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 metap hysis of growing long bones. Proliferation and apoptosis dominate in the pr imary spongiosa subjacent to the growth plate, and differentiation and func tion 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 v ivo regulation by human parathyroid hormone (1-34) (PTH) of apoptosis in bo ne cells of the distal metaphysis of young male rats, Rats were given PTN a t 80 mu g/kg per day, once daily, for 1-28 days. Bone cells rr ere defined for flow cytometry as PTHL-receptor-positive (PTH1R(+)) and growth factor-r eceptor-positive (GFR(+)) cells. Apoptotic cells stained positive for eithe r TdT-mediated dUTP-X nick end labeling (TUNEL) or annexin V (annV(+)) were detected by either flow cytometry or immunohistochemistry Apoptosis was al so assessed at the tissue level by RNAse protection and caspase enzyme acti vity 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 chan ge in the steady-state mRNA levels of selected apoptotic genes. Starting at 3 days, at the tissue level, PTH inhibited activity of caspases, which rec ognize 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 apoptos is in the proliferating zone and terminal trabecular zone may be the result of the increased activation frequency and bone turnover seen with daily PT H treatment. As once-daily PTH increases the number of differentiated osteo blasts, and as these and hematopoietic marrow cells dominate metaphyseal ti ssue, inhibition of caspase activity may contribute to their prolonged surv ival, enabling extension of trabecular bone into the diaphyseal marrow to i ncrease bone mass. (C) 2000 by Elsevier Science Inc. All rights reserved.