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B and Ets Transcription Factors
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, D-60590 Frankfurt am Main, Germany
Address all correspondence and requests for reprints to: Wolfgang Eberhardt, Ph.D., pharmazentrum frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, TheodorStern-Kai 7, D-60590 Frankfurt am Main, Germany. E-mail: w.eberhardt{at}em.uni-frankfurt.de.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B (NF-B) site at -561 to -550 and a region from -511 to -497 bearing a distal activator protein 1 site adjacent to an Ets-binding site are essentially involved in the IL-1ß-mediated transactivation of MMP-9. Inhibition of MMP-9 expression by dexamethasone resides in a promoter region downstream of -597. The IL-1ß-caused increase in DNA binding of both NF-B and Ets-1 immunopositive complexes was substantially suppressed by dexamethasone as shown by EMSA. This was paralleled with a reduced abundance of p65 and Ets-1 proteins in cell nuclei concomitantly with a reduced inhibitor of B (IB) degradation. In addition to NF-B, we suggest a pivotal role for the Ets binding site, in concert with a distal activator protein-1 element, in the transcriptional suppression of cytokine-induced MMP-9 expression by glucocorticoids. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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One common mechanism is thought to arise from mutual interaction between GR and transcriptional activators as reported for activator protein-1 (AP-1) (2), nuclear factor B (NF-B) (3, 4), CCAAT/enhancer binding protein, and signal transducer and activator of transcription 5 (5). All of these transcription factors are essentially involved in the up-regulation of proinflammatory genes, including those for cytokines, chemokines, adhesion molecules, and enzymes. The latter ones involve the matrix metalloproteinases (MMPs), a family of zinc-dependent, neutral proteases degrading specifically components of the extracellular matrix (ECM). MMPs have been implicated in a variety of diseases accompanied with an altered turnover of the ECM (6). In the kidney, a dysregulation of ECM turnover considerably affects the mechanical and functional integrity of the glomerulus, finally leading to the impairment of glomerular filtration (7). Accumulation of ECM proteins, for example, is a hallmark of progressive renal diseases, such as diabetic nephropathy and other conditions leading to glomerulosclerosis (8).
We studied the effects of GCs on cytokine-induced MMP-9 (92-kDa type IV collagenase) expression in rat renal mesangial cells (MCs) because the altered expression of MMP-9 is thought to be a key event in the pathological remodeling of glomerular ECM leading to glomerulosclerosis.
Expression of MMP-9 is regulated by various stimuli including mitogens, growth factors, activators of receptor tyrosine kinases, oncoproteins of the Ras family, phorbol esters, and inflammatory cytokines (9, 10, 11, 12, 13). Recently, we and others have shown that proximal AP-1 and NF-B sites within a 0.6-kb fragment of the MMP-9 promoter region are necessary and sufficient for IL-1ß-dependent MMP-9 promoter activation in rat MCs (11, 14). We now report on an additional functional region located further upstream and encompassing a binding site for Ets transcription factors in close neighborhood to a second AP-1 site. Ets is a member of a transcription factor family identified on the basis of high homology to the v-Ets oncogene. Members of this family are functionally important for angiogenesis and, therefore, suggested to be involved in the pathogenesis of a number of diseases, including rheumatoid arthritis, diabetic retinopathy, and cancer. Ets proteins are functionally highly diverse because they participate in a variety of cellular events including transcriptional regulation, DNA replication, and growth control (15). Ets proteins can mediate transcriptional activation as monomers or in complexes with other transcriptional regulators, e.g. Elk-1 (16). Interestingly, binding sites for Ets are highly conserved in the promoters of different MMPs. Ets-related proteins have been identified as a target for a negative regulation of MMP-1 (collagenase) expression by interaction with the androgen receptor (17). In this study we show that inhibition of MMP-9 expression by GCs is largely due to a diminished transactivation and a reduced binding activity of NF-B and Ets-containing complexes within the 5'-flanking region of the MMP-9 gene independent from a DNA binding to a GRE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, supernatants of MCs under stimulatory conditions contain both gelatinases, MMP-2 and MMP-9, characterized by their different migration properties in the zymogen gel as lytic bands of 70 kDa and 92 kDa, respectively (13). Simultaneous incubation of MCs with dexamethasone substantially reduced the IL-1ß-caused lytic activity of MMP-9. As shown previously, the lytic band migrating at 92-kDa corresponds to the inactive proform of MMP-9 (14). The reduction in the lytical content by dexamethasone is fully reversed upon simultaneous incubation of MCs with dexamethasone and the GR antagonist RU-486, thus demonstrating that the inhibitory effects of dexamethasone are dependent on binding to the GR (Fig. 1A).
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(w.b.), supernatants from untreated control cells contain low levels of MMP-9 protein whereas the amount of MMP-9 protein is much higher in the conditioned media from IL-1ß-stimulated MCs. Similar to the effects observed by zymography, conditioned media derived from dexamethasone-treated MCs showed a significant reduced content of MMP-9 protein (Fig. 1A, lower panel). The conditioned media from MCs cotreated with dexamethasone and RU-486 again contained high MMP-9 protein levels. Therefore, we conclude that the dexamethasone-mediated alterations of zymogen activity predominantly result from changes in the MMP-9 expression levels. Consistent with these observations, dexamethasone did not directly alter MMP-9 activity as monitored by in vitro zymography (data not shown).
Dexamethasone Attenuates Cytokine-Induced MMP-9 mRNA Steady-State Level
Next we performed Northern blot analysis using a cDNA probe from the rat MMP-9 gene (13). MCs were stimulated for 24 h with IL-1ß (2 nM) in the presence of vehicle (control), or dexamethasone (100 nM), with or without RU-486 (1 µM), before RNA extraction. As shown in Fig. 1B, the IL-1ß-caused increase in MMP-9 mRNA steady-state level was almost totally blocked by dexamethasone (from 11.8 ± 3.5-fold to 2.7 ± 0.6-fold induction; mean ± SD, n = 4), whereas coincubation with RU-486 partially reversed the dexamethasone-mediated inhibition.
Furthermore, we found that other glucocorticoids (GCs), including prednisolone, fluocinolone, and hydroxycortisone, potently reduced the cytokine-mediated increase in MMP-9 mRNA level and gelatinolytic content (data not shown). Similar to the different glucocorticoids the mineralocorticoid aldosterone (100 nM) had strong inhibitory effects on the cytokine- induced MMP-9 mRNA level. By contrast, the sex steroids estradiol and testosterone had no effects on either the basal or the cytokine-induced MMP-9 mRNA levels (data not shown).
Cloning of the 5'-Flanking Region of the Rat MMP-9 Gene
To further elucidate the downstream targets of glucocorticoid-mediated inhibition of MMP-9 expression, we cloned a 1.3-kb promoter fragment of the rat MMP-9 gene by "gene walking" using MMP-9 gene-specific antisense primers as described in Materials and Methods. The sequence was subjected to computational analysis using the HUSAR software package (Transfac 3.5). Computer analysis revealed the presence of a TATA box-like sequence preceded by a multitude of putative transcription factor binding site consensus sequences, possibly involved in the regulation of MMP-9 gene transcription by cytokines. These include binding sites for AP-1, NF-B, Myb, peroxisome proliferator-activated receptors, and nuclear factor of IL-6. The sequence and DNA boxes are depicted in Fig. 2, A and B.
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1.3) with the shorter 0.6-kb fragment (pGL-MMP-9-0.6) revealed that pGL-MMP-9-0.6 retained a relative promoter inducibility indistinguishable from that of the long promoter fragment pGL-MMP-9-1.3, although luciferase activities of pGL-MMP-9-0.6 displayed an overall reduced level of total activity (Fig. 2C). These data suggest that elements laying between -1378 and -597 contribute mainly to the basal promoter activity of the MMP-9 gene, but the proximal promoter sequence up to -597 contains the positive elements that confer MMP-9 promoter induction by IL-1ß. Treatment of MCs with dexamethasone resulted in a nearly complete inhibition of cytokine-induced luciferase activities with both MMP-9 promoter constructs (Fig. 2C). RU-486, similar to dexamethasone, had no significant effects on basal promoter activities (data not shown).
Involvement of NF-B and Ets Binding Sites in the Activation of the Rat MMP-9 Gene Promoter by IL-1ß
From the data presented in Fig. 2, we suggest that GC-sensitive regions are mainly positioned in a region downstream from -597. This promoter region, in addition to binding sites for NF-B and AP-1, contains a further binding site for an Ets transcription factor adjacent to a second distal AP-1 response element (Fig. 3A). Adjacent Ets and AP-1 binding sites have also been reported for the promoters of other matrix proteases, including collagenase I, stromelysin (18), and human urokinase plasminogen activator (19). Moreover, Ets has also been identified as a target for the negative regulation of MMP-1 expression by androgens in the human prostate carcinoma cell line DU 145 (17).
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1.3 constructs bearing single point mutations in the binding sites of the candidate transcription factors as indicated in Fig. 3A. A promoter analysis comparing the remaining transcriptional activities of these MMP-9 promoter constructs is summarized in Table 1. We observed that all mutant constructs displayed an overall reduced level in total luciferase activity when compared with the wild-type MMP-9 promoter construct. However, the level of basal promoter activities of all mutants was clearly above background promoter activity as determined by measuring luciferase activity of a promoterless pGL basic reporter gene (Table 1). Previously, we have demonstrated that promoter activation of pGL-MMP-9-0.6 by IL-1ß critically depends on an AP-1 (-87/-81) and a NF-B binding site (-560/-550) (14). Both, AP-1 and NF-B are established targets of GR-mediated suppression of a variety of inflammatory genes (5). As shown in Fig. 3B, disruption of the NF-B site at (-560/-550) within the longer pGL-MMP-9-1.3 construct completely reduced transactivation of pGL-MMP-9-1.3 by IL-1ß. In contrast, disruption of the proximal AP-1 site at (-87/-81) within the 1.3-kb MMP-9 promoter fragment only moderately reduced promoter inducibility by IL-1ß (Fig. 3B). This observation somewhat contrasts with the finding that the same mutation within the 0.6-kb construct completely prevented promoter activation through IL-1ß (14). Obviously, in the context of the long promoter, the proximal AP-1 site is of minor functional importance for promoter activation by IL-1ß, but after deletion of a sequence distal from -597 it may gain impact for promoter activation and constitute IL-1ß responsiveness. Furthermore, the distal AP-1/Ets-region (-511/-497) seems to be the functional relevant AP-1 binding site as mutation of either the Ets or the AP-1 binding site in this composite promoter region results in a complete loss of inducibility by IL-1ß (Fig. 3B). In summary, we conclude that in addition to the NF-B element, the region containing an Ets binding site next to a distal AP-1 element is indispensable for MMP-9 gene activation by IL-1ß.
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B by Dexamethasone Is Paralleled by a Reduced Content of Nuclear p65B to the dexamethasone-mediated suppression of cytokine- induced MMP-9 expression, we performed EMSA using a NF-B consensus oligonucleotide. As shown in Fig. 4A (upper panel), binding of a slow migrating, IL-1ß-inducible complex and of a fast migrating, constitutive bound complex is maximal after 30 min of cytokine treatment. By supershift analysis we found that the faster migrating complex mainly contains p50 subunits because it was strongly supershifted by anti-p50, whereas DNA binding was only weakly affected by an anti-p65- specific antibody (Fig. 4B). In contrast, the slow migrating complex mainly contained p65, as indicated by the complete disappearance of the complex after anti-p65 antibody treatment, but also p50, because p50- specific antisera also impaired the DNA binding. Antibodies against c-Rel, a further member of the NF-B family, had also inhibitory effects on the binding of both complexes but did not cause a prominent supershift (Fig. 4B). Simultaneous treatment of cells with IL-1ß and 100 nM dexamethasone caused a total inhibition of the slower migrating complex and also slightly reduced binding of the faster migrating complex, mainly at the very early time point of 30 min (Fig. 4A, upper panel). Coincubation of cells with IL-1ß plus dexamethasone in the presence of RU-486 reconstituted the cytokine-induced binding of both NF-B complexes (data not shown).
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B-DNA binding were attributable to a reduced nuclear translocation of NF-B. To this end we performed Western blot analysis with nuclear extracts using an anti-p65-specific antibody. Treatment of MCs with IL-1ß is followed by appearance of p65 protein within the nuclear extracts at both time points tested. Surprisingly, dexamethasone suppressed the abundance of p65 protein in MC nuclei predominantly at the early time point of 30 min but not at the later time point of 2 h (Fig. 4A, lower panel). To exclude the possibility of a contamination of nuclear fractions with cytosolic proteins, we checked for the presence of GR in the nuclear extracts from MCs treated in the absence of GC. As demonstrated in Fig. 4A (lower panel), the GR band is only detectable in the extracts from dexamethasone-treated MC preparations, thus proving the purity of the nuclear extracts examined. To further corroborate the data generated by EMSA, we finally tested promoter activities of a reporter gene bearing a tandem of five NF-B consensus motifs in front of the luciferase coding region (Fig. 4C). Treatment of MCs with dexamethasone strongly reduced the IL-1ß-evoked increase in luciferase activity, but had no effects on basal promoter activity when tested alone (Fig. 4C). These data convincingly demonstrate that inhibition of the NF-B-DNA binding, seen in the EMSA, functionally correlates with dexamethasone inhibition of NF-B-driven promoter activity.
Inhibition of Cytokine-Induced Degradation of IB by GCs
Activation and nuclear uptake of NF-B as a necessary prerequisite of gene transcription in many cases depend on the degradation and release of NF-B from the inhibitor of B (IB), which is regulated by the action of IB-kinase. As depicted in Fig. 5A, treatment of MCs with IL-1ß caused a rapid decrease in cytosolic IB
protein levels that was maximal at 30 min after IL-1ß treatment. Degradation of IB was partially prevented by dexamethasone mainly after 30 min of treatment, thus suggesting that the decrease in nuclear p65 protein by dexamethasone is paralleled by an attenuated degradation of IB. However, in none of the experiments was the inhibition of IB degradation complete, thus indicating that additional mechanisms, independent from IB degradation, contribute to the inhibition of NF-B DNA binding.
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B in Rat MCsB protein levels were affected by dexamethasone. Transcriptional induction of IB by GCs, in some cell types, is a further possible facet of cross-talk between GR and NF-B (2). Examining total protein lysates in time-course experiments, we found that dexamethasone at two concentrations (100 nM, 1 µM) caused a marked increase in the total IB protein content, preferentially at 1 h and 5 h of stimulation (Fig. 5B). This increase in total IB level remained for at least 5 h but dropped back to control levels at 24 h. These data suggest that, in rat MCs, activation of IB expression or, alternatively, inhibition of its degradation are candidate mechanisms by which glucocorticoids inhibit the expression of NF-B-dependent genes.
Dexamethasone Impairs the IL-1ß-Caused Binding to AP-1 and Ets Motifs
In addition to NF-B, AP-1 is a second prominent target of GC-mediated gene repression. To further test for a possible contribution of AP-1 to dexamethasone-mediated suppression of MMP-9 expression in our cell culture model, we performed EMSA. Nuclear extracts from MCs were isolated 1 h and 5 h after treatment with the indicated agents and incubated with a radioactive labeled AP-1 consensus oligonucleotide. Treatment with IL-1ß strongly induced DNA binding of a single complex, most clearly in the nuclear extracts prepared after 1 h of stimulation (Fig. 6A). The specificity of this DNA-bound complex was proven by competition analysis using an oligo bearing a mutated AP-1 motif as described previously (14). As shown in Fig. 6A, dexamethasone strongly attenuated the IL-1ß-stimulated binding of this complex to the AP-1 motif but had no effects on basal AP-1 binding. These data indicate that in rat MCs, in addition to NF-B, the cytokine-induced DNA binding of AP-1 is negatively influenced by dexamethasone.
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1.3 (Fig. 3). To further confirm the functionality of this binding site in the transcriptional regulation of MMP-9 gene expression by cytokines and GCs, respectively, nuclear extracts were assayed with a radioactive probe comprising the critical Ets binding site from the promoter region of MMP-9 (Table 2). When compared with the low basal binding affinity to AP-1, the EMSA with the gene-specific Ets probe displayed a strong constitutive DNA binding of two complexes (complex I and II) that was further enhanced by IL-1ß (Fig. 6B).
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, upper panel). These results indicate that glucocorticoids have mixed effects on Ets-binding activity depending on the presence or absence of a simultaneous cytokine stimulus. Furthermore, the modulatory effects on DNA binding were paralleled by a change in the nuclear Ets-1 content, as assessed by Western blot analysis (Fig. 6B, lower panel; w.b.), thus indicating that the changes in DNA binding result from the changes in the amount of Ets-1 protein in the nucleus.
To prove specificity of the DNA-bound complexes, we performed supershift analysis using antibodies specific for Ets or different members of the AP-1 transcription factor family (Fig. 7A, left panel). Binding of the slow migrating complex (complex I) was strongly impaired by addition of an antibody raised against the N-terminal domain of c-Jun, thus documenting the presence of members of the AP-1 transcription factor in the Ets-bound complex I. Interestingly, antibodies raised against Jun B and c-Fos had only very weak inhibitory effects on the DNA binding of both complexes. In contrast, the DNA binding mainly of complex I was strongly impaired by either Ets1/2 or Ets1-specific antibodies, respectively. Because DNA binding is reduced to a comparable extent by c-Jun and Ets-1-specific antibodies, we assume that complex I contains both members of transcriptional activators, whereas complex II contains none of these transcription factors.
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). As shown in Fig. 7A (right panel), addition of a 100-fold molar excess of unlabeled gene-specific wild-type Ets-AP-1 oligonucleotide almost completely reduced the binding of both IL-1ß-induced DNA complexes. Competition with cold oligos bearing a mutation in either the AP-1 or Ets-1 core sequence still led to a significant decrease of 40–50% in DNA binding. Competition capacity was nearly completely lost with an oligo bearing double mutations (Fig. 7A, right panel). These findings corroborate the data obtained by mutation analysis and indicate that a DNA binding to both consensus sites is necessary for a transcriptional activation of MMP-9 by IL-1ß.
Finally, we tested competition capacities of wild-type and mutant Ets consensus oligonucleotides. Addition of a 100-fold excess of cold wild-type Ets oligo (1:100), but not of mutant Ets oligo, caused inhibition of DNA binding of both complexes. Only a high excess of unlabeled mutant-oligo (1:10) was able to compete with DNA binding probably due to unspecific effects (Fig. 7B).
GR Interacts with Nuclear p65 and Ets-1 Proteins
To gain further insight into the mechanism of GC-dependent inhibition of the cytokine-induced activation of NF-B and Ets transcription factors in MCs, we investigated a possible physical interaction between the GR and both cytokine-inducible types of transcription factors by coimmunoprecipitation experiments. First, we studied for the association between GR and p65, by precipitation of nuclear extracts from MCs treated for 1 h with either vehicle (control) or IL-1ß plus dexamethasone with a monoclonal anti-GR antibody. Likewise, immunoblotting of GR immunoprecipitates with a polyclonal anti-GR or a polyclonal anti-p65 antibody revealed a clear coimmunoprecipitation of p65 with GR in the cells treated with IL-1ß and dexamethasone but not in cells exposed to vehicle (Fig. 8A). In addition to the migration properties of p65 and the GR, which run at 65 and 92 kDa, respectively, we separated them in parallel crude nuclear extracts on the same SDS gel as positive controls (far left and right lanes of Fig. 8A).
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). Furthermore, immunoblotting of the GR immunoprecipitates with anti-c-Jun did not reveal any coimmunoprecipitation of the two proteins (data not shown). In summary, these data indicate that Ets and AP-1 motifs within the -511/-497 promoter region of MMP-9 contribute to the DNA binding capacities and are functionally important for the transcriptional activation of the MMP-9 gene by IL-1ß. Furthermore, these results demonstrate that the GR negatively interferes with p65 and Ets-1 through physical interaction also commonly denoted as transrepression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In the present study we demonstrate that reduction of IL-1ß-induced MMP-9 gelatinolytic activity by GCs is mainly caused by an inhibition of MMP-9 gene expression.
Transcriptional regulation of MMP-9 expression by IL-1ß in MCs critically depends on the activation of NF-B and AP-1 transcription factors (11, 14). Both transcription factors were demonstrated to be engaged in a cross-talk with steroid receptors in a way that is independent of DNA binding (2, 5). We have cloned and characterized a promoter region required for IL-1ß-dependent transcription of the rat MMP-9 gene. Sequencing of a 1.3-kb fragment of the 5'-flanking region of the rat MMP-9 gene identified several putative binding sites for regulation of MMP-9 expression by IL-1ß, including binding sites for AP-1, NF-B, and Ets transcription factors. By comparing two MMP-9 promoter fragments, we found that binding sites laying upstream of -597 are not required for promoter activation by IL-1ß. Mutational analysis of the proximal region further implicates a functional role of NF-B-, AP-1-, and Ets-binding sites in the IL-1ß signaling triggering MMP-9 gene expression. EMSA confirmed that in MCs the binding of AP-1, NF-B, and Ets to their cognate binding sites is activated by IL-1ß. Remarkably, the GR displays both stimulatory and inhibitory effects on gene transcription. Because the GC-responsive MMP-9 promoter fragments do not contain a negative GRE, we conclude that suppression of MMP-9 transcription primarily results from a mechanism termed "tethering GRE," i.e. through a direct interaction between the GR and an activating transcription factor, independent of a direct binding of GR to DNA, as was exemplified for AP-1 and NF-B (2, 5, 28). In line with these observations, we show that inhibition of cytokine-induced MMP-9 promoter activity by dexamethasone can be partly accounted for by decreased promoter binding of NF-B and probably is caused by a physical interaction between the GR and p65, as demonstrated by coimmunoprecipitation experiments. By EMSA we demonstrate that the rapid IL-1ß-induced binding of a p50/p65-containing NF-B complex is strongly attenuated in cells treated with dexamethasone. In addition, translocation studies revealed that the inhibitory effects of dexamethasone on p65 translocation are transient when compared with the sustained inhibitory effects observed on MMP-9 transcription, thus suggesting that NF-B is only a transient target of GC action. Suppression of NF-B-mediated gene expression is attributed to multiple mechanisms including inhibition of IB degradation and a decrease in DNA-binding affinity due to attenuation of the trans-acting potential as reported for c-Rel (29). In various cell types, the expression of IB itself has been found to be under the control of NF-B and, in addition, some reports could demonstrate induction of IB expression by GCs, although a functional GRE could not be mapped in the IB promoter (3, 30). In line with these reports, we demonstrate an increase of total IB protein by dexamethasone treatment, indicating a modulation of IB expression or, alternatively, in IB degradation as possible mechanisms of GC-mediated inhibition of gene expression in MCs. In this context it is worth mentioning that glucocorticoids also exert relevant posttranscriptional action on mRNA and protein stability (31). In addition to NF-B, we could identify, by site-directed mutagenesis, a functional binding site for Ets transcription factors that is indispensable for a full transactivation of a 1.3-kb MMP-9 promoter luciferase construct by IL-1ß (Fig. 4). Moreover, EMSA and supershift analysis revealed that Ets-related proteins, namely Ets-1, in combination with c-Jun, are further targets for the negative regulation of MMP-9 expression by GCs in MCs. Interestingly, an arrangement of adjacent binding sites for Ets and AP-1 transcription factors has been found in the promoters of several matrix protease genes, including MMP-1, MMP-3, MMP-9, urokinase-type plasminogen activator, and tissue inhibitor of metalloproteinase-1 (32, 33, 34, 35, 36), therefore suggesting that in addition to AP-1, Ets plays a pivotal role in the regulation of matrix protease expression. The close neighborhood of AP-1- and Ets-binding sites allows for a cooperative binding between both types of transcription factors and is important for the synergistic interaction of the transcription factors (36).
We found by competition assays that both binding sites show a high redundancy because mutation of each binding site retained a similar level of competition capacity. This suggests that, at least in vitro, the bound complexes can similarly occupy both regulatory elements. Moreover, functionality of the 1.3-kb MMP-9 reporter gene construct strongly depends on both sites being intact, thus indicating that a possible interaction between Ets and AP-1 requires a site- specific DNA binding to both corresponding core sequences. Notably, we found that the DNA binding capacity of the low migrating complex bound to the juxtaposed AP-1/Ets sites was significantly reduced by addition of either AP-1/c-Jun or by Ets-1-specific antibodies, indicating that both transcription factors, by simultaneously binding to a composite promoter region, may activate MMP-9 gene transcription. It is commonly assumed that interaction of Ets- and AP-1 transcription factors allows for a highly precise regulation of gene expression, most importantly of genes regulating tissue remodeling (32, 33, 34, 35, 36).
A physical interaction between the GR and Ets-2 is necessary for the functional synergism of transcriptional activation of the rat cytochrome P-450c27 promoter (37). Moreover, a positive integration of Ets in GC-dependent signaling confers the basal and GC induced expression of rat tyrosine aminotransferase (38). To the best of our knowledge, we demonstrate here, for the first time, a negative interference of GC with Ets and thus demonstrate that interference between GR and Ets-dependent pathways can exhibit positive as well as negative effects on gene transcription. Similarly, the interaction of the androgen receptor by interference with Ets and AP-1 allows for a negative regulation of MMP-1, MMP-3, and MMP-7 expression (17). Moreover, we demonstrate here that the inhibitory effects of GCs on Ets binding similar to the positive interference involve a direct interaction of GR with Ets-containing complexes as shown by coimmunoprecipitation studies.
In summary, we conclude that members of the Ets family are an additional important regulatory element in the signaling cascades of cytokine-mediated MMP-9 expression in noninvasive, nontransformed cell types. Moreover, our data indicate that the proximity between AP-1 and Ets binding motifs may determine not only the transcriptional activation by cytokines but allow for additional modulation through GR-mediated signals. The interaction of GCs with multiple signal transduction pathways, therefore, highlights the complex repertoire of regulatory events targeted by a glucocorticoid therapy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell Culture
Rat glomerular MCs were grown in Roswell Park Memorial Institute 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 5 ng/ml insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin. Serum-free preincubations were performed in DMEM supplemented with 0.1 mg/ml of fatty acid-free BSA for 24 h before cytokine treatment. For experiments 3.0–5.0 x 106 of MCs per 10-cm culture dish were used between passages 8 and 19. All supplements were purchased from Life Technologies, Inc./BRL (Eggenstein, Germany). The amount of dead cells was determined by trypan blue exclusion. Cell cytotoxicity was measured as described previously (13).
cDNA Clones and Plasmids
cDNA insert for rat MMP-9 was generated as recently described (13).
A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA clone was generated using internal primers of coding sequence of rat GAPDH mRNA (GenBank/EMBL databases, accession no. NM 017008). A cDNA insert from mouse 18S rRNA was from Ambion, Inc. (Austin, TX).
Cloning of rat MMP-9 Promoter and Transient Transfections
The 5'-flanking region of the rat MMP-9 gene was cloned utilizing the Genome Walker Kit (CLONTECH Laboratories, Inc., Heidelberg, Germany) using internal (upstream) and external (downstream) primers from the rat MMP-9 cDNA (GenBank/EMBL databases, accession no. U36476) as follows:
MMP-9 internal primer: 5'-AGGGGCAGCAAAGCTGTAGCCTAG-3';
MMP-9 external primer: 5'-TTTCAGGTCTCGGGGGAAGACCACATA-3'.
A 1.3-kb fragment from a EcoRV cut library was isolated by PCR under stringent conditions. The fragment was subsequently subcloned into pBluescript-II KS+ and sequenced using the automated sequence analyzer ABI 310 (PE Applied Biosystems, Weiterstadt, Germany). The sequence has been deposited in the GenBank/EMBL databases (accession no. AJ438266). The forward and reverse primer sequences used for subcloning into pGL-III Basic vector coding for beetle luciferase (Promega Corp., Mannheim, Germany) were as follows:
5'-CTCACAGACTCATACGTCCCTTTA-3' (forward) and 5'-TGAGAACCGAAGCTTCT-GGGT-3' (reverse). Introduction of a double-point mutation into the NF-B site (GGAATTCCCCC to GGAATTGGCCC) to generate pGL-MMP-9-NF-B was done, using the following (forward) primer: 5'-GGGTTGCCCCGTGGAATTGGCCCAAATCCTGC-3' (corresponding to a region from -572 to -541). Generation of a double transition within the Ets-binding site (GAGGAA to GAGAGA) to generate pGL-MMP-9 Ets was done using the following (forward) primer: 5'-GGCAGGAGAGAGAGCTGAGTCAAAGACA-3' (corresponding to a region from -518 to -491). Generation of a double transition within a proximal AP-1 binding site (CTGAGTCA to CTGAGTTG) to generate pGL-MMP-9 AP-1 was done using the following (forward) primer: 5'-CACACACCCTGAGTTGGCGTAAGCCTGGAGGG-3' (corresponding to a region from -98 to -65). Mutation of a second, distal lying AP-1 site (CTGAGTCA to CTGAGTTG) to generate pGL-MMP-9 AP-1/wtEts was performed using the following (forward) primer: 5'-GGCAGGAGAGGAAGCTGAGTTGAAGACA-3' (corresponding to a region from -518 to -491). All mutant constructs were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). pNF-B-Luc, a cis-reporting vector containing the luciferase gene driven by a TATA-box joined to five tandem repeats of NF-B binding sites, was obtained from Stratagene. Transient transfections of MCs were performed using the Effectene reagent (QIAGEN, Hilden, Germany). Transfections were performed following the manufacturer’s instructions. The transfections were done as triplicates and repeated at least three times to ensure reproducibility of the results. Transfection with pRL-CMV coding for Renilla luciferase was used for control of transfection efficiencies. Luciferase activities were measured with the dual reporter gene system (Promega Corp., Madison, WI) using an automated chemiluminescence detector (Berthold, Bad Wildbad, Germany).
Northern Blot Analysis
Total cellular RNA was extracted from MCs using the Tri reagent (Sigma, St. Louis, MO). Procedures for RNA hybridization were as described previously (13).
SDS-PAGE Zymography
Assessment of gelatinolytic activity of proteins from cellular supernatants was performed as described previously (13). To exclude the possibility that alterations in gelatinolytic contents were due to differences in cell numbers, we routinely determined total cell numbers under each of the experimental conditions. Proteins with gelatinolytic activity were visualized as areas of lytic activity on an otherwise blue gel. Migration properties of proteins were determined by comparison with that of prestained full range rainbow protein markers (Amersham Pharmacia Biotech, Freiburg, Germany). Photographs of the gels were scanned by an imaging densitometer system from Bio-Rad Laboratories, Inc. (München, Germany).
EMSA
Preparation of crude nuclear extracts from cultured mesangial cells and subsequent EMSA was done as described previously (39). The cytoplasmic fractions were separated by centrifugation and used for detection of IB protein levels. Consensus oligonucleotides for NF-B and AP-1 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Competition experiments were done by coincubation with a 10- to 100-fold excess (10–100 pmol) of unlabeled double-stranded oligonucleotide in the DNA-protein binding reaction. Wild-type and mutant consensus oligonucleotides for competition experiments were from Santa Cruz Biotechnology, Inc. The sequences for wild-type and mutant oligonucleotides coding for gene-specific binding sites are summarized in Table 2.
Polyclonal antibodies used for supershift experiments were purchased from Santa Cruz Biotechnology, Inc. For supershift analysis, 2 µl of the antibody were preincubated overnight before the binding reaction.
Western Blot Analysis
Nuclear cell extracts (20–50 µg) were used for assessing nuclear import of p65. IB protein levels were analyzed using 50–100 µg of total protein from the corresponding cytoplasmic fractions. Total cellular levels of IB and GR protein were analyzed using total cellular extracts (40). Western blot analysis of different fractions was performed as described previously (14). Detection of MMP-9 from cell supernatants was done by trichloroacetic acid precipitation (41).
A polyclonal antibody specific for human MMP-9 was obtained from CHEMICON International (Hofheim, Germany). All other antibodies used in this study were from Santa Cruz Biotechnology, Inc.
Coimmunoprecipitation
Coimmunoprecipitations were performed by using the Seize Primary Immunoprecipitation Kit from Pierce Chemical Co. (Rockford, IL). This kit uses a chemical cross-linking of the primary antibody to avoid the interference with the antibody heavy and light chain bands on the Western blot. According to the manufacturer’s protocol, 50–100 µg of the antibody used for immunoprecipitation were chemically immobilized to a coupling gel that was subsequently packed onto a spin column. Nuclear extracts (250 µg) were subjected to this column and incubated with gentle mixing for several hours in a cold room to allow binding of the antigen to the immobilized antibody. After several wash steps with immunoprecipitation sample buffer containing 0.025 M Tris, 0.15 M NaCl (pH 7.2), the immunoprecipitated complex was eluted from the column by addition of ImmunoPure IgG elution buffer and directly resolved on a 10% SDS-PAGE gel. The proteins were transferred to a nitrocellulose membrane and successively probed with polyclonal antibodies to p65, GR, and Ets-1, respectively. As a positive control for each transcription factor, 100 µg of nuclear extracts from IL-1ß- (p65 and Ets-1) or IL-1ß plus dexamethasone (GR)-treated MCs were directly subjected to the same gel used for resolution of the immunoprecipitated complexes and analyzed by immunoblotting. All antibodies used for immunoprecipitations were obtained from Santa Cruz Biotechnology, Inc.
Migration properties of proteins were determined by comparison with that of prestained full range rainbow protein markers (Amersham Pharmacia Biotech, Arlington Heights, IL).
Statistical Analysis
Results are expressed as means ± SD. The data are presented as x-fold induction compared with control conditions or compared with IL-1ß-stimulated values (#). Statistical analysis was performed using Student’s t test and ANOVA for significance.
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FOOTNOTES |
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Abbreviations: AP-1, Activator protein 1; ECM, extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, glucocorticoid; GR, glucocorticoid receptor; GRE, glucocorticoid responsive element; IB, inhibitor of B; MCs, mesangial cells; MMP-9, metalloproteinase-9; NF-B, nuclear factor B.
Received for publication October 18, 2001. Accepted for publication March 29, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B activity through induction of IB synthesis. Science 270:286–290B and AP-1. Am J Physiol 270:F123–F130
B and activating protein-1 and involves activation of the mitogen-activated protein kinase pathways. J Immunol 165:5788–5797 activation of NF-B requires the phosphorylation of Ser-471 in the transactivation domain of c-Rel. J Biol Chem 275:24383–24391B controls expression of inhibitor IB: evidence for an inducible autoregulatory pathway. Science 259:1912–1915
0.05 compared with control conditions (*) or to IL-1ß-stimulated values (#). P 
