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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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Expression of CaSR mRNA and protein in human MCs.(A) RT-PCR was performed on total RNA extracted from human MCs or MCF-7 cells (a human breast cancer cell line used as a positive control) using primers specific for the human CaSR to detect CaSR mRNA expression. A PCR product of the expected size (424 bp) was observed. In the absence of reverse transcriptase (RT-), no PCR-amplified products were detected. (B) Western blot analysis was performed to detect the protein expression of CaSR in human MCs. A 130 kDa band was found in both human MCs and in the positive control of MCF-7 cells. (C) Immunofluorescence detection of CaSR localization in human MCs with anti-CaSR antibody. Red color indicates labeling with CaSR, and blue color shows DAPI-stained nuclei. Scale bar, 20 µm. At least 50 cells were examined, and the present images represent typical staining pattern for the majority of examined cells.
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pone-0098777-g001: Expression of CaSR mRNA and protein in human MCs.(A) RT-PCR was performed on total RNA extracted from human MCs or MCF-7 cells (a human breast cancer cell line used as a positive control) using primers specific for the human CaSR to detect CaSR mRNA expression. A PCR product of the expected size (424 bp) was observed. In the absence of reverse transcriptase (RT-), no PCR-amplified products were detected. (B) Western blot analysis was performed to detect the protein expression of CaSR in human MCs. A 130 kDa band was found in both human MCs and in the positive control of MCF-7 cells. (C) Immunofluorescence detection of CaSR localization in human MCs with anti-CaSR antibody. Red color indicates labeling with CaSR, and blue color shows DAPI-stained nuclei. Scale bar, 20 µm. At least 50 cells were examined, and the present images represent typical staining pattern for the majority of examined cells.

Mentions: To determine whether CaSR mRNA is expressed in cultured human MCs, RT-PCR was performed using specific primers for CaSR. As shown in Fig. 1A, a PCR product of the expected size (424 bp) was observed. In the absence of reverse transcriptase, no PCR-amplified products were detected, indicating that the tested RNA samples were free of genomic contamination. As a positive control, an RT-PCR product from MCF-7 cells revealed a band of the same size as the human MCs. The expression of CaSR protein in human MCs was explored by Western blot analysis and immunostaining. As shown in Fig. 1B, a 130 kDa band, corresponding with the mature CaSR, was found in both human MCs and in the positive control of MCF-7 cells. Immunostaining showed that CaSR protein was mainly localized at the plasma membrane along with some cytoplasmic localization (Fig. 1C). Taken together, these data demonstrate that CaSR is present in cultured human MCs.


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)

Expression of CaSR mRNA and protein in human MCs.(A) RT-PCR was performed on total RNA extracted from human MCs or MCF-7 cells (a human breast cancer cell line used as a positive control) using primers specific for the human CaSR to detect CaSR mRNA expression. A PCR product of the expected size (424 bp) was observed. In the absence of reverse transcriptase (RT-), no PCR-amplified products were detected. (B) Western blot analysis was performed to detect the protein expression of CaSR in human MCs. A 130 kDa band was found in both human MCs and in the positive control of MCF-7 cells. (C) Immunofluorescence detection of CaSR localization in human MCs with anti-CaSR antibody. Red color indicates labeling with CaSR, and blue color shows DAPI-stained nuclei. Scale bar, 20 µm. At least 50 cells were examined, and the present images represent typical staining pattern for the majority of examined cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098777-g001: Expression of CaSR mRNA and protein in human MCs.(A) RT-PCR was performed on total RNA extracted from human MCs or MCF-7 cells (a human breast cancer cell line used as a positive control) using primers specific for the human CaSR to detect CaSR mRNA expression. A PCR product of the expected size (424 bp) was observed. In the absence of reverse transcriptase (RT-), no PCR-amplified products were detected. (B) Western blot analysis was performed to detect the protein expression of CaSR in human MCs. A 130 kDa band was found in both human MCs and in the positive control of MCF-7 cells. (C) Immunofluorescence detection of CaSR localization in human MCs with anti-CaSR antibody. Red color indicates labeling with CaSR, and blue color shows DAPI-stained nuclei. Scale bar, 20 µm. At least 50 cells were examined, and the present images represent typical staining pattern for the majority of examined cells.
Mentions: To determine whether CaSR mRNA is expressed in cultured human MCs, RT-PCR was performed using specific primers for CaSR. As shown in Fig. 1A, a PCR product of the expected size (424 bp) was observed. In the absence of reverse transcriptase, no PCR-amplified products were detected, indicating that the tested RNA samples were free of genomic contamination. As a positive control, an RT-PCR product from MCF-7 cells revealed a band of the same size as the human MCs. The expression of CaSR protein in human MCs was explored by Western blot analysis and immunostaining. As shown in Fig. 1B, a 130 kDa band, corresponding with the mature CaSR, was found in both human MCs and in the positive control of MCF-7 cells. Immunostaining showed that CaSR protein was mainly localized at the plasma membrane along with some cytoplasmic localization (Fig. 1C). Taken together, these data demonstrate that CaSR is present in cultured human MCs.

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