Inducible overexpression of endoplasmic reticulum chaperone regulators in a mouse liver cell line.

摘要

Reduction of dietary calories while maintaining adequate nutrition, calorie restriction (CR), has been reported to increase the rate of apoptosis in preneoplasitic and normal cells, perhaps accounting for at least a part of its anticancer benefits. Our lab has shown that CR decreases the expression of almost every endoplasmic reticulum (ER) chaperone (such as GRP78) tested in the liver and some other tissues. In addition, reduced ER (or cytoplasmic) chaperone abundance is associated with enhanced apoptosis, reduced cancer incidence and reduced tumor progression. Increased ER (or cytoplasmic) chaperone abundance suppresses apoptosis. To directly investigate the link between CR, ER chaperone levels, and apoptosis, the widely employed tetracycline-controlled gene expression system was used to overexpress and underexpress GRP78 and the ER chaperone regulators IRE1 and ATF6 in a mouse embryonic liver cell line. The inducible cell lines were constructed by first transfecting the mouse liver cells with a vector containing the reverse tet-controlled transactivator (rtTA) driven by a liver-specific promoter. Cells containing the vector were selected, tested and transfected with a second vector containing the target gene under the control of the Tet Response Element (TRE). Cells containing both vectors were then selected, subcloned and characterized.;In the ATF6alpha N-terminal form-overexpressing cells, the N-terminal transgene mRNA and protein levels increased by 20--30-fold in the presence of the inducer doxycycline as compared with the levels in the absence of doxycycline, while the levels of the endogenous full-length ATF6alpha mRNA and protein were not changed. The mRNA and protein levels of ER chaperones GRP78 and GRP94 were induced by 2--4-fold in these cells. The IRE1alpha-overexpressing cells exhibited a 5--6-fold increase in IRE1alpha mRNA levels in the presence of doxycycline. But the increase in the protein levels could only be barely observed. These cells exhibited a mild increase in GRP78 and GRP94 mRNA and protein levels. These cell lines will allow investigations of the link between modulating ER chaperones levels and apoptosis independent of diet and stress in cells, as well as other related studies.