Alterations in molecular chaperones and eIF2alpha during lung endothelial cell apoptosis

Abstract

We have previously demonstrated that inhibition of CAAX carboxyl methylation with AGGC caused redistribution and condensation of the ER molecular chaperones, glucose-regulated protein (GRP)-94 and calnexin; an effect that was attenuated by overexpression of dominant active RhoA. We have also shown that AGGC decreased GRP94 protein level; an effect that was dependent on caspase activity. In the present study, we tested the effects of inhibition of posttranslational processing of CAAX proteins on localization and protein levels of molecular chaperones and phosphorylation and protein level of eIF2alpha. We found that both AGGC, which inhibits CAAX carboxyl methylation, and simvastatin, which inhibits CAAX geranylgeranylation, caused relocalization of GRP94, calnexin, and calreticulin, effects that were not seen during endothelial apoptosis induced by TNF-alpha or ultraviolet (UV) irradiation. These results suggest that posttranslational processing of CAAX proteins is important in maintaining localization of molecular chaperones normally found in the ER. We also noted that AGGC, but not simvastatin, TNF-alpha, or UV irradiation, decreased protein levels of most molecular chaperones. Increased eIF2alpha phosphorylation was observed in the early stages of apoptosis, which was independent of the cause of apoptosis. These results suggest that eIF2alpha phosphorylation is a common early response to apoptosis-inducing stimuli. Interestingly, eIF2alpha protein level was decreased in the late stages of apoptosis induced by AGGC, TNF-alpha, and UV irradiation: an effect that was prevented by caspase inhibition. Thus we speculate that caspase(s)-dependent proteolysis of molecular chaperones and eIF2alpha may be novel signaling pathways of apoptosis. We also speculate that increased eIF2alpha phosphorylation is a defensive response against endothelial cell apoptosis.

Fig. 1.

Relocalization of endoplasmic reticulum (ER) molecular chaperones during endothelial cell apoptosis. PAEC were incubated with vehicle or 10 μM AGGC in serum-free MEM medium for 3 h (A), with vehicle or 25 μM simvastatin in MEM medium containing 10% FBS for 24 h (B), with vehicle or 20 ng/ml of TNF-α in HEPES buffer without CO₂ for 3 h (C), or were exposed to 256-nm wavelength UV light for 0 or 3 min and then incubated in serum-free MEM medium for 3 h (D). Subcellular localization of GRP94, calnexin, and calreticulin were assessed by immunofluorescence microscopy. Representative pictures from three independent experiments for each data set are shown. Arrows indicate perinuclear staining, and arrow heads indicate condensed staining.

Fig. 2.

Alterations in protein levels of molecular chaperones during endothelial apoptosis. PAEC were preincubated with vehicle or 100 μM of zVAD-fmk for 1 h and then incubated with vehicle or 10 μM of AGGC in HEPES buffer without CO₂ in the absence or presence of 100 μM zVAD-fmk for 24 h (A), incubated with vehicle or indicated concentrations of simvastatin in serum-free MEM medium for 48 h (B), preincubated with vehicle or 100 μM of zVAD-fmk for 1 h and then incubated with vehicle or 20 ng/ml of TNF-α in HEPES buffer without CO₂ in the absence or presence of 100 μM of zVAD-fmk for 24 h (C), or preincubated with vehicle or 100 μM of zVAD-fmk for 1 h and then exposed to 256-nm wavelength UV light for 0 (vehicle) or 3 min and then incubated with serum-free MEM medium in the absence or presence of 100 μM of zVAD-fmk for 24 h (D). Protein levels of GRP78, GRP94, calnexin, calreticulin, HSP90, and HSP70 were assessed by immunoblot analysis using equal amounts of total proteins in lysates. Equal loading of proteins was confirmed by staining the blots using Ponceau S solution (data not shown). Representative immunoblots of multiple independent experiments for each data set are shown. A: n = 3–7; B: n = 3; C: n = 3–7; D: n = 3–7.

Fig. 3.

The effects of apoptosis-inducing stimuli on eIF2α phosphorylation. PAEC were incubated with vehicle (V) or 10 μM AGGC (A) in HEPES buffer without CO₂ for indicated times (A), incubated with vehicle (0), 10 and 25μM simvastatin in serum-free MEM medium for indicated times (B), incubated with vehicle (V) or 20 ng/ml of TNF-α (T) in HEPES buffer without CO₂ for indicated times (C), or exposed to 256-nm wavelength UV light for 0 (vehicle, V) or 3 min (UV) and then incubated with serum-free MEM medium for indicated times (D). Phosphorylated eIF2α was assessed by immunoblot analysis using equal amounts of total proteins in lysates. Equal loading of proteins was confirmed by staining the blots using Ponceau S solution (data not shown). The data is presented as the ratio of treatment with apoptotic stimuli (AGGC, simvastatin, UV, and TNF-α) to respective vehicle at each time point. A: n = 4–5; B: n = 4; C: n = 3; D: n = 3. In B, the statistical data represent 10 μM simvastatin. *Significant difference vs. respective vehicle (P < 0.05).

Fig. 4.

The effects of apoptosis-inducing stimuli on eIF2α protein level. PAEC were preincubated with vehicle or 100 μM zVAD-fmk for 1 h and then incubated with vehicle or 10 μM AGGC in HEPES buffer without CO₂ in the absence or presence of 100 μM of zVAD-fmk for 24h (A), preincubated with vehicle or 100 μM zVAD-fmk for 1 h and then incubated with vehicle or 20 ng/ml of TNF-α in HEPES buffer without CO₂ in the absence or presence of 100 μM zVAD-fmk for 24 h (B), or preincubated with vehicle or 100 μM zVAD-fmk for 1 h and then exposed to 256-nm wavelength UV light for 0 (vehicle) or 3 min (UV) and then incubated with serum-free MEM medium in the absence or presence of 100 μM zVAD-fmk for 24 h (C). Protein levels of phosphorylated and total eIF2α were assessed by immunoblot analysis using equal amounts of total proteins in lysates. Equal loading of proteins was confirmed by staining the blots using Ponceau S solution (data not shown). Representative immunoblots of multiple independent experiments for each data set are shown. A: n = 4–8; B: n = 3; C: n = 5–7.

Fig. 5.

Proposed mechanisms: AGGC caused endothelial apoptosis through alterations in molecular chaperones and eIF2α. Solid arrows indicate defined pathways, and dashed arrows indicate speculative pathways.