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S.C. Pflugfelder, H. Kim, D. Li; Doxycycline Inhibits TGF-ß1 Stimulated MMP-9 in Human Corneal Epithelial Cells via Smad and MAPK Pathways . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1404.
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Purpose: To evaluate the effects of doxycycline on the expression of TGF-ß1 stimulated matrix metalloproteinase 9 (MMP-9) in human corneal epithelial cells and on the activation of Smad2, c-Jun N-terminal kinase (JNK) and extracellular-regulated kinase (ERK) signaling intermediates that are induced by TGF-ß1. Methods: Primary human corneal epithelial cells were cultured from limbal explants in 6-well plates until confluence. The cells were then exposed to serum-free media alone (control), or with different concentrations of TGF-ß1 (0.1, 1,10 ng/ml) with or without TGF-ß1 neutralizing mAb (5 µg/ml), SB202190 (10-40 µM) and doxycycline (5-40 µg/ml) for different lengths of time. The conditioned media were collected from cultures treated for 24 hours for zymography and MMP-9 activity assay. The cells treated for 5-60 min were lysed in RIPA buffer and subjected to Western blot with phospho-specific antibodies against Smad2, JNK or ERK. Results: TGF-ß1 dose-dependently increased the production and activity of MMP-9 by human corneal epithelial cells. This stimulation was abolished by 5 µg/ml of TGF-ß1 neutralizing mAb or 10 µg/ml of doxycycline. TGF-ß1 dose-dependently induced phorsphorylated JNK1/2, ERK and Smad2, as early as 5 min, peaking at 15 or 30 min respectively. Doxycycline markedly inhibited the TGF-ß1 induced activation of JNK1/2, ERK1 and Smad2 with activity equal to SB202190. Conclusions: Doxycycline inhibits TGF-ß1 induced MMP-9 production and activity, perhaps through the Smad and MAPK pathways. This finding may explain the reported efficacy of doxycycline in treating MMP-mediated ocular surface disease.
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