Influence of actin cytoskeletal integrity on matrix metalloproteinase-2 activation in cultured human trabecular meshwork cells.
OBJECTIVE
The goal of this study was to investigate the possible link between actin cytoskeletal integrity and the activation of matrix metalloproteinases (MMPs) in trabecular meshwork (TM) cells.


METHODS
Primary human TM (HTM) cells treated with different actin cytoskeleton-interfering agents, including cytochalasin D, latrunculin A, ethacrynic acid (ECA), a Rho kinase inhibitor (Y-27632), and H-7 (serine/threonine kinase inhibitor), were examined for changes in actin cytoskeletal organization by phalloidin staining, MMP-2 activation by gelatin zymography, expression of MT1-MMP by quantitative real-time PCR analysis, levels of tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2), and activation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated protein kinase (ERK) by immunoblotting.


RESULTS
Treatment of HTM cells with cytochalasin D and latrunculin A led to significant activation of MMP-2, p38 MAPK, and ERK1/2, which appeared to correlate with changes in cell morphology and actin depolymerization. Additionally, treatment with these cytoskeleton-disrupting agents elicited increased expression of MT1-MMP in HTM cells, concomitant with a decrease in the levels of secreted TIMP-1 and TIMP-2. In contrast, treatment with ECA, Y-27632, or H-7 triggered changes in cell shape and reduced actin stress fibers in HTM cells but did not exert significant effects on MMP-2 activation or MT1-MMP expression.


CONCLUSIONS
These studies indicate that cytochalasin D- and latrunculin A-induced alteration of actin cytoskeletal integrity in HTM cells is associated with MMP-2 activation, most likely through the upregulation of its activator, MT1-MMP. These data provide a mechanistic connection between actin cytoskeletal organization and MMP-2 activation in TM cells and offer new insights into extracellular matrix remodeling in the aqueous outflow pathway.
Sanka
Krishna
K
Departments of Ophthalmology, Duke University School of Medicine, Durham, North Carolina 27710, USA.
Maddala
Rupalatha
R
Epstein
David L
DL
Rao
P Vasantha
PV
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000199:Actins / Q000378:metabolism*; D000293:Adolescent; D000328:Adult; D000577:Amides / Q000494:pharmacology; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation; D004976:Ethacrynic Acid / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D011725:Pyridines / Q000494:pharmacology; D013552:Swine; D053778:Thiazolidines / Q000494:pharmacology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D014129:Trabecular Meshwork / Q000166:cytology / Q000201:enzymology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000199:Actins; D000577:Amides; D019086:Bridged Bicyclo Compounds, Heterocyclic; D011725:Pyridines; D053778:Thiazolidines; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; C108830:Y 27632; D015638:Cytochalasin D; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020778:Matrix Metalloproteinase 2; D004976:Ethacrynic Acid; C037067:latrunculin A