NUCLEAR EVENTS IN PTH ACTION ON BONE CELLS

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DESCRIPTION (Adapted from the Applicant's Abstract): Parathyroid hormone (PTH) has multiple actions in bone and stimulates both bone resorption and formation. The overall goal of this proposal is to investigate the mechanisms by which PTH stimulates the expression of collagenase in osteoblast-like osteosarcoma cells (UMR 106-01). In this competing renewal application, the investigator will continue studies on the transcription factor(s) which mediate the effects of PTH on the collagenase gene. The investigator has demonstrated that stimulation of collagenase expression by PTH requires new protein synthesis and has localized the indirect effects of PTH on transcription of the collagenase gene to the 151 bp 5'-flanking DNA upstream to the transcription start site. The overall hypothesis is that PTH stimulates collagenase gene expression by means of a novel transcription factor either directly or indirectly. Preliminary data suggests that there is no known cis-acting element or trans-activating factor which accounts for the PTH-specific effects on the collagenase gene. The Specific Aims of the proposal are to: 1) identify nuclear proteins which bind to the tentative PTH response element(s) of the collagenase gene by screening known trans-activating factors using gel mobility shift assays and isolating novel factor(s) by screening an expression cDNA library with an oligonucleotide probe to the cis-acting region(s) or by protein purification using an oligonucleotide affinity column followed by peptide sequencing and cDNA library screening with degenerate oligonucleotide probes; 2) examine the regulation (i.e, PTH responsiveness, cAMP signal transduction dependence, and protein synthesis requirement) of the PTH response element binding proteins using gel shift assays, Western blots (novel factors will require production of polyclonal antibodies), immunoprecipitation, and Northern blots; 3) identify the functional domains of the PTH response element transcription factors using deletion or mutation constructs and transient co-transfection of UMR cells with the collagenase promoter/CAT expression vectors; and 4) determine the role of PTH response element transcription factor in vitro (by measuring the effects of sense and antisense expression constructs on expression of the collagenase gene promoter/CAT vectors) and in vivo by developing transgenic mice with overexpression or "knock out" of the PTH response element binding protein gene.