Restoration of sterol-regulatory-element-binding protein-1c gene expression in HepG2 cells by peroxisome-proliferator-activated receptor-gamma co-activator-1alpha - PubMed (original) (raw)

Restoration of sterol-regulatory-element-binding protein-1c gene expression in HepG2 cells by peroxisome-proliferator-activated receptor-gamma co-activator-1alpha

Hannes Oberkofler et al. Biochem J. 2004.

Abstract

The expression of SREBP-1 (sterol-regulatory-element-binding protein-1) isoforms differs between tissues and cultured cell lines in that SREBP-1a is the major isoform in established cell lines, whereas SREBP-1c predominates in liver and most other human tissues. SREBP-1c is transcriptionally less active than SREBP-1a, but is a main mediator of hepatic insulin action and is selectively up-regulated by LXR (liver X receptor) agonists. LXR-mediated transactivation is co-activated by PGC-1alpha (peroxisome-proliferator-activated receptor-gamma co-activator-1alpha), which displays deficient expression in skeletal-muscle-derived cell lines. In the present paper, we show that PGC-1alpha expression is also deficient in HepG2 cells and in a human brown adipocyte cell line (PAZ6). In transient transfection studies, PGC-1alpha selectively amplified the LXR-mediated transcription from the human SREBP-1c promoter in HepG2 and PAZ6 cells via two LXR-response elements with extensive similarity to the respective murine sequence. Mutational analysis showed that the human LXR-response element-1 (hLXRE-1) was essential for co-activation of LXR-mediated SREBP-1c gene transcription by PGC-1alpha. Ectopic overexpression of PGC-1alpha in HepG2 cells enhanced basal SREBP-1c and, to a lesser extent, -1a mRNA expression, but only SREBP-1c expression was augmented further in an LXR/RXR (retinoic X receptor)-dependent fashion, thereby inducing mRNA abundance levels of SREBP-1c target genes, fatty acid synthase and acetyl-CoA carboxylase. These results indicate that PGC-1alpha contributes to the regulation of SREBP-1 gene expression, and can restore the SREBP-1 isoform expression pattern of HepG2 cells to that of human liver.

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Figures

Figure 1. PGC-1α potentiates LXR-mediated transcription of the SREBP-1c gene

(A) Sequence comparison of two previously characterized LXREs in the mouse SREBP-1c promoter with the respective region of the human SREBP-1c gene identifies LXREs with extensive similarities. (B) Differentiated PAZ6 cells were transiently transfected with the SREBP-1c–Luc reporter plasmid encompassing two putative LXREs (black bars) or co-transfected with a human PGC-1α expression construct (grey bars). SREBP-1c-driven firefly luciferase activity levels were standardized to pRL-CMV driven Renilla luciferase levels used as transfection controls. Cells were incubated with 22(R)HC, or 22(R)HC and 9cRA, 10 μM each. (C) HepG2 cells were used in transient transfection experiments as described above. (D) Differentiated PAZ6 cells incubated with 22(R)HC or with 22(R)HC and 9cRA were transfected with the SREBP-1a–Luc reporter plasmid encompassing approx. 4.5 kb of the human SREBP-1a promoter or co-transfected with PGC-1α. Fold induction refers to basal SREBP-1c–Luc or SREBP-1a–Luc reporter gene expression levels in the absence of PGC-1α expression plasmid and drugs.

Figure 2. The hLXRE-1 binding site is essential for PGC-1α-mediated transcriptional co-activation of the human SREBP-1c gene

(A) Schematic representation of the hLXRE-1mut, hLXRE-2mut and hLXRE-1/2mut constructs. Mutated binding sites are indicated as white boxes. (B) Differentiated PAZ6 cells were transfected with hLXRE-1mut or co-transfected with PGC-1α (grey bars) and incubated with 22(R)HC, or 22(R)HC and 9cRA, 10 μM each. Differentiated PAZ6 cells were transfected with hLXRE-2mut (C) or hLXRE-1/2mut (D) or co-transfected with PGC-1α (grey bars) in the presence or absence of 22(R)HC, or 22(R)HC and 9cRA. Fold induction refers to basal (black bars) hLXRE-1mut, hLXRE-2mut and hLXRE-1/2mut expression levels respectively, in the absence of PGC-1α expression plasmid and drugs.

Figure 3. Ectopic expression of PGC-1α in HepG2 cells potentiates the transcription of SREBP-1c and SREBP-1c target genes

(A) Real-time RT-PCR analyses of SREBP-1a (grey bars)/SREBP-1c (black bars) gene expression in control HepG2 cells stimulated with 22(R)HC, 9cRA, or a combination of 22(R)HC and 9cRA. Results are means±SD for experiments performed in quadruplicate normalized for the expression of ARP (acidic ribosomal protein p0). Fold induction refers to basal SREBP-1a/SREBP-1c expression in the absence of drugs. (B) HepG2 cells were infected with recombinant PGC-1α virions at a MOI (multiplicity of infection) of >100 and stimulated with drugs as indicated. SREBP-1a (grey bars)/SREBP-1c (black bars) gene expression was quantified using real-time RT-PCR. (C) Analyses of gene expression of FAS (black bars) and ACC (grey bars) in HepG2 cells in the absence or presence of 22(R)HC, 9cRA, or a combination of 22(R)HC and 9cRA. Fold induction refers to basal FAS or ACC expression in the absence of drugs. (D) HepG2 cells were infected with recombinant PGC-1α virions, and FAS (black bars) and ACC (grey bars) mRNA abundance was determined as described above.

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Restoration of sterol-regulatory-element-binding protein-1c gene expression in HepG2 cells by peroxisome-proliferator-activated receptor-gamma co-activator-1alpha - PubMed (original) (raw)