Use of GFP Markers to Assess the Effect of PTH

University Of Connecticut Sch Of Med/Dnt, Farmington CT

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Abstract

[unreadable] DESCRIPTION (provided by applicant): The maintenance of bone mass is accomplished by the continuous recruitment of osteoprogenitors to differentiate into mature osteoblasts that secrete the supporting bone matrix. The anabolic effect of intermittent administration of parathyroid hormone (PTH) on bone formation is well known but the underlying mechanism is not fully understood. The objective of this grant application is to identify at the cellular level how PTH acts either to inhibit or increase the number of active osteoblasts using a newly developing transgenic primary culture model. Transgenic mice harboring green fluorescent protein (GFP) marker genes driven by various Col1A1 promoter fragments are used to identify the level of osteoblastic differentiation in real time in primary bone cell culture. Based on the patterns of expression of GFP, three osteoblastic subpopulations are identified: multipotential osteoprogenitor, preosteoblast, and differentiated osteoblast. The hypothesis to be tested is that intermittent PTH acts to increase the number of osteoblastic cells by two distinct mechanisms: (1) to expand the population of multipotential osteoprogenitors and preosteoblasts that can subsequently progress to full differentiation once PTH is removed and (2) to induce a process in which differentiated osteoblasts dedifferentiate and proliferate under the influence of PTH and regain osteoblastic differentiation when PTH is removed. The effect of PTH will be specifically assessed on cells in both early and late lineage of osteoblastic differentiation using the tempo of GFP expression and FACS analysis for cell proliferation and differentiation. Results of this application will not only provide the information to understand the mechanism of anabolic effect of PTH at the cellular level, but also present a new model to examine the progression of osteoprogenitor lineage. [unreadable] [unreadable]