The proinflammatory protein HMGB1 is a substrate of transglutaminase-2 and forms high-molecular weight complexes with autoantigens.

High-mobility group box 1 (HMGB1) is a chromatin-associated protein that, in response to stress or injury, translocates from the nucleus to the extracellular milieu, where it functions as an alarmin. HMGB1's function is in part determined by the complexes (HMGB1c) it forms with other molecules. However, structural modifications in the HMGB1 polypeptide that may regulate HMGB1c formation have not been previously described. In this report, we observed high-molecular weight, denaturing-resistant HMGB1c in the plasma and peripheral blood mononuclear cells of individuals with systemic lupus erythematosus (SLE) and, to a much lesser extent, in healthy subjects. Differential HMGB1c levels were also detected in mouse tissues and cultured cells, in which these complexes were induced by endotoxin or the immunological adjuvant alum. Of note, we found that HMGB1c formation is catalyzed by the protein-cross-linking enzyme transglutaminase-2 (TG2). Cross-link site mapping and MS analysis revealed that HMGB1 can be cross-linked to TG2 as well as a number of additional proteins, including human autoantigens. These findings have significant functional implications for studies of cellular stress responses and innate immunity in SLE and other autoimmune disease.

IL-18 Drives ILC3 Proliferation and Promotes IL-22 Production via NF-κB.

Group 3 innate lymphoid cells (ILC3s) are important regulators of the immune system, maintaining homeostasis in the presence of commensal bacteria, but activating immune defenses in response to microbial pathogens. ILC3s are a robust source of IL-22, a cytokine critical for stimulating the antimicrobial response. We sought to identify cytokines that can promote proliferation and induce or maintain IL-22 production by ILC3s and determine a molecular mechanism for this process. We identified IL-18 as a cytokine that cooperates with an ILC3 survival factor, IL-15, to induce proliferation of human ILC3s, as well as induce and maintain IL-22 production. To determine a mechanism of action, we examined the NF-κB pathway, which is activated by IL-18 signaling. We found that the NF-κB complex signaling component, p65, binds to the proximal region of the IL22 promoter and promotes transcriptional activity. Finally, we observed that CD11c+ dendritic cells expressing IL-18 are found in close proximity to ILC3s in human tonsils in situ. Therefore, we identify a new mechanism by which human ILC3s proliferate and produce IL-22, and identify NF-κB as a potential therapeutic target to be considered in pathologic states characterized by overproduction of IL-18 and/or IL-22.

A Critical Role for CD200R Signaling in Limiting the Growth and Metastasis of CD200+ Melanoma.

CD200 is a cell surface glycoprotein that functions through engaging CD200R on cells of the myeloid lineage and inhibits their functions. Expression of CD200 was implicated in a variety of human cancer cells, including melanoma cells; however, its roles in tumor growth and immunity are not clearly understood. In this study, we used CD200R-deficient mice and the B16 tumor model to evaluate this issue. We found that CD200R-deficient mice exhibited accelerated growth of CD200(+), but not CD200(-), B16 tumors. Strikingly, CD200R-deficient mice receiving CD200(+) B16 cells i.v. exhibited massive tumor growth in multiple organs, including liver, lung, kidney, and peritoneal cavity, whereas the growth of the same tumors in wild-type mice was limited. CD200(+) tumors grown in CD200R-deficient mice contained higher numbers of CD11b(+)Ly6C(+) myeloid cells, exhibited increased expression of VEGF and HIF1α genes with increased angiogenesis, and showed significantly reduced infiltration of CD4(+) and CD8(+) T cells, presumably as the result of reduced expression of T cell chemokines, such as CXCL9 and CXCL16. The liver from CD200R-deficient mice, under metastatic growth of CD200(+) tumors, contained significantly increased numbers of CD11b(+)Gr1(-) myeloid cells and Foxp3(+) regulatory T cells and reduced numbers of NK cells. Liver T cells also had a reduced capacity to produce IFN-γ or TNF-α. Taken together, we revealed a critical role for CD200R signaling in limiting the growth and metastasis of CD200(+) tumors. Thus, targeting CD200R signaling may potentially interfere with the metastatic growth of CD200(+) tumors, like melanoma.

Estrogen-regulated STAT1 activation promotes TLR8 expression to facilitate signaling via microRNA-21 in systemic lupus erythematosus.

Recent studies implicate innate immunity to systemic lupus erythematosus (SLE) pathogenesis. Toll-like receptor (TLR)8 is estrogen-regulated and binds viral ssRNA to stimulate innate immune responses, but recent work indicates that microRNA (miR)-21 within extracellular vesicles (EVs) can also trigger this receptor. Our objective was to examine TLR8 expression/activation to better understand sex-biased responses involving TLR8 in SLE. Our data identify an estrogen response element that promotes STAT1 expression and demonstrate STAT1-dependent transcriptional activation of TLR8 with estrogen stimulation. In lieu of viral ssRNA activation, we explored EV-encapsulated miR-21 as an endogenous ligand and observed induction of both TLR8 and cytokine expression in vitro. Moreover, extracellular miR detection was found predominantly within EVs. Thus, just as a cytokine or chemokine, EV-encapsulated miR-21 can act as an inflammatory signaling molecule, or miRokine, by virtue of being an endogenous ligand of TLR8. Collectively, our data elucidates a novel innate inflammatory pathway in SLE.

The YhhN protein of Legionella pneumophila is a Lysoplasmalogenase.

Lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. We recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as TMEM86B, an integral membrane protein that is a member of the YhhN family found in numerous species of eukaryotes and bacteria. To test the hypothesis that bacterial YhhN proteins also function as lysoplasmalogenase enzymes, we cloned the Lpg1991 gene of Legionella pneumophila, which encodes a 216 amino acid YhhN protein (LpYhhN), and expressed it in Escherichia coli as a C-terminal-GFP-His8-fusion. Membranes were solubilized and the fusion protein was purified by nickel-affinity chromatography, cleaved with Tobacco Etch Virus protease, and subjected to a reverse nickel column to purify the un-tagged LpYhhN. Both the fusion protein and un-tagged LpYhhN exhibit robust lysoplasmalogenase activity, cleaving the vinyl-ether bond of lysoplasmalogen with a Vmax of 12 µmol/min/mg protein and a Km of 45 µM. LpYhhN has no activity on diradyl plasmalogen, 1-alkenyl-glycerol, and monoacylglycerophospho-ethanolamine or monoacylglycerophospho-choline; the pH optimum is 6.5-7.0. These properties are very similar to mammalian TMEM86B. Sequence analysis suggests that YhhN proteins contain eight transmembrane helices, an N-in/C-in topology, and about 5 highly conserved amino acid residues that may form an active site. This work is the first to demonstrate a function for a bacterial YhhN protein, as a vinyl ether bond hydrolase specific for lysoplasmalogen. Since L. pneumophila does not contain endogenous plasmalogens, we hypothesize that LpYhhN may serve to protect the bacterium from lysis by lysoplasmalogen derived from plasmalogens of the host.

The natural product phyllanthusmin C enhances IFN-γ production by human NK cells through upregulation of TLR-mediated NF-κB signaling.

Natural products are a major source for cancer drug development. NK cells are a critical component of innate immunity with the capacity to destroy cancer cells, cancer-initiating cells, and clear viral infections. However, few reports describe a natural product that stimulates NK cell IFN-γ production and unravel a mechanism of action. In this study, through screening, we found that a natural product, phyllanthusmin C (PL-C), alone enhanced IFN-γ production by human NK cells. PL-C also synergized with IL-12, even at the low cytokine concentration of 0.1 ng/ml, and stimulated IFN-γ production in both human CD56(bright) and CD56(dim) NK cell subsets. Mechanistically, TLR1 and/or TLR6 mediated PL-C's activation of the NF-κB p65 subunit that in turn bound to the proximal promoter of IFNG and subsequently resulted in increased IFN-γ production in NK cells. However, IL-12 and IL-15Rs and their related STAT signaling pathways were not responsible for the enhanced IFN-γ secretion by PL-C. PL-C induced little or no T cell IFN-γ production or NK cell cytotoxicity. Collectively, we identify a natural product with the capacity to selectively enhance human NK cell IFN-γ production. Given the role of IFN-γ in immune surveillance, additional studies to understand the role of this natural product in prevention of cancer or infection in select populations are warranted.

A chimeric human-mouse model of Sjögren's syndrome.

Despite recent advances in the understanding of Sjögren's Syndrome (SjS), the pathogenic mechanisms remain elusive and an ideal model for early drug discovery is not yet available. To establish a humanized mouse model of SjS, peripheral blood mononuclear cells (PBMCs) from healthy volunteers or patients with SjS were transferred into immunodeficient NOD-scid IL-2rγ(null) mouse recipients to produce chimeric mice. While no difference was observed in the distribution of cells, chimeric mice transferred with PBMCs from SjS patients produced enhanced cytokine levels, most significantly IFN-γ and IL-10. Histological examination revealed enhanced inflammatory responses in the lacrimal and salivary glands of SjS chimeras, as measured by digital image analysis and blinded histopathological scoring. Infiltrates were primarily CD4+, with minimal detection of CD8+ T-cells and B-cells. These results demonstrate a novel chimeric mouse model of human SjS that provides a unique in vivo environment to test experimental therapeutics and investigate T-cell disease pathology.

MicroRNAs activate natural killer cells through Toll-like receptor signaling.

MicroRNAs (miRNAs) bind to complementary sequences of target mRNAs, resulting in translational repression or target degradation and thus gene silencing. miRNAs are abundant in circulating blood, yet it is not known whether, as a class of regulatory molecules, they interact with human natural killer (NK) cells. Here we found that the treatment of human NK cells with several mature miRNAs in the presence of a low concentration of interleukin-12 induced CD69 expression, interferon-γ production, and degranulation marker CD107a expression. In vivo, infusion of several miRNAs alone in murine peripheral blood also resulted in comparable NK-cell activation, but not T-cell activation. Furthermore, miRNA administration significantly protected mice from tumor development in an NK cell-dependent manner. Mechanistically, we found that miRNA stimulation led to downstream activation of nuclear factor κB (NF-κB), an effect that was blunted by a block in Toll-like receptor 1(TLR1) signaling and attenuated in lymphoma patients. Knockdown of TLR1 resulted in less activation by miRNAs. Collectively, we show that miRNAs have a capacity to selectively activate innate immune effector cells that is, at least in part, via the TLR1-NF-κB signaling pathway. This may be important in the normal host defense against infection and/or malignant transformation.

Lenalidomide-mediated enhanced translation of C/EBPα-p30 protein up-regulates expression of the antileukemic microRNA-181a in acute myeloid leukemia.

Recently, we showed that increased miR-181a expression was associated with improved outcomes in cytogenetically normal acute myeloid leukemia (CN-AML). Interestingly, miR-181a expression was increased in CN-AML patients harboring CEBPA mutations, which are usually biallelic and associate with better prognosis. CEBPA encodes the C/EBPα transcription factor. We demonstrate here that the presence of N-terminal CEBPA mutations and miR-181a expression are linked. Indeed, the truncated C/EBPα-p30 isoform, which is produced from the N-terminal mutant CEBPA gene or from the differential translation of wild-type CEBPA mRNA and is commonly believed to have no transactivation activity, binds to the miR-181a-1 promoter and up-regulates the microRNA expression. Furthermore, we show that lenalidomide, a drug approved for myelodysplastic syndromes and multiple myeloma, enhances translation of the C/EBPα-p30 isoform, resulting in higher miR-181a levels. In xenograft mouse models, ectopic miR-181a expression inhibits tumor growth. Similarly, lenalidomide exhibits antitumorigenic activity paralleled by increased miR-181a expression. This regulatory pathway may explain an increased sensitivity to apoptosis-inducing chemotherapy in subsets of AML patients. Altogether, our data provide a potential explanation for the improved clinical outcomes observed in CEBPA-mutated CN-AML patients, and suggest that lenalidomide treatment enhancing the C/EBPα-p30 protein levels and in turn miR-181a may sensitize AML blasts to chemotherapy.

Estrogen modulation of endosome-associated toll-like receptor 8: an IFNα-independent mechanism of sex-bias in systemic lupus erythematosus.

Females of child-bearing age are more resistant to infectious disease and have an increased risk of systemic lupus erythematosus (SLE). We hypothesized that estrogen-induced gene expression could establish an immunoactivated state which would render enhanced defense against infection, but may be deleterious in autoimmune development. Using peripheral blood mononuclear cells (PBMCs), we demonstrate enhanced responses with immunogen stimulation in the presence of 17ß-estradiol (E2) and gene array analyses reveal toll-like receptor 8 (TLR8) as an E2-responsive candidate gene. TLR8 expression levels are up-regulated in SLE and PBMCs stimulated with TLR8 agonist display a female sex-biased, E2-sensitive response. Moreover, we identify a putative ERα-binding region near the TLR8 locus and blocking ERα expression significantly decreases E2-mediated TLR8 induction. Our findings characterize TLR8 as a novel estrogen target gene that can lower the inflammatory threshold and implicate an IFNα-independent inflammatory mechanism that could contribute to higher SLE incidence in women.

IL-27 enhances the survival of tumor antigen-specific CD8+ T cells and programs them into IL-10-producing, memory precursor-like effector cells.

IL-27 is a member of the IL-12 family of cytokines that is comprised of an IL-12 p40-related protein subunit, EBV-induced gene 3, and a p35-related subunit, p28. IL-27 functions through IL-27R and has been shown to have potent antitumor activity via activation of a variety of cellular components, including antitumor CD8(+) T-cell responses. However, the exact mechanisms of how IL-27 enhances antitumor CD8(+) T-cell responses remain unclear. Here we show that IL-27 significantly enhances the survival of activated tumor antigen-specific CD8(+) T cells in vitro and in vivo, and programs tumor antigen-specific CD8(+) T cells into memory precursor-like effector cells, characterized by upregulation of Bcl-6, SOCS3, Sca-1, and IL-10. While STAT3 activation and the CTL survival-enhancing effects can be independent of CTL IL-10 production, we show here that IL-27-induced CTL IL-10 production contributes to memory precursor cell phenotype induction, CTL memory, and tumor rejection. Thus, IL-27 enhances antitumor CTL responses via programming tumor antigen-specific CD8(+) T cells into a unique memory precursor type of effector cells characterized by a greater survival advantage. Our results have important implications for designing immunotherapy against human cancer.

Inhibition of miRNA associated with a disease-specific signature and secreted via extracellular vesicles of systemic lupus erythematosus patients suppresses target organ inflammation in a humanized mouse model.

Introduction: Distinct, disease-associated intracellular miRNA (miR) expression profiles have been observed in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematous (SLE) patients. Additionally, we have identified novel estrogenic responses in PBMCs from SLE patients and demonstrated that estrogen upregulates toll-like receptor (TLR)7 and TLR8 expression. TLR7 and TLR8 bind viral-derived single-stranded RNA to stimulate innate inflammatory responses, but recent studies have shown that miR-21, mir-29a, and miR-29b can also bind and activate these receptors when packaged and secreted in extracellular vesicles (EVs). The objective of this study was to evaluate the association of EV-encapsulated small RNA species in SLE and examine the therapeutic approach of miR inhibition in humanized mice. Methods: Plasma-derived EVs were isolated from SLE patients and quantified. RNA was then isolated and bulk RNA-sequencing reads were analyzed. Also, PBMCs from active SLE patients were injected into immunodeficient mice to produce chimeras. Prior to transfer, the PBMCs were incubated with liposomal EVs containing locked nucleic acid (LNA) antagonists to miR-21, mir-29a, and miR-29b. After three weeks, blood was collected for both immunophenotyping and cytokine analysis; tissue was harvested for histopathological examination. Results: EVs were significantly increased in the plasma of SLE patients and differentially expressed EV-derived small RNA profiles were detected compared to healthy controls, including miR-21, mir-29a, and miR-29b. LNA antagonists significantly reduced proinflammatory cytokines and histopathological infiltrates in the small intestine, liver, and kidney, as demonstrated by H&E-stained tissue sections and immunohistochemistry measuring human CD3. Discussion: These data demonstrate distinct EV-derived small RNA signatures representing SLE-associated biomarkers. Moreover, targeting upregulated EV-encapsulated miR signaling by antagonizing miRs that may bind to TLR7 and TLR8 reveals a novel therapeutic opportunity to suppress autoimmune-mediated inflammation and pathogenesis in SLE.

Purification, identification, and cloning of lysoplasmalogenase, the enzyme that catalyzes hydrolysis of the vinyl ether bond of lysoplasmalogen.

Lysoplasmalogenase (EC 3.3.2.2 and EC 3.3.2.5) is an enzyme that catalyzes hydrolytic cleavage of the vinyl ether bond of lysoplasmalogen, forming fatty aldehyde and glycerophosphoethanolamine or glycerophosphocholine and is specific for the sn-2-deacylated form of plasmalogen. Here we report the purification, characterization, identification, and cloning of lysoplasmalogenase. Rat liver microsomal lysoplasmalogenase was solubilized with octyl glucoside and purified 500-fold to near homogeneity using four chromatography steps. The purified enzyme has apparent K(m) values of ∼50 µm for both lysoplasmenylcholine and lysoplasmenylethanolamine and apparent V(m) values of 24.5 and 17.5 µmol/min/mg protein for the two substrates, respectively. The pH optimum was 7.0. Lysoplasmalogenase was competitively inhibited by lysophosphatidic acid (K(i) ∼20 µm). The predominant band on a gel at ∼19 kDa was subjected to trypsinolysis, and the peptides were identified by mass spectrometry as Tmem86b, a protein of unknown function. Transient transfection of human embryonic kidney (HEK) 293T cells showed that TMEM86b cDNA yielded lysoplasmalogenase activity, and Western blot analyses confirmed the synthesis of TMEM86b protein. The protein was localized in the membrane fractions. The TMEM86b gene was also transformed into Escherichia coli, and its expression was verified by Western blot and activity analyses. Tmem86b is a hydrophobic transmembrane protein of the YhhN family. Northern blot analyses demonstrated that liver expressed the highest level of Tmem86b, which agreed with tissue distribution of activity. Overexpression of TMEM86b in HEK 293T cells resulted in decreased levels of plasmalogens, suggesting that the enzyme may be important in regulating plasmalogen levels in animal cells.

Reactivation of RASSF1A in breast cancer cells by curcumin.

Reactivation of tumor suppressor genes (TSGs) involved in carcinogenesis by nontoxic bioactive food component represents a promising strategy for cancer chemoprevention. Recently, curcumin has been demonstrated to inhibit a bacterial DNA methyltransferase (M. Sss I) activity, induce global DNA hypomethylation in leukemia cells, and reactivate several hypermethylation silenced genes in lung and prostate cancer cells. Herein, we demonstrated that curcumin can enhance the mRNA and protein levels of ras-association domain family protein 1A (RASSF1A), 1 hypermethylation-silenced TSG, and decrease its promoter methylation in breast cancer cells. Mechanistic study demonstrated that curcumin can decrease DNA methylation activity of nuclear extract and downregulate the mRNA and protein levels of DNMT1 in MCF-7 cells, which may be associated with curcumin-induced disruption of NF-κB/Sp1 complex bound to the promoter region of DNMT1. Altogether, this study reveals a novel molecular mechanism of curcumin as a chemo-preventive agent for breast cancer through hypomethylation reactivation of RASSF1A.

Targeting activation-induced cytidine deaminase overcomes tumor evasion of immunotherapy by CTLs.

Activation-induced cytidine deaminase (AID) is an enzyme essential for the generation of Ab diversity in B cells and is considered to be a general gene mutator. In addition, AID expression was also implicated in the pathogenesis of human B cell malignancies and associated with poor prognosis. In this study, we report that small interfering RNA silencing of AID in plasmacytoma dramatically increased its susceptibility to immunotherapy by CTLs. AID silencing did not decrease the mutation frequencies of tumor Ag gene P1A. Gene-array analysis showed dramatically altered expression of a number of genes in AID-silenced plasmacytoma cells, and upregulation of CD200 was shown to be in favor of tumor eradication by CTLs. Taken together, we demonstrate a novel function of AID in tumor evasion of CTL therapy and that targeting AID should be beneficial in the immunotherapy of AID-positive tumors.

Aromatase-Inhibitor-Induced Musculoskeletal Inflammation Is Observed Independent of Oophorectomy in a Novel Mouse Model.

Aromatase Inhibitors (AIs) block estrogen production and improve survival in patients with hormone-receptor-positive breast cancer. However, half of patients develop aromatase-inhibitor-induced arthralgia (AIIA), which is characterized by inflammation of the joints and the surrounding musculoskeletal tissue. To create a platform for future interventional strategies, our objective was to characterize a novel animal model of AIIA. Female BALB/C-Tg(NFκB-RE-luc)-Xen mice, which have a firefly luciferase NFκB reporter gene, were oophorectomized and treated with an AI (letrozole). Bioluminescent imaging showed significantly enhanced NFκB activation with AI treatment in the hind limbs. Moreover, an analysis of the knee joints and legs via MRI showed enhanced signal detection in the joint space and the surrounding tissue. Surprisingly, the responses observed with AI treatment were independent of oophorectomy, indicating that inflammation is not mediated by physiological estrogen levels. Histopathological and pro-inflammatory cytokine analyses further demonstrated the same trend, as tenosynovitis and musculoskeletal infiltrates were detected in all mice receiving AI, and serum cytokines were significantly upregulated. Human PBMCs treated with letrozole/estrogen combinations did not demonstrate an AI-specific gene expression pattern, suggesting AIIA-mediated pathogenesis through other cell types. Collectively, these data identify an AI-induced stimulation of disease pathology and suggest that AIIA pathogenesis may not be mediated by estrogen deficiency, as previously hypothesized.

Gut dysbiosis is associated with acceleration of lupus nephritis.

The gut microbiota (GM) exerts a strong influence over the host immune system and dysbiosis of this microbial community can affect the clinical phenotype in chronic inflammatory conditions. To explore the role of the GM in lupus nephritis, we colonized NZM2410 mice with Segmented Filamentous Bacteria (SFB). Gut colonization with SFB was associated with worsening glomerulonephritis, glomerular and tubular immune complex deposition and interstitial inflammation compared to NZM2410 mice free of SFB. With SFB colonization mice experienced an increase in small intestinal lamina propria Th17 cells and group 3 innate lymphoid cells (ILC3s). However, although serum IL-17A expression was elevated in these mice, Th17 cells and ILC3s were not detected in the inflammatory infiltrate in the kidney. In contrast, serum and kidney tissue expression of the macrophage chemoattractants MCP-1 and CXCL1 were significantly elevated in SFB colonized mice. Furthermore, kidney infiltrating F4/80+CD206+M2-like macrophages were significantly increased in these mice. Evidence of increased gut permeability or "leakiness" was also detected in SFB colonized mice. Finally, the intestinal microbiome of SFB colonized mice at 15 and 30 weeks of age exhibited dysbiosis when compared to uncolonized mice at the same time points. Both microbial relative abundance as well as biodiversity of colonized mice was found to be altered. Collectively, SFB gut colonization in the NZM2410 mouse exacerbates kidney disease, promotes kidney M2-like macrophage infiltration and overall intestinal microbiota dysbiosis.

MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1.

Aberrant DNA hypermethylation contributes to myeloid leukemogenesis by silencing structurally normal genes involved in hematopoiesis. MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression by targeting protein-coding mRNAs. Recently, miRNAs have been shown to play a role as both targets and effectors in gene hypermethylation and silencing in malignant cells. In the current study, we showed that enforced expression of miR-29b in acute myeloid leukemia cells resulted in marked reduction of the expression of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B at both RNA and protein levels. This in turn led to decrease in global DNA methylation and reexpression of p15(INK4b) and ESR1 via promoter DNA hypomethylation. Although down-regulation of DNMT3A and DNMT3B was the result of a direct interaction of miR-29b with the 3' untranslated regions of these genes, no predicted miR-29b interaction sites were found in the DNMT1 3' untranslated regions. Further experiments revealed that miR-29b down-regulates DNMT1 indirectly by targeting Sp1, a transactivator of the DNMT1 gene. Altogether, these data provide novel functional links between miRNAs and aberrant DNA hypermethylation in acute myeloid leukemia and suggest a potentially therapeutic use of synthetic miR-29b oligonucleotides as effective hypomethylating compounds.

Modulation of DNA methylation by a sesquiterpene lactone parthenolide.

Hypermethylation of 5'-cytosine-guanosine islands of tumor suppressor genes resulting in their silencing has been proposed to be a hallmark of various tumors. Modulation of DNA methylation with DNA methylation inhibitors has been shown to result in cancer cell differentiation or apoptosis and represents a novel strategy for chemotherapy. Currently, effective DNA methylation inhibitors are mainly limited to decitabine and 5-azacytidine, which still show unfavorable toxicity profiles in the clinical setting. Thus, discovery and development of novel hypomethylating agents, with a more favorable toxicity profile, is essential to broaden the spectrum of epigenetic therapy. Parthenolide, the principal bioactive sesquiterpene lactone of feverfew, has been shown to alkylate Cys(38) of p65 to inhibit nuclear factor-kappaB activation and exhibit anti-tumor activity in human malignancies. In this article, we report that parthenolide 1) inhibits DNA methyltransferase 1 (DNMT1) with an IC(50) of 3.5 microM, possibly through alkylation of the proximal thiolate of Cys(1226) of the catalytic domain by its gamma-methylene lactone, and 2) down-regulates DNMT1 expression possibly associated with its SubG(1) cell-cycle arrest or the interruption of transcriptional factor Sp1 binding to the promoter of DNMT1. These dual functions of parthenolide result in the observed in vitro and in vivo global DNA hypomethylation. Furthermore, parthenolide has been shown to reactivate tumor suppressor HIN-1 gene in vitro possibly associated with its promoter hypomethylation. Hence, our study established parthenolide as an effective DNA methylation inhibitor, representing a novel prototype for DNMT1 inhibitor discovery and development from natural structural-diversified sesquiterpene lactones.

SMAD4 promotes TGF-ß-independent NK cell homeostasis and maturation and antitumor immunity.

SMAD4 is the only common SMAD in TGF-ß signaling that usually impedes immune cell activation in the tumor microenvironment. However, we demonstrated here that selective deletion of Smad4 in NK cells actually led to dramatically reduced tumor cell rejection and augmented tumor cell metastases, reduced murine CMV clearance, as well as impeded NK cell homeostasis and maturation. This was associated with a downregulation of granzyme B (Gzmb), Kit, and Prdm1 in Smad4-deficient NK cells. We further unveiled the mechanism by which SMAD4 promotes Gzmb expression. Gzmb was identified as a direct target of a transcriptional complex formed by SMAD4 and JUNB. A JUNB binding site distinct from that for SMAD4 in the proximal Gzmb promoter was required for transcriptional activation by the SMAD4-JUNB complex. In a Tgfbr2 and Smad4 NK cell-specific double-conditional KO model, SMAD4-mediated events were found to be independent of canonical TGF-ß signaling. Our study identifies and mechanistically characterizes unusual functions and pathways for SMAD4 in governing innate immune responses to cancer and viral infection, as well as NK cell development.