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1.  Changes in synovial tissue Jak‐STAT expression in rheumatoid arthritis in response to successful DMARD treatment 
Annals of the Rheumatic Diseases  2006;65(12):1558-1564.
Background
Modulation of Jak‐STAT signalling may provide an effective therapeutic strategy in inflammatory arthritis (IA).
Objective
To examine the effect of successful disease‐modifying antirheumatic drug (DMARD) treatment on the expression of Jak‐STAT in a cohort of patients with active rheumatoid arthritis.
Methods
Synovial tissue biopsy specimens from 16 patients with active rheumatoid arthritis, taken before and after initiation of DMARD treatment, were examined for the presence of janus kinase (Jak)3, signal transducer and activator of transcription (STAT)1, STAT4 and STAT6 expression using immunohistochemistry.
Results
Successful treatment with DMARDs results in reduction in STAT1 expression in the lining, and STAT1 and STAT6 in the sublining of rheumatoid arthritis synovial tissue. Although the overall expression of STAT4 and Jak3 was not significantly altered by DMARD treatment, there was a significant reduction in the expression of the STAT4 and Jak3 bright cells, thought to be an activated dendritic cell subpopulation.
Conclusion
Results show that Jak3, STAT1, STAT4 expression and STAT6 sublining expression decrease in response to successful treatment of rheumatoid arthritis with standard DMARDs. Therefore, altering the expression of these pathways may represent an alternative treatment option, either through promoting up‐regulation of inhibitory pathways, or suppressing inflammatory paths.
doi:10.1136/ard.2005.050385
PMCID: PMC1798468  PMID: 16760256
2.  Characterisation of a dendritic cell subset in synovial tissue which strongly expresses Jak/STAT transcription factors from patients with rheumatoid arthritis 
Annals of the Rheumatic Diseases  2007;66(8):992-999.
Objectives
To characterise the phenotype of the putative dendritic cells strongly expressing Jak3 and STAT4, which have been previously identified in the synovial tissue of patients with active rheumatoid arthritis (RA).
Methods
Synovial biopsy specimens were obtained at arthroscopy from 30 patients with active RA (42 synovial biopsies). Immunohistological analysis was performed using monoclonal antibodies to detect dendritic cell subsets, including activation markers and cytokines relevant to dendritic cell function. Co‐localisation of cell surface markers and cytokines was assessed primarily using sequential sections, with results confirmed by dual immunohistochemistry and immunofluorescence with confocal microscopy.
Results
The dendritic cells identified in RA synovial tissue that strongly express Jak3 also strongly express STAT4 and STAT 6 and are correlated with the presence of serum rheumatoid factor. These cells are not confined to a single dendritic cell subset, with cells having phenotypes consistent with both myeloid‐ and plasmacytoid‐type dendritic cells. The activation status of these dendritic cells suggests that they are maturing or mature dendritic cells. These dendritic cells produce IL12 as well as interferon α and γ.
Conclusions
The close correlation of these dendritic cells with the presence of serum rheumatoid factor, a prognostic factor for worse disease outcome, and the strong expression by these cells of components of the Jak/STAT transcription factor pathway suggest a potential therapeutic target for the treatment of RA.
doi:10.1136/ard.2006.060822
PMCID: PMC1954703  PMID: 17223651
rheumatoid arthritis; myeloid dendritic cells; plasmacytoid dendritic cells; IL12; interferon alpha; interferon gamma
3.  Activation of the STAT1 pathway in rheumatoid arthritis 
Annals of the Rheumatic Diseases  2004;63(3):233-239.
Background: Expression of signal transducer and activator of transcription 1 (STAT1), the mediator of interferon (IFN) signalling, is raised in synovial tissue (ST) from patients with rheumatoid arthritis (RA).
Objectives: To determine the extent to which this pathway is activated by phosphorylation in RA synovium. Additionally, to investigate the cellular basis of STAT1 activation in RA ST.
Methods: ST specimens from 12 patients with RA and 14 disease controls (patients with osteoarthritis and reactive arthritis) were analysed by immunohistochemistry, using antibodies to STAT1, tyrosine phosphorylated STAT1, and serine phosphorylated STAT1. Lysates of cultured fibroblast-like synoviocytes stimulated with IFNß were analysed by western blotting. Phenotypic characterisation of cells expressing STAT1 in RA ST was performed by double immunolabelling for STAT1 and CD3, CD22, CD55, or CD68.
Results: Raised levels of total STAT1 protein and both its activated tyrosine and serine phosphorylated forms were seen in RA synovium as compared with controls. STAT1 was predominantly abundant in T and B lymphocytes in focal inflammatory infiltrates and in fibroblast-like synoviocytes in the intimal lining layer. Raised levels of STAT1 are sustained in cultured RA compared with OA fibroblast-like synoviocytes, and STAT1 serine and tyrosine phosphorylation is rapidly induced upon stimulation with IFNß.
Conclusion: These results demonstrate activation of the STAT1 pathway in RA synovium by raised STAT1 protein expression and concomitantly increased tyrosine (701) and serine (727) phosphorylation. High expression of STAT1 is intrinsic to RA fibroblast-like synoviocytes in the intimal lining layer, whereas activation of the pathway by phosphorylation is an active process.
doi:10.1136/ard.2003.013276
PMCID: PMC1754903  PMID: 14962955
4.  Induction of the cytokine signal regulator SOCS3/CIS3 as a therapeutic strategy for treating inflammatory arthritis 
Journal of Clinical Investigation  2001;108(12):1781-1788.
Immune and inflammatory systems are controlled by multiple cytokines, including ILs and INFs. These cytokines exert their biological functions through Janus tyrosine kinases and STAT transcription factors. One such cytokine, IL-6, has been proposed to contribute to the development of rheumatoid arthritis (RA). We found that STAT3 was strongly tyrosine phosphorylated in synovial tissue of RA patients, but not those with osteoarthritis. Blockade of the IL-6-gp130-JAK-STAT3–signaling pathway might therefore be beneficial in the treatment of RA. We show here that the mRNA for the endogenous cytokine signaling repressor CIS3/SOCS3 is abundantly expressed in RA patients. To determine whether CIS3 is effective in treating experimental arthritis, a recombinant adenovirus carrying the CIS3 cDNA was injected periarticularly into the ankle joints of mice with antigen-induced arthritis or collagen-induced arthritis (CIA). Periarticular injection of CIS3 adenovirus drastically reduced the severity of arthritis and joint swelling compared with control groups. CIS3 was more effective than a dominant-negative form of STAT3 in the CIA model. Thus, induction of CIS3 could represent a new approach for effective treatment of RA.
PMCID: PMC209467  PMID: 11748261
5.  Constitutive activation of signal transducer and activator of transcription 3 regulates expression of vascular endothelial growth factor in human meningioma differentiation 
Purpose
Janus tyrosine kinases (JAKs) and signal transducer and activator of transcription factors (STATs), especially STAT3, are constitutively activated in human cancers. The function of STAT3 in the pathogenesis of meningioma remains unknown. In this study, we investigated the role of JAK1/STAT3 regulating vascular endothelial growth factor (VEGF) expression in the occurrence and progression of human meningioma.
Methods
We detected the expression of JAK1, p-JAK1, STAT3, p-STAT3, and VEGF in human meningioma and normal dura tissues by RT–PCR, Western blot analysis, and immunohistochemistry.
Results
JAK1, p-JAK1, STAT3, p-STAT3, and VEGF showed high expression in grade I and grade II meningioma. The level of STAT3 activation was associated with VEGF expression; all meningioma tumors that expressed p-STAT3 also expressed VEGF. Both frequency of positivity and expression were enhanced with increasing tumor grade; high frequencies and levels were found in grade II tumors, with no expression detected in normal dura tissues (P < 0.05).
Conclusions
VEGF is directly regulated by constitutive STAT3 activity and associated with meningioma differentiation. STAT3 has an important role in the occurrence and development of human meningioma by regulating VEGF expression.
doi:10.1007/s00432-009-0743-9
PMCID: PMC2874489  PMID: 20052595
Constitutive activation; Human meningioma; p-STAT3; STAT3; VEGF
6.  Inhibitory Effect of Curcumol on Jak2-STAT Signal Pathway Molecules of Fibroblast-Like Synoviocytes in Patients with Rheumatoid Arthritis 
Hyperplasia of synovial membrane in rheumatoid arthritis (RA) is a critical pathological foundation for inducing articular injury. The janus kinase and signal transducer and activator of transcription (Jak-STAT) pathway plays a critical role in synovial membrane proliferation induced by platelet-derived growth factor (PDGF). To explore the anti-cell proliferation mechanism of curcumol, a pure monomer extracted from Chinese medical plant zedoary rhizome, the changes of Jak2-STAT1/3 signal pathway-related molecules in synoviocytes were observed in vitro. In this study, the fibroblast-like synoviocytes (FLS) in patients with RA were collected and cultured. The following parameters were measured: cell proliferation (WST-1 assay), cell cycles (fluorescence-activated cell sorting, FACS), STAT1 and STAT3 activities (electrophoretic mobility shift assay, EMSA), and the protein expressions of phosphorylated Jak2, STAT1, and STAT3 (Western blot). It was shown that curcumol could inhibit the RA-FLS proliferation and DNA synthesis induced by PDGF-BB in a dose-dependent manner in vitro. The transcription factors activities of STAT1 and STAT3 were obviously elevated after PDGF-BB stimulation (P < 0.05). Super-shift experiments identified the STAT1 or STAT3 proteins in the complex. Furthermore, the different concentration curcumol could downregulate the DNA binding activities of STAT1 and STAT3 (P < 0.05) and inhibit the phosphorylation of Jak2 while it had no effect on the protein expressions of STAT1 and STAT3. Positive correlations were found between changes of cell proliferation and DNA-binding activities of STAT1 and STAT3, respectively (P < 0.01). In conclusion, curcumol might suppress the FLS proliferation and DNA synthesis induced by PDGF-BB through attenuating Jak2 phosphorylation, downregulating STAT1 and STAT3 DNA-binding activities, which could provide theoretical foundation for clinical treatment of RA.
doi:10.1155/2012/746426
PMCID: PMC3310153  PMID: 22474524
7.  Regulation of Inflammatory Responses in Tumor Necrosis Factor - Activated and Rheumatoid Arthritis Synovial Macrophages by Janus Kinase Inhibitors 
Arthritis and rheumatism  2012;64(12):3856-3866.
Objective
Inhibitors of the Janus kinases (JAKs) have been developed as anti-inflammatory and immunosuppressive agents and are currently undergoing testing in clinical trials. The JAK inhibitors CP-690,550 (tofacitinib) and INCB018424 (ruxolitinib) have demonstrated clinical efficacy in rheumatoid arthritis (RA). However, the mechanisms that mediate the beneficial actions of these compounds are not known. In this study, we examined the effects of both JAK inhibitors on inflammatory and tumor necrosis factor (TNF) responses in human macrophages (MΦs).
Methods
In vitro studies were performed with peripheral blood MΦs from healthy donors treated with TNF and synovial fluid MΦs from patients with RA. Levels of activated signal transducer and activator of transcription (STAT) proteins and other transcription factors were detected by Western blot, and gene expression was measured by real-time polymerase chain reaction. In vivo effects of JAK inhibitors were evaluated in the K/BxN serum-transfer model of arthritis.
Results
JAK inhibitors suppressed activation and expression of STAT1 and downstream inflammatory target genes in TNF-stimulated and RA synovial macrophages. In addition, JAK inhibitors decreased nuclear localization of NF-κB subunits in TNF-stimulated and RA synovial macrophages. CP-690,550 significantly decreased IL6 expression in synovial MΦs. JAK inhibitors augmented nuclear levels of NFATc1 and cJun, followed by increased formation of osteoclast-like cells. CP-690,550 strongly suppressed K/BxN arthritis that is dependent on macrophages but not on lymphocytes.
Conclusion
Our findings demonstrate that JAK inhibitors suppress macrophage activation and attenuate TNF responses, and suggest that suppression of cytokine/chemokine production and innate immunity contributes to the therapeutic efficacy of JAK inhibitors.
doi:10.1002/art.37691
PMCID: PMC3510320  PMID: 22941906
macrophages; TNF; STAT1; rheumatoid arthritis; JAK inhibitors
8.  CP690,550 inhibits oncostatin M-induced JAK/STAT signaling pathway in rheumatoid synoviocytes 
Introduction
Interleukin (IL)-6-type cytokines exert their effects through activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling cascade. The JAK/STAT pathways play an important role in rheumatoid arthritis, since JAK inhibitors have exhibited dramatic effects on rheumatoid arthritis (RA) in clinical trials. In this study, we investigated the molecular effects of a small molecule JAK inhibitor, CP690,550 on the JAK/STAT signaling pathways and examined the role of JAK kinases in rheumatoid synovitis.
Methods
Fibroblast-like synoviocytes (FLS) were isolated from RA patients and stimulated with recombinant oncostatin M (OSM). The cellular supernatants were analyzed using cytokine protein chips. IL-6 mRNA and protein expression were analyzed by real-time PCR method and ELISA, respectively. Protein phosphorylation of rheumatoid synoviocytes was assessed by Western blot using phospho-specific antibodies.
Results
OSM was found to be a potent inducer of IL-6 in FLS. OSM stimulation elicited rapid phosphorylation of STATs suggesting activation of the JAK/STAT pathway in FLS. CP690,550 pretreatment completely abrogated the OSM-induced production of IL-6, as well as OSM-induced JAK/STAT, and activation of mitogen-activated kinases (MAPKs) in FLS.
Conclusions
These findings suggest that IL-6-type cytokines contribute to rheumatoid synovitis through activation of the JAK/STAT pathway in rheumatoid synoviocytes. Inhibition of these pro-inflammatory signaling pathways by CP690,550 could be important in the treatment of RA.
doi:10.1186/ar3333
PMCID: PMC3218881  PMID: 21548952
9.  Intracellular Signaling Pathways in Rheumatoid Arthritis 
Dysfunctional intracellular signaling involving deregulated activation of the Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) and “cross-talk” between JAK/STAT and the stress-activated protein kinase/mitogen-activated protein kinase (SAPK/MAPK) and Phosphatidylinositide-3-Kinase/AKT/mammalian Target of Rapamycin (PI-3K/AKT/mTOR) pathways play a critical role in rheumatoid arthritis. This is exemplified by immune-mediated chronic inflammation, up-regulated matrix metalloproteinase gene expression, induction of articular chondrocyte apoptosis and “apoptosis-resistance” in rheumatoid synovial tissue. An important consideration in the development of novel therapeutics for rheumatoid arthritis will be the extent to which inhibiting these signal transduction pathways will sufficiently suppress immune cell-mediated inflammation to produce a lasting clinical remission and halt the progression of rheumatoid arthritis pathology. In that regard, the majority of the evidence accumulated over the past decade indicated that merely suppressing pro-inflammatory cytokine-mediated JAK/ STAT, SAPK/MAPK or PI-3K/AKT/mTOR activation in RA patients may be necessary but not sufficient to result in clinical improvement. Thus, targeting aberrant enzyme activities of spleen tyrosine kinase, sphingosine kinases-1, -2, transforming growth factor β-activated kinase-1, bone marrow kinase, and nuclear factor-κB-inducing kinase for intervention may also have to be considered.
doi:10.4172/2155-9899.1000160
PMCID: PMC3947501  PMID: 24619558
Apoptosis; Arthritis; Cartilage; Cell survival; Inflammation; Protein kinase; Signal transduction; Small molecule inhibitor; Synovium
10.  A shift from pStat6 to pStat3 predominance is associated with inflammatory bowel disease-associated dysplasia 
Inflammatory Bowel Diseases  2011;18(7):1267-1274.
Background
Activated Stat3 is an important mediator of oncogenesis in the colon. To test the hypothesis that select Stat activation and/or the pattern of Stat activation serves as a marker for early neoplastic transformation, we examined the distribution of activated Stat1(pStat1), Stat6(pStat6) and Stat3(pStat3) in colitis along the continuum of inactive disease to colitis-associated cancer.
Methods
Tissue microarrays were constructed using colonoscopy biopsy and surgical specimens from 67 patients with ulcerative colitis or Crohn’s colitis and 11 controls. 111 sets of samples were analyzed representing normal tissue, active disease, low-grade dysplasia, high-grade dysplasia, and colitis-associated cancer. Immunohistochemistry to detect pStat1, pStat6 and pStat3 in colonic epithelial and mucosal immune cells was then correlated with clinical and pathological data (tumor location, histologic type, grade and lymph node involvement).
Results
In 50% of colitis-associated cancer samples, pStat3 was detected prominently in epithelial cells, where it was routinely associated with intense pStat3 staining in surrounding immune cells. Stat3 activation was greater in low-grade tumors than in high-grade ones (P<0.05). pStat6 expression was more common in normal tissues than in colitis-associated cancer (P<0.05). pStat1 was detected in a small subset of immune cells in patients with chronic inactive and active colitis, low and high grade dysplasia, but not in colitis-associated cancer.
Conclusions
pStat3 may be a marker for neoplastic transformation in patients with colitis. A shift from predominant immune cell Stat6 activation to Stat3 activation accompanies the onset of dysplasia with concomitant increased epithelial cell Stat3 activation in a subset of patients.
doi:10.1002/ibd.21908
PMCID: PMC3266961  PMID: 22021169
Stat3; Th17; Colitis-associated cancer
11.  Regulatory effect of calcineurin inhibitor, tacrolimus, on IL-6/sIL-6R-mediated RANKL expression through JAK2-STAT3-SOCS3 signaling pathway in fibroblast-like synoviocytes 
Introduction
This study investigated whether the calcineurin inhibitor, tacrolimus, suppresses receptor activator of NF-κB ligand (RANKL) expression in fibroblast-like synoviocytes (FLS) through regulation of IL-6/Janus activated kinase (JAK2)/signal transducer and activator of transcription-3 (STAT3) and suppressor of cytokine signaling (SOCS3) signaling.
Methods
The expression of RANKL, JAK2, STAT3, and SOCS3 proteins was assessed by western blot analysis, real-time PCR and ELISA in IL-6 combined with soluble IL-6 receptor (sIL-6R)-stimulated rheumatoid arthritis (RA)-FLS with or without tacrolimus treatment. The effects of tacrolimus on synovial inflammation and bone erosion were assessed using mice with arthritis induced by K/BxN serum. Immunofluorescent staining was performed to identify the effect of tacrolimus on RANKL and SOCS3. The tartrate-resistant acid phosphatase staining assay was performed to assess the effect of tacrolimus on osteoclast differentiation.
Results
We found that RANKL expression in RA FLS is regulated by the IL-6/sIL-6R/JAK2/STAT3/SOCS3 pathway. Inhibitory effects of tacrolimus on RANKL expression in a serum-induced arthritis mice model were identified. Tacrolimus inhibits RANKL expression in IL-6/sIL-6R-stimulated FLS by suppressing STAT3. Among negative regulators of the JAK/STAT pathway, such as CIS1, SOCS1, and SOCS3, only SOCS3 is significantly induced by tacrolimus. As compared to dexamethasone and methotrexate, tacrolimus more potently suppresses RANKL expression in FLS. By up-regulating SOCS3, tacrolimus down-regulates activation of the JAK-STAT pathway by IL-6/sIL-6R trans-signaling, thus decreasing RANKL expression in FLS.
Conclusions
These data suggest that tacrolimus might affect the RANKL expression in IL-6 stimulated FLS through STAT3 suppression, together with up-regulation of SOCS3.
doi:10.1186/ar4162
PMCID: PMC3672788  PMID: 23406906
12.  Intimal lining layer macrophages but not synovial sublining macrophages display an IL-10 polarized-like phenotype in chronic synovitis 
Introduction
Synovial tissue macrophages play a key role in chronic inflammatory arthritis, but the contribution of different macrophage subsets in this process remains largely unknown. The main in vitro polarized macrophage subsets are classically (M1) and alternatively (M2) activated macrophages, the latter comprising interleukin (IL)-4 and IL-10 polarized cells. Here, we aimed to evaluate the polarization status of synovial macrophages in spondyloarthritis (SpA) and rheumatoid arthritis (RA).
Methods
Expression of polarization markers on synovial macrophages, peripheral blood monocytes, and in vitro polarized monocyte-derived macrophages from SpA versus RA patients was assessed by immunohistochemistry and flow cytometry, respectively. The polarization status of the intimal lining layer and the synovial sublining macrophages was assessed by double immunofluorescence staining.
Results
The expression of the IL-10 polarization marker cluster of differentiation 163 (CD163) was increased in SpA compared with RA intimal lining layer, but no differences were found in other M1 and M2 markers between the diseases. Furthermore, no significant phenotypic differences in monocytes and in vitro polarized monocyte-derived macrophages were seen between SpA, RA, and healthy controls, indicating that the differential CD163 expression does not reflect a preferential M2 polarization in SpA. More detailed analysis of intimal lining layer macrophages revealed a strong co-expression of the IL-10 polarization markers CD163 and cluster of differentiation 32 (CD32) but not any of the other markers in both SpA and RA. In contrast, synovial sublining macrophages had a more heterogeneous phenotype, with a majority of cells co-expressing M1 and M2 markers.
Conclusions
The intimal lining layer but not synovial sublining macrophages display an IL-10 polarized-like phenotype, with increased CD163 expression in SpA versus RA synovitis. These differences in the distribution of the polarized macrophage subset may contribute to the outcome of chronic synovitis.
doi:10.1186/ar3796
PMCID: PMC3446447  PMID: 22494514
13.  Involvement of neurotrophins and their receptors in spondyloarthritis synovitis: relation to inflammation and response to treatment 
Annals of the Rheumatic Diseases  2005;64(11):1542-1549.
Objective: To investigate whether expression of the four members of the neurotrophin (NT) family and their four corresponding receptors is related to synovial inflammation in patients with spondyloarthritis (SpA).
Material and Methods: Synovial fluid (SF) and serum NTs and their receptors were measured by ELISA. Immunohistochemistry was used for synovial tissue biopsy specimens from patients with SpA, rheumatoid arthritis, and osteoarthritis (OA). In SpA synovium, immunoreactivity of the receptors trkA and NGFRp75 was also assessed before and after 12 weeks of treatment with the monoclonal anti-tumour necrosis factor α antibody, infliximab.
Results: mRNA transcripts of all NTs and receptors were expressed in the inflamed synovium. At the protein level, brain derived neurotrophic factor and NT-3 were significantly higher in the SF of patients with SpA than in those with OA. In contrast, ELISA of serum samples showed that the highest member in SpA was NT-4. Immunohistochemistry demonstrated that the NT receptors trkA and NGFRp75 were highly expressed in the inflamed synovium of patients with SpA, correlating with vascularity and lymphoid aggregates, respectively. Additionally, immunoreactivity of both receptors was significantly decreased after infliximab treatment.
Conclusions: NTs and their receptors are expressed in inflamed peripheral joints of patients with SpA. Their expression is not constitutive but related to inflammation and they may be involved in the local disease processes.
doi:10.1136/ard.2004.032599
PMCID: PMC1755273  PMID: 15817657
14.  Modulation of Innate and Adaptive Immune Responses by Tofacitinib (CP-690,550) 
Inhibitors of the JAK family of non-receptor tyrosine kinases have demonstrated clinical efficacy in rheumatoid arthritis and other inflammatory disorders; however, the precise mechanisms by which JAK inhibition improves inflammatory immune responses remain unclear. Here we examined the mode of action of tofacitinib (CP-690,550) on JAK/STAT signaling pathways involved in adaptive and innate immune responses. To determine the extent of inhibition of specific JAK/STAT-dependent pathways, we analyzed cytokine stimulation of mouse and human T cells in vitro. We also investigated the consequences of CP-690,550 treatment on Th cell differentiation of naïve murine CD4+ T cells. CP-690,550 inhibited IL-4-dependent Th2 cell differentiation, and interestingly also interfered with Th17 cell differentiation. Expression of IL-23 receptor and of the Th17 cytokines IL-17A, IL-17F and IL-22 were blocked when naïve Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A-production was enhanced when Th17 cells were differentiated in the presence of TGF-β. Moreover, CP-690,550 also prevented activation of STAT1, induction of T-bet and subsequent generation of Th1 cells. In a model of established arthritis, CP-690,550 rapidly improved disease by inhibiting production of inflammatory mediators and suppressing STAT1-dependent genes in joint tissue. Furthermore, efficacy in this disease model correlated with inhibition of both JAK1 and JAK3 signaling pathways. CP-690,550 also modulated innate responses to LPS in vivo through a mechanism likely involving inhibition of STAT1 signaling. Thus, CP-690,550 may improve autoimmune diseases and prevent transplant rejection by suppressing the differentiation of pathogenic Th1 and Th17 cells, as well as innate immune cell signaling.
doi:10.4049/jimmunol.1003668
PMCID: PMC3108067  PMID: 21383241
15.  JAKs go nuclear: Emerging role of nuclear JAK1 and JAK2 in gene expression and cell growth 
The four JAKS comprise a family of intracellular, non-receptor tyrosine kinases that first gained attention as signaling mediators of the type I and type II cytokine receptors. Subsequently, the JAKs were found to be involved in signaling downstream of the insulin receptor, a number of receptor tyrosine kinases, and certain G-protein coupled receptors. Although a number of cytoplasmic targets for the JAKs have been identified, their predominant action was found to be the phosphorylation and activation of the STAT transcription factors. Through the STATs, the JAKs activate gene expression linked to cellular stress, proliferation, and differentiation. The JAKs are especially important in haematopoiesis, inflammation, and immunity, and aberrant JAK activity has been implicated in a number of disorders including rheumatoid arthritis, psoriasis, polycythemia vera, and myeloproliferative diseases. Although once thought to reside strictly in the cytoplasm, recent evidence shows that JAK1 and JAK2 are present in the nucleus of certain cells often under conditions associated with high rates of cell growth. Nuclear JAKs have now been shown to affect gene expression by activating other transcription factors besides the STATs and exerting epigenetic actions, for example, by phosphorylating histone H3. The latter action derepresses global gene expression and has been implicated in leukemogenesis. Nuclear JAKs may have a role as well in stem cell biology. Here we describe recent developments in understanding the noncanonical nuclear actions of JAK1 and JAK2.
doi:10.3109/08977194.2011.614949
PMCID: PMC3595105  PMID: 21892841
16.  Constitutive STAT3 Phosphorylation Contributes to Skeletal Muscle Insulin Resistance in Type 2 Diabetes 
Diabetes  2013;62(2):457-465.
Signal transducer and activator of transcription 3 (STAT3) is involved in cytokine- and nutrient-induced insulin resistance. The role of STAT3 in the development of skeletal muscle insulin resistance and type 2 diabetes (T2D) pathogenesis is incompletely defined. We tested the hypothesis that STAT3 signaling contributes to skeletal muscle insulin resistance in T2D. Protein abundance and phosphorylation of STAT3 signaling molecules were determined in skeletal muscle biopsy specimens from BMI- and age-matched overweight individuals with normal glucose tolerant (NGT) and T2D patients. The direct role of STAT3 in the development of lipid-induced skeletal muscle insulin resistance was determined using small interfering (si)RNA. Phosphorylated STAT3, phosphorylated Janus kinase 2 (JAK2), and suppressor of cytokine signaling 3 (SOCS3) protein abundance was increased in skeletal muscle from T2D patients. STAT3 phosphorylation positively correlated with free fatty acid level and measures of insulin sensitivity in NGT but not T2D patients. Palmitate exposure led to a constitutive phosphorylation of STAT3, increased protein abundance of SOCS3, and development of insulin resistance in L6 myotubes. These effects were prevented by siRNA-mediated STAT3 silencing. In summary, STAT3 is constitutively phosphorylated in skeletal muscle from T2D patients. STAT3 gene silencing prevents lipid-induced insulin resistance in cultured myotubes. Collectively, our results implicate excessive STAT3 signaling in the development of skeletal muscle insulin resistance in T2D.
doi:10.2337/db12-0337
PMCID: PMC3554355  PMID: 23043161
17.  JAK-STAT in lipid metabolism of adipocytes 
JAK-STAT  2013;2(4):e27203.
JAK-STAT signaling pathway plays an important role in the cells’ development and homeostasis. Over the past decades, the studies have identified the role of the JAK-STAT pathway in cell proliferation and apoptosis. Here, we want to discuss that whether and how the JAK-STAT pathway affects the lipid metabolism of adipose tissue. A host of cytokines and hormones can regulate lipid metabolism through activating the JAK-STAT signaling pathway. Activated STATs can regulate lipid metabolism directly by influencing the expression of enzymes. We have summarized the relevant research and articles of JAK-STAT during the recent years. Within this review, we will introduce you the recent research and highlight the unresolved problems in understanding how JAK-STAT signaling pathway contribute to the lipid metabolism in mature adipocytes and preadipocytes. Dysregulation of the JAK-STAT pathway would lead to a multiple metabolism disorders and medicines for this signaling pathway maybe become a new idea for diseases such as metabolic syndrome, especially in children.
doi:10.4161/jkst.27203
PMCID: PMC3906428  PMID: 24498541
JAK-STAT; lipid metabolism; adipocytes; preadipocytes; obesity
18.  The kinase inhibitor tofacitinib in patients with rheumatoid arthritis: latest findings and clinical potential 
Macrophages, T and B cells, and neutrophils concentrate mainly into the synovial tissue of rheumatoid arthritis (RA) patients and produce several inflammatory mediators including cytokines. More recently, small molecule inhibitors of signalling mediators which have intracellular targets (mainly in T and B cells) such as the Janus kinase (JAK) family of tyrosine kinases have been introduced in RA treatment. The JAK family consist of four types: JAK1, JAK2, JAK3 and TyK2. In particular, JAK3 is the only JAK family member that associates with just one cytokine receptor, the common gamma chain, which is exclusively used by the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 critically involved in T and natural killer (NK)-cell development, and B-cell function and proliferation. Tofacitinib is one of the first JAK inhibitors tested and mainly interacts with JAK1 and JAK3. Four phase II (one A and three B dose-ranging) trials in RA patients, lasting from 6 to 24 weeks, achieved significant improvements of American College of Rheumatology 20% improvement criteria (ACR20) and Disease Activity Score in 28 joints using the C-reactive protein level (DAS28-CRP) or erythrocyte sedimentation rate (DAS-ESR; in one study that analysed this), as early as week 2 and sustained at week 24 in two studies. Doses ranged from 1, 3, 5, 10, 15, 20 up to 30 mg and were administered orally twice a day. ACR20 response rates for dosages ≥3 mg were found to be significantly (p ≤ 0.05) greater than those for placebo in all phase II studies. In general, the major adverse effects included liver test elevation, neutropenia, lipid and creatinine elevation and increased incidence of infections. More recently, RA patients randomly assigned to 5 or 10 mg of tofacitinib twice daily, in both 6- and 12-month phase III trials, achieved a significantly higher ACR20 than those receiving placebo. Adverse events occurred more frequently with tofacitinib than with placebo, and included pulmonary tuberculosis and other serious infections. The balance of efficacy and safety of tofacitinib compared with standard of care therapy is bringing this first orally available biological disease-modifying antirheumatic drug (DMARD) a step closer for RA patients.
doi:10.1177/1759720X12470753
PMCID: PMC3582307  PMID: 23515130
biological disease-modifying antirheumatic drug (DMARD); Janus kinase inhibitors; kinases; rheumatoid arthritis; therapy for rheumatoid arthritis
19.  Role of Janus Kinase/Signal Transducers and Activators of Transcription in the Pathogenesis of Pancreatitis and Pancreatic Cancer 
Gut and Liver  2012;6(4):417-422.
In the pathogenesis of pancreatitis, oxidative stress is involved in the activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and cytokine expression. High serum levels of cholecystokinin (CCK) have been reported in patients with acute pancreatitis, and treatment with cerulein, a CCK analogue, induces acute pancreatitis in a rodent model. Recent studies have shown that cerulein-activated nicotinamide adenine dinucleotide phosphate oxidase elicits reactive oxygen species, which trigger the phosphorylation of the JAK1, STAT1, and STAT3 proteins and induce the production of inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, in pancreatic acinar cells. The JAK/STAT pathway also stimulates cell proliferation and malignant transformation and inhibits apoptosis in the pancreas. This review discusses the possible role of the JAK/STAT pathway in the pathogenesis of pancreatitis and pancreatic cancer in response to oxidative stress.
doi:10.5009/gnl.2012.6.4.417
PMCID: PMC3493719  PMID: 23170143
JAK/STAT; Pancreatitis; Pancreatic cancer; Oxidative stress
20.  Activation of the IL-6/JAK/STAT3 signaling pathway in human middle ear cholesteatoma epithelium 
Interleukin-6 (IL-6) is one of the most important cytokines which has been shown to play a critical role in the pathogenesis of cholesteatoma. In this study, we aimed to investigate the expression of interleukin-6 (IL-6) and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in middle ear cholesteatoma epithelium in an effort to determine the role of IL-6/JAK/STAT3 signaling pathway in the pathogenesis of cholesteatoma. Immunohistochemistry was used to examine the expression of IL-6 and p-STAT3 in 25 human middle ear cholesteatoma samples and 15 normal external auditory canal (EAC) epithelium specimens. We also analyzed the relation of IL-6 and p-STAT3 expression levels to the degree of bone destruction in cholesteatoma. We found that the expression of IL-6 and p-STAT3 were significantly higher in cholesteatoma epithelium than in normal EAC epithelium (p<0.05). In cholesteatoma epithelium, a significant positive association was observed between IL-6 and p-STAT3 expression (p<0.05). However, no significant relationships were observed between the degree of bone destruction and the levels of IL-6 and p-STAT3 expression (p>0.05). To conclude, our results support the concept that IL-6/JAK/STAT3 signaling pathway is active and may play an important role in the mechanisms of epithelial hyper-proliferation responsible for cholesteatoma.
PMCID: PMC3925917  PMID: 24551293
Cholesteatoma; IL-6; STAT3
21.  Reversed Expression of the JAK/STAT Pathway Related Proteins Prolactin Receptor and STAT5a in Normal and Abnormal Breast Epithelial Cells 
The JAK/STAT pathway is important for cellular metabolism. One component, STAT5a, is activated in the breast upon prolactin to prolactin receptor (PRLR) binding facilitating the transcription of genes involved in lobule development. STAT5a was previously found to be expressed in most normal breast epithelial cells but not in many in situ or invasive carcinomas except secretory carcinomas which retain STAT5a expression. This report examines the JAK/STAT pathway in the breast through the detection of PRLR and STAT5a. Fifty breast tissues, including benign secretory change, microglandular adenosis, usual and atypical hyperplasia and in situ and invasive ductal carcinoma both usual and secretory, were obtained from the files of the Armed Forces Institute of Pathology. Sections were immunostained with antibodies to PRLR and STAT5a. PRLR was minimally detected on the surface of a few normal breast epithelial cells whereas STAT5a was greatly expressed in over 80% of normal cell nuclei. PRLR was also minimally detected in secretory carcinomas expressing STAT5a. However, the opposite pattern was seen in breast carcinomas lacking STAT5a expression. PRLR was abundantly expressed in these cells. This reversed expression may indicate a JAK/STAT pathway disturbance that could play a role in the initiation or maintenance of an abnormal breast phenotype.
PMCID: PMC3091403  PMID: 21655368
STAT5a; prolactin receptor; immunohistochemistry; breast; secretory
22.  Elevated expression of caspase-3 inhibitors, survivin and xIAP correlates with low levels of apoptosis in active rheumatoid synovium 
Introduction
Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a tumour necrosis factor (TNF) family member capable of inducing apoptosis in many cell types.
Methods
Using immunohistochemistry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and real-time PCR we investigated the expression of TRAIL, TRAIL receptors and several key molecules of the intracellular apoptotic pathway in human synovial tissues from various types of arthritis and normal controls. Synovial tissues from patients with active rheumatoid arthritis (RA), inactive RA, osteoarthritis (OA) or spondyloarthritis (SpA) and normal individuals were studied.
Results
Significantly higher levels of TRAIL, TRAIL R1, TRAIL R2 and TRAIL R4 were observed in synovial tissues from patients with active RA compared with normal controls (p < 0.05). TRAIL, TRAIL R1 and TRAIL R4 were expressed by many of the cells expressing CD68 (macrophages). Lower levels of TUNEL but higher levels of cleaved caspase-3 staining were detected in tissue from active RA compared with inactive RA patients (p < 0.05). Higher levels of survivin and x-linked inhibitor of apoptosis protein (xIAP) were expressed in active RA synovial tissues compared with inactive RA observed at both the protein and mRNA levels.
Conclusions
This study indicates that the induction of apoptosis in active RA synovial tissues is inhibited despite stimulation of the intracellular pathway(s) that lead to apoptosis. This inhibition of apoptosis was observed downstream of caspase-3 and may involve the caspase-3 inhibitors, survivin and xIAP.
doi:10.1186/ar2603
PMCID: PMC2688245  PMID: 19171073
23.  LIF Upregulates Expression of NK-1R in NHBE Cells 
Mediators of Inflammation  2006;2006(5):84829.
Leukemia inhibitory factor (LIF), a cytokine at the interface between neurobiology and immunology, is mainly mediated through JAK/STAT pathway and MAPK/ERK pathway. Evidence suggested LIF is related to the higher expression of neurokinin-1 receptor (NK-1R) in asthma. In this study, the immunohistochemistry stain showed the expressions of NK-1R, LIF, p-STAT3, and p-ERK1/2 in the lung tissues of allergic rats were increased compared with the controls, and the main positive cell type was airway epithelial cell. Normal human bronchial epithelial cells were treated with LIF in the presence or absence of AG490 (JAK2 inhibitor), PD98059 (MEK inhibitor), and the siRNA against STAT3. Western blot and RT-PCR indicated that LIF induced the expression of NK-1R, which was inhibited by the inhibitors mentioned above. No significant interaction was found between JAK/STAT pathway and MAPK/ERK pathway. In summary, bronchial epithelial cell changes in asthma are induced by LIF which promotes the expression of NK-1R, and JAK/STAT pathway and MAPK/ERK pathway may participate in this process.
doi:10.1155/MI/2006/84829
PMCID: PMC1657075  PMID: 17392578
24.  The involvement of Galectins in the modulation of the JAK/STAT pathway in myeloproliferative neoplasia 
Background
In patients with myeloproliferative neoplasia (MPN) the development of fibrosis and increased vessel density correlate with poor prognosis. The JAK2V617F mutation constitutively activates JAK2, which phosphorylates signal transducer activator of transcription (STAT), up-regulating vascular endothelial growth factor (VEGF), which might be responsible for angiogenesis in MPN. Galectins are involved in the development of fibrosis and angiogenesis and might also be involved in activation of the JAK/STAT pathway in MPN.
Methods
106 MPN patients, 36 essential thrombocythemia (ET), 25 polycythemia vera (PV) and 45 primary myelofibrosis (PMF), were analyzed for the expression pattern of galectin-1, galectin-3, pSTAT3, pSTAT5 and MVD by immunostaining of bone marrow biopsy sections followed by automated image analysis. The JAK2 mutational status was analysed through real time PCR in blood samples.
Results
The expression of galectin-1 was significantly higher in all MPN patients compared to normal controls. Galectin-3 was expressed more in PV patients. MVD was significantly higher in all MPN patients and correlated with galectin-1 and pSTAT5 expression. pSTAT5 expression showed a trend of higher expression in patients carrying the JAK2V617F mutation as well as in PV patients. PMF patients and all JAK2V617F positive patients showed a significantly higher pSTAT3 expression compared to control and ET patients.
Conclusion
The findings suggest the involvement of galectin-1 in MPN development, regardless of the subtype. Furthermore involvement of galectin-3 in PV development, pSTAT5 in that of PV and JAK2V617F positive patients and angiogenesis, as well as pSTAT3 is involved in the pathogenesis of PMF.
PMCID: PMC3384397  PMID: 22762031
MPN; myeloproliferative neoplasia; galectin; JAK; STAT; angiogenesis; MVD
25.  Dendritic Cells from Crohn’s Disease Patients Show Aberrant STAT1 and STAT3 Signaling 
PLoS ONE  2013;8(8):e70738.
Abnormalities of dendritic cells (DCs) and STAT proteins have been reported in Crohn’s disease (CD). Studies on JAK/STAT signaling in DCs are, however, lacking in CD. We applied a flowcytometric single-cell-based phosphoepitope assay to evaluate STAT1 and STAT3 pathways in DC subsets from CD patients. In addition, circulating DC counts were determined, together with the activation-related immunophenotype. We found that IL-6- and IFN-α-induced STAT3 phosphorylation and IFN-α-induced STAT1 phosphorylation were impaired in plasmacytoid DCs (pDCs) from CD patients (P = 0.005, P = 0.013, and P = 0.006, respectively). In myeloid DCs (mDCs), IFN-α-induced STAT1 and STAT3 phosphorylation were attenuated (P<0.001 and P = 0.048, respectively), but IL-10-induced STAT3 phosphorylation was enhanced (P = 0.026). IFN-γ-induced STAT1 signaling was intact in both DC subtypes. Elevated plasma IL-6 levels were detected in CD (P = 0.004), which strongly correlated with disease activity (ρ = 0.690, P<0.001) but not with IL-6-induced STAT3 phosphorylation. The numbers of pDCs and BDCA3+ mDCs were decreased, and CD40 expression on CD1c+ mDCs was increased in CD. When elucidating the effect of IL-6 signaling on pDC function, we observed that IL-6 treatment of healthy donor pDCs affected the maturation of and modified the T-cell priming by pDCs, favoring Th2 over Th1 type of response and the expression of IL-10 in T cells. Our results implicate DC signaling in human CD. Reduced IL-6 responsiveness in pDCs, together with the attenuated IFN-α-induced signaling in both DC subtypes, may contribute to the immunological dysregulation in CD patients.
doi:10.1371/journal.pone.0070738
PMCID: PMC3737363  PMID: 23950992

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