p63, a homologue of the tumor suppressor p53, is pivotal for epithelial development, because its loss causes severe epithelial dysgenesis, although no information is so far available on the role of p63 in the thymus. We identified the expression of all p63 isoforms in the developing thymus. The p63 ؊/؊ thymi show severe abnormalities in size and cellularity, even though the organ expresses normal levels of keratins 5 and 8, indicating a p63-independent differentiation of thymic epithelial cells (TEC). TEC were sufficiently developed to allow a significant degree of education to produce CD4/CD8 single-and double-positive T cells. To study the selective contribution of transactivation-active p63 (TAp63) and amino-deleted p63 (⌬Np63) isoforms to the function of the TEC, we genetically complemented p63 ؊/؊ mice by crossing p63 ؉/؊ mice with transgenic mice expressing either TAp63␣ or ⌬Np63␣ under the control of the keratin 5 promoter. Thymic morphology and cellularity were partially restored by complementation with ⌬Np63, but not TAp63, one downstream effector being fibroblast growth factor receptor 2-IIIb (FgfR2-IIIb). Indeed, FgfR2-IIIb is regulated directly by p63, via its interaction with apobec-1-binding protein-1, and its knockout shows thymic defects similar to those observed in p63 ؊/؊ thymi. In addition, expression of Jag2, a component of the Notch signaling pathway known to be required for thymic development, was enhanced by p63 in vivo genetic complementation. Like Jag2 ؊/؊ thymi, p63 ؊/؊ thymi also show reduced ␥␦ cell formation. Therefore, p63, and particularly the ⌬Np63 isoform, is essential for thymic development via enhanced expression of FgfR2 and Jag2. The action of ⌬Np63 is not due to a direct regulation of TEC differentiation, but it is compatible with maintenance of their ''stemness,'' the thymic abnormalities resulting from epithelial failure due to loss of stem cells.epithelia ͉ thymus

Cytotoxic necrotizing factor 1 (CNF1) is a protein toxin produced by some pathogenic strains of Escherichia coli that specifically activates Rho, Rac, and Cdc42 GTPases. We previously reported that this toxin prevents the ultraviolet-Binduced apoptosis in epithelial cells, with a mechanism that remained to be defined. In this work, we show that the proteasomal degradation of the Rho GTPase is necessary to achieve cell death protection, because inhibition of Rho degradation abolishes the prosurvival activity of CNF1. We hypothesize that Rho inactivation allows the activity of Rac to become dominant. This in turn leads to stimulation of the phosphoinositide 3-kinase/Akt/I B kinase/nuclear factor-B prosurvival pathway and to a remarkable modification in the architecture of the mitochondrial network, mainly consisting in the appearance of elongated and interconnected mitochondria. Importantly, we found that Bcl-2 silencing reduces the ability of CNF1 to protect cells against apoptosis and that it also prevents the CNF1-induced mitochondrial changes. It is worth noting that the ability of a bacterial toxin to induce such a remodeling of the mitochondrial network is herein reported for the first time. The possible pathophysiological relevance of this finding is discussed. INTRODUCTIONToday, it is largely acknowledged that apoptosis, besides being an evolutionary conserved form of cell death that plays a pivotal role during development, morphogenesis, and cell homeostasis, is also critically implied in a constantly growing number of diseases (Fadeel and Orrenius, 2005). In fact, apoptosis can be regarded as a widespread strategy exploited by pathogenic bacteria to favor their own survival or spreading in the host , often by producing protein toxins that mediate their long-range cross-talk with host cells. In this context, we have previously reported the ability of a protein toxin from Escherichia coli, namely the cytotoxic necrotizing factor 1 (CNF1), to prevent the ultraviolet-B (UVB)-induced apoptosis and to increase the expression of antiapoptotic Bcl-2 family proteins (Fiorentini et al., 1998). The precise mechanism by which CNF1 allows cells to survive, however, is not yet defined.CNF1 is a protein toxin produced by some pathogenic strains of E. coli mainly involved in extraintestinal infections (Landraud et al., 2000). In eukaryotic cells, CNF1 binds to its receptor, reported to be the receptor of laminin , and it is endocytosed and released into the cytoplasm by an acidic-dependent mechanism (Contamin et al., 2000). Once in the cytoplasm, CNF1 exerts its enzymatic activity that is represented by deamidation of a pivotal glutamine residue of the guanosine triphosphate (GTP)-binding proteins Rho, Rac, and Cdc42 (glutamine 63 of Rho or glutamine 61 of Rac and Cdc42), giving rise to a glutamic acid (Flatau et al., 1997;Schmidt et al., 1997;Lerm et al., 1999). The glutamine residue modified by CNF1 lies in the switch 2 domain of Rho proteins, which is involved in GTP hydrolysis; thus, the modification exerted by CNF1...

Evidence is accumulating that a growing number of bacterial toxins act by modulating the eukaryotic cell cycle machinery. In this context, we provide evidence that a protein toxin named cytotoxic necrotizing factor 1 (CNF1) from uropathogenic Escherichia coli is able to block cell cycle G 2 /M transition in the uroepithelial cell line T24. CNF1 permanently activates the small GTP-binding proteins of the Rho family that, beside controlling the actin cytoskeleton organization, also play a pivotal role in a large number of other cellular processes, including cell cycle regulation. The results reported here show that CNF1 is able to induce the accumulation of cells in the G 2 /M phase by sequestering cyclin B1 in the cytoplasm and down-regulating its expression. The possible role played by the Rho GTPases in the toxin-induced cell cycle deregulation has been investigated and discussed. The activity of CNF1 on cell cycle progression can offer a novel view of E. coli pathogenicity.It is largely recognized that bacterial pathogens use a variety of strategies, including the production of protein toxins, to manipulate host cell functions. In the past decade, evidence has accumulated on the ability of certain bacterial toxins to interfere with the eukaryotic cell cycle machinery, either promoting cell proliferation (30, 44) or inducing cell cycle arrest (7, 25). These protein toxins belong to the family of so-called "cyclomodulins," which actively deregulate the passage of cells throughout the host cell cycle (reviewed in reference 29). In this work, we show that a block in G 2 /M transition can be induced by cytotoxic necrotizing factor 1 (CNF1), a 110-kDa monomeric protein toxin frequently produced by pathogenic Escherichia coli strains associated with urinary tract infections. CNF1 is a chromosomally encoded toxin that activates the small GTP-binding proteins of the Rho family (Rho, Rac, and Cdc42), regulatory proteins that behave as molecular switches, cycling between an active GTP-bound state and an inactive GDP-bound state. CNF1 acts by deamidating a specific glutamine residue in the switch 2 domain that is crucial in GTP hydrolysis (glutamine 63 in Rho [18,36,37] or glutamine 61 in Rac and Cdc42 [24]), thus locking the G protein in the active state. This toxin activity confers new properties on epithelial cells, including the cytoskeleton-dependent ability to behave as professional phagocytes (13,17).Beside controlling the actin cytoskeleton organization (31), the Rho signaling pathways are also intimately involved in transducing mitogenic signals to the translational apparatus and play a pivotal role in different points of cell cycle regulation (28). Indeed, Rho, Rac, and Cdc42 GTPases have been demonstrated to participate in the control of G 1 /S progression, primarily through regulation of expression of cyclins and cyclin-dependent kinase inhibitors (42).Hence, considering the Rho-dependent ability of CNF1 to interfere with cytokinesis, thus causing multinucleation (15), and to inhibit cell differentiation (41) ...

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