ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell– cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architec-ture during inflammatory stressWe gratefully acknowledge the expert technical advice of the Confocal Microscopy, Electron Microscopy, and Genomic facilities of the CBM Severo Ochoa, especially the contribution of Milagros Guerra, from the electron microscopy facility. We thank the staff of the Advanced Light Microscopy and cryo-EM facilities of the CNB-CSIC for their expert technical assistance with the correlative Cryo-SXT. We also thank Dr. Eva Pereiro at ALBA Synchrotron Light Source (Cerdanyola del Vall\u00E8s, Spain) for her expert technical advice, Prof. Nancy Hogg at the Francis Crick Institute (London, UK) for generating and sharing the Icam1_KO mouse, Dr. Luc\u00EDa Cordero Espinoza at the Gurdon Institute (Cambridge, UK) for her technical support in generating liver organoids, and Ana L\u00F3pez Sancha for her technical support with isolating the PBMCs. The work was supported by grants PID2020-119881RB-I00 from AEI (to CC-N, CL-P, NC-A, SB, GdR, JF, and JM) and P2022/BMD-7232 TomoXliver2 (to AC, SB, JMC, and JM), and IND2019/BMD-17139 (to JM) from Comunidad de Madrid. This research work was also funded by the European Commission\u2500NextGenerationEU (Regulation EU 2020/2094), through CSIC\u2019s Global Health Platform (PTI Salud Global). SB is supported by Endocornea, Convenio Colaboraci\u00F3n CSIC, funded by Instituto de Investigaci\u00F3n Fundaci\u00F3n Jim\u00E9nez D\u00EDaz. CM acknowledges support through the grant PID2021-125386NB-I00 funded by MCIN/AEI/10.13039/501100011033/and FEDER 'ERDF A way of making Europe'. CC-N is a recipient of FPI fellowships from MINECO. NC-A is a recipient of an FPU fellowship from MECD. NR-R is supported by funding from the People Programme (Marie Curie Actions) of the European Union\u2019s Seventh Framework Programme (FP7/2007\u20132013) under REA grant agreement no. 608765 and also by Ram\u00F3n y Cajal program, grant RYC2021-031221-I and grant PID2022-137552OA-I00 from AE

Plasmolipin regulates basolateral-to-apical transcytosis of ICAM-1 and leukocyte adhesion in polarized hepatic epithelial cells

Apical localization of Intercellular Adhesion Receptor (ICAM)-1 regulates the adhesion and guidance of leukocytes across polarized epithelial barriers. Here, we investigate the molecular mechanisms that determine ICAM-1 localization into apical membrane domains of polarized hepatic epithelial cells, and their effect on lymphocyte-hepatic epithelial cell interaction. We had previously shown that segregation of ICAM-1 into apical membrane domains, which form bile canaliculi and bile ducts in hepatic epithelial cells, requires basolateral-to-apical transcytosis. Searching for protein machinery potentially involved in ICAM-1 polarization we found that the SNARE-associated protein plasmolipin (PLLP) is expressed in the subapical compartment of hepatic epithelial cells in vitro and in vivo. BioID analysis of ICAM-1 revealed proximal interaction between this adhesion receptor and PLLP. ICAM-1 colocalized and interacted with PLLP during the transcytosis of the receptor. PLLP gene editing and silencing increased the basolateral localization and reduced the apical confinement of ICAM-1 without affecting apicobasal polarity of hepatic epithelial cells, indicating that ICAM-1 transcytosis is specifically impaired in the absence of PLLP. Importantly, PLLP depletion was sufficient to increase T-cell adhesion to hepatic epithelial cells. Such an increase depended on the epithelial cell polarity and ICAM-1 expression, showing that the epithelial transcytotic machinery regulates the adhesion of lymphocytes to polarized epithelial cells. Our findings strongly suggest that the polarized intracellular transport of adhesion receptors constitutes a new regulatory layer of the epithelial inflammatory respons

Estudio de los mecanismos moleculares que median la polarización apical u función de ICAM-1 en células epiteliales hépaticas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 25-06-2020Esta tesis tiene embargado el acceso al texto completo hasta el 25-12-202

Compensatory increase of VE-cadherin expression through ETS1 regulates endothelial barrier function in response to TNFα

VE-cadherin plays a central role in controlling endothelial barrier function, which is transiently disrupted by proinflammatory cytokines such as tumor necrosis factor (TNFα). Here we show that human endothelial cells compensate VE-cadherin degradation in response to TNFα by inducing VE-cadherin de novo synthesis. This compensation increases adherens junction turnover but maintains surface VE-cadherin levels constant. NF-κB inhibition strongly reduced VE-cadherin expression and provoked endothelial barrier collapse. Bacterial lipopolysaccharide and TNFα upregulated the transcription factor ETS1, in vivo and in vitro, in an NF-κB dependent manner. ETS1 gene silencing specifically reduced VE-cadherin protein expression in response to TNFα and exacerbated TNFα-induced barrier disruption. We propose that TNFα induces not only the expression of genes involved in increasing permeability to small molecules and immune cells, but also a homeostatic transcriptional program in which NF-κB- and ETS1-regulated VE-cadherin expression prevents the irreversible damage of endothelial barriers.SAF2017-88187-R and S2017/BMD-3817 TomoXliver (to J.M.), BFU2015–67266-R (to I.C) and Instituto de Salud Carlos III (PI18/01662 to CR, co-funded with European FEDER contribution) and of the Programa de Actividades en Biomedicina de la Comunidad de Madrid-B2017/BMD-3671-INFLAMUNE; An institutional support of Fundación Ramón Areces to the CBMSO and MINEC

Compensatory increase of VE-cadherin expression through ETS1 regulates endothelial barrier function in response to TNF¿

VE-cadherin plays a central role in controlling endothelial barrier function, which is transiently disrupted by proinflammatory cytokines such as tumor necrosis factor (TNFα). Here we show that human endothelial cells compensate VE-cadherin degradation in response to TNFα by inducing VE-cadherin de novo synthesis. This compensation increases adherens junction turnover but maintains surface VE-cadherin levels constant. NF-κB inhibition strongly reduced VE-cadherin expression and provoked endothelial barrier collapse. Bacterial lipopolysaccharide and TNFα upregulated the transcription factor ETS1, in vivo and in vitro, in an NF-κB dependent manner. ETS1 gene silencing specifically reduced VE-cadherin protein expression in response to TNFα and exacerbated TNFα-induced barrier disruption. We propose that TNFα induces not only the expression of genes involved in increasing permeability to small molecules and immune cells, but also a homeostatic transcriptional program in which NF-κB- and ETS1-regulated VE-cadherin expression prevents the irreversible damage of endothelial barriersSAF2017-88187-R and S2017/BMD-3817 TomoXliver (to J.M.), BFU2015–67266-R (to I.C) and Instituto de Salud Carlos III (PI18/01662 to CR, co-funded with European FEDER contribution) and of the Programa de Actividades en Biomedicina de la Comunidad de Madrid-B2017/BMD-3671-INFLAMUNE. S.B.F is supported by Endocornea2, convenio colaboración CSIC, funded by Instituto de Investigación Fundación Jiménez Díaz. An institutional support of Fundación Ramón Areces to the CBMS

Molecular Insights of Cholestasis in MDR2 Knockout Murine Liver Organoids

MDR3 (multidrug resistance 3) deficiency in humans (MDR2 in mice) causes progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 is a lethal disease characterized by an early onset of intrahepatic cholestasis progressing to liver cirrhosis, a preneoplastic condition, putting individuals at risk of hepatocellular carcinoma (HCC). Hepatocyte-like organoids from MDR2-deficient mice (MDR2KO) were used in this work to study the molecular alterations caused by the deficiency of this transporter. Proteomic analysis by mass spectrometry allowed characterization of 279 proteins that were differentially expressed in MDR2KO compared with wild-type organoids. Functional enrichment analysis indicated alterations in three main cellular functions: (1) interaction with the extracellular matrix, (2) remodeling intermediary metabolism, and (3) cell proliferation and differentiation. The affected cellular processes were validated by orthogonal molecular biology techniques. Our results point to molecular mechanisms associated with PFIC3 that may drive the progression to liver cirrhosis and HCC and suggest proteins and cellular processes that could be targeted for the development of early detection strategies for these severe liver diseases.Peer reviewe

Molecular Insights of Cholestasis in MDR2 Knockout Murine Liver Organoids

MDR3 (multidrug resistance 3) deficiency in humans (MDR2 in mice) causes progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 is a lethal disease characterized by an early onset of intrahepatic cholestasis progressing to liver cirrhosis, a preneoplastic condition, putting individuals at risk of hepatocellular carcinoma (HCC). Hepatocyte-like organoids from MDR2-deficient mice (MDR2KO) were used in this work to study the molecular alterations caused by the deficiency of this transporter. Proteomic analysis by mass spectrometry allowed characterization of 279 proteins that were differentially expressed in MDR2KO compared with wild-type organoids. Functional enrichment analysis indicated alterations in three main cellular functions: (1) interaction with the extracellular matrix, (2) remodeling intermediary metabolism, and (3) cell proliferation and differentiation. The affected cellular processes were validated by orthogonal molecular biology techniques. Our results point to molecular mechanisms associated with PFIC3 that may drive the progression to liver cirrhosis and HCC and suggest proteins and cellular processes that could be targeted for the development of early detection strategies for these severe liver diseases

Molecular Insights of Cholestasis in MDR2 Knockout Murine Liver Organoids

MDR3 (multidrug resistance 3) deficiency in humans (MDR2 in mice) causes progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 is a lethal disease characterized by an early onset of intrahepatic cholestasis progressing to liver cirrhosis, a preneoplastic condition, putting individuals at risk of hepatocellular carcinoma (HCC). Hepatocyte-like organoids from MDR2-deficient mice (MDR2KO) were used in this work to study the molecular alterations caused by the deficiency of this transporter. Proteomic analysis by mass spectrometry allowed characterization of 279 proteins that were differentially expressed in MDR2KO compared with wild-type organoids. Functional enrichment analysis indicated alterations in three main cellular functions: (1) interaction with the extracellular matrix, (2) remodeling intermediary metabolism, and (3) cell proliferation and differentiation. The affected cellular processes were validated by orthogonal molecular biology techniques. Our results point to molecular mechanisms associated with PFIC3 that may drive the progression to liver cirrhosis and HCC and suggest proteins and cellular processes that could be targeted for the development of early detection strategies for these severe liver diseases