Expression of hepcidin, the hormone regulating iron homeostasis, is increased by iron overload and decreased by accelerated erythropoiesis or iron deficiency. The purpose of the study was to examine the effect of these stimuli, either alone or in combination, on the main signaling pathway controlling hepcidin biosynthesis in the liver, and on the expression of splenic modulators of hepcidin biosynthesis. Liver phosphorylated SMAD 1 and 5 proteins were determined by immunoblotting in male mice treated with iron dextran, kept on an iron deficient diet, or administered recombinant erythropoietin for four consecutive days. Administration of iron increased liver phosphorylated SMAD protein content and hepcidin mRNA content; subsequent administration of erythropoietin significantly decreased both the iron-induced phosphorylated SMAD proteins and hepcidin mRNA. These results are in agreement with the recent observation that erythroferrone binds and inactivates the BMP6 protein. Administration of erythropoietin substantially increased the amount of erythroferrone and transferrin receptor 2 proteins in the spleen; pretreatment with iron did not influence the erythropoietin-induced content of these proteins. Erythropoietin-treated iron-deficient mice displayed smaller spleen size in comparison with erythropoietin-treated mice kept on a control diet. While the erythropoietin-induced increase in splenic erythroferrone protein content was not significantly affected by iron deficiency, the content of transferrin receptor 2 protein was lower in the spleens of erythropoietin-treated mice kept on iron-deficient diet, suggesting posttranscriptional regulation of transferrin receptor 2. Interestingly, iron deficiency and erythropoietin administration had additive effect on hepcidin gene downregulation in the liver. In mice subjected both to iron deficiency and erythropoietin administration, the decrease of hepcidin expression was much more pronounced than the decrease in phosphorylated SMAD protein content or the decrease in the expression of the SMAD target genes Id1 and Smad7. These results suggest the existence of another, SMAD-independent pathway of hepcidin gene downregulation.

• MeSH
• deficit železa MeSH
• erythropoetin aplikace a dávkování   MeSH
• erytropoéza účinky léků   MeSH
• fosforylace MeSH
• hepcidiny genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• přetížení železem metabolismus   patologie   MeSH
• proteiny Smad genetika   metabolismus   MeSH
• regulace genové exprese účinky léků   MeSH
• železo aplikace a dávkování   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Our objective was to investigate the effect of cholesterol [hypercholesterolemia and 7-ketocholesterol (7K)] on endoglin (Eng) expression and regulation with respect to endothelial or vascular dysfunction in vivo and in vitro. In vivo experiments were performed in 2-mo-old atherosclerosis-prone apolipoprotein E-deficient/LDL receptor-deficient (ApoE-/-/LDLR-/-) female mice and their wild-type C57BL/6J littermates. In in vitro experiments, human aortic endothelial cells (HAECs) were treated with 7K. ApoE-/-/LDLR-/- mice developed hypercholesterolemia accompanied by increased circulating levels of P-selectin and Eng and a disruption of NO metabolism. Functional analysis of the aorta demonstrated impaired vascular reactivity, and Western blot analysis revealed down-regulation of membrane Eng/Smad2/3/eNOS signaling in ApoE-/-/LDLR-/- mice. 7K increased Eng expression via Krüppel-like factor 6 (KLF6), liver X nuclear receptor, and NF-κB in HAECs. 7K-induced Eng expression was prevented by the treatment with 2-hydroxypropyl-β-cyclodextrin; 8-{[5-chloro-2-(4-methylpiperazin-1-yl) pyridine-4-carbonyl] amino}-1-(4-fluorophenyl)-4, 5-dihydrobenzo[g]indazole-3-carboxamide; or by KLF6 silencing. 7K induced increased adhesion and transmigration of monocytic human leukemia promonocytic cell line cells and was prevented by Eng silencing. We concluded that hypercholesterolemia altered Eng expression and signaling, followed by endothelial or vascular dysfunction before formation of atherosclerotic lesions in ApoE-/-/LDLR-/- mice. By contrast, 7K increased Eng expression and induced inflammation in HAECs, which was followed by an increased adhesion and transmigration of monocytes via endothelium, which was prevented by Eng inhibition. Thus, we propose a relevant role for Eng in endothelial or vascular dysfunction or inflammation when exposed to cholesterol.-Vicen, M., Vitverova, B., Havelek, R., Blazickova, K., Machacek, M., Rathouska, J., Najmanová, I., Dolezelova, E., Prasnicka, A., Sternak, M., Bernabeu, C., Nachtigal, P. Regulation and role of endoglin in cholesterol-induced endothelial and vascular dysfunction in vivo and in vitro.

• MeSH
• aorta cytologie   metabolismus   patologie   MeSH
• apolipoproteiny E genetika   MeSH
• aterosklerotický plát etiologie   genetika   metabolismus   MeSH
• beta-cyklodextriny farmakologie   MeSH
• cévní endotel účinky léků   metabolismus   patologie   MeSH
• cholesterol metabolismus   MeSH
• endoglin genetika   metabolismus   MeSH
• hypercholesterolemie komplikace   genetika   metabolismus   MeSH
• indazoly farmakologie   MeSH
• KLF6 metabolismus   MeSH
• kultivované buňky MeSH
• kyseliny isonikotinové farmakologie   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• NF-kappa B metabolismus   MeSH
• oxid dusnatý metabolismus   MeSH
• P-selektin metabolismus   MeSH
• proteiny Smad metabolismus   MeSH
• receptory LDL genetika   MeSH
• synthasa oxidu dusnatého, typ III metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: During cancer progression, epithelial cancer cells can be reprogrammed into mesenchymal-like cells with increased migratory potential through the process of epithelial-mesenchymal transition (EMT), representing an essential step of tumor progression towards metastatic state. AGR2 protein was shown to regulate several cancer-associated processes including cellular proliferation, survival and drug resistance. METHODS: The expression of AGR2 was analyzed in cancer cell lines exposed to TGF-β alone or to combined treatment with TGF-β and the Erk1/2 inhibitor PD98059 or the TGF-β receptor specific inhibitor SB431542. The impact of AGR2 silencing by specific siRNAs or CRISPR/Cas9 technology on EMT was investigated by western blot analysis, quantitative PCR, immunofluorescence analysis, real-time invasion assay and adhesion assay. RESULTS: Induction of EMT was associated with decreased AGR2 along with changes in cellular morphology, actin reorganization, inhibition of E-cadherin and induction of the mesenchymal markers vimentin and N-cadherin in various cancer cell lines. Conversely, induction of AGR2 caused reversion of the mesenchymal phenotype back to the epithelial phenotype and re-acquisition of epithelial markers. Activated Smad and Erk signaling cascades were identified as mutually complementary pathways responsible for TGF-β-mediated inhibition of AGR2. CONCLUSION: Taken together our results highlight a crucial role for AGR2 in maintaining the epithelial phenotype by preventing the activation of key factors involved in the process of EMT.

• MeSH
• buněčná adheze genetika   MeSH
• epitelo-mezenchymální tranzice účinky léků   genetika   MeSH
• genový knockdown MeSH
• kadheriny metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• pohyb buněk genetika   MeSH
• proteiny Smad metabolismus   MeSH
• proteiny genetika   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• signální transdukce účinky léků   MeSH
• transformující růstový faktor beta farmakologie   MeSH
• vimentin metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Inflammatory-mediated pathological processes in the endothelium arise as a consequence of the dysregulation of vascular homeostasis. Of particular importance are mediators produced by stimulated monocytes/macrophages inducing activation of endothelial cells (ECs). This is manifested by excessive soluble pro-inflammatory mediator production and cell surface adhesion molecule expression. Nitro-fatty acids are endogenous products of metabolic and inflammatory reactions that display immuno-regulatory potential and may represent a novel therapeutic strategy to treat inflammatory diseases. The purpose of our study was to characterize the effects of nitro-oleic acid (OA-NO2) on inflammatory responses and the endothelial-mesenchymal transition (EndMT) in ECs that is a consequence of the altered healing phase of the immune response. METHODS: The effect of OA-NO2 on inflammatory responses and EndMT was determined in murine macrophages and murine and human ECs using Western blotting, ELISA, immunostaining, and functional assays. RESULTS: OA-NO2 limited the activation of macrophages and ECs by reducing pro-inflammatory cytokine production and adhesion molecule expression through its modulation of STAT, MAPK and NF-κB-regulated signaling. OA-NO2 also decreased transforming growth factor-β-stimulated EndMT and pro-fibrotic phenotype of ECs. These effects are related to the downregulation of Smad2/3. CONCLUSIONS: The study shows the pleiotropic effect of OA-NO2 on regulating EC-macrophage interactions during the immune response and suggests a role for OA-NO2 in the regulation of vascular endothelial immune and fibrotic responses arising during chronic inflammation. GENERAL SIGNIFICANCE: These findings propose the OA-NO2 may be useful as a novel therapeutic agent for treatment of cardiovascular disorders associated with dysregulation of the endothelial immune response.

• MeSH
• cévní endotel cytologie   účinky léků   metabolismus   MeSH
• endoteliální buňky účinky léků   metabolismus   MeSH
• epitelo-mezenchymální tranzice * MeSH
• kyseliny olejové farmakologie   MeSH
• lidé MeSH
• makrofágy účinky léků   metabolismus   MeSH
• MAP kinasový signální systém MeSH
• myši MeSH
• NF-kappa B metabolismus   MeSH
• proteiny Smad metabolismus   MeSH
• transformující růstový faktor beta farmakologie   MeSH
• transkripční faktory STAT metabolismus   MeSH
• zánět metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Cellular senescence provides a biological barrier against tumor progression, often associated with oncogene-induced replication and/or oxidative stress, cytokine production and DNA damage response (DDR), leading to persistent cell-cycle arrest. While cytokines such as tumor necrosis factor-alpha (TNFα) and interferon gamma (IFNγ) are important components of senescence-associated secretome and induce senescence in, for example, mouse pancreatic β-cancer cell model, their downstream signaling pathway(s) and links with oxidative stress and DDR are mechanistically unclear. Using human and mouse normal and cancer cell models, we now show that TNFα and IFNγ induce NADPH oxidases Nox4 and Nox1, reactive oxygen species (ROS), DDR signaling and premature senescence. Unlike mouse tumor cells that required concomitant presence of IFNγ and TNFα, short exposure to IFNγ alone was sufficient to induce Nox4, Nox1 and DDR in human cells. siRNA-mediated knockdown of Nox4 but not Nox1 decreased IFNγ-induced DDR. The expression of Nox4/Nox1 required Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling and the effect was mediated by downstream activation of transforming growth factor-beta (TGFβ) secretion and consequent autocrine/paracrine activation of the TGFβ/Smad pathway. Furthermore, the expression of adenine nucleotide translocase 2 (ANT2) was suppressed by IFNγ contributing to elevation of ROS and DNA damage. In contrast to mouse B16 cells, inability of TC-1 cells to respond to IFNγ/TNFα by DDR and senescence correlated with the lack of TGFβ and Nox4 response, supporting the role of ROS induced by NADPH oxidases in cytokine-induced senescence. Overall, our data reveal differences between cytokine effects in mouse and human cells, and mechanistically implicate the TGFβ/SMAD pathway, via induction of NADPH oxidases and suppression of ANT2, as key mediators of IFNγ/TNFα-evoked genotoxicity and cellular senescence.

• MeSH
• enzymová indukce účinky léků   MeSH
• interferon gama farmakologie   MeSH
• lidé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• NADPH-oxidasy biosyntéza   genetika   MeSH
• oxidační stres účinky léků   MeSH
• poškození DNA * MeSH
• proteiny Smad metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• signální transdukce účinky léků   MeSH
• stárnutí buněk účinky léků   MeSH
• TNF-alfa farmakologie   MeSH
• transformující růstový faktor beta metabolismus   MeSH
• transkripční faktory STAT metabolismus   MeSH
• translokátor adeninových nukleotidů 2 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily. Its ligand, 1,25-(OH)2D, is a metabolically active hormone derived from vitamin D3. The levels of vitamin D3 are decreased in patients with systemic sclerosis (SSc). Here, we aimed to analyse the role of VDR signalling in fibrosis. METHODS: VDR expression was analysed in SSc skin, experimental fibrosis and human fibroblasts. VDR signalling was modulated by siRNA and with the selective agonist paricalcitol. The effects of VDR on Smad signalling were analysed by reporter assays, target gene analyses and coimmunoprecipitation. The effects of paricalcitol were evaluated in the models of bleomycin-induced fibrosis and fibrosis induced by overexpression of a constitutively active transforming growth factor-β (TGF-β) receptor I (TBRI(CA)). RESULTS: VDR expression was decreased in fibroblasts of SSc patients and murine models of SSc in a TGF-β-dependent manner. Knockdown of VDR enhanced the sensitivity of fibroblasts towards TGF-β. In contrast, activation of VDR by paricalcitol reduced the stimulatory effects of TGF-β on fibroblasts and inhibited collagen release and myofibroblast differentiation. Paricalcitol stimulated the formation of complexes between VDR and phosphorylated Smad3 in fibroblasts to inhibit Smad-dependent transcription. Preventive and therapeutic treatment with paricalcitol exerted potent antifibrotic effects and ameliorated bleomycin- as well as TBRI(CA)-induced fibrosis. CONCLUSIONS: We characterise VDR as a negative regulator of TGF-β/Smad signalling. Impaired VDR signalling with reduced expression of VDR and decreased levels of its ligand may thus contribute to hyperactive TGF-β signalling and aberrant fibroblast activation in SSc.

• MeSH
• bleomycin toxicita   MeSH
• dospělí MeSH
• ergokalciferoly farmakologie   MeSH
• fibroblasty účinky léků   metabolismus   MeSH
• fibróza chemicky indukované   metabolismus   MeSH
• kůže účinky léků   metabolismus   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• mladý dospělý MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• proteiny Smad účinky léků   metabolismus   MeSH
• receptory kalcitriolu agonisté   metabolismus   MeSH
• senioři MeSH
• signální transdukce účinky léků   fyziologie   MeSH
• systémová sklerodermie metabolismus   MeSH
• transformující růstový faktor beta účinky léků   metabolismus   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

• MeSH
• chronická renální insuficience * farmakoterapie   metabolismus   prevence a kontrola   MeSH
• diabetické nefropatie * farmakoterapie   metabolismus   MeSH
• fibroblasty * metabolismus   MeSH
• hypertenze * metabolismus   MeSH
• lidé MeSH
• NF-kappa B * metabolismus   MeSH
• pohyb buněk MeSH
• produkty pokročilé glykace * metabolismus   MeSH
• proliferace buněk MeSH
• proteiny Smad * metabolismus   MeSH
• signální transdukce MeSH
• svaly hladké cévní * fyziologie   MeSH
• transformující růstový faktor beta * metabolismus   MeSH
• vápníkem aktivované draslíkové kanály se střední vodivostí * antagonisté a inhibitory   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

Many cancers arise at sites of infection and inflammation. Cellular senescence, a permanent state of cell cycle arrest that provides a barrier against tumorigenesis, is accompanied by elevated proinflammatory cytokines such as IL1, IL6, IL8 and TNFα. Here we demonstrate that media conditioned by cells undergoing any of the three main forms of senescence, i.e. replicative, oncogene- and drug-induced, contain high levels of IL1, IL6, and TGFb capable of inducing reactive oxygen species (ROS)-mediated DNA damage response (DDR). Persistent cytokine signaling and activated DDR evoke senescence in normal bystander cells, accompanied by activation of the JAK/STAT, TGFβ/SMAD and IL1/NFκB signaling pathways. Whereas inhibition of IL6/STAT signaling had no effect on DDR induction in bystander cells, inhibition of either TGFβ/SMAD or IL1/NFκB pathway resulted in decreased ROS production and reduced DDR in bystander cells. Simultaneous inhibition of both TGFβ/SMAD and IL1/NFκB pathways completely suppressed DDR indicating that IL1 and TGFβ cooperate to induce and/or maintain bystander senescence. Furthermore, the observed IL1- and TGFβ-induced expression of NAPDH oxidase Nox4 indicates a mechanistic link between the senescence-associated secretory phenotype (SASP) and DNA damage signaling as a feature shared by development of all major forms of paracrine bystander senescence.

• MeSH
• buněčné linie MeSH
• bystander efekt účinky léků   MeSH
• etoposid farmakologie   MeSH
• geny ras * MeSH
• interleukin-1 metabolismus   MeSH
• interleukin-6 metabolismus   MeSH
• Janus kinasy metabolismus   MeSH
• kultivační média speciální metabolismus   MeSH
• lidé MeSH
• NADPH-oxidasy metabolismus   MeSH
• NF-kappa B metabolismus   MeSH
• oxidační stres účinky léků   MeSH
• parakrinní signalizace účinky léků   MeSH
• poškození DNA * MeSH
• proliferace buněk * MeSH
• proteiny Smad metabolismus   MeSH
• RNA interference MeSH
• signální transdukce účinky léků   MeSH
• stárnutí buněk účinky léků   MeSH
• transfekce MeSH
• transformující růstový faktor beta metabolismus   MeSH
• transkripční faktory STAT metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Chronic inflammation plays an important role in the initiation and progression of various human diseases including benign prostatic hyperplasia or prostate cancer. Here we show that the proinflammatory cytokine interleukin-6 (IL-6) has prosurvival effects and chronically activates the Jak2/STAT3 signalling pathway in a model of benign prostatic hyperplasia (BPH-1). We demonstrate that the antiinflammatory cytokine transforming growth factor-beta1 (TGF-beta1), which also permanently activates its canonical signalling pathway through SMAD proteins in BPH-1 cells, modifies the effects of IL-6 on cell proliferation. Importantly, TGF-beta1 inhibits IL-6 signal transduction by decreasing the phosphorylation levels of STAT3. This effect is associated with decreased expression of Jak2 at both mRNA and protein levels. Moreover, we showed that TGF-beta1 inhibits IL-6-induced expression of the cancer-associated gene MUC1. These observations demonstrated a novel interaction between TGF-beta1 and IL-6 signalling and suggested another mechanism of how defects in TGF-beta signalling, frequently associated with prostate pathologies, can contribute to the disruption of tissue homeostasis.

• MeSH
• buněčné linie MeSH
• epitelové buňky metabolismus   MeSH
• fosforylace MeSH
• hyperplazie prostaty enzymologie   metabolismus   MeSH
• interleukin-6 antagonisté a inhibitory   farmakologie   MeSH
• Janus kinasa 2 genetika   metabolismus   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• mucin 1 metabolismus   MeSH
• proliferace buněk MeSH
• prostata cytologie   enzymologie   metabolismus   MeSH
• proteiny Smad metabolismus   MeSH
• RNA interference MeSH
• signální transdukce MeSH
• transformující růstový faktor beta1 farmakologie   MeSH
• transkripční faktor STAT3 metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• aorta metabolismus   účinky léků   MeSH
• ateroskleróza * etiologie   genetika   metabolismus   MeSH
• cévní endotel metabolismus   účinky léků   MeSH
• exprese genu * genetika   účinky léků   MeSH
• financování organizované MeSH
• intracelulární signální peptidy a proteiny genetika   izolace a purifikace   metabolismus   MeSH
• lipoproteiny izolace a purifikace   klasifikace   metabolismus   MeSH
• myši MeSH
• proteiny Smad genetika   klasifikace   metabolismus   MeSH
• statiny diagnostické užití   MeSH
• synthasa oxidu dusnatého metabolismus   účinky léků   MeSH
• transformující růstový faktor beta izolace a purifikace   metabolismus   MeSH
• western blotting metody   využití   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH