Reduced methylation of PP2Ac promotes ethanol–induced lipid accumulation through FOXO1 phosphorylation in vitro and in vivo
Introduction
Excessive alcohol consumption leads to alcoholic liver disease (ALD), which is the leading cause of death among all chronic liver diseases (Liangpunsakul et al., 2016). The development of alcoholic fatty liver is the earliest response of the liver to chronic alcohol consumption and can progress to more extensive liver injuries such as steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma (Gao and Bataller, 2011). (Gao and Bataller, 2011). Although this general disease progression is well established, the underlying molecular mechanisms by which ethanol causes liver steatosis are still not completely understood.
Accumulating evidence has indicated that alterations of the methionine cycle and the reduction of the ratio of S-adenosylmethionine (SAM):S-adenosylhomocystein (SAH) play a fundamental role in the pathogenesis of ALD (Kharbanda et al., 2013; Fujii and Kawada, 2014; Williams et al., 2014). A decrease in the SAM:SAH ratio affects hepatic methylation capacity by affecting SAM-dependent methyltransferase reactions, including methylation of protein phosphatase 2A (PP2A) (Sutter et al., 2013; Laxman et al., 2014). PP2A encompasses a large family of Ser/Thr phosphatases that all have a catalytic subunit (C), a structural scaffolding subunit (A), and one of many possible regulatory B subunits that provide substrate specificity to the holoenzyme (Shi, 2009). Fifteen B subunit genes encoding 26 different isoforms give rise to more than 100 combinations of the PP2A holoenzyme, enabling a wide spectrum of biological activities. The PP2A catalytic C subunit (PP2Ac) is methylated on Leu-309 by SAM-dependent leucine carboxyl methyltransferase-1 (LCMT-1) (Bottiglieri et al., 2012). Changes in the methylation state of the C subunit critically modulate the recruitment of specific regulatory B subunits to the (AC) core enzyme, thereby influencing the substrate specificity of the PP2A holoenzyme (Sontag and Sontag, 2014; Sontag et al., 2014). The importance of PP2Ac methylation has been underlined by the observation of its changing methylation status in SAMe and methionine-treated hepatocytes, suggesting a potential role in liver steatosis due to altered interactions with TORC1 (Zubiete-Franco et al., 2016).
Forkhead box O1 (FOXO1) belongs to a family of transcription factors related to stress response pathways. Of note, FOXO1 plays a major role in regulating hepatic lipid metabolism, and the liver is one of its most important sites of action (Deng et al., 2012; Valenti et al., 2008; Tikhanovich et al., 2013). FOXO1 is regulated through reversible phosphorylation, which is catalyzed by kinases and phosphatases. The phosphorylation of FOXO1 leads to nuclear exclusion and thus inhibition of its transcriptional activity (Xie et al., 2012). A growing body of evidence has suggested that PP2A is a physiologically important phosphatase for FOXO1 (Yan et al., 2012, 2008). Of note, a recent study showed that FOXO1 levels were reduced in the livers of patients with alcoholic hepatitis (AH) and in animal models of ALD, supporting the inhibitory effect of ethanol on FOXO1 (Heo et al., 2019). Nonetheless, the question of whether alcohol consumption promotes a change in FOXO1 phosphorylation and, if so, how FOXO1 phosphorylation is related to the progression of ALD have not been explored to date. Given that PP2A is affected by ethanol–induced hypomethylation and that it regulates FOXO1 phosphorylation, we hypothesized that PP2A may be part of the link between ethanol–induced hypomethylation and FOXO1 dysregulation in the progression of ALD.
The present study investigated the mechanisms by which reduced methylation of PP2Ac is related to the phosphorylation FOXO1 in ALD. Using betaine, a methyl donor, we first demonstrated that decreased hepatic methylation capacity is an important factor that causes reduced PP2Ac methylation. Then, we found that ethanol–induced demethylation of PP2Ac contributes to the reduction of PP2A/B55α, leading to hyperphosphorylation of FOXO1 and subsequent accumulation of triglyceride (TG) via upregulation of thioredoxin-interacting protein (TXNIP).
Section snippets
Materials
Antibodies against FOXO1 phosphorylated at Thr24, total FOXO1, the PP2A-A subunit, the PP2A-B subunit, and the PP2A-C subunit were purchased from Cell Signaling Technology (Beverly, Massachusetts). Antibodies against demethylated PP2Ac and PP2A Bα, short interfering RNAs (siRNAs) against PP2A Bα, and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California). Antibodies against TXNIP and methylated PP2Ac (Leu309) were purchased from Abcam (Cambridge,
Ethanol-induced liver lesions are associated with decreased hepatic methylation in mice
We evaluated the extent of hepatic damage in mice fed the Lieber-DeCarli diet for 4 weeks by measuring hepatic TG content, quantifying TUNEL-positive cells, and measuring circulating liver enzyme levels. As shown in Fig. 1A and B, there was a significant accumulation of fat in the livers of mice chronically exposed to the alcohol-containing diet, as determined by histologic examination (H&E staining and Oil red staining) (Fig. 1A) and biochemical measurement of the hepatic lipid content (Fig. 1
Discussion
Ethanol affects methionine metabolism, leading to alterations in the SAM:SAH ratio and consequently decreasing hepatic methylation capacity, thus contributing to the pathogenesis of ALD (Kharbanda, 2013; Fujii and Kawada, 2014; Williams et al., 2014). However, the specific mechanisms underlying these relationships are not fully understood. PP2A plays a crucial role in regulating cellular functions and is the most common eukaryotic serine/threonine phosphatase, representing 0.1–1% of total
Declaration of Competing Interest
The authors declare no competing financial interests.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 81500446), the Natural Science Foundation of Jiangsu Province (Grant No. BK20130397), and the Jiangsu Government Scholarship for Overseas Studies (JS-2019-257).
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