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Cdc42 regulates Fc gamma receptor-mediated phagocytosis through the activation and phosphorylation of Wiskott-Aldrich syndrome protein (WASP) and neural-WASP.

ABSTRACT: Cdc42 is a key regulator of the actin cytoskeleton and activator of Wiskott-Aldrich syndrome protein (WASP). Although several studies have separately demonstrated the requirement for both Cdc42 and WASP in Fc(gamma) receptor (Fc(gamma)R)-mediated phagocytosis, their precise roles in the signal cascade leading to engulfment are still unclear. Reduction of endogenous Cdc42 expression by using RNA-mediated interference (short hairpin RNA [shRNA]) severely impaired the phagocytic capacity of RAW/LR5 macrophages, due to defects in phagocytic cup formation, actin assembly, and pseudopod extension. Addition of wiskostatin, a WASP/neural-WASP (N-WASP) inhibitor showed extensive inhibition of phagocytosis, actin assembly, and cell extension identical to the phenotype seen upon reduction of Cdc42 expression. However, using WASP-deficient bone marrow-derived macrophages or shRNA of WASP or N-WASP indicated a requirement for both WASP and N-WASP in phagocytosis. Cdc42 was necessary for WASP/N-WASP activation, as determined using a conformation-sensitive antibody against WASP/N-WASP and partial restoration of phagocytosis in Cdc42 reduced cells by expression of a constitutively activated WASP. In addition, Cdc42 was required for proper WASP tyrosine phosphorylation, which was also necessary for phagocytosis. These results indicate that Cdc42 is essential for the activation of WASP and N-WASP, leading to actin assembly and phagocytic cup formation by macrophages during Fc(gamma)R-mediated phagocytosis.

SUBMITTER: Park H

PROVIDER: S-EPMC2770938 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The clinical symptomatology in the X-linked Wiskott-Aldrich syndrome (WAS), a combined immunodeficiency and autoimmune disease resulting from WAS protein (WASp) deficiency, reflects the underlying coexistence of an impaired T helper 1 (TH1) immunity alongside intact TH2 immunity. This suggests a role for WASp in patterning T(H) subtype immunity, yet the molecular basis for the TH1-TH2 imbalance in human WAS is unknown. We have discovered a nuclear role for WASp in the transcriptional regulation of the TH1 regulator gene TBX21 at the chromatin level. In primary TH1-differentiating cells, a fraction of WASp is found in the nucleus, where it is recruited to the proximal promoter locus of the TBX21 gene, but not to the core promoter of GATA3 (a TH2 regulator gene) or RORc (a TH17 regulator gene). Genome-wide mapping demonstrates association of WASp in vivo with the gene-regulatory network that orchestrates TH1 cell fate choice in the human TH cell genome. Functionally, nuclear WASp associates with H3K4 trimethyltransferase [RBBP5 (retinoblastoma-binding protein 5)] and H3K9/H3K36 tridemethylase [JMJD2A (Jumonji domain-containing protein 2A)] proteins, and their enzymatic activity in vitro and in vivo is required for achieving transcription-permissive chromatin dynamics at the TBX21 proximal promoter in primary differentiating TH1 cells. During TH1 differentiation, the loss of WASp accompanies decreased enrichment of RBBP5 and, in a subset of WAS patients, also of filamentous actin at the TBX21 proximal promoter locus. Accordingly, human WASp-deficient TH cells, from natural mutation or RNA interference-mediated depletion, demonstrate repressed TBX21 promoter dynamics when driven under TH1-differentiating conditions. These chromatin derangements accompany deficient T-BET messenger RNA and protein expression and impaired TH1 function, defects that are ameliorated by reintroducing WASp. Our findings reveal a previously unappreciated role of WASp in the epigenetic control of T-BET transcription and provide a new mechanism for the pathogenesis of WAS by linking aberrant histone methylation at the TBX21 promoter to dysregulated adaptive immunity.

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Cdc42 regulates Fc gamma receptor-mediated phagocytosis through the activation and phosphorylation of Wiskott-Aldrich syndrome protein (WASP) and neural-WASP.

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