肝醣合成酶激酶-3 於干擾素-γ 訊息傳遞及其生物功能的角色

Abstract

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase which possesses multiple regulatory roles on cell differentiation, proliferation, and apoptosis. GSK-3 is a regulator in interferon-γ (IFN-γ) signaling and is involved in IFN-γ-induced inflammation. Acute hepatic injury is induced by mitogenic concanavalin A (ConA), a lectin (carbohydrate-binding protein) originally extracted from the jack-bean Canavalia ensiformis, has been established as an experimental model of immune hepatic injury. IFN-γ and signal transducer and activator of transcription 1 (STAT1) deficient mice are resistant to ConA-induced immune hepatitis have been widely demonstrated. However, the role of GSK-3 in ConA-induced IFN-γ-mediated immune hepatic injury is not well understood. The aim of this thesis is to examine the pathogenic role of GSK-3 in immune hepatic injury. Results may shed light on the mechanisms by which GSK-3 regulates inflammation and apoptosis in IFN-γ-related liver diseases and serve as an anti-inflammatory therapeutic strategy. In this thesis, the mechanism that activates GSK-3 and the effects of activation on IFN-γ-induced inflammation were investigated. Results showed that treating cells with GSK-3β inhibitor and short interfering RNA showed a decrease on IFN-γ-induced inflammatory responses, including inducible nitric oxide synthase (iNOS)/nitric oxide (NO) biosynthesis and proinflammatory cytokine and chemokine production. Following IFN-γ stimulation, activation of GSK-3β was regulated by two distinct mechanisms, which included okadaic acid (OA)-sensitive phosphatase (PPase)-mediated dephosphorylation at Ser9 and proline-rich tyrosine kinase (Pyk2)-mediated phosphorylation at Tyr216. Inhibiting GSK-3β activated SH2 domain-containing tyrosine phosphatase (SHP2), thereby preventing STAT1 activation in the late stage of IFN-γ stimulation. These results demonstrate that activated GSK-3β synergistically affected IFN-γ-induced STAT1 activation by inhibiting SHP2. Therefore, I further examined the effects of GSK-3 inhibition on IFN-γ mediated immune hepatitis. Immune hepatic injury induced by ConA results primarily from IFN-γ-mediated inflammation, followed by hepatic cell death. GSK-3, which acts proapoptotically and is proinflammatory, is also important for facilitating IFN-γ signaling. ConA stimulation caused GSK-3 activation in the livers of C57BL/6 mice. GSK-3 inhibition reduced ConA hepatic injury, including hepatic necrosis and apoptosis, inflammation, infiltration of T cells and granulocytes, and deregulated expression of intercellular adhesion molecule-1 (ICAM-1). ConA induced hepatic injury in an IFN-γ receptor 1 (IFNGR1)-dependent manner. ConA/IFN-γ induced activation and expression of STAT1 in a GSK-3-dependent manner. GSK-3 facilitated IFN-γ-induced iNOS, but had limited effects on CD95 upregulation and CD95-mediated hepatocyte apoptosis in vitro. Notably, inhibiting GSK-3 decreased ConA-induced IFN-γ production in both wild-type and IFNGR1-deficient C57BL/6 mice. In ConA-activated natural killer T (NKT) cells, GSK-3 was also activated and was required for nuclear translocation of T-box transcription factor Tbx21 (T-bet), a transcription factor of IFN-γ, but it was not required for CD95 ligand expression or activation-induced cell death. Taken together, these data demonstrate the proinflammatory and proapoptotic roles of GSK-3 are indispensable in the pathogenesis of immune hepatic injury, and targeting GSK-3 can be used for the treatment of IFN-γ-related liver diseases.