Matrix metalloproteinase-1 contribution to sarcoma cell invasion

Abstract

Matrix metalloproteinase-1 (MMP-1) activity has been linked to numerous disease processes from arthritis to ulcer. Its proteolytic activity has been implicated inconsistently in different steps of tumourigenesis and metastasis. The discrepancies may be attributable to our limited understanding of MMP-1 production, cellular trafficking, secretion and local activation. Specifically, regulation of MMP-1 directional delivery versus its general extracellular matrix secretion is largely unknown. Inhibition of prenylation by farnesyl transferase inhibitor (FTI-276) decreased extracellular MMP-1 and subsequently reduced invasiveness by 30%. Parallel, stable cell line RNAi knockdown of MMP-1 confirmed its role in cellular invasiveness. The prenylation agonist farnesyl pyrophosphate (FPP) partially normalized FTI-276 inhibited extracellular MMP-1 levels and invasion capacity while transiently delayed its cellular podia distribution. MMP-1 directional delivery to these structures were confirmed by combination of a MMP-1-specific fluorogenic substrate, a MMP1-Ds-Red fusion protein construct expression and DQ-collagen degradation, which demonstrated coupling of directional delivery and activation. MetaMorph analysis of cellular lamellipodia structures indicated that FTI-276 inhibited formation and delivery to these structures. Farnesyl pyrophosphate partially restored lamellipodia area but not MMP-1 delivery under the time frame investigated. These results indicate that MMP-1 directional delivery to podia structures is involved in the invasive activity of sarcoma cells, and this process is prenylation sensitive.

Fig 1

Cell invasion was affected by farnesyl transferase inhibitor. (A) FTI-276 concentration effect on extracellular MMP-1 protein levels. Pan-Ras antibody was used to show that the drug is working and β actin employed as loading control. (B) Cellular invasion through a transmembrane collagen matrix down-regulated by FTI-276. Blanks (no cells) were used as controls. The invasive behaviour of parental 143B cells were significantly affected by FTI and FPP, while their combination partially normalized the activity (FT/FP). Y-axis measures cells that migrated through the transwell as described under the Materials and Methods section in reporter light units (RLU). (C) Extracellular MMP-1 was affected by FTI-276 and FPP. The extracellular MMP-1 protein level is displayed. The cellular FNTA levels demonstrate that the inhibitor does not alter the FTase subunit concentration and pan-Ras antibody was employed to validate efficacy of the inhibitor. Actin is the loading control. (D) Raw digital Western blot images show highly significant differences with P < 0.001 values for all bands analysed, except for the loading control (actin, P ∇ 0.176).

Fig 2

Validation of drug responses by real-time quantitative PCR method. (A) Expressional mRNA levels of target molecules. Relative quantity (RQ log10) values reflect changes from untreated control samples (gene expression levels ∇ 100%, RQ ∇ 1). MMP-1 expression shows increased sensitivity to FTI/FPP combination than to FTI alone. KRAS and FNTA expression levels are minimally affected by FTI and FPP exposure. Statistical analysis (ANOVA two factor without replication) indicates that gene expression was stable with P-values for rows: P ∇ 0.2519 and columns: P ∇ 0.0418. (B) RT-qPCR analysis of MMP and TIMP gene expression changes of the 143B/T6-4, 5, 7 clones are shown with reference to the parental 143B cell line (where all target values are 100%). (C) MMP-1 expression levels were validated by Western blot. (D) Stable MMP-1 RNAi down-regulation affect on trans-membrane invasion was compared between the parental and T6-(4, 5, 7) clones.

Fig 3

Determination of MMP-1 lamellipodia localization. (A) Visual demonstration of objective invadopodia boundary determination through depth-coding. (A) Cellular architecture was outlined by Alexa-647-phalloidin staining (pseudo-coloured white) and aided by nuclear counterstaining with DAPI (B-blue). MMP-1 localization (C-red) and the reflected image (D-turquoise). The reflection image interference rings [E-Merge, labelled with asterisk (_*)] delineate the stretched out regions of cellular bodies (lamellipodia) showing striped wave-like formations of protruding cellular frontlines. To demonstrate this phenomena more clearly, the interferential image was pseudo-coloured according to the height of cellular features (depth-coding, F). The boundaries of lamellipodia region circled (G-white). (B) MMP-1 delivery is FTI-276 sensitive. Fifteen to twenty randomly chosen fields of individual images (representing 60–90 individual cells) were captured of each condition with 63χ oil immersion lens on the Zeiss LSM510 confocal microscope. Blue (DAPI, nucleus), red (MMP-1), pseudo-coloured white (F-actin) and reflected interference images (not shown) were collected. MetaMorph analysis was performed on the extracted red channels for total and partial surface areas and pixel counts to determine the podia formation activity and its partial MMP-1 representation as described. Each cell total surface area and MMP-1 content treated as fixed (100%). From this the lamellipodia formation and partial MMP-1 localization were determined as percentage of total values (surface area and red pixel counts). These percentage results presented as bar graph, where untreated cells provide the reference basis ∇ 100% showing for drugs affecting lamellipodia surface area (bar graph on the left) and MMP-1 loading (bar graph on the right). Statistical analysis shows that the quantization of lamellipodia area and MMP-1 loading are different for and by the presented conditions with P ∇ 4.09E−73 and P ∇ 5.60786E−30 confidence levels, respectively.

Fig 4

MMP-1 directional trafficking into invadopodia regions. (A) 143B cells were seeded in 12-well plates at 3.0 χ 10⁴ concentrations. The live cell-related matrix degradation and MMP1-DsRed fusion construct production was imaged between 24 and 36 hrs from the DNA transfection. Except the DsRed fusion protein (constant fluorescence signal), the DQ-collagen becomes fluorescent upon enzymatic cleavage and degradation (three representative fields are shown, bar: 20 μm). (B) Images of MMP-1-DsRed transfected live cells. The data show that all fluorescent signal (green, red, blue,) demonstrate partial co-localization (inset), with white pixels where all three is simultaneously present (bars 20 μm).

Fig 5

MMP-1 activity is affected by farnesyl transferase inhibitor. (A) The lamellipodia regions were determined with polarized light and plastic optimized dichroic filter (PlasDIC). MMP-1 enzyme activity was detected by quenched Fluorogenic MMP1-Substrate-III (blue fluorescence indicates MMP-1 specific cleavage). The signals collected in the blue channel at ∼λ = 460 nm, and up to 12 images of each condition (∼25–30 cells). Five-pointed star labels MMP-1 activity localized to the podia regions of the cells. (B) Lamellipodia MMP-1 activities were quantized on 20 randomly selected cells in triplicate.