Sodium arsenite and cadmium chloride induction of proteasomal inhibition and HSP accumulation in Xenopus laevis A6 kidney epithelial cells.

Sodium arsenite (NA) and cadmium chloride (CdCl(2)) are relatively abundant environmental toxicants that have multiple toxic effects including carcinogenesis, dysfunction of gene regulation and DNA and protein damage. In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with concentrations of NA (20-30 μM) or CdCl(2) (100-200 μM) that induced HSP30 and HSP70 accumulation also produced an increase in the relative levels of ubiquitinated protein. Actin protein levels were unchanged in these experiments. In time course experiments, the levels of ubiquitinated protein and HSPs increased over a 24h exposure to NA or CdCl(2). Furthermore, treatment of cells with NA or CdCl(2) reduced the relative levels of proteasome chymotrypsin (CT)-like activity compared to control. Interestingly, pretreatment of cells with the HSP accumulation inhibitor, KNK437, prior to NA or CdCl(2) exposure decreased the relative levels of ubiquitinated protein as well as HSP30 and HSP70. A similar finding was made with ubiquitinated protein induced by proteasomal inhibitors, MG132 and celastrol, known to induce HSP accumulation in A6 cells. However, the NA- or CdCl(2)-induced decrease in proteasome CT-like activity was not altered by KNK437 pretreatment. This study has shown for the first time in poikilothermic vertebrates that NA and CdCl(2) can inhibit proteasomal activity and that there is a possible association between HSP accumulation and the mechanism of protein ubiquitination.

Brunt JJ ，Khan S ，Heikkila JJ 《COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY》

Enhanced HSP30 and HSP70 accumulation in Xenopus cells subjected to concurrent sodium arsenite and cadmium chloride stress.

Heat shock proteins (HSPs) are molecular chaperones that aid in protein folding, translocation and in preventing stress-induced protein aggregation. The present study examined the effect of simultaneous sodium arsenite and cadmium chloride treatment on the pattern of HSP30 and HSP70 accumulation in A6 kidney epithelial cells of the frog, Xenopus laevis. Immunoblot analysis revealed that HSP30 and HSP70 accumulation in concurrent stressor treatments were significantly higher than the sum of HSP30 or HSP70 accumulation in individual treatments. This finding suggested a synergistic action between sodium arsenite and cadmium chloride. KNK437 inhibitor studies indicated that the combined stressor-induced accumulation of HSPs may be regulated, at least in part, at the level of transcription. Immunocytochemistry revealed that simultaneous treatment of cells with the two stressors induced HSP30 accumulation primarily in the cytoplasm in a punctate pattern with some dysregulation of F-actin structure. Increased ubiquitinated protein accumulation was observed with combined sodium arsenite and cadmium chloride treatment compared to individual stressors suggesting an impairment of the ubiquitin proteasome degradation system. The addition of a mild heat shock further enhanced the accumulation of HSP30 and HSP70 in response to relatively low concentrations of sodium arsenite plus cadmium chloride.

Khamis I ，Heikkila JJ 《COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY》

Accumulation of heme oxygenase-1 (HSP32) in Xenopus laevis A6 kidney epithelial cells treated with sodium arsenite, cadmium chloride or proteasomal inhibitors.

The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats.

Music E ，Khan S ，Khamis I ，Heikkila JJ ... -  《COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY》

Celastrol can inhibit proteasome activity and upregulate the expression of heat shock protein genes, hsp30 and hsp70, in Xenopus laevis A6 cells.

In eukaryotes, the ubiquitin-proteasome system (UPS) is responsible for the degradation of most proteins. Proteasome inhibition, which has been associated with various diseases, can cause alterations in various intracellular processes including the expression of heat shock protein (hsp) genes. In this study, we show that celastrol, a quinone methide triterpene and anti-inflammatory agent, inhibited proteasome activity and enhanced HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Treatment of cells with celastrol induced the accumulation of ubiquitinated protein and inhibited chymotrypsin-like activity. This was accompanied by a dose- and time-dependent accumulation of HSP30 and HSP70. Celastrol-induced HSP accumulation was mediated by HSF1-DNA binding activity since this response was inhibited by the HSF1 activation inhibitor, KNK437. Simultaneous exposure of cells with celastrol plus either mild heat shock or the proteasome inhibitor, MG132, produced an enhanced accumulation of HSP30 that was greater than the sum of the individual stressors alone. Immunocytochemical analysis revealed that celastrol-induced HSP30 accumulation occurred in the cytoplasm in a granular pattern supplemented with larger circular HSP30 staining structures. HSP30 was also noted in the nucleus with less staining in the nucleolus. In some cells, celastrol induced the collapse of the actin cytoskeleton and conversion to a rounder morphology. In conclusion, this study has shown that celastrol inhibited proteasome activity and induced HSF1-mediated expression of hsp genes in amphibian cells.

Walcott SE ，Heikkila JJ 《-》

Comparison of the effect of heat shock factor inhibitor, KNK437, on heat shock- and chemical stress-induced hsp30 gene expression in Xenopus laevis A6 cells.

Voyer J ，Heikkila JJ 《-》