Doxycycline inhibits TGF-beta1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells.
OBJECTIVE
To evaluate the effects of TGF-beta1 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-beta1 (0.1, 1, or 10 ng/mL), with or without TGF-beta1-neutralizing mAb (5 microg/mL), SP600125 (30 microM), PD98059 (40 microM), SB202190 (20 microM), or doxycycline (5-40 microg/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-beta1 increased expression, production, and activity of MMP-9 by human corneal epithelial cells in a concentration-dependent fashion. TGF-beta1 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-beta1-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-beta1-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.
Kim
Hyun-Seung
HS
Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, USA.
Luo
Lihui
L
Pflugfelder
Stephen C
SC
Li
De-Quan
DQ
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology*; D019573:Epithelium, Corneal / Q000166:cytology / Q000201:enzymology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis* / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051899:Smad2 Protein; D015534:Trans-Activators / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000900:Anti-Bacterial Agents; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; C497782:SMAD2 protein, human; D051899:Smad2 Protein; C508960:TGFB1 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020780:Matrix Metalloproteinase 9; D004318:Doxycycline