Activation of Fibroblast Procollagen α1(I) Transcription by Mechanical Strain Is Transforming Growth Factor-β-dependent and Involves Increased Binding of CCAAT-binding Factor (CBF/NF-Y) at the Proximal Promoter

Abstract

During normal developmental tissue growth and in a number of diseases of the cardiopulmonary system, adventitial and interstitial fibroblasts are subjected to increased mechanical strain. This leads to fibroblast activation and enhanced collagen synthesis, but the underlying mechanisms involved remain poorly understood. In this study, we have begun to identify and characterize mechanical strain-responsive elements in the rat procollagen ␣1(I) (COL1A1) gene and show that the activity of COL1A1 promoter constructs, transiently transfected into cardiac fibroblasts, was increased between 2-and 4-fold by continuous cyclic mechanical strain. This was accompanied by a ϳ3-fold increase in the levels of total active transforming growth factor-␤ (TGF-␤) released into the medium. Inclusion of a panspecific TGF-␤ neutralizing antibody inhibited straininduced COL1A1 promoter activation. Deletion analysis revealed the presence of two potential strain response regions within the proximal promoter, one of which contains an inverted CCAAT-box overlapping a GC-rich element. Both mechanical strain and exogenously added TGF-␤1 enhanced the binding activity of CCAAT-binding factor, CBF/NF-Y, at this site. Moreover, this element was sufficient to confer strain-responsiveness to an otherwise unresponsive SV40 promoter. In summary, this study demonstrates that strain-induced COL1A1 promoter activation in cardiac fibroblasts is TGF-␤-dependent and involves increased binding of CCAAT-binding factor at the proximal promoter. Furthermore, these findings suggest a novel and potentially important TGF-␤ response element in the rat COL1A1 gene.

Expression patterns of Tgfβ1–3 associate with myocardialisation of the outflow tract and the development of the epicardium and the fibrous heart skeleton

“…Both epicardium and epicardial populated structures (i.e., sulcus region between atria and ventricles, AV cushions) are positive for Tgf␤3 and as such might relate to the production of procollagen-1 formation of the fibrous heart skeleton. This assumption is supported by a positive effect of Tgf␤ on the expression of procollagen-1 (Lindahl et al, 2002). In conclusion, the three Tgf␤ isoforms exhibit characteristic spatiotemporal expression patterns in the developing heart, which partly overlap, implying both specific and complementary functions during cardiovascular morphogenesis.…”

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

“…In DNA binding assays, the formation of a CBF/NF-Y complex was unchanged upon CTGF overexpression in isolation. CBF binding activity has been reported to be enhanced by TGF␤ in rat fibroblasts (29) and in explanted fibroblasts from the skin (39) and lungs (Lindahl GE: unpublished observations) of patients with SSc. It is therefore possible either that CTGF is not involved in CBF activation or, perhaps more likely, that other factors in combination with CTGF are needed for induced CBF binding activity in fibrosis.…”

“…Nuclear extracts from cells transfected with either the pCMV-CTGF expression vector or the pCMV-EV empty control vector were prepared as described previously (29). Binding reactions were prepared in EMSA binding buffer (Promega) using 2.5 g nuclear extract and a 5Ј-end 32 P-␥ATP-labeled doublestranded oligonucleotide probe, used on its own or together with either a cold competitor consensus oligonucleotide (Promega or Santa Cruz Biotechnology) or a specific supershift/ blocking antibody (Santa Cruz Biotechnology).…”

Mechanoregulation of gene expression in fibroblasts

“…These include the consensus TATA and CCAAT motifs, which are the potential targets for the action of promoter-specific transcription factors (Chu et al, 1985;De Wet et al, 1987). Transcriptional activation of rat COL1A1 gene in cardiac fibroblasts subjected to mechanical stretching with a 10% strain at 1 Hz, similar to the physiological tension experienced by the cells in the heart, has been reported to be mediated by autocrine stimulation by TGF-β (Lindahl et al, 2002). This activation involves TGF-β response elements in the promoter region of the collagen gene and increased binding of CCAAT-binding factor (CBF/NF-Y) at the proximal promoter.…”

“…Finally, mechanisms that include triggering autocrine or paracrine release of growth factors such as TGF-β by mechanical loads to regulate the transcription of "mechanoresponsive genes" are also presented (Davies et al, 1997;Sadoshima and Izumo, 1997). Mechanical stress stimulates the release of TGF-β (Lindahl et al, 2002;Nakatani et al, 2002;Yang et al, 2004) as described in the previous section, as well as enhances its gene transcription through activation of EGR-1 . These indirect mechanotransduction mechanisms have been explained in detail previously (Chiquet, 1999;Sarasa-Renedo and Chiquet, 2005).…”