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CCAAT/Enhancer-Binding Protein Isoforms and the Regulation of -Smooth Muscle Actin Gene Expression by IL-1¹

Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The role of IL-1 in inflammation is amply documented, but its ability to inhibit myofibroblast differentiation and, in particular, the suppression of -smooth muscle actin (-SMA) gene expression is less well understood. Because IL-1 can induce C/EBP expression, the role of C/EBP isoforms in IL-1 regulation of -SMA gene expression was investigated in rat lung myofibroblasts. The results showed that IL-1 inhibited -SMA expression in a dose-dependent manner, which was associated with stimulation of the expression of both C/EBP isoforms, liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP). However, a greater increase in LIP relative to LAP expression resulted in a reduced LAP/LIP ratio after IL-1 treatment. Transfection with an LAP-expressing plasmid stimulated, whereas an LIP-expressing plasmid inhibited, -SMA expression. Cells from C/EBP-deficient mice had reduced levels of -SMA expression and promoter activity, which failed to respond to IL-1 treatment. Sequence analysis identified the presence of a C/EBP consensus binding sequence in the -SMA promoter, which, when mutated, resulted in diminished promoter activity and abolished its responsiveness to IL-1 treatment. EMSA revealed binding of C/EBP to this C/EBP consensus binding sequence from the -SMA promoter. Finally, IL-1 enhanced the expression of eukaryotic initiation factor 4E, a stimulator of LIP expression, which may account for a mechanism by which IL-1 could alter the LAP/LIP ratio. These data taken together suggest that C/EBP isoforms regulate -SMA gene expression, and that its inhibition by IL-1 was due to preferential stimulation of LIP expression.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Myofibroblasts possess ultrastructural features intermediate between those of fibroblasts and smooth muscle cells and have been defined by their ability to express contractile proteins, particularly -smooth muscle actin (-SMA)³ protein (1, 2, 3). Their derivation from fibroblasts can be demonstrated in vitro (4, 5). The myofibroblast differentiation process appears to be a critical event in the pathogenesis of fibrotic diseases such as pulmonary fibrosis (6, 7, 8, 9, 10, 11). IL-1, one of the major cytokines that mediate inflammatory reactions, represses myofibroblast differentiation and -SMA gene expression (12). Although there is evidence of selective myofibroblast apoptosis in response to IL-1 treatment, the effect on -SMA gene expression per se has not been specifically addressed, especially with respect to the molecular mechanism.

The basic leucine zipper transcription factor, C/EBP, is a trans-acting factor that controls cell differentiation and proliferation in a number cell types (13, 14, 15, 16, 17). Two kinds of C/EBP isoforms, varying in size from 14–38 kDa, have been reported. One is the 38- and 35-kDa full-length liver-enriched activator protein (LAP) isoforms, and the other is the 21- and 14-kDa truncated liver-enriched inhibitory protein (LIP) isoforms (18, 19, 20, 21). The 14-kDa truncated LIP isoform was identified in macrophages (18). Both the p14 and p21 truncated C/EBP (LIP) isoforms retain the C-terminal bZIP and DNA binding domains, but lack the N-terminal transactivation domain (20, 21, 22). Probably due to its higher affinity for its DNA cognate sequences, LIP can attenuate the transcriptional stimulation by LAP in substoichiometric amounts (20). Several reports indicate that the ratio of C/EBP isoforms (i.e., LAP/LIP ratio) present in a cell can be altered by a variety of cellular conditions (20, 22). This is potentially significant, because evidence indicates that the N-terminal truncated C/EBP (LIP) isoforms function as dominant negative inhibitors of the full-length C/EBP (LAP) isoform even when present at substoichiometric levels (i.e., LAP:LIP ratio of 5:1) (20, 22).

In this study the roles of C/EBP isoforms in the regulation of -SMA gene expression were analyzed. Western blot analysis showed that C/EBP isoforms, 21-kDa LIP as well as the 38- and 35-kDa LAP isoforms, were expressed in rat lung fibroblasts. IL-1 treatment preferentially enhanced the nuclear accumulation of LIP and significantly decreased the LAP/LIP ratio. Transient transfection and gene knockout (KO) analysis demonstrated that LAP stimulated, whereas LIP inhibited, expression of the -SMA gene. Promoter scanning identified a C/EBP binding consensus sequence in the -SMA promoter that was shown to be important for -SMA gene expression by site-directed mutagenesis. Binding of C/EBP to this promoter was demonstrated by EMSA. Further study showed that IL-1 enhanced the expression of eukaryotic initiation factor-4E (eIF-4E), which is an activator for expression of the C/EBP inhibitor isoform LIP. These data taken together strongly suggest an important role for C/EBP in mediating the regulation of -SMA gene expression by IL-1.

	Materials and Methods

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Animals and cell culture

C/EBP-deficient male mice (–/–) were gifts from Dr. P. F. Johnson (National Cancer Institute, National Institutes of Health, Bethesda, MD) and were generated as described previously (23). They were mated with wild-type C57BL/6 females for generation of heterozygous progeny (+/–), which were genotyped as previously described (23). Then the male and female heterozygous progeny were mated to generate homozygous C/EBP (–/–) KO and wild type (+/+) littermate control mice. Fibroblasts were isolated from adult murine and rat (Fisher 344) lungs as described previously (7). Briefly, animals were killed, and their lungs were perfused with PBS. The lung tissue was digested in trypsin-EDTA solution until the cells were released. The cells were cultured in complete medium, composed of DMEM supplemented with 10% plasma-derived serum (PDS; Cocalico Biologicals, Reamstown, PA), 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml Fungizone, 1% insulin/transferrin/selenium (Sigma-Aldrich, St. Louis, MO), 5 ng/ml platelet-derived growth factor (R&D Systems, Minneapolis, MN), and 10 ng/ml EGF (R&D Systems, Minneapolis, MN). These cells were confirmed as fibroblasts on the basis of stellate morphology, production of collagen type I, and ability to propagate in this medium.

IL-1 treatment

Cells were cultured overnight in complete medium. They were deprived of growth factors by rinsing twice in PBS and incubating in DMEM containing 0.5% PDS for 24 h. This was followed by addition of IL-1 (R&D Systems) at the indicated concentrations. After 6 and 48 h, cells were harvested for mRNA and protein analyses, respectively, as described below.

-SMA promoter constructs and cDNA clones

The rat -SMA promoter was previously cloned by PCR from rat genomic DNA (7). It was inserted into vector pGL3-basic (Promega) at the SmaI site to form the -SMAp-luc fusion plasmid pGal3-SMAp, which was used as the template for construction of -SMA promoter mutants. A C/EBP binding consensus, TTTGGGAAG, was identified at –67 to –58 from the transcriptional start site in the rat -SMA promoter. In accordance with the -SMA promoter sequence, primers A (5'-TTTGCTCCTTGTTTGCCGGCCGAGTGGGAGGGGATCAGACCAG-3') and B (5'-CTGGTCTGATCCCCTCCCACTCGGCCGGCAAACAAGGAGCAAA-3') were used for construction of the C/EBP binding consensus mutated -SMA promoter mutant -SMAp-luc-C/EBPmut to confirm the functional importance of this C/EBP binding consensus. Primers C (5'-TGCAAACCATGCCTGCAGATGCTTCATGACACTAGC-3') and D (5'-GCAAGTGTCATGAAGGTTCTGCAGGCAGGGTTTGCA-3') were used to construct a control -SMA promoter mutant -SMAp-luc-control with a mutation outside the C/EBP binding consensus. An EagI or a PstI restriction endonuclease site was created in each mutant, respectively, for screening. All mutants were constructed with the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). These mutants were then inserted into vector pGL3-Basic to form fusion plasmid pGal3-SMAp-C/EBPmut and pGal3-SMAp-Control. Rat C/EBP cDNA (35-kDa LAP) expression plasmid pCMV-LAP and C/EBP cDNA (21-kDa LIP) expression plasmid pCMV-LIP were gifts from Dr. J. Schwartz (University of Michigan) and were originally obtained from Dr. U. Schibler (University of Geneva, Geneva, Switzerland) (20).

mRNA analysis

For analysis of -SMA mRNA levels, real-time PCR was used as previously described (24). Results were expressed as 2^–CT with GAPDH used as endogenous control (24). For analysis of eIF-4E mRNA expression, total RNA samples were used for RT-PCR analysis. RT-PCR was undertaken with the SuperScript one-step RT-PCR system (Invitrogen Life Technologies, Gaithersburg, MD) and using the following protocol: one cycle each of 50°C for 30 min and 94°C for 2 min, followed by 26 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 2 min, and finally by one cycle of 72°C for 10 min. To normalize the amounts of input RNA, amplification of the GAPDH mRNA signal was used as an internal control. The following primers were used: eIF-4E, 5'-CCGAATTCAGGAGCGGTTGTGCG-3' and 5'-GAGGCCCCTCAGAAGGTGTCTTC; and GAPDH, 5'-GTCTTCTGAGTGGCAGTGATG-3' and 5'-TCCAGTATGACTCTACCCACG-3'. Equal aliquots of each sample were electrophoresed on 1.5% agarose gels. Bands were visualized by ethidium bromide staining.

Immunoblotting

Cells (7 x 10⁴/well) were plated in six-well plates and treated with IL-1 as described above. The cells were harvested by scraping into Laemmli sample loading buffer. Equal amount of proteins were electrophoresed through 12% SDS-polyacrylamide gels. Where indicated in the case of analysis of LIP and LAP expression, nuclear extracts were used and prepared as previously described (7). Equal loading was confirmed by probing the blots for -tubulin using specific Abs (Santa Cruz Biotechnology, Santa Cruz, CA). The separated protein bands were transferred onto nitrocellulose membranes. Nonspecific binding was blocked with 10% nonfat milk (Bio-Rad, Hercules, CA) in 10 mM Tris-buffered saline containing 0.5% Tween 20). -SMA was detected using anti--SMA mAb (Cymbus Biotechnology, Hampshire, U.K.) at a dilution of 1/2000, an anti-mouse IgG linked to HRP (Amersham Biosciences, Piscataway, NJ), and chemiluminescent substrate LumiGLO (New England Biolabs, Beverly, MA). C/EBP Ab (sc-7962X) against both C/EBP isoforms was purchased from Santa Cruz Biotechnology. The blots were exposed to Hyperfilm ECL film (Amersham Biosciences).

Transfection and reporter gene assay

All transient transfections were performed using the FuGENE 6 reagent (Roche, Indianapolis, IN) according to the manufacturer’s instructions. Supercoiled DNA was isolated with an endotoxin-free column kit (Qiagen, Valencia, CA). Unless otherwise indicated, cells were seeded in six-well plates at a density of 10⁵/well in DMEM containing 10% PDS and incubated at 37°C overnight. Two micrograms of DNA of the construct of interest and 1 µg of plasmid pRL-TK control vector (used for normalization) were cotransfected per culture in DMEM containing 0.5% PDS with or without 10 ng/ml IL-1 treatment. After 48 h, the cells were harvested, and the activity of firefly or Renilla luciferase was measured using the dual luciferase assay system from Promega (Madison, WI). The relative luciferase activity was calculated by normalizing firefly luciferase activity to that of Renilla luciferase. Experiments with each construct were repeated two to four times, and relative activity (fold over promoterless control) was expressed as the mean ± SE.

Nuclear extract preparation

Nuclear extracts were prepared from untreated and IL-1-treated cultures as previously described (7, 9). Briefly, the cultures were rinsed twice with cold PBS and then with Dignam’s buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, and 4 µg/ml leupeptin). The cells were then scraped into buffer A and washed once with the same buffer. The cell pellet was finally resuspended in buffer A and kept on ice for 15 min before brief extraction in 0.6% Nonidet P-40. The extract was vortexed and centrifuged briefly, and the cytoplasmic extract was removed. The nuclei were further extracted in Dignam’s buffer C (20 mM HEPES, 1.5 mM MgCl₂, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, and 4 µg/ml leupeptin) for 20 min on ice. The extracts were centrifuged, and the supernatants were stored at –70°C until used. The protein concentration was tested by the bicinchoninic acid method (Pierce, Rockford, IL).

EMSA

EMSA was conducted as previously described (7). Single-stranded oligonucleotides in the sense and antisense directions were synthesized to detect the binding of C/EBP. The sense probe sequence is 5'-TTTGCTCCTTGTTTGGGAAGCGAGTGGGAGGGGATCAGACCAG-3', corresponding to the region between –78 and –36 from the transcription start site of the -SMA promoter. Another set of primers in which the C/EBP binding consensus was altered with the sense sequence 5'-TTTGCTCCTTGTTTGCCGGCCGAGTGGGAGGGGATCAGACCAG-3' was prepared to confirm the specificity of binding. The oligonucleotides were annealed before labeling with T4 polynucleotide kinase and [-³²P]ATP to detect dsDNA-binding proteins. EMSA reaction mixtures contained 3–5 µg of protein extract, 1.0 µg of poly(dI-dC), 0.1 µg of poly-L-lysine, 0.5–1 ng of labeled probe (20,000–30,000 cpm), and Abs as indicated in a final volume 15 µl of Dignam’s buffer C. Where indicated, the EMSA reaction mixtures were preincubated with Abs on ice for 30 min before probe addition and incubation for another 20 min at room temperature. Samples were then analyzed by electrophoresis on 4% nondenaturing polyacrylamide gels at 100 V in 1x 89 mM Tris-borate and 2 mM EDTA, pH 8.0. After electrophoresis, the gels were dried and exposed to x-ray film for 24 h.

Statistical analysis

This was undertaken as before using ANOVA, followed, where appropriate, by post hoc testing using Scheffé’s test (24). A value of p < 0.05 was used as a criterion for statistical significance in comparisons between any two groups.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of IL-1 on fibroblast C/EBP expression

-SMA expression is a key phenotypic marker of myofibroblast differentiation from fibroblasts (1, 2, 3). The differentiation process is repressed by cytokines such as IL-1, one of the major cytokines that mediate inflammatory reactions (12). However, the mechanism of this inhibition is unclear. The basic leucine zipper transcription factor C/EBP belongs to the C/EBP) family and is reported to be induced by IL-1 in rat liver (25). To investigate whether C/EBP can play a role in the repression of -SMA gene expression by IL-1, the expression of C/EBP in rat lung fibroblasts was analyzed by Western blot analysis. The results showed that control untreated cell nuclei contained detectable levels of the LAP forms (35 and 38 kDa) of C/EBP, with very low levels of the LIP (21-kDa) isoform (Fig. 1). Treatment with IL-1 increased the nuclear levels of both LAP and LIP isoforms in a dose-dependent manner while concomitantly decreasing -SMA expression. However in response to the same doses of IL-1, the increase in LIP (6.5-fold at 50 ng/ml IL-1 treatment) was greater than that in LAP (2.5-fold at 50 ng/ml IL-1 treatment), resulting in a decreasing LAP/LIP ratio (from 6.74 in control cells to 2.34 in cells treated with 50 ng/ml IL-1). Thus, the altered expression ratio between these C/EBP isoforms after IL-1 treatment was associated with alterations in -SMA expression, suggesting a role for these isoforms in mediating the IL-1 signaling in myofibroblast differentiation.

View larger version (58K): [in this window] [in a new window]   FIGURE 1. Effects of IL-1 on C/EBP and -SMA expression. Rat lung fibroblasts were treated with the indicated doses of IL-1 for 48 h, then harvested for Western blotting analysis to evaluate expression of -SMA (upper panel) and C/EBP isoforms (lower panel). Equal amounts of protein were loaded for gel electrophoresis before blotting. All samples for detecting -SMA were from whole-cell lysates, and samples for detecting C/EBP were from nuclear extracts. The different isoforms of C/EBP were indicated by arrows. The numbers beneath each lane of the -SMA blot (upper panel) indicate the relative band intensities (as a percentage of the untreated control band intensity) determined by densitometric scanning, with the untreated control set at 100%. The numbers beneath each lane of the blot of C/EBP isoforms (lower panel) indicate the relative total LAP or LIP band intensities (as a percentage of LIP band intensity in the untreated control) determined by densitometric scanning, with the untreated LIP control set at 100%. The LAP/LIP ratio is also indicated at the bottom row of numbers beneath each lane. For loading control, the blot for -tubulin is shown. Representative blots of -SMA and C/EBP isoforms are shown. This experiment was repeated three times with similar results.

Effects of C/EBP isoforms on -SMA expression

To confirm the role of C/EBP in the regulation of -SMA gene expression by IL-1, LAP and LIP expression plasmids were used to induce their transient expression in rat lung fibroblasts, and the effects on -SMA expression were analyzed by real-time PCR. As expected, transfection with the LIP plasmid caused a significant increase in nuclear LIP protein levels in control cells as well as in cells treated with IL-1 (Fig. 2A, left panel). However, the LIP plasmid-induced increase in IL-1-treated cells was less due to the higher baseline levels of LIP in these cells. Transfection with the LAP plasmid did not affect LIP levels in these cells. Similarly, transfection with the LAP plasmid increased nuclear LAP levels, although the increases were smaller than in LIP levels induced by the LIP plasmid (Fig. 2A, right panel). These were probably due to the already higher endogenous levels of LAP in cells transfected with the empty vector only. Transfection with the LIP plasmid also had no effect on nuclear LAP levels. The effects of these alterations in nuclear LIP or LAP levels on -SMA expression were then analyzed by real-time PCR. The results (Fig. 2B) show that transfection with the LAP expression plasmid caused a significant increase in -SMA expression in both control and IL-1-treated rat lung fibroblasts relative to that in cells transfected with the control empty vector. This was in contrast to the significant reduction in cells transfected with the LIP expression plasmid. Western blot analysis showed similar responses in -SMA protein expression, namely, stimulation by transfection with LAP expression plasmid and inhibition by the LIP expression plasmid, although the stimulation by the LAP plasmid was less in untreated cells than in IL-1-treated cells (Fig. 3).

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Effect of C/EBP isoforms on fibroblast -SMA mRNA levels. Rat lung fibroblasts were transfected for 24 h with empty vector (pCDNA3) or plasmids expressing LIP (pCMV-LIP) or LAP (pCMV-LAP) as indicated. The cells were then treated for 6 h with buffer only (NONE) or with IL-1 (10 ng/ml). At the end of incubation, nuclear extracts were prepared for analysis of LIP and LAP protein by Western blotting (A). Bands corresponding to either isoform were quantitated by densitometric scanning and expressed as a percentage of the signal from untreated cells (NONE) transfected with the empty vector only (pcDNA3). The LIP plasmid caused significant increases in nuclear LIP protein levels in both control and IL-1-treated cells, but had no significant effect on LAP levels (A, left panel). Transfection with the LAP plasmid caused significant increases in LAP protein levels, but not LIP levels (A, right panel). Total RNA was isolated from additional cell samples for analysis of -SMA mRNA levels by real-time PCR (B). Results were expressed as 2–CT, with GAPDH used as the endogenous control. Data are shown as the mean ± SE of triplicate samples. The stimulation by LAP plasmid and the inhibition by LIP plasmid were statistically significant in both untreated and IL-1-treated cells, whereas the effect of IL-1 was significant in all groups.

View larger version (39K): [in this window] [in a new window]   FIGURE 3. Effect of C/EBP on -SMA expression. Plasmids expressing C/EBP isoforms LIP (pCMV-LIP), LAP (pCMV-LAP), or empty vector only (pCDNA3) were transfected into lung fibroblasts. The cells were treated with buffer only (None) or with IL-1 (10 ng/ml). They were then lysed, and the cell extracts were analyzed by Western blotting for -SMA protein expression. Equal amounts of protein were loaded for gel electrophoresis before blotting. A representative blot is shown in A, and the result of densitometric scanning of replicates (mean ± SE; n = 4) is shown in B. Relative to the empty vector (pcDNA3), the stimulatory effects of the LAP plasmid were significant only in IL-1-treated cells, whereas the inhibitory effects of the LIP plasmid were significant in both untreated and IL-1-treated cells.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Effect of C/EBP isoforms on -SMA promoter activity. Fibroblasts were transfected with the -SMA promoter construct (pGal3--SMAp-luc, also abbreviated as -SMAp-luc; left panel) or the same construct mutated at its C/EBP binding consensus sequence (-SMAP-luc-C/EBPmut; right panel), then cotransfected with the indicated individual C/EBP construct (pCMV-LAP for LAP; pCMV-LIP for LIP) or expression vector only (pCDNA3). The cells were then treated with buffer only (NONE) or with 10 ng/ml IL-1 (IL-1) for 48 h, then the cell extracts were harvested and assayed for luciferase activities. The luciferase activity was normalized for each construct to its respective Renilla luciferase control activity, and the results are shown as the fold increase over the promoterless activity. Data represent the mean ± SE of triplicate samples. Transfection of untreated cells with the LAP plasmid caused significant stimulation of wild-type promoter activity, whereas the LIP plasmid caused its significant inhibition (left panel). IL-1-treated cells also responded similarly, but at a significantly lower level of promoter activity. Neither LAP nor LIP plasmid had a significant effect on the mutant promoter activity in untreated or IL-1-treated cells (right panel).

Effects of C/EBP deficiency on -SMA expression

To further confirm the importance of C/EBP isoforms in the regulation of -SMA gene expression, lung fibroblasts from C/EBP homozygous KO (–/–), heterozygous KO (–/+), or wild-type (+/+) mice were analyzed for -SMA expression by Western blotting and -SMA promoter activity. The results of Western blotting analysis showed that the level of lung fibroblast -SMA expression correlated with the level of C/EBP isoform expression in the various mouse strains (Fig. 5). Thus, the level of -SMA expression progressively decreased in cells from wild-type to homozygous KO mice, with intermediate expression in cells from heterozygous KO mice. These gradations in -SMA expression were also noted in cells treated with IL-1. However, in IL-1-treated cells, -SMA expression was decreased relative to their respective untreated control cells, except for the cells from homozygous KO mice, which essentially did not respond to IL-1 treatment.

View larger version (40K): [in this window] [in a new window]   FIGURE 5. Effect of C/EBP deficiency on -SMA expression. Fibroblasts were isolated from C/EBP KO mice (–/–), heterozygous deficient mice (–/+), or wild-type littermates (+/+). They were treated with 10 ng/ml IL-1 or buffer only (NONE) for 48 h. Then the whole cell protein or nuclear extracts were analyzed for -SMA (upper panel) or C/EBP (lower panel) isoforms, respectively, by Western blotting. The numbers beneath each lane of the -SMA blot (upper panel) indicate the relative level of expression (as a percentage of the untreated control) determined by densitometric scanning, with the untreated control set at 100%. For the loading control, the blot for -tubulin is shown.

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Effect of C/EBP deficiency on -SMA promoter activity. Wild-type -SMA promoter construct (pGal3--SMAp-luc, also abbreviated as -SMAp-luc) was transfected into fibroblasts isolated from C/EBP KO mice (–/–), heterozygous deficient mice (–/+), or wild-type (+/+) littermates, then treated with 10 ng/ml IL-1 or buffer only (NONE) for 48 h. The cell extracts were harvested and assayed for luciferase activities. The luciferase activity for each construct was normalized to its respective Renilla luciferase internal control activity and shown as the fold increase over the promoterless activity. Data represent the mean ± SE of triplicate samples. Promoter activity in untreated treated cells from KO mice was significantly lower than that in cells from heterozygous or wild-type mice, whereas heterozygous cells had significantly lower activity than wild-type cells. IL-1 treatment caused a significant reduction in promoter activity only in wild-type and heterozygous cells.

Role of C/EBP binding consensus in the -SMA promoter

In view of the preceding results, the -SMA gene promoter sequence was scanned for a possible C/EBP binding sequence to further confirm direct regulation of the -SMA promoter by this transcription factor. C/EBP is thought to recognize and bind to a TKNNGNAAK (K = T or G, N = A, T, C, or G) consensus sequence to regulate its target gene expression (26). The scanning results identified such a consensus sequence, TTTGGGAAG, at –66 to –58 upstream of the transcription start site in the rat -SMA promoter (Fig. 7A). To determine whether this consensus sequence is actually functional or important for -SMA gene expression, site-directed mutagenesis was used to construct a C/EBP binding consensus mutated -SMA promoter mutant (-SMAp-luc-C/EBPmut) and an -SMA promoter control mutant with altered sequence outside of the C/EBP consensus sequence (-SMAp-luc-control). These -SMA promoter mutants and the wild-type -SMA promoter (-SMAp-luc) were each inserted into promoterless luciferase vector pGL3-basic to drive the transcription of luciferase in the corresponding constructs (Fig. 7A). Upon transfection into rat lung fibroblasts, the wild-type construct showed expected promoter activity that was suppressed by IL-1 treatment (Fig. 7B). The construct mutated at the C/EBP binding consensus sequence (-SMAp-luc-C/EBPmut) showed markedly reduced -SMA promoter activity (<40% of wild-type promoter), which was not further affected by IL-1 treatment. The activity of the control mutant promoter construct (-SMAp-luc-control) was comparable to that for the wild-type control in the absence or the presence of IL-1 treatment. These findings confirmed the importance of the C/EBP consensus sequence in regulation of -SMA gene expression, especially for its inhibition by IL-1.

View larger version (22K): [in this window] [in a new window]   FIGURE 7. Effect of C/EBP binding consensus mutation on -SMA promoter activity. A, A representation map of wild-type -SMA promoter and mutant constructs is shown. The relative locations of the C/EBP binding consensus, TTTGGGAAG, and the site for control mutation are indicated with their nucleotide sequences. Mutated bases in the C/EBP binding site and control constructs are italicized and underlined. These wild-type and mutant constructs were transfected into fibroblasts, and the cells were treated with buffer only (NONE) or with IL-1 (IL-1). Cell extracts were then analyzed for luciferase activities. The normalized luciferase activities in cells transfected with the indicated constructs (letters on the abscissa refer to the constructs listed in A) are shown in B. Results were expressed as the fold increase over the promoterless control mean value and are shown as the mean ± SE of triplicate samples. The activity of the mutant construct (B) was significantly lower than those of the wild-type (A) and control (C) constructs. In contrast to the significant inhibition of both wild-type and control constructs by IL-1 treatment, the activity of the mutant construct was not significantly affected by IL-1.

Binding of C/EBP to the -SMA promoter C/EBP binding consensus

Because C/EBP isoforms and the C/EBP binding consensus sequence in the -SMA promoter appeared to be important for -SMA gene expression, the possibility of direct binding of this transcription factor to the consensus sequence was examined. To evaluate such a direct interaction of C/EBP with the TTTGGGAAG binding consensus in the -SMA promoter, single-stranded sense and antisense oligonucleotides corresponding to this sequence at –78 to –36 of the -SMA promoter were synthesized and annealed as the probe used in EMSAs. The results showed that a complex was formed when the probe was incubated with nuclear extracts from either untreated control or IL-1-treated fibroblasts, although complex formation was dramatically enhanced with extracts from IL-1-treated cells (Fig. 8). Complex formation was specific because competition with a 100-fold excess of unlabeled probe abolished the shifted band. Furthermore, incubation with anti-C/EBP Abs caused the band to supershift, indicative of the presence of C/EBP in the DNA-protein complex. Mutation in the binding consensus abrogated the formation of the DNA-protein complex (data not shown). These results indicated that C/EBP could bind to the C/EBP binding consensus in the -SMA promoter, which was enhanced by treating cells with IL-1. Such binding appears to play a key role in regulation of -SMA gene expression, especially its inhibition by IL-1.

View larger version (58K): [in this window] [in a new window]   FIGURE 8. C/EBP binding to its consensus binding sequence in -SMA promoter. EMSA was used to analyze binding of C/EBP to an oligonucleotide probe containing the C/EBP binding consensus sequence found in the -SMA promoter. The labeled probe was incubated with nuclear extracts from either IL-1-treated (10 ng/ml) or untreated rat lung fibroblasts as indicated, and then were analyzed by gel electrophoresis. The DNA-protein complex is indicated by a solid arrow. Selected samples were pretreated with anti-C/EBP Abs against both LAP and LIP isoforms at the indicated doses before addition of radiolabeled probe. The location of supershifted bands is indicated by an open arrow. Addition of a 100-fold excess of unlabeled probe, as indicated, was used to document the specificity of binding.

IL-1-induced eIF-4E expression

The findings from the above experiments suggested that IL-1 inhibited -SMA gene expression by altering the ratio of C/EBP isoforms that act on the C/EBP binding consensus sequence in the -SMA promoter. eIF-4E, a 25-kDa cap binding protein involved in the initiation of protein translation (27), is known to selectively promote LIP expression (28). To investigate whether IL-1 reduced the LAP/LIP ratio via such a mechanism, the expression of eIF-4E in rat lung fibroblasts was analyzed by RT-PCR and Western blot. The results shown in Fig. 9 indicated that the expression of eIF-4E at both the mRNA and protein levels was stimulated by IL-1 treatment in a dose-dependent manner. As a control, the cellular levels of GAPDH mRNA were found not to be significantly affected by IL-1. Because eIF-4E is known to selectively activate LIP expression (28), its induction by IL-1 suggests a potential role for this factor in mediating reduction of the LAP/LIP ratio by IL-1.

View larger version (74K): [in this window] [in a new window]   FIGURE 9. Effect of IL-1 on eIF-4E expression. Rat lung fibroblasts were treated with the indicated doses of IL-1 for 6 h (for RT-PCR analysis) or 48 h (for Western blotting analysis), and the total RNA or total protein was harvested for RT-PCR (upper panel) or Western blotting (lower panel) analysis to evaluate expression of eIF-4E. The total RNA was analyzed by RT-PCR for eIF-4E and GAPDH mRNA levels. For Western blotting analysis, equal amounts of total protein were loaded per lane, and after blotting, proteins were detected with Abs to eIF-4E. Results from a representative experiment are shown. This experiment was repeated three times with similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We showed that IL-1-induced, dose-dependent reduction in -SMA expression in rat lung fibroblasts was associated with a corresponding dose-dependent increase in the level of C/EBP isoform expression. However, the increase in LIP isoform expression was substantially greater than that for the LAP isoforms, resulting in a reduced LAP/LIP ratio that correlated with the reduction in -SMA expression. These results suggest that LAP/LIP ratio may be important for controlling the -SMA expression by IL-1. This conclusion is supported by transient transfection studies showing that transfection of LAP expression plasmid stimulated, whereas transfection of LIP expression plasmid, inhibited -SMA expression. The importance of C/EBP in regulating -SMA gene expression was further underscored by reduced -SMA expression in C/EBP-deficient cells. In its complete absence in cells from homozygous deficient mice, -SMA expression was approximately reduced by half relative to the level in their wild-type counterparts. Moreover, a C/EBP consensus binding sequence was identified in the -SMA promoter and found to be important for -SMA gene expression by site-directed mutagenesis studies. Thus, the C/EBP isoforms may play a direct role in regulation of the -SMA promoter via binding interactions with this consensus binding sequence, which was confirmed by EMSA in combination with supershift analysis. The importance of this direct C/EBP regulation of the -SMA promoter to IL-1 regulation of -SMA gene expression is demonstrated by the lack of responsiveness to IL-1 in C/EBP-deficient cells and in cells transfected with an -SMA promoter with a mutated C/EBP consensus binding sequence. Thus, the totality of these findings provides adequate proof that C/EBP is important in regulation of -SMA expression, and that IL-1 inhibition of such expression is entirely dependent on this transcription factor as mediated by its LIP isoform, perhaps through dominant negative inhibition of LAP activity. LIP is known to be a dominant negative isoform of LAP with the capacity to control LAP activity at substoichiometric concentrations (20, 22). The mechanism of inhibition by LIP is believed to be due to the greater binding capability of LIP to the C/EBP binding element or consensus sequence, which is 4-fold higher than that of LAP (20). Although LIP can preferentially bind to this element in the -SMA promoter at the expense of LAP, it lacks the activation domain present in LAP and is consequently unable to activate the target gene. Consequently, a decreasing LAP/LIP ratio will result in diminution of the activating effects of LAP (20). Hence, the preferential stimulation of LIP by IL-1 represents a key mechanism by which this inflammatory cytokine inhibits -SMA gene expression.

The mechanism for controlling the LAP/LIP ratio is not fully understood, although there is evidence for eIF-4E-mediated control at the translational level. eIF-4E binds to the 7-methyl guanosine-containing cap of mRNA and facilitates the binding of the eIF-4G and eIF-4A complexes (27, 28, 29). These subunits act as an RNA helicase, resulting in the unwinding of the 5'-untranslated regions of mRNA, the binding by the 40S ribosome subunit and the initiation of protein synthesis (30, 31). eIF-4E is the least abundant initiation factor in most cells, and its activity is increased in response to many extracellular stimuli that stimulate cell growth. Thus, overexpression of eIF4E would be expected to preferentially increase the translation of mRNAs that are poorly translated due to presence of extensive 5' secondary structure. In the case of C/EBP, the LAP and LIP isoforms are believed to be translated from the same mRNA, but with different translation initiation sites (20), with eIF-4E enhancing the expression of the truncated isoforms in an upstream (relative to the LIP translation initiation site) open reading frame-dependent manner (28). As for how IL-1 controls the LAP/LIP ratio, our results indicated that eIF-4E may be involved. In this study we showed for the first time the enhancement of eIF-4E expression by IL-1 treatment, which could account for the preferential increase in LIP expression, resulting in the observed reduced LAP/LIP ratio. Further studies are needed to fully delineate the role of eIF-4E and other potential factors in differential regulation of C/EBP isoform expression by IL-1.

	Acknowledgments

 
We are grateful for the generous gifts of rat C/EBP plasmids from Drs. Jessica Schwartz (University of Michigan) and Ueli Schibler (University of Geneva), and of C/EBP knockout mice from Dr. Peter F. Johnson (National Cancer Institute, National Institutes of Health). The expert technical assistance of Bridget McGarry and Matthew R. Ullenbruch is also gratefully acknowledged.

	Footnotes

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

¹ This work was supported in part by National Institutes of Health Grants HL28737, HL31963, HL52285, and HL56402.

² Address correspondence and reprint requests to Dr. Sem H. Phan, Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-0602. E-mail address: shphan{at}umich.edu

³ Abbreviations used in this paper: -SMA, -smooth muscle actin; eIF-4A, eukaryotic initiation factor 4A; eIF-4E, eukaryotic initiation factor 4E; eIF-4G, eukaryotic initiation factor 4G; KO, knockout; LAP, liver-enriched activator protein; LIP, liver-enriched inhibitory protein; PDS, plasma-derived serum.

Received for publication March 1, 2004. Accepted for publication July 28, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Gabbiani, G., G. B. Ryan, G. Majno. 1971. Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction. Experientia 27:549.[Medline]
2. Grinnell, F.. 1994. Fibroblasts, myofibroblasts, and wound contraction. J. Cell Biol. 124:401.[Free Full Text]
3. Kuhn, C., J. A. McDonald. 1991. The roles of the myofibroblast in idiopathic pulmonary fibrosis: ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis. Am. J. Pathol. 138:1257.[Abstract]
4. Desmouliere, A., G. Gabbiani. 1995. Myofibroblast differentiation during fibrosis. Exp. Nephrol. 3:134.[Medline]
5. Schelling, J. R., S. Sinha, M. Konieczkowski, J. R. Sedor. 2002. Myofibroblast differentiation: plasma membrane microdomains and cell phenotype. Exp. Nephrol. 10:313.[Medline]
6. Gauldie, J., P. J. Sime, Z Xing, B Marr, G. M. Tremblay. 1999. Transforming growth factor- gene transfer to the lung induces myofibroblast presence and pulmonary fibrosis. Curr. Top. Pathol. 93:35.[Medline]
7. Hu, B, Z. Wu, S. H. Phan. 2003. Smad3 mediates transforming growth factor--induced -smooth muscle actin expression. Am. J. Respir. Cell Mol. Biol. 29:397.[Abstract/Free Full Text]
8. Phan, S. H.. 2002. The myofibroblast in pulmonary fibrosis. Chest 122:286S.[Abstract/Free Full Text]
9. Phan, S. H.. 1996. Role of the myofibroblast in pulmonary fibrosis. Kidney Int. 54:(Suppl.):S46.
10. Phan, S. H., K. Zhang, H. Y. Zhang, M. Gharaee-Kermani. 1999. The myofibroblast as an inflammatory cell in pulmonary fibrosis. Curr. Top. Pathol. 93:173.[Medline]
11. Tomasek, J. J., G. Gabbiani, B. Hinz, C. Chaponnier, R. A. Brown. 2002. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat. Rev. Mol. Cell. Biol. 3:349.[Medline]
12. Zhang, H. Y., M. Gharaee-Kermani, S. H. Phan. 1997. Regulation of lung fibroblast -smooth muscle actin expression, contractile phenotype, and apoptosis by IL-1. J. Immunol. 158:1392.[Abstract]
13. Buck, M., V. Poli, P. van der Geer, M. Chojkier, T. Hunter. 1999. Phosphorylation of rat serine 105 or mouse threonine 217 in C/EBP is required for hepatocyte proliferation induced by TGF. Mol. Cell 4:1087.[Medline]
14. Darlington, G. J., S. E. Ross, O. A. MacDougald. 1998. The role of C/EBP genes in adipocyte differentiation. J. Biol. Chem. 273:30057.[Free Full Text]
15. Hadaschik, D., K. Hinterkeuser, M. Oldak, H. J. Pfister, S. Smola-Hess. 2003. The papillomavirus E2 protein binds to and synergizes with C/EBP factors involved in keratinocyte differentiation. J. Virol. 77:5253.[Abstract/Free Full Text]
16. Katz, S., E. Kowenz-Leutz, C. Muller, K. Meese, S. A. Ness, A. Leutz. 1993. The NF-M transcription factor is related to C/EBP and plays a role in signal transduction, differentiation and leukemogenesis of avian myelomonocytic cells. EMBO J. 12:1321.[Medline]
17. Soriano, H. E., T. A. Bilyeu, T. S. Juan, W. Zhao, G. J. Darlington. 1995. DNA binding by C/EBP proteins correlates with hepatocyte proliferation. In Vitro Cell Dev. Biol. Anim. 31:703.[Medline]
18. Baer, M., S. C. Williams, A. Dillner, R. C. Schwartz, P. F. Johnson. 1998. Autocrine signals control CCAAT/enhancer binding protein expression, localization, and activity in macrophages. Blood 92:4353.[Abstract/Free Full Text]
19. Buck, M., H. Turler, M. Chojkier. 1994. LAP (NF-IL-6), a tissue-specific transcriptional activator, is an inhibitor of hepatoma cell proliferation. EMBO J. 13:851.[Medline]
20. Descombes, P., U. Schibler. 1991. A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA. Cell 67:569.[Medline]
21. Ossipow, V., P. Descombes, U. Schibler. 1993. CCAAT/enhancer-binding protein mRNA is translated into multiple proteins with different transcription activation potentials. Proc. Natl. Acad. Sci. USA 90:8219.[Abstract/Free Full Text]
22. Dearth, L. R., J. Hutt, A. Sattler, A. Gigliotti, J. DeWille. 2001. Expression and function of CCAAT/enhancer binding protein (C/EBP) LAP and LIP isoforms in mouse mammary gland, tumors and cultured mammary epithelial cells. J. Cell. Biochem. 82:357.[Medline]
23. Sterneck, E., L. Tessarollo, P. F. Johnson. 1997. An essential role for C/EBP in female reproduction. Genes Dev. 11:2153.[Abstract/Free Full Text]
24. Liu, T., S. M. Dhanasekaran, H. Jin, S. A. Tomlins, A. M. Chinnaiyan, S. H. Phan. 2004. Induction of FIZZ-1 expression in lung injury and fibrosis. Am. J. Pathol. 164:1315.[Abstract/Free Full Text]
25. Magalini, A., G. Savoldi, F. Ferrari, M. Garnier, P. Ghezzi, A. Albertini, D. Di Lorenzo. 1995. Role of IL-1 and corticosteroids in the regulation of the C/EBP-, and genes in vivo. Cytokine 7:753.[Medline]
26. Rosati, M., A. Valentin, D. J. Patenaude, G. N. Pavlakis. 2001. CCAAT-enhancer-binding protein (C/EBP) activates CCR5 promoter: increased C/EBP and CCR5 in T lymphocytes from HIV-1-infected individuals. J. Immunol. 167:1654.[Abstract/Free Full Text]
27. Rhoades, R. E., S. Joshi-Barve, C. Rinker-Schaeffer. 1993. Mechanism of action and regulation of protein synthesis initiation factor 4E: Effects of mRNA discrimination, cellular growth rate, and oncogenesis. Prog. Nucleic Acid Res. Mol. Biol. 46:183.[Medline]
28. Calkhoven, C. F., C. Müller, A. Leutz. 2000. Translational control of C/EBP and C/EBP isoform expression. Genes Dev. 14:1920.[Abstract/Free Full Text]
29. Teraoka, Y., S. Morino, K. Tomoo, T. Ishida. 1996. Mutation of the cysteine residues in human initiation factor 4E: effects on mRNA cap binding ability. Biochem. Biophys. Res. Commun. 228:704.[Medline]
30. Jaramillo, M., T. E. Dever, W. C. Merrick, N. Sonenberg. 1991. RNA unwinding in translation: assembly of helicase complex intermediates comprising eukaryotic initiation factors eIF-4F and eIF-4B. Mol. Cell. Biol. 11:5992.[Abstract/Free Full Text]
31. Pause, A., N. Sonenberg. 1992. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 11:2643.[Medline]

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