Lanfen Chen

Mlkl knockout mice demonstrate the indispensable role of Mlkl in necroptosis

Mixed lineage kinase domain-like protein (Mlkl) was recently found to interact with receptor interacting protein 3 (Rip3) and to be essential for tumor necrosis factor (TNF)-induced programmed necrosis (necroptosis) in cultured cell lines. We have generated Mlkl-deficient mice by transcription activator-like effector nucleases (TALENs)-mediated gene disruption and found Mlkl to be dispensable for normal mouse development as well as immune cell development. Mlkl-deficient mouse embryonic fibroblasts (MEFs) and macrophages both showed resistance to necrotic but not apoptotic stimuli. Mlkl-deficient MEFs and macrophages were indistinguishable from wild-type cells in their ability to activate NF-κB, ERK, JNK, and p38 in response to TNF and lipopolysaccharides (LPS), respectively. Consistently, Mlkl-deficient macrophages and mice exhibited normal interleukin-1β (IL-1β), IL-6, and TNF production after LPS treatment. Mlkl deficiency protects mice from cerulean-induced acute pancreatitis, a necrosis-related disease, but has no effect on polymicrobial septic shock-induced animal death. Our results provide genetic evidence for the role of Mlkl in necroptosis.

Interleukin-1β Induces a Reactive Astroglial Phenotype via Deactivation of the Rho GTPase–Rock Axis

The cytokine interleukin-1β (IL-1β) is critical to the formation of an astrocytic scar after CNS injury, but the mechanisms by which it induces a reactive phenotype remain unresolved. Here, we show that IL-1β regulates the phenotype of astrocytes via deactivation of the Rho GTPase–Rho kinase (ROCK) pathway, which governs cellular morphology and migration via effects on F-actin and its interactions with focal adhesions, nonmuscle myosin, and microvillar adapter proteins of the ezrin–radixin–moesin (ERM) family. We found that IL-1β induced cortical reorganization of F-actin and dephosphorylation of focal adhesion kinase, myosin light chain 2, and myosin phosphatase targeting subunit 1 in primary human astrocytes, and that all of these effects were mimicked by Rho-ROCK pathway blockade. We also found that IL-1β conversely potentiated ERM phosphorylation, and that this effect was mediated via a Rho–ROCK-independent mechanism. Next, we used a rhotekin pulldown assay to confirm directly that IL-1β deactivates Rho, and further demonstrated that a constitutively active Rho construct rescued astrocytes from developing an IL-1β-induced reactive phenotype. These data implicate cytokine regulation of the Rho–ROCK pathway in the generation of a reactive astrogliosis, and we suggest that interventions targeted at this level may facilitate manipulation of the glial scar in inflammatory disorders of the human CNS.

The Nore1B/Mst1 complex restrains antigen receptor-induced proliferation of naïve T cells

The Mst1 and Mst2 protein kinases are the mammalian homologs of hippo, a major inhibitor of cell proliferation in Drosophila. Mst1 is most abundant in lymphoid tissues. Mice lacking Mst1 exhibit markedly reduced levels of the Mst1 regulatory protein Nore1B/RAPL in lymphoid cells, whereas Mst2 abundance is unaltered. Mst1-null mice exhibit normal T cell development but low numbers of mature naïve T cells with relatively normal numbers of effector/memory T cells. In vitro, the Mst1-deficient naïve T cells exhibit markedly greater proliferation in response to stimulation of the T cell receptor whereas the proliferative responses of the Mst1-null effector/memory T cell cohort is similar to wild type. Thus, elimination of Mst1 removes a barrier to the activation and proliferative response of naïve T cells. The levels of Mst1 and Nore1B/RAPL in wild-type effector/memory T cells are approximately 10% those seen in wild-type naïve T cells, which may contribute to the enhanced proliferative responses of the former. Freshly isolated Mst1-null T cells exhibit high rates of ongoing apoptosis, a likely basis for their low numbers in vivo; they also exhibit defective clustering of LFA-1, as previously observed for Nore1B/RAPL-deficient T cells. Among known Mst1 substrates, only the phosphorylation of the cell cycle inhibitory proteins MOBKL1A/B is lost entirely in TCR-stimulated, Mst1-deficient T cells. Mst1/2-catalyzed MOBKL1A/B phosphorylation slows proliferation and is therefore a likely contributor to the anti-proliferative action of Mst1 in naïve T cells. The Nore1B/RAPL-Mst1 complex is a negative regulator of naïve T cell proliferation.

Exacerbation of experimental autoimmune encephalomyelitis in P2X7R-/- mice: evidence for loss of apoptotic activity in lymphocytes.

The purinergic receptor P2X7R is a nucleotide-gated ion channel that has been proposed to function as a major regulator of inflammation. In this study we examined the role of this receptor in regulating inflammation in the CNS by determining the effects of the loss of this receptor (P2X7R−/−) on experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. We show here that P2X7R−/− mice developed more severe clinical and pathological expression of EAE than wild type (WT) controls and that spleen and lymph node cells from P2X7R−/− mice proliferated more vigorously to Ag in vitro. Bone marrow (BM) radiation chimeras revealed that enhanced susceptibility to EAE was detected in chimeric mice of WT host engrafted with P2X7R−/− BM cells, indicating that the genotype of the BM cells regulated disease susceptibility. Coculture of P2X7R−/− macrophages with WT lymphocytes and vice versa showed that enhanced proliferative activity resided within the P2X7R−/− lymphocyte population and correlated with reduced levels of IFN-γ and NO and apoptosis of lymphocytes. mRNA and protein for IFN-γ were also significantly reduced in the CNS of P2X7R−/− mice with EAE. FACS analysis of cells isolated from the CNS showed significantly fewer annexin V/propidium iodide-positive lymphocytes in the CNS of P2X7R−/− mice early in the disease, and TUNEL staining of inflamed CNS tissues supported this result. From these data we conclude that enhanced susceptibility of P2X7R−/− mice to EAE reflects a loss of apoptotic activity in lymphocytes, supporting an important role for this receptor in lymphocyte homeostasis.

Regulation of immune response by P2X7 receptor

The P2X7 receptor is an ATP-gated cation channel that is widely expressed in cells of the immune system. Signal transduction is accompanied by fast influx of Ca2+ and Na+, and efflux of K+. This receptor differs from other members of the P2X family in its relatively low affinity for ATP, the presence of a long C-terminal region that contains several protein-protein interaction motifs, and the activation of two membrane conductance states following receptor ligation. In the immune system, this receptor has been implicated in the processing and release of cytokines such as IL-1 beta, and in the initiation of cell death via both apoptotic and necrotic pathways. As such, it has been proposed to function as a major regulator of inflammation. Consistent with this hypothesis, inactivation of this receptor in mice modulates disease pathogenesis in several animal models of inflammatory and autoimmune diseases. Loss-of-function polymorphisms have also been noted in the human population, and there is accumulating evidence that these polymorphisms are linked to certain diseases. In this article, we review the current status of research in this field, with particular emphasis on the signaling pathways activated by this receptor, the mechanisms involved in the initiation of cell death, and associations with disease states in mice and humans.

The Ets Transcription Factor GABP Is a Component of the Hippo Pathway Essential for Growth and Antioxidant Defense

The transcriptional coactivator Yes-associated protein (YAP) plays an important role in organ-size control and tumorigenesis. However, how Yap gene expression is regulated remains unknown. This study shows that the Ets family member GABP binds to the Yap promoter and activates YAP transcription. The depletion of GABP downregulates YAP, resulting in a G1/S cell-cycle block and increased cell death, both of which are substantially rescued by reconstituting YAP. GABP can be inactivated by oxidative mechanisms, and acetaminophen-induced glutathione depletion inhibits GABP transcriptional activity and depletes YAP. In contrast, activating YAP by deleting Mst1/Mst2 strongly protects against acetaminophen-induced liver injury. Similar to its effects on YAP, Hippo signaling inhibits GABP transcriptional activity through several mechanisms. In human liver cancers, enhanced YAP expression is correlated with increased nuclear expression of GABP. Therefore, we conclude that GABP is an activator of Yap gene expression and a potential therapeutic target for cancers driven by YAP.

Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity

Mitochondria need to be juxtaposed to phagosomes for the synergistic production of ample reactive oxygen species (ROS) in phagocytes to kill pathogens. However, how phagosomes transmit signals to recruit mitochondria has remained unclear. Here we found that the kinases Mst1 and Mst2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activated the GTPase Rac to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for the recruitment of mitochondria to phagosomes. Inactive forms of Rac, including the human Rac2D57N mutant, disrupted the TRAF6-ECSIT complex by sequestering TRAF6 and substantially diminished ROS production and enhanced susceptibility to bacterial infection. Our findings demonstrate that the TLR-Mst1-Mst2-Rac signaling axis is critical for effective phagosome-mitochondrion function and bactericidal activity.Summary Mitochondria need to be juxtaposted to phagosomes to synergistically produce ample reactive oxygen species (ROS) in phagocytes for pathogens killing. However, how phagosomes transmit signal to recruit mitochondria remains unclear. Here, we report that the kinases Mst1 and Mst2 function to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activate Rac GTPase to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for mitochondrial recruitment to phagosomes. Inactive forms of Rac, including the human Rac2D57N mutant, disrupt the TRAF6-ECSIT complex by sequestering TRAF6, and severely dampen ROS production and greatly increase susceptibility to bacterial infection. These findings demonstrate the TLR-Mst1-Mst2-Rac signalling axis to be critical for effective phagosome-mitochondrion function and bactericidal activity.

Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Inhibits Proximal TCR Signaling by Targeting ZAP-70

The long cytoplasmic tail (CT) isoforms of carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1) are expressed on activated human T cells and possess two ITIM motifs in the CT. These isoforms of CEACAM1 are inhibitory for T cell responses initiated by the TCR/CD3 complex with the inhibition dependent upon the ITIMs of CEACAM1 and Src homology 2 domain-containing phosphatase 1 (SHP-1). However, the mechanism by which this inhibition occurs in T cells is unknown. We demonstrate here that the Src family kinase, Lck, and the ability of CEACAM1 to bind homophilically are required for the ITIM phosphorylation of CEACAM1 that is a prerequisite for CEACAM1 association with SHP-1. We further show that CEACAM1 associates with and recruits SHP-1 to the TCR/CD3 complex leading to decreased phosphorylation of CD3-ζ and ZAP-70 and consequently decreased activation of the elements downstream of ZAP-70. This is physiologically relevant because extinction of SHP-1 expression or blockade of homophilic binding by CEACAM1 using a Fab that specifically recognizes the homophilic binding region of human CEACAM1 increases the cytolytic function initiated by the TCR/CD3 complex. These studies show that long CT isoforms of CEACAM1 orchestrate an inhibitory program that abrogates extremely proximal events downstream of the TCR/CD3 complex by focusing on the activation of ZAP-70.

Pharmacological targeting of kinases MST1 and MST2 augments tissue repair and regeneration

Manipulation of the Hippo signaling pathway with a reversible and selective small-molecule inhibitor of Hippo kinase MST1/2 provides a therapeutic option for tissue injury and repair. Drug-induced regeneration Popping a pill to repair an organ may eventually become reality. Turning away from conventional scaffolds, materials, and cell-based regenerative medicine strategies, Fan and colleagues sought a small molecule that could specifically target a critical signaling molecule in the Hippo pathway. Loss of kinases in this pathway, MST1/2, increases cell proliferation during development; thus, the authors hypothesized that inhibiting their activity in mature organs could help repair any damage. They discovered a drug, XMU-MP-1, that blocked MST1/2 activity and found that it promoted liver repair and regeneration in four different mouse models of acute and chronic injuries, including acetaminophen-induced injury, which is a common cause of liver failure worldwide. Such a pharmacological strategy could make tissue regeneration easier for many, compared to complex biomaterial and cell therapies. Tissue repair and regenerative medicine address the important medical needs to replace damaged tissue with functional tissue. Most regenerative medicine strategies have focused on delivering biomaterials and cells, yet there is the untapped potential for drug-induced regeneration with good specificity and safety profiles. The Hippo pathway is a key regulator of organ size and regeneration by inhibiting cell proliferation and promoting apoptosis. Kinases MST1 and MST2 (MST1/2), the mammalian Hippo orthologs, are central components of this pathway and are, therefore, strong target candidates for pharmacologically induced tissue regeneration. We report the discovery of a reversible and selective MST1/2 inhibitor, 4-((5,10-dimethyl-6-oxo-6,10-dihydro-5H-pyrimido[5,4-b]thieno[3,2-e][1,4]diazepin-2-yl)amino)benzenesulfonamide (XMU-MP-1), using an enzyme-linked immunosorbent assay–based high-throughput biochemical assay. The cocrystal structure and the structure-activity relationship confirmed that XMU-MP-1 is on-target to MST1/2. XMU-MP-1 blocked MST1/2 kinase activities, thereby activating the downstream effector Yes-associated protein and promoting cell growth. XMU-MP-1 displayed excellent in vivo pharmacokinetics and was able to augment mouse intestinal repair, as well as liver repair and regeneration, in both acute and chronic liver injury mouse models at a dose of 1 to 3 mg/kg via intraperitoneal injection. XMU-MP-1 treatment exhibited substantially greater repopulation rate of human hepatocytes in the Fah-deficient mouse model than in the vehicle-treated control, indicating that XMU-MP-1 treatment might facilitate human liver regeneration. Thus, the pharmacological modulation of MST1/2 kinase activities provides a novel approach to potentiate tissue repair and regeneration, with XMU-MP-1 as the first lead for the development of targeted regenerative therapeutics.

Transcriptional Profiling of γδ T Cells Identifies a Role for Vitamin D in the Immunoregulation of the Vγ9Vδ2 Response to Phosphate-Containing Ligands

Vitamin D is a steroid hormone that, in addition to its well-characterized role in calcium/phosphate metabolism, has been found to have regulatory properties for immune system function. The nuclear vitamin D receptor is widely expressed in tissues, but has also been shown to be regulated by hormones, growth factors, and cytokines. In this study we show that activation of human Vδ2Vγ9 T cells by nonpeptidic monoalkyl phosphates such as isopentenyl pyrophosphate leads to the up-regulation of the vitamin D receptor via a pathway that involves the classical isoforms of protein kinase C. We further show that this receptor is active by demonstrating that the ligand 1α,25-dihydroxyvitamin D3 (vitD3) significantly inhibits in a dose-dependent fashion phospholigand-induced γδ T cell expansion, IFN-γ production, and CD25 expression. We also show that vitD3 negatively regulates signaling via Akt and ERK and, at high concentrations, potentiates Ag-induced cell death. As such, these data provide further support for the immunoregulatory properties of vitamin D, and suggest that the ability of vitD3 to negatively regulate the proinflammatory activity of γδ T cells may contribute to the protection this vitamin affords against inflammatory and autoimmune disorders dependent upon Th1-type responses.