Unphosphorylated HSP 27 ( HSPB 1 ) regulates the translation initiation process via a direct association with eIF 4 E in osteoblasts

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 (T3)-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 T3 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 T3 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 T3 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. Introduction Heat-shock proteins (HSPs) are induced by a variety of physiological and environmental stresses, such as heat stress (1). As molecular chaperones, HSPs facilitate the refolding of non-native proteins, or assist in their elimination via chaperone-mediated autophagy or via the ubiquitin proteasome system. HSPs have recently been classified into seven families; HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), HSPD/E (HSP60/HSP10), CCT (TRiC), DNAJ (HSP40) and HSPB (small HSP) (1,2). HSP27 (HSPB1) belongs to the HSPB family, with monomeric molecular masses ranging from 15 to 30 kDa. The primary structure of HSP27 is highly homologous to other small HSPs, including αB-crystallin and HSP20, which all contain amino acid sequences known as ̔α-crystallin domains̓ (3). Although HSP27 is ubiquitously expressed in human cells and tissues, its functions have mainly been studied in skeletal, smooth and cardiac muscles, which have higher expression levels (1,4). The HSP27 expression level is significantly changed under various cellular conditions (1). It is generally recognized that the functions of HSP27 are regulated by post‐translational modifications, such as phosphorylation. HSP27, which normally exists as an unphosphorylated oligomer, possesses three phosphorylatable serine residues (Ser-15, Ser-78 and Ser-82) (5,6). Once HSP27 is phosphorylated, a conformational change occurs from the aggregated form to the dimer (5-7). Phosphorylated HSP27 has been previously shown to suppress the growth of hepatocellular carcinoma via inhibition of extracellular signal-regulated kinase (8). However, the exact role of phosphorylated HSP27 has not yet been clarified. Bone metabolism is strictly regulated through continuous bone remodeling in order to maintain the structural bone integrity and mineral homeostasis (9). Bone remodeling is mainly comprised of two functional events, osteoblastic bone formation and osteoclastic bone resorption (10). It has been reported that the outcome for osteosarcoma patients with overexpression of HSP27 is poorer (11). With regard to the functions of HSP27 in osteoblasts, it is reportedly involved in the balance between differentiation and apoptosis (12,13). The HSP27 protein is barely detectable in osteoblasts under unstimulated conditions (14). In our previous study (15), we Unphosphorylated HSP27 (HSPB1) regulates the translation initiation process via a direct association with eIF4E in osteoblasts GEN KUROYANAGI1,2, HARUHIKO TOKUDA2,3, NAOHIRO YAMAMOTO1,2, RIE MATSUSHIMA-NISHIWAKI1, OSAMU KOZAWA2 and TAKANOBU OTSUKA1 1Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi 467-8601; 2Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194; 3Department of Clinical Laboratory, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511, Japan Received January 19, 2015; Accepted June 30, 2015 DOI: 10.3892/ijmm.2015. 2274 Correspondence to: Dr Osamu Kozawa, Department of Pharmacology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan E-mail: okkasugai@yahoo.co.jp