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

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.

[1]  S. Wiemann,et al.  WNT11/ROR2 signaling is associated with tumor invasion and poor survival in breast cancer , 2021, Journal of Experimental & Clinical Cancer Research.

[2]  J. Marc,et al.  Roles of Non-Canonical Wnt Signalling Pathways in Bone Biology , 2021, International journal of molecular sciences.

[3]  Jiang Chen,et al.  Effect of inorganic phosphate on migration and osteogenic differentiation of bone marrow mesenchymal stem cells , 2021, BMC developmental biology.

[4]  S. Bhadada,et al.  Role of Phosphate in Biomineralization , 2020, Calcified Tissue International.

[5]  Yi Gong,et al.  Inorganic phosphate-osteogenic induction medium promotes osteogenic differentiation of valvular interstitial cells via the BMP-2/Smad1/5/9 and RhoA/ROCK-1 signaling pathways. , 2020, American journal of translational research.

[6]  M. McKee,et al.  Biological stenciling of mineralization in the skeleton: Local enzymatic removal of inhibitors in the extracellular matrix. , 2020, Bone.

[7]  Y. Takashi,et al.  Phosphate-sensing and regulatory mechanism of FGF23 production , 2020, Journal of Endocrinological Investigation.

[8]  D. Newby,et al.  Osteocalcin Regulates Arterial Calcification Via Altered Wnt Signaling and Glucose Metabolism , 2019, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[9]  A. Dyakonov,et al.  Osteogenic Differentiation of Human Adipose Tissue-Derived MSCs by Non-Toxic Calcium Poly(ethylene phosphate)s , 2019, International journal of molecular sciences.

[10]  T. Yu,et al.  Knockout of TLR4 promotes fracture healing by activating Wnt/β-catenin signaling pathway. , 2019, Pathology, research and practice.

[11]  Cun-Yu Wang,et al.  Wnt1 inhibits vascular smooth muscle cell calcification by promoting ANKH expression. , 2019, Journal of molecular and cellular cardiology.

[12]  Yingzi He,et al.  Wnt/β-catenin interacts with the FGF pathway to promote proliferation and regenerative cell proliferation in the zebrafish lateral line neuromast , 2019, Experimental & Molecular Medicine.

[13]  K. Ozono,et al.  Roles of Phosphate in Skeleton , 2019, Front. Endocrinol..

[14]  C. Bergwitz,et al.  Role of phosphate sensing in bone and mineral metabolism , 2018, Nature Reviews Endocrinology.

[15]  N. Udagawa,et al.  Non-canonical Wnt signals regulate cytoskeletal remodeling in osteoclasts , 2018, Cellular and Molecular Life Sciences.

[16]  B. Boyan,et al.  Role of Wnt11 during Osteogenic Differentiation of Human Mesenchymal Stem Cells on Microstructured Titanium Surfaces , 2018, Scientific Reports.

[17]  Lucas C. Reineke,et al.  FGFR1-activated Translation of WNT Pathway Components with Structured 5' UTRs is Vulnerable to Inhibition of EIF4A-dependent Translation Initiation , 2018 .

[18]  J. Tardif,et al.  Role of Noncanonical Wnt Signaling Pathway in Human Aortic Valve Calcification , 2017, Arteriosclerosis, thrombosis, and vascular biology.

[19]  J. Millán,et al.  Alkaline Phosphatase and Hypophosphatasia , 2015, Calcified Tissue International.

[20]  K. Tang,et al.  Mechanical Tension Promotes the Osteogenic Differentiation of Rat Tendon-derived Stem Cells Through the Wnt5a/Wnt5b/JNK Signaling Pathway , 2015, Cellular Physiology and Biochemistry.

[21]  Nathaniel S. Hwang,et al.  Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling , 2014, Proceedings of the National Academy of Sciences.

[22]  Rajendra Prasad,et al.  Alkaline Phosphatase: An Overview , 2013, Indian Journal of Clinical Biochemistry.

[23]  Roland Baron,et al.  WNT signaling in bone homeostasis and disease: from human mutations to treatments , 2013, Nature Medicine.

[24]  C. Niehrs.  The complex world of WNT receptor signalling , 2012, Nature Reviews Molecular Cell Biology.

[25]  T. Katada,et al.  Negative regulation of wnt11 expression by Jnk signaling during zebrafish gastrulation , 2010, Journal of cellular biochemistry.

[26]  Yusuke Nakamura,et al.  Orphan receptor tyrosine kinase ROR2 as a potential therapeutic target for osteosarcoma , 2009, Cancer science.

[27]  Z. Massy,et al.  High extracellular inorganic phosphate concentration inhibits RANK–RANKL signaling in osteoclast‐like cells , 2008, Journal of cellular physiology.

[28]  R. Rosenberg,et al.  Expression of osteopontin, a target gene of de‐regulated Wnt signaling, predicts survival in colon cancer , 2007, International journal of cancer.

[29]  M. Yavropoulou,et al.  The role of the Wnt signaling pathway in osteoblast commitment and differentiation. , 2007, Hormones.

[30]  M. Asashima,et al.  Maternal Wnt11 Activates the Canonical Wnt Signaling Pathway Required for Axis Formation in Xenopus Embryos , 2005, Cell.

[31]  C. Marcelle,et al.  Wnt regulation of chondrocyte differentiation , 2002, Journal of Cell Science.

[32]  Han G. Brunner,et al.  Mutation of the gene encoding the ROR2 tyrosine kinase causes autosomal recessive Robinow syndrome , 2000, Nature Genetics.

[33]  Jihua Li,et al.  Wnt and Notch signaling pathways in calcium phosphate-enhanced osteogenic differentiation: A pilot study. , 2019, Journal of biomedical materials research. Part B, Applied biomaterials.