The vinculin gene codes for a cytoskeletal protein, found in focal adhesion plaques and in cell-cell adherens junctions. Vinculin was inactivated by homologous recombination using a targeting vector ...in embryonic stem (ES) cells. The heterozygous ES cells were introduced into mice by established procedures to produce heterozygous animals that were normal and fertile. No homozygous vinculinâ/â embryos were born and analyses during the gestational period showed that the vinculin null embryos were small and abnormal from day E8 but some survived until E10. The most prominent defect was lack of midline fusion of the rostral neural tube, producing a cranial bilobular appearance and attenuation of cranial and spinal nerve development. Heart development was curtailed at E9.5, with severely reduced and akinetic myocardial and endocardial structures. Mutant embryos were 30â40% smaller, somites and limbs were retarded and ectodermal tissues were sparse and fragile. Fibroblasts (MEF) isolated from mutant embryos were shown to have reduced adhesion to fibronectin, vitronectin, laminin and collagen compared to wild-type levels. In addition, migration rates over these substrata were two-fold higher and the level of focal adhesion kinase (FAK) activity was three-fold higher. We conclude that vinculin is necessary for normal embryonic development, probably because of its role in the regulation of cell adhesion and locomotion, cell behaviors essential for normal embryonic morphogenesis, although specific roles in neural and cardiac development cannot be ruled out.
PU.1 is a member of the ets family of transcription factors and is expressed exclusively in cells of the hematopoietic lineage. Mice homozygous for a disruption in the PU.1 DNA binding domain are ...born alive but die of severe septicemia within 48 h. The analysis of these neonates revealed a lack of mature macrophages, neutrophils, B cells and T cells, although erythrocytes and megakaryocytes were present. The absence of lymphoid commitment and development in null mice was not absolute, since mice maintained on antibiotics began to develop normal appearing T cells 3–5 days after birth. In contrast, mature B cells remained undetectable in these older mice. Within the myeloid lineage, despite a lack of macrophages in the older antibiotic‐treated animals, a few cells with the characteristics of neutrophils began to appear by day 3. While the PU.1 protein appears not to be essential for myeloid and lymphoid lineage commitment, it is absolutely required for the normal differentiation of B cells and macrophages.
We report that keratin 8 (mK8) gene disruption causes colorectal hyperplasia in FVB/N mice. The intestinal lesions affect uniformly the cecum, colon, and rectum but not the small intestine. The ...elongation of the crypts is accompanied by an inflammation of the lamina propria and submucosa. Hepatic, renal, and pancreatic functions tested in clinical assays are within nonpathological range, suggesting that the major defect lies in colonic epithelial cells. Still, small but consistent elevation in the hepatic enzymes alanine (AST) and asparate (ALT) aminotransferase are observed, along with a 70% increase in spleen weight. No homozygous mouse line has been established, because of a markedly reduced fertility of the mK8-/- females. Previously, we reported that the mK8- targeted mutation causes embryonic lethality in (C57B1/6x129Sv) mice. This strong effect of the genetic background on the mK8- mutant phenotype emphasizes the importance of using several inbred mouse strains to reveal the polygenic contribution to mutant phenotypes. Our results demonstrate that genetic modifiers of K8/K18 filament functions, with profound effects on embryogenesis and gut functional integrity, are differentially active in the FVB/N and C57B1/6 genetic backgrounds. More importantly, the increase in mK8-/- gut epithelial cell number, rather than cell disruption, contrasts with the known function of epidermal keratins in providing mechanical strength.
Keratin 8 (K8) and keratin 18 (K18) are the most common and characteristic members of the large intermediate filament gene family expressed in 'simple' or single layer epithelial tissues of the body. ...Their persistent expression in tumor cells derived from these epithelia has led to the wide spread use of keratin monoclonal antibodies as aids in the detection and identification of carcinomas. Oncogenes which activate ras signal transduction pathways stimulate expression of the K18 gene through transcription factors including members of the AP-1 (jun and fos) and ETS families. The persistent expression of K8 and K18 may reflect the integrated transcriptional activation of such transcription factors and, in the cases of ectopic expression, an escape from the suppressive epigenetic mechanisms of DNA methylation and chromatin condensation. Comparison of the mechanisms of transcriptional control of K18 expression with expression patterns documented in both normal and pathological conditions leads to the proposal that persistent K8 and K18 expression is a reflection of the action of multiple different oncogenes converging on the nucleus through a limited number of transcription factors to then influence the expression of a large number of genes including these keratins. Furthermore, correlation of various tumor cell characteristics including invasive behavior and drug sensitivity with K8 and K18 expression has stimulated consideration of the possible functions of these proteins in both normal development and in tumorigenesis. Recent developments in the analysis of the functions of these intermediate filament proteins provide new insights into diverse functions influenced by K8 and K18.
Vinculin, a major constituent of focal adhesions and zonula adherens junctions, is thought to be involved in linking the microfilaments to areas of cell-substrate and cell-cell contacts. To test the ...role of vinculin in cell adhesion and motility, we used homologous recombination to generate F9 embryonal carcinoma and embryonic stem cell clones homozygous for a disrupted vinculin gene. When compared to wild-type cells, vinculin-mutant cells displayed a rounder morphology and a reduced ability to adhere and spread on plastic or fibronectin. Decreased adhesion of the mutant cells was associated with a reduction in lamellipodial extensions, as observed by time-lapse video microscopy. The locomotive activities of control F9 and the vinculin-null cells were compared in two assays. Loss of vinculin resulted in a 2.4-fold increase in cell motility. These results demonstrate an important role for vinculin in determining cell shape, adhesion, surface protrusive activity, and cell locomotion.
Keratin 8 (mK8) and its partner keratin 18 (mK18) are the first intermediate filament proteins expressed during mouse embryogenesis. They are found in most extraembryonic and embryonic simple ...epithelia, including trophectoderm, visceral yolk sac, gastrointestinal tract, lungs, mammary glands, and uterus. We report that a targeted null mutation in the mK8 gene causes mid-gestational lethality. Mutant embryos are growth retarded and suffer from internal bleeding, with an abnormal accumulation of erythrocytes in fetal livers. The mK8- phenotype has 94% penetrance, with a few mice surviving into adulthood. We suggest that mK8/mK18 filaments are important for the integrity of the fetal liver, like specialized human epidermal keratins for the integrity of the epidermis. This phenotype in mice differs from the reported function of simple epithelium keratins in Xenopus at the gastrulation stage. In mice, mK8 fulfills a vital function at 12 days postcoitum.
Simple epithelial tissues such as liver and pancreas express keratins 8 (K8) and 18 (K18) as their major intermediate filament proteins. K8 and K18 null mice and transgenic mice that express mutant ...K18 (K18C) manifest several hepatocyte abnormalities and demonstrate that K8/18 are important in maintaining liver tissue and cell integrity, although other potential functions remain uncharacterized. Here, we report an additional abnormal liver phenotype, which is similar in K8 null, K18 null, and K18C mouse models. Liver histologic examination showed large polynuclear areas that lacked cell membranes, desmosomal structures, and filamentous actin. Similar, but less prominent, areas were observed in the pancreas. The parenchyma outside the polynuclear areas displayed irregular sinusoidal structures and markedly enlarged nuclei. Most K8 null hepatocytes were positive for the proliferating cell nuclear antigen (PCNA) with a doubled DNA content in comparison with the predominantly PCNA-negative wild-type hepatocytes. The distribution of the 14-3-3zeta protein was also altered in K8 null mice. Taken together, our results indicate that absence of keratin filaments causes disturbances in cell-cycle regulation, driving cells into the S-G2 phase and causing aberrant cytokinesis. These effects could stem from disturbed functions of K8/18-dependent cell-cycle regulators, such as the signaling integrator, 14-3-3. (HEPATOLOGY 2001;34:1174-1183.)
Abstract
Background
Disruption of the colon epithelial barrier is a characteristic of ulcerative colitis (UC), allowing luminal microbes to come into contact with intestinal immune cells resulting in ...inflammation. All approved UC treatments are anti-inflammatory drugs and because they do not directly heal the epithelial barrier, the intestinal immune system continues to be exposed to gut microbes, resulting in low remission rates. Physiological Wnt signaling is fundamental to intestine epithelial homeostasis and renewal. Modulation of Wnt signaling might present an opportunity to achieve histological remission, which is associated with an improved clinical course, in UC. Previously, in vivo overexpression or administration of exogenous R-Spondin (RSPO), which amplifies Wnt signaling by increasing the amount of Wnt receptors on the cell surface, was reported to show effects on the intestine epithelium and helped regenerate intestine epithelium in various injury models. However, RSPO was also reported, to induce hyperplasia in normal intestine epithelium. Wnt signaling may also be modulated with recently developed Wnt mimetics which mimic endogenous Wnt proteins and activate downstream β-catenin signaling upon engaging with receptors of Wnts. The objective of the current study was to compare the effect of these two approaches in an acute DSS model.
Methods
Utilizing a mouse acute DSS colitis model, we compared the epithelial healing effects of a Surrozen proprietary Wnt mimetic, R2M3-26, RSPO2, and combination treatments of R2M3-26 and RSPO2. Animals received 4% (wt/vol) dextran sulfate sodium (DSS) in drinking water for 7 days followed by 1% DSS in drinking water from day 8. Protein treatments began on day 4. Disease activity, body weight (BW), fecal score, and occult blood were measured daily. Animals were terminated on day 10 for colon and small intestine histology as well as a serum cytokine panel.
Results
DSS damage to the colon epithelium was visible by H&E stain at day 4 and day 7 and continued to progress. We observed reduced mRNA expression of Wnt target genes and Wnt ligands in the colon, suggesting compromised Wnt signaling in the tissue. R2M3-26, RSPO2, and the combination treatments were efficacious in improving Disease Activity Index (DAI) and reducing serum cytokine levels. However, both RSPO treatments and combination treatments caused hyperproliferation of epithelium in the small intestine and the colon, consistent with what was reported in normal mice.
Conclusion
The Surrozen Wnt mimetic, R2M3-26, repaired the DSS damaged colon epithelium without causing hyperproliferation of epithelial cells in the intestine, making it a potential therapeutic platform for IBD.
Keratin 8 (K8)-deficient adult mice develop a severe disease of the gastrointestinal tract characterized mainly by colorectal hyperplasia and inflammation. Given that hepatocytes contain K8/K18 ...heteropolymers only, this animal model was used to assess the contribution of these simple epithelium keratins to hepatocyte structural and functional integrity. Homozygous mutant (HMZ), heterozygous, and wild-type (WT) mice were examined for hepatocyte structural and metabolic features and their survival to partial hepatectomy. Except for the presence of few necrotic foci, no other tissular or cellular alterations were observed in nonhepatectomized HMZ mouse livers; glycogen and lipid peroxidation levels were essentially normal, but a small reduction in bile flow was observed. In response to a single pentobarbital injection, HMZ mice had longer sleeping times than heterozygous and WT mice. After a two-thirds partial hepatectomy under pentobarbital anesthesia, all HMZ mice died within a few hours, whereas those anesthetized with ether survived for 1 to 2 days. One hour after hepatectomy after pentobarbital anesthesia, many hepatocytes contained erythrocytes and large vacuoles in the cytoplasm, which suggests damage at the plasma membrane level in response to a sudden increase in portal blood flow. In line with these findings, an uptake of trypan blue by HMZ but not WT mouse hepatocytes was observed during a 10 ml/minute perfusion via the portal vein with a dye-supplemented buffer. Subsequent cellular dispersion led to viable WT mouse hepatocytes but largely nonviable HMZ mouse hepatocytes. Better viability was obtained at lower perfusion rates. Partially hepatectomized heterozygous mice developed liver steatosis, a condition that was not associated with a change in K8 content but perhaps linked to the presence of the neo gene. Transgenic HMZ mouse rescue experiments with a full-length K8 gene confirmed that the phenotypic alterations observed in partially hepatectomized HMZ mice were caused by the disruption of the K8 gene. Taken together, these findings demonstrate that simple epithelium keratins are essential for the maintenance of hepatocyte structural and functional integrity.
The function and regulation of keratin 8 (K8) and 18 (K18), intermediate filament (IF) proteins of the liver, are not fully understood. We employed the liver damage induced by microcystin‐LR (MC‐LR), ...a liver‐specific inhibitor of type‐1 and type‐2A protein phosphatases, in normal and in keratin assembly–incompetent mouse strains as a model to elucidate the roles of IF phosphorylation in situ. The mouse strains used were wild‐type (wt) mice and mice with abnormal filament assembly, caused by a targeted null mutation of the K8 gene or caused by expression of a point‐mutated dominant negative human K18. In vivo 32P‐labeled wt mice, subsequently injected with a lethal dose of MC‐LR, showed hyperphosphorylation, disassembly, and reorganization of K8/K18, in particular K18, indicating high phosphate turnover on liver keratins in situ. At lethal doses, the keratin assembly–incompetent mice displayed liver lesions faster than wt mice, as indicated histopathologically and by liver‐specific plasma enzyme elevations. The histological changes included centrilobular hemorrhage in all mouse strains. The assembly‐incompetent mice showed a marked vacuolization of periportal hepatocytes. Indistinguishable MC‐LR–induced reorganization of microfilaments was observed in all mice, indicating that this effect on microfilaments is not dependent on the presence of functional K8/K18 networks. At sublethal doses of MC‐LR, all animals had the same potential to recover from the liver damage. Our study shows that K8/K18 filament assembly is regulated in vivo by serine phosphorylation. The absence or occurrence of defective K8/K18 filaments render animals more prone to liver damage, which supports the previously suggested roles of keratin IFs in maintenance of structural integrity.
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