Fibrosis contributes to tissue repair, but excessive fibrosis disrupts organ function. Alagille syndrome (ALGS, caused by mutations in JAGGED1) results in liver disease and characteristic fibrosis. Here, we show that Jag1Ndr/Ndr mice, a model for ALGS, recapitulate ALGS-like fibrosis. Single-cell RNA-seq and multi-color flow cytometry of the liver revealed immature hepatocytes and paradoxically low intrahepatic T cell infiltration despite cholestasis in Jag1Ndr/Ndr mice. Thymic and splenic regulatory T cells (Tregs) were enriched and Jag1Ndr/Ndr lymphocyte immune and fibrotic capacity was tested with adoptive transfer into Rag1-/- mice, challenged with dextran sulfate sodium (DSS) or bile duct ligation (BDL). Transplanted Jag1Ndr/Ndr lymphocytes were less inflammatory with fewer activated T cells than Jag1+/+ lymphocytes in response to DSS. Cholestasis induced by BDL in Rag1-/- mice with Jag1Ndr/Ndr lymphocytes resulted in periportal Treg accumulation and three-fold less periportal fibrosis than in Rag1-/- mice with Jag1+/+ lymphocytes. Finally, the Jag1Ndr/Ndr hepatocyte expression profile and Treg overrepresentation were corroborated in patients' liver samples. Jag1-dependent hepatic and immune defects thus interact to determine the fibrotic process in ALGS.

• MeSH
• Alagillův syndrom patologie   genetika   MeSH
• buněčná diferenciace * MeSH
• hepatocyty * metabolismus   patologie   MeSH
• jaterní cirhóza * patologie   genetika   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• protein jagged-1 * metabolismus   genetika   MeSH
• regulační T-lymfocyty imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

[Figure: see text].

• Publikační typ
• časopisecké články MeSH

ADAM10 and ADAM17 are proteases that affect multiple signalling pathways by releasing molecules from the cell surface. As their substrate specificities partially overlaps, we investigated their concurrent role in liver regeneration and fibrosis, using three liver-specific deficient mouse lines: ADAM10- and ADAM17-deficient lines, and a line deficient for both proteases. In the model of partial hepatectomy, double deficient mice exhibited decreased AKT phosphorylation, decreased release of EGFR activating factors and lower shedding of HGF receptor c-Met. Thus, simultaneous ablation of ADAM10 and ADAM17 resulted in inhibited EGFR signalling, while HGF/c-Met signalling pathway was enhanced. In contrast, antagonistic effects of ADAM10 and ADAM17 were observed in the model of chronic CCl4 intoxication. While ADAM10-deficient mice develop more severe fibrosis manifested by high ALT, AST, ALP and higher collagen deposition, combined deficiency of ADAM10 and ADAM17 surprisingly results in comparable degree of liver damage as in control littermates. Therefore, ADAM17 deficiency is not protective in fibrosis development per se, but can ameliorate the damaging effect of ADAM10 deficiency on liver fibrosis development. Furthermore, we show that while ablation of ADAM17 resulted in decreased shedding of TNF RI, ADAM10 deficiency leads to increased levels of soluble TNF RI in serum. In conclusion, hepatocyte-derived ADAM10 and ADAM17 are important regulators of growth receptor signalling and TNF RI release, and pathological roles of these proteases are dependent on the cellular context.

• MeSH
• fibróza metabolismus   MeSH
• játra * metabolismus   patologie   MeSH
• kultivované buňky MeSH
• membránové proteiny fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nemoci jater * metabolismus   patologie   MeSH
• primární buněčná kultura MeSH
• protein ADAM10 fyziologie   MeSH
• protein ADAM17 fyziologie   MeSH
• regenerace jater * MeSH
• sekretasy fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Plectin, a highly versatile cytolinker protein, provides tissues with mechanical stability through the integration of intermediate filaments (IFs) with cell junctions. Here, we hypothesize that plectin-controlled cytoarchitecture is a critical determinant of the intestinal barrier function and homeostasis. Mice lacking plectin in an intestinal epithelial cell (IEC; PleΔIEC) spontaneously developed colitis characterized by extensive detachment of IECs from the basement membrane (BM), increased intestinal permeability, and inflammatory lesions. Moreover, plectin expression was reduced in the colons of ulcerative colitis (UC) patients and negatively correlated with the severity of colitis. Mechanistically, plectin deficiency in IECs led to aberrant keratin filament (KF) network organization and the formation of dysfunctional hemidesmosomes (HDs) and intercellular junctions. In addition, the hemidesmosomal α6β4 integrin (Itg) receptor showed attenuated association with KFs, and protein profiling revealed prominent downregulation of junctional constituents. Consistent with the effects of plectin loss in the intestinal epithelium, plectin-deficient IECs exhibited remarkably reduced mechanical stability and limited adhesion capacity in vitro. Feeding mice with a low-residue liquid diet that reduced mechanical stress and antibiotic treatment successfully mitigated epithelial damage in the PleΔIEC colon.

• MeSH
• desmozomy genetika   metabolismus   MeSH
• dospělí MeSH
• keratiny metabolismus   MeSH
• kolitida metabolismus   prevence a kontrola   MeSH
• kolon patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• modely nemocí na zvířatech MeSH
• myši knockoutované MeSH
• myši MeSH
• plektin genetika   metabolismus   MeSH
• senioři MeSH
• střevní sliznice metabolismus   patologie   MeSH
• ulcerózní kolitida metabolismus   prevence a kontrola   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• myši MeSH
• senioři MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

• MeSH
• aktiny metabolismus   MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• cytoskelet metabolismus   MeSH
• extracelulární matrix metabolismus   MeSH
• hepatocyty metabolismus   patologie   MeSH
• kolagen metabolismus   MeSH
• kultivované buňky MeSH
• mikrofilamenta MeSH
• mikrotubuly metabolismus   MeSH
• myši MeSH
• žluč metabolismus   MeSH
• žlučové kanálky metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND & AIMS: Plectin, a highly versatile cytolinker protein, controls intermediate filament cytoarchitecture and cellular stress response. In the present study, we investigate the role of plectin in the liver under basal conditions and in experimental cholestasis. METHODS: We generated liver-specific plectin knockout (PleΔalb) mice and analyzed them using two cholestatic liver injury models: bile duct ligation (BDL) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding. Primary hepatocytes and a cholangiocyte cell line were used to address the impact of plectin on keratin filament organization and stability in vitro. RESULTS: Plectin deficiency in hepatocytes and biliary epithelial cells led to aberrant keratin filament network organization, biliary tree malformations, and collapse of bile ducts and ductules. Further, plectin ablation significantly aggravated biliary damage upon cholestatic challenge. Coincidently, we observed a significant expansion of A6-positive progenitor cells in PleΔalb livers. After BDL, plectin-deficient bile ducts were prominently dilated with more frequent ruptures corresponding to an increased number of bile infarcts. In addition, more abundant keratin aggregates indicated less stable keratin filaments in PleΔalb hepatocytes. A transmission electron microscopy analysis revealed a compromised tight junction formation in plectin-deficient biliary epithelial cells. In addition, protein profiling showed increased expression of the adherens junction protein E-Cadherin, and inefficient upregulation of the desmosomal protein desmoplakin in response to BDL. In vitro analyses revealed a higher susceptibility of plectin-deficient keratin networks to stress-induced collapse, paralleled by elevated activation of p38 MAP kinase. CONCLUSION: Our study shows that by maintaining proper keratin network cytoarchitecture and biliary epithelial stability, plectin plays a critical role in protecting the liver from stress elicited by cholestasis. LAY SUMMARY: Plectin is a cytolinker protein capable of interconnecting all three cytoskeletal filament systems and linking them to plasma membrane-bound junctional complexes. In liver, the plectin-controlled cytoskeleton mechanically stabilizes epithelial cells and provides them with the capacity to adapt to increased bile pressure under cholestasis.

• MeSH
• cholestáza metabolismus   patologie   MeSH
• epitel metabolismus   patologie   MeSH
• hepatocyty metabolismus   patologie   MeSH
• játra abnormality   metabolismus   patologie   MeSH
• keratiny metabolismus   MeSH
• MAP kinasový signální systém MeSH
• mitogenem aktivované proteinkinasy p38 metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• plektin nedostatek   genetika   metabolismus   MeSH
• stabilita proteinů MeSH
• žlučové ústrojí abnormality   metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The complex architecture of the liver biliary network represents a structural prerequisite for the formation and secretion of bile as well as excretion of toxic substances through bile ducts. Disorders of the biliary tract affect a significant portion of the worldwide population, often leading to cholestatic liver diseases. Cholestatic liver disease is a condition that results from an impairment of bile formation or bile flow to the gallbladder and duodenum. Cholestasis leads to dramatic changes in biliary tree architecture, worsening liver disease and systemic illness. Recent studies show that the prevalence of cholestatic liver diseases is increasing. The availability of well characterized animal models, as well as development of visualization approaches constitutes a critical asset to develop novel pathogenetic concepts and new treatment strategies.

• MeSH
• cholestáza diagnostické zobrazování   patofyziologie   MeSH
• lidé MeSH
• mikroskopie elektronová rastrovací metody   MeSH
• modely nemocí na zvířatech * MeSH
• teoretické modely MeSH
• žlučové cesty diagnostické zobrazování   fyziologie   patofyziologie   MeSH
• žlučové ústrojí diagnostické zobrazování   fyziologie   patofyziologie   MeSH
• zobrazování trojrozměrné metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Matrix metalloproteinases (MMPs), responsible for extracellular matrix remodelling and processing of numerous soluble and cell-surface proteins, appear to play important roles in pathogenesis of gastrointestinal diseases. MMPs influence migration of inflammatory cells, mucosal destruction, matrix deposition and degradation. In this study, we analysed the expression of MMP-19 in the main forms of gastrointestinal diseases including inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease, and colorectal carcinoma. We identified prominent MMP-19 expression in unaffected areas of intestinal epithelia and macrophages but not in other cells or tissues. Abundant expression of MMP-19 was also found in the endothelium of blood and lymphatic vessels of inflamed intestinal tissue. High MMP-19 immunoreactivity was also associated with macrophages in inflamed areas and myenteric plexuses. In comparison to the intestinal epithelium, all these cell types and compartments appeared to express MMP-19 irrespective of the disease pathogenesis and progression. Intestinal epithelia exhibited striking differential immunoreactivity for MMP-19. While immunoreactivity of monoclonal antibody recognizing the propeptide domain declined in virtually all IBD and colorectal carcinoma samples, other polyclonal antibodies against the hinge region and propetide domain did not show such an obvious decrease. Additional Western blotting analysis revealed that the antibodies against MMP-19 recognize differently processed forms of this MMP. The disappearance of immunoreactivity of the monoclonal anti-propeptide domain antibody does not mean down-regulation of MMP-19, but processing of the immature form. As this processing likely leads to the activation of this MMP, the differential staining pattern may be an important sign of disease progression.

• MeSH
• dospělí MeSH
• gastrointestinální nemoci enzymologie   patologie   MeSH
• HCT116 buňky MeSH
• kolon enzymologie   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metaloproteinasy secernované do matrix metabolismus   MeSH
• mladý dospělý MeSH
• posttranslační úpravy proteinů * MeSH
• progrese nemoci * MeSH
• protilátky metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• senioři MeSH
• střevní sliznice enzymologie   patologie   MeSH
• tenké střevo enzymologie   patologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Liver fibrosis is characterized by the deposition and increased turnover of extracellular matrix. This process is controlled by matrix metalloproteinases (MMPs), whose expression and activity dynamically change during injury progression. MMP-19, one of the most widely expressed MMPs, is highly expressed in liver; however, its contribution to liver pathology is unknown. The aim of this study was to elucidate the role of MMP-19 during the development and resolution of fibrosis by comparing the response of MMP-19-deficient (MMP19KO) and wild-type mice upon chronic liver CCl(4)-intoxication. We show that loss of MMP-19 was beneficial during liver injury, as plasma ALT and AST levels, deposition of fibrillar collagen, and phosphorylation of SMAD3, a TGF-ß1 signaling molecule, were all significantly lower in MMP19KO mice. The ameliorated course of the disease in MMP19KO mice likely results from a slower rate of basement membrane destruction and ECM remodeling as the knockout mice maintained significantly higher levels of type IV collagen and lower expression and activation of MMP-2 after 4 weeks of CCl(4)-intoxication. Hastened liver regeneration in MMP19KO mice was associated with slightly higher IGF-1 mRNA expression, slightly increased phosphorylation of Akt kinase, decreased TGF-ß1 mRNA levels and significantly reduced SMAD3 phosphorylation. In addition, primary hepatocytes isolated from MMP19KO mice showed impaired responsiveness towards TGF-ß1 stimulation, resulting in lower expression of Snail1 and vimentin mRNA. Thus, MMP-19-deficiency improves the development of hepatic fibrosis through the diminished replacement of physiological extracellular matrix with fibrotic deposits in the beginning of the injury, leading to subsequent changes in TGF-ß and IGF-1 signaling pathways.

• MeSH
• chronická nemoc MeSH
• hepatocyty cytologie   MeSH
• insulinu podobný růstový faktor I metabolismus   MeSH
• jaterní cirhóza chemicky indukované   enzymologie   MeSH
• metaloproteinasy secernované do matrix genetika   MeSH
• modely nemocí na zvířatech MeSH
• myši knockoutované MeSH
• myši MeSH
• otrava chloridem uhličitým enzymologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• progrese nemoci MeSH
• proliferace buněk MeSH
• signální transdukce MeSH
• transformující růstový faktor beta metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Charcot-Marie-Tooth (CMT) neuropathy is the most common inherited neuromuscular disorder. CMT is genetically very heterogeneous. Mutations in the SH3TC2 gene cause Charcot-Marie-Tooth neuropathy type 4C (CMT4C), a demyelinating form with autosomal recessive inheritance. In this study, two novel splice site mutations in the SH3TC2 gene have been studied (c.279G → A, c.3676-8G → A). Mutation c.279G → A was detected on one allele in two unrelated families with CMT4C in combination with a known pathogenic mutation (c.2860 C →T in one family, c.505T → C in the other) on the second allele of SH3TC2 gene. Variant c.3676-8G → A was detected in two patients from unrelated families on one allele of the SH3TC2 gene in combination with c.2860C →T mutation on the other allele. Several in silico tests were performed and exon trap experiments were undertaken in order to prove the effect of both mutations on proper splicing of SH3TC2. Fragments of SH3TC2 were subcloned into pET01 exon trap vector (Mobitec) and transfected into COS-7 cells. Aberrant splicing was predicted in silico for both mutations, which was confirmed by exon trap analysis. For c.279G → A mutation, 19 bases from intron 3 are retained in cDNA. The mutation c.3676-8G→ A produces a novel splice acceptor site for exon 17 and complex changes in splicing were observed. We present evidence that mutations c.279G → A and c.3676-8G →A in the SH3TC2 gene cause aberrant splicing and are therefore pathogenic and causal for CMT4C.

• MeSH
• Cercopithecus aethiops MeSH
• Charcotova-Marieova-Toothova nemoc genetika   patologie   patofyziologie   MeSH
• COS buňky MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace genetika   MeSH
• nervové vedení genetika   MeSH
• počítačová simulace * MeSH
• proteiny genetika   MeSH
• referenční hodnoty MeSH
• transfekce MeSH
• zdraví rodiny MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH