Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond–Blackfan anaemia and 5q- syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anaemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor (RNH1) is an ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 to E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knock down in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.
Vijaykumar Chennupati, Diogo F.T. Veiga, Kendle M. Maslowski, Nicola Andina, Aubry Tardivel, Eric Chi-Wang Yu, Martina Stilinovic, Cedric Simillion, Michel A. Duchosal, Manfredo Quadroni, Irene Roberts, Vijay G. Sankaran, H. Robson MacDonald, Nicolas Fasel, Anne Angelillo-Scherrer, Pascal Schneider, Trang Hoang, Ramanjaneyulu Allam
Nonalcoholic fatty liver disease (NAFLD) is a major risk factor for liver cancer; therefore, its prevention is an important clinical goal. Ablation of phosphatase and tensin homolog (PTEN) or the protein kinase Hippo signaling pathway induces liver cancer via activation of AKT or the transcriptional regulators YAP/TAZ, respectively; however, the potential for crosstalk between the PTEN/AKT and Hippo/YAP/TAZ pathways in liver tumorigenesis has thus far remained unclear. Here, we have shown that deletion of both PTEN and SAV1 in the liver accelerates the development of NAFLD and liver cancer in mice. At the molecular level, activation of YAP/TAZ in the liver of Pten–/– Sav1–/– mice amplified AKT signaling through the upregulation of insulin receptor substrate 2 (IRS2) expression. Both ablation of YAP/TAZ and activation of the Hippo pathway could rescue these phenotypes. A high level of YAP/ TAZ expression was associated with a high level of IRS2 expression in human hepatocellular carcinoma (HCC). Moreover, treatment with the AKT inhibitor MK-2206 or knockout of IRS2 by AAV-Cas9 successfully repressed liver tumorigenesis in Pten–/– Sav1–/– mice. Thus, our findings suggest that Hippo signaling interacts with AKT signaling by regulating IRS2 expression to prevent NAFLD and liver cancer progression and provide evidence that impaired crosstalk between these 2 pathways accelerates NAFLD and liver cancer.
Sun-Hye Jeong, Han-Byul Kim, Min-Chul Kim, Ji-min Lee, Jae Ho Lee, Jeong-Hwan Kim, Jin-Woo Kim, Woong-Yang Park, Seon-Young Kim, Jae Bum Kim, Haeryoung Kim, Jin-Man Kim, Hueng-Sik Choi, Dae-Sik Lim
Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18–/– AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18–/– mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.
Beiyun Zhou, Per Flodby, Jiao Luo, Dan R. Castillo, Yixin Liu, Fa-Xing Yu, Alicia McConnell, Bino Varghese, Guanglei Li, Nyam-Osor Chimge, Mitsuhiro Sunohara, Michael N. Koss, Wafaa Elatre, Peter Conti, Janice M. Liebler, Chenchen Yang, Crystal N. Marconett, Ite A. Laird-Offringa, Parviz Minoo, Kunliang Guan, Barry R. Stripp, Edward D. Crandall, Zea Borok
The mechanisms that mediate durable protection against Staphylococcus aureus skin reinfections are unclear, as recurrences are common despite high antibody titers and memory T cells. Here, we developed a mouse model of S. aureus skin reinfection to investigate protective memory responses. In contrast with WT mice, IL-1β–deficient mice exhibited poor neutrophil recruitment and bacterial clearance during primary infection that was rescued during secondary S. aureus challenge. The γδ T cells from skin-draining LNs utilized compensatory T cell–intrinsic TLR2/MyD88 signaling to mediate rescue by trafficking and producing TNF and IFN-γ, which restored neutrophil recruitment and promoted bacterial clearance. RNA-sequencing (RNA-seq) of the LNs revealed a clonotypic S. aureus–induced γδ T cell expansion with a complementarity-determining region 3 (CDR3) aa sequence identical to that of invariant Vγ5+ dendritic epidermal T cells. However, this T cell receptor γ (TRG) aa sequence of the dominant CDR3 sequence was generated from multiple gene rearrangements of TRGV5 and TRGV6, indicating clonotypic expansion. TNF- and IFN-γ–producing γδ T cells were also expanded in peripheral blood of IRAK4-deficient humans no longer predisposed to S. aureus skin infections. Thus, clonally expanded γδ T cells represent a mechanism for long-lasting immunity against recurrent S. aureus skin infections.
Carly A. Dillen, Bret L. Pinsker, Alina I. Marusina, Alexander A. Merleev, Orly N. Farber, Haiyun Liu, Nathan K. Archer, Da B. Lee, Yu Wang, Roger V. Ortines, Steven K. Lee, Mark C. Marchitto, Shuting S. Cai, Alyssa G. Ashbaugh, Larissa S. May, Steven M. Holland, Alexandra F. Freeman, Loren G. Miller, Michael R. Yeaman, Scott I. Simon, Joshua D. Milner, Emanual Maverakis, Lloyd S. Miller
Tuberous sclerosis complex (TSC) is a dominantly inherited disease, caused by hyperactivation of the mTORC1 pathway and characterized by the development of hamartomas and benign tumors, also in the brain. Among the neurological manifestations associated with TSC, the tumor progression of static subependymal nodules (SENs) into subependymal giant cell astrocytomas (SEGAs) is one of the major causes of morbidity and shortened life expectancy. To date, mouse modeling has failed in reproducing these two lesions. Here we report that simultaneous hyperactivation of mTORC1 and Akt pathways by codeletion of Tsc1 and Pten, selectively in postnatal neural stem cells (pNSCs), is required for the formation of bona fide SENs and SEGAs. Notably, both lesions closely recapitulate the pathognomonic morphological and molecular features of the corresponding human abnormalities. The establishment of long-term expanding pNSC lines from mouse SENs and SEGAs made possible the identification of mTORC2 as one of the mediators conferring tumorigenic potential to SEGA pNSCs. Of note, in spite of concurrent Akt hyperactivation in mouse brain lesions, single mTOR inhibition by rapamycin was sufficient to strongly impair mouse SEGA growth. This study provides the first evidence that, concomitant with mTORC1 hyperactivation, sustained activation of Akt and mTORC2 in pNSCs is a mandatory step for the induction of SENs and SEGAs and, at the same time, makes available an unprecedented NSC-based in vivo/in vitro model to be exploited for identifying actionable targets in TSC.
Paola Zordan, Manuela Cominelli, Federica Cascino, Elisa Tratta, Pietro L. Poliani, Rossella Galli
Myc activation is a primary oncogenic event in many human cancers; however, these transcription factors are difficult to inhibit pharmacologically, suggesting that Myc-dependent downstream effectors may be more tractable therapeutic targets. Here we show that Myc overexpression induces endoplasmic reticulum (ER) stress and engages the IRE1α-XBP1 pathway through multiple molecular mechanisms in a variety of c-Myc- and N-Myc-dependent cancers. In particular, Myc-overexpressing cells require IRE1α-XBP1 signaling for sustained growth and survival in vitro and in vivo, dependent on elevated stearoyl-CoA-desaturase 1 (SCD1) activity. Pharmacological and genetic XBP1 inhibition induces Myc-dependent apoptosis, which is alleviated by exogenous unsaturated fatty acids. Of note, SCD1 inhibition phenocopies IRE1α RNase activity suppression in vivo. Furthermore, IRE1α inhibition enhances the cytotoxic effects of standard chemotherapy drugs used to treat c-Myc-overexpressing Burkitt’s lymphoma, suggesting that inhibiting the IRE1α-XBP1 pathway is a useful general strategy for treatment of Myc-driven cancers.
Hong Xie, Chih-Hang Anthony Tang, Jun H. Song, Anthony Mancuso, Juan R. Del Valle, Jin Cao, Yan Xiang, Chi V. Dang, Roy Lan, Danielle J. Sanchez, Brian Keith, Chih-Chi Andrew Hu, M. Celeste Simon
Profound hyperphagia is a major disabling feature of Prader-Willi syndrome (PWS). Characterization of the mechanisms that underlie PWS-associated hyperphagia has been slowed by the paucity of animal models with increased food intake or obesity. Mice with a microdeletion encompassing the Snord116 cluster of noncoding RNAs encoded within the Prader-Willi minimal deletion critical region have previously been reported to show growth retardation and hyperphagia. Here, consistent with previous reports, we observed growth retardation in Snord116+/–P mice with a congenital paternal Snord116 deletion. However, these mice neither displayed increased food intake nor had reduced hypothalamic expression of the proprotein convertase 1 gene PCSK1 or its upstream regulator NHLH2, which have recently been suggested to be key mediators of PWS pathogenesis. Specifically, we disrupted Snord116 expression in the mediobasal hypothalamus in Snord116fl mice via bilateral stereotaxic injections of a Cre-expressing adeno-associated virus (AAV). While the Cre-injected mice had no change in measured energy expenditure, they became hyperphagic between 9 and 10 weeks after injection, with a subset of animals developing marked obesity. In conclusion, we show that selective disruption of Snord116 expression in the mediobasal hypothalamus models the hyperphagia of PWS.
Joseph Polex-Wolf, Brian Y.H. Lam, Rachel Larder, John Tadross, Debra Rimmington, Fàtima Bosch, Verónica Jiménez Cenzano, Eduard Ayuso, Marcella K.L. Ma, Kara Rainbow, Anthony P. Coll, Stephen O’Rahilly, Giles S.H. Yeo
Recent findings have highlighted the role of microglia in orchestrating normal development and refining neural network connectivity in the healthy CNS. Microglia are not only vital cells in maintaining CNS homeostasis, but also respond to injury, infection, and disease by undergoing proliferation and changes in transcription and morphology. A better understanding of the specific role of microglia in responding to viral infection is complicated by the presence of nonmicroglial myeloid cells with potentially overlapping function in the healthy brain and by the rapid infiltration of hematopoietic myeloid cells into the brain in diseased states. Here, we used an inhibitor of colony-stimulating factor 1 receptor (CSF1R) that depletes microglia to examine the specific roles of microglia in response to infection with the mouse hepatitis virus (MHV), a neurotropic coronavirus. Our results show that microglia were required during the early days after infection to limit MHV replication and subsequent morbidity and lethality. Additionally, microglia depletion resulted in ineffective T cell responses. These results reveal nonredundant, critical roles for microglia in the early innate and virus-specific T cell responses and for subsequent host protection from viral encephalitis.
D. Lori Wheeler, Alan Sariol, David K. Meyerholz, Stanley Perlman
Acute graft-versus-host disease (GVHD) represents a severe, T cell–driven inflammatory complication following allogeneic hematopoietic cell transplantation (allo-HCT). GVHD often affects the intestine and is associated with a poor prognosis. Although frequently detectable, proinflammatory mechanisms exerted by intestinal tissue–infiltrating Th cell subsets remain to be fully elucidated. Here, we show that the Th17-defining transcription factor basic leucine zipper transcription factor ATF-like (BATF) was strongly regulated across human and mouse intestinal GVHD tissues. Studies in complete MHC-mismatched and minor histocompatibility–mismatched (miHA-mismatched) GVHD models revealed that BATF-expressing T cells were functionally indispensable for intestinal GVHD manifestation. Mechanistically, BATF controlled the formation of colon-infiltrating, IL-7 receptor–positive (IL-7R+), granulocyte-macrophage colony-stimulating factor–positive (GM-CSF+), donor T effector memory (Tem) cells. This T cell subset was sufficient to promote intestinal GVHD, while its occurrence was largely dependent on T cell–intrinsic BATF expression, required IL-7–IL-7R interaction, and was enhanced by GM-CSF. Thus, this study identifies BATF-dependent pathogenic GM-CSF+ effector T cells as critical promoters of intestinal inflammation in GVHD and hence putatively provides mechanistic insight into inflammatory processes previously assumed to be selectively Th17 driven.
Evelyn Ullrich, Benjamin Abendroth, Johanna Rothamer, Carina Huber, Maike Büttner-Herold, Vera Buchele, Tina Vogler, Thomas Longerich, Sebastian Zundler, Simon Völkl, Andreas Beilhack, Stefan Rose-John, Stefan Wirtz, Georg F. Weber, Sakhila Ghimire, Marina Kreutz, Ernst Holler, Andreas Mackensen, Markus F. Neurath, Kai Hildner
Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12–/–) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor–mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of αMβ2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12–/– mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12–/– hosts was sufficient to correct the neutrophil migration defect in F12–/– mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.
Evi X. Stavrou, Chao Fang, Kara L. Bane, Andy T. Long, Clément Naudin, Erdem Kucukal, Agharnan Gandhi, Adina Brett-Morris, Michele M. Mumaw, Sudeh Izadmehr, Alona Merkulova, Cindy C. Reynolds, Omar Alhalabi, Lalitha Nayak, Wen-Mei Yu, Cheng-Kui Qu, Howard J. Meyerson, George R. Dubyak, Umut A. Gurkan, Marvin T. Nieman, Anirban Sen Gupta, Thomas Renné, Alvin H. Schmaier