Suppressor Of Cytokine Signaling Research Articles

SOCS5 knockdown suppresses metastasis of hepatocellular carcinoma by ameliorating HIF-1α-dependent mitochondrial damage

The Pringle maneuver (PM) is widely used during hepatocellular carcinoma (HCC) resection. However, it inevitably leads to ischemia and hypoxia, which promotes tumor metastasis. In this study, immunohistochemical staining of specimens from 130 HCC patients revealed that long-time PM significantly affected the prognosis of patients with high expression of suppressor of cytokine signaling 5 (SOCS5), but did not affect the prognosis of patients with low expression of SOCS5. The TCGA database showed that patients with high expression of SOCS5 had higher hypoxia scores, and it was proved that SOCS5 could promote the expression of hypoxia-inducible factor 1 subunit alpha (HIF-1α) protein by clinical tissue samples, cell experiments, lung metastases, and subcutaneous tumorigenesis experiments. Then, we used CoCl2 to construct a hypoxia model, and confirmed that SOCS5 knockdown resisted hypoxia-induced mitochondrial damage by inhibiting the expression of HIF-1α, thereby inhibiting the invasion and migration of HCC cells by immunofluorescence, electron microscopy, migration, invasion, and other experiments. We performed rescue experiments using LY294002 and rapamycin and confirmed that the knockdown of SOCS5-inhibited HCC cell invasion and migration by inhibiting the PI3K/Akt/mTOR/HIF-1α signaling axis. More importantly, we obtained consistent conclusions from clinical, cellular, and animal studies that the hypoxia-induced invasion and migration ability of SOCS5-inhibited HCC were weaker than that of normal HCC. In conclusion, we identified a novel role for SOCS5 in regulating HIF-1α-dependent mitochondrial damage and metastasis through the PI3K/Akt/mTOR pathway. The development of a SOCS5-specific inhibitor, an indirect inhibitor of HIF-1α, might be effective at controlling PM-induced tumor micrometastases during HCC resection.

Methylation of the suppressor of Cytokine Signaling 1 Gene (SOCS1) in Philadelphia-negative myeloproliferative neoplasms.

Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activation is initiated by mutations in the JAK2 gene. This activation is in turn, a vital pathogenetic mechanism in myeloproliferative neoplasms (MPNs). However, several factors affect the pathogenesis of MPNs other than the JAK2 gene mutations, such as the downregulation of cytokine signaling (SOCS) proteins, which are potent inhibitors of the JAK/STAT pathway. Therefore, we hypothesized that the regulation of SOCS protein system might be a possible pathogenetic mechanism of MPNs through activating the JAK/STAT pathway. Our study aimed to investigate the status of the Suppressors of cytokine signaling 1 (SOCS1) in 125 MPNs specimens at the level of mutated points. The acquired mutations, aberrant expression, and/or CpG island hypermethylation of SOCS1 were analyzed among Philadelphia-negative myeloproliferative neoplasm patients. SOCS1 was identified in 20.0% of all patients with Philadelphia-negative myeloproliferative neoplasm. At the diagnosis, the prevalence of methylation was 41.0% for Polycythaemia Vera (PV), 27.7% for Essential Thrombocythaemia (ET), and 6.6% for Primary Myelofibrosis (PMF). The methylation was not detected in 20 healthy adult people. A significant association was found between disease groups (p=.077). The presence of methylated SOCS1 was found to be significantly correlated with age (p=.005), total RBCs count (p=.019), hemoglobin (Hb) concentration (p=.002), and Hematopoietic cell transplant (HCT) (p=.007) in PV patients. However, the presence of methylated SOCS1 was found to be significantly associated with age (p=.012), total RBCs count (p=.022), Hb concentration (p=.024), HCT (p=.033), and platelets count (p=.037) in ET patients. Moreover, the presence of methylated SOCS1 was significantly associated with Hb concentration (p=.046) and HCT (p=.040) in PMF patients. We concluded that the activation of the JAK/STAT signaling pathway in alternative or with JAK2 mutations leads to SOCS1 hypermethylation, which could represent a potential therapeutic target.

Downregulation of miR-122 by porcine reproductive and respiratory syndrome virus promotes viral replication by targeting SOCS3

MicroRNAs are small non-coding RNA that regulate host anti-viral immune response. In this study, we used high-throughput sequencing to identify miRNAs that were differentially expressed upon PRRSV infection in porcine alveolar macrophages. We observed that the expression level of miR-122 was decreased upon PRRSV infection. Over-expression of miR-122 remarkably suppressed PRRSV replication, while blockage of endogenous miR-122 enhanced PRRSV replication. Moreover, over-expression of miR-122 reduced the protein level of porcine suppressor of cytokine signaling 3 (SOCS3), a negative regulator of JAK-STAT signaling, resulting in enhanced production of type Ⅰ IFN. Further analysis revealed that miR-122 decreased the expression of SOCS3 at the post-transcription level by targeting the 3’ UTR region of SOCS3 mRNA. In conclusion, this study demonstrates that the expression of miR-122 was reduced during PRRSV infection. miR-122 impaired PRRSV replication by promoting the production of type I interferon. Our study may provide new insights into understanding PRRSV immune evasion mechanisms.

Ulinastatin affects focal cerebral ischemia-reperfusion injury via SOCS1-mediated JAK2/STAT3 signalling pathway.

Cerebral ischemia results in loss of cerebral blood flow, which contributes to neuronal damage, neurocognitive impairment, as well as learning and memory difficulties. Although reperfusion is necessary to restore the blood supply to the brain, it also leads to several detrimental effects on the brain. The purpose of this study was to assess the effects of ulinastatin (UTI) on preventing focal cerebral ischemia/reperfusion-induced injury (FCIRI). First, a rat model of FCIRI was established and treated with UTI. The effects of UTI on FCIRI in rats were evaluated using Morris water maze assay, triphenyl tetrazolium chloride staining, TUNEL, western blot assay, and enzyme-linked immunosorbent assay analysis. UTI was found to improve the learning memory ability, reduce infarction area, inhibit apoptosis and decrease inflammation in FCIRI rats. Messenger RNA microarray analysis of hippocampal tissues revealed that suppressor of cytokine signalling-1 (SOCS1) was the downstream target of UTI in FCIRI. SOCS1 depletion impaired the protective effect of UTI on FCIRI in rats. SOCS1 blocked the activation of the JAK2/STAT3 pathway. JAK2 inhibitor caused the JAK2/STAT3 pathway deficit, hence reversing the effect of sh-SOCS1 on FCIRI in rats. Taken together, our results demonstrate that UTI alleviated FCIRI in rats, which was, to some extent, related to SOCS1-mediated JAK2/STAT3 pathway.

Role of IFNγ in pathogenesis of SARS-CoV-2 infection

To date, there is no consensus explaining the relationship between varying concentrations of IFNγ and the severity of infection caused by SARS-CoV-2. The aim of this article was to analyze and formulate conclusions from the selected studies and publications, which, in sum, provide a potentially reasonable view on the role of IFNγ in COVID-19 pathogenesis. This article highlights current data on the immunological role of IFNγ which affects differentiation of naive T helper cells, acting as a polarizing factor. It activates the major histocompatibility complex (MHC) class I and II, by increasing the expression of MHC I/II subunits, inhibiting replication of the viral particles by initiating activation of interferon-stimulated genes followed by subsequent synthesis of antiviral proteins. Moreover, IFNγ activates the production of cytokines by T cells, enhancing cytotoxic activity of the T killers. IFNγ exerts immunostimulatory and immunomodulatory effects via STAT1, SOCS1 and PIAS genes, thus regulating activation of the JAK-STAT signaling pathway. A number of studies were considered where the patterns of changes in serum IFNγ concentration were examined in viral infections and SARS-CoV-2. We performed a systemic analysis of the results of studies that showed a relationship between high concentrations of IFNγ and COVID-19 severity. In a number of studies, the significantly high levels of IFNγ in COVID-19 patients were often associated with a poor outcome of the disease. The median values of the IFNγ concentration in severe COVID-19 were found to be significantly higher compared to the results obtained in the cases of moderate severity. It shows an increase, in parallel with viral load in the nasopharyngeal samples upon worsening of the clinical condition. Based on the data on the decreased IFNγ concentrations in convalescent patients, the mechanism of antagonism between IFNγ and IL-4 is considered, where the decreases serum concentrations of IFNγ along with increasing level of IL-4 may be an indirect proof of normal adaptive immune response with subsequent development of antibodies to SARS-CoV-2 and gradual elimination of the virus from the body. Moreover, the evidence is discussed that the patients harboring some parasitic infections (Toxoplasma gondii, Cryptosporidium, Blastocystis hominis, Giardia duodenalis, Entamoeba histolytica) with persistently elevated level of IFNγ are at reduced risk for severe course of COVID-19.

Exploring novel independent prognostic biomarkers for hepatocellular carcinoma based on TCGA and GEO databases.

Hepatocellular carcinoma (HCC) has become the fifth most common cancer globally, with the second-highest mortality rate and poor survival outcomes. In our research, we aimed to use The Cancer Genome Atlas and gene expression omnibus databases to identify potential genetic biomarkers to predict and improve the survival rate of HCC patients. In GSE60502, GSE76427, and GSE84402, we performed differential expression analysis to obtain differentially expressed genes (DEGs). In the The Cancer Genome Atlas database, the FPKM expression profile was subjected to weighted gene co-expression analysis to obtain modules closely related to HCC. We received common genes by intersecting the genes in the module with the differential genes. Then, we fused the common genes' expression profiles, survival time, and survival status for univariate, Least Absolute Shrinkage and Selection Operator, and multivariate COX regression analysis to obtain prognostic genes. Predictive genes were performed in K-M survival analysis and combined with clinical data for independent predictive analysis. After differential expression analysis, GSE60502 obtained 1107 DEGs, GSE76427 obtained 424 DEGs, and GSE84402 obtained 1668 DEGs. Through weighted gene co-expression analysis analysis, we can see that the blue and brown modules were closely associated with HCC. After single and multivariate COX regression analysis, we found that suppressor of cytokine signaling 2 (SOCS2) and SERPINF2 were independent prognostic genes for HCC. After survival analysis, HCC patients with high expression of SOCS2 and SERPINF2 had a longer survival time. These 2 genes in normal liver tissues were higher than in HCC at the transcriptional level. SOCS2 and SERPINF2 were new independent prognostic genes of HCC. So, they may provide new treatment methods and measures for diagnosing HCC.

Epigenetic and genetic investigation of SOCS-1 gene in patients with multiple myeloma.

The suppressor of cytokine signaling-1 (SOCS-1) functions to induce an appropriate immune response and is an essential physiological regulator of interferon signaling. DNA methylation involves adding a methyl group to the carbon 5 position of cytosine. Besides comparing SOCS-1 gene methylation status between patients with multiple myeloma (MM) and healthy controls, this study also aimed to demonstrate the effect of SOCS-1 gene distribution and the effect of methylation of SOCS-1 on progression-free survival (PFS) and overall survival (OS). This study included 120 patients diagnosed with MM between January 2018 and 2020 and 80 healthy individuals. The distribution of the SOCS-1 genotypes was statistically compared between MM patients and healthy controls. Additionally, the statistically significant effects of these genotypes on survival were examined. The CA/CA genotype of SOCS-1 was significantly higher in healthy controls (P=0.001), while the Del/Del genotype was significantly higher in patients with MM (P=0.034). The percent methylated reference (PMR) value of the SOCS-1 gene was significantly higher in the healthy controls (median, 43.48; range, 2.76‒247.75; P=0.001). Patients with a PMR value of ≥43.48 were 3.125 times more likely to develop progression than those with a PMR value of ＜43.48. The effects of SOCS-1 polymorphisms on the pathogenesis of.

Novel Hydroxypyridine Compound Protects Brain Cells against Ischemic Damage In Vitro and In Vivo.

A non-surgical pharmacological approach to control cellular vitality and functionality during ischemic and/or reperfusion-induced phases of strokes remains extremely important. The synthesis of 2-ethyl-6-methyl-3-hydroxypyridinium gammalactone-2,3-dehydro-L-gulonate (3-EA) was performed using a topochemical reaction. The cell-protective effects of 3-EA were studied on a model of glutamate excitotoxicity (GluTox) and glucose-oxygen deprivation (OGD) in a culture of NMRI mice cortical cells. Ca2+ dynamics was studied using fluorescent bioimaging and a Fura-2 probe, cell viability was assessed using cytochemical staining with propidium iodide, and gene expression was assessed by a real-time polymerase chain reaction. The compound anti-ischemic efficacy in vivo was evaluated on a model of irreversible middle cerebral artery (MCA) occlusion in Sprague-Dawley male rats. Brain morphological changes and antioxidant capacity were assessed one week after the pathology onset. The severity of neurological disorder was evaluated dynamically. 3-EA suppressed cortical cell death in a dose-dependent manner under the excitotoxic effect of glutamate and ischemia/reoxygenation. Pre-incubation of cerebral cortex cells with 10-100 µM 3-EA led to significant stagnation in Ca2+ concentration in a cytosol ([Ca2+]i) of neurons and astrocytes suffering GluTox and OGD. Decreasing intracellular Ca2+ and establishing a lower [Ca2+]i baseline inhibited necrotic cell death in an acute experiment. The mechanism of 3-EA cytoprotective action involved changes in the baseline and ischemia/reoxygenation-induced expression of genes encoding anti-apoptotic proteins and proteins of the oxidative status; this led to inhibition of the late irreversible stages of apoptosis. Incubation of brain cortex cells with 3-EA induced an overexpression of the anti-apoptotic genes BCL-2, STAT3, and SOCS3, whereas the expression of genes regulating necrosis and inflammation (TRAIL, MLKL, Cas-1, Cas-3, IL-1β and TNFa) were suppressed. 3-EA 18.0 mg/kg intravenous daily administration for 7 days following MCA occlusion preserved rats' cortex neuron population, decreased the severity of neurological deficit, and spared antioxidant capacity of damaged tissues. 3-EA demonstrated proven short-term anti-ischemic activity in vivo and in vitro, which can be associated with antioxidant activity and the ability to target necrotic and apoptotic death. The compound may be considered a potential neuroprotective molecule for further pre-clinical investigation.

High-fat diet induced cyclophilin B enhances STAT3/lncRNA-PVT1 feedforward loop and promotes growth and metastasis in colorectal cancer

High-fat diet (HFD) has been implicated to promote colorectal cancer (CRC). Recently, oncogene Cyclophilin B (CypB) is reported to be induced by cholesterol. However, the role of CypB in CRC carcinogenesis and metastasis associated with HFD remains unknown. In the present study, we showed that HFD-induced CypB enhances proliferation and metastasis through an inflammation-driven circuit, including Signal Transducer and Activator of Transcription 3 (STAT3)-triggered transcription of lncRNA-PVT1, and its binding with CypB that promotes activation of STAT3. CypB was found to be upregulated in CRC, which was correlated with elevated body mass index and poor prognosis. HFD induced CypB expression and proinflammatory cytokines in colon of mice. Besides, CypB restoration facilitated growth, invasion and metastasis in CRC cells both in vitro and in vivo. Moreover, RIP sequencing data identified lncRNA-PVT1 as a functional binding partner of CypB. Mechanistically, PVT1 increased the phosphorylation and nuclear translocation of STAT3 in response to IL-6, through directly interaction with CypB, which impedes the binding of Suppressors Of Cytokine Signalling 3 (SOCS3) to STAT3. Furthermore, STAT3 in turn activated PVT1 transcription through binding to its promoter, forming a regulatory loop. Finally, this CypB/STAT3/PVT1 axis was verified in TCGA datasets and CRC tissue arrays. Our data revealed that CypB linked HFD and CRC malignancy by enhancing the CypB/STAT3/PVT1 feedforward axis and activation of STAT3.

Circulating microRNA203 and its target genes' role in psoriasis pathogenesis.

Numerous microRNAs (miRNAs) have been found to have an aberrant expression in the peripheral blood or psoriasis patients' lesions. Psoriasis was shown to have the abnormal expression of microRNA-203 (miR-203). It is a skin-specific signal that governs cellular proliferation in a protein kinase C-dependent manner and is mostly generated by keratinocytes. This work evaluated the expression levels of the circulating miR-203 target genes SOCS3, SOCS6, TP63, TNF-, IL8, and IL24 in psoriasis patients. Using a relative quantitation PCR technique, we determined the expression levels of miR-203 and its target genes (SOCS3, SOCS6, TP63, TNF-, IL8, and IL24) in the plasma of 120 psoriatic patients and matched healthy controls. The disease characteristics of the patients were then correlated with the expression results. We also conducted numerous enrichment analyses for the diseases, functions, and pathways connected to the under-researched biomarkers. Compared to healthy controls, psoriatic patients had significantly increased levels of miR-203 expression; 7.1 (4.4-9.9). In contrast, psoriatic patients had significantly lower expression of all the examined genes compared to healthy controls. Regarding all the study biomarkers, the receiver operating characteristic (ROC) curve analysis demonstrated significant sensitivity and specificity for differentiating between psoriatic patients and healthy controls. According to the results of the disease matching score generated by miR-203 and its target genes, psoriasis was ranked first with a score of 4.45. The third-place finisher with a value of 3.98, it also demonstrated that miR-203 and its target genes are connected to various skin disorders. Our results show that miR-203 contributes to psoriasis pathogenesis not only locally in skin lesions but also in circulation, indicating that it may contribute to the systemic symptoms of the illness. MiR-203 overexpression in psoriasis suggests that miR-203 may be involved in an anti-inflammatory response because it targets both SOCS gene family members and pro-inflammatory cytokines.

Involvement of the IL-6 Signaling Pathway in the Anti-Anhedonic Effect of the Antidepressant Agomelatine in the Chronic Mild Stress Model of Depression.

Neuroinflammation has emerged as an important factor in the molecular underpinnings of major depressive disorder (MDD) pathophysiology and in the mechanism of action of antidepressants. Among the inflammatory mediators dysregulated in depressed patients, interleukin (IL)-6 has recently been proposed to play a crucial role. IL-6 activates a signaling pathway comprising the JAK/STAT proteins and characterized by a specific negative feedback loop exerted by the cytoplasmic protein suppressor of cytokine signalling-3 (SOCS3). On these bases, here, we explored the potential involvement of IL-6 signaling in the ability of the antidepressant drug agomelatine to normalize the anhedonic-like phenotype induced in the rat by chronic stress exposure. To this aim, adult male Wistar rats were subjected to the chronic mild stress (CMS) paradigm and chronically treated with vehicle or agomelatine. The behavioral evaluation was assessed by the sucrose consumption test, whereas molecular analyses were performed in the prefrontal cortex. We found that CMS was able to stimulate IL-6 production and signaling, including SOCS3 gene and protein expression, but the SOCS3-mediated feedback-loop inhibition failed to suppress the IL-6 cascade in stressed animals. Conversely, agomelatine treatment normalized the stress-induced decrease in sucrose consumption and restored the negative modulation of the IL-6 signaling via SOCS3 expression and activity. Our results provide additional information about the pleiotropic mechanisms that contribute to agomelatine’s therapeutic effects.

Effects of suppressor of cytokine signaling 3 (SOCS3) on the development of colon cancer via regulation of HIF-1α

Purpose: To investigate the influence of suppressor of cytokine signaling 3 (SOCS3) on rats with colon cancer (CC).
 Methods: Sprague-Dawley (SD) rats were randomly divided into CC group and control group. CC models were constructed. The expression of SOCS3 in CC tissues was determined by quantitative real time-polymerase chain reaction (qRT-PCR). Hematoxylin-eosin staining (H&E) was used to examine colon tissue morphology, while immunohistochemistry (IHC) staining assay was performed to determine the expression of SOCS3 protein in colon tissues. The content of HIF-1α, phosphorylated phosphatidylinositol 3-hydroxy kinase (p-PI3K), and phosphorylated protein kinase B (p-AKT) proteins was determined by Western blotting (WB).
 Results: Compared with that in the control group, the number of tumors in the CC group was significantly increased (p < 0.05). Protein and messenger ribonucleic acid (mRNA) expressions of SOCS3 were down-regulated in CC group (p < 0.05), while protein expressions of p-PI3K, p-AKT and HIF-1α were significantly elevated in CC group (p < 0.05).
 Conclusion: SOCS3 is poorly expressed in CC rats, and promotes the expression of HIF-1α by activating PI3K/AKT signaling pathway. The findings, thus, provide a probable strategy for management of colon cancer.

Epigenome-wide association study of biomarkers of liver function identifies albumin-associated DNA methylation sites among male veterans with HIV.

Background: Liver disease (LD) is an important cause of morbidity and mortality for people with HIV (PWH). The molecular factors linked with LD in PWH are varied and incompletely characterized. We performed an epigenome-wide association study (EWAS) to identify associations between DNA methylation (DNAm) and biomarkers of liver function-aspartate transaminase, alanine transaminase, albumin, total bilirubin, platelet count, FIB-4 score, and APRI score-in male United States veterans with HIV. Methods: Blood samples and clinical data were obtained from 960 HIV-infected male PWH from the Veterans Aging Cohort Study. DNAm was assessed using the Illumina 450K or the EPIC 850K array in two mutually exclusive subsets. We performed a meta-analysis for each DNAm site measured by either platform. We also examined the associations between four measures of DNAm age acceleration (AA) and liver biomarkers. Results: Nine DNAm sites were positively associated with serum albumin in the meta-analysis of the EPIC and 450K EWAS after correcting for multiple testing. Four DNAm sites (cg16936953, cg18942579, cg01409343, and cg12054453), annotated within the TMEM49 and four of the remaining five sites (cg18181703, cg03546163, cg20995564, and cg23966214) annotated to SOCS3, FKBP5, ZEB2, and SAMD14 genes, respectively. The DNAm site, cg12992827, was not annotated to any known coding sequence. No significant associations were detected for the other six liver biomarkers. Higher PhenoAA was significantly associated with lower level of serum albumin (β = -0.007, p-value = 8.6 × 10-4, CI: -0.011116, -0.002884). Conclusion: We identified epigenetic associations of both individual DNAm sites and DNAm AA with liver function through serum albumin in men with HIV. Further replication analyses in independent cohorts are warranted to confirm the epigenetic mechanisms underlying liver function and LD in PWH.

Inflammasome Activation Dampens Type I IFN Signaling to Strengthen Anti-Toxoplasma Immunity.

Innate immunity acts as the first line of defense against pathogen invasion. During Toxoplasma gondii infection, multiple innate immune sensors are activated by invading microbes or pathogen-associated molecular patterns (PAMPs). However, how inflammasome is activated and its regulatory mechanisms during T. gondii infection remain elusive. Here, we showed that the infection of PRU, a lethal type II T. gondii strain, activates inflammasome at the early stage of infection. PRU tachyzoites, RNA and soluble tachyzoite antigen (STAg) mainly triggered the NLRP3 inflammasome, while PRU genomic DNA (gDNA) specially activated the AIM2 inflammasome. Furthermore, mice deficient in AIM2, NLRP3, or caspase-1/11 were more susceptible to T. gondii PRU infection, and the ablation of inflammasome signaling impaired antitoxoplasmosis immune responses by enhancing type I interferon (IFN-I) production. Blockage of IFN-I receptor fulfilled inflammasome-deficient mice competent immune responses as WT mice. Moreover, we have identified that the suppressor of cytokine signaling 1 (SOCS1) is a key negative regulator induced by inflammasome-activated IL-1β signaling and inhibits IFN-I production by targeting interferon regulatory factor 3 (IRF3). In general, our study defines a novel protective role of inflammasome activation during toxoplasmosis and identifies a critical regulatory mechanism of the cross talk between inflammasome and IFN-I signaling for understanding infectious diseases. IMPORTANCE As a key component of innate immunity, inflammasome is critical for host antitoxoplasmosis immunity, but the underlying mechanisms are still elusive. In this study, we found that inflammasome signaling was activated by PAMPs of T. gondii, which generated a protective immunity against T. gondii invasion by suppressing type I interferon (IFN-I) production. Mechanically, inflammasome-coupled IL-1β signaling triggered the expression of negative regulator SOCS1, which bound to IRF3 to inhibit IFN-I production. The role of IFN-I in anti-T. gondii immunity is little studied and controversial, and here we also found IFN-I is harmful to host antitoxoplasmosis immunity by using knockout mice and recombinant proteins. In general, our study identifies a protective role of inflammasomes to the host during T. gondii infection and a novel mechanism by which inflammasome suppresses IFN-I signaling in antitoxoplasmosis immunity, which will likely provide new insights into therapeutic targets for toxoplasmosis and highlight the cross talk between innate immune signaling in infectious diseases prevention.

Protein interaction, cytotoxic, transcriptomic and proteomic responses to structurally distinct EPAC1 activators in HUVECs

The N-acylsulfonamide derivative, I942, represents the first non-cyclic nucleotide partial agonist of EPAC1. This was soon followed by the identification of the I942 analogues, PW0381, PW0521 and PWO577 and a series of benzofuran oxoacetic acid EPAC1 activators, SY006, SY007 and SY009. Protein interaction, cytotoxicity and EPAC1 activation assays applied here identify PWO577 and SY007 as being effective EPAC1 binders that are well tolerated in HUVECs at concentrations greater than 100 μM and up to 48 h incubation and are effective activators of transfected EPAC1 in U2OS cells. Using RNAseq in HUVECs we show that PWO577 and SY007 regulate approximately 11,000 shared genes, with only few differential gene changes being “off-target”. The genes significantly regulated by both PWO577 and SY007 included a subset of genes normally associated with endothelial activation, including ICAM1, MMP1 and CCL2. Of these, only the expression of MMP1 was markedly increased at the protein level, as determined by LC–MS-based proteomics. Both PWO577 and SY007 suppressed IL-6-induced STAT3 activation and associated downstream gene expression, including inhibition of SOCS3, STAT3, IL6ST and JAK3 genes. Together these results demonstrate the utility of structurally distinct, specific and non-toxic EPAC1 activators. Future modifications will be aimed at eliminating the few noted off-target effects.

Unravelling the role of SOCS3 in aortic valve stenosis

Abstract Background Calcific aortic valve stenosis (CAVD) is the most prevalent heart valve disease worldwide and, in advanced stages, leads to clinical deterioration and poor prognosis. Until now, no medical treatment is available. Thought to be a merely degenerative disease, we now know that disease initiation and progression are actively regulated by immune cell infiltration, chronic inflammation, osteogenic differentiation of valvular interstitial cells (VIC) and endothelial-to-mesenchymal transition (EndMT) of valvular endothelial cells (VEC). Suppressor of Cytokine Signaling 3 (SOCS3) is known to be a key regulator of inflammation. It promotes the polarisation of macrophages to an inflammatory phenotype and thus, contributes to disease progression in atherosclerosis. In CAVD, not only inflammation but also heterotopic bone formation leads to the thickening and obstruction of the aortic valve cusps and as SOCS3 is one of the main regulators of physiological bone formation, it could play a critical role in the progression of CAVD. Purpose We hypothesize that SOCS3 plays a crucial role during calcification of the aortic valve through regulation of EndMT, calcification of valvular interstitial cells and macrophage polarization. Methods and results In initial screening experiments, we investigated SOCS3-protein expression in explanted human aortic valves from patients undergoing surgical aortic valve replacement. SOCS3 is expressed in both stenotic and non-stenotic valve tissues. Immunofluorescence-staining of human aortic valves shows the co-localisation of SOCS3 with the interstitial cell-marker Vimentin and SOCS1, which is known to regulate macrophage-polarisation together with SOCS3 (Fig. 1). Staining of the macrophage-marker CD68 revealed its co-localisation with calcified areas of the aortic valve cusp. We validated our findings in our in vitro model of VIC calcification using two different calcifying conditions. Upregulation of RUNX2 and BMP2 verified successful osteogenic differentiation of VICs. During osteogenic differentiation for 7 days, SOCS3 and SOCS1 are significantly upregulated (Fig. 2A). We were able to induce EndMT by stimulating VEC with either TNFα or TGFβ/IL1β in vitro. We show that EndMT leads to the loss of endothelial cell markers like eNOS and VWF and to an upregulation of interstitial cell markers (such as Vimentin and α-SMA) and markers of EndMT (SNAI2). During EndMT, we were also able to observe a significant upregulation of SOCS1 and SOCS3 after 7 days (Fig. 2B). Ongoing knockdown experiments will help to elucidate the role of SOCS1 and SOCS3 during initiation and progression of CAVD. Conclusion We aim towards a better understanding of SOCS3 and its role in inflammation and calcification as hallmarks of the pathogenesis of CAVD. Since Zoledronic acid has been shown to induce a decreased expression of SOCS3 in macrophages, this might present as a possible treatment strategy to target SOCS3 therapeutically. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Deutsche Forschungsgemeinschaft

Harnessing tumorous flaws for immune supremacy: is miRNA-155 the weak link in breast cancer progression?

With the advent of immune checkpoint blockade (ICB) therapy, treatment strategies for late-stage cancers have seen a radical advancement. In this issue of the JCI, Wang et al. characterize the functional role of miR-155 in breast cancer and its potential in harnessing the efficacy of immunotherapy. The study reports that high expression levels of miR-155 in breast cancer cells downregulated suppressor of cytokine signaling 1 (SOCS1), increased the phosphorylated STAT1 (pSTAT1)/pSTAT3 ratio, and thereby stimulated chemoattractants for tumor infiltration of effector T cells. Moreover, miR-155 overexpression set the stage for ICB therapy via increased programmed death ligand 1 (PD-L1) expression on cancer cells and enhanced immunological memory response via the release of miR-155–containing extracellular vesicles. Collectively, these data suggest that miR-155 is a strong candidate as a prognostic biomarker for ICB therapy.

Opening KATP channels induces inflammatory tolerance and prevents chronic pain

Current treatments for chronic pain are unsatisfactory, therefore, new therapeutics are urgently needed. Our previous study indicated that KATP channel openers have analgesic effects, but the underlying mechanism has not been elucidated. We speculated that KATP channel openers might increase suppressor of cytokine signaling (SOCS)-3 expression to induce inflammatory tolerance and attenuate chronic pain. Postoperative pain was induced by plantar incision to establish a chronic pain model. Growth arrest–specific 6 (Gas6)−/− and Axl−/− mice were used for signaling studies. The microglia cell line BV-2 was cultured for the in vitro experiments. The KATP channel opener significantly attenuated incision-induced mechanical allodynia in mice associated with the upregulated expression of SOCS3. Opening KATP channels induced the expression of SOCS3 in the Gas6/Axl signaling pathway in microglia, inhibited incision-induced mechanical allodynia by activating the Gas6/Axl-SOCS3 signaling pathway, and induced inflammatory tolerance to relieve neuroinflammation and postoperative pain. We demonstrated that opening of the KATP channel opening activated Gas6/Axl/SOCS3 signaling to induce inflammatory tolerance and relieve chronic pain. We explored a new target for anti-inflammatory and analgesic effects by regulating the innate immune system and provided a theoretical basis for clinical preemptive analgesia.

Tumor necrosis factor-α promotes lipolysis and reduces insulin sensitivity by activating nuclear factor kappa B and c-Jun N-terminal kinase in primary bovine adipocytes.

Sustained lipolysis and insulin resistance increase the risk of metabolic dysfunction in dairy cows during the transition period. Proinflammatory cytokines are key regulators of adipose tissue metabolism in nonruminants, but biological functions of these molecules in ruminants are not well known. Thus, the objective of this study was to investigate whether tumor necrosis factor-α (TNF-α) could affect insulin sensitivity and lipolysis in bovine adipocytes as well as the underlying mechanisms. Bovine adipocytes (obtained from the omental and mesenteric adipose depots) isolated from 5 Holstein female calves (1 d old) with similar body weight (median: 36.9 kg, range: 35.5-41.2 kg) were differentiated and used for (1) treatment with different concentrations of TNF-α (0, 0.1, 1, or 10 ng/mL) for 12 h; (2) pretreatment with 10 μM lipolytic agonist isoproterenol (ISO) for 3 h, followed by treatment with or without 10 ng/mL TNF-α for 12 h; and (3) pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 (20 μM for 2 h) and nuclear factor kappa B (NF-κB) inhibitor BAY 11-7082 (10 μM for 1 h) followed by treatment with or without 10 ng/mL TNF-α for 12 h. The TNF-α increased glycerol content in supernatant, decreased triglyceride content and insulin-stimulated phosphorylation of protein kinase B suggesting activation of lipolysis and impairment of insulin sensitivity. The TNF-α reduced cell viability, upregulated mRNA abundance of Caspase 3 (CASP3), an apoptosis marker, and increased activity of Caspase 3. In addition, increased phosphorylation of NF-κB and JNK, upregulation of mRNA abundance of interleukin-6 (IL-6), TNFA, and suppressor of cytokine signaling 3 (SOCS3) suggested that TNF-α activated NF-κB and JNK signaling pathways. Furthermore, ISO plus TNF-α-activated NF-κB and JNK signaling pathway to a greater extent than TNF-α alone. Combining TNF-α and ISO aggravated TNF-α-induced apoptosis, insulin insensitivity and lipolysis. In the absence of TNF-α, inhibition of NF-κB and JNK did not alter glycerol content in supernatant, triglyceride content or insulin-stimulated phosphorylation of protein kinase B. In the presence of TNF-α, inhibition of NF-κB and JNK alleviated TNF-α-induced apoptosis, insulin insensitivity and lipolysis. Overall, TNF-α impairs insulin sensitivity and induces lipolysis and apoptosis in bovine adipocytes, which may be partly mediated by activation of NF-κB and JNK. Thus, the data suggested that NF-κB and JNK are potential therapeutic targets for alleviating lipolysis dysregulation and insulin resistance in adipocytes.

Leptin receptor in osteocytes promotes cortical bone consolidation in female mice.

Bone strength is partially determined during cortical bone consolidation, a process comprising coalescence of peripheral trabecular bone and its progressive mineralisation. Mice with genetic deletion of suppressor of cytokine signalling 3 (Socs3), an inhibitor of STAT3 signalling, exhibit delayed cortical bone consolidation, indicated by high cortical porosity, low mineral content, and low bone strength. Since leptin receptor (LepR) is expressed in the osteoblast lineage and is suppressed by SOCS3, we evaluated whether LepR deletion in osteocytes would rectify the Dmp1cre.Socs3fl/fl bone defect. First, we tested LepR deletion in osteocytes by generating Dmp1cre.LepRfl/fl mice and detected no significant bone phenotype. We then generated Dmp1cre.Socs3fl/fl.LepRfl/fl mice and compared them to Dmp1cre.Socs3fl/fl controls. Between 6 and 12 weeks of age, both Dmp1cre.Socs3fl/fl.LepRfl/fl and control (Dmp1cre.Socs3fl/fl) mice showed an increasing proportion of more heavily mineralised bone, indicating some cortical consolidation with time. However, at 12 weeks of age, rather than resolving the phenotype, delayed consolidation was extended in female Dmp1cre.Socs3fl/fl.LepRfl/fl mice. This was indicated in both metaphysis and diaphysis by greater proportions of low-density bone, lower proportions of high-density bone, and greater cortical porosity than Dmp1cre.Socs3fl/fl controls. There was also no change in the proportion of osteocytes staining positive for phospho-STAT3, suggesting the effect of LepR deletion in Dmp1cre.Socs3fl/fl mice is STAT3-independent. This identifies a new role for leptin signalling in bone which opposes our original hypothesis. Although LepR in osteocytes has no irreplaceable physiological role in normal bone maturation, when STAT3 is hyperactive, LepR in Dmp1Cre-expressing cells supports cortical consolidation.