Parathyroid Hormone-1 Research Articles

Receptor activity-modifying protein modulation of parathyroid hormone-1 receptor function and signaling.

Receptor activity-modifying proteins (RAMPs) are known to modulate the pharmacology and function of several G-protein-coupled receptors (GPCRs), including the parathyroid hormone 1 receptor (PTH1R). However, the precise effects of different RAMPs on PTH1R signalling and trafficking remain poorly understood. This study investigated the impact of RAMP2 and RAMP3 on PTH1R function using a range of PTH and PTH-related protein (PTHrP)-derived ligands. We employed FRET imaging to assess PTH1R interactions with RAMPs. Cell surface expression of PTH1R was evaluated in the presence of RAMPs. PTH1R-mediated cAMP accumulation, β-arrestin recruitment, and calcium signalling were measured in response to various ligands. Antibody-capture scintillation proximity assays were used to examine G-protein activation patterns. PTH1R preferentially interacted with RAMP2 and, to a lesser extent, RAMP3, but not with RAMP1. RAMP3 co-expression reduced cell surface expression of PTH1R. RAMP2 significantly enhanced PTH1R-mediated signalling responses to PTH (1-34), PTHrP (1-34), PTH (1-84), and PTH (1-17) analogue ZP2307, while RAMP3 co-expression attenuated or abolished these responses. Full-length PTHrP analogues exhibited lower potency and efficacy than PTHrP (1-34) in activating PTH1R. RAMP2 increased the potency and/or efficacy of these analogues, whereas RAMP3 reduced these responses. RAMP2 differentially modulated G-protein activation by PTH1R in a ligand-dependent manner, with PTH (1-34) and PTHrP (1-34) inducing distinct patterns of G-protein subtype activation. These findings highlight the complex role of RAMPs in regulating PTH1R signalling and trafficking, revealing differential effects of RAMP2 and RAMP3 on receptor function. The data suggest that targeting the PTH1R/RAMP2 complex may be a promising strategy for developing novel bone anabolic therapies by leveraging biased agonism and functional selectivity. Further research using physiologically relevant models is needed to elucidate the therapeutic potential of this approach.

Indications for initiation of drug therapy and modern therapy protocols in patients with osteoporosis

Introduction. Pharmacotherapy and physical therapy in patients with osteoporosis are aimed at increasing bone density and reducing the risk of fall in order to prevent fractures. Medications approved for the treatment of osteoporosis reduce the risk of fracture, either by reducing bone resorption or by stimulating bone formation. Bisphosphonates are most widely used antiresorptive agents that lower bone turnover markers to premenopausal levels and reduce fracture rates. Bisphosphonates bind to bone minerals and have a long-lasting effect. Long-term, continuous use of oral bisphosphonates is usually interspersed with drug breaks of 1-2 years to reduce the risk of atypical femoral fractures. Denosumab is a monoclonal antibody that also acts as an antiresorptive and it targets receptor activators of nuclear factor-?B ligand thus inhibiting the formation and function of osteoclasts. Denosumab is administered as a subcutaneous injection every 6 months. Anti-fracture effects of denosumab are similar to those of bisphosphonates, but there is a marked loss of antiresorptive effect 7 months after the last dose, which may lead to recurrent vertebral fractures. Anabolic drugs work by stimulating bone formation. Teriparatide and abaloparatide bind to the parathyroid hormone-1 receptor and are given as daily subcutaneous injection for up to 2 years. Romosozumab is a monoclonal antibody that targets sclerostin, stimulates bone formation and inhibits resorption. The effects of anabolics are transient, so it is necessary to switch to antiresorptive medications. Conclusion. It is a matter of great importance to determine the optimal strategy for cycles of anabolics, antiresorptive drugs and therapy-free periods.

Characteristics of Parathyroid hormone-1 Receptor Agonists and Antagonists

Aim: Parathyroid hormone-1 receptor (PTH1R) is a member of B G protein-coupled receptors. The agonistic activation of the PTH1R results in the production and secretion of osteoclast-stimulating cytokines while antagonists may be used to treat bone metastases, hypercalcemia, cachexia and hyperparathyroidism. Results: We built pharmacophore models and investigated the characteristics of PTH1R agonists and antagonists. The agonist model consists of three hydrophobic points, one hydrogen bond acceptor and one positive ionizable point. The antagonist model consists of one hydrogen bond donor and three hydrophobic points. Conclusion: The features of the two models are similar, but the hydrogen bond acceptor, which is the main difference between PTH1R agonists and antagonists, suggests it may be essential for the agonist.

G protein-coupled receptors as anabolic drug targets in osteoporosis.

Osteoporosis is a progressive bone disorder characterised by imbalance between bone building (anabolism) and resorption (catabolism). Most therapeutics target inhibition of osteoclast-mediated bone resorption, but more recent attention in early drug discovery has focussed on anabolic targets in osteoblasts or their precursors. Two marketed agents that display anabolic properties, strontium ranelate and teriparatide, mediate their actions via the G protein-coupled calcium-sensing and parathyroid hormone-1 receptors, respectively. This review explores their activity, the potential for improved therapeutics targeting these receptors and other putative anabolic GPCR targets, including Smoothened, Wnt/Frizzled, relaxin family peptide, adenosine, cannabinoid, prostaglandin and sphingosine-1-phosphate receptors.

CORRELATION OF PARATHYROID HORMONE-1 RECEPTOR EXPRESSION TO BONE METASTASIS OF BREAST CARCINOMA PATIENTS

Background: Bone metastasis is a common complication of metastasis of breast cancer and it is a unique pathobiology process. The Parathyroid Hormone-related Peptide (PTHrP) is a protein which has an important role in breast cancer cells to invade and infiltrate bones or bone marrow and accelerate angiogenetic process. The objective of this study is to reveal the relationship of PTHrP receptor named Parathyroid Hormone-1 Receptor (PTH1R) expression to bone metastasis in breast cancer patients. Methods: This was an analytical cross-sectional study, applying a non probability consecutive sampling. Samples were divided into two groups, i.e. one group of breast cancer metastasis to bone (+others) and another group with non-bone metastasis. Patients were collected from an existed data base (from medical record, cancer register, histopathology), since 2007. The specimen paraffin blocks were re-examined using IHC technique for PTHrP receptor. The data were analyzed and tested with Chi-Square (X 2 ), otherwise it would be tested using Fisher Exact Test. Each group would be allocated minimal of 17 patients/samples. Results: The Chi-Square test failed to show the association between PTH1R expression in breast cancer patients with bone metastasis ( p =0.295). The relative prevalence result for positive PTH1R expression was 1.48. There was no proof that positive PTH1R expression was an associated factor for bone metastasis (95% confidence interval). Conclusion : PTH1R expression is not a factor associated with bone metastasis in breast cancer patients.

Editorial (Hot Topic: New Insight into the RAMP-based Family B GPCRs and their Peptide Ligands)

Editorial (Hot Topic: New Insight into the RAMP-based Family B GPCRs and their Peptide Ligands)

Parathyroid Hormone–Related Protein Enhances Human β-Cell Proliferation and Function With Associated Induction of Cyclin-Dependent Kinase 2 and Cyclin E Expression

OBJECTIVEInducing human β-cell growth while enhancing function is a major goal in the treatment of diabetes. Parathyroid hormone–related protein (PTHrP) enhances rodent β-cell growth and function through the parathyroid hormone-1 receptor (PTH1R). Based on this, we hypothesized that PTH1R is expressed in human β-cells and that PTHrP has the potential to enhance human β-cell proliferation and/or function.RESEARCH DESIGN AND METHODSPTH1R expression, β-cell proliferation, glucose-stimulated insulin secretion (GSIS), and expression of differentiation and cell-cycle genes were analyzed in human islets transduced with adenoviral PTHrP constructs or treated with PTHrP peptides. The effect of overexpression of late G1/S cell cycle molecules was also assessed on human β-cell proliferation.RESULTSWe found that human β-cells express PTH1R. More importantly, overexpression of PTHrP causes a significant approximately threefold increase in human β-cell proliferation. Furthermore, the amino terminus PTHrP(1-36) peptide is sufficient to increase replication as well as expression of the late G1/S cell-cycle proteins cyclin E and cyclin-dependent kinase 2 (cdk2) in human islets. Notably, PTHrP(1-36) also enhances GSIS. Finally, overexpression of cyclin E alone, but not cdk2, augments human β-cell proliferation, and when both molecules are expressed simultaneously there is a further marked synergistic increase in replication.CONCLUSIONSPTHrP(1-36) peptide enhances human β-cell proliferation as well as function, with associated upregulation of two specific cell-cycle activators that together can induce human β-cell proliferation several fold. The future therapeutic potential of PTHrP(1-36) for the treatment of diabetes is especially relevant given the complementary therapeutic efficacy of PTHrP(1-36) in postmenopausal osteoporosis.

Communication between epithelial and stromal cells

Epithelial-stromal interactions are critical for the development of organs, such as the mammary gland. The mammary mesenchyme is required for the initiation of mammary ductal branching morphogenesis in females and the destruction of the mammary epithelial bud in males. For both functions the mammary mesenchymal cells must be activated by PTHrP (parathyroid hormone-related protein). By binding to the PTH1 (parathyroid hormone 1) receptor PTHrP triggers the expression of the androgen receptor and activates LEF1 and β-catenin, components of the Wnt signaling pathway (Dunbar et al., 1999; Dunbar et al., 1998; Foley et al., 2001). Such paracrine ligand/receptor interactions are a classical way by which the epithelial and stromal compartments communicate. Other factors that mediate crosstalk are Ihh (Indian hedgehog), IGF-I (insulin growth factor-I) and GH (growth hormone). Ihh is produced by mammary epithelial cells and interacts with Ptc-1 (patched-1) in stromal cells. Mice haploinsufficient for Ptc-1 exhibit hyperplasia and dysplasia of mammary ducts (Lewis et al., 1999). Epithelial GH activates the GH receptor in stromal cells which, in response, produce IGF-I which acts back on epithelial cells by activating their IGF-I receptor (Wiseman and Werb, 2002). Both factors, GH and IGF-I, are required for normal mammary gland development. Another paracrinely acting ligand is TGFβ (transforming growth factor β). Produced by the mammary epithelium TGFβ interacts with the TGFβ receptor in adjacent fibroblasts which, in turn, cause a decelerated ductal growth into the mammary fat path (Crowley et al., 2005). In addition to fibroblasts, macrophages are involved in mammary gland development. Macrophages attracted by CSF-1 (colony stimulating factor-1) to the mammary gland promote ductal invasion during puberty (Gouon-Evans et al., 2000).

Anabolic effect of human synthetic parathyroid hormone-1–34 on cancellous bone in the spontaneous hypertensive rat

Anabolic effect of human synthetic parathyroid hormone-1–34 on cancellous bone in the spontaneous hypertensive rat

Discovery and Characterization of Novel, Potent, Non-Peptide Parathyroid Hormone-1 Receptor Antagonists

A 1,3,4-benzotriazepine was identified as a suitable lead in our effort toward obtaining a non-peptide parathyroid hormone-1 receptor (PTH1R) antagonist. A process of optimization afforded derivatives displaying nanomolar PTH1R affinity, a representative example of which behaved as a PTH1R antagonist in cell-based cyclic adenosine monophosphate (cAMP) assays, with selectivity over PTH2 receptors.

Abnormal Renovascular Parathyroid Hormone-1 Receptor in Hypertension: Primary Defect or Secondary to Angiotensin II Type 1 Receptor Activation?

We previously reported that PTHrP-induced renal vasodilation is impaired in mature spontaneously hypertensive rats (SHR) through down-regulation of the type 1 PTH/PTHrP receptor (PTH1R), a feature that contributes to the high renal vascular resistance in SHR. Here we asked whether this defect represents a prime determinant in genetic hypertension or whether it is secondary to angiotensin II (Ang II) and/or the mechanical forces exerted on the vascular wall. We found that the treatment of SHR with established hypertension by the Ang II type 1 receptor antagonist, losartan, reversed the down-regulation of PTH1R expression in intrarenal small arteries and restored PTHrP-induced vasodilation in ex vivo perfused kidneys. In contrast, the PTH1R deregulation was not found in intrarenal arteries isolated from prehypertensive SHR. Moreover, this defect, which is not seen in extrarenal vessels (aorta, mesenteric arteries) from mature SHR appeared kidney specific in accordance with the acknowledged enrichment of interstitial Ang II in this organ and its enhancement in SHR. In deoxycorticosterone-acetate-salt rats, an Ang II-independent model of hypertension, renovascular PTH1R expression and related vasodilation were not altered. In SHR-derived renovascular smooth muscle cells (RvSMCs), the PTH1R was spontaneously down-regulated and its transcript destabilized, compared with Wistar RvSMCs, both effects being antagonized by losartan. Exogenous Ang II elicited down-regulation of PTH1R mRNA in RvSMCs from Wistar rats. Together, these data demonstrate that Ang II acts via the Ang II type 1 receptor to destabilize PTH1R mRNA in the renal vessel in the SHR model of genetic hypertension. This process is kidney specific and independent from blood pressure increase.

Large-scale purification and characterization of human parathyroid hormone-1 receptor stably expressed in HEK293S GnTI − cells

Human parathyroid hormone-1 receptor (hPTHR1) belongs to class II of the G protein-coupled receptor (GPCR) family, whose members all contain a seven-transmembrane helix domain. The receptor regulates bone metabolism through interactions with its ligand, human parathyroid hormone (hPTH). For structural studies of the hPTHR1/hPTH complex, we constructed a mammalian cell line to stably express recombinant hPTHR1 in large-scale. The receptor was solubilized with dodecyl maltoside and purified with affinity chromatography. The purified receptor displayed restricted N-glycosylation as expected. Functionality was demonstrated: the hPTHR1 retained affinity for bPTH-(1–34) and specifically cross-linked to a radioiodinated bPTH-(1–34) analog. This work describes an approach for preparing milligram-scale quantities of receptor for elucidation of the structural biology of this seven-transmembrane GPCR.

The parathyroid hormone-related protein system: more data but more unsolved questions.

The present review focuses on recent studies that might be considered as the most relevant advances in the parathyroid hormone-related protein field, with special emphasis on proven functions in renovascular and cardiovascular systems, in physiological as well as pathological conditions. Thus, the questions as to whether and how parathyroid hormone-related protein intervenes in vascular development and homeostasis and in vascular diseases such as hypertension, atherosclerosis, restenosis and heart failure have begun to be unraveled. Since its discovery from hypercalcemia-associated tumors in 1987, it has become clear that parathyroid hormone-related protein is a ubiquitously expressed poly-hormone and plays crucial roles in normal life. The early lethality to parathyroid hormone-related protein knockout mice emphasizes the crucial roles of the protein in development but has limited the use of these models. However, data accumulated from transgenic animals overexpressing the protein in particular cells have provided considerable support to its physiological and pathological relevance. The recent demonstration that nascent parathyroid hormone-related protein not only follows the secretory pathways, but also directly translocates to the nucleus, is beginning to uncover new actions for the protein in a number of physiological systems such as bone, mammary gland and vascular smooth muscle, as well as in pathological situations, such as cancer, osteoporosis, sepsis, atherosclerosis and hypertension. The development of mice with conditionally deleted parathyroid hormone-related protein or parathyroid hormone-1 receptor alleles will allow the creation of cell- or tissue-specific parathyroid hormone-related protein knockout mice which will greatly facilitate the determination of the biological relevance of this protein in a specific cell or tissue type, particularly in the cardiovascular system.

Protein–protein interactions regulate PTH1 receptor signalling

Association of Na+–H+ exchanger regulatory factors (NHERFs) with the parathyroid hormone 1 (PTH1) receptor via a PDZ-domain interaction leads to formation of molecular complexes that enhance Gi/o-mediated phospholipase C (PLC) activation but reduce Gs-mediated signalling.Evidence is accumulating that hormone receptors are present in the cells as part of multimolecular complexes that determine the function of the receptor. Mahon et al. [1xNa+/H+ exchanger regulatory factor 2 directs parathyroid hormone 1 receptor signalling. Mahon, M.J. et al. Nature. 2002; 417: 858–861Crossref | PubMed | Scopus (228)See all References][1] have studied interactions of the C-terminal tail of the PTH1 receptor with other proteins, because earlier data indicated that the C-terminal tail modulates PTH1 receptor signalling. The authors performed yeast two-hybrid screens using the C-terminal tail of the PTH1 receptor as bait. In a human kidney cDNA library, they found that a fragment of Na+–H+ exchanger regulatory factor 2 (NHERF2) interacts strongly with the C-terminal tail of the PTH1 receptor. Because NHERF has been shown to interact with other hormone receptors, and it has two PDZ (PSD95, discs large protein, ZO1) domains that interact with well-defined C-terminal motifs, in addition to an ERM (ezrin, radixin, moesin)-binding motif, the authors have analysed the biological importance of this interaction. In vitro analyses revealed that the C-terminal end of the PTH1 receptor binds to the second PDZ domain (PDZ2) of NHERF2, and a similar interaction is detected with NHERF1. In PS120 Chinese hamster fibroblast cells expressing PTH1 receptor and NHERF2, coprecipitation of these molecules was observed. Most importantly, PTH-induced cAMP stimulation is inhibited when NHERF2 is coexpressed with the PTH1 receptor (from 10–15-fold stimulation without NHERF2 to only 2-fold stimulation with NHERF2), whereas the inositol phosphate (IP) response was markedly increased with NHERF2 coexpression (from 2–4-fold to 20–25-fold). The authors concluded that stimulation of IP production was mediated by Gi/o proteins, because the response was pertussis-toxin sensitive. Because all four isoenzymes of PLCβ, which mediate the hormone-induced generation of IP, contain consensus C-terminal PDZ domain interaction motifs, the authors have used pull-down assays to show that NHERF2 can form multiprotein complexes consisting of PLCβ1 bound to PDZ1 and PTH1 receptor bound to PDZ2. However, it remains to be elucidated whether other subtypes of PLCβ (e.g. PLCβ2) produce similar interactions. Furthermore, the authors have compared the PTH1 receptor-mediated signal generation in two cell lines. They show that in ECV304 cells (a human endothelium-derived cell line with abundant NHERF1 and 2 expression), PTH predominantly increases IP production, whereas in ROS 17/2.8 cells (a rat osteoblastic cell line with no detectable NHERF expression), PTH causes more than a 30-fold increase in cAMP production without a detectable effect on IP signal generation. The appearance of PLC-mediated signalling after transient transfection of NHERF2 into ROS17/2.8 cells was indirectly detected using an AP1-responsive luciferase reporter gene.These data demonstrate that expression of NHERFs leads to formation of molecular complexes that regulate the signalling of the PTH1 receptor. This might be particularly important in polarized cells, because the basal and luminal membranes often have different NHERF expression, and suggests that assembly of receptor complexes with NHERF and possibly other PDZ-domain-containing proteins could be a general mechanism for site-specific regulation of secondary messenger generation.

Specificity and stability of a new PTH1 receptor antagonist, mouse TIP(7–39)

Parathyroid hormone 1 (PTH1) receptor antagonists might be of benefit in hypercalcemia of malignancy (HHM) and hyperparathyroidism. We previously identified bovine tuberoinfundibular peptide (7–39) (bTIP(7–39)) as a high-affinity PTH1 receptor antagonist. Mouse TIP(7–39) is an antagonist (rPTH1 K B=44 nM, rPTH2=940 nM) that is more potent than other known PTH1 receptor antagonists: bTIP(7–39) (210 nM), PTH-related protein (PTHrP)(7–34) (640 nM), and bPTH(7–34) (>3000 nM). Plasma proteases slowly ( t 1/2=81 min) inactivated [ 125 I ]mTIP(7–39). Intravenous plasma [ 125 I ]mTIP(7–39) was bi-phasically cleared (radioactivity t 1/2=2.9 min (70%) and 120 min (30%), binding activity t 1/2=3.6 min (92%), and t 1/2=21 min (8%)). Loss of unlabeled mTIP(7–39) (250 μg/kg i.v.) receptor binding was similar. mTIP(7–39)’s high-affinity should facilitate animal evaluation of effects of PTH1 receptor antagonism.

Molecular Mechanisms of Ligand Recognition by Parathyroid Hormone 1 (PTH1) and PTH2 Receptors.

The mammalian parathyroid hormone (PTH) / PTH receptor family includes PTH1 and PTH2 receptors and three related ligands (PTH, PTH-related protein (PTHrP) an d tuberoinfundibular peptide of 39 residues (TIP39)). Here we comparatively and systematically review the pharmacological properties of PTH receptors and ligands, structure of the ligands, and molecular mechanisms of receptor-ligand interaction. The PTH1 receptor is activated by PTH and PTHrP but not by TIP39. The PTH2 receptor is activated by TIP39 but not by PTHrP. PTH strongly activates the human PTH2 receptor but is a weak partial agonist for rat and zebrafish PTH2 receptors. Receptor-G-protein interaction increases the receptor binding selectivity of PTHrP and TIP39. Despite different primary structures, the secondary structures of PTH, PTHrP and TIP39 are quite similar. Each ligand contains an N-terminal and a C-terminal alpha-helix in secondary structure-inducing conditions. Receptor-bound ligand structure is less well-characterized. The orientation of receptor-ligand interaction is highly similar for PTH and PTHrP binding to the PTH1 receptor and TIP39 interaction with the PTH2 receptor. Ligands bind according to a 'two-site' mechanism, in which the C-terminal portion of the ligand binds the extracellular N-terminal domain of the receptor (N-interaction), and the N-terminal ligand portion binds to the juxtamembrane receptor domain (J-interaction). The N-interaction provides most of the PTH1-receptor binding energy for PTH and PTHrP but provides less energy for PTH2 receptor-TIP39 interaction. The J-interaction stimulates G-protein activation. For the PTH-PTH1 receptor interaction, the efficacy-generating component of the J-interaction is independent of the N-domain of the receptor and C-terminal portion of the ligand. This finding suggests that it might be possible to design low molecular-weight PTH1 receptor agonists, which could be bone anabolic agents and used for the treatment of osteoporosis.

Parathyroid Hormone-1 Receptor Down-Regulation in Kidneys from Rats with Chronic Renal Failure

Although secondary hyperparathyroidism is a common complication of chronic renal failure, few studies have examined the characteristics of parathyroid hormone (PTH) binding to the kidney or the regulation of the PTH receptor in chronic renal disease. In this study we measured PTH binding to the PTH-1 receptor in renal cortical membranes from normal rats and from rats with experimentally induced chronic renal failure. In normal rats, analysis of saturation binding experiments using ¹²⁵I PTH-related peptide (chicken, cPTHrP) revealed apparent K_d and B_max values of 1.16 ± 0.14 nmol/l and 338 ± 22.7 fmol/mg, respectively. Three weeks following induction of renal failure there was no change in the affinity of the PTH-1 receptor (K_d = 1.51 ± 0.24 nmol/l) but the B_max was reduced by 45% (183 ± 32.5). In normal rats which had undergone thyroparathyroidectomy, the K_d was unchanged (1.17 ± 0.09) while the B_max increased to 459 ± 31 fmol/mg. We conclude that chronic renal failure is accompanied by a downregulation of renal PTH-1 receptors.

3',5'-Cyclic adenosine monophosphate activation in osteoblastic cells: effects on parathyroid hormone-1 receptors and osteoblastic differentiation in vitro.

PTH has anabolic and catabolic effects in bone through activation of the PTH-1 (PTH/PTHrP) receptor and the cAMP/protein kinase A pathway. The effects of agents that regulate cAMP in nontransformed osteoblasts in relation to cell differentiation have not been described. The purpose of this study was to determine the effects of PTH fragments with differing cAMP-stimulating activity, and nonPTH cAMP regulators on PTH-1 receptor expression and activity, and osteoblast differentiation in vitro using MC3T3-E1 and primary rat calvarial cells. PTH (1-34), but not PTH (53-84), (7-34), or PTHrP (107-139) treatment (24 h) resulted in down-regulation of steady-state messenger RNA for the PTH-1 receptor. Forskolin (a stimulator of cAMP accumulation) also down regulated the PTH-1 receptor, whereas 9-(tetrahydro-2-furyl) adenine (THFA) (an inhibitor of adenylyl cyclase) had no effect. Similarly, PTH (1-34) treatment for 48 h abolished PTHrP binding to cell surface receptors; however, neither the PTH analogs nor the cAMP regulating agents altered PTH binding or numbers of binding sites on osteoblastic cells. Basal levels of cAMP were reduced in cultured cells treated for 6 days with PTH (7-34) or THFA compared with controls. In contrast, PTH-stimulated cAMP levels were significantly increased in cultures treated with PTH (7-34) and THFA for 6 days during osteoblast differentiation and were decreased in cultures treated with PTH (1-34) and forskolin compared with controls. To evaluate effects of the cAMP pathway on osteoblast differentiation, cultures were treated continuously with PTH analogs and cAMP regulators during an 18-day differentiation regime, total RNA was isolated at multiple time points, and Northern blot analysis for osteocalcin (OCN) was performed. THFA and PTH (7-34)-treated cultures had increased OCN expression; whereas, PTH (1-34) and forskolin reduced OCN expression. Interestingly, PTH (7-34) and THFA-treated cultures had increased mineralized nodule formation, in contrast to PTH (1-34) and forskolin treatment, which reduced nodule formation. Similarly, calcium accumulation in cultures was significantly increased in the PTH (7-34) and THFA-treated cultures and reduced in the PTH (1-34) and forskolin-treated cultures. These data demonstrate that agents that increase cAMP down regulate PTH-1 receptor messenger RNA and inhibit osteoblast differentiation in vitro. Agents that reduce or block adenylyl cyclase or cAMP activity do not alter PTH-1 receptor expression or binding, but have striking effects on promoting osteoblast differentiation. We conclude that many effects of PTH on osteoblasts may be mimicked or antagonized by agents that alter cAMP activity and bypass the PTH-1 receptor.

3',5'-Cyclic Adenosine Monophosphate Activation in Osteoblastic Cells: Effects on Parathyroid Hormone-1 Receptors and Osteoblastic Differentiation in Vitro

PTH has anabolic and catabolic effects in bone through activation of the PTH-1 (PTH/PTHrP) receptor and the cAMP/protein kinase A pathway. The effects of agents that regulate cAMP in nontransformed osteoblasts in relation to cell differentiation have not been described. The purpose of this study was to determine the effects of PTH fragments with differing cAMP-stimulating activity, and nonPTH cAMP regulators on PTH-1 receptor expression and activity, and osteoblast differentiation in vitro using MC3T3-E1 and primary rat calvarial cells. PTH (1–34), but not PTH (53–84), (7–34), or PTHrP (107–139) treatment (24 h) resulted in down-regulation of steady-state messenger RNA for the PTH-1 receptor. Forskolin (a stimulator of cAMP accumulation) also down regulated the PTH-1 receptor, whereas 9-(tetrahydro-2-furyl) adenine (THFA) (an inhibitor of adenylyl cyclase) had no effect. Similarly, PTH (1–34) treatment for 48 h abolished PTHrP binding to cell surface receptors; however, neither the PTH analogs nor the cAMP regulating agents altered PTH binding or numbers of binding sites on osteoblastic cells. Basal levels of cAMP were reduced in cultured cells treated for 6 days with PTH (7–34) or THFA compared with controls. In contrast, PTH-stimulated cAMP levels were significantly increased in cultures treated with PTH (7–34) and THFA for 6 days during osteoblast differentiation and were decreased in cultures treated with PTH (1–34) and forskolin compared with controls. To evaluate effects of the cAMP pathway on osteoblast differentiation, cultures were treated continuously with PTH analogs and cAMP regulators during an 18-day differentiation regime, total RNA was isolated at multiple time points, and Northern blot analysis for osteocalcin (OCN) was performed. THFA and PTH (7–34)-treated cultures had increased OCN expression; whereas, PTH (1–34) and forskolin reduced OCN expression. Interestingly, PTH (7–34) and THFA-treated cultures had increased mineralized nodule formation, in contrast to PTH (1–34) and forskolin treatment, which reduced nodule formation. Similarly, calcium accumulation in cultures was significantly increased in the PTH (7–34) and THFA-treated cultures and reduced in the PTH (1–34) and forskolin-treated cultures. These data demonstrate that agents that increase cAMP down regulate PTH-1 receptor messenger RNA and inhibit osteoblast differentiation in vitro. Agents that reduce or block adenylyl cyclase or cAMP activity do not alter PTH-1 receptor expression or binding, but have striking effects on promoting osteoblast differentiation. We conclude that many effects of PTH on osteoblasts may be mimicked or antagonized by agents that alter cAMP activity and bypass the PTH-1 receptor.

Sublocalization of the mouse parathyroid hormone-like peptide gene to distal chromosome 6 by interspecific backcross analysis and [formula omitted] hybridization

Parathyroid hormone 1 (PTH1) receptor antagonists might be of benefit in hypercalcemia of malignancy (HHM) and hyperparathyroidism. We previously identified bovine tuberoinfundibular peptide (7–39) (bTIP(7–39)) as a high-affinity PTH1 receptor antagonist. Mouse TIP(7–39) is an antagonist (rPTH1 KB=44 nM, rPTH2=940 nM) that is more potent than other known PTH1 receptor antagonists: bTIP(7–39) (210 nM), PTH-related protein (PTHrP)(7–34) (640 nM), and bPTH(7–34) (>3000 nM). Plasma proteases slowly (t1/2=81 min) inactivated [125I]mTIP(7–39). Intravenous plasma [125I]mTIP(7–39) was bi-phasically cleared (radioactivity t1/2=2.9 min (70%) and 120 min (30%), binding activity t1/2=3.6 min (92%), and t1/2=21 min (8%)). Loss of unlabeled mTIP(7–39) (250 μg/kg i.v.) receptor binding was similar. mTIP(7–39)’s high-affinity should facilitate animal evaluation of effects of PTH1 receptor antagonism.