Acidic Extracellular pH Induces Matrix Metalloproteinase-9 Expression in Mouse Metastatic Melanoma Cells through the Phospholipase D-Mitogen-activated Protein Kinase Signaling

Abstract

The extracellular pH (pHe) of tumor tissues is often acidic, which can induce the expression of several proteins. We previously showed that production of matrix metalloproteinase-9 (MMP-9) was induced by culturing cells at acidic pHe (5.4-6.5). Here we have investigated the signal transduction pathway by which acidic pHe induces MMP-9 expression. We found that acidic pHe (5.9) activated phospholipase D (PLD), and inhibition of PLD activity by 1-butanol and Myr-ARF6 suppressed the acidic pHe-induced MMP-9 expression. Exogenous PLD, but not phosphatidylinositol-specific PLC or PLA2, mimicked MMP-9 induction by acidic pHe. Western blot analysis revealed that acidic pHe increased the steady-state levels of phosphorylated extracellular signal-regulated kinases 1/2 and p38 and that the PLD inhibitors suppressed these increases. Using 5'-deletion mutant constructs of the MMP-9 promoter, we found that the acidic pHe-responsive region was located at nucleotide -670 to -531, a region containing the NF kappa B binding site. A mutation into the NF kappa B binding site reduced, but not completely, the acidic pHe-induced MMP-9 promoter activity, and NF kappa B activity was induced by acidic pHe. Pharmacological inhibitors specific for mitogen-activated protein kinase kinase 1/2 (PD098059) and p38 (SB203580) attenuated the acidic pHe-induced NF kappa B activity and MMP-9 expression. These data suggest that PLD, mitogen-activated protein kinases (extracellular signal-regulated kinases 1/2 and p38), and NF kappa B mediate the acidic pHe signaling to induce MMP-9 expression. A transcription factor(s) other than NF kappa B may also be involved in the MMP-9 expression.