Phosphate promotes osteogenic differentiation through non-canonical Wnt signaling pathway in human mesenchymal stem cells

Rui, Shumin; Kubota, Takuo; Ohata, Yasuhisa; Yamamoto, Kenichi; Fujiwara, Makoto; Takeyari, Shinji; Ozono, Keiichi

Affiliations

¹Department of Pediatrics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

²Department of Statistical Genetics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

³The 1st. Department of Oral and Maxillofacial Surgery, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan

Abstract and keywords

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.

Keywords: Phosphate; Non-canonical Wnt signaling pathway; WNT5b; Osteogenic differentiation; Human mesenchymal stem cells

Links

DOI: 10.1016/j.bone.2022.116525

PubMed: 35987514

WoS: 000863197700008

History

Received: 2022-06-23

Revised: 2022-08-14

Accepted: 2022-08-15

Online: 2022-08-18

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