Post-transcriptional regulation of retroviral vector-transduced low density lipoprotein receptor activity.

Abstract

We have reported the use of a retroviral vector to introduce the low density lipoprotein (LDL) receptor gene into receptor-deficient Watanabe heritable hyperlipidemic (WHHL) rabbit fibroblasts (Miyanohara, A., M. F. Sharkey, D. Steinberg, J. L. Witztum, and T. Friedmann. 1988. Proc. Natl. Acad. Sci. USA. 85: 6538-6542). Because the cDNA for the LDL receptor did not contain the 5' sterol regulatory element that confers sterol-mediated inhibition of LDL receptor transcription, we did not anticipate that LDL receptor activity transduced by this vector would be sterol-responsive. However, we now demonstrate sterol-mediated down-regulation of receptor protein in the infected cells by a mechanism that appears to be mediated at a post-transcriptional level. Down-regulation of LDL receptor activity occurred when infected WHHL cells were preincubated with either LDL or cholesterol plus 25-hydroxycholesterol. Identically organized vectors bearing cDNAs encoding irrelevant genes such as firefly luciferase or bacterial beta-galactosidase exhibited no sterol regulation of reporter gene activity. Insulin receptor activity in WHHL fibroblasts and in WHHL fibroblasts infected with the LDL receptor retroviral vector was also unchanged by sterol. Transfection of LDL receptor-deficient Chinese hamster ovary (CHO) cells with a nonretroviral vector containing the same LDL receptor cDNA also resulted in sterol responsiveness of the transduced LDL receptor. These experiments suggest that the effect of sterol was specific for the LDL receptor transcript. Transgene LDL receptor mRNA levels from the infected cells were unaffected by sterol, indicating that the sterol-mediated reduction in LDL receptor activity did not result from alterations in steady state mRNA levels. These data suggest the existence of post-transcriptional level mechanisms that are responsible for sterol regulation of expression of the transduced LDL receptor gene.