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 down-regulated, 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 a MMP-9 expression is regulated by the ERK1/2 and p38 MAPK in oral keratinocytes, and the that a of this of the oral 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 down-regulated, 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 a MMP-9 expression is regulated by the ERK1/2 and p38 MAPK in oral keratinocytes, and the that a of this of the oral squamous cell oral squamous cell extracellular kinase kinase extracellular oral squamous cell extracellular kinase kinase extracellular is the of the oral oral OSCC is by and the that OSCC a of and a of suggesting that metastatic is with and in of OSCC that the including MMP-9 in and of activity MMP-9 in OSCC with of and is enhanced in highly invasive and metastatic suggesting a between MMP-9 activity and metastatic of that MMP-9 is in invasive OSCC with the invasive the of MMP-9 expression enhanced invasive behavior whereas blocking MMP-9 expression in in an of OSCC the that MMP-9 is an important of the invasive in a of the expression of is by in the of is a calcium the oral with calcium in the and extracellular calcium keratinocyte in of a between MMP-9 expression and expression of MMP-9 with the keratinocyte the of MMP-9 regulation and regulation of MMP-9 expression a role in keratinocyte behavior and MMP-9 expression is correlated with invasive and metastatic MMP-9 gene expression in OSCC evaluated. of the that extracellular calcium induced a dose-dependent increase in MMP-9 expression in immortalized normal and premalignant oral keratinocytes, but not in highly invasive OSCC Differential activation of kinase signaling was also induced by calcium. p38 MAPK activity was down-regulated, whereas extracellular activity was enhanced. Pharmacologic inhibition of p38 MAPK activity or expression of a catalytically inactive mutant of the upstream kinase increased the 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 a MMP-9 expression is regulated by the ERK1/2 and p38 MAPK in oral keratinocytes, and the that a of this of the oral and and the p38 MAPK p38 and MAPK enhanced was and keratinocyte was was was and was was oral a of normal oral and OSCC cell lines and by of and in keratinocyte with with the and in keratinocyte with with the and in calcium and with calcium or with of MMP-9 in the with and or was a in and in with of of MMP-9 in the also by the was with the was by MMP-9 and and MMP-9 and and of the MMP-9 and was and was the and in the of was in a that the in the of and was and and of by of with MMP-9 the was by of the or the mutant of was by of in calcium with of MMP-9 keratinocyte the of the or the mutant of was with the MMP-9 the with the and calcium and by and in calcium with the the in and by and the with ERK1/2 expression or with ERK1/2 of MAPK and with with or calcium. in and and with kinase in and two in kinase and the kinase by the of of kinase of a and of was and was by the of by and the of was by MMP-9 in MMP-9 expression keratinocyte the effect of calcium on MMP-9 expression in immortalized normal or premalignant oral and OSCC and was evaluated. in calcium and with of calcium by of MMP-9 by and normal and premalignant oral and increased MMP-9 expression in calcium MMP-9 expression was not in and MMP-9 expression was in and was by an increase in MMP-9 in calcium in and whereas the MMP-9 in and the MMP-9 expression was enhanced gene the of of the MMP-9 lines with MMP-9 and with a of calcium by and a dose-dependent increase in activation of the MMP-9 was with calcium in normal and premalignant oral keratinocytes, in a increase in activity and whereas calcium not MMP-9 activity in and and a increase in MMP-9 was and the of an increase in MMP-9 in calcium in but not in extracellular calcium and Together that normal or premalignant oral extracellular calcium in MMP-9 of MMP-9 and and in calcium with calcium and MMP-9 expression was by of calcium MMP-9 and with a MMP-9 the with calcium the and by and the of calcium MMP-9 and and an with calcium was and MMP-9 and and on and by ERK1/2 and p38 that MMP-9 expression regulated by ERK1/2 and that ERK1/2 MMP-9 gene expression the MMP-9 expression was an the effect of calcium on ERK1/2 activation was in calcium by with calcium of and the and ERK1/2 by of ERK1/2 in extracellular calcium in and and by and that calcium enhanced ERK1/2 by with effect and ERK1/2 expression increase in was in calcium in and and calcium ERK1/2 in normal and premalignant oral and in calcium by with calcium the and of on and with the of ERK1/2 or with ERK1/2 expression by and enhanced of of the was an p38 MAPK signaling also in the regulation of MMP-9 expression p38 MAPK a of including and the effect of extracellular calcium on p38 MAPK the of the p38 kinase a was an kinase by in or calcium and with or in of the in activity was by a in of by in calcium and a of p38 kinase and was in with calcium p38 MAPK and with or calcium in the or of and with an and in kinase an was by MMP-9 by ERK1/2 and p38 in regulation of MMP-9 expression and in extracellular calcium ERK1/2 and p38 MAPK activity and the of MAPK activity MMP-9 expression was evaluated. with the MMP-9 and with the ERK1/2 activation by the upstream kinase or with the p38 MAPK with calcium and inhibition of ERK1/2 activation MMP-9 activity whereas blocking p38 MAPK activity a increase in of the gene that p38 MAPK is a regulator of MMP-9 in normal and premalignant oral of ERK1/2 activation a in MMP-9 activity in and activation was in p38 MAPK inhibition inhibition of p38 MAPK activity effect on activation of the MMP-9 calcium and p38 MAPK MMP-9 with MMP-9 and with or with extracellular calcium the and by and the of with the of the effect of p38 MAPK on MMP-9 expression with inhibition and of the upstream p38 MAPK with the MMP-9 and the mutant by with calcium with the the p38 MAPK of a catalytically inactive mutant enhanced MMP-9 gene expression the mutant MMP-9 and expression was the p38 MAPK the that p38 MAPK negatively MMP-9 gene expression in negatively MMP-9 with a MMP-9 and with or the with calcium an and by and with the of of ERK1/2 and p38 MAPK the reciprocal regulation of MMP-9 expression activation of the ERK1/2 and p38 MAPK in normal and premalignant oral keratinocytes, the signaling between was evaluated. with of the p38 MAPK with calcium of p38 MAPK activity a dose-dependent increase in ERK1/2 in and and effect of on ERK1/2 was by with the the effect inhibition of p38 MAPK activity not ERK1/2 in and and the of ERK1/2 was by with ERK1/2 and that blocking p38 MAPK activity MMP-9 reciprocal activation of with the MMP-9 and with the p38 MAPK in the or of the with calcium inhibition of p38 MAPK activity enhanced MMP-9 in and and with the this demonstrating that p38 MAPK activity MMP-9 gene expression an normal and premalignant oral keratinocytes, p38 MAPK inhibition effect on MMP-9 activation in and and inhibition of ERK1/2 activation MMP-9 and this effect was in the of the p38 MAPK and Together a of MMP-9 gene expression reciprocal between MAPK signaling that a in p38 MAPK activity MMP-9 gene expression reciprocal of ERK1/2 of p38 MAPK inhibition on ERK1/2 and and with or in with with calcium and of was on and with the of ERK1/2 or with ERK1/2 of p38 MAPK inhibition on MMP-9 and with a MMP-9 with or with and with calcium. and by and the of regulation of MMP-9 expression an important role in normal keratinocyte of MMP-9 expression is in squamous cell of the oral with enhanced expression in of increased of MMP-9 the invasive is by in a OSCC of the of MMP-9 expression inhibition of ERK1/2 activation demonstrating the of this MAPK MMP-9 regulation in in extracellular calcium of the oral and MAPK the effect of calcium on MMP-9 gene expression was of the that extracellular calcium a dose-dependent increase in MMP-9 expression in premalignant oral keratinocytes, but not in highly invasive OSCC also induced activation of ERK1/2 with a of p38 MAPK of p38 MAPK activity or enhanced the MMP-9 gene expression, suggesting that p38 MAPK is a regulator of MMP-9 that calcium gene expression or that calcium activation in a squamous cell of the oral of activity MMP-9 expression in and was in with the that calcium regulation of MMP-9 is normal or premalignant the oral cell lines and two is that OSCC normal on MMP-9 premalignant calcium regulated MMP-9 expression the of gene of this was that calcium MMP-9 expression in normal was the of gene with demonstrating that the effect of calcium is a MMP-9 was regulated by MMP-9 expression was correlated with enhanced ERK1/2 in premalignant oral keratinocytes, and of a mutant of MMP-9 activity not is in with a number of ERK1/2 the of MMP-9 gene and MMP-9 expression in was by ERK1/2 activation and by with of increased MMP-9 expression in whereas catalytically inactive MMP-9 expression in in the a was between p38 MAPK activity and MMP-9 gene expression in oral a between the ERK1/2 and p38 MAPK by that or inhibition of p38 activity in ERK1/2 activation Together a of MMP-9 gene expression reciprocal between MAPK signaling that a in p38 MAPK activity MMP-9 gene expression reciprocal of ERK1/2 is with that MMP-9 activity in is regulated by ERK1/2 and negatively regulated by p38 kinase activity of with the p38 MAPK increased ERK1/2 and this was by the that is also between ERK1/2 and p38 MAPK in in extracellular calcium by the extracellular or activation of this activation of including ERK1/2 that activation with the also MMP-9 gene expression not suggesting that signaling the MMP-9 expression in premalignant oral calcium is a regulator of keratinocyte function, that in calcium in the extracellular also of this of of the oral squamous cell oral squamous cell extracellular kinase kinase extracellular oral squamous cell extracellular kinase kinase extracellular is the of the oral oral OSCC is by and the that OSCC a of and a of suggesting that metastatic is with and in of OSCC that the including MMP-9 in and of activity MMP-9 in OSCC with of and is enhanced in highly invasive and metastatic suggesting a between MMP-9 activity and metastatic of that MMP-9 is in invasive OSCC with the invasive the of MMP-9 expression enhanced invasive behavior whereas blocking MMP-9 expression in in an of OSCC the that MMP-9 is an important of the invasive in OSCC. a of the expression of is by in the of is a calcium the oral with calcium in the and extracellular calcium keratinocyte in of a between MMP-9 expression and expression of MMP-9 with the keratinocyte the of MMP-9 regulation and Because regulation of MMP-9 expression a role in keratinocyte behavior and MMP-9 expression is correlated with invasive and metastatic MMP-9 gene expression in OSCC evaluated. of the that extracellular calcium induced a dose-dependent increase in MMP-9 expression in immortalized normal and premalignant oral keratinocytes, but not in highly invasive OSCC Differential activation of kinase signaling was also induced by calcium. p38 MAPK activity was down-regulated, whereas extracellular activity was enhanced. Pharmacologic inhibition of p38 MAPK activity or expression of a catalytically inactive mutant of the upstream kinase increased the 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 a MMP-9 expression is regulated by the ERK1/2 and p38 MAPK in oral keratinocytes, and the that a of this of the oral and and the p38 MAPK p38 and MAPK enhanced was and keratinocyte was was was and was was oral a of normal oral and OSCC cell lines and by of and in keratinocyte with with the and in keratinocyte with with the and in calcium and with calcium or with of MMP-9 in the with and or was a in and in with of of MMP-9 in the also by the was with the was by MMP-9 and and MMP-9 and and of the MMP-9 and was and was the and in the of was in a that the in the of and was and and of by of with MMP-9 the was by of the or the mutant of was by of in calcium with of MMP-9 keratinocyte the of the or the mutant of was with the MMP-9 the with the and calcium and by and in calcium with the the in and by and the with ERK1/2 expression or with ERK1/2 of MAPK and with with or calcium. in and and with kinase in and two in kinase and the kinase by the of of kinase of a and of was and was by the of by and the of was by and and the p38 MAPK p38 and MAPK enhanced was and keratinocyte was was was and was was oral a of normal oral and OSCC cell lines and by of and in keratinocyte with with the and in keratinocyte with with the and in calcium and with calcium or with calcium. of MMP-9 in the with and or was a in and in with of of MMP-9 in the also by the MMP-9 was with the was by MMP-9 and and MMP-9 and and of the MMP-9 and was and was the and in the of was in a that the in the of and was and and of by of with MMP-9 MMP-9 the was by of the or the mutant of was by of in calcium with of MMP-9 keratinocyte the of the or the mutant of was with the MMP-9 the with the and calcium and by and in calcium ERK1/2 with the the in and by and the with ERK1/2 expression or with ERK1/2 of p38 MAPK and with with or calcium. in and and with kinase in and two in kinase and the kinase by the of of kinase of a and of was and was by the of by and the of was by MMP-9 in MMP-9 expression keratinocyte the effect of calcium on MMP-9 expression in immortalized normal or premalignant oral and OSCC and was evaluated. in calcium and with of calcium by of MMP-9 by and normal and premalignant oral and increased MMP-9 expression in calcium MMP-9 expression was not in and MMP-9 expression was in and was by an increase in MMP-9 in calcium in and whereas the MMP-9 in and the MMP-9 expression was enhanced gene the of of the MMP-9 lines with MMP-9 and with a of calcium by and a dose-dependent increase in activation of the MMP-9 was with calcium in normal and premalignant oral keratinocytes, in a increase in activity and whereas calcium not MMP-9 activity in and and a increase in MMP-9 was and the of an increase in MMP-9 in calcium in but not in extracellular calcium and Together that normal or premalignant oral extracellular calcium in MMP-9 calcium MMP-9 and with a MMP-9 the with calcium the and by and the of calcium MMP-9 and and an with calcium was and MMP-9 and and on and by ERK1/2 and p38 that MMP-9 expression regulated by ERK1/2 and that ERK1/2 MMP-9 gene expression the MMP-9 expression was an the effect of calcium on ERK1/2 activation was in calcium by with calcium of and the and ERK1/2 by of ERK1/2 in extracellular calcium in and and by and that calcium enhanced ERK1/2 by with effect and ERK1/2 expression increase in was in calcium in and and calcium ERK1/2 in normal and premalignant oral and in calcium by with calcium the and of on and with the of ERK1/2 or with ERK1/2 expression by and enhanced of of the was an p38 MAPK signaling also in the regulation of MMP-9 expression p38 MAPK a of including and the effect of extracellular calcium on p38 MAPK the of the p38 kinase a was an kinase by in or calcium and with or in of the in activity was by a in of by in calcium and a of p38 kinase and was in with calcium p38 MAPK and with or calcium in the or of and with an and in kinase an was by MMP-9 by ERK1/2 and p38 in regulation of MMP-9 expression and in extracellular calcium ERK1/2 and p38 MAPK activity and the of MAPK activity MMP-9 expression was evaluated. with the MMP-9 and with the ERK1/2 activation by the upstream kinase or with the p38 MAPK with calcium and inhibition of ERK1/2 activation MMP-9 activity whereas blocking p38 MAPK activity a increase in of the gene that p38 MAPK is a regulator of MMP-9 in normal and premalignant oral of ERK1/2 activation a in MMP-9 activity in and activation was in p38 MAPK inhibition inhibition of p38 MAPK activity effect on activation of the MMP-9 calcium and p38 MAPK MMP-9 with MMP-9 and with or with extracellular calcium the and by and the of with the of the effect of p38 MAPK on MMP-9 expression with inhibition and of the upstream p38 MAPK with the MMP-9 and the mutant by with calcium with the the p38 MAPK of a catalytically inactive mutant enhanced MMP-9 gene expression the mutant MMP-9 and expression was the p38 MAPK the that p38 MAPK negatively MMP-9 gene expression in negatively MMP-9 with a MMP-9 and with or the with calcium an and by and with the of of ERK1/2 and p38 MAPK the reciprocal regulation of MMP-9 expression activation of the ERK1/2 and p38 MAPK in normal and premalignant oral keratinocytes, the signaling between was evaluated. with of the p38 MAPK with calcium of p38 MAPK activity a dose-dependent increase in ERK1/2 in and and effect of on ERK1/2 was by with the the effect inhibition of p38 MAPK activity not ERK1/2 in and and the of ERK1/2 was by with ERK1/2 and that blocking p38 MAPK activity MMP-9 reciprocal activation of with the MMP-9 and with the p38 MAPK in the or of the with calcium inhibition of p38 MAPK activity enhanced MMP-9 in and and with the this demonstrating that p38 MAPK activity MMP-9 gene expression an normal and premalignant oral keratinocytes, p38 MAPK inhibition effect on MMP-9 activation in and and inhibition of ERK1/2 activation MMP-9 and this effect was in the of the p38 MAPK and Together a of MMP-9 gene expression reciprocal between MAPK signaling that a in p38 MAPK activity MMP-9 gene expression reciprocal of ERK1/2 of p38 MAPK inhibition on ERK1/2 and and with or in with with calcium and of was on and with the of ERK1/2 or with ERK1/2 of p38 MAPK inhibition on MMP-9 and with a MMP-9 with or with and with calcium. and by and the of MMP-9 in MMP-9 expression keratinocyte the effect of calcium on MMP-9 expression in immortalized normal or premalignant oral and OSCC and was evaluated. in calcium and with of calcium by of MMP-9 by and normal and premalignant oral and increased MMP-9 expression in calcium MMP-9 expression was not in and MMP-9 expression was in and was by an increase in MMP-9 in calcium in and whereas the MMP-9 in and the MMP-9 expression was enhanced gene the of of the MMP-9 lines with MMP-9 and with a of calcium by and a dose-dependent increase in activation of the MMP-9 was with calcium in normal and premalignant oral keratinocytes, in a increase in activity and whereas calcium not MMP-9 activity in and and a increase in MMP-9 was and the of an increase in MMP-9 in calcium in but not in extracellular calcium and Together that normal or premalignant oral extracellular calcium in MMP-9 ERK1/2 and p38 that MMP-9 expression regulated by ERK1/2 and that ERK1/2 MMP-9 gene expression the MMP-9 expression was an the effect of calcium on ERK1/2 activation was in calcium by with calcium of and the and ERK1/2 by of ERK1/2 in extracellular calcium in and and by and that calcium enhanced ERK1/2 by with effect and ERK1/2 expression increase in was in calcium in and and p38 MAPK signaling also in the regulation of MMP-9 expression p38 MAPK a of including and the effect of extracellular calcium on p38 MAPK the of the p38 kinase a was an kinase by in or calcium and with or in of the in activity was by a in of by in calcium and a of p38 kinase and was in with MMP-9 by ERK1/2 and p38 in regulation of MMP-9 expression and in extracellular calcium ERK1/2 and p38 MAPK activity and the of MAPK activity MMP-9 expression was evaluated. with the MMP-9 and with the ERK1/2 activation by the upstream kinase or with the p38 MAPK with calcium and inhibition of ERK1/2 activation MMP-9 activity whereas blocking p38 MAPK activity a increase in of the gene that p38 MAPK is a regulator of MMP-9 in normal and premalignant oral of ERK1/2 activation a in MMP-9 activity in and activation was in p38 MAPK inhibition inhibition of p38 MAPK activity effect on activation of the MMP-9 calcium the effect of p38 MAPK on MMP-9 expression with inhibition and of the upstream p38 MAPK with the MMP-9 and the mutant by with calcium with the the p38 MAPK of a catalytically inactive mutant enhanced MMP-9 gene expression the mutant MMP-9 and expression was the p38 MAPK the that p38 MAPK negatively MMP-9 gene expression in of ERK1/2 and p38 MAPK the reciprocal regulation of MMP-9 expression activation of the ERK1/2 and p38 MAPK in normal and premalignant oral keratinocytes, the signaling between was evaluated. with of the p38 MAPK with calcium of p38 MAPK activity a dose-dependent increase in ERK1/2 in and and effect of on ERK1/2 was by with the the effect inhibition of p38 MAPK activity not ERK1/2 in and and the of ERK1/2 was by with ERK1/2 and that blocking p38 MAPK activity MMP-9 reciprocal activation of with the MMP-9 and with the p38 MAPK in the or of the with calcium inhibition of p38 MAPK activity enhanced MMP-9 in and and with the this demonstrating that p38 MAPK activity MMP-9 gene expression an normal and premalignant oral keratinocytes, p38 MAPK inhibition effect on MMP-9 activation in and and inhibition of ERK1/2 activation MMP-9 and this effect was in the of the p38 MAPK and Together a of MMP-9 gene expression reciprocal between MAPK signaling that a in p38 MAPK activity MMP-9 gene expression reciprocal of ERK1/2 regulation of MMP-9 expression an important role in normal keratinocyte of MMP-9 expression is in squamous cell of the oral with enhanced expression in of increased of MMP-9 the invasive is by in a OSCC of the of MMP-9 expression inhibition of ERK1/2 activation demonstrating the of this MAPK MMP-9 regulation in in extracellular calcium of the oral and MAPK the effect of calcium on MMP-9 gene expression was of the that extracellular calcium a dose-dependent increase in MMP-9 expression in premalignant oral keratinocytes, but not in highly invasive OSCC also induced activation of ERK1/2 with a of p38 MAPK of p38 MAPK activity or enhanced the MMP-9 gene expression, suggesting that p38 MAPK is a regulator of MMP-9 that calcium gene expression or that calcium activation in a squamous cell of the oral of activity MMP-9 expression in and was in with the that calcium regulation of MMP-9 is normal or premalignant the oral cell lines and two is that OSCC normal on MMP-9 premalignant calcium regulated MMP-9 expression the of gene of this was that calcium MMP-9 expression in normal was the of gene with demonstrating that the effect of calcium is a MMP-9 was regulated by MMP-9 expression was correlated with enhanced ERK1/2 in premalignant oral keratinocytes, and of a mutant of MMP-9 activity not is in with a number of ERK1/2 the of MMP-9 gene and MMP-9 expression in was by ERK1/2 activation and by with of increased MMP-9 expression in whereas catalytically inactive MMP-9 expression in in the a was between p38 MAPK activity and MMP-9 gene expression in oral a between the ERK1/2 and p38 MAPK by that or inhibition of p38 activity in ERK1/2 activation Together a of MMP-9 gene expression reciprocal between MAPK signaling that a in p38 MAPK activity MMP-9 gene expression reciprocal of ERK1/2 is with that MMP-9 activity in is regulated by ERK1/2 and negatively regulated by p38 kinase activity of with the p38 MAPK increased ERK1/2 and this was by the that is also between ERK1/2 and p38 MAPK in in extracellular calcium by the extracellular or activation of this activation of including ERK1/2 that activation with the also MMP-9 gene expression not suggesting that signaling the MMP-9 expression in premalignant oral calcium is a regulator of keratinocyte function, that in calcium in the extracellular also of this of of the oral regulation of MMP-9 expression an important role in normal keratinocyte of MMP-9 expression is in squamous cell of the oral with enhanced expression in of increased of MMP-9 the invasive is by in a OSCC of the of MMP-9 expression inhibition of ERK1/2 activation demonstrating the of this MAPK MMP-9 regulation in in extracellular calcium of the oral and MAPK the effect of calcium on MMP-9 gene expression was of the that extracellular calcium a dose-dependent increase in MMP-9 expression in premalignant oral keratinocytes, but not in highly invasive OSCC also induced activation of ERK1/2 with a of p38 MAPK of p38 MAPK activity or enhanced the MMP-9 gene expression, suggesting that p38 MAPK is a regulator of MMP-9 that calcium gene expression or that calcium activation in a squamous cell of the oral of activity MMP-9 expression in and was in with the that calcium regulation of MMP-9 is normal or premalignant the oral cell lines and two is that OSCC normal on MMP-9 premalignant calcium regulated MMP-9 expression the of gene of this was that calcium MMP-9 expression in normal was the of gene with demonstrating that the effect of calcium is a MMP-9 was regulated by MMP-9 expression was correlated with enhanced ERK1/2 in premalignant oral keratinocytes, and of a mutant of MMP-9 activity not is in with a number of ERK1/2 the of MMP-9 gene and MMP-9 expression in was by ERK1/2 activation and by with of increased MMP-9 expression in whereas catalytically inactive MMP-9 expression in in the a was between p38 MAPK activity and MMP-9 gene expression in oral a between the ERK1/2 and p38 MAPK by that or inhibition of p38 activity in ERK1/2 activation Together a of MMP-9 gene expression reciprocal between MAPK signaling that a in p38 MAPK activity MMP-9 gene expression reciprocal of ERK1/2 is with that MMP-9 activity in is regulated by ERK1/2 and negatively regulated by p38 kinase activity of with the p38 MAPK increased ERK1/2 and this was by the that is also between ERK1/2 and p38 MAPK in in extracellular calcium by the extracellular or activation of this activation of including ERK1/2 that activation with the also MMP-9 gene expression not suggesting that signaling the MMP-9 expression in premalignant oral calcium is a regulator of keratinocyte function, that in calcium in the extracellular also of this of of the oral