PTH (13-34) (HUMAN)

Osteocalcin (OC) is a bone-specific extracellular matrix protein expressed by mature osteoblasts during late stages of differentiation. Previous studies have shown that forskolin, an activator of adenylate cyclase, stimulated OC production. Because PTH has been shown to activate several intracellular signal transduction pathways including cAMP, inositol phosphate and intracellular calcium mobilization, we investigated whether PTH action on cAMP accumulation leads to OC promoter activation. The rat OC promoter (1095 bp) was cloned into the promoterless luciferase gene reporter vector. The transcriptional activity of the rat OC promoter was evaluated after transfection of SaOS-2, an osteosarcoma cell line, with the OC promoter followed by treatment with PTH. Maximal OC promoter activity was observed within 4-8 h after the addition of 10(-8) M PTH, whereas very little induction was seen after 24 and 48 h of treatment. The induction of OC promoter activity by PTH was concentration dependent. PTH analogs (PTH 1-84, PTH 1-34, and PTH 1-31) that stimulate intracellular cAMP accumulation, induced OC promoter activity, whereas other PTH analogs (PTH 3-34, PTH 7-34, PTH 13-34, and PTH 53-84) that do not stimulate cAMP production had no effect on OC promoter activation. Furthermore, PTH activation of the OC promoter was significantly enhanced in the presence of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Inactivation of cAMP-dependent protein kinase A activity by either a selective protein kinase A inhibitor, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5 isoquinolinesulfonamide), or antisense oligonucleotide directed against the regulatory subunit of cAMP-dependent protein kinase A, led to a corresponding loss of OC promoter activation by PTH. 5' deletion analysis of the OC promoter demonstrated that the promoter (1095 bp) exhibited the greatest response to PTH, whereas the -198 bp construct of the OC promoter, containing only one cAMP response element and OC box, was no longer responsive. The constructs with further deletions (-120, -92, and -74) retained PTH responsiveness, but to a lesser extent. In summary, our results indicate that PTH activation of the OC promoter is a rapid event and mediated by the cAMP-dependent protein kinase A pathway. Although the novel cAMP response region overlapping the OC box is required for activation, full activation may require several cis-acting cAMP response elements or other response elements.

Antibodies to a urea-trichloroacetic acid extract [hPTH-(TCA)] of human parathyroid tumors and to the synthetic NH(2)-terminal fragments of human parathyroid hormone hPTH-(1-12) and -(1-34) were developed in goats to characterize immunochemically various PTH preparations and to estimate immunoreactive PTH (iPTH) in human sera. They were quantitated on the basis of their capacity to bind [(131)I]-hPTH-(1-12), [(131)I]hPTH-(1-34) or [(131)I]bovine PTH (bPTH-(1-84)). The quality of the antibodies was assessed by reference to inhibition of their interaction with labeled peptides by synthetic hPTH comprising 34 NH(2)-terminal amino acid residues or fragments thereof [hPTH-(1-12), -(13-34), -(18-34), -(25-34), -(18-24)] or by the Sephadex G-100-purified full-length peptide hPTH-(1-84) [hPTH-(1-84)G-100]. The synthetic peptides used in this work correspond in their structure to the NH(2)-terminal amino acid sequence 1-34, as elucidated by Brewer and collaborators (1972. Proc. Natl. Acad. Sci. U. S. A.69: 3583-3588). Inhibition studies were also carried out with bPTH-(1-34) and bPTH-(1-84). Anti-hPTH-(TCA) exhibited specificities directed to determinants in the COOH-terminal and NH(2)-terminal part of hPTH-(1-84) and exhibited cross-reactivity with bPTH-(1-84). Anti-hPTH-(1-34), on the other hand, showed immunological specificities mainly directed to antigenic determinants located in the COOH-terminal half of hPTH-(1-34). In addition, some reactivity with the NH(2)-terminal hPTH-(1-12) and with the extractive full-length peptides of human and bovine origin was observed. Antibodies to hPTH-(1-12) cross-reacted with hPTH-(1-34) and -(1-84)G-100.IPTH WAS RADIOIMMUNOLOGICALLY DETERMINED IN HUMAN SERA BY THE FOLLOWING SYSTEMS: (a) [(131)I]bPTH-(1-84), anti-hPTH-(TCA) and hPTH-(1-84)G-100 as standard; (b) [(131)I]hPTH-(1-34), anti-hPTH-(1-34) and hPTH-(1-34) as standard. With system (a), COOH-terminal fragments of hPTH-(1-84) having a molecular weight of approximately 7,000 were detected, and there was an almost total discrimination of serum iPTH levels in normal and in hyperparathyroid subjects. With system (b), on the other hand, several molecular species of iPTH were detected, including a component larger than hPTH-(1-84) and others similar to hPTH-(1-84) and to a fragment co-eluting with the NH(2)-terminal fragment hPTH-(1-34). When serum iPTH was assayed in system (b), there was a large overlap of iPTH levels in control subjects and in patients with primary hyperparathyroidism.

Most of what we know on PTH bioactivity has been associated with the first 34 amino acids of the PTH structure acting on the type I PTH/PTHrP receptor, leaving little place to the carboxyl-terminal structure. This reality has dictated the evolution of the PTH assay. The first generation of PTH assays has permitted the description of circulating PTH immunoreactivity and of its acute regulation by calcium concentration. Most assays reacted with the dominant forms of circulating PTH, PTH fragments devoid of bioactivity. This was believed to limit their clinical performance, particularly in the diagnosis of hypercalcemic disorders and the evaluation of secondary hyperparathyroidism and/or bone diseases associated with chronic renal failure. This brought up the development of a 2nd generation of PTH assays, the Intact (I) PTH assay. These assays were initially demonstrated to react only with hPTH(1-84), the bioactive form of the hormone. They greatly improved the differential diagnosis of hypercalcemic disorders, facilitated studies of parathyroid function in renal failure patients but were still limited in their capacity to dissociate the various bone diseases associated with chronic renal failure. Eventually, it was demonstrated that these assays, which used 13-34 epitopes, reacted with large C-PTH fragments having a partially preserved amino-terminal (N) structure, also called non-(1-84) PTH. These fragments accounted for up to 50% of I-PTH immunoreactivity in renal failure patients. hPTH(7-84), a surrogate of non-(1-84) PTH fragments, was demonstrated to cause hypocalcemia and to antagonize hPTH(1-34) and hPTH(1-84) calcemic effect in vivo and to inhibit bone resorption in vitro via a C-PTH receptor, different from the type I PTH/PTHrP receptor. This suggested a dual control of calcium concentration via N- and C-PTH molecular forms. This also explained why the ratio of C-PTH fragments/I-PTH was so well regulated both acutely and chronically in various experimental conditions. The fact that I-PTH assays detected circulating PTH molecular forms with biological effects opposite to those of hPTH(1-84) was believed to explain their limitations, particularly in renal failure, and prompted the evolution of a third generation of PTH assays. The last is based on a 1-4 epitope to reveal PTH(1-84) and not hPTH(7-84). It also permits an indirect evaluation of non-(1-84) PTH fragments by subtracting a 3rd generation PTH value from a 2nd generation PTH value and the calculation of a PTH(1-84)/non-(1-84) PTH ratio. The combination of a third generation PTH value with the PTH(1-84)/non-(1-84) PTH ratio value has in some studies improved the differential diagnosis of bone diseases associated with renal failure. But more studies are required to see whether PTH(1-84)/PTH fragment ratios will improve the clinical performance of PTH concentrations used alone.