TGFβ₁regulates Scleraxis expression in primary cardiac myofibroblasts by a Smad-independent mechanism

Abstract

In cardiac wound healing following myocardial infarction (MI), relatively inactive resident cardiac fibroblasts phenoconvert to hypersynthetic/secretory myofibroblasts that produce large quantities of extracellular matrix (ECM) and fibrillar collagen proteins. Our laboratory and others have identified TGF␤ 1 as being a persistent stimulus in the chronic and inappropriate wound healing phase that is marked by hypertrophic scarring and eventual stiffening of the entire myocardium, ultimately leading to the pathogenesis of heart failure following MI. Ski is a potent negative regulator of TGF␤/Smad signaling with known antifibrotic effects. Conversely, Scleraxis is a potent profibrotic basic helix-loop-helix transcription factor that stimulates fibrillar collagen expression. We hypothesize that TGF␤ 1 induces Scleraxis expression by a novel Smad-independent pathway. Our data support the hypothesis that Scleraxis expression is induced by TGF␤ 1 through a Smad-independent pathway in the cardiac myofibroblast. Specifically, we demonstrate that TGF␤ 1 stimulates p42/44 (Erk1/2) kinases, which leads to increased Scleraxis expression. Inhibition of MEK1/2 using U0126 led to a sequential temporal reduction of phosphop42/44 and subsequent Scleraxis expression. We also found that adenoviral Ski expression in primary myofibroblasts caused a significant repression of endogenous Scleraxis expression at both the mRNA and protein levels. Thus we have identified a novel TGF␤ 1-driven, Smad-independent, signaling cascade that may play an important role in regulating the fibrotic response in activated cardiac myofibroblasts following cardiac injury.

A primer on current progress in cardiac fibrosis

“…In our various studies, any interference with scleraxis function -whether by gene deletion, knockdown, use of a dominant negative, or alteration of post-translational modifications -attenuated evidence of fibroblast to myofibroblast transition and reduced expression of collagen, other ECM components or myofibroblast markers (Bagchi and Czubryt 2012;Bagchi et al 2016a;Bagchi et al 2016b;Bagchi et al 2016c;Espira et al 2009;Roche et al 2016). This effect occurred regardless of whether the upstream inducer of fibrosis was TGFβ, Smad3 or mechanical stretch, suggesting that scleraxis acts as a signaling nexus upon which multiple pro-fibrotic pathways descend.…”

“…It has long been known that plating primary fibroblasts onto firm substrates, including even tissue culture plastic ware, induces rapid conversion to myofibroblasts (Hinz 2009). Physical force, acting through integrins, up-regulates multiple pro-fibrotic genes including α-SMA and fibrillar collagens І and ІІІ (Carver et al 1991;Roche et al 2016). The resulting increase in ECM synthesis may represent an attempt to resist excessive extracellular forces via the construction of stronger matrix.…”

“…TGFβ also appears to be capable of inducing scleraxis expression via a Smad-independent non-canonical pathway, possibly through c-Jun which has also been implicated in fibrosis (Zeglinski et al 2016). Given the central roles of these pathways in fibrosis, it is thus not surprising that scleraxis expression increases significantly in the cardiac infarct scar, suggesting it may play a key causative role in the fibrotic process in vivo (Espira et al 2009).…”

“…Given the central roles of these pathways in fibrosis, it is thus not surprising that scleraxis expression increases significantly in the cardiac infarct scar, suggesting it may play a key causative role in the fibrotic process in vivo (Espira et al 2009). Mechanical stretch also induces scleraxis expression, and knockdown of scleraxis attenuates the ability of stretch to cause fibroblast to myofibroblast conversion (Roche et al 2016). However, while stretch causes transactivation of the scleraxis promoter, the precise mechanism and transcriptional regulator involved is yet to be elucidated.…”

“…One approach is knockdown via adenovirus-mediated gene delivery of a suitable, specific shRNA. We have successfully employed this approach in vitro to nearly eliminate scleraxis levels and attenuate scleraxis-dependent ECM gene expression (Bagchi et al 2016a;Bagchi et al 2016b;Roche et al 2016). A potential refinement for this approach would be to drive the expression of the shRNA D r a f t with a cell type-specific gene promoter, such as that for TCF21, which would facilitate delivery to cardiac fibroblasts but not to other cells of the myocardium, thus reducing off-target effects (Acharya et al 2012).…”