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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.  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
3.  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
4.  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
5.  Ectopic Lymphoid Structures Support Ongoing Production of Class-Switched Autoantibodies in Rheumatoid Synovium 
PLoS Medicine  2009;6(1):e1.
Background
Follicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium.
Methods and Findings
Using immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Iγ-Cμ circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis.
Conclusions
Our demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell–depleting therapies.
Costantino Pitzalis and colleagues show that lymphoid structures in synovial tissue of patients with rheumatoid arthritis support production of anti-citrullinated peptide antibodies, which continues following transplantation into SCID mice.
Editors' Summary
Background.
More than 1 million people in the United States have rheumatoid arthritis, an “autoimmune” condition that affects the joints. Normally, the immune system provides protection against infection by responding to foreign antigens (molecules that are unique to invading organisms) while ignoring self-antigens present in the body's own tissues. In autoimmune diseases, this ability to discriminate between self and non-self fails for unknown reasons and the immune system begins to attack human tissues. In rheumatoid arthritis, the lining of the joints (the synovium) is attacked, it becomes inflamed and thickened, and chemicals are released that damage all the tissues in the joint. Eventually, the joint may become so scarred that movement is no longer possible. Rheumatoid arthritis usually starts in the small joints in the hands and feet, but larger joints and other tissues (including the heart and blood vessels) can be affected. Its symptoms, which tend to fluctuate, include early morning joint pain, swelling, and stiffness, and feeling generally unwell. Although the disease is not always easy to diagnose, the immune systems of many people with rheumatoid arthritis make “anti-citrullinated protein/peptide antibodies” (ACPA). These “autoantibodies” (which some experts believe can contribute to the joint damage in rheumatoid arthritis) recognize self-proteins that contain the unusual amino acid citrulline, and their detection on blood tests can help make the diagnosis. Although there is no cure for rheumatoid arthritis, the recently developed biologic drugs, often used together with the more traditional disease-modifying therapies, are able to halt its progression by specifically blocking the chemicals that cause joint damage. Painkillers and nonsteroidal anti-inflammatory drugs can reduce its symptoms, and badly damaged joints can sometimes be surgically replaced.
Why Was This Study Done?
Before scientists can develop a cure for rheumatoid arthritis, they need to know how and why autoantibodies are made that attack the joints in this common and disabling disease. B cells, the immune system cells that make antibodies, mature in structures known as “germinal centers” in the spleen and lymph nodes. In the germinal centers, immature B cells are exposed to antigens and undergo two genetic processes called “somatic hypermutation” and “class-switch recombination” that ensure that each B cell makes an antibody that sticks as tightly as possible to just one antigen. The B cells then multiply and enter the bloodstream where they help to deal with infections. Interestingly, the inflamed synovium of many patients with rheumatoid arthritis contains structures that resemble germinal centers. Could these ectopic (misplaced) lymphoid structures, which are characterized by networks of immune system cells called follicular dendritic cells (FDCs), promote autoimmunity and long-term inflammation by driving the production of autoantibodies within the joint itself? In this study, the researchers investigate this possibility.
What Did the Researchers Do and Find?
The researchers collected synovial tissue from 55 patients with rheumatoid arthritis and used two approaches, called immunohistochemistry and real-time PCR, to investigate whether FDC-containing structures in synovium expressed an enzyme called activation-induced cytidine deaminase (AID), which is needed for both somatic hypermutation and class-switch recombination. All the FDC-containing structures that the researchers found in their samples expressed AID. Furthermore, these AID-containing structures were surrounded by mature B cells making ACPAs. To test whether these B cells were derived from AID-expressing cells resident in the synovium rather than ACPA-expressing immune system cells coming into the synovium from elsewhere in the body, the researchers transplanted synovium from patients with rheumatoid arthritis under the skin of a special sort of mouse that largely lacks its own immune system. Four weeks later, the researchers found that the transplanted human lymphoid tissue was still making AID, that the level of AID expression correlated with the amount of human ACPA in the blood of the mice, and that the B cells in the transplant were proliferating.
What Do These Findings Mean?
These findings show that the ectopic lymphoid structures present in the synovium of some patients with rheumatoid arthritis are functional and are able to make ACPA. Because ACPA may be responsible for joint damage, the survival of these structures could, therefore, be involved in the development and progression of rheumatoid arthritis. More experiments are needed to confirm this idea, but these findings may explain why drugs that effectively clear B cells from the bloodstream do not always produce a marked clinical improvement in rheumatoid arthritis. Finally, they suggest that AID might provide a new target for the development of drugs to treat rheumatoid arthritis.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0060001.
This study is further discussed in a PLoS Medicine Perspective by Rene Toes and Tom Huizinga
The MedlinePlus Encyclopedia has a page on rheumatoid arthritis (in English and Spanish). MedlinePlus provides links to other information on rheumatoid arthritis (in English and Spanish)
The UK National Health Service Choices information service has detailed information on rheumatoid arthritis
The US National Institute of Arthritis and Musculoskeletal and Skin Diseases provides Fast Facts, an easy to read publication for the public, and a more detailed Handbook on rheumatoid arthritis
The US Centers for Disease Control and Prevention has an overview on rheumatoid arthritis that includes statistics about this disease and its impact on daily life
doi:10.1371/journal.pmed.0060001
PMCID: PMC2621263  PMID: 19143467
6.  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
7.  MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia  
PLoS Medicine  2006;3(7):e270.
Background
The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR).
Methods and Findings
DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9–4.0 × 10 12/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis.
Conclusions
Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD.
Editors' Summary
Background.
Myelofibrosis with myeloid metaplasia (MF) is one of a group of chronic blood disorders, known as chronic myeloproliferative disorders. These disorders sometimes turn into acute leukemia. The main abnormality in myelofibrosis is for the bone marrow to become filled with fibrous (scar) tissue (hence the name myelofibrosis), which stops it from producing normal blood cells efficiently. In addition, the white blood cells that remain are abnormal (that is, metaplastic). The clinical effect of these abnormalities are that patients are anemic (they have low numbers of red cells), are more likely to get infections because of the abnormal white cells which cannot fight infections normally, and may bleed more easily because of a lack of the platelets that help the blood to clot. Scientists who study this disorder believe that the disease starts from just one abnormal cell, which divides to replace all the other cells—that is, all the abnormal cells are part of one clone.
Why Was This Study Done?
In two similar diseases, polycythemia vera (in which the bone marrow produces too many red blood cells) and essential thrombocytosis (in which the bone marrow produces too many platelets), and in some patients with MF, scientists have found genetic changes which seem to trigger these diseases. However, there are some patients with MF in which no abnormal gene has been found. The scientists here wanted to look at other genes to see if they could find any changes that might trigger MF.
What Did the Researchers Do and Find?
They decoded the DNA sequence of three genes that are known to be involved in how blood cells develop for 45 patients with MF. They looked at DNA from white blood cells, and also from normal cheek cells for comparison. They found that in four of the 45 patients the DNA in the bone marrow, but not the cheek, carried a mutation in a gene for the thrombopoietin receptor (also called MPL). This gene is necessary for the cells that make platelets to grow correctly. The mutation was not present in any samples from patients with diseases related to MF, nor in 270 normal samples. The mutation that was identified was at position 515 in the MPL gene sequence, hence the name MPLW515L—the W and the L are the shorthand way of indicating exactly which change occurred. The change meant that the gene became abnormally active. The researchers tested the effect of the abnormal gene by putting it into cells grown in culture in the laboratory; they found that it made the cells grow more than was normal. In addition, when cells with the abnormal gene were put into mice, the mice developed a blood disorder similar to that seen in humans with MF.
What Do These Findings Mean?
It seems likely that the genetic change that has been identified here is responsible for the MF that develops in some patients. The MPL gene is known to be part of a pathway of genes that control how certain blood cells develop. However, it is not yet clear exactly how the genetic change found here causes the blood cells to grow abnormally, or how it causes the other clinical effects of MF. Further work will also need to be done to see if it is possible to develop drugs that can act on this gene mutation, or on the other genes that it affects so as to return the cells to normal.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030270.
• MedlinePlus, a Web site of the US National Library of Health, has pages of information on myelofibrosis and related diseases
• The National Cancer Institute, which funds research into many cancers, has information for patients on myelofibrosis, including information on clinical trials
• The MPD Foundation has information for patients with myelofibrosis and related diseases
Activation of JAK-STAT signaling via a mutation - MPLW515L- in the thrombopoietin receptor seems to have a role in the pathogenesis of some patients with myelofibrosis.
doi:10.1371/journal.pmed.0030270
PMCID: PMC1502153  PMID: 16834459
8.  Local and Systemic Alterations in Signal Transducers and Activators of Transcription (STAT) Associated with Human Abdominal Aortic Aneurysms 
The Journal of Surgical Research  2011;176(1):321-328.
Introduction
Signal transducers and activators of transcription (STAT) proteins are transcription factors that when activated, by phosphorylation, regulate gene expression and cellular activity. The aim of this study was to evaluate the local and systemic expression and activation of STAT proteins associated with abdominal aortic aneurysms (AAA).
Methods
Expression and activation of STAT proteins were assessed in aortic wall samples obtained from patients undergoing repair of AAA (N=9) and from non-aneurysmal (NA) donors (N=17). Aortic samples were evaluated for mRNA and protein expression for STAT1, 2, 3, 4, 5a, and 5b using RT-PCR and immunoblot (WB) assays and normalized to β-actin (expressed as arbitrary units). STAT activation was assessed with WB assays using phosphorylated (p)-STAT-specific antibodies. Alterations in STAT activation were calculated by normalizing p-STAT proteins to corresponding total STAT levels. Immunohistochemistry was performed on AAA and NA samples using the total and pSTAT antibodies.Systemic alterations in STAT activation were assessed by evaluating circulating leukocytes for the presence of p-STAT from patients with AAA (AAA, N=8), repaired aneurysm (RA, N=8), or age/gender matched controls with no AAA (CT, N=8). Flow cytometry was performed to assess for circulating levels of STAT1 (pY701), STAT3 (pY705), and STAT5a (pY694) in monocytes, granulocytes, and lymphocytes. Assessments were made at baseline and in response to in vitro stimulation with IFN-gamma (50 ng/mL) or IL-6 (100 ng/mL). Results were analyzed using Student’s T-test and are expressed as mean±SEM.
Results
In AAA tissue compared to NA, STAT-1 (1.08±0.09 v. 0.62±0.07), -2 (0.98±0.07 v. 0.55±0.08), and -4 (0.89±0.12 v. 0.35±0.11) mRNA levels were elevated (P<0.01, all). Corresponding increases in STAT protein were only observed for STAT1 (2.77±0.93 v. 0.93±0.08, P<0.05). Increases in activation were observed in AAA compared to NA in p-STAT2 (0.77±0.1 v. 0.1±0.02, P<0.01), p-STAT3 (1.6±0.3 v. 0.2±0.06, P<0.02) and p-STAT5 (0.57±0.03 v. 0.2±0.03, P<0.05) levels. Phosphorylated STAT1, 2, 3, and 5 were observed in inflammatory cells invading the AAA adventitia. In addition, STAT3 was observed in the media of AAA and NA, but pSTAT3 was only observed in the media of AAA. There were no differences in baseline levels of p-STAT-positive circulating leukocytes. IFN-gamma stimulation decreased STAT-5a (pY694)-positive CT lymphocytes to 40±13% of baseline, but had no effect on AAA or RA lymphocytes (116±35%, 102±19%, respectively; P=0.01). STAT-5a (pY694)-positive CT granulocytes also decreased to 62±18% of baseline compared to AAA or RA granulocytes (122±25%, 126±17%, respectively; P=0.01). Alterations in STAT1 (pY701) and STAT3 (pY705) were not observed in leukocytes following cytokine stimulation.
Conclusions
STAT proteins are important regulators of transcriptional activity and have been linked to cardiovascular disease. The present data suggest that altered levels of phosphorylated STATs are associated with AAA. Understanding their role may provide further insight into the mechanisms of AAA formation and allow for the development of medical treatment options.
doi:10.1016/j.jss.2011.05.041
PMCID: PMC3197955  PMID: 21764069
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.  IL-4 AND IL-13 EMPLOY DISCRETE SIGNALING PATHWAYS FOR TARGET GENE EXPRESSION IN ALTERNATIVELY ACTIVATED MONOCYTES/MACROPHAGES 
Monocytes/macrophages are innate immune cells that play a crucial role in the resolution of inflammation. In presence of Th2 cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13), they display an anti-inflammatory profile and this activation pathway is known as alternative activation. In this study we compare and differentiate pathways mediated by IL-4 and IL-13 activation of human monocytes/macrophage. Here we report differential regulation of IL-4 and IL-13 signaling in monocytes/macrophages starting from IL-4/IL-13 cytokine receptors to Jak-Stat-mediated signaling pathways that ultimately control expression of several infl1ammatory genes. Our data demonstrate that while the receptor-associated tyrosine kinases Jak2 and Tyk2 are activated after the recruitment of IL-13 to its receptor (containing IL-4Rα and IL-13Rα1), IL-4 stimulates Jak1 activation. We further show that Jak2 is upstream of Stat3 activation and Tyk2 controls Stat1 and Stat6 activation in response to IL-13 stimulation. In contrast, Jak1 regulates Stat3 and Stat6 activation in IL-4-induced monocytes. Our results further reveal that while IL-13 utilizes both IL-4Rα-Jak2-Stat3 and IL-13Rα1-Tyk2-Stat1/Stat6 signaling pathways, IL-4 can only use the IL-4Rα-Jak1-Stat3/Stat6 cascade to regulate the expression of some critical inflammatory genes including 15-lipoxygenase (15-LO), monoamine oxidase A (MAO-A) and scavenger receptor CD36. Moreover, we demonstrate here that IL-13 and IL-4 can uniquely affect the expression of particular genes like dual specificity phosphatase 1 (DUSP1) and tissue inhibitor of metalloprotease-3 (TIMP3) and do so through different Jak kinaes. As evidence of differential regulation of gene function by IL-4 and IL-13, we further report that MAO-A-mediated reactive oxygen species (ROS) generation is influenced by different Jak kinases. Collectively, these results have major implications for understanding the mechanism and function of alternatively activated monocytes/macrophages by IL-4 and IL-13 and add novel insights into the pathogenesis and potential treatment of different inflammatory diseases.
doi:10.1016/j.freeradbiomed.2012.10.553
PMCID: PMC3534796  PMID: 23124025
Monocytes; cytokines; Inflammation; Gene regulation; ROS; alternative activation
11.  On-target JAK2/STAT3 inhibition slows disease progression in orthotopic xenografts of human glioblastoma brain tumor stem cells 
Neuro-Oncology  2012;15(2):198-207.
Background
Glioblastoma multiforme (GBM) is characterized by an aggressive clinical course, therapeutic resistance, and striking molecular heterogeneity. GBM-derived brain tumor stem cells (BTSCs) closely model this molecular heterogeneity and likely have a key role in tumor recurrence and therapeutic resistance. Emerging evidence indicates that Janus kinase (JAK)2/signal transducer and activator of transcription (STAT)3 is an important mediator of tumor cell survival, growth, and invasion in a large group of GBM. Here, we used a large set of molecularly heterogeneous BTSCs to evaluate the translational potential of JAK2/STAT3 therapeutics.
Methods
BTSCs were cultured from GBM patients and MGMT promoter methylation, and the mutation statuses of EGFR, PTEN, and TP53 were determined. Endogenous JAK2/STAT3 activity was assessed in human GBM tissue, BTSCs, and orthotopic xenografts by immunohistochemistry and Western blotting. STAT3 short hairpin (sh)RNA, cucurbitacin-I, and WP1066 were used to inhibit JAK2/STAT3 activity in vitro and in vivo.
Results
The JAK2/STAT3 pathway was demonstrated to be highly activated in human GBM, molecularly heterogeneous BTSCs derived from these tumors, and BTSC xenografts. STAT3 shRNA knockdown or cucurbitacin-I and WP1066 administration resulted in on-target JAK2/STAT3 inhibition and dramatically reduced BTSC survival regardless of endogenous MGMT promoter methylation or EGFR, PTEN, and TP53 mutational status. BTSC orthotopic xenografts maintained the high levels of activated JAK2/STAT3 seen in their parent human tumors. Intraperitoneal WP1066 reduced intratumoral JAK2/STAT3 activity and prolonged animal survival.
Conclusion
Our study demonstrates the in vitro and in vivo efficacy of on-target JAK2/STAT3 inhibition in heterogeneous BTSC lines that closely emulate the genomic and tumorigenic characteristics of human GBM.
doi:10.1093/neuonc/nos302
PMCID: PMC3548588  PMID: 23262510
JAK2/STAT3; brain tumor stem cells; glioblastoma; molecular therapeutics
12.  Resistance of Cancer Cells to Targeted Therapies Through the Activation of Compensating Signaling Loops 
The emergence of low molecular weight kinase inhibitors as “targeted” drugs has led to remarkable advances in the treatment of cancer patients. The clinical benefits of these tumor therapies, however, vary widely in patient populations and with duration of treatment. Intrinsic and acquired resistance against such drugs limits their efficacy. In addition to the well studied mechanisms of resistance based upon drug transport and metabolism, genetic alterations in drug target structures and the activation of compensatory cell signaling have received recent attention. Adaptive responses can be triggered which counteract the initial dependence of tumor cells upon a particular signaling molecule and allow only a transient inhibition of tumor cell growth. These compensating signaling mechanisms are often based upon the relief of repression of regulatory feedback loops. They might involve cell autonomous, intracellular events or they can be mediated via the secretion of growth factor receptor ligands into the tumor microenvironment and signal induction in an auto- or paracrine fashion. The transcription factors Stat3 and Stat5 mediate the biological functions of cytokines, interleukins and growth factors and can be considered as endpoints of multiple signaling pathways. In normal cells this activation is transient and the Stat molecules return to their non-phosphorylated state within a short time period. In tumor cells the balance between activating and de-activating signals is disturbed resulting in the persistent activation of Stat3 or Stat5. The constant activation of Stat3 induces the expression of target genes, which cause the proliferation and survival of cancer cells, as well as their migration and invasive behavior. Activating components of the Jak-Stat pathway have been recognized as potentially valuable drug targets and important principles of compensatory signaling circuit induction during targeted drug treatment have been discovered in the context of kinase inhibition studies in HNSCC cells [1]. The treatment of HNSCC with a specific inhibitor of c-Src, initially resulted in reduced Stat3 and Stat5 activation and subsequently an arrest of cell proliferation and increased apoptosis. However, the inhibition of c-Src only caused a persistent inhibition of Stat5, whereas the inhibition of Stat3 was only transient. The activation of Stat3 was restored within a short time period in the presence of the c-Src inhibitor. This process is mediated through the suppression of P-Stat5 activity and the decrease in the expression of the Stat5 dependent target gene SOCS2, a negative regulator of Jak2. Jak2 activity is enhanced upon SOCS2 downregulation and causes the reactivation of Stat3. A similar observation has been made upon inhibition of Bmx, bone marrow kinase x-linked, activated in the murine glioma cell lines Tu-2449 and Tu-9648. Its inhibition resulted in a transient decrease of P-Stat3 and the induction of a compensatory Stat3 activation mechanism, possibly through the relief of negative feedback inhibition and Jak2 activation. These observations indicate that the inhibition of a single tyrosine kinase might not be sufficient to induce lasting therapeutic effects in cancer patients. Compensatory kinases and pathways might become activated and maintain the growth and survival of tumor cells. The definition of these escape pathways and their preemptive inhibition will suggest effective new combination therapies for cancer.
doi:10.2174/1574362409666140206221931
PMCID: PMC4095943  PMID: 25045345
Drug resistance; signaling redundancy and compensation; targeted tumor therapy.
13.  Adipose Tissue-Derived Stem Cells Secrete CXCL5 Cytokine with Neurotrophic Effects on Cavernous Nerve Regeneration 
The journal of sexual medicine  2010;8(2):437-446.
Introduction
Previously we reported that paracrine actions likely mediated the therapeutic effects of adipose tissue-derived stem cells (ADSC) on a rat model of cavernous nerve (CN) injury.
Aim
To identify potential neurotrophic factors in ADSC’s secretion, test the most promising one, and identify the molecular mechanism of its neurotrophic action.
Methods
Rat major pelvic ganglia (MPG) were cultured in conditioned media of ADSC and penile smooth muscle cells (PSMC). Cytokine expression in these two media was probed with a cytokine antibody array. CXCL5 cytokine was quantified in these two media by enzyme-linked immunosorbent assay (ELISA). Activation of JAK/STAT by CXCL5 was tested in neuroblastoma cell lines BE(2)C and SH-SY5Y as well as in Schwann cell line RT4-D6P2T by western blot. Involvement of CXCL5 and JAK/STAT in ADSC-conditioned medium’s neurotrophic effects was confirmed with anti-CXCL5 antibody and JAK inhibitor AG490, respectively.
Main Outcome Measures
Neurotrophic effects of ADSC and PSMC-conditioned media were quantified by measuring neurite length in MPG cultures. Secretion of CXCL5 in these two media was quantified by ELISA. Activation of JAK/STAT by CXCL5 was quantified by densitometry on western blots for STAT1 and STAT3 phosphorylation.
Results
MPG neurite length was significantly longer in ADSC than in PSMC-conditioned medium. CXCL5 was secreted 8 times higher in ADSC than in PSMC-conditioned medium. Anti-CXCL5 antibody blocked the neurotrophic effects of ADSC-conditioned medium. CXCL5 activated JAK/STAT concentration-dependently from 0 to 50 ng/ml in RT4-D6P2T Schwann cells. At 50 ng/ml, CXCL5 activated JAK/STAT time-dependently, peaking at 45 min. AG490 blocked these activities as well as the neurotrophic effects of ADSC-conditioned medium.
Conclusions
CXCL5 was secreted by ADSC at a high level, promoted MPG neurite growth, and activated JAK/STAT in Schwann cells. CXCL5 may contribute to ADSC’s therapeutic efficacy on CN injury-induced ED.
doi:10.1111/j.1743-6109.2010.02128.x
PMCID: PMC3176296  PMID: 21114767
Adipose tissue-derived stem cells; CXCL5 cytokine; cavernous nerve regeneration; erectile dysfunction; JAK/STAT
14.  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
15.  Marburg Virus Evades Interferon Responses by a Mechanism Distinct from Ebola Virus 
PLoS Pathogens  2010;6(1):e1000721.
Previous studies have demonstrated that Marburg viruses (MARV) and Ebola viruses (EBOV) inhibit interferon (IFN)-α/β signaling but utilize different mechanisms. EBOV inhibits IFN signaling via its VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. In contrast, MARV infection inhibits IFNα/β induced tyrosine phosphorylation of STAT1 and STAT2. MARV infection is now demonstrated to inhibit not only IFNα/β but also IFNγ-induced STAT phosphorylation and to inhibit the IFNα/β and IFNγ-induced tyrosine phosphorylation of upstream Janus (Jak) family kinases. Surprisingly, the MARV matrix protein VP40, not the MARV VP24 protein, has been identified to antagonize Jak and STAT tyrosine phosphorylation, to inhibit IFNα/β or IFNγ-induced gene expression and to inhibit the induction of an antiviral state by IFNα/β. Global loss of STAT and Jak tyrosine phosphorylation in response to both IFNα/β and IFNγ is reminiscent of the phenotype seen in Jak1-null cells. Consistent with this model, MARV infection and MARV VP40 expression also inhibit the Jak1-dependent, IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. Finally, expression of MARV VP40 is able to prevent the tyrosine phosphorylation of Jak1, STAT1, STAT2 or STAT3 which occurs following over-expression of the Jak1 kinase. In contrast, MARV VP40 does not detectably inhibit the tyrosine phosphorylation of STAT2 or Tyk2 when Tyk2 is over-expressed. Mutation of the VP40 late domain, essential for efficient VP40 budding, has no detectable impact on inhibition of IFN signaling. This study shows that MARV inhibits IFN signaling by a mechanism different from that employed by the related EBOV. It identifies a novel function for the MARV VP40 protein and suggests that MARV may globally inhibit Jak1-dependent cytokine signaling.
Author Summary
The closely related members of the filovirus family, Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic disease in humans with high fatality rates. Infected individuals exhibit dysregulated immune responses which appear to result from several factors, including virus-mediated impairment of innate immune responses. Previous studies demonstrated that both MARV and EBOV block the type I interferon-induced Jak-STAT signaling pathway. For EBOV, the viral protein VP24 mediates the inhibitory effects by interfering with the nuclear translocation of activated STAT proteins. Here, we show that MARV uses a distinct mechanism to block IFN signaling pathways. Our data revealed that MARV blocks the phosphorylation of Janus kinases and their target STAT proteins in response to type I and type II interferon and interleukin 6. Surprisingly, the observed inhibition is not achieved by the MARV VP24 protein, but by the matrix protein VP40 which also mediates viral budding. Over-expression studies indicate that MARV VP40 globally antagonizes Jak1-dependent signaling. Further, we show that a MARV VP40 mutant defective for budding retains interferon antagonist function. Our results highlight a basic difference between EBOV and MARV, define a new function for MARV VP40 and reveal new targets for the development of anti-MARV therapies.
doi:10.1371/journal.ppat.1000721
PMCID: PMC2799553  PMID: 20084112
16.  Benzoxathiol derivative BOT-4-one suppresses L540 lymphoma cell survival and proliferation via inhibition of JAK3/STAT3 signaling 
Experimental & Molecular Medicine  2011;43(5):313-321.
Persistently activated JAK/STAT3 signaling pathway plays a pivotal role in various human cancers including major carcinomas and hematologic tumors, and is implicated in cancer cell survival and proliferation. Therefore, inhibition of JAK/STAT3 signaling may be a clinical application in cancer therapy. Here, we report that 2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo [1,3]oxathiol-4-one (BOT-4-one), a small molecule inhibitor of JAK/STAT3 signaling, induces apoptosis through inhibition of STAT3 activation. BOT-4-one suppressed cytokine (upd)-induced tyrosine phosphorylation and transcriptional activity of STAT92E, the sole Drosophila STAT homolog. Consequently, BOT-4-one significantly inhibited STAT3 tyrosine phosphorylation and expression of STAT3 downstream target gene SOCS3 in various human cancer cell lines, and its effect was more potent in JAK3-activated Hodgkin's lymphoma cell line than in JAK2-activated breast cancer and prostate cancer cell lines. In addition, BOT-4-one-treated Hodgkin's lymphoma cells showed decreased cell survival and proliferation by inducing apoptosis through down-regulation of STAT3 downstream target anti-apoptotic gene expression. These results suggest that BOT-4-one is a novel small molecule inhibitor of JAK3/STAT3 signaling and may have therapeutic potential in the treatment of human cancers harboring aberrant JAK3/STAT3 signaling, specifically Hodgkin's lymphoma.
doi:10.3858/emm.2011.43.5.035
PMCID: PMC3104254  PMID: 21499010
BOT-4-one; cancer; JAK; small molecule inhibitor, apoptosis; STAT3
17.  Therapeutic Potential of AZD1480 for the Treatment of Human Glioblastoma 
Molecular cancer therapeutics  2011;10(12):2384-2393.
Aberrant activation of the Janus Kinase (JAK)/Signal Activator of Transcription (STAT) pathway has been implicated in glioblastoma (GBM) progression. To develop a therapeutic strategy to inhibit STAT-3 signaling, we have evaluated the effects of AZD1480, a pharmacological inhibitor of JAK1 and JAK2. In this study, the in vitro efficacy of AZD1480 was tested in human and murine glioma cell lines. AZD1480 treatment effectively blocks constitutive and stimulus-induced JAK1, JAK2 and STAT-3 phosphorylation in both human and murine glioma cells, and leads to a decrease in cell proliferation and induction of apoptosis. Furthermore, we utilized human xenograft GBM samples as models for the study of JAK/STAT-3 signaling in vivo, since human GBM samples propagated as xenografts in nude mice retain both the hallmark genetic alterations and the invasive phenotype seen in vivo. In these xenograft tumors, JAK2 and STAT-3 are constitutively active, but levels vary among tumors, which is consistent with the heterogeneity of GBMs. AZD1480 inhibits constitutive and stimulus-induced phosphorylation of JAK2 and STAT-3 in these GBM xenograft tumors in vitro, downstream gene expression and inhibits cell proliferation. Furthermore, AZD1480 suppresses STAT-3 activation in the glioma-initiating cell population in GBM tumors. In vivo, AZD1480 inhibits the growth of subcutaneous tumors and increases survival of mice bearing intracranial GBM tumors by inhibiting STAT-3 activity, indicating that pharmacological inhibition of the JAK/STAT-3 pathway by AZD1480 should be considered for study in the treatment of patients with GBM tumors.
doi:10.1158/1535-7163.MCT-11-0480
PMCID: PMC3237864  PMID: 22027691
AZD1480; glioma; STAT-3; JAK1; JAK2
18.  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
19.  Adenosine Blocks IFN-γ-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation1 
Macrophages are activated by IFN-γ, a proinflammatory and proatherogenic cytokine that mediates its downstream effects primarily through STAT1. IFN-γ signaling induces phosphorylation of two STAT1 residues: Tyr701 (Y701), which facilitates dimerization, nuclear translocation, and DNA binding; and Ser727 (S727), which enables maximal STAT1 transcription activity. Immunosuppressive molecules such as adenosine in the cellular microenvironment can reduce macrophage inflammatory and atherogenic functions through receptor-mediated signaling pathways. We hypothesized that adenosine achieves these protective effects by interrupting IFN-γ signaling in activated macrophages. This investigation demonstrates that adding adenosine to IFN-γ-stimulated murine RAW 264.7 and human THP-1 macrophages results in unique modulation of STAT1 serine and tyrosine phosphorylation events. We show that adenosine inhibits IFN-γ-induced STAT1 S727 phosphorylation by >30% and phosphoserine-mediated transcriptional activity by 58% but has no effect on phosphorylation of Y701 or receptor-associated JAK tyrosine kinases. Inhibition of the adenosine A3 receptor with a subtype-specific antagonist (MRS 1191 in RAW 264.7 cells and MRS 1220 in THP-1 cells) reverses this adenosine suppressive effect on STAT1 phosphoserine status by 25–50%. Further, RAW 264.7 A3 receptor stimulation with Cl-IB-MECA reduces IFN-γ-induced STAT1 transcriptional activity by 45% and STAT1-dependent gene expression by up to 80%. These data suggest that A3 receptor signaling is key to adenosine-mediated STAT1 modulation and anti-inflammatory action in IFN-γ-activated mouse and human macrophages. Because STAT1 plays a key role in IFN-γ-induced inflammation and foam cell transformation, a better understanding of the mechanisms underlying STAT1 deactivation by adenosine may improve preventative and therapeutic approaches to vascular disease.
doi:10.4049/jimmunol.0900331
PMCID: PMC2916018  PMID: 19846878
20.  Division of labor by dual feedback regulators controls JAK2/STAT5 signaling over broad ligand range 
Quantitative analysis of time-resolved data in primary erythroid progenitor cells reveals that a dual negative transcriptional feedback mechanism underlies the ability of STAT5 to respond to the broad spectrum of physiologically relevant Epo concentrations.
A mathematical dual feedback model of the Epo-induced JAK2/STAT5 signaling pathway was calibrated with extensive time-resolved quantitative data sets from immunoblotting, mass spectrometry and qRT–PCR experiments in primary erythroid progenitor cells.We show that the amount of nuclear phosphorylated STAT5 integrated for 60 min post Epo stimulation directly correlates with the fraction of surviving cells 24 h later.CIS and SOCS3 were identified as the most relevant transcriptional feedback regulators of JAK2/STAT5 signaling in primary erythroid progenitor cells. Applying the model, we revealed that CIS-mediated inhibitory effects are most important at low ligand concentrations, whereas SOCS3 inhibition is more effective at high ligand doses.The distinct modes of inhibition of CIS and SOCS3 at various Epo concentrations provide a strategy for achieving control of JAK2/STAT5 signaling over the entire range of physiological Epo concentrations.
Cells interpret information encoded by extracellular stimuli through the activation of intracellular signaling networks and translate this information into cellular decisions. A prime example for a system that is exposed to extremely variable ligand concentrations is the erythroid lineage. The key regulator Erythropoietin (Epo) facilitates continuous renewal of erythrocytes at low basal levels but also secures compensation in case of, e.g., blood loss through an up to 1000-fold increase in hormone concentration. The Epo receptor (EpoR) is expressed on erythroid progenitor cells at the colony forming unit erythroid (CFU-E) stage. Stimulation of these cells with Epo leads to rapid but transient activation of receptor and JAK2 phosphorylation followed by phosphorylation of the latent transcription factor STAT5. Although STAT5 is known to be an essential regulator of survival and differentiation of erythroid progenitor cells, a quantitative link between the dynamic properties of STAT5 signaling and survival decisions remained unknown. STAT5-mediated responses in CFU-E cells are modulated by multiple attenuation mechanisms that operate on different time scales. Fast-acting mechanisms such as depletion of Epo by rapid receptor turnover and recruitment of the phosphatase SHP-1 control the initial signal amplitude at the receptor level. Transcriptional feedback regulators such as suppressor of cytokine signaling (SOCS) family members CIS and SOCS3 operate at a slower time scale. Despite the ample knowledge of the individual components involved, only little is known about the specific contributions of these regulators in controlling dynamic properties of STAT5 in response to a broad range of input signals. Therefore, dynamic pathway modeling is required to understand the complex regulatory network of feedback regulators.
To address these questions, we established a dual negative feedback model of JAK2/STAT5 signaling in primary erythroid progenitor cells isolated from mouse fetal livers. We provide a large data set of JAK2/STAT5 signaling dynamics employing quantitative immunoblotting, mass spectrometry and quantitative RT–PCR measured under different perturbation conditions to calibrate our model (Figure 3). The structure of our model was constructed to comprise the minimal number of parameters necessary to explain the data. Thereby, we aimed at a model with fully identifiable parameters that are essential to obtain high predictive power. Parameter identifiability was analyzed by the profile likelihood approach. Applying this method, we could establish a dual negative feedback model of JAK2-STAT5 signaling with structurally and in most cases practically identifiable parameters.
A major bottle-neck in combining signal transduction events with cellular phenotypes is the discrepancy in the time scale and stimuli concentrations that are applied in the different experiments. The sensitivity of biochemical assays to determine phosphorylation events within minutes or hours after stimulation is usually lower than the threshold of sensitivity in assays to determine the physiological response after one or more days. Facilitated by the model, we were able to compute the integrated response of JAK2/STAT5 signaling components for experimentally unaddressable Epo concentrations. Our results demonstrate that the integrated response of pSTAT5 in the nucleus accurately correlates with the experimentally determined survival of CFU-E cells. This provides a quantitative link of the dependency of primary CFU-E cells on pSTAT5 activation dynamics. By correlation analysis, we could identify the early signaling phase (⩽1 h) of STAT5 to be the most predictive for the fraction of surviving cells, which was determined ∼24 h later. Thus, we hypothesize that as a general principle in apoptotic decisions, ligand concentrations translated into kinetic-encoded information of early signaling events downstream of receptors can be predictive for survival decisions 24 h later.
After the first hour of stimulation, it is important to constrain signaling to a residual steady-state level. Constitutive phosphorylation of the JAK2/STAT5 pathway has a crucial role in the onset of polycythemia vera (PV), a disease associated with Epo-independent erythroid differentiation. The two identified transcriptional feedback proteins, CIS and SOCS3, are responsible for adjusting the phosphorylation level of STAT5 after 1 h of stimulation. Since the Epo input signal can vary over a broad range of ligand concentrations, we asked how CIS and SOCS3 can facilitate control of STAT5 long-term phosphorylation levels over the entire physiological relevant hormone concentrations. By using model simulations, we revealed that the two feedbacks are most effective at different Epo concentration ranges. Predicted by our mathematical model, the major role of CIS in modulating STAT5 phosphorylation levels is at low, basal Epo concentrations, whereas SOCS3 is essential to control the STAT5 phosphorylation levels at high Epo doses (Figure 6). As a potential molecular mechanism of this dose-dependent inhibitory effect, we could identify the quantity of pJAK2 relative to pEpoR that increases with higher Epo concentrations. Since SOCS3 can inhibit JAK2 directly via its KIR domain to attenuate downstream STAT5 activation, SOCS3 becomes more effective with the relative increase of JAK2 activation. Hence, CIS and SOCS3 act in a concerted manner to ensure tight regulation of STAT5 responses over the broad physiological range of Epo concentrations.
In summary, our mathematical approach provided new insights into the specific function of feedback regulation in STAT5-mediated life or death decisions of primary erythroid cells. We dissected the roles of the transcriptionally induced proteins CIS and SOCS3 that operate as dual feedback with divided function thereby facilitating the control of STAT5 activation levels over the entire range of physiological Epo concentrations. The detailed understanding of the molecular processes and control distribution of Epo-induced JAK/STAT signaling can be further applied to gain insights into alterations promoting malignant hematopoietic diseases.
Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. The specific contributions of individual feedback regulators, however, remain unclear. Based on extensive time-resolved data sets in primary erythroid progenitor cells, we established a dynamic pathway model to dissect the roles of the two transcriptional negative feedback regulators of the suppressor of cytokine signaling (SOCS) family, CIS and SOCS3, in JAK2/STAT5 signaling. Facilitated by the model, we calculated the STAT5 response for experimentally unobservable Epo concentrations and provide a quantitative link between cell survival and the integrated response of STAT5 in the nucleus. Model predictions show that the two feedbacks CIS and SOCS3 are most effective at different ligand concentration ranges due to their distinct inhibitory mechanisms. This divided function of dual feedback regulation enables control of STAT5 responses for Epo concentrations that can vary 1000-fold in vivo. Our modeling approach reveals dose-dependent feedback control as key property to regulate STAT5-mediated survival decisions over a broad range of ligand concentrations.
doi:10.1038/msb.2011.50
PMCID: PMC3159971  PMID: 21772264
apoptosis; erythropoietin; mathematical modeling; negative feedback; SOCS
21.  Signal pathways in astrocytes activated by cross-talk between of astrocytes and mast cells through CD40-CD40L 
Background
Astrocytes, which play an active role in chronic inflammatory diseases like multiple sclerosis, exist close to mast cells with which they share perivascular localization. We previously demonstrated the possibility that astrocytes and mast cells interact in vitro and in vivo. This study aimed to investigate the signaling pathways and the role for astrocytes in the interaction of astrocytes and mast cells.
Methods
We co-cultured human U87 glioblastoma (U87) and human mast cell-1 (HMC-1) cell lines, and mouse cerebral cortices-derived astrocytes and mouse bone marrow-derived mast cells (BMMCs). Intracellular Ca2+ ([Ca2+]i) was measured by confocal microscopy; CD40 siRNA by Silencer Express Kit; small GTPases by GTP-pull down assay; PKCs, MAPKs, CD40, CD40L, Jak1/2, STAT1, TNF receptor 1 (TNFR1) by Western blot; NF-κB and AP-1 by EMSA; cytokines by RT-PCR. An experimental allergic encephalomyelitis (EAE) model was induced using myelin oligodendrocyte glycoprotein (MOG) peptide and pertussis toxin in mice. Co-localization of TNFR1 and astrocytes in EAE brain tissues was determined by immunohistochemistry.
Results
Each astrocyte co-culture had increases in [Ca2+]i levels, release of cytokines and chemokines; activities of Rho-family GTPases, NF-κB/AP-1/STAT1727, and Jack1/2, STAT1701. These effects were inhibited by anti-CD40 antibody or CD40 siRNA, and signaling pathways for Jak1/2 were inhibited by anti-TNFR1 antibody. EAE score, expression of TNFR1, and co-localization of TNFR1 and astrocytes were enhanced in brain of the EAE model. Anti-CD40 antibody or 8-oxo-dG pretreatment reduced these effects in EAE model.
Conclusions
These data suggest that astrocytes activated by the CD40-CD40L interaction in co-culture induce inflammatory cytokine production via small GTPases, and the secreted cytokines re-activate astrocytes via Jak/STAT1701 pathways, and then release more cytokines that contribute to exacerbating the development of EAE. These findings imply that the pro-inflammatory mediators produced by cell-to-cell cross-talk via interaction of CD40-CD40L may be as a promising therapeutic target for neurodegenerative diseases like MS.
doi:10.1186/1742-2094-8-25
PMCID: PMC3068960  PMID: 21410936
22.  NSC114792, a novel small molecule identified through structure-based computational database screening, selectively inhibits JAK3 
Molecular Cancer  2010;9:36.
Background
Human or animals lacking either JAK3 or the common gamma chain (γc) expression display severe combined immunodeficiency disease, indicating the crucial role of JAK3 in T-cell development and the homeostasis of the immune system. JAK3 has also been suggested to contribute to the pathogenesis of tumorigenesis. Recent studies identified activating JAK3 mutations in patients with various hematopoietic malignancies, including acute megakaryoblastic leukemia. Importantly, functional analyses of some of those JAK3 mutations have been shown to cause lethal hematopoietic malignancies in animal models. These observations make JAK3 an ideal therapeutic target for the treatment of various human diseases. To identify novel small molecule inhibitors of JAK3, we performed structure-based virtual screen using the 3D structure of JAK3 kinase domain and the NCI diversity set of compounds.
Results
We identified NSC114792 as a lead compound. This compound directly blocked the catalytic activity of JAK3 but not that of other JAK family members in vitro. In addition, treatment of 32D/IL-2Rβ cells with the compound led to a block in IL-2-dependent activation of JAK3/STAT5 but not IL-3-dependent activation of JAK2/STAT5. Consistent with the specificity of NSC114792 for JAK3, it selectively inhibited persistently-activated JAK3, but failed to affect the activity of other JAK family members and other oncogenic kinases in various cancer cell lines. Finally, we showed that NSC114792 decreases cell viability by inducing apoptosis through down-regulating anti-apoptotic gene expression only in cancer cells harboring persistently-active JAK3.
Conclusions
NSC114792 is a lead compound that selectively inhibits JAK3 activity. Therefore, our study suggests that this small molecule inhibitor of JAK3 can be used as a starting point to develop a new class of drugs targeting JAK3 activity, and may have therapeutic potential in various diseases that are caused by aberrant JAK3 activity.
doi:10.1186/1476-4598-9-36
PMCID: PMC2830973  PMID: 20149240
23.  The JAK-STAT Transcriptional Regulator, STAT-5, Activates the ATM DNA Damage Pathway to Induce HPV 31 Genome Amplification upon Epithelial Differentiation 
PLoS Pathogens  2013;9(4):e1003295.
High-risk human papillomavirus (HPV) must evade innate immune surveillance to establish persistent infections and to amplify viral genomes upon differentiation. Members of the JAK-STAT family are important regulators of the innate immune response and HPV proteins downregulate expression of STAT-1 to allow for stable maintenance of viral episomes. STAT-5 is another member of this pathway that modulates the inflammatory response and plays an important role in controlling cell cycle progression in response to cytokines and growth factors. Our studies show that HPV E7 activates STAT-5 phosphorylation without altering total protein levels. Inhibition of STAT-5 phosphorylation by the drug pimozide abolishes viral genome amplification and late gene expression in differentiating keratinocytes. In contrast, treatment of undifferentiated cells that stably maintain episomes has no effect on viral replication. Knockdown studies show that the STAT-5β isoform is mainly responsible for this activity and that this is mediated through the ATM DNA damage response. A downstream target of STAT-5, the peroxisome proliferator-activated receptor γ (PPARγ) contributes to the effects on members of the ATM pathway. Overall, these findings identify an important new regulatory mechanism by which the innate immune regulator, STAT-5, promotes HPV viral replication through activation of the ATM DNA damage response.
Author Summary
Over 120 types of human papillomavirus (HPV) have been identified, and approximately one-third of these infect epithelial cells of the genital mucosa. A subset of HPV types are the causative agents of cervical and other anogenital cancers. The infectious life cycle of HPV is dependent on differentiation of the host epithelial cell, with viral genome amplification and virion production restricted to differentiated suprabasal cells. While normal keratinocytes exit the cell cycle upon differentiation, HPV-positive suprabasal cells are able to re-enter S-phase to mediate productive replication. HPV induces an ATM-dependent DNA damage response in differentiating cells that is essential for viral genome amplification. Our studies describe an important mechanism by which human papillomaviruses activate a member of the JAK/STAT innate immune signaling pathway to induce the ATM DNA damage pathway. This is necessary for differentiation-dependent productive viral replication. HPVs must suppress the transcription of one member of the JAK/STAT pathway, STAT-1, while at the same time activating STAT-5 to regulate genome amplification in suprabasal cells. The E7 protein activates STAT-5 leading to induction of ATM phosphorylation through the PPARγ pathway. Our study identifies important links between innate immune signaling, the ATM DNA damage pathway and productive HPV replication that may lead to the characterization of new targets for the development of therapeutics to treat HPV-induced infections.
doi:10.1371/journal.ppat.1003295
PMCID: PMC3616964  PMID: 23593005
24.  Brevilin A, a Novel Natural Product, Inhibits Janus Kinase Activity and Blocks STAT3 Signaling in Cancer Cells 
PLoS ONE  2013;8(5):e63697.
Signal abnormalities in human cells usually cause unexpected consequences for individual health. We focus on these kinds of events involved in JAK-STAT signal pathways, especially the ones triggered by aberrant activated STAT3, an oncoprotein which participates in essential processes of cell survival, growth and proliferation in many types of tumors, as well as immune diseases. By establishing a STAT3 signal based high-throughput drug screening system in human lung cancer A549 cells, we have screened a library from natural products which contained purified compounds from medicinal herbs. One compound, named Brevilin A, exhibited both strong STAT3 signal inhibition and STAT3 signal dependent cell growth inhibition. Further investigations revealed that Brevilin A not only inhibits STAT3 signaling but also STAT1 signaling for cytokines induced phosphorylation of STAT3 and STAT1 as well as the expression of their target genes. In addition, we found Brevilin A could attenuate the JAKs activity by blocking the JAKs tyrosine kinase domain JH1. The levels of cytokine induced phosphorylation of STATs and other substrates were dramatically reduced by treatment of Brevilin A. The roles of Brevilin A targeting on JAKs activity indicate that Brevilin A may not only be used as a STAT3 inhibitor but also a compound blocking other JAK-STAT hyperactivation. Thus, these findings provided a strong impetus for the development of selective JAK-STAT inhibitors and therapeutic drugs in order to improve survival of patients with hyperactivated JAKs and STATs.
doi:10.1371/journal.pone.0063697
PMCID: PMC3660600  PMID: 23704931
25.  Grape Seed Proanthocyanidin Extract–Mediated Regulation of STAT3 Proteins Contributes to Treg Differentiation and Attenuates Inflammation in a Murine Model of Obesity-Associated Arthritis 
PLoS ONE  2013;8(11):e78843.
Grape seed proanthocyanidin extract (GSPE) is a natural flavonoid that exerts anti-inflammatory properties. Obesity is an inflammatory condition and inflammatory cells and their secretion of pro-inflammatory molecules contribute to the pathogenesis of obesity. Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by inflammation of joints lined by synovium. Previously, we demonstrated that obesity augmented arthritis severity in collagen induced arthritis (CIA), a murine model of human RA. Here, we investigated whether oral administration of GSPE showed antiobesity and anti-arthritic effects in high-fat diet-induced obese (DIO) mice and in obese CIA mice, respectively. The pathophysiologic mechanisms by which GSPE attenuates weight gain and arthritis severity in vivo were also investigated. In DIO mice, GSPE administration significantly inhibited weight gain, reduced fat infiltration in liver and improved serum lipid profiles. The antiobesity effect of GSPE was associated with increased populations of regulatory T (Treg) cells and those of decreased Th17 cells. Decrease of Th17 cells was associated with significant inhibition of their key transcriptional factors, pSTAT3Tyr705 and pSTAT3Ser727. On the contrary, GSPE-induced Treg induction was associated with enhanced pSTAT5 expression. To identify the anti-arthritis effects of GSPE, GSPE was given orally for 7 weeks after type II collagen immunization. GSPE treatment significantly attenuated the development of autoimmune arthritis in obese CIA model. In line with DIO mice, GSPE administration decreased Th17 cells and reciprocally increased Treg cells by regulating STAT proteins in autoimmune arthritis model. The expressions of pro-inflammatory cytokines and nitrotyrosine in synovium were significantly inhibited by GSPE treatment. Taken together, GSPE functions as a reciprocal regulator of T cell differentiation – suppression of Th17 cells and induction of Tregs in both DIO and obese CIA mice. GSPE may act as a therapeutic agent to treat immunologic diseases related with enhanced STAT3 activity such as metabolic disorders and autoimmune diseases.
doi:10.1371/journal.pone.0078843
PMCID: PMC3818494  PMID: 24223854

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