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Balogh P.,Semmelweis University | Magyar M.,Semmelweis University | Szabo A.,Semmelweis University | Mullner N.,Semmelweis University | And 3 more authors.
European Journal of Cell Biology | Year: 2015

We previously showed that intraperitoneal administration of Freund's adjuvant treatment resulted in acute peritonitis and TGF-β was found to be one of the main organizers of the subsequent EMT in mesothelial cells. In the present study, we investigated whether TGF-β signaling molecules are present in mesothelial cells and how their compartmentalization pattern changes with the dynamics of inflammatory events in vivo. In addition, we tried to evaluate the turnover of endosomal compartments concomitant with the internalization of signaling molecules and examine whether caveola-mediated internalization might play a role in the termination of TGF-β signaling. Using immunocytochemical approach, we could detect TβRII in EEA1 positive compartments and as the inflammation progressed, at D3, the receptor appeared in caveolin-1 positive intracellular structures as well. The latter event was accompanied by the appearance of negative regulatory protein, Smad7 in caveolae. We also found EEA1 and caveolin-1 double positive vesicular structures that were corresponded to forming MVBs affirmed by our immuno-electron microscopical results. Fine structural, morphometric and immunoblot analysis proved that Cd63 positive multivesicular body (MVB) formation was significantly increased by D3 and the IP results confirmed that TβRII as well as caveolin-1 were strongly associated with these endosomal compartments at this time. In contrast, by the termination of inflammation, by D5, caveolin-1 was found to be associated with late endosomal marker, Rab7 and entirely degraded from the system. Despite the limitations of an in vivo system, our results provide both morphological and biochemical data about the endosomal compartments involved in the internalization of TβRII upon inflammatory stimuli. Furthermore, our study implies the possible role of caveola-mediated endocytosis in the attenuation of TGF-β signaling and highlight the significance of endosomal compartments via which caveolae might meet the classical endocytic pathway under in vivo inflammatory conditions. © 2015 Elsevier GmbH. Source


Balogh P.,Semmelweis University | Magyar M.,Semmelweis University | Szabo A.,Semmelweis University | Mullner N.,Semmelweis University | And 3 more authors.
European journal of cell biology | Year: 2015

We previously showed that intraperitoneal administration of Freund's adjuvant treatment resulted in acute peritonitis and TGF-β was found to be one of the main organizers of the subsequent EMT in mesothelial cells. In the present study, we investigated whether TGF-β signaling molecules are present in mesothelial cells and how their compartmentalization pattern changes with the dynamics of inflammatory events in vivo. In addition, we tried to evaluate the turnover of endosomal compartments concomitant with the internalization of signaling molecules and examine whether caveola-mediated internalization might play a role in the termination of TGF-β signaling. Using immunocytochemical approach, we could detect TβRII in EEA1 positive compartments and as the inflammation progressed, at D3, the receptor appeared in caveolin-1 positive intracellular structures as well. The latter event was accompanied by the appearance of negative regulatory protein, Smad7 in caveolae. We also found EEA1 and caveolin-1 double positive vesicular structures that were corresponded to forming MVBs affirmed by our immuno-electron microscopical results. Fine structural, morphometric and immunoblot analysis proved that Cd63 positive multivesicular body (MVB) formation was significantly increased by D3 and the IP results confirmed that TβRII as well as caveolin-1 were strongly associated with these endosomal compartments at this time. In contrast, by the termination of inflammation, by D5, caveolin-1 was found to be associated with late endosomal marker, Rab7 and entirely degraded from the system. Despite the limitations of an in vivo system, our results provide both morphological and biochemical data about the endosomal compartments involved in the internalization of TβRII upon inflammatory stimuli. Furthermore, our study implies the possible role of caveola-mediated endocytosis in the attenuation of TGF-β signaling and highlight the significance of endosomal compartments via which caveolae might meet the classical endocytic pathway under in vivo inflammatory conditions. Copyright © 2015 Elsevier GmbH. All rights reserved. Source


Balogh P.,Semmelweis University | Szabo A.,Semmelweis University | Liko I.,Has Se Lendulet Hereditary Endocrine Tumors Research Group | Patocs A.,Has Se Lendulet Hereditary Endocrine Tumors Research Group | L.Kiss A.,Semmelweis University
Cell and Tissue Research | Year: 2015

Following Freund’s adjuvant-induced acute inflammation, the regeneration of rat mesothelium is accompanied by the reduction of cell organelles. The aim of the present study is to test whether autophagy may play a role in the recovery process of mesothelial cells by eliminating accumulated cell organelles and also to investigate the presence of potential inducers and molecular transmitters of the process. Control and treated (from day 2 to day 11; D2–D11) mesothelial cells (n = 16 samples/group) obtained from male rats were isolated and phenotypically characterized. Morphological studies included light and electron microscopy. Biochemical studies performed on tissue samples as well as isolated cells were used to evaluate the dynamics of autophagy and also to detect the expression levels of TNF-α, LC3B, estrogen receptors (ER-α and GPR30) and Erk1/2. Gene expression was measured by individual Taqman assays on quantitative RT-PCR. Protein expression study was performed by Western blotting and immunolabeling. Estradiol concentration was measured both in peritoneal fluid and plasma samples in control and treated animals (n = 3–10 animals per group). Our conventional electron microscopic and morphometric results showed a progressive autophagosome formation with a peak by the termination of inflammation (D5). Subsequently, autophagolysosome formation dominated between D6 and D8 with a concomitant expression of LC3B proved by immunoblotting. We further observed the reduction of cell compartments by D11 parallel with the morphological restitution of mesothelium. Estradiol showed a sustained level in the peritoneal fluid but not in plasma samples between D3 and D11 compared to levels obtained from untreated animals. The mRNA expression of TNF-α was increased between D2 and D11 compared to control. Western blot analysis showed a constitutive expression of GPR30, while ER-α could not be detected between D6 and D11. Erk1/2 was activated by phosphorylation with a peak at D6. Considering our present in vivo results, we hypothesize that the facilitated autophagy might play an important role in the removal of cytoplasmic organelles during the recovery of mesothelium, while our results also suggest that the detected peritoneal estradiol as well as TNF-α may contribute to this process. © 2015, Springer-Verlag Berlin Heidelberg. Source


Labadi A.,University of Pecs | Grassi E.S.,University of Milan | Gellen B.,University of Szeged | Kleinau G.,Charite - Medical University of Berlin | And 11 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2015

Context: Congenital hypothyroidism (CH) is one of the most common inborn endocrine disorders with genetic background. Despite the well-established newborn CH screening program in Hungary, no systematic examination of the underlying genetic alterations has been performed as yet. Objective: We aimed to explore TSH receptor (TSHR) mutations in a cohort of Hungarian patients with CH. Patients: Eighty-five unrelated patients with permanent primary CH, all diagnosed at newborn screening, were selected. Main Outcome Measures: Coding exons of the TSHR gene were sequenced and evaluated together with the thyroid-specific clinical parameters. Functional features of the novel mutations were experimentally examined, and their comparative molecular models were built. Results: In four patients (one heterozygous and three compound heterozygous), seven TSHR mutations were identified. Among these, N4321.50D and P4492.39L are novel missense alterations. Importantly, theN4321.50 residueis highlyconservedamongGprotein-coupledreceptors,anditsfunctionhas not been examined yet in human glycoprotein hormone receptors. Our results indicate that the N4321.50D mutation disrupts important, architecture-stabilizing intramolecular interactions and ultimately leads to the complete intracellular retention of the receptor. On the other hand, P4492.39 is located in the intracellular part of the receptor, which is important inGprotein coupling. The P4492.39L mutation results in signaling impairment, with a more profound effect on the Gq/11 pathway. Conclusion: TSHR mutations are common among Hungarian patients with CH. The novel genetic alterations revealed an important structural role of the N4321.50 and the P4492.39 residues in receptor expression and signaling, respectively. Copyright © 2015 by the Endocrine Society. Source

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