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

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 1ß 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.

Age-dependent loss of hepatic SIRT1 enhances NLRP3 inflammasome signaling and impairs capacity for liver fibrosis resolution.

Our studies indicate that the longevity factor SIRT1 is implicated in metabolic disease; however, whether and how hepatocyte-specific SIRT1 signaling is involved in liver fibrosis remains undefined. We characterized a functional link of age-mediated defects in SIRT1 to the NLRP3 inflammasome during age-related liver fibrosis. In multiple experimental murine models of liver fibrosis, we compared the development of liver fibrosis in young and old mice, as well as in liver-specific SIRT1 knockout (SIRT1 LKO) mice and wild-type (WT) mice. Liver injury, fibrosis, and inflammation were assessed histologically and quantified by real-time PCR analysis. In a model of hepatotoxin-induced liver fibrosis, old mice displayed more severe and persistent liver fibrosis than young mice during liver injury and after injury cessation, as characterized by inhibition of SIRT1, induction of NLRP3, infiltration of macrophages and neutrophils, activation of hepatic stellate cells (HSCs), and excessive deposition and remodeling of the extracellular matrix. Mechanistically, deletion of SIRT1 in hepatocytes resulted in NLRP3 and IL-1ß induction, pro-inflammatory response, and severe liver fibrosis in young mice, mimicking the ability of aging to impair the resolution of established fibrosis. In an aging mouse model, chronic-plus-binge alcohol feeding-induced liver fibrosis was attenuated by treatment with MCC950, a selective NLRP3 inhibitor. NLRP3 inhibition ameliorated alcoholic liver fibrosis in old mice by repressing inflammation and reducing hepatocyte-derived danger signaling-ASK1 and HMGB1. In conclusion, age-dependent SIRT1 defects lead to NLRP3 activation and inflammation, which in turn impairs the capacity to resolve fibrosis during aging.

Guanabenz Treatment Accelerates Disease in a Mutant SOD1 Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons. The mechanisms leading to motor neuron degeneration in ALS are unclear. However, there is evidence for involvement of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in ALS, notably in mutant SOD1 mediated models of ALS. Stress induced phosphorylation of the eIF2 alpha subunit by eukaryotic translation initiation factor 2-alpha kinase 3 Perk activates the UPR. Guanabenz is a centrally acting alpha2 adrenergic receptor agonist shown to interact with a regulatory subunit of the protein phosphatase, Pp1/Gadd34, and selectively disrupt the dephosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eif2alpha). Here we demonstrate that guanabenz is protective in fibroblasts expressing G93A mutant SOD1 when they are exposed to tunicamycin mediated ER stress. However, in contrast to other reports, guanabenz treatment accelerated ALS-like disease progression in a strain of mutant SOD1 transgenic ALS mice. This study highlights challenges of pharmacological interventions of cellular stress responses in whole animal models of ALS.

Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

RhoBTB2 is a substrate of the mammalian Cul3 ubiquitin ligase complex.

Rhobtb2 is a candidate tumor suppressor located on human chromosome 8p21, a region commonly deleted in cancer. Rhobtb2 is homozygously deleted in 3.5% of primary breast cancers, and gene expression is ablated in approximately 50% of breast and lung cancer cell lines. RhoBTB2 is an 83-kD, atypical Rho GTPase of unknown function, comprising an N-terminal Rho GTPase domain and two tandem BTB domains. In this report, we demonstrate that RhoBTB2 binds to the ubiquitin ligase scaffold, Cul3, via its first BTB domain and show in vitro and in vivo that RhoBTB2 is a substrate for a Cul3-based ubiquitin ligase complex. Moreover, we show that a RhoBTB2 missense mutant identified in a lung cancer cell line is neither able to bind Cul3 nor is it regulated by the ubiquitin/proteasome system, resulting in increased RhoBTB2 protein levels in vivo. We suggest a model in which RhoBTB2 functions as a tumor suppressor by recruiting proteins to a Cul3 ubiquitin ligase complex for degradation.