Real-time visualization of MMP-13 promoter activity in transgenic mice.
Cutaneous wound repair involves extracellular matrix degradation, cell migration, matrix resynthesis and tissue remodeling. In the rodent, transcriptional regulation of collagenase-3 (MMP-13) most likely plays a role in these processes. Therefore, we isolated and characterized a 1.76-kb 5'-flanking region of the mouse MMP-13 gene. Assay of promoter activity by transient transfection of HT1080 cells and primary mouse skin fibroblasts allowed identification of several functional regions of the 5'-flanking DNA. Expression of luciferase reporter constructs in these cells was induced by phorbol myristate acetate (PMA), but not by transforming growth factor-beta(2) (TGF-beta(2)). To study the regulation of MMP-13 in cutaneous wound healing, we generated transgenic mouse lines harboring the firefly luciferase reporter gene under control of a 660-bp mouse MMP-13 promoter which showed maximal response. MMP-13 mRNA levels in transgenic lung fibroblasts increased 1.5-2.6-fold after PMA challenge. MMP-13 promoter activity in wounds was visualized and quantified in vivo as luciferase bioluminescence. MMP-13 expression was present at day 1 and maximal at day 18 post-wounding. Luciferase activity progressed from the wound margin towards the center of the wound. In situ hybridization showed the same spatial and temporal patterns for the luciferase and endogenous MMP-13 mRNA. Both signals localized predominantly to dermal fibroblasts at the wound periphery but not to granulation tissue or to keratinocytes. These results suggested that MMP-13 participated in the wound healing of acute wounds, and it was a significant factor in long-term remodeling of wound connective tissue in rodent skin.
Wu
Nanjun
N
Department of Pathology, Vanderbilt University School of Medicine, C-3321 Medical Center North, Nashville, TN 37232-2561, USA.
Opalenik
Susan
S
Liu
Jinhua
J
Jansen
E Duco
ED
Giro
Maria Gabriella
MG
Davidson
Jeffrey M
JM
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017364:Collagenases / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D017403:In Situ Hybridization; D008156:Luciferases / Q000235:genetics; D008168:Lung / Q000166:cytology / Q000201:enzymology; D053509:Matrix Metalloproteinase 13; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014947:Wounds and Injuries
D012333:RNA, Messenger; D008156:Luciferases; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; C507081:Mmp13 protein, mouse