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Calcium sensing receptor modulates extracellular calcium entry and proliferation via TRPC3/6 channels in cultured human mesangial cells.

Meng K, Xu J, Zhang C, Zhang R, Yang H, Liao C, Jiao J - PLoS ONE (2014)

Bottom Line: Interestingly, the CaSR activation-induced increase in [Ca2+]i results not only from intracellular Ca2+ release from internal stores but also from canonical transient receptor potential (TRPC)-dependent Ca2+ influx.Further experiments indicate that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of receptor-operated calcium channels, significantly enhances the CaSR activation-induced [Ca2+]i increase.Moreover, under conditions in which intracellular stores were already depleted with thapsigargin (TG), CaSR agonists also induced an increase in [Ca2+]i, suggesting that calcium influx stimulated by CaSR agonists does not require the release of calcium stores.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology, The Second Affiliated Hospital, Harbin Medical University, Harbin, China.

ABSTRACT
Calcium-sensing receptor (CaSR) has been demonstrated to be present in several tissues and cells unrelated to systemic calcium homeostasis, where it regulates a series of diverse cellular functions. A previous study indicated that CaSR is expressed in mouse glomerular mesangial cells (MCs), and stimulation of CaSR induces cell proliferation. However, the signaling cascades initiated by CaSR activation in MCs are currently unknown. In this study, our data demonstrate that CaSR mRNA and protein are expressed in a human mesangial cell line. Activating CaSR with high extracellular Ca2+ concentration ([Ca2+]o) or spermine induces a phospholipase C (PLC)-dependent increase in intracellular Ca2+ concentration ([Ca2+]i). Interestingly, the CaSR activation-induced increase in [Ca2+]i results not only from intracellular Ca2+ release from internal stores but also from canonical transient receptor potential (TRPC)-dependent Ca2+ influx. This increase in Ca2+ was attenuated by treatment with a nonselective TRPC channel blocker but not by treatment with a voltage-gated calcium blocker or Na+/Ca2+ exchanger inhibitor. Furthermore, stimulation of CaSR by high [Ca2+]o enhanced the expression of TRPC3 and TRPC6 but not TRPC1 and TRPC4, and siRNA targeting TRPC3 and TRPC6 attenuated the CaSR activation-induced [Ca2+]i increase. Further experiments indicate that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of receptor-operated calcium channels, significantly enhances the CaSR activation-induced [Ca2+]i increase. Moreover, under conditions in which intracellular stores were already depleted with thapsigargin (TG), CaSR agonists also induced an increase in [Ca2+]i, suggesting that calcium influx stimulated by CaSR agonists does not require the release of calcium stores. Finally, our data indicate that pharmacological inhibition and knock down of TRPC3 and TRPC6 attenuates the CaSR activation-induced cell proliferation in human MCs. With these data, we conclude that CaSR activation mediates Ca2+ influx and cell proliferation via TRPC3 and TRPC6 in human MCs.

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TRPC3 and TRPC6 are involved in high [Ca2+]o-mediated cell proliferation.Cell proliferation was measured by a Cell Proliferation ELISA BrdU kit. After starvation for 24-free medium, cells were incubated in the same medium supplemented with different [Ca2+]o (1–5 mM) in the presence or absence of various inhibitors for 24 h. Untreated cells cultured in medium containing 1 mM Ca2+ were used as a control. (A) Incubation of cells for 24 h with 3 mM and 5 mM [Ca2+]o increase proliferation of human MCs, respectively, compared with 1 mM [Ca2+]o (Ctl) (*p<0.05, **p<0.01 vs. Ctl, n = 3). (B) Pretreatment with 10 µM NPS2390 significantly reduces the 5 mM [Ca2+]o-induced cell proliferation (**p<0.01 vs. Ctl, # p<0.05 vs. 5 mM [Ca2+]o without NPS2390, n = 3). Incubation with NPS2390 alone do not affect cell proliferation at 1 mM [Ca2+]o (p>0.05, n = 3). (C) The [Ca2+]o-mediated cell proliferation is significantly inhibited by pretreatment with 50 µM SKF96365 or 100 µM 2-APB (**p<0.01 vs. Ctl, #p<0.05 vs. 5 mM [Ca2+]o without inhibitors, n = 3). (D) Transfection of TRPC3 siRNA and TRPC6 siRNA significantly attenuate the promotion of proliferation by 5 mM [Ca2+]o (**p<0.01 vs. Scr+1 mM [Ca2+]o, #p<0.05 vs. Scr+5 mM [Ca2+]o), respectively, compared with scramble siRNA treated with 5 mM [Ca2+]o. Cells were transfected with TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA followed by treatment with 5 mM [Ca2+]o for 24 h. Untransfected cells cultured in medium containing 1 mM Ca2+ were used as a control. Data are shown as the means ± SEMs.
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pone-0098777-g010: TRPC3 and TRPC6 are involved in high [Ca2+]o-mediated cell proliferation.Cell proliferation was measured by a Cell Proliferation ELISA BrdU kit. After starvation for 24-free medium, cells were incubated in the same medium supplemented with different [Ca2+]o (1–5 mM) in the presence or absence of various inhibitors for 24 h. Untreated cells cultured in medium containing 1 mM Ca2+ were used as a control. (A) Incubation of cells for 24 h with 3 mM and 5 mM [Ca2+]o increase proliferation of human MCs, respectively, compared with 1 mM [Ca2+]o (Ctl) (*p<0.05, **p<0.01 vs. Ctl, n = 3). (B) Pretreatment with 10 µM NPS2390 significantly reduces the 5 mM [Ca2+]o-induced cell proliferation (**p<0.01 vs. Ctl, # p<0.05 vs. 5 mM [Ca2+]o without NPS2390, n = 3). Incubation with NPS2390 alone do not affect cell proliferation at 1 mM [Ca2+]o (p>0.05, n = 3). (C) The [Ca2+]o-mediated cell proliferation is significantly inhibited by pretreatment with 50 µM SKF96365 or 100 µM 2-APB (**p<0.01 vs. Ctl, #p<0.05 vs. 5 mM [Ca2+]o without inhibitors, n = 3). (D) Transfection of TRPC3 siRNA and TRPC6 siRNA significantly attenuate the promotion of proliferation by 5 mM [Ca2+]o (**p<0.01 vs. Scr+1 mM [Ca2+]o, #p<0.05 vs. Scr+5 mM [Ca2+]o), respectively, compared with scramble siRNA treated with 5 mM [Ca2+]o. Cells were transfected with TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA followed by treatment with 5 mM [Ca2+]o for 24 h. Untransfected cells cultured in medium containing 1 mM Ca2+ were used as a control. Data are shown as the means ± SEMs.

Mentions: Because a previous study has reported that [Ca2+]o mediates mouse MC proliferation via activation of CaSR, we investigated the role of TRPC3 and TRPC6 in [Ca2+]o-mediated cell proliferation. As shown in Fig. 10A, incubation of cells for 24 hours with 3 mM and 5 mM [Ca2+]o significantly increased proliferation by 35.95% and 66.24%, respectively, compared with 1 mM [Ca2+]o (p<0.05 and p<0.01, n = 3). The cell viability was not affected under our experimental conditions (data not shown). Consistent with previous reports, the promotion of cell proliferation by [Ca2+]o appeared to act through CaSR stimulation because pretreatment of cells with 10 µM NPS2390 significantly inhibited [Ca2+]o-mediated cell proliferation (Fig. 10B). Incubation with NPS2390 alone did not affect cell proliferation at 1 mM [Ca2+]o. The [Ca2+]o-mediated cell proliferation was significantly inhibited by pretreatment with TRPC channel blockers, 50 µM SKF96365 and 100 µM 2-APB (Fig. 10C). Furthermore, transfection of TRPC3 siRNA and TRPC6 siRNA significantly attenuated the promotion of proliferation by [Ca2+]o, respectively, compared with scramble RNA (Fig. 10D). Taken together, these data indicate that TRPC3 and TRPC6 play a role in cell proliferation induced by CaSR stimulation.


Calcium sensing receptor modulates extracellular calcium entry and proliferation via TRPC3/6 channels in cultured human mesangial cells.

Meng K, Xu J, Zhang C, Zhang R, Yang H, Liao C, Jiao J - PLoS ONE (2014)

TRPC3 and TRPC6 are involved in high [Ca2+]o-mediated cell proliferation.Cell proliferation was measured by a Cell Proliferation ELISA BrdU kit. After starvation for 24-free medium, cells were incubated in the same medium supplemented with different [Ca2+]o (1–5 mM) in the presence or absence of various inhibitors for 24 h. Untreated cells cultured in medium containing 1 mM Ca2+ were used as a control. (A) Incubation of cells for 24 h with 3 mM and 5 mM [Ca2+]o increase proliferation of human MCs, respectively, compared with 1 mM [Ca2+]o (Ctl) (*p<0.05, **p<0.01 vs. Ctl, n = 3). (B) Pretreatment with 10 µM NPS2390 significantly reduces the 5 mM [Ca2+]o-induced cell proliferation (**p<0.01 vs. Ctl, # p<0.05 vs. 5 mM [Ca2+]o without NPS2390, n = 3). Incubation with NPS2390 alone do not affect cell proliferation at 1 mM [Ca2+]o (p>0.05, n = 3). (C) The [Ca2+]o-mediated cell proliferation is significantly inhibited by pretreatment with 50 µM SKF96365 or 100 µM 2-APB (**p<0.01 vs. Ctl, #p<0.05 vs. 5 mM [Ca2+]o without inhibitors, n = 3). (D) Transfection of TRPC3 siRNA and TRPC6 siRNA significantly attenuate the promotion of proliferation by 5 mM [Ca2+]o (**p<0.01 vs. Scr+1 mM [Ca2+]o, #p<0.05 vs. Scr+5 mM [Ca2+]o), respectively, compared with scramble siRNA treated with 5 mM [Ca2+]o. Cells were transfected with TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA followed by treatment with 5 mM [Ca2+]o for 24 h. Untransfected cells cultured in medium containing 1 mM Ca2+ were used as a control. Data are shown as the means ± SEMs.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048219&req=5

pone-0098777-g010: TRPC3 and TRPC6 are involved in high [Ca2+]o-mediated cell proliferation.Cell proliferation was measured by a Cell Proliferation ELISA BrdU kit. After starvation for 24-free medium, cells were incubated in the same medium supplemented with different [Ca2+]o (1–5 mM) in the presence or absence of various inhibitors for 24 h. Untreated cells cultured in medium containing 1 mM Ca2+ were used as a control. (A) Incubation of cells for 24 h with 3 mM and 5 mM [Ca2+]o increase proliferation of human MCs, respectively, compared with 1 mM [Ca2+]o (Ctl) (*p<0.05, **p<0.01 vs. Ctl, n = 3). (B) Pretreatment with 10 µM NPS2390 significantly reduces the 5 mM [Ca2+]o-induced cell proliferation (**p<0.01 vs. Ctl, # p<0.05 vs. 5 mM [Ca2+]o without NPS2390, n = 3). Incubation with NPS2390 alone do not affect cell proliferation at 1 mM [Ca2+]o (p>0.05, n = 3). (C) The [Ca2+]o-mediated cell proliferation is significantly inhibited by pretreatment with 50 µM SKF96365 or 100 µM 2-APB (**p<0.01 vs. Ctl, #p<0.05 vs. 5 mM [Ca2+]o without inhibitors, n = 3). (D) Transfection of TRPC3 siRNA and TRPC6 siRNA significantly attenuate the promotion of proliferation by 5 mM [Ca2+]o (**p<0.01 vs. Scr+1 mM [Ca2+]o, #p<0.05 vs. Scr+5 mM [Ca2+]o), respectively, compared with scramble siRNA treated with 5 mM [Ca2+]o. Cells were transfected with TRPC3 siRNA, TRPC6 siRNA or scrambled siRNA followed by treatment with 5 mM [Ca2+]o for 24 h. Untransfected cells cultured in medium containing 1 mM Ca2+ were used as a control. Data are shown as the means ± SEMs.
Mentions: Because a previous study has reported that [Ca2+]o mediates mouse MC proliferation via activation of CaSR, we investigated the role of TRPC3 and TRPC6 in [Ca2+]o-mediated cell proliferation. As shown in Fig. 10A, incubation of cells for 24 hours with 3 mM and 5 mM [Ca2+]o significantly increased proliferation by 35.95% and 66.24%, respectively, compared with 1 mM [Ca2+]o (p<0.05 and p<0.01, n = 3). The cell viability was not affected under our experimental conditions (data not shown). Consistent with previous reports, the promotion of cell proliferation by [Ca2+]o appeared to act through CaSR stimulation because pretreatment of cells with 10 µM NPS2390 significantly inhibited [Ca2+]o-mediated cell proliferation (Fig. 10B). Incubation with NPS2390 alone did not affect cell proliferation at 1 mM [Ca2+]o. The [Ca2+]o-mediated cell proliferation was significantly inhibited by pretreatment with TRPC channel blockers, 50 µM SKF96365 and 100 µM 2-APB (Fig. 10C). Furthermore, transfection of TRPC3 siRNA and TRPC6 siRNA significantly attenuated the promotion of proliferation by [Ca2+]o, respectively, compared with scramble RNA (Fig. 10D). Taken together, these data indicate that TRPC3 and TRPC6 play a role in cell proliferation induced by CaSR stimulation.

Bottom Line: Interestingly, the CaSR activation-induced increase in [Ca2+]i results not only from intracellular Ca2+ release from internal stores but also from canonical transient receptor potential (TRPC)-dependent Ca2+ influx.Further experiments indicate that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of receptor-operated calcium channels, significantly enhances the CaSR activation-induced [Ca2+]i increase.Moreover, under conditions in which intracellular stores were already depleted with thapsigargin (TG), CaSR agonists also induced an increase in [Ca2+]i, suggesting that calcium influx stimulated by CaSR agonists does not require the release of calcium stores.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology, The Second Affiliated Hospital, Harbin Medical University, Harbin, China.

ABSTRACT
Calcium-sensing receptor (CaSR) has been demonstrated to be present in several tissues and cells unrelated to systemic calcium homeostasis, where it regulates a series of diverse cellular functions. A previous study indicated that CaSR is expressed in mouse glomerular mesangial cells (MCs), and stimulation of CaSR induces cell proliferation. However, the signaling cascades initiated by CaSR activation in MCs are currently unknown. In this study, our data demonstrate that CaSR mRNA and protein are expressed in a human mesangial cell line. Activating CaSR with high extracellular Ca2+ concentration ([Ca2+]o) or spermine induces a phospholipase C (PLC)-dependent increase in intracellular Ca2+ concentration ([Ca2+]i). Interestingly, the CaSR activation-induced increase in [Ca2+]i results not only from intracellular Ca2+ release from internal stores but also from canonical transient receptor potential (TRPC)-dependent Ca2+ influx. This increase in Ca2+ was attenuated by treatment with a nonselective TRPC channel blocker but not by treatment with a voltage-gated calcium blocker or Na+/Ca2+ exchanger inhibitor. Furthermore, stimulation of CaSR by high [Ca2+]o enhanced the expression of TRPC3 and TRPC6 but not TRPC1 and TRPC4, and siRNA targeting TRPC3 and TRPC6 attenuated the CaSR activation-induced [Ca2+]i increase. Further experiments indicate that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of receptor-operated calcium channels, significantly enhances the CaSR activation-induced [Ca2+]i increase. Moreover, under conditions in which intracellular stores were already depleted with thapsigargin (TG), CaSR agonists also induced an increase in [Ca2+]i, suggesting that calcium influx stimulated by CaSR agonists does not require the release of calcium stores. Finally, our data indicate that pharmacological inhibition and knock down of TRPC3 and TRPC6 attenuates the CaSR activation-induced cell proliferation in human MCs. With these data, we conclude that CaSR activation mediates Ca2+ influx and cell proliferation via TRPC3 and TRPC6 in human MCs.

Show MeSH
Related in: MedlinePlus