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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.

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Inhibiting mTORC2 down-regulated TRPC6 protein expression.(A–D) In a time- and concentration-dependent manner, the TRPC6 protein levels displayed no significant difference between the control group and the rapamycin treatment group. (E, F) In podocytes exposed to ku0063794 for 2, 4, 8, 12, or 24 hours, the TRPC6 protein levels were decreased in a time-dependent manner. (G, H) The TRPC6 protein levels were decreased in a concentration-dependent manner following treatment with 0.5, 1, 2, 3, or 4 µmol/l ku0063794 compared to the control group. (I, J) The targeted siRNAs specifically knocked down raptor or rictor, accompanied by a decrease in the levels of the downstream target p-p70s6k or p-Akt, respectively. TRPC6 protein expression was decreased in the rictor siRNA treatment group compared to the control group. No difference was detected between the raptor siRNA and control siRNA treatment groups. Podocin protein expression was significantly decreased in rictor siRNA group while there was no difference in raptor siRNA group compared with control siRNA group. (*p<0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control; ns no statistical significance; n = 3.)
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pone-0112972-g003: Inhibiting mTORC2 down-regulated TRPC6 protein expression.(A–D) In a time- and concentration-dependent manner, the TRPC6 protein levels displayed no significant difference between the control group and the rapamycin treatment group. (E, F) In podocytes exposed to ku0063794 for 2, 4, 8, 12, or 24 hours, the TRPC6 protein levels were decreased in a time-dependent manner. (G, H) The TRPC6 protein levels were decreased in a concentration-dependent manner following treatment with 0.5, 1, 2, 3, or 4 µmol/l ku0063794 compared to the control group. (I, J) The targeted siRNAs specifically knocked down raptor or rictor, accompanied by a decrease in the levels of the downstream target p-p70s6k or p-Akt, respectively. TRPC6 protein expression was decreased in the rictor siRNA treatment group compared to the control group. No difference was detected between the raptor siRNA and control siRNA treatment groups. Podocin protein expression was significantly decreased in rictor siRNA group while there was no difference in raptor siRNA group compared with control siRNA group. (*p<0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control; ns no statistical significance; n = 3.)

Mentions: In podocytes exposed to 50 nmol/l rapamycin for 2, 4, 8, 12, or 24 hours or to 10, 50, 100, 500, or 1000 nmol/l rapamycin for 24 hours, no difference in TRPC6 protein expression was detected between the control and rapamycin treatment groups (Figure 3A–D). To evaluate the effect of mTORC2 signaling on TRPC6 expression, the podocytes were treated with ku0063794. As shown in Figure 3E–H, TRPC6 protein expression was significantly decreased in a time- and concentration-dependent manner. These data revealed that inhibition of the mTORC2 signaling pathway was involved in the decreased TRPC6 protein expression in podocytes. To completely clarify this result, we investigated TRPC6 protein expression using targeted siRNA knockdown. When siRNA specific for raptor, a component of the mTORC1 complex, was employed, TRPC6 protein expression was not different between the control siRNA and raptor siRNA groups. Transfection with siRNA specific for rictor, a component of mTORC2, caused a significant decrease in TRPC6 protein expression (Figure 3I–J). Based on these data, blocking the mTORC2 signaling pathway decreases TRPC6 protein expression in podocytes.


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)

Inhibiting mTORC2 down-regulated TRPC6 protein expression.(A–D) In a time- and concentration-dependent manner, the TRPC6 protein levels displayed no significant difference between the control group and the rapamycin treatment group. (E, F) In podocytes exposed to ku0063794 for 2, 4, 8, 12, or 24 hours, the TRPC6 protein levels were decreased in a time-dependent manner. (G, H) The TRPC6 protein levels were decreased in a concentration-dependent manner following treatment with 0.5, 1, 2, 3, or 4 µmol/l ku0063794 compared to the control group. (I, J) The targeted siRNAs specifically knocked down raptor or rictor, accompanied by a decrease in the levels of the downstream target p-p70s6k or p-Akt, respectively. TRPC6 protein expression was decreased in the rictor siRNA treatment group compared to the control group. No difference was detected between the raptor siRNA and control siRNA treatment groups. Podocin protein expression was significantly decreased in rictor siRNA group while there was no difference in raptor siRNA group compared with control siRNA group. (*p<0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control; ns no statistical significance; n = 3.)
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pone-0112972-g003: Inhibiting mTORC2 down-regulated TRPC6 protein expression.(A–D) In a time- and concentration-dependent manner, the TRPC6 protein levels displayed no significant difference between the control group and the rapamycin treatment group. (E, F) In podocytes exposed to ku0063794 for 2, 4, 8, 12, or 24 hours, the TRPC6 protein levels were decreased in a time-dependent manner. (G, H) The TRPC6 protein levels were decreased in a concentration-dependent manner following treatment with 0.5, 1, 2, 3, or 4 µmol/l ku0063794 compared to the control group. (I, J) The targeted siRNAs specifically knocked down raptor or rictor, accompanied by a decrease in the levels of the downstream target p-p70s6k or p-Akt, respectively. TRPC6 protein expression was decreased in the rictor siRNA treatment group compared to the control group. No difference was detected between the raptor siRNA and control siRNA treatment groups. Podocin protein expression was significantly decreased in rictor siRNA group while there was no difference in raptor siRNA group compared with control siRNA group. (*p<0.05 vs. control; **p<0.01 vs. control; ***p<0.001 vs. control; ns no statistical significance; n = 3.)
Mentions: In podocytes exposed to 50 nmol/l rapamycin for 2, 4, 8, 12, or 24 hours or to 10, 50, 100, 500, or 1000 nmol/l rapamycin for 24 hours, no difference in TRPC6 protein expression was detected between the control and rapamycin treatment groups (Figure 3A–D). To evaluate the effect of mTORC2 signaling on TRPC6 expression, the podocytes were treated with ku0063794. As shown in Figure 3E–H, TRPC6 protein expression was significantly decreased in a time- and concentration-dependent manner. These data revealed that inhibition of the mTORC2 signaling pathway was involved in the decreased TRPC6 protein expression in podocytes. To completely clarify this result, we investigated TRPC6 protein expression using targeted siRNA knockdown. When siRNA specific for raptor, a component of the mTORC1 complex, was employed, TRPC6 protein expression was not different between the control siRNA and raptor siRNA groups. Transfection with siRNA specific for rictor, a component of mTORC2, caused a significant decrease in TRPC6 protein expression (Figure 3I–J). Based on these data, blocking the mTORC2 signaling pathway decreases TRPC6 protein expression in podocytes.

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