Abstract

BackgroundEndoplasmic reticulum (ER) stress induces ER expansion. The expansion of the intracisternal space of the ER was found in macrophages associated with human atherosclerotic lesions. We also previously reported that palmitate induces cisternal ER expansion and necrosis in RAW 264.7 cells. In this study, we report on an investigation of the likely mechanism responsible for this palmitate-induced cisternal ER expansion in a mouse macrophage cell line, RAW 264.7 cells.MethodsRAW 264.7 cells were pre-treated with the designated inhibitor or siRNA, followed by treatment with palmitate. Changes in the ER structure were examined by transmission electron microscopy. The induction of ER stress was confirmed by an increase in the extent of phosphorylation of PERK, the expression of BiP and CHOP, and the splicing of XBP-1 mRNA. Phospholipid staining was performed with the LipidTOX Red phospholipidosis detection reagent. Related gene expressions were detected by quantitative real time-RT-PCR or RT-PCR.ResultsPalmitate was found to induce ER stress and cisternal ER expansion. In addition, palmitate-induced cisternal ER expansion was attenuated by ER stress inhibitors, such as 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid (TUDCA). The findings also show that palmitate induced-mRNA expression of CCTα, which increases phospholipid synthesis, was attenuated by the down-regulation of XBP-1, a part of ER stress. Furthermore, palmitate-induced phospholipid accumulation and cisternal ER expansion were attenuated by the down-regulation of XBP-1 or CCTα.ConclusionsThe findings reported herein indicate that palmitate-induced cisternal ER expansion is dependent on the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells.

Highlights

  • IntroductionWe previously reported that palmitate induces cisternal Endoplasmic reticulum (ER) expansion and necrosis in RAW 264.7 cells

  • Endoplasmic reticulum (ER) stress induces ER expansion

  • Palmitate induces cisternal ER expansion in RAW 264.7 cells We previously reported that treatment with palmitate induced necrotic cell death [7]

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Summary

Introduction

We previously reported that palmitate induces cisternal ER expansion and necrosis in RAW 264.7 cells. We report on an investigation of the likely mechanism responsible for this palmitate-induced cisternal ER expansion in a mouse macrophage cell line, RAW 264.7 cells. Palmitate has been reported to induce the production of intracellular reactive oxygen species (ROS) generation, ER stress is one of features shown in lipid-laden macrophages of atherosclerotic plaques [8,9,10]. ER stress is generally thought to induce ER expansion via unfolded protein response (UPR)-mediated lipid biosynthesis and the subsequent enlargement of the ER, which, in turn, alleviates ER stress [11]. We hypothesized that palmitate-induced cisternal ER expansion may be mediated by XBP-1/CCTα-mediated phospholipid accumulation in macrophages

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