Ribozyme-mediated inhibition of caspase-12 activity reduces apoptosis induced by endoplasmic reticulum stress in primary mouse hepatocytes.

Apoptosis via endoplasmic reticulum (ER) stress has been reported in many cell lines. ER stress plays an important role in many liver diseases and caspase-12 is the central player in ER stress-induced apoptosis. We conducted an investigation to determine whether catalytic cleavage of caspase-12 mRNA by hammerhead ribozymes can protect liver cells from apoptosis induced by ER stress. Thapsigargin (TG) was used to induce primary mouse hepatocytes apoptosis to establish the experimental system of ER stress- mediated apoptosis. The effective ribozyme-Rz138 selected in vitro was embedded in eukaryotic expression vector and transfected into cultured cells. The activity of Rz138 in primary mouse hepatocytes was determined by testing the expression of caspase-12 mRNA and procaspase-12 protein in ribozyme treated cells compared with the control. The anti-apoptotic effect was assayed by the nuclear morphological features of primary mouse hepatocytes stained with Hoechst 33258 under the fluorescence microscope. Primary mouse hepatocytes were incubated with 4 micromol/l TG, the percentage of apoptotic cells increased along with the treatment time, obvious apoptosis was observed after 4 micromol/l TG treatment for 30 h. Expression of caspase-12 mRNA and procaspase-12 protein were decreased significantly in hepatocytes transfected with pRz138 compared with those untransfected. The percentage of apoptotic cells was also decreased in pRz138 treated cells measured by staining with Hoechst 33258. Rz138, as a specific inhibitor of caspase-12 can down-regulate the expression of caspase-12 in primary mouse hepatocytes and protect the cells from apoptosis induced by TG. These results further elucidated the new treatment for diseases associated with ER stress-mediated apoptosis.