Dataset Information

Differential regulation of extracellular signal-regulated protein kinases (ERKs) 1 and 2 by cAMP and dissociation of ERK inhibition from anti-mitogenic effects in rabbit vascular smooth muscle cells.

ABSTRACT: The inhibition of extracellular signal-regulated protein kinases (ERKs) is implicated in the negative regulation of vascular smooth muscle cell (VSMC) mitogenesis by cAMP-elevating agents and transforming growth factor beta(1) (TGF-beta(1)). These factors inhibited rabbit aortic VSMC mitogenesis induced by platelet-derived growth factor (PDGF)-BB by preventing the entry of cells into S-phase. cAMP-elevating agents partly inhibited the late phase (1-4 h) of activation of ERKs 1 and 2 induced by PDGF-BB without inhibiting the early phase of activity (5-15 min) and had no effect on activity induced by basic fibroblast growth factor (bFGF). In contrast, cAMP elevation caused a marked inhibition of early ERK activation induced by angiotensin II and thrombin. TGF-beta(1) had no inhibitory effect on ERK activation induced by PDGF-BB or bFGF. The inhibition of PDGF-BB-stimulated DNA synthesis by either forskolin/3-isobutyl-1-methylxanthine (IBMX) or TGF-beta(1) was not decreased when the agents were added up to 8 h after growth factor. In contrast, the selective ERK kinase inhibitor PD98059 was a weak inhibitor of DNA synthesis; a combination of PD98059 and forskolin/IBMX had an additive inhibitory effect on DNA synthesis. Forskolin/IBMX inhibited the growth factor-induced expression of c-myc mRNA and cyclin D(1) protein, and enhanced the protein expression of p27(kip1). TGF-beta(1) had no effect on the expression of c-myc or p27(kip1) and weakly attenuated the expression of cyclin D(1). These findings support the conclusion that the suppression of VSMC mitogenesis by cAMP and TGF-beta(1) is independent of ERK inhibition. Anti-mitogenic effects of cAMP might be primarily mediated by events in late G(1).

SUBMITTER: Cospedal R

PROVIDER: S-EPMC1220478 | biostudies-other | 1999 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:INTRODUCTION:We have previously demonstrated that transforming growth factor-? (TGF-?) in the presence of elevated levels of Smad3, its primary signaling protein, stimulates rat vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. The mechanism is partly 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 evaluated the role of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) in TGF-?-induced VSMC proliferation. METHODS:Cultured rat aortic VSMCs were incubated with TGF-? 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 small interfering RNA (siRNA). 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. RESULTS:In cultured VSMCs, TGF-? induced activation and phosphorylation of ERK MAPK in a time-dependent and concentration-dependent manner. Overexpression of the signaling protein Smad3 enhanced TGF-?-induced activation of ERK MAPK, whereas inhibition of Smad3 with a siRNA blocked ERK MAPK phosphorylation in response to TGF-?. These data suggest that Smad3 acts as a signaling intermediate between TGF-? and ERK MAPK. Inhibition of ERK MAPK activation with PD98059 completely blocked the ability of TGF-?/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 proliferating cell nuclear antigen. CONCLUSIONS:Our findings demonstrate a mechanism through which TGF-? stimulates VSMC proliferation. Although TGF-? has been traditionally identified as an inhibitor of proliferation, our data suggest that TGF-? enhances VSMC proliferation through a Smad3/ERK MAPK signaling pathway. These findings at least partly explain the mechanism by which TGF-? enhances intimal hyperplasia. Knowledge of this pathway provides potential novel targets that may be used to prevent restenosis.

Project description:The present study was designed to investigate the role of endogenous sulfur dioxide (SO2) in vascular smooth muscle cell (VSMC) proliferation, and explore the possible role of cross-talk between cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) pathways in this action. By cell counting, growth curve depict, flow cytometry and bromodeoxyuridine (BrdU) labeling assays, we found that SO2 inhibited VSMC proliferation by preventing cell cycle progression from G1 to S phase and by reducing DNA synthesis. SO2 synthase aspartate aminotransferase (AAT1 and AAT2) overexpression significantly inhibited serum-induced proliferating cell nuclear antigen (PCNA) protein expression in VSMCs, demonstrated by western blot analysis. Moreover, overexpression of AAT1 or AAT2 markedly reduced incorporation of BrdU in serum-treated VSMCs. By contrast, either AAT1 or AAT2 knockdown significantly exacerbated serum-stimulated VSMC proliferation. Thus, both exogenous- and endogenous-derived SO2 suppressed serum-induced VSMC proliferation. However, annexin V-propidium iodide (PI) staining and cell cycle analysis demonstrated that SO2 did not influence VSMC apoptosis in the serum-induced proliferation model. In a platelet-derived growth factor (PDGF)-BB-stimulated VSMC proliferation model, SO2 dephosphorylated the active sites of Erk1/2, MAPK kinase 1/2 and RAF proto-oncogene serine/threonine-protein kinase (c-Raf) induced by PDGF-BB. However, the inactivation of the three kinases of the Erk/MAPK pathway was not due to the separate interferences on them by SO2 simultaneously, but a consequence of the influence on the upstream activity of the c-Raf molecule. Hence, we examined the cAMP/PKA pathway, which could inhibit Erk/MAPK transduction in VSMCs. The results showed that SO2 could stimulate the cAMP/PKA pathway to block c-Raf activation, whereas the Ser259 site on c-Raf had an important role in SO2-induced suppression of Erk/MAPK pathway. The present study firstly demonstrated that SO2 exerted a negative regulation of VSMC proliferation via suppressing the Erk/MAPK pathway mediated by cAMP/PKA signaling.

Dataset Information

Differential regulation of extracellular signal-regulated protein kinases (ERKs) 1 and 2 by cAMP and dissociation of ERK inhibition from anti-mitogenic effects in rabbit vascular smooth muscle cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure