The involement of Wnt signaling in osteoblastic cell behavior

Wnt (wingless and int-related proteins) are a family of secreted cysteine-rich glycoproteins, expressed in a variety of tissues in developing embryos and adult tissues, thought to be implicated in oncogenesis, embryonic development, and regeneration of adult tissues. In our study, Wnt5a was found to be one of the predominant Wnts that are expressed during osteoblastic differentiation of hMSCs in vitro and during hMSC-directed ectopic osteogenesis in the severe combined immunodeficient (SCID) mouse host. RT-PCR further revealed that hWNT5A and its receptor Frizzled family member 5 (hFZD5) was up-regulated during osteoblastic differentiation. The function of Wnt5a was further evaluated using knock-out model. Wnt5a-/- calvarial cells showed significantly slower cell proliferation when compared to Wnt5a+/- and wild type cells. Gene expression profiles of the Wnt5a-/- calvarial cells as compared to wild type cells were evaluated using microarray analysis. 255 genes exhibited at least 2-fold changes in expression. In addition, genes including Runx2, osterix, and alkaline phosphatase (ALP) were shown to be down-regulated in Wnt5a-/- cells. The results suggested that Wnt5a plays a role in osteogenesis. The low density lipoprotein receptor-related protein 5 (LRP5) is a key determinant of bone mass. In this study, constitutively active LRP5 was constructed by deletion of the extracellular domain of LRP5 (LRP5ΔN). The LRP5 signaling and the effects of an intracellular domain single nucleotide polymorphism (SNP: p.V1525A) on osteoblast differentiation and mineralization were studied. Expression of LRP5ΔN-V, which carries the allele p.1525V, induced higher β-catenin/TCF-LEF activity compared to LRP5ΔN-A, which carries the allele p.1525A. In a yeast two-hybrid assay, LRP5ΔN-V also demonstrated a stronger interaction with AXIN than LRP5ΔN-A. Expression of either of the alleles did not change cell proliferation. However, cells expressing LRP5ΔN-V showed increased ALP activity and bone nodule formation compared to cells transfected with empty vector or LRP5ΔN-A after osteogenic supplement (OS) treatment. Cells expressing LRP5ΔN-V revealed significantly increased bone sialoprotein (BSP) and osteocalcin (OCN) mRNA levels with OS treatment. LRP5ΔN-V expressing cells demonstrated positive interaction with BMP-2 signaling of transcription at the SBE-luc promoter. The mRNA levels of Runx2 and Osterix were not affected by this SNP