A growing body of evidence supports an important role for the endocannabinoid system as a regulator of appetite, body weight, and systemic metabolism, which is overactive in obesity and type 2 diabetes. While initial attempts to target this system using the cannabinoid receptor inverse agonist rimonabant were successful in producing modest weight loss and improving obesity-related metabolic complications in humans, adverse central nervous system side effects precluded introduction of this drug into clinical practice. However, new data, presented by Tam and colleagues in this issue of the JCI, demonstrate that selective blockade of peripheral cannabinoid receptors may be a novel successful therapeutic approach.
Basophils are the least abundant granulocytes found in the circulation. Until recently, their functions were poorly understood. In the past few years, the list of basophil functions in the context of immunity has dramatically increased. Thus, the need for basophil-deficient animal models to confirm these findings is imperative. In this issue of the JCI, Wada and colleagues introduce the first mouse model in which basophils are conditionally ablated in vivo. Using this model, they then uncover a nonredundant role for basophils in acquired immunity against tick infection.
The use of niacin to improve plasma lipid levels and reduce risk of myocardial infarction is limited by noxious skin effects that result from stimulation of G protein–coupled receptor 109A (GPR109A) in skin immune cells. Niacin causes vasodilation, manifest as rubor (redness) of the head and neck, providing a visible sign associated with other, more bothersome skin complaints. The working theory is that niacin provokes Langerhans cells to produce prostaglandin D2 (PGD2), stimulating vascular DP1 receptors to cause vasodilation. In this issue of the JCI, Hanson and colleagues raise a serious challenge to this paradigm in showing that the major player in vasodilation is the keratinocyte, which produces PGE2, stimulating EP2/4 receptors, shifting the role of the Langerhans/PGD2/DP1 pathway to that of an accomplice. They also show that the antipsoriasis drug monomethyl fumarate, itself a GPR109A agonist, provokes vasodilation through the same cells. These efforts bring us one step closer to solving a key limitation of an important cardioprotective drug and reveal that the skin response to niacin is much more complicated than previously thought.
Richard L. Dunbar, Joel M. Gelfand
Targeted cancer therapeutics can be effective when patients are preselected to maximize the chance of response. Increasingly, molecular markers such as oncogenic DNA mutations are being exploited to help guide patient preselection. These DNA lesions can predict for either a positive or negative response to a given drug. Finding such predictive biomarkers is an ongoing challenge. New work by Di Nicolantonio and colleagues in this issue of the JCI demonstrates that PI3K catalytic α subunit (PIK3CA) mutations can sensitize cancer cells to the mammalian target of rapamycin (mTOR) inhibitor everolimus. In addition, they show that the concurrent presence of PIK3CA mutations and mutations in either KRAS or BRAF predict for resistance to this drug. These data suggest that mTOR inhibitors currently in use will be ineffective against cancers that have a mutation in either KRAS or BRAF despite having PI3K/AKT/mTOR pathway activation.
Morassa Mohseni, Ben Ho Park
Class 3 semaphorins (Sema3s) regulate axon guidance, angiogenesis, tumor growth, and tumor metastasis. Neuropilins (NRPs; NRP1 and NRP2) are the cell surface receptors for the Sema3s. However, to signal, interaction of Sema3s and NRPs with plexins is obligatory. In this issue of the JCI, Casazza and colleagues report data that challenge the conventional wisdom about the role of Sema3s in tumor metastasis. As a rule, Sema3B and Sema3F, for example, are inhibitors of tumor angiogenesis, progression, and metastasis. However, Casazza et al. found that Sema3E inhibited tumor growth but atypically promoted invasiveness and metastasis. This metastatic potential was dependent on Plexin D1 expression but was independent of NRP expression. Of clinical importance, Sema3E and Plexin D1 were found to be upregulated in human colon cancer, liver metastasis, and melanoma progression.
Michael Klagsbrun, Akio Shimizu
Febrile seizures are a common childhood seizure disorder and a defining feature of genetic epilepsy with febrile seizures plus (GEFS+), a syndrome frequently associated with Na+ channel mutations. Here, we describe the creation of a knockin mouse heterozygous for the C121W mutation of the β1 Na+ channel accessory subunit seen in patients with GEFS+. Heterozygous mice with increased core temperature displayed behavioral arrest and were more susceptible to thermal challenge than wild-type mice. Wild-type β1 was most concentrated in the membrane of axon initial segments (AIS) of pyramidal neurons, while the β1(C121W) mutant subunit was excluded from AIS membranes. In addition, AIS function, an indicator of neuronal excitability, was substantially enhanced in hippocampal pyramidal neurons of the heterozygous mouse specifically at higher temperatures. Computational modeling predicted that this enhanced excitability was caused by hyperpolarized voltage activation of AIS Na+ channels. This heat-sensitive increased neuronal excitability presumably contributed to the heightened thermal seizure susceptibility and epileptiform discharges seen in patients and mice with β1(C121W) subunits. We therefore conclude that Na+ channel β1 subunits modulate AIS excitability and that epilepsy can arise if this modulation is impaired.
Verena C. Wimmer, Christopher A. Reid, Suzanne Mitchell, Kay L. Richards, Byron B. Scaf, Bryan T. Leaw, Elisa L. Hill, Michel Royeck, Marie-Therese Horstmann, Brett A. Cromer, Philip J. Davies, Ruwei Xu, Holger Lerche, Samuel F. Berkovic, Heinz Beck, Steven Petrou
Limb-girdle muscular dystrophy type 2A (LGMD2A) is a genetic disease that is caused by mutations in the calpain 3 gene (CAPN3), which encodes the skeletal muscle–specific calpain, calpain 3 (also known as p94). However, the precise mechanism by which p94 functions in the pathogenesis of this disease remains unclear. Here, using p94 knockin mice (termed herein p94KI mice) in which endogenous p94 was replaced with a proteolytically inactive but structurally intact p94:C129S mutant protein, we have demonstrated that stretch-dependent p94 distribution in sarcomeres plays a crucial role in the pathogenesis of LGMD2A. The p94KI mice developed a progressive muscular dystrophy, which was exacerbated by exercise. The exercise-induced muscle degeneration in p94KI mice was associated with an inefficient redistribution of p94:C129S in stretched sarcomeres. Furthermore, the p94KI mice showed impaired adaptation to physical stress, which was accompanied by compromised upregulation of muscle ankyrin-repeat protein-2 and hsp upon exercise. These findings indicate that the stretch-induced dynamic redistribution of p94 is dependent on its protease activity and essential to protect muscle from degeneration, particularly under conditions of physical stress. Furthermore, our data provide direct evidence that loss of p94 protease activity can result in LGMD2A and molecular insight into how this could occur.
Koichi Ojima, Yukiko Kawabata, Harumi Nakao, Kazuki Nakao, Naoko Doi, Fujiko Kitamura, Yasuko Ono, Shoji Hata, Hidenori Suzuki, Hiroyuki Kawahara, Julius Bogomolovas, Christian Witt, Coen Ottenheijm, Siegfried Labeit, Henk Granzier, Noriko Toyama-Sorimachi, Michiko Sorimachi, Koichi Suzuki, Tatsuya Maeda, Keiko Abe, Atsu Aiba, Hiroyuki Sorimachi
Semaphorin 3E (Sema3E) is a secreted molecule implicated in axonal path finding and inhibition of developmental and postischemic angiogenesis. Sema3E is also highly expressed in metastatic cancer cells, but its mechanistic role in tumor progression was not understood. Here we show that expression of Sema3E and its receptor Plexin D1 correlates with the metastatic progression of human tumors. Consistent with the clinical data, knocking down endogenous expression of either Sema3E or Plexin D1 in human metastatic carcinoma cells hampered their metastatic potential when injected into mice, while tumor growth was not markedly affected. Conversely, overexpression of exogenous Sema3E in cancer cells increased their invasiveness, transendothelial migration, and metastatic spreading, although it inhibited tumor vessel formation, resulting in reduced tumor growth in mice. The proinvasive and metastatic activity of Sema3E in tumor cells was dependent on transactivation of the Plexin D1–associated ErbB2/Neu oncogenic kinase. In sum, Sema3E–Plexin D1 signaling in cancer cells is crucially implicated in their metastatic behavior and may therefore be a promising target for strategies aimed at blocking tumor metastasis.
Andrea Casazza, Veronica Finisguerra, Lorena Capparuccia, Andrea Camperi, Jakub M. Swiercz, Sabrina Rizzolio, Charlotte Rolny, Claus Christensen, Andrea Bertotti, Ivana Sarotto, Mauro Risio, Livio Trusolino, Jurgen Weitz, Martin Schneider, Massimilano Mazzone, Paolo M. Comoglio, Luca Tamagnone
Hypoxia-inducible factor 1α (HIF-1α) and HIF-2α display unique and sometimes opposing activities in regulating cellular energy homeostasis, cell fate decisions, and oncogenesis. Macrophages exposed to hypoxia accumulate both HIF-1α and HIF-2α, and overexpression of HIF-2α in tumor-associated macrophages (TAMs) is specifically correlated with high-grade human tumors and poor prognosis. However, the precise role of HIF-2α during macrophage-mediated inflammatory responses remains unclear. To fully characterize cellular hypoxic adaptations, distinct functions of HIF-1α versus HIF-2α must be elucidated. We demonstrate here that mice lacking HIF-2α in myeloid cells (Hif2aΔ/Δ mice) are resistant to lipopolysaccharide-induced endotoxemia and display a marked inability to mount inflammatory responses to cutaneous and peritoneal irritants. Furthermore, HIF-2α directly regulated proinflammatory cytokine/chemokine expression in macrophages activated in vitro. Hif2aΔ/Δ mice displayed reduced TAM infiltration in independent murine hepatocellular and colitis-associated colon carcinoma models, and this was associated with reduced tumor cell proliferation and progression. Notably, HIF-2α modulated macrophage migration by regulating the expression of the cytokine receptor M-CSFR and the chemokine receptor CXCR4, without altering intracellular ATP levels. Collectively, our data identify HIF-2α as an important regulator of innate immunity, suggesting it may be a useful therapeutic target for treating inflammatory disorders and cancer.
Hongxia Z. Imtiyaz, Emily P. Williams, Michele M. Hickey, Shetal A. Patel, Amy C. Durham, Li-Jun Yuan, Rachel Hammond, Phyllis A. Gimotty, Brian Keith, M. Celeste Simon
Progression of prostate cancer following castration is associated with increased androgen receptor (AR) expression and signaling despite AR blockade. Recent studies suggest that these activities are due to the generation of constitutively active AR splice variants, but the mechanisms by which these splice variants could mediate such effects are not fully understood. Here we have identified what we believe to be a novel human AR splice variant in which exons 5, 6, and 7 are deleted (ARv567es) and demonstrated that this variant can contribute to cancer progression in human prostate cancer xenograft models in mice following castration. We determined that, in human prostate cancer cell lines, ARv567es functioned as a constitutively active receptor, increased expression of full-length AR (ARfl), and enhanced the transcriptional activity of AR. In human xenografts, human prostate cancer cells transfected with ARv567es cDNA formed tumors that were resistant to castration. Furthermore, the ratio of ARv567es to ARfl expression within the xenografts positively correlated with resistance to castration. Importantly, we also detected ARv567es frequently in human prostate cancer metastases. In summary, these data indicate that constitutively active AR splice variants can contribute to the development of castration-resistant prostate cancers and may serve as biomarkers for patients who are likely to suffer from early recurrence and are candidates for therapies directly targeting the AR rather than ligand.
Shihua Sun, Cynthia C.T. Sprenger, Robert L. Vessella, Kathleen Haugk, Kathryn Soriano, Elahe A. Mostaghel, Stephanie T. Page, Ilsa M. Coleman, Holly M. Nguyen, Huiying Sun, Peter S. Nelson, Stephen R. Plymate
The cell death receptor Fas plays a role in the establishment of fulminant hepatitis, a major cause of drug-induced liver failure. Fas activation elicits extrinsic apoptotic and hepatoprotective signals; however, the mechanisms by which these signals are integrated during disease are unknown. Tissue inhibitor of metalloproteinases 3 (TIMP3) controls the critical sheddase a disintegrin and metalloproteinase 17 (ADAM17) and may dictate stress signaling. Using mice and cells lacking TIMP3, ADAM17, and ADAM17-regulated cell surface molecules, we have found that ADAM17-mediated ectodomain shedding of TNF receptors and EGF family ligands controls activation of multiple signaling cascades in Fas-induced hepatitis. We demonstrated that TNF signaling promoted hepatotoxicity, while excessive TNF receptor 1 (TNFR1) shedding in Timp3–/– mice was protective. Compound Timp3–/–Tnf–/– and Timp3–/–Tnfr1–/– knockout conferred complete resistance to Fas-induced toxicity. Loss of Timp3 enhanced metalloproteinase-dependent EGFR signaling due to increased release of the EGFR ligands TGF-α, amphiregulin, and HB-EGF, while depletion of shed amphiregulin resensitized Timp3–/– hepatocytes to apoptosis. Finally, adenoviral delivery of Adam17 prevented acetaminophen-induced liver failure in a clinically relevant model of Fas-dependent fulminant hepatitis. These findings demonstrate that TIMP3 and ADAM17 cooperatively dictate cytokine signaling during death receptor activation and indicate that regulated metalloproteinase activity integrates survival and death signals during acute hepatotoxic stress.
Aditya Murthy, Virginie Defamie, David S. Smookler, Marco A. Di Grappa, Keisuke Horiuchi, Massimo Federici, Maria Sibilia, Carl P. Blobel, Rama Khokha
Gait disorders and postural instability, which are commonly observed in elderly patients with Parkinson disease (PD), respond poorly to dopaminergic agents used to treat other parkinsonian symptoms. The brain structures underlying gait disorders and falls in PD and aging remain to be characterized. Using functional MRI in healthy human subjects, we have shown here that activity of the mesencephalic locomotor region (MLR), which is composed of the pedunculopontine nucleus (PPN) and the adjacent cuneiform nucleus, was modulated by the speed of imagined gait, with faster imagined gait activating a discrete cluster within the MLR. Furthermore, the presence of gait disorders in patients with PD and in aged monkeys rendered parkinsonian by MPTP intoxication correlated with loss of PPN cholinergic neurons. Bilateral lesioning of the cholinergic part of the PPN induced gait and postural deficits in nondopaminergic lesioned monkeys. Our data therefore reveal that the cholinergic neurons of the PPN play a central role in controlling gait and posture and represent a possible target for pharmacological treatment of gait disorders in PD.
Carine Karachi, David Grabli, Frédéric A. Bernard, Dominique Tandé, Nicolas Wattiez, Hayat Belaid, Eric Bardinet, Annick Prigent, Hans-Peter Nothacker, Stéphane Hunot, Andreas Hartmann, Stéphane Lehéricy, Etienne C. Hirsch, Chantal François
Activating transcription factor 4 (ATF4) is a critical transcription factor for osteoblast (OBL) function and bone formation; however, a direct role in osteoclasts (OCLs) has not been established. Here, we targeted expression of ATF4 to the OCL lineage using the Trap promoter or through deletion of Atf4 in mice. OCL differentiation was drastically decreased in Atf4–/– bone marrow monocyte (BMM) cultures and bones. Coculture of Atf4–/– BMMs with WT OBLs or a high concentration of RANKL failed to restore the OCL differentiation defect. Conversely, Trap-Atf4-tg mice displayed severe osteopenia with dramatically increased osteoclastogenesis and bone resorption. We further showed that ATF4 was an upstream activator of the critical transcription factor Nfatc1 and was critical for RANKL activation of multiple MAPK pathways in OCL progenitors. Furthermore, ATF4 was crucial for M-CSF induction of RANK expression on BMMs, and lack of ATF4 caused a shift in OCL precursors to macrophages. Finally, ATF4 was largely modulated by M-CSF signaling and the PI3K/AKT pathways in BMMs. These results demonstrate that ATF4 plays a direct role in regulating OCL differentiation and suggest that it may be a therapeutic target for treating bone diseases associated with increased OCL activity.
Huiling Cao, Shibing Yu, Zhi Yao, Deborah L. Galson, Yu Jiang, Xiaoyan Zhang, Jie Fan, Binfeng Lu, Youfei Guan, Min Luo, Yumei Lai, Yibei Zhu, Noriyoshi Kurihara, Kenneth Patrene, G. David Roodman, Guozhi Xiao
Studies in mice and humans have revealed that the T cell, immunoglobulin, mucin (TIM) genes are associated with several atopic diseases. TIM-1 is a type I membrane protein that is expressed on T cells upon stimulation and has been shown to modulate their activation. In addition to a recently described interaction with dendritic cells, TIM-1 has also been identified as a phosphatidylserine recognition molecule, and several protein ligands have been proposed. Our understanding of its activity is complicated by the possibility that TIM-1 possesses multiple and diverse binding partners. In order to delineate the function of TIM-1, we generated monoclonal antibodies directed to a cleft formed within the IgV domain of TIM-1. We have shown here that antibodies that bind to this defined cleft antagonize TIM-1 binding to specific ligands and cells. Notably, these antibodies exhibited therapeutic activity in a humanized SCID model of experimental asthma, ameliorating inflammation, and airway hyperresponsiveness. Further experiments demonstrated that the effects of the TIM-1–specific antibodies were mediated via suppression of Th2 cell proliferation and cytokine production. These results demonstrate that modulation of the TIM-1 pathway can critically influence activated T cells in a humanized disease model, suggesting that TIM-1 antagonists may provide potent therapeutic benefit in asthma and other immune-mediated disorders.
Sanchaita Sriwal Sonar, Yen-Ming Hsu, Melanie Lynn Conrad, Gerard R. Majeau, Ayse Kilic, Ellen Garber, Yan Gao, Chioma Nwankwo, Gundi Willer, Jan C. Dudda, Hellen Kim, Véronique Bailly, Axel Pagenstecher, Paul D. Rennert, Harald Renz
The renin-angiotensin-aldosterone system (RAAS) is a key hormonal system regulating blood pressure. However, expression of RAAS components has recently been detected in immune cells, and the RAAS has been implicated in several mouse models of autoimmune disease. Here, we have identified Ang II as a paracrine mediator, sustaining inflammation in the CNS in the EAE mouse model of MS via TGF-β. Ang II type 1 receptors (AT1Rs) were found to be primarily expressed in CNS-resident cells during EAE. In vitro, astrocytes and microglia responded to Ang II treatment by inducing TGF-β expression via a pathway involving the TGF-β–activating protease thrombospondin-1 (TSP-1). TGF-β upregulation in astrocytes and microglia during EAE was blocked with candesartan (CA), an inhibitor of AT1R. Treatment of EAE with CA ameliorated paralysis and blunted lymphocyte infiltration into the CNS, outcomes that were also seen with genetic ablation of AT1Ra and treatment with an inhibitor of TSP-1. These data suggest that AT1R antagonists, frequently prescribed as antihypertensives, may be useful to interrupt this proinflammatory, CNS-specific pathway in individuals with MS.
Tobias V. Lanz, Zhaoqing Ding, Peggy P. Ho, Jian Luo, Ankur N. Agrawal, Hrishikesh Srinagesh, Robert Axtell, Hui Zhang, Michael Platten, Tony Wyss-Coray, Lawrence Steinman
Cerebral cavernous malformation is a common human vascular disease that arises due to loss-of-function mutations in genes encoding three intracellular adaptor proteins, cerebral cavernous malformations 1 protein (CCM1), CCM2, and CCM3. CCM1, CCM2, and CCM3 interact biochemically in a pathway required in endothelial cells during cardiovascular development in mice and zebrafish. The downstream effectors by which this signaling pathway regulates endothelial function have not yet been identified. Here we have shown in zebrafish that expression of mutant ccm3 proteins (ccm3Δ) known to cause cerebral cavernous malformation in humans confers cardiovascular phenotypes identical to those associated with loss of ccm1 and ccm2. CCM3Δ proteins interacted with CCM1 and CCM2, but not with other proteins known to bind wild-type CCM3, serine/threonine protein kinase MST4 (MST4), sterile 20–like serine/threonine kinase 24 (STK24), and STK25, all of which have poorly defined biological functions. Cardiovascular phenotypes characteristic of CCM deficiency arose due to stk deficiency and combined low-level deficiency of stks and ccm3 in zebrafish embryos. In cultured human endothelial cells, CCM3 and STK25 regulated barrier function in a manner similar to CCM2, and STKs negatively regulated Rho by directly activating moesin. These studies identify STKs as essential downstream effectors of CCM signaling in development and disease that may regulate both endothelial and epithelial cell junctions.
Xiangjian Zheng, Chong Xu, Annarita Di Lorenzo, Benjamin Kleaveland, Zhiying Zou, Christoph Seiler, Mei Chen, Lan Cheng, Jiping Xiao, Jie He, Michael A. Pack, William C. Sessa, Mark L. Kahn
Mechanistic target of rapamycin (MTOR) plays a critical role in the regulation of cell growth and in the response to energy state changes. Drugs inhibiting MTOR are increasingly used in antineoplastic therapies. Myocardial MTOR activity changes during hypertrophy and heart failure (HF). However, whether MTOR exerts a positive or a negative effect on myocardial function remains to be fully elucidated. Here, we show that ablation of Mtor in the adult mouse myocardium results in a fatal, dilated cardiomyopathy that is characterized by apoptosis, autophagy, altered mitochondrial structure, and accumulation of eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1). 4E-BP1 is an MTOR-containing multiprotein complex-1 (MTORC1) substrate that inhibits translation initiation. When subjected to pressure overload, Mtor-ablated mice demonstrated an impaired hypertrophic response and accelerated HF progression. When the gene encoding 4E-BP1 was ablated together with Mtor, marked improvements were observed in apoptosis, heart function, and survival. Our results demonstrate a role for the MTORC1 signaling network in the myocardial response to stress. In particular, they highlight the role of 4E-BP1 in regulating cardiomyocyte viability and in HF. Because the effects of reduced MTOR activity were mediated through increased 4E-BP1 inhibitory activity, blunting this mechanism may represent a novel therapeutic strategy for improving cardiac function in clinical HF.
Denghong Zhang, Riccardo Contu, Michael V.G. Latronico, Jianlin Zhang, Roberto Rizzi, Daniele Catalucci, Shigeki Miyamoto, Katherine Huang, Marcello Ceci, Yusu Gu, Nancy D. Dalton, Kirk L. Peterson, Kun-Liang Guan, Joan Heller Brown, Ju Chen, Nahum Sonenberg, Gianluigi Condorelli
Sirtuins are a phylogenetically conserved NAD+-dependent protein deacetylase/ADP-ribosyltransferase family implicated in diverse biological processes. Several family members localize to mitochondria, the function of which is thought to determine the developmental potential of preimplantation embryos. We have therefore characterized the role of sirtuins in mouse preimplantation development under in vitro culture conditions. All sirtuin members were expressed in eggs, and their expression gradually decreased until the blastocyst stage. Treatment with sirtuin inhibitors resulted in increased intracellular ROS levels and decreased blastocyst formation. These effects were recapitulated by siRNA-induced knockdown of Sirt3, which is involved in mitochondrial energy metabolism, and in Sirt3–/– embryos. The antioxidant N-acetyl-L-cysteine and low-oxygen conditions rescued these adverse effects. When Sirt3-knockdown embryos were transferred to pseudopregnant mice after long-term culture, implantation and fetal growth rates were decreased, indicating that Sirt3-knockdown embryos were sensitive to in vitro conditions and that the effect was long lasting. Further experiments revealed that maternally derived Sirt3 was critical. Sirt3 inactivation increased mitochondrial ROS production, leading to p53 upregulation and changes in downstream gene expression. The inactivation of p53 improved the developmental outcome of Sirt3-knockdown embryos, indicating that the ROS-p53 pathway was responsible for the developmental defects. These results indicate that Sirt3 plays a protective role in preimplantation embryos against stress conditions during in vitro fertilization and culture.
Yumiko Kawamura, Yasunobu Uchijima, Nanao Horike, Kazuo Tonami, Koichi Nishiyama, Tomokazu Amano, Tomoichiro Asano, Yukiko Kurihara, Hiroki Kurihara
Activating mutations in Ras proteins are present in about 30% of human cancers. Despite tremendous progress in the study of Ras oncogenes, many aspects of the molecular mechanisms underlying Ras-induced tumorigenesis remain unknown. Through proteomics analysis, we previously found that the protein Gankyrin, a known oncoprotein in hepatocellular carcinoma, was upregulated during Ras-mediated transformation, although the functional consequences of this were not clear. Here we present evidence that Gankyrin plays an essential role in Ras-initiated tumorigenesis in mouse and human cells. We found that the increased Gankyrin present following Ras activation increased the interaction between the RhoA GTPase and its GDP dissociation inhibitor RhoGDI, which resulted in inhibition of the RhoA effector kinase Rho-associated coiled coil–containing protein kinase (ROCK). Importantly, Gankyrin-mediated ROCK inhibition led to prolonged Akt activation, a critical step in activated Ras–induced transformation and tumorigenesis. In addition, we found that Gankyrin is highly expressed in human lung cancers that have Ras mutations and that increased Gankyrin expression is required for the constitutive activation of Akt and tumorigenesis in these lung cancers. Our findings suggest that Gankyrin is a key regulator of Ras-mediated activation of Akt through inhibition of the downstream RhoA/ROCK pathway and thus plays an essential role in Ras-induced tumorigenesis.
Jiang-Hong Man, Bing Liang, Yue-Xi Gu, Tao Zhou, Ai-Ling Li, Tao Li, Bao-Feng Jin, Bing Bai, Hai-Ying Zhang, Wei-Na Zhang, Wei-Hua Li, Wei-Li Gong, Hui-Yan Li, Xue-Min Zhang
The cytokine TGF-β acts as a tumor suppressor in normal epithelial cells and during the early stages of tumorigenesis. During malignant progression, cancer cells can switch their response to TGF-β and use this cytokine as a potent oncogenic factor; however, the mechanistic basis for this is poorly understood. Here we demonstrate that downregulation of disabled homolog 2 (DAB2) gene expression via promoter methylation frequently occurs in human squamous cell carcinomas (SCCs) and acts as an independent predictor of metastasis and poor prognosis. Retrospective microarray analysis in an independent data set indicated that low levels of DAB2 and high levels of TGFB2 expression correlate with poor prognosis. Immunohistochemistry, reexpression, genetic knockout, and RNAi silencing studies demonstrated that downregulation of DAB2 expression modulated the TGF-β/Smad pathway. Simultaneously, DAB2 downregulation abrogated TGF-β tumor suppressor function, while enabling TGF-β tumor-promoting activities. Downregulation of DAB2 blocked TGF-β–mediated inhibition of cell proliferation and migration and enabled TGF-β to promote cell motility, anchorage-independent growth, and tumor growth in vivo. Our data indicate that DAB2 acts as a tumor suppressor by dictating tumor cell TGF-β responses, identify a biomarker for SCC progression, and suggest a means to stratify patients with advanced SCC who may benefit clinically from anti–TGF-β therapies.
Adèle Hannigan, Paul Smith, Gabriela Kalna, Cristiana Lo Nigro, Clare Orange, Darren I. O’Brien, Reshma Shah, Nelofer Syed, Lindsay C. Spender, Blanca Herrera, Johanna K. Thurlow, Laura Lattanzio, Martino Monteverde, Meghan E. Maurer, Francesca M. Buffa, Jelena Mann, David C.K. Chu, Catharine M.L. West, Max Patridge, Karin A. Oien, Jonathan A. Cooper, Margaret C. Frame, Adrian L. Harris, Louise Hiller, Linda J. Nicholson, Milena Gasco, Tim Crook, Gareth J. Inman
Personalized cancer medicine is based on the concept that targeted therapies are effective on subsets of patients whose tumors carry specific molecular alterations. Several mammalian target of rapamycin (mTOR) inhibitors are in preclinical or clinical trials for cancers, but the molecular basis of sensitivity or resistance to these inhibitors among patients is largely unknown. Here we have identified oncogenic variants of phosphoinositide-3-kinase, catalytic, α polypeptide (PIK3CA) and KRAS as determinants of response to the mTOR inhibitor everolimus. Human cancer cells carrying alterations in the PI3K pathway were responsive to everolimus, both in vitro and in vivo, except when KRAS mutations occurred concomitantly or were exogenously introduced. In human cancer cells with mutations in both PIK3CA and KRAS, genetic ablation of mutant KRAS reinstated response to the drug. Consistent with these data, PIK3CA mutant cells, but not KRAS mutant cells, displayed everolimus-sensitive translation. Importantly, in a cohort of metastatic cancer patients, the presence of oncogenic KRAS mutations was associated with lack of benefit after everolimus therapy. Thus, our results demonstrate that alterations in the KRAS and PIK3CA genes may represent biomarkers to optimize treatment of patients with mTOR inhibitors.
Federica Di Nicolantonio, Sabrina Arena, Josep Tabernero, Stefano Grosso, Francesca Molinari, Teresa Macarulla, Mariangela Russo, Carlotta Cancelliere, Davide Zecchin, Luca Mazzucchelli, Takehiko Sasazuki, Senji Shirasawa, Massimo Geuna, Milo Frattini, José Baselga, Margherita Gallicchio, Stefano Biffo, Alberto Bardelli
Ticks are ectoparasitic arthropods that can transmit a variety of microorganisms to humans and animals during blood feeding, causing serious infectious disorders, including Lyme disease. Acaricides are pharmacologic agents that kill ticks. The emergence of acaricide-resistant ticks calls for alternative control strategies for ticks and tick-borne diseases. Many animals develop resistance to ticks after repeated infestations, but the nature of this acquired anti-tick immunity remains poorly understood. Here we investigated the cellular and molecular mechanisms underlying acquired resistance to Haemaphysalis longicornis ticks in mice and found that antibodies were required, as was IgFc receptor expression on basophils but not on mast cells. The infiltration of basophils at tick-feeding sites occurred during the second, but not the first, tick infestation. To assess the requirement for basophil infiltration to acquired tick resistance, mice expressing the human diphtheria toxin receptor under the control of the mast cell protease 8 (Mcpt8) promoter were generated. Diphtheria toxin administration to these mice selectively ablated basophils. Diphtheria toxin–mediated basophil depletion before the second tick infestation resulted in loss of acquired tick resistance. These data provide the first clear evidence, to our knowledge, that basophils play an essential and nonredundant role in antibody-mediated acquired immunity against ticks, which may suggest new strategies for controlling tick-borne diseases.
Takeshi Wada, Kenji Ishiwata, Haruhiko Koseki, Tomoyuki Ishikura, Tsukasa Ugajin, Naotsugu Ohnuma, Kazushige Obata, Ryosuke Ishikawa, Soichiro Yoshikawa, Kaori Mukai, Yohei Kawano, Yoshiyuki Minegishi, Hiroo Yokozeki, Naohiro Watanabe, Hajime Karasuyama
Insufficient insulin secretion and reduced pancreatic β cell mass are hallmarks of type 2 diabetes (T2DM). Here, we confirm that a previously identified polymorphism (rs2295490/Q84R) in exon 2 of the pseudokinase-encoding gene tribbles 3 (TRB3) is associated with an increased risk for T2DM in 2 populations of people of mixed European descent. Carriers of the 84R allele had substantially reduced plasma levels of C-peptide, the product of proinsulin processing to insulin, suggesting a role for TRB3 in β cell function. Overexpression of TRB3 84R in mouse β cells, human islet cells, and the murine β cell line MIN6 revealed reduced insulin exocytosis, associated with a marked reduction in docked insulin granules visualized by electron microscopy. Conversely, knockdown of TRB3 in MIN6 cells restored insulin secretion and expression of exocytosis genes. Further analysis in MIN6 cells demonstrated that TRB3 interacted with the transcription factor ATF4 and that this complex acted as a competitive inhibitor of cAMP response element-binding (CREB) transcription factor in the regulation of key exocytosis genes. In addition, the 84R TRB3 variant exhibited greater protein stability than wild-type TRB3 and increased binding affinity to Akt. Mice overexpressing TRB3 84R in β cells displayed decreased β cell mass, associated with reduced proliferation and enhanced apoptosis rates. These data link a missense polymorphism in human TRB3 to impaired insulin exocytosis and thus increased risk for T2DM.
Chong Wee Liew, Jacek Bochenski, Dan Kawamori, Jiang Hu, Colin A. Leech, Krzysztof Wanic, Maciej Malecki, James H. Warram, Ling Qi, Andrzej S. Krolewski, Rohit N. Kulkarni
Abetalipoproteinemia (ABL) is a rare Mendelian disorder of lipid metabolism due to genetic deficiency in microsomal triglyceride transfer protein (MTP). It is associated with defects in MTP-mediated lipid transfer onto apolipoprotein B (APOB) and impaired secretion of APOB-containing lipoproteins. Recently, MTP was shown to regulate the CD1 family of lipid antigen-presenting molecules, but little is known about immune function in ABL patients. Here, we have shown that ABL is characterized by immune defects affecting presentation of self and microbial lipid antigens by group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 molecules. In dendritic cells isolated from ABL patients, MTP deficiency was associated with increased proteasomal degradation of group 1 CD1 molecules. Although CD1d escaped degradation, it was unable to load antigens and exhibited functional defects similar to those affecting the group 1 CD1 molecules. The reduction in CD1 function resulted in impaired activation of CD1-restricted T and invariant natural killer T (iNKT) cells and reduced numbers and phenotypic alterations of iNKT cells consistent with central and peripheral CD1 defects in vivo. These data highlight MTP as a unique regulator of human metabolic and immune pathways and reveal that ABL is not only a disorder of lipid metabolism but also an immune disease involving CD1.
Sebastian Zeissig, Stephanie K. Dougan, Duarte C. Barral, Yvonne Junker, Zhangguo Chen, Arthur Kaser, Madelyn Ho, Hannah Mandel, Adam McIntyre, Susan M. Kennedy, Gavin F. Painter, Natacha Veerapen, Gurdyal S. Besra, Vincenzo Cerundolo, Simon Yue, Sarah Beladi, Samuel M. Behar, Xiuxu Chen, Jenny E. Gumperz, Karine Breckpot, Anna Raper, Amanda Baer, Mark A. Exley, Robert A. Hegele, Marina Cuchel, Daniel J. Rader, Nicholas O. Davidson, Richard S. Blumberg
Pubertal onset, initiated by pulsatile gonadotropin-releasing hormone (GnRH), only occurs in a favorable, anabolic hormonal milieu. Anabolic factors that may signal nutritional status to the hypothalamus include the growth factors insulin and IGF-1. It is unclear which hypothalamic neuronal subpopulation these factors affect to ultimately regulate GnRH neuron function in puberty and reproduction. We examined the direct role of the GnRH neuron in growth factor regulation of reproduction using the Cre/lox system. Mice with the IR or IGF-1R deleted specifically in GnRH neurons were generated. Male and female mice with the IR deleted in GnRH neurons displayed normal pubertal timing and fertility, but male and female mice with the IGF-1R deleted in GnRH neurons experienced delayed pubertal development with normal fertility. With IGF-1 administration, puberty was advanced in control females, but not in females with the IGF-1R deleted in GnRH neurons, in control males, or in knockout males. These mice exhibited developmental differences in GnRH neuronal morphology but normal number and distribution of neurons. These studies define the role of IGF-1R signaling in the coordination of somatic development with reproductive maturation and provide insight into the mechanisms regulating pubertal timing in anabolic states.
Sara A. DiVall, Tameeka R. Williams, Sarah E. Carver, Linda Koch, Jens C. Brüning, C. Ronald Kahn, Fredric Wondisford, Sally Radovick, Andrew Wolfe
The antidyslipidemic drug nicotinic acid and the antipsoriatic drug monomethyl fumarate induce cutaneous flushing through activation of G protein–coupled receptor 109A (GPR109A). Flushing is a troublesome side effect of nicotinic acid, but may be a direct reflection of the wanted effects of monomethyl fumarate. Here we analyzed the mechanisms underlying GPR109A-mediated flushing and show that both Langerhans cells and keratinocytes express GPR109A in mice. Using cell ablation approaches and transgenic cell type–specific GPR109A expression in Gpr109a–/– mice, we have provided evidence that the early phase of flushing depends on GPR109A expressed on Langerhans cells, whereas the late phase is mediated by GPR109A expressed on keratinocytes. Interestingly, the first phase of flushing was blocked by a selective cyclooxygenase-1 (COX-1) inhibitor, and the late phase was sensitive to a selective COX-2 inhibitor. Both monomethyl fumarate and nicotinic acid induced PGE2 formation in isolated keratinocytes through activation of GPR109A and COX-2. Thus, the early and late phases of the GPR109A-mediated cutaneous flushing reaction involve different epidermal cell types and prostanoid-forming enzymes. These data will help to guide new efficient approaches to mitigate nicotinic acid–induced flushing and may help to exploit the potential antipsoriatic effects of GPR109A agonists in the skin.
Julien Hanson, Andreas Gille, Sabrina Zwykiel, Martina Lukasova, Björn E. Clausen, Kashan Ahmed, Sorin Tunaru, Angela Wirth, Stefan Offermanns
Hypermethylation-mediated tumor suppressor gene silencing plays a crucial role in tumorigenesis. Understanding its underlying mechanism is essential for cancer treatment. Previous studies on human N-α-acetyltransferase 10, NatA catalytic subunit (hNaa10p; also known as human arrest-defective 1 [hARD1]), have generated conflicting results with regard to its role in tumorigenesis. Here we provide multiple lines of evidence indicating that it is oncogenic. We have shown that hNaa10p overexpression correlated with poor survival of human lung cancer patients. In vitro, enforced expression of hNaa10p was sufficient to cause cellular transformation, and siRNA-mediated depletion of hNaa10p impaired cancer cell proliferation in colony assays and xenograft studies. The oncogenic potential of hNaa10p depended on its interaction with DNA methyltransferase 1 (DNMT1). Mechanistically, hNaa10p positively regulated DNMT1 enzymatic activity by facilitating its binding to DNA in vitro and its recruitment to promoters of tumor suppressor genes, such as E-cadherin, in vivo. Consistent with this, interaction between hNaa10p and DNMT1 was required for E-cadherin silencing through promoter CpG methylation, and E-cadherin repression contributed to the oncogenic effects of hNaa10p. Together, our data not only establish hNaa10p as an oncoprotein, but also reveal that it contributes to oncogenesis through modulation of DNMT1 function.
Chung-Fan Lee, Derick S.-C. Ou, Sung-Bau Lee, Liang-Hao Chang, Ruo-Kai Lin, Ying-Shiuan Li, Anup K. Upadhyay, Xiaodong Cheng, Yi-Ching Wang, Han-Shui Hsu, Michael Hsiao, Cheng-Wen Wu, Li-Jung Juan
Distinct populations of leptin-sensing neurons in the hypothalamus, midbrain, and brainstem contribute to the regulation of energy homeostasis. To assess the requirement for leptin signaling in the hypothalamus, we crossed mice with a floxed leptin receptor allele (Leprfl) to mice transgenic for Nkx2.1-Cre, which drives Cre expression in the hypothalamus and not in more caudal brain regions, generating LeprNkx2.1KO mice. From weaning, LeprNkx2.1KO mice exhibited phenotypes similar to those observed in mice with global loss of leptin signaling (Leprdb/db mice), including increased weight gain and adiposity, hyperphagia, cold intolerance, and insulin resistance. However, after 8 weeks of age, LeprNkx2.1KO mice maintained stable adiposity levels, whereas the body fat percentage of Leprdb/db animals continued to escalate. The divergence in the adiposity phenotypes of Leprdb/db and LeprNkx2.1KO mice with age was concomitant with increased rates of linear growth and energy expenditure in LeprNkx2.1KO mice. These data suggest that remaining leptin signals in LeprNkx2.1KO mice mediate physiological adaptations that prevent the escalation of the adiposity phenotype in adult mice. The persistence of severe adiposity in LeprNkx2.1KO mice, however, suggests that compensatory actions of circuits regulating growth and energy expenditure are not sufficient to reverse obesity established at an early age.
Laurence E. Ring, Lori M. Zeltser
Organic anion transporting polypeptides (OATPs) are uptake transporters for a broad range of endogenous compounds and xenobiotics. To investigate the physiologic and pharmacologic roles of OATPs of the 1A and 1B subfamilies, we generated mice lacking all established and predicted mouse Oatp1a/1b transporters (referred to as Slco1a/1b–/– mice, as SLCO genes encode OATPs). Slco1a/1b–/– mice were viable and fertile but exhibited markedly increased plasma levels of bilirubin conjugated to glucuronide and increased plasma levels of unconjugated bile acids. The unexpected conjugated hyperbilirubinemia indicates that Oatp1a/1b transporters normally mediate extensive hepatic reuptake of glucuronidated bilirubin. We therefore hypothesized that substantial sinusoidal secretion and subsequent Oatp1a/1b-mediated reuptake of glucuronidated compounds can occur in hepatocytes under physiologic conditions. This alters our perspective on normal liver functioning. Slco1a/1b–/– mice also showed drastically decreased hepatic uptake and consequently increased systemic exposure following i.v. or oral administration of the OATP substrate drugs methotrexate and fexofenadine. Importantly, intestinal absorption of oral methotrexate or fexofenadine was not affected in Slco1a/1b–/– mice. Further analysis showed that rifampicin was an effective and specific Oatp1a/1b inhibitor in controlling methotrexate pharmacokinetics. These data indicate that Oatp1a/1b transporters play an essential role in hepatic reuptake of conjugated bilirubin and uptake of unconjugated bile acids and drugs. Slco1a/1b–/– mice will provide excellent tools to study further the role of Oatp1a/1b transporters in physiology and drug disposition.
Evita van de Steeg, Els Wagenaar, Cornelia M.M. van der Kruijssen, Johanna E.C. Burggraaff, Dirk R. de Waart, Ronald P.J. Oude Elferink, Kathryn E. Kenworthy, Alfred H. Schinkel
Obesity and its metabolic consequences are a major public health concern worldwide. Obesity is associated with overactivity of the endocannabinoid system, which is involved in the regulation of appetite, lipogenesis, and insulin resistance. Cannabinoid-1 receptor (CB1R) antagonists reduce body weight and improve cardiometabolic abnormalities in experimental and human obesity, but their therapeutic potential is limited by neuropsychiatric side effects. Here we have demonstrated that a CB1R neutral antagonist largely restricted to the periphery does not affect behavioral responses mediated by CB1R in the brains of mice with genetic or diet-induced obesity, but it does cause weight-independent improvements in glucose homeostasis, fatty liver, and plasma lipid profile. These effects were due to blockade of CB1R in peripheral tissues, including the liver, as verified through the use of CB1R-deficient mice with or without transgenic expression of CB1R in the liver. These results suggest that targeting peripheral CB1R has therapeutic potential for alleviating cardiometabolic risk in obese patients.
Joseph Tam, V. Kiran Vemuri, Jie Liu, Sándor Bátkai, Bani Mukhopadhyay, Grzegorz Godlewski, Douglas Osei-Hyiaman, Shinobu Ohnuma, Suresh V. Ambudkar, James Pickel, Alexandros Makriyannis, George Kunos
Development of a vaccine that targets blood-stage malaria parasites is imperative if we are to sustainably reduce the morbidity and mortality caused by this infection. Such a vaccine should elicit long-lasting immune responses against conserved determinants in the parasite population. Most blood-stage vaccines, however, induce protective antibodies against surface antigens, which tend to be polymorphic. Cell-mediated responses, on the other hand, offer the theoretical advantage of targeting internal antigens that are more likely to be conserved. Nonetheless, few of the current blood-stage vaccine candidates are able to harness vigorous T cell immunity. Here, we present what we believe to be a novel blood-stage whole-organism vaccine that, by combining low doses of killed parasite with CpG-oligodeoxynucleotide (CpG-ODN) adjuvant, was able to elicit strong and cross-reactive T cell responses in mice. Our data demonstrate that immunization of mice with 1,000 killed parasites in CpG-ODN engendered durable and cross-strain protection by inducing a vigorous response that was dependent on CD4+ T cells, IFN-γ, and nitric oxide. If applicable to humans, this approach should facilitate the generation of robust, cross-reactive T cell responses against malaria as well as antigen availability for vaccine manufacture.
Alberto Pinzon-Charry, Virginia McPhun, Vivian Kienzle, Chakrit Hirunpetcharat, Christian Engwerda, James McCarthy, Michael F. Good
Pulmonary metastasis remains the leading ca use of death for cancer patients. Opportunities to improve treatment outcomes for patients require new methods to study and view the biology of metastatic progression. Here, we describe an ex vivo pulmonary metastasis assay (PuMA) in which the metastatic progression of GFP-expressing cancer cells, from a single cell to the formation of multicellular colonies, in the mouse lung microenvironment was assessed in real time for up to 21 days. The biological validity of this assay was confirmed by its prediction of the in vivo behavior of a variety of high- and low-metastatic human and mouse cancer cell lines and the discrimination of tumor microenvironments in the lung that were most permissive to metastasis. Using this approach, we provide what we believe to be new insights into the importance of tumor cell interactions with the stromal components of the lung microenvironment. Finally, the translational utility of this assay was demonstrated through its use in the evaluation of therapeutics at discrete time points during metastatic progression. We believe that this assay system is uniquely capable of advancing our understanding of both metastasis biology and therapeutic strategies.
Arnulfo Mendoza, Sung-Hyeok Hong, Tanasa Osborne, Mohammed A. Khan, Kirk Campbell, Joseph Briggs, Ananth Eleswarapu, Lauren Buquo, Ling Ren, Stephen M. Hewitt, El-H. Dakir, Susan Garfield, Renard Walker, Glenn Merlino, Jeffrey E. Green, Kent W. Hunter, Lalage M. Wakefield, Chand Khanna
Preterm infants are highly susceptible to life-threatening infections that are clinically difficult to detect, such as late-onset septicemia and necrotizing enterocolitis (NEC). Here, we used a proteomic approach to identify biomarkers for diagnosis of these devastating conditions. In a case-control study comprising 77 sepsis/NEC and 77 nonsepsis cases (10 in each group being monitored longitudinally), plasma samples collected at clinical presentation were assessed in the biomarker discovery and independent validation phases. We validated the discovered biomarkers in a prospective cohort study with 104 consecutively suspected sepsis/NEC episodes. Proapolipoprotein CII (Pro-apoC2) and a des-arginine variant of serum amyloid A (SAA) were identified as the most promising biomarkers. The ApoSAA score computed from plasma apoC2 and SAA concentrations was effective in identifying sepsis/NEC cases in the case-control and cohort studies. Stratification of infants into different risk categories by the ApoSAA score enabled neonatologists to withhold treatment in 45% and enact early stoppage of antibiotics in 16% of nonsepsis infants. The negative predictive value of this antibiotic policy was 100%. The ApoSAA score could potentially allow early and accurate diagnosis of sepsis/NEC. Upon confirmation by further multicenter trials, the score would facilitate rational prescription of antibiotics and target infants who require urgent treatment.
Pak Cheung Ng, Irene Ling Ang, Rossa Wai Kwun Chiu, Karen Li, Hugh Simon Lam, Raymond Pui On Wong, Kit Man Chui, Hon Ming Cheung, Eddy Wing Yin Ng, Tai Fai Fok, Joseph Jao Yiu Sung, Yuk Ming Dennis Lo, Terence Chuen Wai Poon
J. Anthony G. Scott, W. Abdullah Brooks, J.S. Malik Peiris, Douglas Holtzman, E. Kim Mulholland