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Mary Ann Stepp, Sonali Pal-Ghosh, Tomas Blanco, Ahdeah Pajoohesh-Ganji, Gauri Tadvalkar, Arpitha Parthasarathy, James D. Zieske; MMP9 Cleavage Of The β4 Integrin Ectodomain Leads To Recurrent Epithelial Erosions In Mice. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4792.
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Epithelial debridement of the mouse cornea leaves an intact basement membrane and beginning two weeks after debridement wounds close, spontaneous recurrent epithelial erosions develop. These studies were initiated to determine the mechanisms behind these erosions.
Manual debridement or keratectomy wounding was performed on BALB/c and/or C5Bl6 mice. At different times after wounding, corneas were fixed for whole mounts and confocal microscopy, or corneal epithelium was harvested and extracts of corneal epithelial cells used for either QPCR or immunoblots. Mouse epidermal cell cultures and human HCLE (need to write out) were used to obtain extracts for experiments to assess the sensitivity of integrins to constitutive and PMA-induced MMP-mediated cleavage. Extracts from unwounded and wounded corneas were also used to show that MMP9 and β4 integrin proteins associate. Confocal microscopy was performed for detection of the β4 integrin ectodomain and MMP9 at erosions sites.
β4, α3, and β1 integrins are cleaved by several MMPs. Chronic elevation of MMP9 mRNA and protein is induced after debridement wounds. β4 integrin is cleaved over time in epithelial cell extracts derived from debridement wounded corneas and cleavage can be blocked or reduced in unwounded and wounded extracts, respectively, by inhibiting MMPs. Phorbol ester treatment of keratinocytes induces shedding of β4 and α6 but not α3 integrin within 20 minutes consistent with the MMP-induced shedding of cell surface α6β4 integrin. β4 integrin and MMP9 associate in extracts from control and wounded corneal epithelia. Confocal imaging with 3-D reconstruction shows that MMP9 localizes to erosion sites in vivo where the ectodomain of β4 integrin is absent.
This report is the first to show that the β4 integrin ectodomain is a target for cleavage by MMP9 in vivo under pathological conditions.
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