The expression level of the liver low density lipoprotein receptor (LDLR) is one of the most important regulators of human plasma LDL cholesterol (LDL-c). Increased hepatic LDLR expression results in an improved clearance of LDL-c from circulation, hence directly reducing the risk of coronary heart disease. Currently, in the research area of LDLR, the transcriptional regulation of the LDLR gene has been extensively investigated and most of the pharmaceutical interventions are aimed to increase LDLR gene transcription, although indirectly, in order to lower plasma LDL-c. However, the posttranscriptional modulations of LDLR are much understudied and the mechanisms controlling LDLR mRNA turnover are largely unknown. By conducting a screening of 700 compounds isolated from Chinese herbs, we have identified Berberine (BBR), a compound originally isolated from the Chinese herb Huanglian, as a novel upregulator of hepatic LDLR. BBR strongly increased the LDLR mRNA and protein expression in human hepatoma-derived cells and it also effectively reduced the serum LDL-c in hypercholesterolemic patients when tested in a placebo-controlled clinical study. Further investigations reveal that BBR increases the LDLR expression by a unique posttranscriptional mechanism involving LDLR mRNA stabilization through its 3'-untranslated region (3'UTR). We have now further localized the BBR-responsive regulatory sequences to a 905 nt segment in the 5'proximal region of LDLR 3'UTR. The overall objectives of this new proposal are to fully elucidate the molecular and cellular mechanisms by which BBR regulates LDL mRNA stability in cell culture and in animal models.
The specific aims of this new proposal are to: 1) identify the BBRresponsive cis-regulatory elements residing in the LDLR mRNA 3'UTR through a series of mutational and functional analyses of a panel of reporter constructs that contain the wild-type and mutated cis-regulatory sequences of the 905 nt BBR-responsive region of the 3'UTR; 2) characterize the mRNA-binding proteins that specifically interact with the BBR-responsive elements; and 3) demonstrate the in vivo activities of BBR in stabilizing the hepatic LDLR mRNA and the consequential LDL-c lowering in animal models. The successful accomplishment of these specific aims will have profound impacts to the current research area of hypercholesterolemia. A clear understanding at the molecular level of how LDLR mRNA stability is regulated in terms of cis-acting regulatory sequences and trans-acting factors will provide an important foundation for exploiting the new therapeutic approach that increases liver LDLR expression through mRNA stabilization.
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