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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1591-1598

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1591-1598.)
© 1995 American Heart Association, Inc.

Articles

Transcriptional Activation of the Macrophage-Colony Stimulating Factor Gene by Minimally Modified LDL

Involvement of Nuclear Factor-{kappa}B

Tripathi B. Rajavashisth; Hisashi Yamada; Nikhilesh K. Mishra

From the Divisions of Endocrinology and Medical Genetics, Department of Medicine (T.B.R., N.K.M.), Harbor-UCLA Medical Center, Torrance, Calif, and the Jikei University School of Medicine, Department of Medicine (H.Y.), Aoto Hospital, Katsushika-ku, Tokyo, Japan.

Correspondence to Dr Tripathi B. Rajavashisth, Division of Endocrinology, RB-1, Harbor-UCLA Medical Center, 1124 W Carson St, Torrance, CA 90502-2064.


*    Abstract
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Abstract Minimally modified LDL (MM-LDL), obtained by mild iron oxidation or prolonged storage at 4°C, has been shown to induce the expression of macrophage-colony stimulating factor (M-CSF) in cultured aortic endothelial cells. To examine whether other cell types also respond to MM-LDL, we investigated its effect on the expression of M-CSF mRNA in mouse L-cells and human aortic smooth muscle cells. Both L-cells and human aortic smooth muscle cells showed increased levels of M-CSF mRNA in response to 10 to 200 µg/mL MM-LDL in a dose-dependent manner. This allowed us to use mouse L-cells as a model to study the mechanism involved in MM-LDL–mediated increase in M-CSF mRNA. Nuclear run-on assays showed that M-CSF gene transcription was activated by MM-LDL. In the present study, we identified specific elements that conferred MM-LDL–mediated transcriptional activation of the human M-CSF gene. Chimeric constructs containing sequential deletions in the 5'-promoter region of the M-CSF gene linked to a reporter chloramphenicol acetyltransferase (CAT) gene were transfected into mouse L-cells. The human M-CSF promoter region extending upstream from the transcription start site to nucleotide -406 showed maximum induction of CAT activity by MM-LDL. Induction of CAT activity was drastically reduced, with a deletion plasmid lacking the promoter region -406 to -344. A functional nuclear factor (NF)–{kappa}B binding site present in this critical region was required for MM-LDL–mediated induction of CAT activity since an internal deletion construct lacking this element showed significant loss of transcriptional activation. Similar results also were obtained with the use of bovine aortic endothelial cells, suggesting that part of the mechanism is shared in different cell types. Gel shift assays with bovine aortic endothelial cell nuclear extracts revealed that this element binds to MM-LDL–inducible nuclear protein(s) that exhibited DNA binding specificity of NF-{kappa}B and cross-reacted to NF-{kappa}B–specific antibodies. Taken together, these results are consistent with the involvement of NF-{kappa}B in the transcriptional activation of the human M-CSF gene by MM-LDL.


Key Words: transcriptional activation • macrophage-colony stimulating factor • lipoproteins • nuclear factor-{kappa}B • atherogenesis


*    Introduction
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 *Introduction
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M-CSF (or CSF-1) plays an important role in the differentiation, proliferation, and survival of mononuclear phagocytes by binding to a specific cell surface receptor encoded by the proto-oncogene c-fms.1 2 3 4 Recent studies recognize that M-CSF also can function as a growth factor for cells such as placental trophoblasts and bone osteoclasts.2 5 Besides its role as a growth and survival factor, M-CSF functions as a chemotactic agent for monocytes and can regulate the effector functions of mature monocytes and macrophages.2 6 M-CSF modulates inflammatory response by stimulating the production of several cytokines and growth factors.7 A single M-CSF gene in haploid genome exhibits a complex expression of alternatively spliced transcripts in a highly tissue-specific manner.8 9 10 11 The multiple M-CSF transcripts encode at least two distinct proteins, secreted as glycoprotein and proteoglycan forms, and a third membrane-bound form with cell-surface biological activity.11 12 13

Atherosclerotic lesions derived from humans and rabbits contain increased levels of M-CSF mRNA and immunoreactive protein,14 15 16 suggesting that M-CSF contributes to the pathogenesis of atherosclerosis by affecting the growth, function, and survival of lesional monocytes and macrophages. Several experimental findings support this suggestion. Macrophages proliferate within atherosclerotic lesions.17 18 M-CSF stimulates proliferation of monocyte precursors and is necessary for the survival of macrophages in culture and in vivo.1 2 3 4 5 M-CSF may regulate systemic lipoprotein metabolism and local lipid processing by cells in the vessel wall. M-CSF lowers plasma cholesterol levels in humans, nonhuman primates, and hypercholesterolemic rabbits.19 20 21 M-CSF enhances the clearance of apolipoprotein B-100–containing lipoproteins through both LDL receptor–dependent and LDL receptor–independent pathways in rabbits,22 stimulates uptake and degradation of acetylated LDL and cholesterol esterification in human monocyte-derived macrophages,23 and modulates lipoprotein lipase secretion in macrophages.24 The ability of M-CSF to stimulate the uptake and degradation of modified lipoproteins by upregulating scavenger receptor may lead to the removal of oxidized lipoproteins from the extracellular space and the generation of foam cells.23 25 Production of M-CSF may be critical in promoting the survival of lipid-loaded foam cells observed in early and advanced stages of atherosclerosis.

There is accumulating evidence that oxidized lipoproteins, identified in atherosclerotic plaques and early lesions, play an important role in atherogenesis. We have previously shown that MM-LDL, prepared by either mild iron oxidation or prolonged cold storage, activates cultured endothelial cells, causing a rapid and large induction of M-CSF expression.14 Our studies suggest that lipoprotein metabolism can influence M-CSF expression in the artery wall.14 Supporting this observation were in vivo findings derived from the effects of modified LDL in the whole animal. Intravenous injection of MM-LDL caused increased levels of M-CSF activity in the serum of mice.26 Diet-induced hyperlipidemia in swine was associated with the enhanced monocyte progenitor cells in bone marrow, which was accompanied by increased levels of colony stimulating activity in the serum.27 Collectively, these results indicate that increased expression of M-CSF in response to oxidatively modified LDL may be a key step in the early stages of atherogenesis. Understanding of the mechanism through which oxidatively modified LDL induces the expression of M-CSF may provide insights into the early events leading to the generation of atheromatous lesions.

This report investigated the mechanism involved in MM-LDL–mediated induction of M-CSF gene expression. Our results indicate that MM-LDL activates the promoter of human M-CSF gene, resulting in an increased rate of transcription. Mouse L-cells derived from subcutaneous areolar and adipose tissues were used as a model because they produced increased M-CSF in response to MM-LDL, as observed in endothelial cells14 and smooth muscle cells,15 and could be easily transfected for studies of the M-CSF promoter. Major findings obtained with the use of L-cells were verified in BAECs. We present data demonstrating that an NF-{kappa}B–binding cis-DNA element present in the promoter of human M-CSF gene participates in the MM-LDL response. This element has been used to examine the nature of transcription factor, the understanding of which may prove useful in further analysis of the mechanism involved in MM-LDL–mediated M-CSF gene regulation in human vessel wall cells.


*    Methods
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 *Methods
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Cell Cultures
Mouse L-cells were purchased from American Type Culture Collection, Rockville, Md. L-cells were cultured in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum (FCS). HASMCs derived from aortic specimens of human donors were kindly provided by Dr Mahamad Navab, Division of Cardiology, UCLA. HASMCs were grown essentially as described.28 BAECs were derived from fresh aorta and maintained in DMEM in the presence of 10% FCS.29 Preparation and treatment of MM-LDL were performed as previously described.14 30

Preparation of RNA and Northern Blot Analyses
Total cellular RNA was isolated by lysis of cells in guanidinium isothiocyanate, phenol-chloroform extraction, and ethanol precipitation.31 Each RNA preparation (15 µg) was denatured and electrophoresed through a 1.2% formaldehyde agarose gel followed by blotting onto nylon filters and UV cross-linking. Filters were hybridized with isolated M-CSF cDNA probe.14 32 The blots were washed, autoradiographed, and rehybridized with an {alpha}-tubulin cDNA probe as an internal control. Quantitative results of the assays were obtained by laser densitometry of autoradiograms.

Nuclear Run-on Assays
The nuclear run-on transcription assays were performed according to a published procedure.33 Nuclei from untreated and MM-LDL–treated cells were incubated in a reaction mixture containing 10 mmol/L Tris, pH 8.0, 20% glycerol, 0.15 mol/L KCl, 1.5 mmol/L MgCl2, 5 mmol/L dithiothreitol, and 250 units RNAsin (Promega) supplemented with 0.5 mmol/L each of CTP, ATP, and GTP and 0.250 mCi {alpha}-[32P]UTP (New England Nuclear). Radiolabeled nuclear RNA was purified by DNAse I and proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. Relative amounts of incorporation of label into specific RNAs were estimated by DNA–excess filter hybridization. Linearized and denatured plasmids carrying mouse M-CSF and ß-actin DNAs were slot-blotted onto nylon filters. Filters were probed with an equal amount of radiolabeled RNA probes as described.14 34 The blots were washed and autoradiographed. Quantitative results of the assays were obtained both by counting of individual hybridized slots and densitometry of autoradiograms.

Plasmid Constructs, Transfection, and CAT Assays
We used previously published reporter CAT constructs containing sequential deletion of the 5'-region of human M-CSF gene promoter.35 All constructions were confirmed by DNA sequencing.34 Transfection of plasmid DNA into mouse L-cells and BAECs was performed by the calcium phosphate coprecipitation method36 with the use of 25 µg of appropriate plasmids purified by CsCl banding. In all transfections, 5 µg of a reference plasmid pCH110 (Pharmacia) that expresses ß-galactosidase was used to monitor the transfection efficiency. Forty-eight hours after transfection, cells were washed with phosphate-buffered saline (PBS) and were either untreated or treated with MM-LDL. Cells were harvested and lysed by three cycles of rapid freeze-thawing or by use of the reporter lysis buffer (Promega). The protein content of extracts was determined by the Bio-Rad protein assay kit. Analyses of CAT and ß-galactosidase activity in equal amounts of lysate were performed as described.37 38 39 CAT activity was quantified by scintillation counting and normalized to that of ß-galactosidase activity. Induction of CAT activity by MM-LDL is reported as the average from at least three separate experiments and represents the ratio of CAT activity from MM-LDL–treated cells versus untreated cells. In each transfection set, p0CAT, which contained no promoter, and pSV2CAT, which contained the CAT gene linked to SV40 early promoter, were used as negative and positive control plasmids, respectively.

Nuclear Extracts and Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from BAECs that were untreated or treated with 200 µg/mL MM-LDL for 6 hours.40 Aliquots of nuclear extracts were quick-frozen in liquid nitrogen and stored at -70°C. Double-stranded DNA probes were prepared by annealing synthetic oligonucleotides that span {kappa}B sites in the human M-CSF promoter. DNA probes were radiolabeled by using [{gamma}-32P]ATP and T4 polynucleotide kinase. Protein-DNA complexes were analyzed by following the standard gel shift protocol.41 Briefly, binding reactions were performed by incubating 10 µg of nuclear extract with 2 µg poly[dI.dC] and 10 µg BSA in binding buffer (10 mmol/L Tris, pH 7.5, 50 mmol/L NaCl, 1 mmol/L dithiothreitol, 1 mmol/L EDTA and 5% glycerol) in a volume of 20 µL for 20 minutes at room temperature. Approximately 20 000 cpm (0.4 ng) of probe then was added and allowed to bind at room temperature for 20 minutes. The reaction mixture was resolved by electrophoresis through 5% polyacrylamide gels in a low ionic strength buffer (25 mmol/L Tris, 25 mol/L boric acid, 1 mmol/L EDTA, pH 7.5). Gels were dried and visualized by autoradiography. For competition experiments, conditions were identical except that appropriate competitor oligonucleotides were added at 100-fold molar excess to the reaction mixture before addition of nuclear extracts. Supershift assay was performed by incubating binding the reaction mixed with 2 µL of (100 µg/mL) antibody raised against the p65 subunit of NF-{kappa}B (Santo Cruz Biotechnology) for 20 minutes at room temperature.


*    Results
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 up arrowMethods
 *Results
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MM-LDL Induces Expression of M-CSF mRNA in Nonendothelial Cells
To determine whether nonendothelial cell types share the characteristics of MM-LDL–mediated induction of M-CSF expression in endothelial cells, we treated mouse L-cells and HASMCs with varying concentrations of MM-LDL. Fig 1ADown presents quantitative data obtained from the Northern blot assays of the total RNA extracted from mouse L-cells exposed for 6 hours with 10 to 200 µg/mL MM-LDL. As previously observed in the aortic endothelial cells,14 mouse L-cells responded to MM-LDL in a dose-dependent manner. The level of M-CSF mRNA was enhanced more than 10-fold when the cells were stimulated by 100 or 200 µg/mL MM-LDL. A plateau in M-CSF mRNA expression was reached at a concentration of MM-LDL at 50 to 200 µg/mL. Similarly, HASMCs also responded to MM-LDL, although to a lesser extent than L-cells, and showed increased levels of M-CSF mRNA in response to a higher concentration of MM-LDL (Fig 1BDown). The level of M-CSF mRNA increased over 6-fold when HASMCs were treated with 200 µg/mL MM-LDL. These data indicate that in addition to endothelial cells, nonendothelial cells such as L-cells or HASMCs also can express increased levels of M-CSF mRNA in response to MM-LDL.



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  		Figure 1. Induction of M-CSF mRNA by MM-LDL in (A) mouse L-cells and (B) cultured HASMCs. Mouse L-cells were grown in DMEM supplemented with 10% FCS. Primary cultures of HASMCs (passages 3-7) were derived as previously described.28 Cells were treated with indicated amounts of MM-LDL. The preparation of MM-LDL and treatment of cells with MM-LDL were performed as described.14 Quantitative results of the Northern blots of M-CSF mRNA were obtained by laser densitometry of the autoradiograms. Densitometric units were normalized with the {alpha}-tubulin mRNA. Results represent mean±SD of three separate Northern blot analyses.

MM-LDL Activates the Rate of M-CSF mRNA Synthesis in Mouse L Cells
Increased levels of M-CSF mRNA in MM-LDL–stimulated L-cells and HASMCs could result from enhanced transcription and RNA processing or reduced degradation. To examine whether an increase in levels of M-CSF mRNA in response to MM-LDL was associated with the increased rate of M-CSF gene transcription, we performed transcription run-on assays on isolated nuclei. The results shown in Fig 2ADown indicate a basal level of M-CSF gene transcription in L-cells that was induced about 10-fold when cells were stimulated with MM-LDL. ß-Actin gene transcription remained unchanged in response to MM-LDL. To verify that RNA polymerase II is responsible for the M-CSF gene transcription and that our assay is specific for nascent transcription, run-on assays were performed in the presence of {alpha}-amanitin (2.5 µg/mL). Fig 2BDown shows that {alpha}-amanitin inhibited the incorporation of radionucleotide into M-CSF mRNA by more than 90%, suggesting that variation in the steady state levels of mRNA in response to MM-LDL was largely due to increased transcription of the M-CSF gene by RNA polymerase II.



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  		Figure 2. Induction of M-CSF gene transcription by MM-LDL. A, Nuclei from L-cells grown in the absence or presence of 200 µg/mL MM-LDL for 6 hours were isolated for preparation of radiolabeled nuclear RNA.33 Equal amounts of linearized plasmids carrying vector pSP65, mouse M-CSF, and ß-actin DNA were slot-blotted onto nylon filters. Filters then were probed with equal amounts of [32P]-labeled nuclear RNA. B, Nuclear run-on assay on the MM-LDL–stimulated L-cell nuclei was carried out in the absence and presence of {alpha}-amanitin.

MM-LDL Activates the Promoter of Human M-CSF Gene
To further understand the molecular mechanism involved in MM-LDL–induced M-CSF gene transcription, we analyzed the regulatory structure of the human M-CSF gene promoter. Inspection of the M-CSF promoter sequence with the use of the current list of transcription factor binding sites revealed the presence of several putative cis-regulatory elements that may participate in regulating M-CSF gene transcription. Besides the cis-DNA elements that were identified previously,8 35 42 we found motifs located between the transcription start site and nucleotide -565 that could recognize AP-1, NF-IL6, NF-1, and interferon-{alpha}–responsive and interferon-{gamma}–responsive transcription factors. Two sites resembling recently described shear stress–responsive elements (SSRE) also were identified upstream to the purine and pyrimidine stretch at positions -140 and -133. A schematic illustration of the M-CSF promoter and location of putative cis-DNA elements is shown in Fig 3Down.



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  		Figure 3. Schematic illustration of the M-CSF promoter and location of putative cis-acting DNA elements. nt refers to the nucleotide. Start of the transcription is indicated by +1; start of translation (ATG codon) by +178. TATA box sequence is shown at -26; positions of other putative cis-DNA elements are shown. DNA-binding proteins are activator protein-1 (AP-1), promoter specific transcription factor-1 (Sp-1), nuclear factor-{kappa}B (NF-{kappa}B), nuclear factor -1 (NF-1), interferon {alpha}- and {gamma}-responsive elements (IRE-{alpha}/{gamma}), nuclear factor -IL6 (NF-IL6), and shear stress–responsive elements (SSRE). (RY)48 indicates the stretch of repeating purine-pyrimidine nucleotides.

To determine the MM-LDL inducibility of M-CSF gene promoter, we examined its functional potential on a reporter gene in mouse L-cells. Plasmids containing the entire M-CSF gene promoter region fused directly upstream to the bacterial CAT gene were transfected into mouse L-cells by the calcium phosphate coprecipitation method.36 L-cells were similar to aortic endothelial cells in their response to MM-LDL and were easy to transfect. When inserted downstream to a reporter gene, the 5'-flanking sequence of the human M-CSF gene promotes expression of CAT activity in mouse L-cells. In addition, we observed a 10-fold increase in CAT activity after treatment with 50 to 200 µg/mL MM-LDL, indicating that this sequence also contains the region required for upregulation of M-CSF gene expression in response to MM-LDL (Fig 4ADown). A control reporter plasmid containing CAT gene linked to SV40 early promoter (pSV2CAT) did not respond to MM-LDL, suggesting that induction of CAT activity requires specific elements in the human M-CSF gene promoter.



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  		Figure 4. A, Analysis of MM-LDL–induced M-CSF promoter activity by MM-LDL in mouse L-cells. Human M-CSF promoter–CAT reporter plasmid p-565CAT and control plasmid pSV2CAT were used to transfect mouse L-cells by calcium phosphate coprecipitation method.36 Forty-eight hours after transfection, the cells were split into two groups; one was treated with indicated amounts of MM-LDL and the other was left untreated. Harvested cells then were used to prepare cellular extracts and were assayed for transient expression of CAT activity.37 38 B, Deletion analysis of the human M-CSF promoter in mouse L-cells. Eight M-CSF promoter–CAT constructs including the parental plasmid p-565 CAT are shown diagrammatically on the left. Relative CAT activity of M-CSF promoter constructs in the absence and presence of MM-LDL is indicated on the right. The fold induction was calculated as a ratio of the CAT activity from MM-LDL–induced cells to that from uninduced cells. Results given for each construct are the average from at least three separate transfections.

To map the region required for activation by MM-LDL, plasmids containing CAT reporter gene driven by sequentially deleted 5'-regions of the human M-CSF promoter were transiently transfected into L-cells for MM-LDL induction assays. The results of functional testings of the mutant plasmids in mouse L-cells are shown in Fig 4BUp. Deletion of 75 and 159 nucleotide from the 5'-end of the plasmid p-565CAT did not affect the extent of induction by MM-LDL. The increase of CAT activity from basal level with mutant plasmids p-490CAT and p-406CAT was comparable to the parent plasmid p-565CAT. However, activity was reduced from 10-fold to 3-fold with the deletion plasmid p-343CAT, suggesting that the critical element responsible for maximum induction must reside between -406 and -343. Deletion extending from nucleotide -343 to -248 in the plasmid p-248CAT showed similar loss of induction as in p-343CAT. Further deletions from -248 toward -9 in plasmid p-95CAT and p-9CAT showed a total loss of MM-LDL response, suggesting that the promoter region between -344 and -249 contains elements that mediate a minimal MM-LDL response. The functional specificity of the region -406 to -343 was further determined by expression analysis of plasmid that lacks this region internally. The internal deletion mutant p(-416/-344)CAT showed reduced inducible activity as in p-343CAT, providing additional evidence about the activator function of region -406 to -344 in MM-LDL–mediated upregulation of human M-CSF gene transcription.

To ascertain whether results obtained from the use of mouse L-cells also apply to cells of the vessel wall, we performed transient transfection studies in low-passage BAECs with selected human M-CSF promoter deletion constructs. Plasmids p-565CAT, p-248CAT, p-95CAT, p-9CAT, and p(-416/-344)CAT, along with pCH110, were introduced into BAECs by the calcium phosphate coprecipitation method (Fig 5Down). Although the degree of basal expression varied, the overall pattern of MM-LDL inducibility of the deletion mutants resembled that seen in mouse L-cells (Fig 4BUp), indicating that activator region -406 to -344 contains the element that recognizes the activator protein in both L-cells and BAECs. An examination of the nucleotide sequence corresponding to the human M-CSF promoter region (-406 to -344) revealed the presence of two elements that can be classified as {kappa}B elements (Fig 6ADown). One element, {kappa}B-1 (CCCTGAAAGG), in an inverted orientation, extends from -368 to -377 and is identical to the {kappa}B core sequence first identified in the immunoglobulin {kappa} light chain enhancer as the binding site for the NF-{kappa}B transcription factor.43 The other element, {kappa}B-2 (GGGATTTTCA), extends from -397 to -388 and differs at position 10 from the consensus {kappa}B core sequence.44 Functional analyses of these {kappa}B sequences have shown that {kappa}B-1 sequence CCCTGAAAGG is essential for TNF-{alpha}–mediated activation of M-CSF gene transcription in HL-60 cells.35 This suggests that MM-LDL–mediated induction of the M-CSF gene might also involve, in part, the M-CSF {kappa}B-1 sequence.



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  		Figure 5. MM-LDL induces expression of human M-CSF promoter activity in BAECs. Selected M-CSF promoter–CAT constructs were transfected into BAECs by calcium phosphate coprecipitation method.36 Cells were incubated 60 hours after transfection in the absence ({square}) and presence ({blacksquare}) of MM-LDL. Relative CAT activities are expressed as values compared with that obtained with unstimulated p-565CAT. Results represent mean±SD of three separate transfections.



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  		Figure 6. A, Nucleotide sequence of the critical activator region of human M-CSF promoter required for the induction by MM-LDL. Putative {kappa}B elements are underlined and highlighted. B, Electrophoretic mobility shift assays with DNA probes containing human M-CSF promoter {kappa}B-1 binding site. End-labeled 22-mer DNA probes containing native human M-CSF promoter (TGGAGGGA-AAGTCCCTTGGGAC) were incubated with nuclear extracts derived from the BAECs unstimulated (lane 1) or stimulated (lanes 2-6) with MM-LDL. The position of NF-{kappa}B nucleoprotein complex that specifically binds the probe is shown by an arrow. Competition assays were carried out with a 100-fold molar excess of unlabeled oligonucleotides containing immunoglobulin {kappa}B (AGTTGAGGGGA-CTTTCCCAGGC) sequence (lane 5), a mutated version of M-CSF {kappa}B-1 (TGGAGGGAAAagatCTTGGGAC) sequence (lane 6), or a same-size unrelated sequence (lane 4). Supershifted immunoreactive band using antibody raised against p65 subunit of NF-{kappa}B is indicated by an arrow.

MM-LDL Activates NF-{kappa}B Binding to the M-CSF {kappa}B Element
To establish that increased M-CSF gene transcription was due to activated NF-{kappa}B transcription factor, we performed electrophoretic mobility shift assays on human M-CSF {kappa}B sequences. Radiolabeled double-stranded DNA probes containing M-CSF {kappa}B-1 and {kappa}B-2 sequences were allowed to bind nuclear proteins obtained from BAECs grown in the absence or presence of MM-LDL. Specific DNA-protein complexes were identified with {kappa}B-1 sequence using nuclear extracts prepared from MM-LDL–treated BAECs (Fig 6BUp, lane 2). Under the assay conditions used, the {kappa}B-2 sequence failed to bind specific nuclear proteins (not shown). {kappa}B-1–shifted bands shown with an arrow indicate the position of NF-{kappa}B protein–DNA complex. While these bands were not visible in nuclear extract prepared from unstimulated BAECs (lane 1), their intensity increased when nuclear extract was prepared from MM-LDL–treated BAECs (lane 2). The signal of the shifted band was specifically inhibited in the presence of unlabeled {kappa}B oligonucleotide competitor derived from immunoglobulin {kappa} chain enhancer (lane 5) but not with unrelated DNA sequence (lane 4) or unlabeled mutant M-CSF {kappa}B-1mut (lane 6). In addition, this complex showed immunological reactivity with an antibody raised against the p65 subunit of NF-{kappa}B. When included in the binding reaction, the antibody interacted only with NF-{kappa}B complex, producing a supershift (lane 3). Together these results indicate that increased M-CSF gene transcription by MM-LDL occurs by activation of NF-{kappa}B transcription factor in BAECs.


*    Discussion
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 *Discussion
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This study shows that nonendothelial cell types such as mouse L-cells and HASMCs respond to MM-LDL in a similar manner as aortic endothelial cells. Exposure of mouse L-cells to increasing concentrations of MM-LDL increased levels of M-CSF mRNA, suggesting that these cells could be used as a model to investigate the mechanism involved in MM-LDL–induced expression of M-CSF mRNA. Nuclear run-on assays on isolated L-cell nuclei revealed that change in the steady state levels of M-CSF mRNA resulted from increased M-CSF gene transcription. To investigate the mechanism involved in this transcriptional activation, we performed a functional analysis of human M-CSF gene promoter by transient expression assays. When inserted downstream to a reporter CAT gene, the human M-CSF gene promoter contained elements that augmented expression of CAT activity in mouse L-cells. In addition, there was more than a 10-fold increase in CAT activity when cells were stimulated with MM-LDL. Plasmids containing sequential deletion of 5'-flanking sequence of human M-CSF gene showed that promoter region -406 to -344 was required for the maximum activation of M-CSF gene transcription. We verified our major findings derived from the use of L-cells in BAECs by transient expression assays of selected M-CSF promoter–CAT constructs. Although there was variation in the basal level expression, a very similar pattern of inducibility by MM-LDL was apparent, suggesting that the same promoter region is commonly used in both L-cells and BAECs. Curiously, in both these cell types, we consistently observed a minimal induction by MM-LDL with plasmids containing M-CSF promoter region -343 to -95, indicating the presence of additional cis-DNA element(s) that might associate with NF-{kappa}B and modulate the MM-LDL response.

Yamada et al35 have reported that promoter region -406 to -344 confers TNF-{alpha} inducibility to the M-CSF gene transcription in HL-60 cells. These investigators further established that a functional {kappa}B element present in this region was essential for TNF-{alpha} stimulation. Deletion of this element substantially reduced MM-LDL response indicating involvement of this site in the transcriptional activation of the human M-CSF gene. Supporting this conclusion were the previous studies on the time course of MM-LDL suggesting that MM-LDL and TNF-{alpha} share the signaling pathways to induce endothelial cell expression of M-CSF.14 In addition, studies designed to understand the signaling pathway of MM-LDL indicated that MM-LDL–induced inflammatory response was mediated by cAMP, which activates NF-{kappa}B.45 More direct support for the involvement of NF-{kappa}B transcription factor in the regulation of M-CSF came from the recent findings obtained by transient cotransfection of expression plasmids encoding p65 and/or p50 subunits of NF-{kappa}B with the p-565CAT and p-248CAT in BAECs.46 Cotransfection of the p-565CAT–containing functional {kappa}B site, with p65 and p50, resulted in a 12-fold increase in promoter activity compared with only a 3-fold increase for the non–{kappa}B-containing p-248CAT, implying that the increase in the activity of NF-{kappa}B factor can directly induce the transcription of M-CSF gene.

Evidence for the involvement of NF-{kappa}B in MM-LDL–mediated induction of M-CSF gene transcription also was supported by mobility shift and supershift assays. With the DNA–probe containing {kappa}B site, we observed NF-{kappa}B–specific shifted bands. The treatment of BAECs with MM-LDL was associated with the increased levels of DNA binding as measured by gel shift assay. The band was specific to the NF-{kappa}B, since the presence of unlabeled synthetic oligonucleotides containing the native immunoglobulin {kappa}B sequences effectively inhibited the binding of M-CSF {kappa}B-1 sequences in the nuclear extract prepared from MM-LDL–stimulated BAECs. Unrelated DNA and mutated M-CSF {kappa}B-1 ({kappa}B-1mut) sequences in which nucleotides within the core {kappa}B sites were changed failed to compete. The identity of this complex caused by binding of NF-{kappa}B was further established by performing supershift assays with antibodies specific to p65 subunits of NF-{kappa}B. In summary, these results indicate that NF-{kappa}B is involved in specific and potent induction of human M-CSF gene transcription by MM-LDL.

The NF-{kappa}B family of proteins are ubiquitously expressed pleiotropic transcription factors that have been shown to activate expression of a number of genes involved in inflammation, cellular growth control, and immune function.43 44 47 48 Numerous studies have demonstrated that activation of NF-{kappa}B is necessary for the control of a variety of genes that are rapidly induced by stimuli such as cytokines (TNF-{alpha} and IL-1), mitogens, phorbol esters, lipopolysaccharide, double-stranded RNA, and viral agents.43 44 The rapid activation of NF-{kappa}B in response to these stimuli occurs through its dissociation from a cytoplasmic inhibitory protein designated as I{kappa}B and its subsequent translocation to the nucleus, where it binds specific cis-DNA sequences and in cooperation with other regulatory factor(s) activates gene transcription. Activation of NF-{kappa}B may be mediated by multiple signal transduction pathways; for example, cytokines and mitogens can activate NF-{kappa}B by binding to their specific receptor and activating secondary messenger pathways involving protein kinase A or C activity. A number of studies have indicated that diverse stimuli that activate NF-{kappa}B may act through a common intracellular pathway involving oxidative stress caused by increased synthesis of reactive oxygen intermediates.47 48 It was proposed recently that activation of NF-{kappa}B as a result of increased synthesis of reactive oxygen intermediates in response to a diverse group of atherogenic agents including oxidized lipids may play an important role in the initiation of atherosclerotic lesions.48 Whether MM-LDL–mediated activation of NF-{kappa}B also may involve synthesis of reactive oxygen intermediates is not known. It is clear, however, from studies by Parhami et al45 that MM-LDL–induced activation of NF-{kappa}B involves elevated levels of cAMP in cultured endothelial cells, suggesting the presence of a distinct signaling pathway. This observation may be of importance in terms of the pathological relevance of MM-LDL to atherosclerosis. It led to the conclusion that elevated levels of cAMP in response to MM-LDL may selectively induce or inhibit specific genes whose protein products favor monocytic but not neutrophilic or lymphocytic infiltration in the vessel wall. The results revealed that high levels of cAMP in response to MM-LDL activate NF-{kappa}B and induce the expression of genes for monocyte chemoattractant protein-1 and M-CSF that may be required in the recruitment and growth of monocytes.45 Our current observation that MM-LDL by activating NF-{kappa}B upregulates M-CSF gene expression supports this conclusion. A recent finding that oxidized phospholipid fraction of MM-LDL induces selective monocyte binding to endothelial cells provides further support to the inflammatory and atherogenic potential of MM-LDL.49 Preliminary studies have revealed that oxidized phospholipids derived from MM-LDL may be the specific components that activate the DNA binding activity of endothelial NF-{kappa}B (Judith Berliner, personal communication). Considering that the activation of the NF-{kappa}B family of proteins also occurs in response to other atherogenic agents, this family of transcription factors may represent a common link among diverse groups of atherogenic stimuli. It may explain in part the previous observations that a number of genes that might play important roles in atherogenesis contain in their promoters elements identical to functional NF-{kappa}B binding sites.43 44 47 48

Conclusions
The results of the present study indicate that NF-{kappa}B performs a crucial role in the transcriptional regulation of the M-CSF gene by MM-LDL. These results agree with the concept that activation of NF-{kappa}B represents a common mechanism underlying the induction of a number of genes that show increased expression in response to MM-LDL. Results from our studies also suggest that there may be individual variations from cell to cell. Regulatory elements that confer MM-LDL response at a minimal level indicate that transcriptional activation of the M-CSF gene by MM-LDL may involve a complex interaction of additional element(s) acting in concert with transcription factor NF-{kappa}B. In light of the variations from cell to cell, results obtained using mouse L-cells and BAECs need further verification in human artery wall cells.


*    Selected Abbreviations and Acronyms
 
BAECs = bovine aortic endothelial cells
CAT = chloramphenicol acetyltransferase
HASMCs = human aortic smooth muscle cells
M-CSF = macrophage-colony stimulating factor
MM-LDL = minimally modified low-density lipoprotein
NF = nuclear factor


*    Acknowledgments
 
This work was supported in part by a Grant-in-Aid Award to T.B.R. from the American Heart Association, Greater Los Angeles Affiliate, and by National Institutes of Health grant HL-51980. We wish to thank Dr Judith Berliner for providing MM-LDL preparations, Dr W.-N. Paul Lee for helpful advice, and Jaishree Mehta and Khiem Doan for expert technical assistance. We are grateful to Drs Peter Libby, James Liao, and Steve Clinton for critical readings of the manuscript. The authors thank Dr Maria Muszynski, DVM, for help with bovine endothelial cell culture experiments.

Received May 19, 1995; accepted August 2, 1995.


*    References
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  1. Stanley ER, Guilbert LJ, Tushinski RJ, Bartelmez SH. CSF-1: a mononuclear phagocyte lineage specific hemopoetic growth factor. J Cell Biochem. 1983;21:151-159. [Medline] [Order article via Infotrieve]
  2. Sherr CJ, Rettenmier CW, Roussel MF. Macrophage colony-stimulating factor, CSF-1, and its proto-oncogene-encoded receptor. Cold Spring Harb Symp Quant Biol. 1988;53:521-530.
  3. Tushinski RJ, Olive IT, Guilbert LJ, Tynan PW, Warner JR, Stanley ER. Survival of mononuclear phagocytes depends on a lineage-specific growth factor that the differentiated cell selectively destroys. Cell. 1982;28:71-77. [Medline] [Order article via Infotrieve]
  4. Sherr CJ, Rettenmier CW, Sacca R, Roussel MF, Look AT, Stanley ER. The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor CSF-1. Cell. 1985;41:665-676. [Medline] [Order article via Infotrieve]
  5. Kodama H, Yamasaki A, Nose M, Ohgame Y, Abe M, Kumegawa M, Suda T. Congenital osteoclast deficiency in osteopetrotic (op/op) mice is cured by injections of macrophage colony-stimulating factor. J Exp Med. 1991;173:269-272. [Abstract/Free Full Text]
  6. Wang JM, Griffin JD, Rambaldi A, Chen ZG, Mantovani AI. Induction of monocyte migration by recombinant macrophage-colony stimulating factor. J Immunol. 1988;141:575-579. [Abstract]
  7. Warren MK, Ralph P. Macrophage growth factor CSF-1 stimulates human monocyte production of interferon, tumor necrosis factor, and colony stimulating activity. J Immunol. 1986;137:2281-2285. [Abstract]
  8. Ladner MB, Martin GA, Noble JA, Nikoloff DM, Tal R, Kawasaki ES, White TJ. Human CSF-1: gene structure and alternative splicing of mRNA precursors. EMBO J. 1987;6:2693-2698. [Medline] [Order article via Infotrieve]
  9. Rajavashisth TB, Eng R, Shadduck RK, Waheed A, Ben-Avram CM, Shively JE, Lusis AJ. Cloning and tissue-specific expression of mouse macrophage-colony stimulating factor mRNA. Proc Natl Acad Sci U S A. 1987;84:1157-1161. [Abstract/Free Full Text]
  10. Cerretti DP, Wignall J, Anderson D, Tushinski RJ, Gallis BM, Stya M, Gillis S, Urdal DL, Cosman D. Human macrophage-colony stimulating factor: alternative RNA and protein processing from a single gene. Mol Immunol. 1988;25:761-770. [Medline] [Order article via Infotrieve]
  11. Kawasaki ES, Ladner MB, Wang AM, Van Arsdell J, Warren MK, Coyne MY, Schweickart VL, Lee MT, Wilson KJ, Boosman A, Stanley ER, Ralph P, Mark DF. Molecular cloning of a complementary DNA encoding human macrophage-specific colony stimulating factor (CSF-1). Science. 1985;230:291-296. [Abstract/Free Full Text]
  12. Suzu S, Ohtsuki T, Yanai N, Takatsu Z, Kawashima T, Takaku F, Nagata N, Motoyashi K. Identification of a high molecular weight macrophage colony-stimulating factor as a glycosaminoglycan-containing species. J Biol Chem. 1992;267:4345-4348. [Abstract/Free Full Text]
  13. Rettenmier CW, Roussel, MF, Ashmun AR, Ralph P, Price K, Sherr CJ. Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and down modulation of CSF-1 receptors in NIH3T3 cells transformed by cotransfection of the human CSF-1 and c-fms. Mol Cell Biol. 1987;8:2378-2387.
  14. Rajavashisth TB, Andalibi A, Territo MC, Berliner JA, Navab M, Fogelman AM, Lusis AJ. Induction of endothelial cell expression of granulocyte and macrophage colony-stimulating factors by modified low-density lipoproteins. Nature. 1990;344:254-257. [Medline] [Order article via Infotrieve]
  15. Clinton SK, Underwood R, Hayes L, Sherman ML, Kufe DW, Libby P. Macrophage colony-stimulating factor gene expression in vascular cells and in experimental and human atherosclerosis. Am J Pathol. 1990;140:301-316. [Abstract]
  16. Rosenfeld ME, Yla-Herttuala S, Lipton BA, Ord VA, Witztum JL, Steinberg D. Macrophage colony-stimulating factor mRNA and protein in atherosclerotic lesions of rabbits and humans. Am J Pathol. 1992;140:291-300. [Abstract]
  17. Gordon D, Reidy MA, Benditt EP, Schwartz SM. Cell proliferation in human coronary arteries. Proc Natl Acad Sci U S A. 1990;87:4600-4604. [Abstract/Free Full Text]
  18. Rosenfeld ME, Ross R. Macrophage and smooth muscle cell proliferation in atherosclerotic lesion of WHHL and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis. 1990;10:680-687. [Abstract/Free Full Text]
  19. Motoyoshi K, Takaku F. Serum cholesterol-lowering activity of human monocyte colony stimulating factor. Lancet. 1989;2:326-327. [Medline] [Order article via Infotrieve]
  20. Stoudemire JB, Garnick MB. Effects of recombinant human macrophage colony-stimulating factor on plasma cholesterol levels. Blood. 1991;77:750-755. [Abstract/Free Full Text]
  21. Schaub RG, Bree MP, Hayes LL, Rudd MA, Rabbani L, Loscalzo J, Clinton SK. Recombinant human macrophage colony-stimulating factor reduces plasma cholesterol and carrageenan granuloma foam cell formation in Watanabe heritable hyperlipidemic rabbits. Arterioscler Thromb. 1994;14:70-76. [Abstract/Free Full Text]
  22. Shimano H, Yamada N, Ishibashi S, Harada K, Matsumoto A, Mori N, Inaba T, Motoyoshi K, Itakura H, Takaku F. Human monocyte colony-stimulating factor enhances the clearance of lipoproteins containing apolipoprotein B-100 via both low-density lipoprotein receptor-dependent and -independent pathways in rabbits. J Biol Chem. 1990;265:12869-12875. [Abstract/Free Full Text]
  23. Ishibashi S, Inaba T, Shimano H, Harada K, Inoue I, Mokuno H, Mori N, Gotoda T, Takaku F, Yamada N. Monocyte colony-stimulating factor enhances uptake and degradation of acetylated low-density lipoproteins and cholesterol esterification in human monocyte-derived macrophages. J Biol Chem. 1990;265:14109-14117. [Abstract/Free Full Text]
  24. Mori N, Gotoda T, Ishibashi S, Shimano H, Harada K, Inaba T, Takaku F, Yazaki Y, Yamada N. Effects of human recombinant macrophage-colony-stimulating factor on the secretion of lipoprotein lipase from macrophages. J Biol Chem. 1991;11:1315-1321.
  25. Kodama T, Freeman M, Rohrer L, Zabrecky J, Matsudaira P, Kreiger M. Type I macrophage scavenger receptor contains {alpha}-helical and collagen-like coiled coils. Nature. 1990;343:531-535. [Medline] [Order article via Infotrieve]
  26. Liao F, Berliner JA, Mehrabian M, Navab M, Demer LL, Lusis AJ, Fogelman AM. Minimally modified low density lipoprotein is biologically active in vivo in mice. J Clin Invest. 1991;87:2253-2257.
  27. Averill LE, Meagher RC, Gerrity R. Enhanced monocyte progenitor cell proliferation in bone marrow of hyperlipidemic swine. Am J Pathol. 1989;135:369-377. [Abstract]
  28. Navab M, Hough GP, Stevenson LW, Drinkwater DC, Laks H, Fogelman AM. Monocyte migration into the subendothelial space of a coculture of adult human aortic endothelial and smooth muscle cells. J Clin Invest. 1988;82:1853-1863.
  29. Gimbrone MA Jr. In: Spaet TH, ed. Progress in Hemostasis and Thrombosis. Vol 3. New York, NY: Grune and Stratton Inc; 1990:1-28.
  30. Berliner JA, Territo MC, Sevanian A, Ramin J, Kim JA, Bombshad B, Esterson M, Fogelman AM. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions. J Clin Invest. 1990;85:1260-1266.
  31. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]
  32. Feinberg AP, Vogelstein B. A method for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983;132:6-13. [Medline] [Order article via Infotrieve]
  33. Marzluff WF, Huang RCC. In: Hames BD, Higgins SJ, eds. Transcription and Translation: A Practical Approach. Oxford, England: RL Press; 1982:89-129.
  34. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1982.
  35. Yamada H, Iwase S, Mohri M, Kufe D. Involvement of a nuclear factor-{kappa}B like protein in induction of the macrophage colony-stimulating factor gene by tumor necrosis factor. Blood. 1991;78:1988-1995. [Abstract/Free Full Text]
  36. Graham FL, van der Eb AB. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973;52:456-467. [Medline] [Order article via Infotrieve]
  37. Gorman CM, Moffat LM, Howard BH. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982;2:1044-1051. [Abstract/Free Full Text]
  38. Seed B, Sheens JY. A simple phase-extraction assay for chloramphenicol acyltransferase activity. Gene. 1988;67:271-275. [Medline] [Order article via Infotrieve]
  39. Herbomel P, Bourachot B, Yaniv M. Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell. 1984;39:653-662. [Medline] [Order article via Infotrieve]
  40. Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983;11:1475-1489. [Abstract/Free Full Text]
  41. Fried M, Crothers DM. Equilibration and kinetics of lac repressor-operator interaction by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981;9:6505-6525. [Abstract/Free Full Text]
  42. Harrington MA, Edenburg HJ, Saxman S, Pedigo LM, Daub R, Broxmeyer HE. Cloning and characterization of the murine promoter for the colony-stimulating factor-1-encoding gene. Gene. 1992;102:165-170.
  43. Sen R, Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell. 1986;46:705-716. [Medline] [Order article via Infotrieve]
  44. Lenardo MJ, Baltimore D. NF-{kappa}B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell. 1989;58:227-229. [Medline] [Order article via Infotrieve]
  45. Parhami F, Fang ZT, Fogelman AM, Andalibi A, Territo MC, Berliner JA. Minimally modified low density lipoprotein-induced inflammatory responses in endothelial cells are mediated by cyclic adenosine monophosphate. J Clin Invest. 1993;92:471-478.
  46. Peng HB, Rajavashisth TB, Libby P, Liao JK. Nitric oxide inhibits macrophage-colony stimulating factor gene transcription in vascular endothelial cells. J Biol Chem. 1995;270:17050-17055. [Abstract/Free Full Text]
  47. Schreck R, Alberman K, Baeurele PA. Nuclear factor {kappa}B: an oxidative stress-responsive transcription factor of eukaryotic cells. Free Radic Res Comm. 1992;17:221-237. [Medline] [Order article via Infotrieve]
  48. Collins T. Endothelial nuclear factor-{kappa}B and the initiation of the atherosclerotic lesion. Lab Invest. 1993;68:499-508. [Medline] [Order article via Infotrieve]
  49. Watson AD, Navab M, Hama SY, Sevanian A, Prescott SM, Stafforini DM, McIntyre TM, La Du BN, Fogelman, AM, Berliner JA. Effect of platelet activating factor-acetylhydrolase on the formation and action of minimally oxidized low density lipoprotein. J Clin Invest. 1995;95:774-782.




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