QUANTITATION OF MATRIX METALLOPROTEINASES IN CULTURED RAT ASTROCYTES USING THE POLYMERASE CHAIN-REACTION WITH A MULTI-COMPETITOR CDNA STANDARD

Matrix metalloproteinases (MMPs) are a family of Zn2+ endopeptidases t hat are expressed in many inflammatory conditions and that contribute to connective tissue breakdown and the release of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha). There is emerging evidence that MMPs have a role in inflammatory disorders of the centra l nervous system (CNS) such as multiple sclerosis. However, little is known about the expression of MMPs by inflamed tissue within the CNS o r by the glia, neurones, and leucocytes which participate in the infla mmatory response. To address this issue we have developed a polymerase chain reaction (PCR)-based method for the quantitation of rat MMP mRN A levels, which we have applied to astrocyte cultures with and without inflammatory stimulation. The technique relies on a competition react ion in which a synthetic standard cDNA is co-amplified with the target cDNA in the same PCR reaction. Standard multi-competitor cDNAs, conta ining priming sites for nine MMPs, and two housekeeping genes were con structed. We have shown that MMP activity is increased over three-fold in neonatal rat astrocyte cultures following stimulation with lipopol ysaccharide (LPS). At the mRNA level, MT-MMP-1, 72 kDa gelatinase, and stromelysin-3 were constitutively expressed and unaffected by LPS tre atment, whereas 92 kDa gelatinase, and stromelysin-l were strongly ind uced (1,000-fold). Stromelysin-2, rat collagenase, and macrophage meta lloelastase were modestly upregulated by LPS treatment. Matrilysin was not expressed. This technique is suitable for quantifying MMP express ion in the cells which contribute to inflammation in the CNS and could also be applied directly to tissue samples from animal models of dise ase. (C) 1996 Wiley-Liss, Inc.