Heterozygous midnolin knockout attenuates severity of nonalcoholic fatty liver disease in mice fed a Western-style diet high in fat, cholesterol, and fructose.

Although midnolin has been studied for over 20 years, its biological roles in vivo remain largely unknown, especially due to the lack of a functional animal model. Indeed, given our recent discovery that the knockdown of midnolin suppresses liver cancer cell tumorigenicity and that this antitumorigenic effect is associated with modulation of lipid metabolism, we hypothesized that knockout of midnolin in vivo could potentially protect from nonalcoholic fatty liver disease (NAFLD) which has become the most common cause of chronic liver disease in the Western world. Accordingly, in the present study, we have developed and now report on the first functional global midnolin knockout mouse model. Although the overwhelming majority of global homozygous midnolin knockout mice demonstrated embryonic lethality, heterozygous knockout mice were observed to be similar to wild-type mice in their viability and were used to determine the effect of reduced midnolin expression on NAFLD. We found that global heterozygous midnolin knockout attenuated the severity of NAFLD in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism. Collectively, our results support a role for midnolin in regulating cholesterol/lipid metabolism in the liver. Thus, midnolin may represent a novel therapeutic target for NAFLD. Finally, our observation that midnolin was essential for survival underscores the broad importance of this gene beyond its role in liver biology.NEW & NOTEWORTHY We have developed and now report on the first functional global midnolin knockout mouse model. We found that global heterozygous midnolin knockout attenuated the severity of nonalcoholic fatty liver disease (NAFLD) in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism.

Midnolin Regulates Liver Cancer Cell Growth In Vitro and In Vivo.

Hepatocellular carcinoma (HCC) ranks worldwide as one of the most lethal cancers. In spite of the vast existing knowledge about HCC, the pathogenesis of HCC is not completely understood. Discovery of novel genes that contribute to HCC pathogenesis will provide new insights for better understanding and treating HCC. The relatively obscure gene midnolin has been studied for over two decades; however, its biological roles are largely unknown. Our study is the first to demonstrate the functional significance of midnolin in HCC/cancer: Midnolin expression correlates with poor prognosis in HCC patients, and suppression of midnolin severely inhibits tumorigenicity of HCC cells in vitro and in mice and disrupts retinoic acid/lipid metabolism in these cells.

Targeting the CBP/ß-Catenin Interaction to Suppress Activation of Cancer-Promoting Pancreatic Stellate Cells.

BACKGROUND: Although cyclic AMP-response element binding protein-binding protein (CBP)/ß-catenin signaling is known to promote proliferation and fibrosis in various organ systems, its role in the activation of pancreatic stellate cells (PSCs), the key effector cells of desmoplasia in pancreatic cancer and fibrosis in chronic pancreatitis, is largely unknown. METHODS: To investigate the role of the CBP/ß-catenin signaling pathway in the activation of PSCs, we have treated mouse and human PSCs with the small molecule specific CBP/ß-catenin antagonist ICG-001 and examined the effects of treatment on parameters of activation. RESULTS: We report for the first time that CBP/ß-catenin antagonism suppresses activation of PSCs as evidenced by their decreased proliferation, down-regulation of "activation" markers, e.g., α-smooth muscle actin (α-SMA/Acta2), collagen type I alpha 1 (Col1a1), Prolyl 4-hydroxylase, and Survivin, up-regulation of peroxisome proliferator activated receptor gamma (Ppar-γ) which is associated with quiescence, and reduced migration; additionally, CBP/ß-catenin antagonism also suppresses PSC-induced migration of cancer cells. CONCLUSION: CBP/ß-catenin antagonism represents a novel therapeutic strategy for suppressing PSC activation and may be effective at countering PSC promotion of pancreatic cancer.

An Adversarial DNA N6-Methyladenine-Sensor Network Preserves Polycomb Silencing.

Adenine N6 methylation in DNA (6mA) is widespread among bacteria and phage and is detected in mammalian genomes, where its function is largely unexplored. Here we show that 6mA deposition and removal are catalyzed by the Mettl4 methyltransferase and Alkbh4 dioxygenase, respectively, and that 6mA accumulation in genic elements corresponds with transcriptional silencing. Inactivation of murine Mettl4 depletes 6mA and causes sublethality and craniofacial dysmorphism in incross progeny. We identify distinct 6mA sensor domains of prokaryotic origin within the MPND deubiquitinase and ASXL1, a component of the Polycomb repressive deubiquitinase (PR-DUB) complex, both of which act to remove monoubiquitin from histone H2A (H2A-K119Ub), a repressive mark. Deposition of 6mA by Mettl4 triggers the proteolytic destruction of both sensor proteins, preserving genome-wide H2A-K119Ub levels. Expression of the bacterial 6mA methyltransferase Dam, in contrast, fails to destroy either sensor. These findings uncover a native, adversarial 6mA network architecture that preserves Polycomb silencing.

Erasure of Tet-Oxidized 5-Methylcytosine by a SRAP Nuclease.

Enzymatic oxidation of 5-methylcytosine (5mC) in DNA by the Tet dioxygenases reprograms genome function in embryogenesis and postnatal development. Tet-oxidized derivatives of 5mC such as 5-hydroxymethylcytosine (5hmC) act as transient intermediates in DNA demethylation or persist as durable marks, yet how these alternative fates are specified at individual CpGs is not understood. Here, we report that the SOS response-associated peptidase (SRAP) domain protein Srap1, the mammalian ortholog of an ancient protein superfamily associated with DNA damage response operons in bacteria, binds to Tet-oxidized forms of 5mC in DNA and catalyzes turnover of these bases to unmodified cytosine by an autopeptidase-coupled nuclease. Biallelic inactivation of murine Srap1 causes embryonic sublethality associated with widespread accumulation of ectopic 5hmC. These findings establish a function for a class of DNA base modification-selective nucleases and position Srap1 as a determinant of 5mC demethylation trajectories during mammalian embryonic development.

Stearoyl-CoA Desaturase Promotes Liver Fibrosis and Tumor Development in Mice via a Wnt Positive-Signaling Loop by Stabilization of Low-Density Lipoprotein-Receptor-Related Proteins 5 and 6.

BACKGROUND & AIMS: Stearoyl-CoA desaturase (SCD) synthesizes monounsaturated fatty acids (MUFAs) and has been associated with the development of metabolic syndrome, tumorigenesis, and stem cell characteristics. We investigated whether and how SCD promotes liver fibrosis and tumor development in mice. METHODS: Rodent primary hepatic stellate cells (HSCs), mouse liver tumor-initiating stem cell-like cells (TICs), and human hepatocellular carcinoma (HCC) cell lines were exposed to Wnt signaling inhibitors and changes in gene expression patterns were analyzed. We assessed the functions of SCD by pharmacologic and conditional genetic manipulation in mice with hepatotoxic or cholestatic induction of liver fibrosis, orthotopic transplants of TICs, or liver tumors induced by administration of diethyl nitrosamine. We performed bioinformatic analyses of SCD expression in HCC vs nontumor liver samples collected from patients, and correlated levels with HCC stage and patient mortality. We performed nano-bead pull-down assays, liquid chromatography-mass spectrometry, computational modeling, and ribonucleoprotein immunoprecipitation analyses to identify MUFA-interacting proteins. We examined the effects of SCD inhibition on Wnt signaling, including the expression and stability of low-density lipoprotein-receptor-related proteins 5 and 6 (LRP5 and LRP6), by immunoblot and quantitative polymerase chain reaction analyses. RESULTS: SCD was overexpressed in activated HSC and HCC cells from patients; levels of SCD messenger RNA (mRNA) correlated with HCC stage and patient survival time. In rodent HSCs and TICs, the Wnt effector ß-catenin increased sterol regulatory element binding protein 1-dependent transcription of Scd, and ß-catenin in return was stabilized by MUFAs generated by SCD. This loop required MUFA inhibition of binding of Ras-related nuclear protein 1 (Ran1) to transportin 1 and reduced nuclear import of elav-like protein 1 (HuR), increasing cytosolic levels of HuR and HuR-mediated stabilization of mRNAs encoding LRP5 and LRP6. Genetic disruption of Scd and pharmacologic inhibitors of SCD reduced HSC activation and TIC self-renewal and attenuated liver fibrosis and tumorigenesis in mice. Conditional disruption of Scd2 in activated HSCs prevented growth of tumors from TICs and reduced the formation of diethyl nitrosamine-induced liver tumors in mice. CONCLUSIONS: In rodent HSCs and TICs, we found SCD expression to be regulated by Wnt-ß-catenin signaling, and MUFAs produced by SCD provided a forward loop to amplify Wnt signaling via stabilization of Lrp5 and Lrp6 mRNAs, contributing to liver fibrosis and tumor growth. SCD expressed by HSCs promoted liver tumor development in mice. Components of the identified loop linking HSCs and TICs might be therapeutic targets for liver fibrosis and tumors.

Wnt Pathway Stabilizes MeCP2 Protein to Repress PPAR-γ in Activation of Hepatic Stellate Cells.

PPAR-γ is essential for differentiation of hepatic stellate cells (HSC), and its loss due to epigenetic repression by methyl-CpG binding protein 2 (MeCP2) causes HSC myofibroblastic activation mediated in part via Wnt pathway, the key cellular event in liver fibrosis. Decreased miR-132 was previously proposed to promote MeCP2 protein translation for Ppar-γ repression in activated HSC (aHSC). The present study aimed to test this notion and to better understand the mechanisms of MeCP2 upregulation in aHSC. MeCP2 protein is increased on day 3 to 7 as HSC become activated in primary culture on plastic, but this is accompanied by increased but not reduced miR-132 or miR-212 which is also expected to target MeCP2 due to its similar sequence with miR-132. The levels of these mRNAs are decreased 40~50% in aHSCs isolated from experimental cholestatic liver fibrosis but increased 6-8 fold in aHSC from hepatotoxic liver fibrosis in rats. Suppression of either or both of miR132 and miR212 with specific anti-miRNA oligonucleotides (anti-oligo), does not affect MeCP2 protein levels in aHSCs. The Wnt antagonist FJ9 which inhibits HSC activation, increases miR-132/miR-212, reduces MeCP2 and its enrichment at 5' Ppar-γ promoter, and restores Ppar-γ expression but the anti-oligo do not prevent Ppar-γ upregulation. The pan-NADPH oxidase (NOX) inhibitor diphenyleneiodonium (DPI) also reduces both MeCP2 and stabilized non-(S33/S37/Thr41)-phospho ß-catenin and reverts aHSC to quiescent cells but do not affect miR-132/miR-212 levels. Wnt antagonism with FJ9 increases MeCP2 protein degradation in cultured HSC, and FJ9-mediated loss of MeCP2 is rescued by leupeptin but not by proteasome and lysozome inhibitors. In conclusion, canonical Wnt pathway increases MeCP2 protein due to protein stability which in turn represses Ppar-γ and activates HSC.

Mechanisms of clonal evolution in childhood acute lymphoblastic leukemia.

Childhood acute lymphoblastic leukemia (ALL) can often be traced to a pre-leukemic clone carrying a prenatal genetic lesion. Postnatally acquired mutations then drive clonal evolution toward overt leukemia. The enzymes RAG1-RAG2 and AID, which diversify immunoglobulin-encoding genes, are strictly segregated in developing cells during B lymphopoiesis and peripheral mature B cells, respectively. Here we identified small pre-BII cells as a natural subset with increased genetic vulnerability owing to concurrent activation of these enzymes. Consistent with epidemiological findings on childhood ALL etiology, susceptibility to genetic lesions during B lymphopoiesis at the transition from the large pre-BII cell stage to the small pre-BII cell stage was exacerbated by abnormal cytokine signaling and repetitive inflammatory stimuli. We demonstrated that AID and RAG1-RAG2 drove leukemic clonal evolution with repeated exposure to inflammatory stimuli, paralleling chronic infections in childhood.

Self-enforcing feedback activation between BCL6 and pre-B cell receptor signaling defines a distinct subtype of acute lymphoblastic leukemia.

Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR(+) ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR(+) ALL.

Alcohol directly stimulates epigenetic modifications in hepatic stellate cells.

BACKGROUND & AIMS: Alcohol is a primary cause of liver disease and an important co-morbidity factor in other causes of liver disease. A common feature of progressive liver disease is fibrosis, which results from the net deposition of fibril-forming extracellular matrix (ECM). The hepatic stellate cell (HSC) is widely considered to be the major cellular source of fibrotic ECM. We determined if HSCs are responsive to direct stimulation by alcohol. METHODS: HSCs undergoing transdifferentiation were incubated with ethanol and expression of fibrogenic genes and epigenetic regulators was measured. Mechanisms responsible for recorded changes were investigated using ChIP-Seq and bioinformatics analysis. Ethanol induced changes were confirmed using HSCs isolated from a mouse alcohol model and from ALD patient's liver and through precision cut liver slices. RESULTS: HSCs responded to ethanol exposure by increasing profibrogenic and ECM gene expression including elastin. Ethanol induced an altered expression of multiple epigenetic regulators, indicative of a potential to modulate chromatin structure during HSC transdifferentiation. MLL1, a histone 3 lysine 4 (H3K4) methyltransferase, was induced by ethanol and recruited to the elastin gene promoter where it was associated with enriched H3K4me3, a mark of active chromatin. Chromatin immunoprecipitation sequencing (ChIPseq) revealed that ethanol has broad effects on the HSC epigenome and identified 41 gene loci at which both MML1 and its H3K4me3 mark were enriched in response to ethanol. CONCLUSIONS: Ethanol directly influences HSC transdifferentiation by stimulating global changes in chromatin structure, resulting in the increased expression of ECM proteins. The ability of alcohol to remodel the epigenome during HSC transdifferentiation provides mechanisms for it to act as a co-morbidity factor in liver disease.

Integrative epigenomic analysis identifies biomarkers and therapeutic targets in adult B-acute lymphoblastic leukemia.

UNLABELLED: Genetic lesions such as BCR-ABL1, E2A-PBX1, and MLL rearrangements (MLLr) are associated with unfavorable outcomes in adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Leukemia oncoproteins may directly or indirectly disrupt cytosine methylation patterning to mediate the malignant phenotype. We postulated that DNA methylation signatures in these aggressive B-ALLs would point toward disease mechanisms and useful biomarkers and therapeutic targets. We therefore conducted DNA methylation and gene expression profiling on a cohort of 215 adult patients with B-ALL enrolled in a single phase III clinical trial (ECOG E2993) and normal control B cells. In BCR-ABL1-positive B-ALLs, aberrant cytosine methylation patterning centered around a cytokine network defined by hypomethylation and overexpression of IL2RA(CD25). The E2993 trial clinical data showed that CD25 expression was strongly associated with a poor outcome in patients with ALL regardless of BCR-ABL1 status, suggesting CD25 as a novel prognostic biomarker for risk stratification in B-ALLs. In E2A-PBX1-positive B-ALLs, aberrant DNA methylation patterning was strongly associated with direct fusion protein binding as shown by the E2A-PBX1 chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq), suggesting that E2A-PBX1 fusion protein directly remodels the epigenome to impose an aggressive B-ALL phenotype. MLLr B-ALL featured prominent cytosine hypomethylation, which was linked with MLL fusion protein binding, H3K79 dimethylation, and transcriptional upregulation, affecting a set of known and newly identified MLL fusion direct targets with oncogenic activity such as FLT3 and BCL6. Notably, BCL6 blockade or loss of function suppressed proliferation and survival of MLLr leukemia cells, suggesting BCL6-targeted therapy as a new therapeutic strategy for MLLr B-ALLs. SIGNIFICANCE: We conducted the first integrative epigenomic study in adult B-ALLs, as a correlative study to the ECOG E2993 phase III clinical trial. This study links for the first time the direct actions of oncogenic fusion proteins with disruption of epigenetic regulation mediated by cytosine methylation. We identify a novel clinically actionable biomarker in B-ALLs: IL2RA (CD25), which is linked with BCR-ABL1 and an inflammatory signaling network associated with chemotherapy resistance. We show that BCL6 is a novel MLL fusion protein target that is required to maintain the proliferation and survival of primary human adult MLLr cells and provide the basis for a clinical trial with BCL6 inhibitors for patients with MLLr.

BCL6 enables Ph+ acute lymphoblastic leukaemia cells to survive BCR-ABL1 kinase inhibition.

Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.

The Fer tyrosine kinase regulates interactions of Rho GDP-Dissociation Inhibitor α with the small GTPase Rac.

BACKGROUND: RhoGDI proteins are important regulators of the small GTPase Rac, because they shuttle Rac from the cytoplasm to membranes and also protect Rac from activation, deactivation and degradation. How the binding and release of Rac from RhoGDI is regulated is not precisely understood. RESULTS: We report that the non-receptor tyrosine kinase Fer is able to phosphorylate RhoGDIα and form a direct protein complex with it. This interaction is mediated by the C-terminal end of RhoGDIα. Activation of Fer by reactive oxygen species caused increased phosphorylation of RhoGDIα and pervanadate treatment further augmented this. Tyrosine phosphorylation of RhoGDIα by Fer prevented subsequent binding of Rac to RhoGDIα, but once a RhoGDIα-Rac complex was formed, the Fer kinase was not able to cause Rac release through tyrosine phosphorylation of preformed RhoGDIα-Rac complexes. CONCLUSIONS: These results identify tyrosine phosphorylation of RhoGDIα by Fer as a mechanism to regulate binding of RhoGDIα to Rac.

BCL6 is critical for the development of a diverse primary B cell repertoire.

BCL6 protects germinal center (GC) B cells against DNA damage-induced apoptosis during somatic hypermutation and class-switch recombination. Although expression of BCL6 was not found in early IL-7-dependent B cell precursors, we report that IL-7Ralpha-Stat5 signaling negatively regulates BCL6. Upon productive VH-DJH gene rearrangement and expression of a mu heavy chain, however, activation of pre-B cell receptor signaling strongly induces BCL6 expression, whereas IL-7Ralpha-Stat5 signaling is attenuated. At the transition from IL-7-dependent to -independent stages of B cell development, BCL6 is activated, reaches expression levels resembling those in GC B cells, and protects pre-B cells from DNA damage-induced apoptosis during immunoglobulin (Ig) light chain gene recombination. In the absence of BCL6, DNA breaks during Ig light chain gene rearrangement lead to excessive up-regulation of Arf and p53. As a consequence, the pool of new bone marrow immature B cells is markedly reduced in size and clonal diversity. We conclude that negative regulation of Arf by BCL6 is required for pre-B cell self-renewal and the formation of a diverse polyclonal B cell repertoire.

Differential regulation of Streptococcus pneumoniae-induced human MUC5AC mucin expression through distinct MAPK pathways.

Human epithelial mucin, the major glycoprotein component of mucus, plays a critical role in host innate defense response against invading microbes by facilitating the mucociliary clearance. Excess mucin production, however, overwhelms the mucociliary clearance, resulting in not only defective mucosal defense but also conductive hearing loss in the middle ear and mucus obstruction in the airway. Indeed, mucus overproduction is a hall-mark of otitis media (OM) and chronic obstructive pulmonary diseases (COPD). Thus, tight regulation of mucin production plays an important role in maintaining an appropriate balance between beneficial and detrimental outcomes. We previously reported that Streptococcus pneumoniae (S. pneumoniae) up-regulates MUC5AC mucin expression via a positive MAPK ERK1/2 and a negative JNK1/2 signaling pathway. However, the signaling components including the up-stream activators and the down-stream transcription factors involved in these two path-ways remain largely unknown. In the present study, we showed that positive regulation of MUC5AC mucin expression by ERK1/2 is dependent on Ras-Raf-1 signaling pathway, whereas the negative regulation of MUC5AC expression by JNK1/2 is dependent on MEKK3. Moreover, transcriptional factor AP-1 acts as a key regulator for both of the positive and negative regulation of MUC5AC mucin expression as evidenced by mutagenesis analysis of two AP-1 sites in the promoter region of human MUC5AC mucin gene. Ras-Raf1-ERK1/2-dependent AP-1 activation positively regulates MUC5AC mucin induction by S. pneumoniae, whereas MEKK3-JNK1/2-dependent AP-1 activation negatively regulates it. Therefore, our data unveiled a novel signaling mechanism underlying the tight regulation of MUC5AC mucin induction by S. pneumoniae and may lead to the development of new therapeutic strategy for reducing mucus overproduction in both OM and COPD.

Streptococcus pneumoniae synergizes with nontypeable Haemophilus influenzae to induce inflammation via upregulating TLR2.

BACKGROUND: Toll-like receptor 2 (TLR2) plays a critical role in mediating inflammatory/immune responses against bacterial pathogens in lung. Streptococcus pneumoniae (S. pneumoniae) and nontypeable Haemophilus influenzae (NTHi) were previously reported to synergize with each other to induce inflammatory responses. Despite the relatively known intracellular signaling pathways involved in the synergistic induction of inflammation, it is still unclear if both bacterial pathogens also synergistically induce expression of surface TLR2. RESULTS: Here we provide direct evidence that S. pneumoniae synergizes with NTHi to upregulate TLR2 expression in lung and middle ear of the mice. Pneumolysin (PLY) appears to be the major virulence factor involved in this synergism. Moreover, S. pneumoniae PLY induces TLR2 expression via a TLR4-MyD88-NF-kappaB-dependent signaling pathway. Interestingly, tumor suppressor CYLD acts as a negative regulator of S. pneumoniae-induced TLR2 up-regulation via negative-crosstalk with NF-kappaB signaling. CONCLUSION: Our study thus provides novel insights into the regulation of TLR2 expression in mixed bacterial infections.

Activity of the Bcr GTPase-activating domain is regulated through direct protein/protein interaction with the Rho guanine nucleotide dissociation inhibitor.

The cycling of Rac GTPases, alternating between an active GTP- and an inactive GDP-bound state, is controlled by guanine nucleotide exchange factors, GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). Little is known about how these controlling activities are coordinated. Studies using null mutant mice have demonstrated that Bcr and Abr are two physiologically important GAPs for Rac. Here, we report that in the presence of RhoGDIalpha, Bcr is unable to convert Rac-GTP to Rac-GDP because RhoGDI forms a direct protein complex with Bcr. Interestingly, RhoGDIalpha binds to the GAP domain in Bcr and Abr, a domain that also binds to Rac-GTP and catalyzes conversion of the bound GTP to GDP on Rac. The presence of activated Rac diminished the Bcr/RhoGDIalpha interaction. Moreover, a Bcr mutant that lacks the ability to promote hydrolysis of Rac-GTP bound to its GAP domain did not bind to RhoGDIalpha in cell lysates, indicating that binding of RhoGDIalpha and Rac-GTP to the Bcr GAP domain is mutually exclusive. Our results provide the first identification of a protein that regulates BcrGAP activity.

TGF-beta induces p65 acetylation to enhance bacteria-induced NF-kappaB activation.

Transforming growth factor-beta (TGF-beta) family members are multifunctional growth factors involved in regulating diverse biological processes. Despite the critical role for TGF-beta in regulating cell proliferation, differentiation, migration and development, its role in regulating NF-kappaB-dependent inflammatory response still remains unclear. Here, we show that TGF-beta1 induces acetylation of NF-kappaB p65 subunit to synergistically enhance bacterium nontypeable Haemophilus influenzae-induced NF-kappaB activation and inflammatory response in vitro and in vivo. The TGF-beta1-induced acetylation of p65 is mediated via a Smad3/4-PKA-p300-dependent signaling pathway. Acetylation of p65 at lysine 221 by TGF-beta1 is critical for synergistic enhancement of bacteria-induced DNA-binding activity, NF-kappaB activation, NF-kappaB-dependent transcription of TNF-alpha and IL-1beta and interstitial polymorphonuclear neutrophil infiltration in vitro and in vivo. These studies provide new insights into the novel regulation of NF-kappaB by TGF-beta signaling.

Synergistic activation of NF-kappaB by nontypeable H. influenzae and S. pneumoniae is mediated by CK2, IKKbeta-IkappaBalpha, and p38 MAPK.

In review of the past studies on NF-kappaB regulation, most of them have focused on investigating how NF-kappaB is activated by a single inducer at a time. Given the fact that, in mixed bacterial infections in vivo, multiple inflammation inducers, including both nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae, are present simultaneously, a key issue that has yet to be addressed is whether NTHi and S. pneumoniae simultaneously activate NF-kappaB and the subsequent inflammatory response in a synergistic manner. Here, we show that NTHi and S. pneumoniae synergistically induce NF-kappaB-dependent inflammatory response via activation of multiple signaling pathways in vitro and in vivo. The classical IKKbeta-IkappaBalpha and p38 MAPK pathways are involved in synergistic activation of NF-kappaB via two distinct mechanisms, p65 nuclear translocation-dependent and -independent mechanisms. Moreover, casein kinase 2 (CK2) is involved in synergistic induction of NF-kappaB via a mechanism dependent on phosphorylation of p65 at both Ser536 and Ser276 sites. These studies bring new insights into the molecular mechanisms underlying the NF-kappaB-dependent inflammatory response in polymicrobial infections and may lead to development of novel therapeutic strategies for modulating inflammation in mixed infections for patients with otitis media and chronic obstructive pulmonary diseases.

Protective role of tissue transglutaminase in the cell death induced by TNF-alpha in SH-SY5Y neuroblastoma cells.

Tissue transglutaminase (tTGase) regulates various biological processes, including extracellular matrix organization, cellular differentiation, and apoptosis. Here we report the protective role of tTGase in the cell death that is induced by the tumor necrosis factor alpha (TNF-alpha) and ceramide, a product of the TNF-alpha signaling pathway, in human neuroblastoma SH-SY5Y cells. Treatment with retinoic acid (RA) induced the differentiation of the neuroblastoma cells with the formation of extended neurites. Immunostaining and Western blot analysis showed the tTGase expression by RA treatment. TNF-alpha or C(2) ceramide, a cell permeable ceramide analog, induced cell death in normal cells, but cell death was largely inhibited by the RA treatment. The inhibition of tTGase by the tTGase inhibitors, monodansylcadaverine and cystamine, eliminated the protective role of RA-treatment in the cell death that is caused by TNF-alpha or C(2)-ceramide. In addition, the co-treatment of TNF-alpha and cycloheximide decreased the protein level of tTGase and cell viability in the RA-treated cells, supporting the role of tTGase in the protection of cell death. DNA fragmentation was also induced by the co-treatment of TNF-alpha and cycloheximide. These results suggest that tTGase expressed by RA treatment plays an important role in the protection of cell death caused by TNF-alpha and ceramide.