| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Division of Endocrinology (J.T.-H., N.C., S.Y., D.K., P.R., E.M.B.), Diabetes and Hypertension, Department of Medicine and Membrane Biology Program, Brigham and Women’s Hospital and Harvard Medical School, 02115 Boston, Massachusetts; and Osteoporosis and Bone Metabolic Unit (J.T.-H., P.S.), Department of Clinical Biochemistry and Endocrinology, Copenhagen University Hospital, Hvidovre DK-2650, Denmark
Address all correspondence and requests for reprints to: Jacob Tfelt-Hansen, Division of Endocrinology, Diabetes and Hypertension, 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 |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Humoral hypercalcemia of malignancy (HHM) is a syndrome seen in breast, lung, testicular, and kidney cancer (11, 12, 13). The cancer cells produce a hormone/cytokine that activates the osteoblast, which in turn stimulates the osteoclast to degrade bone matrix. In the vast majority of cases of HHM, the hormone inducing the hypercalcemia is PTH-related peptide (PTHrP) (14). TGF-ß, IGF-I and -II, platelet-derived growth factor, and Ca2+ are factors released from the bone into the blood when the bone matrix is degraded. These factors have all been shown to be capable of stimulating PTHrP secretion by cancer cells (14, 15). The increased PTHrP will then activate bone degradation, leading to a vicious cycle. Using rat testis H-500 Leydig cancer cells, an in vitro model for HHM, we previously showed that the CaR mediates calcium-stimulated secretion of PTHrP (15).
PTHrP was originally discovered as an important HHM-inducing factor, but its role is not limited to the malignant state per se (16). PTHrP is now recognized as a major player in the regulation of cell growth and differentiation, promotion of smooth muscle relaxation, and stimulation of transplacental calcium transport. The effects of PTHrP on the regulation of cell growth vary from cell type to cell type, as PTHrP has been shown to be mitogenic in some cells and antiproliferative in others (17, 18). Our previous observations showed that calcium induces the secretion of PTHrP via the CaR in H-500 cells. Here we report that high Ca2+, acting via the CaR, is mitogenic, which involves the PI3K and p38 MAPK pathways. In addition we report, for the first time, that high Ca2+ via the CaR protects H-500 cells against apoptosis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cell culture
The Rice H-500 rat Leydig cell tumor was obtained from the National Cancer Institute-Frederick Cancer Research and Development Center Division of Cancer Treatment Tumor Repository (Frederick, MD). Male Fischer 344 rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 200–220 g (10 wk of age) were used for all experiments. 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 (FBS; 100 U/ml) and penicillin-streptomycin (100 µg/ml) 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 experiments within the first 20 passages. All cell culture reagents were purchased from GIBCO-BRL (Grand Island, NY) with the exception of FBS, which was obtained from Gemini Bio-Products (Calabasas, CA). Rats were handled in accordance with local institutional guidelines.
Proliferation assay
H-500 cells were trypsinized from a 75-cm2 flask maintained in growth medium and then seeded in 24-well plates at a density of 105/well in 0.5 ml of growth medium. The cells were cultured for 72 h and then serum starved for 4 h, after which calcium (0.5–7.5 mM) alone or with NPS R-467, NPS S-467, PD98059, SB203580, or LY294002 was added along with [3H]thymidine (1 µl/ml; 50 µCi/ml), and the cells were cultured again for 24 h. Incorporation of [3H]thymidine was measured by removing the medium and lysing the cells with 0.5 ml of 10% trichloroacetic acid. The resultant DNA pellet was dissolved in 0.5 ml of 200 mM NaOH.
Western blot analysis
For the determination of AKT (Ser473) phosphorylation, monolayers of H-500 cells were grown on six-well plates. Cells were incubated for 18 h in serum-free, Ca2+-free DMEM containing 4 mM L-glutamine, 0.2% BSA, and 0.5 mM CaCl2. The medium was removed and replaced with the same medium supplemented with 7.5 mM CaCl2, as described in Results. At the end of the incubation period, the medium was removed, the cells were washed twice with ice-cold PBS containing 1 mM sodium vanadate and 25 mM NaF, and then 100 µl of ice-cold lysis buffer was added at 0, 5, 15, 30, 60, and 120 min (20 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 25 mM NaF; 1% Triton X-100; 10% glycerol; 1 mM dithiothreitol; 1 mM sodium orthovanadate; 50 mM glycerophosphate; and a cocktail of protease inhibitors). The protease inhibitors were aprotinin, leupeptin, soybean trypsin inhibitor, pepstatin, and calpain inhibitor (10 µg/ml of each), all from frozen stocks, as well as 100 µg/ml Pefabloc. The sodium orthovanadate, NaF, and Pefabloc were freshly prepared on the day of the experiment. The cells were scraped into the lysis buffer, sonicated for 5 sec, and then centrifuged at 6000 x g for 5 min at 4 C. Equal amounts of the supernatant protein (100 µg) were separated by SDS-PAGE. The separated proteins were electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) and incubated with blocking solution (10 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Triton X-100; and 0.25% BSA) containing 5% dry milk for at least 1 h at room temperature. AKT phosphorylation was detected by immunoblotting using an 18-h incubation with a 1:1000 dilution of a rabbit polyclonal antiserum specific for phospho-AKT. Blots were washed for five 15-min periods at room temperature (1% PBS, 1% Triton X-100, and 0.3% dry milk) and then incubated for 1 h with a secondary goat antirabbit, peroxidase-linked antiserum (1:2000) in blocking solution. Blots were then washed again (5 x 15 min), and bands were visualized by chemiluminescence according to the manufacturer’s protocol (Supersignal, Pierce). The same membrane was used after stripping (Restore Western Blot Stripping, Pierce) to measure non-phospho-AKT. Protein concentrations were measured with the Micro BCA protein kit (Pierce). The phosphorylation of AKT was determined by scanning the Western blots to a JPEG file and then quantifying them using the NIH software ImageJ 1.28u.
PTHrP release
The effects of extracellular Ca2+ (Ca2+o) as well as MAPK and protein kinase C inhibitors on PTHrP release were determined by seeding cells in 96-well plates (1 x 104 cells/well) in 0.1 ml of growth medium. After 48 h, the growth medium was removed and replaced with 0.1 ml of Ca2+-free DMEM containing 4 mM L-glutamine, 0.2% BSA, 100 U/ml penicillin-100 µg/ml streptomycin, and 0.5 mM CaCl2. Two hours later, this medium was removed and replaced with 0.225 ml of the same medium or medium supplemented with additional CaCl2 (to a final concentration of 2.5, 5.0, or 7.5 mM) for 6 h. In other experiments, the medium was supplemented either with the kinase inhibitors described in Results or with 7.5 mM CaCl2 together with the same inhibitors. Six hours later, the conditioned medium was removed for determination of PTHrP release. The 6-h incubation time was chosen after carrying out a time-course experiment examining the effects of low and high calcium on PTHrP release at 4, 6, and 24 h. The fold increase in PTHrP release with high extracellular calcium did not vary over the first 24 h. We chose the 6-h time point for subsequent experiments because it yielded PTHrP values falling on the linear portion of the PTHrP assay, whereas at 4 and 24 h, PTHrP was at the lower or upper portion of the curve, respectively.
PTHrP was measured in conditioned medium using a two-site immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA) that detects PTHrP (1–72) with a sensitivity of approximately 0.3 pmol/liter. PTHrP assays were initiated immediately after removal of the conditioned medium from cultures to minimize degradation of the peptide resulting from freeze-thawing and other manipulations. Standard curves of PTHrP concentrations were generated with the addition of recombinant PTHrP (1–86) to the treatment medium used in the study (i.e. unconditioned Ca2+-free DMEM containing 0.5 mM CaCl2). Calcium and AKT inhibitor alone had no effect in the PTHrP assay.
RT-PCR
One-step RT-PCR (kit from QIAGEN, Santa Clarita, CA) was used for determining the presence of PTH receptor type 1 (PTHR1) transcript(s) in H-500 cells using a pair of primers that would yield a 411-bp product spanning from nucleotides 181–592 of the rat PTHR1 cDNA (NM_020073). Primer sequences are 1) sense, 5'-CAG ATT TTC CTG CTG CAC CG-3'; 2) antisense, 5'-TGA ACT TGA GGC ACT CGC TGT-3'. In brief, we used the following procedure for RT-PCR: 2 µg total RNA was mixed with a master cocktail containing RT-PCR buffer, sense and antisense PTHR1 primers, dNTPs, RNase inhibitor, and an enzyme mixture containing reverse transcriptase (Omniscript and Sensiscript) and HotStart Taq DNA polymerase at the concentrations recommended by the manufacturer (QIAGEN) to a final volume of 50 µl. The temperature-cycle protocol was as follows: 30 min at 50 C for the RT reaction followed by denaturation and activation of HotStart DNA polymerase for15 min at 95 C and PCR amplification (30 sec at 94 C, 30 sec at 55 C, and 1 min at 72 C for 40 cycles). A final extension for 10 min at 72 C was performed after the end of 40 cycles. To eliminate amplification taking place from contaminating genomic DNA, we omitted RT as a negative control for the RT-PCR. RT-PCR products were fractionated on 1.5% agarose gels. The presence of a 411-bp amplified product was indicative of a positive PCR arising from the presence of a PTHR1-related sequence within the cDNA.
Terminal uridine deoxynucleotidyl nick end labeling (TUNEL) assay
The TUNEL reaction was performed to detect apoptosis. Cells were plated on coverslips and treated as described for the proliferation assay. We stained the cells with an in situ cell-death detection kit, (Roche Diagnostics, Indianapolis, IN) following the manufacturer’s recommendations. Briefly, cells were washed with PBS once and fixed with 4% formalin for 5 min. After fixation, the coverslips were dried and then stored at -80 C. After the cells were washed with PBS, they were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. Slides were rinsed twice with PBS and then incubated for 60 min at 37 C with terminal deoxynucleotidyl transferase (TdT) enzyme in reaction buffer. The slides were rinsed three times with PBS and mounted with Vectashield (Vector Laboratories, Burlingame, CA). Samples were analyzed by confocal and fluorescence microscopy. TUNEL-positive nuclei were detected by the bright color in condensed or ruptured nuclei. Two researchers blinded to the identities of the samples counted the apoptotic cells as well as the total number of cells in six different fields from three independent experiments.
Statistics
The data are presented as the mean ± SEM of the indicated number of experiments. Data were analyzed by one-way ANOVA followed by Fisher’s projected least significant difference test. A P value of <0.05 was considered to indicate a statistically significant difference.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Similar results were seen using the bromodeoxyuridine ELISA, another method of assessing nucleotide incorporation as a measure of proliferation (data not shown). For cells to progress from the G1 to the S phase, the presence of cyclin D1 is critical (19). Using Northern analysis, we observed abundant expression of cyclin D1 mRNA at low and high calcium concentrations at 18 h, although there was no difference in the level of cyclin D1 between the two treatments (data not shown).
|
).
|
). In contrast, the p38 MAPK inhibitor decreased proliferation at both low and high calcium to 66 ± 9% and 92 ± 23%, respectively, compared with 100 ± 4% and 162 ± 6% with low and high calcium alone (P < 0.05). The PI3K inhibitor LY294002 decreased only high calcium-induced proliferation to 93 ± 7% compared with high calcium alone (e.g. 162 ± 6%) (P < 0.05).
|
is a representative Western blot with specific antibodies to phosphorylated AKT (p-AKT) and total AKT showing calcium-induced increase in phosphorylation of AKT at 15, 30, and 60 min. Four Western blots were scanned to quantify the p-AKT bands (Fig. 4B). At 15, 30, and 60 min, the intensity of p-AKT was increased by 1.41 ± 0.1-, 2.07 ± 0.28-, and 1.92 ± 0.11-fold, respectively, compared with baseline p-AKT at 0 min (P < 0.05). We showed in our previous report that the CaR mediates the calcium-induced PTHrP release in these cells (15). Because a PI3K inhibitor abolished proliferation induced by high calcium concentration, we next investigated whether PI3K is involved in PTHrP secretion. LY294002 (10 µM) inhibited a 741 ± 50% increase of PTHrP release due to high Ca2+o (7.5 mM) to 438 ± 21% (P < 0.05) but had no effect on PTHrP release at low Ca2+o (0.5 mM) (Fig. 5). These results indicate that PI3K stimulates both CaR-mediated proliferation and PTHrP release.
|
|
). Because the H-500 cells produce very high levels of PTHrP basally without Ca2+o stimulation, we used the PTH (7–34) peptide (24), a PTH receptor antagonist, to block a potential autocrine action of PTHrP. Addition of either 0.1 or 1 µM PTH (7–34) did not alter cellular proliferation observed at high Ca2+o (7.5 mM), 161 ± 11% (Fig. 6B), demonstrating thereby that the effect of the CaR on proliferation is direct and not mediated by an autocrine mode of PTHrP action. In addition, 1 µM PTH (7–"34") did not have any effect at the basal Ca2+o concentration (0.5 mM).
|
shows two representative photographs of TUNEL-stained H-500 cells at low or high Ca2+o. Six fields from three independent experiments of TUNEL-stained cells were quantified by two investigators blinded to the treatments, and 52.2 ± 8.6% of the H-500 cells were TUNEL positive at 0.5 mM calcium, whereas 29.8 ± 4.4% of the cells were TUNEL-positive at 7.5 mM calcium (P < 0.05). The addition of the PTH receptor antagonist PTH (7–34) (0.1 µM) to cells grown with high calcium did not reverse the protective effect of high Ca2+o against serum withdrawal-induced apoptosis (25.6 ± 5.5% TUNEL-positive cells). This confirmed our hypothesis that high Ca2+o protects cells against apoptosis, which might be the mechanism underlying its mitogenic effect. Therefore, we next studied whether the effect of high Ca2+o on cell survival was CaR mediated. We observed that H-500 cells under serum-free conditions had 23.3 ± 5.5% apoptotic cells in the presence of 3 µM NPS R-467 plus 0.5 mM Ca2+o compared with 52.2 ± 8.6% apoptotic cells at 0.5 mM Ca2+o alone (P < 0.05). On the other hand, cells treated with the less potent enantiomer NPS S-467 (3 µM plus 0.5 mM Ca2+o) had a lesser protective action (35.3 ± 8.3% TUNEL-positive cells) that was not statistically significantly different from the number of apoptotic cells observed at 0.5 mM Ca2+o alone.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, CaR-induced proliferation was found to be dependent on p38 MAPK and AKT but not on ERK. Because we have shown that p38 is activated in H-500 cells by high Ca2+o (15), we used Western blot analysis to determine whether high Ca2+o activates AKT in H-500 cells. Constitutive AKT activation is believed to promote proliferation and increased cell survival and thereby contribute to cancer progression (29). In a heterologous system, HEK-293 cells stably transfected with the CaR (HEK-CaR), the receptor was found not to activate AKT despite protecting cells from apoptosis (30). Our study in H-500 cells where the CaR is endogenously expressed clearly demonstrates that CaR-induced proliferation is mediated by the AKT pathway. A similar observation has been reported in a study using ovarian surface epithelial cells where the CaR activated AKT in an ERK-independent manner (31).
Differential involvement of ERK1/2, AKT, and p38 MAPK has been widely reported in diverse cell systems in response to various stimuli. For example, in myoblasts, insulin-induced proliferation was found to take place through the AKT and ERK1/2 pathways but not p38 MAPK, whereas insulin’s antiapoptotic effects were mediated only through the PI3K/AKT pathway (32). In a study examining the antiapoptotic effects of epidermal growth factor and TNF-
in hepatocytes, the prosurvival effect of epidermal growth factor took place through the AKT and ERK1/2 pathways but not through p38; in contrast, the prosurvival effect of TNF- occurred through ERK1/2 and p38 MAPK but not the AKT pathway (33). The reason that activation of these pathways leads to different outcomes depending on the cell type and the stimuli that they are exposed to is currently poorly understood. The inhibition of basal proliferation by the p38 inhibitor is only 34% (100 to 66/100), whereas the inhibition of the high calcium-induced proliferation is nearly 100% because the p38 inhibitor reduces the proliferation to 92% of the basal level. The difference between these two numbers is presumably the residual CaR-mediated effect. Therefore, p38 seems to contribute to basal (e.g. low calcium) proliferation but to have a more dramatic inhibitory effect on high calcium-induced proliferation.
The fact that the PI3K inhibitor LY294002 abolished high Ca2+o-induced proliferation in H-500 cells, whereas it only partially reduced high Ca2+o-induced PTHrP release, suggests that high Ca2+o-elicited proliferation and PTHrP release use different pathways to some extent. This observation is further supported by the fact that the MEK1 inhibitor PD98059 had no effect on Ca2+o-induced proliferation in these cells. Previous work from our laboratory has documented an effect of PD98059 on calcium-induced PTHrP release in H-500 cells (15). The finding that PD98059 had no effect on proliferation is also interesting in the light of results showing that the ERK1/2 pathway mediates the CaR effect on proliferation in fibroblasts, primary human osteoblasts, and ovarian surface epithelial cells (7, 34, 35).
Our previous work showed that PTHrP release is induced by Ca2+o through the CaR (15). Therefore, an important question we have addressed in this work is whether the CaR acts directly on proliferation or indirectly through PTHrP. First we observed that mRNA for the PTHR1 is present in H-500 cells. Then we demonstrated, using the PTHR1 antagonist, PTH (7–34), that the CaR-induced proliferation does not result from an autocrine effect of PTHrP. PTHrP has a nuclear localization signal and has been reported to enhance proliferation of PC3 prostate cancer cells in an intracrine fashion (17). It has been further reported that PTHrP, acting in an intracrine manner, can also protect MCF-7 breast cancer cells against serum starvation-induced apoptosis (18). Therefore, although we show that blocking the PTHR did not change the CaR-induced proliferation and protection against apoptosis, we cannot rule out that an intracrine action of PTHrP mediates one or more of the effects of calcium on H-500 cells.
Because the CaR induces proliferation through PI3K/AKT, a pathway originally discovered as a survival pathway, we investigated whether high Ca2+o could protect the H-500 cells from apoptosis induced by serum deprivation. By using TUNEL staining, we observed more apoptotic cells after 72 h of serum deprivation than in cells grown in RPMI 1640 media containing 10% FBS (data not shown). Statistical analyses revealed that action of high calcium and NPS R-467 in the presence of 0.5 mM calcium was protective against apoptosis in H-500 cells. The effect of elevated Ca2+o in preventing apoptosis has been shown with Ca2+o and other polyvalent CaR agonists in AT-3 prostate cancer cells (36). Here we unequivocally demonstrate the effect of the CaR in preventing apoptosis in an endogenously CaR-expressing cell system. This suggests that in the setting of hypercalcemia of malignancy, the CaR could play an important role in determining whether cancer cells divide or die.
In conclusion, we report that high extracellular Ca2+o induces cell proliferation in H-500 cells, a model for HHM, through the CaR. Our results also reveal that the p38 MAPK and PI3K but not the ERK1/2 pathways are involved in high Ca2+o-induced proliferation. High Ca2+o also activates the prosurvival protein AKT by phosphorylating it. Lastly we show that the CaR protects H-500 cells from apoptosis induced by serum deprivation. Thus, hypercalcemia in the H-500 cell model of HHM could contribute to tumor growth through a CaR-mediated increase in cell proliferation, protection against apoptosis, and increase in PTHrP release. Our data show that high extracellular Ca2+o, acting via its cell surface receptor, CaR, exerts direct effects on proliferation and prevention of apoptosis in H-500 cells, without the involvement of PTHrP acting through its cell surface receptor, PTHR1. Therefore, antagonizing the CaR could be a therapeutic strategy for interrupting the vicious cycle observed in HHM.
|
Footnotes |
|---|
Abbreviations: AKT, Protein kinase B; Ca2+o, extracellular calcium; CaR, calcium-sensing receptor; FBS, fetal bovine serum; HHM, humoral hypercalcemia of malignancy; MEK, MAPK kinase; p-AKT, phosphorylated AKT; PI3K, phosphatidylinositol 3-kinase; PTHR1, PTH receptor type 1; PTHrP, PTH-related peptide; TUNEL, terminal uridine deoxynucleotidyl nick end labeling.
Received June 13, 2003.
Accepted for publication November 17, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-mediated rat hepatocyte survival and apoptosis. Hepatology 31:420–427[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  A. Maiti, N. C. Hait, and M. J. Beckman Extracellular Calcium-sensing Receptor Activation Induces Vitamin D Receptor Levels in Proximal Kidney HK-2G Cells by a Mechanism That Requires Phosphorylation of p38{alpha} MAPK J. Biol. Chem., January 4, 2008; 283(1): 175 - 183. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Peiris, I. Pacheco, C. Spencer, and R. J. MacLeod The extracellular calcium-sensing receptor reciprocally regulates the secretion of BMP-2 and the BMP antagonist Noggin in colonic myofibroblasts Am J Physiol Gastrointest Liver Physiol, March 1, 2007; 292(3): G753 - G766. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Liao, A. Schneider, N. S. Datta, and L. K. McCauley Extracellular calcium as a candidate mediator of prostate cancer skeletal metastasis. Cancer Res., September 15, 2006; 66(18): 9065 - 9073. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Chattopadhyay Effects of calcium-sensing receptor on the secretion of parathyroid hormone-related peptide and its impact on humoral hypercalcemia of malignancy Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E761 - E770. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Tfelt-Hansen, J. L. Hansen, S. Smajilovic, E. F. Terwilliger, S. Haunso, and S. P. Sheikh Calcium receptor is functionally expressed in rat neonatal ventricular cardiomyocytes Am J Physiol Heart Circ Physiol, March 1, 2006; 290(3): H1165 - H1171. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S Corbetta, L Vicentini, S Ferrero, A Lania, G Mantovani, D Cordella, P Beck-Peccoz, and A Spada Activity and function of the nuclear factor kappaB pathway in human parathyroid tumors Endocr. Relat. Cancer, December 1, 2005; 12(4): 929 - 937. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Tfelt-Hansen, A. Ferreira, S. Yano, D. Kanuparthi, J. R. Romero, E. M. Brown, and N. Chattopadhyay Calcium-sensing receptor activation induces nitric oxide production in H-500 Leydig cancer cells Am J Physiol Endocrinol Metab, June 1, 2005; 288(6): E1206 - E1213. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yano, R. Mentaverri, D. Kanuparthi, S. Bandyopadhyay, A. Rivera, E. M. Brown, and N. Chattopadhyay Functional Expression of {beta}-Chemokine Receptors in Osteoblasts: Role of Regulated upon Activation, Normal T Cell Expressed and Secreted (RANTES) in Osteoblasts and Regulation of Its Secretion by Osteoblasts and Osteoclasts Endocrinology, May 1, 2005; 146(5): 2324 - 2335. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Tfelt-Hansen, S. Yano, S. Bandyopadhyay, R. Carroll, E. M. Brown, and N. Chattopadhyay 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 Endocrinology, September 1, 2004; 145(9): 4222 - 4231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Chattopadhyay, S. Yano, J. Tfelt-Hansen, P. Rooney, D. Kanuparthi, S. Bandyopadhyay, X. Ren, E. Terwilliger, and E. M. Brown Mitogenic Action of Calcium-Sensing Receptor on Rat Calvarial Osteoblasts Endocrinology, July 1, 2004; 145(7): 3451 - 3462. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
