Negative regulation of parathyroid hormone (PTH)-activated phospholipase C by PTH/PTH-related peptide receptor phosphorylation and protein kinase A

PTH binding to the PTH/PTHrP receptor activates adenylate cyclase/protein kinase A (PKA) and phospholipase C (PLC) pathways and increases receptor phosphorylation. The mechanisms regulating PTH activation of PLC signaling are poorly understood. In the current study, we explored the role of PTH/PTHrP receptor phosphorylation and PKA in PTH activation of PLC. When treated with PTH, LLCPK-1 cells stably expressing a green fluorescent protein (GFP)-tagged wild-type (WT) PTH/PTHrP receptor show a small dose-dependent increase in PLC signaling as measured by inositol trisphosphate accumulation assay. In contrast, PTH treatment of LLCPK-1 cells stably expressing a GFP-tagged receptor mutated in its carboxyl-terminal tail so that it cannot be phosphorylated (PD-GFP) results in significantly higher PLC activation (P<0.001). The effects of PTH on PLC activation are dose dependent and reach maximum at the 100 nm PTH dose. When WT receptor-expressing cells are pretreated with H89, a specific inhibitor of PKA, PTH activation of PLC signaling is enhanced in a dose-dependent manner. H89 pretreatment in PD-GFP cells causes a further increase in PLC activation in response to PTH treatment. Interestingly, PTH and forskolin (adenylate cyclase/PKA pathway activator) treatment causes an increase in PLCbeta3 phosphorylation at the Ser1105 inhibitory site and that increase is blocked by the PKA inhibitor, H89. Expression of a mutant PLCbeta3 in which Ser1105 was mutated to alanine (PLCbeta3-SA), in WT or PD cells increases PTH stimulation of inositol 1,4,5-trisphosphate formation. Altogether, these data suggest that PTH signaling to PLC is negatively regulated by PTH/PTHrP receptor phosphorylation and PKA. Furthermore, phosphorylation at Ser1105 is demonstrated as a regulatory mechanism of PLCbeta3 by PKA.     

C-terminal parathyroid hormone-related protein (PTHrP) (107-139) stimulates intracellular Ca(2+) through a receptor different from the type 1 PTH/PTHrP receptor in osteoblastic osteosarcoma UMR 106 cells.

Studies were undertaken to determine whether PTH-related protein (PTHrP) (107-139) mobilizes [Ca(2+)](i) in osteoblastic osteosarcoma UMR 106 cells. PTHrP (107-139), in a manner similar to PTHrP (107-111), induced a rapid [Ca(2+)](i) response in these cells that was dose dependent (EC(50) of approximately 0.1 pM) and more efficient than that of PTHrP (1-36) (EC(50) of approximately 1 nM). This effect of PTHrP (107-139) was abrogated by micromolar doses of verapamil or nifedipine. However, it was unaffected by 10 microM U73122 (a phospholipase C inhibitor), 100 microg/ml heparin (an inositol 1,4,5-trisphosphate receptor inhibitor), or 400 ng/ml pertussis toxin (a G(i) inhibitor), which inhibited the [Ca(2+)](i) response to PTHrP (1-36), or by either 25 nM bisindolylmaleimide I (BIM), a protein kinase (PK) C inhibitor, or 1 microM phorbol-12-myristate-13-acetate preincubation (22 h). PTHrP (107-139) and PTHrP (1-36), at 100 nM, desensitized the [Ca(2+)](i) response to a second challenge with the same peptide, but not with the other peptide in these cells. PTHrP (7-34), a type 1 PTH/PTHrP receptor (PTH1R) antagonist, decreased the effect of PTHrP (1-36) on [Ca(2+)](i). In contrast, PTHrP (107-111), but neither PTHrP (109-138) nor PTHrP (7-34), abolished this effect of PTHrP (107-139). Both PTHrP (107-139) and PTHrP (1-36), added together at submaximal doses, induced a higher [Ca(2+)](i) response. Moreover, PTHrP (107-139) increased the efficacy of PTHrP (1-36) on [Ca(2+)](i), but decreased its induced increase in PKA activity in these cells. Verapamil or nifedipine (at 50 microM) or 25 nM BIM, but not 25 microM adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer, a PKA inhibitor, abolished the PTHrP (107-139)-induced increase in interleukin 6 messenger RNA (assessed by RT, followed by PCR) in UMR 106 cells. This peptide also increased c-fos messenger RNA in these cells; an effect inhibited by BIM, but unaffected by either verapamil or EGTA. These findings support the existence of high-affinity receptors for PTHrP (107-139), associated with an induced Ca(2+) influx, different from the PTH1R in UMR 106 cells. The present results suggest that PTHrP could affect bone turnover by interacting with the PTH1R and other yet unknown receptors in bone cells through complex mechanisms.     

Parathyroid hormone activates mitogen-activated protein kinase in opossum kidney cells

Many G protein-coupled receptor agonists activate p42/p44 mitogen-activated protein kinase (MAPK), using signaling pathways that are a function of receptor, G protein-coupled, and effector complement. In opossum kidney (OK) cells, activation of endogenous PTH receptors caused a time- (peak within 15-30 min, sustained for approximately 2 h) and dose-dependent (EC50 approximately 3 x 10(-10) M) activation of MAPK. Immunoblot analysis with an activation- specific MAPK antibody indicated that PTH activated both p42 and p44 MAPK. Epidermal growth factor (EGF) also activated p42 and p44MAPK in a time- (peak at 5 min, return to basal within 2 h) and dose-dependent (EC50 approximately 3 ng/ml) fashion. PTH-dependent MAPK activation was mimicked by the protein kinase C activator (PKC) phorbol myristate acetate (PMA), and the protein kinase A activators 8 bromo-cAMP (8-Br-cAMP) and forskolin but was not affected by pertussis toxin pretreatment. PMA or 8-Br-cAMP pretreatment blocked MAPK activation by reexposure to each kinase activator but caused no significant reduction in MAPK activation by PTH. MAPK activation by PTH, EGF, and 8-Br-cAMP was inhibited by the MAPK kinase inhibitor PD98059 and an EGF receptor (EGFR)-selective inhibitor tyrphostin AG1478. AG1478 also blocked MAPK activation by insulin-like growth factor-1 and platelet-derived growth factor. EGF and PTH caused time- and AG1478-sensitive phosphorylation of the EGFR, but EGFR desensitization did not affect MAPK activation by PTH. EGF, PMA, and low doses of PTH (10(12) to 10(-9) M) stimulated while 8-Br-cAMP and high doses of PTH (10(-8) to 10(-6) M) inhibited [3H]thymidine uptake. These data demonstrate that PTH activates MAPK and suggest that PKC, protein kinase A, and the EGFR play roles in PTH signaling. The biphasic effect of PTH on DNA synthesis suggests that MAPK activation by the hormone leads to distinct cellular responses.     

Increased osteoblastic c-fos expression by parathyroid hormone requires protein kinase A phosphorylation of the cyclic adenosine 3',5'-monophosphate response element-binding protein at serine 133

PTH induces c-fos expression rapidly and transiently in osteoblastic cells and requires the activity of the cAMP response element-binding protein (CREB). Here we provide evidence that protein kinase A (PKA) is the enzyme responsible for phosphorylating CREB at serine 133 (S133) and that this event is required for PTH-induced c-fos expression. PTH increases the level of phosphorylation of CREB at S133 in a time- and dose-dependent manner, correlating with the time and level of activation of PKA in response to PTH. PTH-(1-34) and -(1-31), each known to activate the cAMP pathway, induced the phosphorylation of CREB and increased the levels of c-fos messenger RNA, whereas PTH-(3-34), -(13-34), and -(28-48) could not. Specific inhibitors of calcium/calmodulin-dependent protein kinases and protein kinase C could not inhibit CREB phosphorylation or c-fos expression in response to PTH; however, H-89, a specific inhibitor of PKA, could do so in a dose-dependent manner. In addition, PTH-induced c-fos promoter activity was completely inhibited in a dose-dependent fashion by transfection of the heat-stable inhibitor of PKA. Taken together, these data provide strong evidence that PKA is the enzyme responsible for phosphorylating CREB at S133 in response to PTH and that PKA activity is required for PTH-induced c-fos expression.     

The mutual regulation of arginine-vasopressin and PTHrP secretion in dissociated supraoptic neurons

PTHrP is detected in the supraoptic nucleus (SON) and paraventricular nucleus. We have recently demonstrated that PTHrP(1-34) is involved in AVP release and synthesis in the SON in vivo and in vitro. PTHrP and AVP, which act on blood vessels, may interact by autocrine and paracrine mechanisms in the central nervous system. The present study was undertaken to determine the mutual regulation of AVP and PTHrP secretion in dissociated magnocellular neurons of the SON. Both AVP and PTHrP existed in the dissociated SON neurons by immunohistochemistry. PTHrP(1-34) stimulated AVP secretion from the cells dose dependently, but PTHrP(7-34) and PTH(1-34) did not. PTHrP(1-34)-stimulated AVP secretion was associated with cAMP generation. PTHrP(1-34)-induced cAMP generation was inhibited by a 100-fold molar excess of PTHrP(7-34) but not by that of PTH(1-34). PTHrP(1-34) also stimulated AVP mRNA expression in the cells. These results are consistent with our previous observations that PTHrP(1-34) is involved in AVP secretion through a receptor distinct from type I PTH/PTHrP receptor. Next, AVP stimulated dose-dependent PTHrP release from the dissociated SON neurons. The AVP-induced PTHrP release was suppressed by both OPC-21268 (V(1a) receptor antagonist) and dP[Thy(Me)(2)]AVP (V(1a)/V(1b) receptor antagonist) but not by OPC-31260 (V(2) receptor antagonist). AVP increased PKC activity dose dependently but not cAMP generation in the SON neurons. The AVP-stimulated PTHrP release was blocked by staurosporine (PKC inhibitor), nicardipine (L-type calcium channel blocker) or omega-agatoxin IVA (N type). Furthermore, AVP stimulated PTHrP mRNA expression for 12 h in the SON neurons. These results indicate that AVP caused increases in PTHrP secretion and its mRNA levels through V(1a) and/or V(1b) receptors in the SON neurons. Our observations, taken together, suggest that PTHrP stimulates AVP secretion into the extracellular space of the SON, which in turn leads to further secretion of AVP and PTHrP by an autocrine/paracrine mechanism.     

The intracellular signal transduction systems of PTH and its effects on bone resorption and formation

PTH binds to PTH/PTHrP receptor, one of seventh transmembrane receptor, which activates cAMP/PKA system through Gs protein and causes an activation of PKC and an elevation of cytosolic calcium through Gq protein. PTH affects bone cells through these dual second messenger signaling systems(cAMP/PKA and Ca/PKC system). Recent studies revealed that PTH stimulates bone resorption as well as bone formation mainly through cAMP/PKA system. Further work is necessary to clarify the downstream of the signal, such as CREB and AP-1.     

Role of signal transduction in internalization of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein

For G protein-coupled receptors, limited information is available on the role of agonist binding or of the second-messenger products of receptor signaling on receptor endocytosis. We explored this problem using the opossum PTH/PTH-related protein (PTHrP) receptor, a prototypical Class II G protein-coupled receptor, as a model. In one approach, we evaluated the endocytic properties of mutated forms of the opossum PTH/PTHrP receptor that we had previously shown to be impaired in their ability to initiate agonist-induced signaling when expressed in COS-7 cells. A point mutation in the third cytoplasmic loop (K382A) that severely impairs PTH/PTHrP receptor signaling significantly reduced internalization, whereas two mutant receptors that displayed only partial defects in signaling were internalized normally. To explore more directly the role of second-messenger pathways, we used a cleavable biotinylation method to assess endocytosis of the wild-type receptor stably expressed in human embryonic kidney (HEK) 293 cells. A low rate of constitutive internalization was detected (<5% over a 30-min incubation at 37 C); the rate of receptor internalization was enhanced about 10-fold by the receptor agonists PTH(1-34) or PTHrP(1-34), whereas the receptor antagonist PTH(7-34) had no effect. Forskolin treatment produced a minimal increase in constitutive receptor endocytosis, and the protein kinase (PK)-A inhibitor H-89 failed to block agonist-stimulated endocytosis. Similarly, activation of PK-C, by treatment with phorbol 12-myristate 13-acetate, elicited only a minimal increase in constitutive receptor endocytosis; and blockade of the PK-C pathway, by treatment with a bisindolylmaleimide, failed to inhibit agonist-induced receptor endocytosis. Immunofluorescence confocal microscopic studies of PTH/PTHrP receptor internalization confirmed the results using receptor biotinylation. These findings suggest that: 1) agonist binding is required for the efficient endocytosis of the PTH/PTHrP receptor; 2) receptor activation (agonist-induced receptor conformational change) and/or coupling to G proteins plays a critical role in receptor internalization; and 3) activation of PK-A and PK-C is neither necessary nor sufficient for agonist-stimulated receptor internalization.     

Mutation of phenylalanine-34 of parathyroid hormone disrupts NHERF1 regulation of PTH type I receptor signaling

Internalization of the PTH type I receptor (PTH1R) is regulated in a cell- and ligand-specific manner. We previously demonstrated that the sodium/proton exchanger regulatory factor type 1 (NHERF1; EBP50) is pivotal in determining the range of peptides that internalize the PTH1R. Antagonist PTH fragments can internalize the PTH1R in some kidney and bone cell models. PTH(7-34), which binds to, but does not activate, the PTH1R, internalizes the PTH1R in kidney distal tubule (DT) cells, where NHERF1 is not expressed. The effect of antagonist PTHrP peptides has not, to this point, been assessed. PTH1R internalization was measured by real-time confocal fluorescence microscopy of DT cells stably expressing 10⁵ EGFP-tagged PTH1R/cell (1). PTHrP(7-34) internalized the PTH1R in a manner indistinguishable from PTH(7-34). Introduction of NHERF1 into DT cells, however, blocked PTH(7-34)—, but not PTHrP(7-34)—, induced PTH1R internalization. To delineate the sequences within PTHrP that determine whether PTH1R internalization is affected by NHERF1, chimeric PTH/PTHrP fragments were tested for their ability to induce PTH1R internalization. PTH(7-21)/PTHrP (22-34), PTH(7-32)/PTHrP(33-34), and PTH(7-33)/PTHrP(34) at 1 μM each internalized the PTH1R 50–70% in a NHERF1-independent manner. When the C terminus of PTHrP was replaced with homologous amino acids from PTH, NHERF1 inhibited PTH1R internalization. It was determined that simply mutating F34 to A in PTH induced PTH1R internalization in a NHERF1-independent manner. None of the chimeric peptides activated the PTH1R but all effectively competed for 1 nM PTH(1-34) in cyclic AMP assays. In addition, all chimeric peptides competed for radiolabeled PTH(1-34) in binding assays in DT cells. PTH(1-34) and PTHrP(7-34), but not PTH(7-34), efficiently recruited β-arrestin1 to plasma membrane PTH1Rs. We, therefore, conclude that PTH(1-34) and PTHrP(7-34) induce a conformational change in the PTH1R that promotes arrestin binding and dissociates NHERF1 from PTH1R internalization.     

甲状旁腺激素调控人成骨样细胞膜型基质金属蛋白酶-1表达

目的 探讨甲状旁腺激素(PTH)对人成骨样MG-63细胞膜型基质金属蛋白酶-1(MT1-MMP)表达的影响及调节骨代谢作用的机制。方法 用PTH(1-34)干预MG-63细胞培养，Northern杂交及Western杂交检测MT1-MMP mRNA与蛋白质表达水平。结果 PTH(1-34)10^-9mmol／L～10^-7mmol／L浓度对MG-63细胞MT1-MMP mRNA及蛋白质表达有抑制作用，并呈剂量依赖性。10^-8mmol／L干预在2～48h内对抑制MT1-MMP mRNA及蛋白质表达抑制有时间依赖性。蛋白激酶A(PKA)抑制剂H-89阻断PTH(1-34)抑制MT1-MMP表达的效应，而蛋白激酶A(PKA)激动剂Forskolin抑制MT1-MMP表达。结论 PTH具有抑制成骨样细胞MT1-MMP表达的作用，其作用途径可能有PKA信号转导途径参与。     

Parathyroid hormone-related protein regulates osteoclast inhibitory lectin expression via multiple signaling pathways in osteoblast-like cells

Osteoclast inhibitory lectin (OCIL) is a recently identified inhibitor of osteoclast formation. A variety of osteotropic factors regulate OCIL expression in osteoblastic cells, however, little information is available to date concerning how this gene is controlled. Using real-time RT-PCR, we examined the regulation of OCIL expression by PTHrp and the signaling pathways used. We demonstrated in rat osteoblast-like UMR-106 cells, rat calvarial primary osteoblastic cells, and murine MC3T3-E1 cells, PTHrp(1–34) increased OCIL expression. In UMR-106 cells, the increase began and reached maximum later than RANKL induction and OPG suppression. cAMP/PKA signaling activators PTH(1–31), forskolin and dibutyryl cAMP (db-cAMP), and calcium ionophore A23187 all increased OCIL levels. In contrast, PKC activator phorbol-12-myristate-13-acetate reduced OCIL expression in short term but induced OCIL mRNA in long term. PKA inhibitor KT5720, mitogen-activated protein kinase (MAPK) cascade inhibitor PD98059, calmodulin antagonist W-7, and Ca²⁺/calmodulin-dependent protein kinase II (CaMK II) inhibitor KN-62 all significantly blunted PTHrp-stimulated OCIL expression. Moreover, PD98059 blocked the stimulation of OCIL by FSK or db-cAMP but not that by A23187. In primarily cultured osteoblasts, the PTHrp induction of OCIL was blocked by KT5720, W-7, and PD98059 as well. The data established that PTHrp(1–34) regulates OCIL expression in vitro through cAMP/PKA, Ca²⁺/CaMK II, and MAPK signaling pathways. Fang Zheng and Hui Liang contributed equally to this work.     

The protein kinase-C activation domain of the parathyroid hormone.

The PTH activates both adenylate cyclase and a mechanism that increases membrane-associated protein kinase-C (PKC) activity. To define the hormone's PKC activation domain we have used a panel of PTH fragments and ROS 17/2 rat osteosarcoma cells as the target cells. PTH equally and maximally increased PKC activity in ROS 17/2 cell membranes at physiological concentrations between 1-50 pM and 5-50 nM, but not at intermediate concentrations or concentrations above 50 nM. The PKC-stimulating picomolar concentrations of PTH did not stimulate adenylate cyclase in ROS 17/2 cells, while the PKC-stimulating nanomolar concentrations of the hormone did stimulate adenylate cyclase, with an EC50 of 1-2 nM. Very high concentrations of PTH, such as 100 nM, that did not increase membrane PKC activity were still able to maximally stimulate adenylate cyclase. PTH fragments lacking the N-terminal amino acids needed for adenylate cyclase activation increased membrane PKC activity, and the PKC activation domain was found to lie within the 28-34 region of the PTH molecule. This was confirmed by showing that optimally effective picomolar concentrations of the human PTH-(28-34) fragment itself were able to increase membrane-associated PKC activity to the same extent as the optimally effective picomolar concentrations of the intact PTH-(1-84) or the larger PTH-(1-34) or PTH-(3-34) fragments.     

Regulation of PTH/PTH-related protein receptor expression by endogenous PTH-related protein in the rat osteosarcoma cell line ROS 17/2.8

We have utilized clonal lines of the rat osteoblastic cell line ROS 17/2.8 stably transfected with full-length parathyroid hormone-related protein (PTHrP) cDNA in a sense or an antisense orientation to examine the effects of alteration in the production of endogenous PTHrP on expression of the PTH/PTHrP receptor. In the stably transfected clonal cell lines, changes in PTH/PTHrP receptor expression were evaluated by Northern blot analysis, whole-cell ligand binding of ¹²⁵I-[Tyr³⁶] PTHrP (1–36), and exogenous PTHrP (1–34)-stimulated cyclic adenosine monophosphate (cAMP) accumulation. Compared to control (vector-transfected) cells, PTHP-overproducing (sense-transfected) cells exhibited a marked decrease in the expression of PTH/PTHrP receptor mRNA and PTHrP ligand binding, as well as a corresponding decrease in the PTHrP (1–34)-stimulated cAMP response. By contrast, the antisense-transfected cells showed a marked increase in expression of PTH/PTHrP receptor mRNA and PTHrP (1–34) ligand binding, but a significant increase in the PTHrP (1–34)-stimulated cAMP response was not detected. Using antisense-transfected ROS cells, PTH/PTHrP receptor mRNA expression and ¹²⁵I-[Tyr³⁶] PTHrP (1–36) binding were downregulated by treatment for 24 h with exogenous PTHrP (1–36), forskolin, or dibutyryl cAMP. The findings extend those of earlier studies showing receptor downregulation by exogenous PTH by indicating that endogenous PTHrP, as well as circulating PTH, may help regulate receptor production; and suggesting that even very low concentrations of the peptide may influence receptor production.     

Parathyroid hormone(1-34) and parathyroid hormone-related protein(1-34) stimulate calcium release from human syncytiotrophoblast basal membranes via a common receptor

The placental syncytiotrophoblast is the site for mineral and nutrient exchange across the maternal-fetal interface. It has been proposed that parathyroid hormone-related protein (PTHrP) is a key factor in the maintenance of a maternal-fetal calcium gradient. Using simultaneously prepared microvillous (maternal facing) and basal (fetal facing) syncytiotrophoblast membranes from term human placentae (n=8), we determined the relative contribution of PTH(1-34), PTHrP(1-34) and PTHrP(67-94) to the regulation of syncytiotrophoblast calcium efflux. The vesicles had correct right-side-out membrane orientation and specific markers validated the fractionation of microvillous and basal membrane vesicles. Calcium efflux was studied by preloading vesicles with calcium-45 in the presence of calcium and magnesium and then incubating the vesicles at 37 degrees C for 15 min with the peptides. In basal membranes, PTHrP(1-! 34) significantly stimulated calcium efflux at a dose of 12.5 nmol/l, whereas PTH(1-34)-stimulated efflux was significant at 50 nmol/l (P<0.05, ANOVA). This efflux was significantly reduced in the presence of the PTH/PTHrP receptor antagonist (PTHrP(7-34)). Midmolecule PTHrP(67-94) had no significant effect on basal membrane calcium efflux. PTH(1-34), PTHrP(1-34) or PTHrP(67-94) had no significant effects on MVM calcium efflux. This study, using the human syncytiotrophoblast in vitro membrane system, demonstrated that PTHrP(1-34) and PTH(1-34) stimulate calcium transport across the basal, but not microvillous, syncytiotrophoblast membrane vesicles, mediated via the PTH/PTHrP receptor.