Limits...
Mammalian target of rapamycin complex 2 signaling pathway regulates transient receptor potential cation channel 6 in podocytes.

Ding F, Zhang X, Li X, Zhang Y, Li B, Ding J - PLoS ONE (2014)

Bottom Line: Rapamycin displayed no effect on the TRPC6 mRNA or protein expression levels or TRPC6-dependent calcium influx in podocytes.Furthermore, knockdown of raptor did not affect TRPC6 expression or function, whereas rictor knockdown suppressed TRPC6 protein expression and TRPC6-dependent calcium influx in podocytes.These findings indicate that the mTORC2 signaling pathway regulates TRPC6 in podocytes but that the mTORC1 signaling pathway does not appear to exert an effect on TRPC6.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Peking University First Hospital, Beijing, China.

ABSTRACT
Transient receptor potential cation channel 6 (TRPC6) is a nonselective cation channel, and abnormal expression and gain of function of TRPC6 are involved in the pathogenesis of hereditary and nonhereditary forms of renal disease. Although the molecular mechanisms underlying these diseases remain poorly understood, recent investigations revealed that many signaling pathways are involved in regulating TRPC6. We aimed to examine the effect of the mammalian target of rapamycin (mTOR) complex (mTOR complex 1 [mTORC1] or mTOR complex 2 [mTORC2]) signaling pathways on TRPC6 in podocytes, which are highly terminally differentiated renal epithelial cells that are critically required for the maintenance of the glomerular filtration barrier. We applied both pharmacological inhibitors of mTOR and specific siRNAs against mTOR components to explore which mTOR signaling pathway is involved in the regulation of TRPC6 in podocytes. The podocytes were exposed to rapamycin, an inhibitor of mTORC1, and ku0063794, a dual inhibitor of mTORC1 and mTORC2. In addition, specific siRNA-mediated knockdown of the mTORC1 component raptor and the mTORC2 component rictor was employed. The TRPC6 mRNA and protein expression levels were examined via real-time quantitative PCR and Western blot, respectively. Additionally, fluorescence calcium imaging was performed to evaluate the function of TRPC6 in podocytes. Rapamycin displayed no effect on the TRPC6 mRNA or protein expression levels or TRPC6-dependent calcium influx in podocytes. However, ku0063794 down-regulated the TRPC6 mRNA and protein levels and suppressed TRPC6-dependent calcium influx in podocytes. Furthermore, knockdown of raptor did not affect TRPC6 expression or function, whereas rictor knockdown suppressed TRPC6 protein expression and TRPC6-dependent calcium influx in podocytes. These findings indicate that the mTORC2 signaling pathway regulates TRPC6 in podocytes but that the mTORC1 signaling pathway does not appear to exert an effect on TRPC6.

Show MeSH

Related in: MedlinePlus

ku0063794 but not rapamycin decreased the TRPC6 mRNA levels in podocytes.(A) ku0063794 (2 µmol/l) or rapamycin (50 nmol/l) was applied to evaluate the effects of mTORC1 and mTORC2 on TRPC6 in podocytes. Application of ku0063794 for 24 h caused a significant decrease in the TRPC6 mRNA levels. However, no significant difference was detected between the control group and rapamycin treatment group. (B) The TRPC6 real-time PCR product was examined via agarose gel electrophoresis. Lane 1: 100 bp DNA marker; lane 2: control cells treated with DMSO for 24 hours; lanes 3 and 4: cells treated for 24 hours with 50 nmol/l rapamycin or 2 µmol/l ku0063794, respectively. (C) The TRPC6 mRNA levels were decreased in a concentration-dependent manner following treatment with 2, 3 or 4 µmol/l ku0063794. No significant difference between the control group and the 0.5 or 1 µmol/l ku0063794 treatment group was detected. (D) The TRPC6 mRNA levels were decreased following application of 3 µmol/l ku0063794 for 8, 12, or 24 h compared to the control treatment. There was no significant difference between the control treatment and ku0063794 treatment for 2 or 4 h. (*p<0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control; ns no statistical significance; n = 3.)
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231083&req=5

pone-0112972-g002: ku0063794 but not rapamycin decreased the TRPC6 mRNA levels in podocytes.(A) ku0063794 (2 µmol/l) or rapamycin (50 nmol/l) was applied to evaluate the effects of mTORC1 and mTORC2 on TRPC6 in podocytes. Application of ku0063794 for 24 h caused a significant decrease in the TRPC6 mRNA levels. However, no significant difference was detected between the control group and rapamycin treatment group. (B) The TRPC6 real-time PCR product was examined via agarose gel electrophoresis. Lane 1: 100 bp DNA marker; lane 2: control cells treated with DMSO for 24 hours; lanes 3 and 4: cells treated for 24 hours with 50 nmol/l rapamycin or 2 µmol/l ku0063794, respectively. (C) The TRPC6 mRNA levels were decreased in a concentration-dependent manner following treatment with 2, 3 or 4 µmol/l ku0063794. No significant difference between the control group and the 0.5 or 1 µmol/l ku0063794 treatment group was detected. (D) The TRPC6 mRNA levels were decreased following application of 3 µmol/l ku0063794 for 8, 12, or 24 h compared to the control treatment. There was no significant difference between the control treatment and ku0063794 treatment for 2 or 4 h. (*p<0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control; ns no statistical significance; n = 3.)

Mentions: To determine which mTOR signaling pathway modulates the TRPC6 mRNA levels, podocytes were exposed to 50 nmol/l rapamycin or 2 µmol/l ku0063794. As shown in Figure 2A, rapamycin displayed no effect on the TRPC6 mRNA levels; however, ku0063794 caused a significant decrease in the TRPC6 mRNA levels. The specificity of the real-time RT-PCR amplification process for TRPC6 was confirmed by the appearance of a single band at 157 bp via gel electrophoresis (Figure 2B). Melting curve analysis confirmed the specificity of the TRPC6 transcripts in the podocytes. To further confirm this result, in podocytes exposed to 0.5, 1, 2, 3 or 4 µmol/l ku0063794 for 24 hours, the TRPC6 mRNA levels were decreased in a concentration-dependent manner (Figure 2C). In addition, the TRPC6 mRNA levels were decreased in a time-dependent manner when the podocytes were exposed to ku0063794 (3 µmol/l) for 2, 4, 8, 12 or 24 hours (Figure 2D).


Mammalian target of rapamycin complex 2 signaling pathway regulates transient receptor potential cation channel 6 in podocytes.

Ding F, Zhang X, Li X, Zhang Y, Li B, Ding J - PLoS ONE (2014)

ku0063794 but not rapamycin decreased the TRPC6 mRNA levels in podocytes.(A) ku0063794 (2 µmol/l) or rapamycin (50 nmol/l) was applied to evaluate the effects of mTORC1 and mTORC2 on TRPC6 in podocytes. Application of ku0063794 for 24 h caused a significant decrease in the TRPC6 mRNA levels. However, no significant difference was detected between the control group and rapamycin treatment group. (B) The TRPC6 real-time PCR product was examined via agarose gel electrophoresis. Lane 1: 100 bp DNA marker; lane 2: control cells treated with DMSO for 24 hours; lanes 3 and 4: cells treated for 24 hours with 50 nmol/l rapamycin or 2 µmol/l ku0063794, respectively. (C) The TRPC6 mRNA levels were decreased in a concentration-dependent manner following treatment with 2, 3 or 4 µmol/l ku0063794. No significant difference between the control group and the 0.5 or 1 µmol/l ku0063794 treatment group was detected. (D) The TRPC6 mRNA levels were decreased following application of 3 µmol/l ku0063794 for 8, 12, or 24 h compared to the control treatment. There was no significant difference between the control treatment and ku0063794 treatment for 2 or 4 h. (*p<0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control; ns no statistical significance; n = 3.)
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231083&req=5

pone-0112972-g002: ku0063794 but not rapamycin decreased the TRPC6 mRNA levels in podocytes.(A) ku0063794 (2 µmol/l) or rapamycin (50 nmol/l) was applied to evaluate the effects of mTORC1 and mTORC2 on TRPC6 in podocytes. Application of ku0063794 for 24 h caused a significant decrease in the TRPC6 mRNA levels. However, no significant difference was detected between the control group and rapamycin treatment group. (B) The TRPC6 real-time PCR product was examined via agarose gel electrophoresis. Lane 1: 100 bp DNA marker; lane 2: control cells treated with DMSO for 24 hours; lanes 3 and 4: cells treated for 24 hours with 50 nmol/l rapamycin or 2 µmol/l ku0063794, respectively. (C) The TRPC6 mRNA levels were decreased in a concentration-dependent manner following treatment with 2, 3 or 4 µmol/l ku0063794. No significant difference between the control group and the 0.5 or 1 µmol/l ku0063794 treatment group was detected. (D) The TRPC6 mRNA levels were decreased following application of 3 µmol/l ku0063794 for 8, 12, or 24 h compared to the control treatment. There was no significant difference between the control treatment and ku0063794 treatment for 2 or 4 h. (*p<0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control; ns no statistical significance; n = 3.)
Mentions: To determine which mTOR signaling pathway modulates the TRPC6 mRNA levels, podocytes were exposed to 50 nmol/l rapamycin or 2 µmol/l ku0063794. As shown in Figure 2A, rapamycin displayed no effect on the TRPC6 mRNA levels; however, ku0063794 caused a significant decrease in the TRPC6 mRNA levels. The specificity of the real-time RT-PCR amplification process for TRPC6 was confirmed by the appearance of a single band at 157 bp via gel electrophoresis (Figure 2B). Melting curve analysis confirmed the specificity of the TRPC6 transcripts in the podocytes. To further confirm this result, in podocytes exposed to 0.5, 1, 2, 3 or 4 µmol/l ku0063794 for 24 hours, the TRPC6 mRNA levels were decreased in a concentration-dependent manner (Figure 2C). In addition, the TRPC6 mRNA levels were decreased in a time-dependent manner when the podocytes were exposed to ku0063794 (3 µmol/l) for 2, 4, 8, 12 or 24 hours (Figure 2D).

Bottom Line: Rapamycin displayed no effect on the TRPC6 mRNA or protein expression levels or TRPC6-dependent calcium influx in podocytes.Furthermore, knockdown of raptor did not affect TRPC6 expression or function, whereas rictor knockdown suppressed TRPC6 protein expression and TRPC6-dependent calcium influx in podocytes.These findings indicate that the mTORC2 signaling pathway regulates TRPC6 in podocytes but that the mTORC1 signaling pathway does not appear to exert an effect on TRPC6.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Peking University First Hospital, Beijing, China.

ABSTRACT
Transient receptor potential cation channel 6 (TRPC6) is a nonselective cation channel, and abnormal expression and gain of function of TRPC6 are involved in the pathogenesis of hereditary and nonhereditary forms of renal disease. Although the molecular mechanisms underlying these diseases remain poorly understood, recent investigations revealed that many signaling pathways are involved in regulating TRPC6. We aimed to examine the effect of the mammalian target of rapamycin (mTOR) complex (mTOR complex 1 [mTORC1] or mTOR complex 2 [mTORC2]) signaling pathways on TRPC6 in podocytes, which are highly terminally differentiated renal epithelial cells that are critically required for the maintenance of the glomerular filtration barrier. We applied both pharmacological inhibitors of mTOR and specific siRNAs against mTOR components to explore which mTOR signaling pathway is involved in the regulation of TRPC6 in podocytes. The podocytes were exposed to rapamycin, an inhibitor of mTORC1, and ku0063794, a dual inhibitor of mTORC1 and mTORC2. In addition, specific siRNA-mediated knockdown of the mTORC1 component raptor and the mTORC2 component rictor was employed. The TRPC6 mRNA and protein expression levels were examined via real-time quantitative PCR and Western blot, respectively. Additionally, fluorescence calcium imaging was performed to evaluate the function of TRPC6 in podocytes. Rapamycin displayed no effect on the TRPC6 mRNA or protein expression levels or TRPC6-dependent calcium influx in podocytes. However, ku0063794 down-regulated the TRPC6 mRNA and protein levels and suppressed TRPC6-dependent calcium influx in podocytes. Furthermore, knockdown of raptor did not affect TRPC6 expression or function, whereas rictor knockdown suppressed TRPC6 protein expression and TRPC6-dependent calcium influx in podocytes. These findings indicate that the mTORC2 signaling pathway regulates TRPC6 in podocytes but that the mTORC1 signaling pathway does not appear to exert an effect on TRPC6.

Show MeSH
Related in: MedlinePlus