Capacitative calcium influx and proliferation of human osteoblastic-like MG-63 cells

Adult bone tissue is continuously being remodelled and bone mass is maintained by a balance between osteoclastic bone resorption and osteoblastic bone formation. Alteration of osteoblastic cell proliferation may account in part for lack of balance between these two processes in bone loss of osteoporosis. There is calcium (Ca2+) control in numerous cellular functions; however, involvement of capacitative Ca2+ entry (CCE) in proliferation of bone cells is less well investigated. OBJECTIVES: The study described here was aimed to investigate roles of CCE in the proliferation of osteoblast-like MG-63 cells. MATERIALS AND METHODS: Pharmacological characterizations of CCE were undertaken in parallel, with evaluation of the expression of transient receptor potential canonical (TRPC) channels and of cell proliferation. RESULTS: Intracellular Ca2+ store depletion by thapsigargin induced CCE in MG-63 cells; this was characterized by a rapid transient increase of intracellular Ca2+ followed by significant CCE, induced by conditions that stimulated cell proliferation, namely serum and platelet-derived growth factor. Inhibitors of store-operated Ca2+ channels (2-APB and SKF-96365) prevented CCE, while voltage-dependent Ca2+ channel blockers had no effect. Expression of various TRPC channels was shown in the cells, some having been shown to be responsible for CCE. Voltage-dependent Ca2+ channel blockers had no effect on osteoblast proliferation while thapsigargin, 2-APB and SKF-96395, inhibited it. Cell cycle analysis showed that 2-APB and SKF-96395 lengthen the S and G2/M phases, which would account for the reduction in cell proliferation. CONCLUSIONS: Our results indicate that CCE, likely attributed to the activation of TRPCs, might be the main route for Ca2+ influx involved in osteoblast proliferation

Lower Blood Calcium Associates with Unfavorable Prognosis and Predicts for Bone Metastasis in NSCLC

Ionized calcium was involved in various cellular signal pathways,and regulates many cellular processes, including those relevant to tumorigenesis. We hypothesis that imbalance of calcium homeostasis is correlated with development of lung carcinomas. We collected the clinical data of 1084 patients with non small cell lung cancer (NSCLC) treated in Shandong Provincial Hospital, Shandong University. Logistic regression was used to determine the association between calcium levels and clinical characteristics, and COX regression and Kaplan-Meier model were applied to analyze risk factors on overall survival. Blood electrolytes were tested before treatment; and nearly 16% patients with NSCLC were complained with decreased blood calcium, which is more frequent than that in other electrolytes. Further, Multivariate logistic regression analysis disclosed that there were significant correlation between blood calcium decrease and moderate and poor differentiation (P = 0.012, OR = 1.926 (1.203–4.219)), squamous cell carcinoma (P = 0.024, OR = 1.968(1.094–3.540)), and bone metastasis (P = 0.032, OR = 0.396(0.235–0.669)). In multivariate COX regression analysis, advanced lymph node stage and decreased blood calcium were significantly and independent, unfavorable prognostic factors (P<0.001). Finally, the Kaplan-Meier Survival curve revealed that blood calcium decrease was associated with shorter survival (Log-rank; χ2 = 26.172,P<0.001). Our finding indicates that lower blood calcium levels are associated with a higher risk of unfavorable prognosis and bone metastasis of NSCLC

Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells

<p>Abstract</p> <p>Background</p> <p>Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. Nuclear translocation of nuclear factor of activated T-cells (NFAT) is thought to be involved in PASMC proliferation and PAH. Increase in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a prerequisite for NFAT nuclear translocation. Elevated [Ca²⁺]_iin PASMC of PAH patients has been demonstrated through up-regulation of store-operated Ca²⁺channels (SOC) which is encoded by the transient receptor potential (TRP) channel protein. Thus we investigated if: 1) up-regulation of TRPC1 channel expression which induces enhancement of SOC-mediated Ca²⁺influx and increase in [Ca²⁺]_iis involved in hypoxia-induced PASMC proliferation; 2) hypoxia-induced promotion of [Ca²⁺]_ileads to nuclear translocation of NFAT and regulates PASMC proliferation and TRPC1 expression; 3) the anti-proliferative effect of sildenafil is mediated by inhibition of this SOC/Ca²⁺/NFAT pathway.</p> <p>Methods</p> <p>Human PASMC were cultured under hypoxia (3% O₂) with or without sildenafil treatment for 72 h. Cell number and cell viability were determined with a hemocytometer and MTT assay respectively. [Ca²⁺]_iwas measured with a dynamic digital Ca²⁺imaging system by loading PASMC with fura 2-AM. TRPC1 mRNA and protein level were detected by RT-PCR and Western blotting respectively. Nuclear translocation of NFAT was determined by immunofluoresence microscopy.</p> <p>Results</p> <p>Hypoxia induced PASMC proliferation with increases in basal [Ca²⁺]_iand Ca²⁺entry via SOC (SOCE). These were accompanied by up-regulation of TRPC1 gene and protein expression in PASMC. NFAT nuclear translocation was significantly enhanced by hypoxia, which was dependent on SOCE and sensitive to SOC inhibitor SKF96365 (SKF), as well as cGMP analogue, 8-brom-cGMP. Hypoxia-induced PASMC proliferation and TRPC1 up-regulation were inhibited by SKF and NFAT blocker (VIVIT and Cyclosporin A). Sildenafil treatment ameliorated hypoxia-induced PASMC proliferation and attenuated hypoxia-induced enhancement of basal [Ca²⁺]_i, SOCE, up-regulation of TRPC1 expression, and NFAT nuclear translocation.</p> <p>Conclusion</p> <p>The SOC/Ca²⁺/NFAT pathway is, at least in part, a downstream mediator for the anti-proliferative effect of sildenafil, and may have therapeutic potential for PAH treatment.</p

The Increased Activity of TRPV4 Channel in the Astrocytes of the Adult Rat Hippocampus after Cerebral Hypoxia/Ischemia

The polymodal transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRP channel family, is a calcium-permeable cationic channel that is gated by various stimuli such as cell swelling, low pH and high temperature. Therefore, TRPV4-mediated calcium entry may be involved in neuronal and glia pathophysiology associated with various disorders of the central nervous system, such as ischemia. The TRPV4 channel has been recently found in adult rat cortical and hippocampal astrocytes; however, its role in astrocyte pathophysiology is still not defined. In the present study, we examined the impact of cerebral hypoxia/ischemia (H/I) on the functional expression of astrocytic TRPV4 channels in the adult rat hippocampal CA1 region employing immunohistochemical analyses, the patch-clamp technique and microfluorimetric intracellular calcium imaging on astrocytes in slices as well as on those isolated from sham-operated or ischemic hippocampi. Hypoxia/ischemia was induced by a bilateral 15-minute occlusion of the common carotids combined with hypoxic conditions. Our immunohistochemical analyses revealed that 7 days after H/I, the expression of TRPV4 is markedly enhanced in hippocampal astrocytes of the CA1 region and that the increasing TRPV4 expression coincides with the development of astrogliosis. Additionally, adult hippocampal astrocytes in slices or cultured hippocampal astrocytes respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate (4αPDD) by an increase in intracellular calcium and the activation of a cationic current, both of which are abolished by the removal of extracellular calcium or exposure to TRP antagonists, such as Ruthenium Red or RN1734. Following hypoxic/ischemic injury, the responses of astrocytes to 4αPDD are significantly augmented. Collectively, we show that TRPV4 channels are involved in ischemia-induced calcium entry in reactive astrocytes and thus, might participate in the pathogenic mechanisms of astroglial reactivity following ischemic insult

G protein-coupled receptor-mediated calcium signaling in astrocytes

Astrocytes express a large variety of G~protein-coupled receptors (GPCRs) which mediate the transduction of extracellular signals into intracellular calcium responses. This transduction is provided by a complex network of biochemical reactions which mobilizes a wealth of possible calcium-mobilizing second messenger molecules. Inositol 1,4,5-trisphosphate is probably the best known of these molecules whose enzymes for its production and degradation are nonetheless calcium-dependent. We present a biophysical modeling approach based on the assumption of Michaelis-Menten enzyme kinetics, to effectively describe GPCR-mediated astrocytic calcium signals. Our model is then used to study different mechanisms at play in stimulus encoding by shape and frequency of calcium oscillations in astrocytes.Comment: 35 pages, 6 figures, 1 table, 3 appendices (book chapter

Calcium mobilization via intracellular ion channels, store organization and mitochondria in smooth muscle

In smooth muscle, Ca2+ release from the internal store into the cytoplasm occurs via inositol trisphosphate (IP3R) and ryanodine receptors (RyR). The internal Ca2+ stores containing IP3R and RyR may be arranged as multiple separate compartments with various IP3R and RyR arrangements, or there may be a single structure containing both receptors. The existence of multiple stores is proposed to explain several physiological responses which include the progression of Ca2+ waves, graded Ca2+ release from the store and various local responses and sensitivities. We suggest that, rather than multiple stores, a single luminally-continuous store exists in which Ca2+ is in free diffusional equilibrium throughout. Regulation of Ca2+ release via IP3R and RyR by the local Ca2+ concentration within the stores explains the apparent existence of multiple stores and physiological processes such as graded Ca2+ release and Ca2+ waves. Close positioning of IP3R on the store with mitochondria or with receptors on the plasma membrane creates ‘IP3 junctions’ to generate local responses on the luminally-continuous store

STIM2 contributes to enhanced store-operated Ca2+ entry in pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension

Pulmonary vasoconstriction and vascular remodeling are two major causes for elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with idiopathic pulmonary arterial hypertension (IPAH). An increase in cytosolic free Ca2+concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation, which causes pulmonary vascular remodeling. Store-operated Ca2+ entry (SOCE), induced by depletion of stored Ca2+ in the sarcoplasmic reticulum (SR), can increase [Ca2+]cyt in PASMC, independent of other means of Ca2+ entry. Stromal interaction molecule (STIM) proteins, STIM1 and STIM2, were both recently identified as sensors for store depletion and also signaling molecules to open store-operated Ca2+ channels. We previously reported that SOCE was significantly enhanced in PASMC from IPAH patients compared to PASMC from normotensive control subjects. Enhanced SOCE plays an important role in the pathophysiological changes in PASMC associated with pulmonary arterial hypertension. In this study, we examine whether the expression levels of STIM1 and STIM2 are altered in IPAH-PASMC compared to control PASMC, and whether these putative changes in the STIM1 and STIM2 expression levels are responsible for enhanced SOCE and proliferation in IPAH-PASMC. Compared to control PASMC, the protein expression level of STIM2 was significantly increased in IPAH-PASMC, whereas STIM1 protein expression was not significantly changed. In IPAH-PASMC, the small interfering RNA (siRNA)-mediated knockdown of STIM2 decreased SOCE and proliferation, while knockdown of STIM2 in control PASMC had no effect on either SOCE or proliferation. Overexpression of STIM2 in the control PASMC failed to enhance SOCE or proliferation. These data indicate that enhanced protein expression of STIM2 is necessary, but not sufficient, for enhanced SOCE and proliferation of IPAH-PASMC