RNAse protection assays for the simultaneous and semiquantitative analysis of multiple murine matrix metalloproteinase (MMP) and MMP inhibitor mRNAs.
Matrix metalloproteinases (MMPs) are a family of proteinases that play a major role in the metabolic degradation of extracellular matrix proteins. In order to examine the expression pattern of different MMP or MMP-inhibitor genes two RNase protection assays (RPAs) were developed that allow the simultaneous and semiquantitative assessment of their respective mRNAs. Probes for the detection of MMPs stromelysin 1, 2 and 3, matrilysin, metalloelastase, gelatinase A and B, collagenase and membrane type MMP (MT1-MMP) were included in the first RPA probe set, while probes for tissue inhibitor of matrix metalloproteinase (TIMP) 1, 2, 3 and alpha 2-macroglobulin (alpha 2-M) were included in the second probe set (inhibitor of matrix metalloproteinase-IMP set). Titration experiments revealed that this method allows the detection of MMP and inhibitor mRNAs present in at least 0.03 microgram of spleen poly(A)+ RNA. Both RPA sets were further evaluated by analyzing the expression of MMP and IMP genes in brain, kidney, spleen and liver in a murine model for endotoxemia after intraperitoneal LPS injection. Control animals showed an organ-specific constitutive expression of one or more MMPs and a high expression of TIMPs. Following LPS injection, an organ-specific upregulation or induction of MMP and TIMP RNA species was found. This change was most pronounced in the spleen, while liver, kidney and brain showed minor or no changes in MMP expression. An IMP upregulation was detected in all organs. These RPA probe sets provide a valuable tool for the simultaneous assessment of MMP and IMP gene expression under physiological and pathological conditions.
Pagenstecher
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A
Department of Neuropharmacology, Scripps Research Institute, La Jolla, CA 92037, USA.
Stalder
A K
AK
Campbell
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IL
D000818:Animals; D017364:Collagenases / Q000235:genetics; D015342:DNA Probes / Q000138:chemical synthesis; D004591:Electrophoresis, Polyacrylamide Gel; D018093:Gelatinases / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics*; D008297:Male; D019278:Matrix Metalloproteinase 3 / Q000235:genetics; D020783:Matrix Metalloproteinase 7; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D009928:Organ Specificity / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D012260:Ribonucleases / Q000378:metabolism*; D019714:Tissue Inhibitor of Metalloproteinases
D015342:DNA Probes; D006023:Glycoproteins; D012333:RNA, Messenger; D019714:Tissue Inhibitor of Metalloproteinases; D012260:Ribonucleases; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D020783:Matrix Metalloproteinase 7