In vivo regulation of apoptosis in metaphyseal trabecular bone of young rats by synthetic human parathyroid hormone (1-34) fragment.

Osteoblast differentiation and function can be studied in situ in the metaphysis of growing long bones. Proliferation and apoptosis dominate in the primary spongiosa subjacent to the growth plate, and differentiation and function dominate in the proximal metaphysis. Apoptosis of osteocytes dominates at the termination of the trabeculae in diaphyseal marrow. As parathyroid hormone regulates all phases of osteoblast development, we studied the in vivo regulation by human parathyroid hormone (1-34) (PTH) of apoptosis in bone cells of the distal metaphysis of young male rats. Rats were given PTH at 80 microg/kg per day, once daily, for 1-28 days. Bone cells were defined for flow cytometry as PTH1-receptor-positive (PTH1R(+)) and growth factor-receptor-positive (GFR(+)) cells. Apoptotic cells stained positive for either TdT-mediated dUTP-X nick end labeling (TUNEL) or annexin V (annV(+)) were detected by either flow cytometry or immunohistochemistry. Apoptosis was also assessed at the tissue level by RNAse protection and caspase enzyme activity assays. PTH increased apoptotic osteoblasts in the proliferating zone and apoptotic osteocytes in the terminal trabecular zone, by 40%-60% within 2-6 days of PTH treatment, but values became equivalent to controls after 21-28 days of treatment. This transient increase was confirmed in PTH1R(+), GFR(+) bone cells isolated by flow cytometry. There was no detectable change in the steady-state mRNA levels of selected apoptotic genes. Starting at 3 days, at the tissue level, PTH inhibited activity of caspases, which recognize the DEVD peptide substrate (caspases 2, 3, and/or 7), but not those caspases recognizing LEHD or YVAD peptide sequences. We speculate that the localized and tissue level effects of PTH on apoptosis can be explained on the basis of its anabolic effect on bone. The transient increase in apoptosis in the proliferating zone and terminal trabecular zone may be the result of the increased activation frequency and bone turnover seen with daily PTH treatment. As once-daily PTH increases the number of differentiated osteoblasts, and as these and hematopoietic marrow cells dominate metaphyseal tissue, inhibition of caspase activity may contribute to their prolonged survival, enabling extension of trabecular bone into the diaphyseal marrow to increase bone mass.