Lessons from Keratin 18 Knockout Mice: Formation of Novel Keratin Filaments, Secondary Loss of Keratin 7 and Accumulation of Liver-specific Keratin 8-Positive Aggregates

Here, we report on the analysis of keratin 18 null mice. Unlike the ablation of K8, which together with K18 is expressed in embryonic and simple adult epithelia, K18 null mice are viable, fertile, and show a normal lifespan. In young K18 null mice, hepatocytes were completely devoid of keratin filaments. Nevertheless, typical desmosomes were formed and maintained. Old K18 null mice, however, developed a distinctive liver pathology with abnormal hepatocytes containing K8-positive aggregates. These stained positively for ubiquitin and MM120-1 and were identified as Mallory bodies, one hallmark of human alcoholic hepatitis. This is the first demonstration that the ablation of one keratin leads to the accumulation of its single partner. Another striking finding was the absence or drastic down regulation of K7 in several tissues despite its ongoing transcription. Moreover, K18 null mice revealed new insights in the filament-forming capacity of the tail-less K19 in vivo. Due to the unexpected secondary loss of K7, only K8/19 are expressed in the uterine epithelium of K18 null mice. Immunoelectron microscopy of this tissue demonstrated the presence of typical K8/19 IF, thus highlighting in vivo that K19 is a fully competent partner for K8.

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Formation of Mallory Body-like Inclusions and Cell Death Induced by Deregulated Expression of Keratin 18

Molecular Biology of the Cell ◽

10.1091/mbc.01-10-0510 ◽

2002 ◽

Vol 13 (10) ◽

pp. 3441-3451 ◽

Cited By ~ 23

Author(s):

Ikuo Nakamichi ◽

Shigetsugu Hatakeyama ◽

Keiichi I. Nakayama

Keyword(s):

Cell Death ◽

Important Determinant ◽

Cellular Model ◽

Microtubule Network ◽

Cytoplasmic Inclusions ◽

Mallory Bodies ◽

Keratin 18 ◽

Intracellular Aggregates

Mallory bodies (MBs) are cytoplasmic inclusions that contain keratin 8 (K8) and K18 and are present in hepatocytes of individuals with alcoholic liver disease, nonalcoholic steatohepatitis, or benign or malignant hepatocellular neoplasia. Mice fed long term with griseofulvin are an animal model of MB formation. However, the lack of a cellular model has impeded understanding of the molecular mechanism of this process. Culture of HepG2 cells with griseofulvin has now been shown to induce both the formation of intracellular aggregates containing K18 as well as an increase in the abundance of K18 mRNA. Overexpression of K18 in HepG2, HeLa, or COS-7 cells also induced the formation of intracellular aggregates that stained with antibodies to ubiquitin and with rhodamine B (characteristics of MBs formed in vivo), eventually leading to cell death. The MB-like aggregates were deposited around centrosomes and disrupted the microtubular array. Coexpression of K8 with K18 restored the normal fibrous pattern of keratin distribution and reduced the toxicity of K18. In contrast, an NH2-terminal deletion mutant of K8 promoted the formation of intracellular aggregates even in the absence of K18 overexpression. Deregulated expression of K18, or an imbalance between K8 and K18, may thus be an important determinant of MB formation, which compromises the function of centrosomes and the microtubule network and leads to cell death.

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The Hierarchic Order of Intermediate Filament Structure and Assembly

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100120308 ◽

1985 ◽

Vol 43 ◽

pp. 722-725

Author(s):

U. Aebi ◽

E.C. Glavaris ◽

R. Eichner

Keyword(s):

Tissue Specificity ◽

Structural Model ◽

Eukaryotic Cells ◽

Cdna Sequencing ◽

Filament Structure ◽

Keratin Filaments ◽

Isoelectric Points ◽

Immunological Crossreactivity

Five different classes of intermediate-sized filaments (IFs) have been identified in differentiated eukaryotic cells: vimentin in mesenchymal cells, desmin in muscle cells, neurofilaments in nerve cells, glial filaments in glial cells and keratin filaments in epithelial cells. Despite their tissue specificity, all IFs share several common attributes, including immunological crossreactivity, similar morphology (e.g. about 10 nm diameter - hence ‘10-nm filaments’) and the ability to reassemble in vitro from denatured subunits into filaments virtually indistinguishable from those observed in vivo. Further more, despite their proteinchemical heterogeneity (their MWs range from 40 kDa to 200 kDa and their isoelectric points from about 5 to 8), protein and cDNA sequencing of several IF polypeptides (for refs, see 1,2) have provided the framework for a common structural model of all IF subunits.

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Sirt1 deacetylates and stabilizes p62 to promote hepato-carcinogenesis

Cell Death and Disease ◽

10.1038/s41419-021-03666-z ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Lifeng Feng ◽

Miaoqin Chen ◽

Yiling Li ◽

Muchun Li ◽

Shiman Hu ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Overall Survival ◽

Cell Growth ◽

Knockout Mice ◽

Liver Tumors ◽

Underlying Mechanism ◽

Carcinoma Cells ◽

Conditional Knockout

Abstractp62/SQSTM1 is frequently up-regulated in many cancers including hepatocellular carcinoma. Highly expressed p62 promotes hepato-carcinogenesis by activating many signaling pathways including Nrf2, mTORC1, and NFκB signaling. However, the underlying mechanism for p62 up-regulation in hepatocellular carcinoma remains largely unclear. Herein, we confirmed that p62 was up-regulated in hepatocellular carcinoma and its higher expression was associated with shorter overall survival in patients. The knockdown of p62 in hepatocellular carcinoma cells decreased cell growth in vitro and in vivo. Intriguingly, p62 protein stability could be reduced by its acetylation at lysine 295, which was regulated by deacetylase Sirt1 and acetyltransferase GCN5. Acetylated p62 increased its association with the E3 ligase Keap1, which facilitated its poly-ubiquitination-dependent proteasomal degradation. Moreover, Sirt1 was up-regulated to deacetylate and stabilize p62 in hepatocellular carcinoma. Additionally, Hepatocyte Sirt1 conditional knockout mice developed much fewer liver tumors after Diethynitrosamine treatment, which could be reversed by the re-introduction of exogenous p62. Taken together, Sirt1 deacetylates p62 at lysine 295 to disturb Keap1-mediated p62 poly-ubiquitination, thus up-regulating p62 expression to promote hepato-carcinogenesis. Therefore, targeting Sirt1 or p62 is a reasonable strategy for the treatment of hepatocellular carcinoma.

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Further pharmacological evaluation of a novel synthetic peptide bradykinin B2 receptor agonist

Biological Chemistry ◽

10.1515/hsz-2012-0295 ◽

2013 ◽

Vol 394 (3) ◽

pp. 353-360 ◽

Cited By ~ 9

Author(s):

Martin Savard ◽

Julie Labonté ◽

Céléna Dubuc ◽

Witold Neugebauer ◽

Pedro D’Orléans-Juste ◽

...

Keyword(s):

Knockout Mice ◽

Synthetic Peptide ◽

Receptor Agonist ◽

Rabbit Lung ◽

Hypotensive Action ◽

Duration Of Action ◽

Selective Agonist ◽

Comparable Magnitude

Abstract We recently identified a novel human B2 receptor (B2R) agonist [Hyp3,Thi5,NChg7,Thi8]-bradykinin (NG291) with greater in vitro and in vivo potency and duration of action than natural bradykinin (BK). Here, we further examined its stability and selectivity toward B2R. The hypotensive, antithrombotic, and profibrinolytic functions of NG291 relative to BK and its analogue ([Hyp3,Thi5,(4-Me)Tyr8(ΨCH2NH)Arg9]-BK) (RMP-7) were also tested. Contraction assays using isolated mouse stomachs (containing kinin B1R, B2R, and kininase I- and II-like activities) showed that NG291 is a more potent contractant than BK and is inhibited by HOE-140 (B2R antagonist) but unaffected by R954 (B1R antagonist), whereas both decreased the potency of BK. In stomach tissues from B2R knockout mice, BK maintained its activity via B1R, whereas NG291 had no contractile effect, indicating that it was selective for B2R. Unlike BK, NG291 was not degraded by rabbit lung ACE. Comparing intravenously administered BK and NG291 revealed that NG291 exhibited more potent and prolonged hypotensive action and greater antithrombotic and profibrinolytic activities. These effects were of comparable magnitude to RMP-7 and were absent in B2R knockout mice. We concluded that NG291 is a novel biostable B2R-selective agonist that may prove suitable for investigating the (pre)clinical cardioprotective efficacy of B2R activation.

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Iron absorption in hepcidin1 knockout mice

British Journal Of Nutrition ◽

10.1017/s0007114510005507 ◽

2011 ◽

Vol 105 (11) ◽

pp. 1583-1591 ◽

Cited By ~ 11

Author(s):

Patarabutr Masaratana ◽

Abas H. Laftah ◽

Gladys O. Latunde-Dada ◽

Sophie Vaulont ◽

Robert J. Simpson ◽

...

Keyword(s):

Knockout Mice ◽

Iron Absorption ◽

Fe Deficiency ◽

Acute Effects ◽

Regulatory Peptide ◽

Fe Uptake

Hepcidin, the Fe-regulatory peptide, has been shown to inhibit Fe absorption and reticuloendothelial Fe recycling. The present study was conducted to explore the mechanism of in vivo Fe regulation through genetic disruption of hepcidin1 and acute effects of hepcidin treatment in hepcidin1 knockout (Hepc1− / − ) and heterozygous mice. Hepcidin1 disruption resulted in significantly increased intestinal Fe uptake. Hepcidin injection inhibited Fe absorption in both genotypes, but the effects were more evident in the knockout mice. Hepcidin administration was also associated with decreased membrane localisation of ferroportin in the duodenum, liver and, most significantly, in the spleen of Hepc1− / − mice. Hypoferraemia was induced in heterozygous mice by hepcidin treatment, but not in Hepc1− / − mice, 4 h after injection. Interestingly, Fe absorption and serum Fe levels in Hepc1− / − and heterozygous mice fed a low-Fe diet were not affected by hepcidin injection. The present study demonstrates that hepcidin deficiency causes increased Fe absorption. The effects of hepcidin were abolished by dietary Fe deficiency, indicating that the response to hepcidin may be influenced by dietary Fe level or Fe status.

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PRMT5 Prevents Dilated Cardiomyopathy via Suppression of Protein O-GlcNAcylation

Circulation Research ◽

10.1161/circresaha.121.319456 ◽

2021 ◽

Author(s):

Zhenhua Li ◽

Jingping Xu ◽

Yao Song ◽

Chong Xin ◽

Lantao Liu ◽

...

Keyword(s):

Dilated Cardiomyopathy ◽

Knockout Mice ◽

Rna Splicing ◽

Gene Knockout ◽

Major Type ◽

Integrated Analysis ◽

Arginine Methyltransferase ◽

Potential Strategy ◽

Glcnac Transferase

Rationale: Protein O-GlcNAcylation is dynamically regulated by two key enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Excessive protein O-GlcNAcylation contributes to dilated cardiomyopathy (DCM), but its regulatory mechanisms are not fully understood. The protein arginine methyltransferase 5 (PRMT5) is the major type II arginine methyltransferase, which plays critical physiological roles by symmetrically dimethylating various downstream targets including proteins involved in RNA splicing. However, its function in regulating protein O-GlcNAcylation and DCM is unexplored. Objective: To elucidate the physiological function of PRMT5 and the mechanism underlying its role in regulating cardiac O-GlcNAcylation and homeostasis. Methods and Results: Conditional gene knockout was used to study the in vivo function of Prmt5 in regulating cardiac homeostasis. An integrated analysis of transcriptomic and metabolomic profiles was performed to investigate the molecular mechanism. Adeno-associated virus 9 (AAV9)-mediated gene delivery in the mouse was used to study the protein O-GlcNAcylation in Prmt5 deficiency-induced DCM. PRMT5 mRNA was decreased in human DCM hearts, and cardiomyocyte-specific Prmt5 deletion in mice resulted in DCM and heart failure. Transcriptomic and metabolomic profiling identified increased O-GlcNAcylation in the hearts of Prmt5-knockout mice. Mechanistically, Prmt5 deletion suppressed O-GlcNAcase (OGA) expression by inhibiting the transcription of Oga and triggering its aberrant splicing. Consistently, a positive correlation of PRMT5 and OGA was identified in human DCM hearts. Notably, gene therapy with AAV9 encoding the correctly spliced Oga normalized the cardiac protein O-GlcNAcylation levels and partially rescued the dilation and dysfunction of the hearts in Prmt5-knockout mice. Conclusions: Our data demonstrate a novel function of PRMT5 in regulating protein O-GlcNAcylation to maintain cardiac homeostasis, suggesting that targeting the PRMT5-OGA axis could be a potential strategy for treating DCM.

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Vascular Endothelin-B Receptor System In Vivo Plays a Favorable Inhibitory Role in Vascular Remodeling After Injury Revealed by Endothelin-B Receptor–Knockout Mice

Circulation ◽

10.1161/01.cir.0000032004.56585.2a ◽

2002 ◽

Vol 106 (15) ◽

pp. 1991-1998 ◽

Cited By ~ 65

Author(s):

Nobuyuki Murakoshi ◽

Takashi Miyauchi ◽

Yoshihiko Kakinuma ◽

Takashi Ohuchi ◽

Katsutoshi Goto ◽

...

Keyword(s):

Knockout Mice ◽

Vascular Remodeling ◽

Receptor System ◽

Endothelin B Receptor ◽

Inhibitory Role ◽

Endothelin B

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Preoperative administration of the 5-HT4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons

Gut ◽

10.1136/gutjnl-2018-317263 ◽

2018 ◽

Vol 68 (8) ◽

pp. 1406-1416 ◽

Cited By ~ 15

Author(s):

Nathalie Stakenborg ◽

Evelien Labeeuw ◽

Pedro J Gomez-Pinilla ◽

Sebastiaan De Schepper ◽

Raymond Aerts ◽

...

Keyword(s):

Knockout Mice ◽

Postoperative Ileus ◽

Intestinal Inflammation ◽

Electrical Field Stimulation ◽

Enteric Neurons ◽

Postoperative Treatment ◽

Wild Type ◽

Clinical Recovery ◽

Preoperative Administration

ObjectivesVagus nerve stimulation (VNS), most likely via enteric neurons, prevents postoperative ileus (POI) by reducing activation of alpha7 nicotinic receptor (α7nAChR) positive muscularis macrophages (mMφ) and dampening surgery-induced intestinal inflammation. Here, we evaluated if 5-HT4 receptor (5-HT4R) agonist prucalopride can mimic this effect in mice and human.DesignUsing Ca2+ imaging, the effect of electrical field stimulation (EFS) and prucalopride was evaluated in situ on mMφ activation evoked by ATP in jejunal muscularis tissue. Next, preoperative and postoperative administration of prucalopride (1–5 mg/kg) was compared with that of preoperative VNS in a model of POI in wild-type and α7nAChR knockout mice. Finally, in a pilot study, patients undergoing a Whipple procedure were preoperatively treated with prucalopride (n=10), abdominal VNS (n=10) or sham/placebo (n=10) to evaluate the effect on intestinal inflammation and clinical recovery of POI.ResultsEFS reduced the ATP-induced Ca2+ response of mMφ, an effect that was dampened by neurotoxins tetrodotoxin and ω-conotoxin and mimicked by prucalopride. In vivo, prucalopride administered before, but not after abdominal surgery reduced intestinal inflammation and prevented POI in wild-type, but not in α7nAChR knockout mice. In humans, preoperative administration of prucalopride, but not of VNS, decreased Il6 and Il8 expression in the muscularis externa and improved clinical recovery.ConclusionEnteric neurons dampen mMφ activation, an effect mimicked by prucalopride. Preoperative, but not postoperative treatment with prucalopride prevents intestinal inflammation and shortens POI in both mice and human, indicating that preoperative administration of 5-HT4R agonists should be further evaluated as a treatment of POI.Trial registration numberNCT02425774.

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Direct evidence of iNOS-mediated in vivo free radical production and protein oxidation in acetone-induced ketosis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00015.2008 ◽

2008 ◽

Vol 295 (2) ◽

pp. E456-E462 ◽

Cited By ~ 28

Author(s):

Krisztian Stadler ◽

Marcelo G. Bonini ◽

Shannon Dallas ◽

Danielle Duma ◽

Ronald P. Mason ◽

...

Keyword(s):

Lipid Peroxidation ◽

Free Radical ◽

Nadph Oxidase ◽

Knockout Mice ◽

Protein Oxidation ◽

Ketone Bodies ◽

Free Radical Production ◽

Radical Production ◽

Inos Knockout Mice

Diabetic patients frequently encounter ketosis that is characterized by the breakdown of lipids with the consequent accumulation of ketone bodies. Several studies have demonstrated that reactive species are likely to induce tissue damage in diabetes, but the role of the ketone bodies in the process has not been fully investigated. In this study, electron paramagnetic resonance (EPR) spectroscopy combined with novel spin-trapping and immunological techniques has been used to investigate in vivo free radical formation in a murine model of acetone-induced ketosis. A six-line EPR spectrum consistent with the α-(4-pyridyl-1-oxide)- N-t-butylnitrone radical adduct of a carbon-centered lipid-derived radical was detected in the liver extracts. To investigate the possible enzymatic source of these radicals, inducible nitric oxide synthase (iNOS) and NADPH oxidase knockout mice were used. Free radical production was unchanged in the NADPH oxidase knockout but much decreased in the iNOS knockout mice, suggesting a role for iNOS in free radical production. Longer-term exposure to acetone revealed iNOS overexpression in the liver together with protein radical formation, which was detected by confocal microscopy and a novel immunospin-trapping method. Immunohistochemical analysis revealed enhanced lipid peroxidation and protein oxidation as a consequence of persistent free radical generation after 21 days of acetone treatment in control and NADPH oxidase knockout but not in iNOS knockout mice. Taken together, our data demonstrate that acetone administration, a model of ketosis, can lead to protein oxidation and lipid peroxidation through a free radical-dependent mechanism driven mainly by iNOS overexpression.

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JAZF1 heterozygous knockout mice show altered adipose development and metabolism

Cell & Bioscience ◽

10.1186/s13578-021-00625-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jain Jeong ◽

Soyoung Jang ◽

Song Park ◽

Wookbong Kwon ◽

Si-Yong Kim ◽

...

Keyword(s):

Adipose Tissue ◽

Knockout Mice ◽

Metabolic Disorders ◽

Adipocyte Differentiation ◽

Zinc Finger Protein ◽

Tissue Mass ◽

In Vivo Models ◽

Brown Adipose

Abstract Background Juxtaposed with another zinc finger protein 1 (JAZF1) is associated with metabolic disorders, including type 2 diabetes mellitus (T2DM). Several studies showed that JAZF1 and body fat mass are closely related. We attempted to elucidate the JAZF1 functions on adipose development and related metabolism using in vitro and in vivo models. Results The JAZF1 expression was precisely regulated during adipocyte differentiation of 3T3-L1 preadipocyte and mouse embryonic fibroblasts (MEFs). Homozygous JAZF1 deletion (JAZF1-KO) resulted in impaired adipocyte differentiation in MEF. The JAZF1 role in adipocyte differentiation was demonstrated by the regulation of PPARγ—a key regulator of adipocyte differentiation. Heterozygous JAZF1 deletion (JAZF1-Het) mice fed a normal diet (ND) or a high-fat diet (HFD) had less adipose tissue mass and impaired glucose homeostasis than the control (JAZF1-Cont) mice. However, other metabolic organs, such as brown adipose tissue and liver, were negligible effect on JAZF1 deficiency. Conclusion Our findings emphasized the JAZF1 role in adipocyte differentiation and related metabolism through the heterozygous knockout mice. This study provides new insights into the JAZF1 function in adipose development and metabolism, informing strategies for treating obesity and related metabolic disorders.

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