Pulmonary Center and Department of Biochemistry, Boston University School of Medicine, and Boston Department of Veterans Affairs Medical Center, Boston, Massachusetts 02118
Submitted 1 May 2003 ; accepted in final form 21 July 2003
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ABSTRACT |
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lung; fibroblasts; emphysema; nuclear factor-B
We previously reported (3, 18) that IL-1 reduces the steady-state levels for elastin mRNA in neonatal rat lung fibroblasts by a mechanism that involves activation of NF-
B and subsequent decreases in the rate of elastin gene transcription. Activation of NF-
B by a variety of inflammatory or stress-related events is usually followed by nuclear translocation and transactivation or inhibition of target genes. NF-
B can also affect gene expression indirectly via interactions with other transcription factors. IL-1
activates NF-
B by inducing phosphorylation of I
B with subsequent release and nuclear translocation of NF-
B (10, 31). We found that IL-1
treatment induced a large and sustained increase in intranuclear NF-
B in neonatal lung fibroblasts (18). Transfection of expression plasmids encoding the NF-
B p65 subunit, but not the p50 subunit, decreased the transcriptional activity of the elastin promoter and levels of elastin mRNA. Our results (18) together with other reports (30) indicate that NF-
B, particularly the p65 component, can interfere with the transcription of certain matrix components including elastin and
1(I) collagen. We now demonstrate that IL-1
-induced activation of NF-
B requires production of CCAAT/enhancer-binding protein (C/EBP)
isoforms to downregulate elastin mRNA expression.
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EXPERIMENTAL PROCEDURES |
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Plasmid constructs. The rat elastin promoter sequence from -1 to -118 bp was cloned into a luciferase basic reporter PGL-2 (PGL-2/118; Promega) as described previously (18). C/EBP cDNA fragments were generated by PCR from the C/EBP
LAP pBabe viral vector (kindly provided by Dr. S. Farmer) with 5'-end primers for LAP, 5'-CGCGGATCCCCACCAT-GGAAGTGGCCAACTT-3', and LIP, 5'-CGCGGATCCCCACCATGGCGGCCG-GCTT-3', based on published sequences (13) and the 3'-end primer 5'-GCAGT-GACCCGCCGAGGCCAGCAGC-3'. The resulting open reading frame (ORF) sequences for LAP and LIP without stop codon was cloned into V5-tagged pcDNA TOPO (Invitrogen). A Kozak sequence was introduced into C/EBP
expression vectors (13). All constructs were verified by DNA sequencing.
Transient transfection and luciferase assay. Transient transfection was performed with PGL-2/118 linked to firefly luciferase as a reporter gene (1 µg), C/EBP expression vectors (1 µg), and pEGFP-N1 (0.5 µg; Clontech) or pRLTK expressing Renilla luciferase as an internal control (0.5 µg; Promega) by LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol. At 48 h after transfection, cells were harvested in lysis buffer (100 µl). Firefly and Renilla luciferase (10 µl of lysate) activity was determined with the Dual-Luciferase Reporter system (Promega) and by scintillation counting. Firefly luciferase values were normalized to Renilla luciferase values and expressed as relative firefly/Renilla luciferase activity. The percentage of green fluorescent protein (GFP)-positive cells was assessed to monitor transfection efficiency. Experiments were performed in triplicate and repeated at least three times. In some cases, transfected cells were used for immunoprecipitation with anti-V5 antibody. For Northern analysis, LIC were seeded into 100-mm dishes and transfected with 8 µg of empty vector (pcDNA 3.1) or C/EBP
expression vectors (4 and 8 µg of pcDNA LAP or pcDNA LIP) with LipofectAMINE 2000. After 48 h, total RNA was isolated for Northern blot analysis.
RNA isolation and Northern blot analysis. Total RNAs were prepared with the RNeasy mini kit (Qiagen) according to the manufacturer's protocol. Equal amounts of RNAs (10 µg/lane) were electrophoresed in 1% agarose gel with formaldehyde and transferred to Hybond-N+ membrane (Amersham). RNA membrane was ultraviolet cross-linked and sequentially hybridized with 32P-labeled cDNA probes for elastin, p56, C/EBP, or oligonucleotides that bind the 18S or 28S ribosome subunits in Rapid Hybridization Buffer (Amersham), followed by washing twice in 2x SSC-0.1% SDS at room temperature (RT) for 15 min and once in 0.1x SSC-0.1% SDS at 65°C for cDNA probes and at 37°C for oligonucleotide probes for 20 min. The 18S- and 28S-specific oligonucleotides were synthesized by IDT (Coralville, IA).
Preparation of nuclear extracts and electrophoretic mobility shift assay. Nuclear extracts were prepared on ice at 4°C according to Dignam et al. (11) with modifications, and electrophoretic mobility shift assay (EMSA) was performed as previously described (18). Elastin promoter fragments generated by restriction enzyme digestion were gel purified and end labeled by 3' recessed end fill-in with [-32P]dCTP and Klenow (New England Biolabs). Nuclear extracts (20 µg) were incubated with 1 x 105 cpm of labeled elastin promoter fragments (1 ng). To identify non-zinc finger DNA binding proteins, 1 or 10 mM EDTA-EGTA was included in the reaction buffer. To perform supershift experiments, we used an anti-C/EBP
antibody (C-19, 4 µg; Santa Cruz Biotechnology) that was generated against the leucine zipper region of C/EBP
at the COOH terminus and recognizes both LAP and LIP (36). For competition studies, 100 ng of unlabeled double-stranded DNA oligonucleotides was preincubated before addition of 32P-labeled elastin promoter fragments. DNA-nucleoprotein complexes were resolved on a preelectrophoresed 4% nondenaturing polyacrylamide gel (acrylamide-bisacrylamide, 40:1) in 1x TBE (45 mM Tris-borate, 1 mM EDTA) at 150 V for 2.5 h. The double-stranded DNA oligonucleotide sequences (5' to 3'; sense strand) used are shown below. C/EBP
(TGCAGATTGCGCAATCTGCA; Santa Cruz Biotechnology) and Sp1 (ATTCGATCGGGGCGGG-GCGAGC; Promega) oligonucleotides correspond to their consensus sequences, respectively. The GCAAT (-70TACCAGGCAGC-AATTACGCTTTGGGGA-44) and the ATAAA (-55AC-GCTTTGGGGATAAAACGAG-GCGCAG-29) oligonucleotides encoding a C/EBP binding element and a RNA polymerase recognition element of rat elastin promoter, respectively, were synthesized by IDT. Boldfaced sequences are composed of the consensus binding motifs.
Western blot analysis and immunoprecipitation. Nuclear extracts (30 µg) were resolved by 4-12% gradient of SDS-PAGE (Invitrogen) and transferred to nitrocellulose membrane (0.2 µm; Schleicher & Schuell). Milk (0.5%)-blocked membranes were incubated with anti-p65 antibody (1:1,000; Santa Cruz Biotechnology) or anti-C/EBP antibody (1:1,000; Santa Cruz Biotechnology) overnight at 4°C with shaking. After incubation with the specific secondary horseradish peroxidase-conjugated antibody (1:2,500 dilution; Amersham) at RT for 1 h, membrane was extensively washed and proteins were detected with an enhanced chemiluminescence (ECL) kit (Amersham).
For immunoprecipitation studies, LIC were harvested in 0.5 ml of ice-cold lysis buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 5 µg/ml leupeptin). The cell lysates were cleared by centrifugation, and supernatants were precleared with protein A/G Sepharose (Santa Cruz Biotechnology) before immunoprecipitation with 2 µg of anti-V5 antibodies (Invitrogen) and 50 µl of protein A/G Sepharose for 24 h at 4°C. The immunoprecipitates were extensively washed, separated by 4-12% gradient SDS-PAGE (Invitrogen), and transferred to nitrocellulose membranes (0.2 µm; Schleicher & Schuell). C/EBP-V5 fusion proteins were detected with anti-V5 antibodies (Invitrogen).
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RESULTS |
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IL-1 treatment decreased the activity of the proximal rat elastin promoter (PGL-2/118) in transient transfection assays using neonatal rat fibroblasts (18). We reported (18) that Sp1, Sp3, and another unidentified zinc finger transcriptional factor bound to the elastin promoter region between -66 to -118 bp were unchanged by IL-1
treatment. To identify other potential IL-1
-regulated transcription factors involved in the downregulation of elastin mRNA, we examined the binding of nuclear proteins to a larger promoter fragment (-1 to -118 bp). Proteins binding to the promoter fragment (-1 to -118 bp) were identified by EMSA using nuclear extracts isolated from IL-1
-treated or untreated neonatal lung fibroblasts. With the use of this larger promoter fragment, several nuclear proteins were induced by IL-1
(Fig. 2A). To better resolve these complexes, we used EDTA-EGTA to disrupt the binding of zinc finger proteins (Fig. 2A). Three protein complexes were identified that were up-regulated by IL-1
(designated as C1, C2, and C3). Deletion studies confirmed that these IL-1
-induced proteins bind to the proximal elastin promoter (-1 to -66 bp; Fig. 2B). Computer analysis (38) of the proximal fragment sequence (-1to -118) indicated that the GCAAT sequence located at -56 to -62 bp may function as a putative binding site for C/EBP-related proteins.
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To determine whether proteins binding to the proximal elastin promoter were related to C/EBP, we performed competition studies using oligonucleotides encoding the C/EBP binding sequence found in the elastin proximal promoter, the consensus sequences for C/EBP or Sp1, or the binding sequence in the elastin promoter for the RNA polymerase II ATAAA (Fig. 3). The oligonucleotides were incubated with nuclear extracts before addition of the radiolabeled elastin promoter fragment (-1 to -66 bp) in EMSA. We found that C/EBP-encoding oligonucleotides, but not Sp1- or ATAAA-encoding oligonucleotides, abolished the binding of three proteins induced by IL-1 treatment (Fig. 3, lanes 6-13). The three C/EBP complexes were supershifted by anti-C/EBP
antibodies (Fig. 3, lane 5) but not anti-C/EBP
antibodies (data not shown). Previous studies demonstrated that this electrophoretic pattern represents the dimerization of two C/EBP isoforms consisting of LAP and the truncated fragment termed LIP [the dimers were designated as C1 (LAP-LAP), C2 (LAP-LIP), and C3 (LIP-LIP)] (26-28, 37, 39, 40).
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To examine the effect of IL-1 treatment on C/EBP
mRNA and protein levels, we performed Northern and Western blot analyses. We found that IL-1
induced large increases in the levels of both p65 and C/EBP
mRNAs (Fig. 4A). Western blot analysis of nuclear extracts from LIC demonstrated that IL-1
treatment increased levels of C/EBP
proteins in the nucleus (Fig. 4B). The identity of the proteins was confirmed as LAP and LIP with nuclear extracts from 3T3.L1 murine cell lines that constitutively express LAP and LIP (13). The full-length C/EBP
proteins from rat and mouse differ by three amino acid substitutions and generate C/EBP
isoforms with the same mechanisms (5, 6, 8, 28). The addition of CHX abolished both LIP and LAP protein expression (Fig. 4B).
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To examine the functional role of IL-1-induced C/EBP
proteins on the transcriptional activity of the elastin promoter, we carried out cotransfection experiments. PGL-2/118 elastin luciferase reporter was cotransfected with pcDNA expression vectors for the transcriptionally active LAP or the transcriptional repressor LIP into neonatal lung fibroblasts (9, 29, 36). We found that cotransfection with LAP or LIP decreased luciferase activity of PGL-2/118 by 26% and 50%, respectively (Fig. 5A, top). Immunoprecipitation with anti-V5 antibody indicated that LAP and LIP were expressed in transfected neonatal lung fibroblasts (Fig. 5A, bottom). We transfected LAP or LIP pcDNA expression plasmids into neonatal lung fibroblasts and examined endogenous elastin mRNA levels by Northern blot analysis. We found that LAP decreased endogenous elastin mRNA after transfection at a high dose of plasmid DNA, whereas LIP decreased elastin mRNA after transfection at a low dose (Fig. 5B).
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We used helenalin, a specific inhibitor of NF-B activation, to examine whether IL-1
-induced C/EBP expression is dependent on NF-
B activation. Helenalin irreversibly alkylates the p65 subunit and thereby inhibits the association of p65 with promoter elements (15, 23). The addition of helenalin (10 µM) inhibited IL-1
-induced increases in C/EBP mRNA and decreases in elastin mRNA (Fig. 6).
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DISCUSSION |
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C/EBP is a basic leucine zipper transcription factor encoded by a single intronless gene that generates several isoforms (full-length LAP 38 kDa, LAP 35 kDa, LIP 20 kDa and a smaller 16-kDa form) that bind to DNA as homodimers or heterodimers (9). The isoforms may arise by two potential mechanisms depending on the cell type and culture conditions (2, 9). The truncated isoform (LIP) can be generated by a leaky ribosomal scanning mechanism with initiation of translation at the third AUG. The regulation of LIP translation may involve RNA binding proteins and the influence of a short ORF (sORF) located in the 5' region of the C/EBP
mRNA (33). The LAP isoform contains an activation domain, a DNA binding domain, and a leucine zipper domain. The truncated protein (LIP) is generated from the downstream AUG containing only the DNA binding domain and leucine zipper domain and inhibits transcription either by competing with the LAP isoforms for binding sites or by interfering with the binding of the transcriptional apparatus (12). In addition, formation of heterodimers of LIP with LAP decreases transcriptional activation (9, 20). The C/EBP fragments are also formed by a proteolytic cleavage mechanism in certain cell types (2). This mechanism requires nuclear localization and is increased during certain extraction procedures for isolation of nuclear proteins (2). We did not detect LIP in nuclear proteins isolated from a stable preadipocyte cell line (13) with a mutated third AUG in the C/EBP
gene, indicating that our isolation procedure for nuclear proteins did not result in proteolytic production of LIP.
Overexpression of LAP and LIP in a transient transfection assay decreased elastin promoter activity and elastin mRNA levels. Deletion analysis and competition studies with oligonucleotides encoding portions of the proximal elastin promoter indicate that C/EBP binds to the GCAAT sequence located at -56 to -62 bp in the elastin promoter. Because we could not detect C/EBP isoforms in untreated cultures, LIP likely inhibits elastin transcription by blocking the binding of RNA polymerase II as shown for phosphoenolpyruvate carboxykinase gene promoter (12). Transfection of LAP also decreased the activity of the elastin promoter, but to a lesser extent than LIP. Western blot analysis of nuclear lysates indicates that both LAP and LIP are generated after transfection of LAP at a higher dose.
Our results suggest that the inhibitory activity of C/EBP expression on elastin promoter activity is the result of binding of LIP-LIP homodimers and LAP-LIP heterodimers. IL-1
treatment induced larger amounts of LAP than LIP (Fig. 4B). However, only low levels of LAP-LAP DNA binding complexes were formed (Figs. 2 and 3). Acetylation of C/EBP
proteins decreases the formation of the transcriptional activator LAP-LAP but not transcriptional repressors LAP-LIP and LIP-LIP (39). These results suggest that IL-1
-induced C/EBP
proteins may be acetylated in rat neonatal lung fibroblasts. Overexpressed LAP may be acetylated and fail to form the active LAP-LAP homodimer.
IL-1 increases C/EBP mRNA levels in liver and kidney in vivo (1, 24) and in rat chondrogenic (26) and osteoblastic (14) cells in vitro. IL-1
also affects C/EBP activity by altering phosphorylation on serine in hepatocytes (35). We find that IL-1
treatment increases the levels of C/EBP
mRNA and protein. This induction is mediated by NF-
B. In rat hepatoma cells, NF-
B increased the activity of the rat C/EBP
promoter (25). Together, our results indicate that IL-1
-induced downregulation of elastin mRNA involves an initial activation of NF-
B. Translocation of the p65 subunit into the nucleus induced expression of C/EBP
mRNA and the subsequent expression of C/EBP
proteins. These isoforms, especially LIP, bind to the proximal elastin promoter and decrease transcriptional activity. This pathway can be inhibited by blocking either activation of NF-
B or production of C/EBP
isoforms. Regulation of elastin production by IL-1
during inflammatory reactions may contribute to the impaired elastin resynthesis that results in airway enlargement (19, 21).
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DISCLOSURES |
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FOOTNOTES |
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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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