditioned by non-apoptotic ECs. Fibroblasts exposed to SSC in vitro showed cardinal features of myofibroblast differentiation with increased stress fiber formation and expression of aSMA. Caspase-3 silencing in ECs prevented the release of mediators favoring myofibroblast differentiation. To identify the fibrogenic factor(s) released by ECs, the protein contents of media conditioned by either apoptotic or non-apoptotic ECs were compared using SDS-PAGE-liquid chromatography (LC)-tandem mass spectrometry (MS/MS) and two-dimensional LC-MS/MS. Connective tissue growth factor (CTGF) was the only fibrogenic protein found increased in SSC. Pan-caspase inhibition with ZVAD-FMK or caspase-3 silencing in ECs confirmed that CTGF was released downstream of caspase-3 activation. The fibrogenic signaling signatures of SSC and CTGF on fibroblasts in vitro were similarly Pyk2-, Src-family kinases-and PI3K dependent, but TGF-b-independent. CTGF-immunodepleted SSC failed to induce myofibroblast differentiation in vitro and skin fibrosis in vivo. These results identify caspase-3 activation in ECs as a novel inducer of CTGF release and fibrogenesis.

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“…However, outside autoimmunity, only a few genes have been associated with SSc, with CTGF ( CCN2 ) being the most studied 44, 45. We performed a genetic association study in 2 independent cohorts of SSc patients as a model of vascular and pulmonary pathology.…”

Section: Discussionmentioning

confidence: 99%

Molecular Basis for Dysregulated Activation of NKX2‐5 in the Vascular Remodeling of Systemic Sclerosis

Dritsoula

Guerra

Fonseca

et al. 2018

Arthritis & Rheumatology

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Objective NKX2‐5 is a homeobox transcription factor that is required for the formation of the heart and vessels during development, with significant postnatal down‐regulation and reactivation in disease states, characterized by vascular remodeling. The purpose of this study was to investigate mechanisms that activate NKX2‐5 expression in diseased vessels, such as systemic sclerosis (scleroderma; SSc)–associated pulmonary hypertension (PH), and to identify genetic variability that potentially underlies susceptibility to specific vascular complications.MethodsWe explored NKX2‐5 expression in biopsy samples from patients with SSc‐associated PH and in pulmonary artery smooth muscle cells (PASMCs) from patients with scleroderma. Disease‐associated putative functional single‐nucleotide polymorphisms (SNPs) at the NKX2-5 locus were cloned and studied in reporter gene assays. SNP function was further examined through protein–DNA binding assays, chromatin immunoprecipitation assays, and RNA silencing analyses.ResultsIncreased NKX2‐5 expression in biopsy samples from patients with SSc‐associated PH was localized to remodeled vessels and PASMCs. Meta‐analysis of 2 independent scleroderma cohorts revealed an association of rs3131917 with scleroderma (P = 0.029). We demonstrated that disease‐associated SNPs are located in a novel functional enhancer, which increases NKX2-5 transcriptional activity through the binding of GATA‐6, c‐Jun, and myocyte‐specific enhancer factor 2C. We also characterized an activator/coactivator transcription‐enhancer factor domain 1 (TEAD1)/Yes‐associated protein 1 (YAP1) complex, which was bound at rs3095870, another functional SNP, with TEAD1 binding the risk allele and activating the transcription of NKX2-5.Conclusion NKX2-5 is genetically associated with scleroderma, pulmonary hypertension, and fibrosis. Functional evidence revealed a regulatory mechanism that results in NKX2-5 transcriptional activation in PASMCs through the interaction of an upstream promoter and a novel downstream enhancer. This mechanism can act as a model for NKX2‐5 activation in cardiovascular disease characterized by vascular remodeling.

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A Polymorphism in theCTGFPromoter Region Associated with Systemic Sclerosis

Abstract: The G-945C substitution represses CTGF transcription, and the -945G allele is significantly associated with susceptibility to systemic sclerosis.

Cited by 182 publications

References 35 publications

Pivotal role of connective tissue growth factor in lung fibrosis: MAPK‐dependent transcriptional activation of type I collagen

Pivotal role of connective tissue growth factor in lung fibrosis: MAPK‐dependent transcriptional activation of type I collagen

Caspase-3-mediated secretion of connective tissue growth factor by apoptotic endothelial cells promotes fibrosis

Molecular Basis for Dysregulated Activation of NKX2‐5 in the Vascular Remodeling of Systemic Sclerosis

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