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First published online 22 February 2005
doi: 10.1242/jcs.01708

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3ß1 integrin regulates MMP-9 mRNA stability in immortalized keratinocytes: a novel mechanism of integrin-mediated MMP gene expression

Center for Cell Biology and Cancer Research, Albany Medical College, MC-165, 47 New Scotland Avenue, Albany, New York 12208, USA

View larger version (99K): [in a new window]   Fig. 4. Culture on fibronectin and/or vitronectin does not restore MMP-9 expression in 3ß1-deficient MK–/– cells. (A) Total RNA was isolated from MK+/+ cells or MK–/– cells grown on surfaces coated with LN-5 ECM (L), LN-5 ECM plus 20 µg/ml fibronectin (L+F), 20 µg/ml fibronectin (F), 20 µg/ml fibronectin plus 15 µg/ml vitronectin (F+V), or 15 µg/ml vitronectin (V). RT-PCR was performed to compare MMP-9 and ß-actin mRNA levels. (B) Parallel cultures of MK+/+ cells (a, b, e, f) or MK–/– cells (c, d, g, h) were grown on coverslips coated with 20 µg/ml fibronectin (a-d) or 20 µg/ml fibronectin plus 15 µg/ml vitronectin (e-h). F-actin was stained with TRITC-conjugated phalloidin (b, d, f, h); the corresponding phase-contrast image is shown for each field (a, c, e, g). Arrows in d and h indicate spindle-shaped cells. Bar, 20 µm.

View larger version (17K): [in a new window]   Fig. 5. MMP-9 promoter activation by RasV12 is MEK/ERK-dependent but 3ß1-independent. (A) MMP-9 promoter activity: MK+/+ cells (+/+), MK–/– cells (–/–) and MK–/– cells expressing human 3 (3) were grown on LN-5 ECM and transfected with an MMP-9 promoter/luciferase reporter plasmid. 24 hours later, cells were assayed for luciferase expression as described in the text. Normalized luciferase signals are plotted as the percentage of control levels seen in MK+/+ cells. Data are presented as the mean±s.e.m.; n=3 in two separate experiments. (B) MK+/+ cells (MK+/+), MK–/– cells (MK–/–), or MK–/– cells transfected with 3 (3) grown on LN-5 ECM were infected with adenovirus expressing either HA-tagged RasV12 (Ras) or ß-galactosidase (C); MOI=10. Cells were then transfected with the MMP-9 promoter/luciferase reporter plasmid in the presence or absence of 10 µM U0126, as indicated and assayed for luciferase expression as in A. Normalized luciferase signals are plotted relative to the control levels seen in the absence of U0126, which is set to 1.0 for each cell type (C, –). Data are presented as the mean±s.e.m.; n=3 in two separate experiments.

View larger version (40K): [in a new window]   Fig. 1. MEK/ERK signaling is required for MMP-9 expression in MK cells. Wild-type MK+/+ cells were grown on laminin-5-rich extracellular matrix (LN-5 ECM) in serum-free medium for 36 hours in the presence or absence of the MEK inhibitor U0126 (10 µM), as indicated. MMP-9 levels in the culture medium were assayed by gelatin zymography (top panel). Position of the 105 kDa murine pro-MMP-9 (mMMP-9) is indicated on the left. S lane, positions of human MMP-9 (hMMP-9) and MMP-2 (hMMP-2) purified standards are indicated. ERK activation was assayed by immunoblot of the corresponding cell lysates for phospho-ERK (pERK panel). Blots were stripped and reprobed for total ERK (ERK panel).

View larger version (25K): [in a new window]   Fig. 2. Activation of MEK/ERK signaling by oncogenic RasV12 induces MMP-9 in an 3ß1-dependent manner. Wild-type MK+/+ cells (MK+/+) or 3ß1-deficient MK–/– cells (MK–/–) grown on LN-5 ECM were infected with adenovirus expressing either HA-tagged RasV12 (RasV12) or ß-galactosidase (control); MOI=70. Cells were then grown in serum-free medium for an additional 36 hours in the presence or absence of 10 µM U0126, as indicated. Levels of secreted MMP-9 in the culture medium were assayed by gelatin zymography (top panels); murine MMP-9 and human MMP-9 standard are indicated. To confirm RasV12 expression, equal protein from corresponding cell lysates was immunoblotted with anti-HA tag (RasV12 panel). Parallel blots with anti-phospho-ERK confirmed ERK activation in RasV12-infected cells and inhibition of ERK activation by treatment with U0126 (pERK panel).

View larger version (25K): [in a new window]   Fig. 3. 3ß1 is required for MMP-9 mRNA expression. (A) MK+/+ cells (+/+ lane), MK–/– cells (–/– lane) and MK–/– cells stably transfected with the human 3 subunit (–/–, 3 lane) or the parental expression vector (–/–, V lane) were cultured on LN-5 ECM for 4 days in serum-containing medium. Total RNA was isolated and assayed by northern blotting with cDNA probes for murine MMP-9 (top panel) or A50 as a control (bottom panel). Two distinct MMP-9 mRNA transcripts of 3.5 kb and 2.7 kb are indicated. (B) RT-PCR was performed using RNA collected as in A as a template. MMP-9 mRNA levels (top panel) and ß-actin mRNA levels (bottom panel) are shown for MK+/+ cells (+/+ lane), MK–/– cells (–/– lane) and MK–/– cells transfected with human 3 (3 lane). (C) FACS analysis with the monoclonal antibody P1B5 confirms high levels of 3ß1 surface expression in MK–/– cells transfected with the human 3 integrin subunit (gray peak), but not in untransfected MK–/– cells (white peak).

View larger version (17K): [in a new window]   Fig. 6. RasV12-mediated induction of MMP-9 mRNA accumulation is 3ß1-dependent. (A) MK+/+ and MK–/– cells were infected with adenovirus expressing either ß-galactosidase as a control (– lanes) or RasV12 (+ lanes), then cultured on LN-5 ECM for 48 hours in serum-free medium. Total RNA was isolated and RT-PCR was performed to compare MMP-9 and ß-actin mRNA levels. (B) Signals for MMP-9 mRNA and ß-actin mRNA were quantified; graphs depict relative MMP-9 mRNA levels for each sample, after normalization to ß-actin mRNA. Data are presented as the mean±s.e.m. for three separate experiments; *P<0.05, two-way ANOVA with Newman Keuls post-hoc test.

View larger version (20K): [in a new window]   Fig. 7. 3ß1 promotes stability of MMP-9 mRNA. MK+/+ cells, MK–/– cells, or MK–/– cells transfected with 3 grown on LN-5-ECM were pre-treated with cycloheximide to promote accumulation of MMP-9 mRNA in MK–/– cells, and then grown under serum-free conditions in the presence of actinomycin D to inhibit new transcription (see text for details). Total RNA was isolated at various time points and RT-PCR was performed to monitor turnover of mRNA for MMP-9 (A) and ß-actin (B). Results are plotted as the percentage of mRNA remaining relative to the starting amounts at 0 hour; diamonds, MK+/+ cells; squares, MK–/– cells; open circles, 3-transfected MK–/– cells. Data are presented as the mean±s.e.m. for three separate experiments; *P<0.05, one-way ANOVA with Newman Keuls post-hoc test. Gels show results from a representative experiment.

View larger version (64K): [in a new window]   Fig. 8. A model for regulation of MMP-9 expression by 3ß1. MMP-9 gene transcription can be induced by Ras/MEK/ERK signaling in response to growth factors, cytokines, or oncogenic Ras. Our data reveal a novel mechanism wherein 3ß1 is required for stabilization of MMP-9 mRNA, leading to the accumulation of MMP-9 transcripts and subsequent production of MMP-9 protein in response to ERK-mediated transcriptional induction. In the absence of 3ß1, transcriptional induction by Ras/MEK/ERK does not lead to substantial accumulation of MMP-9 transcript and increased MMP-9 protein owing to the destabilization and rapid decay of MMP-9 mRNA. The 3 and ß1 integrin subunits are indicated.

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