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PTH upregulates the expression of the receptor activator of nuclear factor κB ligand (Rankl) in cells of the osteoblastic lineage, but the precise differentiation stage of the PTH target cell responsible for RANKL-mediated stimulation of bone resorption remains undefined. We report that constitutive activation of PTH receptor signaling only in osteocytes in transgenic mice (DMP1-caPTHR1) was sufficient to increase Rankl expression and bone resorption. Resorption in DMP1-caPTHR1 mice crossed with mice lacking the distal control region regulated by PTH in the Rankl gene (DCR(-/-)) was similar to DMP1-caPTHR1 mice at 1 month of age, but progressively declined to reach values undistinguishable from wild-type (WT) mice at 5 months of age. Moreover, DMP1-caPTHR1 mice exhibited low tissue material density and increased serum alkaline phosphatase activity at 5 month of age, and these indices of high remodeling were partially and totally corrected in compound DMP1-caPTHR1;DCR(-/-) male mice, and less affected in female mice. Rankl expression in bones from DMP1-caPTHR1 mice was elevated at both 1 and 5 months of age, whereas it was high, similar to DMP1-caPTHR1 mice at 1 month, but low, similar to WT levels at 5 months in compound mice. Moreover, PTH increased Rankl and decreased Sost and Opg expression in ex vivo bone organ cultures established from WT mice, but only regulated Sost and Opg expression in cultures from DCR(-/-) mice. PTH also increased RANKL expression in osteocyte-containing primary cultures of calvarial cells, in isolated murine osteocytes, and in WT but not in DCR(-/-) osteocyte-enriched bones. Thus, PTH upregulates Rankl expression in osteocytes in vitro, ex vivo and in vivo, and resorption induced by PTH receptor signaling in the adult skeleton requires direct regulation of the Rankl gene in osteocytes.

The physiology of calcium and the other minerals involved in its metabolism is complex and intimately tied in with the physiology of bone. Five principal humoral factors are involved in maintaining plasma levels of calcium, magnesium and phosphate and coordinating the balance between these and their content in bone. The transmembrane transport of these elements is dependent on a series of complex mechanisms that are controlled by these hormones. The plasma concentration of calcium is initially sensed by a calcium-sensing receptor which then sets up a cascade of events that initially determines parathyroid hormone secretion and eventually results in a specific action within the target organs, mainly bone and kidney. This chapter describes the physiology of these humoral factors and relates them to the pathological processes that give rise to disorders of calcium and bone metabolism. It details the stages in the calcium cascade and describes the effects on the various target organs. The pathology of disorders of bone and calcium metabolism is described in detail in the relevant chapters., (Copyright (c) 2009 S. Karger AG, Basel.)

Osteocytes, former osteoblasts buried within bone, are thought to orchestrate skeletal adaptation to mechanical stimuli. However, it remains unknown whether hormones control skeletal homeostasis through actions on osteocytes. Parathyroid hormone (PTH) stimulates bone remodeling and may cause bone loss or bone gain depending on the balance between bone resorption and formation. Herein, we demonstrate that transgenic mice expressing a constitutively active PTH receptor exclusively in osteocytes exhibit increased bone mass and bone remodeling, as well as reduced expression of the osteocyte-derived Wnt antagonist sclerostin, increased Wnt signaling, increased osteoclast and osteoblast number, and decreased osteoblast apoptosis. Deletion of the Wnt co-receptor LDL related receptor 5 (LRP5) attenuates the high bone mass phenotype but not the increase in bone remodeling induced by the transgene. These findings demonstrate that PTH receptor signaling in osteocytes increases bone mass and the rate of bone remodeling through LRP5-dependent and -independent mechanisms, respectively.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two factors that share amino acid sequence homology and act via a common receptor. In tetrapods, PTH is the main endocrine factor acting in bone and kidney to regulate calcium and phosphate. PTHrP is an essential paracrine developmental factor present in many tissues and is involved in the regulation of ossification, mammary gland development, muscle relaxation, and other functions. Fish apparently lack an equivalent of the parathyroid gland and were long thought to be devoid of PTH. Only in recent years has the existence of PTH-like peptides and their receptors in fish been firmly established. Two forms of PTH, two of PTHrP, and a protein with intermediate characteristics designated PTH-L are encoded by separate genes in teleost fish. Three receptors encoded by separate genes in fish mediate PTH/PTHrP actions, whereas only two receptors have so far been found in terrestrial vertebrates. PTHrP has been more intensively studied than PTH, from lampreys to advanced teleosts. It is expressed in many tissues and is present in high concentration in fish blood. Administration of this peptide alters calcium metabolism and has marked effects on associated gene expression and enzyme activity in vivo and in vitro. This review provides a comprehensive overview of the physiological roles, distribution, and molecular relationships of the piscine PTH-like peptides.

Parathyroid hormone (PTH), an 84-amino acid polypeptide, is a major systemic regulator of calcium homeostasis that activates PTH/PTHrP receptors (PTH1Rs) on target cells. Carboxyl fragments of PTH (CPTH), secreted by the parathyroids or generated by PTH proteolysis in the liver, circulate in blood at concentrations much higher than intact PTH-(1-84) but cannot activate PTH1Rs. Receptors specific for CPTH fragments (CPTHRs), distinct from PTH1Rs, are expressed by bone cells, especially osteocytes. Activation of CPTHRs was previously reported to modify intracellular calcium within chondrocytes. To further investigate the mechanism of action of CPTHRs in osteocytes, cytosolic free calcium concentration ([Ca(2+)](i)) was measured in the PTH1R-null osteocytic cell line OC59, which expresses abundant CPTHRs but no PTH1Rs. [Ca(2+)](i) was assessed by single-cell ratiometric microfluorimetry in fura-2-loaded OC59 cells. A rapid and transient increase in [Ca(2+)](i) was observed in OC59 cells in response to the CPTH fragment hPTH-(53-84) (250 nM). No [Ca(2+)](i) signal was observed in COS-7 cells, in which CPTHR binding also cannot be detected. Neither hPTH-(1-34) nor a mutant CPTH analog, [Ala(55-57)]hPTH-(53-84), that does not to bind to CPTHRs, increased [Ca(2+)](i) in OC59 cells. The [Ca(2+)](i) response to hPTH-(53-84) required the presence of extracellular calcium and was blocked by inhibitors of voltage-dependent calcium channels (VDCCs), including nifedipine (100 nM), omega-agatoxin IVA (10 nM), and omega-conotoxin GVIA (100 nM). We conclude that activation of CPTHRs in OC59 osteocytic cells leads to a rapid increase in influx of extracellular calcium, most likely through the opening of VDCCs.

Hematopoietic stem cells (HSC) are rare primitive cells capable of reconstituting all blood cell lineages throughout the life of an individual. The microenvironment in which stem cells reside is essential for their survival, self-renewal, and differentiation. This microenvironment, or HSC niche, has been difficult to define in bone and bone marrow, but recent studies from our laboratory and others have shown that osteoblasts, the bone-forming cells, are an essential regulatory component of this complex cellular network. We established that parathyroid hormone (PTH), through activation of the PTH/PTHrP receptor (PTH1R) in osteoblastic cells, could alter the HSC niche resulting in HSC expansion in vivo and in vitro and improving dramatically the survival of mice receiving bone marrow transplants. These findings are of great clinical appeal, because they suggest that a strategy aimed at modifying supportive cells in a stem cell niche can expand HSC. While a number of molecules have been found to be important for hematopoietic/osteoblastic interactions, we have focused on the Jagged1/Notch signaling pathway, which was necessary for the PTH-dependent HSC expansion. Since the Jagged1/Notch signaling pathway has been implicated in the microenvironmental control of stem cell self-renewal in several organ systems, definition of Jagged1 modulation, which is currently poorly understood, should provide additional molecular targets for stem cell regulation and advance the understanding of stem cell-microenvironmental interactions.

Since parathyroid hormone (PTH) is the only proven anabolic therapy for bone, it becomes the benchmark by which new treatments will be evaluated. The anabolic effect of PTH is dependent upon intermittent administration, but when an elevated PTH level is maintained even for a few hours it initiates processes leading to new osteoclast formation, and the consequent resorption overrides the effects of activating genes that direct bone formation. Identification of PTH-related protein (PTHrP) production by cells early in the osteoblast lineage, and its action through the PTH1R upon more mature osteoblastic cells, together with the observation that PTHrP+/- mice are osteoporotic, all raise the possibility that PTHrP is a crucial paracrine regulator of bone formation. The finding that concurrent treatment with bisphosphonates impairs the anabolic response to PTH, adds to other clues that osteoclast activity is necessary to complement the direct effect that PTH has in promoting differentiation of committed osteoblast precursors. This might involve the generation of a coupling factor from osteoclasts that are transiently activated by receptor activator of nuclear factor-kappaB ligand (RANKL) in response to PTH. New approaches to anabolic therapies may come from the discovery that an activating mutation in the LRP5 gene is responsible for an inherited high bone mass syndrome, and the fact that this can be recapitulated in transgenic mice, whereas inactivating mutations result in severe bone loss. This has focused attention on the Wnt/frizzled/beta-catenin pathway as being important in bone formation, and proof of the concept has been obtained in experimental models.

Elucidating the bimolecular interface between parathyroid hormone (PTH) and its cognate G protein-coupled receptor (PTHR1) should yield insights into the basis of molecular recognition and the mechanism of ligand-mediated intracellular signaling for a system that is critically important in regulating calcium levels in blood. We used photoaffinity scanning (PAS) to identify key ligand-receptor interactions for residues from the unstructured mid-region domain of PTH-(1-34). Four PTH analogues, containing a single photoreactive p-benzoylphenylalanine (Bpa) residue in position 11, 15, 18, or 21, were found to photo-cross-link within receptor regions [165-176], [183-189], [190-298], and [165-176], respectively. Addition of these mid-region contacts as constraints to our previously proposed model of the PTH-PTHR1 complex and extensive molecular simulation experiments enables substantial refinement of the model. Specifically, (1) the overall receptor-bound conformation of the hormone is not extended, but bent; (2) helix [169-176] of the N-terminal extracellular domain (N-ECD) of the receptor is redirected toward the heptahelical bundle; and (3) the hormone traverses between the top of transmembrane (TM) helices 1 and 2, rather than between TM-7 and TM-1. This significantly alters the model of both the receptor-bound tertiary structure of the hormone and the topological orientation of the C-terminus of the N-ECD in the hormone-receptor bimolecular complex. We propose that the mid-region of PTH-(1-34) has a role in fixing, by extensive contacts with the receptor, the entry of the N-terminal helix of the hormone into the heptahelical bundle between TM-1 and TM-2. This anchorage would orient the amino terminus into position to activate the receptor.

Research on parathyroid hormone (PTH) has undergone four rather distinctive phases, beginning just before the turn of the 20th century. Early debates about the function of the parathyroids were resolved by 1925, when understanding the role of PTH led to comprehending the action of the glands in calcium physiology. Elucidation of the pathophysiology of hormone excess (severe bone loss) and deficiency (hypocalcemia) continued over the following decades. With the advent of advances in chemical and molecular biology, the structure of PTH and its principal receptor (PTHrP-receptor [PTHR1]) were established. Tests with purified hormonal peptide in humans led to the surprising, even paradoxical, finding that PTH can be used pharmacologically to build bone, providing a dramatic therapeutic impact on osteoporosis. These developments have stimulated the field of calcium and bone biology and posed new questions about the role of PTH as well as possible new directions in therapy.

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.

PTH is a major systemic regulator of the concentrations of calcium, phosphate, and active vitamin D metabolites in blood and of cellular activity in bone. Intermittently administered PTH and amino-terminal PTH peptide fragments or analogs also augment bone mass and currently are being introduced into clinical practice as therapies for osteoporosis. The amino-terminal region of PTH is known to be both necessary and sufficient for full activity at PTH/PTHrP receptors (PTH1Rs), which mediate the classical biological actions of the hormone. It is well known that multiple carboxyl-terminal fragments of PTH are present in blood, where they comprise the major form(s) of circulating hormone, but these fragments have long been regarded as inert by-products of PTH metabolism because they neither bind to nor activate PTH1Rs. New in vitro and in vivo evidence, together with older observations extending over the past 20 yr, now points strongly to the existence of novel large carboxyl-terminal PTH fragments in blood and to receptors for these fragments that appear to mediate unique biological actions in bone. This review traces the development of this field in the context of the evolution of our understanding of the "classical" receptor for amino-terminal PTH and the now convincing evidence for these receptors for carboxyl-terminal PTH. The review summarizes current knowledge of the structure, secretion, and metabolism of PTH and its circulating fragments, details available information concerning the pharmacology and actions of carboxyl-terminal PTH receptors, and frames their likely biological and clinical significance. It seems likely that physiological parathyroid regulation of calcium and bone metabolism may involve receptors for circulating carboxy-terminal PTH ligands as well as the action of amino-terminal determinants within the PTH molecule on the classical PTH1R.

There is considerable evidence implicating the cAMP-signaling pathway in the anabolic action of PTH; and to date, all PTH and PTHrp peptides that stimulate cyclic AMP are active in animal models of osteogenesis. We have tested two C-terminally truncated peptides, PTH(1-29) and a modified PTH(1-21) (MPTH(1-21)), in in vitro and in vivo assays of PTH action. Each of the C-terminally truncated peptides was of low nanomolar potency in assays of receptor binding and cAMP stimulation. However, when we tested these peptides for functional response in Saos-2 cells stably transfected with a cyclic AMP response element (CRE) reporter, the C-terminally truncated peptides were two to four times less potent than would be expected from their binding and cAMP-stimulating properties. Furthermore, PTH(1-29), although active, was approximately 20-fold less potent than PTH(1-34) in a rat model of osteogenesis while MPTH(1-21) was inactive. The relative lack of activity of these peptides in vivo suggests that while activation of the cAMP pathway may be important for the anabolic effect of PTH fragments, it is not, of itself, predictive of their in vivo activity.

Recent investigations of parathyroid hormone (PTH) have advanced our understanding of its circulating forms as well as its action. It is now clear that first-generation immunoradiometric assays of so-called intact "PTH" not only measured full-length PTH(1-84) but also recognized large PTH fragments lacking the amino-terminus. New, second generation assays detect only full-length PTH. Under diverse pathological settings, second generation assays display lower levels of PTH (1-84). By measuring full-length PTH (bioactive PTH) and the combined full-length plus amino-terminal PTH fragments, the amount of non-PTH(1-84) in circulation can be estimated. The primary amino-terminal fragment is likely to be PTH(7-84). A considerable controversy surrounds the pathological significance of PTH(7-84) and its relation to adynamic bone disease. While these findings were emerging, other work uncovered the apparent basis by which PTH receptors signal through cAMP in some instances but through Ca/inositol phosphate in others. This signaling switch is dictated by the cytoplasmic adapter protein NHERF1 (EBP50), which is expressed in a cell-selective fashion. Other provocative findings may provide a means of unifying determinations of PTH(7-84) with the effects of NHERF1 on PTH receptor signaling. These latter studies reveal that in cells expressing NHERF1, PTH(7-84) has no effect on PTH receptor signaling or internalization. However, in cells lacking or expressing low levels of NHERF1, PTH(7-84) internalizes the PTH receptor without accompanying activation. Together, these findings suggest that the accumulation of PTH(7-84) in renal failure may lead to PTH resistance by internalizing and down-regulating PTH receptors.

Parathyroid hormone-related protein (PTHrP) is expressed in normal and malignant lung and has roles in development, homeostasis, and pathophysiology of injury and cancer. Its effects in developing lung include regulation of branching morphogenesis and type II cell maturation. In adult lung, PTHrP stimulates disaturated phosphatidylcholine secretion, inhibits type II cell growth, and sensitizes them to apoptosis. In lung cancer, PTHrP may play a role in carcinoma progression, or metastasis. The protein could be a useful marker for assessing lung maturity or type II cell function, predicting risk of injury, and detecting lung cancer. PTHrP-based therapies could also prove useful in lung injury and lung cancer.

G protein-coupled receptors (GPCRs) mediate the action of many hormones, cytokines, and sensory and chemical signals. It is generally thought that receptor desensitization and internalization require occupancy and activation of the GPCR. PTH and PTHrP receptor (PTH1R) belongs to GPCR class B and is the major regulator of extracellular calcium homeostasis. Using kidney distal convoluted tubule cells transfected with a human PTH1R/enhanced green fluorescent protein fusion protein, quantitative, real-time fluorescence microscopy was used to analyze receptor internalization. In these cells, which are the target of the calcium-sparing action of PTH, PTH(1-34) activated adenylyl cyclase (AC) and phospholipase C (PLC) and PTH1R endocytosis. PTH(1-31), however, stimulated AC and PLC but not PTH1R endocytosis. Conversely, PTH(7-34) rapidly stimulated PTH1R internalization without activating AC or PLC. PTH(2-34) and (3-34) caused PTH1R internalization intermediate between PTH(1-34) and (7-34). PTH1R sequestration occurred in a dynamin- and clathrin-dependent manner. Directly activating AC inhibited PTH1R internalization in response to PTH(7-34). PTH1R endocytosis was sensitive to protein kinase C inhibition. PTH(1-34), (7-34), and (1-31) evoked PTH1R phosphorylation. Removal of most of the C terminus of the PTH1R eliminated receptor phosphorylation and the cAMP/protein kinase C sensitivity of internalization. PTH(1-34) and (7-34) internalized the truncated PTH1R with identical kinetics, and the response was unaffected by forskolin. Thus, the PTH1R C terminus contains regulatory sequences that are involved in, but not required for, PTH1R internalization. The results demonstrate that receptor activation and internalization can be selectively dissociated.

The parathyroid hormone-related peptide (PTHrP) was initially identified in the early eighties, as the humoral mediator causing hypercalcaemia associated with malignancy. However, recently PTHrP was also shown to mediate a wide range of local paracrine/autocrine and intracrine functions in various tissues under physiological and pathological conditions. Indeed, PTHrP is a polyhormone, which can act through different receptors, including the type 1 parathyroid hormone (PTH) receptor 1 (PTH-1R). In the cardiovascular system, PTHrP appears to have potent effects on vascular smooth muscle cells and cardiomyocytes, where it participates in different pathological conditions, such as ischemia and heart failure. Therefore, it is conceivable that further studies on the regulation of PTHrP expression, characterization of its autocrine/paracrine/intracrine functions and definition of its intracellular signal transduction pathways in cardiomyocytes and cardiac vascular smooth muscle cells can elucidate the potential role of PTHrP in cardiovascular pathophysiology.

Parathyroid hormone inhibits sodium-phosphate cotransport in proximal renal tubule cells through activation of several kinases. We tested the hypothesis that the activity of these kinases was coordinated by an A kinase anchoring protein (AKAP) by demonstrating that the type II sodium-phosphate cotransporter (NaPi-4) physically associated with an AKAP and that this association was necessary for regulation of phosphate transport by parathyroid hormone. Immunoprecipitation with anti-NaPi-4 antiserum and glutathione S-transferase pull-down with GST-NaPi-4 showed that NaPi-4 associated with AKAP79, protein kinase A catalytic and regulatory subunits, and the parathyroid hormone receptor in opossum kidney cells. When the regulatory subunit of protein kinase A was uncoupled from the AKAP by a competing peptide, parathyroid hormone lost the ability to inhibit phosphate transport. This result was confirmed by co-transfecting HEK293 cells with the sodium-phosphate cotransporter and wild type AKAP, a mutant AKAP79, or the empty vector. 8-Bromo-cAMP was able to inhibit phosphate transport in cells expressing the wild type AKAP79 but not empty vector or mutant AKAP79. We conclude that parathyroid hormone inhibits proximal renal tubule sodium-phosphate cotransport through a signaling complex dependent upon an AKAP.

The N-terminal domain of PTH(1-34) is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated the possibility that the side chains of residues 6 (Gln) and 10 (Gln or Asn) of PTH analogues, which would align on the same face of the predicted alpha-helix, could interact and thereby contribute to the PTH/P1R interaction process. We utilized PTH(1-11), PTH(1-14), and PTH(1-34) analogues substituted with alanine at one or both of these positions and functionally evaluated the peptides in cell lines (HKRK-B7 and HKRK-B28) stably expressing the P1R, as well as in COS-7 cells transiently expressing either the P1R or a P1R construct that lacks the amino-terminal extracellular domain (P1R-DelNt). In HKRK-B7 cells, the single substitutions of Gln(6) --> Ala and Gln(10) --> Ala reduced the cAMP-stimulating potency of [Ala(3),Gln(10),Arg(11)]rPTH(1-11)NH(2) approximately 60- and approximately 2-fold, respectively, whereas the combined Ala(6,10) substitution resulted in a approximately 2-fold gain in potency, relative to the single Ala(6) substitution. Similar effects on P1R-mediated cAMP-signaling potency and P1R-binding affinity were observed for these substitutions in [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]rPTH(1-14)NH(2). Installation of a lactam bridge between the Lys(6) and the Glu(10) side chains of [Ala(3,12),Lys(6),Glu(10),Har(11),Trp(14)]rPTH(1-14)NH(2) increased signaling potency 6-fold, relative to the nonbridged linear analogue. Alanine substitutions at positions 6 and/or 10 of [Tyr(34)]hPTH(1-34)NH(2) did not affect signaling potency nor binding affinity on the intact P1R; however, Ala(6) abolished PTH(1-34) signaling on P1R-DelNt, and this effect was reversed by Ala(10). The overall data support the hypothesis that the N-terminal portion of PTH is alpha-helical when bound to the activation domain of the PTH-1 receptor and they further suggest that intrahelical side chain interactions between residues 6 and 10 of the ligand can contribute to the receptor interaction process.

PTHrP is a key developmental regulatory protein and a potent vasoactive agent. Previous studies have shown that mice lacking either the Pthrp or the PTH type 1 receptor (Pth1r) gene exhibit severe chondrodysplasia. In addition, in most genetic backgrounds, the receptor null mice die prenatally at midgestation, but the cause of death remains elusive. Here we show the loss of the Pth1r gene in C57BL6 mice leads to massive, abrupt cardiomyocyte death and embryonic lethality between embryonic days (E) E11.5 and E12.5. PTH1R mRNA was abundantly expressed in the developing wild-type mouse heart and cardiomyocytes from E11.5 embryos demonstrated acute increases in cAMP and increased Ca(2+)oscillations in response to PTHrP-(1-34)NH(2). Analyses of more than 300 embryos (E8-E14.5) from C57BL6/PTH1R +/- matings showed that PTH1R-/- mice survived until E11 with no obvious defects in any tissue. By E12, only 10% of the PTH1R-/- embryos survived and all PTH1R null mice were dead by E13. Ultrastructural and histological analysis revealed striking mitochondrial abnormalities at E11.5 and precipitous cardiomyocyte death between E12.0 and E12.5, followed by degenerative changes in the liver and massive necrosis of other tissues. No abnormalities were observed in the yolk sac or placenta implicating the heart degeneration as the primary cause of death. Taken together, these findings indicate that the PTH1R is required for the development of normal cardiomyocyte function.

The neuropeptide TIP39 was recently purified from bovine hypothalamus based on the ability of the peptide to activate the parathyroid hormone 2 receptor (PTH2R) ( Nat. Neurosci. 2 (1999) 941). PTH2R is abundantly expressed in the nervous system, and its expression pattern suggests that it may play a role in modulation of pituitary function and in nociception. Towards understanding the physiological role of TIP39 and PTH2R, we cloned human, mouse and rat TIP39 gene. Our results revealed that: (1) the mature peptide is processed from a precursor; (2) TIP39 peptide is highly conserved among species; and (3) TIP39 from all species activates adenylyl cyclase and elevates intracellular calcium levels through PTH2R. We also defined and compared the structure-activity relationship of TIP39 on both activation of adenylyl cyclase and calcium mobilization pathways through PTH2R, finding common and differential determinants of TIP39 that are required for these pathways. Furthermore, we observed that TIP39 elevates intracellular calcium levels in primary dorsal root ganglion neurons whereas the peptide inactive on PTH2R do not, suggesting that TIP39 may activate these neurons important for nociception in vivo through PTH2R-dependent mechanisms.

As the chronic kidney disease patient is being managed for PTH, calcium, phosphate, vitamin D, calcium x phosphate product and bone quality an accurate PTH measurement is essential. Over and under PTH suppressive therapies pose significant risks of mineral metabolism disturbances, osteodystrophies and soft tissue calcifications. Until recently it was thought that there was only one hormone secreted by the parathyroid gland, 1-84 PTH (or CAP). It is now known that there is another hormone secreted by the parathyroid gland (CIP) which is most likely 7-84 PTH. 7-84 PTH has been demonstrated to be an antagonist of 1-84 PTH with inverse biological activities. 7-84 PTH has been demonstrated to be hypocalcemic and able to lower bone turnover through an inhibition of osteoclast formation resulting in an overall inhibition of bone resorption. Whereas, 1-84 PTH operates through the PTH/PTHrp receptor the 7-84 PTH appears to operate through a C terminal PTH receptor. The CAP/CIP ratio decreases in the dialysis patient when calcium increases and vice versa. The 2nd generation "intact" PTH assays measure the sum of CAP plus CIP which render them ineffective at predicting bone turnover (72% predictive) and monitoring PTH suppressive treatments. By contrast the CAP/CIP ratio predicts bone turnover in the dialysis patient with a histologically determined 93% predictability. An elevated CAP/CIP ratio indicates high bone turnover and a decreased CAP/CIP ratio indicates adynamic low bone turnover.

Since its discovery in 1923, the parathyroid hormone (PTH), was thought to be the sole hormone capable of stimulating bone resorption, renal tubular calcium reabsorption, calcitriol synthesis, and urinary excretion of phosphate. However, in 1987, the PTHrP (PTH-related peptide), was demonstrated to share most of the biological actions of PTH through the activation of the same receptor. This receptor was cloned in 1992 and named PTH/PTHrP receptor or PTH-R1. Both, PTH and PTHrP bind with great affinity to PTH-R1 and stimulate a signal transduction system involving different G-proteins, phospholipase C, and adenylate cyclase. A third member of the PTH family, the TIP-39 (tuberoinfundibular peptide), binds and activates another PTH receptor (PTH-R2). There is evidence for other PTH receptors, a PTH-R3, probably specific for PTHrP in keratinocytes, kidney, placenta and a PTH-R4 specific for C-terminal PTH fragments. Activating mutations in the PTH-R1 gene cause Jansen type metaphyseal chondrodysplasia, whereas inactivating mutations are responsible for Blomstrand type rare chondrodysplasia and enchondromatosis. The renal and bone PTH-R1 expression is upregulated in vitamin D deficient rats and by endotoxin, interleukin-2, dexamethasone, T3, and TGF beta. On the contrary, PTH, PTHrP, angiotensin-II, IGF-1, PGE2, vitamin D, and chronic renal failure decrease its expression. In conclusions, the biological implications of the identification and cloning of different PTH receptors are at their beginning. The almost ubiquitous distribution of PTHrP and PTH-R1, the numerous PTHrP and PTH fragments, let us suppose the existence of other PTH-related receptors, and a great complexity of the bone and mineral metabolism.

We examined here the role of second messenger-dependent kinases and beta-arrestins in short-term regulation of the PTH receptor (PTHR) signaling. The inhibition of protein kinase C (PKC) in COS-7 cells transiently expressing PTHR, led to an approximately 2-fold increase in PTH-stimulated inositol phosphate (IP) and cAMP production. The inhibition of protein kinase A increased cAMP production 1.5-fold without affecting IP signaling. The effects of PKC inhibition on PTHR-mediated G(q) signaling were strongly decreased for a carboxy-terminally truncated PTHR (T480) that is phosphorylation deficient. PKC inhibition was associated with a decrease in agonist-stimulated PTHR phosphorylation and internalization without blocking PTH-dependent mobilization of beta-arrestin2 to the plasma membrane. Overexpression of beta-arrestins strongly decreased the PTHR-mediated IP signal, whereas cAMP production was impaired to a much lower extent. The regulation of PTH-stimulated signals by beta-arrestins was impaired for the truncated T480 receptor. Our data reveal mechanisms at, and distal to, the receptor regulating PTHR-mediated signaling pathways by second messenger-dependent kinases. We conclude that regulation of PTHR-mediated signaling by PKC and beta-arrestins are separable phenomena that both involve the carboxy terminus of the receptor. A major role for PKC and beta-arrestins in preferential regulation of PTHR-mediated G(q) signaling by independent mechanisms at the receptor level was established.

Although lower than in brain, the type 2 PTH receptor (PTH2-R) has been shown to be expressed throughout the cardiovascular system. Tuberoinfundibular peptide (TIP) purified from brain is thought to be the endogenous selective ligand of the PTH2-R. In the present studies, TIP and PTH2-R mRNA expressions were evidenced by RT-PCR in rat intrarenal arteries as well as in renovascular smooth muscle cells cultured from these arteries. In the isolated perfused rat kidney (IPK), peptides known to bind to both PTH1- and PTH2-Rs, such as rat PTH (1-34) and the hybrid PTH/PTHrP peptide, [Ile(5), Trp(23)]PTHrP (1-36), failed to exhibit improved vasodilatory effect, compared with human PTHrP (1-36), which binds only to the PTH1-R. Thus, a non-PTH1-R seemed not to be involved in the vasodilatory effects of these peptides. On the other hand, TIP exhibited complex vasoactivity, constricting the IPK at 10 nM and dilating the IPK at 1, 100, and 1000 nM. Moreover, [p-benzoyl-L-Phe(4),Ile(5),Trp(23)]PTHrP (1-36), initially described as a selective PTH2-R antagonist, also displayed a strong vasodilatory effect and therefore could not be used to check that TIP-induced vasoactivity was mediated by the PTH2-R. However, both [p-benzoyl-L-Phe(4),Ile(5),Trp(23)]PTHrP (1-36) and TIP displayed similar or even enhanced vasodilation in IPK in which PTH1-R-induced vasodilation was fully desensitized by sustained exposure to human PTHrP (1-36). Importantly, in IPK desensitized to the vasodilatory action of PTHrP (1-36), the hybrid PTH/PTHrP peptide and rat PTH (1-34), whose vasodilatory responses appeared exclusively PTH1-R dependent in naive IPK, produced a new and strong vasodilation. In conclusion, TIP and PTH2-R mRNAs are expressed in renal vessels and TIP appears as a new vasoactive peptide. Whether TIP interacts with PTH2-R could not be shown. However, these studies reveal the ability of TIP, as well as of other peptides known to bind to the PTH2-R, to dilate renal vessels in a PTH1-R-independent manner. Moreover, results obtained in IPK desensitized to the vasodilatory action of PTHrP (1-36) strongly suggest that TIP, along with PTHrP, might be coordinately involved in the regulation of renal hemodynamics.

During bone formation, multipotential mesenchymal cells proliferate and differentiate into osteoblasts, and subsequently many die because of apoptosis. Evidence suggests that the receptor for parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), the PTH-1 receptor (PTH-1R), plays an important role in this process. Multipotential mesenchymal cells (C3H10T1/2) transfected with normal or mutant PTH-1Rs and MC3T3-E1 osteoblastic cells were used to explore the roles of PTH, PTHrP, and the PTH-1R in cell viability relative to osteoblastic differentiation. Overexpression of wild-type PTH-1R increased cell numbers and promoted osteocalcin gene expression versus inactivated mutant receptors. Furthermore, the effects of PTH and PTHrP on apoptosis were dramatically dependent on cell status. In preconfluent C3H10T1/2 and MC3T3-E1 cells, PTH and PTHrP protected against dexamethasone-induced reduction in cell viability, which was dependent on cAMP activation. Conversely, PTH and PTHrP resulted in reduced cell viability in postconfluent cells, which was also dependent on cAMP activation. Further, the proapoptotic-like effects were associated with an inhibition of Akt phosphorylation. These data suggest that parathyroid hormones accelerate turnover of osteoblasts by promoting cell viability early and promoting cell departure from the differentiation program later in their developmental scheme. Both of these actions occur at least in part via the protein kinase A pathway.

Enchondromas are common benign cartilage tumors of bone. They can occur as solitary lesions or as multiple lesions in enchondromatosis (Ollier and Maffucci diseases). Clinical problems caused by enchondromas include skeletal deformity and the potential for malignant change to chondrosarcoma. The extent of skeletal involvement is variable in enchondromatosis and may include dysplasia that is not directly attributable to enchondromas. Enchondromatosis is rare, obvious inheritance of the condition is unusual and no candidate loci have been identified. Enchondromas are usually in close proximity to, or in continuity with, growth-plate cartilage. Consequently, they may result from abnormal regulation of proliferation and terminal differentiation of chondrocytes in the adjoining growth plate. In normal growth plates, differentiation of proliferative chondrocytes to post-mitotic hypertrophic chondrocytes is regulated in part by a tightly coupled signaling relay involving parathyroid hormone related protein (PTHrP) and Indian hedgehog (IHH). PTHrP delays the hypertrophic differentiation of proliferating chondrocytes, whereas IHH promotes chondrocyte proliferation. We identified a mutant PTH/PTHrP type I receptor (PTHR1) in human enchondromatosis that signals abnormally in vitro and causes enchondroma-like lesions in transgenic mice. The mutant receptor constitutively activates Hedgehog signaling, and excessive Hedgehog signaling is sufficient to cause formation of enchondroma-like lesions.

These studies examine whether PTHrP(1-36), a vasodilator, modulates BP and renal vascular resistance (RVR) in spontaneously hypertensive rat (SHR). Within the kidney of normotensive rats, PTHrP(1-36) was enriched in vessels. In vessels of SHR, PTHrP was upregulated by 40% and type 1 PTH receptor (PTH1R) was downregulated by 65% compared with normotensive rats. To investigate the role of endogenous PTHrP in the regulation of BP and RVR, SHR were subjected to somatic human (h)PTH1R gene delivery. Three weeks after a single intravenous injection of pcDNA1.1 plasmid containing the hPTH1R gene under the control of the cytomegalovirus promoter, hPTH1R mRNA was detected in all of the main organs. Within the kidney, the transgene was enriched in vessels. In the isolated perfused kidney, RVR was reduced by 23% and PTHrP(1-36)-induced vasodilation, which is depressed in SHR, was restored and a vasoconstrictory response to PTH(3-34), a PTH1R antagonist, was revealed. These effects were not observed in control SHR treated with empty plasmid. BP remained unchanged, and plasma renin activity increased by 60%. Thus, in SHR renal vessels, a reduced number of PTH1R contributes to the high RVR, despite the higher expression of vasodilatory PTHrP. Moreover, these studies provide evidence for a direct link between the density of PTH1R and plasma renin activity, which might be responsible for the absence of effect of PTH1R gene delivery on BP in SHR. Overall, PTHrP significantly contributes to the homeostasis of renal and systemic hemodynamics in SHR.

The parathyroid hormone 2 (PTH2) receptor's anatomical distribution suggests that, among other functions, it may be involved in modulation of nociception. We localized PTH2 receptor protein to spinal cord lamina II and showed that it is synthesized by subpopulations of primary sensory neurons and intrinsic spinal cord dorsal horn neurons. Tuberoinfundibular peptide of 39 residues (TIP39) selectively activates the PTH2 receptor. Intraplantar microinjection of TIP39 caused a paw-withdrawal response and intrathecal injection caused scratching, biting, and licking, a nocifensive response. Intrathecal administration of a TIP39 antibody decreased sensitivity in tail-flick and paw-pressure assays. Intrathecal administration of TIP39 potentiated responses in these assays. We determined the sequence of TIP39's precursor and found that mRNA encoding TIP39 and TIP39-like immunoreactivity is concentrated in two brainstem areas, the subparafascicular area and the caudal paralemniscal nucleus. Cells in these areas project to the superficial dorsal horn of the spinal cord. Our data suggest that TIP39 released from supraspinal fibers potentiates aspects of nociception within the spinal cord.

Parathyroid hormone (PTH) is an 84-amino-acid polypeptide hormone functioning as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. PTH and PTH-related protein (PTHrP) indirectly activate osteoclasts resulting in increased bone resorption. During this process, PTH changes the phenotype of the osteoblast from a cell involved in bone formation to one directing bone resorption. In addition to these catabolic effects, PTH has been demonstrated to be an anabolic factor in skeletal tissue and in vitro. As a result, PTH has potential medical application to the treatment of osteoporosis, since intermittent administration of PTH stimulates bone formation. Activation of osteoblasts by PTH results in expression of genes important for the degradation of the extracellular matrix, production of growth factors, and stimulation and recruitment of osteoclasts. The ability of PTH to drive changes in gene expression is dependent upon activation of transcription factors such as the activator protein-1 family, RUNX2, and cAMP response element binding protein (CREB). Much of the regulation of these processes by PTH is protein kinase A (PKA)-dependent. However, while PKA is linked to many of the changes in gene expression directed by PTH, PKA activation has been shown to inhibit mitogen-activated protein kinase (MAPK) and proliferation of osteoblasts. It is now known that stimulation of MAPK and proliferation by PTH at low concentrations is protein kinase C (PKC)-dependent in both osteoblastic and kidney cells. Furthermore, PTH has been demonstrated to regulate components of the cell cycle. However, whether this regulation requires PKC and/or extracellular signal-regulated kinases or whether PTH is able to stimulate other components of the cell cycle is unknown. It is possible that stimulation of this signaling pathway by PTH mediates a unique pattern of gene expression resulting in proliferation in osteoblastic and kidney cells; however, specific examples of this are still unknown. This review will focus on what is known about PTH-mediated cell signaling, and discuss the established or putative PTH-regulated pattern of gene expression in osteoblastic cells following treatment with catabolic (high) or anabolic (low) concentrations of the hormone.

The linear sequence of intact mammalian PTH consists of 84 amino acids, of which only the most amino(N)-terminal portion, i.e. PTH-(1-34), is required for the classical actions of the hormone on mineral ion homeostasis mediated by the type 1 PTH/PTHrP receptor (PTH1R). Like the N-terminus, the carboxyl (C)-terminal sequence of PTH is highly conserved among species, and various circulating PTH C-fragments are generated by peripheral metabolism of intact PTH or are directly secreted, in a calcium-dependent manner, by the parathyroid glands. Certain synthetic PTH C-fragments exert actions on bone and cartilage cells that are not shared by PTH-(1-34), and specific binding of PTH C-peptides has been demonstrated in bone cells in which PTH1R expression was eliminated by gene targeting. The peptide human (h) PTH-(7-84) recently was shown to inhibit the calcemic actions of hPTH-(1-34) or hPTH-(1-84) in parathyroidectomized animals. To determine whether this anticalcemic effect of hPTH-(7-84) in vivo might result from direct actions on bone, we studied its effects on both resorption of intact bone in vitro and formation of osteoclasts in primary cultures of murine bone marrow. Human (h) PTH-(7-84) (300 nM) reduced basal 72-h release of preincorporated (45)Ca from neonatal mouse calvariae by 50% (9.6 +/- 1.9% vs. 17.8 +/- 5.7%; P < 0.001) and similarly inhibited resorption induced by hPTH-(1-84), hPTH-(1-34), 1,25-dihydroxyvitamin D(3) (VitD), PGE(2), or IL-11. In 12-d murine marrow cultures, both hPTH-(7-84) (300 nM) and hPTH-(39-84) (3000 nM) lowered VitD-dependent formation of osteoclast-like cells by 70%. On the contrary, these actions of hPTH-(7-84) were not observed with the PTH1R antagonists hPTH-(3-34)NH(2) and [L(11),D-W(12),W(23),Y(36)]hPTHrP-(7-36)NH(2), which, unlike hPTH-(7-84), did inhibit PTH1R-dependent cAMP accumulation in ROS 17/2.8 cells. We conclude that hPTH-(7-84), acting via receptors distinct from the PTH1R and presumably specific for PTH C-fragments, exerts a direct antiresorptive effect on bone that may be partly due to impaired osteoclast differentiation.

The parathyroid hormone (PTH) and parathyroid hormone type 1 receptor (PTH1-Rc) are major players in regulating blood calcium homeostasis. PTH1-Rc is a G protein-coupled receptor (GPCR) and it is also activated by PTH-related protein (PTHrP), which has potent effects in several target tissues including bone, via endocrine, paracrine, and autocrine mechanisms in prenatal and adult stages of life, and in several diseases. Another layer of complexity is added by the recently discovered PTH2-Rc subtype displaying unique tissue distribution and ligand specificity, and the potential presence in mammals of a third receptor subtype. Understanding of the mechanisms regulating PTH1-Rc gene expression, receptor desensitization, endocytosis, recycling, and intracellular signaling is advancing and provides insights to understand PTH1-Rc's role in both normal and pathophysiology. In addition, development of an experimentally based model of the PTH-PTH1-Rc complex by combining photoaffinity crosslinking, mutagenesis, conformational analysis, and molecular modeling sheds light, at the atomic resolution, on the molecular mechanisms involved in ligand binding, receptor activation, and coupling to intracellular effectors. Taken together, these lines of investigation offer not only a better understanding of GPCR mechanisms of action in general, but also contribute to the treatment of PTH/PTHrP-PTH1-Rc-related disease states and to the rational development of novel mechanism-based drugs to treat them.

We examined the capacity of PTHrP to modulate the terminal differentiation of the preadipocytic cell line, 3T3-L1. These cells express endogenous PTHrP and its receptor, but expression levels were undetectable after differentiation into mature adipocytes. Cells stably overexpressing PTHrP failed to differentiate when induced to undergo adipogenesis and proliferated at a faster rate. MAPK activity was elevated in PTHrP-transfected 3T3-L1 cells, and treatment with the PKA inhibitor H-8 decreased this activity. Inhibition of MAPK kinase with PD098059 permitted terminal differentiation of PTHrP-transfected 3T3-L1 cells to proceed. Although PPAR gamma gene expression levels remained relatively constant in the PTHrP-transfected cells, PPAR gamma phosphorylation was enhanced. Furthermore, the capacity of PPAR gamma to stimulate transcription in the presence of troglitazone was diminished by PTHrP. Expression of the PPAR gamma-regulated adipocyte specific gene aP2 transiently rose and then fell in PTHrP-transfected cells. These results indicate that PTHrP can increase MAPK activity in 3T3-L1 cells via the PKA pathway, thereby enhancing PPAR gamma phosphorylation. This modification can inactivate the transcriptional enhancing activity of PPAR gamma and diminish the expression of adipocyte-specific genes. These studies therefore demonstrate that PTHrP may inhibit the terminal differentiation of preadipocytes and describe a molecular pathway by which this action can be achieved.

The cloning of the so-called 'parathyroid hormone-related protein' (PTHrP) in 1987 was the result of a long quest for the factor which, by mimicking the actions of PTH in bone and kidney, is responsible for the hypercalcemic paraneoplastic syndrome, humoral calcemia of malignancy. PTHrP is distinct from PTH in a number of ways. First, PTHrP is the product of a separate gene. Second, with the exception of a short N-terminal region, the structure of PTHrP is not closely related to that of PTH. Third, in contrast to PTH, PTHrP is a paracrine factor expressed throughout the body. Finally, most of the functions of PTHrP have nothing in common with those of PTH. PTHrP is a poly-hormone which comprises a family of distinct peptide hormones arising from post-translational endoproteolytic cleavage of the initial PTHrP translation products. Mature N-terminal, mid-region and C-terminal secretory forms of PTHrP are thus generated, each of them having their own physiologic functions and probably their own receptors. The type 1 PTHrP receptor, binding both PTH(1-34) and PTHrP(1-36), is the only cloned receptor so far. PTHrP is a PTH-like calciotropic hormone, a myorelaxant, a growth factor and a developmental regulatory molecule. The present review reports recent aspects of PTHrP pharmacology and physiology, including: (a) the identification of new peptides and receptors of the PTH/PTHrP system; (b) the recently discovered nuclear functions of PTHrP and the role of PTHrP as an intracrine regulator of cell growth and cell death; (c) the physiological and developmental actions of PTHrP in the cardiovascular and the renal glomerulo-vascular systems; (d) the role of PTHrP as a regulator of pancreatic beta cell growth and functions, and, (e) the interactions of PTHrP and calcium-sensing receptors for the control of the growth of placental trophoblasts. These new advances have contributed to a better understanding of the pathophysiological role of PTHrP, and will help to identify its therapeutic potential in a number of diseases.

The type 1 parathyroid hormore receptor (PTH1r) belongs to the class II family of G protein-coupled receptors. To delineate the sites in the PTH1r's N-terminal region, and the carboxy-core domain (transmembrane segments + extracellular loops) involved in PTH binding, we have evaluated the functional properties of 27 PTH1-secretin chimeras receptors stably expressed in HEK-293 cells. The wild type and chimeric receptors were analyzed for cell surface expression, binding for PTH and secretin, and functional responsiveness (cAMP induction) toward secretin and PTH. The expression levels of the chimeric receptors were comparable to that of the PTH1r (60-100%). The N-terminal region of PTH1r was divided into three segments that were replaced either singly or in various combinations with the homologous region of the secretin receptor (SECr). Substitution of the carboxy-terminal half (residues 105-186) of the N-terminal region of PTH1r for a SECr homologous segment did not reduced affinity for PTH but abolished signaling in response to PTH. This data indicate that receptor activation is dissociable from high affinity hormone binding in the PTH1r, and that the N-terminal region might play a critical role in the activation process. Further segment replacements in the N-termini focus on residues 105-186 and particularly residues 146-186 of PTH1r as providing critical segments for receptor activation. The data obtained suggest the existence of two distinct PTH binding sites in the PTH1r's N-terminal region: one site in the amino-terminal half (residues 1-62) (site 1) that participates in high-affinity PTH binding; and a second site of lower affinity constituted by amino acid residues scattered throughout the carboxy-terminal half (residues 105-186) (site 2). In the absence of PTH binding to site 1, higher concentrations of hormone are required to promote receptor activation. In addition, elimination of the interaction of PTH with site 2 results in a loss of signal transduction without loss of high-affinity PTH binding. Divers substitutions of the extracellular loops of the PTH1r highlight the differential role of the first- and third extracellular loop in the process of PTH1r activation after hormone binding. A chimera containing the entire extracellular domains of the PTH1r and the transmembrane + cytoplasmic domains of SECr had very low PTH binding affinity and did not signal in response to PTH. Further substitution of helix 5 of PTH1r in this chimera increased affinity for PTH that is close to the PTH affinity for the wild-type PTH1r but surprisingly, did not mediate signaling response. Additional substitutions of PTH1r's helices in various combinations emphasize the fundamental role of helix 3 and helix 6 on the activation process of the PTH1r. Overall, our studies demonstrated that several PTH1r domains contribute differentially to PTH binding affinity and signal transduction mechanism and highlight the role of the N-terminal domain and helix 3 and helix 6 on receptor activation.

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.

Type-1 PTH/PTH-related peptide receptors (PTH1Rs), which activate both adenylyl cyclase and phospholipase C (PLC), control endochondral bone development by regulating chondrocyte differentiation. To directly analyze PTH1R function in such cells, we isolated conditionally transformed clonal chondrocytic cell lines from tibial growth plates of neonatal mice heterozygous for PTH1R gene ablation. Among 104 cell lines isolated, messenger RNAs for PTH1R, collagen II, and collagen X were detected in 28%, 90%, and 29%, respectively. These cell lines were morphologically diverse. Some appeared large, rounded, and enveloped by abundant extracellular matrix; whereas others were smaller, flattened, and elongated. Two PTH1R-expressing clones showed similar PTH1R binding and cAMP responsiveness to PTH and PTH-related peptide but disparate morphologic features, characteristic of hypertrophic (hC1--5) or nonhypertrophic (nhC2--27) chondrocytes, respectively. hC1--5 cells expressed messenger RNAs for collagen II and X, alkaline phosphatase (ALP), and matrix GLA protein, whereas nhC2--27 cells expressed collagen II and Indian hedgehog but not collagen X or ALP. In hC1--5 cells, PTH and cAMP analog, but not phorbol ester, inhibited both ALP and mineralization. PTH1R-null hC1--5 subclones were isolated by in vitro selection and then reconstituted by stable transfection with wild-type PTH1Rs or mutant (DSEL) PTH1Rs defective in PLC activation. ALP and mineralization were inhibited similarly via both forms of the receptor. These results indicate that PLC activation is not required for PTH1R regulation of mineralization or ALP in hypertrophic chondrocytes and are consistent with a major role for cAMP in regulating differentiation of hypertrophic chondrocytes.

PTH and thyroid hormone (T(3)) stimulate anabolic and catabolic processes in bone predominantly by acting on osteoblasts. Both inadequate and excessive secretion of either hormone can result in clinical bone disorders. In addition, T(3) and PTH related peptide (PTHrP) have multiple effects on a wide number of other tissues modulating both cell differentiation and proliferation. To address the question of whether there might be functional mutual regulation of T(3) receptors (TR) and PTH/PTHrP receptors (PTHR), we studied their expression and receptor-mediated intracellular effects in rat osteoblastic osteosarcoma (ROS) 17/2.8 cells. PTHR were up-regulated by T(3) pretreatment (10(-)(10)-10(-)(6) M) in ROS 17/2.8 cells in a dose-dependent manner. T(3) pretreatment increased both PTH-induced cyclic AMP response element binding protein (CREB) phosphorylation and PTH-induced intracellular calcium transients, and further decreased PTH-induced down-regulation of alkaline phosphatase activity, suggesting that there are functional consequences of the PTHR up- regulation. Pretreatment with PTH (10(-)(10)-10(-)(6) M) or PTHrP (10(-)(9) M) for 3-4 days resulted in a dose-dependent up-regulation of TR in ROS 17/2.8 cells. cAMP analogues or a calcium ionophore were able to mimic the effect of PTH on TR binding, suggesting that either the cAMP-signaling pathway or Ca(2+) could be involved in PTH-induced up-regulation of the TR. These observations provide a novel example of mutual interactions between nuclear receptors and membrane receptors and may have significant implications for the regulation of bone remodeling in health and disease.

Normal development of the growth plate requires coordinated proliferation and differentiation of chondrocytes and osteoblasts. In previous work, we have shown that Indian hedgehog (IHH), produced by prehypertrophic and hypertrophic chondrocytes, stimulates production of parathyroid hormone-related protein (PTHrP) by perichondrial and early chondrocytic cells. PTHrP then maintains chondrocytes in a proliferative, less differentiated state. Because this less differentiated state delays the production of IHH, IHH and PTHrP may participate in a negative feedback loop that synchronizes and determines the pace of differentiation of chondrocytes in the growth plate. To establish the roles of physiological levels of PTHrP and IHH, we have now injected PTH/PTHrP receptor (-/-) embryonic stem (ES) cells into normal blastocysts to generate mice with chimeric growth plates. The PTH/PTHrP receptor cells leave the proliferative cycle and differentiate prematurely in the middle of the normal proliferative columns. The columns of wild-type cells are longer than normal and the adjacent bone collar is also longer than normal. Patterns of gene expression and the use of chimeras using PTH/PTHrP receptor (-/-); IHH (-/-) ES cells suggest that modified patterns of IHH and PTHrP synthesis explain these abnormalities. Thus, IHH is a master regulator of both chondrocyte and osteoblast differentiation.

The parathyroid hormone (PTH) family currently includes three peptides and three receptors. PTH regulates calcium homeostasis through bone and kidney PTH1 receptors. PTH-related peptide, probably also through PTH1 receptors, regulates skeletal, pancreatic, epidermal, and mammary gland differentiation and bladder and vascular smooth muscle relaxation and has a CNS role that is under investigation. Tuberoinfundibular peptide of 39 residues (TIP39) was recently purified from bovine hypothalamus based on selective PTH2 receptor activation. PTH2 receptor expression is greatest in the CNS, where it is concentrated in limbic, hypothalamic, and sensory areas, especially hypothalamic periventricular neurons, nerve terminals in the median eminence, superficial layers of the spinal cord dorsal horn, and the caudal part of the sensory trigeminal nucleus. It is also present in a number of endocrine cells. Thus TIP39 and PTH2 receptor-influenced functions may range from pituitary and pancreatic hormone release to pain perception. A third PTH-recognizing receptor has been found in zebrafish., (Copyright 2000 Academic Press.)

Mice with a targeted deletion of parathyroid hormone (PTH)-related peptide (PTHrP) develop a form of dyschondroplasia resulting from diminished proliferation and premature maturation of chondrocytes. Abnormal, heterogeneous populations of chondrocytes at different stages of differentiation were seen in the hypertrophic zone of the mutant growth plate. Although the homozygous null animals die within several hours of birth, mice heterozygous for PTHrP gene deletion reach adulthood, at which time they show evidence of osteopenia. Therefore, PTHrP appears to modulate cell proliferation and differentiation in both the pre and post natal period. PTH/PTHrP receptor expression in the mouse is controlled by two promoters. We recently found that, while the downstream promoter controls PTH/PTHrP receptor gene expression in bone and cartilage, it is differentially regulated in the two tissues. 1alpha,25-dihydroxyvitamin D3 downregulated the activity of the downstream promoter in osteoblasts, but not in chondrocytes, both in vivo and in vitro. Most of the biological activity of PTHrP is thought to be mediated by binding of its amino terminus to the PTH/PTHrP receptor. However, recent evidence suggests that amino acids 87-107, outside of the amino terminal binding domain, act as a nucleolar targeting signal. Chondrocytic cell line, CFK2, transfected with wild-type PTHrP cDNA showed PTHrP in the nucleoli as well as in the secretory pathway. Therefore, PTHrP appears to act as a bifunctional modulator of both chondrocyte proliferation and differentiation, through signal transduction linked to the PTH/PTHrP receptor and by its direct action in the nucleolus.

Parathyroid hormone-related peptide (PTHrP), which frequently causes the humoral hypercalcemia of malignancy syndrome, is an autocrine/paracrine regulator of chondrocyte proliferation and differentiation that acts through the PTH/PTHrP receptor (PTH1R). PTHrP is generated in response to Indian hedgehog (Ihh), which mediates its actions through the membrane receptor patched, but interacts also with hedgehog-interacting protein (Hip). Mice lacking PTHrP show accelerated chondrocyte differentiation, and thus premature ossification of those bones that are formed through an endochondral process, and similar but more-severe abnormalities are observed in PTH1R-ablated animals. The mirror image of these skeletal findings, i.e., a severe delay in chondrocyte differentiation and endochondral ossification, is observed in transgenic mice that overexpress PTHrP under the control of the alpha1(II) procollagen promoter. Severe abnormalities in chondrocyte proliferation and differentiation are also observed in two genetic disorders in humans that are most likely caused by mutations in the PTH1R. Heterozygous PTH1R mutations that lead to constitutively activity were identified in Jansen metaphyseal chondrodysplasia, and homozygous or compound heterozygous mutations that lead to less-active or completely inactive receptors were identified in patients with Blomstrand lethal chondrodysplasia. Based on the growth plate abnormalities observed in these human disorders and in mice with abnormal expression of either PTHrP or the PTH1R, it appears plausible that impaired expression of PTHrP and/or its receptor contributes to the growth abnormalities in children with end-stage renal disease. In fact, mild-to-moderate renal failure leads in animals to a reduction in PTH1R expression in growth plates and impaired growth, but it remains uncertain whether this contributes to altered chondrocyte growth and differentiation.

The receptor for parathyroid hormone (PTH) and PTH-related peptide (PTHrP) is a G-protein-coupled receptor with four potential sites for N-linked glycosylation. The contribution of the oligosaccharide moieties to cell surface expression, ligand binding, and signal transduction was investigated. Site-directed mutagenesis of the rat PTH/PTHrP receptor cDNA was performed at single or combination of the four potential glycosylation sites to determine the effect of the putative carbohydrate chains on the activities of the receptor. The results revealed that all four potential N-glycosylation sites in the PTH/PTHrP receptor are glycosylated. Receptors missing a single or multiple glycosylation consensus but with at least one intact glycosylation site expressed sufficiently and functioned normally. In contrast, the nonglycosylated receptor, in which all four glycosylation sites were mutated, is deficient in these functions. These data indicate important roles for N-linked glycosylation in PTH/PTHrP receptor functions.

Renal osteodystrophy affects all patients with end-stage renal failure, resulting in significant skeletal and extra-skeletal morbidity. The patterns of disease seen in bone are the result of changes in calcium, phosphate, parathyroid hormone (PTH), and vitamin D metabolism, as well as the effects of uremia. Standard histological techniques, however, give little insight into the altered biological activity or mechanisms of disease at the cellular level. In order to examine the cellular abnormalities in renal bone disease we have performed a series of in situ hybridization studies to examine renal bone cell expression of genes for PTH receptor (PTHR1), transforming growth factor beta (TGF-beta) and insulin growth factor 1 (IGF-I). PTHR1 mRNA was expressed predominantly by osteoblasts, but also by resorbing osteoclasts, suggesting that these cells may be stimulated directly by PTH. Semi-quantitative analysis of gene expression showed down-regulation of PTHR1 mRNA by osteoblasts in renal bone compared with normal, fracture and Pagetic bone. This may be important in the pathogenesis of skeletal resistance seen in end-stage renal failure, altering the "threshold" at which PTH has its effects on bone cells. TGF-beta and IGF-I mRNA expression was also decreased, suggesting that synthesis of these factors, postulated to be mediators of PTH, is also downregulated.