3D epithelial cell culture; connexin; fluorescence loss in photobleaching (FLIP); gap junction intercellular communication (GJIC); human papillomavirus (HPV); viral replication; Connexins; Connexin 43; Humans; Connexins/genetics; Connexins/metabolism; Connexin 43/genetics; Connexin 43/metabolism; Human Papillomavirus Viruses; Gap Junctions/metabolism; Epithelium; Cell Communication/physiology; Cell Transformation, Neoplastic; Papillomavirus Infections; Carcinoma, Squamous Cell; Microbiology; Immunology; Microbiology (medical); Infectious Diseases
Abstract :
[en] Human papillomaviruses (HPVs) are highly prevalent commensal viruses that require epithelial stratification to complete their replicative cycle. While HPV infections are most often asymptomatic, certain HPV types can cause lesions, that are usually benign. In rare cases, these infections may progress to non-replicative viral cycles associated with high HPV oncogene expression promoting cell transformation, and eventually cancer when not cleared by host responses. While the consequences of HPV-induced transformation on keratinocytes have been extensively explored, the impact of viral replication on epithelial homeostasis remains largely unexplored. Gap junction intercellular communication (GJIC) is critical for stratified epithelium integrity and function. This process is ensured by a family of proteins named connexins (Cxs), including 8 isoforms that are expressed in stratified squamous epithelia. GJIC was reported to be impaired in HPV-transformed cells, which was attributed to the decreased expression of the Cx43 isoform. However, it remains unknown whether and how HPV replication might impact on the expression of Cx isoforms and GJIC in stratified squamous epithelia. To address this question, we have used 3D-epithelial cell cultures (3D-EpCs), the only model supporting the productive HPV life cycle. We report a transcriptional downregulation of most epithelial Cx isoforms except Cx45 in HPV-replicating epithelia. At the protein level, HPV replication results in a reduction of Cx43 expression while that of Cx45 increases and displays a topological shift toward the cell membrane. To quantify GJIC, we pioneered quantitative gap-fluorescence loss in photobleaching (FLIP) assay in 3D-EpCs, which allowed us to show that the reprogramming of Cx landscape in response to HPV replication translates into accelerated GJIC in living epithelia. Supporting the pathophysiological relevance of our observations, the HPV-associated Cx43 and Cx45 expression pattern was confirmed in human cervical biopsies harboring HPV. In conclusion, the reprogramming of Cx expression and distribution in HPV-replicating epithelia fosters accelerated GJIC, which may participate in epithelial homeostasis and host immunosurveillance.
Disciplines :
Oncology
Author, co-author :
Gallego, Carmen; Inflammation, Microbiome and Immunosurveillance, INSERM UMR-996, Université Paris-Saclay, Orsay, France
Jaracz-Ros, Agnieszka; Inflammation, Microbiome and Immunosurveillance, INSERM UMR-996, Université Paris-Saclay, Orsay, France
Laganà, Marta; Inflammation, Microbiome and Immunosurveillance, INSERM UMR-996, Université Paris-Saclay, Orsay, France
Mercier-Nomé, Françoise; Inflammation, Microbiome and Immunosurveillance, INSERM UMR-996, Université Paris-Saclay, Orsay, France ; US31-UMS3679-Plateforme PHIC, Ingénierie et Plateformes au Service de l'Innovation Thérapeutique (IPSIT), INSERM, CNRS, Université Paris-Saclay, Orsay, France
Domenichini, Séverine; UMS-IPSIT Plateforme MIPSIT, Université Paris-Saclay, CNRS, Inserm, Ingénierie et Plateformes au Service de l'Innovation Thérapeutique, Orsay, France
Fumagalli, Amos; CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, France
Roingeard, Philippe; INSERM U1259, Université de Tours et CHRU de Tours & Plateforme IBiSA des Microscopies, PPF ASB, CHRU de Tours, Tours, France
Herfs, Michael ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques
Pidoux, Guillaume; INSERM UMR-S 1180, Université Paris-Saclay, Orsay, France
Bachelerie, Françoise; Inflammation, Microbiome and Immunosurveillance, INSERM UMR-996, Université Paris-Saclay, Orsay, France
Schlecht-Louf, Géraldine; Inflammation, Microbiome and Immunosurveillance, INSERM UMR-996, Université Paris-Saclay, Orsay, France
Language :
English
Title :
Reprogramming of connexin landscape fosters fast gap junction intercellular communication in human papillomavirus-infected epithelia.
ANR - Agence Nationale de la Recherche [FR] FRM - Fondation pour la Recherche Médicale [FR] UE - Union Européenne [BE]
Funding text :
This work was supported by grants from the ERA-Net Infect-ERA “HPV-MOTIVA” (ANR-15-IFEC-0004-01; FB and GS-L; http://www.infect-era.eu/ ) and MSCA-ITN-2014-ETN - Marie Skłodowska-Curie Innovative Training Networks (ITN-ETN) “ONCOgenic Receptor Network of Excellence and Training” (FB and GS-L; http://www.oncornet.eu/ ). CG was supported by the MSCA-ITN-2014-ETN - Marie Skłodowska-Curie Innovative Training Networks (ITN-ETN) “ONCOgenic Receptor Network of Excellence and Training” (Grant agreement ANR-15-IFEC-0004-01 641833 ONCORNET) and the Fondation pour la Recherche Médicale (FDT201805005700; https://www.frm.org/ ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. AcknowledgmentsThe authors thank Morgan Ocimek for excellent technical support. They especially thank Dr. Paul F. Lambert (WI Institutes for Medical Research, USA) for kindly providing the 3T3 and NIKS cell lines. They greatly acknowledge the Cell and Tissue Imaging (PICT-IBiSA), Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04; https://france-bioimaging.org/about/ ) and thank Vincent Fraisier for his help. The authors thank the Vista research team at Irisa/Inria Rennes for the use of the Motion2D software () through the Mobyle portal. The authors also thank the PLAIMMO facility (Ingénierie et Plateformes au Service de l’Innovation Thérapeutique (IPSIT), https://www.ipsit.universite-paris-saclay.fr ) as well as the financial support of Île-de-France region for this facility.
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