Parathyroid Hormone-related Protein Release Research Articles

Hypoxia-mimicking scaffolds with controlled release of DMOG and PTHrP to promote cartilage regeneration via the HIF-1α/YAP signaling pathway

Efficiently driving chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) while avoiding undesired hypertrophy remains a challenge in the field of cartilage tissue engineering. Here, we report the sequential combined application of dimethyloxalylglycine (DMOG) and parathyroid hormone-related protein (PTHrP) to facilitate chondrogenesis and prevent hypertrophy. To support their delivery, poly(lactic-co-glycolic acid) (PLGA) microspheres were fabricated using a double emulsion method. Subsequently, these microspheres were incorporated onto a poly(l-lactic acid) (PLLA) scaffold with a highly porous structure, high interconnectivity and collagen-like nanofiber architecture to construct a microsphere-based scaffold delivery system. These functional constructs demonstrated that the spatiotemporally controlled release of DMOG and PTHrP effectively mimicked the hypoxic microenvironment to promote chondrogenic differentiation with phenotypic stability in a 3D culture system, which had a certain correlation with the interaction between hypoxia-inducible Factor 1 alpha (HIF-1α) and yes-associated protein (YAP). Subcutaneous implantation in nude mice revealed that the constructs were able to maintain cartilage formation in vivo at 4 and 8 weeks. Overall, this study indicated that DMOG and PTHrP controlled-release PLGA microspheres incorporated with PLLA nanofibrous scaffolds provided an advantageous 3D hypoxic microenvironment for efficacious and clinically relevant cartilage regeneration and is a promising treatment for cartilage injury.

PTH/PTHrP in controlled release hydrogel enhances orthodontic tooth movement by regulating periodontal bone remodaling.

This study aimed to evaluate the effects of local application of parathyroid hormone (PTH) or parathyroid hormone-related protein (PTHrP) on osteogenesis and osteoclastogenesis during orthodontic tooth movement (OTM). Periodontal bone remodeling is the crucial biological process in the OTM that involves both bone resorption and formation, with the former more important as the initiator. PTH or PTHrP both play dual roles in bone remodeling regulation, and the balance may shift to the bone resorption side when they are given continuously, suggesting them as potential candidate medicine for OTM acceleration. A total of 40 rats underwent orthodontic mesialization of the maxillary first molars and received no micro-perforation (MOP), or MOP followed by injection of temperature-sensitive hydrogel containing PTH, PTHrP, or normal saline. The rats were sacrificed after 2-week OTM, except for the relapse groups, which had one more week of observation after removal of the force appliances. The amount of tooth movement, rate of relapse after OTM, and effects on the bone remodeling were assessed through micro-computed tomography (μCT) analysis, alkaline phosphatase (ALP) assay, alizarin red staining, tartrate-resistant acid phosphatase (TRAP) staining, immunohistochemistry (IHC) analysis, Western blot (WB), and quantitative real-time polymerase chain reaction (qRT-PCR). The effects of PTHrP on the osteogenic differentiation of human periodontal ligament cells (hPDLCs) were explored in vitro. The cumulative release of PTH or PTHrP from PECE hydrogels was beyond 75% at 14days in a sustained manner. After the intervention in vivo, the distance of OTM in the PTH (0.78±0.06mm) or PTHrP (0.81±0.04mm) group was significantly larger than that of the MOP only (0.51±0.04mm) or the no MOP (0.46±0.05mm) group. Moreover, PTH injection significantly reduced the rate of relapse after OTM (25.7±4.3%) compared to the control (69.6±6.1%). μCT analysis showed decreased BV/TV, BS/BV, and Tb.N, while increased Tb.Sp of alveolar bone in the PTH or PTHrP group. There were also more TRAP-positive osteoclasts in the PTH or PTHrP group with a significantly enhanced ratio of receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG). The protein expressions of PTH/PTHrP type 1 receptor (PTHR1), alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), and β-catenin were significantly increased in the PTH or PTHrP group, as well as the gene expressions of Pth1r, Bglap, and Alpl. There was no significant difference between the effects of PTH and PTHrP. Nevertheless, inhibition of PTHrP on the osteogenic differentiation of hPDLCs was detected in vitro with decreased expression of OCN, RUNX2, COL-1, and ALP. Local injection of either PTH or PTHrP carried by controlled release PECE hydrogel similarly enhances OTM in rats through regulating periodontal bone remodeling, which deserves further study for potential clinical application.

Of mice and (wo)men: Mouse models of breast cancer metastasis to bone

Of mice and (wo)men: Mouse models of breast cancer metastasis to bone

Dexamethasone downregulates the expression of parathyroid hormone-related protein (PTHrP) in mesenchymal stem cells

Parathyroid hormone-related protein (PTHrP) has been shown to have anabolic effects in women with postmenopausal osteoporosis. PTHrP promotes the recruitment of osteogenic cells and prevents apoptotic death of osteoblasts and osteocytes. The receptor responsible for the effects of PTHrP is the common PTH/PTHrP receptor (PTH1R). Glucocorticoids (GC) are commonly used as drugs to treat inflammatory diseases. Long-term GC treatments are often associated with bone loss which can lead to GC-induced osteoporosis. The aim of this work was to study the effects of the glucocorticoid dexamethasone (Dex) on the expression of PTHrP and PTH1R in adult human mesenchymal stem cells, the progenitor cells of osteoblasts. Adult human mesenchymal stem cells (hMSC) were cultured and differentiated by standard methods. The expression of PTHrP and PTH1R mRNA was assayed by real-time qPCR. The PTHrP release into the culture media was measured by an immunoradiometric assay. Treatment with Dex (10 nM) resulted in an 80% drop in the PTHrP release within 6 h. A 24 h Dex treatment also reduced the expression of PTHrP mRNA by up to 90%. The expression of PTH1R receptor mRNA was simultaneously increased up to 20-fold by 10 nM Dex. The effects of Dex on PTHrP and PTH1R were dose-dependent and experiments with the GC-receptor antagonist mifepristone showed an involvement of GC-receptors in these effects. In addition to the Dex-induced effects on PTHrP and PTH1R, Dex also increased mineralization and the expression of the osteoblast markers Runx2 and alkaline phosphatase. In our studies, we show that dexamethasone decreases the expression of PTHrP and increases the expression of the PTH1R receptor. This could have an impact on PTHrP-mediated anabolic actions on bone and could also affect the responsiveness of circulating PTH. The results indicate that glucocorticoids affect the signalling pathway of PTHrP by regulating both PTHrP and PTH1R expression and these mechanisms could be involved in glucocorticoid-induced osteoporosis.

P40. Calcium-sensing receptor mediates the effect of elevated extracellular calcium concentrations on PTHrP secretion and expression in human lung squamous cell carcinoma lines

Lung squamous cell carcinoma (SCC) is associated with humoral hypercalcemia of malignancy due to over production of parathyroid hormone-related protein (PTHrP). Studies have shown that high [Ca2+]o can stimulate PTHrP release from breast and prostate cancer lines by activation of the calcium-sensing receptor (CaR). The purpose of this study was to investigate if CaR was expressed in and likely mediated high [Ca2+]o – induced PTHrP expression and secretion from the RWGT2, HARA, and BEN Aus SCC cells.

Calcium-sensing receptor activation stimulates parathyroid hormone-related protein secretion in prostate cancer cells: role of epidermal growth factor receptor transactivation

We have previously reported that high extracellular Ca 2+ stimulates parathyroid hormone-related protein (PTHrP) release from human prostate and breast cancer cell lines as well as from H-500 rat leydig cancer cells, an action mediated by the calcium-sensing receptor (CaR). Activating the CaR leads to phosphorylation of mitogen-activated protein kinases (MAPKs) that participate in PTHrP synthesis and secretion. Because the CaR is a G protein-coupled receptor (GPCR), it is likely to transactivate the epidermal growth factor receptor (EGFR) or the platelet-derived growth factor receptor (PDGFR). In this study, we hypothesized that activation of the CaR transactivates the EGFR or PDGFR, and examined whether transactivation affects PTHrP secretion in PC-3 human prostate cancer cells. Using Western analysis, we observed that an increase in extracellular Ca 2+ resulted in delayed activation of extracellular signal-regulated kinase (ERK) in PC-3 cells. Pre-incubation with AG1478 (an EGFR kinase inhibitor) or an EGFR neutralizing antibody inhibited the high Ca 2+-induced phosphorylation of ERK1/2. GM6001, a pan matrix metalloproteinase (MMP) inhibitor, also partially suppressed the ERK activation, but AG1296 (a PDGFR kinase inhibitor) did not. High extracellular Ca 2+ stimulates PTHrP release during a 6-h incubation (1.5- to 2.5- and 3- to 4-fold increases in 3.0 and 7.5 mM Ca 2+, respectively). When cells were preincubated with AG1478, GM6001, or an antihuman heparin-binding EGF (HB-EGF) antibody, PTHrP secretion was significantly inhibited under basal as well as high Ca 2+ conditions, while AG1296 had no effect on PTHrP secretion. Taken together, these findings indicate that activation of the CaR transactivates the EGFR, but not the PDGFR, leading to phosphorylation of ERK1/2 and resultant PTHrP secretion, although CaR-EGFR-ERK might not be the only signaling pathway for PTHrP secretion. This transactivation is most likely mediated by activation of MMP and cleavage of proheparin-binding EGF (proHB-EGF) to HB-EGF.

Effect of nuclear factor-kappa B inhibitors and peroxisome proliferator-activated receptor-gamma ligands on PTHrP release from human fetal membranes

Parathyroid hormone-related protein (PTHrP) has been implicated in many processes during normal and pathological pregnancies. In the human fetal membranes, PTHrP exhibits cytokine-like actions. We have recently shown that inhibitors of the nuclear factor-kappa B (NF-κB) and activators of the peroxisome proliferator-activated receptor (PPAR)-γ signalling pathways down-regulate cytokine release from human gestational tissues. Therefore, the aim of this study was to determine whether NF-κB and PPAR-γ also regulate PTHrP release from human fetal membranes. Human amnion and choriodecidua explants were incubated in the absence (control) or presence of two known NF-κB inhibitors (1, 5 and 10 mM sulphasalazine (SASP) or 5, 10 and 15 mM N-acetyl-cysteine (NAC)), and two PPAR-γ ligands (15 and 30 μM 15-deoxy-Δ 12,14-PGJ 2 (15d-PGJ 2) or 15 and 30 μM troglitazone), under basal conditions. After 18 h incubation, the tissues were collected and NF-κB p65 DNA binding activity in nuclear extracts was assessed by ELISA, and the incubation medium was collected and the release of PTHrP was quantified by RIA. Treatment of amnion and choriodecidual tissues with SASP concentrations greater than 5 mM, 15 mM NAC, 30 μM 15d-PGJ 2 and 30 μM troglitazone significantly reduced the release of PTHrP ( p<0.05). This study demonstrates that PTHrP release from human fetal membranes is regulated by inhibitors of NF-κB, and ligands of PPAR-γ.

Paraneoplastic endocrine syndromes: a review.

The paraneoplastic endocrine syndromes ("ectopic" or "inappropriate" hormone production) comprise a wide array of symptom complexes associated with malignant or less commonly benign neoplasms. Most of the syndromes are associated with the production of peptide hormones, which, in some instances, have autocrine stimulatory effects. Hypercalcemia, the most common paraneoplastic endocrine syndrome, may be due to the systemic release of parathyroid hormone-related protein (PTHrP), factors that may be produced locally (cytokines), or by a combination of these mechanisms. A spectrum of other syndromes may be related to the production of specific hormones or growth factors, including insulin-like growth factor and fibroblast growth factor 23. Molecular mechanisms responsible for the development of these syndromes are poorly understood. Mutational events not only may initiate neoplastic transformation but may also lead to the activation (re-expression) of genes responsible for hormone production. Additionally, epigenetic events such as methylation may also be responsible for the development of these syndromes. It is likely that a multiplicity of genetic and epigenetic events may contribute to the development of paraneoplastic endocrine syndromes.

Ca(2+)-sensing receptor expression and PTHrP secretion in PC-3 human prostate cancer cells.

Prostate cancer metastasizes frequently to bone. Elevated extracellular calcium concentrations ([Ca(2+)](o)) stimulate parathyroid hormone-related protein (PTHrP) secretion from normal and malignant cells, potentially acting via the [Ca(2+)](o)-sensing receptor (CaR). Because prostate cancers produce PTHrP, if high [Ca(2+)](o) stimulates PTHrP secretion via the CaR, this could initiate a mechanism whereby osteolysis caused by bony metastases of prostate cancer promotes further bone resorption. We investigated whether the prostate cancer cell lines LnCaP and PC-3 express the CaR and whether polycationic CaR agonists stimulate PTHrP release. Both PC-3 and LnCaP prostate cancer cell lines expressed bona fide CaR transcripts by Northern analysis and RT-PCR and CaR protein by immunocytochemistry and Western analysis. The polycationic CaR agonists [Ca(2+)](o), neomycin, and spermine each concentration dependently stimulated PTHrP secretion from PC-3 cells, as measured by immunoradiometric assay, with maximal, 3.2-, 3.6-, and 4.2-fold increases, respectively. In addition, adenovirus-mediated infection of PC-3 cells with a dominant negative CaR construct attenuated high [Ca(2+)](o)-evoked PTHrP secretion, further supporting the CaR's mediatory role in this process. Finally, pretreating PC-3 cells with transforming growth factor (TGF)-beta(1) augmented both basal and high [Ca(2+)](o)-stimulated PTHrP secretion. Thus, in PTHrP-secreting prostate cancers metastatic to bone, the CaR could initiate a vicious cycle, whereby PTHrP-induced bone resorption releases [Ca(2+)](o) and TGF-beta stored within bone, further increasing PTHrP release and osteolysis.

Molecular Dissection of the Importin β1-Recognized Nuclear Targeting Signal of Parathyroid Hormone-Related Protein

Produced by various types of solid tumors, parathyroid hormone-related protein (PTHrP) is the causative agent of humoral hypercalcemia of malignancy. The similarity of PTHrP's amino-terminus to that of parathyroid hormone enables it to share some of the latter's signalling properties, but its carboxy-terminus confers distinct functions including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. PTHrP nuclear import occurs via a novel importin β1-mediated pathway. The present study uses several different direct binding assays to map the interaction of PTHrP with importin β using a series of alanine mutated PTHrP peptides and truncated human importin β1 derivatives. Our results indicate that PTHrP amino acids 83–93 (KTPGKKKKGK) are absolutely essential for importin β1 recognition with residues 71–82 (TNKVETYKEQPL) additionally required for high affinity binding; residues 380–643 of importin β1 are required for the interaction. Binding of importin β1 to PTHrP is reduced in the presence of the GTP-bound but not GDP-bound form of the guanine nucleotide binding protein Ran, consistent with the idea that RanGTP binding to importin β is involved in the release of PTHrP into the nucleus following translocation across the nuclear envelope. This study represents the first detailed examination of a modular, non-arginine-rich importin β1-recognized nuclear targeting signal.

Expression of PTHrP and PTHR (PTH/PTHrP-r) mRNAs and polypeptides in bovine ovary and stimulation of bovine blastocyst development in vitro following PTHrP treatment during oocyte maturation.

Parathyroid hormone related protein (PTHrP) and its receptor have well-established roles in the development and regulation of many tissues, including bone and mammary gland. The objectives of this study were: (1) to characterize the distribution of mRNAs encoding parathyroid hormone (PTH)-related protein (PTHrP) and receptor (PTHR) in bovine ovary; (2) to characterize the distribution of PTHrP and PTHR polypeptides in bovine ovary; (3) to examine the influences of PTHrP (1-141) treatment during bovine oocyte maturation in vitro on blastocyst development. mRNAs encoding PTHrP and PTHR were detected by in situ hybridization methods in oocytes, and granulosa cells in all follicles from primordial to large antral. PTHrP and PTHR polypeptides displayed distinct distribution patterns with PTHrP polypeptides primarily confined to oocytes from primordial to large antral follicles. PTHrP polypeptides were detectable but at a reduced level in ovarian stroma and in granulosa and thecal layers. PTHR polypeptides were detected in oocytes of all follicular stages but were predominantly found in ovarian stroma, granulosa and theca follicular layers. Supplementation of serum-free cSOFMaa oocyte maturation medium with PTHrP (1-141) resulted in a concentration-dependent increase in development to the blastocyst stage in vitro. The results suggest that granulosa cells may be a primary site of PTHrP production and release. Oocytes from all follicular stages stained strongly for PTHrP polypeptides and PTHrP enhanced development to the blastocyst stage in vitro.

Parathyroid hormone-related protein(1-34) in gestational fluids and release from human gestational tissues.

Parathyroid hormone-related protein (PTHrP) is present in fetal and gestational tissues, in which its proposed roles include stimulation of epithelial growth and differentiation, vasodilatation of the uteroplacental vasculature, relaxation of uterine muscle and stimulation of placental calcium transport. The aim of this study was to determine whether the release of PTHrP from gestational tissue explants was tissue specific. In addition, PTHrP concentrations were measured in maternal plasma, umbilical artery and vein plasma, and amniotic fluid from term, uncomplicated pregnancies before the onset of labour. PTHrP was detected in low concentrations in the mother, fetus and placental tissue. Amniotic fluid had ten times the PTHrP concentration compared with that in the maternal or fetal circulations. Using late pregnant human gestational tissues in an in vitro explant system, we found that amnion over placenta, choriodecidua, reflected amnion, and placenta released PTHrP into culture medium in progressively greater amounts over 24 h (P<0.05). This release was not associated with a loss of cell membrane integrity, as indicated by measurement of the intracellular enzyme, lactate dehydrogenase, in the incubation media. After 24 h incubation, the fetal membranes released significantly (P<0.05) greater amounts of PTHrP than did the placenta (placenta 3. 7+/-0.5 pmol PTHrP/g protein). Amnion over placenta released significantly more PTHrP (139.3+/- 43.1 pmol PTHrP/g protein) than did reflected amnion (29.0+/-8.3 pmol PTHrP/g protein) (P<0.05). This study unequivocally demonstrated that human gestational tissues release PTHrP and it was concluded that the main contributors to PTHrP in amniotic fluid were the human fetal membranes, particularly amnion over placenta. Fetal membrane-derived and amniotic fluid PTHrP are proposed to have stimulatory effects on epithelial growth and differentiation in fetal lung, gut, skin and hair follicles and paracrine effects on placental vascular tone and calcium transport.

Regulation of Parathyroid Hormone-Related Protein Expression in MCF-7 Breast Carcinoma Cells by Estrogen and Antiestrogens

Expression of parathyroid hormone-related protein (PTHrP) in breast carcinoma is a frequent cause of the paraneoplastic syndrome of hypercalcemia. In response to treatment with estrogen or tamoxifen, some breast cancer patients also develop a transient hypercalcemia. Therefore, the effect of 17β-estradiol (E2), tamoxifen, or its more potent metabolite, 4-hydroxytamoxifen (OH-tamoxifen), on PTHrP expression in an estrogen receptor (ER)-positive breast carcinoma cell line (MCF-7) was evaluated. E2increased PTHrP mRNA levels in MCF-7 cells and stimulated PTHrP(1-86) release in a dose-dependent fashion (10−10–10−6M). Tamoxifen and OH-tamoxifen also stimulated PTHrP release in a concentration-dependent fashion that paralleled their relative ER binding affinities (10−6or 10−8–10−6M, respectively). Combined treatment with the partial estrogen agonist, OH-tamoxifen, and E2decreased E2-stimulated PTHrP secretion in MCF-7 cells to the levels seen with OH-tamoxifen treatment alone. These results suggest that transient estrogen- or tamoxifen-induced hypercalcemia in patients with breast carcinoma may be a PTHrP-mediated effect that is a marker of ER positivity.

Synovium as a source of increased amino-terminal parathyroid hormone-related protein expression in rheumatoid arthritis. A possible role for locally produced parathyroid hormone-related protein in the pathogenesis of rheumatoid arthritis.

Proinflammatory cytokines, including tumor necrosis factor (TNF) and interleukin 1 (IL-1), mediate the joint destruction that characterizes rheumatoid arthritis (RA). Previous studies have shown that parathyroid hormone-related protein (PTHrP) is a member of the cascade of proinflammatory cytokines induced in parenchymal organs during lethal endotoxemia. To test the hypothesis that NH2-terminal PTHrP, a potent bone resorbing agent, could also be a member of the synovial cascade of tissue-destructive cytokines whose expression is induced in RA, PTHrP expression was examined in synovium and synoviocytes obtained from patients with RA and osteoarthritis (OA). PTHrP production, as determined by measurement of immunoreactive PTHrP(1-86) in tissue explant supernatants, was increased 10-fold in RA versus OA synovial tissue. Synovial lining cells and fibroblast-like cells within the pannus expressed both PTHrP and the PTH/PTHrP receptor, findings that were confirmed by in vitro studies of cultured synoviocytes. TNF-alpha and IL-1beta stimulated PTHrP expression in synoviocytes, while dexamethasone and interferon-gamma, agents with some therapeutic efficacy in the treatment of RA, inhibited PTHrP release. Treatment of synoviocytes with PTHrP(1-34) stimulated IL-6 secretion. These results suggest that proinflammatory cytokine-stimulated production of NH2-terminal PTHrP by synovial tissue directly invading cartilage and bone in RA may mediate joint destruction through direct effects on cartilage or bone, or, indirectly, via the induction of mediators of bone resorption in the tumor-like synovium.

PTH-related protein is released into the mother's bloodstream during lactation: evidence for beneficial effects on maternal calcium-phosphate metabolism.

Recent studies have indicated that parathyroid hormone-related protein (PTHrP) may have important actions in lactation, affecting the mammary gland, and also calcium metabolism in the newborn and the mother. However, there are as yet no longitudinal studies to support the notion of an endocrine role of this peptide during nursing. We studied a group of 12 nursing mothers, mean age 32 years, after they had been nursing for an average of 7 weeks (B) and also 4 months after stopping nursing (A). It was assumed that changes occurring between A and B correspond to the effect of lactation. Blood was assayed for prolactin (PRL), PTHrP (two-site immunoradiometric assay with sheep antibody against PTHrP(1-40), and goat antibody against PTHrP(60-72), detection limit 0.3 pmol/l), intact PTH (iPTH), ionized calcium (Ca2+), 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), alkaline phosphatase (alkP), as well as for creatinine (Cr), protein, phosphorus (P), and total calcium (Ca). Fasting 2-h urine samples were analyzed for Ca excretion (CaE) and renal phosphate threshold (TmP/GFR). PRL was significantly higher during lactation than after weaning (39 +/- 10 vs. 13 +/- 9 micrograms/l; p = 0.018) and so was PTHrP (2.8 +/- 0.35 vs. 0.52 +/- 0.04 pmol/l; p = 0.002), values during lactation being above the normal limit (1.3 pmol/l) in all 12 mothers. There was a significant correlation between PRL and PTHrP during lactation (r = 0.8, p = 0.002). Whole blood Ca2+ did not significantly change from A (1.20 +/- 0.02 mmol/l) to B (1.22 +/- 0.02, mmol/l), whereas total Ca corrected for protein (2.18 +/- 0.02 mmol/l) or uncorrected (2.18 +/- 0.02 mmol/l) significantly rose during lactation (2.31 +/- 0.02 mmol/l, p = 0.003 and 2.37 +/- 0.03 mmol/l, p = 0.002, respectively). Conversely, iPTH decreased during lactation (3.47 +/- 0.38 vs. 2.11 +/- 0.35 pmol/l, A vs. B, p = 0.02). Serum-levels of 25(OH)D3 and 1,25(OH)2D3 did not significantly change from A to B (23 +/- 2.3 vs. 24 +/- 1.9 ng/ml and 29.5 +/- 6.0 vs. 21.9 +/- 1.8 pg/ml, respectively). Both TmP/GFR and P were higher during lactation than after weaning (1.15 +/- 0.03 vs. 0.86 +/- 0.05 mmol/l GF, p = 0.003 and 1.25 +/- 0.03 vs. 0.96 +/- 0.05 mmol/l, p = 0.002, respectively) as was alkP (74.0 +/- 7.1 vs. 52.6 +/- 6.9 U/l, p = 0.003). CaE did not differ between A and B (0.015 +/- 0.003 vs. 0.017 +/- 0.003 mmol/l GF, A vs. B, NS). We conclude that lactation is accompanied by an increase in serum PRL. This is associated with a release of PTHrP into the maternal blood circulation. A rise in total plasma Ca ensues, probably in part by increased bone turnover as suggested by the elevation of alkP. PTH secretion falls, with a subsequent rise of TmP/GFR and plasma P despite high plasma levels of PTHrP.

Transforming growth factor β1 and parathyroid hormone-related protein control the secretion of dipeptidyl peptidase II by rat astrocytes

Meninges synthesize parathyroid hormone-related protein (PTHrP) and transforming growth factor (TGFβ1). Both factors control the activity of the glial enzyme dipeptidyl peptidase II (DPP II): TGFβ1 induces the secretion of enzyme activity by cultivated astrocytes in a dose dependent manner. The maximal effect is achieved with a concentration of 10 ng/ml and is dependent on the time of incubation. PTHrP itself has no effect on the release of DPP II activity but considerably reduces the effect of TGFβ1. It is assumed that DPP II influences the course of cicatrization after penetrating injuries of the brain and thus, meninges control glial scarring by the release of TGFβ1 and PTHrP.

Effects of epidermal growth factor on parathyroid hormone-related protein production by mammary epithelial cells

Parathyroid hormone-related protein (PTHrP) plays a prominent role in the pathogenesis of humoral hypercalcemia of malignancy. However, it is also expressed in various nonmalignant tissues, particularly during fetal organogenesis and tissue differentiation. Thus, PTHrP is synthesized in skin, placenta, and mammary gland during lactation. Little is known, however, about the regulation of PTHrP synthesis and release in nontumoral cells. We investigated the regulation of PTHrP production by epidermal growth factor (EGF), a factor of major importance in the development of lactating breast, in primary cultures of rat mammary epithelial cells. EGF stimulated the production of immunoreactive and bioactive PTHrP in a time- and concentration-dependent manner. A 12 h incubation with 10 ng/ml of EGF increased PTHrP production by 36.0 +/- 7.1% (n = 7 experiments, p < 0.01). This was accompanied by an increase in PTHrP mRNA steady-state levels. The production of PTHrP was stimulated by the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA) by 82.9 +/- 9.7% (n = 4 experiments, p < 0.01). The effects of PMA and EGF were additive. The EGF-induced stimulation appeared to be independent of extracellular calcium concentration, prostaglandin, or cAMP synthesis, but may have involved tyrosine kinase-mediated mechanisms. These results indicate that EGF was capable of increasing the production of PTHrP by cultured mammary epithelial cells. They also suggest that factors activating the PKC pathway are involved in the upregulation of PTHrP expression in mammary epithelial cells.

Cyclic AMP increases the release of parathyroid hormone-related protein from a lung-cancer cell line.

Parathyroid hormone-related protein (PTHrP) plays an important role in the pathogenesis of malignant hypercalcemia by stimulating bone resorption and/or renal tubular reabsorption of calcium. In cultured cancer cells, its production can be influenced by various factors or ions, but the regulation of its production is still poorly understood. We investigated the effects of stimulators of cAMP synthesis on PTHrP release by a human lung squamous-carcinoma cell line (BEN). In superfused cells grown on microcarrier beads, PTHrP production was significantly increased after incubation with calcitonin for only 20 min. The release of immunoreactive and bioactive PTHrP was increased by incubating the cells with forskolin, 3-isobutyl-1-methylxanthine or dibutyryl cAMP even in the presence of the protein-synthesis inhibitor cycloheximide for 6 hr. The calcitonin-mediated stimulation was not accompanied by concomitant changes in PTHrP mRNA. The microfilament-disrupter cytochalasin D was shown to enhance the basal and calcitonin-induced production of PTHrP. These results indicate that stimulators of cAMP synthesis enhanced PTHrP release by BEN cells.

Regulation of Proliferation in JEG-3 Cells by a 500-kDa Ca 2+ Sensor and Parathyroid Hormone-Related Protein

JEG-3 cells are derived from human trophoblasts and demonstrated to express a 500-kDa Ca 2+ sensing protein, which elicits biphasic elevations of cytoplasmic Ca 2+ concentrations ([Ca 2+] i) and mediates Ca 2+ regulation of parathyroid hormone-related protein (PTHrP) release from placental cytotrophoblasts. Cytocentrifuged JEG-3 cells were immunostained by monoclonal and polyclonal antiserum toward PTHrP(1-34) and (38-64). Elevation of external Ca 2+ from 0.5 to 3.0 mM induced only a sluggish rise in [Ca 2+] i and no stimulation of cAMP production despite a more than twofold elevation of PTHrP(1-34) release. Monoclonal antibodies recognizing functionally discrepant epitopes of the Ca 2+ sensor protein substantiated uncoupling of this sensor in the Ca 2+-regulated PTHrP release. Exogenous activation of protein kinase C by a phorbol ester strongly augmented the secretion of PTHrP(1-34), whereby uncoupling of the Ca 2+ sensor was partially reversed. This functional differentiation was associated with reduced [ 3H]thymidine incorporation in JEG-3 cells. Proliferation of these cells was inhibited by 71% upon rise of extracellular Ca 2+ from 0.5 to 3.0 mM, and this inhibition was abolished by antibody-mediated interference with the Ca 2+ sensor function. PTHrP(1-86) and PTH(1-34) at concentrations up to 10 −7 M decreased proliferation and stimulated the cAMP content of JEG-3 cells. The findings support concomitant Ca 2+ sensor and PTH/PTHrP receptor expression in JEG-3 cells, and that Ca 2+ inhibits proliferation by actions on the Ca 2+ sensor as well as by stimulation of PTHrP release possibly mediating autocrine growth inhibition.

Enhancing effect of interleukin-2 on production of parathyroid hormone-related protein by adult T-cell leukemia cells.

Leukemic cells from patients with adult T‐cell leukemia (ATL) can produce a calcium‐regulating protein, parathyroid hormone‐related protein (PTHrP). Moreover, it has been reported that ATL cells produce some cytokines besides PTHrP and that these cells respond to the T‐cell growth factors, interleukin‐2 (IL‐2) and interIenkin‐4 (IL‐4). To elucidate whether PTHrP produced by ATL cells is regulated by IL‐2 or IL‐4, we investigated the in vitro effects of IL‐2 and IL‐4 on the release of PTHrP. IL‐2 increased the release of PTHrP into the conditioned medium from leukemic cells in some, but not all, ATL patients; however, IL‐4 did not affect the PTHrP release. PTHrP messenger RNA (mRNA) levels were increased in ATL cells cultured in the presence of IL‐2. These data suggest that IL‐2 plays a role in the regulation of hypercalcemia by enhancing the production of PTHrP in ATL patients.