Rac1, a small GTPase, regulates macrophage MMP (matrix metalloproteinase)-9 in an ERK (extracellular-signal-regulated kinase)- and SP (specificity protein)-1-dependent manner. SP-1 contains a PEST (Pro-Glu-Ser-Thr) domain that may modulate protein stability. We hypothesize that Thr578, Ser586 and/or Ser587 in the PEST domain are required for SP-1 stability and MMP-9 expression secondary to activation of ERK, a serine/threonine kinase. We determined the effects of Rac1 and ERK on MMP-9 expression driven by SP-1WT (wild-type) and the SP-1 mutants T578A, S586A and S587A. Expression of WT and mutant SP-1 increased MMP9 promoter activity in alveolar macrophages. However, constitutively active Rac1 suppressed MMP9 promoter activity in cells expressing SP-1WT, SP-1T578A and SP-1S587A, but not SP-1S586A. Furthermore, constitutive ERK activation, which was inhibited by Rac1, significantly increased MMP9 transcription in cells expressing SP-1WT, but not SP-1S586A. As Rac1 activation and ERK inactivation increased degradation of SP-1WT and not SP-1S586A, the results of the present study suggest that SP-1 stability mediated at Ser586 regulates MMP9 transcription. Ex vivo, alveolar macrophages obtained from patients with asbestosis had less MMP-9 expression that was associated with decreased SP-1 expression and ERK activation. These observations demonstrate that Ser586 in the PEST domain of SP-1 is important for MMP9 gene expression in alveolar macrophages and highlight the importance of these proteins in pulmonary fibrosis.
- extracellular-signal-regulated kinase (ERK)
- matrix metalloproteinase 9 (MMP-9)
- pulmonary fibrosis
- specificity protein 1 (SP-1)
A hallmark of pulmonary fibrosis is aberrant matrix deposition characterized by an imbalance between the deposition and degradation of extracellular matrix proteins. Under normal conditions, there is a dynamic balance between the two processes that is regulated by a family of zinc-dependent endopeptidases known as MMPs (matrix metalloproteinases) . However, under conditions of excessive remodelling of the extracellular matrix, as seen in fibrosis, the precise role of MMPs in the development and maintenance of the pathology is unclear. Although some studies report that MMPs exacerbate fibrosis, others demonstrate their protective role [2–8].
Macrophages are an important cell type in the development of pulmonary fibrosis. Our previous observations suggest that macrophage-derived MMP-9 attenuated asbestos-induced pulmonary fibrosis . In addition, we found that MMP-9 production and activity was regulated at the transcriptional level by the small GTPase Rac1 . Deletion of Rac1 in macrophages significantly enhanced transcription of MMP9 in vitro and in vivo. Furthermore, mice with a conditional deletion of Rac1 in their macrophages were protected from asbestos-induced pulmonary fibrosis [9,10].
SP (specificity protein)-1 is a ubiquitous transcription factor expressed in mammalian cells that regulates the transcription of genes involved in multiple cellular processes [11,12]. SP-1 undergoes several post-transcriptional modifications, such as phosphorylation, ubiquitination, SUMOylation, glycosylation and acetylation, which regulate its transcriptional activity. It is one of many transcription factors that bind to the MMP9 promoter to induce its transcription [9,13]. We previously reported that Rac1 partly inhibits the transcriptional activation of the MMP9 gene at its SP-1-binding site . However, the mechanism of SP-1 regulation in pulmonary fibrosis is not known.
In the present study we addressed mechanisms by which Rac1 inhibited SP-1-mediated transcriptional activation of the MMP9 gene. We identified a PEST (Pro-Glu-Ser-Thr) domain in murine SP-1 containing one threonine (Thr578) and two serine (Ser586 and Ser587) residues that are potential sites of phosphorylation by serine/threonine kinases. As the activation of ERK (extracellular-signal-regulated kinase), a serine/threonine kinase, is inhibited by Rac1 , and phosphorylation within the PEST domain modulates protein stability [14–16], we hypothesized that Rac1 inhibits MMP9 promoter activity secondary to suppression of ERK-mediated stabilization of SP-1 at the threonine and/or serine residues within its PEST domain. Serine to alanine residue mutation of the Ser586 residue, but not the threonine or other serine residues, prevented the modulation of the MMP9 promoter by Rac1 and ERK. In addition, ERK activation increased the stability and abundance of SP-1, an effect that was mediated at the Ser586 residue. Furthermore, the importance of these proteins in pulmonary fibrosis was demonstrated ex vivo. MMP-9 expression was significantly less in alveolar macrophages obtained from patients with asbestosis compared with normal subjects, and was associated with lower levels of SP-1 and active ERK. These observations indicate that Rac1 inhibits MMP9 gene expression in alveolar macrophages by attenuating the activation of ERK and ERK-mediated stabilization of SP-1 at the Ser586 residue within its PEST domain.
U0126, anti-β-actin antibody, goat anti-rabbit IgG–HRP (horseradish peroxidase), goat anti-mouse IgG–HRP and cycloheximide were purchased from Sigma. Anti-SP-1 antibody was from Santa Cruz Biotechnology. Anti-V5 antibody was from Invitrogen. SimpleChIP™ enzymatic ChIP (chromatin immunoprecipitation) kit was from Cell Signaling Technology and ChIP grade V5 was from Abcam.
WT (wild-type) and Rac1-null macrophages have been described previously . The mouse alveolar macrophage cell line, MH-S, was obtained from A.T.C.C. and were maintained according to the manufacturer's instructions. All experiments were performed in medium containing 0.5% serum.
WT and Rac1-null mice have been described previously . All protocols were approved by the University of Iowa Institutional Animal Care and Use Committee. Chrysotile asbestos (100 μg in 50 μl of normal saline) was administered intratracheally to 6–10-week old mice after 2–5 min of anaesthesia that was induced by 3% isoflurane delivered via a Fortec vaporizer (Cyprane). After 28 days, mice were killed with an overdose of isoflurane, and BAL (bronchoalveolar lavage) was performed. BAL cells were used for the determination of total and differential cell counts. BAL fluid was used for the determination of MMP-9 activity.
The Human Subjects Review Board of the University of Iowa Carver College of Medicine approved the protocol for obtaining alveolar macrophages from normal volunteers. Normal volunteers had to meet the following criteria: (i) aged between 18 and 55 years; (ii) no history of cardiopulmonary disease or other chronic disease; (iii) no prescription or non-prescription medication except oral contraceptives; (iv) no recent or current evidence of infection; and (v) lifetime non-smoker. Alveolar macrophages were also obtained from patients with asbestosis. Patients with asbestosis met the following criteria: (i) FEV1 (forced expiratory volume in 1 s) and DLCO (diffusing capacity of the lung for carbon monoxide) at least 50% predicted; (ii) current non-smoker; (iii) no recent or current evidence of infection; and (iv) evidence of restrictive physiology on pulmonary function tests and interstitial fibrosis on chest computed tomography. Fibre-optic bronchoscopy with BAL was performed after subjects received intramuscular atropine, 0.6 mg, and local anaesthesia. Three sub-segments of the lung were lavaged with five 20 ml aliquots of normal saline, and the first aliquot in each was discarded. The percentage of alveolar macrophages was determined using the Wright–Giemsa stain and varied from 90 to 98%.
Plasmids and transfections
The murine SP1 cDNA (NM_013672) was directionally cloned into pcDNA3.1D/V5.His-TOPO (Invitrogen). Serine to alanine residue mutations of SP-1 at Thr578, Ser586 and Ser587 were accomplished using the QuikChange® Lightning Multi Site-Directed Mutagenesis kit (Agilent Technologies). The MMP9 promoter reporter vectors have been described previously . CA (constitutively active) Rac1 (Q61L) in pUSEamp plasmid was from Millipore. CA pCMV-MEK1 [MAPK (mitogen-activated protein kinase)/ERK kinase 1] and dominant-negative pCMV-HA (haemagglutinin)-ERK2 (K/A) plasmids (gifts from Dr Roger Davis, University of Massachusetts, Worcester, MA, U.S.A.) have been described previously . Cells were transfected with Effectene® Transfection Reagent or X-Treme Gene 9 Transfection Reagent according to the manufacturer's instructions. To correct for transfection efficiency, cells were co-transfected with phL-TK Renilla luciferase vector (Promega). Firefly and Renilla luciferase activities were assayed in cell lysates using the Dual Luciferase Reporter Assay Kit (Promega).
Quantitative RT (reverse transcription)–PCR
Total RNA from BAL cells obtained from normal subjects and patients with asbestosis was isolated using TRIzol®, DNase treated, and reverse-transcribed using the reverse transcriptase kit Iscript (Bio-Rad Laboratories). MMP9 and HPRT (hypoxanthine-guanine phosphoribosyltransferase) mRNA transcripts were determined by quantitative real-time PCR using SYBR Green (Bio-Rad Laboratories) and respective primers on an IQ5 real-time PCR machine (Bio-Rad Laboratories). The following primers were used: 5′-GGCAAGGGCGTCGTGGTTCC-3′ (forward) and 5′-TCCGTGGTGCAGGCGGAGTA-3′ (reverse) for human MMP9 and 5′-AGCCCTGGCGTCGTGATTAGTGA-3′ (forward) and 5′-TGTCCCCTGTTGACTGGTCATTACA-3′ (reverse) for human HPRT. Data were calculated by the cycle threshold (ΔΔCT) method. MMP9 mRNA was normalized to HPRT and expressed as arbitrary units.
MH-S cells were transfected with pcDNA3.1 (empty) or pcDNA3.1V5.His vectors expressing SP-1WT, SP-1T578A, SP-1S586A or SP-1S587A. After an overnight incubation, nuclei were isolated and subjected to chromatin digestion using the SimpleChIP™ enzymatic ChIP kit as described previously . The resulting cross-linked chromatin preparations were used for input controls (2% of total) or for immunoprecipitation using anti-V5 (Abcam) or anti-histone 3 (Cell Signaling Technology) as a positive control, or normal rabbit IgG antibody (Cell Signaling Technology) as a negative control. Chromatin–immune complexes were eluted, and the chromatin was subjected to reversal of cross-links followed by DNA purification as described in the protocol. DNA was analysed by quantitative real-time PCR on an IQ5 real-time PCR machine (Bio-Rad Laboratories) using SYBR Green (Bio-Rad Laboratories) and the following primers: 5′-GCTCCCACATGTGTGTGTC-3′ and 5′-CCTAGCTCCAGCAGGCTG-3′ for the murine MMP-9 SP-1 binding site (76-bp product) and RPL30 primers (Cell Signaling Technology) for murine ribosomal protein gene locus (159-bp product). PCR products were resolved on a 2% agarose gel. Results were calculated as arbitrary units relative to empty-vector-transfected cells (control).
EMSAs (electrophoretic mobility-shift assays)
Nuclear proteins were extracted as described previously from MH-S cells transfected with either empty pcDNA3.1, WT SP-1 or mutant SP-1 vectors . The consensus SP-1 (5′-ATTCGATCGGGGCGGGGCGAGC-3′) oligonucleotide was labelled with [γ-32P]ATP (NEN Life Science Products) and allowed to bind to 12.5 μg of nuclear proteins as described previously . Protein–DNA complexes were separated on a 5% non-reducing polyacrylamide gel which was dried and exposed to X-ray film.
Measurement of MMP-9 activity
Total and pro-MMP-9 were determined in BAL fluid isolated from asbestos-exposed mice using mouse quantikine ELISA kits (R&D Systems). Active MMP-9 was calculated as the difference between total and pro-MMP-9.
Statistical comparisons were performed using either an unpaired one-tailed t test or one-way ANOVA followed by Tukey's t test. Values in the Figures are expressed as means±S.E.M, and P<0.05 was considered to be significant.
Rac1 regulates SP-1-mediated MMP9 gene expression at Ser586
Previous observations show that Rac1 partly modulates MMP-9 expression and activity in macrophages by decreasing SP-1 transcriptional activity and nuclear abundance . The mechanism by which this occurs is not known. Stability of proteins is often determined by a PEST domain, a region rich in the amino acids proline (P), glutamic acid (E), serine (S) and threonine (T). A PEST domain score greater than 5 typically targets proteins for degradation . We identified a PEST domain (amino acids 575–595) in murine SP-1 having a score of 6.1 that contains three potential sites of phosphorylation by serine/threonine kinases, Thr578, Ser586 and Ser587 (Figure 1A). ERK-mediated phosphorylation of amino acid residues within the PEST domain is known to stabilize proteins [14,15]. We hypothesized that Rac1 inhibits MMP9 transcription by increasing SP-1 degradation and thereby decreasing its transcriptional activity. To address this, we generated threonine to alanine and serine to alanine residue mutations of SP-1 at Thr578, Ser586 and Ser587 residues and transfected alveolar macrophages with vectors expressing these mutants. SP-1WT and the three SP-1 mutants were predominantly expressed in the nucleus with no detectable levels observed in the cytoplasm (Figure 1B). To examine the effects of the PEST domain of SP-1 on MMP9 promoter activity, macrophages were transfected with SP-1WT or the SP-1 PEST domain mutants together with a reporter plasmid driven by the MMP9 promoter. Cells expressing the SP-1WT and the SP-1 mutants significantly enhanced MMP9 promoter activity compared with cells expressing the empty vector (Figure 1C). As recombinant SP-1 expression increased MMP-9 expression, we next evaluated the effect of Rac1. Macrophages were transfected with SP-1WT or the SP-1 PEST domain mutants, a CA Rac1 vector, and the MMP9 promoter reporter vector. Cells transfected with CA Rac1 significantly decreased SP-1-mediated MMP-9-luciferase in cells expressing SP-1WT (Figure 1D). A similar effect was observed with SP-1T578A and SP-1S587A mutants. In contrast, MMP9 promoter activity was unchanged by CA Rac1 in cells expressing SP-1S586A, suggesting that Ser586 is important for MMP9 gene expression in macrophages.
Recombinant SP-1 binds to the endogenous MMP9 promoter
To investigate whether the difference in MMP9 promoter activity was due to an alteration in DNA binding, we determined the binding of WT and mutant SP-1 to a consensus SP-1 oligonucleotide. Compared with cells transfected with empty pcDNA3.1, all of the SP-1 vectors had an increase in binding to the SP-1 oligonucleotide compared with cells expressing the empty vector, although there was a slight reduction in the binding of the T578A mutant compared with the other vectors (Figure 2A).
To more specifically determine the DNA-binding ability of the SP-1 vectors, we evaluated binding to the endogenous MMP9 promoter. Cells were transfected with an empty vector or with the SP-1WT, SP-1T578A, SP-1S586A or SP-1S587A vectors. Nuclei were isolated from these cells and analysed by ChIP assay using the anti-V5 antibody to pull down chromatin bound to V5-tagged SP-1. Subsequent real-time PCR analysis of purified chromatin DNA with primers specific for the SP-1-binding site on the MMP9 promoter revealed that SP-1WT and the mutant SP-1 vectors bound with greater affinity to the MMP9 promoter relative to cells expressing the empty vector (Figure 2B).
To determine the specificity of SP-1-driven MMP9 promoter activity, MMP9 promoter constructs with the SP-1-binding site either deleted or mutated were evaluated. Macrophages were transfected with the SP-1 vectors together with the MMP9 promoter reporter constructs. WT and mutant SP-1 increased WT MMP9 promoter activity compared with cells expressing the empty vector (Figures 2C and 2D). In contrast, luciferase activity driven by the truncated MMP9 promoter with its SP-1 site deleted (MMP-9SP-1del) was markedly diminished in all cells expressing either empty, WT or SP-1 mutants (Figure 2C). Similar results were obtained with the MMP9 promoter with its SP-1-binding site mutated (MMP-9SP-1mut) (Figure 2D). Promoter activity observed with this vector was dramatically less than the WT MMP9 promoter in all cells, regardless of whether they were transfected with WT or SP-1 mutants. Taken together, these results establish the specificity of recombinant SP-1-driven MMP-9 gene expression.
ERK inhibition by Rac1 modulates SP-1-mediated MMP9 gene expression
To address whether Rac1 inhibited MMP9 transcription secondary to ERK inactivation, we first determined the effect of Rac1 on ERK activation. WT and Rac1-null macrophages were assayed for levels of activated phospho-ERK and we found that phospho-ERK was significantly lower in WT cells compared with Rac1-null macrophages (Figure 3A). As ERK is known to regulate SP-1 transcriptional activity [9,21–23], we asked whether ERK could regulate SP-1-driven MMP9 promoter activity. Macrophages were co-transfected with a MMP9 promoter reporter and either SP-1WT or SP-1S586A in combination with either an empty vector or the CA MEK1, which is the upstream kinase that activates ERK. Compared with cells transfected with the empty vector alone, MEK1 significantly increased the MMP9 promoter activity in cells expressing SP-1WT, but not the SP-1S586A mutant (Figure 3B). To verify the role of ERK, cells were co-transfected with the MMP9 promoter vector together with either SP-1WT or SP-1S586A. After 24 h, cells were incubated with the MEK1 inhibitor U0126 for 6 h. U0126 significantly reduced MMP-9 transcription in cells expressing SP-1WT, but not the SP-1S586A mutant (Figure 3C). Similarly, macrophages transfected with a dominant-negative ERK [pCMV-HA-ERK2 (K/A)] vector decreased MMP9 promoter activity in cells overexpressing SP-1WT, but not in cells transfected with the SP-1S586A mutant (Figure 3D). Taken together, these results strongly suggest that Rac1 regulates MMP9 gene expression by inhibition of ERK, which modulates SP-1 at Ser586.
ERK increases the stability of SP-1 at Ser586
ERK phosphorylation of serine and/or threonine residues within the PEST domain is linked to increased stability of proteins [14,15]. To address whether SP-1 stability can be regulated by ERK, we first determined the rate of SP-1 degradation in WT and Rac1-null macrophages incubated with cycloheximide, a general inhibitor of protein translation, for up to 16 h. The abundance of SP-1 was determined in cells by immunoblot analysis. SP-1 degradation in WT cells decreased in a time-dependent manner in the presence of cycloheximide, whereas there was no evidence of degradation of SP-1 in Rac1-null macrophages (Figure 4A). The t1/2 of native SP-1 in WT cells was approximately 12 h, whereas the t1/2 of native SP-1 in Rac1-null cells was greater than 16 h (Figure 4B). To determine whether the rate of SP-1 degradation was due to ERK inactivation, we inhibited ERK in Rac1-null cells using U0126 and measured SP-1 degradation in the presence of cycloheximide. ERK inhibition significantly decreased the abundance of SP-1 in the absence of protein synthesis in Rac1-null cells, suggesting that ERK activation increases SP-1 stability (Figure 4C). To confirm that ERK acted on SP-1 Ser586, cells were transfected with either SP-1WT or SP-1S586A. Whole-cell lysates were subjected to affinity purification, and the expression of the V5 tag following U0126 exposure was determined. ERK inactivation by U0126 significantly decreased the expression of SP-1WT, but not the expression of SP-1S586A (Figure 4D).
Alveolar macrophages from patients with asbestosis have less MMP-9, SP-1 and active ERK
As Rac1-null mice express high levels of MMP-9 in their alveolar macrophages and do not develop asbestos-induced pulmonary fibrosis , we determined MMP-9 activity in BAL fluid from WT and Rac1-null mice after exposure to chrysotile asbestos. Although there was no significant difference in MMP-9 activity 1 day after exposure, there was a significant increase in the activity in BAL from Rac1-null mice at 28 days post-exposure (Figure 5A). Our previous observations demonstrate that macrophages constitute the majority of cells when fibrosis becomes evident . Therefore these data suggest that increased macrophage-derived MMP-9 expression and activity in Rac1-null mice contribute, in part, to protection from development of pulmonary fibrosis.
To further address the biological significance of MMP-9, we determined MMP-9 expression in alveolar macrophages isolated from patients with asbestosis. Compared with normal subjects, MMP9 mRNA was approximately 16-fold less in alveolar macrophages obtained from patients with asbestosis (Figure 5B). To determine whether our in vitro data was recapitulated in patients, we investigated whether patients with asbestosis have lower levels of SP-1 expression and decreased ERK activation in alveolar macrophages. The expression of SP-1 (Figure 5C) and phosphorylated ERK (Figure 5D) were significantly decreased in alveolar macrophages from patients with asbestosis compared with normal subjects. Taken together, these results demonstrate that Rac1, SP-1 and ERK are important in the regulation of MMP-9 expression in alveolar macrophages.
Asbestosis, which is the most debilitating asbestos-related lung disease, is a prototypical form of pulmonary fibrosis. Despite tight regulatory controls to limit exposure, more than 1.3 million workers are exposed to hazardous levels annually [24,25]. A distinguishing feature of pulmonary fibrosis is aberrant matrix deposition that results from alteration in the balance between matrix deposition and degradation . MMPs have been implicated in the development of fibrosis, but their precise role has not been determined [2–8]. Several studies suggest that MMPs increase pulmonary fibrosis, whereas other studies suggest that inhibition or deletion of MMPs may alleviate the development of fibrosis [3–5,7,27]. We showed previously that Rac1 is required for the development of pulmonary fibrosis in mice exposed to asbestos . As the production of the macrophage-derived MMP-9 is inhibited by Rac1, these results suggest that the effects of Rac1 on fibrosis development are mediated, in part, by an inhibition of MMP-9 production. In the present study we demonstrated that MMP-9 expression is dependent on post-translational regulation of SP-1. Rac1 inhibited MMP9 gene transcription by inhibiting ERK activation and increasing SP-1 degradation. We report a novel finding that ERK activation increased SP-1 stability at Ser586 and, thereby, increased MMP9 promoter activity. These results establish, for the first time, the molecular mechanism by which Rac1 modulates the matrix remodelling enzyme MMP-9 via ERK and SP-1 and suggest that this mechanism could account for the fibrotic phenotype observed in asbestosis (Figure 6).
SP-1 is a ubiquitously expressed transcription factor that belongs to the C2H2-type zinc-finger protein family. It binds to GC-rich motifs on the promoters of various genes to either enhance or repress transcription. It is known to be highly regulated by post-translational modifications, such as phosphorylation, ubiquitination, SUMOylation, acetylation and O-glycosylation that regulate its transcriptional activity [23,28–31]. Phosphorylation modulates the transcriptional activity of SP-1 by regulating its stability [30,32]. Proteins containing a region rich in the amino acids proline (P), glutamine (E) or asparagine (D), serine (S) and threonine (T) are often targeted to be rapidly degraded . We identified a PEST domain in murine SP-1 between amino acids 575 and 595 containing one threonine (Thr578) and two serine (Ser586 and Ser587) residues that are potential sites of phosphorylation by serine/threonine kinases. Phosphorylation within the PEST domain of serine/threonine residues stabilizes proteins, as observed with ERK phosphorylation of serine residues in A170, MKP (MAPK phosphatase)-7 and MCL1 (myeloid cell leukaemia sequence 1) [14–16]. Although multiple kinases phosphorylate SP-1 at its many serine and threonine residues to either increase or decrease its transcriptional activity, none have been shown to phosphorylate SP-1 on residues within its PEST domain . As our previous results demonstrate the involvement of ERK in Rac1-mediated suppression of MMP9 gene expression , we questioned whether ERK altered SP-1 stability and the transcriptional activation of SP-1 by modulating the PEST domain. To our knowledge, our observations are the first to suggest that ERK regulates SP-1 stability within the PEST domain and, consequentially, its transcriptional activation of the MMP9 promoter.
SP-1 has been shown to be phosphorylated by MAPKs, but only on threonine residues [23,32]. JNK phosphorylates SP-1 on the threonine residues Thr278 and Thr739 during mitosis and protects SP-1 from ubiquitination and degradation, which results in increased transcriptional activation of the 12(S)-lipoxygenase gene . ERK phosphorylates SP-1 on Thr453 and Thr739, which is associated with increased VEGF (vascular endothelial growth factor) transcription or decreased FGF (fibroblast growth factor)-mediated PDGFRA (platelet-derived growth factor receptor α) gene expression [21,23]. Our results demonstrate that the Ser586 residue within the PEST domain is another site of ERK action on SP-1. Furthermore, although other serine residues have been shown to mediate SP-1 degradation [30,32], the involvement of the Ser586 residue in SP-1 protein stability has not been reported. SP-1 degradation is often linked to post-translational modifications, such as SUMOylation and/or ubiquitination or phosphorylation [30,32]. Phosphorylation of Ser7 results in SP-1 ubiquitination and degradation , whereas phosphorylation of Ser59 regulates SUMOylation and ubiquitination of SP-1 . Given that PEST domains target proteins for ubiquitination and subsequent degradation, it is likely that phosphorylation of Ser586 regulates proteasomal targeting .
Our observations from the present study do not rule out the role of other regions in the SP-1 protein that modulate MMP9 transcription. The phosphorylation of SP-1 at Ser7 results in ubiquitination and subsequent degradation . We found that MMP9 promoter activity increased with a mutation of Ser7 to alanine (results not shown). In addition, mutation of Lys16 to arginine also enhanced MMP9 promoter activity (results not shown). This is consistent with a previous report that SUMOylation at this residue represses SP-1 transcriptional activity . However, our observations in the present study indicate that CA Rac1 did not alter the MMP-9 expression in cells expressing these SP-1 mutants (results not shown). Interactions between residues on the SP-1 protein have been shown to control protein activity [33,34]. Interestingly, mutations of Thr578, Ser586 and Ser587 increased basal MMP9 gene transcription. It is likely that these residues either modulate one another's effect and/or co-operate with other regions in the protein to regulate the overall functional activity of SP-1.
A novel finding of the present study is that patients with asbestosis were found to have reduced MMP-9 expression in alveolar macrophages compared with normal subjects. Moreover, the modulation of MMP-9 by Rac1 is supported by our recent data showing that alveolar macrophages from patients with asbestosis have increased Rac1 activity compared with normal subjects . This, together with our observation that asbestos-exposed Rac1-null mice have greater MMP-9 expression and activity, supports the notion that macrophage-derived MMP-9 is important in matrix remodelling. Taken together, the present study reveals that SP-1 (Ser586), Rac1 and ERK are potential biomarkers for pulmonary fibrosis.
A. Brent Carter and Shubha Murthy conceived and designed the study. Shubha Murthy, Alan Ryan and A. Brent Carter acquired the data. Shubha Murthy, Alan Ryan and A. Brent Carter analysed and interpreted the data. Shubha Murthy and A. Brent Carter wrote and critically reviewed the paper prior to submission. A. Brent Carter gave final approval for the published version.
This work was supported by the National Institutes of Health [grant numbers ES015981 and ES014871] and a Merit Review from the Department of Veterans Affairs.
Abbreviations: BAL, bronchoalveolar lavage; CA, constitutively active; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility-shift assay; ERK, extracellular-signal-regulated kinase; HA, haemagglutinin; HRP, horseradish peroxidase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; MAPK, mitogen-activated protein kinase; MEK1, MAPK/ERK kinase 1; MMP, matrix metalloproteinase; PEST, Pro-Glu-Ser-Thr; SP-1, specificity protein 1; WT, wild-type
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