This application describes proposed studies on human mesenchymal stem cell (MSC) differentiation and maturation catalyzed by hepatocyte growth factor (HGF) and 1,25-dihydroxyvitamin D (1,25OHD), the most active metabolite of vitamin D. Data shows 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) deltaN-(lacks part of the NH2 terminus) p63. These two forms also have distinct RNA splice variants denoted as TAp63- or deltaNp63a, B, and y, depending mainly on the length of the C-terminus. The TA and deltaN forms of p63 can act in opposition to activate or repress specific activities. The biological significance o 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 MSC differentiation is dependent upon a switch from the upstream p63 promoter (TA, repressor) to the internal p63 promoter (deltaN, activator) mediating bone and cartilage development. This 1,25D/HGF regulated p63 switch results in increases in the deltaN 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 deltaN) during osteoblastic differentiation will be identified using luciferase assays to determine changes in promoter selection. ChIP assays will identify specific binding of 1,25OHD activated VDR to putative response elements on the TA vs deltaNp63 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 knockdown and then by lentiviral stable over-expression of specific variants. Confirmation/validation the role of specifi p63 isoforms/variants in MSC-mediated bone repair in vivo, will be done using an established drill-hole xenograft model of bone repair in athymic nude rats. Lenti-viral over- expression (OX) vectors for TA- and Np63, and the specific variant(s) identified in Specific Aim #1 will be used to produce stable over-expression of p63 variants in MSC. The model involves making a reproducible defect (drill hole) in the third tail vertebral body, and subsequently quantifying the rate of bone healing over time by digital analysis of X-ray images after MSCs (with various modifications to p63) have been placed into the hole. At the end of the study period (8-12 weeks) both CT imaging and immunohistochemical analyses will be done on the samples to further define the changes observed. The third part of the overall studies will be an evaluation / identification of the p63 gene expression and activation effects during MSC chondrogenic and adipogenic differentiation induced by HGF,1,25OHD, and 1,25OHD+HGF using the techniques described above for osteogenic differentiation. These studies are expected to identify a specific form / splice variant of p63 as a major component of the regulation of VDR by 1,25OHD and HGF, during MSC differentiation, supporting a generalized paradigm implicating p63 as a key player during MSC differentiation. Differentiation into other lineages would further strengthen this paradigm, but are beyond the scope of this proposal.
Osteoporosis and osteoarthritis are major causes of frailty in the veteran population. A greater understanding of the regulation of human bone marrow-derived stem cell differentiation and bone and cartilage biology could eventually lead to more effective therapies for these diseases. Specifically, our methodology and information could open the way to facilitate stem cell therapy for veterans with osteoporosis and potentially with cartilage repair in osteoarthritis. Because of the potential for adding new bone to a weakened skeleton, such therapy would be a significant addition to present therapies, which are directed mainly at preserving bone. Because of the problems with cartilage loss/damage in osteoarthritis, understanding the regulation of stem cell differentiation into cartilage-forming cells could also add to present therapies. The work proposed will provide a detailed understanding of how vitamin D and its receptor interact with growth factors and other molecules to guide the stem cells into respective mature cell types that could eventually be used for new therapies.
