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.

Low level electricity increases the secretion of extracellular vesicles from cultured cells

“…However, detailed cellular mechanisms for positive effects by ET onto brain remain largely unclear. Our previous and present studies demonstrated that ET induces activation of intercellular signaling such as Rho GTPase via HSP90 and PKC [22], and also increases the secretion of EV from cultured cells. Although the effect of ET on brain-derived cells are needed to be examined in future, ET-mediated intracellular signaling activation and increase of EV secretion may have the potential to be involved in beneficial effects of tDCS in the brain.…”

“…Since our previous study demonstrated that ET of cultured cells induces endocytosis of macromolecules via activation of Rho GTPase [22], we evaluated the influence of Rho GTPase inhibition on ET-mediated increase in EV secretion by using a Rho GTPase inhibitor, Rhosin hydrochloride [26]. The results showed that increase in the particle number of the collected EVs from 3T3 Swiss Albino cells treated by low level electricity was hardly inhibited by the Rho GTPase inhibitor (Fig.…”

“…Thus, external stimuli that activate intracellular signaling would likely increase exosome secretion from the cells. Our previous reports demonstrated that treatment of cultivated cells with ET (0.34 mA/cm 2 ) activates intracellular signaling and induces endocytosis of extraneous macromolecules such as siRNA and FITC-conjugated dextran (molecular weight: 10,000 and 70,000) [[20], [21], [22]]. Hence, in this study, we investigated whether ET-mediated intracellular signaling activation leads to increased EV secretion from cultured cells.…”

“…In addition, ET could increase EV secretion from not only a cancer cell (B16F1) but also a normal cell (3T3 Swiss Albino), implying that the method using ET may be applied for various types of cells, whereas other stimuli reported previously were investigated only in cancer cell lines except that the method using LLLI was performed in human ECs. Although the detailed mechanisms of ET-mediated EV secretion need to be elucidated, our previous studies reported that ET induces Rho GTPase activation via HSP90 and PKC by Ca 2+ influx, and also phosphorylates Ras-related protein Rab-10, a protein associated with vesicle-mediated transport processes [22]. Since cellular signalings including Rho GTPase [37], the Rab family [19], and increase of intracellular Ca 2+ were reportedly involved in the production and secretion of exosomes [18], ET-mediated activation of those signaling pathways could increase EV secretion.…”

“…Our previous studies reported that low level electric treatment (ET; 0.3–0.5 mA/cm 2 ), which is employed for iontophoresis, a transdermal drug delivery technology, of cultured cells induce cellular uptake of macromolecules such as small interfering RNA (siRNA) and dextran (molecular weight: 10,000 and 70,000) via induction of endocytosis [20,21]. With regard to the mechanisms of ET-mediated endocytosis, we also reported that activation of Rho GTPase via heat shock protein 90 (HSP90) and protein kinase C (PKC) is involved in endocytosis induction [22]. On the other hand, it was previously reported that early endosomes imported by endocytosis are sort to late endosomes and then form multivesicular bodies (MVB), and exosomes are produced in MVB [23].…”