3.a Effects of KCl and Cyclosporin A on the nuclear translocation of TORC proteins The potential of TORC proteins to enhance CREB-directed gene transcription is tightly regulated by cytosolic and nuclear shuttling of TORC. Under resting conditions TORC is phosphorylated and sequestered in the cytoplasm bound by 14-3-3 proteins. Elevated intracellular Ca2+ and cAMP levels induce the dephosphorylation of TORC and its nuclear accumulation (Bittinger et al., 2004; Screaton et al., 2004). The effects of KCl and cyclosporin A (CsA) on the translocation of TORC proteins into the nucleus were analyzed by immunocytochemistry using an antibody recognizing all of the three TORC isoforms.
KCl was used to depolarize the membrane of HIT-T15 cells thereby increasing intracellular Ca2+ levels by activation of the voltage-gated calcium channel of the L-type.
Ca2+ activates the calcium/calmodulin-dependent phosphatase calcineurin which was shown to dephosphorylate TORC, and is potently inhibited by CsA (Bittinger et al., 2004;
Screaton et al., 2004). HIT-T15 cells were treated with 45 mM KCl, 5 µM CsA, or a combination of both. Figure 9A shows typical images of cells that were untreated for control conditions (upper panel), treated with 45 mM KCl (middle panel), and with the combination of 45 mM KCl and 5 µM CsA (lower panel). For a quantitative study 150 cells per group were analyzed with respect to the localization of endogenous TORC proteins.
Figure 9B shows the result of two independent experiments. The data are presented in percentage of cells with nuclear TORC. Statistical analysis by one-way ANOVA revealed specific effects of treatment with p<0.0001. Without treatment 16.66 ± 7.22% of counted cells showed TORC inside the nucleus. Treatment with 45 mM KCl resulted in increased amounts of cells with nuclear TORC with 81.14 ± 2.70% (p<0.015) compared to the control (Figure 9B). The treatment with 5 µM CsA did not affect the translocation of TORC, as in 11.52 ± 1.25% of the cells TORC was detectable in the nucleus (Figure 9B).
Cotreatment of cells with KCl and CsA reduced the amount of cells with nuclear TORC to 22.65 ± 1.12%, compared to treatment with KCl alone (p<0.0025), to levels statistically not different from untreated cells (Figure 9B).
Figure 9: Nuclear translocation of endogenous TORC proteins in HIT-T15 cells upon treatment with KCl and cyclosporin A, analyzed by immunocytochemistry.
Figure 9A: Typical microscopy images of HIT-T15 cells. The nuclei were stained with DAPI, shown in blue on the left. Endogenous TORC proteins were labeled with the panTORC (1-42) antibody and the AlexaFluor®488, shown in green on the right. Co indicates the control condition without treatment on the upper panel. The middle panel shows cells treated for 30 min with 45 mM KCl.
The lower panel demonstrates cells treated with the combination of 45 mM KCl and 5 µM cyclosporin A (CsA).
Figure 9B: Quantitative analysis of the nuclear translocation of endogenous TORC proteins in HIT-T15 cells. The cells were treated for 30 min with KCl and for 90 min with CsA. 150 cells were studied in each group per experiment. The values express the percentage of cells with nuclear TORC. The data are mean values ± SEM of two independent experiments. Statistical analysis was performed by one-way ANOVA, followed by Student’s t-test: ** p<0.025. Treatment with KCl increased the percentage of cells with nuclear TORC. The treatment with CsA alone did not affect the translocation of TORC. CsA strongly reduced the nuclear accumulation of TORC induced by KCl.
3.b Effects of cAMP and lithium on the nuclear translocation of TORC proteins
The nuclear translocation of TORC proteins is also induced by elevated levels of intracellular cAMP. The salt inducible kinase (SIK) phosphorylates TORC under resting conditions. Activation of protein kinase A by cAMP reduces the activity of SIK leading to the nuclear accumulation of TORC (Takemori and Okamoto, 2008). The effects of cAMP and lithium on the nuclear translocation of TORC were investigated by immunocytochemistry as before. HIT-T15 cells were treated with 1 mM or 2 mM 8-bromo-cAMP, with 20 mM LiCl, or a combination of both. Figure 10A shows typical images of cells that were untreated (upper panel), cells that were treated with 2 mM 8-bromo-cAMP (middle panel), and cells that were treated with 20 mM LiCl (lower panel). For the quantitative analysis 150 cells were examined with respect to the localisation of endogenous TORC. The experiment was performed twice. Figure 10B shows the percentage of cells with nuclear TORC. Statistical analysis with one-way ANOVA revealed specific effects of treatment on the translocation of endogenous TORC with p<0.0001.
Under control conditions without treatment 9.62 ± 4.26% of cells showed TORC inside the nucleus (Figure 10B). Treatment with 20 mM LiCl alone did not affect the translocation of TORC. In 13.56 ± 4.09% was TORC detectable inside the nucleus. Treatment with 1 mM or 2 mM 8-bromo-cAMP resulted in an increase of nuclear accumulation of TORC as 66.24 ± 2.75% (p<0.011) and 86.36 ± 3.64% (p<0.007) of the cells showed TORC inside of the nucleus, respectively (Figure 10B). The increase was concentration dependent as revealed by one-way ANOVA with p<0.05. Treatment with LiCl did not further increase the translocation induced by 1 mM or 2 mM 8-bromo-cAMP with 64.37 ± 3.32% and 93.79 ± 1.01% of cells with TORC inside the nucleus, respectively (Figure 10B).
Figure 10: Nuclear translocation of endogenous TORC in HIT-T15 cells upon treatment with lithium and cAMP, analyzed by immunocytochemistry.
Figure 10A: Typical microscopy images of HIT-T15 cells. The nuclei were stained with DAPI, shown in blue on the left. Endogenous TORC proteins were labeled with the panTORC(1-42) antibody and the AlexaFluor®488, shown in green on the right. Co indicates the control condition without treatment on the upper panel. The middle panel shows cells treated for 30 min with 2 mM 8-bromo-cAMP. The lower panel demonstrates cells treated for 90 min with 20 mM LiCl.
Figure 10B: Quantitative analysis of the nuclear translocation of endogenous TORC proteins in HIT-T15 cells. The cells were treated for 90 min with LiCl and for 30 min with 8-bromo-cAMP. 150 cells were examined in each group per experiment. The values express the percentage of cells with nuclear TORC. The data are mean values ± SEM of two independent experiments. Statistical analysis was performed by one-way ANOVA, followed by Student’s t-test: ** p<0.025; *p<0.05.
The treatment with LiCl alone did not affect the nuclear translocation of endogenous TORC in HIT-T15 cells. The treatment with 1 mM or 2 mM 8-bromo-cAMP increased the nuclear accumulation concentration-dependently. The translocation of TORC induced by 8-bromo-cAMP was not affected by cotreatment with LiCl.