title: FGF and ERK signaling coordinately regulate mineralization-related genes and play essential roles in osteocyte differentiation.
abstract: To examine the roles of FGF and ERK MAPK signaling in osteocyte differentiation and function, we performed microarray analyses using the osteocyte cell line MLO-Y4. This experiment identified a number of mineralization-related genes that were regulated by FGF2 in an ERK MAPK-dependent manner. Real-time PCR analysis indicated that FGF2 upregulates Ank, Enpp1, Mgp, Slc20a1, and Dmp1 in MLO-Y4 cells. Consistent with this observation, the selective FGF receptor inhibitor PD173074 decreased Ank, Enpp1, Slc20a1, and Dmp1 mRNA expression in mouse calvaria in organ culture. Since Dmp1 plays a central role in osteocyte differentiation and mineral homeostasis, we further analyzed FGF regulation of Dmp1. Similar to FGF2, FGF23 upregulated Dmp1 expression in MLO-Y4 cells in the presence of Klotho. Furthermore, increased extracellular phosphate levels partially inhibited FGF2-induced upregulation of Dmp1 mRNA expression, suggesting a coordinated regulation of Dmp1 expression by FGF signaling and extracellular phosphate. In MLO-Y4 osteocytes and in MC3T3E1 and primary calvaria osteoblasts, U0126 strongly inhibited both basal expression of Dmp1 mRNA and FGF2-induced upregulation. Consistent with the in vitro observations, real-time PCR and immunohistochemical analysis showed a strong decrease in Dmp1 expression in the skeletal elements of ERK1(-/-); ERK2(flox/flox); Prx1-Cre mice. Furthermore, scanning electron microscopic analysis revealed that no osteocytes with characteristic dendritic processes develop in the limbs of ERK1(-/-); ERK2 (flox/flox); Prx1-Cre mice. Collectively, our observations indicate that FGF signaling coordinately regulates mineralization-related genes in the osteoblast lineage and that ERK signaling is essential for Dmp1 expression and osteocyte differentiation.
authors.affiliation: Department of Orthopaedics, Case Western Reserve University, 2109 Adelbert Road BRB 329, Cleveland, OH 44106, USA.
authors.forename: Ai
authors.initials: A
authors.lastname: Kyono
authors.forename: Nanthawan
authors.initials: N
authors.lastname: Avishai
authors.forename: Zhufeng
authors.initials: Z
authors.lastname: Ouyang
authors.forename: Gary E
authors.initials: GE
authors.lastname: Landreth
authors.forename: Shunichi
authors.initials: S
authors.lastname: Murakami
mesh_terms: D000818:Animals; D002113:Calcification, Physiologic / Q000187:drug effects / Q000235:genetics*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D000089703:Fibroblast Growth Factor-23; D005346:Fibroblast Growth Factors / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007150:Immunohistochemistry; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000235:genetics*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D010011:Osteocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D010710:Phosphates / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011743:Pyrimidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D012886:Skull / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: C513308:Dmp1 protein, mouse; D016326:Extracellular Matrix Proteins; C000717427:FGF23 protein, human; C000717428:Fgf23 protein, mouse; C115711:PD 173074; D010710:Phosphates; D011743:Pyrimidines; D012333:RNA, Messenger; D017468:Receptors, Fibroblast Growth Factor; D016222:Fibroblast Growth Factor 2; D005346:Fibroblast Growth Factors; D000089703:Fibroblast Growth Factor-23; D003513:Cycloheximide; D048049:Extracellular Signal-Regulated MAP Kinases