Transforming growth factor-β₁regulation of C-type natriuretic peptide expression in human vascular smooth muscle cells: dependence on TSC22D1

Abstract

C-type natriuretic peptide (CNP) possesses nitric oxide-like signaling mechanisms and actions in the vasculature, including the inhibition of fibrosis and vascular remodeling through counterregulation of transforming growth factor-β (TGF-β) signaling. The leucine zipper protein transforming growth factor stimulated clone 22 domain 1 (TSC22D1), cloned via its presumed binding to a GC-rich element in the CNP promoter, was the first protein to be described as a CNP transcription factor, but the lack of supporting evidence since its discovery and its lack of a classical DNA-binding site have left in question its role in the regulation of CNP by TGF-β and other factors. To define a specific role for TSC22D1 in CNP transcription, we have examined the effects of the profibrotic growth factors TGF-β1 and PDGF-BB on CNP mRNA expression in cultured human vascular smooth muscle cells (SMC) in which TSC22D1 expression was suppressed with small interfering RNA. Results showed that TGF-β and PDGF-BB significantly increased CNP expression in all three SMC types. Twenty-four-hour TGF-β-induced elevations in CNP were strongly correlated with changes in TSC22D1 mRNA levels, and both genes exhibited their greatest response to TGF-β1 in coronary artery SMC. Furthermore, siRNA suppression of TSC22D1 expression in coronary artery and aortic SMC by ∼90% resulted in 45-65% reductions of both PDGF- and TGF-β-stimulated CNP expression, respectively. These results support a postulated role of TSC22D1 as an enhancer of CNP transcription and suggest that TGF-β-induced upregulation of CNP expression in SMC may be mediated in part by increased transcription of TSC22D1.

Pharmacodynamic responses of plasma and tissue C-type natriuretic peptide to GH: correlation with linear growth in GH-deficient rats

“…The process by which GH stimulates CNP secretion was not addressed in this study, but the failure of short-term GH to affect plasma NTproCNP (or skeletal growth) in rapidly growing healthy lambs despite the large increases in plasma IGF1 suggests that neither GH nor IGF1 directly stimulates CNP gene expression. A range of genes or transcription factors including transforming growth factor b, fibroblast growth factor, PDGF bb, TSC 22 (Suga et al 1992, Mendonca et al 2010) and KLF2 (Parmar et al 2006) are reported to increase CNP mRNA in vascular endothelial cells, but little is known of the mechanisms regulating CNP gene expression within the growth plate. Presumably downstream effectors of GH and IGF1, and possibly GH-induced mechanosensitive pathways (Zhang et al 2011), for example, by stimulating KLF2 (Cameron et al 2009), are involved.…”

Regulation of C-type natriuretic peptide expression

“…Therefore its proposed role as a direct enhancer of CNP transcription has remained in question. Recent studies in our laboratory using siRNA gene silencing techniques have shown that TSC22D1-silencing significantly reduces both PDGF-up-regulated CNP transcript in human aortic SMC and TGF-␤-up-regulated transcript in coronary artery-derived SMC [52]. These results suggest that in human vascular smooth muscle cells, TSC22D1 expression and activity could represent a critical element in a final common pathway leading to increased CNP expression induced by different stimuli.…”

“…Recent studies [31,99] suggest that these protein variants could perform unrelated, or even antagonistic functions in the mammalian cell; e.g., although the short form of TSC22D1 has been described many times as a tumor suppressor, the long form in contrast promotes proliferation, growth and cell survival [99]. Interestingly, in our recent studies of the effects of TSC22D1 silencing on CNP expression in vascular smooth muscle cells, [52], we could only identify a long form of the protein (∼120 kD) in Western blots, yet the downregulation of this protein with TSC22D1 siRNA resulted in reduced production of CNP transcript in these cells.…”

“…One of the first of these factors to be studied was TGF-␤, an enhancer of CNP expression and secretion in endothelial cells [63,83,84] that has since been shown to have a positive effect on CNP transcript levels in rat [92] and human [52] vascular smooth muscle cells. The stimulatory effect of TGF-␤ on vascular CNP production would suggest that this key agent in vascular remodeling after injury could constitute part of a feedback loop limiting TGF-␤-induced fibrosis through the actions of CNP [83].…”

“…Similarly, basic fibroblast growth factor (bFGF) was shown to suppress CNP transcript levels in cultured AoSMC derived from the rat [97], but it elevated CNP transcript levels in cultured smooth muscle cells derived from the human aorta [51]. On the other hand, CNP transcript levels responded positively to basic fibroblast bFGF in smooth muscle cells derived from human aorta, but showed no response to bFGF in human coronary-artery derived smooth muscle cells [52]. Studies such as these underscore the importance of cell phenotype and even species differences in delineating the intracellular transduction pathways controlling CNP production.…”