Endoplasmic reticulum stress-induced CHOP activation mediates the down-regulation of leptin in human neuroblastoma SH-SY5Y cells treated with the oxysterol 27-hydroxycholesterol

Abstract

Epidemiological studies have suggested an inverse relationship between the adipocytokine leptin and the onset of Alzheimer's disease (AD), and leptin supplementation decreases amyloid-β (Aβ) production and tau phosphorylation (p-tau), two major biochemical events that play a key role in the pathogenesis of AD. We have previously shown that the cholesterol oxidized product 27-hydroxycholesterol (27-OHC) inhibits leptin expression, an effect that correlated with increased levels of Aβ and p-tau. We have also shown that 27-OHC induces endoplasmic reticulum (ER) stress, a cellular response that is implicated in AD and confers leptin resistance. However the extent to which ER stress is involved in 27-OHC-induced attenuation in leptin expression has not been determined. In this study we determined the involvement of ER stress in the 27-OHC-induced attenuation of leptin expression in SH-SY5Y human neuroblastoma cells. We demonstrate that 27-OHC-induced ER stress attenuates leptin expression by activating C/EBP Homologous Protein (CHOP) which negatively regulates C/EBPα, a transcription factor required for leptin expression. The molecular chaperone 4-phenylbutyric acid (4-PBA) precludes 27-OHC-evoked ER stress and down-regulation of leptin. Furthermore, we demonstrate that the activation of the transcription factor CHOP in response to ER stress is pivotal in the attenuation of leptin expression as knocking-down CHOP alleviates the attenuation in leptin expression. Our study implicates ER stress as the mechanistic link in the 27-OHC-induced negative regulation of leptin, a hormone that has potential therapeutic effects in AD by reducing Aβ and phosphorylated tau accumulation.

Introduction

Alzheimer's disease (AD) is neuropathologically characterized by the accumulation of β-amyloid (Aβ) peptide as extracellular plaques and the deposition of hyperphosphorylated tau in intracellular neurofibrillary tangles (NFTs). We and others have shown that the adipocytokine leptin is produced endogenously in the brain [1], [2], [3], [4], [5] and can attenuate Aβ production and tau hyperphosphorylation in vivo [6], [7], [8] and in vitro [3], [4], [9], [10], [11]. Epidemiological studies have demonstrated that higher circulating leptin levels are associated with lower risk of dementia including AD [12], and lower circulating levels of leptin have been reported in AD patients [13]. Multiple lines of evidence suggest the involvement of ER stress in AD pathology [14], [15], [16] and in β-amyloid-induced cell death [17], [18], [19]. The endoplasmic reticulum (ER) is a cellular organelle involved in various functions including protein folding, maintenance of Ca²⁺ homeostasis and cholesterol synthesis. Stress to the endoplasmic reticulum triggers a cascade of events that leads to increased expression of the transcription factor C/EBP Homologous Protein (CHOP, also called growth arrest and DNA damage induced gene-153, GADD153 or DDIT3) [20], [21], [22], [23] via the activation of the PERK/eIF2α/ATF4 and ATF6 ER stress signaling pathways [24], [25], [26]. There is evidence that the ER stress induces leptin resistance which culminates in deficient leptin signaling [27], [28]. Leptin expression in the brain is contingent on mTORC1 signaling pathway [3] and requires the activation of the transcription factor CCAAT enhancer binding protein α (C/EBPα) [5]. Evidence suggests that CHOP inhibits the DNA binding activity of C/EBPα and therefore acts as a negative regulator of C/EBPα-mediated transcription [29], [30]. CHOP belongs to the same family of transcription factors as C/EBPα. Notably, CHOP shares significant homology with C/EBPα and this homology renders CHOP and C/EBPα conducive to form heterodimers [29]. However the CHOP-C/EBPα heterodimers are unable to bind to their cognate elements on the promoters of target genes and elicit changes in gene expression [29]. Therefore, CHOP acts as a negative regulator of C/EBPα mediated transcription by sequestering C/EBPα and rendering it effete in binding to the cognate DNA elements in the promoter regions of target genes.

We have recently demonstrated that the cholesterol metabolite 27-hydroxycholesterol (27-OHC) decreases leptin expression levels in the rabbit hippocampus [4]. Previous studies from our laboratory have implicated 27-OHC in increased Aβ42 production and tau hyperphosphorylation in hippocampal organotypic slices [4], [31]. Furthermore, we showed that leptin treatment attenuates the increase in Aβ42 production and tau phosphorylation induced by 27-OHC [4]. We have also shown that 27-OHC induces ER stress and activates CHOP in ARPE-19 cells [32]. As ER stress activates CHOP and given that CHOP negatively regulates C/EBPα, a transcription factor required for leptin expression, we hypothesized that ER stress negatively regulates leptin expression via CHOP induction. We also speculated that 27-OHC attenuates leptin expression by precipitating ER stress and activating CHOP. In this study we determined the potential of ER stress induced by 27-OHC to activate CHOP and subsequently down-regulate C/EBPα-mediated leptin expression in human neuroblastoma SH-SY5Y cells.