In vitro and in vivo antagonists against parathyroid hormone-related protein

Four analogues of parathyroid hormone-related protein (PTHrP), PTHrP(7-34)NH2, (10-34)NH2, (15-34)NH2 and (20-34)NH2, were synthesized and their antagonistic activity against PTHrP(1-34) was examined in vitro and in vivo. In vitro studies revealed that all four analogues antagonized PTHrP-stimulated cyclic AMP production in rat osteosarcoma cells (ROS 17/2.8), and that PTHrP(7-34)NH2 and PTHrP(10-34)NH2 had potent antagonistic activity. In vivo experiments in nude mice also revealed that PTHrP(7-34)NH2 completely inhibited hypercalcemia induced by PTHrP(1-34), indicating that these analogues antagonize the effects of PTHrP(1-34) in vitro and in vivo.     

Parathyroid hormone-related protein (PTHrP) inhibits mitochondrial-dependent apoptosis through CK2

Over the past decade, parathyroid hormone-related protein (PTHrP) has been identified as a key survival factor for cells subjected to apoptotic stimuli. Its anti-apoptotic activity has been attributed to nuclear accumulation of the intact protein, or a synthetic peptide corresponding to its nuclear targeting sequence (NTS), which promotes rapid exit of nutrient deprived cells from the cell cycle. Intracellular PTHrP also inhibited apoptosis by blocking tumor necrosis factor alpha (TNFalpha)-induced apoptosis by blocking signaling from the "death receptor" and preventing damage to the mitochondrial membrane. In both cases, the anti-apoptotic activity was significantly reduced in the presence of a nuclear deficient form of PTHrP with a (88)K/E K/E.K/I(91) mutation in the NTS. The current work was undertaken to determine the mechanism by which nuclear PTHrP blocked mitochondrial-mediated apoptosis. Using sub-cellular fractionation and functional assays we showed that pre-treatment of HEK293 cells with exogenous NTS peptide before inducing apoptosis with TNFalpha was as effective as expression of the full-length protein in inhibiting apoptosis. Inhibition of apoptosis was associated with increased expression of protein kinase casein kinase 2 (CK2) and in sustained CK2 accumulation and activity in the nuclear fraction. In primary chondrogenic cells harvested from the limb buds of PTHrP(+/-) and PTHrP(-/-) embryonic mice, there was a dose-dependent decrease in CK2 expression and activity that correlated with increased susceptibility to apoptosis. Taken together the results indicate that nuclear accumulation of PTHrP effectively inhibits mitochondrial-mediated apoptosis through regulation of the expression, activity, and sub-cellular trafficking of CK2.     

Calcitonin-suppressed expression of parathyroid hormone-related protein in breast cancer cells.

Parathyroid hormone-related protein (PTHrP) is a key factor behind humoral hypercalcemia of malignancy (HHM). It is produced in most breast tumors and may be an important local mediator of skeletal metastases due to breast cancer. PTHrP may mediate local bone destruction in the absence of increased circulating PTHrP. Calcitonin (CT) is used for treatment of HHM, but there are data showing that CT can increase PTHrP expression and secretion in vitro. We have therefore studied the effect of CT on PTHrP gene expression and secretion in MCF-7 breast cancer cells. PTHrP mRNA decreased significantly after 4, 8, and 16 h incubation with 10 nM salmon calcitonin (sCT) when compared with the respective controls. PTHrP mRNA also decreased significantly and dose-dependently after incubation with sCT at 0.1 to 10 nM for 16 h. The PTHrP levels in the conditioned medium also decreased in a similar dose-dependent manner. The adenylate cyclase agonist forskolin lowered the PTHrP mRNA dose-dependently. In cells exposed to varying concentrations of sCT for 15 min, the cAMP levels increased dose-dependently. In conclusion, sCT can suppress PTHrP gene expression in MCF-7 breast cancer cells. The suppressive effect is probably exerted mainly via the cAMP-protein kinase A pathways.     

The activation of cAMP-dependent protein kinase is directly linked to the inhibition of osteoblast proliferation (UMR-106) by parathyroid hormone-related protein

The present study was performed to compare the effect of parathyroid hormone-related protein (PTHrP) on the proliferation of osteoblastic osteosarcoma cells (UMR-106) with that of PTH and characterize the direct involvement of cAMP in the change of osteoblast proliferation by PTHrP. Human(h)PTHrP-(1-34) (10(-11)-10(-7)M) dose-dependently inhibited [3H]thymidine incorporation (TdR) in the same manner as hPTH-(1-34). The simultaneous addition of PTHrP and PTH at a maximal effective dose of 10(-7) M did not cause additive suppressive effect on cell proliferation. Rp-cAMPs, which has been recently shown to act directly as antagonist in the activation of cAMP-dependent protein kinase (PKA), dose-dependently (10(-6)-10(-4)M) antagonized PTHrP-induced suppression of TdR in the same manner as PTH. Present study indicated that PTHrP has the same effect on osteoblast proliferation as PTH and that the activation of PKA is directly linked to the change of osteoblast proliferation by PTHrP.     

Switching of G-protein usage by the calcium-sensing receptor reverses its effect on parathyroid hormone-related protein secretion in normal versus malignant breast cells

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that signals in response to extracellular calcium and regulates parathyroid hormone secretion. The CaR is also expressed on normal mammary epithelial cells (MMECs), where it has been shown to inhibit secretion of parathyroid hormone-related protein (PTHrP) and participate in the regulation of calcium and bone metabolism during lactation. In contrast to normal breast cells, the CaR has been reported to stimulate PTHrP production by breast cancer cells. In this study, we confirmed that the CaR inhibits PTHrP production by MMECs but stimulates PTHrP production by Comma-D cells (immortalized murine mammary cells) and MCF-7 human breast cancer cells. We found that changes in intracellular cAMP, but not phospholipase C or MAPK signaling, correlated with the opposing effects of the CaR on PTHrP production. Pharmacologic stimulation of cAMP accumulation increased PTHrP production by normal and transformed breast cells. Inhibition of protein kinase A activity mimicked the effects of CaR activation on inhibiting PTHrP secretion by MMECs and blocked the effects of the CaR on stimulating PTHrP production in Comma-D and MCF-7 cells. We found that the CaR coupled to Galpha(i) in MMECs but coupled to Galpha(s) in Comma-D and MCF-7 cells. Thus, the opposing effects of the CaR on PTHrP production are because of alternate G-protein coupling of the receptor in normal versus transformed breast cells. Because PTHrP contributes to hypercalcemia and bone metastases, switching of G-protein usage by the CaR may contribute to the pathogenesis of breast cancer.     

Nuclear transport of parathyroid hormone (PTH)-related protein is dependent on microtubules

PTH-related protein (PTHrP) was first discovered as a circulating factor secreted by certain cancers and is responsible for the syndrome of humoral hypercalcemia of malignancy induced by various tumors. The similarity of its N terminus to that of PTH enables PTHrP to share the signaling properties of PTH, but the rest of the molecule possesses distinct functions, including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. PTHrP nuclear import is mediated by importin beta1. In this study we use the technique of fluorescence recovery after photobleaching to demonstrate the ability of PTHrP to shuttle between cytoplasm and nucleus and to visualize directly the transport of PTHrP into the nucleus in living cells. Endogenous and transfected PTHrP was demonstrated to colocalize with microtubule structures in situ using various high-resolution microscopic approaches, as well as in in vitro binding studies, where importin beta1, but not importin alpha, enhanced the microtubular association of PTHrP with microtubules. Significantly, the dependence of PTHrP nuclear import on microtubules was shown by the inhibitory effect of pretreatment with the microtubule-disrupting agent nocodazole on nuclear-cytoplasmic flux. These results indicate that PTHrP nuclear/nucleolar import is dependent on microtubule integrity and are consistent with a direct role for the cytoskeleton in protein transport to the nucleus.     

Parathyroid hormone related protein (PTHrP) inhibits TNFalpha-induced apoptosis by blocking the extrinsic and intrinsic pathways

Parathyroid hormone related protein (PTHrP) is expressed at low levels in many fetal and adult tissues where it plays a central role in regulating cell proliferation, cell death, and tissue homeostasis. In vivo and in vitro, PTHrP has been shown to promote the survival of a variety of cells by regulating expression of the anti-apoptotic protein Bcl2. Additional work has shown that intra-nuclear accumulation of PTHrP in CFK2 (PTH1R positive) and 27m21 (PTH1R negative) condrogenic cells promotes their survival by closing down ribosome biogenesis and promoting quiescence. The current studies were undertaken to examine the role of wild-type PTHrP and a mutant form that cannot translocate to the nucleus in protecting cells from TNFalpha-induced apoptosis. Both forms of the protein were equally effective in blocking the extrinsic pathway by inhibiting expression of the TNF receptor death domain, activating Bid, and promoting cleavage of caspase 8. These observations suggest a direct mechanism of PTHrP action on components of the extrinsic pathway, involving a region of the protein outside of the NTS. PTHrP and M1PTHrP also inhibited the intrinsic pathway by preventing the exchange of anti-apoptotic for pro-apoptotic proteins at the mitochondrial membrane, thus maintaining its integrity and preventing the release of caspase-activating factors into the cytosol. In general, this mitochondrial-related activity was somewhat delayed and was mediated more effectively by PTHrP than by M1PTHrP, suggesting an indirect mechanism of action that might require the presence of an intact NTS.     

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.     

Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways

Parathyroid hormone (PTH)-related peptide (PTHrP) can modulate the proliferation and differentiation of a number of cell types including osteoblasts. PTHrP can activate a G protein-coupled PTH/PTHrP receptor, which can interface with several second-messenger systems. In the current study, we have examined the signaling pathways involved in stimulated type I collagen and alkaline phosphatase expression in the human osteoblast-derived osteosarcoma cells, MG-63. By use of Northern blotting and histochemical analysis, maximum induction of these two markers of osteoblast differentiation occurred after 8 h of treatment with 100 nM PTHrP-(1-34). Chemical inhibitors of adenylate cyclase (H-89) or of protein kinase C (chelerythrine chloride) each diminished PTHrP-mediated type I collagen and alkaline phosphatase stimulation in a dose-dependent manner. These effects of PTHrP could also be blocked by inhibiting the Ras-mitogen-activated protein kinase (MAPK) pathway with a Ras farnesylation inhibitor, B1086, or with a MAPK inhibitor, PD-98059. Transient transfection of MG-63 cells with a mutant form of Galpha, which can sequester betagamma-subunits, showed significant downregulation of PTHrP-stimulated type I collagen expression, as did inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) by wortmannin. Consequently, the betagamma-PI 3-kinase pathway may be involved in PTHrP stimulation of Ras. Collectively, these results demonstrate that, acting via its G protein-coupled receptor, PTHrP can induce indexes of osteoblast differentiation by utilizing multiple, perhaps parallel, signaling pathways.     

Osteolytic activity of Walker carcinosarcoma 256 is due to parathyroid hormone-related protein (PTHrP).

The hypercalcemic Walker carcinosarcoma 256 of the rat is an animal model for humoral hypercalcemia of malignancy. Previous in vivo studies suggested the production of a parathyroid hormone-related protein (PTHrP) by the Walker tumor. Therefore, we have measured immunoreactive PTHrP in serum-free conditioned medium from cells derived from this tumor using an antibody raised against human PTHrP(1-34). Walker tumor cell conditioned medium (WCM) displaced 125I-hPTHrP(1-34) from the antibody in a dose dependent manner, whereas control medium contained no immunoreactive PTHrP. In contrast, we detected no secretion of immunoreactive rat parathyroid hormone (rat PTH) by the Walker tumor cells using a midregional radioimmunoassay for rat PTH. WCM stimulated adenylate cyclase in osteoblast like cells, the dose-response curve paralleling that of hPTHrP(1-34). This effect could be inhibited by the PTH antagonist (8Nle, 18Nle, 34Tyr)bPTH(3-34) and by the addition of anti-hPTHrP(1-34) antibody. Bone resorbing activity of WCM in organ culture (calvaria of fetal rats) was not inhibited by indomethacin and glucocorticoids, suggesting a prostaglandin independent mechanism of osteoclast activation in this model.     

The parathyroid hormone-related protein associated with malignancy is secreted by neuroendocrine tumors

We have demonstrated the production of the PTH-related protein (PTHrP) associated with hypercalcemia of malignancy by human neuroendocrine cell lines that also produce calcitonin gene products and chromogranin A. PTHrP was demonstrable in the cells by immunocytochemistry and immunoassay and Northern analysis of the cells revealed the presence of multiple mRNAs for PTHrP. The cell lines also secreted PTHrP in a regulated fashion, with the most potent secretagogue being phorbol. Thus, PTHrP is secreted by neuroendocrine cells and it may have neuroectodermal lineage. The coexpression of calcitonin gene products and chromogranin A, also neuroendocrine, with PTHRP may influence its secretion and ultimate biological effects in vivo.     

Expression of parathyroid hormone-related protein (PTHrP) in parathyroid tissue under normal and pathological conditions

Parathyroid hormone-related protein (PTHrP), a factor responsible for malignancy associated hypercalcemia, plays a physiological roles such as bone development and placental calcium transport. The expression of PTHrP in adult human parathyroid tissues under normal and pathological conditions was analyzed. By immunohistochemistry, PTHrP expression was detected in 86% of normal parathyroid (12/14 cases), 74% of adenomas (14/19) and 89% of hyperplasia secondary to chronic renal failure (16/18). PTHrP protein was observed mainly in the cytoplasm of oxyphil cells, consistent with the localization of its mRNA demonstrated by in situ hybridization. The rate of PTHrP-positive cells was higher in areas consisting of oxyphil cells than in those of non-oxyphil cells, regardless of whether the parathyroid was normal or pathological. In the normal parathyroid, an age-related increase in PTHrP expression was observed with a relative increase in oxyphil cells, reflecting aging and deterioration of parathyroid tissue. In adenoma, cases with a predominance of oxyphil cells expressed PTHrP, whereas clear cell adenoma did not. In secondary hyperplasia, the rate of PTHrP-expressing cells was higher than in normal parathyroid or adenoma, with varying levels of expression among nodules. We speculate that PTHrP could act through the paracrine/autocrine mechanism to regulate proliferation and differentiation of normal and neoplastic parathyroid cells.     

Negative regulation of parathyroid hormone-related protein expression by steroid hormones

Elevated parathyroid hormone-related protein (PTHrP) is responsible for humoral hypercalcemia of malignancy (HHM), which is of clinical significance in treatment of terminal patients with malignancies. Steroid hormones were known to cause suppression of PTHrP expression. However, detailed studies linking multiple steroid hormones to PTHrP expression are lacking. Here we studied PTHrP expression in response to steroid hormones in four cell lines with excessive PTHrP production. Our study established that steroid hormones negatively regulate PTHrP expression. Vitamin D receptor, estrogen receptor α, glucocorticoid receptor, and progesterone receptor, were required for repression of PTHrP expression by the cognate ligands. A notable exception was the androgen receptor, which was dispensable for suppression of PTHrP expression in androgen-treated cells. We propose a pathway(s) involving nuclear receptors to suppress PTHrP expression.     

Vasopressin and norepinephrine stimulation of inositol phosphate accumulation in rat hepatocytes are modified differently by protein f1nase C and protein kinase A

Rat hepatocytes were maintained in primary monolayer culture for 24 h in the presence of serum. Treatment of hepatocytes with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) for 5-15 min increased membrane-associated protein kinase C activity and concomitantly decreased soluble activity. Membrane protein kinase C activity returned to basal values within 1 h then decreased by more than 50% within 2 h. Prolonged (2-18 h) incubation with PMA did not further decrease protein kinase C activity. Pretreatment of hepatocytes with PMA for 5-15 min had little effect on the subsequent actions of 100 nM vasopressin but abolished the stimulation of inositol phosphate accumulation by 3 nM vasopressin and 20 microM norepinephrine. Long-term exposure (2-18 h) of hepatocytes to 1 microM PMA actually enhanced the effects of vasopressin and 20 microM norepinephrine. The stimulation by norepinephrine (20 microM) of inositol phosphate accumulation was abolished by the alpha 1-adrenergic antagonist prazosin (1 microM), whereas the beta-adrenergic antagonist propranolol (30 microM) had little effect. Addition of 8Br-cAMP (100 microM) or glucagon (10 nM) for 5 min or 8 h had no significant effect alone, but enhanced the subsequent vasopressin stimulation of inositol phosphate accumulation. There was no effect of 8Br-cAMP or glucagon on norepinephrine stimulation of phosphoinositide breakdown. These data indicate that the stimulation of phospholipase C activity in rat hepatocytes by 3 nM vasopressin is enhanced by cyclic AMP-dependent kinase but inhibited by protein kinase C. In contrast, down regulation of protein kinase C markedly enhanced the maximal phosphoinositide response due to both vasopressin and norepinephrine.