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

Abstract

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

Sphingosine 1-Phosphate (S1P) Receptor Agonists Mediate Pro-fibrotic Responses in Normal Human Lung Fibroblasts via S1P2 and S1P3 Receptors and Smad-independent Signaling

“…In contrast, other fibrotic genes, such as CTGF, SERPINE1, thrombospondin 1, CCL2, IL6, SphK1, or hyaluronan synthase 2 were up-regulated by TGF-␤1, S1P, and FTY720-P and to a lower extent by ponesimod. Induction of CTGF especially is of significance because it is known to be one of the major mediators of fibrosis (33,41,42). SphK1 is discussed as a link between TGF-␤1 and S1P signaling, allowing cross-talk between both pathways (7).…”

Transcription Factor ets-2 Plays an Important Role in the Pathogenesis of Pulmonary Fibrosis

“…Yasuoka and colleagues ascertained that the expression of early growth response 1 is up-regulated in lung sections from patients with IPF, and directs the expression of fibronectin in response to the stimulation of fibroblasts by (insulin-like growth factor binding protein 5) (37). Ponticos and colleagues highlighted the relevance of the mitogen-activated protein kinase (MAPK) pathway in the transcriptional activation of Type I collagen (38), to which ets-2 is implicitly related. Furthermore, Boon and colleagues demonstrated that genes in the extracellular signal-related kinase/MAPK pathway are up-regulated in patients with IPF, and these genes include a member of the SWI/SNF family and Ras (39).…”

Lung Pericytes and Resident Fibroblasts

“…Moreover, IGF binding proteins and connective tissue growth factor are important modulators of key pathways for lung development, including WNT, PDGF, and TGFb. 12,30,77 Thus, the gene expression profiles imply that in the mature organ these Col1 þ populations are still operating much of the developmental program used to generate the lung, most plausibly in maintenance and repair.…”