GI24 enhances tumor invasiveness by regulating cell surface membrane-type 1 matrix metalloproteinase

Abstract

GI24, an immunoglobulin superfamily member, has been cloned from a placenta cDNA library as a gene product that promoted activation of matrix metalloproteinase (MMP)-2 mediated by membrane type (MT) 1-MMP. Co-expression of GI24 with MT1-MMP in HEK293T cells increased the cell-surface level of MT1-MMP concomitant with the cleavage of the GI24 at the juxtamembrane site to shed the extracellular domain. HT1080 fibrosarcoma cells stably transfected with the GI24 gene expressed a higher level of MT1-MMP and showed more invasive ability in collagen gel than the control cells. GI24 was cleaved in HT1080 cells, which was blocked by the administration of MMP inhibitor BB94 or transfection of small interfering RNA (siRNA) targeting MT1-MMP. GI24 expression is relatively high in some squamous carcinoma and hepatocarcinoma cell lines. Transfection of siRNA for GI24 into oral squamous carcinoma-derived HSC-4 cells, which express GI24 and MT1-MMP genes reduced the expression of not only GI24 but also MT1-MMP, and attenuated invasive growth in the collagen matrix. These results suggest that GI24 contributes to tumor-invasive growth in the collagen matrix by augmenting cell surface MT1-MMP.

Figure 1

Expression cloning. In the first screening, plasmid DNA aliquots from the human placenta cDNA library were co‐transfected with membrane type 1–matrix metalloproteinase (MT1‐MMP) plasmid into HEK293T cells cultured in 24‐well microplates. At 48 h post‐transfection, cells were incubated with a MMP‐2 sample for 1 h, and cell lysates from each well were subjected to gelatin zymography. Note that MMP‐2 activation to generate the 62‐kDa active form was stimulated in lane 10, as indicated by an arrow (upper panel). In the second screening, single clones of plasmid DNA were examined as described above. Note that MMP‐2 activation was enhanced in lanes 2, 4, 6 and 11, as indicated by arrowheads (lower panel).

Figure 2

GI24 stimulates membrane type 1–matrix metalloproteinase (MT1‐MMP) activity. (A) Control plasmid or expression plasmid for GI24‐HA (400 ng) was co‐transfected with control or MT1‐F plasmid (100 ng) into HEK293T cells plated in 24‐well plates. Twenty four hours after the culture, cells were either incubated with a MMP‐2 sample for gelatin zymography (top panel) or subjected to western blotting with anti‐FLAG M1 antibody to detect the MT1‐MMP active form (α‐FLAG M1), anti‐FLAG M2 antibody to detect active and latent MT1‐MMP (α‐FLAG M2), and anti‐HA antibody to detect GI24‐HA (α‐HA). MT1‐F plasmid (500 ng) was co‐transfected with either control or GI24‐HA plasmid (500 ng) into HEK293T cells cultured in a 6‐well microplate. The cells were labeled and immunoprecipitated by anti‐FLAG‐M2 beads, and the precipitated materials were blotted with IRDye800‐conjugated streptavidin as indicated. NS, non‐specific band. (B) F‐GI24 plasmid (200 ng) was co‐transfected with either control plasmid or expression plasmid for MT‐MMP (400 ng). At 24 h after transfection, the culture medium was replaced with serum‐free medium, and cells were incubated for a further 24 h. Culture supernatants (panel CM) and cell lysates (panels WCL) were then analyzed by western blotting using anti‐FLAG M2 and MT1‐MMP antibody as indicated. (C) GI24‐F plasmid (500 ng) was co‐transfected with either control or MT1‐MMP plasmid (500 ng) into HEK293T cells cultured in a 6‐well microplate, and cells were labeled with biotin at 48 h after transfection. Immunoprecipitation was performed using anti‐FLAG‐M2 beads, and the precipitated materials were blotted with IRDye800‐conjugated streptavidin (top panel). Expression of GI24‐F and MT1‐MMP was confirmed with cell lysates using anti‐FLAG M2 and anti‐MT1‐MMP antibodies as indicated. (D) The recombinant GI24‐GST protein (2 μg) was incubated with or without the recombinant MT1‐MMP catalytic domain (0.2 μg) for 3 h, separated by 12% SDS‐PAGE, and then detected by either Coomassie Brilliant Blue staining (protein staining) or western blotting using anti‐GST antibody (α‐GST). Note that 40 kDa GI24‐GST protein and co‐purified 30 kDa degradation product were cleaved by recombinant MT1‐MMP to generate the 26 kDa fragment.

Figure 3

GI24 enhances invasiveness of HT1080 cells. (A) HT1080 cells transfected with control or GI24‐F plasmid were mock‐treated (lane ‐) or treated with 1 μM BB94 (lanes BB94) or siRNA for membrane type 1–matrix metalloproteinase (MT1‐MMP) (lanes si‐MT1) for 24 h, and subjected to western blotting using anti‐FLAG M2 or anti‐MT1‐MMP antibody. (B) Control HT1080 cells or HT1080/GI24‐F cells were mock‐treated or transfected with si‐RNA for MT1‐MMP, and then immunostained using anti‐FLAG M2 antibody. Scale bar, 10 μm. (C) Parental HT1080 cells (−), HT1080 cells transfected with control plasmid and two independent clones of HT1080/GI24‐F were either incubated with MMP‐2 sample for gelatin zymography or subjected to western blotting using anti‐MT1‐MMP, anti‐FLAG M2 or anti‐tubulin antibody as indicated. (D) Parental HT1080 cells (−), control HT1080 cells and two independent clones of HT1080/GI24‐F (1 × 10⁵ cells) embedded in a 50 μL drop of collagen gel were cultured in 0.5 mL culture medium. Photography was taken 2 and 3 days after the culture. The dotted line represents the border of the collagen gel. Magnification, ×40. The area framed in white is expanded in the bottom panels. Note that HT1080/GI24‐F cells invaded out of the collagen gel and degraded the collagen gel more than the control cells.

Figure 4

GI24 stimulates invasive growth of HSC‐4 cells. (A) GI24 and membrane type 1–matrix metalloproteinase (MT1‐MMP) mRNA levels were compared using quantitative real‐time PCR analysis. For gelatin zymography, cells were incubated with a MMP‐2 sample for 1 h, and then washed with PBS before being dissolved in the sample buffer. (B) HSC‐4 cells were mock‐treated or transfected with control siRNA or siRNA for GI24 or MT1‐MMP, and analyzed by western blotting using anti‐GI24, anti‐MT1‐MMP or anti‐tubulin antibody. Gelatin zymography was performed as described above. (C) HSC‐4 cells were mock‐treated or transfected with control siRNA or siRNA for GI24, and cultured in collagen gel for 6 days. Original magnification, ×100.