Granulocyte Colony-stimulating Factor Promoter Research Articles

Acetylation impacts Fli-1-driven regulation of granulocyte colony stimulating factor.

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.

LPS-Induced G-CSF Expression in Macrophages Is Mediated by ERK2, but Not ERK1.

Granulocyte colony-stimulating factor (G-CSF) selectively stimulates proliferation and differentiation of neutrophil progenitors which play important roles in host defense against infectious agents. However, persistent G-CSF production often leads to neutrophilia and excessive inflammatory reactions. There is therefore a need to understand the mechanism regulating G-CSF expression. In this study, we showed that U0126, a MEK1/2 inhibitor, decreases lipopolysaccharide (LPS)-stimulated G-CSF promoter activity, mRNA expression and protein secretion. Using short hairpin RNA knockdown, we demonstrated that ERK2, and not ERK1, involves in LPS-induced G-CSF expression, but not LPS-regulated expression of TNF-α. Reporter assays showed that ERK2 and C/EBPβ synergistically activate G-CSF promoter activity. Further chromatin immunoprecipitation (ChIP) assays revealed that U0126 inhibits LPS-induced binding of NF-κB (p50/p65) and C/EBPβ to the G-CSF promoter, but not their nuclear protein levels. Knockdown of ERK2 inhibits LPS-induced accessibility of the G-CSF promoter region to DNase I, suggesting that chromatin remodeling may occur. These findings clarify that ERK2, rather than ERK1, mediates LPS-induced G-CSF expression in macrophages by remodeling chromatin, and stimulates C/EBPβ-dependent activation of the G-CSF promoter. This study provides a potential target for regulating G-CSF expression.

Regulation of granulocyte colony stimulating factor by Fli-1, a novel transcriptional activator of inflammatory mediators (CCR3P.213)

Abstract The Fli-1 transcription factor plays a significant role in the pathogenesis of systemic lupus erythematosus (SLE). Fli-1 expression is significantly increased in patients with lupus nephritis and lupus-prone mice with reduced Fli-1 expression (Fli-1+/-) live longer and have significantly less nephritis compared to wild type controls. Recently, 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. In clinical settings, G-CSF increases the inflammatory response and worsens tissue damage. 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 endothelial cells transfected with Fli-1 specific siRNA. The murine G-CSF promoter contains numerous putative Fli-1 binding sites and five regions within the proximal promoter were found to be 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. In conclusion, we have discovered that Fli-1 plays a major role in the regulation of inflammatory cytokines and chemokines and inflammatory disease pathogenesis.

HSF1 regulates expression of G-CSF through the binding element for NF-IL6/CCAAT enhancer binding protein beta

Heat shock factor 1 (HSF1) is the major heat shock transcription factor and plays an essential role in mediating the cellular response to physiological and environmental stress. We found that LPS-induced expression of the granulocyte-colony stimulating factor (G-CSF) gene was upregulated in HSF1 knock-out (HSF1(-/-)) mice using a gene array. In order to determine whether and how HSF1 regulates the induced expression of G-CSF, mRNA, and protein levels of G-CSF were detected by Northern blotting and ELISA, the promoter of G-CSF was analyzed with an online transcription element search system and the transcriptional activity of the G-CSF promoter was analyzed by EMSA and a reporter gene assay. The results showed that transcription and protein secretion of G-CSF induced by LPS are both inhibited by HSF1. Three high affinity binding sites for NF-IL6/CCAAT enhancer binding protein beta, but no heat shock element, were identified in the core promoter of G-CSF. The DNA-binding capability of NF-IL6 to the G-CSF promoter was reinforced by LPS but not influenced by heat shock or HSF1. However, HSF1 was observed to bind to the binding sites of NF-IL6 in the G-CSF promoter. The transcriptional activity of the G-CSF promoter was enhanced by LPS or NF-IL6 and inhibited by HSF1 in a dose dependent manner. We conclude that HSF1 regulates expression of G-CSF through binding to the NF-IL6-binding element.

Inhibition by rapamycin of the lipoteichoic acid-induced granulocyte-colony stimulating factor expression in mouse macrophages

Granulocyte-colony stimulating factor (G-CSF) is a cytokine which involves in anti-inflammation and inflammation as well. Rapamycin is an inhibitor of mTOR which also plays a role in innate immunity. This study investigated the effect of rapamycin on the lipoteichoic acid (LTA)-induced expression of G-CSF in macrophages and its underlying mechanism. Our data show that LTA induced G-CSF expression in RAW264.7 and bone marrow-derived macrophages and that this effect was inhibited by rapamycin. Analysis of the G-CSF 5′ flanking sequence revealed that the −283 to +35 fragment, which contains CSF and octamer elements, was required for maximal promoter activity in response to LTA stimulation. Western blot analyses of proteins that bind to the CSF and octamer element show that LTA increased protein levels of NF-κB, C/EBPβ and Oct-2, and that rapamycin inhibited the LTA-induced increase in Oct-2 protein levels, but not the others. Knockdown of Oct-2 by RNA interference resulted in a decrease in LTA-induced G-CSF mRNA levels. Moreover, forced expression of Oct-2 by transfection with the pCG-Oct-2 plasmid overcame the inhibitory effect of rapamycin on the LTA-induced increase in G-CSF mRNA levels and promoter activity. This study demonstrates that rapamycin reduces G-CSF expression in LTA-treated macrophages by inhibiting Oct-2 expression.

Cyclic AMP increases endogenous granulocyte colony-stimulating factor formation in monocytes and THP-1 macrophages despite attenuated TNF-alpha formation.

The cytokine granulocyte colony-stimulating factor (G-CSF) is in broad clinical use to treat neutropenia, and trials on its use in immunosuppressed conditions and infections are ongoing. To apply G-CSF effectively, it is crucial to understand the regulation and distribution of its endogenous formation. Since G-CSF release is mediated, at least in part, by TNF-alpha formation, we investigated whether drugs suppressing TNF-alpha also impair G-CSF production. Surprisingly, G-CSF formation was enhanced in lipopolysaccharide (LPS)-stimulated blood from a pentoxifylline-treated patient. In the presence of dibutyryl-cAMP, forskolin, tolafentrine or 3-isobutyl-1-methylxanthine, LPS-induced G-CSF formation was enhanced in THP-1 cells, primary monocytes and whole blood. Correspondingly,rp-8-bromo-cAMP suppressed LPS-induced G-CSF release. Addition of prostaglandin E(2) enhanced and indomethacin suppressed G-CSF formation. Reporter gene studies showed that dibutyryl-cAMP enhanced LPS-induced G-CSF promoter activity, indicating a transcriptional up-regulation. Furthermore, disruption of a newly identified putative cAMP-responsive element (CRE) in the G-CSF promoter demonstrated the regulatory role for G-CSF gene transcription. In conclusion, endogenous G-CSF formation critically depends on both TNF-alpha and cyclooxygenase products, exerting effects via cAMP and the CRE in the G-CSF promoter. This might have bearing for drug side effects, putative G-CSF mimetics and our understanding of G-CSF immunobiology.

STIMULATION OF G-CSF GENE EXPRESSION IN THE MACROPHAGE CELL LINE BY CONTACT WITH EXTRACELLULAR MATRIX PROTEINS AND A PRE-B LEUKAEMIA CELL LINE

In order to scrutinize the adherence-dependent interactions for induction of granulocyte colony-stimulating factor (G-CSF) in peripheral monocytes/macrophages, a sensitive reporter gene assay was constructed using the mouse macrophage cell line transfected with the mouse G-CSF promoter region in conjunction with the luciferase gene as a reporter. With this system, lipopolysaccharide (LPS) showed a markedly positive response. Among the extracellular matrix (ECM) proteins, both fibronectin (FN) and vitronectin (VN) markedly induced luciferase activity, but others did so to a much lesser extent. Among the synthetic peptides having Arg-Gly-Asp (RGD) sequences, only FLEPP with multiple RGD significantly induced luciferase activity. Pretreatment of the cells with anti-integrin α6, αM, β1 and β2 monoclonal antibodies (mAbs) significantly reduced the LPS-induced responses and anti-α1, α2 and β3 mAbs to a lesser extent, and anti-α5, α6, αM, β1 and β2 mAbs blocked the FN-induced response. In the cell-to-cell interactions, a significantly positive increase was observed by direct contacting of this cell line with a G-CSF-dependent promyelocytic leukaemia cell line, known to stimulate the induction of G-CSF to the stromal cells. Its effect was mostly blocked by pretreatment with anti-intergrin α5, αL, β1 and β2 and anti-ICAM-1 mAbs. These results indicate that there are several pathways via the cell-to-ECM and cell-to-cell interactions triggering the induction of G-CSF in the macrophages.

Requirement for nuclear factor (NF)-kappa B p65 and NF-interleukin-6 binding elements in the tumor necrosis factor response region of the granulocyte colony-stimulating factor promoter

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor produced by mesenchymal and myeloid cells following activation by inflammatory stimuli. It has previously been shown that a region of the G-CSF promoter, (-200 to -165) containing the decanucleotide CK-1 element and two repeated sequences that resemble nuclear factor (NF)- interleukin-6 (IL-6) binding sites, is required for activation of the G- CSF gene by tumor necrosis factor-alpha (TNF-alpha) and IL-1 beta. We now show that the NF-kappa B p65 protein can bind to and activate this TNF response region. There are several unusual features of this p65 interaction with the TNF response region. First, NF-kappa B p65 but not the related NF-kappa B p50 binds to the CK-1 element and a p50/65 hybrid protein that relies on the p50 rel homology domain for DNA binding does not transactivate the TNF response region. Second, p65 transactivation of this region is cell specific and requires not only its own binding site but also the NF-IL6 consensus sites. NF-IL6 also binds to the TNF response region of the G-CSF promoter. Electrophoretic mobility shift studies show that p65 and NF-IL6 can bind cooperatively to the TNF response region. The ability of this region to respond to TNF-alpha or p65 is correlated with the ability to form the p65/NF-IL6 ternary complex.

Three Essential Promoter Elements Mediate Tumour Necrosis Factor and Interleukin-1 Activation of the Granulocyte-Colony Stimulating Factor Gene

Granulocyte-colony stimulating factor (G-CSF) is a haemopoietic growth factor produced by mesenchymal cells but not T lymphocytes after stimulation with specific cytokines or mitogens. A 330 bp promoter fragment of the human G-CSF gene induced reporter gene expression in human embryonic lung fibroblasts in response to tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta). The same promoter fragment was not active in Jurkat T cells nor did it respond to phorbol ester in either cell type. At least three distinct elements, the CK-1 sequence, a decanucleotide present in haemopoietic growth factor genes, an NF-IL-6 consensus sequence and a consensus octamer sequence, were essential in the G-CSF promoter for TNF-alpha and IL-1 beta response. Mutation of any of these sequences abolished promoter function. In contrast, mutation of two other consensus protein binding sequences, i.e. a Pu-1 site and a CK-2-like sequence, did not eliminate promoter function. Both the CK-1 and octamer sequences acted independently as TNF-alpha and IL-1 beta responsive elements upstream of a heterologous promoter. The response of the octamer sequence and the 330 bp promoter but not the CK-1 sequence was greater with IL-1 beta than TNF-alpha reflecting a similar response of the endogenous gene.

Three individual regulatory elements of the promoter positively activate the transcription of the murine gene encoding granulocyte colony-stimulating factor

At least three regulatory elements GPE1, GPE2 and GPE3 (G-CSF promoter elements) controlling the gene (G-CSF) encoding granulocyte colony-stimulating factor (G-CSF) are indispensable for the constitutive expression of the G-CSF gene in human CHU-2 cells and for its lipopolysaccharide(LPS)-inducible expression in macrophages. The enhancer activities of each regulatory element were examined with or without the SV40 enhancer element placed downstream from the reporter gene. A GPE1 tetramer mediated the constitutive expression in CHU-2 cells, and the LPS-inducible expression in macrophage cell lines, while the GPE2 element was active in CHU-2 and LPS-treated macrophage cell lines only in combination with the SV40 enhancer. A GPE3 tetramer had efficient enhancer activity in CHU-2 cells but not in macrophage cell lines without the SV40 enhancer. In combination with the SV40 enhancer, GPE3 worked as an LPS-inducible enhancer element in macrophage BAM3 cells. Gel retardation assay indicated that the CHU-2 and the macrophage cells contained nuclear factors which specifically bound to each GPE sequence.

Regulatory elements responsible for inducible expression of the granulocyte colony-stimulating factor gene in macrophages.

Granulocyte colony-stimulating factor (G-CSF) plays an essential role in granulopoiesis during bacterial infection. Macrophages produce G-CSF in response to bacterial endotoxins such as lipopolysaccharide (LPS). To elucidate the mechanism of the induction of G-CSF gene in macrophages or macrophage-monocytes, we have examined regulatory cis elements in the promoter of mouse G-CSF gene. Analyses of linker-scanning and internal deletion mutants of the G-CSF promoter by the chloramphenicol acetyltransferase assay have indicated that at least three regulatory elements are indispensable for the LPS-induced expression of the G-CSF gene in macrophages. When one of the three elements was reiterated and placed upstream of the TATA box of the G-CSF promoter, it mediated inducibility as a tissue-specific and orientation-independent enhancer. Although this element contains a conserved NF-kappa B-like binding site, the gel retardation assay and DNA footprint analysis with nuclear extracts from macrophage cell lines demonstrated that nuclear proteins bind to the DNA sequence downstream of the NF-kappa B-like element, but not to the conserved element itself. The DNA sequence of the binding site was found to have some similarities to the LPS-responsive element which was recently identified in the promoter of the mouse class II major histocompatibility gene.

Regulatory Elements Responsible for Inducible Expression of the Granulocyte Colony-Stimulating Factor Gene in Macrophages

Granulocyte colony-stimulating factor (G-CSF) plays an essential role in granulopoiesis during bacterial infection. Macrophages produce G-CSF in response to bacterial endotoxins such as lipopolysaccharide (LPS). To elucidate the mechanism of the induction of G-CSF gene in macrophages or macrophage-monocytes, we have examined regulatory cis elements in the promoter of mouse G-CSF gene. Analyses of linker-scanning and internal deletion mutants of the G-CSF promoter by the chloramphenicol acetyltransferase assay have indicated that at least three regulatory elements are indispensable for the LPS-induced expression of the G-CSF gene in macrophages. When one of the three elements was reiterated and placed upstream of the TATA box of the G-CSF promoter, it mediated inducibility as a tissue-specific and orientation-independent enhancer. Although this element contains a conserved NF-kappa B-like binding site, the gel retardation assay and DNA footprint analysis with nuclear extracts from macrophage cell lines demonstrated that nuclear proteins bind to the DNA sequence downstream of the NF-kappa B-like element, but not to the conserved element itself. The DNA sequence of the binding site was found to have some similarities to the LPS-responsive element which was recently identified in the promoter of the mouse class II major histocompatibility gene.