Cell biology
Abstract
The AP-1 transcription factor c-Jun is required for Ras-driven tumorigenesis in many tissues and is considered as a classical proto-oncogene. To determine the requirement for c-Jun in a mouse model of K-RasG12D–induced lung adenocarcinoma, we inducibly deleted c-Jun in the adult lung. Surprisingly, we found that inactivation of c-Jun, or mutation of its JNK phosphorylation sites, actually increased lung tumor burden. Mechanistically, we found that protein levels of the Jun family member JunD were increased in the absence of c-Jun. In c-Jun–deficient cells, JunD phosphorylation was increased, and expression of a dominant-active JNKK2-JNK1 transgene further increased lung tumor formation. Strikingly, deletion of JunD completely abolished Ras-driven lung tumorigenesis. This work identifies JunD, not c-Jun, as the crucial substrate of JNK signaling and oncogene required for Ras-induced lung cancer.
Authors
E. Josue Ruiz, Linxiang Lan, Markus Elmar Diefenbacher, Eva Madi Riising, Clive Da Costa, Atanu Chakraborty, Joerg D. Hoeck, Bradley Spencer-Dene, Gavin Kelly, Jean-Pierre David, Emma Nye, Julian Downward, Axel Behrens
Abstract
Congenital microcephaly (MCPH) is a neurodevelopmental disease associated to mutations in genes encoding proteins involved in centrosomal and chromosomal dynamics during mitosis. Detailed MCPH pathogenesis at the cellular level is still elusive given the diversity of MCPH genes and lack of comparative in vivo studies. By generating a series of CRISPR/Cas9-mediated genetic knockouts we report here that, whereas defects in spindle pole proteins (ASPM, MCPH5) result in mild microcephaly during development, lack of centrosome (CDK5RAP2, MCPH3) or centriole (CEP135, MCPH8) regulators induces delayed chromosome segregation and chromosomal instability in neural progenitors (NPs). Our novel mouse model of MCPH8 suggests that Cep135 deficiency results in centriole duplication, TP53 activation and cell death of NPs. Trp53 ablation in a Cep135-deficient background prevents cell death, but not microcephaly, and leads to subcortical heterotopias, a malformation seen in MCPH8 patients. These results suggest that microcephaly in some MCPH patients can arise from the lack of adaptation to centriole defects in NPs and may lead to architectural defects if chromosomally unstable cells are not eliminated during brain development.
Authors
José González-Martínez, Andrzej W. Cwetsch, Diego Martínez-Alonso, Luis R. López-Sainz, Jorge Almagro, Anna Melati, Jesús Gómez, Manuel Pérez-Martínez, Diego Megías, Jasminka Boskovic, Javier Gilabert-Juan, Osvaldo Graña Castro, Alessandra Pierani, Axel Behrens, Sagrario Ortega, Marcos Malumbres
Abstract
Necroptosis has emerged as a potential mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, we found that markers of necroptosis, including high mobility group box 1 release and phosphorylation of mixed lineage kinase domain-like protein (p-MLKL), were markedly induced in the late stage of cigarette smoking–induced (CS-induced) emphysema in mouse lung tissue as well as in lung epithelial cells and organoids with higher dosage of or more prolonged exposure to cigarette smoking extract (CSE). Apoptotic signals were also detected and maximally induced in the early stage of CS-exposed mice and CSE-treated epithelial cells. Inhibition of apoptosis by Z-VAD, a pan-caspase inhibitor, switched the cellular stress to enhanced necroptosis in lung epithelial cells and organoids treated with CSE. Depletion or inhibition of receptor-interacting protein kinase 3 (RIP3) or MLKL attenuated the CSE-induced cell death, suggesting that necroptosis contributes to CSE-induced cell death. Silencing or inhibition of RIP1 had no protective effect, indicating a RIP1-independent RIP3 activation pathway. CSE-induced necroptosis released more damage-associated molecular patterns and evoked greater engulfment but slower clearance by bone marrow–derived macrophages, leading to enhanced expression of proinflammatory cytokines Tnfα and Il6. Finally, our in vivo data verified that inhibition of necroptosis by RIP3 inhibitor GSK’872 protected mice from CS-induced emphysema and suppressed the lung inflammation. In conclusion, we provide evidence that necroptosis contributes to the pathogenesis of COPD. Targeting RIP3 and its downstream pathway may be an effective therapy for COPD.
Authors
Dongshi Chen, Alyssa D. Gregory, Xiaoyun Li, Jianxin Wei, Christine L. Burton, Gregory Gibson, Stephen J. Scott, Claudette M. St. Croix, Yingze Zhang, Steven D. Shapiro
Abstract
Mutations in the gene (SFTPC) encoding surfactant protein C (SP-C) are associated with interstitial lung disease in children and adults. To assess the natural history of disease, we knocked-in a familial, disease-associated SFTPC mutation, L188Q [L184Q (LQ) in mice], into the mouse Sftpc locus. Translation of the mutant proprotein, proSP-CLQ, exceeded that of proSP-CWT in neonatal alveolar type 2 epithelial (AT2) cells and was associated with transient activation of oxidative stress and apoptosis leading to impaired expansion of AT2 cells during postnatal alveolarization. Differentiation of AT2 to AT1 cells was also inhibited in ex vivo organoid culture of AT2 cells isolated from LQ mice; importantly, treatment with antioxidant promoted alveolar differentiation. Upon completion of alveolarization, SftpcLQ expression was downregulated leading to resolution of chronic stress responses; however, the failure to restore AT2 cell numbers resulted in a permanent loss of AT2 cells that was linked to decreased regenerative capacity in the adult lung. Collectively, these data support the hypothesis that susceptibility to disease in adult LQ mice is established during postnatal lung development and provide a potential explanation for the delayed onset of disease in patients with familial pulmonary fibrosis.
Authors
Sneha Sitaraman, Emily P. Martin, Cheng-Lun Na, Shuyang Zhao, Jenna Green, Hitesh Deshmukh, Anne-Karina T. Perl, James P. Bridges, Yan Xu, Timothy E. Weaver
Abstract
β3-Adrenergic receptors (β3-ARs) are the predominant regulators of rodent brown adipose tissue (BAT) thermogenesis. However, in humans, the physiological relevance of BAT and β3-AR remains controversial. Herein, using primary human adipocytes from supraclavicular neck fat and immortalized brown/beige adipocytes from deep neck fat from 2 subjects, we demonstrate that the β3-AR plays a critical role in regulating lipolysis, glycolysis, and thermogenesis. Silencing of the β3-AR compromised genes essential for thermogenesis, fatty acid metabolism, and mitochondrial mass. Functionally, reduction of β3-AR lowered agonist-mediated increases in intracellular cAMP, lipolysis, and lipolysis-activated, uncoupling protein 1–mediated thermogenic capacity. Furthermore, mirabegron, a selective human β3-AR agonist, stimulated BAT lipolysis and thermogenesis, and both processes were lost after silencing β3-AR expression. This study highlights that β3-ARs in human brown/beige adipocytes are required to maintain multiple components of the lipolytic and thermogenic cellular machinery and that β3-AR agonists could be used to achieve metabolic benefit in humans.
Authors
Cheryl Cero, Hannah J. Lea, Kenneth Y. Zhu, Farnaz Shamsi, Yu-Hua Tseng, Aaron M. Cypess
Abstract
Patients with chronic kidney disease (CKD) and end stage renal disease suffer from increased cardiovascular events and cardiac mortality. Prior studies have demonstrated a portion of this enhanced risk can be attributed to the accumulation of microbiota-derived toxic metabolites, with most studies focusing on the sulfonated form of p-cresol (PCS). However, unconjugated p-cresol (uPC) itself was never assessed due to rapid and extensive first pass metabolism that results in negligible serum concentrations of uPC. These reports thus failed to consider the host exposure to uPC prior to hepatic metabolism. In the current study, we not only measured the impact of altering the intestinal microbiota on lipid accumulation in coronary arteries, but also examined macrophage lipid uptake and handling pathways in response to uPC. We found atherosclerotic-prone mice fed a high fat diet exhibited significantly higher coronary artery lipid deposits upon receiving fecal material from CKD mice. Furthermore, treatment with uPC increased total cholesterol, triglycerides, hepatic, and aortic fatty deposits in non-CKD mice. Studies employing an in vitro macrophage model demonstrated uPC exposure increased apoptosis where PCS did not. Additionally, uPC exhibited higher potency than PCS to stimulate low density lipoprotein (LDL) uptake and only uPC induced endocytosis and pinocytosis-related genes. Pharmacological inhibition of varying cholesterol influx and efflux systems indicated that uPC increased macrophage LDL uptake by activating macropinocytosis. Overall, these findings indicate uPC itself has a distinct impact on macrophage biology that may contribute to increased cardiovascular risk in patients with CKD.
Authors
Lee D. Chaves, Sham Abyad, Amanda M. Honan, Mark A. Bryniarski, Daniel I. McSkimming, Corrine M. Stahura, Steven C. Wells, Donna M. Ruszaj, Marilyn E. Morris, Richard J. Quigg, Rabi Yacoub
Abstract
Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have been used extensively to model inherited heart diseases, but hiPSC-CM models of ischemic heart disease are lacking. Here our objective was to generate an hiPSC-CM model of ischemic heart disease. To this end, hiPSCs were differentiated to functional hiPSC-CMs and then purified using either a simulated ischemia media or by using magnetic antibody-based purification targeting the non-myocyte population for depletion from the cell population. Flow cytometry analysis confirmed that each purification approach generated hiPSC-CM cultures of >94% cTnT+ cells. Following purification hiPSC-CMs were re-plated as confluent syncytial monolayers for electrophysiological phenotype analysis and protein expression by Western blotting. Metabolic selected hiPSC-CM monolayers’ phenotype recapitulated many of the functional and structural hallmarks of ischemic cardiomyocytes, including: elevated diastolic calcium, diminished calcium transient amplitude, prolonged action potential duration, depolarized resting membrane potential, hypersensitivity to chemotherapy induced cardiotoxicity, depolarized mitochondrial membrane potential, depressed SERCA2a expression, reduced maximal oxygen consumption rate and abnormal response to β1-adrenergic receptor stimulation. These findings indicate that metabolic selection of hiPSC-CMs generates cell populations with phenotype like what is well known to occur in the setting of ischemic heart failure, and thus provides a novel opportunity for study of human ischemic heart disease.
Authors
Justin Davis, Ahmad Chouman, Jeffery Creech, Andre Monteiro da Rocha, Daniela Ponce-Balbuena, Eric N. Jimenez Vazquez, Ruthann Nichols, Andrey Lozhkin, Nageswara R. Madamanchi, Katherine F. Campbell, Todd J. Herron
Abstract
Lung cancer with oncogenic KRAS makes up a significant proportion of lung cancers and is accompanied by a poor prognosis. Recent advances in understanding the molecular pathogenesis of lung cancer with oncogenic KRAS have enabled the development of drugs, yet mutated KRAS remains undruggable. We performed small-molecule library screening and identified verteporfin, a yes-associated protein 1 (YAP1) inhibitor; verteporfin treatment markedly reduced cell viability in KRAS-mutant lung cancer cells in vitro and suppressed KRAS-driven lung tumorigenesis in vivo. Comparative functional analysis of verteporfin treatment and YAP1 knockdown with siRNA revealed that the cytotoxic effect of verteporfin was at least partially independent of YAP1 inhibition. A whole-transcriptome approach revealed the distinct expression profiles in KRAS-mutant lung cancer cells between verteporfin treatment and YAP1 knockdown and identified the selective involvement of the ER stress pathway in the effects of verteporfin treatment in KRAS-mutant lung cancer, leading to apoptotic cell death. These data provide novel insight to uncover vulnerabilities in KRAS-driven lung tumorigenesis.
Authors
Iwao Shimomura, Naoaki Watanabe, Tomofumi Yamamoto, Minami Kumazaki, Yuji Tada, Koichiro Tatsumi, Takahiro Ochiya, Yusuke Yamamoto
Abstract
Most colorectal cancers (CRCs) are moderately-differentiated or well-differentiated, a status that is preserved even in metastatic tumors. However, the molecular mechanisms underlying CRC differentiation remain to be elucidated. Herein, we unravel a novel post-transcriptional regulatory mechanism via a previously unappreciated LIN28B-CDX2 signaling axis that plays a critical role in mediating CRC differentiation. Owing to a large number of mRNA targets, the mRNA-binding protein LIN28B has diverse functions in development, metabolism, tissue regeneration and tumorigenesis. Our RNA-binding protein immunoprecipitation (RIP) assay revealed LIN28B directly binds CDX2 mRNA, which is a pivotal homeobox transcription factor in normal intestinal epithelial cell identity and differentiation. Furthermore, LIN28B overexpression results in enhanced CDX2 expression to promote both differentiation in subcutaneous xenograft tumors generated from CRC cells and metastatic tumor colonization through mesenchymal-epithelial transition in CRC liver metastasis mouse models. Chromatin immunoprecipitation (ChIP) sequence for CDX2 identified Alpha-Methylacyl-CoA racemase (AMACR) as a novel transcriptional target of CDX2 in the context of LIN28B overexpression. We also found AMACR enhances intestinal alkaline phosphatase (ALPi) activity, which is known as a key component of intestinal differentiation, through the upregulation of butyric acid. Overall, we demonstrate that LIN28B promotes CRC differentiation through CDX2-AMACR axis.
Authors
Kensuke Suzuki, Yasunori Masuike, Rei Mizuno, Uma M Sachdeva, Priya Chatterji, Sarah F. Andres, Wenping Sun, Andres J Klein-Szanto, Sepideh Besharati, Helen E Remotti, Michael P Verzi, Anil K. Rustgi
Abstract
Mutations in LAMB2, encoding laminin β2, cause Pierson syndrome and occasionally milder nephropathy without extrarenal abnormalities. The most deleterious missense mutations that have been identified affect primarily the N-terminus of laminin β2. On the other hand, those associated with isolated nephropathy are distributed across the entire molecule, and variants in the β2 LEa-LF-LEb domains are exclusively found in cases with isolated nephropathy. Here we report the clinical features of mild isolated nephropathy associated with 3 LAMB2 variants in the LEa-LF-LEb domains (p.R469Q, p.G699R, and p.R1078C) and their biochemical characterization. Although Pierson syndrome missense mutations often inhibit laminin β2 secretion, the 3 recombinant variants were secreted as efficiently as WT. However, the β2 variants lost pH dependency for heparin binding, resulting in aberrant binding under physiologic conditions. This suggests that the binding of laminin β2 to negatively charged molecules is involved in glomerular basement membrane (GBM) permselectivity. Moreover, the excessive binding of the β2 variants to other laminins appears to lead to their increased deposition in the GBM. Laminin β2 also serves as a potentially novel cell-adhesive ligand for integrin α4β1. Our findings define biochemical functions of laminin β2 variants influencing glomerular filtration that may underlie the pathogenesis of isolated nephropathy caused by LAMB2 abnormalities.
Authors
Yamato Kikkawa, Taeko Hashimoto, Keiichi Takizawa, Seiya Urae, Haruka Masuda, Masumi Matsunuma, Yuji Yamada, Keisuke Hamada, Motoyoshi Nomizu, Helen Liapis, Masataka Hisano, Yuko Akioka, Kenichiro Miura, Motoshi Hattori, Jeffrey H. Miner, Yutaka Harita
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