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Doxycycline inhibits TGF-beta1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells.
Invest Ophthalmol Vis Sci. 2005 Mar; 46(3):840-8.IO

Abstract

PURPOSE

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.

Authors+Show Affiliations

Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15728539

Citation

Kim, Hyun-Seung, et al. "Doxycycline Inhibits TGF-beta1-induced MMP-9 Via Smad and MAPK Pathways in Human Corneal Epithelial Cells." Investigative Ophthalmology & Visual Science, vol. 46, no. 3, 2005, pp. 840-8.
Kim HS, Luo L, Pflugfelder SC, et al. Doxycycline inhibits TGF-beta1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells. Invest Ophthalmol Vis Sci. 2005;46(3):840-8.
Kim, H. S., Luo, L., Pflugfelder, S. C., & Li, D. Q. (2005). Doxycycline inhibits TGF-beta1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells. Investigative Ophthalmology & Visual Science, 46(3), 840-8.
Kim HS, et al. Doxycycline Inhibits TGF-beta1-induced MMP-9 Via Smad and MAPK Pathways in Human Corneal Epithelial Cells. Invest Ophthalmol Vis Sci. 2005;46(3):840-8. PubMed PMID: 15728539.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Doxycycline inhibits TGF-beta1-induced MMP-9 via Smad and MAPK pathways in human corneal epithelial cells. AU - Kim,Hyun-Seung, AU - Luo,Lihui, AU - Pflugfelder,Stephen C, AU - Li,De-Quan, PY - 2005/2/25/pubmed PY - 2005/4/14/medline PY - 2005/2/25/entrez SP - 840 EP - 8 JF - Investigative ophthalmology & visual science JO - Invest Ophthalmol Vis Sci VL - 46 IS - 3 N2 - PURPOSE: 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. SN - 0146-0404 UR - https://www.unboundmedicine.com/medline/citation/15728539/Doxycycline_inhibits_TGF_beta1_induced_MMP_9_via_Smad_and_MAPK_pathways_in_human_corneal_epithelial_cells_ L2 - https://iovs.arvojournals.org/article.aspx?doi=10.1167/iovs.04-0929 DB - PRIME DP - Unbound Medicine ER -