Desmoglein-3 (Dsg3), the Pemphigus Vulgaris (PV) antigen (PVA), plays an essential role in keratinocyte cell-cell adhesion and regulates various signaling pathways involved in the progression and metastasis of cancer where it is upregulated. We show here that expression of Dsg3 impacts on the expression and function of p53, a key transcription factor governing the responses to cellular stress. Dsg3 depletion increased p53 expression and activity, an effect enhanced by treating cells with UVB, mechanical stress and genotoxic drugs, whilst increased Dsg3 expression resulted in the opposite effects. Such a pathway in the negative regulation of p53 by Dsg3 was Dsg3 specific since neither E-cadherin nor desmoplakin knockdown caused similar effects. Analysis of Dsg3-/- mouse skin also indicated an increase of p53/p21WAF1/CIP1 and cleaved caspase-3 relative to Dsg3+/- controls. Finally, we evaluated whether this pathway was operational in the autoimmune disease PV in which Dsg3 serves as a major antigen involved in blistering pathogenesis. We uncovered increased p53 with diffuse cytoplasmic and/or nuclear staining in the oral mucosa of patients, including cells surrounding blisters and the pre-lesional regions. This finding was verified by in vitro studies where treatment of keratinocytes with PV sera, as well as a characterized pathogenic antibody specifically targeting Dsg3, evoked pronounced p53 expression and activity accompanied by disruption of cell-cell adhesion. Collectively, our findings suggest a novel role for Dsg3 as an anti-stress protein, via suppression of p53 function, and this pathway is disrupted in PV.
- MeSH
- desmoglein 3 nedostatek metabolismus MeSH
- desmozomy metabolismus MeSH
- fyziologický stres * MeSH
- kaspasa 3 metabolismus MeSH
- keratinocyty metabolismus MeSH
- kultivované buňky MeSH
- kůže metabolismus MeSH
- leupeptiny farmakologie MeSH
- lidé MeSH
- myši MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- pemfigus krev imunologie patologie MeSH
- proteolýza MeSH
- protilátky imunologie MeSH
- psi MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53β are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53β, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53β overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53β expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases.
- MeSH
- alternativní sestřih MeSH
- Alzheimerova nemoc metabolismus patologie MeSH
- amyotrofická laterální skleróza metabolismus patologie MeSH
- astrocyty cytologie účinky léků metabolismus MeSH
- autofagie účinky léků MeSH
- genetické vektory genetika metabolismus MeSH
- interleukin-6 genetika metabolismus MeSH
- kokultivační techniky MeSH
- kultivované buňky MeSH
- leupeptiny farmakologie MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- mozek metabolismus patologie MeSH
- nádorový supresorový protein p53 antagonisté a inhibitory genetika metabolismus MeSH
- neurony cytologie metabolismus MeSH
- neuroprotekce fyziologie MeSH
- protein - isoformy antagonisté a inhibitory genetika metabolismus MeSH
- RNA interference MeSH
- sekvestosom 1 antagonisté a inhibitory genetika metabolismus MeSH
- serin-arginin sestřihové faktory antagonisté a inhibitory genetika metabolismus MeSH
- stárnutí buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Porcine oocyte-cumulus complexes (OCCs) form an expanded cumulus extracellular matrix (ECM) in response to gonadotropins during meiotic maturation. Essential components of ECM are hyaluronan (HA), tumor necrosis factor α-induced protein 6 (TNFAIP6) and heavy chains (HC) of interalpha-trypsin inhibitor. To form expanded cumulus ECM, intermediate complexes (TNFAIP6-HC) must bind to HA to allow HC transfer onto HA. Protein turnover by the ubiquitin-proteasome pathway is poorly characterized in this process. It is known that the specific proteasomal inhibitor MG132 prevents cumulus expansion and formation of ECM. To determine whether inhibition of proteasomal proteolysis with MG132 affects cumulus cell steroidogenesis and expression of the cumulus expansion-related components (hyaluronan synthase type 2, HAS2, TNFAIP6) we cultured porcine OCCs and granulosa cells (GCs) in a medium supplemented with FSH/LH. Methods performed included real-time reverse transcription PCR, immunofluorescence and RIAs. The expression of TNFAIP6 and HAS2 transcripts increased significantly after the stimulation of OCCs and GCs with FSH/LH. In contrast, treatment with MG132 reduced the expression of TNFAIP6 and HAS2. Hyaluronan was detected with biotinylated HA-binding proteins within FSH/LH-stimulated expanded OCCs but not in those treated with MG132. Progesterone production, although increased almost three times after OCCs stimulation with FSH/LH, was significantly suppressed by MG132. The FSH/LH-stimulated a 40-fold increase in progesterone secretion by GCs was inhibited in the presence of MG132. In conclusion, MG132 affects progesterone secretion and expression of cumulus expansion-related components by cumulus and GCs, suggesting the requirement of ubiquitin-proteasome pathway-regulated protein turnover for formation of ECM during cumulus expansion in the preovulatory period in the pig.
- MeSH
- extracelulární matrix účinky léků metabolismus MeSH
- inhibitory cysteinových proteinas farmakologie MeSH
- inhibitory proteasomu MeSH
- kumulární buňky účinky léků metabolismus MeSH
- kvantitativní polymerázová řetězová reakce veterinární MeSH
- leupeptiny farmakologie MeSH
- messenger RNA biosyntéza genetika MeSH
- molekuly buněčné adheze biosyntéza MeSH
- oocyty účinky léků metabolismus MeSH
- prasata MeSH
- progesteron biosyntéza MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Small ubiquitin-related modifiers 1, 2 and 3 (SUMO-1, -2, -3), members of the ubiquitin-like protein family, can be conjugated to various cellular proteins. Conjugates of SUMO-2 and SUMO-3 (SUMO-2/3) accumulate in cells exposed to various stress stimuli or to MG132 treatment. Although the proteins modified by SUMO-2/3 during heat shock or under MG132 treatment have been identified, the significance of this modification remains unclear. Our data show that the inhibition of translation by puromycin or cycloheximide blocks both the heat shock and MG132 induced accumulation of SUMO-2/3 conjugates in HEK 293T and U2OS cells. However, the heat shock induced accumulation of SUMO-2/3 conjugates was restored by proteasome inhibition, which suggests that the inhibition of translation did not abolish SUMOylation itself. Furthermore, we show that some of the proteins truncated due to the treatment by low concentration of puromycin are SUMOylated in HEK 293T cells. We suggest that the SUMO-2/3 conjugates accumulating under the heat shock or MG132 treatment result largely from new protein synthesis and that portion of them is incorrectly folded.
- MeSH
- benzochinony farmakologie MeSH
- biologické modely MeSH
- cykloheximid farmakologie MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- inhibitory syntézy proteinů farmakologie MeSH
- leupeptiny farmakologie MeSH
- lidé MeSH
- makrocyklické laktamy farmakologie MeSH
- malé modifikační proteiny související s ubikvitinem metabolismus MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- proteosyntéza účinky léků MeSH
- puromycin farmakologie MeSH
- reakce na tepelný šok účinky léků MeSH
- sumoylace účinky léků MeSH
- ubikvitiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The toxic modes of action of non-dioxin-like polychlorinated biphenyls (PCBs) in liver cells are still only partially understood. Several recent studies have indicated that PCBs may interfere with cell membrane protein functions. Therefore, we analyzed in the present study the effects of di-ortho-substituted 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) on proteins involved in the formation of adherens junctions in a model of rat liver progenitor cells - WB-F344 cell line. PCB 153, at micromolar concentrations, induced a gradual degradation of E-cadherin, beta-catenin or plakoglobin (gamma-catenin) proteins. This effect was not due to changes in gene expression, as PCB 153 had no effect on mRNA levels of the above mentioned proteins. Moreover, apart from the reduction of total beta-catenin pool, PCB 153 also decreased levels of the active beta-catenin form, dephosphorylated at residues Ser37 and Thr41, which is the key co-activator of Wnt-induced TCF/LEF-dependent gene expression. Therefore, we also evaluated the impact of PCB 153 on expression of Axin2, a known transcriptional target of canonical Wnt signaling. PCB 153 reduced basal Axin2 mRNA levels and it inhibited induction of Axin2 expression by recombinant mouse Wnt3a. Nevertheless, PCB 153 had no effect on phosphorylation of glycogen synthase kinase-3beta (GSK-3beta), which is supposed to target beta-catenin for its proteasomal degradation. This suggested that GSK-3beta activity is not modulated by PCB 153 and, consequently, not involved in the observed PCB 153-induced decrease of both total and active beta-catenin levels. Protein levels of E-cadherin and beta-catenin were partially restored with lysosomal inhibitor leupeptin, thus suggesting a possible role of lysosomes in the observed degradation of adherens junction proteins. Taken together, the present data suggest that PCB 153 may interfere with functions of adherens junction proteins involved in both cell-to-cell communication and intracellular signaling. Such mechanisms might be involved in the effects of non-dioxin-like PCBs contributing to liver tumor promotion.
- MeSH
- adhezní spoje metabolismus účinky léků MeSH
- beta-katenin antagonisté a inhibitory biosyntéza genetika MeSH
- buněčné linie MeSH
- epitelové buňky cytologie metabolismus účinky léků MeSH
- fosforylace účinky léků MeSH
- gama-katenin biosyntéza genetika metabolismus MeSH
- genetická transkripce účinky léků MeSH
- inhibitory cysteinových proteinas farmakologie MeSH
- játra cytologie metabolismus účinky léků MeSH
- kadheriny biosyntéza genetika metabolismus MeSH
- kinasa 3 glykogensynthasy metabolismus MeSH
- kmenové buňky cytologie metabolismus účinky léků MeSH
- krysa rodu rattus MeSH
- leupeptiny farmakologie MeSH
- lyzozomy metabolismus MeSH
- messenger RNA biosyntéza genetika MeSH
- polychlorované bifenyly toxicita MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- represorové proteiny biosyntéza genetika metabolismus MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
One of the toxic effects of non-dioxin-like polychlorinated biphenyls (NDL-PCBs) is the acute inhibition of gap junctional intercellular communication (GJIC), an event possibly associated with tumor promotion. The model NDL-PCB-2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153)-induces a sustained GJIC inhibition in rat liver epithelial WB-F344 cells. As this effect might be related to deregulation of connexin 43 (Cx43) synthesis, trafficking, or degradation, we investigated the impact of PCB 153 on these events. Although PCB 153 had no effect on Cx43 mRNA levels, it induced a gradual loss of Cx43 protein and significantly decreased the amount of gap junction plaques in plasma membrane. PCB 153 contributed to extracellular signal-regulated kinases 1 and 2 (ERK1/2)-dependent accumulation of hyperphosphorylated Cx43-P3 form, thus indicating that ERK1/2 activation by PCB 153 might contribute to its effects on Cx43 internalization or degradation. Inhibition of either proteasomes or lysosomes with their specific inhibitors largely restored total Cx43 protein levels, thus suggesting that both proteasomes and lysosomes may participate in the PCB 153-enhanced Cx43 internalization and degradation. However, neither the proteasomal nor the lysosomal inhibitors restored normal GJIC or number/size of gap junction plaques. Finally, PCB 153 also interfered with restoration of gap junction plaques following the inhibition of Cx43 transport to plasma membrane. Taken together, multiple modes of action seem to contribute to downregulation of Cx43 in PCB 153-treated rat liver epithelial cells. The enhanced degradation of Cx43, together with persistent inhibition of GJIC, might contribute to tumor-promoting effects of NDL-PCBs.
- MeSH
- analýza rozptylu MeSH
- buněčná membrána účinky léků MeSH
- buněčné linie MeSH
- extracelulárním signálem regulované MAP kinasy antagonisté a inhibitory metabolismus MeSH
- inhibitory proteasomu MeSH
- játra metabolismus MeSH
- konexin 43 genetika metabolismus MeSH
- krysa rodu rattus MeSH
- leupeptiny farmakologie MeSH
- lyzozomy metabolismus účinky léků MeSH
- metabolické sítě a dráhy účinky léků MeSH
- mezerový spoj metabolismus účinky léků MeSH
- mezibuněčná komunikace účinky léků MeSH
- polychlorované bifenyly farmakologie MeSH
- proteasomový endopeptidasový komplex účinky léků metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Cellular signaling by glucocorticoid receptor and aryl hydrocarbon receptor is restricted by microtubules interfering agents (MIAs). This leads to down-regulation of drug metabolizing enzymes and drug interactions. Here we investigated the effects of all-trans-retinoic acid (ATRA) and MIAs, i.e. colchicine, nocodazole and taxol on the regulation of retinoic acid receptor (RAR) genes in primary cultures of rat hepatocytes. ATRA (1microM) down-regulated RARalpha and RARgamma mRNAs (decrease 23% and 41%, respectively) whereas it up-regulated RARbeta mRNA (4.3-fold induction). All MIAs diminished the expression of RARs in dose-dependent manner; the potency of MIAs increased in order NOC
- MeSH
- financování organizované MeSH
- genetická transkripce účinky záření MeSH
- HeLa buňky cytologie metabolismus účinky léků MeSH
- inhibitory proteasomu MeSH
- katalýza účinky léků MeSH
- kolchicin farmakologie MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- leupeptiny farmakologie MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- mikrotubuly účinky léků MeSH
- modulátory tubulinu farmakologie MeSH
- proteasomový endopeptidasový komplex MeSH
- receptory kyseliny retinové genetika metabolismus MeSH
- regulace genové exprese účinky záření MeSH
- termodynamika MeSH
- transglutaminasy metabolismus MeSH
- tretinoin farmakologie MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
Proteasome inhibitors are novel therapeutic agents which may be used in treatment of cancer and other severe disorders. We studied the effect of proteasome inhibitor MG-132 on protein and amino acid metabolism. In MG-132-treated rats we observed a significant decrease in proteasome-dependent proteolysis in skeletal muscle and an increase in whole-body protein turnover (i.e., increase in whole-body proteolysis and protein synthesis). Proteasome-dependent proteolysis was activated in the liver and kidney, protein synthesis increased in skeletal muscle, liver, and kidney. Insignificant changes were found in jejunum and colon. MG-132 administration induced a significant increase in concentration of several amino acids in blood plasma and their decrease in jejunum and colon. We conclude that administration of MG-132 affects both protein anabolic and protein catabolic pathways via the direct effect on proteasome-dependent proteolysis and indirect effect on proteolysis and protein synthesis via unidentified mediators.
- MeSH
- aminokyseliny metabolismus MeSH
- financování organizované MeSH
- inhibitory proteasomu MeSH
- krysa rodu rattus MeSH
- leupeptiny farmakologie MeSH
- metabolická clearance účinky léků MeSH
- orgánová specificita MeSH
- potkani Wistar MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- proteom metabolismus MeSH
- tkáňová distribuce MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
