Controlling LDL Receptor Expression by Regulating mRNA Decay Kinetics

Abstract

Low density lipoprotein (LDL) is the most abundant cholesterol transport vehicle in plasma, and remains a major prognostic indicator of atherosclerotic risk. Hepatic LDL receptors are essential for limiting circulating LDL levels, since cholesterol internalized by the liver can be excreted via conjugation to bile salts. This relationship is clearly seen in familial hypercholesterolemic patients, where depletion or lack of functional LDL receptors dramatically increases circulating LDL, accelerating development of atherosclerosis and associated cardiovascular disease. The focus of this study is to characterize post-transcriptional mechanisms regulating hepatic expression of LDL receptors. In this study, we have found that TPA-induced stabilization of receptor mRNA is accompanied by activation of several mitogen activated protein kinase (MAPK) pathways in HepG2 cells. Initial screens with pharmacological inhibitors of specific MAPK pathways indicated that TPA-induced stabilization of LDL receptor mRNA requires the JNK, but not ERK or p38MAPK, pathways. An essential role for JNK activation in stabilization of receptor mRNA was further confirmed by siRNA knockdown experiments. Adenoviral transduction of a constitutively active mutant of MKK7, the JNK-activating kinase, further confirmed that JNK activation was sufficient to stabilize LDL receptor mRNA in the HepG2 model system. Finally, prolonged induction of JNK activity increased the steady-state levels of LDL receptor mRNA and protein, and significantly enhanced cellular LDL-binding activity. Perturbation of the hepatic JNK pathway may represent a novel anti-atherosclerotic therapeutic target, by enhancing the potential of liver cells to internalize and excrete plasma LDL cholesterol. In our efforts to identify the TPA responsive element within the LDL receptor mRNA, we demonstrated that multiple sequence elements within an Alu-rich region in the 3' untranslated region is responsible for TPA-induced stabilization. At present, no trans-acting factors have been identified that modulate mRNA decay kinetics through any of the Alu repetitive elements scattered throughout the human genome. However, preliminary findings described in this work supported by several observations reported in the literature raise the possibility that microRNA (miRNA)-mediated mechanisms might contribute to the regulated stability of LDL receptor mRNA through the Alu-rich region.