Calcium-induced Matrix Metalloproteinase 9 Gene Expression Is Differentially Regulated by ERK1/2 and p38 MAPK in Oral Keratinocytes and Oral Squamous Cell Carcinoma

Abstract

Matrix metalloproteinases (MMPs) play an important role in the invasive behavior of a number of cancers including oral squamous cell cancer (OSCC), and increased expression of MMP-9 is correlated with invasive and metastatic OSCC. Because calcium is an important regulator of keratinocyte function, the effect of modulating extracellular calcium on MMP-9 expression in OSCC cell lines was evaluated. Increasing extracellular calcium induced a dose-dependent increase in MMP-9 expression in immortalized normal and premalignant oral keratinocytes, but not in two highly invasive OSCC cell lines. Differential activation of MAPK signaling was also induced by calcium. p38 MAPK activity was downregulated, whereas ERK1/2 activity was enhanced. Pharmacologic inhibition of p38 MAPK activity or expression of a catalytically inactive mutant of the upstream kinase MAPK kinase 3 (MKK3) increased the calcium induced MMP-9 gene expression, demonstrating that p38 MAPK activity negatively regulated this process. Interestingly blocking p38 MAPK activity enhanced ERK1/2 phosphorylation, suggesting reciprocal regulation between the ERK1/2 and p38 MAPK pathways. Together these data support a model wherein calcium-induced MMP-9 expression is differentially regulated by the ERK1/2 and p38 MAPK pathways in oral keratinocytes, and the data suggest that a loss of this regulatory mechanism accompanies malignant transformation of the oral epithelium.Oral squamous cell carcinoma (OSCC) 1 is the most common malignancy of the oral cavity causing more deaths than any other oral disease (1, 2). Although OSCC is characterized by local, regional, and distant spread, the biochemical factors that underlie dissemination are poorly understood (1, 3). OSCC tumors demonstrate a progressive lack of basement membrane staining, more diffuse invasion, and a higher frequency of lymph node metastasis, suggesting that basement membrane loss reflects metastatic potential (4 -7). This is consistent with immunohistochemical and in situ hybridization studies of human OSCC tumors that implicate enzymes belonging to the matrix metalloproteinase (MMP) family, including MMP-9 (92-kDa gelatinase B), in basement membrane proteolysis and tissue invasion. The presence of gelatinolytic activity attributable to activated MMP-9 in OSCC biopsy specimens correlates with immunohistochemical staining of frozen sections and is enhanced in both highly invasive and metastatic cases, suggesting a correlation between MMP-9 activity and metastatic potential (8,9). Additional studies of human tumors have shown that MMP-9 is up-regulated in diffuse invasive OSCC specimens, with predominant localization to the invasive front (4, 10). At the cellular level, modulation of MMP-9 expression via phorbol ester treatment leads to enhanced invasive behavior (8 -10), whereas blocking MMP-9 expression in vivo reduces invasion in an orthotopic murine model of OSCC (11). These data support the hypothesis that MMP-9 is an important determinant of the invasive phenotype in OSCC.Oral keratinocytes express a...

α3β1 integrin regulates MMP-9 mRNA stability in immortalized keratinocytes: a novel mechanism of integrin-mediated MMP gene expression

“…Similarly, the ability of TGFβ to induce MMP-9 expression in prostate cancer cells is acquired during cellular transformation (Sehgal et al, 1996;Sehgal and Thompson, 1999). In addition, changes in calcium-mediated regulation of MMP-9 expression occur during malignant transformation of oral keratinocytes (Mukhopadhyay et al, 2004). Thus, it is clear that certain signaling pathways that regulate MMP-9 expression are activated or altered during cellular immortalization or malignant progression.…”

“…mitogen-activated protein kinases (MAPKs) p38 and/or extracellular signal-regulated kinase (ERK) (Johansson et al, 2000;McCawley et al, 1998;Mukhopadhyay et al, 2004;Westermarck and Kahari, 1999;Zeigler et al, 1999). In addition, several studies have demonstrated the importance of cell adhesion in regulating the expression of MMP-9 or other MMPs in immortalized epithelial cells or carcinoma-derived cells (DiPersio et al, 2000;Lochter et al, 1999;Morini et al, 2000;Thomas et al, 2001).…”

Differential growth factor regulation of N-cadherin expression and motility in normal and malignant oral epithelium

“…SCC9 and SCC25 cells were obtained from American Type Culture Collection (ATCC) Rheinwald and Beckett, 1981), whereas UMSCC1 cells were generously provided by Ernst Lengyel (University of Chicago, IL) (Juarez et al, 1993;Lengyel et al, 1995). SCC68 cells and tertimmortalized normal oral keratinocytes (OKF4 and OKF6 cells) were kindly provided by James G. Rheinwald (Brigham and Women's Hospital, Harvard Institutes of Medicine, Boston, MA) (Dickson et al, 2000;Mukhopadhyay et al, 2004). SCC9, SCC25 and UMSCC1 cells were routinely maintained in DMEM and Ham's F-12 medium (1:1) supplemented with 10% fetal calf serum and 100 units/ml penicillin and 100 μg/ml streptomycin.…”

Vascular endothelial growth factor–regulated ovarian cancer invasion and migration involves expression and activation of matrix metalloproteinases

“…[24][25][26][27] Twenty microliters of each reaction mixture were analyzed by 1.0% agarose gels in the presence of 0.5 lg/ml ethidium bromide, and DNA was visualized under UV light. Results from real-time PCR were presented as threshold cycles normalized to that of the GAPDH gene according to the method of Overbergh et al 28 It reversibly represented the number of cDNA copies of an individual gene.…”