Human leucine zipper protein sLZIP induces migration and invasion of cervical cancer cells via expression of matrix metalloproteinase-9

Abstract

Extracellular proteolysis mediates tissue homeostasis. In cancer, altered proteolysis leads to abnormal tumor growth, inflammation, tissue invasion, and metastasis. Matrix metalloproteinase-9 (MMP-9) represents one of the most prominent proteinases associated with inflammation and tumorigenesis. The recently identified human transcription factor sLZIP is a member of the leucine zipper transcription factor family. Although sLZIP is known to function in ligand-induced transactivation of the glucocorticoid receptor, its exact functions and target genes are not known. In this study, we investigated the role of sLZIP in MMP-9 expression and its involvement in cervical cancer development. Our results show that sLZIP increased the expression of MMP-9 at both the mRNA and protein levels and the proteolytic activity of MMP-9 in HeLa and SiHa cells. sLZIP also increased the transcriptional activity of MMP-9 by binding directly to the cAMP-responsive element of the MMP-9 promoter region. Involvement of sLZIP in MMP-9 expression was further supported by the fact that ME-180 cells expressing sLZIP siRNA were refractory to MMP-9 expression. Results from wound healing and invasion assays showed that sLZIP enhanced both the migration and invasion of cervical cancer cells. The increased migration and invasion of HeLa and SiHa cells that were induced by sLZIP were abrogated by inhibition of the proteolytic activity of MMP-9. These results indicate that sLZIP plays a critical role in MMP-9 expression and is probably involved in invasion and metastasis of cervical cancer.

FIGURE 1.

sLZIP induces MMP-9 expression in cervical cancer cells. A, various cancer cells were transfected with the sLZIP expression vector. After 24 h, the sLZIP and MMP-9 transcripts were assessed by semiquantitative RT-PCR using specific primers. GAPDH was amplified as an endogenous control. B, total RNA extracts prepared from HeLa, SiHa, and ME-180 cells were used for semiquantitative RT-PCR and quantitative real-time PCR with specific primers. GAPDH was amplified as an endogenous control. C, HeLa, SiHa, and ME-180 cells were transfected with the indicated amounts of sLZIP or si-sLZIP, and the mRNA level of MMP-9 was determined by RT-PCR. Scrambled siRNA (sc-siRNA; 50 nm) was used as a negative control.

FIGURE 2.

sLZIP enhances MMP-9 proteolytic activity in cervical cancer cells. A, HeLa, SiHa, and ME-180 cells were cotransfected with the indicated amounts of sLZIP expression plasmid or si-sLZIP and the MMP-9-luciferase reporter gene plasmid (0.2 μg). The luciferase activity was determined after 24 h of transfection. The luciferase activity was normalized to β-galactosidase activity, and the experiments were performed in triplicate. Data are expressed as the mean ± S.D. and are presented as the relative luciferase activity. Scrambled siRNA (sc-siRNA; 50 nm) was used as a negative control. Wb, Western blot. B and C, cervical cancer cells were transfected with the indicated amounts of sLZIP or si-sLZIP. The MMP-9 protein levels in the supernatants were determined using an ELISA (B), and the proteolytic activity of MMP-9 was determined by gelatin zymographic analysis (C). Results are shown as the mean ± S.D. of three experiments performed in duplicate.

FIGURE 3.

sLZIP binds directly to the CRE region within the MMP-9 promoter. A, HeLa cells were transfected with the 5′-deletion constructs of the MMP-9-luciferase (Luc) reporter gene plasmid (0.1 μg) and sLZIP (0.5 μg). After 24 h, cells were lysed, and the luciferase activity was determined. The luciferase activity was normalized to β-galactosidase activity, and the experiments were performed in triplicate. Data are expressed as the mean ± S.D. and are presented as the relative luciferase activity. B, EMSA was performed using a purified His-sLZIP protein and individual ³²P-labeled probes containing various putative transcription factor-binding motifs on the 5′-flanking regions of MMP-9. C, an oligonucleotide containing the sLZIP-binding element in the MMP-9 promoter (the proximal CRE region) was incubated with 3, 5, and 10 μg of His-sLZIP protein. For competition, unlabeled oligonucleotides were added at 50- and 100-fold excesses. Arrows indicate the shift corresponding to the position of the His-sLZIP-DNA complex. The His-sLZIP protein was prepared and subjected to EMSA for the DNA binding activity of sLZIP with normal (wild-type (wt)) and mutated (mt) ³²P-labeled oligonucleotides. D, HeLa cells were transfected with the mock and sLZIP plasmids, cross-linked, lysed, and immunoprecipitated with anti-HA antibody and rabbit IgG as a negative control. The precipitated DNA was subjected to PCR with primers specific for the off-target region (−426/−300) or the target region (−181/−29). One aliquot of input DNA was used as a positive control. E, in vivo occupancy of HA-sLZIP at the proximal MMP-9 promoter was shown by real-time PCR. Statistical significance was assessed using unpaired t-tests (p < 0.01). *, p = 0.01.

FIGURE 4.

sLZIP enhances wound healing, migration, and invasion in cervical cancer cells. HeLa (A), SiHa (B), and ME-180 cells (C) were transfected with the mock and sLZIP plasmids (2 μg) or si-sLZIP (50 nm). Representative images of wound healing were taken at the time of the scratch and after 24 h of the wound scratch. The level of cell migration into the wound scratch was quantified as the percentage of wound healing. Data are presented as the mean ± S.D. D, HeLa and SiHa cells were transfected with the mock and sLZIP plasmids (2 μg). After 24 h, cells were seeded in 96-well plates for the indicated time points. Cell proliferation was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Data are presented as the percentage of proliferation relative to 0 day. All measurements were performed in triplicate. E, HeLa and SiHa cells were transfected with the indicated amounts of sLZIP expression plasmid, and after 24 h, Matrigel invasion under normal growth conditions was measured. Data are presented as the percentage of invasion relative to control inserts. *, p = 0.01; **, p = 0.001.

FIGURE 5.

sLZIP CM increases migration and invasion of cervical cancer cells. A, schematic illustration for preparing CM from sLZIP-transfected HeLa cells. B, HeLa cells attached to cell culture plates were wounded and incubated with control CM or sLZIP CM. The wound healing was determined at the time points indicated. Data are presented as the mean ± S.D. C, HeLa cells were incubated with the CM mixture containing 30% control CM and 70% normal medium or with the CM mixture containing sLZIP CM (10 and 30%). Migrated cells were stained with Diff-Quik and counted for quantitative analysis. The quantification of penetrated cells is represented as the mean of three independent experiments. *, p = 0.01; **, p = 0.001. D, ME-180 cells were transfected with scrambled siRNA (sc-siRNA), si-sLZIP, and si-MMP-9, and conditioned media were prepared. ME-180 cells were incubated with scrambled siRNA CM, si-sLZIP CM, and si-MMP-9 CM. After cells were scratched with a pipette tip, the wound area was quantified by measuring the cell-free area for the indicated time points. The level of cell migration into the wound scratch was quantified as the percentage of wound healing. Data are presented as the mean ± S.D.

FIGURE 6.

MMP-9 is involved in sLZIP-induced cell mobility. A, HeLa cells were scratched with a pipette tip and incubated with control CM or sLZIP CM, followed by treatment with various inhibitors (5 nm MMP-9 inhibitor, 1.7 μm MMP-2 inhibitor, and 500 mm EDTA) and anti-MMP-9 antibody for 36 h. TIMP1 CM was prepared from TIMP1-transfected cells. HeLa cells were incubated with the CM mixture containing 30% control CM, 30% TIMP1 CM, and 40% normal medium and with the CM mixture containing 30% sLZIP CM, 30% TIMP1 CM, and 40% normal medium for 36 h. The wound area was quantified by measuring the cell-free area. Cell migration into the wound area is represented as the percentage of recovery relative to 0 h. B, HeLa cells were treated with dimethyl sulfoxide (DMSO), EDTA (500 mm), MMP-9 inhibitor (5 nm), MMP-2 inhibitor (1.7 μm), and anti-MMP-9 antibody. Matrigel invasion was measured under control CM or sLZIP CM conditions. HeLa cells were cultured in the upper part of a Transwell coated with Matrigel. After 24 h, cells on the bottom side of the filter were counted. Statistical significance was assessed using unpaired t-tests. *, p = 0.01; **, p = 0.001.