Human Osteoprogenitor Control by Hepatocyte Growth Factor and Vitamin D

Summary

Principal Investigator: Guy Howard
Abstract: DESCRIPTION (provided by applicant): 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 [unreadable]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.
Funding Period: 2012-07-01 - 2016-06-30
more information: NIH RePORT

Detail Information

Research Grants31

  1. Alcohol Effects on SDF1-Mediated Stem Cell Homing Following Bone Fracture Injury
    John J Callaci; Fiscal Year: 2013
    ....
  2. Function and Regulation of CD-RAP
    Linda J Sandell; Fiscal Year: 2013
    ..As new transcription factors arise, specific mechanism of activity will be deciphered using our CD-RAP and COL2A1 gene models. ..
  3. Differential Effects of BMPs on the Healing of Craniofacial Defects
    Russell R Reid; Fiscal Year: 2013
    ..abstract_text> ..
  4. BIOGENESIS OF EXTRACELLULAR MATRIX
    Bjorn Reino Olsen; Fiscal Year: 2013
    ....
  5. Combined Effect of Noggin Suppression and Nell-1 on Bone Regeneration
    Min Lee; Fiscal Year: 2013
    ..In this aim, we will develop non-viral gene delivery/scaffolding systems that release Nell-1 and Noggin-siRNA and will test whether they can effectively regenerate bone in a segmental femoral defect model. ..
  6. Generation of human chondroprogenitor cells for cartilage restoration
    Denis Evseenko; Fiscal Year: 2013
    ..In summary, this application will serve not only to address immediate and long term scientific questions in the field of chondrogenesis, but also the career development of Dr Evseenko into a successful independent researcher. ..
  7. Cadherin Mediated Cell-Cell Interactions in the Bone Microenvironment
    Roberto Civitelli; Fiscal Year: 2013
    ..Understanding the role of cadherins in bone biology is essential to gain a full picture of the molecular network by which bone development and homeostasis are controlled. ..
  8. Mechanisms Underlying Hormonal Regulation of Fracture Repair
    Dolores M Shoback; Fiscal Year: 2013
    ..An Advisory Committee consisting of local experts in bone biology along with our outside collaborators will provide external review of each project and the PP overall on an annual basis. ..
  9. Sp7 Mediated Control of Runx2 Function for Osteoblast Differentiation
    Amjad Javed; Fiscal Year: 2013
    ..Knowledge obtained from this study will provide molecular insights into components of bone regulatory complex that can be targeted for innovative therapy to improve cartilage and bone formation and repair. ..
  10. Indian Hedgehog Signaling in Osteoblast Differentiation
    Fanxin Long; Fiscal Year: 2013
    ..Research results from this study will provide a molecular framework for developing novel bone-enhancing pharmaceutics. ..
  11. Signaling in Inflammation, Stress, and Tumorigenesis
    GEORGE ROBERT STARK; Fiscal Year: 2013
    ..abstract_text> ..
  12. Smad4/B-Catenin Signaling Cross-Talk for Osteoblastogenesis
    Roberto Civitelli; Fiscal Year: 2013
    ....
  13. Multilineage regulation of mesenchymal stem cell differentiation by microRNAs
    Glyn Palmer; Fiscal Year: 2013
    ..This should improve our current understanding of mesenchymal stem cell differentiation and aid future cell-based therapies for musculoskeletal repair. ..
  14. Defining a myofibroblast/pericyte as a mesenchymal progenitor cell
    Ivo Kalajzic; Fiscal Year: 2013
    ..The knowledge developed in this grant will provide the foundation for future studies aimed at use of mesenchymal progenitor cells as therapeutic tools for osteoporosis and genetic disorders of bone. ..
  15. Regulation of osteoblast number by PTH
    Robert L Jilka; Fiscal Year: 2013
    ....
  16. Tissue engineering application of endochondral ossification for bone regeneration
    CHELSEA SHIELDS BAHNEY; Fiscal Year: 2013
    ..abstract_text> ..
  17. Mechanical Control of Mesenchymal Stem Cell Lineage Allocation
    JANET E RUBIN; Fiscal Year: 2013
    ..We will look at direct effects to prevent adipogenesis (inhibition of PPAR? expression) and indirect [unreadable]- catenin inhibition of PPAR? responses. ..
  18. Impact of Amyloid on the Aging Brain
    Reisa A Sperling; Fiscal Year: 2013
    ..This PPG brings together an exceptional multidisciplinary team of clinical, statistical, cognitive neuroscience, imaging, and laboratory investigators dedicated to exploring the impact of amyloid on the aging brain. ..
  19. Enhanced Osteoinduction and Angioinduction via Polymer/CaO2 Bone Tissue Engineeri
    Cato T Laurencin; Fiscal Year: 2013
    ..We hypothesize the bioactivity of calcium peroxide will significantly improve bone tissue regeneration in vivo over growth factor loaded poly(lactide-co-glycolide) scaffolds. ..
  20. The Role of BMP2 in the Regenerative Effects of MSC in Fracture Repair
    TIMOTHY JOSEPH MYERS; Fiscal Year: 2013
    ....
  21. BBP (Bone Morphogenetic Protein Binding Peptide) and Bone Healing
    SAMUEL SCOTT MURRAY; Fiscal Year: 2013
    ..We will also determine how the native bone protein spp24 (secreted phosphoprotein-24 kDa) inhibits BMP/TGF-b-dependent tumor cell growth. ..