Sphingosine 1‐phosphate induces EGFR expression via Akt/NF‐κB and ERK/AP‐1 pathways in rat vascular smooth muscle cells

Abstract

Sphingosine 1-phosphate (S1P) has been shown to regulate expression of several genes in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating epidermal growth factor receptor (EGFR) expression by S1P in aortic VSMCs remain unclear. Western blotting and RT-PCR analyses showed that S1P induced EGFR mRNA and protein expression in a time- and concentration-dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and phosphatidylinositide 3-kinase (PI3K; wortmannin), and transfection with dominant negative mutants of ERK and Akt, respectively. These results suggested that S1P-induced EGFR expression was mediated through p42/p44 MAPK and PI3K/Akt pathways in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt which was attenuated by U0126 and wortmannin, respectively. Furthermore, S1P-induced EGFR upregulation was blocked by a selective NF-kappaB inhibitor helenalin. Immunofluorescent staining and reporter gene assay revealed that S1P-induced activation of NF-kappaB was blocked by wortmannin, but not by U0126, suggesting that activation of NF-kappaB was mediated through PI3K/Akt. Moreover, S1P-induced EGFR expression was inhibited by an AP-1 inhibitor curcumin and tanshinone IIA. S1P-stimulated AP-1 subunits (c-Jun and c-Fos mRNA) expression was attenuated by U0126 and wortmannin, suggesting that MEK and PI3K/ERK cascade linking to AP-1 was involved in EGFR expression. Upregulation of EGFR by S1P may exert a phenotype modulation of VSMCs. This hypothesis was supported by pretreatment with AG1478 or transfection with shRNA of EGFR that attenuated EGF-stimulated proliferation of VSMCs pretreated with S1P, determined by XTT assay. These results demonstrated that in VSMCs, activation of Akt/NF-kappaB and ERK/AP-1 pathways independently regulated S1P-induced EGFR expression in VSMCs. Understanding the mechanisms involved in S1P-induced EGFR expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis.

Enhanced expression of Gqα and PLC-β1 proteins contributes to vascular smooth muscle cell hypertrophy in SHR: role of endogenous angiotensin II and endothelin-1

“…AP-1 and NF-B are important transcription factors that play a crucial role in cardiovascular pathophysiology (16,21). To investigate the implication of AP-1 and NF-B in enhanced expression of G q ␣/PLC-␤1 proteins in VSMC from 16-wk-old SHR, we examined the effects of curcumin and helenalin, inhibitors of AP-1 and NF-B, respectively (5,22,36), on the enhanced expression of G q ␣ and PLC-␤1 in VSMC from 16-wk-old SHR. Results shown in Fig.…”

Thrombin Induces EGF Receptor Expression and Cell Proliferation via a PKC(δ)/c-Src-Dependent Pathway in Vascular Smooth Muscle Cells

“…25,26 More recently, we have shown that sphingosine 1-phosphate stimulates ERK1/2 and PI3K/Akt phosphorylation associated with EGFR expression in rat VSMCs. 27 In addition, we have also demonstrated that bradykinin stimulates ERK1/2 activation via transactivation of c-Src and EGFR leading to VSMC proliferation. 28 However, the mechanisms underlying thrombin-induced EGFR expression via transactivation of c-Src/EGFR/PI3K/Akt/ERK1/2 linked to cell proliferation are not completely defined in VSMCs.…”

“…PCR fragments were analyzed as previously described. 27 These primer sets specifically recognize only the genes of interest as indicated by amplification of a single band of the expected size (502 bp for EGFR and 514 bp for ␤-actin) and direct sequence analysis of the PCR products.…”

Sphingosine 1-phosphate regulates matrix metalloproteinase-9 expression and breast cell invasion through S1P3–Gαq coupling

“…S1P-induced cell motility is regulated by the p38 MAPK, ERK and Rho pathways in human primary fibroblast-like synoviocytes (Zhao et al, 2008). S1P can also stimulate the activation of PI3K-Akt and ERKs in human dermal MVECs cells (Hsieh et al, 2008;Heller et al, 2008).…”