PTH/PTH-related Peptide Research Articles

Calcium fluxes at the bone/plasma interface: Acute effects of parathyroid hormone (PTH) and targeted deletion of PTH/PTH-related peptide (PTHrP) receptor in the osteocytes.

Calcium ion concentration ([Ca2+]) in the systemic extracellular fluid, ECF-[Ca2+], is maintained around a genetically predetermined set-point, which combines the operational level of the kidney and bone/ECF interfaces. The ECF-[Ca2+] is maintained within a narrow oscillation range by the regulatory action of Parathyroid Hormone (PTH), Calcitonin, FGF-23, and 1,25(OH)2D3. This model implies two correction mechanisms, i.e. tubular Ca2+ reabsorption and osteoclast Ca2+ resorption. Although their alterations have an effect on the ECF-[Ca2+] maintenance, they cannot fully account for rapid correction of the continuing perturbations of plasma [Ca2+], which occur daily in life. The existence of Ca2+ fluxes at quiescent bone surfaces fulfills the role of a short-term error correction mechanism in Ca2+ homeostasis. To explore the hypothesis that PTH regulates the cell system responsible for the fast Ca2+ fluxes at the bone/ECF interface, we have performed direct real-time measurements of Ca2+ fluxes at the surface of ex-vivo metatarsal bones maintained in physiological conditions mimicking ECF, and exposed to PTH. To further characterize whether the PTH receptor on osteocytes is a critical component of the minute-to-minute ECF-[Ca2+] regulation, metatarsal bones from mice lacking the PTH receptor in these cells were tested ex vivo for rapid Ca2+ exchange. We performed direct real-time measurements of Ca2+ fluxes and concentration gradients by a scanning ion-selective electrode technique (SIET). To validate ex vivo measurements, we also evaluated acute calcemic response to PTH in vivo in mice lacking PTH receptors in osteocytes vs littermate controls. Our data demonstrated that Ca2+ fluxes at the bone-ECF interface in excised bones as well as acute calcemic response in the short-term were unaffected by PTH exposure and its signaling through its receptor in osteocytes. Rapid minute-to-minute regulation of the ECF-[Ca2+] was found to be independent of PTH actions on osteocytes. Similarly, mice lacking PTH receptor in osteocytes, responded to PTH challenge with similar calcemic increases.

Parathyroid Hormone Signaling in Osteocytes

ABSTRACTOsteocytes are the most abundant cell type in bone and play a central role in orchestrating skeletal remodeling, in part by producing paracrine‐acting factors that in turn influence osteoblast and osteoclast activity. Recent evidence has indicated that osteocytes are crucial cellular targets of parathyroid hormone (PTH). Here, we will review the cellular and molecular mechanisms through which PTH influences osteocyte function. Two well‐studied PTH target genes in osteocytes are SOST and receptor activator of NF‐κB ligand (RANKL). The molecular mechanisms through which PTH regulates expression of these two crucial target genes will be discussed. Beyond SOST and RANKL, PTH/PTH‐related peptide (PTHrP) signaling in osteocytes may directly influence the way osteocytes remodel their perilacunar environment to influence bone homeostasis in a cell‐autonomous manner. Here, I will highlight novel, additional mechanisms used by PTH and PTHrP to modulate bone homeostasis through effects in osteocytes. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Parathyroid Hormone (PTH)/PTH-related Peptide Type 1 Receptor (PPR) Signaling in Osteocytes Regulates Anabolic and Catabolic Skeletal Responses to PTH

Parathyroid hormone (PTH) is the only Food and Drug Administration-approved anabolic agent to treat osteoporosis; however, the cellular targets of PTH action in bone remain controversial. PTH modulates bone turnover by binding to the PTH/PTH-related peptide (PTHrP) type 1 receptor (PPR), a G-protein-coupled receptor highly expressed in bone and kidneys. Osteocytes, the most abundant cells in adult bone, also express PPR. However, the physiological relevance of PPR signaling in osteocytes remains to be elucidated. Toward this goal, we generated mice with PPR deletion in osteocytes (Ocy-PPRKO). Skeletal analysis of these mice revealed a significant increase in bone mineral density and trabecular and cortical bone parameters. Osteoblast activities were reduced in these animals, as demonstrated by decreased collagen type I α1 mRNA and receptor activator of NF-κB ligand (RANKL) expression. Importantly, when subjected to an anabolic or catabolic PTH regimen, Ocy-PPRKO animals demonstrated blunted skeletal responses. PTH failed to suppress SOST/Sclerostin or induce RANKL expression in Ocy-PPRKO animals compared with controls. In vitro, osteoclastogenesis was significantly impaired in Ocy-PPRKO upon PTH administration, indicating that osteocytes control osteoclast formation through a PPR-mediated mechanism. Taken together, these data indicate that PPR signaling in osteocytes is required for bone remodeling, and receptor signaling in osteocytes is needed for anabolic and catabolic skeletal responses.

Perspective: Cell–Cell Adhesion and Signaling Through Cadherins: Connecting Bone Cells in Their Microenvironment

Perspective: Cell–Cell Adhesion and Signaling Through Cadherins: Connecting Bone Cells in Their Microenvironment

Expression of the PTH/PTHrP receptor in chondrogenic cells during the repair of full-thickness defects of articular cartilage

We studied the accumulation of parathyroid hormone (PTH)/PTHrP receptor-positive mesenchymal cells using double immunohistochemistry and examined whether this correlated with the subsequent regeneration of 3-mm-diameter full-thickness defects of articular cartilage. Cylindrical full-thickness articular cartilage defects (3 mm) were artificially created in the femoral trochlea of male adolescent Japanese white rabbits (n = 210) with a hand-drill. Recombinant human PTH(1-84) was then administered into the defect cavities with an osmotic pump for either 2 or 4 weeks post-injury. Following PTH treatment, the repair processes in the cartilage defects were histologically examined. Double immunostaining analyses for the PTH/PTH-related peptide (PTHrP) receptor and proliferating cell nuclear antigen (PCNA) in the regenerating tissues were then performed. Activation of PTH/PTHrP receptor signaling by hPTH(1-84) results in the inhibition of chondrogenic differentiation in full-thickness articular cartilage defects. At the conclusion of the 2-week PTH treatment, the defect cavities were filled with undifferentiated mesenchymal cells, which were similar to the controls. In addition, almost all of these cells localized at the center of the injuries were both PTH/PTHrP receptor- and PCNA-positive. In contrast, after prolonged PTH treatment for 4 weeks, there was no indication of a cartilaginous repair response and cells that had migrated to the defect cavities were found to have irreversibly lost expression of the PTH/PTHrP receptor. The chondrogenic capacity of cells that had migrated to the area of these defect cavities is closely associated with their ability to express the PTH/PTHrP receptor. Moreover, these cells maintain their chondrogenic potential within only a limited time-span of 2 weeks.

Multiple Mechanisms Are Involved in Inhibition of Osteoblast Differentiation by PTHrP and PTH in KS483 Cells

We examined the mechanism by which PTHrP and PTH inhibit KS483 osteoblastic differentiation. We show that PTHrP and PTH inhibit differentiation downstream of early BMP signaling and downregulated components of the hedgehog (Hh) signaling cascade. In addition, PTHrP and PTH repressed RunX2 and osx expression. Overexpression of either gene, however, could not relieve PTHrP and PTH's inhibitory actions. Our data suggest that multiple parallel mechanisms are involved in the inhibition of osteoblast differentiation and matrix mineralization by PTHrP and PTH. PTH-related peptide (PTHrP) and PTH are potent inhibitors of osteoblast differentiation in vitro by as yet unexplained mechanisms. We treated murine bone marrow stromal cells and the mesenchymal progenitor cell line KS483 with PTHrP and PTH in combination with either BMPs or hedgehog (Hh) and measured early and late markers of osteoblast differentiation and studied the expression of RunX2 and Osterix (osx). In addition, we examined the PTHrP and PTH response in stable KS483 cells overexpressing either RunX2 or osx. PTHrP and PTH inhibited BMP- and Hh-induced osteogenesis downstream of early BMP signaling and by downregulation of components of the Hh signaling cascade. PTHrP and PTH prevented the upregulation of RunX2 expression associated with osteoblast differentiation in an indirect response. However, PTHrP and PTH could still inhibit differentiation, and particularly matrix mineralization, of cells expressing RunX2. In addition, PTHrP and PTH potently downregulated osx expression only in mature osteoblasts in an intermediate early response, but osx overexpression could not relieve the inhibitory effects of PTHrP and PTH on matrix mineralization. Our data suggest that, besides transcriptional repression of RunX2 and osx, other mechanisms in parallel with or downstream of RunX2 and osx are involved in the inhibition of osteoblast differentiation and matrix mineralization by PTHrP and PTH in vitro.

Potential Role of Pancreatic and Enteric Hormones in Regulating Bone Turnover

Potential Role of Pancreatic and Enteric Hormones in Regulating Bone Turnover

Chondrocyte-Specific Knockout of the G Protein Gsα Leads to Epiphyseal and Growth Plate Abnormalities and Ectopic Chondrocyte Formation

G(s)alpha is a ubiquitously expressed G protein alpha-subunit that couples receptors to adenylyl cyclase. Mice with chondrocyte-specific ablation of the G(s)alpha gene had severe epiphyseal and growth plate abnormalities and ectopic cartilage formation within the metaphyseal region of the tibia. These results show that G(s)alpha negatively regulates chondrocyte differentiation and is the critical signaling mediator of the PTH/PTH-rP receptor in growth plate chondrocytes. G(s)alpha is a ubiquitously expressed G protein alpha-subunit that mediates signaling through G protein-coupled receptors to activate the cAMP/protein kinase A signaling pathway. Although studies suggest an important role for G(s)alpha in regulating growth plate development, direct in vivo results examining this role are lacking. The G(s)alpha gene was ablated in murine cartilage by mating mice with loxP sites surrounding the G(s)alpha promoter and first exon with collagen 2a1 promoter-Cre recombinase transgenic mice. Skeletal tissues were studied by gross and microscopic pathology, and gene expression was determined by in situ hybridization. Mice with complete chondrocyte-specific G(s)alpha deficiency (homozygotes) died within minutes after birth and had severe epiphyseal and growth plate defects with shortening of the proliferative zone and accelerated hypertrophic differentiation of growth plate chondrocytes, a phenotype similar to that of PTH/PTH-related peptide (PTHrP) receptor knockout mice. Indian hedgehog and PTH/PTHrP receptor expression in prehypertrophic chondrocytes was unaffected in mutant mice. PTHrP expression in periarticular cartilage was increased in the mutant mice, probably because of the closer proximity of Ihh-secreting chondrocytes to the periarticular zone. In addition, these mice developed ectopic cartilage at the anterior side of the metaphyseal region in the tibia. Mice with partial G(s)alpha deficiency (heterozygotes) exhibited no phenotype. These results show that G(s)alpha negatively regulates chondrocyte differentiation and is the critical signaling mediator of the PTH/PTHrP receptor in epiphyseal and growth plate chondrocytes.

Parathyroid Hormone Receptor Trafficking Contributes to the Activation of Extracellular Signal-regulated Kinases but Is Not Required for Regulation of cAMP Signaling

Agonist-mediated activation of the type 1 parathyroid hormone receptor (PTH1R) results in several signaling events and receptor endocytosis. It is well documented that arrestins contribute to desensitization of both G(s)- and G(q)-mediated signaling and mediate PTH1R internalization. However, whether PTH1R trafficking directly contributes to signaling remains unclear. To address this question, we investigated the role of PTH1R trafficking in cAMP signaling and activation of extracellular signal-regulated kinases ERK1/2 in HEK-293 cells. Dominant negative forms of dynamin (K44A-dynamin) and beta-arrestin1 (beta-arrestin1-(319-418)) abrogated PTH1R internalization but had no effect on cAMP signaling; neither acute cAMP production by PTH nor desensitization and resensitization of cAMP signaling were affected. Therefore, PTH1R trafficking is not necessary for regulation of cAMP signaling. PTH-(1-34) induced rapid and robust activation of ERK1/2. A PTHrP-based analog ([p-benzoylphenylalanine1, Ile5,Arg(11,13),Tyr36]PTHrP-(1-36)NH2), which selectively activates the G(s)/cAMP pathway without inducing PTH1R endocytosis, failed to stimulate ERK1/2 activity. Inhibition of PTH1R endocytosis by K44A-dynamin dampened ERK1/2 activation in response to PTH-(1-34) by 69%. Incubation with the epidermal growth factor receptor inhibitor AG1478 reduced ERK1/2 phosphorylation further. In addition, ERK1/2 phosphorylation occurred following internalization of a PTH1R mutant induced by PTH-(7-34) in the absence of G protein signaling. Collectively, these data indicate that PTH1R trafficking and G(q) (but not G(s)) signaling independently contribute to ERK1/2 activation, predominantly via transactivation of the epidermal growth factor receptor.

Increased expression of G11α in osteoblastic cells enhances parathyroid hormone activation of phospholipase C and AP‐1 regulation of matrix metalloproteinase‐13 mRNA

In osteoblasts parathyroid hormone (PTH) stimulates the PTH/PTH-related peptide (PTHrP) receptor (PTH1R) that couples via G(s) to adenylyl cyclase stimulation and via G(11) to phospholipase C (PLC) stimulation. We have investigated the effect of increasing G(11)alpha levels in UMR 106-01 osteoblastic cells by transient transfection with cDNA encoding G(11)alpha on PTH stimulation of PLC and protein kinase C (PKC) as well as PTH regulation of mRNA encoding matrix metalloproteinase-13 (MMP-13). Transfection with G(11)alpha cDNA resulted in a 5-fold increase in PTH-stimulated PLC activity with no change in PTH-stimulated adenylyl cyclase. PTH-induced translocation of PKC-betaI, -delta, and -zeta to the cell membrane and PKC-zeta to the nucleus was also increased. Increased G(11)alpha protein resulted in increased stimulation of MMP-13 mRNA levels at all doses of PTH. There was a 2.5 +/- 0.35 fold increase in maximal PTH-stimulation of c-jun mRNA and smaller but significant increases in c-fos accompanied by increased basal and PTH-stimulated AP-1 binding in cells expressing increased G(11)alpha. Runx-2 mRNA and protein levels were not significantly increased by increased G(11)alpha expression. The increase in PTH stimulation of c-jun, c-fos, and MMP-13 in G(11)alpha-transfected cells were all blocked by bisindolylmaleimide I, a selective inhibitor of PKC. These results demonstrate that regulation of the PLC pathway through the PTH1R is significantly increased by elevating expression of G(11)alpha in osteoblastic cells. This leads to increased PTH stimulation of MMP-13 expression by increased stimulation of AP-1 factors c-jun and c-fos.

Decreased mRNA Expression of the PTH/PTHrP Receptor and Type II Sodium-Dependent Phosphate Transporter in the Kidney of Rats Fed a High Phosphorus Diet Accompanied with a Decrease in Serum Calcium Concentration

This study investigates the phosphorus (P) homeostasis in the process of an altered parathyroid hormone (PTH) action in the kidney of rats fed a high P diet. Four-week-old male Wistar strain rats were fed diets containing five different P levels (0.3, 0.6, 0.9, 1.2 and 1.5%) for 21 days. The serum PTH concentration and urinary excretion of P were elevated with increasing dietary P level. Compared to rats fed the 0.3% P diet, the serum calcium (Ca) concentration remained unchanged, while the serum 1,25(OH)(2)D(3) concentration and urinary excretion of cAMP were elevated with increasing dietary P level in rats fed the high P diets containing 0.6-0.9% P. On the other hand, a lower serum Ca concentration was observed in rats fed the high P diets containing 1.2% or greater P. The serum 1,25(OH)(2)D(3) concentration remained unchanged in rats fed the high P diets containing 1.2% or greater P, comparison with rats fed the 0.3% P diet. The urinary excretion of cAMP and PTH/PTH-related peptide (PTHrP) receptor and type II sodium-dependent phosphate transporter (NaPi-2) mRNA in the kidney were both decreased in rats fed the high P diets containing 1.2% or greater P. In conclusion, a high P diet with subsequent decrease in serum Ca concentration suppressed the PTH action in the kidney due to PTH/PTHrP receptor mRNA down-regulation. Furthermore, an increase in the urinary excretion of P might have been caused by decreased NaPi-2 mRNA expression without the effects of PTH and 1,25(OH)(2)D(3).

Effect of high phosphorus diet on phosphorus metabolism in parathyroidectomized rats

To determine the parathyroid hormone (PTH) action on kidney and bone by high phosphorus (P) diet, this study investigated PTH/PTH-related peptide (PTHrP) receptor mRNA expression in 6-week-old parathyroidectomized (PTX) rats received constant amount of PTH. To maintain serum PTH levels equally to sham operated rats, PTX rats were constantly exposed to rPTH (1-34) and fed a control diet (0.3% P) and a high P diet (1.2% P) for 7 days, respectively. There were no significant differences in serum PTH (1-34) concentration in rats fed the control diet. In sham groups, serum PTH concentrations, both (1-84) and (1-34) fragments, were increased in rats fed the high P diet than in rats fed the control diet. Urinary excretions of P and C-terminal telopeptides of type I collagen were significantly increased in both PTX and sham rats by the high P diet. PTH/PTHrP receptor mRNA expression in kidney and femur was not changed in both PTX and sham rats by the high P diet. In conclusion, high P diet did not change PTH action in PTX rats and increased urinary excretion of P and bone resorption regardless of PTH action.

Direct Inhibition of Indian Hedgehog Expression by Parathyroid Hormone (PTH)/PTH-Related Peptide and Up-Regulation by Retinoic Acid in Growth Plate Chondrocyte Cultures

Indian hedgehog (Ihh) is highly expressed in prehypertrophic chondrocytes in vivo and has been proposed to regulate the proliferation and maturation of chondrocytes and bone collar formation in the growth plate. In high-density cultures of rabbit growth-plate chondrocytes, Ihh mRNA was also expressed at the highest level in the prehypertrophic stage. To explore endogenous factors that regulate Ihh expression in chondrocytes, we examined the effects of various growth factors on Ihh mRNA expression in this system. Retinoic acid (RA) and bone morphogenetic protein-2 enhanced Ihh mRNA expression, whereas PTH/PTH-related peptide (PTHrP) markedly suppressed Ihh expression. RA at more than 10−8 M induced the expression of Ihh and Patched 1 (Ptc1) within 3 h, before it increased the type X collagen mRNA level at 6–24 h. Cycloheximide blocked the up-regulation of Ihh by RA, indicating the requirement of de novo protein synthesis for this stimulation. These findings suggest that RA is involved in the up-regulation of Ihh during endochondral bone formation. In contrast to RA, PTH (1–84) at 10−7 M abolished the mRNA expression of Ihh and Ptc1 within 2–4 h, before it suppressed the expression of type X collagen at 12–24 h. The inhibition of Ihh expression by PTH (1–84) did not require de novo protein synthesis. PTH (1–34), PTHrP (1–34), and (Bu)2cAMP also suppressed Ihh expression. On the other hand, Ihh has been reported to induce PTHrP synthesis in the perichondrium. Consequently, the direct inhibitory action of PTH/PTHrP on Ihh appears to be a negative feedback mechanism that prevents excess PTHrP accumulation in cartilage.

ADAMTS-1: A Cellular Disintegrin and Metalloprotease with Thrombospondin Motifs Is a Target for Parathyroid Hormone in Bone

PTH stimulates bone formation in animals and humans, and the expressions of a number of genes have been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon we used differential display RT-PCR and screened for genes that are selectively expressed in osteoblast-enriched femoral metaphyseal primary spongiosa of young male rats after a single s.c. injection of human PTH-(1-38) (8 microg/100 g). We show that one of the messenger RNAs that is up-regulated in bone is ADAMTS-1, a new member of the ADAM (A disintegrin and metalloprotease) gene family containing thrombospondin type I motifs. ADAMTS-1 consists of multiple domains common to ADAM family of proteins, including pro-, metalloprotease-like, and disintegrin-like domains. However, unlike other ADAMs, ADAMTS-1 does not possess a transmembrane or cytoplasmic domain and is a secreted protein. Northern blot analysis confirmed that ADAMTS-1 was up-regulated in both metaphyseal (14- to 35-fold) and diaphyseal (4.2-fold) bone 1 h after PTH-(1-38) injection and returned to control levels by 24 h. We also analyzed the regulation of ADAMTS-1 in response to various PTH/PTH-related peptide (PTHrP) analogs and found that PTH-(1-31) and PTHrP-(1-34), which activate the protein kinase A (PKA) pathway, induce ADAMTS-1 expression 1 h after injection, whereas PTH-(3-34) and PTH-(7-34), which do not activate the PKA pathway, did not regulate expression. To investigate the effect of other osteotropic agents, we analyzed ADAMTS-1 expression after a single dose of PGE2 (6 mg/kg) and found that it was up-regulated 1 h after injection and returned to control levels by 6 h. In vitro ADAMTS-1 is expressed in primary osteoblasts and osteoblastic cell lines, but was not detectable in osteoclasts generated from macrophage colony-stimulating factor/receptor activator of NF-kappaB ligand/transforming growth factor-beta1-treated bone marrow cells. Treatment of UMR 106 osteosarcoma cells with PTH, PGE2, forskolin, or (Bu)2cAMP increased ADAMTS-1 expression 7-, 4-, 5-, and 5-fold, respectively. Also, in vitro treatment with 1alpha,25-dihydroxyvitamin D3 increased ADAMTS-1 expression 3-fold. Tissue distribution analysis showed that ADAMTS-1 is expressed at high levels in many tissues, including the heart, lung, liver, skeletal muscle, and kidney. Taken together, these results demonstrate that ADAMTS-1 is specifically up-regulated in bone and osteoblasts by the osteotropic agents PTH, PTHrP, and PGE2 possibly via the cAMP/PKA pathway. We speculate that the rapid and transient increase in ADAMTS-1 expression may contribute to some of the effects of PTH on bone turnover.

Dissection of differentially regulated (G+C)-rich promoters of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene.

The PTH/PTH-related peptide (PTHrP) receptor (PTHR) is required for normal skeletal development, and a wide array of physiological responses mediated by PTH and PTHrP. We have previously identified three promoters, P1-P3, which control human PTHR gene transcription. P2 and P3 are (G+C)-rich, function in a number of tissues, lie within the same CpG island, and display many hallmarks of housekeeping promoters. However, they are differentially regulated during development as P2, but not P3, functions in fetal tissues. Here, we have used both stably and transiently transfected human osteoblast-like cells to delineate regions of P2 and P3 required for promoter activity. Deletion analyses performed in stably transfected cells indicated that sequences extending from -91 to -12 relative to the transcription start site were required for function of the P2 promoter. No negative regulatory elements were detected in P2. In contrast, deletion of an A-rich region of P3 extending from -147 to -115 was required for optimal basal activity, suggesting that this sequence acts as a repressor of P3. Strikingly, however, whereas the A-rich region also functioned as a negative element when inserted upstream of the (G+C)-rich P2 promoter, it enhanced expression from the thymidine kinase promoter, suggesting that its function depends on other transcription factors bound to promoter sequences. Fine deletion of P3 sequences proximal to -115 implicated Spl motifs and downstream initiation sites in P3 function. These studies indicate that function of P2 and P3 is controlled by ubiquitously expressed transcription factors and raise the possibility that P3 activity is repressed during fetal development.

Nuclear localization of the type 1 PTH/PTHrP receptor in rat tissues.

The localization of PTH/PTH-related peptide (PTHrP) receptor (PTHR) has traditionally been performed by autoradiography. Specific polyclonal antibodies to peptides unique to the PTHR are now available, which allow a more precise localization of the receptor in cells and tissues. We optimized the IHC procedure for the rat PTHR using 5-microm sections of paraffin-embedded rat kidney, liver, small intestine, uterus, and ovary. Adjacent sections were analyzed for the presence of PTHR mRNA (by in situ hybridization) and PTHrP peptide. A typical pattern of staining for both receptor protein and mRNA was observed in kidney in cells lining the proximal tubules and collecting ducts. In uterus and gut, the receptor and its mRNA are present in smooth muscle layers (PTHrP target) and in glandular cuboidal cells and surface columnar epithelium. This suggests that PTH, or more likely PTHrP, plays a role in surface/secretory epithelia that is as yet undefined. In the ovary, PTHR was readily detectable in the thecal layer of large antral follicles and oocytes, and was present in the cytoplasm and/or nucleus of granulosa cells, regions that also contained receptor transcripts. PTHR protein and mRNA were found in the liver in large hepatocytes radiating outward from central veins. Immunoreactive cells were also present around the periphery of the liver but not within two or three cell layers of the surface. Clear nuclear localization of the receptor protein was present in liver cells in addition to the expected cytoplasmic/peripheral staining. PTHR immunoreactivity was present in the nucleus of some cells in every tissue examined. RT-PCR confirmed the presence of PTHR transcripts in these same tissues. Examination of the hindlimbs of PTHR gene-ablated mice showed no reaction to this antibody, whereas hindlimbs from their wild-type littermates stained positively. The results emphasize that the PTHR is highly expressed in diverse tissues and, in addition, show that the receptor protein itself can be localized to the cell nucleus. Nuclear localization of the receptor suggests that there is a role for PTH and/or PTHrP in the regulation of nuclear events, either on the physical environment (nucleoskeleton) or directly on gene expression.

Parathyroid hormone-parathyroid hormone-related peptide receptor expression and function in otosclerosis.

The aim of this study was to investigate the possibility that an abnormality related to parathyroid hormone (PTH) action is involved in the increased bone turnover observed in otosclerosis. To do so, expression and function of the PTH-PTH-related peptide (PTHrP) receptor were studied in the involved tissue (stapes) and compared with that in control bone sample obtained from the external auditory canal (EAC) in the same patient in 10 cases of otosclerosis and in 1 case of osteogenesis imperfecta. PTH-PTHrP receptor expression was studied by RT-PCR of RNA prepared from cultured cells in three patients and RNA directly extracted from bone samples in four patients. PTH-PTHrP receptor function was assessed by measuring the stimulation of cAMP production by 0.8, 8, and 80 nM PTH in bone cell cultures in seven cases. Results showed that PTH-PTHrP receptor mRNA expression in the otosclerotic stapes was lower than that in EAC samples (P < 0.05), whereas it was higher in stapes than that in EAC in the case of osteogenesis imperfecta. cAMP production after PTH stimulation was lower in bone cells cultured from otosclerotic stapes compared with that in cells cultured from EAC (range of increase in stimulation: 0.8-4.5 and 1.5-7 in stapes and EAC bone cells, respectively, P < 0.05). In contrast, the stimulation of cAMP production by forskolin was not significantly different in otosclerotic stapes and EAC bone cells (range of increase in stimulation: 20.7-83.1 and 4.9-99.8 in stapes and EAC, respectively, P > 0.05). These results show a lower stimulation of cAMP production in response to PTH associated with a lower PTH-PTHrP receptor mRNA expression in pathological stapes from patients with otosclerosis compared with that in control EAC samples. This difference supports the hypothesis that an abnormal cellular response to PTH contributes to the abnormal bone turnover in otosclerosis.

Epitope tag mapping of the extracellular and cytoplasmic domains of the rat parathyroid hormone (PTH)/PTH-related peptide receptor.

The PTH/PTH-related peptide (PTHrP) receptor is predicted to span the plasma membrane seven times with an amino-terminal extracellular extension and a cytoplasmic carboxyl-terminal tail. To assess this prediction, we inserted 10- or 9-amino acid epitope tags from c-myc or hemophilus influenza hemaglutinin (HA), which are recognized by the monoclonal antibodies 9E10 and 12Ca5, respectively, in different extracellular and cytoplasmic regions of the receptor and examined the immunoreactivity of the epitopes in intact and permeabilized cells. The data show that the epitopes were well tolerated when introduced into the E2 region of the extracellular amino-terminus (E2-myc and E2-HA), in the first extracellular loop (EL1), in the second and third cytoplasmic loops (CL2c and CL3), or in the carboxyl-terminal tail (T-myc). Receptors tagged at these locations were well expressed, bound PTH with high affinity, and increased cAMP accumulation with a good efficiency. Receptors tagged in the second and third extracellular loops (EL2c and EL3c) or the first cytoplasmic loop (CL1c) bound the PTH radioligand with a low affinity, stimulated cAMP accumulation with a low efficiency, and had low expression levels. The receptors tagged on presumed extracellular regions, E2-myc, E2-HA, EL1, EL2c, and EL3c, were readily detected on the surface of intact cells with the monoclonal antibody against the epitope tag. In contrast, receptors tagged with the c-myc epitope in the cytoplasmic loops (CL1c, CL2c, and CL3) or in the carboxyl-terminal tail (T-myc) did not show any 9E10 binding in intact cells. These receptors, however, were well expressed on the cell surface, as detected by the binding of the monoclonal antibody, 12Ca5, to the HA tag that was introduced into the E2 region of these constructs. The c-myc epitopes, however, became accessible after permeabilization of the cell membrane. In conclusion, these data provide experimental evidence for the sidedness of the extracellular and cytoplasmic domains of the PTH/PTHrP receptor.

Multiple regions of ligand discrimination revealed by analysis of chimeric parathyroid hormone 2 (PTH2) and PTH/PTH-related peptide (PTHrP) receptors.

PTH and PTH-related peptide (PTHrP) bind to the PTH/PTHrP receptor and stimulate cAMP accumulation with similar efficacy. Only PTH activates the PTH2 receptor. To examine the structural basis for this selectivity, we analyzed receptor chimeras in which the amino terminus and third extracellular domains of the two receptors were interchanged. All chimeric receptors bound radiolabeled PTH with high affinity. Transfer of the PTH2 receptor amino terminus to the PTH/PTHrP receptor eliminated high-affinity PTHrP binding and significantly decreased activation by PTHrP. A PTH/PTHrP receptor N terminus modified by deletion of the nonhomologous E2 domain transferred weak PTHrP interaction to the PTH2 receptor. Introduction of the PTH2 receptor third extracellular loop into the PTH/PTHrP receptor increased the EC50 for PTH and PTHrP, while preserving high-affinity PTH binding and eliminating high-affinity PTHrP binding. Similarly, transfer of the PTH/PTHrP receptor third extracellular loop preserved high-affinity PTH binding by the PTH2 receptor but decreased its activation. Return of Gln440 and Arg394, corresponding residues in the PTH/PTHrP and PTH2 receptor third extracellular loops, to the parent residue restored function of these receptors. Simultaneous interchange of wild-type amino termini and third extracellular loops eliminated agonist activation but not binding for both receptors. Function was restored by elimination of the E2 domain in the receptor with a PTH/PTHrP receptor N terminus and return of Gln440/Arg394 to the parent sequence in both receptors. These data suggest that the amino terminus and third extracellular loop of the PTH2 and PTH/PTHrP receptors interact similarly with PTH, and that both domains contribute to differential interaction with PTHrP.

Identification, functional characterization, and developmental expression of two nonallelic parathyroid hormone (PTH)/PTH-related peptide receptor isoforms in Xenopus laevis (Daudin).

Complementary DNAs encoding two nonallelic PTH/PTH-related peptide (PTHrP) receptor (PPR) isoforms, xPPR-A and xPPR-B, were isolated from a kidney complementary DNA library of the tetraploid African clawed frog Xenopus laevis. Both isoforms differ in their coding region by 19 amino acids, and lack the region corresponding to the mammalian exon E2. When expressed in mammalian COS-7 cells, both receptor isoforms bound radiolabeled PTH-(1-34) and PTHrP-(1-36) analogs with comparable affinity, and both unlabeled peptides equivalently stimulated the accumulation of cAMP. xPPR-A also mediated inositol phosphate turnover in COS cells and stimulated channel-mediated current changes in voltage clamp experiments after injection into oocytes. Using ribonuclease protection analysis, significant xPPR-A messenger RNA expression was first detected in neurula stage embryos, which subsequently increased approximately 30-fold during tadpole development. Expression reached a maximum at the metamorphotic climax, when isoform B also became detectable at significant levels, and subsequently declined in postmetamorphotic froglets. In the adult frog, xPPR-A was prominently expressed in lung, brain, small bowel, and skin, whereas isoform B was highest in lung, heart, and brain. Using an xPPR-A antisense riboprobe for in situ hybridization, expression appeared during metamorphosis at all sites of chondrogenesis, specifically in the maturing zone of the amphibian growth plate. xPPR-A expression was also seen in a subpopulation of mononuclear cells, possibly representing osteoblasts that line perichondral bone and diaphyseal bone trabeculae. Our findings suggest that xPPRs serve a prominent role in amphibian skeletal development and possibly other functions during embryonal and early larval development.