title: Successful induction of sclerostin in human-derived fibroblasts by 4 transcription factors and its regulation by parathyroid hormone, hypoxia, and prostaglandin E2.
abstract: Sclerostin, coded by SOST, is a secretory protein that is specifically expressed in osteocytes and suppresses osteogenesis by inhibiting WNT signaling. The regulatory mechanism underlying SOST expression remains unclear mainly due to the absence of an adequate human cell model. Thus, we herein attempted to establish a cell model of human dermal fibroblasts in order to investigate the functions of sclerostin. We selected 20 candidate transcription factors (TFs) that induce SOST expression by analyzing gene expression patterns in the human sarcoma cell line, SaOS-2, between differentiation and maintenance cultures using microarrays. An effective set of TFs to induce SOST expression was sought by their viral transduction into fibroblasts, and a combination of four TFs: ATF3, KLF4, PAX4, and SP7, was identified as the most effective inducer of SOST expression. Quantitative PCR demonstrated that the expression levels of SOST in fibroblasts treated with the 4 TFs were 199- and 1439-fold higher than those of the control after 1-week and 4-week cultures, respectively. The level of sclerostin in the conditioned medium, as determined by ELISA, was 21.2pmol/l 4weeks after the transduction of the 4 TFs. Interestingly, the production of Dickkopf1 (DKK1), another secreted inhibitor of WNT signaling, was also increased by transduction of these 4 TFs. Parathyroid hormone (PTH) significantly suppressed the induced SOST by 38% and sclerostin by 82% that of the vehicle. Hypoxia increased the induced SOST by 62% that of normoxia. Furthermore, prostaglandin E2 (PGE2) increased SOST expression levels to 16-fold those of the vehicle. In conclusion, the efficient induction of SOST expression and sclerostin production was achieved in human dermal fibroblasts by the transduction of ATF3, KLF4, PAX4, and SP7, and the induced SOST and sclerostin were regulated by PTH, hypoxia, and PGE2. This model may contribute to elucidating the regulatory mechanisms underlying SOST expression and advancing drug development for metabolic bone diseases.
authors: Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan; First Department of Oral and Maxillofacial Surgery, Osaka University Graduate School of Dentistry, Osaka 565-0871, Japan.
authors: Makoto
authors: M
authors: Fujiwara
authors: Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
authors: Takuo
authors: T
authors: Kubota
authors: Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
authors: Wei
authors: W
authors: Wang
authors: Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
authors: Yasuhisa
authors: Y
authors: Ohata
authors: Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
authors: Kohji
authors: K
authors: Miura
authors: Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
authors: Taichi
authors: T
authors: Kitaoka
authors: DNA-chip Development Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
authors: Daisuke
authors: D
authors: Okuzaki
authors: Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan; Department of Pediatrics, JCHO Osaka Hospital, Osaka 553-0003, Japan.
authors: Noriyuki
authors: N
authors: Namba
authors: Department of Bone and Mineral Research, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka 594-1101, Japan.
authors: Toshimi
authors: T
authors: Michigami
authors: Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
authors: Yasuji
authors: Y
authors: Kitabatake
authors: Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan. Electronic address: keioz@ped.med.osaka-u.ac.jp.
authors: Keiichi
authors: K
authors: Ozono
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D015687:Cell Hypoxia / Q000187:drug effects; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned / Q000494:pharmacology; D015232:Dinoprostone / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005819:Genetic Markers; D057809:HEK293 Cells; D006801:Humans; D000090062:Kruppel-Like Factor 4; D020411:Oligonucleotide Array Sequence Analysis; D010011:Osteocytes / Q000187:drug effects / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism*; D014161:Transduction, Genetic; D060449:Wnt Signaling Pathway / Q000187:drug effects
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D019485:Bone Morphogenetic Proteins; D017077:Culture Media, Conditioned; D005819:Genetic Markers; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D010281:Parathyroid Hormone; C422903:SOST protein, human; D014157:Transcription Factors; D015232:Dinoprostone
keywords: Human fibroblasts; Hypoxia; Parathyroid hormone; Prostaglandin E2; SOST; Sclerostin