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Shumin Rui, Takuo Kubota, Yasuhisa Ohata, Kenichi Yamamoto, Makoto Fujiwara, Shinji Takeyari, Keiichi Ozono, Phosphate promotes osteogenic differentiation through non-canonical Wnt signaling pathway in human mesenchymal stem cells, Bone, 2022, 164, 16525., Background: Phosphate is indispensable in osteogenesis and mineralization. However, mechanisms by which phosphate enhances osteogenic differentiation are not fully understood. In this study, we studied the effect of phosphate on osteogenic differentiation as well as signaling pathways induced by phosphate in the process. Method: Induced human bone marrow-derived mesenchymal stem cells differentiation into osteoblasts by the change of media containing β-glycerophosphate (GP), 1 mM inorganic phosphate, or 3 mM inorganic phosphate (Pi). The differentiation of osteoblasts was verified by the expression of osteoblast differentiation markers and calcium deposition. RNA sequencing was performed to assess transcriptome in the early stage of osteogenic differentiation. Results: Osteogenic differentiation and mineralization were promoted in the 3 mM Pi group compared to those in the GP and 1 mM Pi groups on day 7 of culture. RNA sequencing revealed that the gene expressions involved in osteogenesis and the components in the Wnt signaling pathway was increased in 3 mM Pi group compared with those in the GP on day 7. Analysis with qPCR and Western blot suggested upregulation of components in the non-canonical Wnt signaling pathway, including WNT5b and phosphorylated-c-Jun in the 3 mM Pi group on day 7. WNT11 mRNA expression was increased in the 2 induction groups on day 7. Inhibition of WNT5b by siRNA experiment attenuated the components in non-canonical Wnt signaling expression, including WNT5b, WNT11 and ROR2 mRNA expression and phosphorylated-c-Jun protein expression. In addition, osteogenic differentiation and mineralization were partly decreased in 3 mM Pi group on day 7 by the inhibition of WNT5b. Conclusion: Pi promoted osteogenic differentiation through the up-regulation of the non-canonical Wnt signaling pathway, including WNT5b, WNT11, p-c-Jun/c-Jun, in the early stage of differentiation. These findings provide a new perspective into the association of Pi and the non-canonical Wnt signaling pathway during osteogenic differentiation.

X-linked hypophosphatemic rickets (XLH) is an inheritable type of rickets caused by inactivating variants in the phosphate regulating endopeptidase homolog X-linked (PHEX) gene, which results in the overproduction of fibroblast growth factor 23 (FGF23). The mechanism by which PHEX impairment leads to FGF23 overproduction is unknown. Because little is known regarding the genotype-phenotype correlation in Japanese XLH, we summarized the available clinical and genetic data and analyzed the genotype-phenotype relationships using 3-dimensional (3D) structure modeling to clarify the XLH pathophysiology. We retrospectively reviewed the clinical features and performed genetic analysis of 39 Japanese patients with XLH from 28 unrelated pedigrees carrying any known or novel PHEX variant. To predict changes in the 3D structure of mutant PHEX, we constructed a putative 3D model of each mutant and evaluated the effect of structural alteration by genotype-phenotype correlation analysis. Genetic analysis revealed 23 PHEX variants, including eight novel variants. They were associated with high i-FGF23 levels, hypophosphatemia, phosphaturia, high alkaline phosphatase levels, and short stature. No gene dosage effect or genotype-phenotype correlation was observed when truncating and non-truncating variants were compared. However, the conservation of the zinc-binding site and cavity in PHEX had an impact on the elevation of i-FGF23 levels. Via genotype-phenotype relationship analysis using 3D modeling, we showed that the zinc-binding site and cavity in PHEX can play a critical role in its function. These findings provide new genetic clues for investigating the function of PHEX and the pathogenesis of XLH.

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.