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• W3204103158 abstract "•DRAK2 is markedly upregulated in the livers of both patients and mice with NAFLD/NASH•DRAK2 regulates RNA alternative splicing through SRPK1-mediated SRSF6 phosphorylation•DRAK2 disturbs mitochondrial function in hepatic steatosis via RNA splicing machinery•Suppression of DRAK2 in hepatocytes ameliorates NAFLD/NASH progression Nonalcoholic steatohepatitis (NASH) is an advanced stage of nonalcoholic fatty liver disease (NAFLD) with serious consequences that currently lacks approved pharmacological therapies. Recent studies suggest the close relationship between the pathogenesis of NAFLD and the dysregulation of RNA splicing machinery. Here, we reveal death-associated protein kinase-related apoptosis-inducing kinase-2 (DRAK2) is markedly upregulated in the livers of both NAFLD/NASH patients and NAFLD/NASH diet-fed mice. Hepatic deletion of DRAK2 suppresses the progression of hepatic steatosis to NASH. Comprehensive analyses of the phosphoproteome and transcriptome indicated a crucial role of DRAK2 in RNA splicing and identified the splicing factor SRSF6 as a direct binding protein of DRAK2. Further studies demonstrated that binding to DRAK2 inhibits SRSF6 phosphorylation by the SRSF kinase SRPK1 and regulates alternative splicing of mitochondrial function-related genes. In conclusion, our findings reveal an indispensable role of DRAK2 in NAFLD/NASH and offer a potential therapeutic target for this disease. Nonalcoholic steatohepatitis (NASH) is an advanced stage of nonalcoholic fatty liver disease (NAFLD) with serious consequences that currently lacks approved pharmacological therapies. Recent studies suggest the close relationship between the pathogenesis of NAFLD and the dysregulation of RNA splicing machinery. Here, we reveal death-associated protein kinase-related apoptosis-inducing kinase-2 (DRAK2) is markedly upregulated in the livers of both NAFLD/NASH patients and NAFLD/NASH diet-fed mice. Hepatic deletion of DRAK2 suppresses the progression of hepatic steatosis to NASH. Comprehensive analyses of the phosphoproteome and transcriptome indicated a crucial role of DRAK2 in RNA splicing and identified the splicing factor SRSF6 as a direct binding protein of DRAK2. Further studies demonstrated that binding to DRAK2 inhibits SRSF6 phosphorylation by the SRSF kinase SRPK1 and regulates alternative splicing of mitochondrial function-related genes. In conclusion, our findings reveal an indispensable role of DRAK2 in NAFLD/NASH and offer a potential therapeutic target for this disease. In recent years, with the increasing consumption of energy-dense diets and sedentary lifestyles, NAFLD has become one of the most common chronic liver diseases worldwide, which influences 25% of the world’s population (Younossi et al., 2019Younossi Z. Tacke F. Arrese M. Chander Sharma B. Mostafa I. Bugianesi E. Wai-Sun Wong V. Yilmaz Y. George J. Fan J. Vos M.B. Global perspectives on nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.Hepatology. 2019; 69: 2672-2682Crossref PubMed Scopus (543) Google Scholar; Zhou et al., 2020Zhou J. Zhou F. Wang W. Zhang X.J. Ji Y.X. Zhang P. She Z.G. Zhu L. Cai J. Li H. Epidemiological features of NAFLD from 1999 to 2018 in China.Hepatology. 2020; 71: 1851-1864Crossref PubMed Scopus (114) Google Scholar). NAFLD encompasses a disease spectrum ranging from simple steatosis to NASH, and eventually NASH cirrhosis, which is a major risk factor for hepatocellular carcinoma (HCC) (Oh et al., 2020Oh T.G. Kim S.M. Caussy C. Fu T. Guo J. Bassirian S. Singh S. Madamba E.V. Bettencourt R. Richards L. et al.A universal gut-microbiome-derived signature predicts cirrhosis.Cell Metab. 2020; 32: 901Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar; Wong et al., 2015Wong R.J. Aguilar M. Cheung R. Perumpail R.B. Harrison S.A. Younossi Z.M. Ahmed A. Nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the United States.Gastroenterology. 2015; 148: 547-555Abstract Full Text Full Text PDF PubMed Scopus (1094) Google Scholar). Due to the complexity and our limited understanding of the pathophysiology of NAFLD, there is still a lack of approved pharmacotherapies for this pandemic medical condition (Chen et al., 2019bChen Z. Yu Y. Cai J. Li H. Emerging molecular targets for treatment of nonalcoholic fatty liver disease.Trends Endocrinol. Metab. 2019; 30: 903-914Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar; Wong et al., 2016Wong V.W. Chitturi S. Wong G.L. Yu J. Chan H.L. Farrell G.C. Pathogenesis and novel treatment options for non-alcoholic steatohepatitis.Lancet Gastroenterol. Hepatol. 2016; 1: 56-67Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar; Xu et al., 2021Xu H. Tian Y. Tang D. Zou S. Liu G. Song J. Zhang G. Du X. Huang W. He B. et al.An endoplasmic reticulum stress-microRNA-26a feedback circuit in NAFLD.Hepatology. 2021; 73: 1327-1345Crossref PubMed Scopus (12) Google Scholar). Thus, further studies are urgent to identify novel therapeutic targets for NAFLD treatment. Most eukaryotic pre-mRNAs contain noncoding sequences (introns) that need to be removed to generate the correct concatenations of exonic sequences via alternative splicing (AS), a mechanism through which one single gene can produce multiple mRNAs and structurally different proteins and may affect more than 70% of human genes (Berasain et al., 2010Berasain C. Goñi S. Castillo J. Latasa M.U. Prieto J. Avila M.A. Impairment of pre-mRNA splicing in liver disease: mechanisms and consequences.World J. Gastroenterol. 2010; 16: 3091-3102Crossref PubMed Scopus (31) Google Scholar; Licatalosi and Darnell, 2010Licatalosi D.D. Darnell R.B. RNA processing and its regulation: global insights into biological networks.Nat. Rev. Genet. 2010; 11: 75-87Crossref PubMed Scopus (494) Google Scholar). Alterations in mRNA splicing are important causes of disease, and the alterations in this pathway have been implicated in many human diseases (Bonnal et al., 2020Bonnal S.C. López-Oreja I. Valcárcel J. Roles and mechanisms of alternative splicing in cancer - implications for care.Nat. Rev. Clin. Oncol. 2020; 17: 457-474Crossref PubMed Scopus (143) Google Scholar; Del Río-Moreno et al., 2019Del Río-Moreno M. Alors-Pérez E. González-Rubio S. Ferrín G. Reyes O. Rodríguez-Perálvarez M. Sánchez-Frías M.E. Sánchez-Sánchez R. Ventura S. López-Miranda J. et al.Dysregulation of the splicing machinery is associated to the development of nonalcoholic fatty liver disease.J. Clin. Endocrinol. Metab. 2019; 104: 3389-3402Crossref PubMed Scopus (27) Google Scholar; Estus et al., 2019Estus S. Shaw B.C. Devanney N. Katsumata Y. Press E.E. Fardo D.W. Evaluation of CD33 as a genetic risk factor for Alzheimer’s disease.Acta Neuropathol. 2019; 138: 187-199Crossref PubMed Scopus (40) Google Scholar; Fredericks et al., 2015Fredericks A.M. Cygan K.J. Brown B.A. Fairbrother W.G. RNA-binding proteins: splicing factors and disease.Biomolecules. 2015; 5: 893-909Crossref PubMed Scopus (43) Google Scholar; Singh and Cooper, 2012Singh R.K. Cooper T.A. Pre-mRNA splicing in disease and therapeutics.Trends Mol. Med. 2012; 18: 472-482Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). In recent years, high-throughput array-based techniques evaluating gene expression profiles have provided a growing body of evidence pinpointing an obvious association between several metabolism-related elements and the AS processes (Kaminska et al., 2014Kaminska D. Hämäläinen M. Cederberg H. Käkelä P. Venesmaa S. Miettinen P. Ilves I. Herzig K.H. Kolehmainen M. Karhunen L. et al.Adipose tissue INSR splicing in humans associates with fasting insulin level and is regulated by weight loss.Diabetologia. 2014; 57: 347-351Crossref PubMed Scopus (28) Google Scholar; Kaminska and Pihlajamäki, 2013Kaminska D. Pihlajamäki J. Regulation of alternative splicing in obesity and weight loss.Adipocyte. 2013; 2: 143-147Crossref PubMed Google Scholar; Lee et al., 2017Lee G. Zheng Y. Cho S. Jang C. England C. Dempsey J.M. Yu Y. Liu X. He L. Cavaliere P.M. et al.Post-transcriptional regulation of de novo lipogenesis by mTORC1-S6K1-SRPK2 signaling.Cell. 2017; 171: 1545-1558.e18Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar; Medina and Krauss, 2013Medina M.W. Krauss R.M. Alternative splicing in the regulation of cholesterol homeostasis.Curr. Opin. Lipidol. 2013; 24: 147-152Crossref PubMed Scopus (16) Google Scholar; Pihlajamäki et al., 2011Pihlajamäki J. Lerin C. Itkonen P. Boes T. Floss T. Schroeder J. Dearie F. Crunkhorn S. Burak F. Jimenez-Chillaron J.C. et al.Expression of the splicing factor gene SFRS10 is reduced in human obesity and contributes to enhanced lipogenesis.Cell Metab. 2011; 14: 208-218Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In particular, a few studies have shown that RNA splicing factors changed obviously with the development of NAFLD, and a growing number of splicing factors have been confirmed to play important roles in NAFLD (Del Río-Moreno et al., 2019Del Río-Moreno M. Alors-Pérez E. González-Rubio S. Ferrín G. Reyes O. Rodríguez-Perálvarez M. Sánchez-Frías M.E. Sánchez-Sánchez R. Ventura S. López-Miranda J. et al.Dysregulation of the splicing machinery is associated to the development of nonalcoholic fatty liver disease.J. Clin. Endocrinol. Metab. 2019; 104: 3389-3402Crossref PubMed Scopus (27) Google Scholar; Kumar et al., 2019Kumar D. Das M. Sauceda C. Ellies L.G. Kuo K. Parwal P. Kaur M. Jih L. Bandyopadhyay G.K. Burton D. et al.Degradation of splicing factor SRSF3 contributes to progressive liver disease.J. Clin. Invest. 2019; 129: 4477-4491Crossref PubMed Scopus (35) Google Scholar; López-Vicario et al., 2014López-Vicario C. González-Périz A. Rius B. Morán-Salvador E. García-Alonso V. Lozano J.J. Bataller R. Cofán M. Kang J.X. Arroyo V. et al.Molecular interplay between Δ5/Δ6 desaturases and long-chain fatty acids in the pathogenesis of non-alcoholic steatohepatitis.Gut. 2014; 63: 344-355Crossref PubMed Scopus (88) Google Scholar; Zhang et al., 2020Zhang Z. Zong C. Jiang M. Hu H. Cheng X. Ni J. Yi X. Jiang B. Tian F. Chang M.W. et al.Hepatic HuR modulates lipid homeostasis in response to high-fat diet.Nat. Commun. 2020; 11: 3067Crossref PubMed Scopus (20) Google Scholar). These studies have illustrated the existence of a close relationship between splicing machinery dysregulation and NAFLD development, which will help to further clarify the pathogenesis of NAFLD and provide a novel alternative therapeutic target for NAFLD. DRAK2, also known as STK17B, is a serine/threonine kinase belonging to the death-associated protein kinase (DAPK) family. DRAK2, which was originally identified as a nuclear apoptosis promotor by Sanjo et al. in 1998, is abundantly expressed in the thymus, lymph nodes, B cells, and trachea and mildly expressed in the liver and pancreas. Previous studies have revealed that DRAK2 plays important roles in T cell survival and differentiation, islet survival, and apoptosis (Farag and Roh, 2019Farag A.K. Roh E.J. Death-associated protein kinase (DAPK) family modulators: current and future therapeutic outcomes.Med. Res. Rev. 2019; 39: 349-385Crossref PubMed Scopus (41) Google Scholar; Sanjo et al., 1998Sanjo H. Kawai T. Akira S. DRAKs, novel serine/threonine kinases related to death-associated protein kinase that trigger apoptosis.J. Biol. Chem. 1998; 273: 29066-29071Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar; Wang et al., 2017Wang S. Xu L. Lu Y.T. Liu Y.F. Han B. Liu T. Tang J. Li J. Wu J. Li J.Y. et al.Discovery of benzofuran-3(2H)-one derivatives as novel DRAK2 inhibitors that protect islet β-cells from apoptosis.Eur. J. Med. Chem. 2017; 130: 195-208Crossref PubMed Scopus (16) Google Scholar). Drak2−/− mice are resistant to experimental autoimmune encephalitis and type 1 diabetes (McGargill et al., 2004McGargill M.A. Wen B.G. Walsh C.M. Hedrick S.M. A deficiency in Drak2 results in a T cell hypersensitivity and an unexpected resistance to autoimmunity.Immunity. 2004; 21: 781-791Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, McGargill et al., 2008McGargill M.A. Choy C. Wen B.G. Hedrick S.M. Drak2 regulates the survival of activated T cells and is required for organ-specific autoimmune disease.J. Immunol. 2008; 181: 7593-7605Crossref PubMed Scopus (16) Google Scholar). Intriguingly, Mao et al. demonstrated that DRAK2 expression is rapidly induced in islet β cells after free fatty acid (FFA) stimulation, and that such upregulation of DRAK2 is accompanied by increased β cell apoptosis, and DRAK2 transgenic mice have been found to present compromised glucose tolerance in a diet-induced obesity model (Mao et al., 2008Mao J. Luo H. Wu J. Drak2 overexpression results in increased beta-cell apoptosis after free fatty acid stimulation.J. Cell. Biochem. 2008; 105: 1073-1080Crossref PubMed Scopus (15) Google Scholar). Moreover, Lan et al. found that DRAK2 is significantly upregulated in HCC cell lines and specimens and that patients with ectopic DRAK2 expression were characterized by poor clinicopathological features (Lan et al., 2018Lan Y. Han J. Wang Y. Wang J. Yang G. Li K. Song R. Zheng T. Liang Y. Pan S. et al.STK17B promotes carcinogenesis and metastasis via AKT/GSK-3β/Snail signaling in hepatocellular carcinoma.Cell Death Dis. 2018; 9: 236Crossref PubMed Scopus (28) Google Scholar). According to mounting epidemiological evidence, NAFLD is rapidly becoming a leading underlying factor in the etiologies of many cases of HCC (Anstee et al., 2019Anstee Q.M. Reeves H.L. Kotsiliti E. Govaere O. Heikenwalder M. From NASH to HCC: current concepts and future challenges.Nat. Rev. Gastroenterol. Hepatol. 2019; 16: 411-428Crossref PubMed Scopus (429) Google Scholar). Thus, we were interested in investigating the role of DRAK2 in the development and progression of NAFLD. In this study, we found that DRAK2 expression was markedly upregulated in livers from NAFLD/NASH patients and mouse NAFLD/NASH models. Overexpression of DRAK2 in the liver exacerbated hepatic steatosis and inflammation in a high-fat diet (HFD)-induced mouse NAFLD model, while hepatocyte-specific DRAK2 deficiency in mice protected against HFD-induced hepatic steatosis and high-fat/high-cholesterol plus high fructose (HFF) diet-induced hepatic steatohepatitis. Mechanistically, we identified that DRAK2 directly interacts with serine/arginine (SR)-rich splicing factor 6 (SRSF6) and prevents its phosphorylation by inhibiting its interaction with SRSF protein kinase 1 (SRPK1). Further transcriptomic AS analysis revealed that the phosphorylation of SRSF6 is necessary for the normal splicing forms of many mitochondrial function-related genes to maintain the mitochondria function during NAFLD progression. These findings provide a novel mechanism underlying NAFLD/NASH progression through the dysregulation of mitochondrial function-related gene AS via DRAK2-SRSF6 signaling and suggest that targeting this mechanism may be a promising approach for NAFLD/NASH treatment. To investigate the involvement of DRAK2 in the progression from normal liver to NAFLD/NASH, we first measured DRAK2 expression in representative human liver samples from normal individuals and individuals with NAFLD/NASH by immunohistochemistry (IHC; n = 10 per group) staining. The baseline metabolic parameters of these individuals are compared in Table S1. The IHC results showed that DRAK2 protein levels were significantly higher in the livers of NAFLD/NASH patients than the individuals without NAFLD, and there was a positive correlation between hepatic DRAK2 expression and NAFLD severity measured by the NAFLD activity score (NAS) (Figures 1A, 1B , and S1A; Table S1). In line with our findings in humans, DRAK2 protein expression significantly increased with NAFLD progression in the livers of HFD-fed mice and HFF-diet-fed mice compared with those of normal chow (NC)-fed mice (Figure 1C). Furthermore, livers from leptin receptor-deficient (db/db) mice were examined, and DRAK2 expression was found to be higher in the livers of db/db mice than the corresponding nonsteatotic controls (Figure S1B). Moreover, in vitro experiments demonstrated that DRAK2 expression increased in a concentration-dependent and time-dependent manner in mouse primary hepatocytes upon exposure to palmitic acid (PA; Figure 1D). Consistent with the changes in DRAK2 protein levels, DRAK2 mRNA levels were elevated in the livers of mice with simple steatosis and NASH as well as in PA-treated primary hepatocytes (Figure S1C). To further confirm the correlation of hepatic DRAK2 expression with NAFLD progression, we generated a DRAK2 expression vector using adeno-associated virus (AAV) type 8 (AAV-DRAK2). Western blotting demonstrated that tail vein injection of AAV-DRAK2 successfully elevated DRAK2 expression in the liver, but not in other tissues (Figure S1D). Compared to AAV-vector controls, mice with DRAK2 overexpression in the liver had higher body weights (Figure S1E) and more severe hepatic steatosis in response to HFD consumption, as evidenced by the results of hematoxylin and eosin (H&E; Figure 1E) staining, Oil Red O staining (Figure 1F), hepatic triglyceride (TG) concentrations (Figure 1G, left), and the findings of reduced mRNA expression levels of fatty acid β-oxidation genes but no difference in lipogenesis genes (Figure S1G). However, hepatic cholesterol concentrations did not differ (Figure 1G, right). AAV-DRAK2 mice also had greater inflammatory responses than AAV-vector mice in response to HFD consumption, as indicated by CD68 IHC (Figure 1H) and the mRNA expression levels of proinflammatory genes (Figure S1H) in livers. Furthermore, the level of serum biomarkers of liver injury, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), was also substantially increased in HFD-fed AAV-DRAK2 mice (Figure 1I). Transmission electron microscopy (TEM) analysis shows a significant decrease in mitochondrial cristae density, which indicates a mitochondrial structure destruction in HFD-fed AAV-DRAK2 mice compared with the control AAV-vector mice (Figure 1J). Additionally, the expression of mitochondrial function-related genes was lower in the livers of HFD-fed AAV-DRAK2 mice than the corresponding control mice (Figure S1I). However, no significant difference was observed under NC conditions (Figures 1G, 1I, and S1E–S1I), suggesting that DRAK2 sensitively responds to metabolic stress instead of physiological homeostasis. In parallel, we generated a DRAK2 shRNA using AAV type 8 (AAV-shDrak2) to knock down DRAK2 specifically in the liver, but not in other tissues (Figure S1J). AAV-shDrak2 mice exhibited lower body weights and liver weights than the AAV-scramble mice after 16 weeks of HFD consumption (Figure S1K). HFD-induced hepatic steatosis was also improved in AAV-shDrak2 mice (Figures S1L and S1M). The serum AST and ALT levels indicated that liver injury was significantly improved in AAV-shDrak2 mice compared to AAV-scramble controls (Figure S1N). In addition, TEM analysis revealed that the HFD-induced mitochondrial structure destruction was significantly repaired in AAV-shDrak2 mice (Figure S1O). This striking correlation of DRAK2 expression with NAFLD development reveals a role of DRAK2 in the progression of this disease. To further confirm the function of hepatocyte DRAK2 in NAFLD pathogenesis, we generated hepatocyte-specific Drak2-knockout (Drak2-HKO) mice. Western blotting demonstrated that DRAK2 was successfully knocked out in the primary hepatocytes of Drak2-HKO mice compared to those of Drak2-Flox mice, but not in other tissues (Figure S2A). Consistently, Drak2-HKO strain exhibited lower body weights than Drak2-Flox mice after 16 weeks of HFD consumption (Figure S2B). Compared to Drak2-Flox mice, Drak2-HKO mice had reduced hepatic steatosis in response to HFD consumption, as shown by H&E staining (Figure S2C), Oil Red O staining (Figures S2D and S2E), and hepatic TG concentrations (Figure S2F, left). Hepatic TC concentrations did not differ between the two groups (Figure S2F, right). Drak2-HKO mice also had increased mRNA expression levels of fatty acid β-oxidation genes but no differences in lipogenesis genes or fatty acid uptake gene (Cd36) in response to HFD consumption (Figure S2G). The infiltration of inflammatory cells to liver, as evidenced by CD68 IHC (Figure S2H) and proinflammatory genes expression (Figure S2I), was reduced in the HFD-fed Drak2-HKO mice compared with the corresponding control mice. Drak2-HKO mice also exhibited substantially reduced serum AST and ALT levels after HFD consumption (Figure S2J). TEM analysis revealed that HFD-induced decreased mitochondrial cristae density, indicating a structure destruction, was significantly repaired in Drak2-HKO mice (Figure S2K). Moreover, the expression of mitochondrial function-related genes was markedly higher in the livers of Drak2-HKO mice than in those of Drak2-Flox mice (Figure S2L). Intriguingly, we found that the mRNA level changes of mtDNA encoded genes were more marked than the nuclear encoded mitochondrial function-related genes. These data demonstrate that DRAK2 promotes HFD-induced NAFLD progression. However, HFD feeding alone does not cause significant liver injury or fibrosis; therefore, it is inadequate for investigation of the role of DRAK2 in NASH. To address the function of hepatocyte DRAK2 in NASH in vivo, we subjected Drak2-HKO and Drak2-Flox mice to an NC or HFF diet. Following HFF diet feeding, the Drak2-HKO strain exhibited comparable body weights and lower liver weights compared to the Drak2-Flox controls (Figure 2A). Drak2-HKO mice exhibited ameliorated liver steatosis, as evidenced by H&E staining (Figure 2B), NAS score (Figure 2C), hepatic TG (Figure 2D), and Oil Red O staining (Figure 2E). Consistently, compared to Drak2-Flox controls, Drak2-HKO mice exhibited marked upregulation of hepatic genes involved in fatty acid β-oxidation but no differences in genes related to lipogenesis or fatty acid uptake (Cd36) (Figure 2F). Mice with DRAK2-deficient hepatocytes also had reduced inflammatory responses to HFF diet consumption, as evidenced by the CD68 IHC (Figure 2G) and mRNA expression levels of cytokines (Figure 2H) in the liver. Collagen deposition as revealed by Sirius Red staining (Figure 2I) and hepatic hydroxyproline content (Figure 2J), as well as the mRNA expression levels of fibrogenesis-related genes (Figure 2K) in the liver, were lower in the HFF-diet-fed Drak2-HKO mice than Drak2-Flox mice, which indicated the remission of hepatic fibrosis. Hepatocyte apoptosis is at the center of the transition from hepatic steatosis to NASH. TUNEL staining showed that hepatocyte apoptosis induced by the HFF diet was markedly reduced in Drak2-HKO mice compared with Drak2-Flox mice (Figure 2L). Consistently, the serum levels of ALT and AST, the biomarkers of liver injury, were significantly lower in Drak2-HKO than in Drak2-Flox mice following HFF diet feeding (Figure 2M). Importantly, TEM analysis (Figure 2N) and qPCR (Figure 2O) experiments both indicated the mitochondrial function damage induced by the HFF diet was ameliorated in Drak2-HKO mouse livers compared with Drak2-Flox mouse livers. Consistently, the mRNA level changes of mtDNA encoded genes were more marked than the nuclear encoded mitochondrial function-related genes, which indicated that mtDNA replication and transcription were involved in the role of DRAK2 in mitochondrial function. In conclusion, these findings raise the possibility that increased DRAK2 signaling contributes to steatosis-to-NASH progression and that this contribution is associated with mitochondrial function. To explore the mechanism underlying the effect of DRAK2 on lipid metabolism in hepatocytes, we used DRAK2 small interfering RNA (siDrak2) to knock down and ectopic HA-tagged DRAK2 (HA-Drak2) to overexpress DRAK2 in hepatocytes (Figures S3A and S3B). Nile Red staining (Figures 3A–3C ) and Oil Red O staining (Figures S3C–S3F) revealed that the palmitic acid/oleic acid (PAOA)-induced hepatocyte lipid accumulation in the control group was markedly ameliorated in the DRAK2-knockdown group and exacerbated in the DRAK2-overexpression group. Consistent with the in vivo results, there was no significant difference in BSA-treated groups, and qPCR assays showed a marked upregulation of hepatic genes involved in fatty acid β-oxidation but no differences in genes related to lipogenesis in the PA-treated DRAK2-knockdown group compared with its corresponding control (Figure S3G). Additionally, 22b, a specific inhibitor of DRAK2 reported by Gao et al., 2014Gao L.J. Kovackova S. Sála M. Ramadori A.T. De Jonghe S. Herdewijn P. Discovery of dual death-associated protein related apoptosis inducing protein kinase 1 and 2 inhibitors by a scaffold hopping approach.J. Med. Chem. 2014; 57: 7624-7643Crossref PubMed Scopus (26) Google Scholar, was utilized. The results of Oil Red O staining also showed the inhibitor intervention of DRAK2 could ameliorate the PAOA-induced hepatocyte lipid accumulation (Figure S3H). Using [1-14C] palmitic acid (sodium salt) and [1,2-14C] acetic acid (sodium salt) isotope tracer assays, we found that PA-induced impairment of hepatocyte fatty acid oxidation was obviously ameliorated in the DRAK2-knockdown group and aggravated in the DRAK2-overexpression group, but no significant difference in hepatocyte lipogenesis was observed (Figures 3D and 3E). These results suggest that DRAK2 plays a negative role in mitochondrial function in response to metabolic stress. To confirm the role of DRAK2 in mitochondrial function, we used TEM to analyze mitochondrial structure. Notably, the PA-induced impairment in mitochondrial structure was significantly repaired in DRAK2-knockdown clones compared to negative control clones (Figure 3F). Additionally, cell respiration assays revealed that markedly less PA-induced hepatocyte mitochondrial function damage occurred in the DRAK2-knockdown group than in the control group (Figures 3G and 3H). Treatment with the reported DRAK2 autophosphorylation inhibitor 22b also improved hepatocyte mitochondrial function compared with the control group (Figure S3I). Also, qPCR assays showed that the PA-induced mRNA level reduction of mitochondrial function-related genes, encoded by both nuclear DNA and mtDNA, was elevated in DRAK2-knockdown hepatocytes (Figure 3I); this finding was further confirmed by the fact that mtDNA copy number in DRAK2-knockdown hepatocytes (Figure 3J) and HFF-diet-induced Drak2-HKO mouse livers (Figure 3K) were elevated compared with the control treatment, which suggests the role of DRAK2 in mtDNA replication. Taken together, these in vitro findings suggest that DRAK2 plays important roles in the development and progression of NAFLD by impairing mitochondrial function. To explore how DRAK2, as a serine/threonine kinase, regulates mitochondrial function, especially the system-wide phosphorylation changes, we conducted quantitative analysis of phosphorylome in Drak2-Flox and Drak2-HKO mouse primary hepatocytes, treated with BSA or PA (Figures 4A and S4A). In total, 5,237 high-confidence phosphosites (location probability > 0.75) were identified, of which 84 phosphosites were significantly changed (p < 0.05, fold change > 1.2) after DRAK2 knockout in BSA condition (Figure S4B; Tables S2A and S2B). Notably, pathway enrichment assays showed that these significantly changed phosphoproteins were enriched in RNA splicing-related functions (Figure S4C). After treatment with PA, more drastic protein phosphorylation changes (175 differential phosphosites in total, 8,006 substrates, p < 0.05, fold change > 1.2) were observed (Figure 4B; Tables S2C and S2D). Approximately 9% (12/130) of the phosphoproteins were associated with RNA splicing, including SRSF splicing factors and SRRM matrix proteins. Gene Ontology (GO) analysis confirmed that RNA splicing and other mRNA-related pathways were among the top enriched biological processes (Figure 4C). To further investigate the potential regulatory network of DRAK2, we conducted immunoprecipitation and mass spectrometry analysis (IP-MS) to identify its potential interacting partners. HEK293T cell lines were used to overexpress HA-tagged DRAK2. After precipitation with an anti-HA antibody, whole-cell lysates were eluted for in-gel MS assays (Figure 4D). The intensity-based absolute quantification (iBAQ) values of the identified proteins in MaxQuant software were used for label-free quantification. Proteins with a 2-fold abundance higher or only in the DRAK2 transfection group identified in two or more biological replicates were considered as potential interaction proteins of DRAK2 (STAR Methods). We obtained 109 protein candidates in this experiment. Notably, protein interaction network analysis showed these proteins were also highly enriched in RNA splicing pathways (Figures 4D and 4E). In particular, several SRSF splicing factors were found to be involved in these events, including SRSF6, SRSF10, SRSF1, SRSF9, and SRSF3 (Table S2E). We further confirmed the direct binding of DRAK2 with SRSF6 via coIP analysis. As expected, DRAK2 efficiently co-immunoprecipitated with SRSF6 in both hepatocytes and HA-DRAK2/Flag-SRSF6 transfected HEK293T cells (Figure 4F). As a serine/threonine kinase, DRAK2 might regulate the RNA AS mediated by SRSF splicing factors through phosphorylation, so we tested whether DRAK2 could regulate the phosphorylation level of SRSF6. Western blotting demonstrated that SRSF6 became hyperphosphorylated in the livers of HFD-fed Drak2-HKO mice compared with those of Drak2-Flox mice, as detected by a pan-phospho-SR antibody (MAb104 antibody; Figure 4G). We next used MS analysis to detect whether SRSF6 could be direc" @default.
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• W3204103158 title "DRAK2 aggravates nonalcoholic fatty liver disease progression through SRSF6-associated RNA alternative splicing" @default.
• W3204103158 cites W1556285913 @default.
• W3204103158 cites W1563462044 @default.
• W3204103158 cites W1784206158 @default.
• W3204103158 cites W1980548655 @default.
• W3204103158 cites W1987374058 @default.
• W3204103158 cites W1992980640 @default.
• W3204103158 cites W2013275758 @default.
• W3204103158 cites W2021944979 @default.
• W3204103158 cites W2026657651 @default.
• W3204103158 cites W2029824528 @default.
• W3204103158 cites W2037115052 @default.
• W3204103158 cites W2048613643 @default.
• W3204103158 cites W2050002679 @default.
• W3204103158 cites W2054224668 @default.
• W3204103158 cites W2058970225 @default.
• W3204103158 cites W2064375926 @default.
• W3204103158 cites W2066231767 @default.
• W3204103158 cites W2067740038 @default.
• W3204103158 cites W2071628986 @default.
• W3204103158 cites W2096465161 @default.
• W3204103158 cites W2106391842 @default.
• W3204103158 cites W2115746733 @default.
• W3204103158 cites W2136467350 @default.
• W3204103158 cites W2139977516 @default.
• W3204103158 cites W2169456326 @default.
• W3204103158 cites W2169695761 @default.
• W3204103158 cites W2273379630 @default.
• W3204103158 cites W2316221567 @default.
• W3204103158 cites W2490682853 @default.
• W3204103158 cites W2564221522 @default.
• W3204103158 cites W2589639399 @default.
• W3204103158 cites W2765883724 @default.
• W3204103158 cites W2770225202 @default.
• W3204103158 cites W2775343812 @default.
• W3204103158 cites W2789333397 @default.
• W3204103158 cites W2811337037 @default.
• W3204103158 cites W2889932786 @default.
• W3204103158 cites W2922562530 @default.
• W3204103158 cites W2927034673 @default.
• W3204103158 cites W2931372771 @default.
• W3204103158 cites W2937573806 @default.
• W3204103158 cites W2941033601 @default.
• W3204103158 cites W2968872536 @default.
• W3204103158 cites W2969087302 @default.
• W3204103158 cites W2977274448 @default.
• W3204103158 cites W2979923810 @default.
• W3204103158 cites W3003865038 @default.
• W3204103158 cites W3016273280 @default.
• W3204103158 cites W3025153879 @default.
• W3204103158 cites W3034934436 @default.
• W3204103158 cites W3036486236 @default.
• W3204103158 cites W3107925397 @default.
• W3204103158 cites W4251445305 @default.
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