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Doxycycline Inhibits TGF-ß1–Induced MMP-9 via Smad and MAPK Pathways in Human Corneal Epithelial Cells

¹From the Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas; and the ²Department of Ophthalmology, College of Medicine, The Catholic University of Korea, UiJong-Bu City, Korea.

PURPOSE. To evaluate the effects of TGF-ß1 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-ß1 (0.1, 1, or 10 ng/mL), with or without TGF-ß1–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-ß1 increased expression, production, and activity of MMP-9 by human corneal epithelial cells in a concentration-dependent fashion. TGF-ß1 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-ß1–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-ß1–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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