Parathyroid hormone-related protein: biochemistry and molecular biology

This article critically reviews the current state of knowledge regarding the recently identified and cloned novel hormone parathyroid hormone-related protein (PTHrP). PTHrP is produced by tumors associated with the syndrome of humoral hypercalcemia of malignancy giving rise to the parathyroid hormone (PTH)-like symptoms characteristic of the syndrome. Areas that will be reviewed include identification, purification and cloning, localization, actions, and significance of PTHrP in cancers and normal physiology. The structure and regulation of the PTHrP gene that may be ancestrally related to the PTH gene will also be discussed. Studies in vivo and in vitro with synthetic and recombinant PTHrP sequences and antibodies developed against them have established that the PTH-like actions of PTHrP are mediated via the N-terminal sequences, which show some limited sequence homology with PTH. Evidence for PTH and non-PTH-like actions of PTHrP in normal physiology, which implicate a role for PTHrP in fetal and neonatal development, is also presented.     

Akt promotes increased mammalian cell size by stimulating protein synthesis and inhibiting protein degradation

Expression of constitutively active Akt3 was found to increase the size of MCF-7 cells approximately twofold both in vitro and in vivo. A regulatable version of Akt1 (MER-Akt) was also found capable of inducing a twofold increase in the size of H4IIE rat hepatoma cells. Rapamycin, a specific inhibitor of mTOR function, was found to inhibit the Akt-induced increase in cell size by 70%, presumably via inhibition of the Akt-induced increase in protein synthesis. To determine whether Akt could be inhibiting protein degradation, thereby contributing to its ability to induce an increase in cell size, we conducted protein degradation experiments in the H4IIE cell line. Activation of MER-Akt was found to inhibit protein degradation to a degree comparable to insulin treatment. The effects of these two agents on protein degradation were not additive, thereby suggesting that they were acting on a similar pathway. An inhibitor of the phosphatidylinositol 3-kinase pathway, LY-294002, blocked both insulin- and Akt-induced inhibition of protein degradation, again consistent with the hypothesis that both agents were acting on the same pathway. In contrast, rapamycin did not block the ability of either agent to inhibit protein degradation. These results indicate that Akt increases cell size through both mTOR-dependent and -independent pathways and that the latter involves inhibition of protein degradation. These studies are also consistent with the hypothesis that insulin's ability to regulate protein degradation is to a large extent mediated via Akt.     

Parathyroid hormone-related protein and lung injury after pulmonary thromboendarterectomy

Agouti-related protein (AGRP) is one of two naturally occurring antagonists of G-Protein coupled receptors (GPCRs) identified to date, and has been physiologically implicated in regulating food intake, body weight, and energy homeostasis. AGRP has been identified in vitro, as competitively antagonizing the brain melanocortin-4 (MC4R) and melanocortin-3 (MC3R) receptors, and when over expressed in transgenic mice, results in an obese phenotype. Emerging data propose that AGRP has additional targets in the hypothalamus and/or physiologically functions via a mechanism in addition to competitive antagonism of alpha-MSH at the brain melanocortin receptors. We report data herein supporting an alternative mechanism for AGRP involvement in feeding behavior. A constitutively active MC4R has been generated which possess EC(50) values for melanocortin agonists (alpha-MSH, NDP-MSH, and MTII) and a pA2 value for the synthetic peptide antagonist SHU9119 identical to the wildtype receptor, but increases basal activity to 50% maximal response. AGRP possesses inverse agonist activity at this constitutively active MC4R. These data support the hypothesis for an additional physiological mechanism for AGRP action in feeding behavior and energy homeostasis.     

Parathyroid hormone-related protein production by primary cultures of mammary epithelial cells.

Parathyroid hormone-related protein (PTHrP) plays a major role in the pathogenesis of malignant hypercalcemia, but has also been found in fetal and adult non-neoplastic tissues. Among them, lactating mammary gland was shown to produce PTHrP, and high levels of PTHrP were measured in milk. However, the regulation of PTHrP production by breast cells is still unknown. Primary cultures of mammary cells isolated from rat lactating glands were grown on collagen gels in an insulin/epidermal growth factor (EGF)-supplemented medium. Under these conditions, mammary cells displayed an epithelial phenotype and their number increased more than twofold after 1 week in culture. At that time, the cells were capable of producing immunoreactive PTHrP (range: 25 to 150 pg/10(5) cells x 24 h) and PTH-like bioactivity, as indicated by a 60% increase in cyclic adenosine monophosphate (cAMP) production induced by mammary epithelial cell conditioned medium in the PTH-responsive osteoblast-like UMR-106 cell line. When cell proliferation was hindered by lowering plating density, by removing medium supplements, or by adding transforming growth factor (TGF)-beta, a well-known autocrine inhibitor of mammary epithelial cell growth. PTHrP production was increased. In contrast, the omission of EGF or addition of specified anti-EGF antibodies decreased PTHrP production. In conclusion, primary cultures of mammary epithelial cells isolated from lactating rat were shown for the first time to produce PTHrP in vitro. This production was higher in the presence of EGF and could be modulated by cell growth rate.     

Parathyroid hormone-related protein (107-139) increases human osteoblastic cell survival by activation of vascular endothelial growth factor receptor-2

Parathyroid hormone-related protein (PTHrP) (107-139), in contrast to the N-terminal fragment PTHrP (1-36), has been shown to interact with the vascular endothelial growth factor (VEGF) system to modulate human osteoblast differentiation. In this study, we evaluated whether this interaction might affect human osteoblastic cell survival. Pre-incubation with PTHrP (107-139) for 1-24 h dose-dependently (0.1-100 nM) inhibited dexamethasone- or etoposide-induced cell death in human osteoblastic MG-63 cells and human osteoblast-like cells from trabecular bone. This effect, but not that elicited by PTHrP (1-36), was abolished by the VEGF receptor (VEGFR)-2 inhibitors SU5614 and SU1498 or VEGFR-2 siRNA transfection in these cells. PTHrP (107-139), but not PTHrP (1-36), at 100 nM, rapidly (within 2 min) increased VEGFR-2 tyrosine-phosphorylation in MG-63 cells; an effect unaffected by several inhibitors of metalloproteinases, neutralizing VEGF(165) or VEGFR-2 antibodies, or the VEGF binding inhibitor CBO-PP1. The latter two antagonists also failed to affect (125)I-[Tyr(116)] PTHrP (107-115) binding to these cells. Consistent with its effect on VEGFR-2 activation, PTHrP (107-139) rapidly induced extracellular signal-regulated kinase (ERK) 1/2 and Akt activaton, and both ERK and phosphatidylinsositol-3 kinase (PI3K) inhibitors abolished its pro-survival effect in human osteoblastic cells. In addition, SU5614 and the latter two types of inhibitors abrogated Runx2 activation by this peptide in MG-63 cells. Transfection with a dominant-negative Runx2 construct abolished the pro-survival effect of PTHrP (107-139), associated with a decrease in Bcl-2/Bax protein ratio. Our findings demonstrate that PTHrP (107-139) interacts with VEGFR-2 to promote human osteoblastic cell survival by a mechanism involving Runx2 activation.     

Involvement of rRNA synthesis in the enhanced survival and recovery of protein synthesis seen in thermotolerance

Although acquired thermotolerance has been linked to the induction of heat shock proteins, the molecular mechanism(s) by which cells become resistant to heat is unknown. The present study shows a strong correlation between the survival of cells following heat shock and the rate of recovery of protein, total RNA, and rRNA synthesis. Increasing exposure of CHO cells to 45 degrees C was found to decrease survival and cause a lengthening delay in these synthetic processes. The same reciprocal correlation was seen in thermotolerant cells. As thermotolerance develops, more cells survive a heat challenge and the delay in synthesis decreases. These data argue that enhanced recovery of protein and RNA synthesis is one factor which plays a key role in thermotolerance. The involvement of rRNA synthesis was further investigated by using actinomycin D at 0.1 microgram m1(-1), a concentration at which rRNA synthesis is selectively inhibited. When the drug was present during the recovery from a challenge heat treatment, the survival of thermotolerant cells was approximately 3-fold lower than expected from the mild toxicity of the drug. As this could not be accounted for by an interaction of the drug with the response of cells to single heat treatments, it is concluded that the drug inhibits the expression of thermotolerance in cells which would otherwise express a full degree of thermotolerance. The time and concentration dependence of this effect indicates that the drug acts though inhibition of rRNA synthesis. Therefore, enhanced recovery of RNA synthesis, presumably rRNA synthesis, is identified as one of the mechanisms responsible for enhanced survival of thermotolerant cells following heat shock.     

Parathyroid hormone-related protein in preeclampsia: a linkage between maternal and fetal failures

Preeclampsia is a disorder associated with pregnancy that affects both the mother and the fetus. Typical features of the disease are maternal hypertension, proteinuria, and edema as well as fetal growth retardation. Although the etiological details are still being debated, a consensus exists that the starting point is deficient placentation in the first half of pregnancy. The crucial early steps are reduced trophoblast invasiveness and enhanced apoptotic death. In the present review, we demonstrate that parathyroid hormone-related protein is involved not only in the maternal and fetal failures but also in the etiological aspects of the disease. We hypothesize that reduced local production of the peptide is a major causative event.     

Parathyroid hormone-related protein (PTHrP) in cartilaginous and bony fish tissues.

Tissues from a range of fish were examined for the presence of parathyroid hormone-related protein (PTHrP) to investigate PTHrP protein distribution and PTHrP gene expression in jawless fish, cartilaginous fish, and bony fish. Immunoreactive PTHrP was localized using antisera to N-terminal and mid-molecule regions of human PTHrP and PTHrP gene expression examined using a digoxigenin labeled riboprobe to a conserved region of the mammalian PTHrP gene. In all of the fish studied, PTHrP protein and messenger RNA (mRNA) were localized to the skin, kidney, and skeletal muscle, following the pattern seen in higher vertebrates. Additional sites of localization for both protein and mRNA included gill, nerve cord, and pituitary, as well as developing dermal denticles and rectal gland in the elasmobranch species. The sites of PTHrP distribution indicate that PTHrP may have roles in ionoregulation as well as growth and differentiation in fish, as has been suggested in higher vertebrates. The results imply that the distribution of PTHrP is widespread in fish and that there is homology between the PTHrP molecules found in humans and fish. The conservation of localization and possible similarity of the PTHrP molecules between tetrapods and fish suggests that PTHrP has a number of fundamental roles in vertebrates. J. Exp. Zool. 284:541-548, 1999.     

Parathyroid hormone and parathyroid hormone-related protein inhibit phasic contraction of pig duodenal smooth muscle

Parathyroid hormone (PTH) and a newly discovered PTH-related protein (PTHrP), which has amino-terminal homology with PTH, are potent relaxants of rat gastrointestinal tissues. Since their gastrointestinal relaxant effects have been described only in the rat, we examined their actions in another mammalian species in order to evaluate whether the relaxant property was more generally applicable. Longitudinal smooth muscle strips were obtained from the pig duodenum. The mucosa was removed, the strips were mounted in a tissue chamber, and changes in phasic contraction were detected with a force-displacement transducer and recorded using a polygraph. Acetylcholine-induced phasic contraction was inhibited rapidly in a dose-related manner by [Nle8,18,Tyr34]-bPTH-(1-34)-amide, or hPTHrP-(1-34). The IC50 values for these peptides were 2.6 nM and 6.1 nM, respectively. The maximal effect of both peptides was observed at 60 nM with an 84% decrease of the acetylcholine-induced contraction. At 400 nM, the PTH antagonist, [Nle8,18,Tyr34]-bPTH-(3-34)-amide, had no effect by itself. However, the same 400 nM concentration of this peptide totally blocked the decrease in phasic contraction induced by 10 nM of the bPTH-(1-34) analogue or hPTHrP-(1-34). Our results show that receptors for PTH or PTHrP are present in the muscular layer of the pig duodenum and that activation of these receptors inhibits the phasic contraction of the tissue. Furthermore, the ability of PTH-related peptides to relax gastrointestinal smooth muscle is not restricted to the rat.     

Parathyroid hormone and parathyroid hormone-related protein exert both pro- and anti-apoptotic effects in mesenchymal cells

During bone formation, multipotential mesenchymal cells proliferate and differentiate into osteoblasts, and subsequently many die because of apoptosis. Evidence suggests that the receptor for parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), the PTH-1 receptor (PTH-1R), plays an important role in this process. Multipotential mesenchymal cells (C3H10T1/2) transfected with normal or mutant PTH-1Rs and MC3T3-E1 osteoblastic cells were used to explore the roles of PTH, PTHrP, and the PTH-1R in cell viability relative to osteoblastic differentiation. Overexpression of wild-type PTH-1R increased cell numbers and promoted osteocalcin gene expression versus inactivated mutant receptors. Furthermore, the effects of PTH and PTHrP on apoptosis were dramatically dependent on cell status. In preconfluent C3H10T1/2 and MC3T3-E1 cells, PTH and PTHrP protected against dexamethasone-induced reduction in cell viability, which was dependent on cAMP activation. Conversely, PTH and PTHrP resulted in reduced cell viability in postconfluent cells, which was also dependent on cAMP activation. Further, the proapoptotic-like effects were associated with an inhibition of Akt phosphorylation. These data suggest that parathyroid hormones accelerate turnover of osteoblasts by promoting cell viability early and promoting cell departure from the differentiation program later in their developmental scheme. Both of these actions occur at least in part via the protein kinase A pathway.     

Parathyroid hormone-related protein induces G1 phase growth arrest of vascular smooth muscle cells

In this study, we investigated the mechanisms responsible for the growth-inhibitory action of parathyroid hormone-related protein (PTHRP) in A10 vascular smooth muscle cells (VSMC). Fluorescence-activated cell sorting analysis of serum-stimulated VSMC treated with PTHRP or dibutyryl-cAMP (DBcAMP) demonstrated an enrichment of cells in G1 and a reduction in the S phase. Measurement of DNA synthesis in platelet-derived growth factor-stimulated VSMC treated with DBcAMP revealed that cells became refractory to growth inhibition by 12-16 h, consistent with blockade in mid-G1. cAMP treatment blunted the serum-induced rise in cyclin D1 during cell cycle progression without altering levels of the cyclin-dependent kinase cdk4 or cyclin E and its associated kinase, cdk2. Exposure of cells to PTHRP or cAMP resulted in a reduction in retinoblastoma gene product (Rb) phosphorylation. Immunoblotting of extracts from cAMP-treated cells with antibodies to cdk inhibitors revealed a striking increase in p27(kip1) abundance coincident with the G1 block. Immunoprecipitation with an anti-cyclin D1 antibody of cell lysates prepared from cAMP-treated cells followed by immunoblotting with antisera to p27(kip1) disclosed a threefold increase in p27(kip1) associated with cyclin D1 compared with lysates treated with serum alone. We conclude that PTHRP, by increasing intracellular cAMP, induces VSMC cycle arrest in mid-G1. This occurs secondary to a suppression in cyclin D1 and induction of p27(kip1) expression, which in turn inhibits Rb phosphorylation.     

Inhibition of protein synthesis by anti-5.8 S rRNA oligodeoxyribonucleotides

To examine the role of the 5.8 S rRNA in ribosome function, oligodeoxyribonucleotides, complementary to chemically accessible sequences, were incubated with rabbit reticulocyte or wheat germ extracts undergoing protein synthesis in vitro. Significant and reproducible inhibitions were observed with several different oligonucleotides, the most inhibitory being specific for the universally conserved GAAC sequence. Mutant or heterologous sequences were substantially less inhibitory, results which clearly implicate the 5.8 S rRNA in the inhibitory process and are consistent with the possibility that the 5.8 S rRNA plays an important role in the binding of tRNA.     

Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release

Sympathetic neurons require nerve growth factor for survival and die by apoptosis in its absence. Key steps in the death pathway include c-Jun activation, mitochondrial cytochrome c release, and caspase activation. Here, we show that neurons rescued from NGF withdrawal-induced apoptosis by expression of dominant-negative c-Jun do not release cytochrome c from their mitochondria. Furthermore, we find that the mRNA for BIM(EL), a proapoptotic BCL-2 family member, increases in level after NGF withdrawal and that this is reduced by dominant-negative c-Jun. Finally, overexpression of BIM(EL) in neurons induces cytochrome c redistribution and apoptosis in the presence of NGF, and neurons injected with Bim antisense oligonucleotides or isolated from Bim(-/-) knockout mice die more slowly after NGF withdrawal.     

Albumin promotes neuronal survival by increasing the synthesis and release of glutamate

It is well known that the presence of albumin within the brain and the CSF is developmentally regulated. However, the physiological relevance of this phenomenon is not well established. We have previously shown that albumin specifically increases the flux of glucose and lactate through the pyruvate dehydrogenase reaction in astrocytes. Here we show that, in neurones, albumin also increases the oxidation of glucose and lactate through the pyruvate dehydrogenase-catalysed reaction, the final purpose of this being the synthesis of glutamate. Thus, in neurones, the presence of albumin strongly increased the synthesis and release of glutamate to the extracellular medium. Our results also suggest that glutamate release caused by albumin is designed to promote neuronal survival. Thus, under culture conditions in which neurones die by apoptosis, the presence of albumin promoted neuronal survival and maintained the differentiation programme of these cells, as judged by the expression of the axonal protein, GAP-43. The effect of albumin on neuronal survival was counteracted by the presence of DNQX, an antagonist of non-NMDA-glutamate receptors, suggesting that the glutamate synthesized and released due to the presence of albumin is responsible for neuronal survival. In addition, the effect of albumin seemed to depend on the activity of the NGF receptor, TrkA, suggesting that the glutamate synthesized and released due to the presence of albumin promotes neuronal survival through the activity of TrkA.     

Parathyroid hormone-related protein. Evidence for secretion of a novel mid-region fragment by three different cell types.

The cDNA-predicted amino acid sequence of parathyroid hormone-related protein (PTHrP) contains multiple basic amino acid motifs, suggesting that PTHrP undergoes extensive post-translational processing prior to secretion. The secretory forms of the peptide are currently unknown. To identify these secretory forms, medium was harvested from three cell types: human renal carcinoma (SKRC-1) cells, human keratinocytes, and rat insulinoma cells stably transfected with the cDNA for PTHrP(1-141) (RIN-141 cells). Amino-terminal species were immunopurified using an anti-PTHrP(1-36) column, and mid-region species using an anti-PTHrP(37-74) column. PTHrP peptides in medium and in cell extracts were further resolved by reverse phase high performance liquid chromatography (RP-HPLC) and identified using region-specific immunoassays. SKRC-1 and RIN-141 cells secreted three distinct amino-terminal species and a novel, non-amino-terminal, mid-region fragment. Sequence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the RIN-141 cell mid-region fragment begins at amino acid 38 of the cDNA-predicted sequence and is approximately 70 amino acids in length. Comparison of RP-HPLC elution patterns suggests that SKRC-1 cells and keratinocytes secrete a similar or identical mid-region fragment. Immunofluorescence studies revealed a Golgi pattern for the amino-terminal species and a secretory granule pattern for the mid-region fragment. These studies indicate that 1) multiple PTHrP species are secreted, including a novel mid-region fragment; 2) Arg37 serves as a cleavage site in at least three cell types; 3) PTHrP(1-36) is likely to be an authentic secretory form of PTHrP; and 4) the mid-region fragment appears to be packaged into secretory granules. The marked interspecies conservation of this mid-region PTHrP suggests that it will have important biological functions.     

Metabolic regulation of protein N-alpha-acetylation by Bcl-xL promotes cell survival

Previous experiments suggest a connection between the N-alpha-acetylation of proteins and sensitivity of cells to apoptotic signals. Here, we describe a biochemical assay to detect the acetylation status of proteins and demonstrate that protein N-alpha-acetylation is regulated by the availability of acetyl-CoA. Because the antiapoptotic protein Bcl-xL is known to influence mitochondrial metabolism, we reasoned that Bcl-xL may provide a link between protein N-alpha-acetylation and apoptosis. Indeed, Bcl-xL overexpression leads to a reduction in levels of acetyl-CoA and N-alpha-acetylated proteins in the cell. This effect is independent of Bax and Bak, the known binding partners of Bcl-xL. Increasing cellular levels of acetyl-CoA by addition of acetate or citrate restores protein N-alpha-acetylation in Bcl-xL-expressing cells and confers sensitivity to apoptotic stimuli. We propose that acetyl-CoA serves as a signaling molecule that couples apoptotic sensitivity to metabolism by regulating protein N-alpha-acetylation.     

Tamoxifen elicits its anti-estrogen effects in growth plate chondrocytes by inhibiting protein kinase C

17 beta-Estradiol (E(2)) regulates growth plate cartilage cells via classical nuclear receptor mechanisms, as well as by direct effects on the chondrocyte membrane. These direct effects are stereospecific, causing a rapid increase in protein kinase C (PKC) specific activity, are only found in cells from female rats and are mimicked by E(2)-bovine serum albumin (BSA), which cannot penetrate the cell membrane. E(2) and E(2)-BSA stimulate alkaline phosphatase specific activity and proteoglycan sulfation in female rat costochondral cartilage cell cultures, but traditional nuclear receptors do not appear to be involved. This study examined the effect of the anti-estrogen tamoxifen on these markers of chondrocyte differentiation; the gender-specificity of tamoxifen's effect on PKC, if tamoxifen has an effect on vitamin D metabolite-stimulated PKC, which is mediated via specific membrane receptors (1,25-mVDR; 24,25-mVDR) and whether the effect of tamoxifen is mediated by nuclear estrogen receptors. Tamoxifen dose-dependently inhibited the effect of E(2)-BSA on PKC, alkaline phosphatase and proteoglycan sulfation in confluent cultures of female resting zone (RC) cells and growth zone (GC) (prehypertrophic/upper hypertrophic zones) cells, suggesting that its action is at the membrane and not cell maturation-dependent. Neither the estrogen receptor (ER) antagonist ICI 182780 nor the ER agonist diethylstilbesterol affected E(2) or E(2)-BSA-stimulated PKC in female chondrocytes. Tamoxifen also inhibited the increase in PKC activity due to 1 alpha,25-(OH)(2)D(3) or 24R,25-(OH)(2)D(3) in growth plate cells derived from either female or male rats. Inhibition of PKC by tamoxifen may be a general property of membrane receptors involved in rapid responses to hormones.     

Parathyroid hormone-related protein is induced during lethal endotoxemia and contributes to endotoxin-induced mortality in rodents.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) is a ubiquitous and highly conserved vasoactive peptide whose role and regulation in normal physiology remain an enigma. Recently, we demonstrated that low-dose endotoxin (LPS) induces intrasplenic, but not systemic, levels of PTHrP; and that tumor necrosis factor, a pro-inflammatory cytokine, is the major mediator of this effect. We have therefore hypothesized that, with higher, lethal doses of endotoxin, PTHrP could be induced in multiple tissues to such a degree that it could contribute to the lethality of septic shock. MATERIALS AND METHODS: Northern blot analysis was used to measure PTHrP mRNA levels in vital organs of rats after administration of a near lethal dose (5 mg/250 g) of LPS (or vehicle alone). Plasma levels of PTHrP were also measured by immunoradiometric assay. The ability of the immunoglobulin fraction of two different PTHrP(1-34) antisera to protect from LPS-induced lethality was also studied in mice using survival analysis. RESULTS: In response to a near-lethal dose of endotoxin, PTHrP mRNA levels increased acutely in every vital organ examined (spleen, lung, heart, kidney, and liver). Circulating levels of PTHrP also increased, peaking 2 hr after administration of high-dose endotoxin. Passive immunization of mice with anti-PTHrP(1-34) antibody 6 hr prior to administration of a lethal dose of LPS protected mice from endotoxin-induced death (p < 0.00005). CONCLUSIONS: These results suggest that PTHrP belongs to the cascade of pro-inflammatory cytokines induced during lethal endotoxemia that is responsible for the toxic effects of LPS.