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