Coexpression of PTHrP and PTH/PTHrP receptor in a myoepithelial cell line derived from normal human breast

Parathyroid hormone-related peptide (PTHrP) is the cause of humoral hipercalcaemia of malignancy syndrome (HHM). It is known that the peptide as well as its receptors are widely distributed in many normal organs and tissues, where it influences an array of diverse functions which are realized through paracrine or autocrine pathway. PTHrP is present in large amounts in lactating mammary gland but its function is not fully elucidated. In this study, production of parathyroid hormone-related peptide (PTHrP) by the Hs578Bst cell line corresponding to mammary myoepithelial cells was examined by immunocytochemistry. Using RNA extracted from these cells we analyzed expression of mRNA for PTHrP and for the PTH/PTHrP receptor by RT-PCR. The obtained results demonstrated that Hs578Bst cells produced PTHrP and synthesized mRNA for PTHrP and PTH/PTHrP type I receptor. It provides evidence that myoepithelial cells are target cells for PTHrP. The data support that PTHrP may be an important autocrine/paracrine factor, involved in the regulation of myoepithelial cell function as well as in growth and differentiation of the mammary gland.     

Parathyroid hormone receptor internalization is independent of protein kinase A and phospholipase C activation

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) binding to their common receptor stimulates second messenger accumulation, receptor phosphorylation, and internalization. LLC-PK(1) cells expressing a green fluorescent protein-tagged PTH/PTHrP receptor show time- and dose-dependent receptor internalization. The internalized receptors colocalize with clathrin-coated pits. Internalization is stimulated by PTH analogs that bind to and activate the PTH/PTHrP receptor. Cell lines expressing a mutant protein kinase A regulatory subunit that is resistant to cAMP and/or a mutant receptor (DSEL mutant) that does not activate phospholipase C internalize their receptors normally. In addition, internalization of the wild-type receptor and the DSEL mutant is stimulated by the PTH analog [Gly(1),Arg(19)]hPTH-(1-28), which does not stimulate phospholipase C. Forskolin, IBMX, and the active phorbol ester, phorbol-12-myristate-13-acetate, did not promote receptor internalization or increase PTH-induced internalization. These data indicate that ligand-induced internalization of the PTH/PTHrP receptor requires both ligand binding and receptor activation but does not involve stimulation of adenylate cyclase/protein kinase A or phospholipase C/protein kinase C.     

Important role for the V-type H(+)-ATPase and the Golgi apparatus in the recycling of PTH/PTHrP receptor

Our previous studies demonstrated that a green fluorescent protein-tagged parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor stably expressed in LLCPK-1 cells undergoes agonist-dependent internalization into clathrin-coated pits. The subcellular localization of the internalized PTH/PTHrP receptor is not known. In the present study, we explored the intracellular pathways of the internalized PTH/PTHrP receptor. Using immunofluorescence and confocal microscopy, we show that the internalized receptors localize at a juxtanuclear compartment identified as the Golgi apparatus. The receptors do not colocalize with lysosomes. Furthermore, whereas the internalized receptors exhibit rapid recycling, treatment with proton pump inhibitors (bafilomycin-A1 and concanamycin A) or brefeldin A, Golgi disrupting agents, reduces PTH/PTHrP receptor recycling. Together, these data indicate an important role for the vacuolar-type hydrogen-ATPase and the Golgi apparatus in postendocytic PTH/PTHrP receptor recovery.     

Characterization of an element within the rat parathyroid hormone/parathyroid hormone-related peptide receptor gene promoter that enhances expression in osteoblastic osteosarcoma 17/2.8 cells.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) mediate their actions via a common G-protein-coupled receptor. High levels of PTH/PTHrP receptor expression have been detected in many tissues including bone and kidney. This study has demonstrated specific PTH/PTHrP receptor expression from the U3 promoter in the osteoblastic osteosarcoma ROS 17/2.8 cell line, which expresses the endogenous PTH/PTHrP receptor, compared to rat 2 fibroblasts which do not express the endogenous PTH/PTHrP receptor gene. Transient transfection studies revealed cell-specific expression of a construct containing 4391 bp of DNA upstream of exon U3 of the PTH/PTHrP receptor gene fused to a luciferase reporter gene. Deletion mapping of the 5' region of U3 revealed that a construct containing 206 bp upstream of U3 confers cell-specific expression. These data suggest that cell-specific expression in ROS 17/2.8 involves cell-specific elements within the PTH/PTHrP receptor promoter.     

Eliminating phosphorylation sites of the parathyroid hormone receptor type 1 differentially affects stimulation of phospholipase C and receptor internalization

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTH1R) belongs to family B of seven-transmembrane-spanning receptors and is activated by PTH and PTHrP. Upon PTH stimulation, the rat PTH1R becomes phosphorylated at seven serine residues. Elimination of all PTH1R phosphorylation sites results in prolonged cAMP accumulation and impaired internalization in stably transfected LLC-PK1 cells. The present study explores the role of individual PTH1R phosphorylation sites in PTH1R signaling through phospholipase C, agonist-dependent receptor internalization, and regulation by G protein-coupled receptor kinases. By means of transiently transfected COS-7 cells, we demonstrate that the phosphorylation-deficient (pd) PTH1R confers dramatically enhanced coupling to G(q/11) proteins upon PTH stimulation predominantly caused by elimination of Ser(491/492/493), Ser(501), or Ser(504). Reportedly, impaired internalization of the pd PTH1R, however, is not dependent on a specific phosphorylation site. In addition, we show that G protein-coupled receptor kinase 2 interferes with pd PTH1R signaling to G(q/11) proteins at least partially by direct binding to G(q/11) proteins.     

Expression of PTHrP and the PTH/PTHrP receptor in growing red deer antler

Antler growth is highly co-ordinated, so that trabecular bone and antler skin (velvet) develop together, at a rapid rate and in a manner reminiscent of their development in the fetus. Parathyroid hormone-related peptide (PTHrP) is expressed in both bone and skin, and is therefore a candidate to effect co-ordination between these tissues. The aim of this study was to localize the expression of PTHrP and its principal receptor, the parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrPR), in antler ("spiker") of one-year-old red deer. Using immunohistochemistry and in situ hybridization, intense and overlapping expression of PTHrP and its receptor was seen in developing osseocartilaginous structures and in the underlying layers of velvet epidermis. PTHrP was located on both the cell surface and within the nuclei. Our results strongly suggest that PTHrP, acting via the PTH/PTHrPR and possibly other intracrine mechanisms, plays a central role in the co-ordinated regulation of cell division and differentiation of developing antler bone and skin.     

Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor

The regulatory role of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) signaling has been implicated in embryonic skeletal development. Here, we studied chondrogenic differentiation of the mouse embryonal carcinoma-derived clonal cell line ATDC5 as a model of chondrogenesis in the early stages of endochondral bone development. ATDC5 cells retain the properties of chondroprogenitor cells, and rapidly proliferate in the presence of 5% FBS. Insulin (10 micrograms/ml) induced chondrogenic differentiation of the cells in a postconfluent phase through a cellular condensation process, resulting in the formation of cartilage nodules, as evidenced by expression of type II collagen and aggrecan genes. We found that differentiated cultures of ATDC5 cells abundantly expressed the high affinity receptor for PTH (Mr approximately 80 kD; Kd = 3.9 nM; 3.2 x 10(5) sites/cell). The receptors on differentiated cells were functionally active, as evidenced by a PTH-dependent activation of adenylate cyclase. Specific binding of PTH to cells markedly increased with the formation of cartilage nodules, while undifferentiated cells failed to show specific binding of PTH. Northern blot analysis indicated that expression of the PTH/PTHrP receptor gene became detectable at the early stage of chondrogenesis of ATDC5 cells, preceding induction of aggrecan gene expression. Expression of the PTH/PTHrP receptor gene was undetectable in undifferentiated cells. The level of PTH/PTHrP receptor mRNA was markedly elevated parallel to that of type II collagen mRNA. These lines of evidence suggest that the expression of functional PTH/PTHrP receptor is associated with the onset of chondrogenesis. In addition, activation of the receptor by exogenous PTH or PTHrP significantly interfered with cellular condensation and the subsequent formation of cartilage nodules, suggesting a novel site of PTHrP action.     

Role of asparagine-linked oligosaccharides in the function of the rat PTH/PTHrP receptor

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.     

PTHrP, PTH, and the PTH/PTHrP receptor in endochondral bone development

Endochondral bone development is a fascinating story of proliferation, maturation, and death. An understanding of this process at the molecular level is emerging. In particular, significant advances have been made in understanding the role of parathyroid-hormone-related peptide (PTHrP), parathyroid hormone (PTH), and the PTH/PTHrP receptor in endochondral bone development. Mutations of the PTH/PTHrP receptor have been identified in Jansen metaphyseal chondrodysplasia, Blomstrand's lethal chondrodysplasia, and enchondromatosis. Furthermore, genetic manipulations of the PTHrP, PTH, and the PTH/PTHrP receptor genes, respectively, have demonstrated the critical role of these proteins in regulating both the switch between proliferation and differentiation of chondrocytes, and their replacement by bone cells. A future area of investigation will be the identification of downstream effectors of PTH, PTHrP, and PTH/PTHrP receptor activities. Furthermore, it will be of critical importance to study how these proteins cooperate and integrate with other molecules that are essential for growth plate development.     

The parathyroid hormone family of peptides: structure, tissue distribution, regulation, and potential functional roles in calcium and phosphate balance in fish

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.     

Megalin antagonizes activation of the parathyroid hormone receptor

Parathyroid hormone (PTH) is predominantly cleared from the circulation by glomerular filtration and degradation in the renal proximal tubules. Here, we demonstrate that megalin, a multifunctional endocytic receptor in the proximal tubular epithelium, mediates the uptake and degradation of PTH. Megalin was purified from kidney membranes as the major PTH-binding protein and shown in BIAcore analysis to specifically bind full-length PTH and amino-terminal PTH fragments (Kd 0.5 microM). Absence of the receptor in megalin knockout mice resulted in 4-fold increased levels of amino-terminal PTH fragments in the urine. In F9 cells expressing both megalin and the PTH/PTH-related peptide receptor (PTH/PTHrP receptor), uptake and lysosomal degradation of the hormone was mediated through megalin. Blocking megalin-mediated clearance of PTH resulted in 3-fold increased stimulation of the PTH/PTHrP receptor. These data provide evidence that megalin is involved in the renal catabolism of PTH and potentially antagonizes PTH/PTHrP receptor activity in the proximal tubular epithelium.     

Recent studies on the biological action of parathyroid hormone (PTH)-related peptide (PTHrP) and PTH/PTHrP receptor in cartilage and bone

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.     

Estrogen stimulates PTHrP but not PTH/PTHrP receptor gene expression in the kidney of ovariectomized rat

The aim of the present study was to test the hypothesis that the decreased renal tubular reabsorption of calcium observed in estrogen deficiency is associated with a local regulation of either PTHrP or PTH/PTHrP receptor genes in the kidney. Rats were randomly sham-operated (S) or ovariectomized receiving either vehicule (OVX) or 4 μg E2/kg/day (OVX+E4) or 40 μg E2/kg/d (OVX+E40) during 14 days using alzet minipumps. Plasma PTH and calcium levels were lower in untreated OVX animals than in all other groups (P < 0.01). Plasma PTH was higher in OVX+E40 than in OVX+E4 (P < 0.05). PTHrP mRNA expression in the kidney was unaffected by ovariectomy but was increased in OVX+E40 (0.984 ± 0.452 for PTHrP/GAPDH mRNAs expression vs. 0.213 ± 0.078 in sham, P < 0.01). PTH/PTHrP receptor mRNA expression and the cAMP response of renal membranes to PTH were unaffected by ovariectomy and estrogen substitution. In conclusion, renal PTHrP and PTH/PTHrP receptor mRNAs are not modified by ovariectomy. However, 17β-estradiol increases renal expression of PTHrP mRNA without evident changes in its receptor expression and function. This may help to explain the pharmacological action of estrogen in the kidney, especially how it prevents the renal leak of calcium in postmenopausal women. J. Cell. Biochem. 70:84–93, 1998. © 1998 Wiley-Liss, Inc.     

Parathyroid hormone related peptide can function as an autocrine growth factor in human renal cell carcinoma

Parathyroid hormone related peptide (PTHrP) has been implicated in the cause of the hypercalcemia associated with a number of malignant tumours. The data presented here suggests that PTHrP (in addition to its known role of mediating hypercalcemia) may be involved in the autocrine regulation of growth of some tumours. Polyclonal PTHrP antiserum almost totally inhibited the growth of a human renal cell carcinoma cell line, known to secrete PTHrP, in vitro and growth was significantly inhibited by the competitive PTH antagonist PTH (3-34)NH2.     

A PTH/PTHrP receptor antagonist blocks the hypercalcemic response to estradiol-17beta

Estradiol (E(2)) increases circulating calcium and phosphate levels in fish, thus acting as a hypercalcemic and hyperphosphatemic factor during periods of high calcium requirements, such as during vitellogenesis. Since parathyroid hormone (PTH)-related protein (PTHrP) has been shown to be calciotropic in fish, we hypothesized that the two hormones could be mediating the same process. Sea bream (Sparus auratus) juveniles receiving a single intraperitoneal injection of piscine PTHrP(1-34) showed an elevation in calcium plasma levels within 24 h. In contrast, injections of the PTH/PTHrP receptor antagonist PTHrP(7-34) decreased circulating levels of calcium in the same period. Intraperitoneal implants of estradiol-17beta (E(2); 10 microg/g) evoked significant increases of circulating plasma levels of calcium and phosphorus and a sustained increases of circulating plasma levels of PTHrP. However, a combined treatment of E(2) and PTHrP(7-34) evoked a markedly lower calcium response compared with E(2) alone. We conclude that PTHrP or a related peptide that binds the PTH/PTHrP receptor mediates, at least in part, the hypercalcemic effect of E(2) in calcium and phosphate balance in fish.     

Second messenger signaling in the regulation of cytosolic pH and DNA synthesis by parathyroid hormone (PTH) and PTH-related peptide in osteoblastic osteosarcoma cells: role of Na+/H+ exchange.

The present study was performed to investigate the regulation of cytosolic pH (pHi) and DNA synthesis by parathyroid hormone(PTH) and PTH-related peptide (PTHrP) in osteoblasts, using osteoblastic osteosarcoma cells, UMR-106 which possessed PTH-responsive dual signal transduction systems (cAMP-dependent protein kinase (PKA) and calcium/protein kinase C [Ca/PKC]) and amiloride-inhibitable Na+/H+ exchange system. Both human (h)PTH-(1-34) and hPTHrP-(1-34) caused a progressive decrease in pHi and the inhibition of [3H]thymidine incorporation (TdR) to the same degree in a dose-dependent manner with a minimal effective dose of 10(-10) M. Dibutyryl cAMP (10(-4) M and Sp-cAMPS (10(-4) M), a direct stimulator of PKA also caused a progressive decrease in pHi, and calcium ionophores (A23187 and ionomycin, 10(-6) M) caused a transient decrease in pHi. Pretreatment with amiloride (0.3 mM) mostly blocked dbcAMP- and Sp-cAMPS-induced decrease in pHi but did not affect calcium ionophore-induced decrease in pHi. In the presence of amiloride, PTH and PTHrP caused a transient decrease in pHi, which was similar to the pattern of calcium ionophore-induced change in pHi. Amiloride did not affect the inhibition of TdR by PTH or PTHrP as well as that by cAMP analogues or calcium ionophores. The present study indicated that PTH and PTHrP caused cytosolic acidification through PKA-inhibited Na+/H+ exchange and increased cytosolic calcium-induced pathway and that the regulation of DNA synthesis by PTH and PTHrP was not via Na+/H+ exchange system.     

Development of a photoreactive parathyroid hormone antagonist to probe antagonist-receptor bimolecular interaction.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert their calciotropic activities by binding to a specific seven-transmembrane-helix-containing G protein-coupled receptor mainly located in bone and kidney cells. In order to map in detail the nature of hormone-receptor interaction, we are employing 'photoaffinity scanning' of the bimolecular interface. To this end, we have developed photoreactive benzophenone (BP)-containing PTH analogs which can be specifically and efficiently cross-linked to the human (h) PTH/PTHrP receptor. In this report, we describe the photocross-linking of a BP-containing PTH antagonist, [Nle8,18,D-2-Nal12,Lys13(epsilon-BP),2-Nal23,Tyr34]bPT H(7-34)NH2 (ANT) to the recombinant hPTH/PTHrP receptor stably expressed in human embryonic kidney cells (HEK-293, clone C-21). This photoreactive antagonist has high affinity for the hPTH/PTHrP receptor and inhibits agonist-induced cyclase activity and intracellular calcium release. The photo-induced cross-linking of the radioiodinated antagonist (125I-ANT) to the recombinant hPTH/PTHrP receptor followed by SDS-PAGE analysis reveals a single radiolabeled band of approximately 85kDa, similar to that observed after cross-linking of a radioiodinated BP-containing agonist. The formation of this covalent 125I-ANT - hPTH/PTHrP receptor conjugate is competed dose-dependently by a variety of unlabelled PTH- and PTHrP-derived agonists and antagonists. This is the first report of a specific and efficient photocross-linking of a radioiodinated PTH antagonist to the hPTH/PTHrP receptor. Therefore, it provides the opportunity to study directly the nature of the bimolecular interaction of PTH antagonist with the hPTH/PTHrP receptor.