The NLRP3 inflammasome is a protein complex responsible for caspase-1–dependent maturation of the proinflammatory cytokines IL-1β and IL-18. Gain-of-function missense mutations in NLRP3 cause the disease spectrum known as the cryopyrin-associated periodic syndromes (CAPS). In this study, we generated Nlrp3-knockin mice on various KO backgrounds including Il1b/Il18-, caspase-1–, caspase-11– (Casp1/11-), and Tnf-deficient strains. The Nlrp3L351P Il1b–/– Il18–/– mutant mice survived and grew normally until adulthood and, at 6 months of age, exhibited marked splenomegaly and leukophilia. Injection of these mice with low-dose LPS resulted in elevated serum TNF levels compared with Nlrp3L351P Casp1/11–/– mice and Il1b–/– Il18–/– littermates. Treatment of Nlrp3A350V mice with the TNF inhibitor etanercept resulted in all pups surviving to adulthood, with normal body and spleen/body weight ratios. Nlrp3A350V Tnf–/– mice showed a similar phenotypic rescue, with marked reductions in serum IL-1β and IL-18, reduced myeloid inflammatory infiltrate in the skin and spleen, and substantial decreases in splenic mRNA expression of both inflammasome components (Nlrp3, Pycard, pro-Casp1) and pro-cytokines (Il1b, Il18). Likewise, we observed a reduction in the expression of both pro-Casp1 and pro-Il1b in cultured Nlrp3A350V Tnf–/– BM-derived DCs. Our data show that TNF is an important transcriptional regulator of NLRP3 inflammasome components in murine inflammasomopathies. Moreover, these results may have therapeutic implications for CAPS patients with partial responses to IL-1–targeted therapies.
Matthew D. McGeough, Alexander Wree, Maria E. Inzaugarat, Ariela Haimovich, Casey D. Johnson, Carla A. Peña, Raphaela Goldbach-Mansky, Lori Broderick, Ariel E. Feldstein, Hal M. Hoffman
Abnormal activity of the renin-angiotensin-aldosterone system plays a causal role in the development of hypertension, atherosclerosis, and associated cardiovascular events such as myocardial infarction, stroke, and heart failure. As both a vasoconstrictor and a proinflammatory mediator, angiotensin II (Ang II) is considered a potential link between hypertension and atherosclerosis. However, a role for Ang II–induced inflammation in atherosclerosis has not been clearly established, and the molecular mechanisms and intracellular signaling pathways involved are not known. Here, we demonstrated that the RhoA GEF Arhgef1 is essential for Ang II–induced inflammation. Specifically, we showed that deletion of Arhgef1 in a murine model prevents Ang II–induced integrin activation in leukocytes, thereby preventing Ang II–induced recruitment of leukocytes to the endothelium. Mice lacking both LDL receptor (LDLR) and Arhgef1 were protected from high-fat diet–induced atherosclerosis. Moreover, reconstitution of Ldlr–/– mice with Arhgef1-deficient BM prevented high-fat diet–induced atherosclerosis, while reconstitution of Ldlr–/– Arhgef1–/– with WT BM exacerbated atherosclerotic lesion formation, supporting Arhgef1 activation in leukocytes as causal in the development of atherosclerosis. Thus, our data highlight the importance of Arhgef1 in cardiovascular disease and suggest targeting Arhgef1 as a potential therapeutic strategy against atherosclerosis.
Maria Luigia Carbone, Gilliane Chadeuf, Sandrine Heurtebise-Chrétien, Xavier Prieur, Thibault Quillard, Yann Goueffic, Nathalie Vaillant, Marc Rio, Laure Castan, Maxim Durand, Céline Baron-Menguy, Julien Aureille, Juliette Desfrançois, Angela Tesse, Raul M. Torres, Gervaise Loirand
p120-Catenin (p120) functions as a tumor suppressor in intestinal cancer, but the mechanism is unclear. Here, using conditional p120 knockout in Apc-sensitized mouse models of intestinal cancer, we have identified p120 as an “obligatory” haploinsufficient tumor suppressor. Whereas monoallelic loss of p120 was associated with a significant increase in tumor multiplicity, loss of both alleles was never observed in tumors from these mice. Moreover, forced ablation of the second allele did not further enhance tumorigenesis, but instead induced synthetic lethality in combination with Apc loss of heterozygosity. In tumor-derived organoid cultures, elimination of both p120 alleles resulted in caspase-3–dependent apoptosis that was blocked by inhibition of Rho kinase (ROCK). With ROCK inhibition, however, p120-ablated organoids exhibited a branching phenotype and a substantial increase in cell proliferation. Access to data from Sleeping Beauty mutagenesis screens afforded an opportunity to directly assess the tumorigenic impact of p120 haploinsufficiency relative to other candidate drivers. Remarkably, p120 ranked third among the 919 drivers identified. Cofactors α-catenin and epithelial cadherin (E-cadherin) were also among the highest scoring candidates, indicating a mechanism at the level of the intact complex that may play an important role at very early stages of of intestinal tumorigenesis while simultaneously restricting outright loss via synthetic lethality.
Sarah P. Short, Jumpei Kondo, Whitney G. Smalley-Freed, Haruna Takeda, Michael R. Dohn, Anne E. Powell, Robert H. Carnahan, Mary K. Washington, Manish Tripathi, D. Michael Payne, Nancy A. Jenkins, Neal G. Copeland, Robert J. Coffey, Albert B. Reynolds
Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.
Alexia T. Kedves, Scott Gleim, Xiaoyou Liang, Dennis M. Bonal, Frederic Sigoillot, Fred Harbinski, Sneha Sanghavi, Christina Benander, Elizabeth George, Prafulla C. Gokhale, Quang-De Nguyen, Paul T. Kirschmeier, Robert J. Distel, Jeremy Jenkins, Michael S. Goldberg, William C. Forrester
Food allergy occurs due to IgE- and mast cell–dependent intestinal inflammation. Previously, we showed that histamine-releasing factor (HRF), a multifunctional protein secreted during allergy, interacts with a subset of IgE molecules and that the HRF dimer activates mast cells in an HRF-reactive IgE-dependent manner. In this study, we investigated whether HRF plays any role in food allergy. Specifically, we determined that prophylactic and therapeutic administration of HRF inhibitors that block HRF-IgE interactions reduces the incidence of diarrhea and mastocytosis in a murine model of food allergy. Food allergy–associated intestinal inflammation was accompanied by increased secretion of the HRF dimer into the intestine in response to proinflammatory, Th2, and epithelial-derived cytokines and HRF-reactive IgE levels at the elicitation phase. Consistent with these data, patients with egg allergy had higher blood levels of HRF-reactive IgE compared with individuals that were not hypersensitive. Successful oral immunotherapy in egg-allergy patients and food-allergic mice reduced HRF-reactive IgE levels, thereby suggesting a pathological role for HRF in food allergy. Together, these results suggest that antigen and HRF dimer amplify intestinal inflammation by synergistically activating mast cells and indicate that HRF has potential as a therapeutic target in food allergy.
Tomoaki Ando, Jun-ichi Kashiwakura, Naoka Itoh-Nagato, Hirotaka Yamashita, Minato Baba, Yu Kawakami, Shih Han Tsai, Naoki Inagaki, Kiyoshi Takeda, Tsutomu Iwata, Naoki Shimojo, Takao Fujisawa, Mizuho Nagao, Kenji Matsumoto, Yuko Kawakami, Toshiaki Kawakami
Conventional therapies for breast cancer brain metastases (BCBMs) have been largely ineffective because of chemoresistance and impermeability of the blood-brain barrier. A comprehensive understanding of the underlying mechanism that allows breast cancer cells to infiltrate the brain is necessary to circumvent treatment resistance of BCBMs. Here, we determined that expression of a long noncoding RNA (lncRNA) that we have named lncRNA associated with BCBM (Lnc-BM) is prognostic of the progression of brain metastasis in breast cancer patients. In preclinical murine models, elevated Lnc-BM expression drove BCBM, while depletion of Lnc-BM with nanoparticle-encapsulated siRNAs effectively treated BCBM. Lnc-BM increased JAK2 kinase activity to mediate oncostatin M– and IL-6–triggered STAT3 phosphorylation. In breast cancer cells, Lnc-BM promoted STAT3-dependent expression of ICAM1 and CCL2, which mediated vascular co-option and recruitment of macrophages in the brain, respectively. Recruited macrophages in turn produced oncostatin M and IL-6, thereby further activating the Lnc-BM/JAK2/STAT3 pathway and enhancing BCBM. Collectively, our results show that Lnc-BM and JAK2 promote BCBMs by mediating communication between breast cancer cells and the brain microenvironment. Moreover, these results suggest targeting Lnc-BM as a potential strategy for fighting this difficult disease.
Shouyu Wang, Ke Liang, Qingsong Hu, Ping Li, Jian Song, Yuedong Yang, Jun Yao, Lingegowda Selanere Mangala, Chunlai Li, Wenhao Yang, Peter K. Park, David H. Hawke, Jianwei Zhou, Yan Zhou, Weiya Xia, Mien-Chie Hung, Jeffrey R. Marks, Gary E. Gallick, Gabriel Lopez-Berestein, Elsa R. Flores, Anil K. Sood, Suyun Huang, Dihua Yu, Liuqing Yang, Chunru Lin
Regulation of skeletal muscle development and organization is a complex process that is not fully understood. Here, we focused on amphiphysin 2 (BIN1, also known as bridging integrator-1) and dynamin 2 (DNM2), two ubiquitous proteins implicated in membrane remodeling and mutated in centronuclear myopathies (CNMs). We generated Bin1–/– Dnm2+/– mice to decipher the physiological interplay between BIN1 and DNM2. While Bin1–/– mice die perinatally from a skeletal muscle defect, Bin1–/– Dnm2+/– mice survived at least 18 months, and had normal muscle force and intracellular organization of muscle fibers, supporting BIN1 as a negative regulator of DNM2. We next characterized muscle-specific isoforms of BIN1 and DNM2. While BIN1 colocalized with and partially inhibited DNM2 activity during muscle maturation, BIN1 had no effect on the isoform of DNM2 found in adult muscle. Together, these results indicate that BIN1 and DNM2 regulate muscle development and organization, function through a common pathway, and define BIN1 as a negative regulator of DNM2 in vitro and in vivo during muscle maturation. Our data suggest that DNM2 modulation has potential as a therapeutic approach for patients with CNM and BIN1 defects. As BIN1 is implicated in cancers, arrhythmia, and late-onset Alzheimer disease, these findings may trigger research directions and therapeutic development for these common diseases.
Belinda S. Cowling, Ivana Prokic, Hichem Tasfaout, Aymen Rabai, Frédéric Humbert, Bruno Rinaldi, Anne-Sophie Nicot, Christine Kretz, Sylvie Friant, Aurélien Roux, Jocelyn Laporte
Endothelial cells (ECs) are components of the hematopoietic microenvironment and regulate hematopoietic stem and progenitor cell (HSPC) homeostasis. Cytokine treatments that cause HSPC trafficking to peripheral blood are associated with an increase in dipeptidylpeptidase 4/CD26 (DPP4/CD26), an enzyme that truncates the neurotransmitter neuropeptide Y (NPY). Here, we show that enzymatically altered NPY signaling in ECs caused reduced VE-cadherin and CD31 expression along EC junctions, resulting in increased vascular permeability and HSPC egress. Moreover, selective NPY2 and NPY5 receptor antagonists restored vascular integrity and limited HSPC mobilization, demonstrating that the enzymatically controlled vascular gateway specifically opens by cleavage of NPY by CD26 signaling via NPY2 and NPY5 receptors. Mice lacking CD26 or NPY exhibited impaired HSPC trafficking that was restored by treatment with truncated NPY. Thus, our results point to ECs as gatekeepers of HSPC trafficking and identify a CD26-mediated NPY axis that has potential as a pharmacologic target to regulate hematopoietic trafficking in homeostatic and stress conditions.
Pratibha Singh, Jonathan Hoggatt, Malgorzata M. Kamocka, Khalid S. Mohammad, Mary R. Saunders, Hongge Li, Jennifer Speth, Nadia Carlesso, Theresa A. Guise, Louis M. Pelus
Primary congenital glaucoma (PCG) is a leading cause of blindness in children worldwide and is caused by developmental defects in 2 aqueous humor outflow structures, Schlemm’s canal (SC) and the trabecular meshwork. We previously identified loss-of-function mutations in the angiopoietin (ANGPT) receptor TEK in families with PCG and showed that ANGPT/TEK signaling is essential for SC development. Here, we describe roles for the major ANGPT ligands in the development of the aqueous outflow pathway. We determined that ANGPT1 is essential for SC development, and that Angpt1-knockout mice form a severely hypomorphic canal with elevated intraocular pressure. By contrast, ANGPT2 was dispensable, although mice deficient in both Angpt1 and Angpt2 completely lacked SC, indicating that ANGPT2 compensates for the loss of ANGPT1. In addition, we identified 3 human subjects with rare ANGPT1 variants within an international cohort of 284 PCG patients. Loss of function in 2 of the 3 patient alleles was observed by functional analysis of ANGPT1 variants in a combined in silico, in vitro, and in vivo approach, supporting a causative role for ANGPT1 in disease. By linking ANGPT1 with PCG, these results highlight the importance of ANGPT/TEK signaling in glaucoma pathogenesis and identify a candidate target for therapeutic development.
Benjamin R. Thomson, Tomokazu Souma, Stuart W. Tompson, Tuncer Onay, Krishnakumar Kizhatil, Owen M. Siggs, Liang Feng, Kristina N. Whisenhunt, Tammy L. Yanovitch, Luba Kalaydjieva, Dimitar N. Azmanov, Simone Finzi, Christine E. Tanna, Alex W. Hewitt, David A. Mackey, Yasmin S. Bradfield, Emmanuelle Souzeau, Shari Javadiyan, Janey L. Wiggs, Francesca Pasutto, Xiaorong Liu, Simon W.M. John, Jamie E. Craig, Jing Jin, Terri L. Young, Susan E. Quaggin
Molybdenum cofactor deficiency (MoCD) is an autosomal recessive inborn error of metabolism characterized by neurodegeneration and death in early childhood. The rapid and progressive neurodegeneration in MoCD presents a major clinical challenge and may relate to the poor understanding of the molecular mechanisms involved. Recently, we reported that treating patients with cyclic pyranopterin monophosphate (cPMP) is a successful therapy for a subset of infants with MoCD and prevents irreversible brain damage. Here, we studied S-sulfocysteine (SSC), a structural analog of glutamate that accumulates in the plasma and urine of patients with MoCD, and demonstrated that it acts as an N-methyl D-aspartate receptor (NMDA-R) agonist, leading to calcium influx and downstream cell signaling events and neurotoxicity. SSC treatment activated the protease calpain, and calpain-dependent degradation of the inhibitory synaptic protein gephyrin subsequently exacerbated SSC-mediated excitotoxicity and promoted loss of GABAergic synapses. Pharmacological blockade of NMDA-R, calcium influx, or calpain activity abolished SSC and glutamate neurotoxicity in primary murine neurons. Finally, the NMDA-R antagonist memantine was protective against the manifestation of symptoms in a tungstate-induced MoCD mouse model. These findings demonstrate that SSC drives excitotoxic neurodegeneration in MoCD and introduce NMDA-R antagonists as potential therapeutics for this fatal disease.
Avadh Kumar, Borislav Dejanovic, Florian Hetsch, Marcus Semtner, Debora Fusca, Sita Arjune, Jose Angel Santamaria-Araujo, Aline Winkelmann, Scott Ayton, Ashley I. Bush, Peter Kloppenburg, Jochen C. Meier, Guenter Schwarz, Abdel Ali Belaidi