The synthesis of serine from glucose is a key metabolic pathway supporting cellular proliferation in healthy and malignant cells. Despite this, the role that this aspect of metabolism plays in germinal center biology and pathology is not known. Here, we performed a comprehensive characterization of the role of the serine synthesis pathway in germinal center B cells and lymphomas derived from these cells. We demonstrated that upregulation of a functional serine synthesis pathway is a metabolic hallmark of B-cell activation and the germinal center reaction. Inhibition of phosphoglycerate dehydrogenase (PHGDH), the first and rate limiting enzyme in this pathway, led to defective germinal formation and impaired high-affinity antibody production. In addition, overexpression of enzymes involved in serine synthesis was a characteristic of germinal center B-cell derived lymphomas, with high levels of expression being predictive of reduced overall survival in diffuse large B cell lymphoma. Inhibition of PHGDH induced apoptosis in lymphoma cells reducing disease progression. These findings establish PHGDH as a critical player in humoral immunity and a clinically relevant target in lymphoma.
Annalisa D'Avola, Nathalie Legrave, Mylène Tajan, Probir Chakravarty, Ryan L. Shearer, Hamish W. King, Katarina Kluckova, Eric C. Cheung, Andrew J. Clear, Arief S. Gunawan, Lingling Zhang, Louisa K. James, James I. MacRae, John G. Gribben, Dinis P. Calado, Karen H. Vousden, John C. Riches
T cell immunoglobulin mucin domain-containing protein 3 (Tim-3) negatively regulates innate and adaptive immunity in cancer. To identify the mechanisms of Tim-3 in cancer immunity, we evaluated the effects of Tim-3 blockade in human and mouse melanoma. Here, we show that human PD-1+Tim-3+ CD8+ tumor-infiltrating lymphocytes (TILs) upregulate phosphatidylserine (PS), a receptor for Tim-3, and acquire cell surface myeloid markers from antigen presenting cells (APCs) through transfer of membrane fragments called trogocytosis. Tim-3 blockade acted on Tim-3+ APCs in a PS-dependent fashion to disrupt the trogocytosis of activated tumor antigen-specific CD8+ T cells and PD-1+Tim-3+ CD8+ TILs isolated from melanoma patients. Tim-3 and PD-1 blockades cooperated to disrupt trogocytosis of CD8+ TILs in two melanoma mouse models, decreasing tumor burden and prolonging survival. Deleting Tim-3 in dendritic cells but not on CD8+ T cells impeded the trogocytosis of CD8+ TILs in vivo. Trogocytosed CD8+ T cells presented tumor peptide-major histocompatibility complexes and became the target of fratricide T cell killing, which was reversed by Tim-3 blockade. Our findings have uncovered a mechanism used by Tim-3 to limit antitumor immunity.
Ornella Pagliano, Robert M. Morrison, Joe-Marc Chauvin, Hridesh Banerjee, Diwakar Davar, Quanquan Ding, Tokiyoshi Tanegashima, Wentao Gao, Saranya Rani Chakka, Richelle DeBlasio, Ava Lowin, Kevin Kara, Mignane Ka, Bochra Zidi, Rada Amin, Itay Raphael, Shuowen Zhang, Simon C. Watkins, Cindy Sander, John M. Kirkwood, Marcus Bosenberg, Ana C. Anderson, Vijay K. Kuchroo, Lawrence P. Kane, Alan J. Korman, Arvind Rajpal, Sean M. West, Minhua Han, Christine Bee, Xiaodi Deng, Xiao Min Schebye, Pavel Strop, Hassane M. Zarour
Pregnancy is associated with substantial physiological changes of the heart, and disruptions in these processes can lead to peripartum-cardiomyopathy (PPCM). The molecular processes that cause physiological and pathological changes in the heart during pregnancy are not well characterized. Here, we show that mTORc1 was activated in pregnancy to facilitate cardiac enlargement that was reversed after delivery in mice. mTORc1 activation in pregnancy was negatively regulated by the mRNA-destabilizing-protein ZFP36L2 through its degradation of Mdm2 mRNA and P53 stabilization, leading to increased SESN2 and REDD1 expression. This pathway impeded uncontrolled cardiomyocytes hypertrophy during pregnancy, and mice with cardiac-specific Zfp36l2 deletion developed rapid cardiac dysfunction after delivery, while prenatal treatment of these mice with rapamycin improved post-partum cardiac function. Collectively, these data provide a novel pathway for the regulation of mTORc1 through mRNA stabilization of a P53 ubiquitin ligase. This pathway was critical for normal cardiac growth during pregnancy, and its reduction led to PPCM-like adverse remodeling in mice.
Hidemichi Kouzu, Yuki Tatekoshi, Hsiang-Chun Chang, Jason S. Shapiro, Warren A. McGee, Adam De Jesus, Issam Ben-Sahra, Zoltan Arany, Jonathan Leor, Chunlei Chen, Perry J. Blackshear, Hossein Ardehali
Pericyte-mediated capillary constriction decreases cerebral blood flow in stroke after an occluded artery is unblocked. The determinants of pericyte tone are poorly understood. We show that a small rise in cytoplasmic Ca2+ concentration ([Ca2+]i) in pericytes activates chloride efflux through the Ca2+-gated anion channel TMEM16A, thus depolarizing the cell and opening voltage-gated calcium channels. This mechanism strongly amplifies the pericyte [Ca2+]i rise and capillary constriction evoked by contractile agonists and ischemia. In a rodent stroke model, TMEM16A inhibition slows the ischemia-evoked pericyte [Ca2+]i rise, capillary constriction and pericyte death, reduces neutrophil stalling and improves cerebrovascular reperfusion. Genetic analysis implicates altered TMEM16A expression in poor patient recovery from ischemic stroke. Thus, pericyte TMEM16A is a crucial regulator of cerebral capillary function, and a potential therapeutic target for stroke and possibly other disorders of impaired microvascular flow, such as Alzheimer’s disease and vascular dementia.
Nils Korte, Zeki Ilkan, Claire L. Pearson, Thomas Pfeiffer, Prabhav Singhal, Jason R. Rock, Huma Sethi, Dipender Gill, David Attwell, Paolo Tammaro
Proliferation of latently-infected CD4+ T cells with replication-competent proviruses is an important mechanism contributing to HIV persistence during antiretroviral therapy (ART). One approach to target this latent cell expansion is to inhibit the mechanistic target of rapamycin (mTOR), a regulatory kinase involved with cell growth, metabolism and proliferation. Here we determined the effects of chronic mTOR inhibition with rapamycin +/- T cell activation in SIV-infected rhesus macaques (RM) on ART. Rapamycin perturbed the expression of multiple genes and signaling pathways important for cellular proliferation and substantially decreased the frequencies of proliferating CD4+ memory T cells (TM) in blood and tissues. However, levels of cell-associated SIV DNA and SIV RNA were not markedly different between rapamycin-treated RM relative to controls during ART. T cell activation with an anti-CD3LALA antibody induced increases in SIV RNA in plasma of RM on rapamycin, consistent with SIV production. However, upon ART cessation, both rapamycin + CD3LALA-treated and control-treated RM rebounded in less than 12 days, with no difference in the time to viral rebound or post-ART viral load set points. These results indicate that while rapamycin can decrease the proliferation of CD4+ TM, chronic mTOR inhibition alone or in combination with T cell activation, was not sufficient to disrupt the stability of the SIV reservoir.
Benjamin D. Varco-Merth, William Brantley, Alejandra Marenco, Derick D. Duell, Devin N. Fachko, Brian Richardson, Kathleen Busman-Sahay, Danica Shao, Walter Flores, Kathleen Engelman, Yoshinori Fukazawa, Scott W. Wong, Rebecca L. Skalsky, Jeremy Smedley, Michael K Axthelm, Jeffrey D. Lifson, Jacob D. Estes, Paul T. Edlefsen, Louis Picker, Cheryl M.A. Cameron, Timothy J. Henrich, Afam A. Okoye
The loss function of cerebral cavernous malformation (CCM) genes leads to most CCM lesions characterized by enlarged leaking vascular lesions in the brain. Although we previously showed that NOGOB receptor (NGBR) knockout in endothelial cells (ECs) results in cerebrovascular lesions in the mouse embryo, the molecular mechanism by which NGBR regulates CCM1/2 expression has not been elucidated. Here, we show that temporal genetic depletion of Ngbr in ECs at both postnatal and adult stages results in CCM1/2 expression deficiency and cerebrovascular lesions such as enlarged vessels, blood-brain barrier (BBB) hyperpermeability, and cerebral hemorrhage. To reveal the molecular mechanism, we used RNA-seq analysis to examine changes in the transcriptome. Surprisingly, we found that acetyltransferase HBO1 and histone acetylation were downregulated in NGBR deficient ECs. The mechanistic studies elucidated that NGBR is required for maintaining the expression of CCM1/2 in ECs via HBO1-mediated histone acetylation. ChIP-qPCR data further demonstrated that loss of NGBR impairs the binding of both HBO1 and acetylated H4K5/K12 on the promotor of CCM1 and CCM2 genes. Our findings on epigenetic regulation of CCM1 and CCM2 that modulated by NGBR and HBO1-mediated histone H4 acetylation provide a perspective on the pathogenesis of sporadic CCMs.
Zhi Fang, Xiaoran Sun, Xiang Wang, Ji Ma, Thomas Palaia, Ujala Rana, Benjamin Miao, Louis Ragolia, Wenquan Hu, Qing Robert Miao
Gastric carcinogenesis is mediated by complex interactions among Helicobacter pylori, host, and environmental factors. We now demonstrate that H. pylori augments gastric injury in INS-GAS mice under iron deficient conditions. Mechanistically, these phenotypes were not driven by alterations in the gastric microbiota; however, discovery-based and targeted metabolomics revealed that bile acids were significantly altered in H. pylori-infected mice with iron deficiency, with significant upregulation of deoxycholic acid (DCA), a carcinogenic bile acid. Severity of gastric injury was further augmented when H. pylori-infected mice were treated with DCA, and, in vitro, DCA increased translocation of the H. pylori oncoprotein CagA into host cells. Conversely, bile acid sequestration attenuated H. pylori-induced injury under conditions of iron deficiency. To translate these findings into human populations, the association between bile acid-sequestrant use and gastric cancer risk was evaluated in a large human cohort. Among 416,885 individuals, a significant dose-dependent reduction in risk was associated with cumulative bile acid-sequestrant use. Further, expression of the bile acid receptor TGR5 paralleled the severity of carcinogenic lesions in humans. These data demonstrate that increased H. pylori-induced injury within the context of iron deficiency is tightly linked to altered bile acid metabolism, which may promote gastric carcinogenesis.
Jennifer M. Noto, M. Blanca Piazuelo, Shailja C. Shah, Judith Romero-Gallo, Jessica L. Hart, Chao Di, James D. Carmichael, Alberto G. Delgado, Alese E. Halvorson, Robert A. Greevy, Jr., Lydia E. Wroblewski, Ayushi Sharma, Annabelle B. Newton, Margaret M. Allaman, Keith T. Wilson, M. Kay Washington, M. Wade Calcutt, Kevin L. Schey, Bethany P. Cummings, Charles R. Flynn, Joseph P. Zackular, Richard M. Peek, Jr.
A disequilibrium between immunosuppressive regulatory T cells (Tregs) and inflammatory interleukin (IL)-17-producing Th17 cells is a hallmark of autoimmune diseases, including multiple sclerosis (MS). However, the molecular mechanisms underlying Treg and Th17 imbalance in central nervous system (CNS) autoimmunity remain largely unclear. Identifying factors which drive this imbalance is of high clinical interest. Here, we report a major disease-promoting role for microRNA-92a (miR-92a) in CNS autoimmunity. MiR-92a was elevated in experimental autoimmune encephalomyelitis (EAE), and its loss attenuated EAE. Mechanistically, miR-92a mediated EAE susceptibility in a T cell-intrinsic manner by restricting Treg induction and suppressive capacity, while supporting Th17 responses, by directly repressing the transcription factor, Foxo1. Although miR-92a did not directly alter Th1 differentiation, it appeared to indirectly promote Th1 cells by inhibiting Treg responses. Correspondingly, miR-92a inhibitor therapy ameliorated EAE by concomitantly boosting Treg cell responses and dampening inflammatory T cell responses. Analogous to mice, miR-92a was elevated in MS patient CD4+ T cells, and miR-92a silencing in patient T cells promoted Treg development whereas it limited Th17 differentiation. Together, our results demonstrate that miR-92a drives CNS autoimmunity by sustaining the Treg/Th17 imbalance and implicate miR-92a as a potential therapeutic target for MS.
Mai Fujiwara, Radhika Raheja, Lucien P. Garo, Amrendra K. Ajay, Ryoko Kadowaki-Saga, Sukrut H. Karandikar, Galina Gabriely, Rajesh Krishnan, Vanessa Beynon, Anu Paul, Amee Patel, Shrishti Saxena, Dan Hu, Brian C. Healy, Tanuja Chitnis, Roopali Gandhi, Howard L. Weiner, Gopal Murugaiyan
Whereas immobility is a common cause of muscle atrophy, the mechanism underlying this causality is unclear. We here show that KLF15 and IL-6 are up-regulated in skeletal muscle of limb-immobilized mice and that mice with KLF15 deficiency in skeletal muscle or with systemic IL-6 deficiency are protected from immobility-induced muscle atrophy. A newly developed Ca2+ bioimaging revealed that the Ca2+ concentration ([Ca2+]i) of skeletal muscle is reduced to below the basal level by immobilization, which is associated with the down-regulation of Piezo1. Acute disruption of Piezo1 in skeletal muscle induced Klf15 and Il6 expression as well as muscle atrophy, which was prevented by antibodies to IL-6. A role for the Piezo1/KLF15/IL-6 axis in immobility-induced muscle atrophy was validated by human samples. Our results thus uncover a paradigm for Ca2+ signaling in that a decrease in [Ca2+]i from the basal level triggers a defined biological event.
Yu Hirata, Kazuhiro Nomura, Daisuke Kato, Yoshihisa Tachibana, Takahiro Niikura, Kana Uchiyama, Tetsuya Hosooka, Tomoaki Fukui, Keisuke Oe, Ryosuke Kuroda, Yuji Hara, Takahiro Adachi, Koji Shibasaki, Hiroaki Wake, Wataru Ogawa
The striatin-interacting phosphatase and kinase (STRIPAK) complexes integrate extracellular stimuli to result in intracellular activities. Previously, we discovered STRIPAK to be a key machinery responsible for loss of the Hippo tumor suppressor signal in cancer. Here, we identified the Hippo-STRIPAK complex to be an essential player for the control of DNA double-strand break (DSB) repair and genomic stability. Specifically, the MST1/2 kinases were found, independent of the classical Hippo signaling, to directly phosphorylate ZMYND8 and hence result in suppression of DNA repair in the nucleus. In response to genotoxic stress, the cGAS-STING pathway was determined to relay nuclear DNA damage signals to the dynamic assembly of Hippo-STRIPAK via a TBK1-induced structural stabilization of the SIKE1-SLMAP arm. As such, STRIPAK-mediated MST1/2 inactivation was found to increase the DSB repair capacity of cancer cells and to endow these cells with resistance to radio/chemotherapy and PARP inhibition. Importantly, targeting the STRIPAK assembly with each of three distinct peptide inhibitors efficiently recovered the kinase activity of MST1/2 to suppress DNA repair and re-sensitize cancer cells to PARPi in both animal and patient-derived tumor models. Overall, our findings not only uncovered a previously unrecognized role for STRIPAK in modulating DSB repair, but also provided translational implications of co-targeting STRIPAK and PARP for a new type of synthetic lethality anti-cancer therapy.
Liwei An, Zhifa Cao, Pingping Nie, Hui Zhang, Zhenzhu Tong, Fan Chen, Yang Tang, Yi Han, Wenjia Wang, Zhangting Zhao, Qingya Zhao, Yuqin Yang, Yuanzhi Xu, Gemin Fang, Lei Shi, Huixiong Xu, Haiqing Ma, Shi Jiao, Zhaocai Zhou
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