Dendritic cells (DCs) are the most potent antigen-presenting cells and fine-tune the immune response. We have investigated hypoxia's effects on the differentiation and maturation of DCs from human monocytes in vitro, and have shown that it affects DC functions. Hypoxic immature DCs (H-iDCs) significantly fail to capture antigens through down-modulation of the RhoA/Ezrin-Radixin-Moesin pathway and the expression of CD206. Moreover, H-iDCs released higher levels of CXCL1, VEGF, CCL20, CXCL8, and CXCL10 but decreased levels of CCL2 and CCL18, which predict a different ability to recruit neutrophils rather than monocytes and create a proinflammatory and proangiogenic environment. By contrast, hypoxia has no effect on DC maturation. Hypoxic mature DCs display a mature phenotype and activate both allogeneic and specific T cells like normoxic mDCs. This study provides the first demonstration that hypoxia inhibits antigen uptake by DCs and profoundly changes the DC chemokine expression profile and may have a critical role in DC differentiation, adaptation, and activation in inflamed tissues.
Hypoxia is a condition of low oxygen tension occurring in inflammatory tissues. Dendritic cells (DC) are professional antigen-presenting cells whose differentiation, migration, and activities are intrinsically linked to the microenvironment. DCs will home and migrate through pathologic tissues before reaching their final destination in the lymph node. We studied the differentiation of human monocytes into immature DCs (iDCs) in a hypoxic microenvironment. We generated iDC in vitro under normoxic (iDCs) or hypoxic (Hi-DCs) conditions and examined the hypoxia-responsive element in the promoter, gene expression, and biochemical KEGG pathways. Hi-DCs had an interesting phenotype represented by up-regulation of genes associated with cell movement/migration. In addition, the Hi-DC cytokine/receptor pathway showed a dichotomy between down-regulated chemokines and up-regulated chemokine receptor mRNA expression. We showed that CCR3, CX3CR1, and CCR2 are hypoxia-inducible genes and that CCL18, CCL23, CCL26, CCL24, and CCL14 are inhibited by hypoxia. A strong chemotactic response to CCR2 and CXCR4 agonists distinguished Hi-DCs from iDCs at a functional level. The hypoxic microenvironment promotes the differentiation of Hi-DCs, which differs from iDCs for gene expression profile and function. The most prominent characteristic of Hi-DCs is the expression of a mobility/migratory rather than inflammatory phenotype. We speculate that Hi-DCs have the tendency to leave the hypoxic tissue and follow the chemokine gradient toward normoxic areas where they can mature and contribute to the inflammatory process. (Mol Cancer Res 2008;6(2):175 -85)
The Dbl family guanine nucleotide exchange factors (GEFs) contain a region of sequence similarity consisting of a catalytic Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. PH domains are involved in the regulated targeting of signaling molecules to plasma membranes by protein-protein and/or protein-lipid interactions. Here we show that Dbl PH domain binding to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate results in the inhibition of Dbl GEF activity on Rho family GTPase Cdc42. Phosphatidylinositol 4,5-bisphosphate binding to the PH domain significantly inhibits the Cdc42 interactive activity of the DH domain suggesting that the DH domain is subjected to the PH domain modulation under the influence of phosphoinositides (PIPs). We generated Dbl mutants unable to interact with PIPs. These mutants retained GEF activity on Cdc42 in the presence of PIPs and showed a markedly enhanced activating potential for both Cdc42 and RhoA in vivo while displaying decreased cellular transforming activity. Immunofluorescence analysis of NIH3T3 transfectants revealed that whereas the PH domain localizes to actin stress fibers and plasma membrane, the PH mutants are no longer detectable on the plasma membrane. These results suggest that modulation of PIPs in both the GEF catalytic activity and the targeting to plasma membrane determines the outcome of the biologic activity of Dbl.
ACKnoWLEdgEmEntSWe are grateful to the following for supplies of antisera or plasmids, Art Alberts, P. Aspenstrom, Louise Anderson, Francis Barr and Sam Crouch, and to Kathryn Ayscough for comments on the manuscript. The work was funded by an MRC Career Establishment Grant to S.J.W., J.R.H. was in receipt of a BBSRC studentship and A.E. was supported by grants from the Italian Association for Cancer Research, Compagnia S. Paolo, and Ministero della Salute. ABStRACtDystroglycan is an essential laminin binding cell adhesion molecule, which is also an adaptor for several SH2 domain-containing signaling molecules and as a scaffold for the ERK-MAP kinase cascade. Loss of dystroglycan function is implicated in muscular dystrophies and the aetiology of epithelial cancers. We have previously demonstrated a role for dystroglycan and ezrin in the formation of filopodia structures. Here we demonstrate the existence of a dystroglycan:ezrin:Dbl complex that is targeted to the membrane by dystroglycan where it drives local Cdc42 activation and the formation of filopodia. Deletion of an ezrin binding site in dystroglycan prevented the association with ezrin and Dbl and the formation of filopodia. Furthermore, expression of the dystroglycan cytoplasmic domain alone had a dominant-negative effect on filopodia formation and Cdc42 activation by sequestering ezrin and Dbl away from the membrane. Depletion of dystroglycan inhibited Cdc42-induced filopodia formation. For the first time we also demonstrate co-localization of Cdc42 and dystroglycan at the tips of dynamic filopodia.
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