Xian K. Zhang

Decreased expression of the Ets family transcription factor Fli-1 markedly prolongs survival and significantly reduces renal disease in MRL/lpr mice

Increased Fli-1 mRNA is present in PBLs from systemic lupus erythematosus patients, and transgenic overexpression of Fli-1 in normal mice leads to a lupus-like disease. We report in this study that MRL/lpr mice, an animal model of systemic lupus erythematosus, have increased splenic expression of Fli-1 protein compared with BALB/c mice. Using mice with targeted gene disruption, we examined the effect of reduced Fli-1 expression on disease development in MRL/lpr mice. Complete knockout of Fli-1 is lethal in utero. Fli-1 protein expression in heterozygous MRL/lpr (Fli-1+/−) mice was reduced by 50% compared with wild-type MRL/lpr (Fli-1+/+) mice. Fli-1+/− MRL/lpr mice had significantly decreased serum levels of total IgG and anti-dsDNA Abs as disease progressed. Fli-1+/− MRL/lpr mice had significantly increased splenic CD8+ and naive T cells compared with Fli-1+/+ MRL/lpr mice. Both in vivo and in vitro production of MCP-1 were significantly decreased in Fli-1+/− MRL/lpr mice. The Fli-1+/− mice had markedly decreased proteinuria and significantly lower pathologic renal scores. At 48 wk of age, survival was significantly increased in the Fli-1+/− MRL/lpr mice, as 100% of Fli-1+/− MRL/lpr mice were alive, in contrast to only 27% of Fli-1+/+ mice. These findings indicate that Fli-1 expression is important in lupus-like disease development, and that modulation of Fli-1 expression profoundly decreases renal disease and improves survival in MRL/lpr mice.

The Transcription Factor Fli-1 Modulates Marginal Zone and Follicular B Cell Development in Mice

Fli-1 belongs to the Ets transcription factor family and is expressed primarily in hematopoietic cells, including most cells active in immunity. To assess the role of Fli-1 in lymphocyte development in vivo, we generated mice that express a truncated Fli-1 protein, lacking the C-terminal transcriptional activation domain (Fli-1ΔCTA). Fli-1ΔCTA/Fli-1ΔCTA mice had significantly fewer splenic follicular B cells, and an increased number of transitional and marginal zone B cells, compared with wild-type controls. Bone marrow reconstitution studies demonstrated that this phenotype is the result of lymphocyte intrinsic effects. Expression of Igα and other genes implicated in B cell development, including Pax-5, E2A, and Egr-1, are reduced, while Id1 and Id2 are increased in Fli-1ΔCTA/Fli-1ΔCTA mice. Proliferation of B cells from Fli-1ΔCTA/Fli-1ΔCTA mice was diminished, although intracellular Ca2+ flux in B cells from Fli-1ΔCTA/Fli-1ΔCTA mice was similar to that of wild-type controls after anti-IgM stimulation. Immune responses and in vitro class switch recombination were also altered in Fli-1ΔCTA/Fli-1ΔCTA mice. Thus, Fli-1 modulates B cell development both centrally and peripherally, resulting in a significant impact on the in vivo immune response.

A role for Fli-1 in B cell proliferation: implications for SLE pathogenesis

Transgenic overexpression of Fli-1 in normal mice leads to SLE-like disease and increased expression was reported in SLE-affected human and murine lymphocytes. Reducing Fli-1 expression in MRL/lpr mice decreased antibody production, proteinuria, renal pathology, and mortality. Compared to those with wild-type expression of Fli-1, we report here that proliferative responses of Fli-1-deficient naïve B cells to several mitogens were reduced in lupus-prone and control mice. Expression of mitogen receptors, including BCR, TLR4, and TLR9, was not significantly impacted in Fli-1-deficient naïve B cells. IL12a transcripts were upregulated and NFAT transcripts were downregulated in Fli-1-deficient MRL/lpr B cells. These results demonstrate that Fli-1 deficiency affects B cell proliferative responses to mitogens, independent of BCR and TLR expression. IL12a and NFAT, known to influence proliferation, were identified as potential mediators of this effect. This may be a mechanism by which overexpression of Fli-1 contributes to B cell hyperactivity and subsequent SLE pathogenesis.

Fli-1 transcription factor affects glomerulonephritis development by regulating expression of monocyte chemoattractant protein-1 in endothelial cells in the kidney.

Expression of transcription factor Fli-1 is implicated in the development of glomerulonephritis. Fli-1 heterozygous knockout (Fli1(+/-)) NZM2410 mice, a murine model of lupus, had significantly improved survival and reduced glomerulonephritis. In this study, we found that infiltrated inflammatory cells were significantly decreased in the kidneys from Fli-1(+/-) NZM2410 mice. The expression of monocyte chemoattractant protein-1 (MCP-1) was significantly decreased in kidneys from Fli-1(+/-) NZM2410 mice. The primary endothelial cells isolated from the kidneys of Fli-1(+/-) NZM2410 mice produced significantly less MCP-1. In endothelial cells transfected with specific Fli-1 siRNA the production of MCP-1 was significantly reduced compared to cells transfected with negative control siRNA. By Chromatin Immunoprecipitation (ChIP) assay, we further demonstrated that Fli-1 directly binds to the promoter of the MCP-1 gene. Our data indicate that Fli-1 impacts glomerulonephritis development by regulating expression of inflammatory chemokine MCP-1 and inflammatory cell infiltration in the kidneys in the NZM2410 mice.

Impact of Fli-1 transcription factor on autoantibody and lupus nephritis in NZM2410 mice

The transcription factor Fli‐1 is implicated in the pathogenesis of both murine and human lupus. Increased levels of Fli‐1 mRNA were present in the peripheral blood lymphocytes from lupus patients; furthermore, transgenic overexpression of Fli‐1 in normal mice resulted in the development of a lupus‐like disease. Lupus nephritis is a major cause of death in both lupus patients as well as in animal models. In this study, we generated Fli‐1 heterozygous knockout (Fli‐1+/‐) NZM2410 mice (of which the wild‐type is a widely used lupus murine model) that expressed decreased levels of Fli‐1 and investigated the impact of Fli‐1 expression on lupus nephritis development and survival. Ninety‐three per cent of the Fli‐1+/− NZM2410 mice survived to the age of 52 weeks compared to only 35% of wild‐type NZM2410 mice. Autoantibodies, including anti‐dsDNA and anti‐glomerular basement antigen, in Fli‐1+/− NZM2410 mice were statistically significantly lower when compared to wild‐type NZM2410 mice at the ages of 30 and 34 weeks. Total B cell and activated B cell populations in the spleens from Fli‐1+/− NZM2410 mice were decreased significantly compared to wild‐type NZM2410 mice. Fli‐1+/− NZM2410 mice also had remarkably diminished proteinuria and decreased renal pathological scores when compared with wild‐type NZM2410 mice. Expression of early growth response 1 (Egr‐1) was decreased significantly in the kidneys from Fli‐1+/− NZM2410 mice when compared to wild‐type littermates. Our data indicate that expression of Fli‐1 plays an important role in lupus disease development in NZM2410 mice.

Thrombocytopenia in Mice Lacking the Carboxy-Terminal Regulatory Domain of the Ets Transcription Factor Fli1

ABSTRACT Targeted disruption of the Fli1 gene results in embryonic lethality. To dissect the roles of functional domains in Fli1, we recently generated mutant Fli1 mice that express a truncated Fli1 protein (Fli1ΔCTA) that lacks the carboxy-terminal regulatory (CTA) domain. Heterozygous Fli1ΔCTA mice are viable, while homozygous mice have reduced viability. Early postnatal lethality accounts for 30% survival of homozygotes to adulthood. The peripheral blood of these viable Fli1ΔCTA/Fli1ΔCTA homozygous mice has reduced platelet numbers. Platelet aggregation and activation were also impaired and bleeding times significantly prolonged in these mutant mice. Analysis of mRNA from total bone marrow and purified megakaryocytes from Fli1ΔCTA/Fli1ΔCTA mice revealed downregulation of genes associated with megakaroyctic development, including c-mpl, gpIIb, gpIV, gpIX, PF4, NF-E2, MafG, and Rab27B. While Fli1 and GATA-1 synergistically regulate the expression of multiple megakaryocytic genes, the level of GATA-1 present on a subset of these promoters is reduced in vivo in the Fli1ΔCTA/Fli1ΔCTA mice, providing a possible mechanism for the impared transcription observed. Collectively, these data showed for the first time a hemostatic defect associated with the loss of a specific functional domain of the transcription factor Fli1 and suggest previously unknown in vivo roles in megakaryocytic cell differentiation.

A Critical Role of the Transcription Factor Fli‐1 in Murine Lupus Development by Regulation of Interleukin‐6 Expression

The Fli‐1 transcription factor is implicated in the pathogenesis of systemic lupus erythematosus (SLE), both in humans and in animal models. Dysregulation of interleukin‐6 (IL‐6) is also associated with SLE. The purpose of this study was to investigate whether Fli‐1 directly regulates the expression of IL‐6.

The Fli-1 Transcription Factor Regulates the Expression of CCL5/RANTES

The friend leukemia insertion site 1 (Fli-1) transcription factor, an Ets family member, is implicated in the pathogenesis of systemic lupus erythematosus in human patients and murine models of lupus. Lupus-prone mice with reduced Fli-1 expression have significantly less nephritis, prolonged survival, and decreased infiltrating inflammatory cells into the kidney. Inflammatory chemokines, including CCL5, are critical for attracting inflammatory cells. In this study, decreased CCL5 mRNA expression was observed in kidneys of lupus-prone NZM2410 mice with reduced Fli-1 expression. CCL5 protein expression was significantly decreased in endothelial cells transfected with Fli-1–specific small interfering RNA compared with controls. Fli-1 binds to endogenous Ets binding sites in the distal region of the CCL5 promoter. Transient transfection assays demonstrate that Fli-1 drives transcription from the CCL5 promoter in a dose-dependent manner. Both Ets1, another Ets family member, and Fli-1 drive transcription from the CCL5 promoter, although Fli-1 transactivation was significantly stronger. Ets1 acts as a dominant-negative transcription factor for Fli-1, indicating that they may have at least one DNA binding site in common. Systematic deletion of DNA binding sites demonstrates the importance of the sites located within a 225-bp region of the promoter. Mutation of the Fli-1 DNA binding domain significantly reduces transactivation of the CCL5 promoter by Fli-1. We identified a novel regulator of transcription for CCL5. These results suggest that Fli-1 is a novel and critical regulator of proinflammatory chemokines and affects the pathogenesis of disease through the regulation of factors that recruit inflammatory cells to sites of inflammation.

Decreased expression of Fli-1 in bone marrow-derived haematopoietic cells significantly affects disease development in Murphy Roths Large/lymphoproliferation (MRL/lpr) mice

The transcription factor Fli‐1 is implicated in the pathogenesis of both murine and human lupus. Decreased expression of Fli‐1 in heterozygous (Fli‐1+/−) Murphy Roths Large (MRL)/lpr mice resulted in significantly lower kidney pathological scores and markedly increased survival. In this study, bone marrow (BM) transplantation was used to investigate the role of decreased expression of Fli‐1 in haematopoietic versus non‐haematopoietic cell lineages in autoimmune disease development. Wild‐type (WT) MRL/lpr that received BM from Fli‐1+/− MRL/lpr mice had statistically significantly lower autoantibodies, less proteinuria, reduced renal disease and prolonged survival compared to WT MRL/lpr mice that received BM from WT MRL/lpr mice. Although not statistically significant, Fli‐1+/− MRL/lpr mice that received BM from WT MRL/lpr mice also had lower autoantibodies and improved survival compared to WT MRL/lpr mice that received BM from WT MRL/lpr mice. Our data indicate that expression of Fli‐1 in haematopoietic cell lineages has a significant effect on disease development in MRL/lpr mice.

Fli-1 controls transcription from the MCP-1 gene promoter, which may provide a novel mechanism for chemokine and cytokine activation

Regulation of proinflammatory cytokines and chemokines is a primary role of the innate immune response. MCP-1 is a chemokine that recruits immune cells to sites of inflammation. Expression of MCP-1 is reduced in primary kidney endothelial cells from mice with a heterozygous knockout of the Fli-1 transcription factor. Fli-1 is a member of the Ets family of transcription factors, which are evolutionarily conserved across several organisms including Drosophilla, Xenopus, mouse and human. Ets family members bind DNA through a consensus sequence GGAA/T, or Ets binding site (EBS). Fli-1 binds to EBSs within the endogenous MCP-1 promoter by ChIP assay. In this study, transient transfection assays indicate that the Fli-1 gene actively promotes transcription from the MCP-1 gene promoter in a dose-dependent manner. Mutation of the DNA binding domain of Fli-1 demonstrated that Fli-1 activates transcription of MCP-1 both directly, by binding to the promoter, and indirectly, likely through interactions with other transcription factors. Another Ets transcription factor, Ets-1, was also tested, but failed to promote transcription. While Ets-1 failed to drive transcription independently, a weak synergistic activation of the MCP-1 promoter was observed between Ets-1 and Fli-1. In addition, Fli-1 and the NFκB family member p65 were found to interact synergistically to activate transcription from the MCP-1 promoter, while Sp1 and p50 inhibit this interaction. Deletion studies identified that EBSs in the distal and proximal MCP-1 promoter are critical for Fli-1 activation from the MCP-1 promoter. Together, these results demonstrate that Fli-1 is a novel regulator of the proinflammatory chemokine MCP-1, that interacts with other transcription factors to form a complex transcriptional mechanism for the activation of MCP-1 and mediation of the inflammatory response.