Actions of Parathyroid Hormone Ligand Analogues in Humanized PTH1R Knockin Mice.

Rodent models are commonly used to evaluate parathyroid hormone (PTH) and PTH-related protein (PTHrP) ligands and analogues for their pharmacologic activities and potential therapeutic utility toward diseases of bone and mineral ion metabolism. Divergence, however, in the amino acid sequences of rodent and human PTH receptors (rat and mouse PTH1Rs are 91% identical to the human PTH1R) can lead to differences in receptor-binding and signaling potencies for such ligands when assessed on rodent vs human PTH1Rs, as shown by cell-based assays in vitro. This introduces an element of uncertainty in the accuracy of rodent models for performing such preclinical evaluations. To overcome this potential uncertainty, we used a homologous recombination-based knockin (KI) approach to generate a mouse (in-host strain C57Bl/6N) in which complementary DNA encoding the human PTH1R replaces a segment (exon 4) of the murine PTH1R gene so that the human and not the mouse PTH1R protein is expressed. Expression is directed by the endogenous mouse promoter and hence occurs in all biologically relevant cells and tissues and at appropriate levels. The resulting homozygous hPTH1R-KI (humanized) mice were healthy over at least 10 generations and showed functional responses to injected PTH analog peptides that are consistent with a fully functional human PTH1R in target bone and kidney cells. The initial evaluation of these mice and their potential utility for predicting behavior of PTH analogues in humans is reported here.

Serum PTH, PTH1R/ATF4 pathway, and the sRANKL/OPG system in bone as a new link between bone growth, cross-sectional geometry, and strength in young rats with experimental chronic kidney disease.

The progression of chronic kidney disease (CKD) in children is associated with deregulated parathyroid hormone (PTH), growth retardation, and low bone accrual. PTH can cause both catabolic and anabolic impact on bone, and the activating transcription factor 4 (ATF4), a downstream target gene of PTH, is related to its anabolic effect. Osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) are PTH-dependent cytokines, which may play an important role in the regulation of bone remodeling. This study aimed to evaluate the impact of endogenous PTH and the bone RANKL/OPG system on bone growth, cross-sectional geometry and strength utilizing young, nephrectomized rats. The parameters of cross-sectional geometry were significantly elevated in rats with CKD during the three-month experimental period compared with the controls, and they were strongly associated with serum PTH levels and the expression of parathyroid hormone 1 receptor (PTH1R)/ATF4 genes in bone. Low bone soluble RANKL (sRANKL) levels and sRANKL/OPG ratios were also positively correlated with cross-sectional bone geometry and femoral length. Moreover, the analyzed geometric parameters were strongly related to the biomechanical properties of femoral diaphysis. In summary, the mild increase in endogenous PTH, its anabolic PTH1R/ATF4 axis and PTH-dependent alterations in the bone RANKL/OPG system may be one of the possible mechanisms responsible for the favorable impact on bone growth, cross-sectional geometry and strength in young rats with experimental CKD.

Bone-remodeling transcript levels are independent of perching in end-of-lay white leghorn chickens.

Osteoporosis is a bone disease that commonly results in a 30% incidence of fracture in hens used to produce eggs for human consumption. One of the causes of osteoporosis is the lack of mechanical strain placed on weight-bearing bones. In conventionally-caged hens, there is inadequate space for chickens to exercise and induce mechanical strain on their bones. One approach is to encourage mechanical stress on bones by the addition of perches to conventional cages. Our study focuses on the molecular mechanism of bone remodeling in end-of-lay hens (71 weeks) with access to perches. We examined bone-specific transcripts that are actively involved during development and remodeling. Using real-time quantitative PCR, we examined seven transcripts (COL2A1 (collagen, type II, alpha 1), RANKL (receptor activator of nuclear factor kappa-B ligand), OPG (osteoprotegerin), PTHLH (PTH-like hormone), PTH1R (PTH/PTHLH type-1 receptor), PTH3R (PTH/PTHLH type-3 receptor), and SOX9 (Sry-related high mobility group box)) in phalange, tibia and femur. Our results indicate that the only significant effect was a difference among bones for COL2A1 (femur > phalange). Therefore, we conclude that access to a perch did not alter transcript expression. Furthermore, because hens have been used as a model for human bone metabolism and osteoporosis, the results indicate that bone remodeling due to mechanical loading in chickens may be a product of different pathways than those involved in the mammalian model.

Kinetics and dynamics in the G protein-coupled receptor signaling cascade.

We describe optical and microscopy methods based on Förster resonance energy transfer, fluorescence recovery after photobleaching, and imaging cross-correlation spectroscopy that permit to determine kinetic and dynamic properties of key reactions involved G protein-coupled receptor (GPCR) signaling from the initial ligand binding step to the generation of the second messenger, cAMP. Well suited to determine rate-limiting reactions taking place along a GPCR signaling cascade in live cells, these techniques have also uncovered new concepts in GPCR signaling as well as many interesting mechanistic subtleties by which GPCRs transmit neurotransmitter and hormone signals into cells.

Parathyroid hormone-related protein activates Wnt signaling to specify the embryonic mammary mesenchyme.

Parathyroid hormone-related protein (PTHrP) regulates cell fate and specifies the mammary mesenchyme during embryonic development. Loss of PTHrP or its receptor (Pthr1) abolishes the expression of mammary mesenchyme markers and allows mammary bud cells to revert to an epidermal fate. By contrast, overexpression of PTHrP in basal keratinocytes induces inappropriate differentiation of the ventral epidermis into nipple-like skin and is accompanied by ectopic expression of Lef1, ß-catenin and other markers of the mammary mesenchyme. In this study, we document that PTHrP modulates Wnt/ß-catenin signaling in the mammary mesenchyme using a Wnt signaling reporter, TOPGAL-C. Reporter expression is completely abolished by loss of PTHrP signaling and ectopic reporter activity is induced by overexpression of PTHrP. We also demonstrate that loss of Lef1, a key component of the Wnt pathway, attenuates the PTHrP-induced abnormal differentiation of the ventral skin. To characterize further the contribution of canonical Wnt signaling to embryonic mammary development, we deleted ß-catenin specifically in the mammary mesenchyme. Loss of mesenchymal ß-catenin abolished expression of the TOPGAL-C reporter and resulted in mammary buds with reduced expression of mammary mesenchyme markers and impaired sexual dimorphism. It also prevented the ectopic, ventral expression of mammary mesenchyme markers caused by overexpression of PTHrP in basal keratinocytes. Therefore, we conclude that a mesenchymal, canonical Wnt pathway mediates the PTHrP-dependent specification of the mammary mesenchyme.

Functional characterization and evolution of PTH/PTHrP receptors: insights from the chicken.

BACKGROUND: The parathyroid hormone (PTH)-family consists of a group of structurally related factors that regulate calcium and bone homeostasis and are also involved in development of organs such as the heart, mammary gland and immune system. They interact with specific members of family 2 B1 G-protein coupled receptors (GPCRs), which have been characterised in teleosts and mammals. Two PTH/PTHrP receptors, PTH1R and PTH2R exist in mammals and in teleost fish a further receptor PTH3R has also been identified. Recently in chicken, PTH-family members involved in calcium transport were characterized and specific PTHRs are suggested to exist although they have not yet been isolated or functionally characterized. The aim of this study is to further explore the evolution and function of the vertebrate PTH/PTHrP system through the isolation, phylogenetic analysis and functional characterization of the chicken receptors. RESULTS: Two PTHRs were isolated in chicken and sequence comparison and phylogenetic analysis indicate that the chicken receptors correspond to PTH1R and PTH3R, which emerged prior to the teleost/tetrapod divergence since they are present in cartilaginous fish. The vertebrate PTH2R receptor and its ligand TIP39 have been lost from bird genomes. Chicken PTH1R and PTH3R have a divergent and widespread tissue expression and are also evident in very early embryonic stages of development. Receptor stimulation studies using HEK293 cells stably expressing the chicken PTH1R and PTH3R and monitoring cAMP production revealed they are activated by chicken 1-34 N-terminal PTH-family peptides in a dose dependent manner. PTH-L and PTHrP were the most effective peptides in activating PTH1R (EC(50) = 7.7 nM and EC(50) = 22.7 nM, respectively). In contrast, PTH-L (100 nM) produced a small cAMP accumulation on activation of PTH3R but PTHrP and PTH (EC(50) = 2.5 nM and EC(50) = 22.1 nM, respectively) readily activated the receptor. PTHrP also stimulated intracellular Ca(2+) accumulation on activation of PTH1R but not PTH3R. CONCLUSION: Two PTHR homologues of the vertebrate PTH1R and PTH3R were isolated and functionally characterized in chicken. Their distinct pattern of expression during embryo development and in adult tissues, together with their ligand preference, suggests that they have acquired specific functions, which have contributed to their maintenance in the genome. PTH2R and its activating ligand, TIP39, are absent from bird genomes. Nonetheless identification of putative PTH2R and TIP39 in the genome of an ancient agnathan, lamprey, suggests the PTH/PTHrP ligand and receptor family was already present in an early basal paraphyletic group of vertebrates and during the vertebrate radiation diverged via gene/genome duplication and deletion events. Knowledge of the role PTH/PTHrP system in early vertebrates will help to establish evolution of function.

Parathyroid hormone-related protein: an update.

PTHrP was identified as a cause of hypercalcemia in cancer patients 25 yr ago. In the intervening years, we have learned that PTHrP and PTH are encoded by related genes that are part of a larger "PTH gene family." This evolutionary relationship permits them to bind to the same type 1 PTH/PTHrP receptor, which explains why humoral hypercalcemia of malignancy resembles hyperparathyroidism. This review will outline basic facts about PTHrP biology and its normal physiological functions, with an emphasis on new findings of the past 5-10 yr. The medical and research communities first became aware of PTHrP because of its involvement in a common paraneoplastic syndrome. Now, research into the basic biology of PTHrP has suggested previously unrecognized connections to a variety of disease states such as osteoporosis, osteoarthritis, and breast cancer and has highlighted how PTHrP itself might be used in therapy for osteoporosis and diabetes. Therefore, the story of this remarkable protein is a paradigm for translational research, having gone from bedside to bench and now back to bedside.

Parathyroid hormone 1 (1-34) acts on the scales and involves calcium metabolism in goldfish.

The effect of fugu parathyroid hormone 1 (fugu PTH1) on osteoblasts and osteoclasts in teleosts was examined with an assay system using teleost scale and the following markers: alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts. Synthetic fugu PTH1 (1-34) (100pg/ml-10ng/ml) significantly increased ALP activity at 6h of incubation. High-dose (10ng/ml) fugu PTH1 significantly increased ALP activity even after 18h of incubation. In the case of TRAP activity, fugu PTH1 did not change at 6h of incubation, but fugu PTH1 (100pg/ml-10ng/ml) significantly increased TRAP activity at 18h. Similar results were obtained for human PTH (1-34), but there was an even greater response with fugu PTH1 than with human PTH. In vitro, we demonstrated that both the receptor activator of the NF-κB ligand in osteoblasts and the receptor activator NF-κB mRNA expression in osteoclasts increased significantly by fugu PTH1 treatment. In an in vivo experiment, fugu PTH1 induced hypercalcemia resulted from the increase of both osteoblastic and osteoclastic activities in the scale as well as the decrease of scale calcium contents after fugu PTH1 injection. In addition, an in vitro experiment with intramuscular autotransplanted scale indicated that the ratio of multinucleated osteoclasts/mononucleated osteoclasts in PTH-treated scales was significantly higher than that in the control scales. Thus, we concluded that PTH acts on osteoblasts and osteoclasts in the scales and regulates calcium metabolism in goldfish.

Differential effects of intermittent PTH(1-34) and PTH(7-34) on bone microarchitecture and aortic calcification in experimental renal failure.

PTH(1-84) and PTH(7-84) are elevated in chronic kidney disease (CKD). These peptides, as their shorter analogs PTH(1-34) and PTH(7-34) both promote PTH receptor (PTH1R) internalization but only PTH(1-34) and PTH(1-84) activate the receptor. Here, we examined the effects of intermittent administration of PTH(1-34) and PTH(7-34) on mineral ion metabolism, bone architecture, and vascular calcification in rats with experimental CKD. CKD with or without parathyroidectomy (PTX) was established by 5/6 nephrectomy (NPX) in rats. Animals were divided into 4 groups: Sham PTX+ sham NPX (Sham); PTX+ sham NPX (PTX); Sham PTX+NPX (NPX); PTX+NPX (PTX/NPX). Rats were treated with single daily doses of 40 microg/kg PTH(1-34), PTH(7-34), or vehicle. Creatinine was higher in NPX and Ca lower in PTX and PTX/NPX groups than in Sham or NPX rats. Plasma phosphate was higher in PTX, NPX and PTX/NPX than in Sham rats. PTH(1-34) was more hypercalcemic than PTH(7-34) in PTX rats. Fractional bone volume in rats treated with PTH(1-34) increased significantly in all groups compared to that of vehicle treatment. In addition, trabecular number, thickness and volumetric bone density increased in rats treated with PTH(1-34). In contrast, PTH(1-34) diminished vascular calcification. Bone and renal PTH1R mRNA expression was reduced as much or more in PTX/NPX rats as in NPX alone, whereas PTH(7-34) had no effect on PTH1R expression. Renal but not bone PTH1R mRNA increased in response to PTH(1-34). These findings suggest that PTH(1-34) exerts greater hypercalcemic and anabolic effects in parathyroidectomized and/or nephrectomized rats than does PTH(7-34). There was no evidence for significant bone or vascular actions of PTH(7-34). We conclude that PTH(1-34) protects against vascular calcification and bone demineralization in experimental renal failure.

Altered selectivity of parathyroid hormone (PTH) and PTH-related protein (PTHrP) for distinct conformations of the PTH/PTHrP receptor.

PTH and PTHrP use the same G protein-coupled receptor, the PTH/PTHrP receptor (PTHR), to mediate their distinct biological actions. The extent to which the mechanisms by which the two ligands bind to the PTHR differ is unclear. We examined this question using several pharmacological and biophysical approaches. Kinetic dissociation and equilibrium binding assays revealed that the binding of [(125)I]PTHrP(1-36) to the PTHR was more sensitive to GTPgammaS (added to functionally uncouple PTHR-G protein complexes) than was the binding of [(125)I]PTH(1-34) ( approximately 75% maximal inhibition vs. approximately 20%). Fluorescence resonance energy transfer-based kinetic analyses revealed that PTHrP(1-36) bound to the PTHR more slowly and dissociated from it more rapidly than did PTH(1-34). The cAMP signaling response capacity of PTHrP(1-36) in cells decayed more rapidly than did that of PTH(1-34) (t(1/2) = approximately 1 vs. approximately 2 h). Divergent residue 5 in the ligand, Ile in PTH and His in PTHrP, was identified as a key determinant of the altered receptor-interaction responses exhibited by the two peptides. We conclude that whereas PTH and PTHrP bind similarly to the G protein-coupled PTHR conformation (RG), PTH has a greater capacity to bind to the G protein-uncoupled conformation (R(0)) and, hence, can produce cumulatively greater signaling responses (via R(0)-->RG isomerization) than can PTHrP. Such conformational selectivity may relate to the distinct modes by which PTH and PTHrP act biologically, endocrine vs. paracrine, and may help explain reported differences in the effects that the ligands have on calcium and bone metabolism when administered to humans.

Constitutively active parathyroid hormone receptor signaling in cells in osteoblastic lineage suppresses mechanical unloading-induced bone resorption.

Multiple signaling pathways participate in the regulation of bone remodeling, and pathological negative balance in the regulation results in osteoporosis. However, interactions of signaling pathways that act comprehensively in concert to maintain bone mass are not fully understood. We investigated roles of parathyroid hormone receptor (PTH/PTHrP receptor) signaling in osteoblasts in unloading-induced bone loss using transgenic mice. Hind limb unloading by tail suspension reduced bone mass in wild-type mice. In contrast, signaling by constitutively active PTH/PTHrP receptor (caPPR), whose expression was regulated by the osteoblast-specific Col1a1 promoter (Col1a1-caPPR), suppressed unloading-induced reduction in bone mass in these transgenic mice. In Col1a1-caPPR transgenic (Tg) mice, hind limb unloading suppressed bone formation parameters in vivo and mineralized nodule formation in vitro similarly to those observed in wild-type mice. In addition, serum osteocalcin levels and mRNA expression levels of type I collagen, Runx2 and Osterix in bone were suppressed by unloading in both wild-type mice and Tg mice. However, in contrast to unloading-induced enhancement of bone resorption parameters in wild-type mice, Col1a1-caPPR signaling suppressed, rather than enhanced, osteoclast number and osteoclast surface as well as urinary deoxypyridinoline excretion upon unloading. Col1a1-caPPR signaling also suppressed mRNA expression levels of RANK and c-fms in bone upon unloading. Although the M-CSF and monocyte chemoattractant protein 1 (MCP-1) mRNA levels were enhanced in control Tg mice, these levels were suppressed in unloaded Tg mice. These results indicated that constitutive activation of PTH/PTHrP receptor signaling in osteoblastic cells suppresses unloading-induced bone loss specifically through the regulation of osteoclastic activity.

Effects of overexpression of basic helix-loop-helix transcription factor Dec1 on osteogenic and adipogenic differentiation of mesenchymal stem cells.

We recently reported that forced expression of basic helix-loop-helix transcription factor Dec1 accelerated chondrogenic differentiation of mesenchymal stem cells (MSC) in pellet cultures (Shen, M., Yoshida, E., Yan, W., Kawamoto, T., Suardita, K., Koyano, Y., Fujimoto, K., Noshiro, M., Kato, Y., 2002. Basic helix-loop-helix protein DEC1 promotes chondrocyte differentiation at the early and terminal stages. J. Biol. Chem. 277, 50112-50120). Since MSC have multilineage differentiation potential, we investigated the roles of Dec1 in osteogenic and adipogenic differentiation of human bone marrow-derived MSC. After osteogenic induction of MSC in medium containing dexamethasone, beta-glycerophosphate, and ascorbic acid, Dec1 expression gradually increased from day 5 to day 14, while expression levels of Dec1 mRNA markedly decreased on days 3 and 7 after adipogenic induction. Infection with adenovirus expressing Dec1 raised mRNA levels of several bone characteristic molecules such as osteopontin, PTH receptor, and alkaline phosphatase, even in the absence of the osteogenic induction medium, although it had little effect on Runx2 expression or calcification. In the osteogenic induction medium, Dec1 overexpression enhanced the expression of osteopontin and alkaline phosphatase and induced matrix calcification. Knockdown of Dec1 with siRNA suppressed the expression of osteoblastic phenotype by the induced MSC. Using MSC cultures, we also confirmed that forced expression of Dec1 suppressed adipogenic differentiation. These findings suggest that Dec1 modulates osteogenic differentiation of MSC by inducing the expression of several, but not all, bone-related genes.

[Hereditary skeletal dysplasias and FGFR3 and PTHR1 signaling pathways]. / Dysplasies osseuses héréditaires et voies de signalisation associées aux récepteurs FGFR3 et PTHR1.

Skeletal development is a highly sophisticated process involving, as a first step, migration and condensation of mesenchymal cells into osteoprogenitor cells. These cells further differentiate into chondrocytes and osteoblasts through multiple differentiation stages requiring a set of specific transcriptional factors. Defective endochondral ossification in human is associated with a large number of inherited skeletal dysplasias caused by mutations in genes encoding extracellular matrix components, growth factors and their receptors, signaling molecules and transcription factors. This review summarizes some of the recent findings on a series of chondrodysplasias caused by mutations in FGFR3 and PTHR1, two receptors expressed in the cartilage growth plate and mediating two main signaling pathways. Data from human diseases and relevant animal models provide new clues for understanding how signaling molecules and their interaction with key transcription factors control and regulate the development and growth of long bones.

Extracellular calcium and parathyroid hormone-related peptide signaling modulate the pace of growth plate chondrocyte differentiation.

An adequate supply of Ca2+ is critical for normal growth plate development. Previous studies suggest that changes in extracellular [Ca2+] ([Ca2+]e) modulate the function of chondrocytes with high [Ca2+]e promoting cell differentiation. In contrast, signal transduction by the PTH/PTHrP type I receptor (PTH1R) slows down chondrocyte differentiation. This study addressed whether changes in [Ca2+]e modulate the differentiation of mouse growth plate chondrocytes by interacting with PTHrP/PTH1Rs. Raising [Ca2+]e from 0.5-3.0 mM dose-dependently promoted the development of mouse growth plate chondrocytes as indicated by decreases in proteoglycan accumulation and in the expression of early differentiation marker genes and by increases in mineral deposition and in the expression of markers of terminal differentiation. The effects of high [Ca2+]e on gene expression and matrix synthesis were blunted by incubating cells with PTHrP and vice versa. High [Ca2+]e also suppressed the expression of PTH1Rs. Chronic stimulation of PTHrP/PTH1R signaling by adenoviral expression of constitutively active human PTH1Rs (223hPTH1Rs) reduced the effects of high [Ca2+]e on proteoglycan synthesis and gene expression. Similar results were seen when we treated cells with forskolin or 8-bromo-cAMP. Taken together, these data support the idea that the pace of chondrocyte differentiation depends on a balance of interactions between PTHrP/PTH1R and extracellular Ca2+ signaling and that high [Ca2+]e promote cell differentiation potentially by reducing the availability of PTH1Rs and the level of cAMP-dependent signal transduction.

Bioactive N-terminal undecapeptides derived from parathyroid hormone: the role of alpha-helicity.

The N-terminal 1-34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses in bone characteristic of the native intact PTH. Recent studies have demonstrated that N-terminal fragments presenting the principal activating domain such as PTH(1-11) and PTH(1-14) with helicity-enhancing substitutions yield potent analogues with PTH(1-34)-like activity. To further investigate the role of alpha-helicity on biological potency, we designed and synthesized by solid-phase methodology the following hPTH(1-11) analogues substituted at positions 1 and/or 3 by the sterically hindered and helix-promoting C(alpha)-tetrasubstituted alpha-amino acids alpha-amino isobutyric acid (Aib), 1-aminocyclopentane-1-carboxylic acid (Ac(5)c) and 1-aminocyclohexane-1-carboxylic acid (Ac(6)c): Ac(5)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (I); Aib-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (II); Ac(6)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (III); Aib-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (IV); Aib-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (V); S-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (VI), S-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (VII); Ac(5)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (VIII); Ac(6)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (IX); Ac(5)c-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (X); Ac(6)c-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (XI). All analogues were biologically evaluated and conformationally characterized in 2,2,2-trifluoroethanol (TFE) solution by circular dichroism (CD). Analogues I-V, which cover the full range of biological activity observed in the present study, were further conformationally characterized in detail by nuclear magnetic resonance (NMR) and computer simulations studies. The results of ligand-stimulated cAMP accumulation experiments indicated that analogues I and II are active, analogues III, VI and VII are very weakly active and analogues IV, V, VIII-XI are inactive. The most potent analogue, I exhibits biological activity 3500-fold higher than that of the native PTH(1-11) and only 15-fold weaker than that of the native sequence hPTH(1-34). Remarkably, the two most potent analogues, I and II, and the very weakly active analogues, VI and VII, exhibit similar helix contents. These results indicate that the presence of a stable N-terminal helical sequence is an important but not sufficient condition for biological activity.

Expression of parathyroid hormone-related protein in ameloblastomas.

Parathyroid hormone-related protein (PTHrP) was first discovered as a causative protein for hypercalcemia, which is often seen in the malignant tumor. PTHrP binds to the parathyroid hormone 1 receptor (PTH1R) for signal transduction. PTHrP-PTH1R interactions were associated with bone resorption. The present study, therefore, sought to clarify the expression of PTHrP, parathyroid hormone (PTH) and PTH1R in ameloblastoma, using RT-PCR (N = 8), immunohistochemistry (N = 23) and ELISA (N = 11) techniques. PTHrP and B-actin mRNA were detected in the all samples. Expression of PTHrP was also seen in all of the 23 cases in ameloblastoma by immunohistochemistry. There was a significant difference in PTHrP concentration by ELISA between typical unicystic type and solid type including unicystic type 3 (p = 0.0427). Only one exhibited the weak expression of PTH1R mRNA. PTH1R was observed on osteoblasts in bone around the tumor but no expression was observed on ameloblastoma cells in tumor parenchyma by immunohistochemistry. PTH was not detected in ameloblastoma by RT-PCR, immunohistochemistory as well as ELISA. In addition, hypercalcemia and increase of serum PTHrP level was observed in one case of 8 ameloblastomas. It was suggested that PTHrP level may be associated with local bone infiltration and hypercalcemia in ameloblastoma.

Regulation of PTH1 receptor expression by uremic ultrafiltrate in UMR 106-01 osteoblast-like cells.

BACKGROUND: Homologous down-regulation/desensitization of the parathyroid hormone receptor (PTH1R)/adenylate cyclase system has been demonstrated in uremia, and may contribute to parathyroid hormone (PTH) resistance; however, additional studies have shown that parathyroidectomy fails to normalize the down-regulation of the PTH1R. The present studies were designed to test directly, in vitro, the hypothesis that factors circulating in the uremic environment, other than PTH, decrease the response of osteoblastic cells to PTH. METHODS: Studies were conducted in confluent cultures of UMR 106-01 osteoblast-like cells. Uremic ultrafiltrate (UUF) was obtained from patients on hemodialysis. Cells were exposed to media containing 50% uremic ultrafiltrate for periods of up to 72 hours. Control cultures were exposed to a buffered salt solution containing a comparable ionic composition to that of the UUF. PTH-stimulated cyclic adenosine monophosphate (cAMP) generation was determined by radioimmunoassay (RIA), PTH binding and PTH1R mRNA levels were determined by radioligand binding and Northern analysis, respectively. RESULTS: PTH-stimulated cAMP generation from cultures treated with uremic ultrafiltrate for 48 hours was 1385.8 +/- 183.2 pmol/culture/5 minutes, whereas control cultures generated 2389.5 +/- 271 pmol cAMP/culture/5 minutes (P < 0.05). PTH binding was decreased by 30% in cultures incubated with UUF as compared to controls. The decrease in binding induced by UUF was accompanied by a decrease in PTH1R mRNA levels. CONCLUSION: These findings demonstrate that factors present in UUF decrease PTH-stimulated cAMP generation by a mechanism that involves a decrease in the levels of PTH1R mRNA levels. Thus, the skeletal resistance to PTH in the setting of chronic kidney disease, may be explained, at least in part, by circulating factors other than PTH.

Functional importance of Myc-associated zinc finger protein for the human parathyroid hormone (PTH)/PTH-related peptide receptor-1 P2 promoter constitutive activity.

The aim of the present study was to analyze the functional importance for the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) gene P2 promoter activity of the putative proximal Myc-associated zinc finger protein (MAZ) site localized at position bp -45 to -39 bp, taking advantage of a G/A mutation identified at position -40 in the human sequence. Wild-type 'full-length' (1285P2) and truncated (760P2) promoter sequences were inserted upstream to the luciferase basic (pLucB) and enhancer (pLucE) reporter gene expression vectors. Transient transfections in osteoblast-like SaOS-2 cells and renal cells (RC.SV3A2) showed that the -40 G/A mutation significantly impaired transcriptional activity of wild-type 1285P2-pLucB and 760P2-pLucE promoter constructs. Further truncation of the P2 sequence demonstrated that the sequence -109/-37 bp was essential for promoter activity. Co-transfection with a MAZ expression vector did not modify the wild-type 1285P2-pLucB construct reporter activity but significantly increased 2-fold the mutated construction activity (P<0.05). Electrophoretic mobility shift assays using SaOS-2 nuclear extracts and a double-stranded DNA fragment encompassing the -45 to -39 putative MAZ site (ds-MAZ-oligo) disclosed two specific DNA-protein complexes. Complex II (fast moving) had a lower affinity for the mutated MAZ motif than for the wild-type MAZ motif while complex I (slow moving) had the same affinity for both wild-type or mutated MAZ sequences. Competition studies with Sp1 consensus oligonucleotide (ds-Sp1-oligo) markedly reduced complex I intensity, with a concomitant increase in that of complex II. Finally, ribonuclease protection assays showed that P2-specific PTHR1 mRNA transcript expression was significantly decreased in SaOS-2 cells transfected with ds-MAZ-oligo as compared with that for control (P<0.001) and ds-Sp1-oligo (P<0.05). Taken together, our studies suggest that the putative -45 to -39 MAZ-binding site regulates the constitutive activity of human PTHR1 P2 promoter.

Parathyroid hormone (PTH) down-regulates PTH/PTH-related protein receptor gene expression in UMR-106 osteoblast-like cells via a 3',5'-cyclic adenosine monophosphate-dependent, protein kinase A-independent pathway.

Parathyroid hormone (PTH) regulates osteoblast function via a G protein-linked PTH/PTH-related protein (PTHrP) receptor. We have studied the mechanisms of PTH/PTHrP receptor gene repression by PTH in UMR-106 osteoblast-like cells. Inhibition of PTH/PTHrP receptor mRNA expression by rat (r) PTH(1-34) and Insulin-like growth factor-I (IGF-I) at 10(-7)M was significant at 1 h and 3 h, and maximal at 2 h and 6 h. A maximal decrease in receptor mRNA abundance by rPTH(1-34) and IGF-I was maintained for 24 h. Inhibition of receptor gene expression by rPTH(1-34) was mimicked in UMR-106 cells by the addition of forskolin (an adenylyl cyclase activator), or 8-(4-chlorophenylthio)-adenine 3',5'-cyclic monophosphate (8-pCPTcAMP; a cAMP analogue). Although H89, a selective protein kinase A (PKA) inhibitor, completely inhibited PKA activity stimulated by rPTH(1-34), forskolin or 8-pCPTcAMP, suppression of PTH/PTHrP receptor mRNA synthesis induced by these substances in UMR-106 cells was not affected by H89. In primary osteoblast cultures, rPTH(1-34) inhibited synthesis of PTH/PTHrP receptor mRNA irrespective of H89. The down-regulation effect of rPTH(1-34) was also unaltered by PD98059 (an extracellularly regulated kinase 1/2 mitogen-activated protein kinase pathway inhibitor). Pretreatment with cycloheximide, a protein synthesis inhibitor, did not alter the inhibition of PTH/PTHrP receptor mRNA expression by rPTH(1-34), indicating that receptor mRNA suppression does not require new protein synthesis. Transcriptional activation of PTH/PTHrP receptor gene promoter (U3P or U4P)-luciferase constructs was decreased by rPTH(1-34), forskolin and 8-pCPTcAMP irrespective of H89. Thus, PTH transcriptionally down-regulates PTH/PTHrP receptor gene expression in osteoblast-like cells via a cAMP-dependent, PKA-independent pathway.