ABSTRACT
Endotoxin (bacterial lipopolysaccharide [LPS]) causes fatal septic shock via the Toll-like receptor 4 (TLR-4) protein present on innate immunity effector cells, which activates nuclear factor kappa B (NF-kappaB), inducing proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha). An early step in this process involves nuclear sequestration of the p65-RelA NF-kappaB subunit, enabling transcriptional activation of target inflammatory cytokine genes. Here, we analyzed the role of the nuclear zinc finger protein Gfi1 in the TLR response using primary bone marrow-derived macrophages. We show that upon LPS stimulation, expression of Gfi1 is induced with kinetics similar to those of nuclear translocation of p65 and that Gfi1 interacts with p65 and inhibits p65-mediated transcriptional transactivation by interfering with p65 binding to target gene promoter DNA. Gfi1-deficient macrophages show abnormally high mRNA levels of the TNF-alpha gene and many other p65 target genes and a higher rate of TNF promoter occupancy by p65 than wild-type cells after LPS stimulation, suggesting that Gfi1 functions as an antagonist of NF-kappaB activity at the level of promoter binding. Our findings identify a new function of Gfi1 as a general negative regulator of the endotoxin-initiated innate immune responses, including septic shock and possibly other severe inflammatory diseases.
Subject(s)
DNA-Binding Proteins/metabolism , Inflammation/immunology , Inflammation/metabolism , Toll-Like Receptors/metabolism , Transcription Factor RelA/antagonists & inhibitors , Transcription Factors/metabolism , Animals , Base Sequence , Cell Line , DNA/genetics , DNA/metabolism , DNA Primers/genetics , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Humans , Immunity, Innate/drug effects , Inflammation/etiology , Lipopolysaccharides/toxicity , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Shock, Septic/etiology , Shock, Septic/immunology , Shock, Septic/metabolism , Signal Transduction , Transcription Factors/antagonists & inhibitors , Transcription Factors/deficiency , Transcription Factors/genetics , Transcriptional Activation , Tumor Necrosis Factor-alpha/genetics , Zinc FingersABSTRACT
"Neutropenia" refers to deficient numbers of neutrophils, the most abundant type of white blood cell. Two main forms of inherited neutropenia are cyclic neutropenia, in which neutrophil counts oscillate with a 21-day frequency, and severe congenital neutropenia, in which static neutropenia may evolve at times into leukemia. Mutations of ELA2, encoding the protease neutrophil elastase, can cause both disorders. Among other genes, severe congenital neutropenia can also result from mutations affecting the transcriptional repressor Gfi1, one of whose genetic targets is ELA2, suggesting that the two act through similar mechanisms. In order to identify components of a common pathway regulating neutrophil production, we conducted yeast two-hybrid screens with Gfi1 and neutrophil elastase and detected a novel protein, PFAAP5 (also known as N4BP2L2), interacting with both. Expression of PFAAP5 allows neutrophil elastase to potentiate the repression of Gfi1 target genes, as determined by reporter assays, RNA interference, chromatin immunoprecipitation, and impairment of neutrophil differentiation in HSCs with PFAAP5 depletion, thus delineating a mechanism through which neutrophil elastase could regulate its own synthesis. Our findings are consistent with theoretical models of cyclic neutropenia proposing that its periodicity can be explained through disturbance of a feedback circuit in which mature neutrophils inhibit cell proliferation, thereby homeostatically regulating progenitor populations.
Subject(s)
DNA-Binding Proteins , Gene Expression Regulation , Leukocyte Elastase/metabolism , Neutropenia/metabolism , Repressor Proteins/metabolism , Transcription Factors , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Differentiation , Cell Proliferation , Chromatin Immunoprecipitation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Genes, Reporter , HL-60 Cells , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/physiology , Humans , Leukocyte Elastase/genetics , Neutropenia/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Repressor Proteins/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Two-Hybrid System TechniquesABSTRACT
Growth factor independent-1 (Gfi1) is a zinc finger protein with a SNAG-transcriptional repressor domain. Ajuba is a LIM domain protein that shuttles between the cytoplasm and the nucleus. Ajuba functions as a co-repressor for synthetic Gfi1 SNAG-repressor domain-containing constructs, but a role for Ajuba co-repression of the cognate DNA bound Gfi1 protein has not been defined. Co-immunoprecipitation of synthetic and endogenous proteins and co-elution with gel filtration suggest that an endogenous Ajuba.Gfi1.HDAC multiprotein complex is possible. Active histone deacetylase activity co-immunoprecipitates with Ajuba or Gfi1, and both proteins depend upon histone deacetylases for full transcriptional repression activity. Ajuba LIM domains directly bind to Gfi1, but the association is not SNAG domain-dependent. ChIP analysis and reciprocal knockdown experiments suggest that Ajuba selectively functions as a co-repressor for Gfi1 autoregulation. The data suggest that Ajuba is utilized as a corepressor selectively on Gfi1 target genes.
