Effect of an Inhibitor of HSP70, YM-1, on Hikeshi Knockout Cells

Abstract

Hikeshi is a protein that mediates the heat stress-induced nuclear import of heat shock protein 70 (HSP70: HSPA1 and HSPA8). Dysfunction of Hikeshi in humans can cause serious hereditary diseases, but the cellular function of Hikeshi is not fully understood. Previously, we reported that depletion of Hikeshi resulted in different effects in two human cell lines following proteotoxic stress. Depletion of Hikeshi reduced the survival of HeLa cancer cells after proteotoxic stress. However, Hikeshi-knockout (KO) hTERT-RPE1 cells, immortalized with telomerase reverse transcriptase, acquired resistance against proteotoxic stress, which was accompanied by increased p21 (WAF1 /CIP1, CDKN1A) expression. p21 is a cell-cycle inhibitor and a direct p53-regulated target gene. Here, we investigated the effect of Hikeshi depletion and inhibition of HSP70 molecular chaperone function on cellular signaling in HeLa and hTERT-RPE1 cells. Functional modulation of HSP70 with the inhibitor YM-1 caused cell death in the HeLa cells but resulted in growth arrest of the hTERT-RPE1 cells. Further, YM-1 treatment dramatically up-regulated p53 and p21 proteins in hTERT-RPE1 cells and down-regulated FoxM1 and survivin, which are regulators of cell cycle progression, in both hTERT-RPE1 cells and HeLa cells. Our results showed that regardless of the presence or absence of Hikeshi, the p53-p21 pathway becomes active when hTERT-RPE1 non-cancer cells are treated with YM-1, which contributes to protection against cell death. Hikeshi might function as an upstream regulator of HSP70, which affects activation of the p53-p21 pathway, especially during and after proteotoxic stress. Hikeshi, HSP70, YM-1, p53, p21 Hikeshi was identified as the heat stress-induced nuclear import carrier of HSP70 (Hsc70 /HSPA8 and Hsp70/HSPA1) 1) . Depletion of Hikeshi in HeLa cells inhibits the nuclear import of HSP70 in response to heat stress, resulting in persistence of heat-shock transcription factor 1 (HSF1) activation and reduction in cell viability. Recently, we investigated the effect of Hikeshi depletion on the survival of two human cell lines after proteotoxic stress and found cell-specific impacts against the stress response in HeLa and hTERT-RPE1 (human retinal pigment epithelial cells immortalized with telomerase reverse transcriptase) cells 2) . In contrast with the HeLa cells, Hikeshi-knockout (KO) hTERT-RPE1 cells became more resistant to the proteotoxic stress compared with wild-type hTERT-RPE1 cells. Depletion of Hikeshi in hTERT-RPE1 cells resulted in Effect of a HSP70 inhibitor YM-1 K.M.Z. Rahman et al.