title: Short-term moderate hypothermia stimulates alkaline phosphatase activity and osteocalcin expression in osteoblasts by upregulating Runx2 and osterix in vitro.
abstract: Exposure of Normal Human Osteoblast cells (NHOst) to a period of hypothermia may interrupt their cellular functions, lead to changes in bone matrix and disrupt the balance between bone formation and resorption, resulting in bone loss or delayed fracture healing. To investigate this possibility, we exposed NHOst cells to moderate (35 °C) and severe (27 °C) hypothermia for 1, 12, 24 and 72 h. The effects of hypothermia with respect to cell cytoskeleton organization, metabolic activity and the expression of cold shock chaperone proteins, osteoblast transcription factors and functional markers, were examined. Our findings showed that prolonged moderate hypothermia retained the polymerization of the cytoskeletal components. NHOst cell metabolism was affected differently according to hypothermia severity. The osteoblast transcription factors Runx2 and osterix were necessary for the transcription and translation of bone matrix proteins, where alkaline phosphatase (Alp) activity and osteocalcin (OCN) bone protein were over expressed under hypothermic conditions. Consequently, bone mineralization was stimulated after exposure to moderate hypothermia for 1 week, indicating bone function was not impaired. The cold shock chaperone protein Rbm3 was significantly upregulated (p<0.001) during the cellular stress adaption under hypothermic conditions. We suggest that Rbm3 has a dual function: one as a chaperone protein that stabilizes mRNA transcripts and a second one in enhancing the transcription of Alp and Ocn genes. Our studies demonstrated that hypothermia permitted the in vitro maturation of NHOst cells probably through an osterix-dependent pathway. For that reason, we suggest that moderate hypothermia can be clinically applied to counteract heat production at the fracture site that delays fracture healing.
authors.affiliation: Institute of Medical Molecular Biotechnology & Faculty of Medicine, Universiti Teknologi MARA, 47000 Sungai Buloh, Selangor, Malaysia. Electronic address: aisha.mohddin@gmail.com.
authors.forename: M D
authors.initials: MD
authors.lastname: Aisha
authors.affiliation: Institute of Medical Molecular Biotechnology & Faculty of Medicine, Universiti Teknologi MARA, 47000 Sungai Buloh, Selangor, Malaysia; DDH, Universiti of Teknologi MARA, Shah Alam, Selangor, Malaysia. Electronic address: noras011@salam.uitm.edu.my.
authors.forename: M N K
authors.initials: MN
authors.lastname: Nor-Ashikin
authors.affiliation: DDH, Universiti of Teknologi MARA, Shah Alam, Selangor, Malaysia. Electronic address: sharaniza_abrahim@salam.uitm.edu.my.
authors.forename: A B
authors.initials: AB
authors.lastname: Sharaniza
authors.affiliation: Center for Pathology Diagnostic and Research Laboratories, Clinical Training Center, Universiti Teknologi MARA, 47000 Sungai Buloh, Selangor, Malaysia; DDH, Universiti of Teknologi MARA, Shah Alam, Selangor, Malaysia. Electronic address: hapizah@salam.uitm.edu.my.
authors.forename: H M
authors.initials: HM
authors.lastname: Nawawi
authors.affiliation: Institute of Medical Molecular Biotechnology & Faculty of Medicine, Universiti Teknologi MARA, 47000 Sungai Buloh, Selangor, Malaysia; DDH, Universiti of Teknologi MARA, Shah Alam, Selangor, Malaysia. Electronic address: marina@salam.uitm.edu.my.
authors.forename: M Y
authors.initials: MY
authors.lastname: Kapitonova
authors.affiliation: Institute of Medical Molecular Biotechnology & Faculty of Medicine, Universiti Teknologi MARA, 47000 Sungai Buloh, Selangor, Malaysia; DDH, Universiti of Teknologi MARA, Shah Alam, Selangor, Malaysia. Electronic address: gabriele@salam.uitm.edu.my.
authors.forename: G R A
authors.initials: GR
authors.lastname: Froemming
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002113:Calcification, Physiologic; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007035:Hypothermia; D008928:Mitochondria / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D012333:RNA, Messenger; C493709:RUNX2 protein, human; D000076182:Sp7 Transcription Factor; C478163:SP7 protein, human; D014157:Transcription Factors; D015675:Osteocalcin; D000469:Alkaline Phosphatase
keywords: Cell cytoskeleton; Chaperone protein; Fracture healing; Hypothermia; Osteoblasts; Transcription factor