MiR-19a: an effective regulator of SOCS3 and enhancer of JAK-STAT signalling.
ABSTRACT: Suppressors of cytokine signalling (SOCS) proteins are classic inhibitors of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Many cytokines and pathogenic mediators induce expression of SOCS, which act in a negative feedback loop to inhibit further signal transduction. SOCS mRNA expression is regulated by DNA binding of STAT proteins, however, their post-transcriptional regulation is poorly understood. microRNAs (miRNAs) are small non-coding RNAs that bind to complementary sequences on target mRNAs, often silencing gene expression. miR-19a has been shown to regulate SOCS1 expression during mutiple myeloma and be induced by the anti-viral cytokine interferon-(IFN)-?, suggesting a role in the regulation of the JAK-STAT pathway. This study aimed to identify targets of miR-19a in the JAK-STAT pathway and elucidate the functional consequences. Bioinformatic analysis identified highly conserved 3'UTR miR-19a target sequences in several JAK-STAT associated genes, including SOCS1, SOCS3, SOCS5 and Cullin (Cul) 5. Functional studies revealed that miR-19a significantly decreased SOCS3 mRNA and protein, while a miR-19a antagomir specifically reversed its inhibitory effect. Furthermore, miR-19a-mediated reduction of SOCS3 enhanced IFN-? and interleukin (IL)-6 signal transduction through STAT3. These results reveal a novel mechanism by which miR-19a may augment JAK-STAT signal transduction via control of SOCS3 expression and are fundamental to the understanding of inflammatory regulation.
Project description:Activation of Janus kinase/signal transducers and activators of transcription (JAK/STAT) is an important mechanism by which hyperglycemia contributes to renal damage, suggesting that modulation of this pathway may prevent renal and vascular complications of diabetes. Here, we investigated the involvement of suppressors of cytokine signaling (SOCS) as intracellular negative regulators of JAK/STAT activation in diabetic nephropathy. In a rat model, inducing diabetes resulted in JAK/STAT activation and increased expression of SOCS1 and SOCS3. In humans, we observed increased expression of glomerular and tubulointerstitial SOCS proteins in biopsies of patients with diabetic nephropathy. In vitro, high concentrations of glucose activated JAK/STAT/SOCS in human mesangial and tubular cells. Overexpression of SOCS reversed the glucose-induced activation of the JAK/STAT pathway, expression of STAT-dependent genes (chemokines, growth factors, and extracellular matrix proteins), and cell proliferation. In vivo, intrarenal delivery of adenovirus expressing SOCS1 and SOCS3 to diabetic rats significantly improved renal function and reduced renal lesions associated with diabetes, such as mesangial expansion, fibrosis, and influx of macrophages. SOCS gene delivery also decreased the activation of STAT1 and STAT3 and the expression of proinflammatory and profibrotic proteins in the diabetic kidney. In summary, these results provide direct evidence for a link between the JAK/STAT/SOCS axis and hyperglycemia-induced cell responses in the kidney. Suppression of the JAK/STAT pathway by increasing intracellular SOCS proteins may have therapeutic potential in diabetic nephropathy.
Project description:The use of bone marrow-derived human multipotent stromal cells (hMSC) in cell-based therapies has dramatically increased in recent years, as researchers have exploited the ability of these cells to migrate to sites of tissue injury, inflammation, and tumors. Our group established that hMSC respond to "danger" signals--by-products of damaged, infected or inflamed tissues--via activation of Toll-like receptors (TLRs). However, little is known regarding downstream signaling mediated by TLRs in hMSC.We demonstrate that TLR3 stimulation activates a Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 1 pathway, and increases expression of suppressor of cytokine signaling (SOCS) 1 and SOCS3 in hMSC. Our studies suggest that each of these SOCS plays a distinct role in negatively regulating TLR3 and JAK/STAT signaling. TLR3-mediated interferon regulatory factor 1 (IRF1) expression was inhibited by SOCS3 overexpression in hMSC while SOCS1 overexpression reduced STAT1 activation. Furthermore, our study is the first to demonstrate that when TLR3 is activated in hMSC, expression of CXCR4 and CXCR7 is downregulated. SOCS3 overexpression inhibited internalization of both CXCR4 and CXCR7 following TLR3 stimulation. In contrast, SOCS1 overexpression only inhibited CXCR7 internalization.These results demonstrate that SOCS1 and SOCS3 each play a functionally distinct role in modulating TLR3, JAK/STAT, and CXCR4/CXCR7 signaling in hMSC and shed further light on the way hMSC respond to danger signals.
Project description:Macrophage phenotype plays a crucial role in the pathogenesis of Leishmanial infection. Pro-inflammatory cytokines signals through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway that functions in parasite killing. Suppression of cytokine signaling (SOCS) is a well-known negative feedback regulator of the JAK/STAT pathway. However, change in the expression levels of SOCSs in correlation with the establishment of infection is not well understood. IL6 is a pleotropic cytokine that induces SOCS1 and SOCS3 expression through JAK-STAT signaling. Mathematical modeling of the TLR2 and IL6 signaling pathway has established the immune axis of SOCS1 and SOCS3 functioning in macrophage polarization during the early stage of Leishmania major infection. The ratio has been quantified both in silico and in vitro as 3:2 which is required to establish infection during the early stage. Furthermore, phosphorylated STAT1 and STAT3 have been established as an immunological cross talk between TLR2 and IL6 signaling pathways. Using synthetic biology approaches, peptide based immuno-regulatory circuits have been designed to target the activity of SOCS1 which can restore pro-inflammatory cytokine expression during infection. In a nutshell, we explored the potential of synthetic biology to address and rewire the immune response from Th2 to Th1 type during the early stage of leishmanial infection governed by SOCS1/SOCS3 immune axis.
Project description:In host innate immunity, type I interferons (IFN-I) are major antiviral molecules, and coronaviruses have evolved diverse strategies to counter the IFN-I response during infection. Transmissible gastroenteritis virus (TGEV), a member of the Alphacoronavirus family, induces endoplasmic reticulum (ER) stress and significant IFN-I production after infection. However, how TGEV evades the IFN-I antiviral response despite the marked induction of endogenous IFN-I has remained unclear. Inositol-requiring enzyme 1 ? (IRE1?), a highly conserved ER stress sensor with both kinase and RNase activities, is involved in the IFN response. In this study, IRE1? facilitated TGEV replication via downmodulating the host microRNA (miR) miR-30a-5p abundance. miR-30a-5p normally enhances IFN-I antiviral activity by directly targeting the negative regulators of Janus family kinase (JAK)-signal transducer and activator of transcription (STAT), the suppressor of cytokine signaling protein 1 (SOCS1), and SOCS3. Furthermore, TGEV infection increased SOCS1 and SOCS3 expression, which dampened the IFN-I antiviral response and facilitated TGEV replication. Importantly, compared with mock infection, TGEV infection in vivo resulted in decreased miR-30a-5p levels and significantly elevated SOCS1 and SOCS3 expression in the piglet ileum. Taken together, our data reveal a new strategy used by TGEV to escape the IFN-I response by engaging the IRE1?-miR-30a-5p/SOCS1/3 axis, thus improving our understanding of how TGEV escapes host innate immune defenses.IMPORTANCE Type I interferons (IFN-I) play essential roles in restricting viral infections. Coronavirus infection induces ER stress and the interferon response, which reflects different adaptive cellular processes. An understanding of how coronavirus-elicited ER stress is actively involved in viral replication and manipulates the host IFN-I response has remained elusive. Here, TGEV inhibited host miR-30a-5p via the ER stress sensor IRE1?, which led to the increased expression of negative regulators of JAK-STAT signaling cascades, namely, SOCS1 and SOCS3. Increased SOCS1 or SOCS3 expression impaired the IFN-I antiviral response, promoting TGEV replication. These findings enhance our understanding of the strategies used by coronaviruses to antagonize IFN-I innate immunity via IRE1?-mediated manipulation of the miR-30a-5p/SOCS axis, highlighting the crucial role of IRE1? in innate antiviral resistance and the potential of IRE1? as a novel target against coronavirus infection.
Project description:The SOCS family of proteins are negative-feedback inhibitors of signalling induced by cytokines that act via the JAK/STAT pathway. SOCS proteins can act as ubiquitin ligases by recruiting Cullin5 to ubiquitinate signalling components; however, SOCS1, the most potent member of the family, can also inhibit JAK directly. Here we determine the structural basis of both these modes of inhibition. Due to alterations within the SOCS box domain, SOCS1 has a compromised ability to recruit Cullin5; however, it is a direct, potent and selective inhibitor of JAK catalytic activity. The kinase inhibitory region of SOCS1 targets the substrate binding groove of JAK with high specificity and thereby blocks any subsequent phosphorylation. SOCS1 is a potent inhibitor of the interferon gamma (IFN?) pathway, however, it does not bind the IFN? receptor, making its mode-of-action distinct from SOCS3. These findings reveal the mechanism used by SOCS1 to inhibit signalling by inflammatory cytokines.
Project description:The Janus kinase and signal transducer and activator of transcription (JAK-STAT) pathway genes along with suppressors of cytokine signalling (SOCS) family genes play a crucial role in controlling cytokine signals in the mammary gland and thus mammary gland development. Mammary gene expression studies showed differential expression patterns for all the JAK-STAT pathway genes. Gene expression studies using qRT-PCR revealed differential expression of SOCS2, SOCS4, and SOCS5 genes across the lactation cycle in dairy cows. Using genotypes from 1,546 Australian Holstein-Friesian bulls, a statistical model for an association analysis based on SNPs within 500 kb of JAK-STAT pathway genes, and SOCS genes alone was constructed. The analysis suggested that these genes and pathways make a significant contribution to the Australian milk production traits. There were 24 SNPs close to SOCS1, SOCS3, SOCS5, SOCS7, and CISH genes that were significantly associated with Australian Profit Ranking (APR), Australian Selection Index (ASI), and protein yield (PY). This study supports the view that there may be some merit in choosing SNPs around functionally relevant genes for the selection and genetic improvement schemes for dairy production traits.
Project description:<h4>Aims</h4>The suppressors of cytokine signalling (SOCS) are identified inhibitors of cytokine and growth factor signalling that act via the Janus kinase (JAK) signal transducers and activators of transcription (STAT) pathways. Aberrant JAK/STAT signalling promotes progression from hypertrophy to heart failure. Little information is available concerning the role of SOCS in the transition from hypertrophy to heart failure. To this aim, we investigated the effects of SOCS1 overexpression obtained by in vivo adeno-associated gene transfer using an aortopulmonary cross-clamping technique in a chronic pressure-overload cardiac rat model.<h4>Methods and results</h4>Rats were randomized into four groups: sham-operated (n = 18), aortic banding (AB) (n = 18), AB + viral vector encoding for haemoagglutinin (AB + HA, n = 16), and AB + viral vector encoding for SOCS1 (AB + SOCS1, n = 18). Echocardiographic and haemodynamic measurements were performed 15 weeks after banding. While SOCS3 was upregulated during the hypertrophic phase, SOCS1 transcript levels increased significantly between 15 and 20 weeks. Remodelling was markedly worse in AB + SOCS1, showed larger left ventricular internal dimensions (+16%), higher end-diastolic pressures (+57%) and wall stress (+45%), and reduced fractional shortening (-32%) compared with AB + HA; apoptotic rate was increased three-fold and the gp130 pathway was inhibited. Ex vivo experiments showed that mechanical stretch upregulated SOCS1 expression, which was in turn attenuated by tumour necrosis factor-? (TNF-?) inhibition.<h4>Conclusion</h4>Enhanced SOCS1 myocardial signalling is associated with accelerated transition from hypertrophy to failure in an established model of pressure overload. SOCS1 may represent an attractive target for the prevention of heart failure progression.
Project description:In mammals, local production of tumor necrosis factor ? (TNF?) inhibits growth hormone (GH)-induced IGF-I expression at tissue level and contributes to GH resistance caused by sepsis/endotoxemia and inflammation. Although the loss of GH responsiveness can be mediated by a parallel rise in SOCS expression, the signaling mechanisms for TNF?-induced SOCS expression at the hepatic level have not been characterized and the comparative aspects of the phenomenon, especially in lower vertebrates, are still unknown. Recently, type II SOCS, including SOCS1-3 and CISH, have been cloned in grass carp and shown to act as the feedback repressors for GH signaling via JAK2/STAT5 pathway. To shed light on the mechanisms for TNF?-induced GH resistance in fish model, grass carp TNF? was cloned and confirmed to be a single-copy gene expressed in various tissues including the liver. In carp hepatocytes, incubation with the endotoxin LPS induced TNF? expression with parallel rises in SOCS1-3 and CISH mRNA levels. Similar to LPS, TNF? treatment could block GH-induced IGF-I/-II mRNA expression and elevate SOCS1, SOCS3, and CISH transcript levels. However, TNF? was not effective in altering SOCS2 expression. In parallel experiment, LPS blockade of IGF-I/-II signals caused by GH could be partially reverted by TNF? receptor antagonism. At hepatocyte level, TNF? induction also triggered rapid phosphorylation of I?B?, MEK1/2, ERK1/2, MKK3/6, P38 MAPK, Akt, JAK2, and STAT1,3,5, and TNF?-induced SOCS1, SOCS3, and CISH mRNA expression could be negated by inhibiting the IKK/NF?B, MAPK, PI3K/Akt, and JAK/STAT cascades. Our findings, as a whole, suggest that local production of TNF? may interfere with IGF-I/-II induction by GH in the carp liver by up-regulation of SOCS1, SOCS3, and CISH via IKK/NF?B, MAPK, PI3K/Akt, and JAK/STAT-dependent mechanisms, which may contribute to GH resistance induced by endotoxin in carp species.
Project description:The biological basis of essential thrombocythemia (ET) patients lacking known mutations is still unknown. MicroRNAs (miRNA) regulate hematopoietic differentiation and are deregulated in several hematopoietic malignancies. However, miRNA expression in ET patients has been poorly explored. We performed miRNA profiling in platelets from 19 ET patients and 10 healthy controls. Hierarchical cluster analysis showed two well-separated clusters between patients and controls, indicating that ET platelets had a characteristic 70-miRNA signature (P<0.0001), 68 of which were downregulated. According to the mutational status, three differentially expressed miRNAs, miR-15a (P=0.045), miR-150 (P=0.001) and miR-519a (P=0.036), were identified. A 40-miRNA signature was identified characterizing JAK2V617F-positive ET patients. Eight genes, whose interaction with the miRNAs could activate the JAK/STAT pathway were identified. An inverse correlation was observed between miRNAs expression and their target genes for SOCS1 and miR-221, SOCS3 and miR-221, SOCS3 and miR-203, and PTPN11 and miR-23a. All three miRNAs were upregulated in JAK2V617F-negative ET patients. SOCS1 and SOCS3 were validated as targets of miR-221 and miR-203, respectively. In summary, our study shows that platelets from JAK2V617F-negative ET patients harbor a specific miRNA signature that can participate in the modulation of the JAK/STAT pathway through regulation of key genes as SOCS1 and SOCS3.
Project description:Interferon-alpha (pegylated interferon and ribavirin) is used as standard-of-care therapeutic for chronic hepatitis C virus infection. Besides good cure in some patients other patients do not benefit from the treatment dependent on the virus type and host factors. One class of putative effector proteins is the family of Suppressors of cytokine signalling (SOCS). They act in a classical negative feedback-loop against the action of interferons and many other cytokines. It has been proven that some of them, in particular SOCS1 and SOCS3, inhibit the expression of interferon induced antiviral proteins. Their mode of action depends on the signal they are interfering with. In relation to the interferon-gamma pathway, they are thought to act on the interferon-alpha receptors by masking its recognition site for the Janus kinases (JAK), by blocking the kinase activity of the JAKs and coincidentally hindering STAT molecules from binding to the kinases. They are also thought to ubiquitinate the JAKs resulting in their proteosomal degradation. The function of SOCS proteins in suppressing the interferon-alpha pathway has not yet been characterized exhaustively. This study should unveil links to understand the resistance in interferon-alpha therapy. As results we got almost complete silencing of JAK-STAT signaling in SOCS1 over-expressing cells and tissue-dependent partially suppressed gene induction in SOCS3 over-expressing cell lines. Two human cancer cell lines (ME-15, HuH-7) were stably transfected with pcDNA3.1-SOCS plasmids in presence of geneticin and daughter cell lines were generated after singularization of cells. Next, original cell lines as well as SOCS1 and SOCS3 over-expressing cell lines were treated with 1000 U/ml interferon-alpha for 4 or 24 hours or in normal culture medium. Cells lines obtained from SOCS4 plasmid transfections were screened as additional control. Gene expression levels of cell cultured in control (0 for 4 hours, 2 for 24 hours) or interferon-alpha supplemented medium for 4 hours (1) or 24 hours (4) were analyzed. mRNA abundance was measured in triplicates using 12x8-sample commercial Illumina microarrays (HumanRef 8, version 3) and scanner system (iScan) as well as reagents recommend by Illumina (Illumina® TotalPrep Kit).