1. ¹Zhejiang Respiratory Drugs Research Laboratory of State Foods and Drugs Administration of China, Medical School of Zhejiang University, Hangzhou 310058, China
2. ²Department of Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
3. ³Department of Pharmacology, Medical School of Zhejiang University, Hangzhou 310058, China

Correspondence: Professor Q-M Xie, Zhejiang Respiratory Drugs Research Laboratory of State Foods and Drugs Administration of China, Medical School of Zhejiang University, No.388 Yuhangtang Road, Hangzhou 310058, China. Tel: +86 571 8820 8231; Fax: +86 571 8820 8231; E-mail: xieqm@zju.edu.cn

Received 11 December 2006; Revised 10 May 2007; Accepted 28 May 2007; Published online 22 June 2007.

Abstract

Sodium-dependent vitamin C transporter (SVCT) 2-mediated L-ascorbic acid (AA) uptake is required in osteoblast-like differentiation of MC3T3-E1 cells, and prostaglandin E2 (PGE2) is among the most important local factors in bone formation, but the detailed mechanism by which PGE2 induces osteoblast differentiation remains obscure. We revealed that PGE2 induced AA uptake and osteoblast-like differential markers including alkaline phosphatase, collagen, osteocalcin expression, and mineralization in MC3T3-E1 cells. Inhibition of AA uptake by SVCT2 short isoform functioning as a dominant-negative mutant not only robustly attenuated PGE2-induced markers expression and mineralization, but also decreased their basal levels. However, upregulation of AA uptake resulted from PGE2-induced plasma membrane translocation of cytoplasm SVCT2, and this effect was abolished by pretreatment with EP4 receptor antagonist, AH-23848B or cAMP-dependent protein kinase A (PKA) inhibitor, H-89. Moreover, we showed SVCT2 physically interacted with PKA in immunoprecipitates, and PKA phosphorylated SVCT2 in vitro and in intact cells at Ser402 and Ser639 sites; however, mutation of Ser402 or/and Ser639 in SVCT2 severely diminished SVCT2 translocation in response to PGE2. Together, these results suggest that PGE2-induced SVCT2 plasma membrane translocation through EP4 receptor and subsequent phosphorylation of SVCT2 at Ser402 and Ser639 sites by PKA results in an increase of AA uptake and consequent promotion of osteoblast-like differentiation in MC3T3-E1 cells.