Proteasome inhibition induces hsp30 and hsp70 gene expression as well as the acquisition of thermotolerance in Xenopus laevis A6 cells

Abstract

Previous studies have shown that inhibiting the activity of the proteasome leads to the accumulation of damaged or unfolded proteins within the cell. In this study, we report that proteasome inhibitors, lactacystin and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), induced the accumulation of ubiquitinated proteins as well as a dose-and time-dependent increase in the relative levels of heat shock protein (HSP)30 and HSP70 and their respective mRNAs in Xenopus laevis A6 kidney epithelial cells. In A6 cells recovering from MG132 exposure, HSP30 and HSP70 levels were still elevated after 24 h but decreased substantially after 48 h. The activation of heat shock factor 1 (HSF1) may be involved in MG132-induced hsp gene expression in A6 cells since KNK437, a HSF1 inhibitor, repressed the accumulation of HSP30 and HSP70. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. Immunocytochemical studies determined that HSP30 was localized primarily in the cytoplasm of lactacystin-or MG132-treated cells. In some cells treated with higher concentrations of MG132 or lactacystin, we observed in the cortical cytoplasm (1) relatively large HSP30 staining structures, (2) colocalization of actin and HSP30, and (3) cytoplasmic areas that were devoid of HSP30. Lastly, MG132 treatment of A6 cells conferred a state of thermotolerance such that they were able to survive a subsequent thermal challenge.

Expression and localization of the Xenopus laevis small heat shock protein, HSPB6 (HSP20), in A6 kidney epithelial cells

“…5A). The heat shock, MG132 and cadmium chloride treatments employed in this study were previously determined to induce the accumulation of HSP70 and HSP30 in A6 kidney epithelial cells (Gauley et al, 2008;Young and Heikkila, 2010;Khamis and Heikkila, 2013). Incubation of A6 cells at 33°C did not significantly alter the relative level of HSPB6 above the level of untreated control cells.…”

“…Also, increased levels of HSPB6 were reported in dog and rat heart due to congestive heart failure or exercise and also in rat bladder in response to partial urethral ligation (Batts et al, 2006;Boluyt et al, 2006;Dohke et al, 2006). While the mechanism(s) associated with the increase in X. laevis HSPB6 levels in response to cadmium or MG132 is unclear, it is likely that their enhanced levels may be due, at least in part, to a reduction in HSPB6 degradation since both cadmium and MG132 were found to inhibit the ubiquitin-proteasome system in Xenopus A6 cells (Young and Heikkila, 2010;Brunt et al, 2012;Khan et al, 2012;Khamis and Heikkila, 2013;Khan et al, 2015).…”

“…Maintenance and treatment of cells X. laevis A6 kidney epithelial cells (CCL-102) were purchased from American Type Culture Collection (Rockville, MD, USA) and grown at 22°C in T75 cm 2 BD Falcon tissue culture flasks (VWR International, Mississauga, ON, Canada) employing 70% Leibovitz (L)-15 media supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin (all from Sigma-Aldrich, Oakville, ON, Canada). Cells were heat shocked in a 33°C water bath for 2 h and allowed to recover at 22°C for 2 h. Other flasks of cells were treated with either 30 μM MG132 (carbobenzoxy-L-leucyl-L-leucinal dissolved in DMSO; Sigma-Aldrich; Young and Heikkila, 2010) or 100 μM cadmium chloride for 16 h at 22°C (Khamis and Heikkila, 2013). In previous studies, all of the stressor treatments indicated above were shown to induce the accumulation of HSP70 and HSP30 in A6 cells (Gauley et al, 2008;Young and Heikkila, 2010;Khamis and Heikkila, 2013).…”

“…Cells were heat shocked in a 33°C water bath for 2 h and allowed to recover at 22°C for 2 h. Other flasks of cells were treated with either 30 μM MG132 (carbobenzoxy-L-leucyl-L-leucinal dissolved in DMSO; Sigma-Aldrich; Young and Heikkila, 2010) or 100 μM cadmium chloride for 16 h at 22°C (Khamis and Heikkila, 2013). In previous studies, all of the stressor treatments indicated above were shown to induce the accumulation of HSP70 and HSP30 in A6 cells (Gauley et al, 2008;Young and Heikkila, 2010;Khamis and Heikkila, 2013). After the different treatments, media was removed and the cells were rinsed with 2 mL of 65% Hanks Balanced Salt Solution (HBSS).…”

Flavanonol Taxifolin Attenuates Proteasome Inhibition-Induced Apoptosis in Differentiated PC12 Cells by Suppressing Cell Death Process

“…These results suggest that the protective effect of taxifolin on differentiated PC12 cells appears to be attributed to its protective effect on the lipid and protein components from oxidative insults. Proteasome inhibition increases the intracellular levels of Hsp70 by inducing expression of Hsp70 gene [45,46]. Heat shock response contributes to establish a cytoprotective state in a wide variety of human diseases, including inflammation, cancer, aging and neurodegenerative disorders [41,43].…”