Inhibition of the Otub1/c-Maf axis by the herbal acevaltrate induces myeloma cell apoptosis.

BACKGROUND: The oncogenic transcript factor c-Maf is stabilized by the deubiquitinase Otub1 and promotes myeloma cell proliferation and confers to chemoresistance. Inhibition of the Otub1/c-Maf axis is a promising therapeutic target, but there are no inhibitors reported on this specific axis. METHODS: A luciferase assay was applied to screen potential inhibitors of Otub1/c-Maf. Annexin V staining/flow cytometry was applied to evaluate cell apoptosis. Immunoprecipitation was applied to examine protein ubiquitination and interaction. Xenograft models in nude mice were used to evaluate anti-myeloma activity of AVT. RESULTS: Acevaltrate (AVT), isolated from Valeriana glechomifolia, was identified based on a bioactive screen against the Otub1/c-Maf/luciferase system. AVT disrupts the interaction of Otub1/c-Maf thus inhibiting Otub1 activity and leading to c-Maf polyubiquitination and subsequent degradation in proteasomes. Consistently, AVT inhibits c-Maf transcriptional activity and downregulates the expression of its target genes key for myeloma growth and survival. Moreover, AVT displays potent anti-myeloma activity by triggering myeloma cell apoptosis in vitro and impairing myeloma xenograft growth in vivo but presents no marked toxicity. CONCLUSIONS: The natural product AVT inhibits the Otub1/c-Maf axis and displays potent anti-myeloma activity. Given its great safety and efficacy, AVT could be further developed for MM treatment. Video Abstract.

Prostaglandin E2 inhibits Tr1 cell differentiation through suppression of c-Maf.

Prostaglandin E2 (PGE2), a major lipid mediator abundant at inflammatory sites, acts as a proinflammatory agent in models of inflammatory/autoimmune diseases by promoting CD4 Th1/Th17 differentiation. Regulatory T cells, including the IL-10 producing Tr1 cells counterbalance the proinflammatory activity of effector Th1/Th17 cells. Tr1 cell differentiation and function are induced by IL-27, and depend primarily on sustained expression of c-Maf in addition to AhR and Blimp-1. In agreement with the in vivo proinflammatory role of PGE2, here we report for the first time that PGE2 inhibits IL-27-induced differentiation and IL-10 production of murine CD4+CD49b+LAG-3+Foxp3- Tr1 cells. The inhibitory effect of PGE2 was mediated through EP4 receptors and induction of cAMP, leading to a significant reduction in c-Maf expression. Although PGE2 reduced IL-21 production in differentiating Tr1 cells, its inhibitory effect on Tr1 differentiation and c-Maf expression also occurred independent of IL-21 signaling. PGE2 did not affect STAT1/3 activation, AhR expression and only marginally reduced Egr-2/Blimp-1 expression. The effect of PGE2 on CD4+CD49b+LAG-3+ Tr1 differentiation was not associated with either induction of Foxp3 or IL-17 production, suggesting a lack of transdifferentiation into Foxp3+ Treg or effector Th17 cells. We recently reported that PGE2 inhibits the expression and production of IL-27 from activated conventional dendritic cells (cDC) in vivo and in vitro. The present study indicates that PGE2 also reduces murine Tr1 differentiation and function directly by acting on IL-27-differentiating Tr1 cells. Together, the ability of PGE2 to inhibit IL-27 production by cDC, and the direct inhibitory effect on Tr1 differentiation mediated through reduction in c-Maf expression, represent a new mechanistic perspective for the proinflammatory activity of PGE2.

Epidermal differentiation gene regulatory networks controlled by MAF and MAFB.

Numerous regulatory factors in epidermal differentiation and their role in regulating different cell states have been identified in recent years. However, the genetic interactions between these regulators over the dynamic course of differentiation have not been studied. In this Extra-View article, we review recent work by Lopez-Pajares et al. that explores a new regulatory network in epidermal differentiation. They analyze the changing transcriptome throughout epidermal regeneration to identify 3 separate gene sets enriched in the progenitor, early and late differentiation states. Using expression module mapping, MAF along with MAFB, are identified as transcription factors essential for epidermal differentiation. Through double knock-down of MAF:MAFB using siRNA and CRISPR/Cas9-mediated knockout, epidermal differentiation was shown to be impaired both in-vitro and in-vivo, confirming MAF:MAFB's role to activate genes that drive differentiation. Lopez-Pajares and collaborators integrated 42 published regulator gene sets and the MAF:MAFB gene set into the dynamic differentiation gene expression landscape and found that lncRNAs TINCR and ANCR act as upstream regulators of MAF:MAFB. Furthermore, ChIP-seq analysis of MAF:MAFB identified key transcription factor genes linked to epidermal differentiation as downstream effectors. Combined, these findings illustrate a dynamically regulated network with MAF:MAFB as a crucial link for progenitor gene repression and differentiation gene activation.

MYB controls erythroid versus megakaryocyte lineage fate decision through the miR-486-3p-mediated downregulation of MAF.

The transcription factor MYB has a key role in hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that MYB controls erythroid versus megakaryocyte lineage decision by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which MYB affects lineage fate decision, we performed the integrative analysis of miRNA and mRNA changes in MYB-silenced human primary CD34+ HPCs. Among the miRNAs with the highest number of predicted targets, we focused our studies on hsa-miR-486-3p by demonstrating that MYB controls miR-486-3p expression through the transactivation of its host gene, ankyrin-1 (ANK1) and that miR-486-3p affects HPCs commitment. Indeed, overexpression and knockdown experiments demonstrated that miR-486-3p supports the erythropoiesis while restraining the megakaryopoiesis. Of note, miR-486-3p also favors granulocyte differentiation while repressing the macrophage differentiation. To shed some light on the molecular mechanisms through which miR-486-3p affects HPCs lineage commitment, we profiled the gene expression changes upon miR-486-3p overexpression in CD34+ cells. Among the genes downregulated in miR-486-3p-overexpressing HPCs and computationally predicted to be miR-486-3p targets, we identified MAF as a miR-486-3p target by 3'UTR luciferase reporter assay. Noteworthy, MAF overexpression was able to partially reverse the effects of miR-486-3p overexpression on erythroid versus megakaryocyte lineage choice. Moreover, the MYB/MAF co-silencing constrained the skewing of erythroid versus megakaryocyte lineage commitment in MYB-silenced CD34+ cells, by restraining the expansion of megakaryocyte lineage while partially rescuing the impairment of erythropoiesis. Therefore, our data collectively demonstrate that MYB favors erythropoiesis and restrains megakaryopoiesis through the transactivation of miR-486-3p expression and the subsequent downregulation of MAF. As a whole, our study uncovers the MYB/miR-486-3p/MAF axis as a new mechanism underlying the MYB-driven control of erythroid versus megakaryocyte lineage fate decision.

Inhibition of Small Maf Function in Pancreatic ß-Cells Improves Glucose Tolerance Through the Enhancement of Insulin Gene Transcription and Insulin Secretion.

The large-Maf transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) has been found to be crucial for insulin transcription and synthesis and for pancreatic ß-cell function and maturation. However, insights about the effects of small Maf factors on ß-cells are limited. Our goal was to elucidate the function of small-Maf factors on ß-cells using an animal model of endogenous small-Maf dysfunction. Transgenic (Tg) mice with ß-cell-specific expression of dominant-negative MafK (DN-MafK) experiments, which can suppress the function of all endogenous small-Mafs, were fed a high-fat diet, and their in vivo phenotypes were evaluated. Phenotypic analysis, glucose tolerance tests, morphologic examination of ß-cells, and islet experiments were performed. DN-MafK-expressed MIN6 cells were also used for in vitro analysis. The results showed that DN-MafK expression inhibited endogenous small-Maf binding to insulin promoter while increasing MafA binding. DN-MafK Tg mice under high-fat diet conditions showed improved glucose metabolism compared with control mice via incremental insulin secretion, without causing changes in insulin sensitivity or MafA expression. Moreover, up-regulation of insulin and glucokinase gene expression was observed both in vivo and in vitro under DN-MafK expression. We concluded that endogenous small-Maf factors negatively regulates ß-cell function by competing for MafA binding, and thus, the inhibition of small-Maf activity can improve ß-cell function.

Role of Blimp-1 in programing Th effector cells into IL-10 producers.

Secretion of the immunosuppressive cytokine interleukin (IL) 10 by effector T cells is an essential mechanism of self-limitation during infection. However, the transcriptional regulation of IL-10 expression in proinflammatory T helper (Th) 1 cells is insufficiently understood. We report a crucial role for the transcriptional regulator Blimp-1, induced by IL-12 in a STAT4-dependent manner, in controlling IL-10 expression in Th1 cells. Blimp-1 deficiency led to excessive inflammation during Toxoplasma gondii infection with increased mortality. IL-10 production from Th1 cells was strictly dependent on Blimp-1 but was further enhanced by the synergistic function of c-Maf, a transcriptional regulator of IL-10 induced by multiple factors, such as the Notch pathway. We found Blimp-1 expression, which was also broadly induced by IL-27 in effector T cells, to be antagonized by transforming growth factor (TGF) ß. While effectively blocking IL-10 production from Th1 cells, TGF-ß shifted IL-10 regulation from a Blimp-1-dependent to a Blimp-1-independent pathway in IL-27-induced Tr1 (T regulatory 1) cells. Our findings further illustrate how IL-10 regulation in Th cells relies on several transcriptional programs that integrate various signals from the environment to fine-tune expression of this critical immunosuppressive cytokine.

Host factor transcriptional regulation contributes to preferential expression of HIV type 1 in IL-4-producing CD4 T cells.

HIV type 1 (HIV-1) replicates preferentially in IL-4-producing CD4 T cells for unclear reasons. We show increased HIV-1 expression is irrespective of viral tropism for chemokine receptors as previously suggested, but rather transcription of the HIV-1 long terminal repeat (LTR) is increased in IL-4-producing CD4 T cells. Increased expression of HIV-1 message is also confirmed in IL-4-producing CD4 T cells from HIV-1-infected individuals ex vivo. In exploring a transcriptional mechanism, we identify a novel c-maf (required for IL-4 expression) transcription factor binding site just upstream of the dual NF-κB/NFAT binding sites in the proximal HIV-1 LTR. We demonstrate that c-maf binds this site in vivo and synergistically augments HIV-1 transcription in cooperation with NFAT2 and NF-κB p65, but not NFAT1 or NF-κB p50. Conversely, small interfering RNA inhibition of c-maf reduces HIV-1 transcription in IL-4-producing T cells. Thus, c-maf increases HIV-1 expression in IL-4-producing CD4 T cells by binding the proximal HIV-1 LTR and augmenting HIV-1 transcription in partnership with NFAT2 and NF-κB p65 specifically. This has important implications for selective targeting of transcription factors during HIV-1 infection because, over the course of HIV-1 progression/AIDS, IL-4-producing T cells frequently predominate and substantially contribute to disease pathology.

c-Maf suppresses human T-cell leukemia virus type 1 Tax by competing for CREB-binding protein.

Latent infection of human T-cell leukemia virus type 1 (HTLV-1) is considered to be preferentially associated with CCR4(+) CD4(+) T cells. Here we report that c-Maf, one of the critical transcription factors for Th2 differentiation, suppresses the transcriptional activity of HTLV-1 Tax by competing for CREB-binding protein. Notably, c-maf expression is selectively induced in a fraction of CCR4(+) CD4(+) T cells upon activation. Furthermore, c-Maf significantly decreases Tax-induced HTLV-1 envelope gp46 gene expression from an infectious HTLV-1 molecular clone and tax expression in a cell-free HTLV-1 infection system. Collectively, c-Maf may play a role in latent infection of HTLV-1 in CCR4(+) CD4(+) T cells by negatively regulating Tax activity.