Secretion of Matrix Metalloproteinase-2 and -9 after Mechanical Trauma Injury in Rat Cortical Cultures and Involvement of MAP Kinase

Abstract

Matrix metalloproteinases (MMP) are involved in the pathophysiology of brain injury. We recently showed that knockout mice deficient in MMP-9 expression were protected against traumatic brain injury. However, the cellular sources of MMP activity after trauma remain to be fully defined. In this study, we investigated the hypothesis that resident brain cells secrete MMP after mechanical trauma injury in vitro, and mitogen-activated protein (MAP) kinase signal transduction pathways are involved in this response. Rat primary cortical neurons, astrocytes, and co-cultures were subjected to needle scratch mechanical injury, and levels of MMP-2 and MMP-9 in conditioned media were assayed by zymography. MMP-2 and MMP-9 were increased in cortical astrocytes and co-cultures, whereas only MMP-2 was increased in neurons. Western blots showed that phosphorylated extracellular signal regulated kinase (ERK1/2) and p38 were rapidly upregulated in co-cultures after mechanical injury. No change in phosphorylated c-jun N-terminal kinase (JNK) was observed. In-gel kinase assays confirmed this lack of response in the JNK pathway. Treatment with either 10 μM of U0126 (a MAP kinase/ERK1/2 kinase inhibitor) or 10 μM of SB203580 (a p38 inhibitor) had no detectable effect on MMP-2 and MMP-9 levels after mechanical injury. However, combination treatment with both inhibitors significantly reduced secretion of MMP-9. Herein, we demonstrate that (1) resident brain cells secrete MMP after mechanical injury, (2) astrocytes are the main source of MMP-9 activity, and (3) ERK and p38 MAP kinases are upregulated after mechanical injury, and mediate the secretion of MMP-9.