Choong-Eun Lee

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, p21WAF1/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.

Proto-oncogene FBI-1 (Pokemon/ZBTB7A) Represses Transcription of the Tumor Suppressor Rb Gene via Binding Competition with Sp1 and Recruitment of Co-repressors

FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at bp –308 to –188 of the Rb promoter region. The Rb promoter also contains two Sp1 binding sites: GC-box 1 (bp –65 to –56) and GC-box 2 (bp –18 to –9), the latter of which is also bound by FBI-1. We found that FRE3 (bp –244 to –236) is also a Sp1 binding element. FBI-1 represses transcription of the Rb gene not only by binding to the FREs, but also by competing with Sp1 at the GC-box 2 and the FRE3. By binding to the FREs and/or the GC-box, FBI-1 represses transcription of the Rb gene through its POZ-domain, which recruits a co-repressor-histone deacetylase complex and deacetylates histones H3 and H4 at the Rb gene promoter. FBI-1 inhibits C2C12 myoblast cell differentiation by repressing Rb gene expression.

IFN-γ and IFN-α Posttranscriptionally Down-Regulate the IL-4-Induced IL-4 Receptor Gene Expression

As Th1 and Th2 cytokines, IFN-γ/α and IL-4 counterregulate diverse immune functions. In particular, IFN-γ and IFN-α have been reported to markedly suppress the IL-4-induced IgE production and type II IgE receptor (FcεRII/CD23) expression. Because modulation of IL-4R may be an important mechanism in the regulation of IL-4 response, we have investigated the effect of IFN-γ/α on IL-4R expression and signal transduction mechanisms involved in this process. In human mononuclear cells and B cells isolated from tonsil or peripheral blood, IL-4 up-regulates IL-4R(α) expression at surface protein and mRNA levels, and the IL-4-induced IL-4R(α) is significantly down-regulated by both IFN-γ and IFN-α to a similar extent. The inhibitory effects of IFN-γ/α on the IL-4R mRNA expression require a lag period of about 8 h, and are sensitive to cycloheximide treatment, which suggests that the suppressive effect of IFNs on IL-4R gene expression is a secondary response requiring de novo synthesis of IFN-induced factors. Under such conditions that the inhibitory effects of IFNs are observed, IFNs do not affect the IL-4-induced STAT6 activation and IL-4R transcription, as analyzed by EMSA and nuclear run-on assays, respectively. Subsequently, mRNA stability studies have indicated that the action of IFN-γ/α is primarily mediated by an accelerated decay of IL-4-induced IL-4R mRNA. Thus, it appears that, as already shown in the case of the IL-4-induced FcεRII regulation, posttranscriptional inhibition of IL-4-inducible genes by mRNA destabilization is a common mechanism by which type I and II IFNs antagonize the IL-4 response in human immune cells.

Proto-oncogene FBI-1 (Pokemon) and SREBP-1 Synergistically Activate Transcription of Fatty-acid Synthase Gene (FASN)

FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation.

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.

A Novel POK Family Transcription Factor, ZBTB5, Represses Transcription of p21CIP1 Gene

Transcriptional repression through chromatin remodeling and histone deacetylation has been postulated as a driving force for tumorigenesis. We isolated and characterized a novel POZ domain Krüppel-like zinc finger transcription repressor, ZBTB5 (zinc finger and BTB domain-containing 5). Serial analysis of gene expression (SAGE) analysis showed that ZBTB5 expression is higher in retinoblastoma and muscle cancer tissues. Immunocytochemistry showed that ZBTB5 was localized to the nucleus, particularly nuclear speckles. ZBTB5 directly repressed transcription of cell cycle arrest gene p21 by binding to the proximal GC-box 5/6 elements and the two distal p53-responsive elements (bp −2323 ∼ −2299; bp −1416 ∼ −1392). Chromatin immunoprecipitation assays showed that ZBTB5 and p53 competed with each other in occupying the p53 binding elements. ZBTB5 interacted with co-repressor-histone deacetylase complexes such as BCoR (BCL-6-interacting corepressor), NCoR (nuclear receptor corepressor), and SMRT (silencing mediator for retinoid and thyroid receptors) via its POZ domain. These interactions resulted in deacetylation of histones Ac-H3 and Ac-H4 at the proximal promoter, which is important in the transcriptional repression of p21. MTT (3-(4,5-di meth yl thi azol-2-yl)-2,5-diphenyltetrazolium bromide) assays and fluorescent-activated cell sorter analysis revealed that ZBTB5 stimulated both cell proliferation and cell cycle progression, significantly increasing the number of cells in S-phase. Overall, our data suggest that ZBTB5 is a potent transcription repressor of cell cycle arrest gene p21 and a potential proto-oncogene stimulating cell proliferation.

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

BackgroundIFN-γ 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-γ. The mechanism by which thioredoxin is induced by IFN-γ 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.ResultsHuman thioredoxin was selectively induced by IFN-γ in monocytic and T cell lines. In monocytic cells, the induction of thioredoxin gene expression by IFN-γ was dose-dependent, and both the mRNA and protein levels were increased by 2~3 fold within 4 to 24 h hours of IFN-γ treatment. The thioredoxin induction by IFN-γ was insensitive to cycloheximide treatment, suggesting that it is a primary response gene induced by IFN-γ. Subsequent analysis of the signaling pathways indicated that the Jak/Stat, Akt, and Erk pathways play a role in IFN-γ 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-γ in T cells.ConclusionThese data suggest that thioredoxin is an IFN-γ-induced factor that may play a role in developing Th1 immunity and in the maintenance of immune homeostasis upon infection, radiation, and oxidative stress.

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.

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.

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.