Transcription of macrophage IGF-I exon 1 is positively regulated by the 5'-untranslated region and negatively regulated by the 5'-flanking region

Murry W. Wynes1,2 and David W. H. Riches1,2,3

1Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center; and 2Department of Immunology and 3Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado

Submitted 16 September 2004 ; accepted in final form 26 January 2005


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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Idiopathic pulmonary fibrosis (IPF) is an insidious lung disease with no known cure or effective therapy. Macrophage-derived insulin-like growth factor-I (IGF-I) is thought to play a role in the pathogenesis of IPF; however, little is known about the control of IGF-I expression in macrophages. In this report we investigated the cis-regulatory elements that control basal expression using luciferase reporter constructs in RAW 264.7 macrophages. We show that the +95 to +329 region contains elements necessary to direct maximal promoter activity, whereas the +251 to +329 region contains the minimal promoter. Mapping transcriptional start sites for endogenous IGF-I in primary macrophages revealed that the major transcriptional start site is centered at +150, whereas the most 3'-transcriptional start site is centered at +255. Nuclear proteins from primary and RAW 264.7 macrophages bind specifically to the region required for maximal promoter activity (+134 to +173) and to the region required for minimal promoter activity (+267 to +299). Antibody supershift assays indicate that Sp3 bound to the +267 to +299 region. Moreover, mutation of the putative binding site reduced Sp3 binding in EMSAs and increased promoter activity in luciferase reporter gene assays. We also found that the regions from –1711 to –855 and –855 to –337 contain putative macrophage-specific suppressor elements that do not function in HeLa or COS-7 epithelial cell lines. These data support the view that macrophage IGF-I expression is positively regulated by elements located in the 5'-untranslated region and negatively regulated by elements in the 5'-flanking region of the IGF-I gene.

gene regulation; lung; fibrosis; growth factor


IDIOPATHIC PULMONARY FIBROSIS (IPF), an interstitial lung disease of unknown origin, is associated with high morbidity and mortality, and currently there is no effective therapy. Pathologically, IPF is characterized by usual interstitial pneumonia (UIP) with extensive alveolar epithelial cell injury, hyperplasia and hypertrophy of type II pneumocytes, and an intense fibroproliferative response. This leads to excessive deposition of collagen within the lung parenchyma, resulting in pulmonary impairment and death (20, 25). Collagen deposition is linked to the differentiation and accumulation of {alpha}-smooth muscle actin-expressing myofibroblasts organized into so-called "fibroblastic foci," which are thought to represent the leading edge of the fibrotic response (12, 13).

Insulin-like growth factor I (IGF-I), especially macrophage-derived IGF-I, has long been implicated in the pathogenesis of IPF. Originally described as alveolar macrophage-derived growth factor in the lung (4), IGF-I stimulates many responses that are consistent with its proposed role in the pathogenesis and progression of IPF, including acting as a progression factor for fibroblast proliferation (4, 21), stimulating collagen matrix synthesis (8, 9), and promoting the survival of cells of both epithelial and mesenchymal origin (5, 14, 15, 29). Additionally, IGF-I levels in bronchoalveolar lavage fluid have been shown to be significantly increased in IPF patients and patients with other fibrotic lung diseases compared with healthy control subjects (4, 11, 21). Postmortem and open lung biopsy specimens from patients with IPF exhibit elevated levels of IGF-I mRNA, and alveolar macrophages from IPF patients secrete elevated amounts of IGF-I compared with normal subjects (4, 11, 21). Previous work from our laboratory has revealed that IGF-I is also expressed by alveolar epithelial cells and interstitial macrophages in patients with IPF (27). Importantly, the degree of expression of IGF-I by interstitial macrophages was found to be closely associated with disease severity, emphasizing both the significance of interstitial macrophages and the expression of IGF-I by this cell in the progression of IPF. However, little is known about the molecular mechanisms controlling the basal expression of IGF-I in macrophages.

The rodent IGF-I gene is a single copy gene spread out over >80 kb and contains six exons (6, 23, 26). Its structure is highly conserved with the human IGF-I gene (22). Transcription can initiate from either exon 1 or 2, each being regulated by a structurally and functionally distinct promoter (1, 10, 16). Promoter 1 regulates exon 1 expression and promoter 2 regulates exon 2. Exons 1 and 2 encode alternative leader signal peptides and 5'-untranslated regions. Arkins et al. (2) have shown that macrophages exclusively use exon 1 and not exon 2 for transcription initiation, typical of extrahepatic IGF-I mRNA. The 5'-untranslated and -flanking regions of IGF-I exon 1 are highly conserved between mouse, rat, and human, with nearly 95% conservation of nucleotide sequence over this region. This high degree of homology within the 5'-untranslated region suggests similar regulatory mechanisms of IGF-I exon 1 expression between species. Promoter 1 lacks any recognizable core promoter transcriptional control elements such as TATA and CCAAT boxes (1, 10, 16). Neither does it resemble the GC-rich promoters found in housekeeping genes. A sequence resembling the "initiator" sequence in the TATA-less mouse terminal deoxynucleotidyl transferase gene (24), which directs transcription to a single site, is present. However, this does not appear to be functional in IGF-I exon 1, as evidenced by the multiple transcriptional start sites dispersed over ~350 bp (1, 10, 16).

Although previous studies have shown that macrophages are an important source of IGF-I in the lung, little is known about the mechanisms that regulate IGF-I expression in macrophages. Therefore, in this study we investigated the cis-regulatory regions within the IGF-I exon 1 promoter that are required for basal expression in macrophages. We show that elements located in the 5'-untranslated region positively regulate transcriptional activity in macrophages, whereas sequences located in the 5'-flanking region confer negative transcriptional regulation.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from BioWhittaker (Walkersville, MD) and Atlanta Biologicals (Norcross, GA), respectively. RAW 264.7 cells were obtained from Dr. W. Murphy (University of Kansas, Kansas City, KS). HeLa cells were purchased from the American Type Culture Collection (Rockville, MD). Rat IGF-I exon 1 promoter-pGL2 luciferase constructs were kindly provided by Dr. Peter Rotwein (University of Oregon School of Medicine). The firefly luciferase reporter assay system, pGL3 Basic and Promoter luciferase reporter vector, primer extension kit, and restriction enzymes were purchased from Promega (Madison, WI). [{alpha}-32P]UTP (800 Ci/mmol, 10 mCi) and [{alpha}-32P]ATP (>3,000 Ci/mmol) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Primers and oligonucleotides were synthesized by Genelink (Thornwood, NY). The mRNA isolation [poly(A) pure], in vitro transcription (MAXIscript), and RNase protection assay (RPAIII) kits were purchased from Ambion (Austin, TX). The Oligotex mRNA isolation kit was purchased from Qiagen (Valencia, CA). Opti-MEM, TRIzol reagent, and LipofectAMINE Plus reagent were purchased from GIBCO-BRL Life Technologies (Grand Island, NY). The Sp1 (sc-59), Sp3 (sc644), and Ets-1 (sc-350) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Quikchange site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA).

Cell Culture

RAW 264.7 cells were seeded in six-well culture dishes at 3 x 106 cells/plate with 4 ml/well of RPMI 1640 supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% (vol/vol) FBS and cultured under a 5% CO2 atmosphere. HeLa cells were seeded in six-well culture dishes at 3 x 106 cells/plate with 4 ml/well of DMEM supplemented as above and cultured under a 5% CO2 atmosphere. Monolayers of mouse bone marrow-derived macrophages (BMDM) were prepared as previously described (30).

Construction of Reporter Vectors

The region from –1711 to +329 of the rat IGF-I exon 1 promoter/5'-untranslated region, or smaller fragments thereof, were cloned into the MluI and XhoI sites of the pGL3 vector. The sequence from XhoI to the NcoI site, the beginning of the coding region for luciferase, was removed to ensure that this region was not artifactually affecting transcription. The +1 is the most 5'-transcriptional start described by Hall et al. (10) and Peter Rotwein, the provider of the pGL2 IGF-I promoter construct used as the PCR template to make the inserts for these vectors. The +330 is the translational start site for exon 1 of IGF-I and is not included in these vectors. Site-directed mutations in the reporter constructs were made by following the manufacturer’s instructions for Quikchange site-directed mutagenesis (Stratagene) and were verified by sequencing.

Transfection of Reporter Constructs

Plasmid DNA was transiently transfected into cell lines using the LipofectAMINE Plus method as described in the instructions from GIBCO-BRL Life Technologies. Cells were seeded in six-well culture dishes at 0.5 x 106 cells/well for 24 h and were transfected with 0.22 pmol/well of DNA. Forty-eight hours posttransfection, cells were lysed with 400 µl of 1x passive lysis buffer, and we analyzed 30 µl of the lysate for luciferase activity by adding it to 200 µl of luciferase substrate followed by measurement in a MoonLight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) for 20 s. A portion of the cell lysate was used to determine total protein concentration using the Bradford assay. The luciferase assays were normalized to total protein.

Poly(A) RNA Isolation

Poly(A) RNA was isolated from BMDM by two methods. The first method employed was the Poly(A) Pure kit (Ambion). The manufacturer’s protocol was followed. The second method involved using the Oligotex kit (Qiagen) on total RNA previously isolated using the TRIzol method. The protocol of the manufacturer was followed.

Primer Extension

The primer, corresponding to the anti-sense strand of the first 30 bases of the coding region for mouse IGF-I exon 1, was end-labeled with [{gamma}-32P]ATP (3,000 Ci/mmol, 10 mCi/ml). The final concentration of the primer was adjusted to 100 fmol/µl. Poly(A) RNA (0.3 µg in 5 µl of water) was mixed with 1 µl of the labeled primer and 5 µl of 2x avian myeloblastosis virus (AMV) primer extension buffer to anneal the primer to the RNA. The samples were incubated at 60°C for 30 min and then placed at room temperature for 10 min. The primer extension reaction was then completed by extending the annealed primer with the addition of 9 µl containing 1.6 µl water, 5 µl 2x AMV primer extension buffer, 1.4 µl 40 mmol sodium pyrophosphate, and 1 µl (1 unit) AMV reverse transcriptase. The reaction was mixed and incubated at 42°C for 30 min after which time 20 µl of loading dye were added. Half of the sample was electrophoresed through a 5% (29:1) denaturing (8 M urea) Tris-borate-buffered polyacrylamide gel at 350–400 V. After drying the gel on Whatman paper, we visualized the primer extension products by autoradiography.

RPA

Mouse exon 1, 5'-untranslated region, and 5'-flank (the first 30 bases of the coding region and 630 bases upstream of that) were cloned into the KpnI and PstI sites of Promega’s pGEM4Z vector, which contains a T7 RNA polymerase promoter downstream of the multiple cloning region. The linearized vector was agarose gel purified, and the anti-sense probe was synthesized by using the MAXIscript in vitro transcription kit from Ambion. The full-length radiolabeled probe was PAGE purified. The probe was then immediately used in the RPA for which the RPAIII kit from Ambion was employed. Briefly, 40,000 cpm of labeled probe were mixed with 0.6 µg of poly(A) RNA for each treatment condition. Two control reactions, each containing probe but yeast RNA instead of sample RNA, were included. One-tenth volume of 5 M NH4OAc and 2.5 volumes EtOH were added to all the samples, mixed, and incubated at –70°C for 15 min. The samples were then centrifuged (14,000 g, 15 min, 4°C), after which time the supernatant was discarded, and the pellets were air-dried for 5 min. The pellets were resuspended in 10 µl of hybridization buffer III, heated to 90°C for 3 min, and transferred to 42°C for incubation overnight. RNase digestion buffer III (150 µl) containing 0.375 units of RNase A and 15 units of RNase T1 were added to each sample and to one of the control samples. The other control sample received 150 µl of RNase digestion buffer III lacking RNase. All samples were incubated at 37°C for 30 min and terminated with 225 µl of RNase inactivation/precipitation III solution. Yeast RNA (2 µl, 5 mg/ml stock) were added to each tube, as carrier, along with 150 µl of EtOH, and the samples were precipitated. The resulting pellet was resuspended in 10 µl of gel loading buffer II and vortexed vigorously. The samples were incubated at 90°C for 5 min and electrophoresed through a Tris-borate-buffered 5% (29:1) denaturing (8 M urea) polyacrylamide gel at 350–400 V. The gel was dried onto Whatman paper and autoradiography was performed.

Electrophoretic Mobility Shift Assay

Nuclear protein isolation. Macrophages (~1 x 107) were washed with PBS, scraped into 6 ml of PBS, and centrifuged (750 g, 10 min, 4°C). The cell pellet was resuspended in 500 µl of buffer A [10 mM HEPES, pH 7.8, containing 10 mM NaCl, 0.5 mM EDTA, 1.5 mM MgCl2, 2 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 2 mM sodium orthovanadate, and 2 mM sodium fluoride] and allowed to swell on ice for 10 min. The cells were lysed with 500 µl of buffer B [1.2% (vol/vol) Nonidet P-40 (NP-40) in buffer A], vortexed for 10 s, and centrifuged (2,500 g, 10 min, 4°C). The nuclear pellet was resuspended in 500 µl of buffer A, centrifuged (5,000 g, 5 min, 4°C), and resuspended in 50–100 µl of buffer C [buffer A containing 10% (vol/vol) glycerol and 0.41 M NaCl]. The nuclei were incubated on ice for 30 min, with occasional shaking. After centrifugation (20,000 g, 10 min, 4°C), the supernatant containing the eluted nuclear proteins was stored at –70°C until use.

Probe preparation. Table 1 shows the sequences of the oligonucleotides used in the electrophoretic mobility shift assasys (EMSAs). Equimolar amounts of the sense and corresponding anti-sense oligos were annealed by boiling and gradual cooling. After radiolabeling, the probes were electrophoresed through a Tris-glycine-buffered 6% (29:1) nondenaturing polyacrylamide gel to purify the labeled probe from single-stranded oligos and unincorporated nucleotides. The labeled probes were diluted to 7,500 cpm/µl in probe dilution buffer (20 mM HEPES, pH 7.9, containing 0.1 M KCl and 1 mM MgCl2) and frozen at –20°C.


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Table 1. Sequences of probes used in EMSA analysis

 
Binding reaction and electrophoresis. The reaction contained 20 mM HEPES, pH 7.9, 50 mM KCl, 2.5 mM MgCl2, 1 mM DTT, 2% (vol/vol) glycerol, 37.5 µg/ml poly(dI-dC), 0.1 mg/ml BSA, 0.1% (vol/vol) NP-40, 2 µl probe (15,000 cpm), and 3–5 µg nuclear extract. For the supershift assays, 1 µg of antibody was added. The reactions were incubated on ice for 30 min; 10 µl of the binding reaction were loaded onto a Tris-glycine-buffered 6% (29:1) nondenaturing polyacrylamide gel and run at 250 V for ~2 h at 4°C. The gel was dried onto on Whatman paper and autoradiography was performed.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Regions Within the Exon 1 Promoter of IGF-I Regulate Positive Transcriptional Activity in Macrophages

To determine the cis-regulatory elements in the IGF-I exon 1 promoter that are required for basal expression in macrophages, we created a series of IGF-I promoter-luciferase constructs using the sequence upstream of rat IGF-I exon 1, where +1 is the most 5'-transcriptional start described by Hall et al. (10) and +330 is the translational start site. We transfected these constructs into the macrophage cell line RAW 264.7 and measured luciferase activity (Fig. 1A). The –1711 to +329 construct did not exhibit any luciferase activity above that of the empty vector. However, following sequential 5'-deletions, promoter activity increased. The –433 to +329 and –122 to +329 constructs promoted luciferase activity approximately four- and eightfold above background, respectively. These data suggest that there are suppressive elements within the distal 5'-flanking region of IGF-I that mask activity located further downstream.



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Fig. 1. The region from +95 to +329 of IGF-I exon 1 controls maximal promoter activity, whereas +251 to +329 controls minimal promoter activity in macrophages. A and B: scale schematic representation of the regions upstream of IGF-I exon 1 used in the promoter-luciferase constructs and the corresponding luciferase activity in RAW 264.7 macrophages transfected for 48 h with 0.22 pmol of the pGL3-based constructs. Shown is the mean fold increase ± SE (n > 3), relative to the empty vector.

 
Further deletions were made to focalize the promoter. Deletions made between –122 and +95 revealed no differences in expression relative to the –122 to +329 construct (data not shown), whereas the +95 to +329 construct promoted luciferase activity 13.3-fold over the empty vector control (Fig. 1B) and 22-fold over the –1711 to +329 vector (compare Fig. 1, A to B). Additionally, Fig. 1B shows that after 5'-deletions to the +95 to +329 construct were made, activity began to drop. Deleting the region between +95 and +173 reduced luciferase activity to 6.4-fold, roughly half that seen with the +95 to +329 vector. A modest increase in activity was observed when the region from +173 to +251 was deleted (9.4-fold over vector), although this was less than the +95 to +329 construct. A final 5'-deletion of the region from +251 to +299 further reduced luciferase activity to near background. Based on these data, the region from +95 to +329 appears to direct maximal promoter activity and the region from +251 to +329 constitutes the minimal promoter for IGF-I expression in the macrophage cell line RAW 264.7. Because the region from +95 to +173 contains an element necessary for maximal expression, we examined whether this region is sufficient for maximal expression. Deletion of the region +173 to +329 from the +95 to +329 construct resulted in a 76% reduction in luciferase activity (Fig. 1B). This demonstrates that the region necessary for maximal expression is not sufficient to regulate maximal promoter activity and suggests that this region may work in concert with the minimal promoter to achieve maximal basal promoter activity. We then examined the minimal promoter to determine whether the region from +251 to +299, which is necessary for minimal promoter activity, is sufficient for minimal promoter activity. To test this, the region from +299 to +329 was deleted from the +251 to +329 vector. As shown in Fig. 1B, this deletion reduced the luciferase activity to that of the empty vector (9.4- to 1.3-fold). This demonstrates that the region from +251 to +299 is not sufficient for minimal promoter activity and that the entire region from +251 to +329 is required.

Having defined the minimal and maximal promoter regions for IGF-I in RAW 264.7 cells, we next determined whether these regions are specific to RAW 264.7 cells. We determined luciferase activity in an alveolar macrophage cell line, MH-S, and an epithelial cell line, HeLa. In the MH-S cells the pattern of expression was virtually identical to that of the RAW 264.7 cells (data not shown). IGF-I exon 1 promoter activity in the HeLa cell line was also similar to the pattern of expression in the RAW 264.7 cells (Fig. 2). However, the region from +251 to +329 directed maximal expression and also comprised the minimal promoter. The regions from +95 to +173 and +251 to +299 were not sufficient to promote luciferase activity, suggesting that the region between +299 and +329 was required in conjunction with one of these other regions to promote activity. Collectively, these data demonstrate that the region from +251 to +329 contains the minimal promoter for IGF-I exon 1, as shown in two macrophage cell lines and one epithelial cell line, and that there is no cell-specific regulation with regard to the minimal promoter. However, the region from +95 to +329 regulates maximal IGF-I exon 1 expression in macrophages but not in epithelial cells, implying possible cell-specific regulation.



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Fig. 2. Regions within the exon 1 promoter from the IGF-I gene regulate luciferase activity in the HeLa epithelial cell line. Scale schematic representation of the regions upstream of IGF-I exon 1 used in the promoter-luciferase constructs and the corresponding luciferase activity in HeLa cells transfected for 48 h with 0.22 pmol of the pGL3-based constructs. Shown is the fold increase relative to the empty vector and is representative of 3 experiments.

 
Primary Macrophages Use Multiple Transcriptional Start Sites for IGF-I, and the Major Site Is in the Maximal Promoter Region

To verify that transcription could be initiated in the regions defined as the maximal and minimal promoter elements and to rule out the possibility of transfection artifact, we determined the transcriptional start site usage for the endogenous IGF-I gene in primary mouse macrophages. Others have shown, in other species and cell types, that IGF-I has multiple transcriptional start sites. Therefore, our expectation was to find multiple start sites, including a major start site within the region from +95 to +173. In addition, a minor transcriptional start site was expected in the +251 to +299 region. Figure 3A shows the results from primer extension analysis on mRNA isolated from primary macrophages. The size of the extended primer was 210 bases. Because the primer was 30 bases long (Fig. 3C), the major transcriptional start site maps to approximately +150 (180 bases upstream of the translation start site). Due to the low specific activity of the probe used for the primer extension we also used RNase protection assays on mRNA isolated from primary mouse macrophages. Figure 3B shows that several bands of various lengths were protected from RNase degradation after annealing macrophage mRNA to a radiolabeled probe containing the first 30 bases of the coding region and extending upstream another 630 bases (Fig. 3C). A major band (Fig. 3, lane 3) was observed at ~215 bases, therefore mapping the major start site to approximately +145, consistent with the primer extension. A band of intermediate intensity (Fig. 3B, lane 3) was detected at 260 bases corresponding to a transcriptional start site in the +95 to +173 region, at +100. Finally, bands of weaker intensity were detected at ~105 bases (Fig. 3B, lane 3), corresponding to minor transcriptional start sites located at ~+255. These data thus demonstrate that the major transcriptional start site initiates in the region defined as being required for maximal promoter activity and a minor transcriptional start site lies in the region defined as the minimal promoter.



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Fig. 3. Multiple transcriptional starts are used for IGF-I exon 1 in primary mouse macrophages. A: primer extension analysis. Lane 1: 10-bp ladder. Lane 2: product of primer extension (solid arrowhead), using unstimulated mouse macrophage mRNA and a radiolabeled probe (antisense sequence of the 1st 30 bases of the coding region for IGF-I exon 1). Representative blot of 3 experiments. B: RNase protection assay (RPA) analysis. Lane 1: probe (anti-sense sequence of the first 30 bases of the coding region for IGF-I exon 1 and the 630 bases upstream) and tRNA with no RNase. Lane 2: probe and tRNA with RNase. Lane 3: protected products following annealing the radiolabeled probe to unstimulated mouse macrophage mRNA and the addition of RNase. Solid arrowhead marks the major transcriptional start site, whereas the open arrowhead and arrows denote the minor transcriptional start sites. Lane 4: 10-bp ladder. Representative blot of 3 experiments. C: diagram. Schematic representation of the location of the primer and probe used for primer extension and RPA, respectively. These are shown relative to the IGF-I exon 1 translational start site.

 
Macrophage Nuclear Proteins Specifically Bind to Regions Required for Maximal and Minimal Promoter Activity

Having defined the cis-regulatory elements required for expression of IGF-I in macrophages, we next determined whether nuclear proteins from macrophages bound these regions. Figure 4A shows the relative location and sequence for EMSA probes: +134 to +173 probe (probe 1) and +267 to +299 probe (probe 2). Incubation of these two radiolabeled probes with nuclear proteins from RAW 264.7 macrophages resulted in the formation of multiple DNA/protein complexes (Fig. 4B, left). A similar banding pattern was observed when nuclear proteins from primary macrophages were incubated with the same probes (Fig. 4B, right). To determine the specificity of binding, we added a 50-fold molar excess of unlabeled probe to the reaction. Figure 4C shows that binding to each of the radiolabeled probes was competed away by unlabeled (cold) probe. These results suggest that the cis-regions required for maximal and minimal promoter activity of IGF-I in macrophages specifically bind macrophage nuclear proteins.



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Fig. 4. Macrophage nuclear proteins specifically bind to regions required for maximal and minimal promoter activity. A: diagram. Scale schematic representation of the noncoding region of IGF-I exon 1 and the relative location of the probes used for EMSA analysis. Also shown are the upper strand sequences (5'–3') for both probes. B: EMSA analysis. Left: RAW 264.7 macrophage nuclear proteins incubated with the probes shown in A above, whereas the right panel shows primary macrophage nuclear proteins incubated with these probes. Arrows mark the multiple DNA/protein complexes. Results are representative of ≥3 experiments. C: EMSA analysis with unlabeled probe. RAW 264.7 macrophage nuclear proteins incubated with the probes shown in A above with or without a 50-molar excess of unlabeled (cold) probe. Arrows mark the multiple DNA/protein complexes. Results are representative of 3 experiments.

 
Sp3 Binds to a Region Required for Minimal Promoter Activity of IGF-I in Macrophages

Next we investigated the composition of the complexes that bound to the +267 to +299 probe. Although degenerate, the sequence between +287 and +295 appears to be an Sp protein binding GC box (Fig. 5B). Therefore, using EMSA and supershift analysis, we examined whether Sp1 and/or Sp3 were capable of binding the +267 to +299 probe. Figure 5A shows that complex II contains Sp3, as indicated by the supershifted band and the loss of intensity of complex II. Neither the Sp1 antibody nor the Ets-1 antibody, used as a control, resulted in supershifting, suggesting that neither of these trans-acting factors bound to this probe. Because Sp3 and Sp1 bind to similar sequences, as a control we verified that the Sp1 antibody was capable of binding Sp1 and supershifting a DNA/Sp1 complex using RAW 264.7 macrophage nuclear proteins and an Sp consensus probe. Figure 5A (right) shows that, compared with complex formation in the absence of antibody, the Sp1 antibody supershifted the DNA/Sp1 complex, as evidenced by the shifting of the upper complex. Using a similar approach we also tested the Sp3 antibody with the consensus Sp probe. It can be seen that the lower complex generated from the incubation of macrophage nuclear proteins with the Sp consensus probe contains Sp3 (Fig. 5A, right). Ets-1 and nonimmune IgG antibodies were used as a controls. Thus, in conjunction with Western blot data (not shown), we show that macrophages express Sp1 and Sp3 but only Sp3 binds the +267 to +299 IGF-I probe.



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Fig. 5. Sp3 binds to a region (+267 to +299) required for minimal promoter activity of IGF-I in macrophages. A: EMSA and antibody supershift analysis. Left: RAW 264.7 macrophage nuclear proteins incubated with the +267 to +299 probe either alone or with antibodies specific for Sp1, Sp3, or Ets-1. I and II, upper and lower complexes, respectively. The solid arrowhead marks the supershifted band. Right: RAW 264.7 macrophage nuclear proteins incubated with an Sp consensus probe either alone or with antibodies specific for Sp1, Sp3 Ets-1, or nonimmune IgG. Solid and open arrows mark the lower and upper complexes, respectively. Arrowheads indicate the supershifted bands; open Sp1 and solid Sp3. Results representative of ≥3 experiments. B: sequence of wild-type (WT) and mutant probes. Sequence of the +267 to +299 WT probe with the putative degenerate GC box (Sp binding site) underlined. Sequence of the two +267 to +299 mutant probes (Mut A and B) with the 3 bases that have been mutated in each are underlined. C: EMSA analysis with mutant probes. RAW 264.7 macrophage nuclear proteins incubated with either the +267 to +299 WT probe (probe 2), +267 to +299 Mut A probe (probe 2A), or the +267 to +299 Mut B probe (probe 2B). The upper and lower complexes are denoted by I and II, respectively. Results are representative of ≥3 experiments.

 
To verify that Sp3 was binding the degenerate GC box, +288 to +294 within the +267 to +299 probe, we made two separate mutations within this putative GC box (Fig. 5B). Using these three probes, EMSA analysis showed that mutating TCC to TTC (Fig. 5B) abolished the formation of both complexes I and II seen with the wild-type +267 to +299 probe (Fig. 5C), whereas mutation of GCC to ATT only limits the formation of complex II. Collectively these results suggest that 1) complex II contains Sp3, 2) Sp3 binds the putative GC box, and 3) either set of mutations eliminates the ability of Sp3 to bind in the degenerate GC box.

Mutation of the Sp3 Binding Site in the IGF-I Promoter Increases Transcriptional Activity in Macrophages

We next examined whether the Sp3 binding sequence was functional in the IGF-I promoter-luciferase constructs. The same mutations that are shown in Fig. 5B were made in both the +251 to +329 and +95 to +329 constructs defined to contain the cis-elements required for minimal and maximal promoter activity, respectively, in macrophages (Fig. 1B). Figure 6 shows that when either mutation was made in either construct, luciferase activity was increased (P < 0.05). These data suggest that Sp3 is acting as a transcriptional suppressor for IGF-I expression in macrophages in the region required for minimal expression.



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Fig. 6. Mutation of the Sp3 binding site in the IGF-I promoter increases transcriptional activity in macrophages. Graph of the luciferase activity in RAW 264.7 macrophages transfected with 0.22 pmol of the pGL3-based constructs shown, for 48 h. Mut A and B are the same as those shown in Fig. 5, B and C. Shown is the mean fold increase ± SE (n = 3) relative to the WT construct. *P < 0.05 relative to WT parental construct.

 
Regions in the 5'-Flanking Region of IGF-I Exon 1 Suppress Transcription in Macrophages

We next investigated the suppressive regions of the 5'-flanking region of IGF-I exon 1 located between –1711 and –122, as suggested in Fig. 1A. To define the regions responsible for suppressive activity, internal deletions of the IGF-I exon 1 promoter-luciferase constructs were engineered. The region between –855 and –122 was deleted, thereby leaving the region from –1711 to –855 juxtaposed to the region –122 to +329. As shown in Fig. 7A, the region from –1711 to –855 was sufficient to suppress luciferase activity of the –122 to +329 construct, from eightfold over control down to 0.6-fold of control. This figure also demonstrates that a construct containing the region from –1711 to –855 alone expressed less luciferase activity than the empty vector (a 90% reduction) and also less than the –1711 to +329 construct (an 83% reduction). This suggests that the –1711 to –855 region suppressed the background activity normally seen with the empty vector.



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Fig. 7. The regions from –1711 to –855 and from –855 to –337 in the 5'-untranslated region of IGF-I exon 1 suppress promoter activity. A and B: scale schematic representation of the regions upstream of IGF-I exon 1 used in the promoter-luciferase constructs and the corresponding luciferase activity in RAW 264.7 macrophages transfected for 48 h with 0.22 pmol of the pGL3-based constructs. Shown is the mean fold increase ± SE (n > 3) relative to the empty vector.

 
We also engineered a construct that maintained the regions from –855 to –337 and –82 to +329 but had the intervening sequence deleted. Figure 7B shows that, similar to the –1711 to –855 region, the region from –855 to –337 was sufficient to suppress the normal sevenfold activity of the –82 to +329 construct down to 0.8-fold relative to empty vector. Also, similar to the –1711 to –855 region, the –855 to –337 alone construct expressed less luciferase activity than the empty vector and the –855 to +329 construct.

Having shown that the regions from –1711 to –855 and from –855 to –337 suppress the IGF-I exon 1 promoter in RAW 264.7 macrophages, we next examined whether this suppressive activity was also present in other cell types. The macrophage cell lines MH-S and NR8383 and the epithelial cell lines HeLa and COS-7 were transfected with the IGF-I exon 1 constructs. The regions from –1711 to –855 and –855 to –337 suppressed luciferase activity in a similar manner in the MH-S and NR8383 macrophages as they did in the RAW 264.7 macrophages (data not shown). In contrast, luciferase activity was not suppressed in either the HeLa (Fig. 8) or COS-7 (data not shown) epithelial cell lines. In both these cell lines the –1711 to +329 construct promoted activity fivefold over the empty vector. Collectively, these data suggest that the suppressive elements in the 5'-flanking region of IGF-I exon 1 may function in a cell type-specific manner.



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Fig. 8. The 5'-flanking region of IGF-I exon 1 does not suppress promoter activity in the HeLa epithelial cell line. Scale schematic representation of the regions upstream of IGF-I exon 1 used in the promoter-luciferase constructs and the corresponding luciferase activity in HeLa cells transfected for 48 h with 0.22 pmol of the pGL3-based constructs. Shown is the fold increase relative to the empty vector and is representative of 3 experiments.

 
Because the 5'-flanking region suppressed the ability of IGF-I exon 1 to promote luciferase activity in RAW 264.7 macrophages and other macrophages, we examined the hypothesis that the 5'-flanking region would suppress luciferase activity driven by another promoter. To test this we employed the pGL3 luciferase vector that contains the SV40 promoter. The regions –1711 to –855 and –855 to –337 were cloned upstream of the SV40 promoter and luciferase activity was measured after transfection into RAW 264.7 macrophages. The 5'-flanking region of IGF-I exon 1 did not suppress the activity driven by the SV40 promoter (data not shown). The lack of suppression may be due to strength of the SV40 promoter, and therefore the suppressive elements may only work on weaker promoters or the elements may only suppress TATA-less promoters, like the IGF-I promoter.


    DISCUSSION
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This study is the first to determine the cis-regulatory regions required for IGF-I expression in macrophages and the first to examine IGF-I transcriptional start site usage in macrophages. Here we show that regions in the 5'-untranslated region promote IGF-I transcription, whereas the region upstream of IGF-I exon 1 contains elements that repress expression in macrophages. The region from +95 to +329 is required for maximal basal promoter activity, whereas the region from +251 to +329 constitutes the minimal promoter. These data are consistent with the transcriptional start site usage for endogenous IGF-I in primary macrophages where the major start site and a minor site are in the region from +95 to +173. This region was shown to be necessary but not sufficient for maximal promoter activity and fits with our data showing that when this region is deleted and, hence, the major and a minor start site are removed, much of the promoter activity is lost. The remaining promoter activity seen with the minimal promoter region, +251 to +329, can be attributed to the minor transcriptional start sites centered at +255. Once these are removed, as in the +299 to +329 construct, promoter activity is lost presumably due to the lack of cis-elements and transcriptional start sites. However, there are elements in the +299 to +329 region that are necessary for promoter or translation efficiency, as evidenced by the fact that whenever this region is deleted from the +95 to +329 or the +251 to +329 constructs, luciferase activity is lost. Consistent with these conclusions, macrophage nuclear proteins were found to specifically bind within the regions required for maximal and minimal promoter activity. Sp3, but not Sp1, was found to bind in the minimal promoter and was found to negatively regulate IGF-I promoter activity in macrophages. We have also shown that there are at least two regions in the 5'-flanking region, –1711 to –855 and –855 to –337, that can suppress IGF-I promoter activity in macrophages but not in epithelial cells. The fact that the constructs containing these regions expressed luciferase in epithelial cells but not in macrophage cell lines suggests macrophage specific suppression.

To our knowledge, no other reports have been published using the IGF-I promoter luciferase system in macrophages. However, studies have examined transcriptional regulation of IGF-I using the luciferase reporter system in other cell types, including the liver. Pao et al. (18) identified six DNase I footprints downstream of the major transcriptional start site, and Zhu et al. (31) examined the cis-elements and trans-acting factors that were part of footprint V. It was reported that both Sp1 and Sp3 bound the sequence CCTGCCCA (31) in contrast to our finding of exclusive occupancy by Sp3. Although the reason for the discordance is unclear, our data clearly support the notion that in macrophages: 1) Sp1 is not responsible for basal regulation of IGF-I and 2) Sp3 serves to dampen the basal regulation. These conclusions are bolstered by the data that Sp1, although expressed by macrophages, does not bind the IGF-I promoter whereas Sp3 does. We have also observed that overexpression of Sp1 does not increase IGF-I promoter activity in luciferase reporter gene assays (data not shown). Mutation of the Sp3 binding site not only inhibits Sp3 binding but increases luciferase activity, consistent with the known function for Sp3 as a transcriptional repressor (3, 7).

The regulation of IGF-I in osteoblasts is qualitatively similar to the data presented here in macrophages. Pash et al. (19) reported that maximal promoter activity was observed with the –123 to +372 construct [these base numbers have been changed by 10 bases from the original report to be consistent with the numbering system used by Hall et al. (10) and herein], which includes the region required for maximal promoter activity defined in the present study (+95 to +329). In addition, McCarthy et al. (17) reported that the region from +195 to +329 is required for minimal promoter activity. Other studies have also suggested that the 5'-flanking region contains sequences that suppress IGF-I promoter activity in some cell types such as primary dermal fibroblasts (16), C6 glioma (16, 28), OVCAR-3 (28), and GH3 cells (28). Consistent with our results in HeLa cells, transfection of the longest IGF-I luciferase reporter construct into the neuroepithelioma cell line SK-N-MC resulted in a high amount of luciferase activity (10). Collectively these findings suggest that IGF-I expression via promoter 1 may be differentially regulated in different cell types.

In summary, the findings from the present study provide novel insights into both positive and negative regulation of IGF-I expression by macrophages. With the growing data to suggest that both macrophages and IGF-I may play a role in the initiation and/or progression of IPF/UIP, understanding the mechanisms by which IGF-I is regulated in macrophages is of importance. Only when basal regulation is understood can one undertake the problem of the apparent dysregulated expression of IGF-I in macrophages from IPF patients. These data open up the possibility that the 5'-flanking region of IGF-I may be a target through which IGF-I expression could be manipulated and controlled in macrophages without affecting epithelial cell expression.


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This work was supported by National Heart, Lung, and Blood Institute’s Public Health Service Grants HL-68628, HL-55549, and HL-65326. M. W. Wynes was supported by National Institute of Allergy and Infectious Diseases Institutional T-32 Training Grant AI-00048.


    ACKNOWLEDGMENTS
 
The authors acknowledge Linda Remigio for outstanding technical assistance. We thank Dr. Stephen Frankel and Amanda Kostyk for critical reading of the manuscript and Dr. James Hagman for continued interest and intellectual input into this work.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. W. H. Riches, Program in Cell Biology, Dept. of Pediatrics, Neustadt Rm. D405, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206 (E-mail: richesd{at}njc.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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