Transforming growth factor-β increases vascular smooth muscle cell proliferation through the Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinases pathways

Abstract

Introduction We have previously demonstrated that TGF-β in the presence of elevated levels of its primary signaling protein, Smad3, stimulates rat vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. Moreover, we have shown that the mechanism in part, is through the nuclear exportation of phosphorylated cyclin-dependent kinase inhibitor p27. The objective of this study is to clarify the downstream pathways through which Smad3 produces its proliferative effect. Specifically, we evaluate the role of the ERK mitogen-activated protein kinase (ERK MAPK) in TGF-β-induced VSMC proliferation. Methods Cultured rat aortic VSMCs were incubated with TGF-β at varying concentrations and times, and phosphorylated ERK was measured by Western blotting. Smad3 was enhanced in VSMCs using an adenovirus expressing Smad3 or inhibited with ansiRNA. For in vivo experiments, Male Sprague-Dawley rats underwent carotid balloon injury followed by intraluminal infection with an adenovirus expressing Smad3. Arteries were harvested at 3 days and subjected to immunohistochemistry for Smad3, phospho-ERK MAPK and Proliferating Cell Nuclear Antigen (PCNA). Results In cultured VSMCs, TGF-β induced activation and phosphorylation of ERK MAPK in a time and concentration-dependent manner. Overexpression of the signaling protein, Smad3 enhanced TGF-β-induced activation of ERK MAPK whereas inhibition of Smad3 with ansiRNA blocked ERK MAPK phosphorylation in response to TGF-β. These data suggest that Smad3 acts as a signaling intermediate between TGF-β and ERK MAPK. Inhibition of ERK MAPK activation with PD98059 completely blocked the ability of TGF-β/Smad3 to stimulate VSMC proliferation, demonstrating the importance of ERK MAPK in this pathway. Immunoprecipitation of phospho-ERK MAPK and blotting with Smad3 revealed a physical association, suggesting that activation of ERK MAPK by Smad3 requires a direct interaction. In an in vivo rat carotid injury model, overexpression of Smad3 resulted in an increase in phosphorylated ERK MAPK as well as increased VSMC proliferation as measured by PCNA. Conclusion Our findings demonstrate a mechanism through which TGF-β stimulates VSMC proliferation. Although TGF-β has been traditionally identified as an inhibitor of proliferation, our data suggest that through a Smad3/ERK MAPK signaling pathway, TGF-β enhances VSMC proliferation. These findings explain at least in part, the mechanism by which TGF-β enhances intimal hyperplasia. Knowledge of this pathway provides potential novel targets that may be used to prevent restenosis.

AMP-activated protein kinase inhibits transforming growth factor-β-mediated vascular smooth muscle cell growth: implications for a Smad-3-dependent mechanism

“…In VSMCs, TGF-␤ has historically been considered antiproliferative (2, 14 -16); however, other findings suggest that TGF-␤ and its downstream Smads stimulate growth in primary VSMCs (9,18,19,(25)(26)(27). Adding to this uncertainty, in cultured cells TGF-␤1 is suggested to switch between growth stimulation and growth suppression depending on concentration (3,18) and cell density (9).…”

Calreticulin Regulates Neointima Formation and Collagen Deposition following Carotid Artery Ligation

“…The role of TGF-β in VSMC proliferation is more controversial. Halloran et al demonstrated that TGF-β inhibits human arterial SMC proliferation [10], whereas others showed that TGF-β stimulates VSMC proliferation through Smad3 and ERK-dependent pathways [11, 12]. …”

Vascular Smooth Muscle Cell Phenotypic Changes in Patients With Marfan Syndrome

“…6,14,17,18 However, TGF-β has bifunctional effects because it can also increase VSMC proliferation and migration. [19][20][21] Nonetheless, it is believed that VSMC differentiation and proliferation are not necessarily mutually exclusive, which would facilitate VSMC response and adaptation to a wide variety of pathophysiological conditions. Deregulation in VSMC differentiation occurs in cardiovascular pathologies, such as atherosclerosis and intimal hyperplasia, as well as in aortic aneurysms, [22][23][24][25][26] but its occurrence and relevance in the pathogenesis and progression of human Marfan aortic aneurysms has not been explored in detail.…”