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

Mast cells and ionizing radiation induce a synergistic expression of inflammatory genes in endothelial cells by a Mechanism involving p38α MAP Kinase and (p65) NF-κB activation

International audienceVascular endothelium is a key compartment involved in the development of normal tissue toxicity associated with cancer radiation therapy, i.e., acute inflammation and late fibrosis. Radiation-induced endothelial cell activation has been extensively studied, and activated endothelial cells are characterized by increased expression of inflammatory mediators and adhesion molecules, and activation of the coagulation and thrombosis pathways. However, little is known about the role of vascular endothelium interaction with resident immune cells, such as mast cells on its response to irradiation. Here, we report that endothelial exposure to mast cell conditioned medium and irradiation induces a synergistic expression of many inflammatory genes including interleukin-6 and interleukin-8, CXCL2 and E-selectin. This synergy is blocked by the histamine H1 receptor antagonist mepyramine and partially mimicked by exogenous histamine addition before irradiation. Using pharmacological and molecular inhibition approaches, we show the p38a MAP kinase and p65 (NF-κB) dependence of the synergy. Moreover, our data show a link between both pathways, with p65 (NF-κB) being downstream of p38. These data highlight the possible exacerbation of the radiation-induced endothelial inflammatory response by its interactions with immune cells. It also suggest that p38α MAP kinase and p65 (NF-κB) inhibition in vascular endothelium may limit excessive tissue inflammation induced by radiation therapy, and thereby limit the associated acute and late tissue damage. © 2012 by Radiation Research Society

Bioactive Lipids Lysophosphatidic Acid and Sphingosine 1-Phosphate Mediate Breast Cancer Cell Biological Functions Through Distinct Mechanisms (vol 18, pg 173, 2009)

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are structurally related bioactive lipids with growth factor-like activities. LPA and SIP are naturally produced in vivo by blood platelets upon platelet aggregation and at least in vitro by fibroblasts, adipocytes, and multiple types of tumor cells. Breast cancer cells respond to LPA and S1P. However, their specific actions on breast cancer cell biological functions remain unclear. We therefore conducted an in vitro side-by-side study of these two lipids on breast cancer cells. LPA mediates human breast cancer MDA-BO2 cell proliferation, migration, and invasion through activation of a G JERK1/2-dependent signaling pathway, whereas activation of G(alpha i)/PI3K predominates upon S I P stimulation. In MDA-BO2 cells, LPA but not S I P activities were dependent on active type I insulin-like growth factor and epithelial growth factor receptors. LPA and SIP act directly on endothelial cells to induce angiogenesis. We demonstrate that LPA and SIP have indirect angiogenic properties as judged by induced secretion of angiogenic factors by breast cancer cells primed with these lysophospholipids. SIP, but not LPA, controlled the expression of VEGF-A by breast cancer cells, while LPA, but not S1P, controlled the expression of GM-CSF, Gro-alpha, MCP-1, and IL-6. According to the secretion of these paracrine osteoclastic factors, LPA, but not S1P, stimulates breast cancer cell-induced osteoclastogenesis. These findings suggest that, in vivo, LPA and SIP can coordinate their action on tumor and surrounding cells to induce breast cancer progression both at primary and bone metastatic sites

The TG-interacting factor TGIF1 regulates stress-induced proinflammatory phenotype of endothelial cells

International audienceThe endothelium contributes to the control of the tissue inflammatory response following stress and in particular after exposure to ionizing radiation. We previously showed that the TG-interacting factor 1 (TGIF1) plays a role in radiation-induced normal tissue injury. In this study we hypothesized that this protein could play a role in inflammation. The role of TGIF1 in the stress-induced proinflammatory phenotype was investigated in human endothelial cells. In HUVECs ionizing radiation induces TGIF1 expression as well as a proinflammatory phenotype associated with up-regulation of IL-6, IL-8, CXCL1, MIP-2, and MCP-1. TGIF1 overexpression enhances the radiation-induced proinflammatory phenotype whereas TGIF1 silencing limits both the TNF-α- and radiation-induced overexpression of proinflammatory cytokines. Interestingly, in vivo, in radiation-induced intestinal inflammation in mice, TGIF1 genetic deficiency is associated with a reduced radiation-induced overexpression of proinflammatory molecules. In HUVECs, TNF-α-and radiation-induced NF-κB pathway activation is not influenced by TGIF1 expression, whereas TGIF1 knockdown inhibits both TNF-α- and radiation-induced p38 MAPK pathway activation. This study demonstrates that TGIF1 plays a role in TNF-α-and radiation-induced inflammation and suggests that it could be a target in limiting this event in the vascular compartment. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

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

Plasminogen activator inhibitor-1 controls bone marrow-derived cells therapeutic effect through MMP9 signaling Role in physiological and pathological wound healing

International audienceWe assessed the role of plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinase 9 (MMP9) in wound healing process and in the bone marrow mononuclear cells (BMMNC)-related effects on physiological and pathological wound healing. A full thickness excision wound was created by removal of the skin on the midback of irradiated and nonirradiated animals. Angiogenesis and re-epithelialization were markedly increased in PAI-1-/- mice compared to wild-type (WT) animals. We revealed high MMP activity in tissue of PAI-1-/- animals. Of interest, the wound healing process was reduced in PAI-12/2MMP9-/- animals compared to PAI-1-/- mice, suggesting a key role of MMP9 in beneficial effect of PAI-1 deficiency on wound closure. To unravel the role of PAI-1 in BMMNC relative effects, mice were treated with or without local injection of BMMNC isolated from WT, PAI-1-/-, and PAI-1-/- MMP9-/- animals for 14 days (106 cells, n 5 6 per group). In WT nonirradiated mice, transplantation of BMMNC isolated from PAI-1-/- animals enhanced wound formation when compared with WT BMMNC. BMMNC differentiation into cells with endothelial phenotype was enhanced by PAI-1 deficiency. These effects were abrogated in PAI-1-/-MMP92/2 and MMP92/2 BMMNC. In addition, using chimeric mice, we demonstrated that PAI-1 deficiency environment increased the BMMNC-GFP recruitment to the wound site, whereas this effect was abrogated when using PAI-1-/-MMP92/2 BMMNC. PAI-1 deficiency, at least through MMP9 upregulation, enhanced wound healing and BMMNC therapeutic potential in irradiated and nonirradiated animals. © AlphaMed Press

PAI-1-dependent endothelial cell death determines severity of radiation-induced intestinal injury

International audienceNormal tissue toxicity still remains a dose-limiting factor in clinical radiation therapy. Recently, plasminogen activator inhibitor type 1 (SERPINE1/PAI-1) was reported as an essential mediator of late radiation-induced intestinal injury. However, it is not clear whether PAI-1 plays a role in acute radiation-induced intestinal damage and we hypothesized that PAI-1 may play a role in the endothelium radiosensitivity. In vivo, in a model of radiation enteropathy in PAI-1 -/- mice, apoptosis of radiosensitive compartments, epithelial and microvascular endothelium was quantified. In vitro, the role of PAI-1 in the radiation-induced endothelial cells (ECs) death was investigated. The level of apoptotic ECs is lower in PAI-1 -/- compared with Wt mice after irradiation. This is associated with a conserved microvascular density and consequently with a better mucosal integrity in PAI-1 -/- mice. In vitro, irradiation rapidly stimulates PAI-1 expression in ECs and radiation sensitivity is increased in ECs that stably overexpress PAI-1, whereas PAI-1 knockdown increases EC survival after irradiation. Moreover, ECs prepared from PAI-1 -/- mice are more resistant to radiation-induced cell death than Wt ECs and this is associated with activation of the Akt pathway. This study demonstrates that PAI-1 plays a key role in radiation-induced EC death in the intestine and suggests that this contributes strongly to the progression of radiation-induced intestinal injury. © 2012 Abderrahmani et al