Abstract
Annexin A1 (ANX-A1) is a calcium-dependent membrane-binding protein involved in the modulation of apoptosis and phagocytosis (FASEB J.2003;17:1544). We have previously reported that HDAC inhibitor depsipeptide (FK228) caused marked growth inhibition and apoptosis in t(8;21) Kasumi-1 AML cells with up-regulation of 123 genes (by cDNA array) including ANX-A1 (3.5 fold; Tabe, Blood 2004). By chromatin immunoprecipitation (ChIP) assay, FK228 induced H4 and H3-K9 acetylation in the ANX-A1 promoter with corresponding induction of ANX-A1 mRNA (7.2±1.7 fold, TaqMan RT-PCR) and protein (western blot analysis). The markedly increased ANX-A1 protein localized on the cell membrane of Kasumi-1 cells exposed to FK228 was confirmed by immunofluorecence analysis using confocal microscopy. ANX-A1 membrane localization was diminished by treatment with anti-ANX-A1 mAb. To investigate the contribution of ANX-A1 to FK228-induced apoptosis, we neutralized ANX-A1 by anti-ANX-A1 mAb. This moderately decreased FK228 induced apoptosis (36.0±4.1 vs 26.5±3.7% AnnexinV(+)/PI(+) cells, p=0.01). Similarly, Kasumi-1 cells transfected with siRNA/ANX-A1 were less sensitive to FK228-induced cell death compared with nonsense (N) siRNA transfected cells (siRNA 31.2±3.1% vs NsiRNA 39.5±2.9% annexin(+) cells, p=0.03). These data indicate that the upregulation of endogeneous ANX-A1 (either membrane-binding or secreted form) promotes cell apoptosis in an autocrine fashion. Next, we investigated the functional role of ANX-A1 on leukemia cell phagocytosis. The engulfment of Kasumi-1 cells by cocultured human THP-1 monocyte-derived macrophages was evaluated by cell adherence assay. Compared with untreated cells, the exposure to FK228 induced a dramatic increase in Kasumi-1 cells attachment to macrophages (untreated vs FK228 treated; 57 ± 9 cells vs 196 ± 33 cells/ microscopic fields (0.08 mm2/field), n = 5; p=0.01). FK228-induced cell attachment was completely abrogated in the siRNA/ANX-A1 transfected Kasumi-1 cells (60.5% ± 10.5% decrease; n = 5; p<0.001). Consistently, co-treatment with FK228 and anti-ANX-A1 mAb followed by washout of both compounds resulted in significantl repression of FK228-stimulated engulfment of leukemic cells by macrophages (54.1% ± 3.0% decrease; n = 5; p=0.02). This effect was not further enhanced by adding anti-ANX-A1 mAb to the co-culture medium, suggesting that membrane-associated but not soluble ANX-A1 contributes to leukemia cell engulfment by macrophages. Results presented here demonstrate a novel mechanism of action of HDAC inhibitors in the context of bone marrow microenvironment via histone acetylation, increased expression and externalization of ANX-A1, which provides an “eat-me” signal and mediates phagocytic clearance of apoptotic leukemic cells by macrophages. Our data further suggest that ANX-A1 is silenced via histone deacetylation in leukemic cells, and its re-expression by HDAC inhibitors may stimulate apoptosis in an autocrine fashion while diminishing the inflammatory response through activating phagocytosis in the bone marrow microenvironment.
The intricate role of mast cell proteases and the annexin A1-FPR1 system in abdominal wall endometriosis
