Alterations in mRNA and protein levels of metalloproteinases-2, -9, and -14 and tissue inhibitor of metalloproteinase-2 responses to traumatic skeletal muscle injury.
This study characterizes the temporal relationship of membrane type-1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) expression in skeletal muscle following injury. Tibialis anterior (TA) muscles from 60 mice were exposed and injured by applying a cold steel probe (-79 degrees C) to the muscle for 10 s. Thereafter, TA muscles from uninjured and injured legs were collected at 3, 10, 24, 48, and 72 h postinjury for analysis of local MT1-MMP, TIMP-2, and matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) mRNA and protein content via quantitative RT-PCR, immunoblotting, zymography, and immunofluorescence. All data are expressed as fold change of injured leg vs. uninjured leg. MT1-MMP mRNA levels were decreased significantly at 48 and 72 h postinjury by approximately 9- and 21-fold, respectively (P < 0.01). Both TIMP-2 and MMP-2 mRNA expression significantly decreased in the injured leg by approximately 4- to 10-fold at 10-72 h postinjury (P < 0.01). MMP-9 mRNA expression was significantly increased at 10, 24, and 48 h postinjury by 6- (P < 0.05), 25-, and 12-fold (P < 0.01), respectively. Protein content of latent (63 kDa) MT1-MMP was decreased at 48 and 72 h postinjury by approximately 2-fold (P < 0.01). Content of the soluble (50 kDa) fragment of MT1-MMP was significantly increased by approximately 17-, 25-, and 67-fold at 24 (P < 0.05), 48, and 72 h (P < 0.01) postinjury, respectively. TIMP-2 protein levels diminished from 3 to 48 h postinjury by 1.5-fold to 1.8-fold (P < 0.01), before returning to baseline levels at 72 h postinjury. Zymography revealed visual increases in gelatinase activity in molecular weight regions corresponding to MMP-9 and MMP-2. In conclusion, skeletal muscle injury initiates a sequence of events in the MT1-MMP proteolytic cascade resulting in elevated levels of the soluble (50 kDa) fragment of MT1-MMP, which could enhance pericellular extracellular matrix remodeling.
Barnes
Brian R
BR
United States Army Research Institute of Environmental Medicine, Natick, Massachusetts, USA. bbarnes@arteriocyte.com
Szelenyi
Eric R
ER
Warren
Gordon L
GL
Urso
Maria L
ML
D000818:Animals; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D008297:Male; D053511:Matrix Metalloproteinase 14 / Q000235:genetics / Q000378:metabolism*; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000293:injuries*; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism*; D014947:Wounds and Injuries / Q000201:enzymology
D012333:RNA, Messenger; D019716:Tissue Inhibitor of Metalloproteinase-2; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D053511:Matrix Metalloproteinase 14