ACTIVATION OF CASPASE–12 BY OXIDIZED CHOLESTEROL IN ARPE–19 CELLS

Abstract

Abstract: : Purpose: To investigate the involvement of caspase–12 in apoptosis induced by 7–ketocholesterol in human retinal pigment epithelial (ARPE–19) cells. Methods: ARPE–19 cells were cultured overnight in 60 mm culture dishes with Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and then treated with 20 µg/ml of 7–ketocholesterol for 2 or 24 hours. Cell protein was extracted by lysis of cells with 1X CHAPS buffer and analyzed for activation of caspase–12 by Western blot analysis. Rabbit polyclonal antibody against activated caspase–12 (1:1000) was used for the Western blot. Results: An approximately 42 kDa band of cleaved caspase–12 was seen in ARPE–19 cells treated with 7–ketocholesterol at both 2 hours and 24 hours. There was a slight increase in the amount of activated caspase–12 at 24 hours compared to 2 hours. Cleaved caspase–12 was not seen in the untreated ARPE–19 cells. Conclusions: Previous studies showed that 7–ketocholesterol induced apoptosis in ARPE–19 cells. Caspase–12 is a representative of endoplasmic reticulum stress–induced cell–signaling apoptotic pathway. Our study demonstrated that the endoplasmic reticulum (ER) pathway of apoptosis may be occurring in the ARPE–19 cells as a result of 7–ketocholesterol treatment.

Keywords: retinal pigment epithelium • apoptosis/cell death