JAK/STAT1 Signaling Transduction Pathway In The Pathogenesis And Treatment Of MRL/lpr Lupus Mice

Backgrounds and objectiveCytokines activate multiple intracellular signaling pathways in order to exert their physiological effects. Four signaling pathways have been found to be associated with inflammation process, namely, mitogen-activated protein kinase (MAPK), phosphatidylino-sitol 3'-kinase (PI3), nuclear factor- κ B (NF- κ B), and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. JAK/STAT pathway is one of the most important pathways for cytokine signaling which involves the Janus family of tyrosine kinases and the signal transducers and activators of transcription proteins. Cytokines utilizing this pathway form a ligand-receptor complex, resulting in tyrosine phosphorylation of receptor-associated Janus kinase (JAK), the cytoplasmic portion of the cytokine receptor, and STAT. The phosphorylated STATs form an activated dimer or tetramer, which translocates to the cell nucleus where it binds with a specific DNA sequence and/or other transcription factors to influence "target gene" transcription. Four different JAK kinases have been identified: JAK1, JAK2, JAK3, and TYK2, which may be activated by different cytokines or receptors, a total of seven different STATs have been described to date, and they are STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. Receptor aggregation induced by cytokine binding results in the apposition of JAKs bound to their cytoplasmic domains and activation of their kinase activities by cross-phosphorylation. The JAKs then phosphorylate multiple target proteins, including the receptor cytoplasmic domains, forming a series of tyrosine phosphorylated docking sites for signaling and adaptor molecules. Some of these sites are docking sites for the STATs, which are then phosphorylated on critical C-terminal tyrosines by JAKs. The phosphorylated STATs dissociate from the receptor, form homo- or heterodimers through interactions of their SH2 domains, and are translocated to the nucleus and interact with specific DNA sequences and/or transcription factors to activate gene transcription. Suppressors of cytokine signaling (SOCS) proteins are synthesized following STAT activation. SOCS proteins are classic feedback inhibitors of signaling, by virtue of their SH2 domains, they inhibit JAK activity, block recruitment of signaling molecules to cytokine receptors and promote degradation of cytokine receptors and/or JAKs. SOCS are at very low levels in silent cells, but increased quickly when cells are stimulated by cytokines. Thus, the transcription of SOCS genes means the activation of STAT molecules.Systemic lupus erythematosus (SLE) is the most representative systemic autoimmune disease in humans. It is characterized by the presence of activated T cells and B cells in conjunction with the development of many different auto antibodies, and chronic inflammation that can affect various parts of the body including the skin and kidneys. Although many factors have been proposed to be associated with SLE, such as genetic factors, environmental factors, hormonal action, viruses, and dysregulation of cytokine production, the cause of this complex disease is not well understood. However, a long history of observations has strongly implicated the role of the interferons (IFNs), both type I (IFN-α/β) and type II (IFN-γ), in the pathogenesis of SLE. Several notable observations include: ①increased levels of IFNs are commonly observed in lupus patients, often correlating with disease activity; ②recombinant IFN-α, when administered as a therapeutic reagent to patients with malignancy or hepatitis infection, can induce SLE; ③recent studies using gene expression profiling have shown that most lupus cases demonstrate an up-regulation of IFN-responsive genes, and expression of certain IFN-inducible genes correlated with disease severity; ④ both type I and type II IFNs can promote class switch recombination to Th1-like IgG isotypes (IgG2a, IgG2b, IgG3), pathogenic, complement-fixing isotypes in lupus; ⑤ IFN-α induces maturation of monocytes into highly active antigen-presenting dendritic cells (DCs) which are capable of breaking tolerance and/or at least augmenting humoral autoimmunity; ⑥multiple murine. lupus studies, involving genetically altered and/or therapeutically manipulated animals, have demonstrated the clear importance of IFN-γto autoantibody production and subsequent organ damage. Thus, the IFNs have been heavily implicated in the pathogenesis of SLE and have emerged as a dominant target in the pathogenesis of SLE.The biological effects of IFNs are mediated through the JAK/STAT pathway in which both IFN-α/ β and IFN-γ activate the transcription factor STAT1. Cytokine signaling via the JAK/STAT pathway regulates a number of important biologic processes, including the immune response, cellular growth and differentiation and apoptosis, JAK/STAT pathway have been found to be associated with the pathogenesis of many diseases including tumor, chronic diseases like hepatitis. Recent findings have indicated a key role of STAT1 activation in a number of inflammation-related diseases, and it is likely that STAT1 may represent a new molecular target for the anti-inflammatory treatment. However, little is known about the pathogenic significance of STAT1 in SLE. To better understand the pathogenic significance of JAK-STAT1 in SLE and how the STAT function is regulated, the present study was undertaken to investigate the expression and activation of JAK/STAT1 signaling transduction pathway in common SLE involvement organs including kidney, lung and brain of MRL/lpr mice by assessing the protein level of STAT1 as well as the mRNA levels of SOCS1 and SOCS3.The prognosis of patients with SLE has improved substantially over the past four decades, with a 10-year survival of >90% in some centers. The prognosis heterogeneity associated with SLE can be attributed to gender, age, genetic clinical factors and treatment. Education; earlier intervention, better disease damage control and improved general management of hypertension, co-morbid infections and other clinical risks have contributed to reduced end-organ damage and less morbidity and mortality. Nonetheless, the high renal involvement and subsequent renal failure of this disease remains a challenge. It has been reported that 70% SLE patients demonstrate renal involvement clinically and nearly .100% SLE patients would be affected by nephritis if checked by renal biopsy. Glomerulonephritis remains a severe complication of the renal involvement which is the major cause of morbidity and mortality in SLE. Typically, glucocorticoids and cyclophosphamide(CTX) provide the therapeutic foundation for lupus nephritis. However, the common and severe side effects of the current therapy call for new strategies to improve long-term prognosis and life quality for this disease.Rapamycin (RAPA) is a macrocyclic lactone found in the Streptomyces hygroscopicus organism. Originally identified as an antifungal agent, it was subsequently found to have potent immunosuppressant properties, leading to its approval for the prophylaxis of organ rejection in patients receiving renal transplants. RAPA possesses potent antimicrobial, immunosuppressant, and antitumor properties which believed to be a new effective immunosuppressive agent with less side-effects. Because of their profound immunosuppressive actions, many drugs initially developed for prevention of allograft rejection following organ transplantation, such as Cyclosporin A, mycophenolate mofetil, Leflunomide and FK-506, had been used later as anti-rheumatic drugs. It indicates that RAPA may also be a good therapy for rheumatic diseases. However, the precise mechanisms for RAPA's effects are not fully understood, The antiproliferative actions of rapamycin have been demonstrated to be due to its ability to modulate critical signal transduction pathways that link mitogenic stimuli to the synthesis of proteins required for cell cycle traverse from G1 to S. The immunosuppressive properties of RAPA result from inhibition of leukocyte activity. It blocks T-cell and B-cell proliferation induced by cytokines (IL-1, -2, -3, -4, -6, -7, -12, and -15), alloantigens; and mitogens in a dose-dependent manner. This study aimed to explore the effects of RAPA on the activation of JAK/STAT1 signaling pathway, so as to understand whether the anti-rheumatic effects are associated with the blocking of JAK/STAT1 pathway. We studied the change of JAK/STAT1 signaling pathway after treatment with RAPA so as to reveal that to block signaling transduction pathway would be prospective therapy for SLE treatment. Materials and methodsThe first part studied the activation of JAK/STAT1 signaling transduction pathway in different organs of lupus mice. We included 10 MRL/lpr mice as experiment group and 10 MRL/+ mice used as controls. Mice were sacrificed when they developed spontaneous lupus-like glomerulonephritis. Kidneys were taken out and expression and activation of STAT1 were assessed by Western blotting in extracts of kidney lysates. Cellular localization was determined by immunohistochemistry. Gene expression of the STAT induced feedback inhibitors suppressor of cytokine signaling 1(SOCS1) and SOCS3 was investigated by SYBR green I quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Expression and activation of STAT1 in lung and brain were also examined using the same methods as described above.The second part studied the effects of RAPA on the JAK/STAT1 signaling transduction pathway both in vivo and in vitro. We included two groups with 10 MRL/lpr mice aged up 12-week in each group. Mice were recruited when they developed spontaneous lupus-like glomerulonephritis. Mice in group one were treated with RAPA(0.4 mg/kg/d, intraperitoneal injection, 30d); the other group were free of drugs and received intraperitoneal saline for 30 days. Kidneys were taken out at the same time point for both groups. Expression and activation of STAT1 were assessed by Western blotting. Cellular localization was determined by immunohistochemistry. Gene expression of SOCS1 and SOCS3 was investigated by SYBR green I quantitative real-time RT-PCR. For in vitro study, the activation test of the STAT1 by-interferons was assessed by Western Blot analysis after the addition of IFN- α (1 U/mL, incubate 30min) and IFN- γ (1ng/mL,incubate 30min) in cultured murine mesangial cells; the inhibition test was done by preincubation with JAK-STAT specific inhibitor AG-490 (100 μM) or RAP A (50nM) before the stimulation with interferons.Statistical significance was set at the 0.01 or 0.05 level. Results1. Analysis of p-Tyr-STAT1 levels in the first part showed a significantly higher value in MRL/lpr group relative to the control (P<0.01). Protein level analysis in the second part showed the expression of total STAT1 was also increased in the kidney of MRL/lpr lupus nephritis group (P<0.01).2. Western blot analysis of p-Tyr-STAT1 levels in lung showed a significantly higher value in MRL/lpr group relative to the control, which have significant difference (P<0.05).3. P-STAT1 protein was predominantly abundant in small vessels in the brain of MRL/lpr mice, the p-Tyr-STAT1 levels by western blot showed no difference between the two groups.4. SOCS-1 and SOCS-3 mRNA expression was all increased in kidney, lung and brain5. renal SOCS-1 mRNA expression increased less than pneumonic and cerebral expression6. Rapamycin could reduce the renal phosphorylated STAT1 protein and lower the SOCS-1 gene expression in LN group.7. Stimulation with either IFN-α or IFN-γ rapidly induced phosphorylation of STAT1 in mesangial cells in vitro. IFN-γ induced a rapid and strong increase of STAT1 tyrosine phosphorylation, while IFN-α was much less effective.8. RAPA or AG-490 could block the activation of STAT1 in LN mesangial cells9. The inhibition of IFN-α induced activation was relatively less than that of IFN-γ.Conclusions and significances1. The expression and activation of JAK/STAT signaling transduction pathway were both elevated in the kidney, lung and brain tissues of MRL/lpr mice, which imply that disregulated activation of JAK/STAT1 signaling transduction pathway may be of pathogenic significance in the organ involvement and progression of SLE.2. P-STAT1 protein was predominantly abundant in small vessels in the brain of MRL/lpr mice, suggesting that the vasculitis of small vessels play an important role in the pathophysiology of cerebral lupus.3. Both in vivo and in vitro studies suggest that JAK/STAT1 signaling transduction pathway may play an important role in the pathogenesis of lupus nephritis.4. The pathogenesis of lupus nephritis may also be associated with the down-regulation of SOCS feedback inhibition.5. The pathogenesis for cerebral lupus may be explained by more than one mechanism, nevertheless, the down-regulation of SOCS feedback inhibition may be associated with the pathogenesis of cerebral lupus.6. Rapamycin can lower the expression and activation of JAK/STAT1, which may be one of the mechanisms by which rapamycin treat systemic lupus erythematosus.7. Both IFN-α and IFN-γ induced rapid phosphorylation of STAT1 in vitro in cultured mesangial cells.8. Jak-STAT1 is the main signaling pathway used by IFN-γ in mesangial cells of MRL/lpr mice in vitro while IFN-α may use multiple signaling pathways.9. RAPA and other JAK-STAT inhibitors are promising therapeutic drugs for future interventions in lupus nephritis inflammation.10. Analysis of the intracellular signaling of STAT1 and expression of downstream genes like SOCS responses may reveal important novel aspects in complex inflammatory diseases such as SLE.