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CCAAT/enhancer-binding protein beta (C/EBPβ) knockdown reduces inflammation, ER stress, and apoptosis, and promotes autophagy in oxLDL-treated RAW264.7 macrophage cells

  • MD Khurshidul Zahid
  • Michael Rogowski
  • Christopher Ponce
  • Mahua Choudhury
  • Naima Moustaid-Moussa
  • Shaikh M. RahmanEmail author
Article
  • 18 Downloads

Abstract

Atherosclerosis is associated with deregulated cholesterol metabolism and formation of macrophage foam cells. CCAAT/enhancer-binding protein beta (C/EBPβ) is a transcription factor, and its inhibition has recently been shown to prevent atherosclerosis development and foam cell formation. However, whether C/EBPβ regulates inflammation, endoplasmic reticulum (ER) stress, and apoptosis, in macrophage foam cells and its underlying molecular mechanism remains unknown. Here, we investigated the effect of C/EBPβ knockdown on proteins and genes implicated in inflammation, ER stress, apoptosis, and autophagy in macrophage foam cells. RAW264.7 macrophage cells were transfected with control and C/EBPβ-siRNA and then treated with nLDL and oxLDL. Key proteins and genes involved in inflammation, ER stress, apoptosis, and autophagy were analyzed by western blot and qPCR. We found that short interfering RNA (siRNA)-mediated knockdown of C/EBPβ attenuated atherogenic lipid-mediated induction of proteins and genes implicated in inflammation (P-NFkB-p65, NFkB-p65, and TNFα), ER stress (ATF4 and ATF6), and apoptosis (CHOP, caspase 1, 3, and 12). Interestingly, C/EBPβ knockdown upregulated the expression of autophagy proteins (LC3A/B-II, ATG5) and genes (LC3B, ATG5) but decreased the mammalian target of rapamycin (mTOR) protein phosphorylation and mTORC1 gene expression in oxLDL-loaded RAW264.7 macrophage cells. More importantly, treatment with rapamycin (inhibitor of mTOR) increased expression of proteins implicated in autophagy and cholesterol efflux in oxLDL-loaded RAW 264.7 macrophage cells. The present results suggest that C/EBPβ inactivation regulates macrophage foam cell formation in atherogenesis by reducing inflammation, ER stress, and apoptosis and by promoting autophagy and inactivating mTOR.

Keywords

C/EBPβ Endoplasmic reticulum stress (ERS) Apoptosis Autophagy Macrophage foam cells 

Abbreviations

C/EBPβ

CCAAT/enhancer-binding protein beta

ERS

Endoplasmic reticulum stress

ATG5

Autophagy gene 5

ATF4

Activating transcription factor 4

ATF6

Activating transcription factor 6

LC3-I & II

Microtubule-associated protein 1 light chain 3-I & II

CHOP

CCAAT/enhancer-binding protein (C/EBP) homologous protein

mTOR

Mammalian target of rapamycin

mTORC1

mTOR complex 1

mTORC2

mTOR complex 2

ox-LDL

Oxidized low-density lipoproteins

Notes

Acknowledgements

This research was funded by the American Heart Association (AHA) Beginning Grant In Aid and the Startup Fund (Texas Tech University), USA to SMR.

Author contributions

SMR designed and supervised the research. MKZ, MR, and CP performed the experiments. MKZ, CP, and SMR analyzed the data. MKZ and SMR wrote the manuscript. MC, NMM, MR and SMR revised the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

All the authors declared that they have no conflict of interest.

Supplementary material

11010_2019_3642_MOESM1_ESM.pptx (84 kb)
Supplementary material 1 (PPTX 83 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • MD Khurshidul Zahid
    • 1
  • Michael Rogowski
    • 1
  • Christopher Ponce
    • 2
  • Mahua Choudhury
    • 3
  • Naima Moustaid-Moussa
    • 1
  • Shaikh M. Rahman
    • 1
    Email author
  1. 1.Department of Nutritional Sciences & Obesity Research InstituteTexas Tech UniversityLubbockUSA
  2. 2.Department of MathematicsTexas Tech UniversityLubbockUSA
  3. 3.Department of Pharmaceutical Sciences, Irma Lerma Rangel College of PharmacyTexas A&M, Health Sciences CenterCollege StationUSA

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