Zhenyue Hao

Fas Receptor Expression in Germinal-Center B Cells Is Essential for T and B Lymphocyte Homeostasis

Fas is highly expressed in activated and germinal center (GC) B cells but can potentially be inactivated by misguided somatic hypermutation. We employed conditional Fas-deficient mice to investigate the physiological functions of Fas in various B cell subsets. B cell-specific Fas-deficient mice developed fatal lymphoproliferation due to activation of B cells and T cells. Ablation of Fas specifically in GC B cells reproduced the phenotype, indicating that the lymphoproliferation initiates in the GC environment. B cell-specific Fas-deficient mice also showed an accumulation of IgG1(+) memory B cells expressing high amounts of CD80 and the expansion of CD28-expressing CD4(+) Th cells. Blocking T cell-B cell interaction and GC formation completely prevented the fatal lymphoproliferation. Thus, Fas-mediated selection of GC B cells and the resulting memory B cell compartment is essential for maintaining the homeostasis of both T and B lymphocytes.

Beyond tumor necrosis factor receptor: TRADD signaling in toll-like receptors

Tumor necrosis factor receptor 1-associated death domain protein (TRADD) is the core adaptor recruited to TNF receptor 1 (TNFR1) upon TNFα stimulation. In cells from TRADD-deficient mice, TNFα-mediated apoptosis and TNFα-stimulated NF-κB, JNK, and ERK activation are defective. TRADD is also important for germinal center formation, DR3-mediated costimulation of T cells, and TNFα-mediated inflammatory responses in vivo. TRADD deficiency does not enhance IFNγ-induced signaling. Importantly, TRADD has a novel role in TLR3 and TLR4 signaling. TRADD participates in the TLR4 complex formed upon LPS stimulation, and TRADD-deficient macrophages show impaired cytokine production in response to TLR ligands in vitro. Thus, TRADD is a multifunctional protein crucial both for TNFR1 signaling and other signaling pathways relevant to immune responses.

Effective Destruction of Fas-deficient Insulin-producing β Cells in Type 1 Diabetes

In type 1 diabetes, autoimmune T cells cause destruction of pancreatic β cells by largely unknown mechanism. Previous analyses have shown that β cell destruction is delayed but can occur in perforin-deficient nonobese diabetic (NOD) mice and that Fas-deficient NOD mice do not develop diabetes. However, because of possible pleiotropic functions of Fas, it was not clear whether the Fas receptor was an essential mediator of β cell death in type 1 diabetes. To directly test this hypothesis, we have generated a β cell–specific knockout of the Fas gene in a transgenic model of type 1 autoimmune diabetes in which CD4+ T cells with a transgenic TCR specific for influenza hemagglutinin (HA) are causing diabetes in mice that express HA under control of the rat insulin promoter. Here we show that the Fas-deficient mice develop autoimmune diabetes with slightly accelerated kinetics indicating that Fas-dependent apoptosis of β cells is a dispensable mode of cell death in this disease.

Mule/Huwe1/Arf-BP1 suppresses Ras-driven tumorigenesis by preventing c-Myc/Miz1-mediated down-regulation of p21 and p15

Tumorigenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis, and/or senescence. Many gene products involved in these processes are substrates of the E3 ubiquitin ligase Mule/Huwe1/Arf-BP1 (Mule), but whether Mule acts as an oncogene or tumor suppressor in vivo remains controversial. We generated K14Cre;Mule(flox/flox(y)) (Mule kKO) mice and subjected them to DMBA/PMA-induced skin carcinogenesis, which depends on oncogenic Ras signaling. Mule deficiency resulted in increased penetrance, number, and severity of skin tumors, which could be reversed by concomitant genetic knockout of c-Myc but not by knockout of p53 or p19Arf. Notably, in the absence of Mule, c-Myc/Miz1 transcriptional complexes accumulated, and levels of p21CDKN1A (p21) and p15INK4B (p15) were down-regulated. In vitro, Mule-deficient primary keratinocytes exhibited increased proliferation that could be reversed by Miz1 knockdown. Transfer of Mule-deficient transformed cells to nude mice resulted in enhanced tumor growth that again could be abrogated by Miz1 knockdown. Our data demonstrate in vivo that Mule suppresses Ras-mediated tumorigenesis by preventing an accumulation of c-Myc/Miz1 complexes that mediates p21 and p15 down-regulation.

Ablation of thymic export causes accelerated decay of naïve CD4 T cells in the periphery because of activation by environmental antigen

A model of chemical thymectomy by inducible Rag ablation was used to study peripheral T cell homeostasis. Induction of Rag ablation was efficient and complete, leading to cessation of thymic T cell production within 3–4 weeks. The decay of peripheral T cells became apparent with a delay of an additional 2–3 weeks and was entirely accounted for by loss of naïve T cells, whereas numbers of memory phenotype and regulatory T cells were not decreased. Naïve CD4 T cells decayed with an average half-life of 50 days, whereas naïve CD8 T cells exhibited a considerably longer half-life. The rapid decay of naïve CD4 T cells was not caused by intrinsic survival differences compared with naïve CD8 T cells, but was caused by changes in the lymphopenic environment resulting in higher microbial load and consequential activation. This finding suggests that in lymphopenic conditions involving compromised thymic function replenishment and survival of a naïve CD4 T cell repertoire may be severely curtailed because of chronic activation. Such a scenario might play a role in the aging immune system and chronic viral infection, such as HIV infection, and contribute to loss of CD4 T cells and impaired immune function. As our data show, continued replenishment with cells from the thymus seems to be required to maintain efficient gut mucosal defense.

Estrogen controls the survival of BRCA1-deficient cells via a PI3K–NRF2-regulated pathway

Significance Our establishment of a connection between the phosphatidylinositol 3-kinase (PI3K) and NRF2 pathways provides the basis for the tissue specificity of BRCA1-related cancers. Because BRCA1 is a vital component of the intracellular machinery maintaining genomic stability, BRCA1 functions as a major tumor suppressor in cells of all types. However, BRCA1-related cancers occur overwhelmingly in breasts and ovaries. Our work demonstrates that estrogen (E2) acts via the PI3K–AKT pathway to regulate NRF2 activation in BRCA1-deficient cells, resulting in the induction of antioxidant genes that protect the cell from reactive oxygen species-induced death. BRCA1-deficient cells are thus allowed to survive and may accumulate mutations, such as loss of PTEN, that perpetuate NRF2 activation. Our work supports the emerging clinical strategy of treating BRCA1-related cancers with PI3K inhibitors. Mutations in the tumor suppressor BRCA1 predispose women to breast and ovarian cancers. The mechanism underlying the tissue-specific nature of BRCA1’s tumor suppression is obscure. We previously showed that the antioxidant pathway regulated by the transcription factor NRF2 is defective in BRCA1-deficient cells. Reactivation of NRF2 through silencing of its negative regulator KEAP1 permitted the survival of BRCA1-null cells. Here we show that estrogen (E2) increases the expression of NRF2-dependent antioxidant genes in various E2-responsive cell types. Like NRF2 accumulation triggered by oxidative stress, E2-induced NRF2 accumulation depends on phosphatidylinositol 3-kinase–AKT activation. Pretreatment of mammary epithelial cells (MECs) with the phosphatidylinositol 3-kinase inhibitor BKM120 abolishes the capacity of E2 to increase NRF2 protein and transcriptional activity. In vivo the survival defect of BRCA1-deficient MECs is rescued by the rise in E2 levels associated with pregnancy. Furthermore, exogenous E2 administration stimulates the growth of BRCA1-deficient mammary tumors in the fat pads of male mice. Our work elucidates the basis of the tissue specificity of BRCA1-related tumor predisposition, and explains why oophorectomy significantly reduces breast cancer risk and recurrence in women carrying BRCA1 mutations.

Mutant IDH1 Downregulates ATM and Alters DNA Repair and Sensitivity to DNA Damage Independent of TET2.

Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSCs), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia.

Mule determines the apoptotic response to HDAC inhibitors by targeted ubiquitination and destruction of HDAC2

Histone deacetylases (HDACs) are major epigenetic modulators involved in a broad spectrum of human diseases including cancers. Administration of HDAC inhibitors (HDACis) leads to growth inhibition, differentiation, and apoptosis of cancer cells. Understanding the regulatory mechanism of HDACs is imperative to harness the therapeutic potentials of HDACis. Here we show that HDACi- and DNA damage-induced apoptosis are severely compromised in mouse embryonic fibroblasts lacking a HECT domain ubiquitin ligase, Mule (Mcl-1 ubiquitin ligase E3). Mule specifically targets HDAC2 for ubiquitination and degradation. Accumulation of HDAC2 in Mule-deficient cells leads to compromised p53 acetylation as well as crippled p53 transcriptional activation, accumulation, and apoptotic response upon DNA damage and Nutlin-3 treatments. These defects in Mule-null cells can be partially reversed by HDACis and fully rescued by lowering the elevated HDAC2 in Mule-null cells to the normal levels as in wild-type cells. Taken together, our results reveal a critical regulatory mechanism of HDAC2 by Mule and suggest this pathway determines the cellular response to HDACis and DNA damage.

Glutathione Primes T Cell Metabolism for Inflammation

&NA; Activated T cells produce reactive oxygen species (ROS), which trigger the antioxidative glutathione (GSH) response necessary to buffer rising ROS and prevent cellular damage. We report that GSH is essential for T cell effector functions through its regulation of metabolic activity. Conditional gene targeting of the catalytic subunit of glutamate cysteine ligase (Gclc) blocked GSH production specifically in murine T cells. Gclc‐deficient T cells initially underwent normal activation but could not meet their increased energy and biosynthetic requirements. GSH deficiency compromised the activation of mammalian target of rapamycin‐1 (mTOR) and expression of NFAT and Myc transcription factors, abrogating the energy utilization and Myc‐dependent metabolic reprogramming that allows activated T cells to switch to glycolysis and glutaminolysis. In vivo, T‐cell‐specific ablation of murine Gclc prevented autoimmune disease but blocked antiviral defense. The antioxidative GSH pathway thus plays an unexpected role in metabolic integration and reprogramming during inflammatory T cell responses. Graphical Abstract Figure. No caption available. HighlightsGlutathione (GSH) is not needed for early T cell activation but promotes T cell growthGSH supports mTOR and NFAT activity and drives glycolysis and glutaminolysisGclc‐derived GSH buffers ROS and regulates Myc‐dependent metabolic reprogrammingAblation of Gclc in T cells impairs inflammatory responses in vivo &NA; Upon activation, T cells adapt their metabolism to meet their increased bioenergetic and biosynthetic needs. Activated T cells produce ROS, which trigger the antioxidative GSH response to prevent cellular damage. Mak et al. report that the GSH pathway plays an unexpected role in metabolic integration during inflammatory T cell responses.

The E3 ubiquitin ligase Mule acts through the ATM–p53 axis to maintain B lymphocyte homeostasis

Genetic manipulation reveals that Mule is vital for B cell development, proliferation, and homeostasis as a result of its ability to regulate p53 and ATM.