Effect Of Wnt3a/LRP5 Signaling Pathway To The Proliferation And Differentiation Of ST2 Cells

A tight coupling between bone deposition by osteoblasts and resorption by osteoclasts is required for the growth, development and metabolism of normal skeletal structure. If this delicate balance is disturbed, the density and architecture of bone can become abnormal, resulting in diseases such as osteoporosis. It is well established that bone density and metabolism are predominantly genetically determined. The gene coding low-density lipoprotein receptor-related protein 5 (LRP5) is one of the key regulators of bone density and studied extensively.Loss-of-function mutations in LRP5 lead to osteoporosis, whereas gain-of-function mutations in the same gene result in high bone mass (HBM). LRP5, a single-span transmembrane protein, is a co-receptor of Wnt family, suggesting a crucial role of canonical Wnt/ LRP5 signaling in bone formation. LRP5 is expressed in the osteoblasts lining the endosteal and trabecular bone surfaces, but not in osteoclasts. Studies demonstrate that LRP5 gene determines bone density by osteoblasts, which are derived from mesenchymal stem cells (MSCs). It is proposed that two aspects of the transformation from mesenchymal stem cells to osteoblasts could be controlled by canonical Wnt/LRP5 signaling pathway: proliferation and /or differentiation.To analyze the role of canonical Wnt/LRP5 signaling in mesenchymal osteogenesis, Wnt3a is chosen as a representative of canonical Wnt family and ST2 cell line is chosen as MSCs. The gain of exogenous Wnt3a and its action on ST2 cellsare completed by gathering the culture media from L-cell stably transfected with Wnt3a cDNA as conditioned media (CM) and adding them to ST2 cells. ST2 cells, a clone of stromal cells, are isolated from the bone marrow of BC8 mice and have typical characteristics of MSCs. These multipotential MSCs are able to give rise to osteoblasts under specific induction in vivo or in vitro. The increase of alkaline phosphatase (ALP) activity is a marker of differentiation into osteoblasts.The recombinant constructs that express wild type LRP5, 2 kinds of loss-of-function LRP5 and a gain-of-function LRP5 are transiently transfected into ST2 cells. Expression of LRP5 and recombinant constructs in ST2 cells are observed by RT-PCR, Western Blotting and cell immunofluoresence; Differences of cell proliferation are detected by BrdU cell proliferation assay kit in respond to exogenous Wnt3a plus wild-type or mutative LRP5, and the effect of Wnt3a/LRP5 signaling pathway to ST2 cell proliferation are observed; The effect of Wnt3a/LRP5 signaling pathway and exogenous Wnt3a to cell ALP activity is detected with or without the induction of ST2 cell to osteoblast-like cells by L-ascorbate and β-glycerophosphate sodium; Analysis about the effect of Wnt3a/LRP5 signaling pathway to proliferation and/or differentiation of ST2 cells is done. The results demonstrate that Wnt3a/LRP5 signaling pathway promotes cell proliferation obviously in the transformation of ST2 cells to osteoblasts. Wnt3a promotes differentiation of ST2 cells, more markedly with the induction of ST2 cell to osteoblast-like cells.Analysis about the effect of Wnt3a/LRP5 signaling pathway during the transformation from MSCs to osteoblasts provides insight into the role of this pathway to regulate bone density. This will help to find out pathogenesis of osteoporosis and new effective treatments for it.