Synthetic peptides comprising the amino-terminal sequence of a parathyroid hormone-like protein from human malignancies. Binding to parathyroid hormone receptors and activation of adenylate cyclase in bone cells and kidney

A tumor-derived protein with a spectrum of biologic activities remarkably similar to that of parathyroid hormone (PTH) has recently been purified and its sequence deduced from cloned cDNA. This PTH-like protein (PLP) has substantial sequence homology with PTH only in the amino-terminal 1-13 region and shows little similarity to other regions of PTH thought to be important for binding to receptors. In the present study, we compared the actions of two synthetic PLP peptides, PLP-(1-34)amide and [Tyr36]PLP-(1-36)amide, with those of bovine parathyroid hormone (bPTH)-(1-34) on receptors and adenylate cyclase in bone cells and in renal membranes. Synthetic PLP peptides were potent activators of adenylate cyclase in canine renal membranes (EC50 = 3.0 nM) and in UMR-106 osteosarcoma cells (EC50 = 0.05 nM). Bovine PTH-(1-34) was 6-fold more potent than the PLP peptides in renal membranes, but was 2-fold less potent in UMR-106 cells. A competitive PTH receptor antagonist, [Tyr34]bPTH-(7-34)amide, rapidly and fully inhibited adenylate cyclase stimulation by the PLP peptides as well as bPTH-(1-34). Competitive binding experiments with 125I-labeled PLP peptides revealed the presence of high affinity PLP receptors in UMR-106 cells IC50 = 3-4 nM) and in renal membranes (IC50 = 0.3 nM). There was no evidence of heterogeneity of PLP receptors. Bovine PTH-(1-34) was equipotent with the PLP peptides in binding to PLP receptors. Likewise, PLP peptides and bPTH-(1-34) were equipotent in competing with 125I-bPTH-(1-34) for binding to PTH receptors in renal membranes. Photoaffinity cross-linking experiments revealed that PTH and PLP peptides both interact with a major 85-kDa and minor 55- and 130-kDa components of canine renal membranes. We conclude that PTH and PLP activate adenylate cyclase by binding to common receptors in bone and kidney. The results further imply that subtle differences exist between PTH and PLP peptides in their ability to induce receptor-adenylate cyclase coupling.     

Mutational analysis of the receptor-activating region of human parathyroid hormone.

The first 4 residues of parathyroid hormone (PTH) are highly conserved in evolution and are important for biological activity. We randomly mutated codons 1-4 of human PTH (hPTH) with degenerate oligonucleotides and, after expression in COS cells, screened the mutants for receptor binding and cAMP-stimulating activity using ROS 17/2.8 cells. This survey identified Glu4 and Val2 as important determinants of receptor binding and activation, respectively. Positions 1 and 3 were more tolerant of substitutions indicating that these sites are less vital to hormone function. Activities of synthetic hPTH(1-34) analogs further demonstrated the importance of positions 2 and 4. The binding affinity of [Ala4,Tyr34] hPTH(1-34)NH2 was 100-fold reduced relative to [Tyr34]hPTH(1-34)NH2 (Kd values = 653 +/- 270 and 4 +/- 1 nM, respectively), and [Arg2, Tyr34]hPTH(1-34)NH2 was a weak partial agonist which bound well to the ROS cell receptor (Kd = 31 +/- 10 nM). The Arg2 analog was nearly as potent as PTH(3-34) as an in vitro PTH antagonist in osteoblast derived cells. However, unlike PTH(3-34), [Arg2]PTH was a full agonist in opossum kidney (OK) cells. These observations suggest that the activation domains of the OK and ROS cell PTH receptors are different. Thus, amino-terminal PTH analogs may be useful as probes for distinguishing properties of PTH receptors.     

甲状旁腺素、表皮生长因子及维生素A酸对UMR106细胞EGF受体的调节作用

本文研究了EGF、PTH和RA对UMR106细胞EGF受体的调节作用。结果显示PTH能上调EGF的受体,UMR106细胞经bPTH(1-34)处理3天,EGF受体的相对结合率与对照比较提高了40.3％,每个细胞的EGF受体数目从7.22×10~3增加到1.44×10~4,Kd从2.02×10~(-11)增加到3.68×10~(-11)mol/L。而RA则能下调EGF受体,以RA处理3天,EGF受体数目从7.22×10~3下降到4.28×10~3,Kd则从2.02×10~(-11)增加到4.17×10~(-11)mol/L。提示PTH和RA可能通过调变其EGF受体而分别起到正性和负性生长调节作用。     

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.     

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.     

Molecular cloning and functional characterization of the canine parathyroid hormone/parathyroid hormone related peptide receptor (PTH1)

Parathyroid hormone (PTH) is a major mediator of calcium and phosphate metabolism through its interactions with receptors in kidney and bone. PTH binds with high affinity to PTH1 and PTH2, members of the superfamily of G protein-coupled receptors. In order to clone the canine PTH1 receptor, a canine kidney cDNA library was screened using the human PTH1 receptor cDNA and two clones were further characterized. The longest clone was 2177 bp and contained a single open reading frame of 1785 bp, potentially encoding a protein of 595 amino acids with a predicted molecular weight of 66.4 kD. This open reading frame exhibits >91% identity to the human PTH1 receptor cDNA and >95% identity when the putative canine and human protein sequences are compared. Competition binding following transfection of the canine PTH1 receptor into CHO cells demonstrated specific displacement of ¹²⁵I-human PTH 1-34 by canine PTH 1-34, human PTH 1-34, and canine/human parathyroid hormone related peptide (PTHrP) 1-34. Treatment of canine PTH1 receptor transfected cells, but not mock transfected cells, with these ligands also resulted in increased levels of intracellular cAMP. In contrast, the non-related aldosterone secretion inhibiting factor 1-35 neither bound nor activated the canine PTH1 receptor. Northern blot analysis revealed high levels of PTH1 receptor mRNA in the kidney, with much lower, but detectable, levels in aorta, heart, lung, prostate, testis, and skeletal muscle. Together, these data indicate that we have cloned the canine PTH1 receptor and that it is very similar, both in sequence and in functional characteristics, to the other known PTH1 receptors.     

Quantitative comparison of PTH1R in breast cancer MCF7 and osteosarcoma SaOS-2 cell lines

The aim of the present study was to compare the classical parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptors in MCF7 breast cancer cells with SaOS-2 osteosarcoma cell line. Quantitative binding showed that (125)I-PTHrP-1-34(Tyr) binds with a single binding site in both cells. However (125)I-PTHrP-1-34(Tyr) has higher affinity binding in MCF7 (K(D) = 1.88 +/- 0.08 nM) than in SaOS-2 cells (K(D) = 4.4 +/- 0.185 nM). The competitive binding using 3.3 nM (125)I-PTHrP-1-34(Tyr) with increasing amounts (0.33-33 nM) of unlabelled human PTHrP-1-34, PTHrP-7-34, PTHrP-1-86 His(5)-PTHrP-1-36, His(5)-Phe(23)-PTHrP-1-36 or PTH-1-34 revealed different displacements. In SaOS-2 the PTHrP-7-34 and PTHrP-1-86 caused similar displacement compared with 73% by PTH-1-34 and 70% by PTHrP-1-34. However, in MCF7, PTHrP-7-34, PTHrP-1-86 and PTH-1-34 displaced by 54%, 72% and 67%, respectively, compared to 87% by PTHrP-1-34. The His(5)-Phe(23)-PTHrP-1-36 caused an increase in the K(D) from 2.0 +/- 0.03 nM to 2.75 +/- 0.045 nM in MCF7 cells, but had no significant effect in SaOS-2 cells. The PTH/PTHrP receptor in both cell lines revealed a single 85 KDa band with different intensity. Our results suggest that the PTH/PTHrP receptor in MCF7 cells has higher binding affinity for PTHrP than PTH compared to the receptor in SaOS-2 cells.     

17 beta-estradiol-BSA conjugates and 17 beta-estradiol regulate growth plate chondrocytes by common membrane associated mechanisms involving PKC dependent and independent signal transduction

Nuclear receptors for 17 beta-estradiol (E(2)) are present in growth plate chondrocytes from both male and female rats and regulation of chondrocytes through these receptors has been studied for many years; however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the cell response. E(2) was found to directly affect the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E(2) activates protein kinase C (PKC) in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E(2)-dependent alkaline phosphatase activity and proteoglycan sulfation in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of the present study were: (1) to examine the effect of a cell membrane-impermeable 17 beta-estradiol-bovine serum albumin conjugate (E(2)-BSA) on chondrocyte proliferation, differentiation, and matrix synthesis; (2) to determine the pathway that mediates the membrane effect of E(2)-BSA on PKC; and (3) to compare the action of E(2)-BSA to that of E(2). Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10(-9) to 10(-7) M E(2) or E(2)-BSA and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [(3)H]-thymidine incorporation measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E(2)-BSA in the presence or absence of GDP beta S (inhibitor of G-proteins), GTP gamma S (activator of G-proteins), U73122 or D609 (inhibitors of phospholipase C [PLC]), wortmannin (inhibitor of phospholipase D [PLD]) or LY294002 (inhibitor of phosphatidylinositol 3-kinase). E(2)-BSA mimicked the effects of E(2) on alkaline phosphatase specific activity and proteoglycan sulfation, causing dose-dependent increases in both RC and GC cell cultures. Both forms of estradiol inhibited [(3)H]-thymidine incorporation, and the effect was dose-dependent. E(2)-BSA caused time-dependent increases in PKC in RC and GC cells; effects were observed within three minutes in RC cells and within one minute in GC cells. Response to E(2) was more robust in RC cells, whereas in GC cells, E(2) and E(2)-BSA caused a comparable increase in PKC. GDP beta S inhibited the activation of PKC in E(2)-BSA-stimulated RC and GC cells. GTP gamma S increased PKC in E(2)-BSA-stimulated GC cells, but had no effect in E(2)-BSA-stimulated RC cells. The phosphatidylinositol-specific PLC inhibitor U73122 blocked E(2)-BSA-stimulated PKC activity in both RC and GC cells, whereas the phosphatidylcholine-specific PLC inhibitor D609 had no effect. Neither the PLD inhibitor wortmannin nor the phosphatidylinositol 3-kinase inhibitor LY294022 had any effect on E(2)-BSA-stimulated PKC activity in either RC or GC cells. The classical estrogen receptor antagonist ICI 182780 was unable to block the stimulatory effect of E(2)-BSA on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E(2)-BSA. The specificity of the membrane response to E(2) was also demonstrated by showing that the membrane receptor for 1 alpha,25-(OH)(2)D(3) was not involved. These data indicate that the rapid nongenomic effect of E(2)-BSA on PKC activity in RC and GC cells is dependent on G-protein-coupled PLC and support the hypothesis that many of the effects of E(2) involve membrane-associated mechanisms independent of classical estrogen receptors. (c) 2001 Wiley-Liss, Inc.     

The (1-14) fragment of parathyroid hormone (PTH) activates intact and amino-terminally truncated PTH-1 receptors.

Recent mutagenesis and cross-linking studies suggest that residues in the carboxyl-terminal portion of PTH(1-34) interact with the amino-terminal extracellular domain of the receptor and thereby contribute strongly to binding energy; and that residues in the amino-terminal portion of the ligand interact with the receptor region containing the transmembrane helices and extracellular loops and thereby induce second messenger signaling. We investigated the latter component of this hypothesis using the short amino-terminal fragment PTH(1-14) and a truncated rat PTH-1 receptor (r delta Nt) that lacks most of the amino-terminal extracellular domain. The binding of PTH(1-14) to LLC-PK1 or COS-7 cells transfected with the intact PTH-1 receptor was too weak to detect; however, PTH(1-14) dose-dependently stimulated cAMP formation in these cells over the dose range of 1-100 microM. PTH(1-14) also stimulated cAMP formation in COS-7 cells transiently transfected with r delta Nt, and its potency with this receptor was nearly equal to that seen with the intact receptor. In contrast, PTH(1-34) was approximately 100-fold weaker in potency with r delta Nt than it was with the intact receptor. Alanine scanning of PTH(1-14) revealed that for both the intact and truncated receptors, the 1-9 segment of PTH forms a critical receptor activation domain. Taken together, these results demonstrate that the amino-terminal portion of PTH(1-34) interacts with the juxtamembrane regions of the PTH-1 receptor and that these interactions are sufficient for initiating signal transduction.     

Tumor necrosis factor and interleukin 1 inhibit parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by down-regulating parathyroid hormone receptors.

The effects of the monokines tumor necrosis factor alpha (TNF) and interleukin 1 (IL 1) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TNF and IL 1 incubated with UMR-106 cells for 48 hr each produced concentration-dependent inhibition of PTH-sensitive adenylate cyclase, with maximal inhibition of PTH response (40% for TNF, 24% for IL 1) occurring at 10(-8) M of either monokine. Both monokines also decreased adenylate cyclase stimulation by the tumor-derived PTH-related protein (PTHrP). In contrast, TNF and IL 1 had little or no inhibitory effect on receptor-mediated stimulation of adenylate cyclase by isoproterenol and nonreceptor-mediated enzyme activation by cholera toxin and forskolin; both monokines increased prostaglandin E2 stimulation of adenylate cyclase. Binding of the radioiodinated agonist mono-[125I]-[Nle8,18, Tyr34]bPTH-(1-34)NH2 to UMR-106 cells in the presence of increasing concentrations of unlabeled [Nle8,18, Tyr34]bPTH-(1-34)NH2 revealed a decline in PTH receptor density (Bmax) without change in receptor binding affinity (dissociation constant, Kd) after treatment with TNF or IL 1. Pertussis toxin increased PTH-sensitive adenylate cyclase activity but did not attenuate monokine-induced inhibition of PTH response. In time course studies, brief (1 hr) exposure of cells to TNF or IL 1 during early culture was sufficient to decrease PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. Inhibition of PTH response by monokines was blocked by cycloheximide. The results indicate that TNF and IL 1 impair responsiveness to PTH (and PTHrP) by a time- and protein synthesis-dependent down-regulation of PTH receptors linked to adenylate cyclase.     

Existence of parathyroid hormone binding sites on murine hemopoietic blast cells

We demonstrated that 125I-labeled human parathyroid hormone (1-34;8,18-Nle,34-Tyr)[[125I]hPTH(1-34)] bound specifically to hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor. Half-maximal inhibition of binding was achieved at concentrations of unlabeled hPTH(1-34) of about 5 x 10(-9)M. Insulin and hPTH(39-68) did not compete for PTH binding sites. Specific binding of hPTH(1-34) was detected in neither macrophages nor multinucleated cells (MNC's). Furthermore, treatment of hemopoietic blast cells with hPTH(1-34) stimulated MNC formation, and the range of concentrations (10(-10)-10(-8)M) over which hPTH(1-34) caused these effects was similar to that which inhibited the binding of [125I]hPTH(1-34). These findings suggest the presence of a PTH receptor on osteoclast precursors and the direct effect of PTH on them, resulting in osteoclast-mediated bone resorption.     

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.     

Activation-independent parathyroid hormone receptor internalization is regulated by NHERF1 (EBP50)

Parathyroid hormone (PTH) regulates extracellular calcium homeostasis through the type 1 PTH receptor (PTH1R) expressed in kidney and bone. The PTH1R undergoes beta-arrestin/dynamin-mediated endocytosis in response to the biologically active forms of PTH, PTH-(1-34), and PTH-(1-84). We now show that amino-truncated forms of PTH that do not activate the PTH1R nonetheless induce PTH1R internalization in a cell-specific pattern. Activation-independent PTH1R endocytosis proceeds through a distinct arrestin-independent mechanism that is operative in cells lacking the adaptor protein Na/H exchange regulatory factor 1 (NHERF1) (ezrin-binding protein 50). Using a combination of radioligand binding experiments and quantitative, live cell confocal microscopy of fluorescently tagged PTH1Rs, we show that in kidney distal tubule cells and rat osteosarcoma cells, which lack NHERF1, the synthetic antagonist PTH-(7-34) and naturally circulating PTH-(7-84) induce internalization of PTH1R in a beta-arrestin-independent but dynamin-dependent manner. Expression of NHERF1 in these cells inhibited antagonist-induced endocytosis. Conversely, expression of dominant-negative forms of NHERF1 conferred internalization sensitivity to PTH-(7-34) in cells expressing NHERF1. Mutation of the PTH1R PDZ-binding motif abrogated interaction of the receptor with NHERF1. These mutated receptors were fully functional but were now internalized in response to PTH-(7-34) even in NHERF1-expressing cells. Removing the NHERF1 ERM domain or inhibiting actin polymerization allowed otherwise inactive ligands to internalize the PTH1R. These results demonstrate that NHERF1 acts as a molecular switch that legislates the conditional efficacy of PTH fragments. Distinct endocytic pathways are determined by NHERF1 that are operative for the PTH1R in kidney and bone cells.     

Overexpression of regucalcin enhances its nuclear localization and suppresses L-type Ca2+ channel and calcium-sensing receptor mRNA expressions in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The effect of regucalcin (RC), a regulatory protein in intracellular signaling pathway, on the gene expression of various mineral ion transport-related proteins was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing RC. NRK52E cells (wild-type) and stable RC/pCXN2 transfectant were cultured for 72 h in medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured 24-72 h in a medium containing either vehicle, aldosterone (10(-8) or 10(-7) M), or parathyroid hormone (PTH) (1-34) (10(-8) or 10(-7) M) without BS. RC was markedly localized in the nucleus of transfectants. Overexpression of RC caused a significant increase in rat outer medullary K(+) channel (ROMK) mRNA expression, while it caused a remarkable decrease in L-type Ca(2+) channel and calcium-sensing receptor (CaR) mRNA expressions. Overexpression of RC did not have an effect on epithelial sodium channel (ENaC), Na, K-ATPase (alpha-subunit), Type II Na-Pi cotransporter (NaPi-IIa), angiotensinogen, Na(+)-Ca(2+) exchanger, and glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expressions. Hormonal effect on gene expression, moreover, was examined. Culture with aldosterone (10(-8) or 10(-7) M) caused a significant increase in ENaC, Na, K-ATPase, and ROMK mRNA expressions in the wild-type cells. Those increases were weakened in the transfectants. Culture with PTH (10(-8) or 10(-7) M) significantly decreased NaPi-IIa mRNA expression in the wild-type cells. This effect was not altered in the transfectants. PTH significantly decreased angiotensinogen mRNA expression in the wild-type cells and the transfectants, while aldosterone had no effect. Culture with PTH (10(-8) or 10(-7) M) caused a significant decrease in L-type Ca(2+) channel and CaR mRNA expressions in the wild-type cells, while the hormone significantly increased Na(+)-Ca(2+) exchanger mRNA expression. The effects of PTH on L-type Ca(2+) channel, CaR, and Na(+)-Ca(2+) exchanger mRNA expressions were also seen in the transfectants. This study demonstrates that overexpression of RC caused a remarkable increase in its nuclear localization, and that it has suppressive effects on the gene expression of L-type Ca(2+) channel or CaR, which regulates intracellular Ca(2+) signaling, among various regulator proteins for mineral ions in NRK52E cells.     

Photoaffinity labeling of parathyroid hormone receptors in clonal rat osteosarcoma cells

A photoreactive derivative of a sulfur-free bovine parathyroid hormone (PTH) analogue, [Nle8,N-epsilon-(4-azido-2-nitrophenyl)Lys13,Nle18,Tyr34]bovine PTH-(1-34)-NH2 (NAP-NlePTH), was purified from the products of the reaction of [Nle8,Nle18,Tyr34]bovine PTH-(1-34)-NH2 (NlePTH) with 4-fluoro-3-nitro-phenylazide and was used to identify binding components of the PTH receptor in clonal rat osteosarcoma cells (ROS 17/2.8). The purified analogue, NAP-NlePTH, is a fully active agonist in three different ROS 17/2.8 cell bioassays: 1) specific binding to saturable PTH receptors; 2) stimulation of cyclic AMP accumulation; and 3) inhibition of cellular alkaline phosphatase activity; this analogue gave dose response curves parallel to and 25-33% as potent as its parent molecule, NlePTH. Radioiodinated NAP-NlePTH (125I-labeled NAP-NlePTH) retained maximal receptor-binding potency. Radioligand saturation studies in intact cells showed that the Kd of PTH receptors for the photoligand was slightly less than that for 125I-labeled NlePTH (2.8 and 0.8 nM, respectively), but that the Bmax was essentially identical for both radioligands (8 fmol/10(5) cells). Photoaffinity labeling of ROS 17/2.8 cells revealed several 125I-labeled macromolecular components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One predominant 125I-labeled band, having an apparent Mr of 80,000 daltons (including Mr = 4,347 ligand; hereafter referred to as the Mr = 80,000 protein), was consistently demonstrated in both reducing and nonreducing conditions. Its labeling was completely inhibited by coincubation with NlePTH (10 nM) at 26-fold molar excess to the photoligand, but not by biologically inactive PTH fragments or unrelated hormone. Labeling of several other macromolecular components persisted in the presence of NlePTH (1 microM). Only the labeling of the Mr = 80,000 protein showed saturation kinetics for photoaffinity labeling; the dose of 125I-labeled NAP-NlePTH (0.8 nM) to half-saturate labeling of the Mr = 80,000 protein was close to the Kd (2.8 nM) of specific binding of the photoligand to receptors in intact ROS 17/2.8 cells. Pretreatment of the cells with NlePTH and dexamethasone led to the predicted proportional decrease or increase, respectively, in labeling of the Mr = 80,000 protein. Our data, using a highly purified photoactive derivative of PTH, having carefully defined chemical and biological properties, show a plasma membrane component of Mr = 80,000 in ROS 17/2.8 cells that possesses the affinity, binding capacity, and physiological characteristics of the PTH receptor.     

Normal human osteoclasts formed from peripheral blood monocytes express PTH type 1 receptors and are stimulated by PTH in the absence of osteoblasts

The prevailing view for many years has been that osteoclasts do not express parathyroid hormone (PTH) receptors and that PTH's effects on osteoclasts are mediated indirectly via osteoblasts. However, several recent reports suggest that osteoclasts express PTH receptors. In this study, we tested the hypothesis that human osteoclasts formed in vitro express functional PTH type 1 receptors (PTH1R). Peripheral blood monocytes (PBMC) were cultured on bone slices or plastic culture dishes with human recombinant RANK ligand (RANKL) and recombinant human macrophage colony-stimulating factor (M-CSF) for 16-21 days. This resulted in a mixed population of mono- and multi-nucleated cells, all of which stained positively for the human calcitonin receptor. The cells actively resorbed bone, as assessed by release of C-terminal telopeptide of type I collagen and the formation of abundant resorption pits. We obtained evidence for the presence of PTH1R in these cells by four independent techniques. First, using immunocytochemistry, positive staining for PTH1R was observed in both mono- and multi-nucleated cells intimately associated with resorption cavities. Second, PTH1R protein expression was demonstrated by Western blot analysis. Third, the cells expressed PTH1R mRNA at 21 days and treatment with 10(-7) M hPTH (1-34) reduced PTH1R mRNA expression by 35%. Finally, bone resorption was reproducibly increased by two to threefold when PTH (1-34) was added to the cultures. These findings provide strong support for a direct stimulatory action of PTH on human osteoclasts mediated by PTH1R. This suggests a dual regulatory mechanism, whereby PTH acts both directly on osteoclasts and also, indirectly, via osteoblasts.     

Expression of adenylate cyclase-coupled osseous parathyroid hormone and parathyroid hormone-like peptide receptors in Xenopus oocytes.

Functional parathyroid hormone (PTH) and PTH-like peptide receptors were expressed in Xenopus laevis oocytes after injection of poly(A)+ RNA isolated from the rat osteogenic sarcoma cell line, UMR 106. Increases in cAMP were seen in individual oocytes in response to added bovine (b) PTH-(1-34) (10(-6) M), human (h) PLP-(1-34) (hPLP-(1-34), 10(-6) M), isoproterenol (10(-4) M), and forskolin (10(-4) M). Although both intracellular and extracellular cAMP levels were stimulated approximately 1.5-2-fold by these agonists, intracellular concentrations of cAMP were substantially higher than extracellular concentrations. Peak increases with bPTH-(1-34) occurred after a 30-min incubation with the hormone 48 h after oocyte injection. bPTH-(1-34) caused a concentration-dependent augmentation of cAMP in injected oocytes, and the in vitro antagonist hPLP-(3-34) produced dose-dependent inhibition of both bPTH-(1-34)- and hPLP-(1-34)-stimulated cAMP accumulation. Specific binding of PTH to oocyte membranes was also demonstrated 48 h after oocyte injection with UMR 106 cell mRNA. Following size fractionation of isolated UMR 106 poly(A)+ RNA by sucrose density gradients, mRNA directing the expression of both PTH- and PLP-stimulated cAMP in oocytes appeared in the 3.5-4.9-kilobase fraction. These results demonstrate that adenylate cyclase-coupled osseous PTH and PLP receptors can be expressed after injection of naturally occurring mRNA into Xenopus oocytes, that PTH- and PLP-stimulated increases in cAMP concentrations can be detected in individual oocytes injected with bone cell-derived mRNA, that PTH and PLP appear to cross-react at a common receptor after injection of UMR 106 cell mRNA into oocytes, and that size selection of mRNA encoding the PTH and PLP receptors can be achieved by density gradient centrifugation. These studies, therefore, indicate the potential usefulness of the Xenopus oocyte system in expression cloning of PTH and PLP receptor cDNAs and illustrate the feasibility of employing this system to examine the biology of PTH and PLP receptors.     

Physiological role of vitamin A in growth cartilage cells: low concentrations of retinoic acid strongly promote the proliferation of rabbit costal growth cartilage cells in culture

We have demonstrated that high concentrations of retinoic acid (RA) inhibit expression of the differentiated phenotypes of rabbit costal chondrocytes in culture [M. Takigawa et al. (1980) Proc. Natl. Acad. Sci. U.S. 77, 1481-1485]. In this study we examined the effects of low concentrations of RA on rabbit costal chondrocytes cultured in medium containing vitamin A-deficient serum. In vitamin A-deficient medium, chondrocytes isolated from growth cartilage (GC) proliferated only very slowly, and RA strongly stimulated their proliferation. This stimulatory effect was observable at a concentration of 10(-10) M RA and maximal at a concentration of 10(-8) M. RA at 10(-8) M did not change GC cells from a typical polygonal shape to fibroblast-like cells or inhibit their synthesis of type II collagen. Moreover, RA-treated cells did not synthesize type I collagen. RA inhibited glycosaminoglycan (GAG) synthesis by the cells dose-dependently, but did not change the distribution profile of proteoglycan monomers as determined by glycerol gradient centrifugation. The inhibitory action of RA on GAG synthesis was reversible: after removal of RA from the culture, the rate of GAG synthesis increased within 2 days. In contrast, resting cartilage (RC) cells proliferated well in vitamin A-deficient medium without addition of RA, and RA (10(-8) M) stimulated their proliferation only slightly. Furthermore, the inhibitory effect of RA on GAG synthesis in RC cells was much weaker than that in GC cells. These observations suggest a physiological role of RA in cartilage in stimulating the proliferation of GC cells without causing drastic change in their differentiated phenotypes.     

Visualization of binding sites for bovine parathyroid hormone (PTH 1-84) on cultured kidney cells with a biotinyl-b-PTH (1-84) antagonist

Parathyroid hormone (PTH) receptors have been found in a subpopulation of kidney cells. In this report, we investigated the feasibility of techniques that apply a partial antagonist of PTH conjugated to biotin to localize receptors cytochemically on bovine kidney cortical cells in monolayer culture at the light microscopic level. Biotinylated bovine PTH (1-84) (biotinyl-PTH) was bound to the cultured cells for 1-30 min at 37 degrees C in the amounts of 10(-5) -10(-10) M. In a different set of experiments, the cells were also exposed to a solution containing 10(-6) M biotinylated PTH and an excess of unlabeled PTH, insulin, adrenocorticotropin, or calcitonin for 10 and 30 min at 37 degrees C to test the specificity of the binding. The cells were then fixed in 2.5% glutaraldehyde and stained with the avidin-biotin peroxidase complex (ABC) technique. Diffuse labeling was evident on 30% of the cells in 10 min with concentrations of biotinyl-PTH as low as 10(-8) M. The stain was diffuse, but more intense after 1-10 min in higher concentrations (10(-6) M). If a 15-1500-fold excess of unlabeled PTH was added to the biotinyl-PTH, no staining was observed. The other peptides (insulin, ACTH or calcitonin) had no effect on binding. Longer times in biotinyl-PTH (10(-6) M for 10-30 min) resulted in intense patches of label on the cells resembling caps (in addition to the pale diffuse label). The percentage of labeled cells in the monolayer (30%) did not change with time. These studies show that a partial antagonist of PTH can be used as a cytochemical probe for specific PTH receptors in a subpopulation of cultured cortical kidney cells.