Receptors for the carboxyl-terminal region of PTH(1-84) are highly expressed in osteocytic cells

PTH is a potent systemic regulator of cellular differentiation and function in bone. It acts upon cells of the osteoblastic Lineage via the G protein- coupled type-1 PTH/PTH-related peptide receptor (PTH1R). Carboxyl fragments of intact PTH(1-84) (C-PTH fragments) are cosecreted with it by the parath yroid glands in a calcium-dependent manner and also are generated via prote olysis of the hormone in peripheral tissues. Receptors that recognize C-PTH fragments (CPTHRs) have been described previously in osteoblastic and chon drocytic cells. To directly study CPTHRs in bone cells, we isolated clonal, conditionally transformed cell lines from fetal calvarial bone of mice tha t are homozygous for targeted ablation of the PTH1R gene and transgenically express a temperature-sensitive mutant SV40 T antigen. Cells with the high est specific binding of the CPTHR radio-ligand I-125-[Tyr(34)]hPTH(19-84) e xhibited a stellate, dendritic appearance suggestive of an osteocytic pheno type and expressed 6- to 10-fold more CPTHR sites/cell than did osteoblasti c cells previously isolated from the same bones. In these osteocytic (OC) c ells, expression of mRNAs for CD44, connexin 43, and osteocalcin was high, whereas that for alkaline phosphatase and cbfa-1/osf-2 was negligible. The CPTHR radioligand was displaced completely by hPTH(1-84), hPTH(19-84) and h PTH(24-84) (IC(50)s = 20-50 nM) and by kPTH(39-84) (IC50 = 500 nM) but only minimally (24%) by 10,000 nM hPTH(1-34). CPTHR binding was down-regulated dose dependently by hPTH(1-84), an effect mimicked by ionomycin and active phorbol ester. Human PTH(1-84) and hPTH(39-84) altered connexin 43 expressi on and increased apoptosis in OC cells. Apoptosis induced by PTH(1-84) was blocked by the caspase inhibitor DEVD, We conclude that osteocytes, the mos t abundant cells in bone, may be principal target cells for unique actions of intact PTH(1-84) and circulating PTH C-fragments that are mediated by CP THRs.