Targeting hepatic serine-arginine protein kinase 2 ameliorates alcohol-associated liver disease by alternative splicing control of lipogenesis

Abstract

Background and aims: Lipid accumulation induced by alcohol consumption is not only an early pathophysiological response but also a prerequisite for the progression of alcohol-associated liver disease (ALD). Alternative splicing regulates gene expression and protein diversity; dysregulation of this process is implicated in human liver diseases. However, how the alternative splicing regulation of lipid metabolism contributes to the pathogenesis of ALD remains undefined.

Approach and results: Serine-arginine-rich protein kinase 2 (SRPK2), a key kinase controlling alternative splicing, is activated in hepatocytes in response to alcohol, in mice with chronic-plus-binge alcohol feeding, and in patients with ALD. Such induction activates sterol regulatory element-binding protein 1 and promotes lipogenesis in ALD. Overexpression of FGF21 in transgenic mice abolishes alcohol-mediated induction of SRPK2 and its associated steatosis, lipotoxicity, and inflammation; these alcohol-induced pathologies are exacerbated in FGF21 knockout mice. Mechanistically, SRPK2 is required for alcohol-mediated impairment of serine-arginine splicing factor 10, which generates exon 7 inclusion in lipin 1 and triggers concurrent induction of lipogenic regulators-lipin 1β and sterol regulatory element-binding protein 1. FGF21 suppresses alcohol-induced SRPK2 accumulation through mammalian target of rapamycin complex 1 inhibition-dependent degradation of SRPK2. Silencing SRPK2 rescues alcohol-induced splicing dysregulation and liver injury in FGF21 knockout mice.

Conclusions: These studies reveal that (1) the regulation of alternative splicing by SRPK2 is implicated in lipogenesis in humans with ALD; (2) FGF21 is a key hepatokine that ameliorates ALD pathologies largely by inhibiting SRPK2; and (3) targeting SRPK2 signaling by FGF21 may offer potential therapeutic approaches to combat ALD.

Fig. 1.. Chronic alcohol consumption increases hepatic SRPK2 levels and activity, stimulates SREBP-1 cleavage, and upregulates transcription of SREBP-1-dependent lipogenic genes in a pre-clinical mouse model and in human liver samples.

A-C. Representative immunoblots for SRPK2 levels (A) and kinase activity (B), as assessed by phosphorylation of its downstream targets, including SR proteins (pSRp55) and (pSRp20), in livers from pair-fed mice and chronic-binge alcohol-fed mice. Levels of SRPK2 and SR protein phosphorylation (C) were quantified by densitometry, normalized to those of GAPDH, and presented as the fold change relative to the control. D. Representative Hematoxylin & Eosin (H&E) staining of liver sections in mice. E-F. Measurements of liver and plasma triglyceride concentrations. Liver lipids were extracted, and triglyceride concentrations were measured and expressed as mg of lipid/g of liver tissue. Hepatic triglyceride levels were positively correlated with SRPK2 levels in pair-fed and chronic-binge alcohol-fed mice. G. Representative immunohistochemistry staining of liver sections for lipogenic enzymes including ACLY, FAS, and SCD1. Notably, positive staining for ACLY was much higher in the cytoplasm and nucleus of lipid-rich hepatocytes of chronic-binge alcohol-fed mice. Positive staining for FAS and SCD1 was primarily located in the cytoplasm of lipid-rich hepatocytes in chronic-binge alcohol-fed mice. H-J. Representative immunoblots for nuclear SREBP-1 (nSREBP-1), FAS, and SCD1. The active, nuclear form of SREBP-1 and expression of its target genes, FAS and SCD1, were increased in chronic-binge alcohol-fed mice. K. Real-time qRT-PCR analysis of mRNA levels of lipogenic genes, including SREBP-1c, ACLY, ACC1, FAS, ELOVL6, SCD1, and DGAT2. The data are presented as the mean ± S.E.M., n=6–8 per group. *P<0.05, vs pair-fed mice. All images were acquired using 10X, 20X, or 40X objectives. L-M. Representative immunoblots for SRPK2, pSRp20, and nuclear SREBP-1 in livers of healthy human controls and in patients with ALD. The data are presented as the mean ± S.E.M., n=10 per group. *P<0.05, vs. healthy human controls.

Fig. 2.. Ethanol-induced lipogenesis and triglyceride overproduction are mimicked by SRPK2 overexpression and diminished by SRPK2 knockdown in cultured hepatocytes.

A. Immunoblotting analysis and densitometric quantification of SRPK2 and pSRp20 in primary mouse hepatocytes exposed to ethanol. Primary mouse hepatocytes were incubated with increasing doses of ethanol (50 – 100 mM) for 24 h. B. Immunofluorescent staining of SRPK2 (red) and nuclear staining with DAPI (blue) in AML12 mouse hepatocytes exposed to ethanol (100 mM) for 24 h. C. Immunoblotting analysis and densitometric quantification of lipogenic enzymes including FAS and SCD1 in primary mouse hepatocytes exposed to various doses of ethanol for 24 h. D-E. mRNA levels of SREBP-1c and intracellular triglyceride levels were dose-dependently increased by ethanol exposure in primary mouse hepatocytes. F. Immunoblotting analysis of SRPK2, FAS, and SCD1 in AML12 hepatocytes exposed to increasing doses of ethanol (25 – 100 mM) for 24 h. G. Triglyceride concentrations were increased by exposure of AML12 hepatocytes to ethanol (100 mM) for 24 h. H-L. Overexpression of SRPK2 is sufficient to increase expression of lipogenic enzymes and elevate lipid content in primary mouse hepatocytes. Primary mouse hepatocytes were transduced with an adenovirus expressing either GFP (Ad-GFP) at a multiplicity of infection (MOI) of 5 or an adenovirus encoding SRPK2 (Ad-SRPK2) at different MOIs (1–5). Immunoblotting analysis and densitometric quantification of SRPK2, pSRp20, FAS, or SCD1 were performed. M. Overexpression of SRPK2 is sufficient to increase expression of lipogenic enzymes in AML12 hepatocytes. AML12 cells were transduced with Ad-GFP at an MOI of 5 or Ad-SRPK2 at different MOIs of 1–5. N. Adenovirus-mediated knockdown of SRPK2 abolishes the ability of ethanol to promote lipogenic enzyme expression in AML12 cells. AML12 cells were transduced for 24 h with adenoviruses encoding either short hairpin RNA targeting the SRPK2 gene (Ad-shSRPK2) at an MOI of 10 or shRNA control (Ad-shRNA Control) at an MOI of 10, followed by incubation with ethanol (100 mM) for an additional 24 h. The data are presented as the mean ± S.E.M., n=3, *P<0.05 between two groups. All images were acquired using 40X objectives.

Fig. 3.. Adenovirus-mediated knockdown of hepatic SRPK2 ameliorates chronic-binge alcohol feeding-induced fatty acid synthesis, steatosis, and liver injury in mice.

Mice were administered via tail vein injections of adenoviral vectors encoding shRNA targeting the SRPK2 gene (AdshSRPK2) or shRNA control (AdshRNA). After injection, the mice were fed a Lieber-DeCarli alcohol liquid diet for 10 days, plus one binge of alcohol at the end of experiments. All mice were subsequently sacrificed 9 hours post-binge. A. Immunoblots and densitometric quantification for the abundance of SRPK2 and phosphorylation of SR proteins. Adenovirus-mediated knockdown of hepatic SRPK2 in mice was confirmed by a dramatic decrease in SRPK2 levels and SR protein phosphorylation. B. Alcohol-induced elevation of liver injury, as assessed by plasma ALT levels, was reduced in mice upon SRPK2 knockdown. C. H&E staining for hepatic steatosis in mice. D. Chronic-binge alcohol feeding-induced elevation of hepatic and plasma triglyceride levels in mice were lowered by silencing SRPK2. E-F. Immunoblots (E) and densitometric quantification (F) for nSREBP-1, FAS, and SCD1. G. Real-time qRT-PCR analysis of hepatic lipogenic gene expression. Ad-shSRPK2-injected mice exhibited downregulation of SREBP-1c and its target genes under conditions of chronic-binge alcohol feeding. H. Immunohistochemistry staining of liver sections using antibodies against ACLY, FAS, or SCD1. Upon chronic-binge alcohol feeding, strong positive staining for ACLY, FAS, and SCD1 was visualized mainly in hepatocytes of Ad-shRNA control-injected mice; however, the number and intensity of ACLY⁺, FAS⁺, and SCD1⁺ hepatocytes were reduced by silencing hepatic SRPK2. The data are presented as the mean ± S.E.M., n=6–8 per group. *P<0.05 between two groups. Images were acquired using 20X and 40X objectives.

Fig. 4.. Overexpression of FGF21 in transgenic mice suppresses alcohol-mediated induction of SRPK2 and protects against the progression of ALD.

WT mice and FGF21 overexpression in transgenic (FGF21-Tg) mice were subjected to a Lieber-DeCarli alcohol liquid diet for 10 days plus one binge alcohol feeding at the end of experiments, or a pair-fed control diet. All mice were sacrificed 9 hours post-binge. A. Representative H&E staining of live sections in WT and FGF21-Tg mice. B-C. Hepatic and plasma triglyceride concentrations were lowered in FGF21-Tg mice after chronic-binge alcohol feeding. D-F. Immunoblots and densitometric quantification for SRPK2, phosphorylation of SR proteins, and nSREBP-1. G-H. Real-time qRT-PCR analysis of mRNA levels of SREBP-1 and its target lipogenic genes. FGF21-Tg mice exhibited much lower expression of lipogenic genes than that in WT mice after chronic-binge alcohol feeding. I. Immunohistochemistry staining for ACLY and FAS. Notably, the number and distribution of ACLY⁺ and FAS⁺ hepatocytes were decreased in FGF21-Tg mice following chronic-binge alcohol feeding. J. ALT assays to measure liver injury. K. Immunoblots and densitometric quantification for p10 and p20 fragments of caspase-1. L-M. Real-time qRT-PCR analysis showed that expression of pro-inflammatory regulators and mediators (IL-1β, TNF-α, Cdc11b, CXCL1, MPO, and Ly6g) was much lower in FGF21-Tg mice than in WT mice after chronic-binge alcohol feeding. The data are presented as the mean ± S.E.M., n=6–8 per group. *P<0.05 between two groups. Images were acquired using 20X and 40X objectives.

Fig. 5.. FGF21 overexpression mimics the inhibitive effect of rapamycin on alcohol-induced accumulation of SRPK2 protein in hepatocytes in vitro and in vivo.

A-B. mTORC1 inhibition by rapamycin treatment decreased SRPK2 abundance in AML12 hepatocytes under conditions of nutrient-stimulated activation of mTORC1. AML12 cells were incubated for 24 h in medium containing 2% fetal bovine serum (FBS) or in medium containing 10% FBS (Nutrient-rich condition) in the absence or presence of rapamycin (20 nM). Cell lysates were immunoblotted with antibodies against SRPK2, as well as the phosphorylated forms of mTORC1 downstream effectors, including S6 at Ser235/236 (pS6) and 4E-BP1 at Thr37/46 (p4E-BP1). C-D. mTORC1 inhibition by rapamycin protects hepatocytes from ethanol-induced accumulation of SRPK2 protein. AML12 cells were incubated for 24 h with or without ethanol (100 mM) in the absence and presence of rapamycin (20 nM) under cultured conditions in medium containing 2% FBS. E. Real-time qRT-PCR revealed no significant changes in SRPK2 mRNA when protein levels were decreased in rapamycin-treated AML12 cells. Cells were incubated for 24 h with increasing doses of ethanol (100 mM) in medium containing 2% FBS. The decrease in SRPK2 protein did not appear to be due to a decrease in SRPK2 mRNA levels in rapamycin-treated hepatocytes. F. mTORC1 inhibition by rapamycin reduced ethanol-induced FAS in AML12 cells. G. mTORC1 inhibition by rapamycin promotes the degradation of SRPK2 in AML12 hepatocytes. Cells were chased for the indicated durations with the protein synthesis inhibitor cycloheximide (CHX, 50 μg/ml) in the absence or presence of rapamycin (20 nM). H. Rapamycin treatment ameliorates hepatic steatosis in chronic-binge alcohol-fed mice. Mice fed the alcohol diet were administered daily intraperitoneal injections with either vehicle or rapamycin (1mg/kg/day) for 10 days. The mice were gavaged with a single dose of ethanol (5 g/kg) and then sacrificed 9 hours post-gavage. H&E staining for hepatic steatosis was shown. I. Representative immunoblots of phosphorylation of S6 (pS6) in rapamycin-treated mice. J-K. Immunoblots and densitometric quantification (J) as well as real-time qRT-PCR analysis (K) for SRPK2. Rapamycin treatment decreased SRPK2 induction via chronic-binge alcohol feeding but it did not affect its mRNA expression. L-N. Immunoblots (L-M) and densitometric quantification (N) for FAS and SCD1. O. Rapamycin treatment reduced alcohol-induced liver injury in mice, as assessed by plasma ALT levels. P. FGF21 inhibits alcohol-induced mTORC1 signaling in hepatocytes. AML-12 cells were transduced with or without an adenoviral vector expressing FGF21 at an MOI of 5 and were subsequently incubated for 24 h with ethanol in medium containing 2% FBS. Cell lysates were immunoblotted with antibodies against SRPK2, as well as the phosphorylated forms of mTORC1 downstream effectors, including S6K1 at Thr389, S6 at Ser235/236, and 4E-BP1 at Thr37/46. Q-R. Inhibition of mTORC1 by FGF21 attenuates alcohol-mediated induction of SRPK2 and FAS in AML-12 cells, without affecting SRPK2 mRNA levels. T. Extent of SRPK2 mRNA levels was comparable in the livers of alcohol-fed WT and FGF21-Tg mice. U. FGF21 overexpression in transgenic mice inhibits alcohol-induced activation of mTORC1, as indicated by reduced phosphorylation of S6 at Ser235/236. The data are presented as the mean ± S.E.M., n=6–8 per group. *P<0.05 between two groups. Images were acquired using 20X objectives.

Fig. 6.. FGF21 knockout mice exhibit enhanced SRPK2 activity and develop more severe ALD pathologies.

WT and FGF21 knockout (FGF21 KO) mice were subjected to a Lieber-DeCarli alcohol liquid diet for 10 days plus one binge alcohol feeding at the end of experiments, or a pair-fed control liquid diet. All mice were sacrificed 9 hours post-binge. A. H&E staining of liver sections in both WT and FGF21 KO mice. B-C. Liver and plasma triglyceride levels were higher in FGF21 KO mice than those in WT mice after chronic-binge alcohol feeding. D-E. Immunoblots and densitometric quantification for SRPK2, phosphorylation of SR proteins, and nSREBP-1. F. Real-time qRT-PCR analysis of mRNA levels of SRPK2. G. Immunoblots and densitometric quantification for nSREBP-1. H-I. Real-time qRT-PCR analysis showed that mRNA levels of SREBP-1 and its target genes were increased in FGF21 KO mice after chronic-binge alcohol feeding. J. Immunohistochemistry for ACLY and FAS. Upon chronic-binge alcohol feeding, positive staining for ACLY and FAS was visualized mainly in hepatocytes of WT mice; the distribution and intensity of positively stained hepatocytes were higher in FGF21 KO mice than in WT mice. K. ALT assays to measure liver injury. L. Immunoblots and densitometric quantification for p10 and p20 fragments of caspase-1. M-N. Real-time qRT-PCR analysis of the expression of pro-inflammatory cytokines (IL-1β and TNF-α), as well as neutrophil chemokines (CXCL1) and neutrophil markers (MPO and Ly6g). An inflammatory response was increased in WT mice following chronic-binge alcohol feeding; this induction was further enhanced in FGF21 KO mice. The data are presented as the mean ± S.E.M., n=6–8 per group. *P<0.05 between two groups. Images were acquired using 20X and 40X objectives.

Fig. 7.. Silencing hepatic SRPK2 rescues aberrant regulation of lipid metabolism and alleviates ALD pathologies in FGF21 KO mice.

WT or FGF21 KO mice were injected with either Ad-shSRPK2 or Ad-shRNA control via tail vein injection. Subsequently, adenovirus-injected mice were subjected to a Lieber-DeCarli alcohol liquid diet for 10 days plus one time of alcohol binge feeding at the end of experiments. The mice were sacrificed 9 hours post-binge. A-C. Immunoblots (A-B) and densitometric quantification (C) for SRPK2 and phosphorylation of SR proteins. Adenovirus-mediated hepatic knockdown of SRPK2 in FGF21 KO mice was verified by dramatically decreased SRPK2 and phosphorylation of SR proteins. D. H&E staining showed that alcohol-induced hepatic steatosis was dramatically increased in FGF21 KO mice; However, severe steatosis in FGF21 KO mice was attenuated by silencing SRPK2. E. Hepatic and plasma triglyceride levels in mice. F-G. Immunoblots (F) and densitometric quantification (G) for nSREBP-1, FAS, and SCD1. Upon chronic-binge alcohol feeding, hepatic levels of nSREBP-1 and its target lipogenic enzymes were increased in FGF21 KO mice but these effects were diminished by silencing SRPK2. H. Immunohistochemistry reveals that upon chronic-binge alcohol feeding, the number and distribution of ACLY⁺ and FAS⁺ hepatocytes were higher in FGF21 KO mice than in WT mice. However, the number and intensity of ACLY⁺ and FAS⁺ hepatocytes in FGF21 KO mice were lowered by silencing SRPK2. I. Liver injury was measured by ALT assays. Alcohol-induced liver injury in FGF21 KO mice given an injection of AdshRNA control was restored to normal levels upon SRPK2 knockdown. J-K. Immunoblots (J) and densitometric quantification (K) for cleaved caspase-1. The data are presented as the mean ± S.E.M., n=6–8 per group. *P<0.05 between two groups. Images were acquired using 20X and 40X objectives.

Fig. 8.. Manipulation of SRPK2 and FGF21 modulates dysregulation of SRSF10-mediated lipogenic pre-mRNA splicing associated with ALD.

A-B. Real-time qRT-PCR analysis of mRNA levels of total lipin 1 and different lipin 1 splicing isoforms in pair-fed and alcohol-fed mice. Lipin 1β/α ratio was calculated using their relative mRNA levels. C. Positive correlation between hepatic SRPK2 and lipin 1β levels in pair-fed and alcohol-fed mice. D. Immunoblots for SRSF10 in pair-fed and alcohol-fed mice. E-F. Immunoblots (E) and densitometric quantification (F) for SRSF10 in alcohol-fed mice that were injected with either AdshRNA control or AdshSRPK2. G. Immunohistochemistry for SRSF10. Notably, upon chronic-binge alcohol feeding, positive staining for SRSF10 was visualized mainly in the nucleus of hepatocytes in Ad-shRNA control-injected control mice, but the intensity and distribution of SRSF10⁺ hepatocytes were increased in SRPK2-knockdown mice. H-I. Real-time qRT-PCR analysis of lipin 1, lipin 1β, lipin 1α, as well as lipin 1 β/α ratio in control and SRPK2-knockdown mice. J. Ethanol exposure enhanced the interaction between SRPK2 and SRSF10 in AML-12 hepatocytes. AML12 cells were transduced with Ad-SRPK2 at an MOI of 5 and subsequently incubated for 24 h with or without ethanol (100 mM) in medium containing 2% FBS. Cell lysates were subjected to immunoprecipitation with an anti-SRPK2 antibody; immunoprecipitated proteins and cell lysates were immunoblotted with indicated antibodies. K-L. Immunoblots (K) and densitometric quantification (L) for SRSF10 in WT and FGF21-Tg mice. M-N. Real-time qRT-PCR analysis showed that mRNA levels of lipin 1 and lipin 1β, as well as the lipin 1β/α ratio, were increased by alcohol administration in WT mice, and this induction was attenuated in FGF21-Tg mice. O-P. Real-time qRT-PCR analysis for skipping of exon 7 (lipin 1α) and inclusion of exon 7 (lipin 1β) revealed that rapamycin treatment decreased lipin 1β induction caused by chronic binge alcohol feeding but did not affect mRNA expression levels of lipin 1α. Q-R. Real-time qRT-PCR analysis of mRNA levels of lipin 1 and lipin 1 spliced isoforms as well as the lipin 1β/α ratio in WT and FGF21 KO mice. S. Immunoblots for SRSF10 showed that upon alcohol administration, hepatic SRSF10 was downregulated in AdshRNA-injected FGF21 KO mice and this impairment was rescued by silencing SRPK2. T-U. Real-time qRT-PCR analysis of mRNA levels of lipin 1, lipin 1β, and lipin 1α, as well as the ratio of lipin 1β/lipin 1α. The data are presented as the mean ± S.E.M., n=6–8 per group. *P<0.05 between two groups. Images were acquired using 20X and 40X objectives.