The involvement of JAK/STAT signaling pathway in the treatment of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder in which inflammation and oxidative stress play key etiopathological role. The pathology of PD brain is characterized by inclusions of aggregated α-synuclein (α-SYN) in the cytoplasmic region of neurons. Clinical evidence suggests that stimulation of pro-inflammatory cytokines leads to neuroinflammation in the affected brain regions. Upon neuroinflammation, the Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) signaling pathway, and other transcription factors such as nuclear factor κB (NF-κB), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), mammalian target of rapamycin (mTOR), and toll-like receptors (TLRs) are upregulated and induce the microglial activation, contributing to PD via dopaminergic neuron autophagy. Aberrant activation or phosphorylation of the components of JAK/STAT signaling pathway has been implicated in increased transcription of the inflammation-associated genes and many neurodegenerative disorders such as PD. Interferon gamma (IFN-γ), and interleukine (IL)-6 are two of the most potent activators of the JAK/STAT pathway, and it was shown to be elevated in PD. Stimulation of microglial cell with aggregated α-SYN results in production of nitric oxide (NO), tumor necrosis factor (TNF)-α, and IL-1ß in PD. Dysregulation of the JAK/STAT in PD and its involvement in various inflammatory pathways make it a promising PD therapy approach. So far, a variety of synthetic or natural small-molecule JAK inhibitors (Jakinibs) have been found promising in managing a spectrum of ailments, many of which are in preclinical research or clinical trials. Herein, we provided a perspective on the function of the JAK/STAT signaling pathway in PD progression and gathered data that describe the rationale evidence on the potential application of Jakinibs to improve neuroinflammation in PD.

The Potential Inhibitory Effects of miR-19b on Ocular Inflammation are Mediated Upstream of the JAK/STAT Pathway in a Murine Model of Allergic Conjunctivitis.

Purpose: Thymic stromal lymphopoietin (TSLP) is a pro-allergic cytokine that initiates allergic inflammatory reaction between epithelial and dendritic cells (DCs). miR-19b was reported to suppress TSLP expression. The present study aimed to examine miR-19b expression, regulation, and function in allergic conjunctivitis (AC). Methods: A murine model of experimental AC was induced in BALB/c mice by short ragweed pollen. The serum, eye balls, conjunctiva, and cervical lymph nodes (CLN) were used for the study. Gene expression was determined by RT-PCR, whereas protein production and activation were evaluated by immunostaining, ELISA, and Western blotting. Results: In the murine AC model, miR-19b was aberrantly downregulated, whereas the levels of TSLP and p-STAT3, as well as the number of CD11c+ pSTAT3+ DCs were increased. Moreover, Th2 inflammatory cytokine expression was significantly increased. These severe phenotypes could be counteracted by either applying exogenous miR-19b mimic microRNAs or the JAK/STAT inhibitor CYT387. Moreover, overexpression of miR-19b repressed p-STAT3 expression and the number of CD11c+ cells in AC eye and CLN tissues. Conclusions: These findings suggested that miR-19b reduced ocular surface inflammation by inhibiting Stat3 signaling via TSLP downregulation in a murine AC model. Moreover, the present study further demonstrated the clinical potential of applying miR-19b and anti-JAK/STAT therapies in the treatment of AC.

Cinnamaldehyde Inhibits Inflammation of Human Synoviocyte Cells Through Regulation of Jak/Stat Pathway and Ameliorates Collagen-Induced Arthritis in Rats.

Cinnamaldehyde (Cin), a bioactive cinnamon essential oil from traditional Chinese medicine herb Cinnamomum cassia, has been reported to have multipharmacological activities including anti-inflammation. However, its role and molecular mechanism of anti-inflammatory activity in musculoskeletal tissues remains unclear. Here, we first investigated the effects and molecular mechanisms of Cin in human synoviocyte cells. Then in vivo therapeutic effect of Cin on collagen-induced arthritis (CIA) also studied. Cell Counting Kit CCK-8 assay was performed to evaluate the cell cytotoxicity. Proinflammatory cytokine expression was evaluated using quantitative polymerase chain reaction and ELISA. Protein expression was measured by western blotting. The in vivo effect of Cin (75 mg/kg per day) was evaluated in rats with CIA by gavage administration. Disease progression was assessed by clinical scoring, radiographic, and histologic examinations. Cin significantly inhibited interleukin (IL)-1ß-induced IL-6, IL-8, and tumor necrosis factor-α release from human synoviocyte cells. The molecular analysis revealed that Cin impaired IL-6-induced activation of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1 (STAT1), and STAT3 signaling pathway by inhibiting the phosphorylation of JAK2, STAT1, and STAT3, without affecting NF-κB pathway. Cin reduced collagen-induced swollen paw volume of arthritic rats. The anti-inflammation effects of Cin were associated with decreased severity of arthritis, joint swelling, and reduced bone erosion and destruction. Furthermore, serum IL-6 level was decreased when Cin administered therapeutically to CIA rats. Cin suppresses IL-1ß-induced inflammation in synoviocytes through the JAK/STAT pathway and alleviated collagen-induced arthritis in rats. These data indicated that Cin might be a potential traditional Chinese medicine-derived, disease-modifying, antirheumatic herbal drug. SIGNIFICANCE STATEMENT: In this study, we found that cinnamaldehyde (Cin) suppressed proinflammatory cytokines secretion in rheumatology arthritis synoviocyte cells by Janus kinase/signal transducer and activator of transcription pathway. The in vivo results showed that Cin ameliorated collagen-induced arthritis in rats. These findings indicate that Cin is a potential traditional Chinese medicine-derived, disease-modifying, antirheumatic herbal drug.

ATRA reduces inflammation and improves alveolar epithelium regeneration in emphysematous rat lung.

INTRODUCTION: Pulmonary emphysema characterized by alveolar wall destruction is resultant of persistent chronic inflammation. All-trans retinoic acid (ATRA) has been reported to reverse elastase-induced emphysema in rats. However, the underlying molecular mechanisms are so far unknown. OBJECTIVE: To investigate the therapeutic potential effect of ATRA via the amelioration of the ERK/JAK-STAT pathways in the lungs of emphysematous rats. METHODS: In silico analysis was done to find the binding efficiency of ATRA with receptor and ligands of ERK & JAK-STAT pathway. Emphysema was induced by porcine pancreatic elastase in Sprague-Dawley rats and ATRA was supplemented as therapy. Lungs were harvested for histopathological, genomics and proteomics analysis. RESULTS AND DISCUSSION: In silico docking, analysis confirms that ATRA interferes with the normal binding of ligands (TNF-α, IL6ST) and receptors (TNFR1, IL6) of ERK/JAK-STAT pathways respectively. ATRA restored the histology, proteases/antiproteases balance, levels of inflammatory markers, antioxidants, expression of candidate genes of ERK and JAK-STAT pathways in the therapy group. CONCLUSION: ATRA ameliorates ERK/JAK-STAT pathway in emphysema condition, resulting in alveolar epithelium regeneration. Hence, ATRA may prove to be a potential drug in the treatment of emphysema.

JAK/STAT Blockade Alters Synovial Bioenergetics, Mitochondrial Function, and Proinflammatory Mediators in Rheumatoid Arthritis.

OBJECTIVE: To examine the effects of tofacitinib on metabolic activity, mitochondrial function, and proinflammatory mechanisms in rheumatoid arthritis (RA). METHODS: Ex vivo RA synovial explants and primary RA synovial fibroblasts (RASFs) were cultured with 1 µM tofacitinib. RASF bioenergetics were assessed using an XF24 analyzer, and key metabolic genes were assessed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Mitochondrial function was assessed using specific cell fluorescent probes and by mitochondrial gene arrays. Mitochondrial mutagenesis was quantified using a mitochondrial random mutation capture assay, and lipid peroxidation was quantified by enzyme-linked immunosorbent assay (ELISA). The effect of tofacitinib on spontaneous release of proinflammatory mediators from RA whole tissue synovial explants was quantified by ELISAs/MSD multiplex assays, and metabolic markers were quantified by RT-PCR. Finally, RASF invasion, matrix degradation, and synovial outgrowths were assessed by transwell invasion/Matrigel outgrowth assays and ELISA. RESULTS: Tofacitinib significantly decreased mitochondrial membrane potential, mitochondrial mass, and reactive oxygen species production by RASFs and differentially regulated key mitochondrial genes. Tofacitinib significantly increased oxidative phosphorylation, ATP production, and the maximal respiratory capacity and the respiratory reserve in RASFs, an effect paralleled by a decrease in glycolysis and the genes for the key glycolytic enzymes hexokinase 2 (HK2), glycogen synthase kinase 3α (GSK-3α), lactate dehydrogenase A, and hypoxia-inducible factor 1α. Tofacitinib inhibited the effect of oncostatin M (OSM) on interleukin-6 (IL-6) and monocyte chemotactic protein 1 and reversed the effects of OSM on RASF cellular metabolism. Using RA whole tissue synovial explants, we found that tofacitinib inhibited the key metabolic genes for glucose transporter 1, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, 3'-phosphoinositide-dependent protein kinase 1, HK2, and GSK-3α, the proinflammatory mediators IL-6, IL-8, IL-1ß, intercellular adhesion molecule 1, vascular endothelial growth factor, and TIE-2, and RASF outgrowth from synovial explants, RASF invasion, and matrix metalloproteinase 1 activity. CONCLUSION: This study demonstrates that JAK/STAT signaling mediates the complex interplay between inflammation and cellular metabolism in RA pathogenesis.

Human ß-defensin 3 induces STAT1 phosphorylation, tyrosine phosphatase activity, and cytokine synthesis in T cells.

The AMP hBD-3 stimulates numerous immune effector functions in myeloid cells and keratinocytes, predominantly through the MAPK signaling cascade. In contrast, hBD-3 was reported to neutralize the activation of T cells by antagonizing MAPK signaling initiated by SDF-1α through CXCR4. With the use of complementary proteomic and immunochemical approaches, we investigated possible stimulatory effects of hBD-3 on T cells and demonstrate that hBD-3 induces STAT1 tyrosine phosphorylation within 5 min yet is unable to induce MAPK activation. Inclusion of a PTPase inhibitor increased hBD-3-induced phosphorylation dramatically, suggesting that hBD-3 also stimulates PTPase activity concurrently. The increase in PTPase activity was confirmed by demonstrating that hBD-3 suppresses IFN-γ-induced STAT1 tyrosine phosphorylation but not STAT1 serine and ERK1/2 threonine phosphorylation and stimulates the translocation of SHP-2 into the nucleus within 15 min. The signaling pathways initiated by hBD-3 may lead to the observed enhancement of distinct T cell effector functions during TCR activation, such as the increase in IL-2 and IL-10, but not IFN-γ secretion. Thus, hBD-3 initiates distinct lineage-specific signaling cascades in various cells involved in host defense and induces a concurrent tyrosine kinase and tyrosine phosphatase signaling cascade that may activate simultaneously the targeted T cells and inhibit their response to other immune mediators. Furthermore, these results suggest that this evolutionarily conserved peptide, which exhibits a broad spectrum of antimicrobial and immunomodulatory activities, serves to integrate innate and adaptive immunity.

Kurarinone regulates immune responses through regulation of the JAK/STAT and TCR-mediated signaling pathways.

Sophora flavescens is a medicinal herb that contains flavonoids and quinolizidine alkaloids and has a wide range of biological activities due to its anti-inflammatory, anti-bacterial and anti-cancer properties. We isolated a series of flavonoids from the roots of Sophora flavescens and examined their ability to inhibit immune responses. Among the flavonoids, kurarinone exhibited the strongest inhibitory effect on immune responses. Kurarinone suppressed the differentiation of CD4(+) T cells by inhibiting the expression and production of T-cell lineage-specific master regulators and cytokines. Our results also demonstrated that kurarinone directly suppressed the cytokine-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling and T-cell receptor (TCR) pathways. In two established animal models of chronic inflammatory skin disease, one in which psoriasis-like skin disease was induced by an interleukin 23 (IL-23) injection into mouse ears and another in which 2,4,6-trinitrochlorobenzene (TNCB) application on the abdomens of mice was used to induce contact dermatitis, kurarinone repressed disease development by inhibiting the expression of pro-inflammatory mediators, including cytokines, chemokines and enzyme in murine ear skin. This study provides new evidence that kurarinone may ameliorate chronic inflammatory skin diseases through the suppression of pathogenic CD4(+) T-cell differentiation and the overall immune response.

IL-25 improves IgA levels during parenteral nutrition through the JAK-STAT pathway.

INTRODUCTION: Parenteral nutrition (PN) impairs mucosal immunity and increases the risk of infection in part via lower IgA levels at mucosal surfaces. Transport of immunoglobulin A (IgA) across the mucosa to the gut lumen depends on the epithelial transport protein, polymeric immunoglobulin receptor (pIgR), which is reduced during PN. In vitro, studies demonstrate that IL-4 up-regulates pIgR production via Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. Because IL-4 stimulates IgA and is reduced during PN, we hypothesized that the suppressed pIgR is a result of decreased JAK-1 and STAT-6 phosphorylation. Because IL-4 is mediated by IL-25, we also hypothesized that PN + IL-25 would restore luminal IgA by increasing phosphorylated JAK-1 and STAT-6, resulting in increased tissue pIgR and luminal IgA. METHOD: Experiment 1: 2 days after intravenous cannulation, male Institute of Cancer Research mice were randomized to chow (n = 11) or PN (n = 9). Experiment 2: 2 days after intravenous cannulation, male Institute of Cancer Research mice were randomized to chow (n = 12), PN (n = 10), or PN + 0.7 µg of exogenous IL-25 (n = 11) per day. After 5 days, distal ileum tissue was collected, homogenized, and protein extracted for JAK-STAT expression levels using a phospho-specific antibody microarray. Tissue was homogenized to measure pIgR expression via Western blot or fixed in 4% paraformaldehyde to measure pIgR expression via immunohistochemistry. Small intestinal wash fluid was collected and IgA was quantified using enzyme-linked immunosorbent assay. RESULTS: Experiment 1: PN significantly reduced phosphorylated JAK-1 and STAT-6 compared with chow. PN also decreased the tissue levels of the Th2 cytokines, IL-4 and IL-13, as well as pIgR, and luminal IgA compared with chow. Experiment 2: Exogenous administration of PN + IL-25 increased the phosphorylated JAK-1 and STAT-6 compared with PN alone. IL-25 completely restored expression of IL-13 to chow levels. IL-4, pIgR, IgA, and phosphorylated JAK-1 were significantly increased with IL-25 treatment compared with PN but failed to reach levels measured in chow. STAT-6 was significantly increased with IL-25 treatment compared with chow and PN. CONCLUSIONS: PN significantly decreases the JAK-STAT pathway by reducing levels of phosphorylated STAT-6 and JAK-1. Consistent with our previous work, sIgA, pIgR, and IL-4 decreased with PN, whereas the addition of IL-25 to PN reversed these decreases and demonstrated the role of the JAK-STAT pathway in vivo during PN.

Staurosporine induces megakaryocytic differentiation through the upregulation of JAK/Stat3 signaling pathway.

The induction of megakaryocyte (MK) differentiation is a potent strategy for the clinical treatment of diseases related to blood platelet disorders. Staurosporine (STS) is an inhibitor of protein kinase C (PKC) with an inhibitory effect on cancer cells through apoptosis induction. However, the exact mechanism of STS on MK differentiation is still unclear. The present study assessed the regulatory effect of STS on MK differentiation in both human leukemia cells and mouse bone marrow-derived stem cells. STS not only inhibited the proliferation of both K562 and HEL cell lines, but also induced the cell differentiation into MK lineage, resulting in polyploidy formation, MK-specific markers CD41 and CD61 expression, and platelet factor 4 (PF4) secretions of cells. The induction effect of STS was upregulated through the expression of Stat3, but not PKC. The level of phosphorylated (p)-Stat3 showed an increased expression, translocated to the nucleus, and enhanced the DNA-binding activity in STS-treated cells. Blockage Stat3 and its upstream molecule JAK by Stat3 inhibitor VI and JAK inhibitor I, respectively, demonstrated that the cells obviously reduced the percentage of STS-mediated MK differentiation. Further investigation of the cells with Stat3 siRNA transfection showed that p-Stat3 and MK differentiation was markedly decreased, indicating that Stat3 is an important molecule in inducing MK differentiation. Additionally, the ex vivo assay also confirmed that STS effectively stimulated CFU-MK colony formation and CD61 expression in bone marrow cells. In conclusion, STS is a potent inducer for MK differentiation through the upregulation of JAK/Stat3 signaling pathway and p-Stat3 nuclear translocation.

Inhibition of transcription factors by plant-derived compounds and their implications in inflammation and cancer.

Inflammation is a general term used to describe various pathological processes with diverse causes that can include infection, trauma, or an autoimmune response. Due to its many causes, the inflammatory response involves multiple and varied mediators, including vasoactive amines, free radicals, and both lipidic and peptidic mediators. Medicinal plants and the compounds derived from them are a good source of new and specific inhibitors of the inflammatory process. The past decade has witnessed many important discoveries in this field, with new findings challenging the more traditional views of pharmacologists. Various studies, for example, have demonstrated the positive effects of plant-derived phenolics, which act as anti-oxidants, free radical scavengers, and inhibitors of nitric-oxide synthase, cyclooxygenase, and lipoxygenase. The anti-inflammatory activity of chalcones has been correlated with the induction of heme oxygenase-1 while phlorotannins have been found to inhibit matrix metalloproteinase, which is implicated in arthritis, chronic inflammation, and wrinkle formation. Sesquiterpene lactones have been studied as inhibitors of NF-kappaB activity and the relationship between their chemical structure and pharmacological activity has been clearly established. Recently, cucurbitacins have been described as inhibitors of JAK -STAT and NF-AT functions related to inflammation; they were also found to induce apoptosis of cells involved in the inflammatory response. This review focuses mainly on the effects of natural products on transcription factors, which are the most promising targets for designing new active drugs against inflammation and cancer.

Optimization and utilization of the SureFire phospho-STAT5 assay for a cell-based screening campaign.

The family of signal transducers and activators of transcription (STATs) consists of seven transcription factors that respond to a variety of cytokines, hormones, and growth factors. STATs are activated by tyrosine phosphorylation, which results in their dimerization and translocation into the nucleus where they exert their effect on transcription of regulated target genes. The phosphorylation of STATs is mediated mainly by Janus kinases (JAKs). The JAK/STAT pathway plays a critical role in hematopoietic and immune cell function. Here we focus on one member of the STAT family, STAT5. STAT5 is phosphorylated by several JAKs, including Jak3, Jak2, and Tyk2, in response to interleukin-2, erythropoietin (EPO), and interleukin-22, respectively. Activation of STAT5 is essential to T cell development and has been associated with hematologic malignancies. Therefore, the ability to assess STAT5 phosphorylation is important for discovery efforts targeting these indications. The assay formats available to detect phosphorylated STAT5 (pSTAT5) are relatively low throughput and involve lengthy protocols. These formats include western blot analysis, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. The SureFire (Perkin Elmer, Waltham, MA) pSTAT5 assay is a homogeneous assay that utilizes AlphaScreen (Perkin Elmer) technology to detect pSTAT5 in cell lysates. We have used this assay format to evaluate EPO-induced STAT5 phosphorylation in HEL cells and successfully complete a small-scale screening campaign to identify inhibitors of this event. The results obtained in these studies demonstrate that the SureFire pSTAT5 assay is a robust, reliable assay format that is amenable to high-throughput screening (HTS) applications.

[Gamma interferon: basics aspects, clinic significance and terapeutic uses]. / Interferón gamma: aspectos básicos, importancia clínica y usos terapéuticos.

Interferons are a family of pleiotropic cytokines, their name was assigned because of their anti-replicative viral activity. IFNgamma or immune type II interferon does not share receptors with the type I interferon, its structure is different and its gene is located in different chromosome, although its biologic effects are similar. Along of several years of research, it has been found that IFNgamma enhances the transcription of genes involved in immunomodulation, antiviral responses and antitumoral activities. Regarding to the immune system, IFNgamma increases the cytotoxic and phagocytic activity of macrophages and upregulates the expression of major histocompatibility complex (MHC) class I and class II molecules in dendritics cells and other antigen presenting cells. IFNgamma also promotes the development and differentiation of naive CD4+ T lymphocytes to Th1 helper subset. Indeed, this cytokine has a key role in the control of bacterial, micotic, viral and parasitic infections. Depending of the micro-environment, IFNgamma has a dual role as pro or anti inflammatory cytokine. Novel therapeutic strategies are currently being developed with the aim to enhance the immune response or replace IFNgamma gene abnormal expression with beneficial results in humans, being recombinant IFNgamma safe and well tolerated.

[Influence of moxibustion on JAK-STAT signal transduction pathways of synovial cells in rheumatoid arthritis rabbits].

OBJECTIVE: To observe the effect of moxibustion on Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway of synovial cells in rheumatoid arthritis (RA) rabbits for revealing the underlying mechanism of moxibustion in the treatment of RA. METHODS: A total of 30 rabbits (Japanese Big-ear White species) were randomized (stratified random) into control, model and moxibustion groups with 10 cases in each. RA model was established by injection of Freund's Complete Adjuvant (FCA, 0.5 mL/kg) into the animal's bilateral joint cavities. Moxibustion was applied to bilateral "Shenshu" (BL 23), 5 cones every time, once daily (except Sundays), 3 weeks altogether. The synovial tissue of joint was sampled for analyzing the expression of signal molecules associated with JAK-STAT pathway with gene chip and bioinformation analytical techniques. RESULTS: Compared with normal control group, the perimeters of both knee joints in model group increased significantly from the 3rd day to 21st day after injection of FCA (P < 0.01), while compared with model group, those in moxibustion group decreased considerably from the 6th day on (P < 0.05, 0.01). In comparison with control group, JAK-STAT pathway-associated genes with up-regulated expression in model group were C/EBP beta, CBP, CRP, GATA3, IFNAR1, IFNAR2, IFNGR2, IL-10Rb, INDO, SH2B, STAT3, STAT6, JAK3 and GP130, and those with down-regulated expression were A2M, MIG and IL-2Rr, suggesting an abnormal activation of JAK-STAT pathway; while in comparison with model group, the related gene up-regulated in moxibustion group in the expression was IL22R and those down-regulated were Cyclin D1, C/EBP beta, CRP, GATA3, IFNAR2, INDO, JAK2, JAK3, V-JUN, STAT3, STAT5, SH2B and OSM, showing that moxibustion had an apparent inhibitory effect on AR-induced abnormal activation of some genes as C/EBP beta, GATA3, IFNAR2, INDO, etc. CONCLUSION: Moxibustion can resist inflammation and eliminate swelling in RA rabbits, which may be closely with its effect in inhibiting abnormal activation of JAK-STAT pathway in synovial cells.