Interleukin-6 deficiency modulates testicular function by increasing the expression of suppressor of cytokine signaling 3 (SOCS3) in mice.

Several cytokines have been reported to participate in spermatogenesis, including interleukin-6 (IL6). However, not many studies have been conducted on the loss of Il6 on the male reproductive tract. Nonetheless, there is considerable knowledge regarding the pathological and physiological role of IL6 on spermatogenesis. In this way, this study evaluated the impact of Il6 deficiency on mice testicles in the absence of infection or inflammation. We showed that Il6 deficiency increases daily sperm production, the number of spermatids, and the testicular testosterone and dihydrotestosterone levels. Besides that, mice with a deleted Il6 (IL6KO) showed increased testicular SOCS3 levels, with no changes in pJAK/JAK and pSTAT3/STAT3 ratios. It is worth noting that the aforementioned pathway is not the only pathway to up-regulate SOCS3, nor is it the only SOCS3 target, thus proposing that the increase of SOCS3 in the testis occurs independently of the JAK-STAT signaling in IL6KO mice. Therefore, we suggest that the lack of Il6 drives androgenic production by increasing SOCS3 in the testis, thus leading to an increase in spermatogenesis.

YY1 promotes SOCS3 expression to inhibit STAT3­mediated neuroinflammation and neuropathic pain.

Neuropathic pain is induced by primary injury and dysfunction of the nervous system, and is accompanied by the activation of inflammation signaling pathways. Yin Yang 1 (YY1) is reported to be involved in inflammation; however, its role in the development of neuropathic pain is still unclear. In the present study, a neuropathic pain model was established using the bilateral chronic constriction injury (bCCI) method in rats. The indexes of neuropathic pain were detected, including paw mechanical withdrawal threshold (MWT), paw thermal withdrawal latency (PTWL) and paw frequency in response to cold stimulus, characterizing the symptoms of mechanical allodynia, thermal hyperalgesia and cold hyperalgesia, respectively. YY1 mRNA expression was significantly decreased in the spinal cord cells of bCCI rats. In addition, YY1 was overexpressed in the bCCI rats by intrathecally injecting different doses of the pcDNA­YY1. YY1 reduced rat mechanical allodynia, thermal hyperalgesia and cold hyperalgesia in a dose­dependent manner. Furthermore, YY1 increased the expression of suppressor of cytokine signaling 3 (SOCS3) and suppressed signal transducer and activator of transcription 3 (STAT3)­mediated production of inflammatory factors in a dose­dependent manner. Finally, YY1 were respectively overexpressed and knocked down in primary spinal cord cells. The results revealed that YY1 overexpression promoted SOCS3 expression, increased cell proliferation and suppressed cell apoptosis, and reduced the activation of STAT3 and STAT3­mediated production of inflammatory factors. YY1 knockdown induced the opposite effect to that observed following YY1 overexpression. Furthermore, blockade of SOCS3 by SOCS3­antibody abrogated the effect of YY1 overexpression on the suppression of SOCS3­mediated STAT3 activation and inflammation. In conclusion, YY1 alleviated neuropathic pain by inhibiting the STAT3 signaling pathway, which may be due to the upregulation of SOCS3 expression.

Alteration in global DNA methylation status following preconditioning injury influences axon growth competence of the sensory neurons.

Preconditioning peripheral nerve injury primes the sensory neurons in the dorsal root ganglia (DRGs) to acquire axon regeneration competence. Transcription of a large set of regeneration-associated-genes (RAGs) contributes to the enhanced intrinsic axonal regeneration capacity. However, the mechanism underlying the coordinated upregulation of RAGs orchestrated by preconditioning injury is unclear. We sought to determine potential influence of DNA methylation change on transcriptional activation of RAGs in the L4-L6 DRGs following sciatic nerve injury. Genome-wide sequencing revealed that about 20% of the methylated DNA fragments were differentially methylated, and >3000 genes contained differentially methylated regions. Not only demethylation but also increased methylation was observed to a similar extent. The change in the global DNA methylation did not correlate with the gene expression level of most genes, including the well-documented RAGs. However, pharmacological inhibition or activation of DNA methylation markedly attenuated the axon growth capacity of the preconditioned DRG neurons. Pharmacological perturbation of DNA methylation resulted in simultaneous downregulation of many highly overlapping non-transcription factor RAGs, which was accompanied by a concurrent, robust upregulation of SOCS3 and Serpine1. Overexpression of SOCS3 and Serpine1 in the DRG neurons overrode injury-induced axon growth competence, corroborating their roles as the negative regulators of axon regeneration. We conclude that the injury-induced global alteration of DNA methylome strongly influences the axon growth competence in preconditioned DRG neurons. Our results also suggest a possibility that perturbing DNA methylome changes might lead to the upregulation of negative regulator RAGs thereby attenuating axon growth capacity.

Knockdown of CTCF reduces the binding of EZH2 and affects the methylation of the SOCS3 promoter in hepatocellular carcinoma.

The epigenetic silencing mechanism of suppressor 3 of cytokine signaling (SOCS3) in cancers has not been fully elucidated. Polycomb repressive complexes 2 (PRC2), an important epigenetic regulatory factors, exerts a critical role in repressing the initial phase of gene transcription. Whether PRC2 participates the down- regulation of SOCS3 in Hepatocellular carcinoma (HCC) remains unclear and how does PRC2 be recruited target gene still needs to explore. In this study, Using TCGA HCC dataset, and detecting HCC tissue specimens and cell lines, we found that SOCS3 expression in HCC was inversely related to that of EZH2, and depended on its promoter methylation status. CTCF, vigilin, EZH2 and H3K27me3 were enriched at CTCF and EZH2 binding sites on the methylated SOCS3 gene promoter. The depletion of CTCF did not affect expression of EZH2 and DNMT1, but decrease recruitment of CTCF, vigilin, EZH2 and H3K27me3. Further, knockdown of CTCF led to a loss of methylation of the methylated SOCS3 promoter, which sequentially increased the expression of SOCS3 and decreased the expression of pSTAT3, the downstream effector. These findings suggest that the CTCF dependent recruitment of EZH2 to the SOCS3 gene promoter is likely to participate in the epigenetic silencing of SOCS3 and in regulating its gene expression.

Identification of duplicated suppressor of cytokine signaling 3 (SOCS3) genes in blunt snout bream (Megalobrama amblycephala).

The suppressor of cytokine signaling 3 (SOCS3) negatively regulates the responses of various immune cytokines. In this study, we identified socs3s genes of blunt snout bream. 209- and 216-aa long peptides are encoded by socs3a and socs3b genes, respectively. The socs3s mRNAs are expressed consistently during the entire process of embryonic development. Whole-mount in situ hybridization detected socs3a in the eyes and posterior somites at 12 h post fertilization (hpf), transcribed at the otic vesicle at 24 hpf, and transcribed at the eyes, brain, and otic vesicle at 36 hpf; while the socs3b mRNA was transcribed at the notochord at 12 hpf, expressed in the brain, eyes, and tailbud at 24 hpf, and detected in the brain at 36 hpf. The expression of socs3a is slightly different from that of socs3b in tissues of juvenile and adult blunt snout bream. After recombinant human growth hormone (hGH) treatment, the transcript levels of socs3s of blunt snout bream were increased in gills, spleen, kidney, and gonads. After Aerononas hydrophila infection, the mRNA levels of socs3s of blunt snout bream were significantly increased in the liver, spleen, intestine, and kidney tissues. Blunt snout bream were susceptible to various pathogenic microorganisms, we intraperitoneally injected blunt snout bream with A. hydrophila to explore the immune mechanism of socs3s. These results suggested that socs3s of blunt snout bream plays important roles in the regulation of embryonic development and tissue growth, and that socs3s may also play key roles in regulating the bacterial-induced congenital immune response. Socs3s genes has the potential to be used as targeted genes to improve the immunity against bacteria, which is conducive to the improvement of production and breeding.

Inhibition of the IL-18 Receptor Signaling Pathway Ameliorates Disease in a Murine Model of Rheumatoid Arthritis.

Interleukin (IL)-18 expression in synovial tissue correlates with the severity of joint inflammation and the levels of pro-inflammatory cytokines. However, the role of the IL-18/IL-18 receptor-alpha (Rα) signaling pathway in autoimmune arthritis is unknown. Wild-type (WT) and IL-18Rα knockout (KO) mice were immunized with bovine type II collagen before the onset of arthritis induced by lipopolysaccharide injection. Disease activity was evaluated by semiquantitative scoring and histologic assessment. Serum inflammatory cytokine and anticollagen antibody levels were quantified by an enzyme-linked immunosorbent assay. Joint cytokine and matrix metalloproteinases-3 levels were determined by a quantitative polymerase chain reaction. Splenic suppressors of cytokine signaling (SOCS) were determined by Western blot analysis as indices of systemic immunoresponse. IL-18Rα KO mice showed lower arthritis and histological scores in bone erosion and synovitis due to reductions in the infiltration of CD4+ T cells and F4/80+ cells and decreased serum IL-6, -18, TNF, and IFN-γ levels. The mRNA expression and protein levels of SOCS3 were significantly increased in the IL-18Rα KO mice. By an up-regulation of SOCS, pro-inflammatory cytokines were decreased through the IL-18/IL-18Rα signaling pathway. These results suggest that inhibitors of the IL-18/IL-18Rα signaling pathway could become new therapeutic agents for rheumatoid arthritis.

MiR-203 regulates proliferation and apoptosis of ovarian cancer cells by targeting SOCS3.

OBJECTIVE: Cytokine signal transduction inhibitor 3 (SOCS3) negatively regulates Janus kinases (JAK) - signal transducer and activator of transcription (STAT) pathway. Bioinformatics analysis revealed a targeted relationship between miR-203 and SOCS3 mRNA. This study investigated the role of miR-203 in ovarian cancer cell proliferation and apoptosis. PATIENTS AND METHODS: Ovarian cancer tissues and adjacent tissues were collected to detect the expression of miR-203 and SOCS3. Ovarian cancer HO8910 cells were divided into miR-NC group, miR-203 inhibitor group, and miR-203 mimic group followed by the analysis of the expression of miR-203 and SOCS3 mRNA by quantitative Reverse Transcription-PCR (qRT-PCR), protein expression of p-JAK2 and p-STAT3 by Western blot, cell apoptosis by flow cytometry, and proliferation by 5-Ethynyl-2'-deoxyuridine (EdU) staining chronologically. RESULTS: Compared with adjacent tissues, miR-203 expression was significantly increased in tumor tissues and SCOS3 mRNA expression was decreased. Compared with those with lower miR-203 expression, the prognosis of patients with higher expression of miR-203 was significantly worse. There was a targeted regulatory relationship between miR-203 and SOCS3 mRNA. Compared with IOSE80 cells, miR-203 expression in HO8910 and SKOV3 cells was increased, and its expressions of SOCS3 mRNA and protein were decreased. Compared with miR-NC group, the transfection of miR-203 inhibitor significantly increased SOCS3 expression, and decreased the expression of p-JAK2 and p-STAT3 protein. We draw the conclusion that miR-203 increased cell apoptosis and decreased cell proliferation. However, opposite results were observed after the transfection of miR-203 mimic. CONCLUSIONS: Abnormal miR-203 and SOCS3 expression are related to the pathogenesis of ovarian cancer. MiR-203 affects the proliferation of JAK-STAT pathway and regulates the proliferation and apoptosis of ovarian cancer cells by targeting the inhibition of SOCS3 expression.

Newcastle Disease Virus Nonstructural V Protein Upregulates SOCS3 Expression to Facilitate Viral Replication Depending on the MEK/ERK Pathway.

Newcastle disease virus (NDV) causes serious economic losses to the poultry industry. In our previous study, we found that NDV induced a strong innate immune response in the chicken embryo and bursa of Fabricius (BF). However, the underlying mechanisms by which NDV escapes the host innate immunity are not well-understood. The suppressor of cytokine signaling 3 (SOCS3) inhibits the type I interferon-dependent antiviral signaling pathway by utilizing a feedback loop. In this study, we analyzed the transcriptome data of the chicken embryo and BF infected with NDV and found significant upregulation of SOCS3. Next, we demonstrated that NDV infection and nonstructural V protein induced the up-regulation of SOCS3. Furthermore, we showed that overexpression of SOCS3 facilitated viral replication and reduced the expression of phosphorylation STAT1, MX1, and OASL, while inhibition of SOCS3 with siRNAs reduced virus replication and promoted the expression of phosphorylation STAT1, MX1, and OASL. Finally, we demonstrated that the MEK/ERK signaling pathway was involved in the expression of SOCS3 mediated by NDV infection and V protein transfection, and using specific inhibitor U0126 to block this signaling pathway attenuated SOCS3 expression and inhibited NDV replication through promoting the expression of type I interferon, OASL and MX1. Taken together, these data demonstrate that NDV infection and NDV nonstructural V protein activates the expression of SOCS3 at the mRNA and protein level through a mechanism dependent on the MEK/ERK signaling pathway, which benefits virus replication.

Cilostazol add-on therapy for celecoxib synergistically inhibits proinflammatory cytokines by activating IL-10 and SOCS3 in the synovial fibroblasts of patients with rheumatoid arthritis.

Cilostazol (an inhibitor of phosphodiesterase type III) has potent anti-inflammatory effects, and celecoxib (a COX-2 specific inhibitor) has been reported to improve the unsatisfactory profile of NSAIDs. This study investigated the synergistic anti-arthritic potential of a multitarget-based cotreatment, in which cilostazol was used as an add-on therapy for celecoxib, using the synovial fibroblasts of RA patients (RASFs). Increased COX-2 protein expression and PGE2 synthesis by LPS (1 µg/ml) were significantly and synergistically attenuated by cotreatment with 3 µM cilostazol and 30 µM celecoxib, whereas monotherapy with either cilostazol or celecoxib showed little effects. IL-10 mRNA levels in LPS-treated RASFs were moderately increased by pretreating cilostazol (1-10 µM) or celecoxib (10-50 µM) monotherapy, but 3 µM of cilostazol add-on for 30 µM celecoxib treatment synergistically increased IL-10 mRNA levels and IL-10 release to culture media. Cilostazol and celecoxib cotreatment similarly showed synergistic increase in SOCS3 mRNA levels. Accordingly, LPS-induced increases in IL-1ß and IL-6 mRNA and TNF-α release were significantly and synergistically diminished by cilostazol and celecoxib cotreatment. Moreover, synovial cell proliferation was significantly suppressed by cotreatment. Summarizing, cotreatment with cilostazol and celecoxib exhibited a synergistic increase in IL-10 production and SOCS3 expressions, thereby resulted in synergistic decreases in IL-1ß mRNA, IL-6 mRNA expression and TNF-α synthesis in association with synergistic decreases in COX-2 and PGE2 protein expression in the RA synovial fibroblasts. In conclusion, these observations suggest low concentrations of cilostazol and celecoxib cotreatment may ensure a synergistic anti-arthritic potential.

Protein Expression Analysis in Uterine Cervical Cancer for Potential Targets in Treatment.

Specific markers in lesions of the human uterine cervix cancer (UCC) are still needed for prognostic, diagnostic and/or therapeutic purposes. In this study we evaluated key molecules at protein level between normal epithelium, cervical intraepithelial neoplasia (CIN1-3) and invasive cancer of a group of molecules previously reported at mRNA level. For that purpose, human formalin-fixed paraffin embedded tissue microarrays (TMAs) were constructed containing 205 Mexican tissue core specimens. Immunohistochemistry and quantitative analysis of histological staining was performed against twenty-two distinct proteins for each core and the processing platform ImageJ. In the progression of the disease we found key statistical differences for the proteins SEL1, Notch3 and SOCS3. High expressions of SEL1L, Notch3 and SOCS3 have potential value to increase the prognostic of UCC in combination with markers such as p16INK4a. This study identified key drivers in cervical carcinogenesis that should be evaluated for the development of UCC therapies.

Chronic Intake of Commercial Sweeteners Induces Changes in Feeding Behavior and Signaling Pathways Related to the Control of Appetite in BALB/c Mice.

Nonnutritive sweetener use is a common practice worldwide. Although considered safe for human consumption, accumulating evidence suggests these compounds may affect metabolic homeostasis; however, there is no consensus on the role of frequent sweetener intake in appetite and weight loss. We sought to determine whether frequent intake of commercial sweeteners induces changes in the JAK2/STAT3 signaling pathway in the brain of mice, as it is involved in the regulation of appetite and body composition. We supplemented adult BALB/c mice with sucrose, steviol glycosides (SG), or sucralose, daily, for 6 weeks. After supplementation, we evaluated body composition and expression of total and phosphorylated JAK2, STAT3, and Akt, as well as SOCS3 and ObRb, in brain tissue. Our results show that frequent intake of commercial SG decreases energy intake, adiposity, and weight gain in male animals, while increasing the expression of pJAK2 and pSTAT3 in the brain, whereas sucralose increases weight gain and pJAK2 expression in females. Our results suggest that chronic intake of commercial sweeteners elicits changes in signaling pathways that have been related to the control of appetite and energy balance in vivo, which may have relevant consequences for the nutritional state and long term health of the organism.

Prolactin regulates liver growth during postnatal development in mice.

The liver grows during the early postnatal period first at slower and then at faster rates than the body to achieve the adult liver-to-body weight ratio (LBW), a constant reflecting liver health. The hormone prolactin (PRL) stimulates adult liver growth and regeneration, and its levels are high in the circulation of newborn infants, but whether PRL plays a role in neonatal liver growth is unknown. Here, we show that the liver produces PRL and upregulates the PRL receptor in mice during the first 2 wk after birth, when liver growth lags behind body growth. At postnatal week 4, the production of PRL by the liver ceases coinciding with the elevation of circulating PRL and the faster liver growth that catches up with body growth. PRL receptor null mice ( Prlr-/-) show a significant decrease in the LBW at 1, 4, 6, and 10 postnatal weeks and reduced liver expression of proliferation [cyclin D1 ( Ccnd1)] and angiogenesis [platelet/endothelial cell adhesion molecule 1 ( Pecam1)] markers relative to Prlr+/+ mice. However, the LBW increases in Prlr-/- mice at postnatal week 2 concurring with the enhanced liver expression of Igf-1 and the liver upregulation and downregulation of suppressor of cytokine signaling 2 ( Socs2) and Socs3, respectively. These findings indicate that PRL acts locally and systemically to restrict and stimulate postnatal liver growth. PRL inhibits liver and body growth by attenuating growth hormone-induced Igf-1 liver expression via Socs2 and Socs3-related mechanisms.

SOCS3 Is Upregulated and Targeted by miR30a-5p in Allergic Rhinitis.

BACKGROUND: Suppressor of cytokine signaling 1 (SOCS1) and SOCS3 play important roles in T helper cell differentiation, which is involved with the pathologic mechanisms of allergic rhinitis (AR). The aim of this study was to evaluate the expression of SOCS1 and SOCS3 in AR and find their regulatory microRNAs (miRNAs) to provide a basis for the treatment of AR. METHODS: The expression of SOCS1 and SOCS3 were analyzed by real-time PCR, immunohistochemistry, and Western blot. The correlative regulatory miRNAs were detected by real-time PCR. Luciferase assays and AR mouse model experiments were applied to identify correlative miRNAs that target SOCS3. RESULTS: SOCS1 and SOCS3 mRNA were upregulated in the nasal mucosa and peripheral blood mononuclear cells of AR compared with controls. The expression of SOCS3 protein was significantly increased in the nasal mucosa of AR. The immunohistochemical staining results showed that SOCS3 was similarly localized in the superficial epithelium, submucosal glands, and vascular endothelium in the nasal mucosa of AR subjects and controls. However, SOCS3 protein was especially localized in the inflammatory cells, such as eosinophils, monocytes, and lymphocytes. CONCLUSIONS: SOCS3 was targeted by miR30a- 5p in AR. Further study should be performed to identify the regulatory effect of miR30a-5p in AR, which may provide insights into a new therapeutic strategy.

Lidocaine alleviates morphine tolerance via AMPK-SOCS3-dependent neuroinflammation suppression in the spinal cord.

BACKGROUND: Morphine tolerance is a clinical challenge, and its pathogenesis is closely related to the neuroinflammation mediated by Toll-like receptor 4 (TLR4). In Chinese pain clinic, lidocaine is combined with morphine to treat chronic pain. We found that lidocaine sufficiently inhibited neuroinflammation induced by morphine and improved analgesic tolerance on the basis of non-affecting pain threshold. METHODS: CD-1 mice were utilized for tail-flick test to evaluate morphine tolerance. The microglial cell line BV-2 was utilized to investigate the mechanism of lidocaine. Neuroinflammation-related cytokines were measured by western blotting and real-time PCR. The level of suppressor of cytokine signaling 3 (SOCS3) and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-related signaling pathway was evaluated by western blotting, real-time PCR, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining. RESULTS: Lidocaine potentiated an anti-nociceptive effect of morphine and attenuated the chronic analgesic tolerance. Lidocaine suppressed morphine-induced activation of microglia and downregulated inflammatory cytokines, interleukin-1ß (IL-1ß), and tumor necrosis factor-alpha (TNF-α) via upregulating SOCS3 by activating AMPK. Lidocaine enhanced AMPK phosphorylation in a calcium-dependent protein kinase kinase ß (CaMKKß)-dependent manner. Furthermore, lidocaine decreased the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and inhibited the nuclear factor-κB (NF-κB) in accordance with the inhibitory effects to TLR4. CONCLUSIONS: Lidocaine as a prevalent local anesthetic suppresses morphine tolerance efficiently. AMPK-dependent upregulation of SOCS3 by lidocaine plays a crucial role in the improvement of analgesic tolerance.

High fat diet-induced obesity increases myocardial injury and alters cardiac STAT3 signaling in mice after polymicrobial sepsis.

Little is known about how obesity affects the heart during sepsis and we sought to investigate the obesity-induced cardiac effects that occur during polymicrobial sepsis. Six-week old C57BL/6 mice were randomized to a high fat (HFD) (60% kcal fat) or normal diet (ND) (16% kcal fat). After 6weeks of feeding, mice were anesthetized with isoflurane and polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Plasma and cardiac tissue were obtained for analysis. Echocardiography was performed on a separate cohort of mice at 0 and 18h after CLP. Following 6-weeks of dietary intervention, plasma cardiac troponin I was elevated in obese mice at baseline compared to non-obese mice but troponin increased only in non-obese septic mice. IL-17a expression was 27-fold higher in obese septic mice versus non-obese septic mice. Cardiac phosphorylation of STAT3 at Ser727 was increased at baseline in obese mice and increased further only in obese septic mice. Phosphorylation of STAT3 at Tyr705 was similar in both groups at baseline and increased after sepsis. SOCS3, a downstream protein and negative regulator of STAT3, was elevated in obese mice at baseline compared to non-obese mice. After sepsis non-obese mice had an increase in SOCS3 expression that was not observed in obese mice. Taken together, we show that obesity affects cardiac function and leads to cardiac injury. Furthermore, myocardial injury in obese mice during sepsis may occur through alteration of the STAT3 pathway.

SOCS3 inhibits the pathological effects of IL-22 in non-melanoma skin tumor-derived keratinocytes.

Basal cell carcinomas (BCC) and squamous-cell carcinomas (SCC) are common malignancies in humans, caused by neoplastic transformation of keratinocytes of the basal or suprabasal layers of epidermis, respectively. Tumor-infiltrating lymphocytes (TILs) are frequently found in BCC and SCC, and functionally promote epithelial carcinogenesis. TILs secreting IL-22, in particular, participate to BCC and SCC growth by inducing keratinocyte proliferation and migration, as well as the expression of inflammatory, anti-apoptotic and pro-angiogenic genes.In this study, we identified SOCS3 as a valid candidate to be manipulated for suppressing tumorigenic functions in BCC and SCC. We found that SOCS3 and SOCS1 expression was reduced in vivo, in tumor lesions of BCC and SCC, as compared to other skin inflammatory conditions such as psoriasis, despite the high number of IL-22-secreting TILs. Moreover, IL-22 was not able to induce in vitro the transcriptional expression of SOCS3 in BCC-or SCC-derived keratinocytes, contrarily to healthy cells. Aimed at rescuing SOCS3 activity in these tumor contexts, a SOCS3-derived peptide, named KIR-ESS, was synthesized, and its ability in suppressing IL-22-induced responses was evaluated in healthy and transformed keratinocytes. We found that KIR-ESS peptide efficiently suppressed the IL-22 molecular signaling in keratinocytes, by acting on STAT3 and Erk1/2 cascade, as well as on the expression of STAT3-dependent downstream genes. Interestingly, after treatment with peptide, both healthy and transformed keratinocytes could no longer aberrantly proliferate and migrate in response to IL-22. Finally, treatment of athymic nude mice bearing SCC xenografts with KIR-ESS peptide concomitantly reduced tumor growth and activated STAT3 levels. As a whole, these data provides the rationale for the use in BCC and SCC skin tumors of SOCS3 mimetics, being able to inhibit the deleterious effects of IL-22 in these contexts.

The influence of chronic IL-6 exposure, in vivo, on rat Achilles tendon extracellular matrix.

When compared to placebo, acetaminophen (APAP) reduces tendon stiffness and collagen cross-linking. APAP also enhances the exercise-induced increase in peritendinous levels of IL-6. Elevated levels of IL-6 are associated with tendinopathy, thus we hypothesized that chronic, elevated peritendinous IL-6 would alter tendon extracellular matrix (ECM). IL-6 (∼3000pgml-1) was injected (3dwk-1 for 8-wks) into the Achilles peritendinous region of male Wistar rats (n=16) with the opposite leg serving as a sham. Fractional synthesis rates (FSR) were determined using deuterium oxide. Collagen (hydroxyproline) and hydroxylysl pyridinoline (HP) cross-linking were analyzed by HPLC. ECM and IL-6 related genes were evaluated using qRT-PCR. Relative to sham, collagen (Col) 1a1 but not Col3a1 expression was suppressed (47%) in tendons exposed to IL-6 (p<0.05). Lysyl oxidase (LOX) and MMP-1 expression were also reduced (37%) in IL-6 treated tendons (p<0.05). Relative to sham the expression of MMP-2, -3, -9, and TIMP-1 were not altered by IL-6 treatment (p>0.05). Interleukin-6 receptor subunit beta precursor (IL6st) was lower (16%) in IL-6 treated tendons when compared to sham (p<0.05). Suppressor of cytokine signaling 3 (Socs3), signal transducer and activator of transcription 3 (STAT3), and protein inhibitor of activated STAT 1 (Pias1) were not altered by IL-6 exposure (p>0.05). Neither collagen nor cross-linking content were altered by IL-6 (p>0.05). Additionally, IL-6 treatment did not alter tendon FSR. Chronic treatment with physiologically relevant levels of IL-6 suppresses expression of Col1a1 and LOX while also altering expression of select MMPs but does not alter Achilles tendon collagen synthesis.

SOCS3 Immunohistochemical Expression Seems to Support the 2005 and 2014 International Society of Urological Pathology (ISUP) Modified Gleason Grading System.

BACKGROUND: In the 2014, The International Society of Urological Pathology (ISUP) consensus conference update the grading of prostate, last revised in 2005. In this study we evaluate the SOCS3 immunohistochemical protein expression in different Gleason prostatic adenocarcinoma: classical Gleason grade 3, classical Gleason grade 3 upgraded to Gleason grade 4 according to the ISUP modifications and classical and modified Gleason grade 4. The major conclusions were: (i) Cribriform glands should be assigned a Gleason pattern 4, regardless of morphology; (ii) Glomeruloid glands should be assigned a Gleason pattern 4, regardless of morphology; (iii) Grading of mucinous carcinoma of the prostate should be based on its underlying growth pattern rather than all as pattern 4; and (iv) Intraductal carcinoma of the prostate without invasive carcinoma should not assigned Gleason grade and a comment about aggressive carcinoma probably associated should be made. In a recent report we analyzed the methylathion status of cytokine signaling (SOCS) proteins 3 (SOCS3) gene and the consequences of promoter hypermethylation on mRNA and protein expression in a collection of prostate cancer and benign prostate hyperplasia (BPH) and for the first time we demonstrated that a hypermethylation of SOCS3 with a significant reduction of its mRNA and protein expression identifies a subgroup of prostate cancer with a more aggressive behavior. Moreover we demonstrated that the immunohystochemical analysis of SOCS3 protein expression in prostatic cancer biopsies may provide a useful and easier method than SOCS3 methylation analysis to individuate in cancer with intermediate-high grade Gleason score a subgroup of prostate cancer with a more aggressive behavior. METHODS: A total of 148 radical prostatectomy with diagnosis of prostatic acinar adenocarcinoma were stratified into three different categories on the basis of Gleason grade: (i) Twenty-six prostatic adenocarcinoma with classical and modified Gleason grade 3; (ii) Fifty seven prostatic adenocarcinoma with classical Gleason grade 3 upgraded to Gleason grade 4 by 2005 and 2014 ISUP Consensus Conference; and (iii) Sixty five prostatic adenocarcinoma with classical and modified Gleason grade 4. Immunohistochemical analysis for SOCS3 was performed and SOCS3 staining intensity were evaluated by two pathologists in three different ways on the basis of the intensity of cytoplasmatic staining: positive (intense cytoplasmatic staining in more than 50% of neoplastic cells) (+), negative (absence of cytoplasmatic staining in more than 50% of neoplastic cells) (-), weakly positive (weak cytoplasmatic staining in more than 50% of neoplastic cells (+/-). RESULTS: In the group of prostatic adenocarcinoma Gleason grade 3 we found that SOCS3 positivity (+) were observed in 19 out of 26 cases (73.1%); in 5 out of 26 prostatic adenocarcinoma the neoplastic glands showed weak intensity SOCS3 staining (+/-) (19.2%), while in only two cases we found SOCS-3 negativity (-) (7.7%); in the group of cases with prostatic adenocarcinoma with Gleason grade 4, 16 out 65 cases (24.6%) showed SOCS3 positivity (+); 18 out 65 cases (27.7%) SOCS3 weakly positive (+/-), and in 31 cases (47.7%) SOCS3 negative staining (-) were observed. Interestingly, the group of prostatic adenocarcinoma with histological Gleason 3 pattern upgraded to Gleason 4 pattern according to the 2005 and 2014 ISUP modified grading system, showed SOCS3 positivity (+) in 16 out of 57 cases (28%), in 16 out 57 cases (28%) a weakly positive for SOCS3 (+/-) were observed, while 25 cases (44%) showed negative SOCS3 staining (-). CONCLUSIONS: In this study we demonstrated a significant association of SOCS3 positivity (+) with prostatic carcinoma classical Gleason pattern 3 (P < 0.0001), while SOCS3 negative pattern (-) or SOCS3 weakly positive pattern (+/-) were associated to prostatic carcinomas with Gleason pattern 3 upgraded to Gleason pattern 4 (P = 0.0002) and with classical Gleason pattern 4. The significant difference of SOCS3 immunohistochemical expression between classical Gleason grade 3 and Gleason grade 4 upgraded to grade 4 seems to support the definitions and the modifications of Gleason grade 4 of the 2005 and the 2014 International Society of Urological Pathology (ISUP). The hypoexpression of SOCS3 protein in glomeruloid glands could support the hypothesis that from molecular point of view this growth pattern could be different from classical Gleason pattern 3 and biologically more closely to Gleason pattern 4, confirming the conclusions of the 2014 ISUP Conference assigning a Gleason pattern 4 to glomeruloid glands regardless of morphology. Prostate 77: 597-603, 2017. © 2017 Wiley Periodicals, Inc.

Susceptibility of naïve and differentiated PC12 cells to Japanese encephalitis virus infection.

Japanese encephalitis is a mosquito-borne disease caused by Japanese encephalitis virus (JEV) infection. Although JEV infects and replicates in cells with multiple tissue origins, neurons are the preferential cells for JEV infection. Currently, the identities of JEV cell tropism are largely unclear. To gain better insight into the underlying identities of JEV cell tropism, this study was designed to compare the JEV cell tropism with naïve or differentiated PC12 cells. Through nerve growth factor-differentiated PC12 cells, we discovered that JEV efficiently replicated in differentiated PC12 cells rather than naïve cells. Mechanistic studies revealed that viral adsorption/attachment seemed not to be a crucial factor. Supporting data showed that antagonizing postreceptor intracellular signaling of interferons, along with the activation of suppressor of cytokine signaling-3 (SOCS3) expression and protein tyrosine phosphatase activity, were apparent in differentiated PC12 cells after JEV infection. Independent of differentiating inducing agents, the upregulation of SOCS3 expression and protein tyrosine phosphatase activity, as well as preferential JEV tropism, were common in JEV-infected differentiated PC12 cells. Using cultured primary neurons, JEV efficiently replicated in embryonic neurons rather than adult neurons, and the preference was accompanied by higher SOCS3 expression and protein tyrosine phosphatase activity. Given that both SOCS3 and protein tyrosine phosphatases have been implicated in the process of neuronal differentiation, JEV infection seems to not only create an antagonizing strategy to escape host's interferon antiviral response but also takes advantage of cellular machinery to favor its replication. Taken together, current findings imply that dynamic changes within cellular regulators of antiviral machinery could be accompanied by events of neuronal differentiation, thus concurrently playing roles in the control of JEV cell tropism and replication. © 2017 IUBMB Life, 69(2):79-87, 2017.

Potential Involvement of the IL-6/JAK/STAT3 Pathway in the Pathogenesis of Intervertebral Disc Degeneration.

STUDY DESIGN: Laboratory study. OBJECTIVE: To elucidate the potential involvement of the interleukin-6 (IL-6)/Janus kinase (JAK)/signal transducers and activator of transcription (STAT3) pathway in the development of intervertebral disc (IVD) degeneration. SUMMARY OF BACKGROUND DATA: IL-6 plays a crucial role in IVD degeneration; however, the downstream intracellular signaling of IL-6 in the IVD is not fully understood. METHODS: The expression levels of IL-6 and suppressors of cytokine signaling 3 (SOCS3), a target gene of the IL-6/JAK/STAT3 pathway, were evaluated in rat and human degenerated IVD samples. The effects of IL-6 on primary rat annulus fibrosus (AF) cells were analyzed using quantitative PCR, immunocytochemistry, and Western blotting. The potential efficacy of a JAK inhibitor, CP690,550, in neutralizing the effect of IL-6 was evaluated in vitro. RESULTS: A high expression of IL-6 and SOCS3 was observed in both rat and human degenerated IVD samples. In rat AF cells, IL-6 markedly induced the phosphorylation of STAT3 and the expression of cyclooxygenase-2 and matrix metalloprotease-13. CP690,550 significantly suppressed the phosphorylation of STAT3 and offset the catabolic effect of IL-6 in rat AF cells. CONCLUSION: Our results suggest that the IL-6/JAK/STAT3 pathway is involved in the pathogenesis of IVD degeneration and that CP690,550 suppresses the catabolic effect of the IL-6 in the IVD. LEVEL OF EVIDENCE: N/A.