Ser727 Phosphorylation Research Articles

A randomized double blinded placebo controlled clinical trial for the evaluation of green coffee extract on immune health in healthy adults

BackgroundThe immune system functions to protect the host from a broad array of infectious diseases. Here, we evaluated the in vitro immunomodulatory effects of green coffee extract (GCE), and conducted a double-blinded, randomized and placebo-controlled trial among apparently healthy individuals. MethodsWe determined the levels and functions of inflammatory and immune markers viz., phospho-NF-κB p65 ser536, chemotaxis, phagocytosis, TH1/TH2 cytokines and IgG production. We also evaluated several immunological markers such as total leukocyte counts, differential leukocyte counts, NK cell activity, CD4/CD8 ratio, serum immunoglobulin, C-reactive protein (CRP) and pro-inflammatory cytokines (IL-6 and TNF-α). Results and conclusionGCE significantly inhibited LPS-induced NF-κB p65 ser536 phosphorylation, MCP-1-induced chemotaxis and significantly enhanced phagocytosis and IgG production. In addition, GCE modulated PMA/PHA-induced TH1/TH2 cytokine production. Clinical investigations suggested that the expression of CD56 and CD16 was markedly augmented on NK cells following GCE treatment. GCE significantly enhanced IgA production before and after influenza vaccination. Similarly, IL-6, TNF-α and CRP levels were significantly inhibited by GCE. Together, GCE confers several salubrious immunomodulatory effects at different levels attributing to optimal functioning of immune responses in the host. TaxonomyCell biology, Clinical study, Clinical Trial.

Galectin network in osteoarthritis: galectin-4 programs a pathogenic signature of gene and effector expression in human chondrocytes in vitro

Galectin-4 (Gal-4) is a member of the galectin family, which have been identified as galactose-binding proteins. Gal-4 possesses two tandem repeat carbohydrate recognition domains and acts as a cross-linking bridge in sulfatide-dependent glycoprotein routing. We herein document its upregulation in osteoarthritis (OA) in correlation with the extent of cartilage degradation in vivo. Primary human OA chondrocytes in vitro respond to carbohydrate-inhibitable Gal-4 binding with the upregulation of pro-degradative/-inflammatory proteins such as interleukin-1β (IL-1β) and matrix metalloproteinase-13 (MMP-13), as documented by RT-qPCR-based mRNA profiling and transcriptome data processing. Activation of p65 by phosphorylation of Ser536 within the NF-κB pathway and the effect of three p65 inhibitors on Gal-4 activity support downstream involvement of such signaling. In 3D (pellet) cultures, Gal-4 presence causes morphological and biochemical signs of degradation. Taken together, our findings strongly support the concept of galectins acting as a network in OA pathogenesis and suggest that blocking their activity in disease progression may become clinically relevant in the future.

Abstract 13911: Regulation of STAT3 Phosphorylation Through β IV -Spectrin Interaction and Its Role in Concentric versus Eccentric Hypertrophy

Hypertrophic growth is a critical determinant of cardiac function in the development of heart failure. In pressure overload, hypertrophy is divided by early adaptive remodeling defined by concentric growth, while chronic hypertrophy associates with eccentric growth and loss in cardiac output. The mechanisms regulating this transition and the impact each profile has in influencing heart function are largely unknown. Importantly, previous work identified a role for the structural protein β IV -spectrin and its interaction with the transcription factor, STAT3, in coordinating hypertrophic profiles. Specifically, mice expressing truncated β IV -spectrin lacking interaction with the hypertrophic signaling molecule CaMKII ( qv 3J mice) were resistant to β IV -spectrin degradation and presented with sustained concentric hypertrophy and ejection fraction (EF) compared to WT following 6 weeks transaortic constriction (TAC). Alternatively, mutant β IV -spectrin mice ( qv 4J ) lacking the STAT3/β IV -spectrin binding domain had reduced EF and LV dilation, consistent with eccentric remodeling, suggesting STAT3/β IV -spectrin is a key regulator of hypertrophic profiles. To evaluate how STAT3 function was altered, we used the STAT3 activator, lipopolysaccharide, on isolated cardiomyocytes, which identified impaired STAT3 Ser727 phosphorylation in qv 4J myocytes compared to WT. The significance of this phosphorylation site was investigated using STAT3 Ser727 phospho-ablated mice (STAT3-S727A), which resulted in accelerated eccentric hypertrophy and HF in response to TAC. Furthermore, we identified elevated Ser727 phosphorylation in the qv 3J TAC mice, which present with sustained concentric growth, compared to WT mice which transition to eccentric growth. The relationship of STAT3/β IV -spectrin and hypertrophic orientation was further examined by adenoviral overexpression of the hypertrophic driver, MEK1/2, on isolated myocytes. While MEK increased the width of WT myocytes, STAT3-S727A and qv 4J myocytes increased in length. Together, these data identify a novel role for β IV -spectrin in coordinating STAT3 Ser727 phosphorylation to tune specific hypertrophic profiles in response to chronic stress and influencing cardiac function.

IL-13 Derived Type 2 Innate Lymphocytes Ameliorates Cardiomyocyte Apoptosis Through STAT3 Signaling Pathway.

The involvement of cardiomyopathy during sepsis means higher mortality and prolonged length of hospital stay. Many efforts have been made to alleviate the apoptosis of cardiomyocytes in sepsis. The huge potential of IL-13 in tissue repair has attracted increasing attention. In the present study, we used LPS-treated mice or primary cardiomyocytes as a sepsis model to explore the anti-apoptotic ability of IL-13. It was found that an increased level of exogenous IL-13 was beneficial to the recovery of heart function in sepsis, and this anti-apoptotic effect of IL-13 was probably through enhancing the phosphorylation of STAT3 Ser727. In addition, we identified that the heart protective effect of IL-13 was associated with type 2 innate lymphocytes (ILC2). All these findings may provide a potential promising treatment for sepsis-induced cardiomyopathy.

Neutrophil-derived extracellular vesicles modulate the phenotype of naïve human neutrophils.

Neutrophils (PMN) regulate inflammation in many ways, including communication with other immune cells via extracellular vesicles (EVs). EVs released by human neutrophils activated with N-formylmethionyl-leucyl-phenylalanine (fMLF) (PMN-fMLF EVs) had an outside-out orientation and contained functionally important neutrophil plasma membrane proteins, including flavocytochrome b558, and enzymatically active granule proteins, elastase, and myeloperoxidase. Treatment of naïve PMN with PMN-fMLF EVs primed fMLF-stimulated NADPH oxidase activity, increased surface expression of the complement receptors CD11b/CD18 and CD35, the specific granule membrane protein CD66, and flavocytochrome b558 , and promoted phagocytosis of serum-opsonized Staphylococcus aureus. The primed oxidase activity reflected increased surface expression of flavocytochrome b558 and phosphorylation of SER345 in p47phox , two recognized mechanisms for oxidase priming. Taken together, these data demonstrate that stimulated PMN released EVs that altered the phenotype of naïve phagocytes by priming of the NADPH oxidase activity and augmenting phagocytosis, two responses that are integral to optimal PMN host defense.

Rational drug design of benzothiazole-based derivatives as potent signal transducer and activator of transcription 3 (STAT3) signaling pathway inhibitors.

The cumulative evidence supports STAT3, a transcriptional mediator of oncogenic signaling, as a therapeutic target in cancer. The development of STAT3 inhibitors remain an active area of research as no inhibitors have yet to be approved for cancer treatment. In a continuing effort to develop more potent STAT3 inhibitors based on our previously identified hit compound 16w, a series of benzothiazole derivatives with unique binding mode in SH2 domain of STAT3 were designed, synthesized and biologically evaluated. Of note, compound B19 demonstrated excellent activity against IL-6/STAT3 signaling pathway with the IC50 value as low as 0.067μM as determined by a luciferase reporter assay. Moreover, multiple compounds displayed potent antiproliferative activity against MDA-MB-468 and JAK2 mutant HEL cell lines. Further biochemical study using Western blot assay indicated that B19 blocked the phosphorylation of STAT3 at Tyr 705 and Ser 727 and thus suppressed STAT3-mediated gene expression of c-MYC and MCL-1. Simultaneously, it induced cancer cell G2/M phase arrest and apoptosis both in MDA-MB-468 and HEL cell lines. Finally, molecular docking study along with surface plasmon resonance (SPR) and fluorescence polarization (FP) assays disclosed the binding mode of B19 in STAT3 SH2 domain. Taken together, our finding suggests that B19 is a promising therapeutic STAT3 inhibitor for cancer treatment.

METTL3-dependent N6-methyladenosine RNA modification mediates the atherogenic inflammatory cascades in vascular endothelium

Atherosclerosis is characterized by the plaque formation that restricts intraarterial blood flow. The disturbed blood flow with the associated oscillatory stress (OS) at the arterial curvatures and branch points can trigger endothelial activation and is one of the risk factors of atherosclerosis. Many studies reported the mechanotransduction related to OS and atherogenesis; however, the transcriptional and posttranscriptional regulatory mechanisms of atherosclerosis remain unclear. Herein, we investigated the role of N6-methyladenosine (m6A) RNA methylation in mechanotransduction in endothelial cells (ECs) because of its important role in epitranscriptome regulation. We have identified m6A methyltransferase METTL3 as a responsive hub to hemodynamic forces and atherogenic stimuli in ECs. OS led to an up-regulation of METTL3 expression, accompanied by m6A RNA hypermethylation, increased NF-κB p65 Ser536 phosphorylation, and enhanced monocyte adhesion. Knockdown of METTL3 abrogated this OS-induced m6A RNA hypermethylation and other manifestations, while METTL3 overexpression led to changes resembling the OS effects. RNA-sequencing and m6A-enhanced cross-linking and immunoprecipitation (eCLIP) experiments revealed NLRP1 and KLF4 as two hemodynamics-related downstream targets of METTL3-mediated hypermethylation. The METTL3-mediated RNA hypermethylation up-regulated NLRP1 transcript and down-regulated KLF4 transcript through YTHDF1 and YTHDF2 m6A reader proteins, respectively. In the in vivo atherosclerosis model, partial ligation of the carotid artery led to plaque formation and up-regulation of METTL3 and NLRP1, with down-regulation of KLF4; knockdown of METTL3 via repetitive shRNA administration prevented the atherogenic process, NLRP3 up-regulation, and KLF4 down-regulation. Collectively, we have demonstrated that METTL3 serves a central role in the atherogenesis induced by OS and disturbed blood flow.

Calpain activation mediates microgravity-induced myocardial abnormalities in mice via p38 and ERK1/2 MAPK pathways

The human cardiovascular system has adapted to function optimally in Earth's 1G gravity, and microgravity conditions cause myocardial abnormalities, including atrophy and dysfunction. However, the underlying mechanisms linking microgravity and cardiac anomalies are incompletely understood. In this study, we investigated whether and how calpain activation promotes myocardial abnormalities under simulated microgravity conditions. Simulated microgravity was induced by tail suspension in mice with cardiomyocyte-specific deletion of Capns1, which disrupts activity and stability of calpain-1 and calpain-2, and their WT littermates. Tail suspension time-dependently reduced cardiomyocyte size, heart weight, and myocardial function in WT mice, and these changes were accompanied by calpain activation, NADPH oxidase activation, and oxidative stress in heart tissues. The effects of tail suspension were attenuated by deletion of Capns1. Notably, the protective effects of Capns1 deletion were associated with the prevention of phosphorylation of Ser-345 on p47phox and attenuation of ERK1/2 and p38 activation in hearts of tail-suspended mice. Using a rotary cell culture system, we simulated microgravity in cultured neonatal mouse cardiomyocytes and observed decreased total protein/DNA ratio and induced calpain activation, phosphorylation of Ser-345 on p47phox, and activation of ERK1/2 and p38, all of which were prevented by calpain inhibitor-III. Furthermore, inhibition of ERK1/2 or p38 attenuated phosphorylation of Ser-345 on p47phox in cardiomyocytes under simulated microgravity. This study demonstrates for the first time that calpain promotes NADPH oxidase activation and myocardial abnormalities under microgravity by facilitating p47phox phosphorylation via ERK1/2 and p38 pathways. Thus, calpain inhibition may be an effective therapeutic approach to reduce microgravity-induced myocardial abnormalities.

Folic Acid Deficiency Enhances the Tyr705 and Ser727 Phosphorylation of Mitochondrial STAT3 in In Vivo and In Vitro Models of Ischemic Stroke.

Ischemic stroke remains one of the most common causes of death and disability worldwide. The stroke patients with an inadequate intake of folic acid tend to have increased brain injury and poorer prognosis. However, the precise mechanisms underlying the harmful effects of folic acid deficiency (FD) in ischemic stroke is still elusive. Here, we aimed to test the hypothesis that mitochondrial localized STAT3 (mitoSTAT3) expression may be involved in the process of neuronal damage induced by FD in in vivo and in vitro models of ischemic stroke. Our results exhibited that FD increased infarct size and aggravated the damage of mitochondrial ultrastructure in ischemic brains. Meanwhile, FD upregulated the phosphorylation levels of mitoSTAT3 at Tyr705 (Y705) and Ser727 (S727) sites in the rat middle cerebral artery occlusion/reperfusion (MCAO/R) model and oxygen-glucose deprivation followed by reperfusion (OGD/R) N2a cells. Furthermore, the inhibition of JAK2 by AG490 led to a significant decrease in FD-induced phosphorylation of Y705, while S727 phosphorylation was unaffected. Conversely, U0126 and LY294002, which respectively inhibited phosphorylation of ERK1/2 and Akt, partially prevented S727 phosphorylation, but had limited effects on the level of pY705, suggesting that phosphorylation of Y705 and S727 is regulated via independent mechanisms in FD-treated brains.

The Expression of the Cold Shock Protein RNA Binding Motif Protein 3 is Transcriptionally Responsive to Organ Temperature in Mice.

Mild hypothermia, i.e. maintenance of organ temperature by up to 8°C lower than body temperature, is a critical strategy for exerting some functions of the cells and organs normally, and is an useful therapy for recovering properly from some diseases, including myocardial infarction, cardiac arrest, brain injury, and ischemic stroke. Nevertheless, there were no focusses so far on organ temperature and potential responses of gene expression to organ temperature in organs of homeothermic animals that survive under normal conditions. The present study aimed to assess organ temperature in homeothermic animals and evaluate the effect of their organ temperature on the expression of the cold shock protein RNA binding motif protein 3 (RBM3), and to gain insights into the organ temperature-mediated regulation of RBM3 gene transcription via Nuclear factor β-light-chain-enhancer of activated B cells (NF-κB) p65, which had been identified as a transcription factor that is activated by undergoing the Ser276 phosphorylation and promotes the RBM3 gene expression during mild hypothermia. We measured the temperature of several organs, where RBM3 expression was examined, in female and male mice. Next, in male mice, we tested NF-κB p65 expression and its Ser276 phosphorylation in organs that have their lower temperature than body temperature and compared them with those in organs that have their temperature near body temperature. Organ temperature was around 32°C in the brain and reproductive organs, which is lower than the body temperature, and around 37°C in the heart, liver, and kidney, which is comparable to the body temperature. The expression of RBM3 was detected greatly in the brain and reproductive organs with their organ temperature of around 32°C, and poorly in the heart, liver, and kidney with their organ temperature of around 37°C. In accordance with the changes in the RBM3 expression, the NF-κB p65 Ser276 phosphorylation was detected more greatly in the testis and brain with their organ temperature of around 32°C, than in the heart, liver, and kidney with their organ temperature of around 37°C, although the NF-κB p65 expression was unchanged among all the organs tested. Our data suggested that organ temperature lower than body temperature causes the expression of RBM3 in the brain and reproductive organs of mice, and that lower organ temperature causes the NF-κB p65 activation through the Ser276 phosphorylation, resulting in an increase in the RBM3 gene transcription, in the brain and reproductive organs of mice. The study may present the possibility that organ temperature-induced alterations in gene expression are organ specific in homeotherms and the possibility that organ temperature-induced alterations in gene expression are transcriptionally regulated in some organs of homeotherms.

Helicobacter pylori infection induces STAT3 phosphorylation on Ser727 and autophagy in human gastric epithelial cells and mouse stomach

Helicobacter pylori (H. pylori) infection is considered as one of the principal risk factors of gastric cancer. Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) plays an important role in inflammation-associated gastric carcinogenesis. In the canonical STAT3 pathway, phosphorylation of STAT3 on Tyr705 is a major event of STAT3 activation. However, recent studies have demonstrated that STAT3 phosphorylated on Ser727 has an independent function in mitochondria. In the present study, we found that human gastric epithelial AGS cells infected with H. pylori resulted in localization of STAT3 phosphorylated on Ser727 (P-STAT3Ser727), predominantly in the mitochondria. Notably, H. pylori-infected AGS cells exhibited the loss of mitochondrial integrity and increased expression of the microtubule-associated protein light chain 3 (LC3), the autophagosomal membrane-associated protein. Treatment of AGS cells with a mitophagy inducer, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), resulted in accumulation of P-STAT3Ser727 in mitochondria. In addition, the elevated expression and mitochondrial localization of LC3 induced by H. pylori infection were attenuated in AGS cells harboring STAT3 mutation defective in Ser727 phosphorylation (S727A). We also observed that both P-STAT3Ser727 expression and LC3 accumulation were increased in the mitochondria of H. pylori-inoculated mouse stomach.

STAT3 serine phosphorylation is required for TLR4 metabolic reprogramming and IL-1\u03b2 expression

Detection of microbial components such as lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR4) on macrophages induces a robust pro-inflammatory response that is dependent on metabolic reprogramming. These innate metabolic changes have been compared to aerobic glycolysis in tumour cells. However, the mechanisms by which TLR4 activation leads to mitochondrial and glycolytic reprogramming are unknown. Here we show that TLR4 activation induces a signalling cascade recruiting TRAF6 and TBK-1, while TBK-1 phosphorylates STAT3 on S727. Using a genetically engineered mouse model incapable of undergoing STAT3 Ser727 phosphorylation, we show ex vivo and in vivo that STAT3 Ser727 phosphorylation is critical for LPS-induced glycolytic reprogramming, production of the central immune response metabolite succinate and inflammatory cytokine production in a model of LPS-induced inflammation. Our study identifies non-canonical STAT3 activation as the crucial signalling intermediary for TLR4-induced glycolysis, macrophage metabolic reprogramming and inflammation.

Protein kinase Cι promotes UBF1–ECT2 binding on ribosomal DNA to drive rRNA synthesis and transformed growth of non-small-cell lung cancer cells

Epithelial cell-transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor for Rho GTPases that is overexpressed in many cancers and involved in signal transduction pathways that promote cancer cell proliferation, invasion, and tumorigenesis. Recently, we demonstrated that a significant pool of ECT2 localizes to the nucleolus of non-small-cell lung cancer (NSCLC) cells, where it binds the transcription factor upstream binding factor 1 (UBF1) on the promoter regions of ribosomal DNA (rDNA) and activates rDNA transcription, transformed cell growth, and tumor formation. Here, we investigated the mechanism by which ECT2 engages UBF1 on rDNA promoters. Results from ECT2 mutagenesis indicated that the tandem BRCT domain of ECT2 mediates binding to UBF1. Biochemical and MS-based analyses revealed that protein kinase Cι (PKCι) directly phosphorylates UBF1 at Ser-412, thereby generating a phosphopeptide-binding epitope that binds the ECT2 BRCT domain. Lentiviral shRNA knockdown and reconstitution experiments revealed that both a functional ECT2 BRCT domain and the UBF1 Ser-412 phosphorylation site are required for UBF1-mediated ECT2 recruitment to rDNA, elevated rRNA synthesis, and transformed growth. Our findings provide critical molecular insight into ECT2-mediated regulation of rDNA transcription in cancer cells and offer a rationale for therapeutic targeting of UBF1- and ECT2-stimulated rDNA transcription for the management of NSCLC.

Rotenone-induced reactive oxygen species signal the recruitment of STAT3 to mitochondria.

STAT3, a transcription factor involved in various physiological and pathological processes, is also present in mitochondria. Mitochondrial STAT3 regulates complex I activity and reactive oxygen species (ROS) production, yet the mechanisms governing its translocation to mitochondria remain poorly understood. In this study, we show that rotenone-induced ROS triggers the Ser727 phosphorylation of STAT3 and its increased mitochondrial localisation. Furthermore, we show that STAT3-depleted cells display increased ROS levels during rotenone treatment. Targeted expression in mitochondria of wild-type STAT3 - but not S727A mutant - lowers ROS levels, indicating the importance of Ser727 phosphorylation, both in rotenone-induced mitochondrial targeting and quenching of ROS levels. Together, our results demonstrate a novel STAT3-mediated feedback mechanism to maintain redox homeostasis during stress.

Acute Increase in O-GlcNAc Improves Survival in Mice With LPS-Induced Systemic Inflammatory Response Syndrome.

Sepsis is a systemic inflammatory response syndrome (SIRS) resulting from a severe infection that is characterized by immune dysregulation, cardiovascular derangements, and end-organ dysfunction. The modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation) influences many of the key processes that are altered during sepsis, including the production of inflammatory mediators and vascular contractility. Here, we investigated whether O-GlcNAc affects the inflammatory response and cardiovascular dysfunction associated with sepsis. Mice received an intraperitoneal injection of lipopolysaccharide (LPS, 20 mg/Kg) to induce endotoxic shock and systemic inflammation, resembling sepsis-induced SIRS. The effects of an acute increase in O-GlcNAcylation, by treatment of mice with glucosamine (GlcN, 300 mg/Kg, i.v.) or thiamet-G (ThG, 150 μg/Kg, i.v.), on LPS-associated mortality, production and release of cytokines by macrophages and vascular cells, vascular responsiveness to constrictors and blood pressure were then determined. Mice under LPS-induced SIRS exhibited a systemic and local inflammatory response with increased levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α), as well as severe hypotension and vascular hyporesponsiveness, characterized by reduced vasoconstriction to phenylephrine. In addition, LPS increased neutrophil infiltration in lungs and produced significant lethality. Treatment with GlcN and ThG reduced systemic inflammation and attenuated hypotension and the vascular refractoriness to phenylephrine, improving survival. GlcN and ThG also decreased LPS-induced production of inflammatory cytokines by bone marrow-derived macrophages and nuclear transcription factor-kappa B (NF-κB) activation in RAW 264.7 NF-κB promoter macrophages. Treatment of mice with ThG increased O-glycosylation of NF-κB p65 subunit in mesenteric arteries, which was associated with reduced Ser536 phosphorylation of NF-κB p65. Finally, GlcN also increased survival rates in mice submitted to cecal ligation and puncture (CLP), a sepsis model. In conclusion, increased O-GlcNAc reduces systemic inflammation and cardiovascular disfunction in experimental sepsis models, pointing this pathway as a potential target for therapeutic intervention.

Involvement of the NF-κB signaling pathway in proliferation and invasion inhibited by Zwint-1 deficiency in Pancreatic Cancer Cells.

Pancreatic cancer (PC) is an intractable cancer that is difficult to diagnose early and has a 5-year survival rate of less than 8%. ZW10-interacting kinetochore protein (ZWINT) is a crucial gene that contributes to chromosome instability and is essential for spindle assembly and kinetochore-microtubule attachment during meiosis and mitosis. However, the mechanism through which Zwint-1 promotes PC progression is yet to be elucidated. Here, we report that Zwint-1 is highly expressed in clinical PC specimens (based on analysis of the Gene Expression Profiling Interactive Analysis database) and various PC cell lines. Importantly, Zwint-1-deficient PC cells showed reduced nuclear factor-kappa B (NF-κB) (Ser536) phosphorylation along with inhibited proliferation and colony formation due to downregulation of NF-κB-regulated genes such as CCND1, cIAP1/2, and XIAP. In addition, Zwint-1-deficient PC cells showed reduced invasion and migration abilities, and decreased expression levels of the metalloproteinases MMP2 and MMP9. Furthermore, Zwint-1 deficiency arrested the PC cell cycle at the G2/M phase because the chromosomes failed to segregate properly, and the apoptosis rate in these cells gradually increased, accompanied by increased caspase-3 activation and anti-poly (ADP ribose) polymerase cleavage. Apoptosis caused by Zwint-1 deficiency was demonstrated to occur through caspase-dependent pathways based on experiments involving treatment with a pan-caspase inhibitor (Z-VAD-Fmk). Thus, Zwint-1 contributes to cell growth, invasion, and survival through NF-κB signaling pathways, suggesting that it could serve as a PC biomarker and new therapeutic target.

Ghrelin up-regulates cartilage-specific genes via the ERK/STAT3 pathway in chondrocytes of patients with adolescent idiopathic scoliosis

Adolescent idiopathic scoliosis (AIS) is a severe spinal deformity that often occurs during puberty. The occurrence of AIS is suggested to be related to abnormal development of cartilage. Our previous study found increased serum ghrelin levels in AIS patients that may linked to the development of AIS. However, whether ghrelin affects cartilage in AIS patients is unclear. We used quantitative real-time PCR (qRT-PCR) and immunohistochemistry to detect the expression of cartilage-specific genes and the ghrelin receptor, growth hormone secretagogue receptor (GHSR). The mRNA and protein levels of collagen II (COLII), SOX9, AGGRECAN (ACAN) and GHSR were higher in AIS patients than in controls. In addition, the protein levels of GHSR downstream signaling pathway members p-STAT3 (Ser727), and p-ERK1/2 were increased. Furthermore, we treated chondrocytes from AIS patients with 100 nM ghrelin, the cell proliferation assay and Western blotting showed that ghrelin promotes chondrocyte proliferation and enhances COLII, SOX9, ACAN, p-ERK1/2 and p-STAT3 expression, respectively. Interestingly, all these observed alterations were abolished by ghrelin + [D-Lys3]-GHRP-6 (a ghrelin receptor inhibitor) treatment. And after U0126 (an inhibitor of ERK1/2 phosphorylation) treatment, ERK1/2 and STAT3 (Ser727) phosphorylation was simultaneously suppressed indicating that ERK1/2 is an upstream pathway protein of STAT3 (Ser727). In conclusion, ghrelin plays an important role in upregulating cartilage-specific genes on AIS primary chondrocytes by activating ERK/STAT3 signaling pathway.

Epstein-Barr virus tegument protein BGLF2 inhibits NF-κB activity by preventing p65 Ser536 phosphorylation.

Epstein-Barr virus (EBV), a ubiquitous gammaherpesvirus, can regulate the antiviral response of NF-κB signaling, which is critical for cell survival, growth transformation, and virus latency. Here, we showed that tegument protein BGLF2 could inhibit TNF-α-induced NF-κB activity. BGLF2 was shown to interplay with the NF-κB subunits p65 and p50, and the Rel homology domain of p65 was the pivotal region to interact with BGLF2. Nonetheless, BGLF2 did not influence the development of p65-p50 dimerization. Yet, overexpression of BGLF2 inhibited the phosphorylation of p65 Ser536 (but not Ser276) and blocked the nuclear translocation of p65. In addition, knockdown of BGLF2 during EBV lytic replication elevated NF-κB activity and the phosphorylation of p65 Ser536. Taken together, these results suggest that the inhibition of NF-κB activation may serve as a strategy to escape the host's antiviral innate immunity to EBV during its lytic infection.-Chen, T., Wang, Y., Xu, Z., Zou, X., Wang, P., Ou, X., Li, Y., Peng, T., Chen, D., Li, M., Cai, M. Epstein-Barr virus tegument protein BGLF2 inhibits NF-κB activity by preventing p65 Ser536 phosphorylation.

CGRP Deficiency-Mediated Prolactin Upregulation Via Mag Antibodies Contributes to Painless Pathogenesis in IgG4-Related Disease

Abdominal pain is the most common symptom of pancreatitis, but IgG4-related autoimmune pancreatitis is characterized by painless jaundice. This specific pathological phenomenon may be a breakthrough in finding a new pain mechanism. Recently, we found CGRP deficiency, plasma cell neurotropic enrichment, high basal level of prolactin (PRL) expression, and some specific neuro-antibodies in IgG4-related disease (IgG4-RD). MAG (myelin-associated glycoprotein) antibodies were detected in the serum of IgG4-RD patients by immunofluorescence (IFA) using a monkey nerve as a substrate. The specific antibodies were verified and quantified by ELISA. Cytokines were measured in tissues homogenate obtained from CGRP+/- rats by liquid suspension chip technology. A significant increase in the serum PRL level and an imbalanced ratio Th1/Th2 of IgG4-RD were established. Differential proteins in the inflammatory tissues of IgG4-RD were concentrated in the hormone-sensitive pathway. Specifically, the level of the calcitonin gene-related peptide (CGRP) was significantly decreased. Intriguingly, MAG antibodies (IgG, IgG4, and IgM) were detected by IFA and ELISA. In vivo, higher levels of PRL and Th2 cytokines were detected in CGRP+/- rats than in wild-type rats. Similarly, decreasing trends in the concentrations of IgG4-RD, Akt1 (Ser473), and STAT3 (Ser727) phosphorylation were also found in the CGRP+/- rats. Our findings indicate that the presence of CGRP-deficiency-mediated MAG antibodies is a probable molecular basis for the initial events were triggered in painless IgG4-RD via prolactin upregulation. Funding: This work was supported by a grant from the National Natural Science Foundation of China (no.81571613, 81871293 to L.Q.C., and no.81572442 to G.F.). Declaration of Interest: All authors declare that they have no conflicts of interest. Ethical Approval: This research project was approved by the Ethics Committee of the Fujian Medical University, which supervised the study.

Hepatitis B Virus-Induced Imbalance of Inflammatory and Antiviral Signaling by Differential Phosphorylation of STAT1 in Human Monocytes.

It is not clear how hepatitis B virus (HBV) modulates host immunity during chronic infection. In addition to the key mediators of inflammatory response in viral infection, monocytes also express a high-level IFN-stimulated gene, CH25H, upon response to IFN-α exerting an antiviral effect. In this study, the mechanism by which HBV manipulates IFN signaling in human monocytes was investigated. We observed that monocytes from chronic hepatitis B patients express lower levels of IFN signaling/stimulated genes and higher levels of inflammatory cytokines compared with healthy donors. HBV induces monocyte production of inflammatory cytokines via TLR2/MyD88/NF-κB signaling and STAT1-Ser727 phosphorylation and inhibits IFN-α-induced stat1, stat2, and ch25h expression through the inhibition of STAT1-Tyr701 phosphorylation and in an IL-10-dependent, partially autocrine manner. Further, we found that enhancement of STAT1 activity with a small molecule (2-NP) rescued HBV-mediated inhibition of IFN signaling and counteracted the induction of inflammatory cytokines. In conclusion, HBV contributes to the monocyte inflammatory response but inhibits their IFN-α/β responsiveness to impair antiviral innate immunity. These effects are mediated via differential phosphorylation of Tyr701 and Ser727 of STAT1.