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) ?N-(lacks part of the NH2 terminus) p63. These forms also have RNA splice variants denoted as TAp63- or ?Np63?, ?, and ?, depending on the length of the C-terminus. The TA and ?N 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 (?N, activator) mediating bone development. This 1,25D/HGF regulated p63 switch results in increases in the ?N 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 ?N) 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 ?Np63 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 ?CT 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 CT imaging and immunohistochemical analyses will be done on the samples to define the changes observed / bone formed in the drill hole.
Osteoporosis and osteoarthritis are major causes of frailty in the veteran population. Moreover, non- union is a problem in as much as 30% of fractures. A greater understanding of the regulation of human bone marrow-derived stem cell differentiation and bone biology could eventually lead to more effective therapies for these respective diseases / problems. Specifically, our methodology and information could open the way to facilitate stem cell therapy for veterans with osteoporosis and with non-union fractures. Because of the potential for adding new bone to a weakened skeleton, such therapy would add significantly to present therapies -- directed mainly at preserving bone. The work proposed will provide an understanding of how vitamin D and its receptor interact with growth factors / other molecules to guide stem cells into respective mature cell types that could eventually be used for new therapies.
