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.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Accession codes
Abbreviations
- SVCT2:
-
sodium-dependent vitamin C transporter 2
- PGE2:
-
prostaglandin E2
- PKA:
-
cAMP-dependent protein kinase A
- AA:
-
L-ascorbic acid
- ALP:
-
alkaline phosphatase
- Hyp:
-
hydroxyproline
- OCN:
-
osteocalcin
- dnSVCT2:
-
SVCT2 short isoform
References
Franceschi RT . The role of ascorbic acid in mesenchymal differentiation. Nutr Rev 1992; 50: 65–70.
Franceschi RT, Iyer BS, Cui Y . Effect of ascorbic acid on collagen matrix formation and osteoblast differentiation in murine MC3T3-E1 cells. J Bone Miner Res 1994; 9: 843–854.
Franceschi RT, Iyer BS . Relationship between collagen synthesis and expression of the osteoblast phenotype in MC3T3-E1 cells. J Bone Miner Res 1992; 7: 235–246.
Shi S, Kirk M, Kahn AJ . The role of type I collagen in the regulation of osteoblast phenotype. J Bone Miner Res 1996; 11: 1139–1145.
Tsukaguchi H, Tokui T, Mackenzie B, Berger UV, Chen X-Z, Wang Y et al. A family of mammalian Na+-dependent L-ascorbic acid transporters. Nature 1999; 399: 70–75.
Daruwala R, Song J, Koh WS, Rumsey SC, Levine M . Cloning and functional characterization of the human sodium-dependent vitamin C transporters hSVCT1 and hSVCT2. FEBS Lett 1999; 460: 480–484.
Sotiriou S, Gispert S, Cheng J, Wang Y, Chen A, Hoogstraten-Miller S et al. Ascorbic-acid transporter Slc23a1 is essential for vitamin C transport into brain and for perinatal survival. Nat Med 2002; 8: 514–517.
Franceschi RT, Wilson JX, Dixon SJ . Requirement for Na+-dependent ascorbic acid transport in osteoblast function. Am J Physiol 1995; 268: C1430–C1439.
Fujita I, Hirano J, Itoh N, Nakanishi T, Tanaka K . Dexamethasone induces sodium-dependent vitamin C transporter in a mouse osteoblastic cell line MC3T3-E1. Br J Nutr 2001; 86: 145–149.
Wu X, Itoh N, Taniguchi T, Nakanishi T, Tanaka K . Requirement of calcium and phosphate ions in expression of sodium-dependent vitamin C transporter 2 and osteopontin in MC3T3-E1 osteoblastic cells. Biochim Biophys Acta 2003; 1641: 65–70.
Wu X, Itoh N, Taniguchi T, Nakanishi T, Tatsu Y, Yumoto N et al. Zinc-induced sodium-dependent vitamin C transporter 2 expression: potent roles in osteoblast differentiation. Arch Biochem Biophys 2003; 420: 114–120.
Wu X, Itoh N, Taniguchi T, Hirano J, Nakanishi T, Tanaka K . Stimulation of differentiation in sodium-dependent vitamin C transporter 2 overexpressing MC3T3-E1 osteoblasts. Biochem Biophys Res Commun 2004; 317: 1159–1164.
Harada S, Nagy JA, Sullivan KA, Thomas KA, Endo N, Rodan GA et al. Induction of vascular endothelial growth factor expression by prostaglandin E2 and E1 in osteoblasts. J Clin Invest 1994; 93: 2490–2496.
Klein DC, Raisz LG . Prostaglandin: stimulation of bone resorption in tissue culture. Endocrinology 1970; 86: 1436–1440.
Santoro MG, Jaffe BM, Simmons DJ . Bone resorption in vitro and in vivo in PGE-treated mice. Proc Soc Exp Biol Med 1977; 156: 373–377.
Yang R-S, Liu T-K, Shiau S-Y . Increased bone growth by local prostaglandin E2 in rats. Calcif Tissue Int 1993; 52: 57–61.
Mori S, Jee WSS, Li XJ, Chan S, Kimmel DB . Effects of prostaglandin E2 on production of new cancellous bone in the axial skeleton of ovariectomized rats. Bone (Elmsford) 1990; 11: 103–113.
Chyun YS, Raisz LG . Stimulation of bone formation by prostaglandin E2. Prostaglandins 1984; 27: 97–103.
Woodiel FN, Fall PM, Raisz LG . Anabolic effects of prostaglandins in culture fetal rat calvariae: structure-activity relations and signal transduction pathway. J Bone Miner Res 1996; 11: 1249–1255.
Yang RS, Fu WM, Wang SM, Lu KS, Liu TK, Lin-Shiau SY . Prostaglandin-induced morphological changes in osteoblasts. Bone 1998; 22: 629–636.
Hakeda Y, Nakatani Y, Hiramatsu M, Kurihara N, Tsunoi M, Ikeda E et al. Inductive effects of prostaglandins on alkaline phosphatase in osteo-blastic cells, clone MC3T3-E1. J Biochem 1985; 97: 97–104.
Hakeda Y, Nakatani Y, Kurihara N, Ikeda E, Maeda N, Kumegawa M . Prostaglandin E2 stimulates collagen and non-collagen protein synthesis and prolyl hydroxylase activity in osteoblastic clone MC3T3-E1 cells. Biochem Biophys Res Commun 1985; 126: 340–345.
Nagata T, Kaho K, Nishikawa S, Shinohara H, Wakano Y, Ishida H . Effect of prostaglandin E2 on mineralization of bone nodules formed by fetal rat calvarial cells. Calcif Tissue Int 1994; 55: 451–457.
Flanagan AM, Chambers TJ . Stimulation of bone nodule formation in vitro by prostaglandin E1 and E2. Endocrinology 1992; 130: 443–448.
Lutsenko EA, Carcamo JM, Golde DW . A human sodium-dependent vitamin C transporter 2 isoform acts as a dominant negative inhibitor of ascorbic acid transport. Mol Cell Biol 2004; 24: 3150–3156.
Suzawa T, Miyaura C, Inada M, Maruyama T, Sigimoto Y, Ushikubi F et al. The role of prostaglandin E receptor subtypes (EP1, EP2, EP3 and EP4) in bone resorption: an analysis using specific agonist for the respective EPs. Endocrinology 2000; 141: 1554–1559.
Suda M, Tanaka K, Natsui T, Usui T, Tanaka I, Fukushima M et al. Peostaglandin E receptor subtypes in mouse osteoblastic cell line. Endocrinology 1996; 137: 1689–1705.
Hakeda Y, Yoshino T, Nakatani Y, Kurihara N, Maeda N, Kumegawa M . Prostaglandin E2 stimulates DNA synthesis by a cAMP-independent pathway in osteobalst clone MC3T3-E1 cells. J Cell Physiol 1986; 128: 155–161.
Conconi MT, Tommasini M, Baiguera S, Coppi PD, Parnigotto PP, Nussdorfer GG . Effects of prostaglandin E1 and E2 on the growth and differentiation of osteoblast-like cells cultured in vitro. Int J Mol Med 2002; 10: 451–456.
Kajii T, Suzuki K, Yoshikawa M, Imai T, Matsumoto A, Nakamura S . Long-term effects of prostaglandin E2 on the mineralization of a clonal osteoblastic cell line (MC3T3-E1). Arch Oral Biol 1999; 44: 233–241.
Tai H, Miyamura C, Philbeam CC, Tamura T, Ohsugi Y, Koishihara Y et al. Transcriptional induction of cyclooxygenase-2 in osteoblast is involved in interleukin-6 induced osteocalst formation. Endocrinology 1997; 138: 2372–2379.
Ho ML, Chang JK, Chuang LY, Hsu HK, Wang GJ . Effect of nonsteroidal anti-inflammatory drugs and prostaglandins on osteoblastic functions. Biochem Pharmacol 1999; 58: 983–990.
Rumsey SC, Daruwala R, Al-Hasani H, Zamowski MJ, Simpson IA, Levine M . Dehydroascorbic acid transport by GLUT4 in Xenopus oocytes and isolated rat adipocytes. J Biol Chem 2000; 275: 28246–28253.
Bryant NJ, Govers R, James DE . Regulated transport of the glucose transporter GLUT4. Nat Rev Mol Cell Biol 2002; 3: 267–277.
Watson RT, Kanzaki M, Pessin JE . Regulated membrane trafficking of the insulin- responsive glucose transporter 4 in adipocytes. Endocr Rev 2004; 25: 177–204.
Liang WJ, Johnson D, Ma LS, Jarvis SM, Wei-Jun L . Regulation of the human vitamin C transporters expressed in COS-1 cells by protein kinase C. Am J Physiol Cell Physiol 2002; 283: 1696–1704.
Fujino H, Salvi S, Regan JW . Differential regulation of phosphorylation of the cAMP response element-binding protein after activation of EP2 and EP4 prostanoid receptors by prostaglandin E2 . Mol Pharmacol 2005; 68: 251–259.
Takanaga H, Mackenzie B, Hediger MA . Sodium-dependent ascorbic acid transporter family SLC23. Pflugers Arch 2004; 447: 677–682.
Delamere NA, Coca-Prados M, Aggarwal S . Studies on regulation of the ascorbic acid transporter in a cell line derived from rabbit non-pigmented ciliary epithelium. Biochim Biophys Acta 1993; 1149: 102–108.
Shao J, Jung C, Liu C, Sheng H . Prostaglandin E2 stimulates the β-catenin/T cell factor-dependent transcription in colon cancer. J Biol Chem 2005; 280: 26565–26572.
Acknowledgements
X Wu received a postdoctoral fellowship from the Society of Japanese Pharmacopoeia. We thank Drs K Tanaka, N Itoh, and T Nakanishi from Osaka University for valuable advice and Dr MJ Hilton from Washington University in St Louis for discussion of this work, and correcting errors of grammars.
Author information
Authors and Affiliations
Corresponding author
Additional information
Edited by G Cossu
Supplementary Information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd)
Rights and permissions
About this article
Cite this article
Wu, X., Zeng, LH., Taniguchi, T. et al. Activation of PKA and phosphorylation of sodium-dependent vitamin C transporter 2 by prostaglandin E2 promote osteoblast-like differentiation in MC3T3-E1 cells. Cell Death Differ 14, 1792–1801 (2007). https://doi.org/10.1038/sj.cdd.4402190
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/sj.cdd.4402190
Keywords
This article is cited by
-
Examining Mechanisms for Voltage-Sensitive Calcium Channel-Mediated Secretion Events in Bone Cells
Calcified Tissue International (2023)
-
SVCT2 Is Expressed by Cerebellar Precursor Cells, Which Differentiate into Neurons in Response to Ascorbic Acid
Molecular Neurobiology (2018)
-
SVCT2 Overexpression in Neuroblastoma Cells Induces Cellular Branching that is Associated with ERK Signaling
Molecular Neurobiology (2016)
-
Inhibition of phosphodiesterase 5 reduces bone mass by suppression of canonical Wnt signaling
Cell Death & Disease (2014)
-
Phosphatidylserine-Containing Liposomes: Potential Pharmacological Interventions Against Inflammatory and Immune Diseases Through the Production of Prostaglandin E2 After Uptake by Myeloid Derived Phagocytes
Archivum Immunologiae et Therapiae Experimentalis (2011)
