FIG. 4.

Involvement of Akt, ERK1 and -2 and JNK in heat-induced death of fibroblasts. (A) Inhibition of Akt and ERK1 and -2 kinases but not p38 kinase decreases cell viability under moderate heat shock treatment. IMR90 fibroblasts were pretreated for 30 min with p38 kinase inhibitor SB203580 (SB) (10 μM), ERK1 and -2 inhibitor PD98059 (PD) (50 μM), or IP₃ kinase inhibitor wortmannin (WM) (100 nM) or LY294,002 (LY) (50 μM) and were then subjected to heat shock (HS) for 45 or 75 min. Vehicle (0.1% dimethyl sulfoxide) was added as a control to heat-shocked cells, and cell death was measured 24 h later as shown in Fig. 1C. con, control. (B) SB203580 inhibits p38 activity. Fibroblasts were pretreated with SB203580 as described above and were exposed to heat shock (45°C, 75 min). p38 activity was measured immediately after heat shock by an in vitro assay of its downstream kinase (MAPKAP-2), using Hsp27 as a substrate (see Materials and Methods for further details). (C to E) Inhibition of JNK activity suppresses heat-induced cell death. Fibroblasts were infected with an adenovirus expressing dnSEK; 72 h after infection, cells were subjected to heat shock (45°C, 75 min) and expression of dnSEK (C), JNK activity (D), and cell viability (E) were assayed. Expression of dnSEK was assayed by immunoblotting with antibodies to SEK1; JNK activity was measured with antibodies to active (phosphorylated) JNK. Cell viability was assayed as explained for Fig. 1C. The amount of added dnSEK adenovirus shown in panels C and D was 25 relative units (25 × 10⁹ particles per 35-mm plate). This experiment was reproduced three times. Data shown in panels B to E represent typical experiments. Adv, adenovirus; rel, relative.