Induction of the 72-kilodalton heat shock protein and protection from ultraviolet B-induced cell death in human keratinocytes by repetitive exposure to heat shock or 15-deoxy-delta(12,14)-prostaglandin J2

Abstract

It has been demonstrated that hyperthermia protects keratinocytes from ultraviolet B (UVB)-induced cell death in culture and in vivo. This effect is mediated by the antiapoptotic effect of heat shock proteins that are transiently induced after exposure to heat at sublethal temperatures. Consequently, induction of Hsp has been proposed as a novel means of photoprotection. However, in the face of daily UVB exposure of human skin in vivo, this approach would not be useful if keratinocytes become less sensitive to Hsp induction with repeated exposure to the inducing agent. The aim of this study was to investigate whether repeated exposure to hyperthermia or to the stress protein activating cyclopentenone prostaglandin 15-deoxy-delta(12,14)-prostaglandin J2 (15dPGJ2) leads to adaptation of the cells, attenuation of the heat shock response, and abrogation of the protective effect. Normal human epidermal keratinocytes (NHEK) and the carcinoma-derived cell line A431 were exposed to either 42 degrees C or to 15dPGJ2 for 4 hours at 24-hour intervals for 4 consecutive days. The intracellular level of the 72-kDa heat shock protein (Hsp72) was determined by enzyme-linked immunosorbent assay (ELISA). Cells were exposed to UVB from a metal halide source after the last heat or 15dPGJ2 treatment, and survival was determined 24 hours after exposure by a MTT assay. Our results demonstrate that (1) heat shock and 15dPGJ2 are potent inducers of Hsp72 expression and lead to increased resistance to UVB-induced cell death in human keratinocytes; (2) re-exposure to heat shock leads to a superinduction without attenuation of the absolute increase in Hsp72 and of its UVB-protective effect; (3) the UVB tolerance induced by 15dPGJ2 is enhanced by repeated exposure without a further increase of Hsp72; (4) repeated heat shock and 15dPGJ2 up to a concentration of 1 microg/mL have no influence on cell growth over a period of 4 days. We conclude that through repeated exposure to Hsp-inducing factors, stress tolerance can be maintained without additional toxicity in human keratinocytes. These results provide a basis for the development of nontoxic Hsp inducers that can be repeatedly applied without loss of effect.

Fig 1.

Induction of Hsp72 by repetitive heat shock and 15dPGJ₂. Cells were exposed to 42°C for 4 hours (A,B) or to 15dPGJ₂ (C) every 24 hours for 4 consecutive days (indicated by bold sections on the x-axis). Hsp72 was measured by ELISA prior to and after each heat shock. Results are given as multiples of baseline expression and represent means of 3 (A431) and 4 (NHEK) independent experiments ± SEM

Fig 2.

Heat shock induced resistance to UVB. Cells were exposed to single or repeated heat shock or 15dPGJ₂ prior to irradiation with UVB according to the experimental setup shown in D. Survival was determined 24 hours after irradiation by a colorimetric assay. (A) NHEK exposed to single and repeated heat shock; (B) A431 exposed to single and repeated heat shock; (C) NHEK exposed to single and repeated 15dPGJ₂; the UV dose in this experiment was 400 mJ/cm². Values indicate survival rates relative to unirradiated controls and represent mean ± SEM of 3 independent experiments

Fig 3.

Influence of repeated heat shock on total cellular protein. NHEK (A) and A431 (B) were exposed to 42°C for 4 hours every 24 hours for 4 consecutive days (solid lines) or sham treatments (dashed lines). (C) NHED were exposed to 15dPGJ₂ at various concentrations for 4 hours every 24 hours. Total protein content was determined from cell extracts prepared by repeated cycles of freeze-thawing. Results are given as mean ± SEM of 3 independent experiments