A parathyroid hormone-related peptide (PTHrP) has been purified and cloned from tumors associated with the syndrome of humoral hypercalcemia of malignancy. The human PTRrP gene has been isolated and found to be a complex transcriptional unit that encodes three isoform-specific PTHrP mRNAs. In contrast to the PTH gone, to which it is related, the PTHrP gene seems to be widely expressed, and mounting evidence suggests that the actions of the peptide are predominantly if not exclusively local. We propose here four Specific Aims: 1) PTHrP gene expression appears to be subject to tight control Involving both transcriptional and posttranscriptional mechanisms. We plan to examine these mechanisms in a series of three interrelated studies, which include: 1) extending our evidence for methylation as an important mechanism for 'silencing' the gene to the study of Inducible PTHrP expression in F9 embryonal carcinoma cells, 2) functional mapping of the 5'flanking region of the gene with emphasis on the Identification of regions that are important in the rapid-response aspects of its control, and 3) the use of stably integrated PTHrP-globin hybrid genes to target the regions of PTHrP mRNA(s) that are responsible for instability. 2) The exact secretory form(s) of the PTHrP has yet to be established for any cell type. There are several levels of complexity here, including a) the presence in the PTHrP primary structure of multiple candidate sites for posttranscriptional processing (e.g., amidation and O-glycosylation), b) the existence of three PTHrP isoforms which might be subject to differential processing and/or trafficking, and c) the question of whether the PTHrP is secreted by both constitutive and regulated pathways and, if so, whether PTHrP processing is pathway-specific. These complexities have been addressed by expressing the cDNAs individually in constitutive (CHO) and regulated (RIN) secretory cells as sources of material for structural and biological study. Putative amidation of the peptide will be targeted by mutagenesis and putative O-glycosylation by expression in glycosylation-deficient CHO cells. 3) Of the many tissues in which PTHrP is produced, we have chosen to focus in the short to intermediate term on the rat myometrium. We have evidence in this system that the peak in PTHrP nRNA expression at term is due to mechanotransduction (i.e., mechanical stretch) and that myometrial cells are also PTHrP target cells. Our goals are to establish systems in vitro to study the control of PTHrP gene expression, with specific emphasis on stretch, as well as the mechanisms of PTHrP signal transduction. The findings in this system may be germane to other tonic muscle systems such as the atrium.
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