Mechanical force enhances MMP-2 activation via p38 signaling pathway in human retinal pigment epithelial cells.
BACKGROUND
Rhegmatogenous retinal detachment and proliferative vitreoretinopathy (PVR) are eye diseases that are characterized by mechanical stress involving stretching of the retinal pigment epithelial (RPE) cells by the vitreous or the hyperplastic membranes. Here, we assessed whether mechanical force could change the expression of matrix metalloproteinases (MMPs) in RPE cells via the mitogen-activated protein kinase (MAPK) pathway.


METHODS
Collagen-coated magnetite beads and magnetic fields were used to apply tensile forces to cultured RPE cells at focal adhesions. Activation of the MAPK, including extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 were determined over a time course from 5 to 30 min by Western-blot analysis. Activation of p38 was also tested using immunofluorescence staining. The mRNA levels of MMP-2, MMP-9, tissue inhibitor of MMP (TIMP)-2 and fibronectin (FN) were analyzed by RT-PCR. Active MMP-2 and MMP-9 were demonstrated by zymography. MMP-2 secretion was evaluated by enzyme immunoassay.


RESULTS
Stimulation of RPE cells with mechanical stress did not change the total protein expression of the MAPK proteins ERK, JNK, and p38. However, of the three kinases, only active p38 showed an increased protein expression which was also shown by a 2.8-fold increase in immunofluorescence staining at 5 min following mechanical stress stimulation. This increase in active p38 expression was blocked by treating the cells with the p38 inhibitor SB203580. FN mRNA increased 2.4-fold at 15 min and MMP-2 mRNA increased 2.1-fold at 4 h. MMP-2 secretion increased 1.5-fold at 4 h and 1.9-fold at 12 h. The expression of MMP-2 and FN, and the activation and secretion of MMP-2, were inhibited in the presence of SB203580. The mRNA expression of MMP-9 and TIMP-2 did not change throughout.


CONCLUSIONS
This study shows that mechanical stress upregulates MMP-2 and FN expression through activation of the p38 pathway. The increase in MMP-2 levels evoked by mechanical force may contribute to the remodeling of the extracellular matrix around RPE cells, weakening the interlinkage and membrane attachment between RPE cells, and facilitate cellular migration.
Hou
Xu
X
Department of Ophthalmology, Eye Institute of Chinese PLA, Xijing Hospital, The Fourth Military Medical University, Xi'an, China. hxfmmu@gmail.com
Han
Quan-Hong
QH
Hu
Dan
D
Tian
Lei
L
Guo
Chang-Mei
CM
Du
Hong-Jun
HJ
Zhang
Peng
P
Wang
Yu-Sheng
YS
Hui
Yan-Nian
YN
D000328:Adult; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism*; D008855:Microscopy, Electron, Scanning; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000201:enzymology* / Q000648:ultrastructure; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D013314:Stress, Mechanical; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2