The lectin concanavalin-A signals MT1-MMP catalytic independent induction of COX-2 through an IKKgamma/NF-kappaB-dependent pathway

Abstract

The lectin from Canavalia ensiformis (Concanavalin-A, ConA), one of the most abundant lectins known, enables one to mimic biological lectin/carbohydrate interactions that regulate extracellular matrix protein recognition. As such, ConA is known to induce membrane type-1 matrix metalloproteinase (MT1-MMP) which expression is increased in brain cancer. Given that MT1-MMP correlated to high expression of cyclooxygenase (COX)-2 in gliomas with increasing histological grade, we specifically assessed the early proinflammatory cellular signaling processes triggered by ConA in the regulation of COX-2. We found that treatment with ConA or direct overexpression of a recombinant MT1-MMP resulted in the induction of COX-2 expression. This increase in COX-2 was correlated with a concomitant decrease in phosphorylated AKT suggestive of cell death induction, and was independent of MT1-MMP's catalytic function. ConA- and MT1-MMP-mediated intracellular signaling of COX-2 was also confirmed in wild-type and in Nuclear Factor-kappaB (NF-kappaB) p65(-/-) mutant mouse embryonic fibroblasts (MEF), but was abrogated in NF-kappaB1 (p50)(-/-) and in I kappaB kinase (IKK) gamma(-/-) mutant MEF cells. Collectively, our results highlight an IKK/NF-kappaB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of COX-2. That signaling pathway could account for the inflammatory balance responsible for the therapy resistance phenotype of glioblastoma cells, and prompts for the design of new therapeutic strategies that target cell surface carbohydrate structures and MT1-MMP-mediated signaling. Concise summary Concanavalin-A (ConA) mimics biological lectin/carbohydrate interactions that regulate the proinflammatory phenotype of cancer cells through yet undefined signaling. Here we highlight an IKK/NF-kappaB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of cyclooxygenase-2, and that could be responsible for the therapy resistance phenotype of glioblastoma cells.

Keywords: Cancer; Concanavalin-A; Cyclooxygenase; Inflammation; MT1-MMP.

Fig. 1

Concanavalin-A triggers proMMP-2 activation, MT1-MMP and COX-2 expression. a U87 glioblastoma cells were cultured as described in the “Materials and methods” section until they reached ∼75–90% confluence. They were then serum-starved for 24 h prior to the addition of increasing concentrations of concanavalin-A (ConA). Gelatin zymography (upper panel) was performed to assess the extent of proMMP-2 activation levels, as described in the “Materials and methods” section, using conditioned media isolated from each of the serum-starved cells conditions. Cell lysates were isolated and SDS-PAGE performed (20 μg protein/well). MT1-MMP, COX-2, phosphorylated-AKT, and total AKT expression were assessed by Western blotting and immunodetection was performed as described in the “Materials and methods” section. Scanning densitometry data of the (b) MT1-MMP vs COX-2 and (c) pAKT/AKT vs COX-2 autoradiograms were plotted and are from a representative experiment

Fig. 2

Concanavalin-A-induced COX-2 expression is independent of MT1-MMP’s catalytic function. a Cell lysates were isolated from U87 glioma cells that had been treated with or without ConA, 10 μM Ilomastat, or a combination of both. Gelatin zymography (upper panel) was carried out to assess the extent of proMMP-2 activation levels, as described in the “Materials and methods” section, using conditioned media isolated from each of the serum-starved cell conditions. The extent of MT1-MMP and COX-2 expression were assessed as described in the legend to Fig. 1. b Scanning densitometry data of MMP-2, MT1-MMP, COX-2 and GAPDH expression were plotted and are from 3 independent experiments

Fig. 3

Actinonin is unable to antagonize concanavalin-A-induced COX-2 expression. a Gelatin zymography (upper panel) was carried out to assess the extent of proMMP-2 activation levels, as described in the “Materials and methods” section, using conditioned media isolated from serum-starved U87 glioblastoma cells that had been treated with increasing actinonin concentrations in the presence of ConA. The extent of MT1-MMP and COX-2 expression was assessed in cell lysates as described in the legend to Fig. 1. b Scanning densitometry data of the respective MMP-2, MT1-MMP, COX-2 and GAPDH expression were plotted and are from a representative experiment

Fig. 4

Constitutive recombinant MT1-MMP expression triggers COX-2 expression. U87 glioblastoma cells were either Mock-transfected or transfected with a cDNA plasmid encoding recombinant MT1-MMP/GFP (MT1-MMP-Wt). Cells were then incubated in serum-free media in the presence or absence of Ilomastat (Ilo) or Actinonin (Acti). Gelatin zymography was performed to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells (lower panel). Cell lysates were isolated and SDS-PAGE performed (20 μg protein/well), followed by Western blotting and MT1-MMP, COX-2 and GAPDH immunodetection

Fig. 5

Concanavalin-A- and recombinant MT1-MMP-mediated COX-2 induction necessitates an intact NF-κB1 p50 and IKKγ⁻ signaling axis. Wild-type (Wt), p65^−/−, p50^−/−, IKKα^−/−, IKKβ^−/−, and IKKγ^−/− mouse embryonic fibroblasts (MEF) were either treated with ConA (a), Mock-transfected or transfected with a cDNA plasmid encoding MT1-MMP (b). Gelatin zymography was used to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells (upper panels of a and b). Cell lysates were isolated and SDS-PAGE performed (20 μg protein/well), followed by Western blotting and immunodetection of COX-2 and GAPDH