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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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Effects of rapamycin and ku0063794 on downstream substrates of the mTORC1 and mTORC2 signaling pathways.(A–C) Rapamycin decreased the phosphorylation of the mTORC1 downstream target p70S6K in podocytes in a time-dependent manner. However, rapamycin did not decrease the phosphorylation of the mTORC2 downstream target Akt. (D–F) Rapamycin decreased the phosphorylation of p70s6k but not Akt in a concentration-dependent manner. (G–I) In a time-dependent manner, ku0063794, a dual inhibitor of mTORC1 and mTORC2, decreased the levels of the mTORC1 and mTORC2 downstream targets p-p70s6k and p-Akt, respectively. (J–L) In a concentration-dependent manner, ku0063794 at concentrations >1 µmol/l was sufficient to decrease both the p-p70s6k and p-Akt levels. (Abbreviations: con: control; p-p70s6k: phosphorylation of p70s6k; p-Akt: phosphorylation of Akt. *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-g001: Effects of rapamycin and ku0063794 on downstream substrates of the mTORC1 and mTORC2 signaling pathways.(A–C) Rapamycin decreased the phosphorylation of the mTORC1 downstream target p70S6K in podocytes in a time-dependent manner. However, rapamycin did not decrease the phosphorylation of the mTORC2 downstream target Akt. (D–F) Rapamycin decreased the phosphorylation of p70s6k but not Akt in a concentration-dependent manner. (G–I) In a time-dependent manner, ku0063794, a dual inhibitor of mTORC1 and mTORC2, decreased the levels of the mTORC1 and mTORC2 downstream targets p-p70s6k and p-Akt, respectively. (J–L) In a concentration-dependent manner, ku0063794 at concentrations >1 µmol/l was sufficient to decrease both the p-p70s6k and p-Akt levels. (Abbreviations: con: control; p-p70s6k: phosphorylation of p70s6k; p-Akt: phosphorylation of Akt. *P<0.05 vs. control; **P<0.01 vs. control; ***P<0.001 vs. control; ns, no statistical significance; n = 3.)

Mentions: mTORC1 activation results in the phosphorylation of its downstream target p70S6K, whereas mTORC2 activation results in the phosphorylation of Akt at Ser473 [10], [11]. Rapamycin not only affects mTORC1 signaling but also inhibits the assembly of mTORC2, such that prolonged rapamycin treatment suppresses the downstream activity of mTORC2 [12]. Based on a previous study, we chose a rapamycin concentration that does not exert this long-term effect to specifically examine the short-term effects of rapamycin [7]. 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 were used to determine the rapamycin-induced decrease in the phospho-Akt (Ser473) (p-Akt) protein levels. No significant difference was detected in the p-Akt or Akt levels between each group (Figure 1A, B, D and E). However, the phospho-p70s6k (Thr389) (p-p70s6k) protein levels were markedly decreased in all groups (Figure 1A, B, C and F). No specific mTORC2 inhibitor exists; therefore, to evaluate the effects of mTORC2 signaling on TRPC6 expression, the podocytes were treated with ku0063794, a dual inhibitor of mTORC1 and mTORC2 [13]. As shown in Figure 1G–L, ku0063794 decreased the expression of both the downstream target of mTORC1, p-p70s6k, and the downstream target of mTORC2, p-Akt, in a time- and concentration-dependent manner.


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)

Effects of rapamycin and ku0063794 on downstream substrates of the mTORC1 and mTORC2 signaling pathways.(A–C) Rapamycin decreased the phosphorylation of the mTORC1 downstream target p70S6K in podocytes in a time-dependent manner. However, rapamycin did not decrease the phosphorylation of the mTORC2 downstream target Akt. (D–F) Rapamycin decreased the phosphorylation of p70s6k but not Akt in a concentration-dependent manner. (G–I) In a time-dependent manner, ku0063794, a dual inhibitor of mTORC1 and mTORC2, decreased the levels of the mTORC1 and mTORC2 downstream targets p-p70s6k and p-Akt, respectively. (J–L) In a concentration-dependent manner, ku0063794 at concentrations >1 µmol/l was sufficient to decrease both the p-p70s6k and p-Akt levels. (Abbreviations: con: control; p-p70s6k: phosphorylation of p70s6k; p-Akt: phosphorylation of Akt. *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-g001: Effects of rapamycin and ku0063794 on downstream substrates of the mTORC1 and mTORC2 signaling pathways.(A–C) Rapamycin decreased the phosphorylation of the mTORC1 downstream target p70S6K in podocytes in a time-dependent manner. However, rapamycin did not decrease the phosphorylation of the mTORC2 downstream target Akt. (D–F) Rapamycin decreased the phosphorylation of p70s6k but not Akt in a concentration-dependent manner. (G–I) In a time-dependent manner, ku0063794, a dual inhibitor of mTORC1 and mTORC2, decreased the levels of the mTORC1 and mTORC2 downstream targets p-p70s6k and p-Akt, respectively. (J–L) In a concentration-dependent manner, ku0063794 at concentrations >1 µmol/l was sufficient to decrease both the p-p70s6k and p-Akt levels. (Abbreviations: con: control; p-p70s6k: phosphorylation of p70s6k; p-Akt: phosphorylation of Akt. *P<0.05 vs. control; **P<0.01 vs. control; ***P<0.001 vs. control; ns, no statistical significance; n = 3.)
Mentions: mTORC1 activation results in the phosphorylation of its downstream target p70S6K, whereas mTORC2 activation results in the phosphorylation of Akt at Ser473 [10], [11]. Rapamycin not only affects mTORC1 signaling but also inhibits the assembly of mTORC2, such that prolonged rapamycin treatment suppresses the downstream activity of mTORC2 [12]. Based on a previous study, we chose a rapamycin concentration that does not exert this long-term effect to specifically examine the short-term effects of rapamycin [7]. 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 were used to determine the rapamycin-induced decrease in the phospho-Akt (Ser473) (p-Akt) protein levels. No significant difference was detected in the p-Akt or Akt levels between each group (Figure 1A, B, D and E). However, the phospho-p70s6k (Thr389) (p-p70s6k) protein levels were markedly decreased in all groups (Figure 1A, B, C and F). No specific mTORC2 inhibitor exists; therefore, to evaluate the effects of mTORC2 signaling on TRPC6 expression, the podocytes were treated with ku0063794, a dual inhibitor of mTORC1 and mTORC2 [13]. As shown in Figure 1G–L, ku0063794 decreased the expression of both the downstream target of mTORC1, p-p70s6k, and the downstream target of mTORC2, p-Akt, in a time- and concentration-dependent manner.

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