CSF-1 Promoter Research Articles

LARRPM restricts lung adenocarcinoma progression and M2 macrophage polarization through epigenetically regulating LINC00240 and CSF1

BackgroundLong non-coding RNAs (lncRNAs) are critical regulators in lung adenocarcinoma (LUAD). M2-type tumor-associated macrophages (TAMs) also play oncogenic roles in LUAD. However, the involvement of lncRNAs in TAM activation is still largely unknown.MethodsThe expressions of LARRPM, LINC00240 and CSF1 were determined by RT-qPCR. The regulation of LINC00240 and CSF1 by LARRPM was investigated by RNA–protein pull-down, RNA immunoprecipitation, chromatin immunoprecipitation and bisulfite DNA sequencing. In vitro and in vivo gain- and loss-of-function assays were performed to investigate the roles of LARRPM.ResultsThe lncRNA LARRPM was expressed at low levels in LUAD tissues and cells. The low expression of LARRPM was correlated with advanced stage and poor survival of patients with LUAD. Functional experiments revealed that LARRPM suppressed LUAD cell proliferation, migration and invasion, and promoted apoptosis. LARRPM also repressed macrophage M2 polarization and infiltration. Taken together, LARRPM significantly restricted LUAD progression in vivo. Mechanistically, LARRPM bound and recruited DNA demethylase TET1 to the promoter of its anti-sense strand gene LINC00240, leading to a decrease in DNA methylation level of the LINC00240 promoter and transcriptional activation of LINC00240. Functional rescue assays suggested that the lncRNA LINC00240 was responsible for the roles of LARRPM in the malignant behavior of LUAD cells. LARRPM decreased the binding of TET1 to the CSF1 promoter, resulting in increased DNA methylation of the CSF1 promoter and transcriptional repression of CSF1, which is responsible for the roles of LARRPM in macrophage M2 polarization and infiltration. The TAMs educated by LUAD cells exerted oncogenic roles, which was negatively regulated by LARRPM expressed in LUAD cells.ConclusionsLARRPM restricts LUAD progression through repressing both LUAD cell and macrophages. These data shed new insights into the regulation of LUAD progression by lncRNAs and provide data on the potential utility of LARRPM as a target for LUAD treatment.

Crosstalk between macrophage-derived PGE2 and tumor UHRF1 drives hepatocellular carcinoma progression.

Background: Tumor-associated macrophages (TAMs) and dysregulated tumor epigenetics contribute to hepatocellular carcinoma (HCC) progression. However, the mechanistic interactions between TAMs and tumor epigenetics remain poorly understood.Methods: Immunohistochemistry and multiplexed fluorescence staining were performed to evaluate the correlation between TAMs numbers and UHRF1 expression in human HCC tissues. PGE2 neutralizing antibody and COX-2 inhibitor were used to analyze the regulation of TAMs isolated from HCC tissues on UHRF1 expression. Multiple microRNA prediction programs were employed to identify microRNAs that target UHRF1 3'UTR. Luciferase reporter assay was applied to evaluate the regulation of miR-520d on UHRF1 expression. Chromatin immunoprecipitation (ChIP) assays were performed to assess the abundance of H3K9me2 in the KLF6 promoter and DNMT1 in the CSF1 promoter regulated by UHRF1. The functional roles of TAM-mediated oncogenic network in HCC progression were verified by in vitro colony formation assays, in vivo xenograft experiments and analysis of clinical samples.Results: Here, we find that TAMs induce and maintain high levels of HCC UHRF1, an oncogenic epigenetic regulator. Mechanistically, TAM-derived PGE2 stimulates UHRF1 expression by repressing miR-520d that targets the 3'-UTR of UHRF1 mRNA. In consequence, upregulated UHRF1 methylates H3K9 to diminish tumor KLF6 expression, a tumor inhibitory transcriptional factor that directly transcribes miR-520d. PGE2 reduces KLF6 occupancy in the promoter of miR-520d, dampens miR-520d expression, and sustains robust UHRF1 expression. Moreover, UHRF1 promotes CSF1 expression by inducing DNA hypomethylation of the CSF1 promoter and supports TAM accumulation.Conclusions: Capitalizing on studies on HCC cells and tissues, animal models, and clinical information, we reveal a previously unappreciated TAM-mediated oncogenic network via multiple reciprocal enforcing molecular nodes. Targeting this network may be an approach to treat HCC patients.

Angiotensin II induced CSF1 transcription is mediated by a crosstalk between different epigenetic factors in vascular endothelial cells

Endothelium-derived colony stimulating factor (CSF1) plays a key role in a range of human pathologies. Angiotensin II (Ang II) has been documented to stimulate CSF1 transcription although the underlying epigenetic mechanism remains unclear. Here we report that induction of CSF1 transcription by Ang II in vascular endothelial cells paralleled alterations of signature histone modifications surrounding the CSF1 promoter. Specifically, ChIP assays indicated that there was a simultaneous up-regulation of both acetylated H3 and trimethylated H3K4, indicative of transcriptional activation, and down-regulation of dimethyl H3K9, implicated in transcriptional repression, surrounding the proximal CSF1 promoter. Further analysis revealed that silencing of brahma related gene 1 (BRG1), a chromatin remodeling protein, abrogated CSF1 induction by Ang II. In the meantime, BRG1 silencing erased H3 acetylation and H3K4 trimethylation and restored H3K9 dimethylation. Mechanistically, BRG1 interacted with and recruited SET1A, a histone H3K4 methyltransferase, and JMJD1A, a histone H3K9 demethylase, to the CSF1 promoter to alter chromatin structure thereby promoting CSF1 trans-activation in response to Ang II stimulation. Knockdown of either SET1A or JMJD1A blocked CSF1 induction by Ang II. Finally, we demonstrate that the crosstalk between BRG1 and histone modifying enzymes was mediated by the transcription factor AP-1. In conclusion, our data unveil a novel epigenetic mechanism whereby a BRG1-centered complex mediates transcriptional activation of CSF1 by Ang II in vascular endothelial cells.

Identifications of Highly Aggressive Phenotype with SPI1 Overexpression in Pediatric T Cell Acute Lymphoblastic Leukemia/Lymphoma

T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) accounts for 10% to 15% of newly diagnosed cases of childhood acute lymphoblastic leukemia (ALL), arising from the malignant transformation of hematopoietic progenitors primed toward T cell development, as result of a multistep oncogenic process. However, since the prognostic significance of these genetic alterations in pediatric T-ALL is not clear, genetic basis which contributes aggressive phenotype or progression of pediatric T-ALL is still to be elucidated. Therefore, to discover driver genetic events, which involved in the aggressive phenotype of pediatric T-ALL and to identify it's novel prognostic markers, we performed integrated genetic analysis in a large cohort of T-ALL case.Our cohorts included samples from Tokyo Children's Cancer Study Group (TCCSG) and Japan Association of Childhood Leukemia Study (JACLS). Whole transcriptome sequencing (WTS) was performed in 123 cases. Representative recurrent fusion genes were as follows, SIL-TAL1 (n=25), MLL-ENL (n=5), PICALM-MLLT10 (n=5), and NUP214-ABL1 (n=2). Intriguingly, novel recurrent in-frame SPI1 fusions (STMN1-SPI1 n=2; TCF7-SPI1 n=5) were detected, and RT-PCR analysis in additional 60 cases revealed other 2 TCF7-SPI1 fusions. Thus, SPI1 fusions accounted for 4% of pediatric T-ALL/LBL. Expression data of WTS revealed cases with SPI1 fusion showed significantly higher expression of SPI1 compared to cases without SPI1 fusion, implicating that aberrant high expression of SPI1 involved in leukemogenesis.To address the functional activities of SPI1 fusions, we performed luciferase assay using the reporter vector contained the CSF1 promoter region with SPI1 binding site. Transient transfection of Hela cells with the SPI1 fusions expression vectors as well as the wild type SPI1 expression vector showed strikingly high levels of transcription of the reporter genes, as compared to transfection with the empty expression vector, indicating that both SPI1 fusions have transcriptional activities.Next, to analyze the leukemogenic potential of SPI1 fusions in vitro, we transduced fusions cDNA into mouse double negative T-cells. Since p16(CDKN2A) is frequently silenced in T-ALL, we also used p16 null double negative T-cells. Both wild-type and p16 null double negative T-cells expressing SPI1 fusions showed increased cell proliferation compared to the MOCK cells.We further evaluated the impact of SPI1 fusions on T cells differentiation. TCF7-SPI1 or MOCK vector was transduced mononuclear cells isolated from mouse bone marrow. These cells were cultured under stimulating factors, such as IL6 and TPO for 3 days, and then transplanted into the irradiated mouse. Subsequently, 6 week after transplantation, FACS analysis was performed. Of interest, significantly high population of cells expressing TCF7-SPI1 was observed in the immature single positive stage, implicating that their differentiation was impaired at the pre-T cell stage. These results indicate that novel SPI1 fusions have a potential leukemogenic effect in pediatric T-ALL. We defined SPI1 overexpression cases as outliers of SPI1 expression, resulting in extremely poor prognosis (log-rank p = 1.9 ×10-6). Of note, significant poor outcome was confirmed by univariate and multivariate analysis in cases with SPI1 overexpression cases (log-rank p = 9.3 ×10-6, and p = 2.0 ×10-6, respectively).In conclusion, SPI1 fusions expressing cells expanded and they remained at an immature stage, implicating a potential leukemogenic activity of these fusions. Not only the cases with SPI1 fusions, but also the cases with high SPI1 expression without fusions showed extremely poor prognosis, suggesting the prognostic value of aberrant SPI1 expression in pediatric T-ALL. Although it remains unclear, why cases with SPI1 fusions/high SPI1 expression have a poor prognosis, our results indicate that these cases are genetically distinct subgroup from other pediatric T-ALL. DisclosuresKataoka:Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria; Boehringer Ingelheim: Honoraria. Ogawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Nrf2 activation is associated with Z-DNA formation in the human HO-1 promoter

Using a luciferase reporter assay, we previously demonstrated that a Z-DNA-forming sequence of alternating thymine–guanine repeats in the human heme oxygenase-1 gene (HO-1) promoter is involved in nuclear factor erythroid-derived 2 (NF-E2)–related factor 2 (Nrf2)-mediated HO-1 promoter activation. However, the actual Z-DNA formation in this native genomic locus has not been experimentally demonstrated. To detect Z-DNA formation in vivo, we generated a construct containing the Z-DNA-binding domain of human adenosine deaminase acting on double-stranded RNA 1 fused with enhanced green fluorescence protein, designated as the Z-probe. A chromatin immunoprecipitation assay using an anti-GFP antibody showed that the Z-probe detects the well-characterized Z-DNA formation in the CSF1 promoter. Using this detection system, we demonstrated that the glutathione-depleting agent, diethyl maleate, induced Nrf2-dependent Z-DNA formation in the HO-1 promoter, but not in the thioredoxin reductase 1 gene promoter. Moreover, a time course analysis revealed that Z-DNA formation precedes HO-1 transcriptional activation. Concurrent with Z-DNA formation, nucleosome occupancy was reduced, and the recruitment of RNA polymerase II was enhanced in the HO-1 promoter region, suggesting that Z-DNA formation enhances HO-1 gene transcription. Furthermore, Nrf2-induced BRG1 recruitment to the HO-1 promoter temporarily occurred simultaneously with Z-DNA formation. Thus, these results implicate Nrf2-dependent Z-DNA formation in HO-1 transcriptional activation and suggest the involvement of BRG1 in Z-DNA formation.

Abstract 1399: SRC-1 up-regulates CSF-1 expression to potentiate breast cancer metastasis

Abstract Steroid receptor coactivator-1 (SRC-1/NCOA1) has been reported to be overexpressed in a subset of human breast cancer (BC) and its overexpression correlates with Her2 expression, disease recurrence and endocrine therapy resistance. To determine the role and underlying mechanisms of SRC-1 overexpression in BC progression and metastasis, a transgenic mouse model with overexpression of human SRC-1 in mouse mammary epithelial cells was created. In vivo data revealed that overxpression of SRC-1 potentiated BC metastasis to lung in both MMTV/Neu and MMTV/TVA/PyMT tumor models. While SRC-1 overexpression did not obviously accelerate tumor growth in these BC mouse models, the number of tumor cells invaded into blood vessels and metastasis index in lung tissue significantly increased in these mice. In addition to the elevated Twist expression and activation of Her2 and AKT driven by SRC-1 overexpression in the tumors, CSF-1 level was significantly increased in SRC-1 overexpressed tumors. We also found that CSF-1 expression was decreased in two PyMT/SRC-1 KO tumor cell lines compared with the two PyMT/WT control cell lines. Knockdown of SRC-1 in PyMT/WT cells or human BC cells reduced CSF-1 levels, while adenoviral-mediated expression of SRC-1 in PyMT/SRC-1 KO cells or human BC cells significantly promoted CSF-1 expression. Furthermore SRC-1 was found to interact with transcription factors, c-jun and c-fos, to activate the csf-1 promoter. Both SRC-1 and c-fos were found to associate with the csf-1 promoter at AP-1 binding sites in human BC cells. This study provided compelling evidence that overexpression of SRC-1 promotes BC metastasis. Targeting SRC-1 might be a potential therapeutic strategy for controlling BC metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1399. doi:1538-7445.AM2012-1399

L-selectin ligation-induced CSF-1 gene transcription is regulated by AP-1 in a c-Abl kinase-dependent manner

L-selectin is a cell adhesion molecule that plays an important role both in mediating the initial capture and subsequent rolling of leukocytes along the endothelial cells and in the signal transduction for leukocyte activation. In our previous studies, we reported that L-selectin ligation could increase macrophage colony-stimulating factor (CSF)-1 gene transcription, in which c-Abl acts as a crucial cytoplasmic kinase. Here we investigated the function of the nuclear c-Abl kinase in the CSF-1 gene transcriptional events triggered by L-selectin ligation. We determined that c-Abl kinase recruits to the nucleus following L-selectin ligation, and the nuclear c-Abl kinase can phosphorylate c-Jun and regulate activator protein (AP)-1 activity. Furthermore, the activated c-Abl kinase interacts with AP-1 and forms a complex in the CSF-1 promoter region to regulate CSF-1 gene transcription in the L-selectin ligation-activated cells. These results indicate that nuclear c-Abl kinase can activate CSF-1 gene transcription by regulating AP-1 activity in the signaling events induced by L-selectin ligation.

PDGF Up-regulates CSF-1 Gene Transcription in Ameloblast-like Cells

Macrophage colony-stimulating factor (CSF-1) is a key regulatory cytokine for amelogenesis, and ameloblasts synthesize CSF-1. We hypothesized that PDGF stimulates DNA synthesis and regulates CSF-1 in these cells. We determined the effect of PDGF on CSF-1 expression using MEOE-3M ameloblasts as a model. By RT-PCR, MEOE-3M expressed PDGFRs and PDGF A- and B-chain mRNAs. PDGF-BB increased DNA synthesis and up-regulated CSF-1 mRNA and protein in MEOE-3M. Cells transfected with CSF-1 promoter deletion constructs were analyzed. A PDGF-responsive region between -1.7 and -0.795 kb, containing a consensus Pea3 binding motif, was identified. Electrophoretic mobility shift assay (EMSA) showed that PDGF-BB stimulated protein binding to this motif that was inhibited in the presence of anti-Pea3 antibody. Analysis of these data provides the first evidence that PDGF-BB is a mitogen for MEOE-3M and increases CSF-1 protein levels, predominantly by transcription. Elucidation of the cellular pathways that control CSF-1 expression may provide novel strategies for the regulation of enamel matrix formation.

The Human Severe Congenital Neutropenia-Associated Gfi1N382S Mutant Critically Requires Deregulated Expression of CSF1 To Block Granulopoiesis.

Severe Congenital Neutropenia (SCN) is characterized as a deficiency of mature neutrophils, which leads to recurrent bacterial and fungal infections. Mutations in the Ela2 gene predominate in SCN, but Ela2 mutation in mice does not recapitulate SCN. The Growth factor independent-1 (Gfi1) transcriptional repressor regulates Ela2. The N382S mutation in Gfi1 is associated with human SCN. Unlike Ela2, genetic deletion of Gfi1 results in murine neutropenia. Here we show that the human-SCN-associated Gfi1N382S mutant blocks murine granulopoiesis in a Csf1 dependent manner. Gfi1 functions as a rate-limiting granulopoietic molecular switch, and the DNA-binding-defective Gfi1N382S mutant acts as a dominant negative to block granulopoiesis by selectively derepressing a subset of Gfi1 target genes. One gene repressed by Gfi1 and derepressed by Gfi1N382S is the macrophage colony stimulating factor (Csf1). Chromatin immunoprecipitation and electrophoretic mobility shift analyses demonstrate direct binding of Gfi1 to the Csf1 promoter. Importantly, both antibody absorption of Csf1 and genetic deletion of Csf1 prevent the Gfi1N382S-mediated block to granulopoiesis. These data represent the first identified Gfi1 target gene to be critically required for SCN-like phenotypes engendered by Gfi1N382S, and suggest new potential treatment avenues for Gfi1-mutant SCN patients.

Nuclear actin is involved in the regulation of CSF1 gene transcription in a chromatin required, BRG1 independent manner

Actin is an important protein in nucleus and has been implicated in transcription, however, the mechanism of its function in transcription is still not clear. In this article, we studied the role of actin in the regulation of human CSF1 gene transcription. Our results showed that nuclear actin stimulates the activity of CSF1 promoter, and the role in augmenting CSF1 gene transcription requires the formation of chromatin and Z-DNA structure. The ATP binding motifs of nuclear actin are essential for its function in regulating CSF1 gene transcription, and upon actin overexpression, there is an increase in the ATPase activity of nuclear proteins. Further investigation revealed that nuclear actin regulates CSF1 gene transcription in a BRG1 independent manner. Together, these original results have provided evidence for further understanding the mechanism of nuclear actin in regulating gene transcription.

Engagement of PSGL-1 upregulates CSF-1 transcription via a mechanism that may involve Syk

PSGL-1, the optimal selectin ligand demonstrated by in vivo studies to date, is an essential adhesive molecule mediating the rolling of leukocytes on the endothelial cells and the recruitment of leukocytes to the inflamed tissue. Recent studies demonstrated, in addition to its direct role in capture of leukocytes from the bloodstream, PSGL-1 also functions as a signal-transducing receptor and initiates a series of intracellular signal events during the activation of leukocytes. Our present work showed antibody engagement of PSGL-1 upregulated the transcriptional activity of CSF-1 promotor and increased the endogenous expression of CSF-1 mRNA in Jurkat cell. Overexpression of wild-typed non-receptor tyrosine kinase Syk, but not kinase dead mutant of Syk, promoted the upregulation of the transcriptional activity of CSF-1 promoter caused by antibody engagement of PSGL-1. Additionally, special inhibitor of Syk Piceatannol suppressed the increase of CSF-1 mRNA caused by the engagement of PSGL-1. The results suggest that signal transducted by PSGL-1 upregulate the transcriptional activity of CSF-1, and non-receptor tyrosine kinase Syk participates in this pathway.

Analysis of the mouse CSF-1 gene promoter in a transgenic mouse model.

CSF-1 stimulates monocyte and osteoclast populations. However, the molecular mechanisms involved in regulating CSF-1 gene expression are unclear. To identify regulatory regions that control normal CSF-1 gene expression, a -774/+183-bp fragment of the murine CSF-1 promoter was analyzed in vitro and in vivo. Transcriptional activity was high in cultured osteoblasts that express CSF-1 mRNA compared to ARH-77 B cells that lack CSF-1 gene expression. Transient transfection of osteoblasts with promoter deletion constructs showed that the -774-bp fragment conferred the highest transcriptional activity and contained activator and repressor sequences. To assess the ability of the CSF-1 promoter to confer normal tissue expression of CSF-1, transgenic mice containing the -774/+183-bp region driving the E. coli beta-galactosidase (lacZ) reporter gene were generated. beta-Gal analysis of whole tissue extracts showed transgene expression in all tissues tested except liver and kidney. At the cellular level, the pattern of beta-gal expression in the spleen, thymus, bone, lung, and testes of adult transgenic mice mimicked normal endogenous CSF-1 mRNA expression in non-transgenic littermates detected by in situ hybridization. This region also directed appropriate transgene expression to sites in other tissues known to synthesize CSF-1, with the exception of the liver and kidney. These findings indicate that the -774-bp fragment contains cis-acting elements sufficient to direct CSF-1 gene expression in many tissues. CSF-1 promoter/lacZ mice may be useful for studying the transcriptional mechanisms involved in regulating CSF-1 gene expression in tissues throughout development.

Nucleosome sliding: facts and fiction.

Nucleosome sliding is a frequent result of energy-dependent nucleosome remodelling in vitro. This review discusses the possible roles for nucleosome sliding in the assembly and maintenance of dynamic chromatin and for the regulation of diverse functions in eukaryotic nuclei.

Mutation of macrophage colony stimulating factor ( Csf1) causes osteopetrosis in the tl rat

Osteopetrosis results from a heterogeneous group of congenital bone diseases that display inadequate osteoclastic bone resorption. We recently mapped tl (toothless), a mutation that causes osteopetrosis in rats, to a genetic region predicted to include the rat Csf1 gene. In this study, we sequenced the coding sequence of the rat Csf1 gene to determine if a mutation in Csf1 could be responsible for the tl phenotype. Sequencing revealed a 10-base insertion in the coding sequence of mutant animals that produces a frameshift and generates a stop codon early in the mutant Csf1 coding sequence. The 41 amino acid polypeptide predicted to be produced from the Csf1 promoter would have only the first nine amino acids of the wild-type rat protein. These data suggest that osteopetrosis develops in tl/tl rats because they cannot produce functional mCsf, a growth factor required for osteoclast differentiation and activation.

Regulation of CSF1 Promoter by the SWI/SNF-like BAF Complex

The mammalian BAF complex regulates gene expression by modifying chromatin structure. In this report, we identify 80 genes activated and 2 genes repressed by the BAF complex in SW-13 cells. We find that prior binding of NFI/CTF to the NFI/CTF binding site in CSF1 promoter is required for the recruitment of the BAF complex and the BAF-dependent activation of the promoter. Furthermore, the activation of the CSF1 promoter requires Z-DNA-forming sequences that are converted to Z-DNA structure upon activation by the BAF complex. The BAF complex facilitates Z-DNA formation in a nucleosomal template in vitro. We propose a model in which the BAF complex promotes Z-DNA formation which, in turn, stabilizes the open chromatin structure at the CSF1 promoter.

Nuclear factor-kappaB p50 is required for tumor necrosis factor-alpha-induced colony-stimulating factor-1 gene expression in osteoblasts.

Colony-stimulating factor (CSF)-1 is a hematopoietic growth factor that is released by osteoblasts and is recognized to play a critical role in bone remodeling in vivo and in vitro. We have reported that osteoblasts express CSF-1 constitutively and that tumor necrosis factor (TNF)-alpha, a potent bone-resorbing agent, increases CSF-1 gene expression by a transcriptional mechanism. In the present study, we report that an NF-kappaB site in the CSF-1 promoter is required for TNF-alpha-induced CSF-1 expression in osteoblasts. As determined by electrophoretic mobility shift assays, antiserum against the NF-kappaB-binding protein, p50, retarded the mobility of the inducible complex, whereas antisera against p52, p65, c-Rel, Rel B, IkappaB alpha, IkappaB gamma, and Bcl-3 had no effect. To further confirm that p50 is necessary for TNF-alpha-induced CSF-1 expression in osteoblasts, CSF-1 messenger RNA expression from untreated and TNF-alpha-treated osteoblasts, prepared from wild-type and p50 knock-out mice, was examined by Northern analysis. CSF-1 messenger RNA was increased by TNF treatment in wild-type mice but not in NF-kappaB p50 knock-out mice. Our findings support the conclusion that the NF-kappaB subunit p50 is critical for TNF-induced CSF-1 expression in osteoblasts.

Nuclear Factor- B p50 Is Required for Tumor Necrosis Factor- -Induced Colony-Stimulating Factor-1 Gene Expression in Osteoblasts

Colony-stimulating factor (CSF)-1 is a hematopoietic growth factor that is released by osteoblasts and is recognized to play a critical role in bone remodeling in vivo and in vitro. We have reported that osteoblasts express CSF-1 constitutively and that tumor necrosis factor (TNF)-α, a potent bone-resorbing agent, increases CSF-1 gene expression by a transcriptional mechanism. In the present study, we report that an NF-κB site in the CSF-1 promoter is required for TNF-α-induced CSF-1 expression in osteoblasts. As determined by electrophoretic mobility shift assays, antiserum against the NF-κB-binding protein, p50, retarded the mobility of the inducible complex, whereas antisera against p52, p65, c-Rel, Rel B, IκB α, IκB γ, and Bcl-3 had no effect. To further confirm that p50 is necessary for TNF-α-induced CSF-1 expression in osteoblasts, CSF-1 messenger RNA expression from untreated and TNF-α-treated osteoblasts, prepared from wild-type and p50 knock-out mice, was examined by Northern analysis. CSF-1 messenger RNA was increased by TNF treatment in wild-type mice but not in NF-κB p50 knock-out mice. Our findings support the conclusion that the NF-κB subunit p50 is critical for TNF-induced CSF-1 expression in osteoblasts.

Inhibition of colony-stimulating factor-1 promoter activity by the product of the Wilms' tumor locus.

Colony-stimulating factor-1 (CSF-1) is a member of the immediate early gene family, which is expressed in mitogen-stimulated quiescent fibroblasts. The biological effects of CSF-1 are multifaceted and include stimulation of the proliferation and differentiation of myeloid progenitors and activity of circulating monocytes and tissue-specific macrophages. Ablation of circulating levels of biologically active CSF-1 in mice leads to osteopetrosis and sterility, thus implicating a role for CSF-1 in bone remodeling and implantation. Identification of regulatory elements and cognate transcription factors that bind the csf-1 promoter and mediate such diverse expression patterns is of great interest. We identified a sequence element at -273 to -265 (relative to the transcription initiation site) in the murine csf-1 promoter, which contains overlapping consensus sequences for the Wilms' tumor protein (WT1), EGR-1, SP1, and SP3 proteins. WT1 and EGR-1 proteins produced in vitro bound to this sequence, and co-transfection of wt1 with a csf-1-cat reporter plasmid resulted in repression of promoter activity. Interestingly, nuclear extracts prepared from serum-stimulated C3H10T1/2 cells contained predominantly SP1 and SP3 binding activities, which recognized the -273 to -265 site. Thus repression of the csf-1 promoter by WT1 at this site may involve competition between SP1 family transcriptional activators and the WT1 repressor. Colony-stimulating factor-1 may be a physiologically relevant target gene for regulation by the WT1 transcription factor.

Cloning and characterization of the murine promoter for the colony-stimulating factor-1-encoding gene

Colony-stimulating factor-1 (CSF-1 or M-CSF) is required for the growth and differentiation of macrophage progenitors, and for the survival of mature macrophages. Expression of the CSF-1 gene in monocytes and fibroblasts is controlled at both the transcriptional and post-transcriptional levels. To study the molecular mechanisms which mediate changes in CSF-1 expression, the 5′ promoter region of the mouse CSF-1 gene was cloned. A high degree of structural and sequence similarity between the mouse and human CSF-1 genes was observed. A transcription start point was located 182 bp upstream from the start codon. Several sequences homologous to known cis-acting elements were identified in the 5′-flanking region. The CSF-1 promoter region was able to direct expression of a linked reporter gene in C3H10T1/2 mouse embryo fibroblasts. Deletion in the CSF-1 promoter region between bp -774 and -629 resulted in a significant decrease in promoter activity. The identification of a functional promoter for CSF-1 will serve as a valuable tool for studying the regulation of CSF-1 expression.