p63 Regulation of Human Osteoprogenitor Maturation by Hepatocyte Growth Factor & Vitamin D Metabolites

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The proposed studies focus on human mesenchymal stem cell (MSC) differentiation and maturation regulated by hepatocyte growth factor (HGF) and 1,25-dihydroxyvitamin D (1,25OHD), the most active metabolite of vitamin D in facilitating bone repair. These studies will determine the involvement of endogenous HGF in regulating the 1,25OHD upregulation of its receptor VDR. Our publications and preliminary data show that p63, a member of the p53 family of transcription factors, plays a major role in the cooperative actions of HGF and 1,25OHD to up-regulate the vitamin D receptor (VDR) and promote MSC differentiation. Pilot data suggests that the cooperative effects are based on alterations of p63 differential gene expression products resulting from alternative promoter selection and RNA splicing changes. Regulation of p63 isoform gene expression involves two distinct promoters (an upstream promoter and an alternate promoter located in intron 3) and alternative splicing to generate mRNA. Depending on the promoter selected, 2 distinct forms are produced: 1) TA-(transactivation domain containing NH2 terminus) p63 and 2) ΔN-(lacks part of the NH2 terminus) p63. These forms also have RNA splice variants denoted as TAp63- or ΔNp63α, β, and γ, depending on the length of the C-terminus. The TA and ΔN forms of p63 can act in opposition to activate or repress specific activities. The biological significance of the RNA splice variants during stem-cell- mediated events is not clear. The vitamin D receptor (VDR) is an important regulator of MSC differentiation. 1,25OHD (bound to VDR) activates both VDR and p63 gene expression, p63 binds to the VDR promoter and up-regulates VDR gene expression, and HGF stimulation of VDR expression and HGF regulation of MSC osteoblastic differentiation can be blocked by decreasing p63 expression. Thus it is hypothesized that 1,25OHD + HGF regulation of hMSC differentiation is dependent upon a switch from the upstream p63 promoter (TA, repressor) to the internal p63 promoter (ΔN, activator) mediating bone development. This 1,25D/HGF regulated p63 switch results in increases in the ΔN form(s) vs TA form(s), and a relative increase in gamma splice variants compared to alpha and beta splice variants. To test this hypothesis the effects of HGF, 1,25OHD and HGF+1,25OHD on p63 promoter selection/activation (TA vs ΔN) during osteoblastic differentiation will be identified with luciferase assays to see changes in promoter selection. ChIP assays will identify specific binding of 1,25OHD-activated VDR to response elements on the TA vs ΔNp63 promoters, as well as identify p63 isoform(s) binding to the VDR promoter. Identification of changes in splice variants in response to HGF and 1,25OHD will be done by RT-qPCR and western blots, followed by siRNA and specific inhibitor knockdown, and by lentiviral stable over-expression of specific variants. Confirmation/validation of the role of specific p63 isoforms/variants in MSC-mediated bone repair in vivo, will be done using an established “drill-hole” model of bone repair in athymic nude rats. We have developed lentiviral over- expression vectors for TA- and ∆Np63 and the specific variant(s) with which we have produced stable over- expression of p63 variants in MSC. The in vivo model involves a reproducible defect (drill hole) in the third tail vertebral body, and then quantifying bone healing by μCT imaging after MSCs (with various modifications to p63) are placed into the hole. Because HGF is relatively short-lived in vivo, recent studies confirm the safety and efficacy of using a naked-plasmid for HGF to provide effective concentrations HGF in vivo. This efficient transient delivery of exogenous HGF, leads to activation of growth-related signal transduction events and promotion of cell proliferation (but not cancer).This approach is now in phase II clinical trials for peripheral vascular disease. We will use this method of HGF delivery to facilitate increased bone repair together with hMSC as described. At the end of the study period (8-12 weeks) both µCT imaging and immunohistochemical analyses will be done on the samples to define the changes observed / bone formed in the drill hole.