IL-6 triggers lysosomal degradation of LDL-R and enhances LDL-C uptake in vascular endothelial cells via macropinocytosis

Endothelial dysfunction profoundly compromises the barrier function that precludes trans-endothelial entry of low-density lipoprotein cholesterol (LDL-C) into the vessel wall. LDL-C retention in the vessel wall is atherogenic and its flux involves several mechanisms including LDL-receptor (LDL-R) mediated transcytosis, a process that is facilitated by inflammatory stressors. In this study, we aimed to investigate the role of interleukin-6 (IL-6) in regulating LDL-R and LDL-C uptake by vascular endothelial cells. We used commercially available Human umbilical vein endothelial cells (HUVECs) in this study. Flow cytometry, western blotting, qRT-PCR and ELISA were used to investigate expression of LDL-R and Mylip/IDOL. LDL-C uptake and free cholesterol levels in HUVECs was assessed using flowcytometry and mass-spectrometry respectively. We show that HUVECs treated with a combination of IL-6 and soluble IL-6 receptor (sIL-6R) result in a significant reduction in surface expression of LDL-R, an effect that is reversed by soluble gp130Fc – an antagonist of IL-6 trans-singling. Using pharmacological inhibitors and gene silencing techniques, we demonstrate that IL-6 trans-signaling induced downregulation of LDL-R is attained through lysosomal degradation mediated by the E3 ubiquitin ligase Mylip. Conversely, HUVECs treated with IL-6 in combination with sIL-6R exhibit markedly increased uptake of native LDL-C which is also inhibited by sgp130Fc, the actin inhibitor Cytochalasin D and the macropinocytosis inhibitor EIPA. Although stimulation of HUVECs upregulated the expression of scavenger receptors CD36 and CXCL16, their contribution to native LDL-C uptake turned out to be negligible. Collectively, this study highlights the role of IL-6 in the regulation of LDL-R expression and cholesterol homeostasis in vascular ECs. IL-6 trans-signaling downregulates LDL-R yet increases LDL-C uptake via an LDL-R–independent, actin-dependent macropinocytosis pathway.