ABSTRACT
Mesangial cells offer structural support to the glomerular tuft and regulate glomerular capillary flow through their contractile capabilities. These cells undergo phenotypic changes, such as proliferation and mesangial expansion, resulting in abnormal glomerular tuft formation and reduced capillary loops. Such adaptation to the changing environment is commonly associated with various glomerular diseases, including diabetic nephropathy and glomerulonephritis. Thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling in mesangial cells was not examined. This study investigated protease-activated mechanisms regulating mesangial cell calcium waves that may play an essential role in the mesangial proliferation or constriction of the arteriolar cells. Our results indicate that coagulation proteases like thrombin induce synchronized oscillations in cytoplasmic Ca2+ concentration of mesangial cells. The oscillations required PAR1 GPCRs-related activation, but not a PAR4, and were further mediated presumably through store-operated calcium entry and TRPC3 channel activity. Understanding thrombin signaling pathways and their relation to mesangial cells' contractile or synthetic (proliferative) phenotype may play a role in the development of chronic kidney disease and requires further investigation.
ABSTRACT
Lupus nephritis (LN) is a serious complication for many patients who develop systemic lupus erythematosus, which primarily afflicts women. Our studies to identify biomarkers and the pathogenic mechanisms underlying LN will provide a better understanding of disease progression and sex bias, and lead to identification of additional potential therapeutic targets. The glycosphingolipid lactosylceramide (LacCer) and N-linked glycosylated proteins (N-glycans) were measured in urine and serum collected from LN and healthy control (HC) subjects (10 females and 10 males in each group). The sera from the LN and HC subjects were used to stimulate cytokine secretion and intracellular Ca2+ flux in female- and male-derived primary human renal mesangial cells (hRMCs). Significant differences were observed in the urine of LN patients compared to HCs. All major LacCers species were significantly elevated and differences between LN and HC were more pronounced in males. 72 individual N-glycans were altered in LN compared to HC and three N-glycans were significantly different between the sexes. In hRMCs, Ca2+ flux, but not cytokine secretion, was higher in response to LN sera compared to HC sera. Ca2+ flux, cytokine secretion, and glycosphingolipid levels were significantly higher in female-derived compared to male-derived hRMCs. Relative abundance of some LacCers and hexosylceramides were higher in female-derived compared to male-derived hRMCs. Urine LacCers and N-glycome could serve as definitive LN biomarkers and likely reflect renal disease activity. Despite higher sensitivity of female hRMCs, males may experience greater increases in LacCers, which may underscore worse disease in males. Elevated glycosphingolipid metabolism may poise renal cells to be more sensitive to external stimuli.
Subject(s)
Lupus Erythematosus, Systemic , Lupus Nephritis , Humans , Female , Male , Lupus Nephritis/pathology , Biomarkers , Cytokines , Glycosphingolipids , PolysaccharidesABSTRACT
The transcription factor Fli-1, a member of the ETS family of transcription factors, is implicated in the pathogenesis of lupus disease. Reduced Fli-1 expression in lupus mice leads to decreased renal Cxcl10 mRNA levels and renal infiltrating CXCR3+ T cells that parallels reduced renal inflammatory cell infiltration and renal damage. Inflammatory chemokine CXCL10 is critical for attracting inflammatory cells expressing the chemokine receptor CXCR3. The CXCL10/CXCR3 axis plays a role in the pathogenesis of various inflammatory diseases including lupus. Our data here demonstrate that renal CXCL10 protein levels are significantly lower in Fli-1 heterozygous MRL/lpr mice compared to wild-type MRL/lpr mice. Knockdown of Fli-1 significantly reduced CXCL10 secretion in mouse and human endothelial cells, and human mesangial cells, upon LPS or TNFα stimulation. The Fli-1 inhibitor, Camptothecin, significantly reduced CXCL10 production in human monocyte cells upon interferon stimulation. Four putative Ets binding sites in the Cxcl10 promoter showed significant enrichment for FLI-1; however, FLI-1 did not directly drive transcription from the human or mouse promoters, suggesting FLI-1 may regulate CXCL10 expression indirectly. Our results also suggest that the DNA binding domain of FLI-1 is necessary for regulation of human hCXCR3 promotor activity in human T cells and interactions with co-activators. Together, these results support a role for FLI-1 in modulating the CXCL10-CXCR3 axis by directly or indirectly regulating the expression of both genes to impact lupus disease development. Signaling pathways or drugs that reduce FLI-1 expression may offer novel approaches to lupus treatment.
Subject(s)
Endothelial Cells , Proto-Oncogene Protein c-fli-1 , Animals , Humans , Mice , Chemokine CXCL10/genetics , Chemokine CXCL10/metabolism , Endothelial Cells/metabolism , Kidney/pathology , Mice, Inbred MRL lpr , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Protein c-fli-1/metabolism , Receptors, CXCR3/genetics , Receptors, CXCR3/metabolismABSTRACT
The development of nephritis increases the risk of morbidity and mortality in systemic lupus erythematosus (SLE) patients. While standard induction therapies, such as mycophenolate mofetil (MMF) induce clinical remission (i.e., complete response) in approximately 50% of SLE patients with nephritis, many patients fail to respond. Therapeutic response is often not assessed until 6-12 months after beginning treatment. Those patients that fail to respond to treatment continue to accumulate organ damage, thus, there is a critical need to predict which patients will fail therapy before beginning treatment, allowing physicians to optimize therapy. Our previous studies demonstrated elevated urine, but not serum, glycosphingolipids (GSLs) in SLE patients with nephritis compared to SLE patients without nephritis, suggesting the urine GSLs were derived from the kidney. In this study, we measured the GSLs hexosylceramide and lactosylceramide in extracellular vesicles isolated from longitudinal urine samples of LN patients that were treated with MMF for 12 months. GSL levels were significantly elevated in the baseline samples (prior to treatment) of non-responders compared to complete responders. While a few other proteins measured in the whole urine were higher in non-responders at baseline, only GSLs demonstrated a significant ability to discriminate treatment response in lupus nephritis patients.
ABSTRACT
PURPOSE OF REVIEW: Mesangial cells are critical for the proper function of the glomerulus, playing roles in structural support and injury repair. However, they are also early responders to glomerular immune complex deposition and contribute to inflammation and fibrosis in lupus nephritis. This review highlights recent studies identifying signaling pathways and mediators in mesangial cell response to lupus-relevant stimuli. RECENT FINDINGS: Anti-dsDNA antibodies, serum, or plasma from individuals with lupus nephritis, or specific pathologic factors activated multiple signaling pathways. These pathways largely included JAK/STAT/SOCS, PI3K/AKT, and MAPK and led to induction of proliferation and expression of multiple proinflammatory cytokines, growth factors, and profibrotic factors. NFκB activation was a common mediator of response. Mesangial cells proliferate and express a wide array of proinflammatory/profibrotic factors in response to a variety of lupus-relevant pathologic stimuli. While some of the responses are similar, the mechanisms involved appear to be diverse depending on the stimulus. Future studies are needed to fully elucidate these mechanisms with respect to the diverse milieu of stimuli.
Subject(s)
Lupus Nephritis , Mesangial Cells , Antibodies, Antinuclear , Fibrosis , Humans , Lupus Nephritis/pathology , Mesangial Cells/pathology , Phosphatidylinositol 3-KinasesABSTRACT
OBJECTIVE: SLE is a chronic multisystem autoimmune inflammatory disease impacting a number of organs, including the central nervous system (CNS). The pathophysiology of CNS lupus is multifactorial, making diagnosis problematic. Neurocognitive (NC) testing and specific biomarkers to identify the development of neuropsychiatric (NP) symptoms in lupus are needed. Paediatric patients with SLE have high incidence of NP disease . While serum anti-N-methyl-D-aspartate receptor (NMDAR) antibodies have shown promise as a biomarker of NP in adults with SLE, much less is known with regard to paediatric patients with SLE. METHODS: We performed a cross-sectional study in paediatric patients with SLE. Serum NMDAR antibodies were measured and compared with levels in patients with juvenile idiopathic arthritis (JIA). Formal NC testing was performed in accordance with the Childhood Arthritis & Rheumatology Research Alliance neuropsychological core test battery. NC functioning was compared in the two groups and with NMDAR antibody levels. RESULTS: Serum NMDAR antibody levels were significantly higher in paediatric patients with SLE compared with patients with JIA. There were no significant correlations between NMDAR antibody levels and any measure of NC functioning. In an exploratory examination of anti-ribosomal P (RibP) antibody and NC functioning in a subset of patients with SLE, RibP antibody-positive patients exhibited worse scores for Verbal Memory Index and Design Fluency Test Switching compared with RibP antibody-negative patients. A globally significant association between disease status and NC functioning was observed. Specifically, patients with SLE had lower scores compared with patients with JIA for full-scale IQ, letter-word recognition, reading fluency and calculation skills after adjusting for multiple comparisons. CONCLUSION: These collective results suggest that although serum NMDAR may serve as a biomarker, formal NC testing is superior in identifying paediatric patients with SLE with NP manifestations. RibP also may potentially serve as a biomarker of NP manifestations in paediatric patients with SLE. Additional and longitudinal studies are needed.
Subject(s)
Lupus Vasculitis, Central Nervous System , Adolescent , Antibodies, Antinuclear , Autoantibodies , Child , Cross-Sectional Studies , Female , Humans , Male , Receptors, N-Methyl-D-AspartateABSTRACT
The importance of altered glycosphingolipid (GSL) metabolism is increasingly gaining attention as a characteristic of multiple chronic kidney diseases. Previously, we reported elevated levels of GSLs and neuraminidase (NEU) enzyme activity/expression in the urine or kidney of lupus patients and lupus-prone mice, and demonstrated NEU activity mediates the production of cytokines by lupus-prone mouse primary mesangial cells. This mediation occurs in part through TLR4 and p38/ERK MAPK signalling in response to lipopolysaccharide (LPS) and lupus serum (LS). However, the precise role of NEU1, the most abundant NEU in the kidney, is incompletely known. In this study, we investigated the effect of genetically reduced Neu1 levels in vitro and in vivo. Mesangial cells from non-autoimmune prone Neu1+/- C57BL/6 mice had significantly reduced NEU activity, cytokine expression and cytokine secretion in response to LS and LPS, thereby suggesting reducing Neu1 expression may reduce the inflammatory response in lupus nephritis. Disease was assessed in female B6.SLE1/2/3 lupus-prone mice with genetically reduced levels (Neu1+/-) or wild-type levels (Neu1+/+) of Neu1 from 28 to 44 weeks of age along with aged-matched C57BL/6 controls. Renal disease was unexpectedly mild in all B6.SLE1/2/3 mice despite evidence of systemic disease. B6.SLE1/2/3 Neu1+/- mice exhibited significantly reduced levels of renal NEU1 expression and changes in renal α-2,6 linked sialylated N-glycans compared to the Neu1+/+ or healthy C57BL/6 mice, but measures of renal and systemic disease were similar between the B6.SLE1/2/3 Neu1+/+ and Neu1+/- mice. We conclude that NEU1 is the NEU largely responsible for mediating cytokine release by mesangial cells, at least in vitro, but may not be involved in modulating renal GSL levels in vivo or impact onset of nephritis in lupus-prone mice. However, the effect of reduced NEU1 levels on disease may not be appreciated in the mild disease expression in our colony of B6.SLE1/2/3 mice. The impact of the altered renal sialylated N-glycan levels and potential role of NEU1 with respect to established nephritis (late disease) in lupus-prone mice bears further investigation.
Subject(s)
Cytokines/metabolism , Lupus Nephritis/metabolism , Mesangial Cells/metabolism , Neuraminidase/metabolism , Animals , Disease Models, Animal , Female , Kidney/drug effects , Kidney/metabolism , Lipopolysaccharides/pharmacology , MAP Kinase Signaling System/drug effects , Mesangial Cells/drug effects , Mice , Mice, Inbred C57BLABSTRACT
Previously, we demonstrated neuraminidase (NEU) activity or NEU1 expression, specifically, is increased in the kidneys of lupus mice and urine of human patients with nephritis. Additionally, NEU activity mediates IL-6 secretion from lupus-prone MRL/lpr primary mouse mesangial cells (MCs) in response to an IgG mimic. IL-6 mediates glomerular inflammation and promotes tissue damage in patients and mouse strains with lupus nephritis. This study further elucidates the mechanisms by which NEU activity and NEU1 specifically mediates the release of IL-6 and other cytokines from lupus-prone MCs. We demonstrate significantly increased release of multiple cytokines and NEU activity in MRL/lpr MCs in response to serum from MRL/lpr mice (lupus serum). Inhibiting NEU activity significantly reduced secretion of three of those cytokines: IL-6, GM-CSF and MIP1α. Message levels of Il-6 and Gm-csf were also increased in response to lupus serum and reduced when NEU activity was inhibited. Neutralizing antibodies to cell-surface receptors and MAPK inhibitors in lupus serum- or LPS-stimulated MCs indicate TLR4 and p38 or ERK MAP kinase signalling play key roles in the NEU-mediated secretion of IL-6. Significantly reduced IL-6 release was observed in C57BL/6 (B6) Neu1+/+ primary MCs compared with wild-type (Neu1+/+) B6 MCs in response to lupus serum. Additional results show inhibiting NEU activity significantly increases sialic acid-containing N-glycan levels. Together, our novel observations support a role for NEU activity, and specifically NEU1, in mediating release of IL-6 from lupus-prone MCs in response to lupus serum through a TLR4-p38/ERK MAPK signalling pathway that likely includes desialylation of glycoproteins.
Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , Kidney/metabolism , Lupus Erythematosus, Systemic/metabolism , Lupus Nephritis/metabolism , Mesangial Cells/physiology , Neuraminidase/metabolism , Toll-Like Receptor 4/metabolism , Animals , Cells, Cultured , Cytokines/metabolism , Female , Gene Expression Regulation , Humans , Kidney/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Neuraminidase/genetics , Serum/metabolism , Signal TransductionABSTRACT
Glycosphingolipids (GSLs) hexosylceramides and lactosylceramides are elevated in lupus mice and human patients with nephritis. Whereas other renal diseases characterized by increased GSL levels are thought to be a result of upregulated GSL synthesis, our results suggest elevated hexosylceramides and lactosylceramides in lupus nephritis is a result of increased catabolism of ganglioside GM3 due to significantly increased neuraminidase (NEU) activity. Thus, we hypothesized GM3 would be decreased in lupus nephritis kidneys and blocking NEU activity would reduce GSLs and improve disease in lupus mice. Female MRL/lpr lupus mice were treated with water or the NEU inhibitor oseltamivir phosphate at the onset of proteinuria to block GSL catabolism. Age-matched (non-nephritic) female MRL/MpJ lupus mice served as controls. Renal GM3 levels were significantly higher in the nephritic MRL/lpr water-treated mice compared to non-nephritic MRL/MpJ mice, despite significantly increased renal NEU activity. Blocking GSL catabolism increased, rather than decreased, renal and urine GSL levels and disease was not significantly impacted. A pilot study treating MRL/lpr females with GlcCer synthase inhibitor Genz-667161 to block GSL synthesis resulted in a strong significant negative correlation between Genz-667161 dose and renal GSL hexosylceramide and GM3 levels. Splenomegaly was negatively correlated and serum IgG levels were marginally correlated with increasing Genz-667161 dose. These results suggest accumulation of renal GM3 may be due to dysregulation of one or more of the GSL ganglioside pathways and inhibiting GSL synthesis, but not catabolism, may be a therapeutic approach for treating lupus nephritis.
Subject(s)
Glycosphingolipids/metabolism , Lupus Nephritis/drug therapy , Lupus Nephritis/metabolism , Animals , Ceramides/metabolism , Female , G(M3) Ganglioside/metabolism , Kidney/drug effects , Kidney/metabolism , Lactosylceramides/metabolism , Mice , Mice, Inbred MRL lpr , Neuraminidase/metabolism , Oseltamivir/analogs & derivatives , Oseltamivir/therapeutic use , Phosphorous Acids/therapeutic use , Pilot Projects , Proteinuria/drug therapy , Proteinuria/metabolismABSTRACT
Background: Within free-standing academic medical centers, women continue to be underrepresented at upper faculty ranks and in leadership positions. A career development program (CDP) at the Medical University of South Carolina (MUSC) was implemented with the goal of improving the number of women in the upper ranks and in leadership positions. The CDP was initiated in 2013 as a 2-day program. Beginning in 2015, a half-day promotion-focused program was offered alternating with the 2-day program. Materials and Methods: The CDP has served â¼200 women from 2013 to 2017 and was evaluated for reaction and learning through postprogram surveys. Promotion success of â¼160 women who attended at least one of the programs through 2016 was assessed through an additional survey. Promotion information for â¼3000 faculty members during the same 2013-2016 period (post-CDP), as well as a 4-year time period before implementation of the CDP (pre-CDP), was collected using university-level personnel data. Results: The majority of CDP attendees (94%) indicated overall satisfaction with the program and would recommend the program to a colleague. Of the 137 CDP attendees still employed at MUSC in 2017, 50 had applied for promotion and 42 (84%) were successfully promoted. Among all the MUSC faculty, overall and rank-sepcific promotion rates for women and men were similar during the post-CDP time period and there was a significant increase in the promotion rate of women to Full Professor from pre-CDP to post-CDP time periods. Conclusions: CDP attendees were overwhelmingly satisfied with the program and were highly successful in being promoted. Since the overall university promotion rates of women and men were similar during the post-CDP time period and women are currently underrepresented at the upper faculty ranks, parity between men and women will likely not be achievable without additional programs to retain and/or recruit women in the upper ranks.
Subject(s)
Academic Medical Centers/organization & administration , Faculty, Medical/organization & administration , Leadership , Physicians, Women/statistics & numerical data , Staff Development/organization & administration , Adult , Career Choice , Career Mobility , Female , Humans , Middle Aged , Program Development , Program Evaluation , Retrospective Studies , Surveys and Questionnaires , United StatesABSTRACT
The development of nephritis is a leading cause of morbidity and mortality in lupus patients. Although the general pathophysiological progression of lupus nephritis is known, the molecular mediators and mechanisms are incompletely understood. Previously, we demonstrated that the glycosphingolipid (GSL) catabolic pathway is elevated in the kidneys of MRL/lpr lupus mice and human lupus patients with nephritis. Specifically, the activity of neuraminidase (NEU) and expression of Neu1, an enzyme in the GSL catabolic pathway is significantly increased. To better understand the role and mechanisms by which this pathway contributes to the progression of LN, we analyzed the expression and effects of NEU activity on the function of MRL/lpr lupus-prone mesangial cells (MCs). We demonstrate that NEU1 and NEU3 promote IL-6 production in MES13 MCs. Neu1 expression, NEU activity, and IL-6 production are significantly increased in stimulated primary MRL/lpr lupus-prone MCs, and blocking NEU activity inhibits IL-6 production. NEU1 and NEU3 expression overlaps IgG deposits in MCs in vitro and in renal sections from nephritic MRL/lpr mice. Together, our results suggest that NEU activity mediates IL-6 production in lupus-prone MCs possibly through an IgG-receptor complex signaling pathway.
Subject(s)
Interleukin-6/metabolism , Lupus Nephritis/enzymology , Mesangial Cells/enzymology , Neuraminidase/metabolism , Animals , Cell Line , Cell Proliferation , Disease Models, Animal , Female , Glycoside Hydrolase Inhibitors/pharmacology , Immunoglobulin G/pharmacology , Lupus Nephritis/blood , Lupus Nephritis/drug therapy , Lupus Nephritis/pathology , Male , Mesangial Cells/drug effects , Mesangial Cells/pathology , Mice, Inbred MRL lpr , Neuraminidase/antagonists & inhibitors , Neuraminidase/genetics , Receptors, IgG/metabolism , Signal Transduction , Up-RegulationABSTRACT
Fli-1 has emerged as a critical regulator of inflammatory mediators, including MCP-1, CCL5, and IL-6. The cytokine, granulocyte colony stimulating factor (G-CSF) regulates neutrophil precursor maturation and survival, and activates mature neutrophils. Previously, a significant decrease in neutrophil infiltration into the kidneys of Fli-1+/- lupus-prone mice was observed. In this study, a significant decrease in G-CSF protein expression was detected in stimulated murine and human endothelial cells when expression of Fli-1 was inhibited. The murine G-CSF promoter contains numerous putative Fli-1 binding sites and several regions within the proximal promoter are significantly enriched for Fli-1 binding. Transient transfection assays indicate that Fli-1 drives transcription from the G-CSF promoter and mutation of the Fli-1 DNA binding domain resulted in a 94% loss of transcriptional activation. Mutation of a known acetylation site, led to a significant increase in G-CSF promoter activation. The histone acetyltransferases p300/CBP and p300/CBP associated factor (PCAF) significantly decrease Fli-1 specific activation of the G-CSF promoter. Thus, acetylation appears to be an important mechanism behind Fli-1 driven activation of the G-CSF promoter. These results further support the theory that Fli-1 plays a major role in the regulation of several inflammatory mediators, ultimately affecting inflammatory disease pathogenesis.
Subject(s)
Acetylation , Endothelial Cells/physiology , Granulocyte Colony-Stimulating Factor/metabolism , Inflammation/immunology , Neutrophils/physiology , Vascular Endothelial Growth Factor Receptor-1/metabolism , Animals , Gene Expression Regulation/genetics , Granulocyte Colony-Stimulating Factor/genetics , Human Umbilical Vein Endothelial Cells , Humans , Mice , Mutation/genetics , Promoter Regions, Genetic/genetics , Protein Binding , RNA, Small Interfering/genetics , Vascular Endothelial Growth Factor Receptor-1/geneticsABSTRACT
The ETS factor Friend leukemia virus integration 1 (FLI1) is a key modulator of lupus disease expression. Overexpressing FLI1 in healthy mice results in the development of an autoimmune kidney disease similar to that observed in lupus. Lowering the global levels of FLI1 in two lupus strains (Fli1(+/-)) significantly improved kidney disease and prolonged survival. T cells from MRL/lpr Fli1(+/-) lupus mice have reduced activation and IL-4 production, neuraminidase 1 expression, and the levels of the glycosphingolipid lactosylceramide. In this study, we demonstrate that MRL/lpr Fli1(+/-) mice have significantly decreased renal neuraminidase 1 and lactosylceramide levels. This corresponds with a significant decrease in the number of total CD3(+) cells, as well as CD4(+) and CD44(+)CD62L(-) T cell subsets in the kidney of MRL/lpr Fli1(+/-) mice compared with the Fli1(+/+) nephritic mice. We further demonstrate that the percentage of CXCR3(+) T cells and Cxcr3 message levels in T cells are significantly decreased and correspond with a decrease in renal CXCR3(+) cells and in Cxcl9 and Cxcl10 expression in the MRL/lpr Fli1(+/-) compared with the Fli1(+/+) nephritic mice. Our results suggest that reducing the levels of FLI1 in MRL/lpr mice may be protective against development of nephritis in part through downregulation of CXCR3, reducing renal T cell infiltration and glycosphingolipid levels.
Subject(s)
Glycosphingolipids/metabolism , Kidney/physiology , Nephritis/drug therapy , Proto-Oncogene Protein c-fli-1/metabolism , Receptors, CXCR3/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocytes/immunology , Animals , Antigens, CD/metabolism , Cell Movement/drug effects , Chemokine CXCL10/genetics , Chemokine CXCL10/metabolism , Chemokine CXCL9/genetics , Chemokine CXCL9/metabolism , Gene Expression Regulation , Humans , Kidney/drug effects , Lactosylceramides/metabolism , Mice , Mice, Inbred MRL lpr , Mice, Knockout , Nephritis/immunology , Neuraminidase/metabolism , Proto-Oncogene Protein c-fli-1/genetics , Receptors, CXCR3/geneticsABSTRACT
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.
Subject(s)
Chemokine CCL2/genetics , Promoter Regions, Genetic , Proto-Oncogene Protein c-fli-1/metabolism , Animals , Binding Sites/genetics , Humans , Mice , Mutation/genetics , NIH 3T3 Cells , Protein Binding , Proto-Oncogene Protein c-fli-1/genetics , Sp1 Transcription Factor/metabolism , Transcription Factor RelA/metabolism , Transcription, Genetic , Transcriptional Activation/geneticsABSTRACT
Nearly one half of patients with lupus develop glomerulonephritis (GN), which often leads to renal failure. Although nephritis is diagnosed by the presence of proteinuria, the pathology of nephritis can fall into one of five classes defined by different forms of tissue injury, and the mechanisms involved in pathogenesis are not completely understood. Glycosphingolipids are abundant in the kidney, have roles in many cellular functions, and were shown to be involved in other renal diseases. Here, we show dysfunctional glycosphingolipid metabolism in patients with lupus nephritis and MRL/lpr lupus mice. Specifically, we found that glucosylceramide (GlcCer) and lactosylceramide (LacCer) levels are significantly higher in the kidneys of nephritic MRL/lpr lupus mice than the kidneys of non-nephritic lupus mice or healthy controls. This elevation may be, in part, caused by altered transcriptional regulation and/or activity of LacCer synthase (GalT5) and neuraminidase 1, enzymes that mediate glycosphingolipid metabolism. We show increased neuraminidase 1 activity early during the progression of nephritis (before significant elevation of GlcCer and LacCer in the kidney). Elevated levels of urinary LacCer were detected before proteinuria in lupus mice. Notably, LacCer levels were higher in the urine and kidneys of patients with lupus and nephritis than patients with lupus without nephritis or healthy controls. Together, these results show early and significant dysfunction of the glycosphingolipid metabolic pathway in the kidneys of lupus mice and patients with lupus nephritis and suggest that molecules in this pathway may serve as early markers in lupus nephritis.
Subject(s)
Glycosphingolipids/metabolism , Lupus Nephritis/metabolism , Lupus Nephritis/pathology , Neuraminidase/metabolism , Sterol Regulatory Element Binding Protein 1/metabolism , Analysis of Variance , Animals , Biomarkers/analysis , Biopsy, Needle , Disease Models, Animal , Disease Progression , Follow-Up Studies , Humans , Immunoblotting , Immunohistochemistry , Kidney Function Tests , Lupus Nephritis/physiopathology , Mice , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Neuraminidase/genetics , Sensitivity and Specificity , Severity of Illness Index , Sterol Regulatory Element Binding Protein 1/genetics , UrinalysisABSTRACT
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.
Subject(s)
Chemokine CCL5/genetics , DNA/chemistry , Endothelial Cells/immunology , Proto-Oncogene Protein c-fli-1/genetics , Transcriptional Activation/genetics , 3T3 Cells , Animals , Binding Sites/genetics , Binding Sites/immunology , Cell Line , Chemokine CCL5/biosynthesis , DNA/immunology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Endothelial Cells/cytology , Inflammation/genetics , Inflammation/immunology , Kidney/cytology , Kidney/immunology , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Mice , Mice, Transgenic , Nephritis/genetics , Promoter Regions, Genetic , Proto-Oncogene Protein c-ets-1/genetics , Proto-Oncogene Protein c-fli-1/biosynthesis , Proto-Oncogene Protein c-fli-1/immunology , RNA Interference , RNA, Messenger/biosynthesis , RNA, Small Interfering , TransfectionABSTRACT
Systemic Lupus erythematosus (SLE) is an autoimmune disease caused, in part, by abnormalities in cells of the immune system including B and T cells. Genetically reducing globally the expression of the ETS transcription factor FLI1 by 50% in two lupus mouse models significantly improves disease measures and survival through an unknown mechanism. In this study we analyze the effects of reducing FLI1 in the MRL/lpr lupus prone model on T cell function. We demonstrate that adoptive transfer of MRL/lpr Fli1(+/+) or Fli1(+/-) T cells and B cells into Rag1-deficient mice results in significantly decreased serum immunoglobulin levels in animals receiving Fli1(+/-) lupus T cells compared to animals receiving Fli1(+/+) lupus T cells regardless of the genotype of co-transferred lupus B cells. Ex vivo analyses of MRL/lpr T cells demonstrated that Fli1(+/-) T cells produce significantly less IL-4 during early and late disease and exhibited significantly decreased TCR-specific activation during early disease compared to Fli1(+/+) T cells. Moreover, the Fli1(+/-) T cells expressed significantly less neuraminidase 1 (Neu1) message and decreased NEU activity during early disease and significantly decreased levels of glycosphingolipids during late disease compared to Fli1(+/+) T cells. FLI1 dose-dependently activated the Neu1 promoter in mouse and human T cell lines. Together, our results suggest reducing FLI1 in lupus decreases the pathogenicity of T cells by decreasing TCR-specific activation and IL-4 production in part through the modulation of glycosphingolipid metabolism. Reducing the expression of FLI1 or targeting the glycosphingolipid metabolic pathway in lupus may serve as a therapeutic approach to treating lupus.
Subject(s)
Glycosphingolipids/metabolism , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/metabolism , Proto-Oncogene Protein c-fli-1/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Adoptive Transfer , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Disease Models, Animal , Disease Progression , Female , Homeodomain Proteins/metabolism , Humans , Interleukin-4/biosynthesis , Jurkat Cells , Lymphocyte Activation , Mice , Mice, Inbred MRL lpr , Proto-Oncogene Protein c-fli-1/deficiency , Receptors, Antigen, T-Cell/metabolismABSTRACT
Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease that disproportionately affects African American females. The causes of SLE are unknown but postulated to be a combination of genetic predisposition and environmental triggers. Vitamin D deficiency is one of the possible environmental triggers. In this study we evaluated relationships between vitamin D status, cellular aging (telomere length) and anti-telomere antibodies among African American Gullah women with SLE. The study population included African American female SLE patients and unaffected controls from the Sea Island region of South Carolina. Serum 25-hydroxyvitamin D levels were measured using a nonchromatographic radioimmunoassay. Telomere length was measured in genomic DNA of peripheral blood mononuclear cells (PBMCs) by monochrome multiplex quantitative PCR. Anti-telomere antibody levels were measured by enzyme-linked immunosorbent assay (ELISA). Patients with SLE had significantly shorter telomeres and higher anti-telomere antibody titers compared to age- and gender-matched unaffected controls. There was a positive correlation between anti-telomere antibody levels and disease activity among patients and a significant correlation of shorter telomeres with lower 25-hydroxyvitamin D levels in both patients and controls. In follow-up examination of a subset of the patients, the patients who remained vitamin D deficient tended to have shorter telomeres than those patients whose 25-hydroxyvitamin D levels were repleted. Increasing 25-hydroxyvitamin D levels in African American patients with SLE may be beneficial in maintaining telomere length and preventing cellular aging. Moreover, anti-telomere antibody levels may be a promising biomarker of SLE status and disease activity.
Subject(s)
Lupus Erythematosus, Systemic/genetics , Telomere/genetics , Vitamin D Deficiency/genetics , Adult , Black or African American/genetics , Case-Control Studies , Female , Humans , Lupus Erythematosus, Systemic/blood , Middle Aged , Telomere Homeostasis , Vitamin D/blood , Vitamin D Deficiency/bloodABSTRACT
Fucoidan, a sulfated polysaccharide, has significant cytotoxic activity against tumor cells; however, the mechanism(s) of this action remains poorly understood. The present study was designed to determine the in vitro and in vivo effects of fucoidan and their molecular mechanisms. Fucoidan from Cladosiphon okamuranus Tokida cultivated in Okinawa, Japan, delayed tumor growth in Sarcoma 180 (S-180)-bearing mice. However, it failed to inhibit S-180 cell growth in vitro. Activated macrophages are known to have anti-tumor effects. Murine RAW264.7 macrophages stimulated with fucoidan exerted cytotoxicity towards S-180 cells in vitro. This cytotoxicity was associated with nitric oxide (NO) production. Both cytocidal effect and NO production were significantly inhibited by L-NAME, an inhibitor of NO synthase (NOS). Furthermore, activation of nuclear factor-κB was a key step in the transcriptional activation of the inducible NOS gene. Taken together, our results indicate that the anti-tumor activity of fucoidan on S-180 cells is mediated through increased NO production by fucoidan-stimulated macrophages via nuclear factor-κB-dependent signaling pathway.
Subject(s)
Antineoplastic Agents/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Nitric Oxide/metabolism , Polysaccharides/pharmacology , Animals , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Mice , Mice, Inbred BALB C , Mice, Nude , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/biosynthesis , Nitric Oxide Synthase Type II/genetics , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Random Allocation , Sarcoma 180/drug therapy , Sarcoma 180/metabolism , Sarcoma 180/pathology , Signal Transduction/drug effectsABSTRACT
Systemic lupus erythematosus is a prototypic autoimmune disease characterized by autoantibody production and immune complex formation/deposition in target organs such as the kidney. Resultant local inflammation then leads to organ damage. Nephritis, a major cause of morbidity and mortality in patients with lupus, occurs in approximately 50% of lupus patients. In the present review, we provide an overview of the current research and knowledge concerning mechanisms of renal injury in both lupus-prone mouse models and human lupus patients.