Phosphorylation Of Janus Kinase Research Articles

Leptin limits hepatic lipid accumulation and inflammation via vagal activation of the JAK2-STAT3/AMPK pathway

Non-Alcoholic Fatty Liver Disease (NAFLD) begins with hepatic lipid accumulation, and leptin has antisteatosis properties. In this study, we investigated the effects of leptin on hepatic steatosis and inflammation through the vagal pathway independently of the inhibitory effect of food intake. Male Sprague-Dawley rats were matched for food intake after the high-fat diet (HFD)-induced obesity model and were injected intraperitoneally with leptin or leptin + lidocaine for 6 weeks. Control rats received equal volumes of saline. Adipose tissue mass, NAFLD activity scores (NAS), hepatic inflammatory factors, hepatic triglyceride content and hepatic lipid metabolism-related protein levels were evaluated. Leptin ameliorated HFD-induced hepatic lipid accumulation, improved NAS, and decreased tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1) levels in the presence of matched intake. Lidocaine decreased the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) expression in the nucleus tractus solitarius (NTS) and abrogated the leptin-mediated improvement. Leptin increased hypothalamic phosphorylated Janus kinase 2 (p-JAK2) and p-STAT3 expression, as well as the expression of mitochondrial respiratory chain-related genes. Leptin also increased hepatic phosphorylated adenosine 5′-monophosphate-activated protein kinase (p-AMPK) expression and phosphorylation of its downstream target acetyl Co A carboxylase 1 (ACC1), reducing de novo lipogenesis. Our results suggest that leptin ameliorated hepatic lipid accumulation and inflammation by activating the JAK2-STAT3/AMPK pathway through the vagal pathway independently of the inhibitory effect of ingestion. Leptin has the potential to be a drug for early NAFLD treatment.

Tofacitinib Citrate Coordination-Based Dual-Responsive/Scavenge Nanoplatform Toward Regulate Colonic Inflammatory Microenvironment for Relieving Colitis.

Ulcerative colitis is an inflammation of the colon characterized by immune dysregulation and intestinal inflammation. Developing safe oral nanomedicines that suppress intestinal inflammation, while modulating colonic inflammatory microenvironment by scavenging reactive oxygen species (ROS) and hydrogen sulfide (H2S) is crucial for the effective treatment of colitis. Here, the tofacitinib citrate and copper coordination-based nanoparticle (TF-Cu nanoparticle, T-C) to dual-scavenge ROS and H2S by coordination competition is synthesized. Moreover, the coordination of T-C using computer simulation is explored. To enhance the acid stability and inflammatory targeting of T-C, it is encapsulated with hyaluronic acid-modified chitosan, along with a calcium pectinate coating (T-C@HP). Owing to the dual pH/pectinase-responsive characteristics of T-C@HP, the nanoplatform can target inflamed colonic lesions, inhibiting phosphorylated Janus kinase 1. Furthermore, T-C@HP scavenges ROS and H2S, as well as increases NADPH levels, which is investigated by combining biosensor (HyPer7 and iNap1/c) and chemical probes. T-C@HP also alleviates colitis by regulating the colonic inflammatory microenvironment through multiple processes, including the modulation of apoptosis, macrophage polarization, tight junction, mucus layer, and intestinal flora. Complemented by satisfactory anti-inflammatory and biosafety results, this nanoplatform represents a promising, effective, and safe treatment option for colitis patients.

RBP4 promotes denervation-induced muscle atrophy through STRA6-dependent pathway.

Fat infiltration of skeletal muscle has been recognized as a common feature of many degenerative muscle disorders. Retinol binding protein 4 (RBP4) is an adipokine that has been demonstrated to be correlated with the presence and severity of sarcopenia in the elderly. However, the exact role and the underlying mechanism of RBP4 in muscle atrophy remains unclear. Denervation-induced muscle atrophy model was constructed in wild-type and RBP4 knockout mice. To modify the expression of RBP4, mice were received intramuscular injection of retinol-free RBP4 (apo-RBP4), retinol-bound RBP4 (holo-RBP4) or oral gavage of RBP4 inhibitor A1120. Holo-RBP4-stimulated C2C12 myotubes were treated with siRNAs or specific inhibitors targeting signalling receptor and transporter of retinol 6 (STRA6)/Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (STAT3) pathway. Fat accumulation, myofibre cross-sectional area, myotube diameter and the expression of muscle atrophy markers and myogenesis markers were analysed. The expression levels of RBP4 in skeletal muscles were significantly up-regulated more than 2-fold from 7days and sustained for 28days after denervation. Immunofluorescence analysis indicated that increased RBP4 was localized in the infiltrated fatty region in denervated skeletal muscles. Knockout of RBP4 alleviated denervation-induced fatty infiltration and muscle atrophy together with decreased expression of atrophy marker Atrogin-1 and MuRF1 as well as increased expression of myogenesis regulators MyoD and MyoG. By contrast, injection of retinol-bound holo-RBP4 aggregated denervation-induced ectopic fat accumulation and muscle atrophy. Consistently, holo-RBP4 stimulation also had a dose-dependent effect on the reduction of C2C12 myotube diameter and myofibre cross-sectional area, as well as on the increase of Atrogin-1and MuRF1 expression and decrease of MyoD and MyoG expression. Mechanistically, holo-RBP4 treatment increased the expression of its membrane receptor STRA6 (>3-fold) and promoted the phosphorylation of downstream JAK2 and STAT3. Inhibition of STRA6/JAK2/STAT3 pathway either by specific siRNAs or inhibitors could decrease the expression of Atrogin-1 and MuRF1 (>50%) and decrease the expression of MyoD and MyoG (>3-fold) in holo-RBP4-treated C2C12 myotube. RBP4 specific pharmacological antagonist A1120 significantly inhibited the activation of STRA6/JAK2/STAT3 pathway, ameliorated ectopic fat infiltration and protected against denervation-induced muscle atrophy (30% increased myofibre cross-sectional area) in mice. In conclusion, our data reveal that RBP4 promotes fat infiltration and muscle atrophy through a STRA6-dependent and JAK2/STAT3 pathway-mediated mechanism in denervated skeletal muscle. Our results suggest that lowering RBP4 levels might serve as a promising therapeutic approach for prevention and treatment of muscle atrophy.

The role of Pim-1 kinases in inflammatory signaling pathways.

This observational study investigated the regulatory mechanism of Pim-1 in inflammatory signaling pathways. THP-1, RAW 264.7, BV2, and Jurkat human T cell lines were used. None. Lipopolysaccharide (LPS) was used to induce inflammation, followed by PIM1 knockdown. Western blot, immunoprecipitation, immunofluorescence, and RT-PCR assays were used to assess the effect of PIM1 knockdown on LPS-induced inflammation. PIM1 knockdown in macrophage-like THP-1 cells suppressed LPS-induced upregulation of pro-inflammatory cytokines, inducible nitric oxide synthase, cyclooxygenase-2, phosphorylated Janus kinase, signal transducer and activator of transcription 3, extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38, and nuclear factor kappa B p65 (NF-κB p65). It also suppressed upregulation of inhibitor of NF-κB kinase α/β and enhanced the nuclear translocation of NF-κB p65. Moreover, it inhibited the upregulation of Nod-like receptor family pyrin domain-containing 3 (NLRP3) and cleavage of caspase-1 induced by co-treatment of LPS with adenosine triphosphate. Additionally, p-transforming growth factor-β-activated kinase 1 (TAK1) interacted with Pim-1. All three members of Pim kinases (Pim-1, Pim-2, and Pim-3) were required for LPS-mediated inflammation in macrophages; however, unlike Pim-1 and Pim-3, Pim-2 functioned as a negative regulator of T cell activity. Pim-1 interacts with TAK1 in LPS-induced inflammatory responses and is involved in MAPK/NF-κB/NLRP3 signaling pathways. Additionally, considering the negative regulatory role of Pim-2 in T cells, further in-depth studies on their respective functions are needed.

Effect of Selenium nanoparticles on Paraquat-induced-neuroinflammation and oligodendocyte modulation: Implication of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway

BackgroundParaquat (PQ), is an extensively used herbicide and is a well-established powerful neurotoxin. However, the mechanism underlying its neurotoxicity still needs further investigation. Aim of workThe study investigated the pathogenesis of PQ-induced neuroinflammation of the substantia nigra pars compacta (SNPC) and cerebellum and evaluated the potential effect of selenium nanoparticles (SeN) against such neurotoxicity. MethodsThirty-six mice were randomly divided into three groups; Control group, PQ group: mice received PQ 10 mg/kg (i.p), and PQ + SeN group; mice received PQ in addition to oral SeN 0.1 mg/kg. All regimens were administered for 14 days. The mice's brains were processed for biochemical, molecular, histological, and immune-histochemical assessment. ResultsSeN increased the SNPC and cerebellum antioxidants (reduced glutathione, glutathione peroxidase, and superoxide dismutase 1) while decreasing malondialdehyde concentration. Also, SeN increased the anti-inflammatory interleukin (IL)-10 and decreased the pro-inflammatory IL-1β and −6 along with improving the angiogenic nitric oxide and reducing caspase-1. Further, western blots of phosphorylated Janus kinase (JAK2)/signal transducer and activator of transcription3 (STAT3) proteins showed a significant decline. Those improving effects of SeN on SNPC, and cerebellum were supported by the significantly preserved dopaminergic and Purkinje neurons, the enhanced myelin fibers on Luxol fast blue staining, and the marked increase in Olig-2, Platelet-derived growth factor-alpha, and tyrosine hydroxylase immunoreactivity. ConclusionSeN could mitigate PQ-induced neurotoxicity via its antioxidant, anti-inflammatory, and antiapoptotic properties.

Fibroblast Growth Factor 23 Neutralizing Antibody Ameliorates Abnormal Renal Phosphate Handling in Sickle Cell Disease Mice.

We assessed the involvement of fibroblast growth factor 23 (FGF23) in phosphaturia in sickle cell disease (SCD) mice. Control and SCD mice were treated with FGF23 neutralizing antibody (FGF23Ab) for 24 hours. Serum ferritin was significantly increased in SCD mice and was significantly reduced in female but not male SCD mice by FGF23Ab. FGF23Ab significantly reduced increased erythropoietin in SCD kidneys. Serum intact FGF23 was significantly increased in SCD female mice and was markedly increased in SCD male mice; however, FGF23Ab significantly reduced serum intact FGF23 in both genotypes and sexes. Serum carboxy-terminal-fragment FGF23 (cFGF23) was significantly reduced in SCD IgG male mice and was markedly but not significantly reduced in SCD IgG female mice. FGF23Ab significantly increased cFGF23 in both sexes and genotypes. Serum 1,25-dihydroxyvitamin D3 was significantly increased in SCD IgG and was further significantly increased by FGF23Ab in both sexes and genotypes. Significantly increased blood urea nitrogen in SCD was not reduced by FGF23Ab. The urine phosphate (Pi)/creatinine ratio was significantly increased in SCD in both sexes and was significantly reduced by FGF23Ab. Increased SCD kidney damage marker kidney injury molecule 1 was rescued, but sclerotic glomeruli, increased macrophages, and lymphocytes were not rescued by short-term FGF23Ab. FGF23Ab significantly reduced increased phospho-fibroblast growth factor receptor 1, αKlotho, phosphorylated extracellular signal-regulated kinase, phosphorylated serum/glucocorticoid-regulated kinase 1, phosphorylated sodium-hydrogen exchanger regulatory factor-1, phosphorylated janus kinase 3, and phosphorylated transducer and activator of transcription-3 in SCD kidneys. The type II sodium Pi cotransporter (NPT2a) and sodium-dependent Pi transporter PiT-2 proteins were significantly reduced in SCD kidneys and were increased by FGF23Ab. We conclude that increased FGF23/FGF receptor 1/αKlotho signaling promotes Pi wasting in SCD by downregulating NPT2a and PIT2 via modulation of multiple signaling pathways that could be rescued by FGF23Ab.

MiR-452-5p facilitates retinoblastoma cell growth and invasion via the SOCS3/JAK2/STAT3 pathway.

Retinoblastoma (RB) is an intraocular tumor in children. Accumulated evidence confirmsthat microRNAs (miRNAs) exert critical functions in RB. This research aimed to investigate the miR-452-5p function in RB. MiR-452-5p expressions in RB were tested with quantitative real-time polymerase chain reaction (PCR). MiR-452-5p functions in RB were evaluated via Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine assay, flow cytometry, Western blot, and Transwell. MiR-452-5p mechanism in RB was assessed using bioinformatics software Starbase and dual-luciferase reporter gene assay. Meanwhile, miR-452-5p function in RB in vivo was examined by constructing tumor xenografts in nude mice, immunohistochemistry, and Western blot assays. MiR-452-5p was overexpressed in RB tissues and cells, and miR-452-5p expression was positively correlated with RB clinicopathology including the Largest tumor base (mm) and Differentiation. Functionally, miR-452-5p knockdown restrained RB cell proliferation, invasion, epithelial-mesenchymal transition (EMT), and facilitated cell apoptosis. Mechanistically, suppressors of cytokine signaling (SOCS3) knockdown restored the inhibitory effects of miR-452-5p knockdown on RB cells. Meanwhile, in vivo studies further corroborated that miR-452-5p knockdown reduced RB tumor growth, EMT, and accelerated apoptosis in vivo. Also, miR-452-5p knockdown increased SOCS3 protein levels, and decreased phosphorylated Janus kinase 2/Janus kinase 2 (JAK2), phosphorylated signal transducer and activator of transcription 3/signal transducer and activator of transcription 3 (STAT3) in vivo. MiR-452-5p accelerated RB cell growth and invasion by SOCS3/JAK2/STAT3.

Withaferin A suppressed hepatocellular carcinoma progression through inducing IGF2BP3/FOXO1/JAK2/STAT3 pathway-mediated ROS production

Objective This study aimed to investigate the underlying molecular mechanisms of Withaferin A (WA) in hepatocellular carcinoma (HCC). Materials and Methods The gene and protein expression were analyzed using RT-qPCR and western blot, respectively. The proliferation of HCC cells was evaluated by CCK-8 assays. The migrative ability of HCC cells was measured by transwell assays. Results We revealed that WA suppressed the proliferation and migration of HCC cells and inhibited IGF2BP3 (insulin like growth factor 2 mRNA binding protein 3) expression. IGF2BP3 abundance reversed the reactive oxygen species (ROS) accumulation and suppression of HCC cell proliferation and migration induced by WA. Besides, IGF2BP3 suppressed ROS production to promote the growth and migration of HCC cells. Furthermore, we found that IGF2BP3 exerted its tumor-promotive and ROS-suppressive effect on HCC cells by regulating the expression of FOXO1 (forkhead box O1). In addition, IGF2BP3-stimulated activation of JAK2 (Janus kinase 2)/STAT3 (signal transducer and activator of transcription 3) phosphorylation effectively decreased the transcription of FOXO1. FOXO1 abundance decreased the phosphorylation of JAK2 and STAT3 by increasing ROS level, forming a feedback loop for the inhibition of JAK2/STAT3 signaling activated by IGF2BP3. Conclusions WA-induced ROS inhibited HCC cell growth and migration through the inhibition of IGF2BP3 to deactivate JAK2/STAT3 signaling, resulting in increased FOXO1 expression to further stimulate ROS production and inhibit JAK2/STAT3 signaling.

Lp-PLA2 silencing ameliorates inflammation and autophagy in nonalcoholic steatohepatitis through inhibiting the JAK2/STAT3 pathway.

Nonalcoholic steatohepatitis (NASH), a common cause of liver-related morbidity and mortality worldwide, is characterized by inflammation and hepatocellular injury. Our research focuses on lipoprotein-associated phospholipase A2 (Lp-PLA2), an inflammation-related biomarker that has recently garnered interest in the context of NASH due to its potential roles in disease pathogenesis and progression. We established a NASH mouse model using a high-fat diet (HFD) and treated it with sh-Lp-PLA2 and/or rapamycin (an mTOR inhibitor). Lp-PLA2 expression in NASH mice was detected by qRT-PCR. Serum levels of liver function parameters and inflammatory cytokines were detected using corresponding assay kits. We examined pathological changes in liver using hematoxylin-eosin, oil red O, and Masson staining, and observed autophagy through transmission electron microscopy. The protein levels of Lp-PLA2, mTOR, light chain 3 (LC3) II/I, phosphorylated Janus kinase 2 (p-JAK2)/JAK2, and phosphorylated signal transducer and activator of transcription 3 (p-STAT3)/STAT3 were determined by western blotting. Kupffer cells extracted from C57BL/6J mice were treated to replicate NASH conditions and treated with sh-Lp-PLA2, rapamycin, and/or a JAK2-inhibitor to further verify the roles and mechanisms of Lp-PLA2 in NASH. Our data indicate an upregulation of Lp-PLA2 expression in HFD-induced NASH mice. Silencing Lp-PLA2 in NASH mice reduced liver damage and inflammation markers (aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), triglycerides (TG), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6)), while increasing IL-10 levels, an anti-inflammatory cytokine. Additionally, Lp-PLA2 silencing decreased lipid and collagen accumulation and promoted autophagy. The beneficial effects of sh-Lp-PLA2 on NASH were enhanced by rapamycin. Furthermore, Lp-PLA2 silencing resulted in the downregulation of the expression of p-JAK2/JAK2 and p-STAT3/STAT3 in NASH mice. Similar results were observed in Kupffer cells treated under NASH conditions; Lp-PLA2 silencing promoted autophagy and repressed inflammation, effects which were potentiated by the addition of rapamycin or a JAK2-inhibitor. Our findings suggest that silencing Lp-PLA2 promotes autophagy via deactivating the JAK2/STAT3 signaling pathway, thereby restraining NASH progression. This highlights the potential therapeutic value of targeting Lp-PLA2, adding a new dimension to our understanding of NASH pathogenesis and treatment strategies.

Effects of Arctigenin in Proliferation, Migration, and Invasion of Nasopharyngeal Carcinoma 5-8F Cells.

Nasopharyngeal carcinoma (NPC) is a malignant tumor of the nasopharynx. Here, we aimed to understand better the molecular basis for arctigenin (ARG)'s ability to promote NPC 5- 8F cell invasion. We tested the effects of several doses of ARG on 5-8F cells that had been cultured in vitro. We estimated the metabolic activity of cells by The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay. We examined the influence on cell invasion, and migration using Transwell Evaluation. Real-time polymerase chain reaction analysis was used to determine the relative amounts of epidermal growth factor receptor (EGFR), Janus kinase 2 (JAK2) , and transcriptional activator 3 (STAT 3) mRNA expression. Using western blotting, we looked at the level of phosphorylation of specific proteins like EGFR, phosphorylated EGFR, JAK2, and STAT 3. Our findings revealed that ARG inhibited NPC 5-8F cell development in a dose-and time-dependent manner. The invasiveness and mobility of 5-8F cells were significantly suppressed when ARG was overexpressed in a tumor development model. Expression levels of EGFR, JAK2, and STAT 3 mRNA were considerably low in the experimental group. As a consequence of being treated with ARG, lower levels of EGFR, p-EGFR, p-JAK2, and p-STAT3 expression were observed. These results suggest that ARG may prevent NPC 5-8F cells from proliferating, migrating, and invading other tissues. There are a few potential molecular pathways, two of which are the inhibition of EGFR phosphorylation and the reduction of levels of phospho-JAK2 and phospho-STAT3.

Irisin drives macrophage anti-inflammatory differentiation via JAK2-STAT6-dependent activation of PPARγ and Nrf2 signaling.

Irisin is an exercise-induced myokine that alleviates inflammation and obesity. The induction of anti-inflammatory (M2) macrophage is facilitated for treatment of sepsis and associated lung damage. However, whether irisin drives macrophage M2 polarization remains unclear. Here, we found that irisin induced-macrophage anti-inflammatory differentiation in vivo using an LPS-induced septic mice model and in vitro using RAW64.7cells and bone marrow-derived macrophages (BMDMs). Irisin also promoted the expression, phosphorylation, and nuclear translocation of peroxisome proliferator-activated receptor gamma (PPAR-γ) and nuclear factor-erythroid 2-related factor 2 (Nrf2). Inhibition or knockdown of PPAR-γ and Nrf2 abolished irisin-induced accumulation of M2 macrophage markers, such as interleukin (IL)-10 and Arginase 1. Furthermore, dual-luciferase reporter and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays confirmed that STAT6 boosts PPAR-γ and Nrf2 transcription by binding to their DNA promoters in irisin-stimulated macrophages. In contrast, STAT6 shRNA blocked the irisin-induced activation of Pparγ, Nrf2, and related downstream genes. Moreover, the interaction of irisin with its ligand integrin αVβ5 remarkably promoted Janus kinase 2 (JAK2) phosphorylation, while inhibition or knockdown of integrin αVβ5 and JAK2 attenuated the activation of STAT6, PPAR-γ, and Nrf2 signaling. Interestingly, co-immunoprecipitation (Co-IP) assay also revealed that the binding between JAK2 and integrin αVβ5 is critical for irisin-induced macrophage anti-inflammatory differentiation by enhancing the activation of the JAK2-STAT6 pathway. In conclusion, irisin boosted M2 macrophage differentiation by inducing JAK2-STAT6-dependent transcriptional activation of the PPAR-γ-related anti-inflammatory system and Nrf2-related antioxidant genes. The findings of this study suggest that the administration of irisin is a novel and promising therapeutic strategy for infectious and inflammatory diseases.

Erythropoietin: function and therapeutic potential

Erythropoietin (EPO) exerts its effect on erythroid lineage cells through interaction with the EPO receptor (EPOR), the so-called canonical pathway, and through a complex consisting of EPOR and a common cytokine receptor beta subunit (CD131) – a non-canonical pathway for non-hematopoietic cells of the human and animal body. EPO realizes its effects through the launch of a signaling cascade, which begins with the phosphorylation of Janus kinase 2 (JAK2) and then with the involvement of phosphatidylinositol-3 kinase B (PI3K) or Ras-mitogen-activated protein kinase (MAPK) or signal transducers and transcription activators (STAT). EPO exhibits a direct cytoprotective effect through increased CD131 expression and subsequent development of anti-apoptotic and anti-inflammatory effects in target cells. In addition to its use in the treatment of anemia, EPO is increasingly being used in correction of inflammatory and degenerative processes, both in experimental and clinical studies. EPO promotes the engraftment of stem cells, differentiation of mesenchymal stem cells in the connective tissue direction, suppresses the inflammatory response and apoptosis of cells in the lesion. The article includes literature data concerning EPO and its clinical use in inflammatory and degenerative processes, based on data from eLibrary and the National Center for Biotechnological Information (NCBI) for the period 1998–2022.

Effect of Dunaliella salina on myocardial ischemia-reperfusion injury through KEAP1/NRF2 pathway activation and JAK2/STAT3 pathway inhibition

Myocardial ischemia-reperfusion injury (MIRI) is a life-threatening vascular emergency, in which oxidative stress and excessive inflammation reactions play significant roles. Dunaliella salina is rich in natural beta (β)-carotene, which is considered an antioxidant and anti-cancer compound, preventing night blindness, delaying aging, and regulating immunity. Due to its unique nutritional and functional components, D. salina has the potential to be developed as a nutritional health food. At present, it is unknown whether D. salina can rescue MIRI. The present study aimed at investigating whether D. salina has protective effect on MIRI and at exploring its potential mechanism. We developed a Langendorff perfused heart model in mice. The mice were given D. salina by gavage at a dose of 500 mg/kg for 7 days consecutively. It was found that D. salina could improve left ventricle function and reduce the rate of malignant arrhythmia and infarct size (P < 0.01). Furthermore, D. salina administration increased superoxide dismutase and decreased malondialdehyde content in myocardial tissue (P < 0.01). Importantly, real-time polymerase chain reaction and Western blot results showed that D. salina caused nuclear factor (erythroid-derived 2)-like 2 (NRF2) activation and enhanced the expression of antioxidative genes, such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1) (P < 0.05 and P < 0.01, respectively). At the same time, the phosphorylation of Janus kinase (JAK) and signal transducer and activator of transcription 3 (STAT3) was inhibited (P < 0.05 and P < 0.01, respectively), and proinflammatory cytokines interleukin-1 beta (IL-1β) and IL-6 decreased with D. salina administration (P < 0.05). In conclusion, D. salina has a protective effect on MIRI, which may be mediated by promoting kelch-like ECH-associated protein 1 (KEAP1)/NRF2 pathway and inhibiting JAK2/STAT3 signaling pathway.

Sitagliptin Mitigates Diabetic Nephropathy in a Rat Model of Streptozotocin-Induced Type 2 Diabetes: Possible Role of PTP1B/JAK-STAT Pathway.

Diabetic nephropathy (DN) is a microvascular complication of diabetes mellitus. This study examined the therapeutic effects of sitagliptin, a dipeptidyl peptidase inhibitor, on DN and explored the underlying mechanism. Male Wistar albino rats (n = 12) were intraperitoneally administered a single dose of streptozotocin (30 mg/kg) to induce diabetes. Streptozotocin-treated and untreated rats (n = 12) were further divided into normal control, normal sitagliptin-treated control, diabetic control, and sitagliptin-treated diabetic groups (n = 6 in each). The normal and diabetic control groups received normal saline, whereas the sitagliptin-treated control and diabetic groups received sitagliptin (100 mg/kg, p.o.). We assessed the serum levels of DN and inflammatory biomarkers. Protein tyrosine phosphatase 1 B (PTP1B), phosphorylated Janus kinase 2 (P-JAK2), and phosphorylated signal transducer activator of transcription (P-STAT3) levels in kidney tissues were assessed using Western blotting, and kidney sections were examined histologically. Sitagliptin reduced DN and inflammatory biomarkers and the expression of PTP1B, p-JAK2, and p-STAT3 (p < 0.001) and improved streptozotocin-induced histological changes in the kidney. These results demonstrate that sitagliptin ameliorates inflammation by inhibiting DPP-4 and consequently modulating the PTP1B-related JAK/STAT axis, leading to the alleviation of DN.

Phosphorylation of Janus kinase 1 and signal transducer and activator of transcription 3 and 6 in keratinocytes of canine atopic dermatitis.

Canine atopic dermatitis (cAD) is a disease associated with Type 2 helper T (Th2) immune responses in the acute phase of the disease. In humans, keratinocytes are activated by Th2 cytokines via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. However, the activation of keratinocytes by Th2 cytokines in cAD has not yet been demonstrated. To evaluate keratinocyte activation based on the phosphorylation (p) of JAK1, STAT3 and STAT6. Seven dogs with cAD and three healthy dogs. Immunohistochemical analysis was performed to detect pJAK1, pSTAT3 and pSTAT6 in keratinocytes in normal canine skin, and the skin of atopic dogs. In the latter group samples were collected from both primary and secondary lesions, and nonaffected skin. The percentage of pJAK1-positive keratinocytes was significantly higher in primary cAD lesions than in healthy skin (p < 0.05). No significant differences were observed in pSTAT3-positive keratinocytes among the groups. The percentage of pSTAT6-positive keratinocytes was significantly higher in primary and secondary lesions than in healthy skin (p < 0.05, respectively). The novel finding in this study was the activation of keratinocytes as demonstrated by the phosphorylation of JAK1/STATs in lesional and nonlesional cAD skin. These results suggest the potential of not only JAK1, butalso of STAT6 as therapeutic targets for cAD.

Inhibition of MicroRNA-122-5p Relieves Myocardial Ischemia-Reperfusion Injury via SOCS1.

Evidence has shown that microRNA (miR)-122-5p is a diagnostic biomarker of acute myocardial infarction. Here, we aimed to uncover the functions of miR-122-5p in the pathological process of myocardial ischemia-reperfusion injury (MI/RI). An MI/RI model was established by left anterior descending coronary artery ligation in mice. The levels of miR-122-5p, suppressor of cytokine signaling-1 (SOCS1), phosphorylation of Janus kinase 2 (p-JAK2), and signal transducers and activators of transcription (p-STAT3) in the myocardial tissues of mice were measured. Downregulated miR-122-5p or upregulated SOCS1 recombinant adenovirus vectors were injected into mice before MI/RI modeling. The cardiac function, inflammatory response, myocardial infarction area, pathological damage, and cardiomyocyte apoptosis in the myocardial tissues of mice were evaluated. Cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) injury and cardiomyocyte biological function was tested upon transfection of miR-122-5p inhibitor. The target relation between miR-122-5p and SOCS1 was evaluated. miR-122-5p expression and p-JAK2 and p-STAT3 expression were high, and SOCS1 expression was low in the myocardial tissues of MI/RI mice. Decreasing miR-122-5p or increasing SOCS1 expression inactivated the JAK2/STAT3 pathway to alleviate MI/RI by improving cardiac function and reducing inflammatory reaction, myocardial infarction area, pathological damage, and cardiomyocyte apoptosis in mice. Silencing of SOCS1 reversed depleted miR-122-5p-induced cardioprotection for MI/RI mice. In vitro experiments revealed that the downregulation of miR-122-5p induced proliferative, migratory, and invasive capabilities of H/R cardiomyocytes while inhibiting apoptosis. Mechanically, SOCS1 was a target gene of miR-122-5p. Our study summarizes that inhibition of miR-122-5p induces SOCS1 expression, thereby relieving MI/RI in mice.

Inflammation and Treatment-Resistant Depression from Clinical to Animal Study: A Possible Link?

The aim of this study was to investigate the relationship between treatment-resistant depression (TRD) and inflammation in humans and experimental models. For the human study, a retrospective cohort study was conducted with 206 participants; half were on antidepressants for major depressive disorder. The patients were divided into healthy and depressed groups. Inflammation was assessed based on the values of the main inflammatory biomarkers (CRP, WBC and ESR). For the animal experiments, 35 adult male Wistar rats were assigned to stressed and non-stressed groups. Inflammation and stress were induced using lipopolysaccharide and chronic unpredictable mild stress. A 10 mg/kg intraperitoneal injection of fluoxetine (FLX), a known antidepressant, was simultaneously administered daily for 4 weeks. Behavioral tests were performed. The plasma levels of inflammatory and stress biomarkers were measured and were significantly higher in the stressed and non-responsive groups in both studies. This study provides evidence of the link between inflammation and TRD. We further observed a possible link via the Phosphorylated Janus Kinase 2 and Phosphorylated Signal Transducer and Activator of Transcription 3 (P-JAK2/P-STAT3) signaling pathway and found that chronic stress and high inflammation hinder the antidepressant effects of FLX. Thus, non-response to antidepressants could be mitigated by treating inflammation to improve the antidepressant effect in patients with TRD.

Staphylococcal protein A modulates inflammation by inducing interferon signaling in human nasal epithelial cells

Objective and designStaphylococcus aureus (S. aureus) is one of the leading causes of human respiratory tract infections. The function of Staphylococcal protein A (SpA), expressed on the S. aureus bacterial membrane and released in the environment, on human nasal epithelial cells (HNECs) have not been fully elucidated. In this study, we tested the SpA expression in S. aureus from chronic rhinosinusitis patients and investigated the effects of SpA on HNECs inflammation through Interferon Gamma Receptor 1(IFNGR1)/phosphorylated Janus Kinase 2 (p-JAK2) pathway.MethodsRNA profiling was performed to investigate inflammatory activation in a S. aureus chronic rhinosinusitis (CRS) mouse model. SpA release by S. aureus clinical isolates was determined using ELISA. The effect of purified SpA and SpA enriched conditioned media from S. aureus clinical isolates on HNECs cytotoxicity, apoptosis and release of inflammatory cytokines was evaluated using lactate dehydrogenase assays, and flow cytometry. SpA dependent IFNGR1 and p-JAK2 expression were assessed by qPCR, immunofluorescence and western blot in HNECs.Results49 genes were significantly induced in S. aureus CRS mice indicative of activation of interferon signaling. SpA release was significantly higher in S. aureus clinical isolates from chronic rhinosinusitis with nasal polyps (CRSwNP) patients. Purified SpA significantly increased IFNGR1 mRNA and protein expression in HNECs. SpA induced cytotoxic effects and induced the release of Interleukin-6 (IL-6) and IL-8 in an IFNGR1 dependent way.ConclusionSpA induces interferon signaling through activation of the IFNGR1-JAK-2 pathway, which provides an understanding of how S. aureus SpA affects the inflammatory process in the upper airways.

Pharmacological Modulation of the Crosstalk between Aberrant Janus Kinase Signaling and Epigenetic Modifiers of the Histone Deacetylase Family to Treat Cancer.

Hyperactivated Janus kinase (JAK) signaling is an appreciated drug target in human cancers. Numerous mutant JAK molecules as well as inherent and acquired drug resistance mechanisms limit the efficacy of JAK inhibitors (JAKi). There is accumulating evidence that epigenetic mechanisms control JAK-dependent signaling cascades. Like JAKs, epigenetic modifiers of the histone deacetylase (HDAC) family regulate the growth and development of cells and are often dysregulated in cancer cells. The notion that inhibitors of histone deacetylases (HDACi) abrogate oncogenic JAK-dependent signaling cascades illustrates an intricate crosstalk between JAKs and HDACs. Here, we summarize how structurally divergent, broad-acting as well as isoenzyme-specific HDACi, hybrid fusion pharmacophores containing JAKi and HDACi, and proteolysis targeting chimeras for JAKs inactivate the four JAK proteins JAK1, JAK2, JAK3, and tyrosine kinase-2. These agents suppress aberrant JAK activity through specific transcription-dependent processes and mechanisms that alter the phosphorylation and stability of JAKs. Pharmacological inhibition of HDACs abrogates allosteric activation of JAKs, overcomes limitations of ATP-competitive type 1 and type 2 JAKi, and interacts favorably with JAKi. Since such findings were collected in cultured cells, experimental animals, and cancer patients, we condense preclinical and translational relevance. We also discuss how future research on acetylation-dependent mechanisms that regulate JAKs might allow the rational design of improved treatments for cancer patients. SIGNIFICANCE STATEMENT: Reversible lysine-ɛ-N acetylation and deacetylation cycles control phosphorylation-dependent Janus kinase-signal transducer and activator of transcription signaling. The intricate crosstalk between these fundamental molecular mechanisms provides opportunities for pharmacological intervention strategies with modern small molecule inhibitors. This could help patients suffering from cancer.

Tyrosine Phosphorylation of CARM1 Promotes Its Enzymatic Activity and Alters Its Target Specificity in Myeloid Neoplasms with Hyperactivated JAK2

Introduction Coactivator-associated arginine methyltransferase 1 (CARM1) catalyzes the asymmetric dimethylation of arginine residues in histone and non-histone substrates, including transcription factors, RNA splicing factors, and the SWI/SNF chromatin remodeling complex. CARM1 has emerged as an important target in acute myeloid leukemia. We have previously shown that Janus kinase 2 (JAK2) affects the activity of epigenetic modifiers. A V617F mutation in JAK2 genes leads to its hyperactivation. This mutation is a common hallmark of the BCR/ABL1-negative myeloproliferative neoplasms, and also found in patients with acute myeloid leukemia and myelodysplastic syndrome. Here, we find that hyperactivated JAK2, mediated by biallelic V617F mutation, triggers CARM1 phosphorylation, increasing its methyltransferase activity and altering its target specificity. Methods We generated two phospho-tyrosine specific antibodies against CARM1 tyrosine-149 (Y139) and -334 (Y334), and two asymmetric dimethylation-arginine specific antibodies against RUNX1 arginine-223 (R223) and -319 (R319). We also generated isogenic HEL cells carrying homozygous CARM1 non-phosphorylatable mutation (Y149F or Y334F single, or Y149F/Y334F double mutation), using the CRISPR/Cas9 nuclease system. Results We first performed cell-free in vitro kinase assays using recombinant CARM1 protein and JAK2 kinase, and mass spectrometry analysis, which identified Y139 and Y334 as the sites of JAK2 phosphorylation in CARM1. We next compared the levels of CARM1-Y149 and -Y334 phosphorylation in 15 myeloid leukemia cell lines and normal human CD34+ cord blood cells. HEL and UKE-1 cells, that harbor bi-allelic JAK2-V617F mutation, showed the highest level of phosphorylated CARM1-Y149 and -Y334, while SET2 cells with mono-allelic JAK2-V617F mutation and the other cell lines showed abundant CARM1 protein but a lower relative amount of phosphorylated CARM1 protein. We found that the JAK2 activation, through transphosphorylation by JAK2 and TYK2, enhances tyrosine kinase activity against CARM1 using HEL cells with knockout of either JAK1 or TYK2. Having previously shown that CARM1 methylates RUNX1 at R223, in an immunoprecipitation assay, WT-CARM1 was able to pull down RUNX1, while the non-phosphorylatable CARM1 mutants were not. Using mass spectrometry and an in vitro methylation assay, we confirmed R223 and R319 as CARM1 target sites. We examined non-phosphorylatable CARM1 mutation knock-in HEL cells and found that mutation of either site reduces the asymmetric dimethylation of RUNX1 as well as known substrates (BAF155 and PABP1). We observed that CARM1 Y149F/Y334F mutant-expressing HEL cells reduced cell proliferation with G2/M cell cycle arrest and an increase in apoptosis. To identify the transcription networks regulated by CARM1 phosphorylation, we performed RNA-seq on WT-CARM1 and double CARM1 Y149F/Y334F mutant-expressing HEL cells. Gene set enrichment analysis identified that gene sets associated with G2/M cell cycle progression and anti-apoptosis were significantly downregulated in Y149F/Y334F mutation knock-in HEL cells. We next performed ChIP-seq analyses using antibodies against total RUNX1 and against asymmetrically dimethylated R319-RUNX1 in HEL cells expressing knock-in of CARM1 non-phosphorylatable mutation. CARM1 non-phosphorylatable mutation decreased the signals of dimethylated RUNX1-R319 within 5 kb of the transcription start sites for RUNX1 target genes, with less effect on total RUNX1. We next assessed the efficacy of the combination with CARM1 inhibitor (EPZ025654) and JAK2 inhibitor (ruxolitinib). Dual targeting inhibition of JAK2 and CARM1 showed a synergistic inhibition effect on HEL and UKE-1 cells (with abundant phosphor-CARM1), but only an additive effect on SET2 cells (with less amount of phosphor-CARM1). Conclusion CARM1 phosphorylation mediated by hyperactivated JAK2 regulates its methyltransferase activity and is required for the maximal proliferation of myeloid neoplasms. We also demonstrated that dual targeting of JAK2 and CARM1 is more effective than monotherapy in cell lines with phosphor-CARM1. Thus, our results reveal a novel role for CARM1 phosphorylation in myeloid neoplasms.