Stimulation of matrix metalloproteinases by hyperosmolarity via a JNK pathway in human corneal epithelial cells.
OBJECTIVE
To investigate whether exposure of human corneal epithelial cells to hyperosmotic stress activates the c-Jun NH(2)-terminal kinase (JNK) stress-activated protein kinase (SAPK) pathway, and stimulates production of the matrix metalloproteinases (MMPs): gelatinase (MMP-9), collagenases (MMP-1 and -13), and stromelysin (MMP-3).


METHODS
Primary human corneal epithelial cells cultured in normal osmolar medium (312 mOsM) were exposed to media with higher osmolarity (350-500 mOsM) achieved by adding NaCl, with or without SB202190, an inhibitor of the JNK pathway; dexamethasone; or doxycycline for different lengths of time. The conditioned media were collected after 24 hours of exposure for zymography and ELISA. Total RNA was extracted from cultures treated for 6 hours and subjected to semiquantitative RT-PCR. Cells treated for 5 to 60 minutes were lysed in RIPA buffer and subjected to Western blot with phospho (p)-specific antibodies against p-JNK and p-c-Jun. JNK1 activation was also detected with an immunoassay system.


RESULTS
The concentrations of MMP-9, -1, and -3 proteins in 24-hour conditioned media of corneal epithelial cells progressively increased as the media's osmolarity was increased from 312 to 500 mOsM by the addition of NaCl. The concentration of MMP-13 progressively increased to a peak at 450 mOsM. Active p-JNK-1, p-JNK-2, and p-c-Jun were detected by Western blot as early as 5 minutes and peaked at 60 minutes in cells exposed to hyperosmolar media. The levels of p-JNK-1, p-JNK-2, and p-c-Jun correlated positively with the osmolarity of the culture media. The p-JNK inhibitor SB202190 and doxycycline markedly inhibited the stimulation of p-JNK-1, p-JNK-2, and p-c-Jun, as well as MMP-9, -1, -13, and -3 at both the mRNA and protein levels in the cells exposed to hyperosmolar media.


CONCLUSIONS
Expression and production of MMP-9, -1, -13, and -3 by human corneal epithelial cells correlated positively with increasing media osmolarity. This increase was mediated at least in part through activation of the JNK SAPK pathway. Doxycycline, an agent used to treat MMP-mediated ocular surface disease, inhibited the hyperosmolarity-induced MMP production and JNK activation. The relevance of these findings to stimulated production of MMPs by the elevated tear osmolarity in dry eye remains to be determined.
Li
De-Quan
DQ
Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, 6565 Fannin Street, Houston, TX 77030, USA.
Chen
Zhuo
Z
Song
Xiu Jun
XJ
Luo
Lihui
L
Pflugfelder
Stephen C
SC
D000293:Adolescent; D000328:Adult; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002478:Cells, Cultured; D004318:Doxycycline / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000096:biosynthesis / Q000378:metabolism*; D008875:Middle Aged; D009994:Osmolar Concentration; D011725:Pyridines / Q000494:pharmacology
D000893:Anti-Inflammatory Agents; D004791:Enzyme Inhibitors; D007093:Imidazoles; D061965:Matrix Metalloproteinase Inhibitors; D011725:Pyridines; D048031:JNK Mitogen-Activated Protein Kinases; D020782:Matrix Metalloproteinases; D004318:Doxycycline; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole