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DeltaNp63 regulates thymic development through enhanced expression of FgfR2 and Jag2.

ABSTRACT: 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 (DeltaNp63) isoforms to the function of the TEC, we genetically complemented p63(-/-) mice by crossing p63(+/-) mice with transgenic mice expressing either TAp63alpha or DeltaNp63alpha under the control of the keratin 5 promoter. Thymic morphology and cellularity were partially restored by complementation with DeltaNp63, 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 gammadelta cell formation. Therefore, p63, and particularly the DeltaNp63 isoform, is essential for thymic development via enhanced expression of FgfR2 and Jag2. The action of DeltaNp63 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.

SUBMITTER: Candi E

PROVIDER: S-EPMC1924561 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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DeltaNp63 regulates thymic development through enhanced expression of FgfR2 and Jag2.

Candi Eleonora E Rufini Alessandro A Terrinoni Alessandro A Giamboi-Miraglia Alessandro A Lena Anna Maria AM Mantovani Roberto R Knight Richard R Melino Gerry G

Proceedings of the National Academy of Sciences of the United States of America 20070711 29

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 ...[more]

PMID: 17626181

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Project description:Within the thymus, thymic epithelial cells (TECs) provide dedicated thymic stroma microenvironments for T cell development. Because TEC functionality is sensitive to aging and cytoablative therapies, unraveling the molecular elements that coordinate their thymopoietic role has fundamental and clinical implications. Particularly, the selection of CD4 T cells depends on interactions between TCRs expressed on T cell precursors and self-peptides:MHC II complexes presented by cortical TECs (cTECs). Although the macroautophagy/autophagy-lysosomal protein degradation pathway is implicated in CD4 T cell selection, the molecular mechanism that controls the generation of selecting MHC II ligands remains elusive. LAMP2 (lysosomal-associated membrane protein 2) is a well-recognized mediator of autolysosome (AL) maturation. We showed that LAMP2 is highly expressed in cTECs. Notably, genetic inactivation of Lamp2 in thymic stromal cells specifically impaired the development of CD4 T cells that completed positive selection, without misdirecting MHC II-restricted cells into the CD8 lineage. Mechanistically, defects in autophagy in lamp2-deficient cTECs were linked to alterations in MHC II processing, which was associated with a marked reduction in CD4 TCR repertoire diversity selected within the lamp2-deficient thymic stroma. Together, our findings suggest that LAMP2 interconnects the autophagy-lysosomal axis and the processing of selecting self-peptides:MHC II complexes in cTECs, underling its implications for the generation of a broad CD4 TCR repertoire.Abbreviations: AIRE: autoimmune regulator (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy); AL: autolysosome; AP: autophagosome; Baf-A1: bafilomycin A1; B2M: beta-2 microglobulin; CTSL: cathepsin L; CD74/Ii: CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated); CFSE: carboxyfluorescein succinimidyl ester; CFU: colony-forming unit; CLIP: class II-associated invariant chain peptides; cTECs: cortical TECs dKO: double knockout; DN: double negative; DP: double positive; ENPEP/LY51: glutamyl aminopeptidase; FOXP3: forkhead box; P3 IFNG/IFNγ: interferon gamma; IKZF2/HELIOS: IKAROS family zinc finger 2; IL2RA/CD25: interleukin 2 receptor, alpha chain; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; LIP: lymphopenia-induced proliferation; Lm: Listeria monocytogenes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; mTECs: medullary TECs; PRSS16/TSSP: protease, serine 16 (thymus); SELL/CD62L: selectin, lymphocyte; SP: single positive; TCR: T cell receptor; TCRB: T cell receptor beta chain; TECs: thymic epithelial cells; UEA-1: Ulex europaeus agglutinin-1; WT: wild-type.

Project description:Receptor Interacting Protein Kinase 1 (RIPK1) and caspase-8 (Casp8) jointly orchestrate apoptosis, a key mechanism for eliminating developing T cells which have autoreactive or improperly arranged T cell receptors. Mutations in the scaffolding domain of Ripk1 gene have been identified in humans with autoinflammatory diseases like Cleavage Resistant RIPK1 Induced Autoinflammatory (CRIA) and Inflammatory Bowel Disease. RIPK1 protein also contributes to conventional T cell differentiation and peripheral T cell homeostasis through its scaffolding domain in a cell death independent context. Ripk1 deficient mice do not survive beyond birth, so we have studied the function of this kinase in vivo against a backdrop Ripk3 and Casp8 deficiency which allows the mice to survive to adulthood. These studies reveal a key role for RIPK1 in mediating NK1.1 expression, including on thymic iNKT cells, which is a key requirement for thymic stage 2 to stage 3 transition as well as iNKT cell precursor development. These results are consistent with RIPK1 mediating responses to TcR engagement, which influence NK1.1 expression and iNKT cell thymic development. We also used in vivo and in vitro stimulation assays to confirm a role for both Casp8 and RIPK1 in mediating iNKT cytokine effector responses. Finally, we also noted expanded and hyperactivated iNKT follicular helper (iNKTFH) cells in both DKO (Casp8-, Ripk3- deficient) and TKO mice (Ripk1-, Casp8-, Ripk3- deficient). Thus, while RIPK1 and Casp8 jointly facilitate iNKT effector function, RIPK1 uniquely influenced thymic iNKT cell development most likely by regulating molecular responses to T cell receptor engagement. iNKT developmental and functional aberrances were not evident in mice expressing a kinase-dead version of RIPK1 (RIPK1kd), indicating that the scaffolding function of this protein exerts the critical regulation of iNKT cells. Our findings suggest that small molecule inhibitors of RIPK1 could be used to regulate iNKT cell development and effector function to alleviate autoinflammatory conditions in humans.

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DeltaNp63 regulates thymic development through enhanced expression of FgfR2 and Jag2.

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DeltaNp63 regulates thymic development through enhanced expression of FgfR2 and Jag2.

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