Dissertation
Dissertation > Medicine, health > Basic Medical > Pathology > Pathophysiology

Nuclear Receptor FXR Protects Against Hepatocellular Inflammation Via Cish and SOCS3Induction

Author XuZhiZhen
Tutor HeFengTian
School Third Military Medical University
Course Biochemistry and Molecular Biology
Keywords Farnesoid X receptor (FXR) Suppressor of cytokine signaling (SOCS) Signal transducer and activator of transcription (STAT) Hepatocellular inflammation Regulation
CLC R363
Type PhD thesis
Year 2012
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The family of suppressors of cytokine signaling (SOCS) represents eight differentmembers: cytokine inducible SH2-containing protein (CISH) and SOCS1to SOCS7. Theyall share a central SH2domain and a C-terminal SOCS box. They can be induced bynumerous cytokines and then negatively regulate the Janus kinase (JAK)-signal transducerand activator of transcription (STAT) pathway in a classic feedback loop. SOCSs bind tophosphotyrosines on the target protein through the SH2domain, leading to inhibition ofsignal transduction by N-terminal inactivation of JAK, by blocking access of STAT to thereceptor sites, or by SOCS box-targeting bound proteins to proteasomal degradation.Moreover SOCS can suppress the transcriptional activity of activation protein1(AP1) andnuclear factor-kappa B (NF-κB) through inhibition of c-Jun N-terminal kinase (JNK) andP38kinase. SOCSs have emerged as key physiological regulators of cytokine responses,including those that regulate the inflammatory system. The enforced expressions of SOCSsare reported to be beneficial for controlling inflammatory response in some inflammationrelated diseases. Interestingly, some nuclear receptors peroxisome proliferator-activatedreceptor β (PPARβ), PPARγ and estrogen receptor (ER) agonists have been reported toupregulating SOCSs.Another anti-inflammatory nuclear receptor farnesiod X receptor (FXR, NR1H4), is aligand-activated transcription factor highly expressed in liver and other digestive organs.FXR is able to be activated by a wide variety of compounds, like highest affinityendogenous ligand chenodeoxycholic acid (CDCA) and synthetic agonist GW4064.Although originally considered as a bile acid-activated transcription factor that regulatesmetabolism homeostasis, FXR has been proposed as a novel molecular target in thetreatment of hepatic inflammatory diseases. However, the precise mechanisms are poorlyunderstood. So far there is no final conclusion about whether there is a link between theanti-inflammatory action of FXR and the induction of SOCSs. In order to identify therelationship between FXR and SOCSs, researches were carried out as follow: 1. SOCSs express in human hepatocytes.Using RT-PCR, the mRNA levels of SOCSs were found to express in human hepatomacell line HepG2and hepatic cell line L02.2. FXR agonists induce CISH and SOCS3expression in vitro and in vivo.Using RT-PCR, Real-time PCR and Western blot, the transcription and translationleves of cytokine inducible SH2-containing protein (CISH) and SOCS3were found to beupregulated in a dose-dependent manner in human hepatocytes and liver tissues of miceafter treated with FXR agonists.3. FXR enhances the transcriptional activities of the human Cish and Socs3promoterregion.Reporter assay demonstrated that the transcriptional activities of human Cish andSocs3promoter region were significantly increased via pharmacological or geneticactivation of FXR. A GeneBank database search (http://www.nubiscan.unibas.ch/) revealedsome FXR binding consensus sequences in human Cish and Socs3promoter.4. FXR agonists inhibit IL6-stimulated phosphorylation of STAT5and STAT3in humanhepatocytes.Using Western blot and siRNA approach, we found that pretreatment with FXR agonistsin human hepatocytes markedly suppressed IL6-induced STAT5activation, and theinhibitory effect might be associated with induction of CISH.Using Western blot and siRNA approach, we also found that FXR agonists pretreatmentin human hepatocytes markedly inhibited IL6-induced STAT3activation, and the inhibitoryeffect might be associated with upregulation of SOCS3.5. FXR nature agonist CDCA pretreatment attenuates the LPS-induced liverinflammatory injury.The mice were gavaged with CDCA or vehicle alone (soyabean oil) for a week beforeintraperitoneal injected with LPS (15mg/kg) once on the seventh day.(1) Examination ofliver pathology showed that massive necroses and inflammations were present in micetreated with LPS. However CDCA pretreatment decreased not only the formation ofhepatocyte edema, but also the infiltration of inflammatory cells.(2) The levels of serumalanine amino transferase (ALT) and aspartate amino transferase (AST), the markers ofliver function damage, were also significantly increased by LPS, but reduced in mice pretreated with CDCA.(3) Additionally CDCA treatment attenuated the liver dysfunctioncaused by LPS as demonstrated by the Real-time PCR, ELISA and Western blot analyses ofhepatic inflammatory biomarkers such as interleukin6(IL6), tumor necrosis factor (TNFα),and intercellular adhesion molecule-1(ICAM-1).(4) Moreover pretreatment with CDCAmarkedly suppressed STAT5and STAT3activation stimulated by LPS, and these effectswere accompanied by increased expressions of CISH and SOCS3.In summary, our data showed that FXR could upregulate CISH and SOCS3in humanhepatocytes and liver tissues of mice and protected against LPS-induced liver inflammatoryresponse. Moreover we use a siRNA approach to suggest the interesting note that there is alink between anti-inflammatory action of FXR agonists and induction of SOCSs.Modulation of SOCSs expression may represent a novel mechanism through which FXRactivation could selectively affect cytokine bioactivity in inflammatory response. The studyon the effect of FXR on SOCSs will provide useful information with regarding to thephysiological and pathological roles FXR plays in hepatic inflammation, and suggest a newtherapy.

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