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Quantitation of matrix metalloproteinases in cultured rat astrocytes using the polymerase chain reaction with a
G M Wells1, G Catlin, J A Cossins
1Neures Limited, Abingdon, Oxon, United Kingdom.
Abstract:
Matrix metalloproteinases (MMPs) are a family of Zn2+ endopeptidases that 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 central nervous system (CNS) such as multiple sclerosis. However, little is known about the expression of MMPs by inflamed tissue within the CNS or by the glia, neurones, and leucocytes which participate in the inflammatory response. To address this issue we have developed a polymerase chain reaction (PCR)-based method for the quantitation of rat MMP mRNA levels, which we have applied to astrocyte cultures with and without inflammatory stimulation. The technique relies on a competition reaction in which a synthetic standard cDNA is co-amplified with the target cDNA in the same PCR reaction. Standard multi-competitor cDNAs, containing priming sites for nine MMPs, and two housekeeping genes were constructed. We have shown that MMP activity is increased over three-fold in neonatal rat astrocyte cultures following stimulation with lipopolysaccharide (LPS). At the mRNA level, MT-MMP-1, 72 kDa gelatinase, and stromelysin-3 were constitutively expressed and unaffected by LPS treatment, whereas 92 kDa gelatinase, and stromelysin-1 were strongly induced (1,000-fold). Stromelysin-2, rat collagenase, and macrophage metalloelastase were modestly upregulated by LPS treatment. Matrilysin was not expressed. This technique is suitable for quantifying MMP expression in the cells which contribute to inflammation in the CNS and could also be applied directly to tissue samples from animal models of disease.
Insights
Matrix metalloproteinases (MMPs) are key in inflammation and tissue breakdown. A new PCR method quantifies MMPs in astrocytes, revealing significant induction of specific MMPs after inflammatory stimulation.
Area of Science:
- Neuroscience
- Molecular Biology
- Immunology
Background:
- Matrix metalloproteinases (MMPs) are implicated in inflammatory conditions and connective tissue breakdown.
- Emerging evidence suggests MMPs play a role in central nervous system (CNS) inflammatory disorders like multiple sclerosis.
- Limited knowledge exists on MMP expression by CNS-residing cells during inflammation.
Purpose of the Study:
- To develop and apply a quantitative polymerase chain reaction (PCR)-based method for measuring rat MMP mRNA levels.
- To investigate MMP expression in astrocyte cultures stimulated with inflammatory agents.
- To understand the role of MMPs in CNS inflammatory responses.
Main Methods:
- Developed a competitive PCR assay for quantifying rat MMP mRNA.
- Co-amplified synthetic standard cDNA with target cDNA in the same PCR reaction.
- Applied the method to neonatal rat astrocyte cultures stimulated with lipopolysaccharide (LPS).
Main Results:
- MMP activity increased over threefold in LPS-stimulated astrocytes.
- 92 kDa gelatinase and stromelysin-1 mRNA levels were strongly induced (1,000-fold) by LPS.
- MT-MMP-1, 72 kDa gelatinase, and stromelysin-3 were constitutively expressed, while others showed modest upregulation or no expression.
Conclusions:
- The developed PCR technique is suitable for quantifying MMP expression in CNS-involved cells.
- Specific MMPs, like 92 kDa gelatinase and stromelysin-1, are significantly upregulated in astrocytes during inflammation.
- This method can be applied to tissue samples from animal models of CNS diseases.