Research Article
Abstract
Atypical hemolytic uremic syndrome (aHUS) is frequently associated in humans with loss-of-function mutations in complement-regulating proteins or gain-of-function mutations in complement-activating proteins. Thus, aHUS provides an archetypal complement-mediated disease with which to model new therapeutic strategies and treatments. Herein, we show that, when transferred to mice, an aHUS-associated gain-of-function change (D1115N) to the complement-activation protein C3 results in aHUS. Homozygous C3 p.D1115N (C3KI) mice developed spontaneous chronic thrombotic microangiopathy together with hematuria, thrombocytopenia, elevated creatinine, and evidence of hemolysis. Mice with active disease had reduced plasma C3 with C3 fragment and C9 deposition within the kidney. Therapeutic blockade or genetic deletion of C5, a protein downstream of C3 in the complement cascade, protected homozygous C3KI mice from thrombotic microangiopathy and aHUS. Thus, our data provide in vivo modeling evidence that gain-of-function changes in complement C3 drive aHUS. They also show that long-term C5 deficiency is not accompanied by development of other renal complications (such as C3 glomerulopathy) despite sustained dysregulation of C3. Our results suggest that this preclinical model will allow testing of novel complement inhibitors with the aim of developing precisely targeted therapeutics that could have application in many complement-mediated diseases.
Authors
Kate Smith-Jackson, Yi Yang, Harriet Denton, Isabel Y. Pappworth, Katie Cooke, Paul N. Barlow, John P. Atkinson, M. Kathryn Liszewski, Matthew C. Pickering, David Kavanagh, H. Terence Cook, Kevin J. Marchbank
Abstract
Metastasis is the dominant cause of patient death in small-cell lung cancer (SCLC), and a better understanding of the molecular mechanisms underlying SCLC metastasis may potentially improve clinical treatment. Through genome-scale screening for key regulators of mouse Rb1–/– Trp53–/– SCLC metastasis using the pooled CRISPR/Cas9 library, we identified Cullin5 (CUL5) and suppressor of cytokine signaling 3 (SOCS3), two components of the Cullin-RING E3 ubiquitin ligase complex, as top candidates. Mechanistically, the deficiency of CUL5 or SOCS3 disrupted the functional formation of the E3 ligase complex and prevented the degradation of integrin β1, which stabilized integrin β1 and activated downstream focal adhesion kinase/SRC (FAK/SRC) signaling and eventually drove SCLC metastasis. Low expression levels of CUL5 and SOCS3 were significantly associated with high integrin β1 levels and poor prognosis in a large cohort of 128 clinical patients with SCLC. Moreover, the CUL5-deficient SCLCs were vulnerable to the treatment of the FDA-approved SRC inhibitor dasatinib. Collectively, this work identifies the essential role of CUL5- and SOCS3-mediated integrin β1 turnover in controlling SCLC metastasis, which might have therapeutic implications.
Authors
Gaoxiang Zhao, Liyan Gong, Dan Su, Yujuan Jin, Chenchen Guo, Meiting Yue, Shun Yao, Zhen Qin, Yi Ye, Ying Tang, Qibiao Wu, Jian Zhang, Binghai Cui, Qiurong Ding, Hsinyi Huang, Liang Hu, Yuting Chen, Peiyuan Zhang, Guohong Hu, Luonan Chen, Kwok-Kin Wong, Daming Gao, Hongbin Ji
Abstract
Chromosomal integration of genome-intact HIV-1 sequences into the host genome creates a reservoir of virally infected cells that persists throughout life, necessitating indefinite antiretroviral suppression therapy. During effective antiviral treatment, the majority of these proviruses remain transcriptionally silent, but mechanisms responsible for viral latency are insufficiently clear. Here, we used matched integration site and proviral sequencing (MIP-Seq), an experimental approach involving multiple displacement amplification of individual proviral species, followed by near-full-length HIV-1 next-generation sequencing and corresponding chromosomal integration site analysis to selectively map the chromosomal positions of intact and defective proviruses in 3 HIV-1–infected individuals undergoing long-term antiretroviral therapy. Simultaneously, chromatin accessibility and gene expression in autologous CD4+ T cells were analyzed by assays for transposase-accessible chromatin using sequencing (ATAC-Seq) and RNA-Seq. We observed that in comparison to proviruses with defective sequences, intact HIV-1 proviruses were enriched for non-genic chromosomal positions and more frequently showed an opposite orientation relative to host genes. In addition, intact HIV-1 proviruses were preferentially integrated in either relative proximity to or increased distance from active transcriptional start sites and to accessible chromatin regions. These studies strongly suggest selection of intact proviruses with features of deeper viral latency during prolonged antiretroviral therapy, and may be informative for targeting the genome-intact viral reservoir.
Authors
Kevin B. Einkauf, Guinevere Q. Lee, Ce Gao, Radwa Sharaf, Xiaoming Sun, Stephane Hua, Samantha M.Y. Chen, Chenyang Jiang, Xiaodong Lian, Fatema Z. Chowdhury, Eric S. Rosenberg, Tae-Wook Chun, Jonathan Z. Li, Xu G. Yu, Mathias Lichterfeld
Abstract
MAPK4 is an atypical MAPK. Currently, little is known about its physiological function and involvement in diseases, including cancer. A comprehensive analysis of 8887 gene expression profiles in The Cancer Genome Atlas (TCGA) revealed that MAPK4 overexpression correlates with decreased overall survival, with particularly marked survival effects in patients with lung adenocarcinoma, bladder cancer, low-grade glioma, and thyroid carcinoma. Interestingly, human tumor MAPK4 overexpression also correlated with phosphorylation of AKT, 4E-BP1, and p70S6K, independent of the loss of PTEN or mutation of PIK3CA. This led us to examine whether MAPK4 activates the key metabolic, prosurvival, and proliferative kinase AKT and mTORC1 signaling, independent of the canonical PI3K pathway. We found that MAPK4 activated AKT via a novel, concerted mechanism independent of PI3K. Mechanistically, MAPK4 directly bound and activated AKT by phosphorylation of the activation loop at threonine 308. It also activated mTORC2 to phosphorylate AKT at serine 473 for full activation. MAPK4 overexpression induced oncogenic outcomes, including transforming prostate epithelial cells into anchorage-independent growth, and MAPK4 knockdown inhibited cancer cell proliferation, anchorage-independent growth, and xenograft growth. We concluded that MAPK4 can promote cancer by activating the AKT/mTOR signaling pathway and that targeting MAPK4 may provide a novel therapeutic approach for cancer.
Authors
Wei Wang, Tao Shen, Bingning Dong, Chad J. Creighton, Yanling Meng, Wolong Zhou, Qing Shi, Hao Zhou, Yinjie Zhang, David D. Moore, Feng Yang
Abstract
Chronic stress triggers activation of the sympathetic nervous system and drives malignancy. Using an immunodeficient murine system, we showed that chronic stress–induced epinephrine promoted breast cancer stem-like properties via lactate dehydrogenase A–dependent (LDHA-dependent) metabolic rewiring. Chronic stress–induced epinephrine activated LDHA to generate lactate, and the adjusted pH directed USP28-mediated deubiquitination and stabilization of MYC. The SLUG promoter was then activated by MYC, which promoted development of breast cancer stem-like traits. Using a drug screen that targeted LDHA, we found that a chronic stress–induced cancer stem-like phenotype could be reversed by vitamin C. These findings demonstrated the critical importance of psychological factors in promoting stem-like properties in breast cancer cells. Thus, the LDHA-lowering agent vitamin C can be a potential approach for combating stress-associated breast cancer.
Authors
Bai Cui, Yuanyuan Luo, Pengfei Tian, Fei Peng, Jinxin Lu, Yongliang Yang, Qitong Su, Bing Liu, Jiachuan Yu, Xi Luo, Liu Yin, Wei Cheng, Fan An, Bin He, Dapeng Liang, Sijin Wu, Peng Chu, Luyao Song, Xinyu Liu, Huandong Luo, Jie Xu, Yujia Pan, Yang Wang, Dangsheng Li, Peng Huang, Qingkai Yang, Lingqiang Zhang, Binhua P. Zhou, Suling Liu, Guowang Xu, Eric W.-F. Lam, Keith W. Kelley, Quentin Liu
Abstract
Both natural influenza infection and current seasonal influenza vaccines primarily induce neutralizing antibody responses against highly diverse epitopes within the “head” of the viral hemagglutinin (HA) protein. There is increasing interest in redirecting immunity toward the more conserved HA stem or stalk as a means of broadening protective antibody responses. Here we examined HA stem–specific B cell and T follicular helper (Tfh) cell responses in the context of influenza infection and immunization in mouse and monkey models. We found that during infection, the stem domain was immunologically subdominant to the head in terms of serum antibody production and antigen-specific B and Tfh cell responses. Similarly, we found that HA stem immunogens were poorly immunogenic compared with the full-length HA with abolished sialic acid binding activity, with limiting Tfh cell elicitation a potential constraint to the induction or boosting of anti-stem immunity by vaccination. Finally, we confirm that currently licensed seasonal influenza vaccines can boost preexisting memory responses against the HA stem in humans. An increased understanding of the immune dynamics surrounding the HA stem is essential to inform the design of next-generation influenza vaccines for broad and durable protection.
Authors
Hyon-Xhi Tan, Sinthujan Jegaskanda, Jennifer A. Juno, Robyn Esterbauer, Julius Wong, Hannah G. Kelly, Yi Liu, Danielle Tilmanis, Aeron C. Hurt, Jonathan W. Yewdell, Stephen J. Kent, Adam K. Wheatley
Abstract
Energy stress, such as ischemia, induces mitochondrial damage and death in the heart. Degradation of damaged mitochondria by mitophagy is essential for the maintenance of healthy mitochondria and survival. Here, we show that mitophagy during myocardial ischemia was mediated predominantly through autophagy characterized by Rab9-associated autophagosomes, rather than the well-characterized form of autophagy that is dependent on the autophagy-related 7 (Atg) conjugation system and LC3. This form of mitophagy played an essential role in protecting the heart against ischemia and was mediated by a protein complex consisting of unc-51 like kinase 1 (Ulk1), Rab9, receptor-interacting serine/thronine protein kinase 1 (Rip1), and dynamin-related protein 1 (Drp1). This complex allowed the recruitment of trans-Golgi membranes associated with Rab9 to damaged mitochondria through S179 phosphorylation of Rab9 by Ulk1 and S616 phosphorylation of Drp1 by Rip1. Knockin of Rab9 (S179A) abolished mitophagy and exacerbated the injury in response to myocardial ischemia, without affecting conventional autophagy. Mitophagy mediated through the Ulk1/Rab9/Rip1/Drp1 pathway protected the heart against ischemia by maintaining healthy mitochondria.
Authors
Toshiro Saito, Jihoon Nah, Shin-ichi Oka, Risa Mukai, Yoshiya Monden, Yasuhiro Maejima, Yoshiyuki Ikeda, Sebastiano Sciarretta, Tong Liu, Hong Li, Erdene Baljinnyam, Diego Fraidenraich, Luke Fritzky, Peiyong Zhai, Shizuko Ichinose, Mitsuaki Isobe, Chiao-Po Hsu, Mondira Kundu, Junichi Sadoshima
Abstract
Graft-versus-host disease (GVHD) is a complication of hematopoietic stem cell transplantation (HSCT) that affects multiple organs. GVHD-associated intestinal damage can be separated into two distinct phases, initiation and propagation, which correspond to conditioning-induced damage and effector T cell activation and infiltration, respectively. Substantial evidence indicates that intestinal damage induced by pretransplant conditioning is a key driver of GVHD initiation. Here, we aimed to determine the impact of dysregulated intestinal permeability on the subsequent GVHD propagation phase. The initiation phase of GVHD was unchanged in mice lacking long MLCK (MLCK210), an established regulator of epithelial tight junction permeability. However, MLCK210-deficient mice were protected from sustained barrier loss and exhibited limited GVHD propagation, as indicated by reduced histopathology, fewer CD8+ effector T cells in the gut, and improved overall survival. Consistent with these findings, intestinal epithelial MLCK210 expression and enzymatic activity were similarly increased in human and mouse GVHD biopsies. Intestinal epithelial barrier loss mediated by MLCK210 is therefore a key driver of the GVHD propagation. These data suggest that inhibition of MLCK210-dependent barrier regulation may be an effective approach to limiting GVHD progression.
Authors
Sam C. Nalle, Li Zuo, Ma. Lora Drizella M. Ong, Gurminder Singh, Alicia M. Worthylake, Wangsun Choi, Mario Cabrero Manresa, Anna P. Southworth, Karen L. Edelblum, Gregory J. Baker, Nora E. Joseph, Peter A. Savage, Jerrold R. Turner
Abstract
Molecular signaling mechanisms underlying Alzheimer’s disease (AD) remain unclear. Maintenance of memory and synaptic plasticity depend on de novo protein synthesis, dysregulation of which is implicated in AD. Recent studies showed AD-associated hyperphosphorylation of mRNA translation factor eukaryotic elongation factor 2 (eEF2), which results in inhibition of protein synthesis. We tested to determine whether suppression of eEF2 phosphorylation could improve protein synthesis capacity and AD-associated cognitive and synaptic impairments. Genetic reduction of the eEF2 kinase (eEF2K) in 2 AD mouse models suppressed AD-associated eEF2 hyperphosphorylation and improved memory deficits and hippocampal long-term potentiation (LTP) impairments without altering brain amyloid β (Aβ) pathology. Furthermore, eEF2K reduction alleviated AD-associated defects in dendritic spine morphology, postsynaptic density formation, de novo protein synthesis, and dendritic polyribosome assembly. Our results link eEF2K/eEF2 signaling dysregulation to AD pathophysiology and therefore offer a feasible therapeutic target.
Authors
Brenna C. Beckelman, Wenzhong Yang, Nicole P. Kasica, Helena R. Zimmermann, Xueyan Zhou, C. Dirk Keene, Alexey G. Ryazanov, Tao Ma
Abstract
Persistent, unresolved inflammation in adipose tissue is a major contributor to obesity-associated metabolic complications. However, the molecular links between lipid-overloaded adipocytes and inflammatory immune cells in obese adipose tissues remain elusive. Here we identified adipocyte-secreted microRNA-34a (miR-34a) as a key mediator through its paracrine actions on adipose-resident macrophages. The expression of miR-34a in adipose tissues was progressively increased with the development of dietary obesity. Adipose-selective or adipocyte-specific miR-34a–KO mice were resistant to obesity-induced glucose intolerance, insulin resistance, and systemic inflammation, and this was accompanied by a significant shift in polarization of adipose-resident macrophages from proinflammatory M1 to antiinflammatory M2 phenotype. Mechanistically, mature adipocyte-secreted exosomes transported miR-34a into macrophages, thereby suppressing M2 polarization by repressing the expression of Krüppel-like factor 4 (Klf4). The suppressive effects of miR-34a on M2 polarization and its stimulation of inflammatory responses were reversed by ectopic expression of Klf4 in both bone marrow–derived macrophages and adipose depots of obese mice. Furthermore, increased miR-34a expression in visceral fat of overweight/obese subjects correlated negatively with reduced Klf4 expression, but positively with the parameters of insulin resistance and metabolic inflammation. In summary, miR-34a was a key component of adipocyte-secreted exosomal vesicles that transmitted the signal of nutrient overload to the adipose-resident macrophages for exacerbation of obesity-induced systemic inflammation and metabolic dysregulation.
Authors
Yong Pan, Xiaoyan Hui, Ruby Lai Chong Hoo, Dewei Ye, Cyrus Yuk Cheung Chan, Tianshi Feng, Yu Wang, Karen Siu Ling Lam, Aimin Xu
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ISSN: 0021-9738 (print), 1558-8238 (online)