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.     

The effect of 1,25-dihydroxyvitamin D3 on human osteoblast-like osteosarcoma cell: modification of response to PTH

The influence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on adenylate cyclase responsiveness in cultured osteoblastic cells was studied using a human osteosarcoma cell line SaOS-2. 1,25(OH)2D3 treatment had no effect on cell growth, cell protein and alkaline phosphatase activity. 1,25(OH)2D3 did not alter the basal production of cyclic AMP (cAMP) in intact cells, but the cAMP formation in response to parathyroid hormone (PTH), isoproterenol (ISO) and cholera toxin was attenuated by 1,25(OH)2D3. The response to forskolin, however, was unaffected by 1,25(OH)2D3 treatment. Islet activating protein failed to modify these 1,25(OH)2D3 effect. In cell free experiments, 1,25(OH)2D3 showed similar effect--that is, PTH and ISO-stimulated adenylate cyclase activity were attenuated, but forskolin-stimulated adenylate cyclase was unaffected. 1,25(OH)2D3 treatment had no effect on the kinetics of PTH binding to PTH receptor and on the ADP ribosylation of GTP stimulatory binding protein (Gs) in SaOS-2 cells. According to these results, 1,25(OH)2D3 appeared to change the coupling of Gs with adenylate cyclase, but does not affect receptor, Gs and adenylate cyclase themselves, nor GTP inhibitory binding protein.     

Altered EGFR localization and degradation in human breast cancer cells with an amphiregulin/EGFR autocrine loop

The epidermal growth factor receptor (EGFR) and its ligand amphiregulin (AR) have been shown to be co-over expressed in breast cancer. We have previously shown that an AR/EGFR autocrine loop is required for SUM149 human breast cancer cell proliferation, motility and invasion. We also demonstrated that AR can induce these altered phenotypes when expressed in the normal mammary epithelial cell line MCF10A, or by exposure of these cells to AR in the medium. In the present studies, we demonstrate that SUM149 cells and immortalized human mammary epithelial MCF10A cells that over express AR (MCF10A AR) or are cultured in the presence of exogenous AR, express higher levels of EGFR protein than MCF10A cells cultured in EGF. Pulse-chase analysis showed that EGFR protein remained stable in the presence of AR, yet was degraded in the presence of EGF. Consistent with this observation, tyrosine 1045 on the EGFR, the c-cbl binding site, exhibited less phosphorylation following stimulation with AR than following stimulation with EGF. Ubiquitination of the receptor was also dramatically less following stimulation with AR than following stimulation with EGF. Flow cytometry analysis showed that EGFR remained on the cell surface following stimulation with AR but was rapidly internalized following stimulation with EGF. Immunofluorescence and confocal microscopy confirmed the flow cytometry results. EGFR in MCF10A cells cultured in the presence of EGF exhibited a predominantly intracellular, punctate localization. In stark contrast, SUM149 cells and MCF10A cells growing in the presence of AR expressed EGFR predominantly on the membrane and at cell-cell junctions. We propose that AR alters EGFR internalization and degradation in a way that favors accumulation of EGFR at the cell surface and ultimately leads to changes in EGFR signaling.     

Involvement of alkaline phosphatase in the modulation of receptor signaling in osteoblasts: Evidence for a difference between human parathyroid hormone-related protein and human parathyroid hormone

We have previously reported that alkaline phosphatase (ALPase) is functionally involved in calcium uptake by several osteoblast-like cell lines. We have extended these studies to investigate the actions of ALPase on the cAMP response to and the receptor binding of human parathyroid hormone (hPTH) and human parathyroid hormone-related protein (hPTHrP). Pretreatment of human osteoblast-like SaOS-2 cells with human placental ALPase (hpALPase) inhibited the cAMP response to hPTH(1-34) but had no effect on the actions of hPTHrP(1-34) or vasoactive intestinal peptide. The inhibitory effect was reversed by L-Phe-Gly-Gly, an inhibitor of hpALPase. Treatment of SaOS-2 cells with hpALPase modestly reduced the binding of hPTH to 70% of control values, with little or no effect on the binding of hPTHrP. Bovine kidney and calf intestine ALPases were without effect on either the cAMP response or binding of hPTH or hPTHrP in SaOS-2 cells. In rat osteoblast-like ROS 17/2.8 cells, hpALPase had no effect on cAMP production stimulated by hPTH(1-34) or hPTHrP(1-34), arguing against a nonspecific effect of hpALPase. We suggest that, in SaOS-2 cells, the common PTH/PTHrP receptor can differentiate between the agonist activities of hPTH and hPTHrP by a mechanism that is sensitive to hpALPase. © 1994 Wiley-Liss, Inc.     

Quantitative analysis of agonist-dependent parathyroid hormone receptor trafficking in whole cells using a functional green fluorescent protein conjugate.

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent internalization upon activation. The parathyroid hormone (PTH) receptor undergoes endocytosis following prolonged exposure to ligand although the ultimate fate of the receptor following internalization is largely unknown. To investigate compartmentalization of the PTH receptor, we have established a stable cell line expressing a PTH receptor-green fluorescent protein (PTHR-GFP) conjugate and an algorithm to quantify PTH receptor internalization. HEK 293 cells expressing the PTHR-GFP were compared with cells expressing the wild-type PTH receptor in whole-cell binding and functional assays. 125I-PTH binding studies revealed similar Bmax and kD values in cells expressing either the PTHR-GFP or the wild-type PTH receptor. PTH-induced cAMP accumulation was similar in both cell lines suggesting that addition of the GFP to the cytoplasmic tail of the PTH receptor does not alter the ligand binding or G-protein coupling properties of the receptor. Using confocal fluorescence microscopy, we demonstrated that PTH treatment of cells expressing the PTHR-GFP conjugate produced a time-dependent redistribution of the receptor to the endosomal compartment which was blocked by pretreatment with PTH antagonist peptides. Treatment with hypertonic sucrose prevented PTH-induced receptor internalization, suggesting that the PTH receptor internalizes via a clathrin-dependent mechanism. Moreover, co-localization with internalized transferrin showed that PTHR-GFP trafficking utilized the endocytic recycling compartment. Experiments using cycloheximide to inhibit protein synthesis demonstrated that recycling of the PTHR-GFP back to the plasma membrane was complete within 1-2 h of ligand removal and was partially blocked by pretreatment with cytochalasin D, but not nocodazole. We also demonstrated that the PTH receptor, upon recycling to the plasma membrane, is capable of undergoing a second round of internalization, a finding consistent with a role for receptor recycling in functional resensitization.     

Effect of protein and steroidal osteotropic agents on differentiation and epidermal growth factor-mediated growth of the CFK1 osseous cell line.

The effects of factors known to influence bone metabolism were examined using the osseous cell line CFK1. Parathyroid hormone (PTH) and dexamethasone (DEX) appeared to enhance the formation of cell foci of CFK1 cells in culture whereas retinoic acid (RA) caused a marked alteration in individual cell morphology. Bone morphogenetic protein (BMP-2) and PTH increased alkaline phosphatase activity, however, this index of differentiation was suppressed by epidermal growth factor (EGF), DEX, and RA. BMP-2 and EGF each stimulated DNA synthesis in a dose-dependent manner and enhanced cell numbers, but, no synergistic response of EGF and BMP-2 was observed. PTH and DEX failed to significantly alter cell number or EGF-stimulated DNA synthesis or cell proliferation. Although RA treatment of CFK1 cells resulted in a reduction in cell number compared to control, pretreatment with RA enhanced EGF-stimulated DNA synthesis and proliferative effects. At least part of this effect was by increasing the EGF receptor binding capacity of the cells. Furthermore, using cell cycle analysis, addition of EGF stimulated the progression of RA-treated cells into the DNA synthesis (S) phase with a reduced lag time. EGF and BMP-2, therefore, appear to exert a role in the expansion dynamics of the CFK1 population although BMP-2 may also enhance differentiation. PTH and DEX may act primarily to modulate the differentiated function of the CFK1 cells. RA inhibited cell proliferation and may mediate differentiation towards a less established cell population with upregulation of EGF receptors. The CFK1 cell model may, therefore, provide insight into microenvironmental control of growth and differentiation of precursor osseous cells.     

Transactivation of the epidermal growth factor receptor mediates parathyroid hormone and prostaglandin F2 alpha-stimulated mitogen-activated protein kinase activation in cultured transgenic murine osteoblasts

Recent data suggest that G protein-coupled receptors (GPCRs), including those for PTH and prostaglandins (PGs), contribute to the proliferation and differentiation of osteoblasts in vivo. To understand how these signals are transduced, we studied activation of the ERK1/2 MAPK cascade in cultures of differentiating TMOb murine osteoblasts. In TMOb cells, stimulation of endogenous Gs/Gq-coupled PTH receptors, Gq-coupled PGF2 alpha receptors, and Gi/Gq-coupled lysophosphatidic acid receptors, but not Gs-coupled PGE2 receptors, caused a rapid 5- to 10-fold increase in ERK1/2 phosphorylation. GPCR-stimulated ERK1/2 activation coincided with increased tyrosine phosphorylation of epidermal growth factor (EGF) receptors and was blocked by the EGF receptor inhibitor, tyrphostin AG1478, and the metalloprotease inhibitor, batimastat, suggesting that the response involved transactivation of EGF receptors through the proteolytic release of an EGF receptor ligand. To further examine the mechanism of PTH-stimulated EGF receptor transactivation, we employed COS-7 cells expressing the rat PTH receptor. Here, stimulation with PTH(1-34) caused proteolysis of hemagglutinin epitope-tagged heparin binding-EGF, increased tyrosine autophosphorylation of EGF receptors, and AG1478-sensitive ERK1/2 activation. When PTH receptor-expressing COS-7 cells were placed in a mixed culture with cells lacking the PTH receptor but expressing a green fluorescent protein-tagged ERK2, stimulation with PTH(1-34) induced phosphorylation of green fluorescent protein-ERK2 that was abolished by either batimastat or tyrphostin AG1478. These data suggest that autocrine/paracrine cross-talk between EGF receptors and Gi- or Gq/11-coupled GPCRs represents the predominant mechanism of GPCR-mediated activation of ERK1/2 in cultured TMOb osteoblasts.     

Development of a rat parathyroid hormone 2 receptor antagonist

The parathyroid hormone 2 (PTH2) receptor is a Family B G-protein coupled receptor most highly expressed within the brain. Current evidence suggests that tuberoinfundibular peptide of 39 residues (TIP39) is the PTH2 receptor's endogenous ligand. To facilitate investigation of the physiological function of the PTH2 receptor/TIP39 system, we have developed a novel PTH2 receptor antagonist, by changing several residues within the amino terminal domain of TIP39. Histidine(4), tyrosine(5), tryptophan(6), histidine(7)-TIP39 binds the PTH2 receptor with high affinity, has over 30-fold selectivity for the rat PTH2 receptor over the rat PTH1 receptor and displays no detectable agonist activity. This ligand should be useful for in vivo investigation of PTH2 receptor function.     

Disruption of parathyroid hormone and parathyroid hormone-related peptide receptor phosphorylation prolongs ERK1/2 MAPK activation and enhances c-fos expression

Previous studies have demonstrated that parathyroid hormone (PTH) binding to the PTH/PTH-related peptide receptor (PPR) stimulates G protein coupling, receptor phosphorylation, β-arrestin translocation, and internalization of the ligand/receptor complex. The extracellular signal-regulated mitogen-activated protein kinases 1/2 (ERK1/2 MAPK) are downstream effectors of PPR. In the current study, we investigated the role of PPR phosphorylation in the PTH regulation of the ERK1/2 MAPK pathway. Short treatment with PTH (0-40 min) of LLCP-K(1) cells stably expressing a wild-type (WT) or a phosphorylation-deficient (PD) PPR (WT-PPR or PD-PPR cells, respectively) results in similar activation of ERK1/2. Interestingly, PTH stimulation of ERK1/2 in the WT-PPR cells then decreases as a result of longer PTH (60 min) treatment, and inhibition of ERK1/2 by PTH is observed at 90 min. Strikingly, the PD-PPR cells exhibit prolonged ERK1/2 activation up to 90 min of PTH treatment. An ERK1/2-dependent increase in c-fos expression is observed in the PD-PPR cells. Subsequently, c-fos expression in the WT-PPR and PD-PPR cells was markedly attenuated by a specific ERK1/2 pathway inhibitor. Further investigations revealed that PTH treatment causes a robust recruitment of a green fluorescent protein-tagged β-arrestin2 (β-arrestin2-GFP) in the WT-PPR cells. In contrast, β-arrestin2 recruitment was reduced in the PD-PPR cells. Importantly, expression of a receptor phosphorylation-independent β-arrestin2 (R169E) in the PD-PPR cells restored the biphasic effect of PTH on ERK1/2 as in the WT-PPR cells. The study reports a novel role for receptor phosphorylation and β-arrestin2 in the subsequent inhibition of the ERK1/2 pathway and in control of gene expression.     

Stimulation of arachidonic acid metabolism in primary cultures of osteoblast-like cells by hormones and drugs

The effects of parathyroid hormone (PTH), dihydroxycholecalciferol (1,25-(OH)2 D3), thrombin, epidermal growth factor (EGF) and 12-o-tetradecanoylphorbol-13-acetate (PMA) on the biosynthesis and release of arachidonic acid metabolites were studied in primary cultures of osteoblast-like cells isolated from 18-day-old chick embryo calvaria. Cells were labelled with (14C)-arachidonic acid for 30 h. The radioactive eicosanoids were extracted from the cell culture media after a further 30 h stimulation period and analysed on a PRP-1 column by HPLC. The radioactive products were characterized by co-elution of (3H) standard prostanoids. Osteoblasts showed a basal release of the prostanoids 6-keto-PGF1 alpha, TXB2, PGF2 alpha, PGE2, PGD2 and PGB2, the latter being the most abundant one. Indomethacin (10(-5) M) effectively inhibited the basal release, but not that of an as yet unidentified compound. The release of prostanoids was stimulated by PTH (2 U/ml), thrombin (0.4 NIH/ml), EGF (50 ng/ml) and PMA (25 ng/ml), the latter being by far the most potent one. 1,25-(OH)2D3 was found to slightly inhibit the prostanoid release. These results indicate: (1) primary cultures of osteoblasts synthesize several prostaglandins, thromboxane B2 and one unidentified product. (2) the action on bone of PTH and the various drugs tested may be, at least partly, mediated by an increased prostaglandin production by osteoblasts. Clearly this does not apply to 1,25-(OH)2D3.     

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.     

Molecular determinants of tuberoinfundibular peptide of 39 residues (TIP39) selectivity for the parathyroid hormone-2 (PTH2) receptor. N-terminal truncation of TIP39 reverses PTH2 receptor/PTH1 receptor binding selectivity

Tuberoinfundibular peptide of 39 residues (TIP39) and the parathyroid hormone-2 (PTH2) receptor form part of an extended family of related signaling molecules that includes the PTH1 receptor, which responds to PTH and PTH-related protein. TIP39 does not appreciably activate the PTH1 receptor, but in this study it is shown to bind the receptor with moderate affinity (59 nm). In this study, we investigated the molecular determinants of both ligand and receptor for the PTH2 receptor selectivity of TIP39 and quantitatively evaluated the role of molecular elements in the binding of TIP39 to the PTH2 and PTH1 receptors. A chimeric receptor composed of the N-terminal extracellular domain of the PTH1 receptor and the remainder (juxtamembrane domain) of the PTH2 receptor (P2-NP1) was fully activated by TIP39 (E(max) = 98% of the rPTH-(1-34), E(max), EC(50) = 2.0 nm). This receptor chimera bound TIP39 with an equivalent affinity to the wild-type PTH2 receptor (2. 3 and 2.0 nm, respectively). The reciprocal chimeric receptor (P1-NP2) was not activated by TIP39 and bound the ligand with an affinity equivalent to that of the PTH1 receptor. Thus, the juxtamembrane receptor domain specifies the signaling and binding selectivity of TIP39 for the PTH2 receptor over the PTH1 receptor. Removing six N-terminal residues of TIP39 eliminated activation of the PTH2 receptor and reduced binding affinity 70-fold. In contrast, this truncation increased affinity for the PTH1 receptor 10-fold, reversing the PTH2/PTH1 receptor binding selectivity and resulting in a high affinity interaction of TIP-(7-39) with the PTH1 receptor (6 nm). These findings can be explained by a strong interaction between the N-terminal region of TIP39 and the juxtamembrane domain of the PTH2 receptor, with the corresponding domain of the PTH1 receptor acting as a selectivity barrier against high affinity binding of TIP39. As a result, TIP-(7-39) is a highly potent, selective antagonist for the PTH1 receptor.     

Clonal differences in expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase,of 25-hydroxyvitamin D(3)-24-hydroxylase,and of the vitamin D receptor in human colon carcinoma cells: effects of epidermal growth factor and 1alpha,25-dihydroxyvitamin D(3)

Human colon carcinoma cells express 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1alpha,25-dihydroxyvitamin D(3) (1,25-D3), which can be metabolized by 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.     

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.     

Increased biological potency of hexafluorinated analogs of 1,25-dihydroxyvitamin D3 on bovine parathyroid cells

1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) is known to be involved in regulating the proliferation of parathyroid cells and PTH synthesis through reactions involving its nuclear receptor. We evaluated the effects of 1,25-(OH)₂D₃ and its hexafluorinated analog, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D₃ (26,27-F₆-1,25-(OH)₂D₃), on parathyroid cells. The 1,25-(OH)₂D₃ and 26,27-F₆-1,25-(OH)₂D₃ each inhibited [³H]thymidine incorporation and ornithine decarboxylase (ODC) activity, which is important in cell proliferation, in primary cultured bovine parathyroid cells. The inhibitory effect of 26,27-F₆-1,25-(OH)₂D₃ on PTH secretion from parathyroid cells was significantly more potent than that of 1,25-(OH)₂D ₃ between 10⁻¹¹ M and 10⁻⁸ M. Study of 26,27-F₆-1,25-(OH)₂D₃ metabolism in parathyroid cells in vitro elucidated its slower degradation than that of 1,25-(OH)₂D₃. After 48 h of incubation with [1β-³H]26,27-F₆-1,25-(OH)₂D₃, two HPLC peaks, one for [1β-³H]26,27-F₆-1,25-(OH)₂D₃, and a second larger peak for [1β-³H]26,27-F₆-1,23(S),25-(OH)₃D₃, were detected. No metabolites were detected after the same period of incubation with 1,25-(OH)₂[26,27-³H]D₃. We observed that 26,27-F₆-1,23(S),25-(OH)₃D₃ was as potent as 1,25-(OH)₂D₃ in inhibiting the proliferation of parathyroid cells.

Data suggest that the greater biological activity of 26,27-F₆-1,25-(OH)₂D₃ is explained by its slower metabolisms and by the retention of the biological potency of 26,27-F₆-1,25-(OH)₂D₃ even after 23(S)-hydroxylation.     

Role of calcium and cAMP in heterologous up-regulation of the 1,25-dihydroxyvitamin D3 receptor in an osteoblast cell line.

To understand further the mechanism of action of parathyroid hormone (PTH) in the stimulation of the number of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) binding sites in UMR 106-01 cells we studied the role of cAMP and calcium. In addition to PTH other agents known to act via the cAMP signal pathway, prostaglandin E2, forskolin and dibutyryl cAMP, caused an increase in 1,25(OH)2D3 binding. Addition of the adenylate cyclase inhibitor 9-(tetrahydro-2-furyl)adenine resulted in a marked decrease of PTH-stimulated cAMP production but this was not followed by a reduction of 1,25(OH)2D3 receptor up-regulation by PTH. Increasing the intracellular calcium concentration by Bay K 8644 and A23817 independent of an activation of the cAMP signal pathway did not result in an increased 1,25(OH)2D3 binding. The calcium channel blockers nitrendipine and verapamil and chelating extracellular calcium with EGTA all reduced cAMP-mediated stimulation of 1,25(OH)2D3 binding. This reduction was not due to a reduce cAMP production as verapamil even potentiated PTH- and forskolin-stimulated cAMP production in a dose-dependent manner. The present study provides evidence for an interrelated action of calcium and cAMP in the heterologous up-regulation of the 1,25(OH)2D3 receptor. The current data show an interaction between the cAMP and calcium signal pathway at (1) the level of cAMP generation/degradation, and (2) a level located distal in the cascade leading to 1,25(OH)2D3 receptor up-regulation.