Regulated expression of collagenases MMP-1, -8, and -13 and stromelysins MMP-3, -10, and -11 by human corneal epithelial cells.
OBJECTIVE
This study investigated the regulated expression of collagenases (MMP-1, -8, and -13) and stromelysins (MMP-3, -10, and -11) by human corneal epithelial cells treated with IL-1 beta, TNF-alpha, and doxycycline, a medication used to treat ocular surface diseases.


METHODS
Primary human corneal epithelial cell cultures were treated with IL-1 beta or TNF-alpha, with or without their corresponding inhibitors. Total RNA extracted from cells treated for 4 to 24 hours was subjected to semiquantitative RT-PCR and Northern hybridization. Conditioned media from 24-hour-treated cultures were evaluated for MMP production by ELISA and activity assays.


RESULTS
Semiquantitative RT-PCR and Northern hybridization revealed that the mRNAs of MMP-1, -13, -3, -10, and -11 were dose dependently upregulated by IL-1 beta and TNF-alpha, whereas MMP-8 and -14 and tissue inhibitor of metalloproteinase (TIMP)-1 were not altered, in corneal epithelial cells. MMP ELISA and activity assays confirmed this dose-dependent increase in MMP-1, -13, -3, and -10 protein production in conditioned media by IL-1 beta and TNF-alpha. This stimulated production was inhibited by their neutralizing antibodies and by IL-1 receptor antagonist. Doxycycline suppressed stimulated MMP-1, -10, and -13 production at both the mRNA and protein levels.


CONCLUSIONS
This study demonstrated that IL-1 beta and TNF-alpha upregulate collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, and -11) in human corneal epithelial cells. Doxycycline suppresses stimulated MMP-1, -13, and -10 at the mRNA and protein levels, which suggests that collagenases and stromelysins may play a role in the pathogenesis of sterile corneal ulceration and other ocular surface diseases.
Li
De Quan
DQ
Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas 77030, USA. dequanl@bcm.tmc.edu
Shang
Tie Yan
TY
Kim
Hyun-Seung
HS
Solomon
Abraham
A
Lokeshwar
Balakrishna L
BL
Pflugfelder
Stephen C
SC
D015152:Blotting, Northern; D002478:Cells, Cultured; D017364:Collagenases / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000187:drug effects / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism; D053506:Matrix Metalloproteinase 10; D053507:Matrix Metalloproteinase 11; D053509:Matrix Metalloproteinase 13; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism; D020784:Matrix Metalloproteinase 8 / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation
D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D017364:Collagenases; C507079:MMP13 protein, human; D053507:Matrix Metalloproteinase 11; D053509:Matrix Metalloproteinase 13; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D053506:Matrix Metalloproteinase 10; D020784:Matrix Metalloproteinase 8; D020781:Matrix Metalloproteinase 1; D004318:Doxycycline