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Endocrinology Vol. 144, No. 12 5188-5193
Copyright © 2003 by The Endocrine Society

BRIEF COMMUNICATION

Calcium-Sensing Receptor Induces Messenger Ribonucleic Acid of Human Securin, Pituitary Tumor Transforming Gene, in Rat Testicular Cancer

Jacob Tfelt-Hansen, Peter Schwarz, Ernest F. Terwilliger, Edward M. Brown and Naibedya Chattopadhyay

Division of Endocrinology, Diabetes, and Hypertension (J.T.-H., E.M.B., N.C.), Department of Medicine and Membrane Biology Program, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, 02115; Osteoporosis and Bone Metabolic Unit (P.S.), Department of Clinical Biochemistry and Endocrinology, Copenhagen University Hospital Hvidovre, Copenhagen DK-2650, Denmark; and Division of Experimental Medicine (E.F.T.), Beth Israel Deaconess Medical Center and Harvard Institutes of Medicine, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Jacob Tfelt-Hansen, Endocrine-Hypertension Division, Department of Medicine and Membrane Biology Program, Brigham and Women’s Hospital and Harvard Medical School, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail: jtfelt{at}rics.bwh.harvard.edu.

Abstract

Pituitary tumor transforming gene (PTTG), the human ortholog of securin, is an oncogene. Few normal tissues express PTTG, although in the testis, it is more abundantly expressed. In cancer, however, its wide expression has been directly correlated with the proliferation and angiogenesis, although very little is known about the overall regulation of the PTTG gene. In this study, we investigate the role of the calcium-sensing receptor (CaR), a G protein-coupled receptor (GPCR), in regulating PTTG in a widely used model of humoral hypercalcemia of malignancy, the rat H-500 Leydig cell testicular cancer. We show that extracellular calcium (Ca2+o) up-regulates PTTG mRNA. This up-regulation has a rapid onset, starting at 0.5 h, and remains up-regulated until 40 h. The up-regulation was also Ca2+o concentration dependent, with increases (mean ± SE) of 4.22 ± 1.61-fold, 5.11 ± 1.11-fold, and 5.64 ± 1.92-fold at 5, 7.5, and 10 mM calcium, respectively, compared with 0.5 mM Ca2+o. This effect was abolished by overexpression of a dominant-negative CaR (R185Q), thereby confirming that the effect of high Ca2+o is CaR mediated. Another GPCR agonist, ADP, had no effect on PTTG expression. Because PTTG has been reported to induce angiogenesis, we investigated the effect of elevated Ca2+o on vascular endothelial growth factor (VEGF) expression. Indeed high calcium up-regulated VEGF mRNA by 1.59 ± 0.22-fold. In conclusion, we show for the first time that a GPCR, the CaR, stimulates the synthesis of PTTG mRNA in a nonmetastasizing model for humoral hypercalcemia of malignancy and, in the process, might induce angiogenesis via VEGF.

THE MAMMALIAN ORTHOLOG of securin, a protein crucially involved in initiation of sister-chromatid separation, pituitary tumor transforming gene (PTTG), was discovered in rat pituitary adenoma cell lines by differential mRNA display PCR (1, 2). Despite being involved in cell division, PTTG in mammals is not ubiquitously expressed but displays a restrictive expression. Other normal tissues besides pituitary adenomas in which PTTG mRNA is expressed include the testis and fetal liver, as assessed by Northern blot analysis. The extremely abundant expression of PTTG in the testis is stage specific and is greatest in spermatocytes and spermatids during the rat spermatogenic cycle (3). The important role of PTTG in the testis has been revealed by testicular hypoplasia in mice lacking the PTTG gene (4).

Although the expression profile of PTTG is limited to very few tissues, it is expressed and often overexpressed in most cancers including breast, colon, and ovary cancers (5). PTTG has also been shown to induce proliferation and transformation in vitro and promote tumor formation in nude athymic mice containing PTTG-overexpressing NIH-3T3 fibroblasts (1, 6). Another promalignant characteristic of this model of PTTG overexpression is induction of angiogenesis through production of basic fibroblast growth factor (bFGF) (7). Furthermore, a positive correlation has been demonstrated between the levels of PTTG expression and the degree of pituitary tumor invasiveness (8), although our overall understanding of PTTG regulation is scanty. Given the emerging promalignant/oncogenic roles of PTTG and the fact that it is highly abundant in the testis, we sought to study its regulation in H-500 rat primary Leydig cancer cells, a model for humoral hypercalcemia of malignancy (HHM) (9). Upon implantation into adult male rats, H-500 cells vigorously proliferate. The most widely studied aspect of H-500 cells so far is the regulation of secretion of PTHrP, the major mediator of HHM. We and others have shown that elevated extracellular calcium (Ca2+o) via a G protein-coupled calcium-sensing receptor (CaR) up-regulates PTHrP secretion and synthesis through a mechanism involving multiple MAPKs (10, 11, 12). A similar role of the CaR has been reported in other cancers, such as breast and prostate cancer, making it a likely therapeutic target for antagonizing its function using calcilytics (CaR antagonists).

Unlike the breast and prostate tumors, the H-500 Leydig tumor does not metastasize. Instead, it is marked by robust proliferation and angiogenesis at the site of implantation. Here, we report that elevated Ca2+o up-regulates expression of the PTTG and vascular endothelial growth factor (VEGF) genes in H-500 cells. The effect of high Ca2+o on PTTG gene expression is both rapid in onset and sustained. Finally, high Ca2+o-induced PTTG gene expression is mediated by the CaR because the dominant-negative CaR (R185Q) attenuates this effect.

Materials and Methods

Cell culture
The Rice H-500 rat Leydig cell tumor was obtained from the National Cancer Institute–Frederick Cancer Research and Development Center DCT Tumor Repository (Frederick, MD). Male Fischer 344 rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) weighing 200–220 g (age, 10 wk) were used. A fragment of the H-500 tumor or dispersed H-500 cells (106 per rat) were implanted or injected sc, respectively, in each rat, and the tumors were allowed to grow for 8–14 d. The encapsulated tumor was then excised, rinsed several times with cell culture medium (see below), minced into small pieces, and dispersed by repeated pipetting and several passages through a 22-gauge needle. Dispersed H-500 cells were subsequently plated in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin-100 µg/ml streptomycin and grown at 37 C in a humidified 5% CO2 atmosphere. Cells were passaged every 4–5 d using 0.05% trypsin-0.53 mM EDTA and used for experimentation within the first 10 passages. All cell culture reagents were purchased from GIBCO-BRL (Grand Island, NY), with the exception of fetal bovine serum, which was obtained from Gemini Bio-Products (Calabasas, CA). Rats were handled in accordance with local institutional guidelines.

Northern blot analysis
To study whether high Ca2+o affects the expression of PTTG mRNA, we performed Northern blot analysis as previously described (13). In brief, cellular RNA was isolated (14) using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. The RNA recovered was quantitated by spectrophotometry, and aliquots of 20 µg total RNA from H-500 cells incubated at low Ca2+o (0.5 mM) or high Ca2+o (7.5 mM) concentrations were loaded on a formaldehyde agarose gel after denaturation. The gel was stained with ethidium bromide to visualize RNA standards and rRNA so that equal loading of RNA from the various experimental samples could be documented. The RNA was then blotted onto nylon membranes (Duralon; Stratagene, La Jolla, CA). Blots were hybridized with full-length cDNA probe for rat PTTG and washed under high-stringency conditions as described previously (11). Equal loading was also confirmed by reprobing the membranes with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. Specific radioactive signals were analyzed on a Molecular Dynamics, Inc., PhosphorImager (Sunnyvale, CA) with the ImageQuant program.

Infecting H-500 cells with CaR constructs in recombinant adenoassociated virus (rAAV)
High-efficiency gene transfer into H-500 cells was accomplished using a rAAV-based method. The CaR sequence with a naturally occurring, dominant-negative mutation (R185Q), as well as the same vector containing the cDNA for the ß-galactosidase protein (BG), were under the control of a cytomegalovirus immediate-early promoter element and packaged as previously described (15). The BG served as the control for nonspecific effects of rAAV infection. Cells were seeded (1000 cells/well) in 96-well plates in 0.1 ml of growth medium and cultured overnight. About 1000 virus particles/cell (as optimized by pilot studies) were used to infect each well. Cells were washed once with serum-free {alpha}-MEM. Virus particles were then added, and the culture was incubated for 90 min in serum-free medium at 37 C in a cell-culture incubator. Equal volumes of RPMI 1640 containing 20% serum were added to the cells to achieve a final serum concentration of 10%. The cells were then cultured for 48 h, and experiments with low and high calcium concentrations were performed as described in subsequent sections.

Quantitative real-time PCR
To amplify PTTG (NM_022391), VEGF (NM_031836), and GAPDH cDNA, sense and antisense oligonucleotide primers were designed based on the published cDNA sequences using the Primer Express version 2.0.0 (Applied Biosystems, Foster City, CA). Oligonucleotides were obtained from Genosys (Woodlands, TX). The sequences of the primers were as follows: 5'-ATG ACC CTG GCG TGA AGA TTT-3' [PTTG sense, nucleotides (nt) 127–147], 5'-AAG CAG CAA CAG AGA CCA GAG C-3' (PTTG antisense, nt 227–206), 5'-AGC CTT GTT CAG AGC GGA GAA-3' (VEGF sense, nt 500–520), 5'-TAA CTC AAG CTG CCT CGC CTT-3' (VEGF antisense, nt 606–586), 5'-TTC AAT GGC ACA GTC AAG GC-3' (GAPDH sense), and 5'-TCA CCC CAT TTG ATG TTA GCG-3' (GAPDH antisense).

cDNA was synthesized with the Omniscript RT Kit (QIAGEN, Valencia, CA) using 2 µg total RNA in a 20 µl reaction volume. For real-time PCR, the cDNA was amplified using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). The double-stranded DNA-specific dye SYBR Green I was incorporated into the PCR buffer QuantiTech SYBR PCR (QIAGEN) to allow for quantitative detection of the PCR product in a 30-µl reaction volume. The temperature profile of the reaction was 95 C for 10 min, 40 cycles of denaturation at 95 C for 15 sec, annealing at 60 C for 30 sec, and extension at 72 C for 30 sec. An internal housekeeping gene control, GAPDH, was used to normalize differences in RNA isolation, RNA degradation, and the efficiencies of the reverse transcription (RT). The size of the PCR product was first verified on a 1.5% agarose gel, followed by melting curve analysis thereafter.

Statistics
The data are presented as the mean ± SE of the indicated number of experiments. Data were analyzed by either one-way ANOVA followed by Dunnett’s multiple comparison test or Student’s t test when appropriate. P < 0.05 indicates a statistically significant difference.

Results

High Ca2+o induced PTTG expression as assessed by Northern blot analysis with a full-length PTTG cDNA probe (Fig. 1AGo). Previously, we found that the EC50 for PTHrP secretion by the H-500 cells is 5 mM Ca2+o (16). Therefore, we chose 0.5 and 7.5 mM Ca2+o to be our low and high calcium concentrations, respectively. A 6-h incubation of H-500 cells with high (7.5 mM) Ca2+o up-regulated the PTTG signal by 1.67 ± 0.19-fold (P < 0.05) over low (0.5 mM) Ca2+o. We then studied the effect of 50 µg/ml actinomycin D, an inhibitor of transcription, to see whether high Ca2+o-induced up-regulation of PTTG mRNA involves de novo transcription. Figure 1AGo shows that actinomycin D markedly diminished PTTG expression at both low and high Ca2+o, suggesting that calcium induces de novo synthesis of PTTG mRNA.



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  		FIG. 1. Calcium induces PTTG mRNA up-regulation. The cells were plated in a T-75 flask, and, after 3 d, at 70–80% confluency, were starved in serum-free medium for 4 h. The medium was changed, and a serum-free medium with 0.5 or 7.5 mM Ca2+o was added for 6 h. mRNA was then prepared as described in Materials and Methods. A, Northern blot analysis showing the effect of 7.5 mM Ca2+o compared with 0.5 mM Ca2+o. By adding actinomycin D at 50 µg/ml to the medium, the PTTG mRNA was totally obliterated. B, Pooled data from four Northern blot analyses showing up-regulation of PTTG mRNA at 6 h by high Ca2+o compared with low Ca2+o (*, P < 0.05; n = 4). There is no error bar on the bar representing 0.5 mM Ca2+o because we normalized densitometric data obtained from PTTG Northern blot analysis by assigning the value at 0.5 mM Ca2+o as 1.0 in each experiment and expressed the densitometric value of the PTTG band at 7.5 mM Ca2+o as the fold increase over 1.0.

 
Because PTTG is an oncogene, we were interested in the earliest time point of its induction by high Ca2+o. Using real-time RT-PCR, we observed that 7.5 mM Ca2+o induced PTTG mRNA as early as 4 h with 1.89 ± 0.12-fold up-regulation compared with low calcium. mRNA levels of PTTG peaked at 18 h, with a 7.17 ± 2.86-fold higher than 0.5 mM Ca2+o. However, unlike typical early oncogene induction, which declines after a brief period of induction, PTTG induction by high Ca2+o was sustained, with a 2.32 ± 0.47-fold increase at 40 h, the longest period of time examined (Fig. 2AGo).



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  		FIG. 2. Time course for calcium-induced PTTG mRNA and dose-response curve at 18 h. Cells were plated in 60-mm dishes and, after 72 h, at 80–90% confluency, were starved in serum-free medium containing 0.5 mM Ca2+o for 4 h. Cells were then incubated with 0.5 or 7.5 mM Ca2+o for 0.5, 4, 18, or 40 h. mRNA isolation, cDNA synthesis, and real-time PCR were performed as described in Materials and Methods. A, PTTG mRNA is up-regulated after 0.5 h, peaks at 18 h, and remains high at 40 h after stimulation by high Ca2+o compared with low Ca2+o. The data are pooled from five independent experiments. B, Dose dependency for the up-regulation of PTTG mRNA by high Ca2+o (see text for details). The data are from five independent experiments. GAPDH was used to normalize differences in RNA isolation, RNA degradation, and/or the efficiency of the reverse transcription (RT) reaction (*, P < 0.05, n = 5).

 
After determining the time course of PTTG mRNA induction, we studied the Ca2+o dose-response curve for expression of the gene at 18 h. Using real-time PCR, we observed a dose-dependent increases in PTTG/GAPDH mRNA from 2 to 10 mM Ca2+o; the increases were significant at 5, 7.5, and 10 mM Ca2+o (4.22 ± 1.61, 5.11 ± 1.11, and 5.64 ± 1.92-fold, respectively) compared with the mRNA PTTG/GAPDH level at 0.5 mM Ca2+o (Fig. 2BGo).

We then sought to study whether this high Ca2+o-induced increase in PTTG mRNA is mediated by the G protein-coupled CaR. In the cells infected with rAAV expressing BG, Ca2+o dose-responsively stimulated PTTG mRNA, with 2.14 ± 0.22- and 2.75 ± 0.43-fold increases at 3.5 and 7.5 mM, respectively, compared with 0.5 mM calcium. Infecting H-500 cells with a dominant-negative CaR (R185Q) via rAAV resulted in a significant reduction in the Ca2+o-stimulated PTTG mRNA dose-response compared with cells infected with BG at 3.5 and 7.5 mM calcium (Fig. 3Go).



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  		FIG. 3. Dominant-negative CaR abolishes calcium-induced PTTG mRNA. The H-500 cells were infected with either the dominant-negative CaR or the vector expressing the BG protein 48 h before stimulation, as described in Materials and Methods. The cells were then starved for 4 h in serum-free medium, and the mRNA was prepared after incubation of the cells with 0.5, 3.5, or 7.5 mM Ca2+o in serum-free medium for 18 h. The results represent the fold increase compared with basal PTTG expression and are pooled data from three independent experiments. The 3.5 and 7.5 mM Ca2+o-mediated PTTG expression was 2.14 ± 0.22- and 2.75 ± 0.43-fold greater than basal PTTG expression in the H-500 cells infected with BG (a, P < 0.05 compared with low calcium). In cells infected with dominant-negative CaR, no effect was seen at 3.5 and 7.5 mM Ca2+o, a result that is significantly different from the response seen in the cells infected with BG (b and c, P < 0.05, compared with 3.5 and 7.5 mM, respectively, in cells infected with BG). GAPDH was used to normalize differences in RNA isolation, RNA degradation, and/or the efficiency of the RT reaction.

 
Thus, high Ca2+o-induced PTTG mRNA up-regulation is indeed CaR mediated. To assess the specificity of the CaR in inducing this effect, we used ADP, which is the ligand of another GPCR, a purinergic receptor, that, like the CaR, is coupled to G{alpha}q/11. We have previously shown ADP to be an agonist that is linked to G{alpha}q/11 through its receptor in H-500 cells, thereby eliciting intracellular calcium (Ca2+i) release in H-500 cells loaded with the calcium-sensitive dye, fura-2. Treatment of the cells with ADP produced a rapid and transient increase in Ca2+i in H-500 cells (17), thus confirming that the H-500 cells express a functional ADP receptor linked to the phosphatidylinositol-specific phospholipase C pathway that elevates Ca2+i in an agonist-dependent manner. Because ADP is degraded within 15–30 min by ectonuleotidases, we used ADPßS, a nondegradable agonist of the ADP receptor. Because high Ca2+o, acting via the CaR, increased PTTG expression, we next examined whether this is a generalized effect of activation of any GPCR. Our data showed that high Ca2+o significantly increases PTTG mRNA (6.06 ± 2.18-fold increase), whereas, in contrast, cells treated with ADPßS (10-6 M) did not show any change in PTTG mRNA expression (0.84 ± 0.06) (Fig. 4Go).



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  		FIG. 4. ADPßS, a purinergic ligand also coupled to G{alpha}q/11, has no effect on PTTG mRNA. Cells were treated and mRNA preparation, cDNA synthesis, and real-time PCR were performed as described in Fig. 2Go. High Ca2+o induces PTTG mRNA, whereas ADPßS at 10-6 M has no such effect. Data were pooled from three independent experiments. GAPDH was used to normalize differences in RNA isolation, RNA degradation, and/or the efficiency of the RT reaction (a,b, P < 0.05 compared with low calcium and ADPßS).

 
Up-regulation of PTTG has been shown to induce proliferation and angiogenesis (1, 7). Therefore, in H-500 cells, we next investigated whether the high Ca2+o effect exerted by the CaR on PTTG expression also regulated the expression of the potent angiogenic factor, VEGF. Table 1Go shows that high Ca2+o indeed up-regulated the mRNA of VEGF in H-500 cells stimulated for 18 h by 2.89 ± 1.05-fold compared with low calcium.


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  		TABLE 1. Calcium up-regulates mRNA VEGF

 
Discussion

The purpose of this study was to understand the regulation of PTTG expression. Because PTTG is most abundantly expressed in rodent testis and is overexpressed in various cancer cells, H-500 Leydig cancer cells presented the appropriate combination of both of these features. Traditionally, H-500 cells serve as a nonmetastasizing model for HHM. PTHrP acts as the major mediator of this syndrome. Recently, we have unequivocally shown that CaR activation induces PTHrP secretion and synthesis in these cells, which involves participation of multiple MAPKs (11). PTTG, like PTHrP, is regulated through the MAPKs (18). Upon implantation in rats, H-500 cells, like other highly malignant cells, display a vigorous proliferative capacity along with angiogenesis—functions that have been attributed to PTTG. The downstream mediators of PTTG involved in such processes are transactivation of c-myc, the oncogene that induces cellular proliferation, and bFGF, which induces angiogenesis (7, 19, 20).

Here, we have shown that high Ca2+o, acting via the CaR, rapidly induces PTTG expression. Not surprisingly, given that PTTG is a proto-oncogene, its rapid activation by the CaR is not unusual; however, the sustained induction of PTTG by prolonged CaR activation is striking. Our current understanding of the regulation of PTTG expression is that 1) it is inhibited by cyclosporin A and hydrocortisone in T lymphocytes and 2) it is up-regulated by estrogen and bFGF in pituitary adenomas (20, 21). Here, we have shown for the first time that a G{alpha}q/11-coupled GPCR, the CaR, up-regulates PTTG expression in testis-derived rat Leydig cancer cells. This is not a nonspecific event caused by the activation of any GPCR because activation of a functional G{alpha}q/11-coupled purinergic receptor in these cells by ADPßS (a nondegradable form) failed to alter PTTG expression. However, based on this study, it would be premature to conclude that the CaR would uniformly up-regulate PTTG mRNA in other malignancies and especially malignancies with metastatic capacity. Also, the likelihood of a regulatory effect of the CaR on PTTG gene expression could be contingent on high endogenous expression of any tumor cell, such as the one we studied here.

PTTG has been demonstrated to be promalignant because it induces transformation, proliferation, and angiogenesis in various cancer cells. Here, we have shown the proangiogenic potential of high Ca2+o as evidenced by its induction of VEGF mRNA in H-500 cells. It is relevant in this regard to mention that, in a separate study, we demonstrated induction of proliferation of H-500 cells by the CaR (Tfelt-Hansen, J., N. Chattopadhyay, S. Yano, D. Kanuparthi, P. Rooney, P. Schwarz, and E. M. Brown, manuscript submitted), which could plausibly be mediated by induction of PTTG expression. Various oncogenes, such as raf, ras, and src, are known to regulate VEGF (22). Our findings demonstrate, for the first time, that high Ca2+o by up-regulating VEGF may facilitate angiogenesis by induction of endothelial proliferation and vascular permeability, which is pertinent for a HHM model such as H-500 cells. However, whether this effect is direct or mediated via PTTG awaits further study. Finally, our findings may have an important bearing on the pathogenesis of HHM in which high Ca2+o levels and consequent CaR activation contribute to tumor progression. Induction of PTTG by the CaR may be a newly identified effector arm in processes such as proliferation and/or angiogenesis.

Acknowledgments

We thank Paul Rooney and Deephti Kanuparthi for their technical assistance. The PTTG probe was generously supplied to us by Professor Shlomo Melmed.

Footnotes

This work was supported by Grants DK41415, DK48330, and DK52005 (to E.M.B.) from the National Institutes of Health, by NPS Pharmaceuticals and the St. Giles Foundation (to E.M.B.), and by Pfizer/American Federation for Aging Research and NIH Grant AR02215 (to N.C.).

Abbreviations: bFGF, Basic fibroblast growth factor; BG, ß-galactosidase protein; Ca2+i, intracellular calcium; Ca2+o, extracellular calcium; CaR, calcium-sensing receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPCR, G protein-coupled receptor; HHM, humoral hypercalcemia of malignancy; nt, nucleotides; rAAV, recombinant adenoassociated virus; RT, reverse transcription; VEGF, vascular endothelial growth factor.

Received April 24, 2003.

Accepted for publication September 3, 2003.

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Calcium-sensing receptor activation induces nitric oxide production in H-500 Leydig cancer cells
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Expression of Pituitary Tumor Transforming Gene (PTTG) and Its Binding Protein in Human Astrocytes and Astrocytoma Cells: Function and Regulation of PTTG in U87 Astrocytoma Cells
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Mitogenic Action of Calcium-Sensing Receptor on Rat Calvarial Osteoblasts
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Calcium-Sensing Receptor Induces Proliferation through p38 Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase But Not Extracellularly Regulated Kinase in a Model of Humoral Hypercalcemia of Malignancy
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