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Intestinal intraepithelial lymphocytes (IELs) are distributed along the length of the intestine and are considered the frontline of immune surveillance. The precise molecular mechanisms, especially epigenetic regulation, of their development and function are poorly understood. The trimethylation of histone 3 at lysine 27 (H3K27Me3) is a kind of histone modifications and associated with gene repression. Kdm6b is an epigenetic enzyme responsible for the demethylation of H3K27Me3 and thus promotes gene expression. Here we identified Kdm6b as an important intracellular regulator of small intestinal IELs. Mice genetically deficient for Kdm6b showed greatly reduced numbers of TCRαβ+CD8αα+ IELs. In the absence of Kdm6b, TCRαβ+CD8αα+ IELs exhibited increased apoptosis, disturbed maturation and a compromised capability to lyse target cells. Both IL-15 and Kdm6b-mediated demethylation of histone 3 at lysine 27 are responsible for the maturation of TCRαβ+CD8αα+ IELs through upregulating the expression of Gzmb and Fasl. In addition, Kdm6b also regulates the expression of the gut-homing molecule CCR9 by controlling H3K27Me3 level at its promoter. However, Kdm6b is dispensable for the reactivity of thymic precursors of TCRαβ+CD8αα+ IELs (IELPs) to IL-15 and TGF-β. In conclusion, we showed that Kdm6b plays critical roles in the maturation and cytotoxic function of small intestinal TCRαβ+CD8αα+ IELs.

Aims: Human embryonic stem cell derived-cardiovascular progenitor cells (hESC-CVPCs) are a promising cell source for cardiac repair, while the underlying mechanisms need to be elucidated. We recently observed cardioprotective effects of human pluripotent stem cell (hPSC)-CVPCs in infarcted nonhuman primates, but their effects on inflammation during early phase of myocardial infarction (MI) and the contribution of such effect to the cardioprotection are unclear. Results: Injection of hESC-CVPCs into acutely infarcted myocardium significantly ameliorated the functional worsening and scar formation, concomitantly with reduced inflammatory reactions and cardiomyocyte apoptosis as well as increased vascularization. Moreover, hESC-CVPCs modulated cardiac macrophages toward a reparative phenotype in the infarcted hearts, and such modulation was further confirmed in vitro using human cardiovascular progenitor cell (hCVPC)-conditioned medium (hCVPC-CdM) and highly contained interleukin (IL)-4/IL-13. Furthermore, signal transducer and activator of transcription 6 (STAT6) was activated in hCVPC-CdM- and IL-4/IL-13-treated macrophages in vitro and in hESC-CVPC-implanted MI hearts, resulting in the polarization of macrophages toward a reparative phenotype in the post-MI hearts. However, hESC-CVPC-mediated modulation on macrophages and cardioprotection were abolished in STAT6-deficient MI mice. Innovation: This is the first report about the immunoregulatory role played by hESC-CVPCs in the macrophage polarization in the infarcted hearts, its importance for the infarct repair, and the underlying signaling pathway. The findings provide new insight into the mechanism of microenvironmental regulation of stem cell-based therapy during acute MI. Conclusions: Implantion of hESC-CVPCs during the early phase of MI promotes infarct repair via the modulation of macrophage polarization through secreted cytokine-mediated STAT6 activation. The findings suggest a therapeutic potential by modulating macrophage polarization during acute phase of MI.

Summary The mammalian target of rapamycin (mTOR) is a serine-threonine kinase involved in cellular innate immunity, metabolism, and senescence. FK506-binding protein 12 (FKBP12) inhibits mTOR kinase activity via direct association. The FKBP12-mTOR association can be strengthened by the immunosuppressant rapamycin, but the underlying mechanism remains elusive. We show here that the FKBP12-mTOR association is tightly regulated by an acetylation–deacetylation cycle. FKBP12 is acetylated on the lysine cluster (K45/K48/K53) by CREB-binding protein (CBP) in mammalian cells in response to nutrient treatment. Acetyl-FKBP12 associates with CBP acetylated Rheb. Rapamycin recruits SIRT2 with a high affinity for FKBP12 association and deacetylation. SIRT2-deacetylated FKBP12 then switches its association from Rheb to mTOR. Nutrient-activated mTOR phosphorylates IRF3S386 for the antiviral response. In contrast, rapamycin strengthening FKBP12-mTOR association blocks mTOR antiviral activity by recruiting SIRT2 to deacetylate FKBP12. Hence, on/off mTOR activity in response to environmental nutrients relies on FKBP12 acetylation and deacetylation status in mammalian cells., Graphical abstract, Highlights • FKBP12-mTOR association is tightly regulated by an acetylation–deacetylation cycle • SIRT2 is responsible for FKBP12 deacetylation • Acetylation of Rheb is indispensable to mTOR activation • mTOR phosphorylates IRF3 S386 for type-I interferon gene expression, Biochemistry; Protein; Molecular biology

Regulated/activated protein kinase (PRAK) plays a crucial role in modulating biological function. However, the role of PRAK in mediating cardiac dysfunction and metabolic disorders remains unclear. We examined the effects of deletion of PRAK on modulating cardiac function and insulin resistance in mice exposed to a high-fat diet (HFD). Wild-type and PRAK−/− mice at 8 weeks old were exposed to either chow food or HFD for a consecutive 16 weeks. Glucose tolerance tests and insulin tolerance tests were employed to assess insulin resistance. Echocardiography was employed to assess myocardial function. Western blot was used to determine the molecular signaling involved in phosphorylation of IRS-1, AMPKα, ERK-44/42, and irisin. Real time-PCR was used to assess the hypertrophic genes of the myocardium. Histological analysis was employed to assess the hypertrophic response, interstitial myocardial fibrosis, and apoptosis in the heart. Western blot was employed to determine cellular signaling pathway. HFD-induced metabolic stress is indicated by glucose intolerance and insulin intolerance. PRAK knockout aggravated insulin resistance, as indicated by glucose intolerance and insulin intolerance testing as compared with wild-type littermates. As compared with wild-type mice, hyperglycemia and hypercholesterolemia were manifested in PRAK-knockout mice following high-fat diet intervention. High-fat diet intervention displayed a decline in fractional shortening and ejection fraction. However, deletion of PRAK exacerbated the decline in cardiac function as compared with wild-type mice following HFD treatment. In addition, PRAK knockout mice enhanced the expression of myocardial hypertrophic genes including ANP, BNP, and βMHC in HFD treatment, which was also associated with an increase in cardiomyocyte size and interstitial fibrosis. Western blot indicated that deletion of PRAK induces decreases in phosphorylation of IRS-1, AMPKα, and ERK44/42 as compared with wild-type controls. Our finding indicates that deletion of PRAK promoted myocardial dysfunction, cardiac remodeling, and metabolic disorders in response to HFD.

T helper 17 (Th17)‐cell differentiation triggered by interleukin‐6 (IL‐6) via STAT3 activation promotes inflammation in inflammatory bowel disease (IBD) patients. However, leukemia inhibitory factor (LIF), an IL‐6 family cytokine, restricts inflammation by blocking Th17‐cell differentiation via an unknown mechanism. Here, we report that microbiota dysregulation promotes LIF secretion by intestinal epithelial cells (IECs) in a mouse colitis model. LIF greatly activates STAT4 phosphorylation on multiple SPXX elements within the C‐terminal transcription regulation domain. STAT4 and STAT3 act reciprocally on both canonical cis‐inducible elements (SIEs) and noncanonical “AGG” elements at different loci. In lamina propria lymphocytes (LPLs), STAT4 activation by LIF blocks STAT3‐dependent Il17a/Il17f promoter activation, whereas in IECs, LIF bypasses the extraordinarily low level of STAT4 to induce YAP gene expression via STAT3 activation. In addition, we found that the administration of LIF is sufficient to restore microbiome homeostasis. Thus, LIF effectively inhibits Th17 accumulation and promotes repair of damaged intestinal epithelium in inflamed colon, serves as a potential therapy for IBD.

Glutamine metabolism plays an important role in cancer development and progression. Glutaminase C (GAC), the first enzyme in glutaminolysis, has emerged as an important target for cancer therapy and many studies have focused on the mechanism of enhanced GAC expression in cancer cells. However, little is known about the post-translational modification of GAC. Here, we report that phosphorylation is a crucial post-translational modification of GAC, which is responsible for the higher glutaminase activity in lung tumor tissues and cancer cells. We identify the key Ser314 phosphorylation site on GAC that is regulated by the NF-κB-PKCε axis. Blocking Ser314 phosphorylation by the S314A mutation in lung cancer cells inhibits the glutaminase activity, triggers genetic reprogramming, and alleviates tumor malignancy. Furthermore, we find that a high level of GAC phosphorylation correlates with poor survival rate of lung cancer patients. These findings highlight a previously unappreciated mechanism for activation of GAC by phosphorylation and demonstrate that targeting glutaminase activity can inhibit oncogenic transformation.

Histone deacetylases are recently identified to act as key regulators for cardiac pathophysiology and metabolic disorders. However, the function of histone deacetylase (HDAC) in controlling cardiac performance in Type II diabetes and obesity remains unknown. Here, we determine whether HDAC inhibition attenuates high fat diet (HFD)-induced cardiac dysfunction and improves metabolic features. Adult mice were fed with either HFD or standard chow food for 24 weeks. Starting at 12 weeks, mice were divided into four groups randomly, in which sodium butyrate (1%), a potent HDAC inhibitor, was provided to chow and HFD-fed mice in drinking water, respectively. Glucose intolerance, metabolic parameters, cardiac function, and remodeling were assessed. Histological analysis and cellular signaling were examined at 24 weeks following euthanization of mice. HFD-fed mice demonstrated myocardial dysfunction and profound interstitial fibrosis, which were attenuated by HDAC inhibition. HFD-induced metabolic syndrome features insulin resistance, obesity, hyperinsulinemia, hyperglycemia, lipid accumulations, and cardiac hypertrophy, these effects were prevented by HDAC inhibition. Furthermore, HDAC inhibition attenuated myocyte apoptosis, reduced production of reactive oxygen species, and increased angiogenesis in the HFD-fed myocardium. Notably, HFD induced decreases in MKK3, p38, p38 regulated/activated protein kinase (PRAK), and Akt-1, but not p44/42 phosphorylation, which were prevented by HDAC inhibition. These results suggest that HDAC inhibition plays a critical role to preserve cardiac performance and mitigate metabolic disorders in obesity and diabetes, which is associated with MKK3/p38/PRAK pathway. The study holds promise in developing a new therapeutic strategy in the treatment of Type II diabetic-induced heart failure and metabolic disorders. J. Cell. Biochem. 118: 2395-2408, 2017. © 2017 Wiley Periodicals, Inc.

Background LOX-like 1 (LOXL1) is a lysyl oxidase, and emerging evidence has revealed its effect on malignant cancer progression. However, its role in colorectal cancer (CRC) and the underlying molecular mechanisms have not yet been elucidated. Methods LOXL1 expression in colorectal cancer was detected by immunohistochemistry, western blotting and real-time PCR. In vitro, colony formation, wound healing, migration and invasion assays were performed to investigate the effects of LOXL1 on cell proliferation, migration and invasion. In vivo, metastasis models and mouse xenografts were used to assess tumorigenicity and metastasis ability. Molecular biology experiments were utilized to reveal the underlying mechanisms by which LOXL1 modulates the Hippo pathway. Results LOXL1 was highly expressed in normal colon tissues compared with cancer tissues. In vitro, silencing LOXL1 in CRC cell lines dramatically enhanced migration, invasion, and colony formation, while overexpression of LOXL1 exerted the opposite effects. The results of the in vivo experiments demonstrated that the overexpression of LOXL1 in CRC cell lines drastically inhibited metastatic progression and tumour growth. Mechanistically, LOXL1 inhibited the transcriptional activity of Yes-associated protein (YAP) by interacting with MST1/2 and increasing the phosphorylation of MST1/2. Conclusions LOXL1 may function as an important tumour suppressor in regulating tumour growth, invasion and metastasis via negative regulation of YAP activity. Graphical abstract

Summary In mammalian cells, histone deacetylase (HDAC) and Sirtuin (SIRT) are two families responsible for removing acetyl groups from acetylated proteins. Here, we describe protein deacetylation coupled with deacetylimination as a function of lysyl oxidase (LOX) family members. LOX-like 3 (Loxl3) associates with Stat3 in the nucleus to deacetylate and deacetyliminate Stat3 on multiple acetyl-lysine sites. Surprisingly, Loxl3 N-terminal scavenger receptor cysteine-rich (SRCR) repeats, rather than the C-terminal oxidase catalytic domain, represent the major deacetylase/deacetyliminase activity. Loxl3-mediated deacetylation/deacetylimination disrupts Stat3 dimerization, abolishes Stat3 transcription activity, and restricts cell proliferation. In Loxl3 −/− mice, Stat3 is constitutively acetylated and naive CD4 + T cells are potentiated in Th17/Treg cell differentiation. When overexpressed, the SRCR repeats from other LOX family members can catalyze protein deacetylation/deacetylimination. Thus, our findings delineate a hitherto-unknown mechanism of protein deacetylation and deacetylimination catalyzed by lysyl oxidases.

LIF promotes self-renewal of mouse embryonic stem cells (mESCs), and in its absence, the cells differentiate. LIF binds to the LIF receptor (LIFR) and activates the JAK-STAT3 pathway, but it remains unknown how the receptor complex triggers differentiation or self-renewal. Here, we report that the LIFR cytoplasmic domain contains a self-renewal domain within the juxtamembrane region and a differentiation domain within the C-terminal region. The differentiation domain contains four SPXX repeats that are phosphorylated by MAPK to restrict STAT3 activation; the self-renewal domain is characterized by a 3K motif that is acetylated by p300. In mESCs, acetyl-LIFR undergoes homodimerization, leading to STAT3 hypo- or hyper-activation depending on the presence or absence of gp130. LIFR-activated STAT3 restricts differentiation via cytokine induction. Thus, LIFR acetylation and serine phosphorylation differentially promote stem cell self-renewal and differentiation.

Interferon-γ (IFN-γ) triggers macrophage for inflammation response by activating the intracellular JAK−STAT1 signaling. Suppressor of cytokine signaling 1 (SOCS1) and protein tyrosine phosphatases can negatively modulate IFN-γ signaling. Here, we identify a novel negative feedback loop mediated by STAT3−SOCS3, which is tightly controlled by SENP1 via de-SUMOylation of protein tyrosine phosphatase 1B (PTP1B), in IFN-γ signaling. SENP1-deficient macrophages show defects in IFN-γ signaling and M1 macrophage activation. PTP1B in SENP1-deficient macrophages is highly SUMOylated, which reduces PTP1B-induced de-phosphorylation of STAT3. Activated STAT3 then suppresses STAT1 activation via SOCS3 induction in SENP1-deficient macrophages. Accordingly, SENP1-deficient macrophages show reduced ability to resist Listeria monocytogenes infection. These results reveal a crucial role of SENP1-controlled STAT1 and STAT3 balance in macrophage polarization.

Background Histone deacetylases (HDACs) play a critical role in modulating myocardial protection and cardiomyocyte survivals. However, Specific HDAC isoforms in mediating myocardial ischemia/reperfusion injury remain currently unknown. We used cardiomyocyte-specific overexpression of active HDAC4 to determine the functional role of activated HDAC4 in regulating myocardial ischemia and reperfusion in isovolumetric perfused hearts. Methods In this study, we created myocyte-specific active HDAC4 transgenic mice to examine the functional role of active HDAC4 in mediating myocardial I/R injury. Ventricular function was determined in the isovolumetric heart, and infarct size was determined using tetrazolium chloride staining. Results Myocyte-specific overexpressing activated HDAC4 in mice promoted myocardial I/R injury, as indicated by the increases in infarct size and reduction of ventricular functional recovery following I/R injury. Notably, active HDAC4 overexpression led to an increase in LC-3 and active caspase 3 and decrease in SOD-1 in myocardium. Delivery of chemical HDAC inhibitor attenuated the detrimental effects of active HDAC4 on I/R injury, revealing the pivotal role of active HDAC4 in response to myocardial I/R injury. Conclusions Taken together, these findings are the first to define that activated HDAC4 as a crucial regulator for myocardial ischemia and reperfusion injury. Electronic supplementary material The online version of this article (10.1186/s10020-018-0037-2) contains supplementary material, which is available to authorized users.

Cytokine or growth factor activated STAT3 undergoes multiple post-translational modifications, dimerization and translocation into nuclei, where it binds to serum-inducible element (SIE, 'TTC(N3)GAA')-bearing promoters to activate transcription. The STAT3 DNA binding domain (DBD, 320-494) mutation in hyper immunoglobulin E syndrome (HIES), called the HIES mutation (R382Q, R382W or V463Δ), which elevates IgE synthesis, inhibits SIE binding activity and sensitizes genes such as TNF-α for expression. However, the mechanism by which the HIES mutation sensitizes STAT3 in gene induction remains elusive. Here, we report that STAT3 binds directly to the AGG-element with the consensus sequence 'AGG(N3)AGG'. Surprisingly, the helical N-terminal region (1-355), rather than the canonical STAT3 DBD, is responsible for AGG-element binding. The HIES mutation markedly enhances STAT3 AGG-element binding and AGG-promoter activation activity. Thus, STAT3 is a dual specificity transcription factor that promotes gene expression not only via SIE- but also AGG-promoter activity.

Sirt3, as a major mitochondrial nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, is required for mitochondrial metabolic adaption to various stresses. However, how to regulate Sirt3 activity responding to metabolic stress remains largely unknown. Here, we report Sirt3 as a SUMOylated protein in mitochondria. SUMOylation suppresses Sirt3 catalytic activity. SUMOylation-deficient Sirt3 shows elevated deacetylation on mitochondrial proteins and increased fatty acid oxidation. During fasting, SUMO-specific protease SENP1 is accumulated in mitochondria and quickly de-SUMOylates and activates Sirt3. SENP1 deficiency results in hyper-SUMOylation of Sirt3 and hyper-acetylation of mitochondrial proteins, which reduces mitochondrial metabolic adaption responding to fasting. Furthermore, we find that fasting induces SENP1 translocation into mitochondria to activate Sirt3. The studies on mice show that Sirt3 SUMOylation mutation reduces fat mass and antagonizes high-fat diet (HFD)-induced obesity via increasing oxidative phosphorylation and energy expenditure. Our results reveal that SENP1-Sirt3 signaling modulates Sirt3 activation and mitochondrial metabolism during metabolic stress.

CCN family protein 2 (CCN2), also known as connective tissue growth factor, is a secreting protein that modulates multiple cellular events. We previously demonstrated the metastasis-suppressive effect of CCN2 in lung cancer cells. In this study, we investigate the role of CCN2 in anoikis, a form of programmed cell death that is critical in suppressing cancer metastasis. CCN2 binds to the epidermal growth factor receptor (EGFR) and triggers ubiquitination by inhibiting the formation of the β-pix/Cbl complex, resulting in the degradation of EGFR. Binding of CCN2 to EGFR suppresses the phosphorylation of c-Src and extracellular signal-regulated kinase but increases the expression of death-associated protein kinase, which leads to anoikis. Overall, our findings provide evidence validating the use of CCN2 as an anti-metastatic therapy in lung cancer patients, and prospect a potential therapeutic synergy between CCN2 and the anti-EGFR antibody for the treatment of lung cancer.

DNA polymerase η (Polη) implements translesion DNA synthesis but has low fidelity in replication. We have previously shown that Polη plays an important role in the genesis of nontargeted mutations at undamaged DNA sites in cells exposed to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Here, we report that MNNG-induced Polη expression in an interferon regulatory factor 1 (IRF1)-dependent manner in human cells. Mutagenesis analysis showed that four critical residues (Arg-82, Cys-83, Asn-86, and Ser-87) located in the IRF family conserved DNA binding domain-helix α3 were involved in DNA binding and POLH transactivation by IRF1. Furthermore, Polη up-regulation induced by IRF1 was responsible for the increase of mutation frequency in a SupF shuttle plasmid replicated in the MNNG-exposed cells. Interestingly, IRF1 was acetylated by the histone acetyltransferase CBP in these cells. Lys → Arg substitution revealed that Lys-78 of helix α3 was the major acetylation site, and the IRF1-K78R mutation partially inhibited DNA binding and its transcriptional activity. Thus, we propose that IRF1 activation is responsible for MNNG-induced Polη up-regulation, which contributes to mutagenesis and ultimately carcinogenesis in cells.

Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.

The multiple functions of the oncofetal protein survivin are dependent on its selective expression patterns within immunochemically distinct subcellular pools. The mechanism by which survivin localizes to these compartments, however, is only partly understood. Here we show that nuclear accumulation of survivin is promoted by CREB-binding protein (CBP)-dependent acetylation on lysine 129 (129K, Lys-129). We demonstrate a mechanism by which survivin acetylation at this position results in its homodimerization, while deacetylation promotes the formation of survivin monomers that heterodimerize with CRM1 and facilitate its nuclear export. Using proteomic analysis, we identified the oncogenic transcription factor STAT3 as a binding partner of nuclear survivin. We show that acetylated survivin binds to the N-terminal transcriptional activation domain of the STAT3 dimer and represses STAT3 transactivation of target gene promoters. Using multiplex PCR and DNA sequencing, we identified a single-nucleotide polymorphism (A → G) at Lys-129 that exists as a homozygous mutation in a neuroblastoma cell line and corresponds with a defect in survivin nuclear localization. Our results demonstrate that the dynamic equilibrium between survivin acetylation and deacetylation at amino acid 129 determines its interaction with CRM1, its subsequent subcellular localization, and its ability to inhibit STAT3 transactivation, providing a potential route for therapeutic intervention in STAT3-dependent tumors.

Following publication of their article “CCN2 inhibits lung cancer metastasis through promoting DAPK-dependent anoikis and inducing EGFR degradation”, the authors reported an error in Fig.6b. α-Tubulin image of rCCN2 treatment (upper panel in CL1-5) only showed eight lanes, when there should be nine.

Histone deacetylase (HDAC) inhibition modulates dendritic cell (DC) functions and regulates experimental graft-vs-host disease and other immune-mediated diseases. The mechanisms by which HDAC inhibition modulates immune responses remain largely unknown. STAT-3 is a transcription factor shown to negatively regulate DC functions. In this study we report that HDAC inhibition acetylates and activates STAT-3, which regulates DCs by promoting the transcription of IDO. These findings demonstrate a novel functional role for posttranslational modification of STAT-3 through acetylation and provide mechanistic insights into HDAC inhibition-mediated immunoregulation by induction of IDO.

SummaryOrigin recognition complex 2 (ORC2), a subunit of the ORC, is essential for DNA replication initiation in eukaryotic cells. In addition to a role in DNA replication initiation at the G1/S phase, ORC2 has been shown to localize to the centromere during the G2/M phase. Here, we show that ORC2 is modified by small ubiquitin-like modifier 2 (SUMO2), but not SUMO1, at the G2/M phase of the cell cycle. SUMO2-modification of ORC2 is important for the recruitment of KDM5A in order to convert H3K4me3 to H3K4me2, a “permissive” histone marker for α-satellite transcription at the centromere. Persistent expression of SUMO-less ORC2 led to reduced α-satellite transcription and impaired pericentric heterochromatin silencing, which resulted in re-replication of heterochromatin DNA. DNA re-replication eventually activated the DNA damage response, causing the bypass of mitosis and the formation of polyploid cells. Thus, ORC2 sustains genomic stability by recruiting KDM5A to maintain centromere histone methylation in order to prevent DNA re-replication.

AF9 mutations have been implicated in human neurodevelopmental diseases and murine Af9 mediates histone methylation during cortical neuron generation. However, AF9 function and related mechanisms in human neurodevelopment remain unknown. Here we show that AF9 is necessary and sufficient for human embryonic stem cell (hESC) neural differentiation and neurodevelopmental gene activation. The 5-methylcytosine (5mC) dioxygenase TET2, which was identified in an AF9-associated protein complex, physically interacted with AF9. Both AF9 and TET2 co-localized in 5-hydroxymethylcytosine (5hmC)-positive hESC-derived neurons and were required for appropriate hESC neural differentiation. Upon binding to AAC-containing motifs, AF9 recruited TET2 to occupy the common neurodevelopmental gene loci to direct 5mC-to-5hmC conversion, which was followed by sequential activation of neural target genes and hESC neural commitment. These findings define an AF9–TET2 regulatory complex for modulating human neural development and reveal a novel mechanism by which the AF9 recognition specificity and TET2 hydroxylation activity cooperate to control neurodevelopmental gene activation.

Stat3 and its isoforms belong to a family of cytoplasmic transcription factors that affect the synthesis of various proteins. Caspases are cysteinyl-aspartate proteases that function under apoptotic and non-apoptotic conditions. We now report that, in addition to transcriptional splicing, Stat3 fragmentation can be mediated by caspases. Caspase activation in DU145 cells was achieved by staurosporine (STS) exposure, and Western analysis revealed a reduction in full-length Stat3 (fl-Stat3) expression that was caspase-mediated. This proteolytic relationship was further studied by exposing purified Stat3 protein to a mixture of active caspases under cell-free conditions. This demonstrated that caspases directly cleaved Stat3 and Stat3 cleavage was accompanied by the apparent formation of cleavage fragment(s). Stat3 cleavage fragments, reflecting multiple caspase cleavage sites, also were observed in vitro following STS exposure in DU145 cells and in HEK293T cells transfected to express Stat3 truncation mutants. The impact of cleavage on Stat3 transcriptional activity next was assessed and revealed that cleavage of fl-Stat3 was accompanied by reductions in Stat3-DNA binding, Stat3-driven reporter protein (luciferase) activity, and the expression of selected Stat3-dependent genes. Further, reduced Stat3 expression correlated with increased sensitivity to apoptotic stimuli. In concomitant experiments, reporter activity was assessed in Stat3 truncation mutant-expressing HEK293T cells and revealed that, under non-apoptotic conditions, expression of different Stat3 fragments induced differential effects on Stat3-driven luciferase activity. These findings demonstrate that fl-Stat3 undergoes proteolytic processing by caspases that reduces its expression and leads to the formation of cleavage fragments that may modulate Stat3 transcriptional activity.

Ankyrin repeat and SOCS box (ASB) family members have a C-terminal SOCS box and an N-terminal ankyrin-related sequence of variable repeats belonging to the SOCS superfamily. While SH2-domain-bearing SOCS proteins are mainly involved in the negative feedback regulation of the protein tyrosine kinase-STAT pathway in response to a variety of cytokines, the roles of ASB family members remain largely unknown. To investigate ASB functions, we screened for ASB3-interacting factors by using antibody array technology and identified tumor necrosis factor receptor II (TNF-R2) as an ASB3 binding target. ASB3 expression and activities are required for (i) TNF-R2 ubiquitination both in vivo and in vitro, (ii) TNF-R2 proteolysis via the proteasome pathway, and (iii) the inhibition of TNF-R2-mediated Jun N-terminal protein kinase (JNK) activation. While the ankyrin repeats of ASB3 interact with the C-terminal 37 amino acids of TNF-R2, the SOCS box of ASB3 is responsible for recruiting the E3 ubiquitin ligase adaptors Elongins-B/C, leading to TNF-R2 ubiquitination on multiple lysine residues within its C-terminal region. Downregulation of ASB3 expression by a small interfering RNA inhibited TNF-R2 degradation and potentiated TNF-R2-mediated cytotoxicity. The data presented here implicate ASB3 as a negative regulator of TNF-R2-mediated cellular responses to TNF-alpha by direct targeting of TNF-R2 for ubiquitination and proteasome-mediated degradation.

Susceptibility to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) varies greatly among patients in sepsis/septic shock. The genetic and biochemical reasons for the difference are not fully understood. G protein coupled receptor family C group 5 member A (GPRC5A), a retinoic acid target gene, is predominately expressed in the bronchioalveolar epithelium of lung. We hypothesized that Gprc5a is important in controlling the susceptibility to ALI or ARDS. In this study, we examined the susceptibility of wild-type and Gprc5a-knockout (ko) mice to induced ALI. Administration of endotoxin LPS induced an increased pulmonary edema and injury in Gprc5a-ko mice, compared to wild-type counterparts. Consistently, LPS administration induced higher levels of inflammatory cytokines (IL-1β and TNFα) and chemokine (KC) in Gprc5a-ko mouse lungs than in wild-type. The enhanced pulmonary inflammatory responses were associated with dysregulated NF-κB signaling in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs. Importantly, selective inhibition of NF-κB through expression of the super-repressor IκBα in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs alleviated the LPS-induced pulmonary injury, and inflammatory response. Thus, Gprc5a is critical for lung homeostasis, and Gprc5a deficiency confers the susceptibility to endotoxin-induced pulmonary edema and injury, mainly through NF-κB signaling in bronchioalveolar epithelium of lung.

The cytoplasmic region of human epidermal growth factor receptor (EGFR) contains an intrinsic tyrosine kinase (697-955) followed by a 231-residue-long COOH-terminal tail (C-tail), which contains multiple tyrosine residues. To examine the role of the EGFR C-tail in signal transducer and activator of transcription (STAT) activation, a series of EGFR C-tail truncations were constructed. Transient transfection of 293 cells with EGFR lacking the C-tail, i.e. Y974DeltaEGFR or Y992DeltaEGFR, led to EGF-independent or constitutive STAT activation, whereas EGF-dependent STAT activation was restored with truncations made COOH-terminal to the next tyrosine residue, i.e. EGFR-Y1045Delta. Transfection with the-truncated form EGFR-Y954Delta resulted in the loss of STAT activation, suggesting that the sequence between Tyr(974) and Tyr(954) is essential for STAT activation. Phosphopeptide competition analysis revealed multiple tyrosine residues within the C-tail that can act as the docking sites for both Stat1 and Stat3. A region that negatively regulated STAT activation was also identified, extending from Tyr(1114) to Glu(1172), consistent with the ability of this region to recruit a suppressor of cytokine signaling factors SOCS1 and SOCS3. When cotransfected with the full-length EGFR, but not Y992DeltaEGFR, SOCS1 or SOCS3 inhibited STAT activation by EGF in 293 cells. This suggests that both SOCS1 and SOCS3 can negatively regulate EGFR activation, presumably by inducing ubiquitination-dependent EGFR degradation upon ligand binding. These findings may therefore offer clues to how the EGF receptor C-tail regulates STAT activity.

Transcriptional repressor Snail is a master regulator of epithelial–mesenchymal transition (EMT), yet the epigenetic mechanism governing Snail to induce EMT is not well understood. Here, we report that in pancreatic ductal adenocarcinoma (PDAC), elevated levels of the ubiquitin E3 ligase Ring1B and Snail, along with elevated monoubiquitination of H2A at K119 (H2AK119Ub1), are highly correlated with poor survival. Mechanistic investigations identified Ring1B as a Snail-interacting protein and showed that the carboxyl zinc fingers of Snail recruit Ring1B and its paralog Ring1A to repress its target promoters. Simultaneous depletion of Ring1A and Ring1B in pancreatic cancer cells decreased Snail binding to the target chromatin, abolished H2AK119Ub1 modification, and thereby compromised Snail-mediated transcriptional repression and cell migration. We found that Ring1B and the SNAG-associated chromatin modifier EZH2 formed distinct protein complexes with Snail and that EZH2 was required for Snail-Ring1A/B recruitment to the target promoter. Collectively, our results unravel an epigenetic mechanism underlying transcriptional repression by Snail, suggest Ring1A/B as a candidate therapeutic target, and identify H2AK119Ub1 as a potential biomarker for PDAC diagnosis and prognosis. Cancer Res; 74(16); 4353–63. ©2014 AACR.

Background & Aims Constitutive activation of the transcription factors nuclear factor κB (NF-κB) and STAT3 is involved in the development and progression of human colorectal cancer (CRC). Little is known about how these factors become activated in cancer cells. We investigated whether microRNA miR-221 and miR-222 regulate NF-κB and signal transducer and activator of transcription 3 (STAT3) activation in human CRC cell lines. Methods CRC cell lines (HCT116 and RKO) were transfected with miR-221 or miR-222 mimics or inhibitors. The activity levels of NF-κB and STAT3 were measured in dual luciferase reporter assays. We used immunoblot and real-time polymerase chain reaction analyses to measure protein and messenger RNA (mRNA) levels. Cells were analyzed by proliferation, viability, and flow cytometry analyses. Mice were given injections of azoxymethane, followed by dextran sodium sulfate, along with control lentivirus or those expressing mRNAs that bind miR-221 and miR-222 (miR-221/miR-222 sponge). The levels of miR-221 and miR-222 as well as RelA , STAT3 , and PDLIM2 mRNAs were measured in 57 paired CRC and adjacent nontumor tissues from patients. Results In CRC cell lines, mimics of miR-221 and miR-222 activated NF-κB and STAT3, further increasing expression of miR-221 and miR-222. miR-221 and miR-222 bound directly to the coding region of RelA mRNA, increasing its stability. miR-221 and miR-222 also reduced the ubiquitination and degradation of the RelA and STAT3 proteins by binding to the 3′ untranslated region of PDLIM2 mRNA (PDLIM2 is a nuclear ubiquitin E3 ligase for RelA and STAT3). Incubation of CRC cells with miR-221 and miR-222 inhibitors reduced their proliferation and colony formation compared with control cells. In mice with colitis, injection of lentiviruses expressing miR-221/miR-222 sponges led to formation of fewer tumors than injection of control lentiviruses. Human CRC tissues had higher levels of miR-221 and miR-222 than nontumor colon tissues; increases correlated with increased levels of RelA and STAT3 mRNAs. Levels of PDLIM2 mRNA were lower in CRC than nontumor tissues. Conclusions In human CRC cells, miR-221 and miR-222 act in a positive feedback loop to increase expression levels of RelA and STAT3. Antagonism of miR-221 and miR-222 reduces growth of colon tumors in mice with colitis.

SummaryThe ubiquitin-proteasome and autophagy-lysosome systems are major proteolytic pathways, whereas function of the Ub-independent proteasome pathway is yet to be clarified. Here, we investigated roles of the Ub-independent REGγ-proteasome proteolytic system in regulating metabolism. We demonstrate that mice deficient for the proteasome activator REGγ exhibit dramatic autophagy induction and are protected against high-fat diet (HFD)-induced liver steatosis through autophagy. Molecularly, prevention of steatosis in the absence of REGγ entails elevated SirT1, a deacetylase regulating autophagy and metabolism. REGγ physically binds to SirT1, promotes its Ub-independent degradation, and inhibits its activity to deacetylate autophagy-related proteins, thereby inhibiting autophagy under normal conditions. Moreover, REGγ and SirT1 dissociate from each other through a phosphorylation-dependent mechanism under energy-deprived conditions, unleashing SirT1 to stimulate autophagy. These observations provide a function of the REGγ proteasome in autophagy and hepatosteatosis, underscoring mechanistically a crosstalk between the proteasome and autophagy degradation system in the regulation of lipid homeostasis.

Context: Neutrophils are the primary effector cells in the pathogenesis of transfusion-related acute lung injury or multiple organ failure after blood transfusion. Objective: We aimed to investigate the effect of fresh (1 day preparation) and aged (42 day preparation) PRBC-derived plasma on neutrophil morphology, migration and phagocytosis. Materials and methods: We evaluated the production of reactive oxygen species (ROS) and the expression of non-muscle myosin heavy chain IIA (MYH9) in neutrophils treated with PRBC-derived plasma. We used western blots and antibody arrays to evaluate changes in signal transduction pathways in plasma-treated neutrophils. Results: Aged PRBC-derived plasma elicited a stronger oxidative burst in neutrophils when compared with fresh PRBC-derived plasma (p < 0.05). Antibody arrays showed increased phosphorylation of NF-ĸB proteins (p105, p50 and Ikk) in aged PRBC-derived plasma-treated neutrophils. The expression of non-muscle myosin IIA (MYH9), a cytoskeleton protein involved in immune cell migration and morphological change, was also significantly upregulated in neutrophils treated with aged PRBC-derived plasma compared to fresh plasma (p < 0.05). Pretreatment of neutrophils with blebbistatin (a specific type II myosin inhibitor), ascorbic acid (an antioxidant), or staurosporine (a protein tyrosine kinase inhibitor), effectively abrogated the morphological changes, neutrophil migration, and phagocytosis induced by aged PRBC-derived plasma. Conclusion: Upregulation of MYH9 in neutrophils treated with aged PRBC-derived plasma and abrogation of neutrophil migration in blebbistatin-treated neutrophils suggested a functional role of MYH9 in the directional migration of immune cells. Our data help elucidate the cellular and molecular mechanisms of transfusion-related injury.

Histone deacetylases (HDACs) play a critical role in the regulation of gene transcription, cardiac development, and diseases. The aim of this study was to test whether inhibition of HDACs induces myocardial repair and cardiac function restoration through c-kit signaling in mouse myocardial infarction models. Myocardial infarction in wild type Kit(+/+) and Kit(W)/Kit(W-v) mice was created following thoracotomy by applying permanent ligation to the left anterior descending artery. The HDAC inhibitor, trichostatin A (TSA, 0.1 mg/kg), was intraperitoneally injected daily for a consecutive 8 weeks after myocardial infarction. 5-Bromo-2-deoxyuridine (BrdU, 50 mg/kg) was intraperitoneally delivered every other day to pulse-chase label in vivo endogenous cardiac replication. Eight weeks later, inhibition of HDACs in vivo resulted in an improvement in ventricular functional recovery and the prevention of myocardial remodeling in Kit(+/+) mice, which was eliminated in Kit(W)/Kit(W-v) mice. HDAC inhibition promoted cardiac repairs and neovascularization in the infarcted myocardium, which were absent in Kit(W)/Kit(W-v) mice. Re-introduction of TSA-treated wild type c-kit(+) CSCs into Kit(W)/Kit(W-v) myocardial infarction heart restored myocardial functional improvement and cardiac repair. To further validate that HDAC inhibition stimulates c-kit(+) cardiac stem cells (CSCs) to facilitate myocardial repair, GFP(+) c-kit(+) CSCs were preconditioned with TSA (50 nmol/liter) for 24 h and re-introduced into infarcted hearts for 2 weeks. Preconditioning of c-kit(+) CSCs via HDAC inhibition with trichostatin A significantly increased c-kit(+) CSC-derived myocytes and microvessels and enhanced functional recovery in myocardial infarction hearts in vivo. Our results provide evidence that HDAC inhibition promotes myocardial repair and prevents cardiac remodeling, which is dependent upon c-kit signaling.

Thrombin, a G protein-coupled receptor agonist, induced a biphasic expression of cyclin D1 in primary vascular smooth muscle cells. Although both phases of cyclin D1 expression require binding of the newly identified cooperative complex, NFATc1·STAT-3, to its promoter, the second phase, which is more robust, depends on NFATc1-mediated recruitment of p300 onto the complex and the subsequent acetylation of STAT-3. In addition, STAT-3 is tyrosine-phosphorylated in a biphasic manner, and the late phase requires NFATc1-mediated p300-dependent acetylation. Furthermore, interference with acetylation of STAT-3 by overexpression of acetylation null STAT-3 mutant led to the loss of the late phase of cyclin D1 expression. EMSA analysis and reporter gene assays revealed that NFATc1·STAT-3 complex binding to the cyclin D1 promoter led to an enhanceosome formation and facilitated cyclin D1 expression. In the early phase of its expression, cyclin D1 is localized mostly in the cytoplasm and influenced cell migration. However, during the late and robust phase of its expression, cyclin D1 is translocated to the nucleus and directed cell proliferation. Together, these results demonstrate for the first time that the dual function of cyclin D1 in cell migration and proliferation is temperospatially separated by its biphasic expression, which is mediated by cooperative interactions between NFATc1 and STAT-3.

In TNF-treated cells, TNFR1, TNFR-associated death domain protein (TRADD), Fas-associated death domain protein, and receptor-interacting protein kinase proteins form the signaling complex via modular interaction within their C-terminal death domains. In this paper, we report that the death domain SXXE/D motifs (i.e., S381DHE motif of TNFR1-death domain as well as S215LKD and S296LAE motifs of TRADD-death domain) are phosphorylated, and this is required for stable TNFR1–TRADD complex formation and subsequent activation of NF-κB. Phospho-S215LKD and phospho-S296LAE motifs are also critical to TRADD for recruiting Fas-associated death domain protein and receptor-interacting protein kinase. IκB kinase β plays a critical role in TNFR1 phosphorylation of S381, which leads to subsequent T cell migration and accumulation. Consistently, we observed in inflammatory bowel disease specimens that TNFR1 was constitutively phosphorylated on S381 in those inflammatory T cells, which had accumulated in high numbers in the inflamed mucosa. Therefore, SXXE/D motifs found in the cytoplasmic domains of many TNFR family members and their adaptor proteins may serve to function as a specific interaction module for the α-helical death domain signal transduction.

To maintain intracellular redox homeostasis, genes encoding many antioxidants and detoxification enzymes are transcriptionally upregulated upon deleterious oxidative stress through the cis antioxidant responsive elements (AREs) in their promoter regions. Nrf2 is the critical transcription factor responsible for ARE-dependent transcription. We and others have previously demonstrated that Nrf2 is targeted for ubiquitin-mediated degradation by Keap1 in a redox-sensitive manner through modifications of distinct cysteine residues of Keap1. Here, we report that p300/CBP directly acetylates Nrf2 in response to arsenite-induced stress. We have identified multiple acetylated lysine residues within the Nrf2 Neh1 DNA-binding domain. Combined lysine-to-arginine mutations on the acetylation sites, with no effects on Nrf2 protein stability, compromised the DNA-binding activity of Nrf2 in a promoter-specific manner. These findings demonstrated that acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 and established acetylation as a novel regulatory mechanism that functions in concert with Keap1-mediated ubiquitination in modulating the Nrf2-dependent antioxidant response.

Precise spatial and temporal regulation of cell adhesion and de-adhesion is critical for dynamic lymphocyte migration. Although a great deal of information has been learned about integrin lymphocyte function–associated antigen (LFA)-1 adhesion, the mechanism that regulates efficient LFA-1 de-adhesion from intercellular adhesion molecule (ICAM)-1 during T lymphocyte migration is unknown. Here, we show that nonmuscle myosin heavy chain IIA (MyH9) is recruited to LFA-1 at the uropod of migrating T lymphocytes, and inhibition of the association of MyH9 with LFA-1 results in extreme uropod elongation, defective tail detachment, and decreased lymphocyte migration on ICAM-1, without affecting LFA-1 activation by chemokine CXCL-12. This defect was reversed by a small molecule antagonist that inhibits both LFA-1 affinity and avidity regulation, but not by an antagonist that inhibits only affinity regulation. Total internal reflection fluorescence microscopy of the contact zone between migrating T lymphocytes and ICAM-1 substrate revealed that inactive LFA-1 is selectively localized to the posterior of polarized T lymphocytes, whereas active LFA-1 is localized to their anterior. Thus, during T lymphocyte migration, uropodal adhesion depends on LFA-1 avidity, where MyH9 serves as a key mechanical link between LFA-1 and the cytoskeleton that is critical for LFA-1 de-adhesion.

The receptor tyrosine kinases (RTKs) RET, MET, and RON all carry the Met(p+1loop)--Thr point mutation (i.e., 2B mutation), leading to the formation of tumors with high metastatic potential. Utilizing a novel antibody array, we identified constitutive phosphorylation of STAT3 in cells expressing the 2B mutation but not wild-type RET. MET or RON with the 2B mutation also constitutively phosphorylated STAT3. Members of the EPH, the only group of wild-type RTK that carry Thr(p+1loop) residue, are often expressed unexpectedly in different types of cancers. Ectopic expression of wild-type but not Thr(p+1loop)--Met substituted EPH family members constitutively phosphorylated STAT3. In both RTK(Metp+1loop) with 2B mutation and wild-type EPH members the Thr(p+1loop) residue is required for constitutive kinase autophosphorylation and STAT3 recruitment. In multiple endocrine neoplasia 2B (MEN-2B) patients expressing RET(M918T), nuclear enrichment of STAT3 and elevated expression of CXCR4 was detected in metastatic thyroid C-cell carcinoma in the liver. In breast adenocarcinoma cell lines expressing multiple EPH members, STAT3 constitutively bound to the promoters of MUC1, MUC4, and MUC5B genes. Inhibiting STAT3 expression resulted in reduced expression of these metastasis-related genes and inhibited mobility. These findings provide insight into Thr(p+1loop) residue in RTK autophosphorylation and constitutive activation of STAT3 in metastatic cancer cells.

Previously, we demonstrated that antibodies printed on a solid support were able to detect protein-protein interaction in mammalian cells. Here we further developed the antibody array system for detecting proteins with various post-translational modifications in mammalian cells. In this novel approach, immunoprecipitated proteins were labeled with fluorescent dye followed by incubation over antibody arrays. Targeted proteins, captured by the antibodies immobilized on PVDF membrane or glass slide, were detected by means of near infrared fluorescent scanner or fluorescent microscopy. To demonstrate the application of the antibody arrays in protein post-translational modifications, we profiled protein tyrosine phosphorylation, ubiquitination, and acetylation in mammalian cells under different conditions. Our results indicate that antibody array technology can provide a powerful means of profiling a large number of proteins with different post-translational modifications in cells.

Signal transducer and activator of transcription (STAT) proteins are both tyrosine- and serine-phosphorylated, mediating signal transduction and gene regulation. Following gene regulation, STAT activity in the nucleus is then terminated by a nuclear protein phosphatase(s), which remains unidentified. Using novel antibody arrays to screen the Stat1-specific protein phosphatase(s), we identified a SHP-2-Stat1 interaction in the A431 cell nucleus. SHP-2 and Stat1 nuclear localization and their association in response to either epidermal growth factor or interferon-gamma (IFNgamma) were confirmed by immunofluorescent staining and affinity precipitation assays. The SHP-2 C-terminal region containing protein-tyrosine phosphatase activity interacted with the C-terminal SH2 transcriptional activation domain of Stat1. In SHP-2-/- mouse fibroblast cells, Stat1 phosphorylation at both the tyrosine residue Tyr(701) and the serine residue Ser(727) by IFNgamma was enhanced and prolonged. Consistently, purified GST-SHP-2 dephosphorylated Stat1 at both tyrosine and serine residues when immunoprecipitated phospho-Stat1 or a peptide corresponding to the sequence surrounding Tyr(P)(701) or Ser(P)(727) of Stat1 was used as the substrate. Overexpression of SHP-2 in 293T cells inhibited IFNgamma-dependent Stat1 phosphorylation and suppressed Stat1-dependent induction of luciferase activity. Our findings demonstrate that SHP-2 is a dual-specificity protein phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues and plays an important role in modulating STAT function in gene regulation.

Activated tumor necrosis factor alpha (TNF-alpha) receptor 1 (TNFR1) recruits TNFR1-associated death domain protein (TRADD), which in turn triggers two opposite signaling pathways leading to caspase activation for apoptosis induction and NF-kappaB activation for antiapoptosis gene upregulation. Here we show that Stat1 is involved in the TNFR1-TRADD signaling complex, as determined by employing a novel antibody array screening method. In HeLa cells, Stat1 was associated with TNFR1 and this association was increased with TNF-alpha treatment. TNFR1 signaling factors TRADD and Fas-associated death domain protein (FADD) were also found to interact with Stat1 in a TNF-alpha-dependent process. Our in vitro recombinant protein-protein interaction studies demonstrated that Stat1 could directly interact with TNFR1 and TRADD but not with FADD. Interaction between Stat1 and receptor-interacting protein (RIP) or TNFR-associated factor 2 (TRAF2) was not detected. Examination of Stat1-deficient cells showed an apparent increase in TNF-alpha-induced TRADD-RIP and TRADD-TRAF2 complex formation, while interaction between TRADD and FADD was unaffected. As a consequence, TNF-alpha-mediated I-kappaB degradation and NF-kappaB activation were markedly enhanced in Stat1-deficient cells, whereas overexpression of Stat1 in 293T cells blocked NF-kappaB activation by TNF-alpha. Thus, Stat1 acts as a TNFR1-signaling molecule to suppress NF-kappaB activation.