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M. Aga, K. Keller, M. J. Kelley, T. S. Acott; Altered MMP-2 and MMP-14 Localization in Response to Stretch and MEK Inhibitor U0126. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2072.
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Interactions between trabecular meshwork (TM) cells and their extracellular microenvironment in response to mechanical stretch regulate a wide variety of cellular responses including cytoskeletal reorganization, cell-matrix interactions and matrix turnover. Mitogen activated protein (MAP) kinases often modulate extracellular matrix synthesis and matrix metalloproteinases (MMPs). To better understand the regulatory role of MAP kinases in the TM, we evaluated their effects on the expression and localization of MMP-2, MMP-14, and early growth response (EGR-1) in response to mechanical stretch.
Porcine TM cells were subjected to mechanical stretch for either 0-6 hrs or 24 hrs. Cells were pretreated with the MEK inhibitor (U0126) 60 min prior to initiating stretch. Western immunoblot analysis was used to determine MMP-2, MMP-14, and EGR-1 levels. Immunofluorescence of cultured cells and paraffin sections of perfused human anterior segments was used to evaluate changes caused by the presence or absence of U0126.
MMP-2 and MMP-14 increased with mechanical stretch for 24 hrs, and, surprisingly, this was further augmented by addition of U0126. In perfused anterior segments, elevated IOP enhanced MMP-2 and MMP-14 staining, particularly, along the edges of the trabecular beams. Treatment with U0126 resulted in a marked reduction in this staining pattern, replacing it with a more punctuate staining pattern. In cell culture, stretching produced an increase in co-localization of MMP-2 and MMP-14 along the periphery of the cells, at intercellular interdigitations, and in the perinuclear area. U0126 alone also potentiated co-localization of MMP-2 and MMP-14. Additionally, MMP-2 and TIMP-2 co-localization was increased by stretch as well as by U0126. EGR-1 levels following stretch for 5 minutes were higher than control and were sustained for 1hr. However, pretreatment with U0126 completely eliminated this effect.
Stretching TM cells for 24 hrs led to enhanced cellular levels and co-localization of MMP-2, MMP-14, and TIMP-2. The MEK inhibitor, U0126, further augmented MMP-2 and MMP-14 levels and their co-localization. MMP-2 and MMP-14 both have putative binding sites for EGR-1 in their promoters, and mechanical stretching increased EGR-1 levels. This increase was completely abrogated by U0126. This study suggests EGR-1 regulates critical stretch-induced MMPs in the TM via the MEK/ERK pathway.
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