The ER is a sensor for cellular stresses such as hyperglycemia, dyslipoproteinemia, hypoxia, and oxidative stress, and ER stress may play an important role in DR.
28–30 In the current study, we found that HOG-LDL induced phosphorylation of eIF2α and increased expression levels of GRP78, cleaved ATF6, and CHOP, whereas N-LDL was without effect (
Fig. 3). It is interesting to note that in response to HOG-LDL, these molecules were induced at different time points, with phospho-eIF2α being the earliest, followed by GRP78, CHOP, and ATF-6. Consistent with a previous report that CHOP induces the transcription of pro-apoptotic genes and suppresses the transcription of Bcl-2,
47 our data showed increases in the active forms of caspase-3 and PARP, and a decrease of Bcl-2 at 24 hours (i.e., after CHOP activation [at 12 hours]) (
Figs. 1,
3). Bax expression, however, increased at an earlier time point, perhaps via other synergistic pathways. The timing of responses suggests that HOG-LDL first triggers the unfolded protein response, which in turn leads to activation of apoptosis. Although ATF6 was also activated, the late response to increasing protein degradation by the ATF6 pathway could not prevent the deleterious process. Indeed, blockage of ER stress with the inhibitor, 4-PBA, could inhibit HOG-LDL–induced activation of PARP (
Fig. 4C), thereafter reducing apoptosis (
Fig. 4D) and improving cell viability (
Fig. 4B). Of interest, blockade of oxidative stress with NAC attenuated both ER stress and apoptosis induced by HOG-LDL (
Figs. 3B,
4D), suggesting that oxidative stress is an initial step of HOG-LDL–induced ER stress and apoptosis. These results are consistent with the idea that HOG-LDL–induced oxidative stress results in an accumulation of damaged proteins, thereby eliciting the unfolded protein response and subsequent apoptosis. We have observed the same timing in HOG-LDL–treated human capillary pericytes and retinal pigment epithelial cells (Yang S, Wu M, Fu D, Du M, Wilson K, Lyons T, unpublished data, 2012).