Calcium-sensing receptor activation induces nitric oxide production in H-500 Leydig cancer cells

Abstract

Nitric oxide (NO) is a versatile second messenger. NO is produced by Leydig cells, where NO is a negative regulator of steroidogenesis. In cancer cells, NO is thought to have mutagenic and proliferative effects. We have previously shown that the calcium-sensing receptor (CaR) has promalignant effects in rat H-500 Leydig cancer cells, a model for humoral hypercalcemia of malignancy. Calcium, the major physiological ligand of the CaR, is a recognized intracellular cofactor in the process of NO production by virtue of its positive modulation of neuronal and endothelial nitric oxide synthase (NOS), but importantly, not of inducible (i) NOS activity. iNOS activity is regulated by changes in its expression level. Therefore, we investigated whether CaR activation changes iNOS expression. We found that high extracellular calcium (Ca[Formula: see text]) upregulates the level of mRNA for iNOS, whereas no change was seen in neuronal or endothelial NOS, as assessed by microarray and real-time PCR, respectively. The high Ca[Formula: see text]-induced iNOS upregulation was also detected by Northern and Western blotting. By quantitative real-time PCR, we showed that calcium maximally upregulates iNOS at 18 h. The effect of calcium was abolished by overexpression of a dominant-negative CaR (R185Q), confirming that the effect of Ca[Formula: see text] was mediated by the CaR. Cells treated with high calcium had higher NO production than those treated with low calcium, as detected with the NO-specific DAF2-AM dye. This was confirmed in single-cell fluorescence determinations using confocal microscopy. In conclusion, high calcium upregulates the levels of iNOS mRNA and protein as well as NO production in H-500 cells, and the effect of Ca[Formula: see text] on iNOS expression is mediated by the CaR.

Inhibition of epidermal growth factor receptor signalling reduces hypercalcaemia induced by human lung squamous-cell carcinoma in athymic mice

“…Activation of PTHrP gene expression in vivo has been attributed to circulating factors as well as tumour microenvironment-derived cytokines or growth factors. Observations based on several xenograft HHM models suggest that high plasma calcium concentrations result in CaR-mediated activation of PTHrP production (Sanders et al, 2001;Tfelt-Hansen et al, 2003, 2005a. In the bone microenvironment, TGF-b released from bone at sites of osteoclastic resorption activates PTHrP expression in metastatic cancer cells (Kakonen et al, 2002).…”

Calcium receptor stimulates chemotaxis and secretion of MCP-1 in GnRH neurons in vitro: potential impact on reduced GnRH neuron population in CaR-null mice

“…SYBR green chemistry was used to measure the mRNAs of MCP-1, CCR2, and the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (to normalize differences in RNA isolation and the efficiencies of the RT), following a previously optimized protocol (39). The sequences of the primers were as follows: MCP-1/CCL2 (accession no.…”

“…Gene delivery by recombinant adeno-associated virus. A recombinant adeno-associated virus (rAAV)-based gene delivery method was used, following a previously published protocol (9,38,39). Dominant-negative human CaR (R185Q) (1, 2) and the same vector containing the cDNA for the control ␤-galactosidase protein (referred to hereafter as BG) were under the control of a cytomegalovirus immediate-early (CMV-IE) promoter element (47).…”

Effects of calcium-sensing receptor on the secretion of parathyroid hormone-related peptide and its impact on humoral hypercalcemia of malignancy

“…So far, most studies of the role of the CaR in PTHrP secretion and the pathogenesis of HHM have been on Rice H-500 rat Leydig cell tumors (90,(102)(103)(104)(105)(106). This tumor is a xenotransplantable model of HHM but does not display skeletal metastases (101), thus resembling cancers arising from squamous cells and kidney epithelial cells (64,86).…”

“…CaR-dependent upregulation of PTTG may provide a mechanism for enhanced new vessel formation via the production of the recognized angiogenic factor basic (b)FGF (48). High Ca 2ϩ o also regulates a second potent angiogenic factor, VEGF (105), and stimulates inducible nitric oxide synthase and attendant NO production in H-500 cells (103). NO promotes neovascularization in xenograft tumors, which not only enhances tumor growth but also increases invasiveness and metastatic ability (59,107).…”