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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02465

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Upregulation of MMP-9 in MDCK epithelial cell line in response to expression of the Snail transcription factor

¹ Centre d'Oncologia Molecular, IDIBELL-Institut de Recerca Oncològica, Barcelona, Spain
² Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM) and Departamento de Bioquímica (UAM), Madrid, Spain

View larger version (49K): [in a new window]   Fig. 1. Snail increases the expression and secretion of MMP-9, which is relieved by Snail RNA interference (siSnail). (A) Identification of the gelatinolytic enzymes produced by MDCK-CMV and MDCK-Snail cells. The cell conditioned media and cellular extracts were collected, centrifuged and 12 µg of protein analysed by zymography in gelatin-embedded SDS polyacrylamide gels. The enzymes were detected as clear bands and a major band of approximately 110 kDa was identified as gelatinase B or MMP-9 by reference to the migration of standard proteins. A conditioned media from MXT-c1.1 (mouse mammary carcinoma) cells was included as control for gelatinase A or MMP-2. (B) Detection of MMP-9 mRNA by RT-PCR. 100 ng of poly(A)+ mRNA from MDCK-CMV and MDCK-Snail cells was subjected to RT-PCR. The amplification of cyclophillin was used to normalise for loading (lower panel). (C) Immunofluorescence detection of MMP-9 in MCDK-CMV (left) and MDCK-Snail (right) cells. Note the intense fibrillar and granular staining in MDCK-Snail cells. (D) Cells collected from subconfluent cultures of MDCK-CMV and MDCK-Snail cells were lysed, and the expression of MMP-9 was analysed by western blotting using an anti-MMP-9 polyclonal antibody. -Tubulin was used as loading control. (E) Snail silencing blocks the induction of MMP-9 expression. Upper four panels: RT-PCR analysis of MMP-9, Snail and E-cadherin mRNA levels in MDCK-CMV, MDCK-Snail and in three independent stable clones (C1, C3 and C4) generated after siSnail transfection (MDCK-siSnail) in MDCK-Snail cells. GAPDH mRNA levels are shown as loading control. Lower panel: the effects of Snail interference on secreted MMP-9 were analysed by zymography in gelatin-embedded SDS polyacrylamide gels.

View larger version (17K): [in a new window]   Fig. 2. Stable expression of Snail induces MMP-9 promoter activity and is relieved by siSnail. (A) Schematic representation of the MMP-9 promoter and the deletion mutant constructs (I-VI), indicating the position of potential regulatory control elements. (B) Diagram showing the increase in activity of the different constructs transiently expressed in MDCK-CMV cells (white bars) or MDCK-Snail cells (black bars). Luciferase and renilla activities of each promoter construct are relative to that obtained with the pGL2 control plasmid, expressed as the mean±s.d. of three independent experiments. (C) Induction of MMP-9 promoter mediated by Snail is relieved by siSnail. Analysis of -1170 MMP-9 promoter activity (construct I) in control (MDCK-CMV, MDCK-Snail) and in the indicated individual clones obtained after stable transfection of Snail siRNA. Promoter activity was determined as in B. Results represent the mean±s.d. of four independent experiments.

View larger version (17K): [in a new window]   Fig. 3. Transient expression of Snail induces MMP-9 promoter activity and is relieved by siSnail. (A) Activity of the different constructs was analysed in MDCK-CMV cells transiently transfected with Snail cDNA (black bars) or with the empty expression vector (white bars). The effect of Snail is represented for each MMP-9 reporter as fold stimulation of the activity over the empty vector as the mean±s.d. of three independent experiments. (B) The effect of siSnail on MMP-9 promoter activity (construct I) was measured in MDCK-CMV cells growing in T24 plates, after transfection of the indicated amounts of the different vectors and compensated with the empty plasmid. Promoter activity is given as fold stimulation over that with the empty plasmid (mean±s.d. of four independent experiments). (C) Dose-dependent effect of Snail cDNA on the -389 MMP-9 (construct III) promoter activity was analysed in MDCK-CMV cells as described in A. The amount of the Snail cDNA was compensated with empty plasmid up to 1 µg of total DNA. Promoter activity is given as fold stimulation over that obtained in the absence of Snail cDNA. The small panel at the top shows the dose-dependent effect of the empty plasmid on the activity of this promoter construct. Values are the mean±s.d. of three independent experiments.

View larger version (32K): [in a new window]   Fig. 4. Nuclear extracts from MDCK-Snail cells did not bind to the putative E-box element of the MMP-9 promoter but bound to the E-pal element of E-cadherin promoter. (A) Nuclear extracts from MDCK-CMV and MDCK-Snail cells were analysed in band-shift assays using the 32P-labelled E-box wild-type probe of the MMP-9 promoter containing the putative E-box at nt -648 (lanes 2-8), or the E-pal element of the mouse E-cadherin promoter (lanes 1, 9-11). Lanes 1 and 2 show EMSA in which the nuclear extract was not added. The retarded complexes were detected when using the E-pal probe of the E-cadherin promoter and are indicated by a black arrowhead. Incubation of the MDCK-Snail nuclear extracts in the presence of control rabbit IgG or an anti-Snail antibody is shown in lanes 10 and 11, respectively. The complete sequence of the E-box MMP-9 probe and the E-pal E-cadherin probe are shown at the bottom of the figure with position of E-boxes underlined. The gel shown is representative of at least two independent experiments. (B) Nuclear extracts from MDCK-CMV and MDCK-Snail cells were analysed in band-shift assays using as a probe the E-pal element of the E-cadherin promoter. Nuclear extracts were incubated with the 32P-labelled E-pal probe in the presence of 500-fold molar excess of wild-type (lanes 2 and 5) or mutant cold oligonucleotides (lanes 3 and 6). A black arrowhead indicates a retarded complex. The complete sequence of the E-pal probe is shown at the bottom with the position of the mutated nucleotides indicated by asterisks.

View larger version (52K): [in a new window]   Fig. 5. Nuclear extracts from MDCK-Snail cells contain specific NFB-binding complexes. Nuclear extracts from MDCK-CMV and MDCK-Snail cells were analysed in band-shift assays showing the binding of nuclear proteins to the 32P-labeled NFB wild-type probe, in the absence or presence of 500-fold molar excess of wild-type (lanes 3 and 8) or mutant cold oligonucleotides (lanes 4 and 9) or in the presence of anti-p65 (lanes 6 and 11) or control mouse IgG (lanes 5 and 10). Lane 1 shows a control in the absence of the nuclear extract. White arrowheads indicate the complexes detected (i and ii). A black arrowhead indicates the supershifted complex. The complete sequence of the NFB probe is shown at the bottom of the figure. The specific nucleotides mutated in the NFB oligonucleotide are underlined. The gel shown is representative of at least two independent experiments.

View larger version (12K): [in a new window]   Fig. 6. Ets-1 and Sp-1(HA) elements are responsible for the increase in MMP-9 proximal promoter activity in MDCK-Snail cells. (A) Top, schematic representation of the proximal 5' region -97 bp from the initiation transcription site, indicating the position of the potential regulatory control elements Ets-1, Sp-1 low affinity (LA) or high affinity (HA) and TATA-like (grey box). Middle and lower sequences: point mutations and putative Ets-1 and Sp-1(LA or HA) binding sites are indicated in each mutant construction. (B) The wild-type pMMP9-97 construct (construct Vwt) or mutants (construct VM 89/87 and VM 85/82) fused to the luc reporter gene, were co-transfected with TK renilla into MDCK-Snail cells. Luciferase activity of each construction was determined and represented as fold stimulation of the activity over the -97 MMP-9 promoter (construct Vwt) as the mean±s.d. of three independent experiments.

View larger version (35K): [in a new window]   Fig. 7. Nuclear extracts from MDCK-Snail cells contain specific Ets-1 and Sp-1 binding complexes not observed in MDCK-CMV cells. (A) Nuclear extracts from MDCK-CMV and MDCK-Snail cells were analysed by EMSA showing the binding of nuclear proteins to the HA-SP-1 and Ets-1 elements. Nuclear proteins from serum free cultures were incubated with the 32P-labelled wild-type probe encompassing the sequence from nts -85 to -75 of MMP-9 promoter in the absence or presence of 500-fold molar excess of wild-type (lanes 3 and 8) or mutant cold oligonucleotides (lanes 6 and 10). Lanes 1 and 2 show EMSA in which the nuclear extracts were not added. The 32P-labelled mutant probe was used in lanes 5 and 9. Black arrowhead indicates the main complex detected. The complete sequence of the wild-type probe is shown at the bottom of the figure in which the mutated nucleotides are underlined. The gel shown is representative of at least two independent experiments. (B) Nuclear extracts of MDCK-CMV (lane 2) and MDCK-Snail cells (lanes 3-6) were analysed in band-shift assays. The 32P-labelled wild-type (wt) oligonucleotide encompassing the sequence from nts -97 to -75 of the MMP-9 promoter is shown at the bottom. Nuclear extracts were incubated with either an anti-Sp-1 (lane 4) or anti-Ets-1 (lane 5) antibodies, or control rabbit IgG (lane 6). A black arrowhead indicates the main complex detected, the intensity of which is lowered by both antibodies. The gel shown is representative of four independent experiments.

View larger version (19K): [in a new window]   Fig. 8. Induction of MMP-9 promoter activity and gelatinolytic expression by synergistic co-operation of oncogenic RasVal12 and Snail. (A) MDCK-CMV cells were transiently co-transfected with the pMMP9-392luc reporter construct and the indicated combinations of oncogenic Ras (RasVal12), Snail, a dominant negative Ras (RasN17) or the pCDNA3 expression vector (Mock), as well as TK renilla. After transfection, the cells were treated for an additional 5 hours with UO126 inhibitor (10 µM) or DMSO, prior to determining luciferase activity. The data are expressed as the mean±s.d. of the relative normalised luciferase values from three independent experiments. (B) Schematic representation of the proximal 5' region examined in these experiments. The pMMP9-389luc (construct III) contains 389 bp from the initiation transcription site and the positions of potential regulatory control elements are indicated. (C) The effects of Snail and Ras oncogene were analysed in the conditioned media by zymography in gelatin-embedded SDS polyacrylamide gels. MDCK-Snail cells were transiently transfected with oncogenic Ras (RasV12), a dominant negative Ras (RasN17) or the empty plasmid and media were collected after 24 hours. Conditioned medium from MDCK-CMV cells was also included as control.

View larger version (38K): [in a new window]   Fig. 9. MAPK and PI3K signalling pathways are involved in the induction of MMP-9 proximal promoter by Snail. (A) MMP-9 promoter activity was measured in MDCK-CMV cells by using the reporter construct pMMP9-389luc in the absence or presence of the Snail expression vector, and in the presence or absence of the following inhibitors: UO126 (10 µM); wortmannin (40 nM); SB 203580 (10 µM) or JNK inhibitor II (100 nM). Control cells (untreated) were unstimulated and treated with DMSO alone. Promoter activity is expressed relative to the value obtained in MDCK-CMV untreated cells and results are expressed as the mean±s.d. of three independent experiments. (B,C) Effect of Snail on Erk (B) and Akt (C) phosphorylation. Total cellular protein was extracted from MDCK-CMV and MDCK-Snail untreated cells and cells treated with wortmannin (C). 150 µg were analysed by western blotting using specific antibodies for the phosphorylated forms of these protein kinases. -tubulin was used as a loading control. (D) Cell extracts (30 µg) from MDCK-CMV and MDCK-Snail cells were used in western blots to determine Sp-1 phosphorylation status using a specific antibody that recognizes the phosphorylated and unphosphorylated isoforms. -tubulin was used as a loading control. One representative experiment out of five is shown. (E) Nuclear extracts of MDCK-Snail cells cultured for 24 hours in serum-free medium were analysed by EMSA after transient transfection with MEK-DN expression vector or empty plasmid. The 32P-labelled wild-type (wt) oligonucleotide encompassing the sequence from nts -97 to -76 is the same as that in Fig. 7B. The same nuclear extracts were analysed by western blotting using specific antibodies for the phosphorylated forms of Erk protein kinases. Anti-total Erk antiserum was used in the same membrane as a loading control.

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