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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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Blockade of mTORC2 decreased TRPC6-dependent calcium influx in podocytes (confocal microscopy).(A) The podocytes were imaged at 0 seconds before the addition of hyperforin, a specific agonist of TRPC6, to record the baseline fluorescence. The images in lines 2 and 3 were captured at the time point of the maximal fluorescence intensity (12-seconds) and at the last recorded time point (60-seconds) after hyperforin addition, respectively. The podocytes in rows 1, 2, and 3 were exposed to DMSO (control conditions), rapamycin (50 nmol/l) and ku0063794 (3 µmol/l) for 24 h, respectively. (B, E) The images in rows 1, 2, and 3 are magnifications of cells 1, 2, and 3 marked in Figure 4A, respectively, depicting the changes in fluorescence over shorter periods. Cell 1 and cell 2 exhibited similarly increased fluorescence within the first 12 seconds followed by decreased fluorescence beyond 12 seconds after hyperforin addition. Cell 3 exhibited no significant difference in fluorescence following hyperforin addition within 1 minute. (C, F) The calcium level in podocytes was expressed as the relative fluorescence intensity. Each trace represents the mean value that was derived from 3 cells or fields in a single experiment. The results are presented as the mean value of three independent experiments. (D) After treatment of podocytes with siRNA, the images were captured at 3 different time points: 0 s, 12 s and 60 s. The rictor siRNA group exhibited small changes in fluorescence intensity, whereas the control and raptor siRNA group exhibited peak-like changes in fluorescence intensity. (*P<0.05 vs. control; n = 3. Bar = 40 µm.)
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pone-0112972-g004: Blockade of mTORC2 decreased TRPC6-dependent calcium influx in podocytes (confocal microscopy).(A) The podocytes were imaged at 0 seconds before the addition of hyperforin, a specific agonist of TRPC6, to record the baseline fluorescence. The images in lines 2 and 3 were captured at the time point of the maximal fluorescence intensity (12-seconds) and at the last recorded time point (60-seconds) after hyperforin addition, respectively. The podocytes in rows 1, 2, and 3 were exposed to DMSO (control conditions), rapamycin (50 nmol/l) and ku0063794 (3 µmol/l) for 24 h, respectively. (B, E) The images in rows 1, 2, and 3 are magnifications of cells 1, 2, and 3 marked in Figure 4A, respectively, depicting the changes in fluorescence over shorter periods. Cell 1 and cell 2 exhibited similarly increased fluorescence within the first 12 seconds followed by decreased fluorescence beyond 12 seconds after hyperforin addition. Cell 3 exhibited no significant difference in fluorescence following hyperforin addition within 1 minute. (C, F) The calcium level in podocytes was expressed as the relative fluorescence intensity. Each trace represents the mean value that was derived from 3 cells or fields in a single experiment. The results are presented as the mean value of three independent experiments. (D) After treatment of podocytes with siRNA, the images were captured at 3 different time points: 0 s, 12 s and 60 s. The rictor siRNA group exhibited small changes in fluorescence intensity, whereas the control and raptor siRNA group exhibited peak-like changes in fluorescence intensity. (*P<0.05 vs. control; n = 3. Bar = 40 µm.)

Mentions: TRPC6-mediated calcium influx plays an important role in renal diseases; therefore, the difference in TRPC6 function after treatment with pharmacological inhibitors or siRNA knockdown was also investigated in this study. The Fluo-3 AM fluorescence intensity, which indicates the calcium concentration, was evaluated in podocytes. To examine TRPC6-dependent calcium influx, the specific TRPC6 agonist hyperforin was applied in the present study [14]. As shown in Figure 4A–C, treatment with rapamycin (50 nmol/l) for 24 h did not change the hyperforin-induced Fluo-3 AM fluorescence intensity compared to the control. However, the hyperforin-induced Fluo-3 AM fluorescence intensity was significantly altered after treatment with ku0063794 (3 µmol/l) for 24 h compared to the control. Additionally, knockdown of raptor expression did not affect the Fluo-3 AM fluorescence intensity compared to control siRNA transfection, whereas of knockdown of rictor expression induced a significant alteration in the Fluo-3 AM fluorescence intensity compared to control siRNA transfection (Figure 4D–F).In addition, podocin protein expression was examined due to interact with TRPC6 and modulate the activation of TRPC6 [15]. As shown in Figure 3I, the expression of podocin was significantly decreased in the rictor siRNA group.


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)

Blockade of mTORC2 decreased TRPC6-dependent calcium influx in podocytes (confocal microscopy).(A) The podocytes were imaged at 0 seconds before the addition of hyperforin, a specific agonist of TRPC6, to record the baseline fluorescence. The images in lines 2 and 3 were captured at the time point of the maximal fluorescence intensity (12-seconds) and at the last recorded time point (60-seconds) after hyperforin addition, respectively. The podocytes in rows 1, 2, and 3 were exposed to DMSO (control conditions), rapamycin (50 nmol/l) and ku0063794 (3 µmol/l) for 24 h, respectively. (B, E) The images in rows 1, 2, and 3 are magnifications of cells 1, 2, and 3 marked in Figure 4A, respectively, depicting the changes in fluorescence over shorter periods. Cell 1 and cell 2 exhibited similarly increased fluorescence within the first 12 seconds followed by decreased fluorescence beyond 12 seconds after hyperforin addition. Cell 3 exhibited no significant difference in fluorescence following hyperforin addition within 1 minute. (C, F) The calcium level in podocytes was expressed as the relative fluorescence intensity. Each trace represents the mean value that was derived from 3 cells or fields in a single experiment. The results are presented as the mean value of three independent experiments. (D) After treatment of podocytes with siRNA, the images were captured at 3 different time points: 0 s, 12 s and 60 s. The rictor siRNA group exhibited small changes in fluorescence intensity, whereas the control and raptor siRNA group exhibited peak-like changes in fluorescence intensity. (*P<0.05 vs. control; n = 3. Bar = 40 µm.)
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pone-0112972-g004: Blockade of mTORC2 decreased TRPC6-dependent calcium influx in podocytes (confocal microscopy).(A) The podocytes were imaged at 0 seconds before the addition of hyperforin, a specific agonist of TRPC6, to record the baseline fluorescence. The images in lines 2 and 3 were captured at the time point of the maximal fluorescence intensity (12-seconds) and at the last recorded time point (60-seconds) after hyperforin addition, respectively. The podocytes in rows 1, 2, and 3 were exposed to DMSO (control conditions), rapamycin (50 nmol/l) and ku0063794 (3 µmol/l) for 24 h, respectively. (B, E) The images in rows 1, 2, and 3 are magnifications of cells 1, 2, and 3 marked in Figure 4A, respectively, depicting the changes in fluorescence over shorter periods. Cell 1 and cell 2 exhibited similarly increased fluorescence within the first 12 seconds followed by decreased fluorescence beyond 12 seconds after hyperforin addition. Cell 3 exhibited no significant difference in fluorescence following hyperforin addition within 1 minute. (C, F) The calcium level in podocytes was expressed as the relative fluorescence intensity. Each trace represents the mean value that was derived from 3 cells or fields in a single experiment. The results are presented as the mean value of three independent experiments. (D) After treatment of podocytes with siRNA, the images were captured at 3 different time points: 0 s, 12 s and 60 s. The rictor siRNA group exhibited small changes in fluorescence intensity, whereas the control and raptor siRNA group exhibited peak-like changes in fluorescence intensity. (*P<0.05 vs. control; n = 3. Bar = 40 µm.)
Mentions: TRPC6-mediated calcium influx plays an important role in renal diseases; therefore, the difference in TRPC6 function after treatment with pharmacological inhibitors or siRNA knockdown was also investigated in this study. The Fluo-3 AM fluorescence intensity, which indicates the calcium concentration, was evaluated in podocytes. To examine TRPC6-dependent calcium influx, the specific TRPC6 agonist hyperforin was applied in the present study [14]. As shown in Figure 4A–C, treatment with rapamycin (50 nmol/l) for 24 h did not change the hyperforin-induced Fluo-3 AM fluorescence intensity compared to the control. However, the hyperforin-induced Fluo-3 AM fluorescence intensity was significantly altered after treatment with ku0063794 (3 µmol/l) for 24 h compared to the control. Additionally, knockdown of raptor expression did not affect the Fluo-3 AM fluorescence intensity compared to control siRNA transfection, whereas of knockdown of rictor expression induced a significant alteration in the Fluo-3 AM fluorescence intensity compared to control siRNA transfection (Figure 4D–F).In addition, podocin protein expression was examined due to interact with TRPC6 and modulate the activation of TRPC6 [15]. As shown in Figure 3I, the expression of podocin was significantly decreased in the rictor siRNA group.

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