Retinoic acid (RA) produced by intestinal dendritic cells (DCs) imprints gut-homing specificity on lymphocytes and enhances Foxp3+ regulatory T-cell differentiation. The expression of aldehyde dehydrogenase (ALDH) 1A in these DCs is essential for the RA production. However, it remains unclear how the steady-state ALDH1A expression is induced under specific pathogen-free (SPF) conditions. Here, we found that bone marrow-derived dendritic cells (BM-DCs) generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) expressed Aldh1a2, an isoform of Aldh1a, but that fms-related tyrosine kinase 3 ligand-generated BM-DCs did not. DCs from mesenteric lymph nodes (MLN) and Peyer's patches (PP) of normal SPF mice expressed ALDH1A2, but not the other known RA-producing enzymes. Employing a flow cytometric method, we detected ALDH activities in 10–30% of PP-DCs and MLN-DCs. They were CD11chighCD4−/lowCD8αintermediateCD11b−/low F4/80low/intermediateCD45RBlowCD86highMHC class IIhighB220−CD103+. Equivalent levels of aldehyde dehydrogenase activity (ALDHact) and ALDH1A2 expression were induced synergistically by GM-CSF and IL-4 in splenic DCs in vitro. In BM-DCs, however, additional signals via Toll-like receptors or RA receptors were required for inducing the equivalent levels. The generated ALDH1A2+ DCs triggered T cells to express gut-homing receptors or Foxp3. GM-CSF receptor-deficient or vitamin A-deficient mice exhibited marked reductions in the ALDHact in intestinal DCs and the T cell number in the intestinal lamina propria, whereas IL-4 receptor-mediated signals were dispensable. GM-CSF+CD11c−F4/80+ cells existed constitutively in the intestinal tissues. The results suggest that GM-CSF and RA itself are pivotal among multiple microenvironment factors that enable intestinal DCs to produce RA.

Background: Cortactin is a major phosphotyrosyl protein in pp60 v-src -transformed chicken embryo cells. Cortactin binds to actin filament (F-actin) through a unique region which consists of six tandem 37 amino acid repeats, named cortactin repeats. Furthermore cortactin has one src homology 3 (SH3) domain.

Lysophosphatidic acid (LPA) is a serum-derived phospholipid that induces a variety of biological responses in various cells via heterotrimeric G protein-coupled receptors (GPCRsLysophosphatidic acid (LPA) 1 is a serum-derived phospholipid that induces a variety of biological responses in various cells (1)(2)(3)(4)(5). LPA is also the prototypic G protein-coupled receptor (GPCR) ligand that activates MAP kinase, phospholipase C, and small GTPases, etc., via heterotrimeric G proteins (1-5). Three distinct G protein-coupled receptors for LPA have been identified, termed LPA 1 , LPA 2 , and LPA 3 (previously Edg2, Edg4, and Edg7, respectively). LPA signals induce actin rearrangements via the Rho family GTPase, RhoA, Rac1, and Cdc42 (1, 5-6). Rho family GTPases have GDP-bound inactive and GTP-bound active forms, the cycle of which is regulated by Rho guanine nucleotide exchange factors (RhoGEFs) that stimulate the exchange of GDP for GTP (7). Members of the RhoGEF family have a Dbl homology (DH) domain that catalyzes the exchange reaction and a pleckstrin homology (PH) domain immediately C-terminal to the DH domain (8). The PH domain is responsible for both subcellular localization and modulation of DH domain function (8). Recently, it has been recognized that the G 12/13 family mediates signaling from the LPA receptor to RhoA and that RhoGEFs containing regulators of G protein signaling (RGS) domains are involved in these processes (9 -10). RGS domain-containing RhoGEFs have been described p115-RhoGEF, PDZ-RhoGEF, and leukemia-associated RhoGEF (LARG) (11-13). The activation mechanisms of RGS domain-containing RhoGEFs induced by extracellular signals are well known in the case of p115-RhoGEF (14 -15). The RGS domain of p115-RhoGEF stimulates the intrinsic GTPase activity of the G 12 or G 13 ␣ subunit, and activated G 12 or G 13 ␣ subunit binds to the RGS domain of p115-RhoGEF, thereby enhancing its ability to catalyze guanine nucleotide exchange of RhoA. On the other hand, PDZ-RhoGEF and LARG, but not p115-RhoGEF, both have N-terminal PDZ domains. The PDZ domain is known as a modular domain that binds to specific C-terminal peptide sequences of many membrane proteins (16). PDZ domain-containing proteins function as mediators of clustering of neurotransmitter receptors and ion channels, and then are involved in asymmetric distribution of receptors in epithelial cells (17)(18)(19)(20). PDZ-RhoGEF and LARG also bind to the G 12 or G 13 ␣ subunit via RGS domains in a manner similar to p115-RhoGEF, although the molecular mechanisms controlling the GEF activity are not yet fully understood (8,(21)(22)(23). Recently, we and other groups have reported that the PDZ domains of PDZ-RhoGEF and LARG interact directly with the C-terminal domain of Plexin-B1, a Semaphorin-4D (Sema-4D) receptor, and/or the insulin-like growth factor (IGF-1) receptor (24 -29). Stimulation of Sema4D or IGF-I-induced RhoA activation through the complex of Plexin-B1 or IGF-I receptors with PDZ-RhoGEF or LARG. These observations suggest that the PDZ dom...

Thymocyte positive selection is based on protection of immature CD4/CD8 double-positive (DP) thymocytes from apoptosis and their differentiation into CD4 or CD8 single-positive (SP) cells. Intracellular signals essential for positive selection appear to be induced through the TCR and some of the accessory molecules including LFA-1, CD4 and CD8 upon interaction with thymic stromal cells. The signals, however, still remain to be identified. Since physiological levels of glucocorticoids potentially induce or enhance thymocyte apoptosis even in vivo, the signals are likely to inhibit the apoptotic effect of glucocorticoids. We have previously shown that proper cross-linking of TCR-CD3 with LFA-1, CD4 or CD8 inhibited glucocorticoid-induced thymocyte apoptosis in vitro, and that a proper combination of the calcium ionophore, ionomycin and the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), mimicked the inhibitory effect. Here we determined whether this combination of ionomycin and PMA induces differentiation of isolated DP thymocytes from normal and TCR transgenic mice. We found that pretreatment of DP thymocytes with ionomycin and PMA followed by 1 day culture of the cells without the reagents resulted in the differentiation of the cells into CD4 SP and CD4+ CD8lo T cells that have mostly committed to the CD4 lineage. The changes in expression of other differentiation markers were also in good accordance with those associated with positive selection, except the final maturation. The results indicate that moderate and transient increases in intracellular Ca2+ level and PKC activity induce differentiation and commitment of DP thymocytes to the CD4 lineage, and suggested that the biochemical pathway leading to positive selection is based on a similar mechanism.

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