ARTICLE |
Correspondence to: Sherry L. Abboud, Dept. of Pathology, U. of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284. E-mail: abbouds@uthscsa.edu
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Summary |
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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 ß-galactosidase (lacZ) reporter gene were generated. ß-Gal analysis of whole tissue extracts showed transgene expression in all tissues tested except liver and kidney. At the cellular level, the pattern of ß-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.
(J Histochem Cytochem 51:941949, 2003)
Key Words: macrophage colony- stimulating factor, gene expression, transgenic mice
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Introduction |
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COLONY-STIMULATING FACTOR-1 (CSF-1) is a multifunctional protein that stimulates the proliferation, differentiation, and survival of mononuclear phagocytes (
Studies in normal and CSF-1-deficient op/op mice have shown that CSF-1 regulates the development of tissue macrophages and osteoclasts that are involved in perinatal tissue remodeling and organogenesis (
In previous studies, -774 bp with respect to the transcription start site (+1) of the 5' flanking region of the murine CSF-1 gene was sequenced and putative binding sites for transcription factors were identified (
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Materials and Methods |
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Cell Culture and Northern Blot Hybridization
Primary cultures of fetal rat calvarial osteoblasts were prepared from parietal bones obtained from rat pups at 22 days' gestation as we have previously described (-MEM and RPMI 1640 medium, respectively. Total RNA was isolated from confluent cells using the RNAzol B method (Cinna Biotex; Houston, TX). Northern blots were prepared, hybridized with the full-length murine CSF-1 cDNA (Chiron; Emeryville, CA), and washed as we have previously described (
CSF-1 Constructs and Transient Transfection
Deletion constructs of the murine CSF-1 5' flanking region from -774 to -43 bp, with all containing the same 3' end point at +183 bp, were generated by removing promoter fragments from previously constructed promoters fused to the CAT gene (obtained from M. Harrington; Indiana University, Bloomington, IN) (
Generation and Analysis of Transgenic Mice
The -774 /+183-bp fragment of the CSF-1 promoter was subcloned into pBluescript at the XbaI/HindIII site. After restricting with XbaI, the fragment was blunt-ended by Klenow treatment, excised with KpnI, and then inserted into the SmaI/KpnI-digested pUC19/AUGß-gal plasmid (obtained from Dr. Stephen Harris; UTHSC, San Antonio, TX).Transgenic mice were generated by microinjection of fertilized mouse oocytes according to standard methods (
ß-Galactosidase Analysis
Tissues (100 mg each) from 7-week-old transgenic and non-transgenic mice were homogenized in 200 µl of lysis solution and ß-galactosidase (ß-gal) activity in 20 µl of extract was measured using the Galacto-Light Plus chemiluminescent reporter assay (Tropix; Bedford, MA). The ß-gal activity of each extract was corrected for variations in protein concentration using the BCA microassay (Pierce; Rockford, IL). Tissues from six transgenic and non-transgenic mice were analyzed and values represent the level of transgene expressed minus the endogenous background levels detected in non-transgenic controls. Cellular localization of transgene expression in whole tissues of 7-week-old mice was detected by standard ß-gal histochemical staining. After processing in a xylene substitute, Histo-Clear (National Diagnostics; Atlanta, GA), tissues were embedded in paraffin and 20-µm-thick sections were counterstained with 1% neutral fast red or left unstained. Non-transgenic tissues were examined in parallel as controls for endogenous ß-gal activity.
ISH of Non-transgenic Tissues
A 140-bp PstI-DraI fragment of the CSF-1 cDNA inserted into a pGEM3Zf vector was used to generate antisense and sense RNA probes (
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Results |
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Transcriptional Activity of the CSF-1 Promoter In Vitro
Northern blotting analysis in Fig 1A demonstrates that FRC but not ARH-77 cells constitutively express CSF-1 mRNA. FRCs express the expected 4.4- and, to a lesser extent, 2.2- and 1.4-kb transcripts for CSF-1. To examine the transcriptional activity of the -774-bp region and identify potential cis-acting elements involved in controlling CSF-1 gene expression, the cells were transiently transfected with a series of CSF-1 promoter luciferase constructs shown in Fig 1B. CSF-1 transcriptional activity paralleled CSF-1 mRNA expression, with high luciferase activity in FRCs compared to ARH-77 cells. In FRCs, deletion of DNA sequences between -774 and -627 decreased luciferase activity two- to three-fold, indicating the presence of transcriptional activators in this region. Deletion of sequences between -627 and -509 bp increased transcriptional activity, indicating the presence of negative regulatory elements. Fusion plasmids with 152 bp or less progressively diminished luciferase activity, and minimal basal promoter activity was observed with 43-bp CSF-1 promoter construct.
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To confirm potential activator and repressor regions of the promoter, FRCs were transfected with constructs containing distinct promoters: CSF-1 basal promoter (p-43luc), activator region cloned in either the forward [p(-774/-627)-43luc] or reverse [p(-627/-774)-43luc] orientation upstream of the basal promoter, or the activator and repressor regions cloned upstream of the basal promoter [p(-774/-509)-43luc]. Fig 1C shows that the activator in the forward but not the reverse orientation enhanced basal transcriptional activity approximately twofold. The effect of the activator was abrogated by the addition of the repressor region, with promoter activity of p(-774/-509)-43luc close to basal (p-43luc). This confirms the functional activity of the two regions and indicates that the repressor region has a comparatively stronger effect than the positive elements. The inability to detect the repressor in the context of the entire promoter is likely due to the interaction of proteins with DNA sequences between -509 and -43 bp that masked the repressor effect.
Transcriptional Activity of the -774-bp CSF-1 Promoter in Transgenic Mice
To determine if the -774/+183 bp region is also transcriptionally active in osteoblasts and other CSF-1 synthesizing cells within tissues in vivo, transgenic mice containing this same region linked to the lacZ reporter gene were generated. Two transgenic founders were identified, with both showing a similar pattern of transgene expression. These mice were phenotypically normal and one line was studied in detail. Whole tissue extracts were assayed for ß-gal activity. Fig 2 shows transgene expression in the tissues analyzed, with the exception of the liver and kidney. High ß-gal activity was detected in tissues that normally express abundant CSF-1 mRNA levels, such as the heart and brain.
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Transgene expression at the cellular level was analyzed in hematopoietic tissues, bone, and other organs and correlated with endogenous sites of CSF-1 synthesis. The marginal zone and red pulp of the spleen (Fig 3A) showed strong ß-gal staining of cells that were consistent with endothelial and T-cells. Little or no staining was present in lymphoid follicles or germinal centers of the white pulp where B-lymphocytes are concentrated. In the thymus shown in Fig 3D, scattered positive cells that likely represent epithelioreticular cells were identified in the cortical region and, to a lesser extent, in the medulla. Transgene expression in these tissues mimicked the pattern of endogenous CSF-1 mRNA expression in non-transgenic tissues analyzed by ISH (Fig 3B and Fig 3E). In the bone marrow (Fig 3F), ß-gal expression localized to cells along bone trabeculae that likely represent osteoblasts and correlated with previous ISH studies that have identified CSF-1 transcripts in osteoblasts. Fig 3G shows the lung with positive cells scattered in the peribronchial/perivascular region and along the alveolar septae. In the testes (Fig 3I), strong staining was confined to the interstitial compartment between seminiferous tubules that contains Leydig and, to a lesser extent, macrophage cells. By ISH, endogenous CSF-1 transcripts localized to similar sites in the lung and testes (Fig 3H and Fig 3J). In contrast to these tissues, transgene expression was not observed in the liver or kidney (Fig 3K and Fig 3M). The weak ß-gal staining observed in the proximal tubules of transgenic kidneys represents endogenous ß-gal activity because similar staining was also present in non-transgenic kidneys. ISH of the liver and kidney, however, identified CSF-1 transcripts in cells along the hepatic sinusoids and within renal glomeruli (Fig 3L and Fig 3N).
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The -774-bp fragment also directed appropriate transgene expression to cells in other tissues known to synthesize CSF-1. Sections of the brain (Fig 4A) showed strong staining of neuronal cells in the upper granular layer of the cortex. The endocardial surface of the heart (Fig 4B) showed strong staining of the endothelial cells lining the ventricles, atria, and valves. Endothelial cells lining vascular structures in the lung, brain, skin, uterus, and ovary were also positive (Fig 3G, Fig 4A, Fig 4C, Fig 4G, and Fig 4H). In the salivary gland, there was widespread staining in serous acinar cells (Fig 4D), while sections through the gut revealed staining in epithelial cells along the base of gastric crypts (Fig 4E) and in Paneth cells located in the small intestine (Fig 4F). Although staining was also detected in cells at the neck of sebaceous glands (Fig 4C), uterine epithelial cells, and ovarian granulosa cells (Fig 4H), this reflected endogenous ß-gal staining that was also observed in non-transgenic tissues.
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Discussion |
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The present study demonstrates that the -774/+183-bp fragment of the CSF-1 promoter contains cis-acting elements that, alone, are sufficient to direct promoter activity in multiple cell types that normally express CSF-1. In vitro and in vivo transcriptional activity of this fragment correlated in two cell types, with reporter gene expression observed in osteoblasts but not B-cells. Moreover, transgene expression in mice harboring the -774-bp promoter correlated with endogenous gene expression by ISH and with previously reported sites of local CSF-1 production within many tissues.
Analysis of promoter deletion constructs in cultured osteoblasts identified activator and repressor sequences within the -774-bp fragment that may be critical for regulating CSF-1 gene expression. Since the -774-bp fragment conferred the highest transcriptional activity among the deletion constructs tested, its ability to direct CSF-1 gene expression in osteoblasts and other cells types known to synthesize CSF-1 was tested in vivo using transgenic mice. ß-Gal assay showed transgene expression in many tissues that normally express CSF-1 mRNA, except for liver and spleen.
ß-Gal expression in transgenic mice localized to the same cell populations in the spleen, thymus, bone, lung, and testes that were shown to synthesize CSF-1 by ISH. These findings are novel and are the first to localize the sites of CSF-1 gene expression in normal murine adult tissues. In these and other tissues, CSF-1-producing cells appeared to lie close to target cells expressing CSF-1 receptors, including macrophages and/or osteoclasts. In the spleen, CSF-1 transcripts and ß-gal expression were present in cells in the marginal zone and red pulp, sites that are rich in macrophages. There was little or no staining of the white pulp of the spleen, which is composed primarily of B-cells and lacks macrophages. The thymus showed a few positive cells in the cortex and medulla, which normally contains scattered macrophages (
In several other sites, the -774-bp region also recapitulated the expected pattern of normal endogenous gene expression (
Recently, a larger 3.13-kb fragment of the mouse CSF-1 promoter and first intron (3.28 kb) linked to the lacZ gene was reported to drive normal tissue-specific CSF-1 gene expression in transgenic mice, although localization of endogenous CSF-1 transcripts by ISH was not performed (
Our data indicate that the -774-bp fragment contains cis-acting elements that confer CSF-1 gene expression in multiple adult murine tissues. Studies utilizing CSF-1 promoter/lacZ mice will increase our understanding of the local biological effects of CSF-1 on target cells and may provide a model system for elucidating the molecular mechanisms involved in regulating CSF-1 gene transcription during embryonic development.
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Footnotes |
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1 Portions of this work were published in abstract form at the annual meeting of the American Society of Bone and Mineral Research, St Louis, MO, September 30October 4, 1999.
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Acknowledgments |
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Supported in part by funding from the NIH (AR-42306, SLA), Veteran's Administration Merit Award (SLA), the Research Enhancement Award Program (REAP) from the Veterans Administration (S.L.A.), and Department of Defense (DAMD17-99-9400, NGC).
Received for publication April 23, 2002; accepted February 5, 2003.
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