p38 MAP kinase pathway and stromelysin regulation in trabecular meshwork cells.
OBJECTIVE
Increased expression of stromelysin-1 (matrix metalloproteinase [MMP]-3) by the trabecular meshwork (TM) initiates extracellular matrix turnover and increases aqueous humor outflow facility. Tumor necrosis factor (TNF)alpha and interleukin (IL)-1alpha are efficacious inducers of MMP-3 in TM. To facilitate understanding of the regulation of MMP-3, the authors investigated the involvement of p38 MAP kinase pathway proteins in this process.


METHODS
Western immunoblots were used to determine the effects of these cytokines and p38 MAP kinase pathway inhibitors on MMP-3 protein levels, p38 MAP kinase isoforms, and phosphorylation levels in human and porcine TM cells. The effects of a dominant-negative p38 MAP kinase construct on MMP-3 expression were evaluated. Morphologic changes in the cells were also examined.


RESULTS
Both cytokines increased MMP-3 levels. The p38 MAP kinase inhibitor SB202190 diminished MMP-3 induction by TNFalpha at all times and at 24 hours by IL-1alpha but potentiated the IL-1alpha-induced increase in MMP-3 at later times. MMP-3 induction by both cytokines was blocked by dominant-negative p38 MAP kinase constructs. Each cytokine increased phosphorylation of the p38 MAP kinase pathway components and altered TM cell morphology. The p38 inhibitor blocked only the morphologic changes produced by TNFalpha. Human and porcine TM cells expressed p38 alpha, beta, delta, and gamma isoforms, which migrate coincident with bands of specific phosphorylation.


CONCLUSIONS
The effects of p38 inhibitors and the dominant-negative construct on TNFalpha and IL-1alpha induction of MMP-3 demonstrate an essential role for p38 in this signaling process. Differences between p38 inhibitor effects on TNFalpha and IL-1alpha induction of MMP-3 suggest divergent p38 isoform use, as do the morphologic responses. The anomalous p38 inhibitor effect on IL-1alpha induction of MMP-3 and phosphorylation of p38 delta/gamma suggests complex interactions between p38 MAP kinase isoforms and their differential uses by TNFalpha and IL-1alpha in TM.
Kelley
Mary J
MJ
Casey Eye Institute, Oregon Health & Science University, Portland, Oregon 97239-4197, USA. kelleyma@ohsu.edu
Rose
Anastasia
A
Song
Kaili
K
Lystrup
Barbara
B
Samples
John W
JW
Acott
Ted S
TS
D051698:Activating Transcription Factor 2 / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D048430:Cell Shape; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D053582:Interleukin-1alpha / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D048371:MAP Kinase Kinase 3 / Q000378:metabolism; D048669:MAP Kinase Kinase 6 / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism*; D008858:Microscopy, Phase-Contrast; D010766:Phosphorylation; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction; D013552:Swine; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000099190:MAP-Kinase-Activated Kinase 2
D051698:Activating Transcription Factor 2; D004791:Enzyme Inhibitors; D007093:Imidazoles; D053582:Interleukin-1alpha; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D048371:MAP Kinase Kinase 3; D048669:MAP Kinase Kinase 6; D019278:Matrix Metalloproteinase 3; D017346:Protein Serine-Threonine Kinases; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2; C497105:ATF2 protein, human; C482530:MAP2K3 protein, human; C482566:MAP2K6 protein, human; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole