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Proteome from whole epididymis and caput epididymidis of WT and Ocln knockout mice

ABSTRACT: Epithelial tight junction is the frontline barrier for physiological homeostasis and protection of a host and Occludin (Ocln) is a necessary component of the tight junction complex. Male Ocln knockout (Ocln-KO) mice are overall normal but infertile. The cause of this phenotype remains elusive. We found that the infertility of Ocln-mutant males is due to defects in sperm motility and capacitation – the essential functions acquired during sperm maturation in the epididymis – but not spermatogenesis in the testis, which is normal in these mutant mice. Using epididymis as an epithelial model in Ocln-KO male mice, combining transcriptomic and proteomic analyses, we found a new unconventional phagocytosis mechanism that regulates membrane cholesterol and is featured by forming double-membrane phagosome derived from the plasma membranes of epithelial TJs and. This process, which we term paracellular heterophagy or paraphagy, involves the interaction of Ocln at the TJs with the participation of autophagy machinery and a surface receptor for lipophilic cargo endocytosis to promote cholesterol removal from TJ plasma membranes. We also provide evidence that this is a general mechanism applicable to other epithelia, including airway bronchia, renal tubules, digestive intestine and circulation endothelium, but is particularly profound in epididymis in reproductive tract. Thus, we show that paraphagy at TJs by involving Ocln is a novel mechanism which senses the luminal lipophilic signals and modulates cholesterol homeostasis in epithelia.

ORGANISM(S): Mus Musculus

SUBMITTER: Winnie Shum

PROVIDER: PXD024008 | iProX | Fri Feb 05 00:00:00 GMT 2021

REPOSITORIES: iProX

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Knockout mice reveal key roles for claudin-18 in alveolar fluid homeostasis.

Project description:Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJ), where they serve as regulators of paracellular permeability to ions and solutes. Claudin-18, a member of the large claudin family, is highly expressed in lung epithelium. To elucidate the role of claudin-18 in alveolar epithelial barrier function and fluid homeostasis, we generated claudin-18 knockout (C18 KO) mice. Increased alveolar fluid clearance (AFC) observed in C18 KO mice may have accounted for absence of lung edema despite increased alveolar solute permeability compared to wild type (WT) controls. Higher AFC in C18 KO mice was associated with higher Na-K-ATPase activity and increased expression of the Na-K-ATPase β1 subunit compared to WT controls. Consistent with in vivo findings, alveolar epithelial cell (AEC) monolayers derived from C18 KO mice exhibited lower transepithelial electrical resistance (RT) accompanied by increased solute and ion permeability without changes in ion selectivity. Expression of claudin-3 and claudin-4 was markedly increased in whole lung and in freshly isolated AEC from C18 KO mice, while claudin-5 was unchanged. In contrast, occludin, another major component of the TJ complex, was significantly decreased in C18 KO lung. Further analysis revealed rearrangements in the F-actin cytoskeleton in C18 KO MAECM. These findings demonstrate a crucial non-redundant role for claudin-18 in regulation of alveolar epithelial tight junction composition and permeability to ions and solutes. Importantly, increased AFC in C18 KO mice identifies additional roles for claudin-18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties of the alveolar epithelium. Animals with a ubiquitous knockout (C18 KO) were obtained by crossing mice harboring a conditional (floxed) allele of claudin-18 (Cldn18F/F) with CMV-cre deleter mice to delete exons 2 and 3 by Cre/loxP recombination.

2014-04-06 | E-GEOD-48443 | biostudies-arrayexpress

Knockout mice reveal key roles for claudin-18 in alveolar fluid homeostasis.

2014-04-06 | GSE48443 | GEO

The Tight Junction Protein Claudin-2 Controls Mucosal Permeability, Immune Tolerance and Colitis in Mice

Project description:The mucosal epithelium plays a key role in regulating immune homeostasis. Dysregulation of epithelial barrier function is associated with mucosal inflammation. Expression of claudin-2, a pore-forming tight junction protein, is highly upregulated during inflammatory bowel disease (IBD) and, due to its association with epithelial permeability, has been postulated to promote inflammation. Furthermore, claudin-2 also regulates colonic epithelial cell proliferation and intestinal nutrient absorption. However, the precise role of claudin-2 in regulating colonic epithelial and immune homeostasis remains unclear. Here, we demonstrate, using Villin-Claudin-2 transgenic (Cl-2TG) mice, that increased colonic claudin-2 expression unexpectedly protects mice against experimentally induced colitis and colitis-associated cancer. Notably, Cl-2TG mice exhibited increased colon length and permeability as compared with wild type (WT) littermates. However, despite their leaky colon, Cl-2TG mice subjected to experimental colitis were immune compromised, with reduced induction of TLR-2, TLR-4, Myd-88 expression and NF-kB and STAT3 activation. Most importantly, colonic macrophages in Cl-2TG mice exhibited an anergic phenotype. Claudin-2 overexpression also increased colonocyte proliferation and provided protection against colitis-induced colonocyte death. Taken together, our findings have revealed a critical role of claudin-2 in regulating colonic homeostasis, suggesting novel therapeutic strategies for inflammatory conditions of the gastrointestinal tract. 8-10 weeks old male Villin-Claudin-2 transgenic mice and WT littermates were provided either normal drinking water (control) or Dextran Sodium Sulfate (DSS: 4% w/v) for 10 days. 3 replicates each.

2013-09-15 | E-GEOD-46006 | biostudies-arrayexpress

Sperm proteome of CMTM4-deficient mice

Project description:Cmtm4 Knockout mouse was constructed by CRISPR-Cas9 technology, and the KO mice showed male infertility. A proteomic anlaysis of sperm proteins was performed by comparing KO and WT mice.

2019-05-21 | PXD013092 | Pride

Loss-of-function mutations in QRICH2 cause male infertility with multiple morphological abnormalities of the sperm flagella

Project description:Aberrant sperm flagella impair sperm motility and cause male infertility, yet the genes which have been identified in multiple morphological abnormalities of the flagella (MMAF) can only explain the pathogenic mechanisms of MMAF in a small number of cases. Here, we identify and functionally characterize homozygous loss-of-function mutations of QRICH2 in two infertile males with MMAF from two consanguineous families. Remarkably, Qrich2 knock-out (KO) male mice constructed by CRISPR-Cas9 technology present MMAF phenotypes and sterility. To elucidate the mechanisms of Qrich2 functioning in sperm flagellar formation, we perform proteomic analysis on the testes of KO and wild-type mice. Furthermore, in vitro experiments indicate that QRICH2 is involved in sperm flagellar development through stabilizing and enhancing the expression of proteins related to flagellar development. Our findings strongly suggest that the genetic mutations of human QRICH2 can lead to male infertility with MMAF and that QRICH2 is essential for sperm flagellar formation.

2019-11-12 | PXD011730 | Pride

Embryonic exposure to aluminium chloride blocks the onset of spermatogenesis through disturbing the dynamics of testicular tight junctions via upregulating Slc25a5 in offspring

Project description:Studies reveal neurotoxicity, hepatotoxicity, and developmental and reproductive toxicity in mice exposed to aluminium (Al). However, relatively few researches are conducted to clarify the mechanism of the impact of embryonic exposure to aluminium chloride (AlCl3) on the development of the reproductive system in male offspring. Here, pregnant mice were fed AlCl3 by gavage from day 12.5 of gestation. The results showed that embryonic AlCl3 exposure disrupted testicular development and spermatogenesis by damaging the testicular structure, reducing sperm counts, and altering the expression of the tight junction (TJ) proteins between Sertoli cells (SCs). Further in vitro studies showed that AlCl3 treatment stabilized the TJ proteins Occludin and ZO-1 expression by inhibiting the ERK signaling pathway to upregulate the Slc25a5 protein, which prompted ATP production to disrupt cytoskeletal protein homeostasis. Therefore, our study provided a new mechanism for AlCl3 exposure to interfere with testicular development and spermatogenesis and implied that Slc25a5 could be a target of AlCl3 affecting cell metabolism.

2023-09-06 | GSE242190 | GEO

PCSK9 deficiency results in a specific shedding of excess LDLR in female mice only: Role of hepatic cholesterol

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2022-09-22 | GSE205008 | GEO

Effect of Lactobacillus plantarum MB452 on the gene expression of the intestinal epithelial cell line Caco-2

Project description:The hypothesis of this research was that probiotic bacteria that increase intestinal barrier function achieve this, partly, by increasing the expression of the genes involved in tight junction signalling in healthy intestinal epithelial cells. L. plantarum MB452 isolated from the probiotic product VSL#3 was chosen as the test bacterium because it has a robust, repeatable, positive effect tight junction integrity, as measured by the trans-epithelial electrical resistance (TEER) in vitro.

2009-10-07 | GSE18372 | GEO

Expression profile of Ras, MEK and ERK inhibited 16HBE bronchial epithelial cells

Project description:To identify Ras, MEK and ERK target genes which regulate tight junction formation in 16HBE bronchial epithelial cells

2018-12-01 | GSE63229 | GEO

MRP5 and MRP9 Play a Concerted Role in Male Reproduction and Mitochondrial Function

Project description:Multidrug Resistance Proteins (MRPs) are transporters that play critical roles in cancer even though the physiological substrates of these enigmatic transporters are poorly elucidated. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter as mrp-5 mutants are unviable due to their inability to export heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. By contrast, MRP5 in mammals regulates heme levels in the secretory pathway and MRP5 knockout (KO) mice do not show reproductive phenotypes. The closest homolog of MRP5 in mammals is MRP9/ABCC12, which is absent in C. elegans raising the possibility that MRP9 may genetically compensate for MRP5 deficiency. Here, we show that MRP5 and MRP9 double KO mice are viable but reveal significant male reproductive deficits. Although MRP9 is highly expressed in sperm, MRP9 KO mice show reproductive phenotypes only in the absence of MRP5. Both ABCC transporters localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, DKO mice reveal abnormal sperm mitochondria with reduced mitochondrial membrane potential and fertilization rates. Metabolomics show striking differences in metabolite profiles in the DKO testes and RNA-seq show significant alterations in genes related to mitochondria function and retinoic acid metabolism. Targeted functional metabolomics reveal lower retinoic acid levels in the DKO testes and higher levels of triglycerides in the mitochondria. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating male reproductive functions and mitochondrial sufficiency.

2022-01-19 | GSE176740 | GEO

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