Ski drives an acute increase in MMP-9 gene expression and release in primary cardiac myofibroblasts

Abstract

Many etiologies of heart disease are characterized by expansion and remodeling of the cardiac extracellular matrix ( ECM or matrix) which results in cardiac fibrosis. Cardiac fibrosis is mediated in cardiac fibroblasts by TGF ‐ β 1 /R‐Smad2/3 signaling. Matrix component proteins are synthesized by activated resident cardiac fibroblasts known as myofibroblasts ( MFB ). These events are causal to heart failure with diastolic dysfunction and reduced cardiac filling. We have shown that exogenous Ski, a TGF ‐ β 1 /Smad repressor, localizes in the cellular nucleus and deactivates cardiac myofibroblasts. This deactivation is associated with reduction of myofibroblast marker protein expression in vitro, including alpha smooth muscle actin ( α ‐ SMA ) and extracellular domain‐A ( ED ‐A) fibronectin. We hypothesize that Ski also acutely regulates MMP expression in cardiac MFB . While acute Ski overexpression in cardiac MFB in vitro was not associated with any change in intracellular MMP ‐9 protein expression versus LacZ‐treated controls,exogenous Ski caused elevated MMP ‐9 mRNA expression and increased MMP ‐9 protein secretion versus controls. Zymographic analysis revealed increased MMP ‐9‐specific gelatinase activity in myofibroblasts overexpressing Ski versus controls. Moreover, Ski expression was attended by reduced paxillin and focal adhesion kinase phosphorylation ( FAK ‐ Tyr 397) versus controls. As myofibroblasts are hypersecretory and less motile relative to fibroblasts, Ski's reduction of paxillin and FAK expression may reflect the relative deactivation of myofibroblasts. Thus, in addition to its known antifibrotic effects, Ski overexpression elevates expression and extracellular secretion/release of MMP ‐9 and thus may facilitate internal cytoskeletal remodeling as well as extracellular ECM components. Further, as acute TGF ‐ β 1 treatment of primary cardiac MFB is known to cause rapid translocation of Ski to the nucleus, our data support an autoregulatory role for Ski in mediating cardiac ECM accumulation.

Crosstalk between the activated Slit2–Robo1 pathway and TGF‐β1 signalling promotes cardiac fibrosis

“…3 The key pathophysiological basis is the feedback cycle between HF and continuous differentiation of cardiac fibroblasts (CFs) into cardiac myofibroblasts, 4 which have a potent ability to divide and cause ECM overproduction. 5,6 This activation is mediated by the renin-angiotensin system and numerous neurohumoral factors, including angiotensin II (Ang II) and transforming growth factor β1 (TGF-β1). 3,5 Ang II promotes the release of catecholamine and aldosterone, up-regulates the expression of TGF-β1 in fibroblasts, and is a strong promoting factor for cardiac fibrosis.…”

“…The pathogenesis of cardiac fibrosis includes disruption of normal myocardial construction and abnormal extracellular matrix (ECM) accumulation 3 . The key pathophysiological basis is the feedback cycle between HF and continuous differentiation of cardiac fibroblasts (CFs) into cardiac myofibroblasts, 4 which have a potent ability to divide and cause ECM overproduction 5,6 . This activation is mediated by the renin–angiotensin system and numerous neurohumoral factors, including angiotensin II (Ang II) and transforming growth factor β1 (TGF‐β1) 3,5 .…”

SKI activates the Hippo pathway via LIMD1 to inhibit cardiac fibroblast activation

“…Furthermore, SKI is dysregulated and sequestered to the cytoplasm in a post-MI model of cardiac remodeling, suggesting that it contributes to fibroblast phenotype regulation [12]. We found that SKI also modulates MMP-9 expression and releases from primary cardiac fibroblasts, indicating that it may also influence the deposition and removal of ECM during post-MI wound healing [31]. As SKI's effects in cardiac fibroblasts are substantial and robust, it is possible that its functions within the cell extend beyond SMAD inhibition.…”

“…6 (continued) expression of αSMA and ED-An FN [11,12] in primary cardiac myofibroblasts. It was also established that SKI overexpression in primary cardiac myofibroblasts upregulates MMP-9 transcription and secretion [31], and its chronic expression represses autophagy-mediated survival responses which normally enable the pro-fibrotic phenotype to persist [60]. Recently it was found that SKI functions to inhibit pro-fibrotic signaling in cardiac myofibroblasts via SMADindependent MAPK signaling [61], suggesting that its functions are far more pleiotropic than initially described.…”

“…We previously showed that SKI downregulates myofibroblast markers in vitro [31]; however, it remains unknown as to whether SKI may influence the effects of the mechanotransduction on stiff substrate. This is salient, as the Hippo pathway is intimately linked to focal adhesions and the actin cytoskeleton, in that it is activated when the cell experiences less mechanical tension and there is a decrease in F-actin stress fibers [15].…”