The TGF-β/Smad Repressor TG-Interacting Factor 1 (TGIF1) Plays a Role in Radiation-Induced Intestinal Injury Independently of a Smad Signaling Pathway

Despite advances in radiation delivery protocols, exposure of normal tissues during the course of radiation therapy remains a limiting factor of cancer treatment. If the canonical TGF-β/Smad pathway has been extensively studied and implicated in the development of radiation damage in various organs, the precise modalities of its activation following radiation exposure remain elusive. In the present study, we hypothesized that TGF-β1 signaling and target genes expression may depend on radiation-induced modifications in Smad transcriptional co-repressors/inhibitors expressions (TGIF1, SnoN, Ski and Smad7). In endothelial cells (HUVECs) and in a model of experimental radiation enteropathy in mice, radiation exposure increases expression of TGF-β/Smad pathway and of its target gene PAI-1, together with the overexpression of Smad co-repressor TGIF1. In mice, TGIF1 deficiency is not associated with changes in the expression of radiation-induced TGF-β pathway-related transcripts following localized small intestinal irradiation. In HUVECs, TGIF1 overexpression or silencing has no influence either on the radiation-induced Smad activation or the Smad3-dependent PAI-1 overexpression. However, TGIF1 genetic deficiency sensitizes mice to radiation-induced intestinal damage after total body or localized small intestinal radiation exposure, demonstrating that TGIF1 plays a role in radiation-induced intestinal injury. In conclusion, the TGF-β/Smad co-repressor TGIF1 plays a role in radiation-induced normal tissue damage by a Smad-independent mechanism

Rôle du Transforming Growth Factor-ß (TGFß) dans la physiopathologie des cellules musculaires lisses vasculaires (CMLv) (étude in vitro sur la croissance et par la transgénèse sur le développement)

Ce travail éclaire le rôle du TGFß dans la physiopathologie des CMLv in vitro sur des CMLv en culture et in vivo dans des souris transgéniques avec invalidation de la voie de signalisation du TGFß dans les CMLv. In vitro, nous montrons comment le TGFß inhibe ou stimule à faible ou forte densité cellulaire, respectivement, la croissance des CMLv. Le TGFß impose, indépendamment de la densité, un niveau faible mais constant de prolifération et, à forte densité, les CMLv échappent à l inhibition de contact. Par contre, le TGFß n induit l apoptose des CMLv qu à faible densité. Relativement aux cellules non traitées, à faible densité, la proliferation réduite et la forte apoptose inhibent la croissance des CMLv traitées mais, à forte densité, le maintien de la prolifération en l absence d apoptose stimule la croissance des cellules traitées. Ces effets du TGFß sur la prolifération et l apoptose des CMLv reposent sur une régulation non conventionnelle des protéines du cycle cellulaire et de la voie de survie PI3 Kinase/Akt. Dans les souris transgéniques, l invalidation de la voie de signalisation du TGFß dans les CMLv est létale entre E14,5 et E18,5 en raison d anomalies vasculaires systématiques et cardiaques occasionnelles. Dans l aorte thoracique descendante, les fibres élastiques sont soit absentes soit fragmentées et des anévrismes sont observés. L expression de tropoélastine et de MMP-2 n étant pas modifiée, un défaut d assemblage des fibres élastiques, dont le mécanisme reste à être précisé, semble être responsable des lésions observées. Notre travail montre que la voie de signalisation du TGFß dans les CMLv est cruciale pour la régulation de leur croissance et pour la formation d une paroi artérielle fonctionnelleThis work sheds light on the role of TGF-ß1 on vSMC physiopathology in vitro on vSMC cultures and in vivo in transgenic mice invalidated for TGFß-signaling in the vSMCs. In vitro, we show how TGFß inhibits or stimulates at low or high density, respectively, vSMC growth. TGFß dictates, at all cell densities, a low but constant proliferation rate so that, at high density, vSMCs escape from contact inhibition. In contrast, TGFß induces apoptosis only at low density. By comparison with untreated vSMCs, at low density, the reduced proliferation and strong apoptosis inhibit the growth of TGFß-treated cells while, at high density, maintenance of proliferation in the absence of apoptosis stimulates the growth of TGFß-treated vSMCs. These effects of TGFß on vSMC proliferation and apoptosis relie on unconventional regulation of cell-cycle proteins and the cell-survival pathway PI3 Kinase/Akt. In transgenic mice, invalidation of TGFß signaling in vSMCs is lethal between 14.5 and 18.5 dpc due to systematic vascular and occasional cardiac defects. In the descending thoracic aorta, elastic fibers are either absent or fragmented and aneurysms are present. Tropoelastin and MMP-2 expressions being not altered, a defect in elastic fiber assembly, the mechanism of which has to be identified, should be responsible for the defects observed. Our work demonstrates that TGFß signaling pathway in the vSMCs is crucial for the regulation of their growth and proper building of a functional aortic wallLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Thyroxine (T4) Transfer from Blood to Cerebrospinal Fluid in Sheep Isolated Perfused Choroid Plexus: Role of Multidrug Resistance-Associated Proteins and Organic Anion Transporting Polypeptides

Thyroxine (T4) enters the brain either directly across the blood–brain barrier (BBB) or indirectly via the choroid plexus (CP), which forms the blood–cerebrospinal fluid barrier (B-CSF-B). In this study, using isolated perfused CP of the sheep by single-circulation paired tracer and steady-state techniques, T4 transport mechanisms from blood into lateral ventricle CP has been characterized as the first step in the transfer across the B-CSF-B. After removal of sheep brain, the CPs were perfused with 125I-T4 and 14C-mannitol. Unlabeled T4 was applied during single tracer technique to assess the mode of maximum uptake (Umax) and the net uptake (Unet) on the blood side of the CP. On the other hand, in order to characterize T 125 4 protein transporters, steady-state extraction of I-T4 was measured in presence of different inhibitors such as probenecid, verapamil, BCH, or indomethacin. Increasing the concentration of unlabeled-T4 resulted in a significant reduction in Umax%, which was reflected by a complete inhibition of T4 uptake into CP. In fact, the obtained Unet% decreased as the concentration of unlabeled-T4 increased. The addition of probenecid caused a significant inhibition of T4 transport, in comparison to control, reflecting the presence of a carrier mediated process at the basolateral side of the CP and the involvement of multidrug resistance-associated proteins (MRPs: MRP1 and MRP4) and organic anion transporting polypeptides (Oatp1, Oatp2, and Oatp14). Moreover, verapamil, the P-glycoprotein (P-gp) substrate, resulted in ~34% decrease in the net extraction of T4, indicating that MDR1 contributes to T4 entry into CSF. Finally, inhibition in the net extraction of T4 caused by BCH or indomethacin suggests, respectively, a role for amino acid “Lsystem and MRP1/Oatp1 in mediating T4 transfer. The presence of a carrier-mediated transport mechanism for cellular uptake on the basolateral membrane of the CP, mainly P-gp and Oatp2, would account for the efficient T4 transport from blood to CSF. The current study highlights a carrier-mediated transport mechanism for T4 movement from blood to brain at the basolateral side of B-CSF-B/CP, as an alternative route to BBB

Kinetic analyses of mRNA levels of SMAD co-repressors and inhibitors in a mouse model of 19 Gy-localized small intestinal irradiation: mRNA levels of TGIF1, SnoN, Ski and SMAD7 were measured by real time PCR in total intestinal tissues, 5 hours, 1 day, 3 days, 14 days and 42 days after irradiation.

<p>mRNA level of 18S was used as housekeeping gene and dot line indicates value 1 assigned for control sham-operated animals at each time. Results are +/- SEM (n = 6 to 8 mice/group). *p<0.05 versus sham mice.</p

TGIF1 genetic deficiency is not associated with modifications of radiation-induced TGF-β pathway-related molecular profile <i>in vivo</i>.

<p>mRNA levels of (A) TGF-β signaling-related genes and (B) TGF-β/Smad target genes in TGIF1^+/+, TGIF1^+/− and TGIF1^−/− mice 3 days after 19 Gy localized small intestinal irradiation were determined by real time PCR. mRNA level of 18S was used as housekeeping gene and value 1 was assigned to sham-operated TGIF1^+/+ animals. Results are the mean ± SEM with 8 to 10 mice per group. *P<0.05 vs TGIF1^+/+ sham mice. ^#P<0.05 vs TGIF1^+/+ irradiated mice. No significant difference was obtained between TGIF1^+/+, TGIF1^+/− and TGIF1^−/− sham-irradiated animals.</p

Radiation induces activation of the TGF-β pathway in a mouse model of 19 Gy-localized small intestinal irradiation: mRNA levels measured by real time PCR of TGF-β1, SMAD3 and PAI-1 in total intestinal tissues, 5 hours, 1 day, 3 days, 14 days and 42 days after irradiation.

<p>mRNA level of 18S was used as housekeeping gene and dot line indicates value 1 assigned for control sham-operated animals at each time. Results are +/− SEM (n = 6 to 8 mice/group). *p<0.05 versus sham mice.</p

TGIF1 genetic deficiency sensitizes mice to gastrointestinal syndrome and radiation enteropathy.

<p>Kaplan-Meier analyses represent the percent survival of TGIF1^+/+, TGIF1^+/− and TGIF1^−/− mice following (A) 13 Gy total body irradiation and (B) 19 Gy localized small intestinal irradiation. Statistical differences were determined by the log rank test, *p = 0.0033, **p = 0.19, ***p = 0.00065, ^#p = 0.06, ^##p = 0.11, ^###p = 0.000014.</p

Effect of irradiation on mRNA and protein expression of SMAD3, PAI-1 and SMAD co-repressors and inhibitors in HUVEC.

<p>(A) Irradiation induces SMAD pathway and PAI-1 in HUVEC cells. mRNA levels of SMAD3 and PAI-1 in HUVEC 6, 24, 48 and 72 hours after irradiation were measured by real time PCR. mRNA level of GAPDH was used as housekeeping gene and value 1 was assigned to unirradiated cells for each time. Protein levels of Smad3, phospho-Smad3 and PAI-1 were followed by western blot. (B) Radiation dose- and time-dependent mRNA level and protein abundance of TGIF1, SnoN, Ski and Smad7 were measured by real time PCR and western blot. Results are the mean ± SEM of 3 independent experiments realized in triplicates. *P<0.05 vs unirradiated cells.</p