Low Electric Treatment activates Rho GTPase via Heat Shock Protein 90 and Protein Kinase C for Intracellular Delivery of siRNA

Abstract

Low electric treatment (LET) promotes intracellular delivery of naked siRNA by altering cellular physiology. However, which signaling molecules and cellular events contribute to LET-mediated siRNA uptake are unclear. Here, we used isobaric tags in relative and absolute quantification (iTRAQ) proteomic analysis to identify changes in the levels of phosphorylated proteins that occur during cellular uptake of siRNA promoted by LET. iTRAQ analysis revealed that heat shock protein 90 (Hsp90)α and myristoylated alanine-rich C-kinase substrate (Marcks) were highly phosphorylated following LET of NIH 3T3 cells, but not untreated cells. Furthermore, the levels of phosphorylated Hsp90α and protein kinase C (PKC)γ were increased by LET both with siRNA and liposomes having various physicochemical properties used as model macromolecules, suggesting that PKCγ activated partly by Ca²⁺ influx as well as Hsp90 chaperone function were involved in LET-mediated cellular siRNA uptake. Furthermore, LET with siRNA induced activation of Rho GTPase via Hsp90 and PKC, which could contribute to cellular siRNA uptake accompanied by actin cytoskeleton remodeling. Collectively, our results suggested that LET-induced Rho GTPase activation via Hsp90 and PKC would participate in actin-dependent cellular uptake of siRNA.

Figure 1

Characteristics of phosphorylated proteins induced by LET. Protein samples for iTRAQ analysis were extracted from non- and LET-treated cells. Proteins in LET-exposed cells having phosphorylation levels that were increased or decreased by at least 1.2-fold relative to untreated cells and >0.62 in Unused Prot Score were classified as having up- or down-regulation of phosphorylation. (a) The number of up- or down-regulated phosphoproteins in LET samples compared with untreated samples. (b) GO slim analysis classification of 139 up-regulated phosphoproteins by biological process, molecular function and cellular component. (c) STRING analysis of GO term-attached proteins among the 15 phosphoproteins showing the largest amount of upregulation following LET.

Figure 2

Activation of Hsp90α and cellular uptake of macromolecules by LET. (a,b) NIH 3T3 cells were treated with LET in presence of cationic lipo, anionic lipo, PEG-lipo or siRNA. Hsp90α (Thr^5/7) levels were determined by western blotting. β-actin was used as a loading control. Samples from UV-exposed cells were used as a positive control. (a) Typical images from at least 3 individual experiments. Full-length blots/gels are shown in Supplementary Fig. S1. (b) Relative levels of p-Hsp90α/β-actin. Data are shown as the ratio to untreated cells. (c–e) Cells were pretreated for 1 h with 10 μM 17-DMAG prior to LET in the presence of cationic lipo (c), anionic lipo (d), or PEG-lipo (e). (f) Cells were pretreated with 10 μM 17-DMAG or 10 μM 17-DMAG with 50 μM VER-155008 for 1 h prior to LET in the presence of siRNA. (c–f) After incubation for 45 min, the cells were lysed, and the fluorescence intensity of the lysate was measured to evaluate cellular uptake of macromolecules. Data are shown as the fluorescence intensity divided by protein amount. Values represent the means of 3 individual experiments. Bars represent standard deviations. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 3

Phosphorylation of PKCγ and intracellular amount of Ca²⁺ after LET with or without macromolecules. (a,b,d,e) Levels of phosphorylated PKCγ were determined by western blotting with β-actin as a loading control. (a,d) Typical images from at least 3 individual experiments. Full-length blots/gels are shown in Supplementary Figs S2 and S3. (b,e) Relative levels of p-PKCγ/β-actin. Data are shown as the ratio to untreated cells. (c) Ca²⁺ influx was visualized by staining with fluo-4 fluorescent dye immediately after LET with or without macromolecules. Green signals show intracellular Ca²⁺. Scale bar indicates 100 μm. (f) Effect of EGTA on LET-mediated cellular uptake of siRNA. Cells were pretreated with EGTA for 30 min prior to LET in presence of rhodamine-labeled siRNA. After incubation for 45 min, cells were lysed, and fluorescence intensity in the lysate was measured. Values represent the means of 3 individual experiments. Bars represent standard deviations. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 4

Actin cytoskeleton remodeling by LET with siRNA. Cells were treated with LET in the presence of siRNA, followed by actin staining. Actin cytoskeleton remodeling was evaluated by CLSM observation. Red and blue signals indicate rhodamine phalloidin-labelled actin and nuclei, respectively. Scale bars indicate 20 μm.

Figure 5

Activation of Rho GTPase by LET with siRNA. (a) Relative Rho GTPase activity. After 36 h of serum starvation, cells were treated with LET in presence of siRNA, and were lysed immediately after LET. Rho GTPase activity in the lysate was measured by G-Lisa activation assay (Cytoskeleton Inc.). (b,c) Cells pretreated with Rho inhibitor I for 4 h, followed by LET in presence of siRNA. (b) Effect of Rho inhibitor I on actin cytoskeleton remodeling induced by LET with siRNA. Red and blue signals indicate rhodamine phalloidin-labelled actin and nuclei, respectively. Scale bars indicate 20 μm. (c) The effect of Rho inhibitor I on LET-mediated cellular uptake of siRNA. Values represent the means of 3 individual experiments. Bars represent standard deviations. *P < 0.05 and ***P < 0.001.

Figure 6

Mechanism and signaling pathway for LET-mediated cellular uptake of siRNA. LET with siRNA induces the phosphorylation of Hsp90α and PKCγ, followed by the activation of Rho GTPase. The action remodeling induced by Rho GTPase promotes the cellular uptake and vesicular transport of siRNA.