Flightless I homolog negatively regulates ChREBP activity in cancer cells

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Abstract

The glucose-responsive transcription factor carbohydrate responsive element binding protein (ChREBP) plays an important role in regulating glucose metabolism in support of anabolic synthesis in both hepatocytes and cancer cells. In order to further investigate the molecular mechanism by which ChREBP regulates transcription, we used a proteomic approach to identify proteins interacting with ChREBP. We found several potential ChREBP-interacting partners, one of which, flightless I homolog (FLII) was verified to interact and co-localize with ChREBP in HCT116 colorectal cancer and HepG2 hepatocellular carcinoma cells. FLII is a member of the gelsolin superfamily of actin-remodeling proteins and can function as a transcriptional co-regulator. The C-terminal 227 amino acid region of ChREBP containing the DNA-binding domain interacted with FLII. Both the N-terminal leucine-rich repeat (LRR) domain and C-terminal gelsolin homolog domain (GLD) of FLII interacted and co-localized with ChREBP. ChREBP and FLII localized in both the cytoplasm and nucleus of cancer cells. Glucose increased expression and nuclear localization of ChREBP, and had minimal effect on the level and distribution of FLII. FLII knockdown using siRNAs increased mRNA and protein levels of ChREBP-activated genes and decreased transcription of ChREBP-repressed genes in cancer cells. Conversely, FLII overexpression negatively regulated ChREBP-mediated transcription in cancer cells. Our findings suggest that FLII is a component of the ChREBP transcriptional complex and negatively regulates ChREBP function in cancer cells.

Introduction

The glucose-responsive transcription factor ChREBP was discovered as a critical mediator of glucose-dependent induction of glycolytic and lipogenic enzyme genes in hepatocytes (Yamashita et al., 2001). ChREBP (also called MondoB) and MondoA both contain basic helix–loop–helix leucine zipper (bHLHLZ) domains and are the only two known members of the Mondo family transcription factors (Peterson and Ayer, 2011). Mondo family transcription factors can change their subcellular localization and DNA-binding activity according to nutrient levels (Peterson and Ayer, 2011, Havula and Hietakangas, 2012). 14-3-3 also regulates the subcellular localization of Mondo family transcription factors by binding to MondoA and ChREBP and retaining them in the cytosol (Eilers et al., 2002, Merla et al., 2004, Li et al., 2008b, Sakiyama et al., 2008).

It is reported that glucose increases ChREBP expression, promotes its nuclear translocation and binding to carbohydrate response element (ChoRE) of target genes in hepatocytes (Uyeda and Repa, 2006). Several genes encoding key enzymes for glycolysis and lipogenesis have been demonstrated to be direct ChREBP target genes, including liver pyruvate kinase (LPK), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD1) (Yamashita et al., 2001, Dentin et al., 2004, Ishii et al., 2004, Collier et al., 2007, Jeong et al., 2011). More and more genes with glucose-induced expression have been found to be regulated by ChREBP, such as thioredoxin-interacting protein (TXNIP) and fibroblast growth factor 21 (FGF21) (Pang et al., 2009, Cha-Molstad et al., 2009, Iizuka et al., 2009a). In addition to the canonical ChREBP isoform (ChREBP-α), a new isoform of ChREBP named ChREBP-β has been recently identified which is transcribed from a different promoter and plays an important role in regulating fatty acid synthesis in adipose tissue and insulin sensitivity (Herman et al., 2012). Interestingly, ChREBP-α binds the ChoRE in the ChREBP-β promoter and promotes ChREBP-β transcription (Herman et al., 2012). Recent chromatin immunoprecipitation-sequencing analysis to identify direct target genes of ChREBP has discovered that ChREBP may function as a transcriptional repressor as well as an activator (Jeong et al., 2011). Genes negatively regulated by ChREBP include phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase catalytic subunit (G6Pase), two genes encoding rate-limiting enzymes of gluconeogenesis (Jeong et al., 2011). Some earlier studies show that G6Pase transcription can be promoted by ChREBP in responsive to glucose in rat insulinoma cells and rat hepatocytes (Pedersen et al., 2007, Iizuka et al., 2009b).

Recent findings suggest that ChREBP plays a more generalized role in reprogramming metabolism to support glucose-stimulated proliferation in cancer cells (Tong et al., 2009). Knocking down ChREBP expression results in decreased aerobic glycolysis, de novo lipid and nucleotide biosynthesis, stimulation of mitochondrial respiration and reduced proliferation in liver and colorectal cancer cells (Tong et al., 2009). Moreover, ChREBP is an important mediator for glucose-stimulated proliferation in pancreatic beta cells (Metukuri et al., 2012). ChREBP indirectly induces expression of cell cycle regulators such as cyclin D2, cyclin A2 and cyclin E1 which leads to cell cycle progression in pancreatic beta cells (Metukuri et al., 2012).

Post-translational modifications and co-activators have been suggested to play important roles in regulating ChREBP activity in different types of cells. High level of glucose leads to ChREBP multi-site dephosphorylation which stimulates its nuclear import and DNA-binding activity in hepatocytes (Uyeda and Repa, 2006, Tsatsos et al., 2008). Glucose-activated p300 acetylates ChREBP and increases its transcriptional activity in hepatocytes (Bricambert et al., 2010). The acetyltransferase p300 can also serve as the transcriptional co-activator for ChREBP through histone H4 acetylation and recruitment of RNA polymerase II on the promoter of TXNIP in pancreatic beta cells (Cha-Molstad et al., 2009). O-glycosylation and ubiquitination have also been suggested to regulate ChREBP protein level and activity in hepatocytes (Sakiyama et al., 2010, Ido-Kitamura et al., 2012, Guinez et al., 2011). However, it remains largely unknown how the transcriptional activity of ChREBP is regulated in cancer cells.

The FLII gene is the mammalian homolog of the Drosophila melanogaster flightless-I gene which plays important roles in early embryogenesis and structural organization of indirect flight muscle (Campbell et al., 1993). The FLII protein contains an N-terminal LRR domain and a C-terminal GLD domain (Campbell et al., 1993). PI3-kinase and small GTPases may regulate the subcellular localization of FLII between cytoplasm and nucleus in Swiss 3T3 fibroblasts (Davy et al., 2001). In addition to functioning as an actin-remodeling protein, FLII has been found to interact with various proteins important for cellular signaling (Kopecki and Cowin, 2008, Adams et al., 2008). Interestingly, FLII may function as a transcriptional co-regulator which positively or negatively regulates activity of transcription factors. FLII can be recruited by hormone-activated nuclear receptors to the promoters of target genes to serve as a co-activator of the nuclear receptor transcription complex (Lee et al., 2004). FLII negatively regulates beta-catenin-mediated transcription by disrupting the synergy of FLII leucine rich repeat associated protein 1 (FLAP1) with p300 and beta-catenin (Lee and Stallcup, 2006).

In the present study, we aimed to find out how the transcriptional activity of ChREBP was regulated by its interacting proteins in cancer cells. We found that FLII interacted with ChREBP in HCT116 human colorectal cancer cells and HepG2 human heptacellular carcinoma cells. The C-terminal 227 amino acid region of both ChREBP-α and ChREBP-β which contained the DNA-binding domain was responsible for binding to FLII. Both the N-terminal LRR domain and C-terminal GLD domain of FLII interacted and co-localized with ChREBP. In contrast to ChREBP, glucose did not change expression and subcellular localization of FLII in HCT116 and HepG2 cells. Knocking-down FLII expression using siRNAs increased expression of ChREBP-activated genes such as FAS, SCD1 and TXNIP and decreased transcription of ChREBP-repressed genes including G6Pase and PEPCK in HCT116 cells. Ectopically expressing FLII negatively regulated ChREBP-mediated transcription in HCT116 and 293T cells. Cell fractionation and ChIP analysis showed that FLII did not affect the subcellular localization and DNA binding activity of ChREBP in HCT116 cells. Our findings suggest that FLII is a newly-identified component of the ChREBP transcriptional complex and negatively regulates ChREBP activity in cancer cells.

Section snippets

Materials

The canonical ChREBP isoform cDNA (i.e., the human ChREBP-α cDNA) was obtained as described (Tong et al., 2009). We made partial cDNA clones encompassing different regions of ChREBP: ChREBP 1–251, ChREBP 252–625 and ChREBP 626–852. According to published ChREBP-β sequences (Herman et al., 2012), the human ChREBP-β cDNA clone encompassing the entire protein coding sequence was generated. The cDNA encoding the complete coding region of the human FLII gene was purchased from Open Biosystems and

FLII is a component of the ChREBP transcriptional complex

To identify new proteins interacting with ChREBP, we immunoprecipitated 293T cell lysates using the anti-ChREBP antibody and control serum followed by SDS-PAGE and silver-staining analysis (Fig. 1A). One protein band of about 111KD specifically present in the ChREBP immunoprecipitates was identified to be FLII by mass spectrometry (Fig. S1). We next tried to verify the interaction between FLII and ChREBP using co-immunoprecipitation. After co-expressing Flag-tagged FLII (Flag-FLII) and

Discussion

ChREBP has been proven to be a key transcriptional factor for glucose metabolism in normal and cancer cells. Recent findings suggest that post-translational modifications and co-activators play important roles in regulating ChREBP activity in hepatocytes and pancreaticβ-cells (Bricambert et al., 2010, Metukuri et al., 2012). However, little is known about ChREBP-mediated transcriptional regulation in cancer cells. Here we have identified FLII as a new component of the ChREBP transcriptional

Conclusions

Our findings showed that FLII belonged to the ChREBP transcriptional complex and negatively regulated ChREBP function.

Acknowledgements

We thank Dr. Craig Thompson's laboratory (Memorial Sloan-Kettering Cancer Center, USA) for kindly providing cell lines and reagents. We thank Dr. Don Ayer (University of Utah, USA) and Dr. Howard C. Towle (University of Minnesota, USA) for kindly providing the Myc-MondoA plasmid and the 4 × ACC ChoRE-Luc reporter plasmid, respectively. We thank Dr. Xiaoying Li (Ruijin Hospital, Shanghai, China) for kindly providing the estrogen receptor plasmid.

This work was supported in part by National Basic

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    These authors contributed equally to this work.

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