title: Regulation by heat shock protein 27 of osteocalcin synthesis in osteoblasts.
abstract: We have previously reported that various stimuli, including sphingosine 1-phosphate, are able to induce heat shock protein (HSP) 27 in osteoblast-like MC3T3-E1 cells. However, the precise role of HSP27 in bone metabolism has not been satisfactory clarified. In this study, we investigated the effect of HSP27 on osteocalcin synthesis induced by bone morphogenetic protein (BMP)-4 or T₃ in these cells. In MC3T3-E1 cells, pretreatment with sphingosine 1-phosphate, sodium arsenite, or heat stress caused the attenuation of osteocalcin synthesis induced by BMP-4 or T₃ with concurrent HSP27 induction. To further investigate the effect of HSP27, we established stable HSP27-transfected cells. The osteocalcin synthesis was significantly reduced in the stable HSP27-transfected MC3T3-E1 cells and normal human osteoblasts compared with empty-vector transfected cells. On the other hand, anisomycin, a p38 MAPK activator, caused the phosphorylation of HSP27 in both sphingosine 1-phosphate-stimulated untransfected MC3T3-E1 cells and HSP27-transfected MC3T3-E1 cells. An immunofluorescence microscopy study showed that the phosphorylated HSP27 induced by anisomycin concentrated perinuclearly in these cells, in which it colocalized with the endoplasmic reticulum. We also established stable mutant-HSP27-transfected cells. Osteocalcin synthesis induced by either BMP-4 or T₃ was markedly suppressed in the nonphosphorylatable HSP27-overexpressing MC3T3-E1 cells compared with the phosphomimic HSP27-overexpressing cells. In contrast, the matrix mineralization was more obvious in nonphosphorylatable HSP27-overexpressing cells than that in phosphomimic HSP27-overexpressing cells. Taken together, these results strongly suggest that unphosphorylated HSP27 has an inhibitory effect on osteocalcin synthesis, but has a stimulatory effect on mineralization, in osteoblasts.
authors.affiliation: Department of Pharmacology, Gifu University Graduate School of Medicine, Yanagido, Gifu Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kato
authors.forename: Seiji
authors.initials: S
authors.lastname: Adachi
authors.forename: Rie
authors.initials: R
authors.lastname: Matsushima-Nishiwaki
authors.forename: Chiho
authors.initials: C
authors.lastname: Minamitani
authors.forename: Hideo
authors.initials: H
authors.lastname: Natsume
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Katagiri
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Hirose
authors.forename: Jun
authors.initials: J
authors.lastname: Mizutani
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Tokuda
authors.forename: Osamu
authors.initials: O
authors.lastname: Kozawa
authors.forename: Takanobu
authors.initials: T
authors.lastname: Otsuka
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000841:Anisomycin / Q000494:pharmacology; D015153:Blotting, Western; D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D002113:Calcification, Physiologic; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D055551:HSP27 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015675:Osteocalcin / Q000096:biosynthesis*; D010766:Phosphorylation / Q000187:drug effects; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014284:Triiodothyronine / Q000494:pharmacology
chemical_list: D055415:Bone Morphogenetic Protein 4; D055551:HSP27 Heat-Shock Proteins; D008246:Lysophospholipids; D019384:Nucleic Acid Synthesis Inhibitors; D014284:Triiodothyronine; D015675:Osteocalcin; C060506:sphingosine 1-phosphate; D000841:Anisomycin; D013110:Sphingosine