Laminin induces matrix metalloproteinase-9 expression and activation in human cervical cancer cell line (SiHa)

Abstract

Purpose: Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation and migration including tumor development and invasion. Matrix metalloproteinases (MMP) are a family of metalloproteinases capable of digesting ECM and facilitate cell migration. Binding of ECM to integrins initiates signaling cascades modulating expression and activity of different MMPs. The present study investigates whether laminin-mediated signaling modulates matrix metalloproteinases (MMP) expression and activity in human cervical cancer cell (SiHa).

Methods: Western blot, immunocytochemistry, ELISA, zymography, RT-PCR, EMSA and wound-healing assay were used.

Results: Culture of SiHa cells on laminin (LN)-coated surface induces MMP-9 expression and activation. Wound-healing assay showed that SiHa cells migrate much faster on laminin-coated surface than that of control. LN-induced MMP-9 expression and activation was appreciably reduced with treatment of extracellular signal-regulated kinase (ERK) inhibitor, phosphatidylinositol-3-kinase (PI-3K) inhibitor and anti-α2 antibody. Phosphorylation of focal adhesion kinase (FAK), ERK, and PI-3K was increased upon LN stimulation. LN induces nuclear translocation of PI-3K and nuclear factor kappa B (NF-κB). LN increases DNA-binding activity of NF-κB and activator protein-1 (AP-1) to MMP-9 promoter.

Conclusions: Our findings indicate laminin-induced MMP-9 expression and activation possibly via α2β1 integrin-mediated signaling involving FAK, PI-3K, ERK followed by transcriptional upregulation of MMP-9.

Fig. 1

Assay of MMP-9, MMP-7 and TIMP-1. a SiHa cells (600,000/1.5 ml) were grown without (lane 1) or with laminin (25 μg/ml)-coated culture plate for 30 min (lane 2) and for 1 h (Lane 3) in SFCM. Gelatin zymography of SFCM of SiHa cells grown on culture dishes coated with collagen IV (lane 4) and vitronectin (lane 5) for 1 h. Lane 6: MMP-9/MMP-2 marker lane (SFCM of HT-1080 cells grown for 24 h). Gelatinases were concentrated using Gelatin Sepharose 4B beads and subjected to gelatin zymography. The accompanying graph represents the comparative densitometric analysis of band intensities using Image J Launcher (version 1.4.3.67). Data are means ± SEM of three experiments. b SFCM of control and LN-treated SiHa cells were concentrated with gelatin Sepharose 4B beads and western blot was done with anti-MMP-9 antibody. Actin was used as internal control. c SiHa cells were grown in SFCM in absence (C) and in presence of laminin for 1 h (E). The wells of ELISA plate were coated with SFCM of control and LN-treated SiHa cells (in triplicate) and kept at 4°C overnight. ELISA was performed with anti-MMP-9 or anti-TIMP-1 or anti-MMP-7 antibody (1:1,000). The OD was measured at 450 nm. (d) m-RNA expression (RT–PCR) of MMP-9 of SiHa cells grown without (C) or with LN (25 μg/ml)-coated culture dish for 1 h (E). GAPDH primers were used to confirm equal loading. The array represents the comparative densitometric analysis of the band intensities using Image J Launcher (version 1.4.3.67). Data are means ± SEM of three experiments

Fig. 2

Involvement of integrin α2β1, α3β1, ERK and PI-3K in laminin-induced MMP-9 expression. a SiHa cells were grown on coverslip. Immunocytochemistry was preformed with anti-α2 antibody (1:1,000 dilutions). The arrow indicates the localization of α2 on cell surface. b SiHa cells were collected, extracted and 150 μg protein was subjected to western blot analysis with anti-α2 and anti-β1 antibodies (1:1,000 dilutions). The bands correspond to 165 and 130 kDa of α2 and β1, respectively. c SiHa cells were treated with (E) or without (C) 1 μg/ml anti-α2 antibody for 1 h. The microtitre plate wells were coated with laminin (3–12 μg/ml) in triplicate. Cell adhesion assay of both control and experimental sets were preformed. d SiHa cells were grown in presence of anti-α2 antibody (lane 2) anti α3 antibody (lane 3) or ERK inhibitor (PD 98059) (lane 4), PI-3 K inhibitor (LY 294002) (lane 5), MEK inhibitor (U0126) (lane 6) and p38 inhibitor (SB 203580) (lane 7) for 1 h. Lane 1 represents control SiHa cells (without antibody or inhibitor treatment). Both the control and experimental sets were grown on laminin-coated plates (25 μg/ml) for 1 h. SFCM was collected, and gelatin zymography was performed. Lane 8 MMP-9/MMP-2 marker lane (SFCM of HT-1080 cells grown for 24 h). The accompanying graph represents the comparative densitometric analysis of the band intensities using Image J Launcher (version 1.4.3.67). Data are means ± SEM of three experiments

Fig. 3

Effect of laminin on expression and phosphorylation of FAK. a SiHa cells were grown on coverslips without (C) or with laminin (25 μg/ml) for 30 min (E1) and 1 h (E2). Immunocytochemistry was preformed with anti-FAK or anti-phospho-FAK antibody (1:1,000 dilutions; 1.5 h at 37°C). Cells were observed under a fluorescence microscope (40×). b SiHa cells were grown on 25 μg/ml laminin-coated (lane E) or uncoated (lane C) dishes for 1 h. FAK was immunoprecipitated from cell lysate (150 μg protein) using anti-FAK antibody and immunoblotted with anti-FAK antibody (a) or anti-phospho-tyrosine antibody (b). Actin was used as internal control. The accompanying graphs represent the comparative densitometric analysis of the band intensities using Image J Launcher (version 1.4.3.67). Data are means ± SEM of three experiments. c m-RNA expression (RT–PCR) of FAK of SiHa cells grown without (C) or with LN (25 μg/ml)-coated culture dish for 1 h (E). GAPDH primers were used to confirm equal loading. The array represents the comparative quantitative analysis of the band intensities using Image J Launcher (version 1.4.3.67). Data are means ± SEM of three experiments

Fig. 4

Effect of laminin on expression and localization of ILK, ERK, p-ERK, PI-3K, p-PI-3K and NF-κB. SiHa cells were grown on coverslips without (C) or with laminin (25 μg/ml) for 30 min (E1) and 1 h (E2). Immunocytochemistry was preformed with anti-ILK (Panel a), anti-ERK (Panel b), anti-p-ERK (Panel c), anti-PI-3K (Panel d), anti-p-PI-3K (Panel e) and anti-NF-κB (panel f) antibodies (1:1,000 dilution for1½ hrs at 37°C). Coverslips were observed under a fluorescence microscope

Fig. 5

Effect of laminin on ERK, p-ERK, PI-3 K, p-PI-3K and NF-κB. SiHa cells were grown with (lane E) or without (lane C) laminin (25 μg/ml)-coated dishes for 1 h. The cells were collected, extracted and equal amount of protein (150 μg) was subjected to western blot analysis with anti-ERK (a), anti-p-ERK (b), anti-PI-3K (c), anti-p-PI-3K and (d) anti-NF-κB (e) (cytoplasmic extract-upper panel and nuclear extract-lower panel) antibodies (1:1,000 dilution for 1½ h at 37°C). Actin was used as internal control and done in parallel to all blots. The accompanying graphs represent the comparative densitometric analysis of the band intensities using Image J Launcher (version 1.4.3.67). Data are means ± SEM of three experiments

Fig. 6

DNA binding of NF-κB, AP-1 and Sp1 protein on MMP-9 promoter in response to laminin. SiHa cells were grown on laminin-coated or control (without laminin) culture plates for 1 h in SFCM, and nuclear extract were prepared. Oligonucleotides containing NF-κB (a), AP-1 (b) and Sp1 (c) were end labeled with [γ^−32P] ATP and were incubated with nuclear extract from control (lane 2) and laminin treated (lane 3). Then protein-DNA complexes were electrophoresed on 5% polyacrylamide gel, gel was then dried and subjected to autoradiography at −80°C. Lane 1 represents respective AP-1, Sp1 and NF-κB binding sequences without nuclear extract

Fig. 7

Effect of laminin on cell migration of SiHa cell. SiHa cells were grown as a monolayer on culture plates coated with (E) or without (C) LN for 1 h at 37°C. The monolayer was scratched with a sterile pipette tip, followed by washing ×3 with SFCM to remove cellular debris. The wound was recoated with or without LN in SFCM, and cell migration was observed by microscopy and documented by photography at 0, 6, 12, 18 and 24 h. Arrow indicates the migration of cells