Activation of PKA and phosphorylation of sodium-dependent vitamin C transporter 2 by prostaglandin E2 promote osteoblast-like differentiation in MC3T3-E1 cells

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.

Caveolin-1–mediated internalization of the vitamin C transporter SVCT2 in microglia triggers an inflammatory phenotype

“…Although we observed no direct interaction between c-Src and SVCT2 in microglia, the N-terminal domain of SVCT2 interacted with Cav-1, a component of the endocytic machinery that binds to and is phosphorylated by c-Src (56,69,70). Furthermore, SVCT2 internalization depended on c-Src-dependent phosphorylation of Cav-1 at Tyr 14 . These findings suggest the presence of a c-Src-Cav-1-SVCT2 signaling complex in the plasma membrane of Version: Postprint (identical content as published paper) This is a self-archived document from i3S -Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00 microglia and confirmed Cav-1 as a key component of the c-Src-dependent SVCT2 internalization.…”

“…6J). In conclusion, these data show that c-Src-dependent Tyr 14 phosphorylation of Cav-1 in microglia specifically mediated SVCT2 internalization.…”

“…SVCT2 is highly conserved across vertebrates, with human SVCT2 exhibiting 95 to 98% identity with the homologous proteins from rat, mouse, and rabbit (13). The activity and abundance of this transporter can be regulated by several signaling mechanisms, including direct phosphorylation of SVCT2 by protein kinase A (14) or protein kinase C (11,15) and transcriptional regulation of the gene encoding SVCT2 (SLC23A2) by nitric oxide (NO)-cyclic guanosine 3′,5′-monophosphate (CMG)-protein kinase G (PKG)-nuclear factor kB (NF-kB) signaling (16,17). The importance of SVCT2 in the CNS is illustrated by the severe phenotype of homozygous null mice (SVCT2 −/− ), which show extensive brain hemorrhage, have almost no ascorbate in their brains, and die shortly after birth (18).…”

miR-145 and miR-143 Regulate Odontoblast Differentiation through Targeting Klf4 and Osx Genes in a Feedback Loop

“…For induction of odontoblast differentiation, 50 g/ml ascorbic acid, 10 mmol/liter sodium ␤-glycerophosphate, and 10 nmol/liter dexamethasone (Sigma) were added to the medium for the duration of the experiment as described previously (18,32). Von-Kossa staining was carried out following a protocol reported previously (33).…”

<b>Effect of Prostaglandin E₂on Human Dental Follicle Cells during Osteogenic </b><b>Differentiation </b>

“…Local delivery of PGE 2 enhances bone formation at the cortical bone graft junction (15). The pro-osteogenic effect of PGE 2 has been further substantiated by findings from numerous in vitro studies using murine-or rat-derived MSCs and osteoblasts (16,17). The ability of PGE 2 to enhance bone formation has largely been attributed to its stimulatory effects on MSC differentiation (16,18).…”