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Originally published In Press as doi:10.1074/jbc.M602703200 on July 10, 2006

J. Biol. Chem., Vol. 281, Issue 36, 26424-26436, September 8, 2006
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Expression Cloning Identifies Transgelin (SM22) as a Novel Repressor of 92-kDa Type IV Collagenase (MMP-9) Expression*

Rajesh R. Nair{ddagger}, Julian Solway§, and Douglas D. Boyd{ddagger}1

From the {ddagger}Department of Cancer Biology, M.D. Anderson Cancer Center, Houston, Texas 77030 and the §Department of Medicine, University of Chicago, Chicago, Illinois 60637

Received for publication, March 22, 2006 , and in revised form, July 6, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The 92-kDa gelatinase (MMP-9) expression is prerequisite for tissue remodeling in physiology and cancer. However, there are few known regulators of MMP-9 expression. Using an expression cloning strategy, we identified transgelin (SM22), a 22-25-kDa actin-binding protein localized to the cell membrane and cytoplasm, as a novel regulator of MMP-9 expression. Overexpression of a SM22 cDNA in HT1080 cells decreased MMP-9 mRNA/protein levels and diminished in vitro invasion of the latter rescued with exogenous MMP-9. Conversely, small interfering RNA-mediated knockdown of SM22 elevated MMP-9 synthesis, and uterus from SM22-null mice showed strong MMP-9 immunoreactivity compared with wild type animals. The ability of SM22 to repress MMP-9 expression required an intact amino terminus calponin homology domain. MMP-9 expression is driven by ERK signaling and SM22 targeted this pathway as evidenced by (a) the transience in MAPK activation and (b) blunted stimulation of the MMP-9 promoter by a constitutively active MEK expression vector. Progressive deletion analysis located the SM22 responsive region of the MMP-9 promoter to the proximal 90-bp region harboring an AP-1 motif subsequently implicated by site-directed mutagenesis. Furthermore, nuclear extract from the SM22 transfectants showed diminished c-Fos binding to this motif and SM22 expression reduced the activity of an AP-1-driven reporter by 75%. Thus, SM22 adds to a short list of repressors of MMP-9 expression, achieving this by reducing AP-1-dependent trans-activation of the gene by way of compromised ERK activation. Diminished transgelin expression in several cancers may thus partly account for the elevated MMP-9 expression evident in these tumors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The 92-kDa type IV collagenase (MMP-9) contributes to tissue remodeling both in physiology and pathology. In pregnancy, expression of this metalloproteinase by invading trophoblasts is prerequisite for implantation into the maternal decidua (1) during smooth muscle cell replication and migration into the neointima after denuding injury requires MMP-9 expression (2, 3). Similarly, in bone development, migration of osteoclasts into cartilage matrix is dependent on their expression of this metalloproteinase and defective endochondral bone formation is evident in mice null for this metalloproteinase (4). MMP-9 also plays a key role in angiogenesis with knock-out mice manifesting abnormal skeletal growth plate vascularization (5).

In cancer, MMP-9 enhances tumor progression in some, but not all, malignancies. In an earlier study, expression of an anti-MMP-9 ribozyme effectively blocked metastasis of rat sarcoma cells (6), whereas, more recently, in an elegant series of experiments primary tumor growth, angiogenesis, and lung metastasis were diminished in animals null for this collagenase (7). Recent studies also indicate a function of MMP-9 in cell transformation (8) suggesting a role in an early malignant event. Surprisingly, MMP-9 can also suppress tumor progression depending on the tumor type and/or stage with siRNA2 targeting this metalloproteinase yielding increased intravasation and metastasis in the chorioallantoic membrane assay (9).

The original proposal that MMP-9 solely functions in the cleavage of extracellular matrix components including elastin, type III, IV, and V collagen (10) has had to be broadened in light of recent findings. Indeed, it is becoming increasingly evident that MMP-9 is a multifunctional protein that also regulates angiogenesis (11, 12) by generating both pro-(vascular endothelial growth factor and transforming growth factor-beta) and anti-angiogenic (angiostatin and tumstatin) proteins as well as a cryptic pro-migratory epitope (for endothelial cells) from type IV collagen (11-14). Furthermore, transformation of immortalized human mammary epithelial cells by constitutively active Stat-3-C requires MMP-9 and indeed the amounts of the transcription factor and metalloproteinase correlate in primary breast cancer specimens (8).

MMP-9 expression is largely controlled by transcription of the gene encoded on chromosome 20 (15), although mRNA stability (16-18) and translational efficiency (19) also play a role in regulating the amounts of the protein product. Regulation of transcription is achieved via a 2.2-kb upstream regulatory sequence containing binding sites for AP-1, NF-{kappa}B, Sp1, and PEA3/Ets (20-23) and is also dependent on the state of chromatin condensation (24, 25) dictated by recruitment of histone deacetylase 2 and the Brg-1 subunit of the SWI/SNF chromatin remodeling motor to the promoter via c-Fos·JunD complexes. Translation of the 2.5-kb mRNA yields a latent 92-kDa protein subsequently activated by the enzymatic removal of 73 amino acids at the amino terminus (26, 27). The activity of MMP-9, in turn, is titrated by the endogenous tissue inhibitor of metalloproteinases 1-3 (28, 29).

To date, a mere handful of inducers of MMP-9 expression including KGF, HGF, transforming growth factor-beta, tumor necrosis factor-{alpha} (30-33), and the proto-oncogenes c-Src and H-Ras have been identified (20, 23). The Snail transcription factor, stimulatory for an invasive phenotype, also up-regulates MMP-9 transcription (34). Additionally, MMP-9 expression is a target of the Wnt pathway as evidenced by the ability of Frizzled-related protein 3, a secreted Wnt antagonist, to suppress MMP-9 expression and PC-3 prostate cancer cell invasiveness (35). Conversely, the metastases suppressor Kiss-1 is one of the few negative regulators of MMP-9 expression identified to date achieving this repression by down-regulating NF-{kappa}B translocation (36).

However, so far, there have been no genome-wide studies to identify novel regulators of MMP-9 expression in a systematic manner. Considering the important role of MMP-9 in physiology and pathology we have employed expression cloning to identify hitherto unknown regulators of expression of this collagenase. We report herein SM22 (transgelin) as a novel regulator of MMP-9 expression repressing its expression by interfering with ERK activation and AP-1 signaling.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—HT-1080 cells were maintained in McCoys 5A medium supplemented with 10% fetal bovine serum. WI-38 and VA-13 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For stable transfections, cells were transfected at ~80% confluence using Lipofectamine 2000 as described by the manufacturer (Invitrogen). Briefly, the cells were transfected with 8 µg of DNA and 24 µg of Lipofectamine 2000 for 24 h, after which the transfection mixture was replaced with fresh medium. Cells were incubated for another 48 h and then selected with 1 mg/ml of G418. Clones were isolated, expanded, and screened for SM22 expression.

Constructs—The pQE-30 bacterial expression vector harboring the SM22 cDNA fragments (1-201, 1-186, 1-166, and 1-151) was used as template to PCR amplify (with Pfu Turbo) and subclone these fragments into the HindIII/KpnI-digested pEGFP-C1 expression vector. Similarly, the SM22 cDNA fragments encoding amino acids 51-201, 76-201, and 101-201 were generated by PCR and subcloned into the HindIII/KpnI-digested pEGFP-N1 expression vector.

Expression Cloning Strategy—These assays were performed as described previously (37). Briefly, an arrayed human colon cDNA library (LCO-1001, Origene Technologies, Rockville, MD) in which cDNAs were constructed in the pCMV6-XL4 expression vector was employed. DH10B Escherichia coli bacteria were transformed with DNA from the library and seeded on ampicillin LB-agar plates to obtain ~100 colonies per plate and cDNA pooled from each plate.


Figure 1
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  		FIGURE 1.
Expression cloning identifies a putative regulator of MMP-9 expression. Panel A, HT1080 cells were co-transfected with 100 ng of a 2.2-kb MMP-9 promoter-luciferase (or pGL3) plasmid, a CMV6-driven expression vector encoding nothing or individual cDNAs (identified from primary and secondary screens) (700 ng), and pRL-SV40 (1 ng). After 24 h, cells were lysed and luciferase activity was determined. Data, normalized for differences in transfection efficiency, are shown as % change (±S.D. values of three independent experiments) with the value derived from co-transfection with the MMP-9 promoter and the empty expression construct set at 100%. Panel B, same as panel A except that the 1:1 MMP-9-Luc: clone 13-1(b)-ratio indicates 100 ng each of the reporter and expression constructs.

 
In the primary screen cells in a 24-well format were co-transfected using Lipofectamine 2000 (Invitrogen) with a DNA mixture comprised of the cDNA pool (700 ng), the MMP-9 promoter (2.2 kb)-luciferase reporter (100 ng), and 1 ng of an pRL-SV40 internal control. After 24 h, cells were lysed and assayed for luciferase activity using the Dual Luciferase Reporter Assay System (Promega).


Figure 2
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  		FIGURE 2.
Stable SM22 expression in HT1080 cells reduces MMP-9 expression. Panel A, HT1080 cells were transfected with an SM22 expression construct or the empty vector (pEGFP-C1) and G418-resistant clones expanded and analyzed for SM22 protein by Western blotting using a polyclonal anti-SM22 antibody. Panel B, conditioned medium (in serum-free medium) normalized for cell number differences was analyzed by gelatin zymography. Panel C, total RNA from the indicated clones was subjected to multiplex RT-PCR using primers generating the MMP-9 (120 bp) and actin (621 bp) amplicons. +ve control, PCR of a MMP-9 coding sequence plasmid. The data are typical of duplicate experiments.

 
In secondary screens, positive pools were further subdivided with 10 colonies per plate. cDNA sub-pools were prepared and assayed as described above. Finally, in a tertiary screen, single colonies from positive sub-pools (selected in the secondary screen) were identified as described above.

Immunostaining—Uterus was fixed in 10% formalin and embedded in paraffin. Sections were then de-paraffinized, treated with 3% H2O2 in methanol, and then blocked with 5% normal horse serum and 1% normal goat serum. Sections were incubated overnight with a 1:500 rabbit polyclonal anti-mouse MMP-9 antibody (Santa Cruz Biotechnology, CA) or an equivalent amount of an anti-rabbit IgG and subsequently with an anti-rabbit peroxidase-conjugated F(ab)2 fragment. The diaminobenzidine substrate was used to visualize immunoreactivity after counterstaining with hematoxylin.

Mobility Shift Assays—Nuclear extracts and electrophoretic mobility shift assay were carried out as described by us elsewhere (36). Electrophoretic mobility shift assay was performed using 10 µg of nuclear extract, 0.6 µg of poly(dI/dC), and 2 x 104 cpm of a [{gamma}-32P]ATP T4 polynucleotide kinase-labeled oligonucleotide.

Invasion Assays—These were as described by this laboratory previously (36). Cells (25,000) in 10% albumin (serum-free) were seeded on Matrigel-coated porous filter (8 µm) using 10% fetal bovine serum as chemoattractant. After 24 h, cells on the upper aspect of the membrane were removed by scrubbing and the cells on the lower aspect were counted.

Reporter Assays—These were carried out as previously reported (36). Cells were co-transfected (using Lipofectamine 2000) 24 h post-seeding with a promoter-driven luciferase reporter (0.1 µg) and, where indicated, an expression vector (pEGFP-C1) encoding the full-length or truncated SM22 fragments. A SV40 promoter (unless specified otherwise)-driven Renilla luciferase (4 ng) was included as internal control. Cells were washed 24 h later, lysed, and assayed for luciferase activity.

Semiquantitation of MMP-9 mRNA by RT-PCR—Total RNA was isolated from cultured cells or mouse uterus using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA (2 µg) and oligo(dt) primer (1 µg) were heated at 70 °C for 5 min, and chilled. To this was added 1x RT buffer (Promega), 0.5 mM each of four deoxynucleosides, 1 unit/µl RNasin (Promega), 4 mM sodium pyrophosphate (Sigma), and 30 units of avian myeloblastosis virus reverse transcriptase (Promega). The RNA was reverse transcribed at 42 °C, and 2 µl of the products were used as the template for multiplex PCR using MMP-9 and beta-actin primers. The following primers used were were 5'-GAGGTTCGACGTGAAGGCGCAGATG-3' and 5'-CATAGGTCACGTAGCCCACTTGGTC-3' to amplify the human MMP-9 (product size, 120 bp) and beta-actin (product size, 621 bp), 5'-ACACTGTGCCCATCTACGAGG-3' and 5'-AGGGGCCGGACTCGTCATACT-3'. For mouse tissues, primers used were 5'-ATTGGTGAACAGCCTGTATCCT-3' and 5'-CTCCACGGTAGTTTCCATCG-3' to amplify a 251-bp product for the SM22 transcript. The primers for mouse glyceraldehyde-3-phosphate dehydrogenase were 5'-ACCCAGAAGACTGTGGATGG-3' and 5'-CACATTGGGGGTAGGAACAC-3' to amplify a 171-bp product.


Figure 3
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  		FIGURE 3.
Reduced in vitro invasion of SM22-transfected HT1080 cells. The indicated clones (25,000) were seeded on Matrigel-coated porous filters with or without exogenous (33 ng/ml) MMP-9 protein. After 18 h, cells were removed from the top aspect of the filter. The filter was stained with Diff-Quik and cells on the lower aspect of the filter enumerated. The data in panel B represent average ± S.D. of quadruplicate assays.

 
siRNA Targeting of SM22—Transfection efficiency using Lipofectamine 2000 was optimized using siTOX (Dharmacon RNA Technologies, catalog number D-001500-01-05). Cells were transfected with either siGenome SMARTpool reagent targeting SM22 (Dharmacon RNA Technologies, catalog number M-003714-01-0005) or siCONTROL non-targeting siRNA (Dharmacon RNA Technologies, catalog number D-001210-01-05) at a concentration of 100 nM. The cells were incubated for 48 h post-transfection after which MMP-9 and SM22 expression was determined.

Western Blotting—For ERK, MEK, p38, and JNK-1 immunoblotting, cells were serum starved overnight and then treated with 100 nM phorbol 12-myristate 13-acetate for various times. The cells were lysed in a buffer (10 mM Tris-HCl, pH 7.4, 0.5% Nonidet P-40, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin, leupeptin, and pepstatin) on ice for 30 min. Cell lysates were cleared by centrifugation, proteins (15-50 µg) were resolved by polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. After blocking with 5% milk solution, blots were incubated with primary antibodies to phosphorylated JNK-1, p38 (Santa Cruz sc6254 and sc-7973, respectively), total ERKs (Cell Signaling catalog number 4696), and phosphorylated ERKs (Cell Signaling catalog number 9106) and total and phosphorylated MEKs 1 and 2 (Cell Signaling catalog numbers 9122 and 9121, respectively) at 4 °C overnight. Blots were washed with TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) buffer, and then incubated with a horse-radish peroxidase-conjugated secondary antibody. Proteins were visualized with ECL reagents. MMP-9 and SM22 protein were quantified essentially as described above but using polyclonal anti-MMP-9 (Biomol International, LP (catalog number SA-106)) and anti-SM22 antibodies, the latter as described elsewhere (38).

Zymography—Zymography was carried out as described previously (36), using aliquots of conditioned medium corrected for any differences in cell number.

Quantitation—Band intensity was quantified by densitometry using Quantity One Software (version 4.1) (Bio-Rad).

Statistical Analysis—Differences were tested for statistical significance using an unpaired t test and GraphPad, Prism software (version 3.03).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
There has been no genome-wide search for regulators of MMP-9 expression and, to date, only a few regulators have been identified by empirical "guesswork." Therefore to identify novel regulators of MMP-9 expression we employed a cDNA library comprised of 500,000 cDNAs. In this approach, the library was subdivided with primary screenings undertaken with pools of 100 cDNAs. For screening, we used HT1080 cells because it constitutively expresses moderate MMP-9 amounts allowing a search for both activators and repressors of MMP-9 expression.

Identification of SM22 as a Putative Regulator of MMP-9 Expression—HT1080 cells were co-transfected with cDNA pools and a luciferase reporter regulated by 2.2 kb of MMP-9 upstream sequence and subsequently assayed for luciferase activity. This promoter sequence includes all the regulatory elements necessary for appropriate MMP-9 expression (39). Primary and secondary screens led to a subpool (13-1) of 10 cDNAs that diminished MMP-9 promoter activity (data not shown). This subpool was divided into individual clones and in a tertiary screen (Fig. 1A, open bar) clone 13-1(b) repressed MMP-9 promoter activity. DNA sequencing revealed that clone 13-1(b) corresponded to an open reading frame showing 98% homology with the full-length human transgelin (also known as SM22) cDNA coding sequence. MMP-9 promoter activity was decreased in a dose-dependent manner (Fig. 1B) with a 5:1 ratio of expression vector to the MMP-9 reporter reducing luciferase activity by over 60%.

Whereas several putative regulators of MMP-9 expression were identified, we pursued SM22 as a candidate regulator for three reasons. First, there are few known repressors of MMP-9 expression. Second, data mining of expression profiling studies (Oncomine) revealed that SM22 expression is attenuated in some metastatic cancers (e.g. lung and prostate) characterized by their elevated MMP-9 mRNA/protein expression (40-43). Third, little is known as to SM22 function. The transgelin gene (TAGLN) is located on chromosome 11q23.2 and generates a 1.3-kb mRNA. TAGLN expression is repressed by cell transformation (41). The encoded 25- and 22-kDa protein products localize (44) to the cytoplasm and cell membrane (45). SM22 binds actin via its carboxyl terminus residues (38) and has a putative role in actin gelation.

Stable SM22 Expression Represses MMP-9 Expression—To validate the reporter assays, we determined the effect of SM22 on endogenous MMP-9 expression. HT1080 cells were stably transfected with an expression construct bearing the SM22 coding sequence, G418-resistant clones were expanded and analyzed for SM22 protein by Western blotting (Fig. 2A). Three HT1080 clones (1, 4, 6) were positive for SM22 protein, whereas the parental cells and an empty vector control were negative for this protein. The various clones were then analyzed for MMP-9 activity by zymography (Fig. 2B). A gelatinolytic band identical in size (92 kDa) to MMP-9 was evident (Fig. 2B) in conditioned medium from both parental HT1080 and cells harboring the empty vector, whereas the intensity of the band was substantially diminished in the three SM22-expressing clones. In contrast, the 72-kDa gelatinolytic band, representing the product of the MMP-2 gene (46) was unchanged by SM22 expression. To corroborate these data, MMP-9 mRNA levels were semi quantified by multiplex RT-PCR. Again, whereas an amplified product of the predicted size (120 base pairs) was easily detected with the parental and empty vector-transfected HT1080 cells (Fig. 2C), the intensity of the signal was clearly attenuated in the three separate SM22-expressing HT1080 clones (1, 4, 6). To further confirm its role as a suppressor of MMP-9 expression, HT1080 cells bearing the empty vector or clones expressing SM22 were analyzed for in vitro invasion. Expectedly (47), HT1080 cells expressing the empty vector were highly invasive through an extracellular matrix-coated porous filter (Fig. 3A), whereas two independent clones expressing the SM22 cDNA showed about a 50% reduction in this assay. Diminished invasiveness was effectively rescued by the addition of exogenous MMP-9 protein suggesting that the attenuated behavior was largely because of repressed MMP-9 expression. The reduced invasiveness of the SM22-expressing clones was not a consequence of diminished proliferation (data not shown). Thus, HT1080 cells stably overexpressing SM22 show attenuated MMP-9 expression.


Figure 4
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  		FIGURE 4.
Knockdown of SM22 with siRNA up-regulates MMP-9 synthesis. WI-38 cells were transfected with a pool of 4 siRNA (100 nM final concentration) targeting SM22 or a control mismatched siRNA (Cntrl) using Lipofectamine 2000. Panels A and B, after 72 h the cells were analyzed by Western blotting using polyclonal antibodies directed at SM22 or MMP-9. Panel C, one day post-transfection, cells were changed to serum-free medium for a 24-h period, conditioned medium was harvested, adjusted for differences in cell numbers, and subjected to zymography using 1% gelatin. Band intensity was quantified by densitometry. The experiment was performed twice.

 
Suppression of SM22 Expression Up-regulates MMP-9 Activity—To accrue further evidence implicating SM22 as a regulator of MMP-9 expression we determined if its knockdown elevates synthesis of this metalloproteinase. Toward this end, we used normal lung fibroblasts (WI-38) that express SM22 (41). The cells were transfected with a pool of 4 siRNAs targeting SM22 or, as a control, a non-targeting siRNA. SM22 protein levels were clearly reduced in the WI-38 fibroblasts transfected with the siRNA pool targeting SM22 (Fig. 4A). More importantly, zymography and Western blotting indicated up to a 3-fold increase in MMP-9 protein/activity in lysates and conditioned medium from cells treated with the siRNA targeting SM22 (Fig. 4, B and C).


Figure 5
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  		FIGURE 5.
Extinguishing SM22 expression in lung fibroblasts is accompanied by induced MMP-9 expression. Panel A, equal protein from WI-38 fibroblasts, or their SV40-transformed counterparts (VA-13), was analyzed for SM22 protein by Western blotting. Panel B, conditioned medium from the indicated cells was normalized for cell number differences and analyzed for MMP-9 activity by gelatin zymography. Panel C, total RNA was extracted from the indicated cells, and subjected to multiplex PCR using primers specific for the actin and MMP-9 transcripts. The arrow points to the MMP-9 amplicon and the asterisk indicates primers. The MMP-9 (+ve Cntrl) represents amplified product from a plasmid bearing the MMP-9 coding sequence. For panels B and C, the intensity of the 92-kDa gelatinolytic band and the amplified cDNA corresponding to the MMP-9 transcript was quantified by densitometry. Data are typical of duplicate experiments.

 
Because cell transformation by SV40 abolishes SM22 expression (41) MMP-9 induction should also be apparent in the transformed cells. Consequently, we compared MMP-9 expression in the normal human lung fibroblasts (WI-38) and their SV40-transformed counterparts (VA-13). As expected, SM22 protein levels were extinguished in the transformed cells (VA-13) (Fig. 5A) and this observation was paralleled by a 2-fold increase in 92-kDa gelatinase activity (Fig. 5B). The elevated MMP-9 activity in the VA-13 cells, extinguished for SM22 expression, reflected a parallel increase in MMP-9 mRNA (Fig. 5C) as revealed by RT-PCR.

We then determined if MMP-9 expression was up-regulated in SM22 null mice (48). Uterine tissue, which constitutively expresses transgelin (45), was obtained from wild type or null mice for SM22 (Fig. 6A) and stained for MMP-9 protein. Mice were synchronized in their estrus cycle by housing in the same cage. Tissue from 2 independent SM22 knock-out mice (-/-) showed clear MMP-9 immunoreactivity both in the stromal and epithelial compartments (arrows), whereas little immunoreactivity was evident with tissues derived from the wild type (+/+) animals (Fig. 6B). Thus, taken together, these studies indicate SM22 as a bona fide regulator of MMP-9 expression.

The Actin-binding Region of SM22 Is Dispensable with Respect to MMP-9 Repression—How does SM22 regulate MMP-9 expression? SM22 contains a single amino-terminal located calponin-homology domain (49) and calponin-like repeats at the carboxyl terminus. Because its binding to actin is mediated through the SM22 carboxyl terminus (38), we asked whether this region is required for MMP-9 repression and toward this end, we employed expression constructs encoding the full-length (1-201 amino acids) SM22 or truncations thereof (Fig. 7A, left panel). HT1080 cells were co-transfected with these various SM22 expression constructs and a luciferase reporter driven by the MMP-9 promoter. Luciferase assays (Fig. 7B) indicated a strong repression of MMP-9 promoter activity by the full-length SM22 (wild type), whereas an unrelated promoter (thymidine kinase) was unaffected (data not shown). The carboxyl terminus-deleted construct (SM22-(1-151)), unable to bind actin, as determined by actin co-sedimentation (38), was equiactive with the full-length SM22 in repressing MMP-9 promoter activity.


Figure 6
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  		FIGURE 6.
Increased MMP-9 protein levels in uterus derived from SM22 null mice. Panel A, total RNA from mouse uterus was analyzed for SM22 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts by RT-PCR. Panel B, sections of the uterus derived from the indicated mice were fixed and subjected to immunohistochemistry with a rabbit anti-mouse MMP-9 antibody followed by horseradish peroxidase-conjugated anti-rabbit antibody. MMP-9 immunoreactivity (arrows) was visualized with diaminobenzidine. A zoomed image of the indicated boxed area is shown depicting epithelial (E) and stromal (S) compartments.

 
In contrast, amino-terminal deletions generating constructs 51-201, 76-201, and 101-201 (Fig. 7A, right panel), thus lacking an intact calponin homology domain (amino acids 25-132), yielded SM22 fragments far less effective (Fig. 7B) in repressing MMP-9 promoter activity when compared with the full-length (1-201) protein (SM22 wild type). These data would suggest that the region harboring the calponin homology domain, but not the actin-binding region, is required for optimal MMP-9 repression by SM22.

Transient ERK Activation in SM22 Transfectants—The cytoplasmic localization of SM22 (45) coupled with the absence of a nuclear localization signal suggested that SM22 represses MMP-9 expression indirectly. Furthermore, we noted that the ability of SM22 to repress MMP-9 promoter activity depended on the presence of an intact amino-terminal type 3 calponin homology domain (see above). Because the amino-terminal type 3 calponin homology domain physically interacts with ERKs 1 and 2 (49), SM22 may interfere with signal transduction pathways impinging on MMP-9 expression (see "Discussion" also). Indeed, the regulation of MMP-9 expression by the ERK signaling module has been previously reported by several laboratories (50-52). To determine whether SM22 targets the ERK pathway, activated (phosphorylated) forms of ERKs were measured in the HT1080 SM22 transfectants and the vector control by Western blotting (Fig. 8A). Interestingly, whereas phorbol ester activated ERKs 1 and 2 in both the vector and SM22-expressing HT1080 cells, induction in the former was sustained (up to 6 h) while returning to baseline by 2 h in the latter cells. These findings are noteworthy because sustained, but not transient, ERK activation drives MMP-9 expression (51). The absence of activated ERKs at time 0 probably reflects prior serum starvation. Interestingly, the upstream activator of the ERKs, namely, MEKs 1 and 2 showed little difference in their activation in the SM22 transfectants suggesting that the transient ERK activation was not reflective of regulation at this higher level signaling kinase. We also determined if the JNK or p38 signaling modules were targeted by SM22. However, little change in the levels of these dual activity kinases was evident between the SM22 and vector-expressing HT1080 cells (Fig. 8A).

To accrue further evidence that the ERK pathway was blunted by SM22, HT1080 cells bearing either the empty vector or stably expressing transgelin, were transiently co-transfected with the MMP-9 promoter-driven luciferase reporter and an expression vector encoding a constitutively activated MEK (MEK {Delta}N3S218E-S222D) 60-400 times more active in phosphorylating ERKs than the wild type MEK used as control (53, 54). The mutation-activated MEK stimulated a robust increase (~300%) in MMP-9 promoter activity in the HT1080 cells bearing the empty vector (Fig. 8B). In contrast, the % increase in MMP-9 promoter activity achieved with MEK {Delta}N3S218E-S222D was significantly (<0.0001) less with the HT1080 cells stably expressing SM22. These data further support the notion that SM22 interferes with ERK signaling impinging on MMP-9 expression.


Figure 7
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  		FIGURE 7.
The actin-binding domain of SM22 is dispensable with respect to repression of MMP-9 promoter activity. Panel A, SM22 cDNA fragments cloned into the pEGF-C1/N1 expression constructs were digested with HindIII/KpnI and the products were resolved in an agarose gel. The faster migrating bands in each lane represent the SM22 coding sequence. Actin binding affinity was as published (38) with -, +, ++, and +++ indicating undetectable and undetectable 10, 10-20, and >20% SM22 fragment co-sedimenting with actin, respectively. Panel B, the indicated SM22 expression constructs (700 ng) were co-transfected with a luciferase reporter regulated by 2.2 kb of the MMP-9 regulatory sequence (100 ng). After 24 h, cells were lysed and, after normalization for differences in transfection efficiency, assayed for luciferase activity. The data are typical of four independent experiments.

 
SM22 Reduces AP-1 Trans-activation of the MMP-9 Promoter—To determine the transcriptional elements mediating MMP-9 repression by SM22, we transiently transfected HT1080 cells with 5' deleted MMP-9 promoter fragments fused to a luciferase reporter. SM22 reduced transcription by 50% (Fig. 9) from the longest (2.2 kb) MMP-9 promoter fragment. However, this repression was unimpaired by progressive 5' deletions with the shortest (90 bp) and longest (2.2 kb) fragments showing equal sensitivity. This minimal promoter region contains an AP-1 binding motif, a GC box, as well as the KRE-M9 element recognizing the differentiation-repressing factor 1 (55).

Considering the transient ERK signal evident in the SM22 transfectants together with the well established role of the AP-1 motif in regulating expression of this metalloproteinase downstream of this signaling module (50), we performed two experiments to determine the role of this motif in the SM22-dependent MMP-9 repression. First, the effect of mutating the proximal (-79) and distal (-533) (20, 56) AP-1 motifs on MMP-9 repression by SM22 was determined. Interestingly, only the simultaneous mutation of both proximal and distal AP-1 motifs in context of the 670-bp MMP-9 promoter impaired the repressive effect of SM22 (data not shown) arguing that these motifs are redundant with respect to the suppressive effect of transgelin. Second, we determined if transcription factors binding to the proximal AP-1 site was altered in the SM22 transfectants. Nuclear extract from the parental and vector-transfected HT1080 cells gave a shifted band (parentheses) with an oligonucleotide bearing the proximal AP-1 motif (Fig. 10A, lanes 3 and 4). This shifted band represents specific binding because it was abolished with an excess of non-radioactive probe (Fig. 10A, lane 2). More importantly, the intensity of this shifted band was reduced with the three independent SM22 clones (lanes 6-8). In contrast, binding to the MMP-9 promoter-derived KRE-M9 element, located immediately downstream of this AP-1 motif, and which recognizes differentiation repressing factor-1 (55) was unaffected (Fig. 10B) with nuclear extract from the SM22 transfectants.


Figure 8
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  		FIGURE 8.
Transient activation of signaling kinases in SM22 transfectants. Panel A, the indicated cells were serum-starved for 16 h and then treated with phorbol 12-myristate 13-acetate (PMA) (100 nM). At the stated times thereafter, cells were extracted and analyzed for phosphorylated (activated) or total amounts of the indicated kinase by Western blotting. The experiment was performed twice. Panel B, the indicated cells were cotransfected with 100 ng of a MMP-9 promoter fragment (2.2 kb)-firefly luciferase reporter and 700 ng of either the wild type MEK1 or a constitutively active MEK1 expression construct (MEK {Delta}N3S218E-S222D). A beta-actin promoter-driven Renilla reporter (4 ng) was included as internal control. Cells were lysed 24 h post-transfection and luciferase activity was determined. The activity of the MEK wild type was set to 100% and data represent average ± S.D. % values of four independent experiments.

 
Because transient ERK activation (as evident with the SM22 transfectants) is known to decrease the c-Fos protein amount via de-stabilization (57) we performed supershifting experiments (Fig. 10C) using an antibody directed at this AP-1-binding protein. A supershift was evident with nuclear extract derived from the HT1080 cells bearing the vector only (Fig. 10C, lane 4, arrow) at the expense of the retarded band (parentheses) clearly indicating the presence of this DNA-binding protein in the complex. In contrast, the intensity of this supershifted band was greatly diminished with nuclear extract generated from two independent HT1080 clones expressing the exogenous SM22 cDNA (Fig. 10C, lanes 7 and 10, arrow). To accrue further evidence that SM22 was targeting AP-1-dependent regulation of MMP-9 expression, HT1080 cells were co-transfected with a luciferase reporter regulated by 7x tandem AP-1 repeats (Stratagene number 219074) and an expression vector encoding nothing (pEGFP-C1) or the SM22 coding sequence (pEGFP-SM22). Expression of the SM22 cDNA yielded ~75% reduction in promoter activity (Fig. 10D) when compared with that achieved with the empty expression vector a difference that was statistically significant (p < 0.0001). Taken together, these data suggest that SM22 represses MMP-9 expression at least in part via reduced trans-activation of the promoter through its proximal AP-1 motif.


Figure 9
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  		FIGURE 9.
Identification of the SM22-responsive region of the MMP-9 promoter. HT1080 cells were co-transfected with the indicated MMP-9 promoter fragment-luciferase reporter (100 ng) and the SM22 expression construct or the empty vector pEGFP-C1 (700 ng) as described in the legend to Fig. 1. After 24 h, cell lysates were assayed for luciferase activity. Data are expressed as % change (±S.D. of triplicate experiments) with 100% representing the value achieved by co-transfection with the 2.2-kb MMP-9 promoter fragment and the empty expression vector.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Using an expression cloning strategy, we have identified transgelin (SM22) as a novel regulator of MMP-9 expression. SM22 adds to a short list of proteins, which includes the metastases suppressor Kiss-1 (36) and the yet uncharacterized differentiating-repressing factor-1 (55) that attenuate MMP-9 expression. SM22 diminishes MMP-9 expression by blunting ERK activation leading to suppressed trans-activation of the promoter through the proximal AP-1 motifs.

Currently, little is known regarding SM22 function. Certainly, transgelin binds actin (38) and this association may contribute to actin gelation although such a role has been questioned because it does not occur at physiological pH (58). Nevertheless, a truncated SM22 fragment, incapable of binding actin, was equiactive with the full-length protein in repressing MMP-9 promoter activity making it unlikely that diminished MMP-9 expression reflects its capacity to associate with this cytoskeletal protein. More plausible is that transgelin regulates MMP-9 expression via modulation of ERK activation. Indeed, this signaling module impinges on MMP-9 expression in Madin-Darby canine kidney epithelial cells (34), keratinocytes (51), and oral squamous cell carcinoma (50). Whereas sustained (up to 6 h) ERK activation was evident in HT1080 vector controls, phosphorylated ERK levels in the SM22 transfectants were transient, returning to baseline within 2 h. Sustained, but not transient, ERK activation is critical for elevated MMP-9 expression as demonstrated previously with EGF and hepatocyte growth factor-stimulated keratinocytes (51). Thus, SM22-dependent MMP-9 repression likely reflects blunted ERK activation.

How then does SM22 interfere with ERK activation? SM22 contains a type 3 calponin homology domain located within its amino-terminal 132 amino acids and the calponin protein, bearing this domain, physically interacts with ERKs 1 and 2 (59). Indeed, cDNAs encoding truncated SM22 proteins lacking an intact type 3 calponin homology domain were poor repressors of MMP-9 expression when expressed in HT1080 cells raising the possibility that binding of transgelin to these MAPKs somehow blunts their activation. However, our co-immunoprecipitation attempts to show interaction between these proteins failed. An alternate possibility is that transgelin increases the activity and/or amount of MKP-1, or another dual activity phosphatase, yielding the transient ERK activation evident in the SM22 transfectants. Irrespective of the ERK-regulatory mechanism, the transient activation of this MAPK subset was clearly evident in the SM22 transfectants and probably contributes to MMP-9 repression (51). On the other hand, unlike oral keratinocytes (52), it does not appear that p38 (60) signaling contributes to the SM22-dependent MMP-9 repression insofar as the activated form was unaffected by transgelin expression. Likewise, JNK activation, a prerequisite for MMP-9 induction by the Ras oncogene in ovarian cancer cells (50), was also unchanged by transgelin. Presumably, these two pathways are non-redundant with the ERK signaling module and therefore unable to compensate for the deficient signaling through the latter.

Our studies indicated that repressed MMP-9 expression by SM22 was at least partly due to diminished transcription from the MMP-9 promoter. However, we cannot presently exclude the possibility that reduced expression also reflects, in part, a post-transcriptional component. Whereas our reporter assay indicated a 50-60% reduction in promoter activity, enzyme (zymography) and mRNA determinations indicated a more pronounced effect invoking the possibility of post-transcriptional control. Post-transcriptional modulation of MMP-9 expression has been reported previously with transforming growth factor-beta and lipopolysaccharide both mediating MMP-9 induction via stabilization of the transcript (18, 61). In keratinocytes, ERK activation by a mutation-activated Ras yields MMP-9 mRNA stabilization (16), a finding pertinent to our study that implicated transience in ERK activation in the SM22-dependent MMP-9 repression. Thus, it is possible that SM22 represses MMP-9 expression not only by targeting the transcription machinery but also by regulating mRNA stability or possibly even translational efficiency as evident in myc/ras-transformed murine urogenital sinus cells (19).

The MMP-9 promoter contains multiple cis elements regulatory for its expression including Ets, NF-{kappa}B, and Sp1 binding sites residing between -600 and -533 relative to the transcription start site (20). However, our 5' deletion analysis of the promoter revealed that only the proximal 90 base pairs of the MMP-9 promoter was required for the SM22-dependent MMP-9 repression thus arguing against the contribution of these upstream cis elements (20). These findings distinguish the MMP-9 repression by SM22 from that achieved by Kiss-1. This metastases suppressor blocks p65/p50 nuclear translocation and hence trans-activation of the MMP-9 promoter through its NF-{kappa}B site at -600 (36). The 90 base pairs of 5' flanking MMP-9 sequence contains a well characterized AP-1 motif previously shown to mediate MMP-9 induction by diverse stimuli including integrin-linked kinase, oncogenic Ras, Src, myc, and phorbol ester (20, 21, 23, 25, 62). In fact, our observations of a marked reduction in transcription factor (including c-Fos) binding to this element in the SM22 transfectants reflecting ERK-dependent decreased c-Fos protein (57), nuclear-cytoplasmic shuttling (63), or altered expression of a dimerizing partner (64), strongly argue that this site mediates, to a large extent, MMP-9 repression. Nevertheless, the responsive promoter region (-90) also harbors a GC-box located proximal of this AP-1 site that could feasibly also contribute to the SM22-dependent regulation of MMP-9 expression. Indeed this element mediates, at least in part, MMP-9 induction by the Snail transcription factor (34) and v-src (23). Additionally, the proximal MMP-9 sequence also harbors a KRE-M9 element but because no change in transcription factor binding to this motif was evident with nuclear extract from the SM22 transfectants it is unlikely that transgelin represses MMP-9 expression via this binding site unless transcriptional activity of the corresponding DNA-binding protein (differentiation repressing factor-1) is targeted.


Figure 10
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  		FIGURE 10.
Nuclear extract from the SM22 transfectants shows diminished binding to the MMP-9-derived AP-1 consensus sequence. Panels A and B, nuclear extract (10 µg of protein) was subjected to an electrophoretic mobility shift assay using an oligonucleotide (2 x 104 cpm) spanning the proximal AP-1 motif (-79/-73) (panel A) or the KRE-M9 cis element (-66/-57) (panel B) in the MMP-9 promoter. The nucleotide sequence of the corresponding oligonucleotide is indicated with the consensus sequence in bold. Panel C, electrophoretic mobility shift assay was as in panel A with the exception that the anti-c-Fos antibody or IgG (1 µg) was added to the nuclear extract. The experiment was performed twice. Panel D, HT1080 cells were cotransfected with 100 ng of an AP-1-regulated firefly luciferase reporter and 700 ng of either pEGFP or this vector encoding SM22. All transfections included a beta-actin promoter-driven Renilla reporter (4 ng). Cells were lysed 24 h later and luciferase activity was determined. Data are expressed as average ± S.D. values of four independent experiments.

 
SM22 was originally identified as a transformation-sensitive (diminished expression) protein (41, 44) and mRNA levels are down-regulated in resected breast and colon cancers when compared with adjacent non-malignant mucosa (65). Interestingly, these finding parallel a robust expression of MMP-9 in these two malignancies (66-69). Similarly, expression profiling studies revealed 85% lower levels of SM22 in metastatic prostate cancer when compared with the primary tumor (70) and, like breast and colon cancer, MMP-9 expression is elevated in advanced disease (42). These findings raise the possibility that diminished SM22 expression account, at least in part, for the higher levels of this metalloproteinase in these cancers.

In conclusion, using an unbiased expression cloning strategy, we have identified a novel regulator (SM22/transgelin) of MMP-9 expression. SM22 represses MMP-9 promoter activity in a manner independent of its actin binding arguing for a hitherto unknown function for this protein previously recognized only for its actin-binding capacity. The loss of transgelin expression, evident in cancers of diverse origin (40, 70), may contribute to the well often observed elevated levels of MMP-9 in these malignancies.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01DE10845 and CA58311 (to D. D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Box 173, M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-563-4918; Fax: 713-563-5489; E-mail: dboyd{at}mdanderson.org.

2 The abbreviations used are: siRNA, small interfering RNA; RT, reverse transcriptase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; MEK, mitogen-activated protein kinase kinase/extracellular signal-regulated kinase kinase. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Michael Parmacek (University of Pennsylvania) for providing the SM22 null mice.



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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
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