Endoplasmic reticulum stress causes the activation of sterol regulatory element binding protein-2

https://doi.org/10.1016/j.biocel.2007.05.002Get rights and content

Abstract

Background

Sterol regulatory element binding protein-2 (SREBP-2) is a membrane-bound transcription factor that upon proteolytic processing can activate the expression of genes involved in cholesterol biosynthesis and uptake. We as well as others have demonstrated that the accumulation of misfolded proteins within the endoplasmic reticulum (ER), a condition known as ER stress, can dysregulate lipid metabolism by activating the SREBPs. The purpose of this study was to determine the mechanism by which ER stress induces SREBP-2 activation.

Methods and results

HeLa and MCF7 cells were treated with ER stress-inducing agents to determine the effect of ER stress on SREBP-2 cleavage and subsequent cholesterol accumulation. Cells treated with thapsigargin (Tg) exhibit proteolytic cleavage of SREBP-2. Proteolytic cleavage of SREBP-2 induced by Tg occurred independently of caspase activation and was inhibited by the site-1 protease inhibitor AEBSF, suggesting that Tg-induced SREBP-2 cleavage occurs through the conventional site-1/-2 pathway. Treatment of HeLa cells with Tg also led to the accumulation of free cholesterol as measured by Filipin staining.

Conclusions

These results imply that ER stress-induced SREBP-2 activation occurs through the conventional pathway that normally regulates SREBP in accordance with intracellular sterol concentration.

Introduction

Sterol regulatory element binding proteins (SREBPs) are transcription factors synthesized as inactive precursors bound to the endoplasmic reticulum (ER) membrane responsible for the upregulation of genes involved in cholesterol synthesis. SREBP-1a and -2 are the predominant isoforms in most cultured cell lines, whereas SREBP-1c and -2 are found in the majority of tissues (Shimomura, Shimano, Horton, Goldstein, & Brown, 1997). SREBP-1a and -2 are responsible for the upregulation of genes involved in cholesterol synthesis. SREBP-1c is preferentially responsible for the upregulation of genes involved in the fatty acid biosynthesis pathway (Horton, Goldstein, & Brown, 2002). The SREBPs are localized in the ER through their interaction with SREBP cleavage activating protein (SCAP) (Hua, Nohturfft, Goldstein, & Brown, 1996) and Insig (Yabe, Brown, & Goldstein, 2002; Yang et al., 2002). SCAP forms a complex with the SREBPs and is essential for their activation. Cells that lack SCAP require the addition of exogenous free cholesterol for survival (Rawson, DeBose-Boyd, Goldstein, & Brown, 1999). Insig is an ER-resident protein anchor that binds SCAP and maintains the ER localization of the SCAP–SREBP complex. Upon cellular sterol depletion, cholesterol dissociates from the sterol-sensing domain of SCAP, releasing Insig and allowing SCAP–SREBP to exit the ER through COPII vesicle transport (Sun, Li, Goldstein, & Brown, 2005). SCAP transports SREBP to the Golgi where the site-1-serine protease (S1P) and site-2-zinc metalloproteinase (S2P) release the active SREBP transcription factor. Upon sterol depletion and the initiation of the SREBP proteolytic pathway, the active transcription factor translocates to the nucleus causing SREBP-associated gene expression that encodes for enzymes in the cholesterol/triglyceride biosynthesis and uptake pathways (Goldstein, DeBose-Boyd, & Brown, 2006).

In addition to the location of newly synthesized SREBP, the ER is the principal site for folding and maturation of transmembrane, secretory and ER-resident proteins (Lee, 2001). The ER contains a high level of protein chaperones such as GRP78, GRP94, and calreticulin to assist in the correct folding of synthesized proteins and to prevent the accumulation of misfolded proteins. Disruption in ER function that interferes with proper folding and maturation of proteins causes ER stress and initiates the unfolded protein response (UPR), an integrated intracellular signalling pathway that induces temporary translational inhibition followed by upregulation of ER chaperones. The UPR is mediated via three ER-resident sensors: a type-I ER transmembrane protein kinase (IRE-1), activating transcription factor 6 (ATF-6) and the PKR-like ER kinase (PERK). Activation of these three sensors is mediated by the dissociation of GRP78 following ER stress (Kaufman, 2002, Lawrence de Koning et al., 2003; Lee, 2001, Ron, 2002, Rutkowski and Kaufman, 2004). As a result, the UPR enhances cell survival by ensuring that the adverse effects of ER stress are dealt with in a timely and efficient manner. Although the UPR may provide a protective advantage for the cell, prolonged or severe ER stress can result in caspase activation and apoptosis (Feng et al., 2003, Hossain et al., 2003, Morishima et al., 2002; Nakagawa et al., 2000).

We as well as others have reported that conditions that cause ER stress or apoptosis, induce SREBP activation, independent of intracellular cholesterol content (Higgins & Ioannou, 2001; Lee & Ye, 2004; Pai, Brown, & Goldstein, 1996; Wang et al., 1995, Wang et al., 1996, Werstuck et al., 2001). Previous studies also found that caspase-3 and -7 cleave the SREBPs following apoptotic stimuli in a sterol-independent manner (Pai et al., 1996, Wang et al., 1995, Wang et al., 1996). The suggested caspase cleavage site is located on the N-terminal, cytoplasmic side of SREBP (Pai et al., 1996, Wang et al., 1995, Wang et al., 1996). These studies revealed that caspase-3/-7 cleaves SREBP at a site other then S1P/S2P. In support of these findings, Higgins and Ioannou (2001) found that the caspase-induced SREBP cleavage was transcriptionally active, caused expression of reporter constructs, and occurred very early in the apoptotic process (Higgins & Ioannou, 2001). Since conditions that cause ER stress can lead to caspase activation and SREBP cleavage, studies were initiated to determine whether ER stress may cause SREBP activation and subsequent cholesterol accumulation through a caspase-dependent mechanism. In the present study, we demonstrate that ER stress induces SREBP activation and lipid accumulation independent of caspase activation. Our findings also suggest that ER stress-mediated SREBP activation occurs through the conventional S1P/S2P proteolytic pathway.

Section snippets

Cell culture conditions

The human breast adenocarcinoma cell line MCF7, stably transfected to express caspase-3 (MCF7/cas3) or vector control (MCF7/pbabe), was kindly provided by Dr. Damu Tang (Department of Medicine, McMaster University). The human cervical carcinoma cell line HeLa was obtained from the American Type Culture Collection (ATCC; Manassas, VA). Cell lines were cultured in DMEM (ATCC; Manassas, VA) containing 10% fetal bovine serum (FBS), 100 μg/ml penicillin and 100 μg/ml streptomycin. Cell lines were

ER stress agents activate SREBP-2, induce SRE-controlled gene expression and increase intracellular free cholesterol

To determine whether various agents known to induce ER stress could activate SREBP-2, HeLa cells were treated with ER stress-inducing agents, including thapsigargin (Tg), A23187, tunicamycin (Tm) and homocysteine (Hcy). As a positive control for SREBP-2 activation, the lysosomal cholesterol efflux inhibitor U18666A was used to block cholesterol transport to the ER. Although the various ER stress-inducing agents activated SREBP-2 to different degrees, Tg-induced SREBP-2 cleavage was similar to

Discussion

Activation of the SREBP-2 pathway leading to cholesterol biosynthesis and uptake is a well-defined response to decreased membrane cholesterol content. How SREBP-2 is activated under apoptotic (Higgins & Ioannou, 2001; Pai et al., 1996, Wang et al., 1995, Wang et al., 1996) or ER stress conditions (Lee & Ye, 2004; Werstuck et al., 2001) is not well understood. Our findings provide evidence that apoptosis and ER stress activate SREBP-2 by distinctly different mechanisms. We provide evidence that

Acknowledgements

This work was supported in part by research grants to Richard Austin from the Heart and Stroke Foundation of Ontario (NA-6024) and the Canadian Institutes of Health Research (MOP-74477 and MOP-67116), and the Ontario Research and Development Challenge Fund. Richard C. Austin is a Career Investigator of the Heart and Stroke Foundation of Ontario (CI-5959). Stephen Colgan is supported by a graduate studentship from the Canadian Liver Foundation. We thank Dr. Yiannis A. Ioannou (Mount Sinai School

References (33)

Cited by (0)

View full text