TARGETED OVEREXPRESSION OF PTH-RELATED PEPTIDE

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DESCRIPTIONS (Adapted from the Applicant's Abstract): The PTH and PTHrP genes are members of a small family that encode regulatory molecules whose functions seem to be remarkably different. The PTHrP gene is clearly an ideal candidate for modern targeting strategies in vivo, and two such strategies have recently been applied, both revealing very interesting phenotypes. Although these two strategies involved mirror-image approaches (targeted gene disruption, on the one hand, and targeted overexpression, on the other) the PTHrP function(s) uncovered in both experiments may well be fundamentally the same. The present application proposes four targeted overexpression strategies involving the skin, breast, uterus, and cartilage. The emphasis of each experiment (and of the proposal as a whole) is on work in vivo, and only limited resources are requested for derivative experiments in vitro. First, the human keratin-14 (K14) promoter has been used to target PTHrP overexpression to the epidermis generating a phenotype that appears to reflect a primary failure of hair follicle initiation. The application thus proposes to: a) fully characterize the K14-PTHrP mice; b) study autocrine/paracrine signaling mechanisms in a limited number of experiments in vitro; and c) target PTH (1-84), as well as chimeric and/or deleted molecules, to skin in order to study PTHrP structure-function relationships in vivo. Second, the K14-PTHrP mice appear also to manifest a failure in lobuloalveolar mammary development, possibly reflecting activity of the K14 promoter in myoepithelial cells in the breast. The application proposes: a) to characterize fully the mammary defect in the K14-PTHrP mice; and b) to target PTHrP overexpression to the breast using the MMTV promoter/enhancer in syngeneic mice; this approach is intended to permit hormonal manipulation of MMTV-PTHrP expression, and also allow mammary epithelial fat pad transplantation experiments in order to pinpoint PTHrP developmental effects/defects. Thirdly, the application proposes to target PTHrP overexpression to the mouse myometrium and endometrium using the porcine uteroferrin promoter. The associated experiments will focus on:a) putative PTHrP involvement in the process of parturition per se; and b) the role of nitric oxide in mediating PTHrP actions in the myometrium. Fourthly, the application proposes to target PTHrP overexpression to proliferative and prehypertrophic chondrocytes using the mouse type II collagen promoter (pro-alpha-l(ll)). These experiments will focus on: a) apparent PTHrP function(s) in the developmental program of chondrocyte differentiation; and b) a cross- breeding complementation experiment in attempt to reverse or salvage the phenotype in the PTHrP gene-targeted (PTHrP null) mice.