PTH/PTHrP Receptor Research Articles

Constitutively active PTH/PTHrP receptor in odontoblasts alters odontoblast and ameloblast function and maturation

Parathyroid hormone (PTH)-related protein (PTH-rP) is an important autocrine/paracrine attenuator of programmed cell differentiation whose expression is restricted to the epithelial layer in tooth development. The PTH/PTHrP receptor (PPR) mRNA in contrast is detected in the dental papilla, suggesting that PTHrP and the PPR may modulate epithelial–mesenchymal interactions. To explore the possible interactions, we studied the previously described transgenic mice in which a constitutively active PPR is targeted to osteoblastic cells. These transgenic mice have a vivid postnatal bone and tooth phenotype, with normal tooth eruption but abnormal, widened crowns. Transgene mRNA expression was first detected at birth in the dental papilla and, at 1 week postnatally, in odontoblasts. There was no transgene expression in ameloblasts or in other epithelial structures. Prenatally, transgenic molars and incisors revealed no remarkable change. By the age of 1 week, the dental papilla was widened, with disorganization of the odontoblastic layer and decreased dentin matrix. In addition, the number of cusps was abnormally increased, the ameloblastic layer disorganized, and enamel matrix decreased. Odontoblastic and, surprisingly, ameloblastic cytodifferentiation was impaired, as shown by in situ hybridization and electron microscopy. Interestingly, ameloblastic expression of Sonic Hedgehog, a major determinant of ameloblastic cytodifferentiation, was dramatically altered in the transgenic molars. These data suggest that odontoblastic activation of the PPR may play an important role in terminal odontoblastic and, indirectly, ameloblastic cytodifferentiation, and describe a useful model to study how this novel action of the PPR may modulate mesenchymal/epithelial interactions at later stages of tooth morphogenesis and development.

PTH/PTHrP activity and the programming of skeletal development in utero.

There is increasing evidence that nutritional deficiency in utero adversely affects bone development and the risk of developing osteoporosis in later life. Although the mechanisms involved are unknown, circumstantial evidence points to an important role of PTH/PTHrP activity. It is recognized that PTH and PTHrP are critically involved in regulating fetal calcium homeostasis, actions that are mediated at least in part by PPR. As well as playing a central role in the maintenance of calcium homeostasis in the fetus, studies in transgenic mice show that PTH, PTHrP, and PPR exert similar effects on skeletal development in utero, acting to increase the size of the trabecular envelope and decrease that of the cortical envelopes. Taken together, these observations raise the possibility that stimulation of PTH/PTHrP activity in the fetus in response to calcium deficiency acts to increase the size of the trabecular envelope but to reduce that of the cortical envelope. Although any increase in trabecular bone at birth is likely to be relatively transient, a decrease in size of the cortical envelope may have a persistent effect on the trajectory of bone growth in subsequent childhood. Consistent with this proposal, preliminary findings from birth cohort studies suggest that maternal calcium intake and cord blood calcium levels are positively related to bone mass of the offspring as assessed later in childhood. Further studies are justified to determine whether alterations in fetal PTH/ PTHrP activity caused by calcium stress lead to a reduction in size of the cortical envelope at birth that persists into childhood and later adult life and to identify modifiable maternal factors that are responsible for these changes.

Circulating Forms of Parathyroid Hormone: Peeling Back the Onion

Circulating parathyroid hormone (PTH) regulates normal bone and mineral ion homeostasis and is central in the pathogenesis of bone disease in primary and secondary hyperparathyroidism, especially in advanced renal insufficiency (1). The major biological effects of PTH, a linear polypeptide 84 amino acids in length, reflect activation of the G-protein-coupled PTH/PTHrP receptor (PTHR) expressed on target cells in the renal tubules and the osteoblastic lineage of bone (2). Activation of PTHRs requires only the most N-terminal portion of PTH [PTH(1–34) is a full agonist] and is mostly eliminated if the N-terminal serine residue of the human hormone is removed. PTH, secreted by the parathyroid glands primarily in response to changes in blood ionized calcium, undergoes rapid endopeptidic cleavage, mainly by Kupffer cells in the liver (3). This event generates a series of N-truncated “CPTH” fragments (the corresponding N-terminal segments are destroyed in situ) that reenter the circulation and are cleared mainly by glomerular filtration, an important route for PTH clearance as well (4). Chemical analysis of PTH fragments isolated from rat blood after administration of radiolabeled bovine PTH established that hepatic metabolism yields a family of CPTH fragments with NH2 termini ranging between residues 34 and 43, although the methods used likely would not have detected substantially larger CPTH fragments (5). Interestingly, chemically similar CPTH fragments are secreted directly from the parathyroid glands as well, and the ratio of secreted CPTH fragments to intact PTH increases as blood calcium increases (6)(7). CPTH fragments typically circulate at a three- to fivefold molar excess over intact PTH, but this ratio may increase dramatically in end-stage renal disease (ESRD), depending on the severity and chronicity of renal insufficiency and the blood concentrations of calcium, phosphate, and 1,25-dihydroxyvitamin D, all of which regulate PTH secretion (1)(8). Measurement of …

BMP4 promotes chondrocyte proliferation and hypertrophy in the endochondral cranial base.

Defects in the growth and development of the endochondral bones that comprise the cranial base contribute to several craniofacial dysmorphic syndromes. Since Bone Morphogenetic Protein (BMP) signaling regulates chondrocyte differentiation and endochondral ossification in developing long bones, we have tested the hypothesis that BMP signaling also participates in regulating development of the cranial base. During in vivo developmental progression of the cranial base in mice, a burst of skeletal growth and chondrocyte maturation was identified in the perinatal period. Using a novel serum-free organ culture system, cranial base structures were cultured as explants in the presence of BMP4 or noggin, and analyzed for morphological and molecular changes. Growth of perinatal cranial base explants was inhibited by treatment with noggin, a BMP inhibitor. Exogenous BMP4 promoted cartilage growth, matrix deposition and chondrocyte proliferation in a dose dependent manner. Correspondingly, expression level of the cartilage markers Sox9 and collagen type II were also increased. Alkaline phosphatase and collagen type X expression were up-regulated and expressed in ectopic hypertrophic chondrocytes after treatment of the cultures with 100 ng/ml BMP4 for seven days. This increase in chondrocyte hypertrophy was accompanied by increased indian hedgehog (Ihh) and parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) receptor (PPR) expression, but not increased PTHrP expression. We conclude that endogenous BMPs are required to maintain cartilage growth, and exogenous BMP4 can enhance cartilage maturation and induce ectopic chondrocyte hypertrophy in the cranial base. Therefore, appropriate levels of BMP signaling are important for normal cranial base development.

Inverse agonism and the PTH/PTHrP receptor

The PTH/PTHrP receptor (PPR) is a family B G protein-coupled receptor that plays a vital role in calcium homeostasis and skeletal development. We are using photoaffinity cross-linking methods to explore how inverse agonists interact with constitutively active mutant PPRs. The analog [ para-benzoyl-phenylalanine (Bpa) 2]PTHrP(1–36) is a selective inverse agonist for PPR-H223R (altered in transmembrane domain (TMD) 2), while [ d-Bpa 12]PTHrP(5–36) is an inverse agonist for both PPR-H223R and PPR-T410P (altered in TMD 6). In each ligand, the photolabile Bpa group is the key determinant of inverse agonism. The [Bpa 2]PTHrP(1–36) analog cross-linked to two sites in PPR-H223R: a primary site near the extracellular end of TMD 6 and a secondary site within TMD 4, extracellular loop 2 or TMD 5. Mutational studies revealed that the intensity of cross-linking to TMD 6, relative to TMD 4/5, increased as receptor signaling activity increased. Our preliminary mapping studies with [ d-Bpa 12]PTHrP(5–36) indicate that this analog cross-links to a different site, possibly near the boundary of TMD 1 and the N-terminal domain. Thus, there appear to be two different mechanisms by which inverse agonism can be achieved in constitutively active PPRs. The studies also reveal conformational differences between active and inactive state PPRs, at least in the vicinity of residue 2 of the ligand.

Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes.

Chondrocyte proliferation and differentiation requires their attachment to the collagen type II–rich matrix of developing bone. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). To elucidate the molecular mechanisms whereby integrins control these processes, we have specifically inactivated the ILK gene in growth plate chondrocytes using the Cre-lox methodology. Mice carrying an ILK allele flanked by loxP sites (ILK-fl) were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type II promoter. Inactivation of both copies of the ILK-fl allele lead to a chondrodysplasia characterized by a disorganized growth plate and to dwarfism. Expression of chondrocyte differentiation markers such as collagen type II, collagen type X, Indian hedgehog and the PTH-PTHrP receptor was normal in ILK-deficient growth plates. In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates. Cell-based assays showed that integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.

PTHrP and Indian hedgehog control differentiation of growth plate chondrocytes at multiple steps.

In developing murine growth plates, chondrocytes near the articular surface (periarticular chondrocytes) proliferate, differentiate into flat column-forming proliferating cells (columnar chondrocytes), stop dividing and finally differentiate into hypertrophic cells. Indian hedgehog (Ihh), which is predominantly expressed in prehypertrophic cells, stimulates expression of parathyroid hormone (PTH)-related peptide (PTHrP) which negatively regulates terminal chondrocyte differentiation through the PTH/PTHrP receptor (PPR). However, the roles of PTHrP and Ihh in regulating earlier steps in chondrocyte differentiation are unclear. We present novel mouse models with PPR abnormalities that help clarify these roles. In mice with chondrocyte-specific PPR ablation and mice with reduced PPR expression, chondrocyte differentiation was accelerated not only at the terminal step but also at an earlier step: periarticular to columnar differentiation. In these models, upregulation of Ihh action in the periarticular region was also observed. In the third model in which the PPR was disrupted in about 30% of columnar chondrocytes, Ihh action in the periarticular chondrocytes was upregulated because of ectopically differentiated hypertrophic chondrocytes that had lost PPR. Acceleration of periarticular to columnar differentiation was also noted in this mouse, while most of periarticular chondrocytes retained PPR signaling. These data suggest that Ihh positively controls differentiation of periarticular chondrocytes independently of PTHrP. Thus, chondrocyte differentiation is controlled at multiple steps by PTHrP and Ihh through the mutual regulation of their activities.

A novel PTH/PTHrP receptor (PPRc) mutation: The ongoing tale of PPRc and the growth plate becomes more complex

A novel PTH/PTHrP receptor (PPRc) mutation: The ongoing tale of PPRc and the growth plate becomes more complex

PTHrP Novel Roles in Skeletal Biology.

Parathyroid hormone-related peptide (PTHrP) was discovered as the main mediator of humoral hypercalcemia associated with malignancy but is now known to be expressed by a variety of normal fetal and adult tissues. The amino-terminal region of PTHrP reveals limited but significant homology with parathyroid hormone (PTH), resulting in the interaction of either peptide with a common seven-transmembrane spanning G-protein linked receptor termed the PTH/PTHrP receptor. Targeted inactivation of PTHrP and its receptor in mice has established a critical role for this signaling pathway in chondrocyte biology and endochondral bone formation. Animals homozygous for the targeted alleles demonstrate marked skeletal deformities arising from impaired chondrocyte proliferation, premature differentiation and accelerated apoptosis. The complex processes resulting in normal endochondral bone development involve additional factors such as the hedgehog signaling pathway with which PTHrP interacts. PTHrP, like PTH, also binds to receptors on cells of the osteoblast lineage resulting in enhanced bone formation and also, indirectly, augmented osteoclastic bone resorption. The marked premature osteoporosis observed in mice heterozygous for the disrupted Pthrp allele also points to a crucial role for the protein in the maintenance of the adult skeleton. Further studies into this process are likely to reveal new facets of the pathogenesis underlying a variety of metabolic bone diseases and potentially point to new directions for therapeutic interventions.

Characterization of parathyroid hormone/parathyroid hormone-related protein receptor and signaling in hypercalcemic Walker 256 tumor cells.

Parathyroid hormone (PTH)-related protein (PTHrP) is the main factor responsible for humoral hypercalcemia of malignancy. Both PTH and PTHrP bind to the common type I PTH/PTHrP receptor (PTHR), thereby activating phospholipase C and adenylate cyclase through various G proteins, in bone and renal cells. However, various normal and transformed cell types, including hypercalcemic Walker 256 (W256) tumor cells, do not produce cAMP after PTHrP stimulation. We characterized the PTHrP receptor and the signaling mechanism upon its activation in the latter cells. Scatchard analysis of PTHrP-binding data in W256 tumor cells revealed the presence of high affinity binding sites with an apparent K(d) of 17 nM, and a density of 90 000 sites/cell. In addition, W256 tumor cells immunostained with an anti-PTHR antibody, recognizing its extracellular domain. Furthermore, reverse transcription followed by PCR, using primers amplifying two different regions in the PTHR cDNA corresponding to the N- and C-terminal domains, yielded products from W256 tumor cell RNA which were identical to the corresponding products obtained from rat kidney RNA. Consistent with our previous findings on cAMP production, 1 microM PTHrP(1-34), in contrast to 10 microg/ml cholera toxin or 1 microM isoproterenol, failed to affect protein kinase A activity in W256 tumor cells. However, in these cells we found a functional PTHR coupling to G(alpha)(q/11), whose presence was demonstrated in these tumor cell membranes by Western blot analysis. Our findings indicate that W256 tumor cells express the PTHR, which seems to be coupled to G(alpha)(q/11). Taken together with previous data, these results support the hypothesis that a switch from the cAMP pathway to the phospholipase C-intracellular calcium pathway, associated with PTHR activation, occurs in malignant cells.

Regulation of PTHrP and PTH/PTHrP receptor by extracellular Ca2+ concentration and hormones in the breast cancer cell line 8701-BC.

It was previously reported that 8701-BC breast tumour cells express the gene for parathyroid hormone-related peptide (PTHrP) and PTH/PTHrP receptor (PTHrP-R) and release immunoreactive PTHrP (iPTHrP) into the extracellular medium. Since the regulation of PTHrP and PTHrP-R by breast cancer cells has been poorly investigated so far, we have chosen the 8701-BC cell line as a model system to investigate whether alterations in the extracellular Ca2+ concentration ([Ca2+]e) and treatment with some well-known differentiation agents for breast cells, such as dimethyl sulfoxide, hydrocortisone, progesterone, prolactin, all-trans retinoic acid and transforming growth factor-beta1 might (i) modulate quantitatively the release of iPTHrP, (ii) affect the PTHrP promoter usage and mRNA splicing patterns, and (iii) modify the expression of PTHrP-R. The data obtained indicate that 8701-BC cells are potentially able to utilise different start sites and mRNA splicing patterns for PTHrP transcription, and respond to variations of [Ca2+]e and to the addition of two hormones, hydrocortisone and progesterone, with modifications in the extracellular amount of iPTHrP. Moreover, expression of PTHrP-R is also modulated by changes of [Ca2+]e or treatment with hydrocortisone. This indicates that the 8701 -BC cell line is a suitable in vitro model for further studies on the complex molecular regulation of the PTHrP/PTHrP-R pair in breast cancer.

Apoptosis Mediated by Activation of the G Protein-Coupled Receptor for Parathyroid Hormone (PTH)/ PTH-Related Protein (PTHrP)

The present studies were carried out to evaluate the mechanisms by which PTH/PTHrP receptor (PTHR) activation influences cell viability. In 293 cells expressing recombinant PTHRs, PTH treatment markedly reduced the number of viable cells. This effect was associated with a marked apoptotic response including DNA fragmentation and the appearance of apoptotic nuclei. Similar effects were evidenced in response to serum withdrawal or to the addition of tumor necrosis factor (TNFα). Addition of caspase inhibitors or overexpression of bcl-2 partially abrogated apoptosis induced by serum withdrawal. Caspase inhibitors also protected cells from PTH-induced apoptosis, but overexpression of bcl-2 did not. The effects of PTH on cell number and apoptosis were neither mimicked by activators of the cAMP pathway (forskolin, isoproterenol) nor blocked by an inhibitor (H-89). However, elevation of Cai2+ by addition of thapsigargin induced rapid apoptosis, and suppression of Cai2+ by overexpression of the calcium- binding protein, calbindin D28k, inhibited PTH-induced apoptosis. The protein kinase C inhibitor GF 109203X partially inhibited PTH-induced apoptosis. Regulator of G protein signaling 4 (RGS4) (an inhibitor of the activity of the α-subunit of Gq) suppressed apoptotic signaling by the PTHR, whereas the C-terminal fragment of GRK2 (an inhibitor of the activity of the βγ-subunits of G proteins) was without effect. Chemical mutagenesis allowed selection of a series of 293 cell lines resistant to the apoptotic actions of PTH; a subset of these were also resistant to TNFα. These results suggest that 1) apoptosis produced by PTHR and TNF receptor signaling involve converging pathways; and 2) Gq-mediated phospholipase C/Ca2+ signaling, rather than Gs-mediated cAMP signaling, is required for the apoptotic effects of PTHR activation.

Apoptosis Mediated by Activation of the G Protein-Coupled Receptor for Parathyroid Hormone (PTH)/ PTH-Related Protein (PTHrP)

The present studies were carried out to evaluate the mechanisms by which PTH/PTHrP receptor (PTHR) activation influences cell viability. In 293 cells expressing recombinant PTHRs, PTH treatment markedly reduced the number of viable cells. This effect was associated with a marked apoptotic response including DNA fragmentation and the appearance of apoptotic nuclei. Similar effects were evidenced in response to serum withdrawal or to the addition of tumor necrosis factor (TNFalpha). Addition of caspase inhibitors or overexpression of bcl-2 partially abrogated apoptosis induced by serum withdrawal. Caspase inhibitors also protected cells from PTH-induced apoptosis, but overexpression of bcl-2 did not. The effects of PTH on cell number and apoptosis were neither mimicked by activators of the cAMP pathway (forskolin, isoproterenol) nor blocked by an inhibitor (H-89). However, elevation of Ca(i)2+ by addition of thapsigargin induced rapid apoptosis, and suppression of Ca(i)2+ by overexpression of the calcium- binding protein, calbindin D28k, inhibited PTH-induced apoptosis. The protein kinase C inhibitor GF 109203X partially inhibited PTH-induced apoptosis. Regulator of G protein signaling 4 (RGS4) (an inhibitor of the activity of the alpha-subunit of Gq) suppressed apoptotic signaling by the PTHR, whereas the C-terminal fragment of GRK2 (an inhibitor of the activity of the beta(gamma)-subunits of G proteins) was without effect. Chemical mutagenesis allowed selection of a series of 293 cell lines resistant to the apoptotic actions of PTH; a subset of these were also resistant to TNFalpha. These results suggest that 1) apoptosis produced by PTHR and TNF receptor signaling involve converging pathways; and 2) Gq-mediated phospholipase C/Ca2+ signaling, rather than Gs-mediated cAMP signaling, is required for the apoptotic effects of PTHR activation.

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.

Pathophysiologic effects of uremic retention solutes.

The uremic syndrome can be defined as a deterioration of biochemical and physiologic functions, in parallel with the progression of renal failure, resulting in complex and variable symptomatology (1-3). The compounds that accumulate in the uremic blood and tissues during the development of end-stage renal disease (ESRD), directly or indirectly due to a deficient renal clearance, are called uremic retention solutes. These retention solutes may modify biochemical or physiologic func- tions; if they do so, they contribute to the uremic syndrome. Only a few solutes have an established role as uremic toxins. According to Bergstrom, apart from inorganic compounds, urea, oxalic acid, parathyroid hormone (PTH), and b2-micro- globulin conform to the most strict definition of uremic toxins (4). However, this does not preclude a potential toxic role for various other retention solutes (5). The following factors, which are not always considered, might affect uremic solute concentration and their impact on biologic functions. (1) In addition to classical sources of ure- mic solutes such as dietary protein breakdown, alternative sources such as environment, herbal medicines, or psychedelic drugs may play a role in uremic toxicity. (2) Many solutes with toxic capacity enter the body through the intestine. Changes in the composition of intestinal flora, or changes in intestinal production and absorption, might alter their serum concentra- tion. (3) Some uremic solutes interfere with functions that directly affect the biochemical action of other solutes: the expression of PTH receptors, the response to 1,25(OH)2 vita- min D3, as well as the protein binding and breakdown of several other solutes. (4) Most uremic patients are prescribed a host of drugs. Interference of drugs with protein binding and/or tubular secretion of uremic solutes will influence their biologic effect. (5) Lipophilic compounds may be responsible at least in part for functional alterations in uremia. ( 6) The impact of residual renal function on uremic solute retention should not be neglected. (7) The main strategy that has been used up to now to decrease uremic solute concentration is dialysis, but dialysis is nonspecific and removes essential compounds as well. (8) Uremic solutes accumulate not only in the plasma but also in the cells, where most of the biologic activity is exerted. Re- moval of intracellular compounds during dialysis through the cell membrane may be hampered, resulting in multicompart- mental kinetics and inadequate detoxification. It is of note that lower morbidity and mortality are observed in patients submit- ted to long dialysis sessions (6,7). Compounds may be cleared more efficiently with continuous or long-lasting low efficiency strategies, because removal is more gradual. Our views on the uremic syndrome and several uremic solutes have changed substantially during the last decade. Therefore, it was thought timely to summarize the present state of knowledge about the biochemical, physiologic, and/or clin- ical impact of those compounds that have been subjected to relatively thorough evaluation during these last 10 years. Spe- cific attention was also paid to generation and removal pat- terns.

Reduced Blood Pressure and Increased Sensitivity of the Vasculature to Parathyroid Hormone-Related Protein (PTHrP) in Transgenic Mice Overexpressing the PTH/PTHrP Receptor in Vascular Smooth Muscle

PTH-related protein (PTHrP) is produced in vascular smooth muscle, where it is postulated to exert vasorelaxant properties by activation of the PTH/PTHrP type 1 receptor. As a model for studying the actions of locally produced PTHrP in vascular smooth muscle in vivo, we developed transgenic mice that overexpress the PTH/PTHrP receptor (PTHrP-R) in smooth muscle. Oocyte injection with a SMP8-PTHrP-R fusion construct yielded six founder mice. F1 offspring were viable and demonstrated selective overexpression of the SMP8-PTHP-R messenger RNA in smooth muscle-rich tissues. Baseline blood pressure measured in conscious mice by tail sphygmomanometry was significantly lower in the receptor-overexpressing mice than that in controls (117 ± 4 vs. 133 ± 3 mm Hg; P < 0.05). In anesthetized animals, iv infusion of PTHrP-(1–34)NH2 caused a significantly greater reduction in blood pressure and total peripheral resistance in transgenic mice than in control animals. Vascular contractility was studied in paired, isometrically mounted aortas from 9-week-old transgenic and wild-type mice. The force of contraction in response to phenlyephrine was not significantly different between transgenic and wild-type mice. However, PTHrP-(1–34) NH2 relaxed aortic vessel preparations from transgenic mice to a greater extent than in controls (77.1 ± 3% vs. 38.4 ± 4%; P < 0.001). To determine the impact of overexpression of PTH/PTHrP type 1 receptor and its ligand on the development of the cardiovascular system, double transgenic mice were created by crossing SMP8-PTHrP-R transgenic mice with mice overexpressing PTHrP (SMP8-PTHrP). Double transgenic mice died around day E9 with abnormalities in the developing heart. In conclusion, overexpression of PTH/PTHrP type 1 receptor in vascular smooth muscle of transgenic mice reduces blood pressure, probably through sustained activation of the receptor by endogenous ligand. The cardiovascular defects observed in mice overexpressing both PTHrP and its receptor suggest that PTHrP may play a role in the normal development of the cardiovascular system.

Reduced blood pressure and increased sensitivity of the vasculature to parathyroid hormone-related protein (PTHrP) in transgenic mice overexpressing the PTH/PTHrP receptor in vascular smooth muscle.

PTH-related protein (PTHrP) is produced in vascular smooth muscle, where it is postulated to exert vasorelaxant properties by activation of the PTH/PTHrP type 1 receptor. As a model for studying the actions of locally produced PTHrP in vascular smooth muscle in vivo, we developed transgenic mice that overexpress the PTH/PTHrP receptor (PTHrP-R) in smooth muscle. Oocyte injection with a SMP8-PTHrP-R fusion construct yielded six founder mice. F1 offspring were viable and demonstrated selective overexpression of the SMP8-PTHP-R messenger RNA in smooth muscle-rich tissues. Baseline blood pressure measured in conscious mice by tail sphygmomanometry was significantly lower in the receptor-overexpressing mice than that in controls (117 +/- 4 vs. 133 +/- 3 mm Hg; P < 0.05). In anesthetized animals, iv infusion of PTHrP-(1-34)NH2 caused a significantly greater reduction in blood pressure and total peripheral resistance in transgenic mice than in control animals. Vascular contractility was studied in paired, isometrically mounted aortas from 9-week-old transgenic and wild-type mice. The force of contraction in response to phenlyephrine was not significantly different between transgenic and wild-type mice. However, PTHrP-(1-34) NH2 relaxed aortic vessel preparations from transgenic mice to a greater extent than in controls (77.1 +/- 3% vs. 38.4 +/- 4%; P < 0.001). To determine the impact of overexpression of PTH/PTHrP type 1 receptor and its ligand on the development of the cardiovascular system, double transgenic mice were created by crossing SMP8-PTHrP-R transgenic mice with mice overexpressing PTHrP (SMP8-PTHrP). Double transgenic mice died around day E9 with abnormalities in the developing heart. In conclusion, overexpression of PTH/PTHrP type 1 receptor in vascular smooth muscle of transgenic mice reduces blood pressure, probably through sustained activation of the receptor by endogenous ligand. The cardiovascular defects observed in mice overexpressing both PTHrP and its receptor suggest that PTHrP may play a role in the normal development of the cardiovascular system.

PTH-PTHrP receptor in chronic renal failure.

PTH-PTHrP receptor in chronic renal failure.

Mutations activantes du récepteur de la parathormone (PTH) et du PTH related peptide

Parathyroid hormone (PTH) and PTH related peptide (PTHrP) have a common main receptor: type I PTH-PTHrP receptor. PTH expresses its main metabolic actions, and PTHrP part of its autocrine or paracrine actions through this receptor. Jansen chondrodysplasia is a very rare disease mainly characterized by severe metaphyseal growth disorders leading to dwarfism and hypercalcemia. The group of Jüppner from the Massachusetts General Hospital in Boston recently demonstrated mutations on the gene of the type I PTH-PTHrP receptor in five patients with Jansen chondrodysplasia. These mutations result in permanent activation of the receptor responsible for the observed hypercalcemia and bone growth abnormalities due to the disease. Therefore Jansen chondrodysplasia appears as a remarkable clinical model outlining the major role of PTHrP in bone growth regulation.

Elevation of [Ca2+]i of renal proximal tubular cells and down-regulation of mRNA of PTH-PTHrP, Via and AT1 receptors in kidney of diabetic rats

An elevation in intracellular calcium ([Ca2+]i) in rats with chronic renal failure and elevated blood levels of PTH is associated with down-regulation of the mRNA of many proteins. Similarly, in phosphate depleted animals that have normal renal function and low blood levels of PTH, [Ca2+]i is elevated and the mRNA of PTH-PTHrP receptor is down-regulated. The effect of elevation in [Ca2+]i on molecular machinery of many proteins may represent a generalized phenomenon. Diabetes mellitus may also be associated with a rise in [Ca2+]i and therefore down-regulation of the mRNA of proteins may also occur. The present study examined the effect of streptozotocin-induced diabetes mellitus in rats on the [Ca2+]i of the renal proximal tubular cells and on their mRNAs of the PTH-PTHrP, V1a and AT1 receptors. The basal levels of [Ca2+]i of these cells increased significantly (P < 0.01) after one day of diabetes and remained elevated thereafter. There was a significant (r = 0.67, P < 0.01) direct correlation between the [Ca2+]i of the cells and blood levels of glucose up to 350 mg/dl, and the value of [Ca2+]i plateaued with higher concentrations of glucose. Three days of amlodipine therapy prevented and reversed the elevated levels of [Ca2+]i despite marked hyperglycemia. The mRNA of all three receptors in the kidney were down-regulated and this defect was prevented by amlodipine which normalized the [Ca2+]i of the cells. The results show that: (1) the hyperglycemia of IDDM in rats causes a significant elevation in the basal levels of [Ca2+]i of the renal proximal tubular cells and down-regulation of their mRNA of PTH-PTHrP, V1a and AT1 receptors; (2) normalization of the [Ca2+]i of these cells by treatment of the diabetic rats with amlodipine prevented the elevation of [Ca2+]i and the down-regulation of the mRNA of these receptors; (3) these effects occurred in the presence of normal renal function and normal blood of PTH and phosphorus.