Interaction of human parathyroid hormone-related peptide with parathyroid hormone receptors in clonal rat osteosarcoma cells

Synthetic peptides corresponding to the amino-terminal region of the human parathyroid hormone-related peptide (hPTHrp) were used to characterize the interaction of hPTHrp with parathyroid hormone (PTH) receptors in clonal rat osteosarcoma cells (ROS 17/2.8). Both hPTHrp-(1-34) and [Tyr40]hPTHrp-(1-40) showed full agonist activity in stimulating cyclic AMP accumulation in ROS cells; human PTHrp-(1-34) was approximately 2.5-fold as potent as hPTH-(1-34). Both [Tyr-40]hPTHrp-(3-40) and hPTH-(3-34) inhibited the cyclic AMP increase induced by either hPTHrp or PTH with parallel dose-inhibition curves. Binding to intact ROS cells of a 125I-labeled [Tyr40]hPTHrp-(1-40) (125I-[Tyr40]hPTHrp-(1-40)) which retains full biological activity was time- and temperature-dependent and reversible. Binding of 125I-[Tyr40]hPTHrp-(1-40) and 125I-labeled [Nle8, Nle18, Tyr34]bovine PTH-(1-34)NH2 to ROS cells was competed for, to the same extent and with the comparable potency, by either unlabeled hPTHrp or PTH peptides. The binding capacity and affinity of receptors in ROS cells were strikingly similar for hPTHrp and PTH. Affinity cross-linking with either radioligand resulted in high affinity, specific labeling of an apparently identical macromolecule centering at Mr = 80,000, which was detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both reducing and nonreducing conditions. The data indicate that hPTHrp and PTH, their amino-terminal fragments at least, interact with the identical receptors with regard to affinity, capacity, specificity, and physicochemical characteristics in osteoblastic ROS 17/2.8 cells.     

Expression and regulation of parathyroid hormone-related peptide in normal and malignant melanocytes

We examined parathyroidhormone-related peptide (PTHrP) production and regulation in bothnormal human melanocytes and in a human amelanotic melanoma cell line(A375). Northern blot and immunocytochemical analysis demonstrated thatboth cultured A375 cells and normal human melanocytes express PTHrP,but A375 cells expressed much higher levels of the peptide. PTHrPsecretory rate increased at least 10-fold after treatment with 10%fetal bovine serum (100.2 ± 2.8 pmol/10⁶ cells vs.basal <15 pmol/10⁶ cells) in proliferating A375 cells butonly twofold in confluent cells. Treatment of A375 cells withincreasing concentrations of 1,25-dihydroxyvitamin D₃[1,25-(OH)₂D₃] or its low-calcemic analogEB-1089 revealed that EB-1089 was 10-fold more potent than 1,25-(OH)₂D₃ on inhibition of both cellproliferation and PTHrP expression. Furthermore, inoculation of A375cells into the mammary fat pad of female severe combinedimmunodeficiency mice resulted in the development ofhypercalcemia and elevated concentrations of plasma immunoreactivePTHrP in the absence of detectable skeletal metastases. Our study,therefore, demonstrates a stepwise increase in PTHrP expression whencells progress from normal to malignant phenotype and suggests thatEB-1089 should be further evaluated as a therapeutic agent in human melanoma.

     

Purification and characterization of a receptor for human parathyroid hormone and parathyroid hormone-related peptide

The human parathyroid hormone (PTH) receptor (hPTH1R), containing a 9-amino acid sequence of rhodopsin at its C terminus, was transiently expressed in COS-7 cells and solubilized with 0.25% n-dodecyl maltoside. Approximately 18 microg of hPTH1R were purified to homogeneity per mg of crude membranes by single-step affinity chromatography using 1D4, a monoclonal antibody to a rhodopsin epitope. The N terminus of the hPTH1R is Tyr(23), consistent with removal of the 22-amino acid signal peptide. Comparisons of hPTH1R by quantitative immunoblotting and Scatchard analysis revealed that 75% of the receptors in membrane preparations were functional; there was little, if any, loss of functional receptors during purification. The binding affinity of the purified hPTH1R was slightly lower than membrane-embedded hPTH1R (K(d) = 16.5 +/- 1.3 versus 11.9 +/- 1.9 nm), and the purified receptors bound rat [Nle(8,21),Tyr(34)]PTH-(1-34)-NH(2) (PTH-(1-34)), and rat [Ile(5),Trp(23),Tyr(36)]PTHrP-(5-36)-NH(2) with indistinguishable affinity. Maximal displacement of (125)I-PTH-(1-34) binding by rat [alpha-aminoisobutyric acid (Aib)(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH-(1-21)-NH(2) and rat [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]PTH-(1-14)-NH(2) of 80 and 10%, respectively, indicates that both N-terminal and juxtamembrane ligand binding determinants are functional in the purified hPTH1R. Finally, PTH stimulated [(35)S]GTP gamma S incorporation into G alpha(s) in a time- and dose-dependent manner, when recombinant hPTH1R, G alpha(s)-, and beta gamma-subunits were reconstituted in phospholipid vesicles. The methods described will enable structural studies of the hPTH1R, and they provide an efficient and general technique to purify proteins, particularly those of the class II G protein-coupled receptor family.     

Mechanosensitive release of parathyroid hormone-related peptide from coronary endothelial cells

10.1152/ajpheart.00925. 2001.-Parathyroid hormone-related peptide (PTHrP) is expressed throughout the cardiovascular system and is able to dilate vessels. This study investigated whether mechanical forces generated by changes in regional perfusion influence PTHrP release from the coronary vascular bed. Experiments were performed in vitro on saline-perfused rat hearts or isolated coronary endothelial cells exposed to cyclic strain and in vivo in anesthetized pigs. In vitro, PTHrP release from saline-perfused rat hearts was strongly correlated with coronary flow (r = 0.84). Increasing coronary flow from 5 to 10 ml/min increased PTHrP release from 442 +/- 42 to 1,563 +/- 167 pg/min. Increasing the viscosity of the perfusate did not change basal PTHrP release. Increasing flow without a concomitant increase in pressure did not lead to an increase in release rate, but reduction in pressure under flow-constant conditions reduced PTHrP release rate. Cyclic strain induced a strain-dependent release of PTHrP from endothelial cells that was inhibited by the addition of a calcium-chelating agent. In vivo, there was a net release of PTHrP in the coronary circulation and decreases in coronary flow and pressure decreased the PTHrP release rate. Bradykinin in the presence of constant pressure increased PTHrP release, probably by increasing the intracellular calcium concentration in coronary endothelial cells. In summary, mechanical forces evoked by blood flow can trigger a constant PTHrP release.     

Expression of mRNA of parathyroid hormone-related peptide in fetal bones of the rat

Summary Previous studies have indicated that 19-dayold fetal long bones of the rat contain an adenylyl cyclase-stimulating activity antigenically related to parathyroid hormone-related peptide. To ascertain its origin, Northern blotting and in situ hybridization histochemistry were performed. Results demonstrate that mRNA of parathyroid hormone-related peptide is present in RNA extracted from fetal long bones of the rat and that cells responsible for its production are localized in the periosteum. These cells are not mature osteoblasts because they do not synthesize mRNA of osteocalcin. Thus the present study shows that parathyroid hormone-related peptide could be produced locally, at least in part, in the skeleton of fetal rats.     

Parathyroid hormone-related peptide (hPTHrP) and parathyroid hormone-related peptide receptor type 1 (PTHR1) expression in human thymus.

Parathyroid hormone-related peptide (hPTHrP) is expressed in human tissues and regulates cellular proliferation, differentiation, and apoptosis by an autocrine/paracrine loop. In rodent thymus, both parathormone and parathyroid hormone-related peptide (PTHrP) are expressed by thymic epithelial cells (TECs). The present study demonstrated by RT-PCR and immunohistochemistry that hPTHrP and parathyroid hormone-related peptide receptor type 1 (PTHR1) were expressed in human thymus at both RNA and protein levels. hPTHrP was expressed mainly in the thymic medulla by epithelial (cytokeratin-positive), mature dendritic (CD40+/86+) and plasmacytoid interleukin (IL)-3Ralpha1 cells. This protein was also present in some cells forming Hassall's bodies and a few subcapsular and cortical TECs. PTHR1 was expressed by scattered subcapsular and cortical TECs and by rare TECs in the medulla. Thymocytes did not express either hPTHrP or PTHR1. Primary cultures of human TECs revealed the presence of both hPTHrP and PTHR1 mRNAs, confirming the capacity of TECs to synthesize both peptides. Moreover, synthetic (1-39) hPTHrP peptide administered on cultured TECs induced the expression of IL-6 mRNA, suggesting that hPTHrP can regulate thymic functions by inducing in TECs the expression of IL-6, which is involved in the development and maturation of thymocytes.     

The nucleus: A target site for parathyroid hormone-related peptide (PTHrP) action

It is becoming increasingly apparent that parathyroid hormone-related peptide (PTHrP) modulates cellular function in a dual mode of action: first, by binding and activating its cognate cell surface G-protein-coupled receptor and, second, by direct intracellular effects following translocation to the nucleus and/or nucleolus of the target cell. Little is presently known about the mechanisms and events that determine the timing and degree of PTHrP nuclear translocation or the role it may serve in normal or dysregulated cellular function. Clarifying the nuclear actions of PTHrP would add significantly to our present understanding of this protein as a signaling molecule during embryonic development and as an oncoprotein whose expression in many tumors correlates with increased tumor aggressiveness and propensity for metastasis. J. Cell. Biochem. 70:193–199, 1998. © 1998 Wiley-Liss, Inc.     

Parathyroid hormone and parathyroid hormone-related peptide, and their receptors

Parathyroid hormone (PTH) has a central role in the regulation of serum calcium and phosphate, while parathyroid hormone-related peptide (PTHrP) has important developmental roles. Both peptides signal through the same receptor, the PTH/PTHrP receptor (a class B G-protein-coupled receptor). The different biological effects of these ligands result from their modes of regulation and secretion, endocrine vs. paracrine/autocrine. The importance of PTH and PTHrP is evident by the variety of clinical syndromes caused by deficiency or excess production of either peptide, and the demonstration that intermittent injection of PTH increases bone mass, and thus provides a means to treat osteoporosis. This, in turn, has triggered increased interest in understanding the mechanisms of PTH/PTHrP receptor action and the search for smaller peptide or non-peptide agonists that have efficacy at this receptor when administered non-parenterally.     

Clonal rat parathyroid cell line expresses a parathyroid hormone-related peptide but not parathyroid hormone itself

A novel parathyroid hormone-related peptide has been identified in tumors associated with the syndrome of humoral hypercalcemia of malignancy. Subsequently, mRNAs encoding this peptide have been found to be expressed in a number of normal tissues, including the parathyroids. Using Northern blotting, RNase protection, and immunochemical techniques, we examined a clonal rat parathyroid cell line originally developed as a model system for studying parathyroid cell physiology. We found that this line expresses the parathyroid hormone-related peptide but not parathyroid hormone itself. Secretion of the parathyroid hormone-related peptide varied inversely with extracellular calcium concentration, but neither calcium nor 1,25-dihydroxyvitamin D3 appeared to influence steady-state parathyroid hormone-related peptide mRNA levels. This clonal line may prove to be an interesting system for studying the factors responsible for tissue-specific parathyroid hormone and parathyroid hormone-related peptide gene expression.     

Characterisation of ligand binding to the parathyroid hormone/parathyroid hormone-related peptide receptor in MCF7 breast cancer cells and SaOS-2 osteosarcoma cells

Parathyroid hormone-related peptide (PTHrP) and parathyroid hormone (PTH)/PTHrP-receptor, PTH/PTHrP-R, are frequently expressed in mammary carcinomas as well as in bone cells. In this study we compared the ligand binding characteristics of the PTH/PTHrP-R in SaOS-2 human osteosarcoma cells with those in MCF7 breast cancer cells. We used both Scatchard analysis of saturation kinetics for iodinated ligand and the level of expressed receptor protein by visualising the single radio-labelled receptor-ligand complex from isolated membrane preparations from the two cell lines. In MCF7 cells, ligand binding, (receptor number) was increased by prior exposure of the cultured cells to epidermal growth factor (EGF), estradiol (E2), or dexamethasone (DEX), and decreased following calcitriol (1,25 DHCC). In contrast in the SaOS-2 cells, PTH/PTHrP-R number was increased by exposure to E2 and 1,25DHCC and decreased by DEX while EGF had no effect. These data were confirmed when the PTH/PTHrP-R was cross linked with (125)I-PTHrP-1-34(Tyr), and extended by visualising the intensity of the isolated radiolabelled receptor complex by autoradiography following SDS-PAGE at several time points during the treatment.     

Stretch-induced parathyroid hormone-related peptide gene expression in the rat uterus.

We previously observed a peak in parathyroid hormone-related peptide (PTHrP) mRNA expression in preterm rat myometrium and found that this peak was dependent on intrauterine occupancy. We explored the possibility that mechanotransduction might control PTHrP gene expression in the uterus. This was done by developing an intrauterine balloon system that allowed us to reproduce experimentally the mechanical effects of the fetal pup in utero. An increase in PTHrP mRNA in the unoccupied horn of a unilaterally pregnant rat could be elicited as rapidly as 1 h after balloon inflation and was maintained for up to 72 h. The same response was seen in uterine horns from virgin animals and could be reproduced by three different methods of imposing a physical stretch. Balloon-induced stretch also increased mRNA expression in a muscle bath system in vitro. Mechanotransduction appears to be largely, if not entirely, responsible for the preterm peak in PTHrP mRNA expression.     

Purification and characterization of recombinant human parathyroid hormone-related protein

Full-length human parathyroid hormone-related protein (PTHrP-(1-141] as well as a carboxyl-terminal shortened form (PTHrP-(1-108] have been expressed from recombinant DNA-derived clones. These proteins were expressed in Escherichia coli as fusion proteins so that cyanogen bromide cleavage yields the desired product. Both proteins were purified and then characterized by sodium dodecyl sulfate gel electrophoresis, amino-terminal amino acid sequencing, peptide mapping, and mass spectral analysis. Recombinant PTHrP-(1-141), PTHrP-(1-108), synthetic PTHrP-(1-34), and naturally derived PTHrP are all equipotent in the stimulation of cyclic AMP levels in the osteoblast-like cell line UMR 106-01. However, PTHrP-(1-141) and -(1-108) are two to four times more active than PTHrP-(1-34) in the stimulation of plasminogen activator activity from this cell line. PTHrP-(1-141) reacts equipotently with PTHrP-(1-34) in a radioimmunoassay using an antiserum prepared against PTHrP-(1-34). PTHrP-(1-141), -(1-108), and -(1-84) were used as PTHrP-specific mobility standards on sodium dodecyl sulfate gel electrophoresis to determine the approximate length of two forms of naturally derived PTHrP. The data show that PTHrP purified from the lung tumor cell line BEN contains a major form of about 108 amino acids and another form of about 141 amino acids.     

Structure and activities of constrained analogues of human parathyroid hormone and parathyroid hormone-related peptide: implications for receptor-activating conformations of the hormones

Parathyroid hormone (PTH) has a helix-bend-helix structure in solution. Part of the C-terminal helix, residues 21-31, is amphiphilic and forms a critical receptor-binding region. Stabilization of this alpha-helix by lactam formation between residues spaced i, i + 4 on the polar face was previously reported to increase adenylyl cyclase-stimulating (AC) activity if between residues 22 and 26 but to diminish it if between residues 26 and 30 [Barbier et al. (1997) J. Med. Chem. 40, 1373-1380]. This work reports the effects of other cyclizations on the polar face, differing in ring size or position, on alpha-helix conformation, as measured by circular dichroism, and on AC-stimulating activity. All analogues cyclized between residues 22 and 26 had at least a 1. 5-fold increase in activity, suggesting an alpha-helical structure between about residues 21 and 26. Cyclization between residues 25 and 29 or residues 26 and 30 diminished activity by 20-30%, despite stabilizing alpha-helix, suggesting that residues 25-31 bind to the receptor in a helical, but not classical alpha-helical, conformation. Analogues cyclized between residues 13 and 17 had slightly increased activity. A bicyclic analogue, with lactams between residues 13 and 17 and residues 22 and 26, had about the same activity as that cyclized only between 22 and 26. Parathyroid hormone-related peptide (PTHrP) may bind in a manner similar to the common receptor, but hydrophobic moment calculations suggest that it must bind as a tighter helix in order to optimally present its hydrophobic residues to the receptor. Both PTHrP analogues cyclized between either residues 22 and 26 or residues 26 and 30 had more stable alpha-helices but reduced AC activities, consistent with this hypothesis.