title: Unphosphorylated HSP27 (HSPB1) regulates the translation initiation process via a direct association with eIF4E in osteoblasts.
abstract: Heat-shock protein 27 (HSP27/HSPB1) and its phosphorylation are implicated in multiple physiological and pathophysiological cell functions. Our previous study reported that unphosphorylated HSP27 has an inhibitory role in triiodothyronine (T(3))‑induced osteocalcin (OC) synthesis in osteoblasts. However, the mechanisms behind the HSP27‑mediated effects on osteoblasts remain to be clarified. In the present study, to investigate the exact mechanism of HSP27 and its phosphorylation in osteoblasts, the molecular targets of HSP27 were explored using osteoblast‑like MC3T3‑E1 cells. The levels of OC mRNA induced by T(3) in the HSP27‑overexpressing cells did not show any significant differences compared with those in the control empty vector‑transfected cells. Therefore, the interactions between HSP27 and translational molecules were focused on, including eukaryotic translation initiation factor 4E (eIF4E), eIF4G and 4E‑binding protein 1 (4E‑BP1). The HSP27 protein in the unstimulated cells co‑immunoprecipitated with eIF4E, but not eIF4G or 4E‑BP1. In addition, the association of eIF4E with 4E‑BP1 was observed in the HSP27‑overexpressing cells, as well as in the control cells. Under T(3) stimulation, the binding of eIF4E to eIF4G was markedly attenuated in the HSP27‑overexpressing cells compared with the control cells. In addition, the binding of HSP27 to eIF4E in the unstimulated cells was diminished by the phosphorylation of HSP27. In response to T(3) stimulation, the association of eIF4E with eIF4G in the unphosphorylatable HSP27‑overexpressing cells was markedly reduced compared with the phospho‑mimic HSP27‑overexpressing cells. Taken together, these findings strongly suggest that unphosphorylated HSP27 associates with eIF4E in osteoblasts and suppresses the translation initiation process.
authors.affiliation: Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi 467‑8601, Japan.
authors.forename: Gen
authors.initials: G
authors.lastname: Kuroyanagi
authors.affiliation: Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501‑1194, Japan.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Tokuda
authors.affiliation: Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi 467‑8601, Japan.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Yamamoto
authors.affiliation: Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi 467‑8601, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Matsushima-Nishiwaki
authors.affiliation: Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501‑1194, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Kozawa
authors.affiliation: Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi 467‑8601, Japan.
authors.forename: Takanobu
authors.initials: T
authors.lastname: Otsuka
mesh_terms: D000818:Animals; D002460:Cell Line; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism*; D055551:HSP27 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics; D010442:Peptide Chain Initiation, Translational; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D015854:Up-Regulation
chemical_list: D039561:Eukaryotic Initiation Factor-4E; D055551:HSP27 Heat-Shock Proteins; D012333:RNA, Messenger; D015675:Osteocalcin