Dock10 regulates CD23 expression and sustains B-cell lymphopoiesis in secondary lymphoid tissue.

Dock10, a guanine nucleotide exchange factor for the Rho GTPases Rac1 and Cdc42, affects cell morphology, membrane protrusive activity, and cell movement. Dock10 is prominently expressed in lymphoid tissue and upregulated by IL-4 in B cells. To investigate the physiological role of Dock10, WT mice and Dock10 KO mice were used. KO mice showed decreased numbers of B cells in spleen, both follicular B cells and marginal zone B cells, and in peripheral blood, but not in bone marrow. The antiapoptotic effect of IL-4 in vitro, the migratory response to CXCL13 or CCL21 in vitro, and the whole genome expression profile were intact in spleen B cells from KO mice. CD23, the low-affinity receptor for immunoglobulin E, was overexpressed on follicular B cells from KO mice, suggesting that Dock10 negatively regulates membrane CD23 expression. Negative regulation of CD23 expression by Dock10 could play a role in B cell maturation and function.

IL-4 Up-Regulates MiR-21 and the MiRNAs Hosted in the CLCN5 Gene in Chronic Lymphocytic Leukemia.

Interleukin 4 (IL-4) induces B-cell differentiation and survival of chronic lymphocytic leukemia (CLL) cells. MicroRNAs (miRNAs) regulate mRNA and protein expression, and several miRNAs, deregulated in CLL, might play roles as oncogenes or tumor suppressors. We have studied the miRNA profile of CLL, and its response to IL-4, by oligonucleotide microarrays, resulting in the detection of a set of 129 mature miRNAs consistently expressed in CLL, which included 41 differentially expressed compared to normal B cells (NBC), and 6 significantly underexpressed in ZAP-70 positive patients. IL-4 stimulation brought about up-regulation of the 5p and 3p mature variants of the miR-21 gene, which maps immediately downstream to the VMP1 gene, and of the mature forms generated from the miR-362 (3p and 5p), miR-500a (3p), miR-502 (3p), and miR-532 (3p and 5p) genes, which map within the third intron of the CLCN5 gene. Both genes are in turn regulated by IL-4, suggesting that these miRNAs were regulated by IL-4 as passengers from their carrier genes. Their levels of up-regulation by IL-4 significantly correlated with cytoprotection. MiR-21 has been reported to be leukemogenic, associated to bad prognosis in CLL, and the miRNA more frequently overexpressed in human cancer. Up-regulation by IL-4 of miR-21 and the miRNAs hosted in the CLCN5 locus may contribute to evasion of apoptosis of CLL cells. These findings indicate that the IL-4 pathway and the miRNAs induced by IL-4 are promising targets for the development of novel therapies in CLL.

Dock10, a Cdc42 and Rac1 GEF, induces loss of elongation, filopodia, and ruffles in cervical cancer epithelial HeLa cells.

Dock10 is one of the three members of the Dock-D family of Dock proteins, a class of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Its homologs Dock9 and Dock11 are Cdc42 GEFs. Dock10 is required for maintenance of rounded morphology and amoeboid-type movement. Full-length isoforms of Dock10 have been recently cloned. Here, we address GTPase specificity and GEF activity of Dock10. In order of decreasing intensity, Dock10 interacted with nucleotide-free Rac1, Cdc42, and Rac3, and more weakly with Rac2, RhoF, and RhoG. Inducible expression of Dock10 in HeLa epithelial cells promoted GEF activity on Cdc42 and Rac1, and a morphologic change in two-dimensional culture consisting in loss of cell elongation, increase of filopodia, and ruffles. Area in contact with the substrate of cells that spread with non-elongated morphology was larger in cells expressing Dock10. Inducible expression of constitutively active mutants of Cdc42 and Rac1 in HeLa cells also induced loss of elongation. However, Cdc42 induced filopodia and contraction, and Rac1 induced membrane ruffles and flattening. When co-expressed with Dock10, Cdc42 potentiated filopodia, and Rac1 potentiated ruffles. These results suggest that Dock10 functions as a dual GEF for Cdc42 and Rac1, affecting cell morphology, spreading and actin cytoskeleton protrusions of adherent HeLa cells.

The gene expression response of chronic lymphocytic leukemia cells to IL-4 is specific, depends on ZAP-70 status and is differentially affected by an NFκB inhibitor.

Interleukin 4 (IL-4), an essential mediator of B cell development, plays a role in survival of chronic lymphocytic leukemia (CLL) cells. To obtain new insights into the function of the IL-4 pathway in CLL, we analyzed the gene expression response to IL-4 in CLL and in normal B cells (NBC) by oligonucleotide microarrays, resulting in the identification of 232 non-redundant entities in CLL and 146 in NBC (95 common, 283 altogether), of which 189 were well-defined genes in CLL and 123 in NBC (83 common, 229 altogether) (p<0.05, 2-fold cut-off). To the best of our knowledge, most of them were novel IL-4 targets for CLL (98%), B cells of any source (83%), or any cell type (70%). Responses were significantly higher for 54 and 11 genes in CLL and NBC compared to each other, respectively. In CLL, ZAP-70 status had an impact on IL-4 response, since different sets of IL-4 targets correlated positively or negatively with baseline expression of ZAP-70. In addition, the NFκB inhibitor 6-Amino-4-(4-phenoxyphenethylamino)quinazoline, which reversed the anti-apoptotic effect of IL-4, preferentially blocked the response of genes positively correlated with ZAP-70 (e.g. CCR2, SUSD2), but enhanced the response of genes negatively correlated with ZAP-70 (e.g. AUH, BCL6, LY75, NFIL3). Dissection of the gene expression response to IL-4 in CLL and NBC contributes to the understanding of the anti-apoptotic response. Initial evidence of a connection between ZAP-70 and NFκB supports further exploration of targeting NFκB in the context of the assessment of inhibition of the IL-4 pathway as a therapeutic strategy in CLL, especially in patients expressing bad prognostic markers.

Human and mouse DOCK10 splicing isoforms with alternative first coding exon usage are differentially expressed in T and B lymphocytes.

DOCK10 is a member of the dedicator of cytokinesis (DOCK) family of Rho GTPase activators preferentially expressed in lymphocytes. In this paper, we analyzed DOCK10 mRNA diversity produced because of alternative splicing. Alternative first coding exon usage led to 2 main protein-coding transcripts, DOCK10.1 and DOCK10.2. Full-length cDNA clones of both isoforms were obtained from both normal human peripheral blood mononuclear cells and mouse spleen for the first time for human DOCK10.1, mouse DOCK10.1, and mouse DOCK10.2. Human and mouse DOCK10.1 clones corresponded to the protein coding assemblies provided by the National Center for Biotechnology Information as Reference Sequences for DOCK10. Our analysis especially focused on human cDNA clones, of which 63% were alternatively spliced forms involving diverse exons and introns. DOCK10.1 expression was enriched in normal T cells, and DOCK10.2 expression was enriched in normal B cells and chronic lymphocytic leukemia (CLL) B cells. Both isoforms were upregulated in response to interleukin-4 in B cells, both normal and CLL, but not in T cells. Our data suggest that cell-specific mechanisms regulate expression of the alternative first exon variants of DOCK10 in vertebrates.