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Hyun-Seung Kim, Lihui Luo, Stephen C. Pflugfelder, De-Quan Li; Doxycycline Inhibits TGF-β1–Induced MMP-9 via Smad and MAPK Pathways in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2005;46(3):840-848. doi: 10.1167/iovs.04-0929.
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purpose. To evaluate the effects of TGF-β1 and doxycycline on production of gelatinase MMP-9 and activation of Smad, c-Jun N-terminal kinase (JNK), extracellular-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways in human corneal epithelial cells.
methods. Primary human corneal epithelial cells were cultured to confluence. The cells were treated with different concentrations of TGF-β1 (0.1, 1, or 10 ng/mL), with or without TGF-β1–neutralizing mAb (5 μg/mL), SP600125 (30 μM), PD98059 (40 μM), SB202190 (20 μM), or doxycycline (5–40 μg/mL) for different lengths of time. Conditioned media were collected from cultures treated for 24 to 48 hours to evaluate the MMP-9 production by zymography and activity assay. Total RNA was isolated from cells treated for 6 to 24 hours to evaluate MMP-9 expression by semiquantitative RT-PCR and Northern hybridization. Cells treated for 5 to 60 minutes were lysed in RIPA buffer for Western blot with phospho-specific antibodies against Smad2, JNK1/2, ERK1/2, or p38.
results. TGF-β1 increased expression, production, and activity of MMP-9 by human corneal epithelial cells in a concentration-dependent fashion. TGF-β1 also induced activation of Smad2, JNK1/2, ERK1/2, and p38 within 5 to 15 minutes, with peak activation at 15 to 60 minutes. Doxycycline markedly inhibited the TGF-β1–induced production of MMP-9 and activation of the Smad, JNK1/2, ERK1/2, and p38 signaling pathways. Its inhibitory effects were of a magnitude similar to SP600125, PD98059, and SB202190, specific inhibitors of the JNK1/2, ERK1/2, and p38 pathways, respectively.
conclusions. These findings demonstrated that doxycycline inhibits TGF-β1–induced MMP-9 production and activity, perhaps through the Smad and MAPK signaling pathways. These inhibitory effects may explain the reported efficacy of doxycycline in treating MMP-9–mediated ocular surface diseases.
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