Abstract
Activation of matrix metalloproteinase 2 (MMP-2) has been shown to play a significant role in the behavior of cancer cells, affecting both migration and invasion. The activation process requires multimolecular complex formation involving pro-MMP-2, membrane type 1-MMP (MT1-MMP), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Because calcium is an important regulator of keratinocyte function, we evaluated the effect of calcium on MMP regulation in an oral squamous cell carcinoma line (SCC25). Increasing extracellular calcium (0.09-1.2 mm) resulted in a dose-dependent increase in MT1-MMP-dependent pro-MMP-2 activation. Despite the requirement for MT1-MMP in the activation process, no changes in MT1-MMP expression, cell surface localization, or endocytosis were apparent. However, increased generation of the catalytically inactive 43-kDa MT1-MMP autolysis product and decline in the TIMP-2 levels in conditioned media were observed. The decrease in TIMP-2 levels in the conditioned media was prevented by a broad spectrum MMP inhibitor, suggesting that calcium promotes recruitment of TIMP-2 to MT1-MMP on the cell surface. Despite the decline in soluble TIMP-2, no accumulation of TIMP-2 in cell lysates was seen. Blocking TIMP-2 degradation with bafilomycin A1 significantly increased cell-associated TIMP-2 levels in the presence of high calcium. These data suggest that the decline in TIMP-2 is because of increased calcium-mediated MT1-MMP-dependent degradation of TIMP-2. In functional studies, increasing calcium enhanced MMP-dependent cellular migration on laminin-5-rich matrix using an in vitro colony dispersion assay. Taken together, these results suggest that changes in extracellular calcium can regulate post-translational MMP dynamics and thus affect the cellular behavior of oral squamous cell carcinoma.
Highlights
Oral squamous cell carcinoma (OSCC)1 is characterized by local, regional, and distant spread of the disease; the
Because calcium is an important regulator of keratinocyte function, we evaluated the effect of calcium on matrix metalloproteinase (MMP) regulation in an oral squamous cell carcinoma line (SCC25)
Whereas cells cultured in 0.09 mM calcium concentration expressed pro-matrix metalloproteinase 2 (MMP-2) (Fig. 1A, 1st lane), increasing calcium concentration resulted in a dose-dependent MMP-2 activation (Fig. 1A, 2nd to 4th lanes)
Summary
Oral squamous cell carcinoma (OSCC)1 is characterized by local, regional, and distant spread of the disease; the. Increasing extracellular calcium resulted in a dose-dependent increase in pro-MMP-2 activation, accompanied by enhanced MT1-MMP autolytic processing and a decline in the levels of soluble TIMP-2. Similar to the results obtained with SCC25 cells on plastic (Fig. 1A), cells plated on thin layer collagen demonstrated MMP-2 activation with increasing calcium concentration (Fig. 1B, 1st to 3rd lanes).
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