MMP-14 and TIMP-2 overexpression protects against hydroquinone-induced oxidant injury in RPE: implications for extracellular matrix turnover.
OBJECTIVE
To investigate whether overexpression of MMP-14 and/or TIMP-2 would overcome the effect of nonlethal oxidant injury with hydroquinone (HQ) on MMP-2 activity.


METHODS
Human MMP-14 and TIMP2 cDNA were cloned into a mammalian expression vector. Transient transfections were performed on human ARPE-19 cells. The cells were incubated 48 hours after transfection with a nonlethal dose of HQ for either 6 or 18 hours and then were collected for protein determination or RNA isolation. MMP-2 protein and activity were determined by Western blot and zymography. The extracellular matrix (ECM) components type I and type IV collagen and laminin were analyzed by Western blot analysis and real-time PCR.


RESULTS
HQ for 6 hours modestly decreased MMP-2. MMP-2 recovered only after co-overexpression of MMP-14 and TIMP-2, but activity further decreased after HQ for 18 hours. MMP-14 or TIMP-2 overexpression alone contributed as much as the co-overexpression to the recovery of MMP-2 activity. MMP-2 protein seemed not to be altered. Type I collagen and laminin transcriptional levels remained unaffected, whereas type IV collagen transcripts decreased with HQ. Transfection with MMP-14 and/or TIMP-2 contributed to the return of type IV collagen levels to normal. On the other hand, type I and IV collagens and laminin protein accumulated after HQ treatment, an effect prevented by transfection.


CONCLUSIONS
MMP-14 and TIMP2 contribute to the maintenance of adequate levels of MMP-2 activity in ARPE-19 cells after oxidant injury. In addition, changes in ECM components may result as a consequence of MMP-2 activity and may be relevant to the progression of dry AMD.
Alcazar
Oscar
O
Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Miller School of Medicine, Miami, Florida 33136, USA.
Cousins
Scott W
SW
Marin-Castaño
Maria E
ME
D000975:Antioxidants / Q000633:toxicity; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D003001:Cloning, Molecular; D024042:Collagen Type I / Q000378:metabolism; D024141:Collagen Type IV / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D015870:Gene Expression / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D006873:Hydroquinones / Q000633:toxicity*; D007797:Laminin / Q000378:metabolism; D053511:Matrix Metalloproteinase 14 / Q000096:biosynthesis* / Q000235:genetics; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D000975:Antioxidants; D024042:Collagen Type I; D024141:Collagen Type IV; D006873:Hydroquinones; D007797:Laminin; D019716:Tissue Inhibitor of Metalloproteinase-2; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14; C031927:hydroquinone