SOCS1 Represses Fractionated Ionizing Radiation-Induced EMT Signaling Pathways through the Counter-Regulation of ROS-Scavenging and ROS-Generating Systems.

BACKGROUND/AIMS: Fractionated ionizing radiation (FIR) is an anti-cancer protocol widely applied for the treatment of diverse types of cancers to reduce damage to normal cells. However, cancer cells receiving multiple irradiations at low doses during FIR, often develop resistance to the therapy exhibiting malignant features including epithelial to mesenchymal transition (EMT). The present study has been performed to elucidate the mechanism of FIR-induced EMT signaling pathways and to identify a molecular target for radioresistance modulated by suppressors of cytokine signaling (SOCS)1. METHODS: Colorectal cancer cell lines received FIR with a daily dose of 2 Gy for 3 days. Generation of intracellular reactive oxygen species (ROS) and its role in EMT signaling induced by FIR were analyzed in SOCS1 over-expressing and knock-down cells. ROS were measured by DCF fluorescence using flow cytometry. Expression levels of EMT markers and signaling molecules were analyzed by Western blotting and confocal microscopy. RESULTS: FIR induced ROS and changes in EMT markers including down-regulation of E-cadherin with up-regulation of Twist and Snail. Pretreatment of anti-oxidant N-acetyl cysteine (NAC) abrogated the FIR-induced ROS generation and EMT response. Mechanistic studies indicated that the FIR-induced ROS-mediated EMT signaling proceeded through Akt→Src→Erk pathways. In accordance with the anti-ROS function, SOCS1 blocked the FIR-induced EMT and the associated signaling pathways through thioredoxin (Trx1) up-regulation. This is evidenced by the observation that Trx1 ablation in SOCS1 over-expressing cells negated the inhibitory action of SOCS1 by restoring the FIR-induced ROS and EMT markers. In addition, we have obtained data supporting that the FIR-induced ROS is derived from functional mitochondria and NADPH oxidases (Nox), which are both down-regulated by SOCS1. CONCLUSION: The results demonstrate that ROS signal acts as a mediator of the FIR-induced EMT. The data also suggest a potential anti-tumor function of SOCS1 by blocking the FIR therapy-induced resistance through the counter-regulation of ROS generating and scavenging systems.

Systemic PEGylated TRAIL treatment ameliorates liver cirrhosis in rats by eliminating activated hepatic stellate cells.

UNLABELLED: Liver fibrosis is a common outcome of chronic liver disease that leads to liver cirrhosis and hepatocellular carcinoma. No US Food and Drug Administration-approved targeted antifibrotic therapy exists. Activated hepatic stellate cells (aHSCs) are the major cell types responsible for liver fibrosis; therefore, eradication of aHSCs, while preserving quiescent HSCs and other normal cells, is a logical strategy to stop and/or reverse liver fibrogenesis/fibrosis. However, there are no effective approaches to specifically deplete aHSCs during fibrosis without systemic toxicity. aHSCs are associated with elevated expression of death receptors and become sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death. Treatment with recombinant TRAIL could be a potential strategy to ameliorate liver fibrosis; however, the therapeutic application of recombinant TRAIL is halted due to its very short half-life. To overcome this problem, we previously generated PEGylated TRAIL (TRAILPEG ) that has a much longer half-life in rodents than native-type TRAIL. In this study, we demonstrate that intravenous TRAILPEG has a markedly extended half-life over native-type TRAIL in nonhuman primates and has no toxicity in primary human hepatocytes. Intravenous injection of TRAILPEG directly induces apoptosis of aHSCs in vivo and ameliorates carbon tetrachloride-induced fibrosis/cirrhosis in rats by simultaneously down-regulating multiple key fibrotic markers that are associated with aHSCs. CONCLUSION: TRAIL-based therapies could serve as new therapeutics for liver fibrosis/cirrhosis and possibly other fibrotic diseases. (Hepatology 2016;64:209-223).

Role of promyelocytic leukemia zinc finger (PLZF) in cell proliferation and cyclin-dependent kinase inhibitor 1A (p21WAF/CDKN1A) gene repression.

Promyelocytic leukemia zinc finger (PLZF) is a transcription repressor that was initially isolated as a fusion protein with retinoic acid receptor α. PLZF is aberrantly overexpressed in various human solid tumors, such as clear cell renal carcinoma, glioblastoma, and seminoma. PLZF causes cellular transformation of NIH3T3 cells and increases cell proliferation in several cell types. PLZF also increases tumor growth in the mouse xenograft tumor model. PLZF may stimulate cell proliferation by controlling expression of the genes of the p53 pathway (ARF, TP53, and CDKN1A). We found that PLZF can directly repress transcription of CDKN1A encoding p21, a negative regulator of cell cycle progression. PLZF binds to the proximal Sp1-binding GC-box 5/6 and the distal p53-responsive elements of the CDKN1A promoter to repress transcription. Interestingly, PLZF interacts with Sp1 or p53 and competes with Sp1 or p53. PLZF interacts with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylates Ac-H3 and Ac-H4 histones at the CDKN1A promoter, which indicated the involvement of the corepressor·HDACs complex in transcription repression by PLZF. Also, PLZF represses transcription of TP53 and also decreases p53 protein stability by ubiquitination. PLZF may act as a potential proto-oncoprotein in various cell types.

The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation.

The tumour-suppressor gene CDKN1A (encoding p21Waf/Cip1) is thought to be epigenetically repressed in cancer cells. FBI-1 (ZBTB7A) is a proto-oncogenic transcription factor repressing the alternative reading frame and p21WAF/CDKN1A genes of the p53 pathway. FBI-1 interacts directly with MBD3 (methyl-CpG-binding domain protein 3) in the nucleus. We demonstrated that FBI-1 binds both non-methylated and methylated DNA and that MBD3 is recruited to the CDKN1A promoter through its interaction with FBI-1, where it enhances transcriptional repression by FBI-1. FBI-1 also interacts with the co-repressors nuclear receptor corepressor (NCoR), silencing mediator for retinoid and thyroid receptors (SMRT) and BCL-6 corepressor (BCoR) to repress transcription. MBD3 regulates a molecular interaction between the co-repressor and FBI-1. MBD3 decreases the interaction between FBI-1 and NCoR/SMRT but increases the interaction between FBI-1 and BCoR. Because MBD3 is a subunit of the Mi-2 autoantigen (Mi-2)/nucleosome remodelling and histone deacetylase (NuRD)-HDAC complex, FBI-1 recruits the Mi-2/NuRD-HDAC complex via MBD3. BCoR interacts with the Mi-2/NuRD-HDAC complex, DNMTs and HP1. MBD3 and BCoR play a significant role in the recruitment of the Mi-2/NuRD-HDAC complex- and the NuRD complex-associated proteins, DNMTs and HP. By recruiting DNMTs and HP1, Mi-2/NuRD-HDAC complex appears to play key roles in epigenetic repression of CDKN1A by DNA methylation.

Suppressors of cytokine signaling promote Fas-induced apoptosis through downregulation of NF-κB and mitochondrial Bfl-1 in leukemic T cells.

Suppressors of cytokine signaling (SOCS) are known as negative regulators of cytokine- and growth factor-induced signal transduction. Recently they have emerged as multifunctional proteins with regulatory roles in inflammation, autoimmunity, and cancer. We have recently reported that SOCS1 has antiapoptotic functions against the TNF-α- and the hydrogen peroxide-induced T cell apoptosis through the induction of thioredoxin, which protects protein tyrosine phosphatases and attenuates Jaks. In this study, we report that SOCS, on the contrary, promote death receptor Fas-mediated T cell apoptosis. The proapoptotic effect of SOCS1 was manifested with increases in Fas-induced caspase-8 activation, truncated Bid production, and mitochondrial dysfunctions. Both caspase-8 inhibitor c-Flip and mitochondrial antiapoptotic factor Bfl-1 were significantly reduced by SOCS1. These proapoptotic responses were not associated with changes in Jak or p38/Jnk activities but were accompanied with downregulation of NF-κB and NF-κB-dependent reporter gene expression. Indeed, p65 degradation via ubiquitination was accelerated in SOCS1 overexpressing cells, whereas it was attenuated in SOCS1 knockdown cells. With high NF-κB levels, the SOCS1-ablated cells displayed resistance against Fas-induced apoptosis, which was abrogated upon siBfl-1 transfection. The results indicate that the suppression of NF-κB-dependent induction of prosurvival factors, such as Bfl-1 and c-Flip, may serve as a mechanism for SOCS action to promote Fas-mediated T cell apoptosis. SOCS3 exhibited a similar proapoptotic function. Because both SOCS1 and SOCS3 are induced upon TCR stimulation, SOCS would play a role in activation-induced cell death by sensitizing activated T cells toward Fas-mediated apoptosis to maintain T cell homeostasis.

Counter-regulation mechanism of IL-4 and IFN-α signal transduction through cytosolic retention of the pY-STAT6:pY-STAT2:p48 complex.

IFN-α and IL-4 induce Th1 and Th2 responses, respectively, and often display antagonistic actions against each other. To elucidate the molecular mechanism of counter-regulation, we have investigated the signal interception by IFN-α and IL-4, employing a human B-cell line Ramos, sensitive to both cytokines. In these cells, IFN-α effectively inhibited IL-4-induced Fc epsilon receptor II (CD23) expression, whereas IL-4 suppressed IFN-α-mediated IRF7 expression. The counter-regulatory action by IL-4 and IFN-α proceeded with a delayed kinetics requiring 4 h. Notably, IFN-α did not affect the IL-4-induced tyrosine phosphorylation of STAT6, but induced a time-dependent cytoplasmic accumulation of phosphotyrosine(pY)-STAT6 and a corresponding decrease in nuclear pY-STAT6. By confocal analysis and co-immunoprecipitation assays, we demonstrated the colocalization and molecular interaction of IL-4-induced pY-STAT6 with IFN-α-induced pY-STAT2:p48 in the cytosol. In addition, the over-expression of STAT2 or STAT6 induced the concomitant cytosolic accumulation of pY-STAT6 or pY-STAT2, leading to the suppression of IL-4-induced CD23 or IFN-α-induced IRF7 gene expression, respectively. Our data suggest that the signals ensued by IFN-α and IL-4 induce cytoplasmic sequestration of IL-4-activated STAT6 and IFN-α-activated STAT2:p48 in B cells through the formation of pY-STAT6:pY-STAT2:p48 complex, which provides a novel mechanism by which IFN-α and IL-4 cross-regulate their signaling into the nucleus.

SOCS3 Attenuates Dexamethasone-Induced M2 Polarization by Down-Regulation of GILZ via ROS- and p38 MAPK-Dependent Pathways.

Suppressors of cytokine signaling (SOCS) have emerged as potential regulators of macrophage function. We have investigated mechanisms of SOCS3 action on type 2 macrophage (M2) differentiation induced by glucocorticoid using human monocytic cell lines and mouse bone marrow-derived macrophages. Treatment of THP1 monocytic cells with dexamethasone (Dex) induced ROS generation and M2 polarization promoting IL-10 and TGF-ß production, while suppressing IL-1ß, TNF-α and IL-6 production. SOCS3 over-expression reduced, whereas SOCS3 ablation enhanced IL-10 and TGF-ß induction with concomitant regulation of ROS. As a mediator of M2 differentiation, glucocorticoid-induced leucine zipper (GILZ) was down-regulated by SOCS3 and up-regulated by shSOCS3. The induction of GILZ and IL-10 by Dex was dependent on ROS and p38 MAPK activity. Importantly, GILZ ablation led to the inhibition of ROS generation and anti-inflammatory cytokine induction by Dex. Moreover, GILZ knock-down negated the up-regulation of IL-10 production induced by shSOCS3 transduction. Our data suggest that SOCS3 targets ROS- and p38-dependent GILZ expression to suppress Dex-induced M2 polarization.

SOCS1 counteracts ROS-mediated survival signals and promotes apoptosis by modulating cell cycle to increase radiosensitivity of colorectal cancer cells.

As negative regulators of cytokine signaling pathways, suppressors of cytokine signaling (SOCS) proteins have been reported to possess both pro-tumor and anti-tumor functions. Our recent studies have demonstrated suppressive effects of SOCS1 on epithelial to mesenchymal signaling in colorectal cancer cells in response to fractionated ionizing radiation or oxidative stress. The objective of the present study was to determine the radiosensitizing action of SOCS1 as an anti-tumor mechanism in colorectal cancer cell model. In HCT116 cells exposed to ionizing radiation, SOCS1 over-expression shifted cell cycle arrest from G2/M to G1 and promoted radiation-induced apoptosis in a p53-dependent manner with down-regulation of cyclin B and up-regulation of p21. On the other hand, SOCS1 knock-down resulted in a reduced apoptosis with a decrease in G1 arrest. The regulatory action of SOCS1 on the radiation response was mediated by inhibition of radiation-induced Jak3/STAT3 and Erk activities, thereby blocking G1 to S transition. Radiation-induced early ROS signal was responsible for the activation of Jak3/Erk/STAT3 that led to cell survival response. Our data collectively indicate that SOCS1 can promote radiosensitivity of colorectal cancer cells by counteracting ROS-mediated survival signal, thereby blocking cell cycle progression from G1 to S. The resulting increase in G1 arrest with p53 activation then contributes to the promotion of apoptotic response upon radiation. Thus, induction of SOCS1 expression may increase therapeutic efficacy of radiation in tumors with low SOCS1 levels. [BMB Reports 2022; 55(4): 198-203].

Anti-inflammatory mechanisms of suppressors of cytokine signaling target ROS via NRF-2/thioredoxin induction and inflammasome activation in macrophages.

Suppressors of cytokine signaling (SOCS) exhibit diverse antiinflammatory effects. Since ROS acts as a critical mediator of inflammation, we have investigated the anti-inflammatory mechanisms of SOCS via ROS regulation in monocytic/macrophagic cells. Using PMA-differentiated monocytic cell lines and primary BMDMs transduced with SOCS1 or shSOCS1, the LPS/TLR4-induced inflammatory signaling was investigated by analyzing the levels of intracellular ROS, antioxidant factors, inflammasome activation, and pro-inflammatory cytokines. The levels of LPS-induced ROS and the production of pro-inflammatory cytokines were notably down-regulated by SOCS1 and up-regulated by shSOCS1 in an NAC-sensitive manner. SOCS1 up-regulated an ROS-scavenging protein, thioredoxin, via enhanced expression and binding of NRF-2 to the thioredoxin promoter. SOCS3 exhibited similar effects on NRF-2/thioredoxin induction, and ROS downregulation, resulting in the suppression of inflammatory cytokines. Notably thioredoxin ablation promoted NLRP3 inflammasome activation and restored the SOCS1-mediated inhibition of ROS and cytokine synthesis induced by LPS. The results demonstrate that the anti-inflammatory mechanisms of SOCS1 and SOCS3 in macrophages are mediated via NRF-2-mediated thioredoxin upregulation resulting in the downregulation of ROS signal. Thus, our study supports the anti-oxidant role of SOCS1 and SOCS3 in the exquisite regulation of macrophage activation under oxidative stress. [BMB Reports 2020; 53(12): 640-645].

Eukaryotic translation initiator protein 1A isoform, CCS-3, enhances the transcriptional repression of p21CIP1 by proto-oncogene FBI-1 (Pokemon/ZBTB7A).

FBI-1, a member of the POK (POZ and Kruppel) family of transcription factors, plays a role in differentiation, oncogenesis, and adipogenesis. eEF1A is a eukaryotic translation elongation factor involved in several cellular processes including embryogenesis, oncogenic transformation, cell proliferation, and cytoskeletal organization. CCS-3, a potential cervical cancer suppressor, is an isoform of eEF1A. We found that eEF1A forms a complex with FBI-1 by co-immunoprecipitation, SDS-PAGE, and MALDI-TOF Mass analysis of the immunoprecipitate. GST fusion protein pull-downs showed that FBI-1 directly interacts with eEF1A and CCS-3 via the zinc finger and POZ-domain of FBI-1. FBI-1 co-localizes with either eEF1A or CCS-3 at the nuclear periplasm. CCS-3 enhances transcriptional repression of the p21CIP1 gene (hereafter referred to as p21) by FBI-1. The POZ-domain of FBI-1 interacts with the co-repressors, SMRT and BCoR. We found that CCS-3 also interacts with the co-repressors independently. The molecular interaction between the co-repressors and CCS-3 at the POZ-domain of FBI-1 appears to enhance FBI-1 mediated transcriptional repression. Our data suggest that CCS-3 may be important in cell differentiation, tumorigenesis, and oncogenesis by interacting with the proto-oncogene FBI-1 and transcriptional co-repressors.

Identification of human thioredoxin as a novel IFN-gamma-induced factor: mechanism of induction and its role in cytokine production.

BACKGROUND: IFN-gamma is a multifunctional peptide with a potent immune defense function which is also known as a prototypic Th1 cytokine. While screening for genes differentially expressed by Th1 and Th2 cytokines, human thioredoxin was identified as a novel target gene induced by IFN-gamma. The mechanism by which thioredoxin is induced by IFN-gamma and the signaling pathways involved in its induction were analyzed. In addition, the effects of thioredoxin on immune cell survival and cytokine production were examined by thioredoxin over-expression and recombinant thioredoxin treatment. RESULTS: Human thioredoxin was selectively induced by IFN-gamma in monocytic and T cell lines. In monocytic cells, the induction of thioredoxin gene expression by IFN-gamma was dose-dependent, and both the mRNA and protein levels were increased by 2~3 fold within 4 to 24 h hours of IFN-gamma treatment. The thioredoxin induction by IFN-gamma was insensitive to cycloheximide treatment, suggesting that it is a primary response gene induced by IFN-gamma. Subsequent analysis of the signaling pathways indicated that the Jak/Stat, Akt, and Erk pathways play a role in IFN-gamma signaling that leads to thioredoxin gene expression. Thioredoxin was induced by oxidative or radiation stresses, and it protected the immune cells from apoptosis by reducing the levels of reactive oxygen species. Furthermore, thioredoxin modulated the oxidant-induced cytokine balance toward Th1 by counter-regulating the production of IL-4 and IFN-gamma in T cells. CONCLUSION: These data suggest that thioredoxin is an IFN-gamma-induced factor that may play a role in developing Th1 immunity and in the maintenance of immune homeostasis upon infection, radiation, and oxidative stress.

Ras/Erk pathway positively regulates Jak1/STAT6 activity and IL-4 gene expression in Jurkat T cells.

T helper cells can be largely divided into two functional subsets, Th1 and Th2, which are characterized by the cytokines they produce. The mechanism of Th1 versus Th2 cytokine production is thought to involve interaction of TCR-induced signal and cytokine-induced signal, mainly activating the Ras/MAPK and the Jak/STAT pathway, respectively. In order to gain insight into the signal transduction network for Th1 and Th2 differentiation, we have analyzed the functional cross-talk between the Jak/STAT and the Ras/MAPK pathway. In cytokine-producing Jurkat T cells, we have found that IL-4 induces activation of Erk and Akt, and the IL-4-induced STAT6 activity is suppressed by inhibitors of Erk and PI3K. The transfection of daRas into these cells resulted in the up-regulation of specific activity of Jak1/STAT6 with a concomitant increase in Erk and Akt activity, while siRNA-mediated knock-out of Ras resulted in the inhibition of Jak1/STAT6. Furthermore, the IL-4 mRNA expression and IL-4 promoter activity were enhanced by daRas but not by dnRas. The Ras-induced increase of both STAT6 activity and IL-4 mRNA level was effectively blocked by a Mek/Erk inhibitor, suggesting that Ras/Erk pathway positively regulates STAT6 activity and IL-4 transcription. Together, the results indicate that there is a functional cross-talk between Ras/Erk and IL-4/Jak1/STAT6, which contributes to the regulation of IL-4 transcription in T cells.

Surfactin from Bacillus subtilis displays anti-proliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression.

The effect of surfactin on the proliferation of LoVo cells, a human colon carcinoma cell line, was examined. Surfactin strongly blocked the proliferation of LoVo cells by inducing pro-apoptotic activity and arresting the cell cycle, according to several lines of evidence on DNA fragmentation, Annexin V staining, and altered levels of poly (ADP-ribose) polymerase, caspase-3, p21(WAF1/Cip1), p53, CDK2 and cyclin E. The anti-proliferative activity of surfactin was mediated by inhibiting extracellular-related protein kinase and phosphoinositide 3-kinase/Akt activation, as assessed by phosphorylation levels. Therefore, our data suggest that surfactin may have anti-cancer properties as a result of its ability to downregulate the cell cycle and suppress its survival.

Mechanism of suppressors of cytokine signaling 1 inhibition of epithelial-mesenchymal transition signaling through ROS regulation in colon cancer cells: suppression of Src leading to thioredoxin up-regulation.

Reactive oxygen species (ROS) participate in malignant progression of cancers including epithelial-mesenchymal transition (EMT). We have investigated the role of suppressors of cytokine signaling (SOCS)1 as an inhibitor of ROS-induced EMT using colon cancer cell lines transduced with SOCS1 and shSOCS1. Hydrogen peroxide treatment induced EMT features such as elevation of vimentin and Snail with a corresponding reduction of E-cadherin. The EMT markers are significantly decreased upon SOCS1 over-expression while increased under SOCS1 knock-down. SOCS1 inhibited ROS signaling pathways associated with EMT such as Src, Jak, and p65. Of note, strong up-regulation of Src activity in SOCS1-ablated cells was responsible for the elevated signaling leading to EMT, as shSrc or Src inhibitor abolished the shSOCS1-induced promotion of EMT response. Suppression of ROS-inducible EMT markers and invasion in SOCS1 over-expressing cells correlated with significantly low intracellular ROS levels in these cells. Analysis of antioxidant enzymes in SOCS1-transduced cells revealed a selective up-regulation of thioredoxin (Trx1), while thioredoxin ablation restored ROS levels and the associated EMT markers. As a mechanism of thioredoxin up-regulation by SOCS1, inhibition of Src activity promoting nuclear translocation of Nrf-2 is proposed. Taken together, our data strongly indicate that SOCS1 antagonizes EMT by suppressing Src activity, leading to thioredoxin expression and down-regulation of ROS levels in colon cancer cells.

Mechanism of NF-kappaB activation induced by gamma-irradiation in B lymphoma cells : role of Ras.

Nuclear factor (NF)-kappaB is a ubiquitous transcription factor involved in diverse cellular responses to various stimuli, including growth factors and radiation stress. Recently it was reported that gamma-irradiation (gamma-IR) upregulates allergy-associated adhesion molecule CD23 on B cells and monocytes via NF-kappaB activation. In the present study, the mechanism of NF-kappaB activation by gamma-IR was investigated to understand the signaling pathways involved in IR-induced, NF-kappaB-mediated enhancement of CD23 expression. In human B-cell line Ramos, gamma-IR induced a dose-dependent increase of nuclear translocation and transcriptional activity of NF-kappaB. The gamma-IR-induced NF-kappaB activation in these cells was sensitive to a proteosome inhibitor MG132 and an antioxidant, pyrollidine dithiocarbamate (PDTC), which suggests that gamma-IR-induced NF-kappaB activation proceeds via IkappaB gradation and redox regulation. Since Ras was shown to play a role in NF-kappaB-mediated survival and inflammation of cancer cells against radiation, the role of Ras signaling in the gamma-IR-induced NF-kappaB activation in these transformed B cells was examined. Transfection and overexpression of dominant active Ras produced an increase in NF-kappaB activity as shown by DNA binding and transcriptional activities of the kappaB-dependent reporter gene. gamma-IR, however, did not induce Erk activation, nor the gamma-IR-induced kappaB activity that was suppressed by inhibitors of Ras/Raf interaction or MEK/Erk. Importantly, it was noted that Ras significantly augmented both the gamma-IR-induced NF-kappaB activity and the gamma-IR-induced CD23 expression. Together these results suggest that while gamma-IR and Ras both contribute to the upregulation of CD23 expression via NF-kappaB Raf or Erk is not involved in gamma-IR-induced NF-kappaB activation.

Corticosteroid inhibits IL-4 signaling through down-regulation of IL-4 receptor and STAT6 activity.

Corticosteroids are potent anti-inflammatory and immunosuppressive agents which down-regulate cytokine production and action. Yet, contradictory results have been reported for their effects on the interleukin (IL)-4-mediated response. Using type II Fc receptor for IgE/CD23 as a target gene, here we report that corticosteroids at 10(-4)-10(-6) M inhibit the IL-4 signaling pathway in human primary immune cells by down-regulation of the IL-4-induced IL-4 receptor expression and STAT6 activation. Although functional antagonism between steroid receptor and STAT6 for their transcriptional activity has been recently described, this is the first report that steroid inhibits the IL-4-induced STAT6 activity at the level of tyrosine phosphorylation and target DNA binding.

IGF-1 potentiation of IL-4-induced CD23/Fc(epsilon)RII expression in human B cells.

Interleukin-4 (IL-4) is a major cytokine regulating IgE production and IgE response. Neuroendocrine growth factors, such as growth hormone and insulin-like growth factor (IGF-1), also modulate IgE production by human B cells. We report that in both human primary immune cells and established B cell lines, IGF-1 increased expression of the IL-4-induced type II IgE receptor (Fc(epsilon)RII/CD23). This effect was also seen at the mRNA level. IGF-1 increased the activity of signal transducer and activator of transcription 6 (STAT6) in response to IL-4, and activated NF-(kappa)B. STAT6 and NF-(kappa)B are major transcription factors controlling CD23 expression. The tyrosine kinase inhibitor, tyrphostin, abolished both IL-4- and IL-4 plus IGF-1-mediated induction of STAT6 and the subsequent CD23 expression. In contrast, pyrrolidine dithiocarbamate (PDTC), an NF-(kappa)B inhibitor, only suppressed the enhancing effect of IGF-1 on IL-4-induced CD23 expression. Our data suggest that IGF-1 modulates CD23 gene expression by affecting the STAT6 and NF-(kappa)B pathways. Regulation of CD23 by IGF-1 may have an important implication for the immunomodulatory potential of IGF-1, and may provide a new therapeutic target for allergy and other hypersensitivity reactions involving an excessive IgE response.

Increased serine phosphorylation and activation of STAT1 by oncogenic Ras transfection.

Interferon (IFN)-gamma induces the activation of a signal transducer and activator of transcription 1 (STAT1), and regulates the growth response of diverse cell types. Although STAT1 activation is primarily induced upon tyrosine phosphorylation by Jak1 and Jak2 (the IFN-gamma receptor-associated tyrosine kinases), the full activation of STAT1 is thought to involve serine phosphorylation by unidentified protein kinases. As a part of our on-going investigation on the deregulation of STATs in oncogene-transformed cells, we found that STAT1 activation was efficiently induced by IFN-gamma in oncogenic Ras-transformed fibroblasts, compared to the parental fibroblasts. This was shown by target DNA binding activity, nuclear translocation, and tyrosine phosphorylation of STAT1. Using a transient transfection system, we directly demonstrated that Ras-transfection up-regulated the IFN-y-induced STAT1 activation with a concomitant increase in Erk MAPK activity. Notably, the enhanced serine phosphorylation of STAT1 was observed upon Ras-transfection, which was specifically associated with the induction of MAPK, but not Akt activity in these cells. The data suggest that Ras/MAPK module components may positively regulate STAT1 activity by inducing the serine-phosphorylation of STAT1. This would contribute to the enhanced tyrosine phosphorylation, nuclear translocation, and DNA-binding of STAT1 upon exposure of the cells to IFN-gamma in the Ras-transformed cells.

Gamma irradiation up-regulates expression of B cell differentiation molecule CD23 by NF-kappaB activation.

Gamma irradiation (gamma-IR) is reported to have diverse effects on immune cell apoptosis, survival and differentiation. In the present study, the immunomodulatory effect of a low dose gamma-IR (5~10 Gy) was investigated, focusing on the role of NF-kappaB in the induction of the B cell differentiation molecule, CD23/FceRII. In the human B cell line Ramos, gamma-IR not only induced CD23 expression, but also augmented the IL-4-induced surface CD23 levels. While gamma-IR did not cause STAT6 activation in these cells, it did induce both DNA binding and the transcriptional activity of NF-kappaB in the IkappaB degradation-dependent manner. It was subsequently found that different NF-kappaB regulating signals modulated the gamma-IR-or IL-4-induced CD23 expression. Inhibitors of NF-kappaB activation, such as PDTC and MG132, suppressed the gamma-IR-mediated CD23 expression. In contrast, Ras, which potentiates gamma-IR-induced NF-kappaB activity in these cells, further augmented the gamma-IR- or IL-4-induced CD23 levels, The induction of NF-kappaB activation and the subsequent up-regulation of CD23 expression by gamma-IR were also observed in monocytic cells. These results suggest that gamma-IR, at specific dosages, can modulate immune cell differentiation through the activation of NF-kappaB, and this potentially affects the immune inflammatory response that is mediated by cytokines.

Identification of DC21 as a novel target gene counter-regulated by IL-12 and IL-4.

The Th1 vs. Th2 balance is critical for the maintenance of immune homeostasis. Therefore, the genes that are selectively-regulated by the Th1 and Th2 cytokines are likely to play an important role in the Th1 and Th2 immune responses. In order to search for and identify the novel target genes that are differentially regulated by the Th1/Th2 cytokines, the human PBMC mRNAs differentially expressed upon the stimulation with IL-4 or IL-12, were screened by employing the differential display polymerase chain reaction. Among a number of clones selected, DC21 was identified as a novel target gene that is regulated by IL-4 and IL-12. The DC21 gene expression was up-regulated either by IL-4 or IL-12, yet counterregulated by co-treatment with IL-4 and IL-12. DC21 is a dendritic cell protein with an unknown function. The sequence analysis and conserved-domain search revealed that it has two AU-rich motifs in the 3'UTR, which is a target site for the regulation of mRNA stability by cytokines, and that it belongs to the N-acetyltransferase family. The induction of DC21 by IL-12 peaked around 8-12 h, and lasted until 24 h. LY294002 and SB203580 significantly suppressed the IL-12-induced DC21 gene expression, which implies that PI3K and p38/JNK are involved in the IL-12 signal transduction pathway that leads to the DC21 expression. Furthermore, tissue blot data indicated that DC21 is highly expressed in tissues with specialized-resident macrophages, such as the lung, liver, kidney, and placenta. Together, these data suggest a possible role for DC21 in the differentiation and maturation of dendritic cells regulated by IL-4 and IL-12.