TARGETED OVEREXPRESSION OF PTH RELATED PEPTIDE

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Gene manipulation experiments in mice indicate that parathyroid hormone-related protein (PTHrP) functions as a developmental regulatory molecule. For example, overexpression of PTHrP in keratinocytes, mammary epithelial cells and chondrocytes results in a developmental phenotype in each case, while knockout of the gene leads to a chondrodystrophy that is lethal at birth. We have rescued the PTHrP-null via a genetic strategy, and this mouse provides a window on previously unappreciated PTHrP effects in tissues that share a common epithelial-mesenchymal morphogenesis. This phenotype includes failures in mammary epithelial development and tooth eruption and multiple abnormalities in skin. Delivery of PTHrP to these epithelial sites via a keratin-14-PTHrP transgene rescues these dysplastic features. We have also recently identified a neurodegenerative process in the PTHrP-null CNS that is age-related and seems to have excitotoxicity as its basis. Here we propose to create a series of mouse models to enable more detailed study of these regulatory effects in vivo. Aim 1. We will create "tet-off" and "tet-on" systems under the control of the keratin-14 promoter to provide regulated PTHrP expression in proliferative epithelia. This will allow us to study PTHrP effects on branching morphorgenesis during both embryonic and adolescent phases of mammary development, attempt to implicate PTHrP in multiple iterations of tooth movement/osteoclastic resorption, and explore the question of whether PTHrP deficiency in skin is associated with premature aging. Aim 2. We will use homologous recombination to insert a lacZ reporter gene into a PTHrP allele and will generate rescued PTHrP-null mice bearing the reporter as one of the PTHrP-null alleles. This will enable the study of the temporospatial expression of the gene with a sensitivity and fidelity not previously possible, an apt example being the proposed use of this mouse in Aim 3. Aim 3. It is our working hypothesis here that PTHrP functions in an autoprotective feedback loop (depolarization yields PTHrP yields type 1 receptor yields inhibition of L-type calcium channels) whereby neurons can combat long-latency excitotoxicity. The lacZ reporter will enable high resolution study of the neurodegenerative process in lacZ/PTHrP-null double heterozygotes, and we will attempt to rescue/reverse this process genetically via a PTHrP transgene under the control of a pan-neuronal promoter (neuron-specific enolase).