Emerging evidence has shown that open reading frames inside lncRNA could encode micropeptides. However, their roles in cellular energy metabolism and tumor progression remain largely unknown. Here, we identified a 94-amino acid-length micropeptide encoded by lncRNA LINC00467 in colorectal cancer. We also characterized its conservation across higher mammals, localization to mitochondria, and the concerted local functions. This peptide enhanced the ATP synthase construction by interacting with the subunit α and γ (ATP5A and ATP5C), increased ATP synthase activity and mitochondrial oxygen consumption rate, and thereby promoted colorectal cancer cell proliferation. Hence, this micropeptide was termed as “ATP synthase associated peptide” (ASAP). Furthermore, loss of ASAP suppressed patient-derived xenograft growth with attenuated ATP synthase activity and mitochondrial ATP production. Clinically, high expression of ASAP and LINC00467 predicted poor prognosis of colorectal cancer patients. Taken together, our findings revealed a colorectal cancer-associated micropeptide as a vital player in mitochondrial metabolism and provided a therapeutic target for colorectal cancer.
Qiwei Ge, Dingjiacheng Jia, Dong Cen, Yadong Qi, Chengyu Shi, Junhong Li, Lingjie Sang, Luo-jia Yang, Jiamin He, Aifu Lin, Shujie Chen, Liangjing Wang
Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.
Ronald R. Marchelletta, Moorthy Krishnan, Marianne R. Spalinger, Taylaur W. Placone, Rocio Alvarez, Anica Sayoc-Becerra, Vinicius Canale, Ali Shawki, Young Su Park, Lucas H.P. Bernts, Stephen Myers, Michel L. Tremblay, Kim E. Barrett, Evan Krystofiak, Bechara Kachar, Dermot P.B. McGovern, Christopher R. Weber, Elaine M. Hanson, Lars Eckmann, Declan F. McCole
FOXP3+ Tregs are expanded within the inflamed intestine of human Crohn’s disease, yet FOXP3-mediated gene repression within these cells is lost. The polycomb repressive complexes play a role in FOXP3 target gene regulation, but deeper mechanistic insight is incomplete. We have now specifically identified the polycomb-repressive complex 1 (PRC1) family member, BMI1 in the regulation of a proinflammatory enhancer network in both human and murine Tregs. Using human Tregs and lamina propria T cells, we inferred PRC1 to regulate Crohn’s associated gene networks through assays of chromatin accessibility. Conditional deletion of BMI1 in murine FOXP3+ cells led to systemic inflammation. BMI1-deficient Tregs beared a TH1/TH17-like phenotype as assessed by assays of genome wide transcription, chromatin accessibility and proteomic techniques. Finally, BMI1 mutant FOXP3+ cells did not suppress colitis in the adoptive transfer model of human inflammatory bowel disease. We propose that BMI1 plays an important role in enforcing Treg identity in vitro and in vivo. Loss of Treg identity via genetic or transient BMI1 depletion perturbs the epigenome and converts Tregs into Th1/Th17-like proinflammatory cells, a transition relevant to human Crohn’s disease associated CD4+ T cells.
Michelle M. Gonzalez, Adebowale O. Bamidele, Phyllis A. Svingen, Mary R. Sagstetter, Thomas C. Smyrk, Joseph M. Gaballa, Feda H. Hamdan, Robyn Laura Kosinsky, Hunter R. Gibbons, Zhifu Sun, Zhenqing Ye, Asha Nair, Guilherme P. Ramos, Manuel B. Braga Neto, Alexander Q. Wixom, Angela J. Mathison, Steven A. Johnsen, Raul Urrutia, William A. Faubion Jr.
Disordered lysosomal/autophagy pathways initiate and drive pancreatitis, but the underlying mechanisms and links to disease pathology are poorly understood. Here, we show that mannose-6-phosphate (M6P) pathway of hydrolase delivery to lysosomes critically regulates pancreatic acinar cell cholesterol metabolism. Ablation of the Gnptab gene coding for a key enzyme in M6P pathway disrupted acinar cell cholesterol turnover, causing accumulation of non-esterified cholesterol in lysosomes/autolysosomes, its’ depletion in the plasma membrane, and upregulation of cholesterol synthesis and uptake. We found similar dysregulation of acinar cell cholesterol, and a decrease in GNPTAB levels, in both WT experimental pancreatitis and human disease. The mechanisms mediating pancreatic cholesterol dyshomeostasis in Gnptab-/- and experimental models involve disordered endolysosomal system, resulting in impaired cholesterol transport through lysosomes and blockage of autophagic flux. By contrast, in Gnptab-/- liver the endolysosomal system and cholesterol homeostasis were largely unaffected. Gnptab-/- mice developed spontaneous pancreatitis. Normalization of cholesterol metabolism by pharmacologic means alleviated responses of experimental pancreatitis, particularly trypsinogen activation, the disease hallmark. The results reveal the essential role of M6P pathway in maintaining exocrine pancreas homeostasis and function, and implicate cholesterol disordering in the pathogenesis of pancreatitis.
Olga A. Mareninova, Eszter T. Vegh, Natalia Shalbueva, Carli J.M. Wightman, Dustin L. Dillon, Sudarshan Malla, Yan Xie, Toshimasa Takahashi, Zoltan Rakonczay Jr, Samuel W. French, Herbert Y. Gaisano, Frederick Sanford Gorelick, Stephen J. Pandol, Steven J. Bensinger, Nicholas O. Davidson, David W. Dawson, Ilya Gukovsky, Anna S. Gukovskaya
The role of PI3K and Hippo signaling in chronic pancreatitis (CP) pathogenesis is unclear. Therefore, we assessed the involvement of these pathways in CP by examining the PI3K and Hippo signaling components PTEN and SAV1, respectively. We observed significant decreases in pancreatic PTEN and SAV1 levels in 2 murine CP models: repeated caerulein injection and pancreatic ductal ligation. Additionally, pancreas-specific deletion of Pten and Sav1 (DKO) induced CP in mice. Pancreatic connective tissue growth factor (CTGF) was markedly upregulated in both CP models and DKO mice, and pancreatic CCAAT/enhancer-binding protein alpha (CEBPA) expression was downregulated in the CP models. Interestingly, in pancreatic acinar cells (PACs), CEBPA knockdown reduced PTEN and SAV1 and increased CTGF levels in vitro. Furthermore, CEBPA knockdown in PACs induced acinar-to-ductal metaplasia and activation of cocultured macrophages and pancreatic stellate cells. These results were mitigated by CTGF inhibition. CP in DKO mice was also ameliorated by Ctgf gene deletion, and caerulein-induced CP was alleviated by antibody-mediated CTGF neutralization. Finally, we observed significantly decreased PTEN, SAV1, and CEBPA and increased CTGF levels in human CP tissues compared to nonpancreatitis tissues. Taken together, our results indicate that dysregulation of PI3K and Hippo signaling induces CP via CTGF upregulation.
Takeshi Tamura, Takahiro Kodama, Katsuhiko Sato, Kazuhiro Murai, Teppei Yoshioka, Minoru Shigekawa, Ryoko Yamada, Hayato Hikita, Ryotaro Sakamori, Hirofumi Akita, Hidetoshi Eguchi, Randy L. Johnson, Hideki Yokoi, Masashi Mukoyama, Tomohide Tatsumi, Tetsuo Takehara
Rapidly growing tumors often experience hypoxia and nutrient (e.g., glucose) deficiency because of poor vascularization. Tumor cells respond to the cytotoxic effects of such stresses by inducing molecular adaptations that promote clonal selection of a more malignant tumor-initiating cell phenotype, especially in the innermost tumor regions. Here, we report a regulatory mechanism involving fucosylation by which glucose restriction promotes cancer stemness to drive drug resistance and tumor recurrence. Using hepatocellular carcinoma (HCC) as a model, we showed that restricted glucose availability enhanced the PERK-eIF2α-ATF4 signaling axis to drive fucosyltransferase-1 (FUT1) transcription via direct binding of ATF4 to the FUT1 promoter. FUT1 overexpression is a poor prognostic indicator for HCC. FUT1 inhibition could mitigate tumor initiation, self-renewal and drug resistance. Mechanistically, we demonstrated that CD147, ICAM-1, EGFR and EPHA2 are glycoprotein targets of FUT1, where such fucosylation would consequently converge on deregulated AKT-mTOR-4EBP1 signaling to drive cancer stemness. Treatment with an α-(1,2)-fucosylation inhibitor sensitized HCC tumors to sorafenib, a first-line molecular targeted drug used for advanced HCC patients, and reduced the tumor-initiating subset. FUT1 overexpression and/or CD147, ICAM-1, EGFR and EPHA2 fucosylation may be good prognostic markers and therapeutic targets for cancer patients.
Jane H.C. Loong, Tin-Lok Wong, Man Tong, Rakesh Sharma, Lei Zhou, Kai-Yu Ng, Hua-Jian Yu, Chi Han Li, Kwan Man, Chung-Mau Lo, Xin-Yuan Guan, Terence K. Lee, Jing-Ping Yun, Stephanie Kwai Yee Ma
Hepatic ischemia and reperfusion (IR) injury contributes to the morbidity and mortality associated with liver transplantation. microRNAs (miRNAs) constitute a family of noncoding RNAs that regulate gene expression at the posttranslational level through the repression of specific target genes. Here, we hypothesized that miRNAs could be targeted to enhance hepatic ischemia tolerance. A miRNA screen in a murine model of hepatic IR injury pointed us toward the liver-specific miRNA miR122. Subsequent studies in mice with hepatocyte-specific deletion of miR122 (miR122loxP/loxP Alb-Cre+ mice) during hepatic ischemia and reperfusion revealed exacerbated liver injury. Transcriptional studies implicated hypoxia-inducible factor–1α (HIF1α) in the induction of miR122 and identified the oxygen-sensing prolyl hydroxylase domain 1 (PHD1) as a miR122 target. Further studies indicated that HIF1α-dependent induction of miR122 participated in a feed-forward pathway for liver protection via the enhancement of hepatic HIF responses through PHD1 repression. Moreover, pharmacologic studies utilizing nanoparticle-mediated miR122 overexpression demonstrated attenuated liver injury. Finally, proof-of-principle studies in patients undergoing orthotopic liver transplantation showed elevated miR122 levels in conjunction with the repression of PHD1 in post-ischemic liver biopsies. Taken together, the present findings provide molecular insight into the functional role of miR122 in enhancing hepatic ischemia tolerance and suggest the potential utility of pharmacologic interventions targeting miR122 to dampen hepatic injury during liver transplantation.
Cynthia Ju, Meng Wang, Eunyoung Tak, Boyun Kim, Christoph Emontzpohl, Yang Yang, Xiaoyi Yuan, Huban Kutay, Yafen Liang, David R. Hall, Wasim A. Dar, J. Steve Bynon, Peter Carmeliet, Kalpana Ghoshal, Holger K. Eltzschig
A primordial gut-epithelial innate defense response is the release of hydrogen peroxide by dual NADPH oxidase (DUOX). In inflammatory bowel disease (IBD), a condition characterized by an imbalanced gut microbiota-immune homeostasis, DUOX2 isoenzyme is the highest induced gene. Performing multi-omic analyses using 2,872 human participants of a wellness program, we detected a substantial burden of rare protein-altering DUOX2 gene variants of unknown physiologic significance (155 unique variants with allele frequency < 1%; 12.9% carrier rate). We identified a significant association between these rare loss-of-function variants and increased plasma levels of interleukin-17C (FDR=2.6e-5), which is induced also in mucosal biopsies of IBD patients. DUOX2 deficient mice replicated increased IL17C induction in the intestine, with outlier high Il17c expression linked to the mucosal expansion of specific Proteobacteria pathobionts. Integrated microbiota/host gene expression analyses in IBD patients corroborated IL17C as a marker for epithelial activation by gram-negative bacteria. Finally, the impact of DUOX2 variants on IL17C induction provided a rationale for variant stratification in case-control studies that substantiated DUOX2 as an IBD risk gene (pooled OR = 1.54 [95% CI 1.09-2.18]; P = 7.1e-4). Thus, our study identifies an association of deleterious DUOX2 variants with a preclinical hallmark of disturbed microbiota-immune homeostasis that appears to precede the manifestation of IBD.
Helmut Grasberger, Andrew T. Magis, Elisa Sheng, Matthew P. Conomos, Min Zhang, Lea S. Garzotto, Guoqing Hou, Shrinivas Bishu, Hiroko Nagao-Kitamoto, Mohamad El-Zataari, Sho Kitamoto, Nobuhiko Kamada, Ryan Stidham, Yasutada Akiba, Jonathan Kaunitz, Yael Haberman, Subra Kugathasan, Lee A. Denson, Gilbert S. Omenn, John Y. Kao
Omega-3 fatty acids from fish oil reduce triglyceride levels in mammals, yet the mechanisms underlying this effect have not been fully clarified despite the clinical use of omega-3 ethyl esters to treat severe hypertriglyceridemia and reduce cardiovascular disease risk in humans. Here we identified in bile a class of hypotriglyceridemic omega-3 fatty acid-derived N-acyl taurines (NATs) that, after dietary omega-3 fatty acid supplementation, increased to concentrations similar to those of steroidal bile acids. The biliary docosahexaenoic acid (DHA) containing NAT, C22:6 NAT, was increased in human and mouse plasma after dietary omega-3 fatty acid supplementation and potently inhibited intestinal triacylglycerol hydrolysis and lipid absorption. Supporting this observation, genetic elevation of endogenous NAT levels in mice impaired lipid absorption, while selective augmentation of C22:6 NAT levels protected against hypertriglyceridemia and fatty liver. When administered pharmacologically, C22:6 NAT accumulated in bile and reduced high fat diet-induced, but not sucrose-induced, hepatic lipid accumulation in mice, suggesting that C22:6 NAT was a negative feedback mediator that limited excess intestinal lipid absorption. Thus, biliary omega-3 NATs may contribute to the hypotriglyceridemic mechanism of action of fish oil and could influence the design of more potent omega-3 fatty acid-based therapeutics.
Trisha J. Grevengoed, Samuel A. J. Trammell, Jens S. Svenningsen, Mikhail Makarov, Thomas Svava Nielsen, Jens C. B. Jacobsen, Philip C. Calder, Marie E. Migaud, Benjamin Cravatt, Matthew P. Gillum
Hirschsprung disease (HSCR) is the most frequent developmental anomaly of the enteric nervous system with an incidence of 1/5000 live births. Chronic intestinal pseudo-obstruction (CIPO) is less frequent and classified as neurogenic or myogenic. Isolated HSCR has an oligogenic inheritance with RET as the major disease-causing gene, while CIPO is genetically heterogeneous, caused by mutations in smooth muscle-specific genes. Here, we describe a series of patients with developmental disorders including gastrointestinal dysmotility, and investigate the underlying molecular bases. Trio-exome sequencing led to the identification of biallelic variants in ERBB3 and ERBB2 in eight individuals variably associating HSCR, CIPO, peripheral neuropathy and arthrogryposis. Thorough gut histology revealed aganglionosis, hypoganglionosis and intestinal smooth muscle abnormalities. The cell-type-specific ErbB3 and ErbB2 function was further analysed in mouse single-cell RNA sequencing data and in a conditional ErbB3-deficient mouse model, revealing a primary role for ERBB3 in enteric progenitors. The consequences of the identified variants were evaluated using RT-qPCR on patient-derived fibroblasts or immunoblot assays on Neuro-2a cells overexpressing either wild-type or mutant proteins, revealing either decreased expression or altered phosphorylation of the mutant receptors. Our results demonstrate that dysregulation of ERBB3 or ERBB2 leads to a broad spectrum of developmental anomalies including intestinal dysmotility.
Thuy-Linh Le, Louise Galmiche, Jonathan Levy, Pim Suwannarat, Debby M.E.I. Hellebrekers, Khomgrit Morarach, Franck Boismoreau, Tom E.J. Theunissen, Mathilde Lefebvre, Anna Pelet, Jelena Martinovic, Antoinette Gelot, Fabien Guimiot, Amanda Calleroz, Cyril Gitiaux, Marie Hully, Olivier Goulet, Christophe Chardot, Severine Drunat, Yline Capri, Christine Bole-Feysot, Patrick Nitschke, Sandra Whalen, Linda Mouthon, Holly E. Babcock, Robert Hofstra, Irenaeus F.M. de Coo, Anne-Claude Tabet, Thierry J. Molina, Boris Keren, Alice S. Brooks, Hubert J.M. Smeets, Ulrika Marklund, Christopher T. Gordon, Stanislas Lyonnet, Jeanne Amiel, Nadège Bondurand