| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
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 IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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.
|
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).
|
|
regulates -SMA gene expression at the transcriptional level, the -SMA gene promoter was inserted into promoterless vector pGL3-Basic to form -SMAp-Luc fusion plasmid where the transcription of the luciferase gene was driven by the -SMA promoter. This construct was cotransfected into rat lung fibroblasts with the LAP expression plasmid, the LIP expression plasmid, or the empty expression vector. Luciferase activity was then measured as an indicator of -SMA promoter activity. As shown in Fig. 4 (left panel), relative to cotransfection with the empty vector, cotransfection with the LAP plasmid stimulated, whereas that with the LIP plasmid inhibited, luciferase activity. These differences remained evident when the cells were treated with IL-1, although activities were significantly lower than for their respective nontreated counterparts. These results were consistent with the analysis of -SMA mRNA and protein expression and confirmed that the C/EBP isoforms, LAP and LIP, could divergently regulate transcription of the -SMA gene via the C/EBP-binding consensus sequence present in its promoter. The latter was confirmed by the lack of effect of LAP and LIP plasmids on an -SMA promoter construct mutated at this C/EBP-binding consensus sequence (-SMAP-luc-C/EBPmut; Fig. 4, right panel). Thus, an intact C/EBP binding site in the -SMA promoter is necessary for regulation of transcription by LAP/LIP.
|
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.
|
-SMA promoter activity analysis. Thus, promoter activity as monitored by luciferase activity was highest in cells from wild-type mice, intermediate from heterozygotes, and least from homozygous KO mice (Fig. 6). IL-1 treatment again suppressed promoter activity relative to their corresponding control cells, except for the cells from homozygous KO mice that failed to respond to IL-1 treatment. These results provided additional support for a key role for C/EBP in regulation of -SMA gene expression, in particular its suppression by IL-1.
|
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.
|
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.
|
-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.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-SMA expression is a key characteristic of this myofibroblast differentiation from fibroblasts (8, 9, 10). In contrast, IL-1 represses the differentiation process and -SMA expression, partly through selective induction of myofibroblast apoptosis (12). However, the molecular mechanism for this effect on -SMA expression is unclear. In this study we attempted to evaluate IL-1 regulation of -SMA gene expression and identify potential transcription factors and their cognate binding elements in the -SMA promoter. Based on prior evidence of the ability of IL-1 to up-regulate C/EBP expression in liver, we have chosen to evaluate the role of this transcription factor in rat lung fibroblast in this initial study.
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 |
|---|
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 |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
gene transfer to the lung induces myofibroblast presence and pulmonary fibrosis. Curr. Top. Pathol. 93:35.[Medline]
-induced -smooth muscle actin expression. Am. J. Respir. Cell Mol. Biol. 29:397.-smooth muscle actin expression, contractile phenotype, and apoptosis by IL-1. J. Immunol. 158:1392.[Abstract]
is required for hepatocyte proliferation induced by TGF. Mol. Cell 4:1087.[Medline]
and plays a role in signal transduction, differentiation and leukemogenesis of avian myelomonocytic cells. EMBO J. 12:1321.[Medline]
expression, localization, and activity in macrophages. Blood 92:4353. (C/EBP) LAP and LIP isoforms in mouse mammary gland, tumors and cultured mammary epithelial cells. J. Cell. Biochem. 82:357.[Medline]
in female reproduction. Genes Dev. 11:2153. and corticosteroids in the regulation of the C/EBP-, and 
genes in vivo. Cytokine 7:753.[Medline]
(C/EBP) activates CCR5 promoter: increased C/EBP and CCR5 in T lymphocytes from HIV-1-infected individuals. J. Immunol. 167:1654. and C/EBP isoform expression. Genes Dev. 14:1920.This article has been cited by other articles:
|
|
 |
|
  L. Zheng, Z. Zhou, L. Lin, S. Alber, S. Watkins, N. Kaminski, A. M. K. Choi, and D. Morse Carbon Monoxide Modulates {alpha}-Smooth Muscle Actin and Small Proline Rich-1a Expression in Fibrosis Am. J. Respir. Cell Mol. Biol., July 1, 2009; 41(1): 85 - 92. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M B Goldring, M Otero, K Tsuchimochi, K Ijiri, and Y Li Defining the roles of inflammatory and anabolic cytokines in cartilage metabolism Ann Rheum Dis, December 1, 2008; 67(Suppl_3): iii75 - iii82. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Li, E. Bevilacqua, C.-B. Chiribau, M. Majumder, C. Wang, C. M. Croniger, M. D. Snider, P. F. Johnson, and M. Hatzoglou Differential Control of the CCAAT/Enhancer-binding Protein {beta} (C/EBP{beta}) Products Liver-enriched Transcriptional Activating Protein (LAP) and Liver-enriched Transcriptional Inhibitory Protein (LIP) and the Regulation of Gene Expression during the Response to Endoplasmic Reticulum Stress J. Biol. Chem., August 15, 2008; 283(33): 22443 - 22456. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Milam, V. G. Keshamouni, S. H. Phan, B. Hu, S. R. Gangireddy, C. M. Hogaboam, T. J. Standiford, V. J. Thannickal, and R. C. Reddy PPAR-{gamma} agonists inhibit profibrotic phenotypes in human lung fibroblasts and bleomycin-induced pulmonary fibrosis Am J Physiol Lung Cell Mol Physiol, May 1, 2008; 294(5): L891 - L901. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. H. Phan Biology of Fibroblasts and Myofibroblasts Proceedings of the ATS, April 15, 2008; 5(3): 334 - 337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Zhang, Q. A. Truong-Tran, B. Tancowny, K. E. Harris, and R. P. Schleimer Glucocorticoids Enhance or Spare Innate Immunity: Effects in Airway Epithelium Are Mediated by CCAAT/Enhancer Binding Proteins J. Immunol., July 1, 2007; 179(1): 578 - 589. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Dodig, B. Ogunwale, S. Dasarathy, M. Li, B. Wang, and A. J. McCullough Differences in regulation of type I collagen synthesis in primary and passaged hepatic stellate cell cultures: the role of {alpha}5beta1-integrin Am J Physiol Gastrointest Liver Physiol, July 1, 2007; 293(1): G154 - G164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Hoang, A. Trinh, and R. A. Edwards Decreased MAPK- and PGE2-dependent IL-11 production in Gi{alpha}2-/- colonic myofibroblasts Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1511 - G1519. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Hinz, S. H. Phan, V. J. Thannickal, A. Galli, M.-L. Bochaton-Piallat, and G. Gabbiani The Myofibroblast: One Function, Multiple Origins Am. J. Pathol., June 1, 2007; 170(6): 1807 - 1816. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Prudente, E. Flex, E. Morini, F. Turchi, D. Capponi, S. De Cosmo, V. Tassi, V. Guida, A. Avogaro, F. Folli, et al. A Functional Variant of the Adipocyte Glycerol Channel Aquaporin 7 Gene Is Associated With Obesity and Related Metabolic Abnormalities Diabetes, May 1, 2007; 56(5): 1468 - 1474. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Hu, Z. Wu, T. Liu, M. R. Ullenbruch, H. Jin, and S. H. Phan Gut-Enriched Kruppel-Like Factor Interaction with Smad3 Inhibits Myofibroblast Differentiation Am. J. Respir. Cell Mol. Biol., January 1, 2007; 36(1): 78 - 84. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. S. White, R. G. Atrasz, B. Hu, S. H. Phan, V. Stambolic, T. W. Mak, C. M. Hogaboam, K. R. Flaherty, F. J. Martinez, C. D. Kontos, et al. Negative Regulation of Myofibroblast Differentiation by PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10) Am. J. Respir. Crit. Care Med., January 1, 2006; 173(1): 112 - 121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Han, J. D. Ritzenthaler, B. Wingerd, and J. Roman Activation of Peroxisome Proliferator-activated Receptor {beta}/{delta} (PPAR{beta}/{delta}) Increases the Expression of Prostaglandin E2 Receptor Subtype EP4: THE ROLES OF PHOSPHATIDYLINOSITOL 3-KINASE AND CCAAT/ENHANCER-BINDING PROTEIN {beta} J. Biol. Chem., September 30, 2005; 280(39): 33240 - 33249. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
