La cylindromatose familiale est une pathologie qui predispose les patients a developper des tumeurs de la peau appelees cylindromes. Ces patients portent une mutation du gene cyld qui mene au developpement de cylindromes, montrant ainsi la fonction de suppresseur de tumeur de CYLD. CYLD contient un domaine catalytique qui hydrolyse les chaines d’ubiquitines liees par les Lysines en position 63. Des etudes ont egalement montre que le domaine catalytique confere a CYLD un role de regulateur negatif des voies NF-kB, Jun Kinase (JNK) et Wnt. Cependant, la fonction exacte de CYLD dans le developpement des cylindromes reste encore inconnue. Afin de mieux comprendre la fonction de CYLD, differents modeles murins ont ete generes. Des souris presentant une deletion partielle du domaine ubiquitine hydrolase ont ete concues. Contrairement au phenotype leger observe chez les souris n’exprimant pas la proteine, ces souris CYLDdel932 qui expriment CYLD presentent un phenotype drastique puisque les souris homozygotes meurent a la naissance d’un probleme respiratoire. Lors d’experiences d’immunoprecipitations suivies de spectroscopie de masse, CYLD a ete trouve comme interagissant avec la proteine du centrosome CAP350. Durant ce travail de these, nous avons confirme l’interaction endogene de ces deux proteines et nous avons montre que CYLD est presente au centrosome dans les cellules RPE1. De plus, dans des cellules multiciliees, CYLD est egalement presente a l'extremite des cils. Au final, differentes experiences sur les tissus et cellules de ces souris nous ont permis de mettre en evidence un role de CYLD dans la regulation de la ciliogenese
There was an error published in Development 142, 2194-2202.On p. 2200, Ng et al. (2012) was incorrectly cited in place of Liew et al. (2014). The corrected text and reference appear below. The authors apologise to readers for this mistake.IFT27 was recently shown to play a crucial role in facilitating ciliary exit of the BBSome (Eguether et al., 2014; Liew et al., 2014), and Ift27-deficient mouse embryonic fibroblasts are unable to maintain low levels of SMO in the cilia when the Hh pathway is inactive (Eguether et al., 2014).
Background: Human-induced pluripotent stem cell-derived hepatocytes (iHeps) have been shown to have considerable potential in liver diseases, toxicity, and pharmacological studies. However, there is a growing need to obtain iHeps that are truly similar to primary adult hepatocytes in terms of morphological features and functions. We generated such human iHeps, self-assembled as organoids (iHep-Orgs). Methods: iPSC-derived hepatoblasts were self-assembled into spheroids and differentiated into mature hepatocytes modulating final step of differentiation. Results: In about four weeks of culture, the albumin secretion levels and the complete disappearance of α-fetoprotein from iHep-Orgs suggested the acquisition of a greater degree of maturation than those previously reported. The expression of apical transporters and bile acid secretion evidenced the acquisition of complex hepatocyte polarity as well as the development of a functional and well-defined bile canalicular network confirmed by computational analysis. Activities recorded for CYP450, UGT1A1, and alcohol dehydrogenase, response to hormonal stimulation, and glucose metabolism were also remarkable. Finally, iHep-Orgs displayed a considerable ability to detoxify pathological concentrations of lactate and ammonia. Conclusions: With features similar to those of primary adult hepatocytes, the iHep-Orgs thus produced could be considered as a valuable tool for the development and optimization of preclinical and clinical applications.
Drug-induced cholestasis is mostly intrahepatic and characterized by alterations of bile canaliculi dynamics and morphology as well as accumulation of bile acids (BAs) in hepatocytes. However, little information exists on first changes in BA content and profile induced by cholestatic drugs in human liver. In this study, we aimed to analyze the effects of a large set of cholestatic and noncholestatic drugs in presence of physiological serum concentrations and 60-fold higher levels of 9 main BAs on cellular accumulation of BAs using HepaRG hepatocytes. BAs were measured in cell layers (cells + bile canaliculi) and culture media using high-pressure liquid chromatography coupled with tandem mass spectrometry after 24 h-treatment. Comparable changes in total and individual BA levels were observed in cell layers and media from control and noncholestatic drug-treated cultures: unconjugated BAs were actively amidated and lithocholic acid (LCA) was entirely sulfated. In contrast, cellular accumulation of LCA and in addition, of the 2 other hydrophobic BAs, chenodeoxycholic acid and deoxycholic acid, was evidenced only with cholestatic compounds in presence of BA mixtures at normal and 60-fold serum levels, respectively, suggesting that LCA was the first BA to accumulate. Cellular accumulation of hydrophobic BAs was associated with inhibition of their amidation and for LCA, its sulfation. In conclusion, these results demonstrated that cellular accumulation of unconjugated hydrophobic BAs can be caused by various cholestatic drugs in human hepatocytes and suggest that their cellular detection, especially that of LCA, could represent a new strategy for evaluation of cholestatic potential of drugs and other chemicals.
Ciliopathies are associated with wide spectrum of structural birth defects (SBDs), indicating important roles for cilia in development. Here, we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140 , an intraflagellar transport (IFT) protein regulating ciliogenesis. Ift140 -deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula (TEF), randomized heart looping, congenital heart defects (CHDs), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAGGCre-ER deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD were not observed with 4 Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest-mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathies.
Article25 October 2022Open Access Transparent process Loss of primary cilia promotes inflammation and carcinogenesis Conception Paul Conception Paul Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Conceptualization, Formal analysis, Investigation, Methodology Search for more papers by this author Ruizhi Tang Ruizhi Tang orcid.org/0000-0001-6512-845X Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Ciro Longobardi Ciro Longobardi orcid.org/0000-0003-4965-9976 Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Center for Experimental and Molecular Medicine, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands Oncode Institute, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands Contribution: Conceptualization, Formal analysis, Investigation, Methodology Search for more papers by this author Rossano Lattanzio Rossano Lattanzio orcid.org/0000-0001-9803-4476 Department of Innovative Technologies in Medicine & Dentistry, Center for Advanced Studies and Technology (CAST), 'G. d'Annunzio' University of Chieti–Pescara, Chieti, Italy Contribution: Conceptualization, Formal analysis, Investigation, Methodology Search for more papers by this author Thibaut Eguether Thibaut Eguether orcid.org/0000-0003-1891-9253 Centre de Recherche Saint Antoine, Sorbonne Université, INSERM, APHP, Paris, France Contribution: Investigation, Methodology Search for more papers by this author Hulya Turali Hulya Turali Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Julie Bremond Julie Bremond Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Chloé Maurizy Chloé Maurizy orcid.org/0000-0002-5176-7514 Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Monica Gabola Monica Gabola Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Sophie Poupeau Sophie Poupeau Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Andrei Turtoi Andrei Turtoi orcid.org/0000-0003-3813-6635 Tumor Microenvironment and Resistance to Treatment Laboratory, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France Contribution: Investigation, Methodology Search for more papers by this author Emilie Denicolai Emilie Denicolai Cancer Research Center of Marseille (CRCM), Laboratory of Predictive Oncology, Inserm U1068 - CNRS UMR7258 – University of Aix-Marseille UM105 - Paoli Calmettes Institute (IPC), Label "Ligue contre le cancer", Marseille, France Contribution: Investigation, Methodology Search for more papers by this author Maria Concetta Cufaro Maria Concetta Cufaro orcid.org/0000-0002-6457-7795 Department of Innovative Technologies in Medicine & Dentistry, Center for Advanced Studies and Technology (CAST), 'G. d'Annunzio' University of Chieti–Pescara, Chieti, Italy Contribution: Investigation, Methodology Search for more papers by this author Magali Svrcek Magali Svrcek Department of Pathology, AP-HP, Hôpital Saint-Antoine, Sorbonne Université, Paris, France Contribution: Resources, Investigation Search for more papers by this author Philippe Seksik Philippe Seksik orcid.org/0000-0003-3596-9893 Centre de Recherche Saint Antoine, Sorbonne Université, INSERM, APHP, Paris, France Contribution: Resources Search for more papers by this author Vincent Castronovo Vincent Castronovo Metastasis Research Laboratory, GIGA Cancer, University of Liège, Liège, Belgium Contribution: Resources, Investigation Search for more papers by this author Philippe Delvenne Philippe Delvenne Cancer Research Center of Marseille (CRCM), Laboratory of Predictive Oncology, Inserm U1068 - CNRS UMR7258 – University of Aix-Marseille UM105 - Paoli Calmettes Institute (IPC), Label "Ligue contre le cancer", Marseille, France Department of Pathology, University Hospital (CHU), University of Liège, Liège, Belgium Contribution: Resources, Investigation Search for more papers by this author Vincenzo de Laurenzi Vincenzo de Laurenzi Department of Innovative Technologies in Medicine & Dentistry, Center for Advanced Studies and Technology (CAST), 'G. d'Annunzio' University of Chieti–Pescara, Chieti, Italy Contribution: Conceptualization, Investigation Search for more papers by this author Quentin Da Costa Quentin Da Costa orcid.org/0000-0001-8904-9131 Cancer Research Center of Marseille (CRCM), Laboratory of Predictive Oncology, Inserm U1068 - CNRS UMR7258 – University of Aix-Marseille UM105 - Paoli Calmettes Institute (IPC), Label "Ligue contre le cancer", Marseille, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author François Bertucci François Bertucci orcid.org/0000-0002-0157-0959 Cancer Research Center of Marseille (CRCM), Laboratory of Predictive Oncology, Inserm U1068 - CNRS UMR7258 – University of Aix-Marseille UM105 - Paoli Calmettes Institute (IPC), Label "Ligue contre le cancer", Marseille, France Contribution: Resources, Investigation Search for more papers by this author Bénédicte Lemmers Bénédicte Lemmers orcid.org/0000-0002-2353-131X Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Damiana Pieragostino Damiana Pieragostino Department of Innovative Technologies in Medicine & Dentistry, Center for Advanced Studies and Technology (CAST), 'G. d'Annunzio' University of Chieti–Pescara, Chieti, Italy Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Emilie Mamessier Emilie Mamessier orcid.org/0000-0002-3516-0093 Cancer Research Center of Marseille (CRCM), Laboratory of Predictive Oncology, Inserm U1068 - CNRS UMR7258 – University of Aix-Marseille UM105 - Paoli Calmettes Institute (IPC), Label "Ligue contre le cancer", Marseille, France Contribution: Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Carsten Janke Carsten Janke orcid.org/0000-0001-7053-2000 Institut Curie, Paris Sciences et Lettres (PSL) Research University, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR) 3348, Label "Equipe FRM", Orsay, France Université Paris Sud, Université Paris-Saclay, CNRS UMR 3348, Orsay, France Contribution: Conceptualization, Formal analysis, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Valérie Pinet Corresponding Author Valérie Pinet [email protected] orcid.org/0000-0002-4044-6440 Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Michael Hahne Corresponding Author Michael Hahne [email protected] orcid.org/0000-0002-3953-6430 Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Conceptualization, Supervision, Funding acquisition, Validation, Writing - original draft, Writing - review & editing Search for more papers by this author Conception Paul Conception Paul Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Conceptualization, Formal analysis, Investigation, Methodology Search for more papers by this author Ruizhi Tang Ruizhi Tang orcid.org/0000-0001-6512-845X Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Ciro Longobardi Ciro Longobardi orcid.org/0000-0003-4965-9976 Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Center for Experimental and Molecular Medicine, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands Oncode Institute, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands Contribution: Conceptualization, Formal analysis, Investigation, Methodology Search for more papers by this author Rossano Lattanzio Rossano Lattanzio orcid.org/0000-0001-9803-4476 Department of Innovative Technologies in Medicine & Dentistry, Center for Advanced Studies and Technology (CAST), 'G. d'Annunzio' University of Chieti–Pescara, Chieti, Italy Contribution: Conceptualization, Formal analysis, Investigation, Methodology Search for more papers by this author Thibaut Eguether Thibaut Eguether orcid.org/0000-0003-1891-9253 Centre de Recherche Saint Antoine, Sorbonne Université, INSERM, APHP, Paris, France Contribution: Investigation, Methodology Search for more papers by this author Hulya Turali Hulya Turali Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Julie Bremond Julie Bremond Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Chloé Maurizy Chloé Maurizy orcid.org/0000-0002-5176-7514 Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Monica Gabola Monica Gabola Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Sophie Poupeau Sophie Poupeau Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Andrei Turtoi Andrei Turtoi orcid.org/0000-0003-3813-6635 Tumor Microenvironment and Resistance to Treatment Laboratory, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France Contribution: Investigation, Methodology Search for more papers by this author Emilie Denicolai Emilie Denicolai Cancer Research Center of Marseille (CRCM), Laboratory of Predictive Oncology, Inserm U1068 - CNRS UMR7258 – University of Aix-Marseille UM105 - Paoli Calmettes Institute (IPC), Label "Ligue contre le cancer", Marseille, France Contribution: Investigation, Methodology Search for more papers by this author Maria Concetta Cufaro Maria Concetta Cufaro orcid.org/0000-0002-6457-7795 Department of Innovative Technologies in Medicine & Dentistry, Center for Advanced Studies and Technology (CAST), 'G. d'Annunzio' University of Chieti–Pescara, Chieti, Italy Contribution: Investigation, Methodology Search for more papers by this author Magali Svrcek Magali Svrcek Department of Pathology, AP-HP, Hôpital Saint-Antoine, Sorbonne Université, Paris, France Contribution: Resources, Investigation Search for more papers by this author Philippe Seksik Philippe Seksik orcid.org/0000-0003-3596-9893 Centre de Recherche Saint Antoine, Sorbonne Université, INSERM, APHP, Paris, France Contribution: Resources Search for more papers by this author Vincent Castronovo Vincent Castronovo Metastasis Research Laboratory, GIGA Cancer, University of Liège, Liège, Belgium Contribution: Resources, Investigation Search for more papers by this author Philippe Delvenne Philippe Delvenne Cancer Research Center of Marseille (CRCM), Laboratory of Predictive Oncology, Inserm U1068 - CNRS UMR7258 – University of Aix-Marseille UM105 - Paoli Calmettes Institute (IPC), Label "Ligue contre le cancer", Marseille, France Department of Pathology, University Hospital (CHU), University of Liège, Liège, Belgium Contribution: Resources, Investigation Search for more papers by this author Vincenzo de Laurenzi Vincenzo de Laurenzi Department of Innovative Technologies in Medicine & Dentistry, Center for Advanced Studies and Technology (CAST), 'G. d'Annunzio' University of Chieti–Pescara, Chieti, Italy Contribution: Conceptualization, Investigation Search for more papers by this author Quentin Da Costa Quentin Da Costa orcid.org/0000-0001-8904-9131 Cancer Research Center of Marseille (CRCM), Laboratory of Predictive Oncology, Inserm U1068 - CNRS UMR7258 – University of Aix-Marseille UM105 - Paoli Calmettes Institute (IPC), Label "Ligue contre le cancer", Marseille, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author François Bertucci François Bertucci orcid.org/0000-0002-0157-0959 Cancer Research Center of Marseille (CRCM), Laboratory of Predictive Oncology, Inserm U1068 - CNRS UMR7258 – University of Aix-Marseille UM105 - Paoli Calmettes Institute (IPC), Label "Ligue contre le cancer", Marseille, France Contribution: Resources, Investigation Search for more papers by this author Bénédicte Lemmers Bénédicte Lemmers orcid.org/0000-0002-2353-131X Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Damiana Pieragostino Damiana Pieragostino Department of Innovative Technologies in Medicine & Dentistry, Center for Advanced Studies and Technology (CAST), 'G. d'Annunzio' University of Chieti–Pescara, Chieti, Italy Contribution: Formal analysis, Investigation, Methodology Search for more papers by this author Emilie Mamessier Emilie Mamessier orcid.org/0000-0002-3516-0093 Cancer Research Center of Marseille (CRCM), Laboratory of Predictive Oncology, Inserm U1068 - CNRS UMR7258 – University of Aix-Marseille UM105 - Paoli Calmettes Institute (IPC), Label "Ligue contre le cancer", Marseille, France Contribution: Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Carsten Janke Carsten Janke orcid.org/0000-0001-7053-2000 Institut Curie, Paris Sciences et Lettres (PSL) Research University, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR) 3348, Label "Equipe FRM", Orsay, France Université Paris Sud, Université Paris-Saclay, CNRS UMR 3348, Orsay, France Contribution: Conceptualization, Formal analysis, Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Valérie Pinet Corresponding Author Valérie Pinet [email protected] orcid.org/0000-0002-4044-6440 Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Michael Hahne Corresponding Author Michael Hahne [email protected] orcid.org/0000-0002-3953-6430 Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France Contribution: Conceptualization, Supervision, Funding acquisition, Validation, Writing - original draft, Writing - review & editing Search for more papers by this author Author Information Conception Paul1,†, Ruizhi Tang1,†, Ciro Longobardi1,2,3, Rossano Lattanzio4, Thibaut Eguether5, Hulya Turali1, Julie Bremond1, Chloé Maurizy1, Monica Gabola1, Sophie Poupeau1, Andrei Turtoi6, Emilie Denicolai7, Maria Concetta Cufaro4, Magali Svrcek8, Philippe Seksik5, Vincent Castronovo9, Philippe Delvenne7,10, Vincenzo Laurenzi4, Quentin Da Costa7, François Bertucci7, Bénédicte Lemmers1, Damiana Pieragostino4, Emilie Mamessier7, Carsten Janke11,12, Valérie Pinet *,1,‡ and Michael Hahne *,1,‡ 1Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Label "Equipe FRM", Montpellier, France 2Center for Experimental and Molecular Medicine, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands 3Oncode Institute, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands 4Department of Innovative Technologies in Medicine & Dentistry, Center for Advanced Studies and Technology (CAST), 'G. d'Annunzio' University of Chieti–Pescara, Chieti, Italy 5Centre de Recherche Saint Antoine, Sorbonne Université, INSERM, APHP, Paris, France 6Tumor Microenvironment and Resistance to Treatment Laboratory, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France 7Cancer Research Center of Marseille (CRCM), Laboratory of Predictive Oncology, Inserm U1068 - CNRS UMR7258 – University of Aix-Marseille UM105 - Paoli Calmettes Institute (IPC), Label "Ligue contre le cancer", Marseille, France 8Department of Pathology, AP-HP, Hôpital Saint-Antoine, Sorbonne Université, Paris, France 9Metastasis Research Laboratory, GIGA Cancer, University of Liège, Liège, Belgium 10Department of Pathology, University Hospital (CHU), University of Liège, Liège, Belgium 11Institut Curie, Paris Sciences et Lettres (PSL) Research University, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR) 3348, Label "Equipe FRM", Orsay, France 12Université Paris Sud, Université Paris-Saclay, CNRS UMR 3348, Orsay, France † These authors contributed equally to this work as first authors ‡ These authors contributed equally to this work as senior authors *Corresponding author. Tel: +33 4 34 35 96 38; E-mail: [email protected] *Corresponding author. Tel: +33 4 34 35 96 38; E-mail: [email protected] EMBO Reports (2022)23:e55687https://doi.org/10.15252/embr.202255687 PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Primary cilia (PC) are important signaling hubs, and we here explored their role in colonic pathology. In the colon, PC are mostly present on fibroblasts, and exposure of mice to either chemically induced colitis-associated colon carcinogenesis (CAC) or dextran sodium sulfate (DSS)-induced acute colitis decreases PC numbers. We generated conditional knockout mice with reduced numbers of PC on colonic fibroblasts. These mice show increased susceptibility to CAC, as well as DSS-induced colitis. Secretome and immunohistochemical analyses of DSS-treated mice display an elevated production of the proinflammatory cytokine IL-6 in PC-deficient colons. An inflammatory environment diminishes PC presence in primary fibroblast cultures, which is triggered by IL-6 as identified by RNA-seq analysis together with blocking experiments. These findings suggest an activation loop between IL-6 production and PC loss. An analysis of PC presence on biopsies of patients with ulcerative colitis or colorectal cancer (CRC) reveals decreased numbers of PC on colonic fibroblasts in pathological compared with surrounding normal tissue. Taken together, we provide evidence that a decrease in colonic PC numbers promotes colitis and CRC. Synopsis Primary cilia are present on colonic fibroblasts and their decrease in number promotes colitis and colon carcinogenesis. In colitis, primary cilia loss appears to be ultimately linked to IL-6 signaling. Primary cilia (PC) are present on colonic fibroblasts. Induction of either acute colitis or colitis-associated colon carcinogenesis in mice decreases PC numbers. Primary cilia deficiency promotes colonic pathologies. PC numbers on colonic fibroblasts in biopsies of ulcerative colitis or colorectal cancer patients are decreased. Introduction Colorectal cancer (CRC) remains the third most common cause of cancer death in the western world (Malvezzi et al, 2018). CRC is a highly heterogenous disease and frequently diagnosed only after cancer has spread through the colon or rectal wall (stage II), to the lymph nodes (stage III) and/or to distant organs (stage IV), for example, liver, lung, and peritoneum. Most patients receiving standard-of-care treatment relapse within 5 years, highlighting the need to develop novel biomarker-guided therapeutic approaches to improve the treatment of CRC patients. A meta-analysis identified the existence of 4 distinct gene expression–based Consensus Molecular Subtypes (CMS) of CRC carcinoma, that is, immune (CMS1), canonical (CMS2), metabolic (CMS3), and mesenchymal (CMS4) subtypes, among which only CMS2 patients respond well to standard-of-care therapy (Guinney et al, 2015). CMS4 cancers, in which inflammation and fibroblasts are key drivers, have the worst survival rate. Notably, premalignant adenomas can also be stratified according to the CMS subtypes (Fessler et al, 2016). Interventions, which can be made at the early stages of tumor development, would increase the treatment efficiency. This is hampered at present, as the biology of pretumoral lesions in CRC is still poorly understood. It is estimated that only 5% of colonic adenoma progress to carcinoma. Thus, reliable indicators still need to be established, which would allow identifying lesions with high malignant potential. This includes a better understanding of the crosstalk between premalignant cells and cells of the microenvironment, among which are colonic fibroblasts (CF). An underexplored trait in the cellular crosstalk is primary cilia (PC), an antennae-like structure extruding from the surface of many mammalian cells. PC are cellular organelles serving as sensors of environmental signals (Gerdes et al, 2009). They contain a scaffold of nine microtubule (MT) doublets forming a cylinder-like arrangement in the plasma membrane by a basal body that derives from a centriole. Abnormal ciliary function result in so-called ciliopathies, that is, inherited disorders, such as cystic kidney diseases (Ko & Park, 2013). In vitro studies suggest an inflammation counteracting role of PC (Choi et al, 2021) and indeed, certain ciliopathies display inflammation (Song et al, 2017). PC can be also involved in tumor formation and a concept is emerging that there is an important inter- and intra-tumoral variation of PC presence (Eguether & Hahne, 2018; Liu et al, 2018). PC properties can be modulated by post-translational modifications (PTMs) of tubulin including acetylation and glycylation (Wloga et al, 2017). For example, it has been shown that blocking of deacetylation induces cilia restoration and decreases tumor growth of cholangiocarcinoma (Gradilone et al, 2013). We previously described an unexpected role of the tubulin glycylase TTLL3 in the regulation of colon tumorigenesis (Rocha et al, 2014). Specifically, we discovered that TTLL3 is the only glycylase expressed in the colon and that the absence of TTLL3 leads to decreased numbers of colonic PC (Rocha et al, 2014). When exposed to chemically induced colitis-associated colon carcinogenesis, Ttll3−/− mice are more susceptible to tumor formation (Rocha et al, 2014). Importantly, TTLL3 expression levels were significantly downregulated in human primary colorectal carcinomas and metastases as compared to healthy colon tissue, strongly suggesting a pivotal role of TTLL3 in colorectal cancer. Altogether, these findings reveal a correlation between TTLL3, loss of PC, and colon carcinogenesis. However, it cannot be excluded that TTLL3-catalyzed glycylation has nonciliary functions, especially because other substrates than tubulin can be glycylated. For instance, we observed glycylation of additional proteins in glycylase overexpressing cells (fig 1D in Gadadhar et al, 2017). The role of glycylation of these proteins is not yet explored and a contribution during colon carcinogenesis can therefore not be excluded. In this study, we set out to corroborate the involvement of PC in colonic pathologies by employing relevant mouse models and analyzing patients' biopsies. Results Colonic fibroblasts form primary cilia To characterize the presence of PC in the colon we employed a previously described protocol that allows the detection of PC on paraffin-embedded tissues (Coy et al, 2016). Indeed, this protocol allows to overcome the technical limitations we previously faced in detecting PC on cryosections of the colon and identifying which colonic cell subsets display PC (Rocha et al, 2014). Co-staining was performed by combining the established PC marker Arl13b (Caspary et al, 2007) with markers for epithelial or stromal cells including E-cadherin, vimentin, alpha-smooth muscle actin (α-SMA) and CD140a (platelet-derived growth factor receptor alpha; Fig 1A–D). Vimentin is a pan-fibroblast marker, α-SMA identifies myofibroblasts, whereas CD140a characterizes a subpopulation of fibroblasts present on the upper parts of the crypts (Kurahashi et al, 2013; Roulis & Flavell, 2016; Fig 1B–D). While only few epithelial cells displayed detectable PC (Figs 1A and EV1), PC were mostly found on vimentinpositive or CD140apositive stromal cells in the lamina propria (Fig 1B–D) but also on α-SMApositive myofibroblasts (Fig 1D). Figure 1. Primary cilia in the colon are mostly present on stromal cells A–D. PC were identified by immunostaining for Arl13b (in green except in (D); arrowheads in the panel on the right-hand side), and in parallel by identifying epithelial cells with E-cadherin (A), fibroblasts with vimentin (B), and CD140a (C), labeling (all in red), and myofibroblasts by α-SMA stain (D) (in green). Nuclei were stained with DAPI (in blue). Scale bars represent 30, 10, and 3 μm, respectively (from the left to the right). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Primary cilia are expressed by few E-cadherin+ colonic epithelial cellsCo-staining was performed for E-cadherin (red) and for primary cilia with Arl13b (arrowhead). Different z-stack slices are shown to validate the co-expression of Arl13b and E-cadherin (slices 13–19 represent 1.2 μm thickness). Nuclei were stained with DAPI (in blue). Scale bar represents 10 μm. Download figure Download PowerPoint Progressive loss of colonic primary cilia during colon carcinogenesis To determine how PC presence relates to colon carcinogenesis, we quantified the number of PC in a mouse model that mimics colitis-associated colon carcinogenesis (CAC) (Suzuki et al, 2004; Tanaka, 2012). This model depends on the administration of the mutagen azoxymethane (AOM) and the subsequent induction of inflammation with dextran sodium sulfate (DSS) (Fig 2A). DSS is toxic to mucosal epithelial cells in the colon, and the ensuing breakdown of the mucosal barrier leads to inflammation. The CAC mouse model allows to follow the progression from preneoplastic lesions to adenocarcinoma within 60 days. In this model, PC on vimentinpositive cells in tumor lesions was lower as compared to adjacent normal mucosa (Fig 2B and C). Remarkably, colon areas with high-grade dysplasia showed significantly lower numbers of PC than those with low-grade dysplasia. Taken together, CAC is associated with the downregulation of PC, confirming our initial hypothesis. Figure 2. The number of colonic primary cilia decreases during colon carcinogenesis Experimental timeline for azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CAC. Hematoxylin/Eosin staining of paraffin-sections from normal colon and colon of AOM/DSS-treated C57Bl/6 mice as indicated in (A). Representative areas of normal colon and colon with low- and high-grade dysplasia are shown. Features include hyperchromasia, stratification, and elongated cell nuclei in low-grade dysplasia and marked hyperchromasia and increased pleomorphism of nuclei, as well as loss of cell polarity in high-grade dysplasia. Scale bars represent 100 μm. Box-and-whisker plots showing quantitative analysis of PC expression on vimentin+ cells in normal colon, and colon with low- and high-grade dysplasia in mice exposed to the AOM/DSS protocol (analyzing at least 12 fields depicted from four different mice as illustrated in the middle and right panel of (B)). The box plot shows the median (inside line), 25–75 percentiles (box bottom to top), and the Whiskers connect the minimum and the maximum values to the Box. Significance values were determined by two-tailed unpaired t-tests (***p < 10−3). Download figure Download PowerPoint Decreased numbe
Tryptophan, an essential amino acid, and its metabolites are involved in many physiological processes including neuronal functions, immune system, and gut homeostasis. Alterations to tryptophan metabolism are associated with various pathologies such as neurologic, psychiatric disorders, inflammatory bowel diseases (IBD), metabolic disorders, and cancer. It is consequently critical to develop a reliable, quantitative method for the analysis of tryptophan and its downstream metabolites from the kynurenine, serotonin, and indoles pathways. An LC-MS/MS method was designed for the analysis of tryptophan and 20 of its metabolites, without derivatization and performed in a single run. This method was validated for both serum and stool. The comparisons between serum and plasma, collected with several differing anticoagulants, showed significant differences only for serotonin. References values were established in sera and stools from healthy donors. For stool samples, as a proof of concept, the developed method was applied to a healthy control group and an IBD patient group. Results showed significant differences in the concentrations of tryptophan, xanthurenic acid, kynurenic acid, indole-3-lactic acid, and picolinic acid. This method allowed an extensive analysis of the three tryptophan metabolic pathways in two compartments. Beyond the application to IBD patients, the clinical use of this method is wide-ranging and may be applied to other pathological conditions involving tryptophan metabolism, such as neurological, psychiatric, or auto-inflammatory pathologies.
The 13 subtypes of oral-facial-digital syndrome (OFDS) belong to the heterogeneous group of ciliopathies. Disease-causing genes encode for centrosomal proteins, components of the transition zone or proteins implicated in ciliary signaling. A unique consanguineous family presenting with an unclassified OFDS with skeletal dysplasia and brachymesophalangia was explored. Homozygosity mapping and exome sequencing led to the identification of a homozygous mutation in IFT57, which encodes a protein implicated in ciliary transport. The mutation caused splicing anomalies with reduced expression of the wild-type transcript and protein. Both anterograde ciliary transport and sonic hedgehog signaling were significantly decreased in subjects' fibroblasts compared with controls. Sanger sequencing of IFT57 in 13 OFDS subjects and 12 subjects with Ellis-Van Creveld syndrome was negative. This report identifies the implication of IFT57 in human pathology and highlights the first description of a ciliary transport defect in OFDS, extending the genetic heterogeneity of this subgroup of ciliopathies.
