SOCS Proteins Research Articles

Abstract 4653: Intracellular Delivery of SOCS3 Suppresses Pancreatic Cancer Progression

Abstract Background: JAK/STAT signaling, regulated by SOCS family proteins, plays a role in the progression of the inflammation, growth and metastasis of pancreatic adenocarcinoma. SOCS3 suppresses JAK kinase activity and degrades the activated cytokine receptor complex, and its loss contributes to pancreatic cancer development. We have developed advanced macromolecule transduction domains (aMTDs) to deliver recombinant proteins into to cells and tissues. In principle, aMTD enabled SOCS3 could provide an effective treatment for pancreatic tumors that depends on JAK/STAT signaling for growth or survival. Objective: The present study utilized aMTD sequences to develop improved cell-permeable (iCP-) SOCS3 as a protein-based biotherapeutic against pancreatic cancer. Methods: Improved cell-permeable SOCS3 proteins, with and without a 12-amino acid aMTD sequence, were expressed at high yields in E. coli. The proteins also contained a solubilization domain (SD) to enhance the solubility of the recombinant proteins in physiological buffers. Results: The iCP-SOCS3 exhibited high solubility, cell-/tissue-permeability and biological activity in pancreatic cancer cell lines (BxPC3, HPAC and PANC-1) with decreased endogenous SOCS3 levels, hyperactived JAK/STAT signaling and hypermethylated SOCS3 promoter region. The iCP-SOCS3 suppressed cancer-associated phenotypes (i.e., up to 90% inhibition of cell viability, 52% induction of apoptosis, 14.8% inhibition of cell cycle progression, 54% inhibition of migration and 61.5% inhibition of invasion) in pancreatic cancer cells dependent on elevated JAK/STAT signaling, whereas, these effects were not observed in non-cancer cells (H6c7, pancreatic duct epithelial cell) or with proteins lacking the aMTD sequence. In contrast, gemcitabine, a conventional pancreatic cancer therapeutic, showed undesirable cytotoxic effects including decreased cell viability and induced apoptosis in non-cancer cells. Furthermore, iCP-SOCS3 (30 mg/kg) induced tumor regression (-168% at day 18) in PANC-1 CDX model. In BxPC-3 orthotopic CDX model, iCP-SOCS3 suppressed tumor growth by 98% which was significantly higher compared to the suppressed tumor growth induced by gemcitabine (77%). Correlating to the tumor suppression, iCP-SOCS3 significantly decreased the secretion of TNF-α and the expression of p-STAT1, p-STAT3 and VEGF in tumor tissues. Conclusion: These results provide further evidence that SOCS3 can function as a tumor suppressor and intracellular delivery of SOCS3 with iCP-SOCS3 may provide a novel protein therapy against pancreatic cancer. Citation Format: Kuysook Lee, Seulmee Shin, Yunseo Hwang, Youngsil Choi, Daewoong Jo. Intracellular Delivery of SOCS3 Suppresses Pancreatic Cancer Progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4653.

Abstract LB-006: Intracellular protein therapy with SOCS3 utilizing aMTD-enabled macromolecule intracellular transduction technology (MITT)

Abstract Background Macromolecule intracellular transduction technology (MITT), a platform for the discovery/development of new medicinal drugs, is enabled with a new generation of hydrophobic cell-penetrating peptides (CPPs), termed advanced macromolecule transduction domains (aMTDs). Originally derived from the hydrophobic signal peptides of secreted proteins, earlier generations of MTDs were hampered by poor solubility and less efficient cell-permeability. The MITT platform addresses these limitations, first, by the design of synthetic aMTD sequences that incorporate 6 critical factors found to enhance the intracellular delivery of proteins into cells and tissues and second, by the use of humanized solubilization domains to enhance the solubility of aMTD-containing recombinant proteins. Object The goal of this study is to develop and evaluate an aMTD-SOCS3 (suppressor of cytokine signaling 3) protein (iCP-SOCS3) as cell-permeable biotherapeutic to selectively target tumors dependent on JAK/STAT signaling and to compare the cell-/tissue-penetration efficiencies of iCP-SOCS3 with a previous generation of cell-permeable SOCS3 (MTM-SOCS3: CP-SOCS3) enabled by a hydrophobic CPP, termed membrane translocating motif (MTM) derived from the signal peptide of fibroblast growth factor 4 (FGF4). Results Solubility (up to 10 mg/ml) and final yields of E. coli produced iCP-SOCS3 (60 mg/L) were sufficient for clinical development; whereas, CP-SOCS3 suffered from low solubility and yields (0.4 mg/ml and 0.2 mg/L, respectively). Cellular uptake of fluorescent iCP-SOCS3 was enhanced by 25-fold as compared to CP-SOCS3 as assessed by flow cytometry, and delivery and persistence of the protein in a variety of tissues (heart, lung, spleen, kidney, liver and brain) were similarly increased. In addition, aMTD promoted the cell-to-cell transfer of SOCS3 protein, and the transferred iCP-SOCS3 was biochemically active as assessed by inhibition of IFN-γ-induced phosphorylation of STAT1 and STAT3 with concomitant suppression of cancer-associated phenotypes in cultured cells and in mice bearing human tumor xenografts (90% inhibition of proliferation and 84% inhibition of migration in PANC-1 cells, and 85% suppression of pancreatic tumor growth, respectively). Conclusion These results provide further validation of MITT as a platform to develop protein-based biotherapeutics, and suggest the use of iCP-SOCS3 as a novel protein therapy against tumors dependent on JAK/STAT signaling. Citation Format: Youngsil Choi, Seulmee Shin, Kuysook Lee, Daewoong Jo. Intracellular protein therapy with SOCS3 utilizing aMTD-enabled macromolecule intracellular transduction technology (MITT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-006.

Abstract LB-007: Intracellular delivery of SOCS3 suppresses pancreatic cancer progression

Abstract Background JAK/STAT signaling, regulated by SOCS family proteins, plays a role in the progression of the inflammation, growth and metastasis of pancreatic adenocarcinoma. SOCS3 suppresses JAK kinase activity and degrades the activated cytokine receptor complex, and its loss contributes to pancreatic cancer development. We have developed advanced macromolecule transduction domains (aMTDs) to deliver recombinant proteins into to cells and tissues. In principle, aMTD enabled SOCS3 could provide an effective treatment for pancreatic tumors that depends on JAK/STAT signaling for growth or survival. Objective The present study utilized aMTD sequences to develop improved cell-permeable (iCP-) SOCS3 as a protein-based biotherapeutic against pancreatic cancer. Method Improved cell-permeable SOCS3 proteins, with and without a 12-amino acid aMTD sequence, were expressed at high yields in E. coli. The proteins also contained a solubilization domain (SD) to enhance the solubility of the recombinant proteins in physiological buffers. Results The iCP-SOCS3 exhibited high solubility, cell-/tissue-permeability and biological activity in pancreatic cancer cell lines (BxPC3, HPAC and PANC-1) with decreased endogenous SOCS3 levels, hyperactived JAK/STAT signaling and hypermethylated socs3 promoter region. The iCP-SOCS3 suppressed cancer-associated phenotypes (i.e., up to 90% inhibition of cell viability, 52% induction of apoptosis, 14.8% inhibition of cell cycle progression, 54% inhibition of migration and 61.5% inhibition of invasion) in pancreatic cancer cells dependent on elevated JAK/STAT signaling, whereas, these effects were not observed in non-cancer cells (H6c7, pancreatic duct epithelial cell) or with proteins lacking the aMTD sequence. In contrast, gemcitabine, a conventional pancreatic cancer therapeutic, showed undesirable cytotoxic effects including decreased cell viability and induced apoptosis in non-cancer cells. Furthermore, iCP-SOCS3 (30 mg/kg) induced tumor regression (-168% at day 18) in PANC-1 CDX model. In BxPC-3 orthotopic CDX model, iCP-SOCS3 suppressed tumor growth by 98% which was significantly higher compared to the suppressed tumor growth induced by gemcitabine (77%). Correlating to the tumor suppression, iCP-SOCS3 significantly decreased the secretion of TNF-α and the expression of p-STAT1, p-STAT3 and VEGF in tumor tissues. Conclusion These results provide further evidence that SOCS3 can function as a tumor suppressor and intracellular delivery of SOCS3 with iCP-SOCS3 may provide a novel protein therapy against pancreatic cancer. Citation Format: Kuysook Lee, Seulmee Shin, Yunseo Hwang, Youngsil Choi, Daewoong Jo. Intracellular delivery of SOCS3 suppresses pancreatic cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-007.

Correlation of SOCS-1 gene with onset and prognosis of breast cancer.

The aim of the present study is to study the expressions of suppressor of cytokine signaling (SOCS)-1 in the tumor tissues and adjacent normal tissues of patients with breast cancer. The study was also planned to investigate the association of SOCS-1 gene expression with patients' clinical pathology, molecular subtype and prognosis. A total of 60 cases of frozen and paraffin-embedded specimens of tumor tissues and corresponding adjacent normal tissues of patients with breast cancer were selected. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of SOCS-1 messenger RNA (mRNA) in the patients' tumor tissues and adjacent normal tissues. The immunohistochemical method was applied to detect the expressions of SOCS-1 proteins in the patients' breast cancer tissues and adjacent normal tissues. Moreover, the correlations of SOCS-1 protein expressions in breast cancer tissues with patients' pathological parameters, molecular subtypes and prognosis were analyzed in combination with the clinical data. The results of RT-qPCR showed that the SOCS-1 mRNA expression in breast cancer tissues was significantly lower than that in adjacent normal tissues (p<0.01). The immunohistochemical results indicated that the positive expression rate of the SOCS-1 proteins in breast cancer tissues (23.33%) was remarkably lower than that in adjacent normal tissues (88.33%) (p<0.01). The low expression of SOCS-1 in breast cancer tissues was related to lymph node metastasis and clinical staging. The positive expression rates of the luminal A SOCS-1 proteins were the highest (47.62%) (p<0.01). The 5-year overall survival rate of the breast cancer patients was 63.33% (38/60). The univariate survival analysis revealed that the patients with low expression of SOCS-1 had poorer prognosis. In conclusion, the low expression of SOCS-1 plays a key role in the pathogenesis of breast cancer; in particular, it is associated with the lymph node metastasis and clinical staging of the tumor; so, the SOCS-1 expression in breast cancer tissues can be regarded as an important reference for the prognostic estimation of breast cancer.

The Immunohistochemical Analysis of SOCS3 Protein Identifies a Subgroup of Prostatic Cancer Biopsies With Aggressive Behavior.

Recently, we demonstrated that hypermethylation of SOCS3 determines a significant reduction of its mRNA and protein expression and identifies a subgroup of prostate cancer with aggressive behavior. In this paper, our objective was to investigate whether the immunohistochemical expression of the SOCS3 protein could represent an alternative method to molecular analysis for the individualization of aggressive prostate carcinoma. We analyzed the SOCS3 immunohistochemical expression in 65 patients undergoing biopsies at the Institute of Urology of our hospital between September 2011 and October 2011 (median age, 66.4 y; range, 50 to 73 y), and in 35 cases, a subset of 65 cases originally used for the immunohistochemical study, we studied the methylation status of the SOCS3 promoter. We found that the percentage of cases with SOCS3 negativity (-) or with SOCS3 weak staining in <50% of the neoplastic glands (+/-) correlated to the worst prognosis in terms of the Gleason score (P=0.0001; Fisher's exact test), the pT stage (P=0.012; Fisher's exact test), and progression-free survival (P=0.0334; hazard ratio, 0.34; and 95% confidence interval, from 0.1261 to 0.9188). Moreover, some cases with an SOCS3 unmethylated pattern showed SOCS3-negative immunostaining (-) or SOCS3-negative glands with weak cytoplasmatic staining in <50% of the neoplastic glands (+/-). Our data suggest that in prostatic cancer biopsies, the immunohistochemical analysis of SOCS3 protein expression may provide a method that is less expensive and easier to apply than SOCS3 methylation analysis for the distinction of a subgroup of prostate cancer with a more aggressive behavior.

Overexpression of SOCS3 Attenuates Tracheal Allograft Rejection in the Early Phase After Murine Heterotopic Tracheal Transplantation by the Inhibition of Th1 Response

Although Th1 response plays a key role in the development of obliterative bronchiolitis (OB) after lung transplantation, little is known about the effect of inhibiting Th1 response in OB development. SOCS3 protein, a negative regulator of JAK/STAT pathway, has been shown to skew the Th1/Th2 balance toward Th2 cell phenotype. In this study, we hypothesized that the inhibition of Th1 response by overexpressing SOCS3 attenuates the development of OB in a model of murine heterotopic tracheal transplantation (HTT).

노화 흰쥐 전경골근 및 해마의 SOCS3 및 IL15 단백질 발현에 대한 운동트레이닝 효과

노화 흰쥐 전경골근 및 해마의 SOCS3 및 IL15 단백질 발현에 대한 운동트레이닝 효과

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.

New mechanistic insights on the metabolic-disruptor role of chlorpyrifos in apoE mice: a focus on insulin- and leptin-signalling pathways.

Recently, we have provided evidence, suggesting that mice expressing the human apolipoprotein E3 (apoE3) are more prone to develop an obesity-like phenotype and a diabetic profile when subchronically fed a chlorpyrifos (CPF)-supplemented diet. The aim of the current study was to examine the underlying mechanisms through which CPF alters both insulin- and leptin-signalling pathways in an APOE-dependent manner. Both adult apoE3- and E4-targeted replacement and C57BL/6 mice were exposed to CPF at 0 or 2mg/kg body weight/day through the diet for 8 consecutive weeks. We determined the expression of JAK2, p-JAK2, STAT3, p-STAT3, SOCS3, IRS-1, p-IRS-1, AKT, p-AKT, GSK3β, p-GSK3β, and apoE in the liver, as well as hepatic mRNA levels of pon1, pon2, and pon3. CPF markedly disrupted both leptin and insulin homeostasis, particularly in apoE3 mice. Indeed, only CPF-fed apoE3 mice exhibited an increased phosphorylation ratio of STAT3, as well as increased total SOCS3 protein levels. Similarly, the exposure to CPF drastically reduced the phosphorylation ratio of both AKT and GSK3β, especially in apoE3 mice. Overall, CPF reduced the expression of the three pon genes, principally in C57BL/6 and apoE3 mice. These results provide notable mechanistic insights on the metabolic effects of the pesticide CPF, and attest the increased vulnerability of apoE3 carriers to its metabolic-disruptor role.

ПАТОМОРФОЛОГИЧЕСКАЯ ДИАГНОСТИКА ХРОНИЧЕСКОГО АППЕНДИЦИТА (ОБЗОР ЛИТЕРАТУРЫ)

The review is devoted to the problem of diagnostics and differential diagnostics of chronic appendicitis. As it is commonly&#x0D; known, the very existence of chronic appendicitis is still considered controversial. The article describes the current ideas&#x0D; on chronic appendicitis (primary, secondary, residual and recurrent), presents the list of morphological manifestations&#x0D; in the vermiform appendix at chronic inflammation, discovered at routine histological examination: dystrophic changes&#x0D; in the form of atrophy of all layers of appendix with sclerosis or hypotrophy of various degree, up to appendiclausis&#x0D; and disappearance of mucous membrane, and lipomatosis of submucosa. The authors discuss the complexity of diag-&#x0D; nostics predetermined by lack of precise criteria of the disease; show the possibility of evaluation of the expression of&#x0D; cytoadherence molecules for diagnostics of the disease, in particular of VCAM-1 and ICAM-1 for differential diagnostics&#x0D; of chronic appendicitis. The role of suppressor of signal cytokine proteins SOCS3 in chronic inflammation is discussed.&#x0D; The data demonstrate the increase of T-lymphocytes and neurons in chronic appendicitis as an increase of PGP 9.5&#x0D; (panneuronal marker protein gene product 9.5) level. It has been shown, that immunohistochemical staining on p44&#x0D; MAPK has an advantage in differential diagnostics of chronic appendicitis – specific staining of subserous and muscle&#x0D; layer of the wall is observed only at the discussed pathology of the appendix.&#x0D; In spite of multiple studies and acknowledgment of the fact that chronic form of appendicitis is a separate disease,&#x0D; the diagnostics of this pathology still presents quite a problem especially when only routine methods of examination&#x0D; are used. Thus, we found it necessary to introduce additional immunomorphologic methods into the clinical practice.

Conditioned medium from contracting skeletal muscle cells reverses insulin resistance and dysfunction of endothelial cells

ObjectiveObese adipose tissue has been characterized with chronic inflammation associated with elevated secretion of inflammatory cytokines and declined secretion of anti-inflammatory cytokines which can impair endothelial function in an endocrine manner. Adipose tissue hypoxia plays a role in the changes of cytokines. Physical exercise/muscle contraction may help preventing cardiovascular disease through improving insulin resistance and endothelium function. However the mechanism is unclear. Skeletal muscle is an endocrine tissue. Contracting muscles secrete myokines which may play roles in the beneficial effect of exercise. In this study, the conditioned medium from electrical pulse stimulation (EPS) regulated skeletal muscle cells was used to explore the mechanism of contraction on endothelial dysfunction and insulin resistance induced by conditioned medium from hypoxic adipocytes. Methods3T3-L1 adipocytes were incubated under normoxia or hypoxia condition, respectively. The supernatant was collected as adipocyte conditioned medium (CM-N and CM-H). C2C12 mouse skeletal muscle cells were stimulated with EPS for 12 h. The supernatant was collected as muscle cells conditioned medium (CM-EPS). Human umbilical vein endothelial cells (HUVECs) were incubated with adipocyte CM and muscle cells CM together. Macrophages migration to HUVECs was detected with transwell system. The mRNA expressions of E-selectin, ICAM-1, MCP-1 and IL-6 were measured by real-time PCR. The phosphorylation of IKKα/β, NF-κB, Akt, AMPK, eNOS and SOCS3 protein levels were detected by Western blot. Concentration of NO was measured by ELISA kit. HUVECs apoptosis was detected by flow cytometry. ResultsCM-EPS reduced the increase of mRNA expressions of E-selectin, ICAM-1, MCP-1 and IL-6 in HUVECs induced by CN-H. The phosphorylations of IKKα/β and NF-κB, SOCS3 protein level and endothelial cells apoptosis, which were raised by CM-H, were significantly reduced by CM-EPS. CM-EPS reversed the effects of CM-H on Akt and eNOS phosphorylations and NO production in HUVECs. CM-EPS directly stimulated the phosphorylation of AMPK, which caused the following phosphorylation of eNOS in HUVECs. ConclusionIn summary, CM-EPS reversed endothelial cells inflammation, apoptosis, insulin resistance and dysfunction induced by CM-H.

The role and gene expression profile of SOCS3 in colorectal carcinoma.

SOCS3 has been postulated to play a role in the occurrence and progression of malignancies. However, the relationship of SOCS3 with colorectal carcinoma remains poorly understood. The purpose of the study was to explore the role of SOCS3 in colorectal carcinoma and its underlying mechanisms. Protein and mRNA expression of SOCS3 in colorectal carcinoma and normal colorectal mucosa was detected using immunohistochemistry and real-time quantitative PCR. SOCS3 expression was significantly lower in colorectal carcinoma tissue than in normal colorectal mucosa, and was negatively correlated with tumor invasion depth, lymph node metastasis, differentiation degree, and TNM stage. A stably transfected colorectal carcinoma cell line (8348SOCS3) with high expression of SOCS3 was established. The effects of SOCS3 overexpression on the growth, proliferation, invasion and tumor formation of colorectal carcinoma cells were examined by CCK-8 assay, transwell method and tumorigenicity assays in nude mice. Then we found SOCS3 overexpression significantly decreased proliferation and invasion capability of 8348 cells in vitro and in vivo. Furthermore, the effect of SOCS3 overexpression on the gene expression profile of colorectal carcinoma cells was analyzed using human genome arrays. The results revealed 369 genes that were differentially expressed in 8348SOCS3 cells. 193 genes was significantly increased and 176 genes was significantly decreased. Bioinformatics analysis demonstrated that high SOCS3 expression affected multiple signaling pathways in colorectal carcinoma including TGF-β/Smads, NF-κB, and HIF-MAPK pathways. Especially for the TGF-β/Smads pathways, high SOCS3 expression could inhibit TGF-β1 expression and activate Smad4 expression. These data suggested that low expression of SOCS3 was associated with the occurrence and progression of colorectal carcinoma. SOCS3 protein may be a useful indicator for malignancy and prognosis of colorectal carcinoma and also a new target for gene therapy.

Antimicrobial Peptide Epinecidin-1 Modulates MyD88 Protein Levels via the Proteasome Degradation Pathway.

The cationic antimicrobial peptide epinecidin-1 was identified from Epinephelus coioides and possesses multiple biological functions, including antibacterial, antifungal, anti-tumor, and immunomodulatory effects. In addition, epinecidin-1 suppresses lipopolysaccharide (LPS)-induced inflammation by neutralizing LPS and ameliorating LPS/Toll-like receptor (TLR)-4 internalization. However, it is unclear whether the actions of epinecidin-1 depend on the regulation of TLR adaptor protein MyD88 or endogenous TLR signaling antagonists, which include A20, interleukin-1 receptor associated kinase (IRAK)-M, and suppressor of cytokine signaling (SOCS)-1. Our results demonstrate that epinecidin-1 alone does not affect A20, IRAK-M, or SOCS-1 protein levels. However, pre-incubation of epinecidin-1 significantly inhibits LPS-induced upregulation of A20, IRAK-M, and SOCS-1. In addition, epinecidin-1 significantly reduces the abundance of MyD88 protein. Both MG132 (a specific proteasome inhibitor) and Heclin (a specific Smurf E3 ligase inhibitor) are able to abolish epinecidin-1-mediated MyD88 degradation. Thus, our data suggest that epinecidin-1 directly inhibits MyD88 via induction of the Smurf E3 ligase proteasome pathway.

Regulatory effect of decitabine on human acute myeloid leukemia cell line HL-60 and its mechanism

Objective To investigate the effect of decitabine (DAC) on human acute myeloid leukemia (AML) cell line HL-60 and the regulating of natural killer (NK) cell activating receptor (NKG2D) ligands (NKG2DL), and to detect the molecular mechanism of JAK-STAT3-SOCS signaling pathway. Methods The effect of DAC on the proliferation of HL-60 was detected by using CCK-8 assay. The cell apoptosis was analyzed by using Annexin-V/PI double standard method. The expressions of receptor NKG2DL including MICA/B and ULBPs in HL-60 cells were detected by using flow cytometry (FCM). The killing activity of NK cells was analyzed by using carboxy fluorescein diacetate succinimidyl ester (CFSE). The expressions of JAK/STAT3 signaling pathway or molecules including STAT3, its upstream kinases JAK1, JAK2 and the negative regulator of STAT3, SOCS-1, SOCS-3 were examined by Western blot. Methylation level of the SOCS-1, SOCS-3 gene after the treatment of DAC was analyzed by using methylation-sensitive high resolution melting (MS-HRM). Results There was an obvious inhibitory effect of DAC on HL-60 cells. The cell viability of HL-60 treated with 0.2, 0.5, and 1.0 μmol/L DAC for 48 h was decreased by (25±11) %, (39±8) % and (50±7) % (P < 0.01) respectively compared with those cells without DAC treatment. The incidence of apoptosis was (24.77±7.50) %, (27.10±4.48) % and (30.53±3.93) % after DAC treatment for 48h respectively, which were higher than that of untreated cells [(3.11±0.50) %] (P < 0.01). DAC induced a significant up-regulation of MICA/B, ULBP-1, ULBP-3 in HL-60 cells, and enhanced the sensitivity of HL-60 cells to NK cytotoxicity. Western blot results showed that a down-regulating expression of STAT3 and JAK1, JAK2 protein was detected, in addition to the phosphor-STAT3 and phosphor-JAKs in HL-60 cells after DAC treatment, but the expressions of SOCS-1 and SOCS-3 protein were increased. HRM results showed that DAC could inhibit the methylation of SOCS-3 gene. Conclusion DAC can inhibit the proliferation of HL-60 cells, upregulate the expression of NKG2DL and enhance the cytotoxicity of NK targeted to HL-60 cells, which might be related to the activity regulation of intracellular JAK-STAT3-SOCS signaling pathway. Key words: Leukemia, myeloid, acute; Cell proliferation; Apoptosis; Molecular mechanisms of action; Decitabine

Silencing of SOCS-1 and SOCS-3 suppresses renal interstitial fibrosis by alleviating renal tubular damage in a rat model of hydronephrosis.

Our study was performed to elucidate how SOCS-1/3 silencing suppresses renal interstitial fibrosis (RIF) by alleviating renal tubular damage in rat models affected by hydronephrosis. Male Wistar rats were randomly selected to establish hydronephrosis rat model, after which all rats were classified into normal, model, negative control (NC), siRNA-SOCS-1, siRNA-SOCS-3, and siRNA-SOCS-1 + siRNA-SOCS-3 groups. The levels of urine protein, serum creatinine (Scr), and blood urea nitrogen (BUN) were detected. ELISA was performed to determine levels of cystatin (CysC), β2-microglobulin (β2-MG), interleukin (IL)-6, and tumor necrosis factor (TNF)-α. RT-qPCR and Western blotting were used for mRNA and protein expressions of SOCS-1, SOCS-3, α-smooth muscle actin (α-SMA), and transforming Growth Factor (TGF)-β1. Compared with the normal group, the levels of Scr, BUN, urine protein, NAG, CysC, β2-MG, IL-6, and TNF-α were increased in other groups, as well as elevated mRNA and protein expressions of SOCS-1, SOCS-3, α-SMA, and TGF-β1. The siRNA-SOCS-1, siRNA-SOCS-3, and siRNA-SOCS-1 + siRNA-SOCS-3 groups were found with decreased levels of Scr, BUN, urine protein, NAG, CysC, β2-MG, IL-6, and TNF-α, as well as mRNA and protein expressions of SOCS-1, SOCS-3, α-SMA, and TGF-β1, including positive rates of SOCS-1 and SOCS-3 proteins in comparison with the model and NC groups. In comparison with the siRNA-SOCS-1 and siRNA-SOCS-3 groups, the siRNA-SOCS-1 + siRNA-SOCS-3 group exhibited decreased levels of Scr, BUN, urine protein, NAG, CysC, β2-MG, IL-6, and TNF-α. Our study demonstrated that silencing of SOCS-1/3 may suppress RIF by alleviating the renal tubular damage in rat models affected by hydronephrosis.

SOCS3 participates in cholinergic pathway regulation of synovitis in rheumatoid arthritis

ABSTRACTStimulation of the cholinergic inflammatory pathway can attenuate collagen-induced arthritis (CIA) and inhibit synovitis by Janus kinase (JAK) 2 and signal transducer and activator of transcription (STAT) 3 signaling. Suppressor of cytokine signaling (SOCS) protein can also regulate the inflammatory processes and activate JAK/STAT signal transduction, but its involvement in rheumatoid arthritis (RA) has not been demonstrated. This study investigated the effect of SOCS on cholinergic pathway regulation of synovitis in the fibroblast-like synoviocytes (FLSs) of RA and CIA mice. The effects of nicotine on SOCS1 and SOCS3 protein expression in FLSs were assayed by western blotting before and after transfection with a small interfering RNA oligonucleotide (SOCS3-siRNA or control-siRNA). Interleukin-6 was measured by enzyme-linked immunosorbent assay of SOCS3-siRNA and control-siRNA transfected FLS culture supernatants. Histopathological evaluation and immunohistochemical staining of SOCS3 were performed in joint tissue sections of control, CIA model, vagotomy, and nicotine-treated DBA/1 mice. Nicotine increased SOCS3 expression in the FLSs of RA. The inhibitory effect of nicotine on inflammatory factors was abolished by siRNA knockdown of SOCS3 protein expression. Nicotine increased the expression of SOCS3 protein in the synovium and reduced synovitis and bone erosion in CIA mice.

Suppressor of cytokine signaling 1 expression during LPS-induced inflammation and bone loss in rats.

This study aimed to characterize the dynamics of suppressor of cytokine signaling (SOCS1) expression in a rat model of lipopolysaccharide-induced periodontitis. Wistar rats in the experimental groups were injected three times/week with LPS from Escherichia coli on the palatal aspect of the first molars, and control animals were injected with vehicle (phosphate-buffered saline). Animals were sacrificed 7, 15, and 30 days after the first injection to analyze inflammation (stereometric analysis), bone loss (macroscopic analysis), gene expression (qRT-PCR), and protein expression/activation (Western blotting). The severity of inflammation and bone loss associated with LPS-induced periodontitis increased from day 7 to day 15, and it was sustained through day 30. Significant (p < 0.05) increases in SOCS1, RANKL, OPG, and IFN-γ gene expression were observed in the experimental group versus the control group at day 15. SOCS1 protein expression and STAT1 and NF-κB activation were increased throughout the 30-day experimental period. Gingival tissues affected by experimental periodontitis express SOCS1, indicating that this protein may potentially downregulate signaling events involved in inflammatory reactions and bone loss and thus may play a relevant role in the development and progression of periodontal disease.

Relationship of SOCS3 and TGF-β with IDO expression in early pregnancy chorionic villi and decidua.

We aimed to investigate the expression of suppressors cytokine signaling (SOCS)-3, transforming growth factor (TGF)-β and indoleamine 2,3-dioxygense (IDO) and to analyse the relationship of SOCS3 and TGF-β with IDO expression in early pregnancy chorionic villi and decidua in the maternal-fetal interface. Western blot analysis and immunohistochemical method were used to detect the expression of TGF-β, SOCS3 and IDO in chorionic villi and decidua tissues of normal pregnant women. SOCS3, TGF-β and IDO protein was identified in chorionic villi and decidua tissues of normal pregnant women and there was a negative correlation between the expression of IDO and SOCS3, but TGF-β expression was positively correlated with IDO expression. The levels of IDO expression in the decidua from normal pregnancies were significantly higher than those in chorionic villi, while the expression of SOCS3 was no significant difference between decidua and chorionic villi. In normal physiological state of pregnancy, SOCS3 and TGF-β may be involved in the regulation of immune tolerance by positive or negative regulation of IDO expression at maternal fetal interface.

SOCS3 expression in SF1 cells regulates adrenal differentiation and exercise performance.

Many hormones/cytokines are secreted in response to exercise and cytokine signaling may play a pivotal role in the training adaptations. To investigate the importance of cytokine signaling during vertical ladder climbing, a resistance exercise model, we produced mice lacking SOCS3 protein exclusively in steroidogenic factor-1 (SF1) cells (SF1 Socs3 KO mice). SF1 expression is found in steroidogenic cells of the adrenal cortex and gonads, as well as in neurons of the ventromedial nucleus of the hypothalamus. Histological markers of the fetal adrenal zone (or X-zone in rodents) were still present in adult males and postpartum SF1 Socs3 KO females, suggesting a previously unrecognized effect of SOCS3 on the terminal differentiation of the adrenal gland. This change led to a distinct distribution of lipid droplets along the adrenal cortex. Under basal conditions, adult SF1 Socs3 KO mice exhibited similar adrenal weight, and plasma ACTH and corticosterone concentrations. Nonetheless, SF1 Socs3 KO mice exhibited a blunted ACTH-induced corticosterone secretion. The overall metabolic responses induced by resistance training remained unaffected in SF1 Socs3 KO mice, including changes in body adiposity, glucose tolerance and energy expenditure. However, training performance and glucose control during intense resistance exercise were impaired in SF1 Socs3 KO mice. Furthermore, a reduced counter-regulatory response to 2-deoxy-d-glucose was observed in mutant mice. These findings revealed a novel participation of SOCS3 regulating several endocrine and metabolic aspects. Therefore, cytokine signaling in SF1 cells exerts an important role to sustain training performance possibly by promoting the necessary metabolic adjustments during exercise.

M6A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways.

N6 -methyladenosine (m6A) is the most common and abundant messenger RNA modification, modulated by ‘writers’, ‘erasers’ and ‘readers’ of this mark 1,2. In vitro data have shown that m6A influences all fundamental aspects of mRNA metabolism, mainly mRNA stability, to determine stem cell fates 3,4. However, its in vivo physiological function in mammals and adult mammalian cells is still unknown. Here we show that deletion of m6A ‘writer’ protein METTL3 in mouse T cells disrupts T cell homeostasis and differentiation. In a lymphopenic mouse adoptive transfer model, naive Mettl3 deficient T cells failed to undergo homeostatic expansion and remarkably remained in the naïve state up through 12 weeks, thereby preventing colitis. Consistent with these observations, the mRNAs of SOCS family genes encoding STAT- signaling inhibitory proteins, Socs1, Socs3 and Cish, were marked by m6A, exhibited slower mRNA decay and increased mRNAs and protein expression levels in Mettl3 deficient naïve T cells. This increased SOCS family activity consequently inhibited IL-7 mediated STAT5 activation and T cell homeostatic proliferation and differentiation. We also found that m6A plays important roles for inducible degradation of Socs mRNAs in response to IL-7 signaling in order to reprogram Naïve T cells for proliferation and differentiation. Our study elucidates for the first time the in vivo biological role of m6A modification in T cell mediated pathogenesis and reveals a novel mechanism of T cell homeostasis and signal-dependent induction of mRNA degradation.