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S. He, G. Prasanna, T. Yorio; Endothelin–1–Mediated Signaling in the Expression of Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Metalloproteinases (TIMPs) in Astrocytes . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2607.
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© ARVO (1962-2015); The Authors (2016-present)
Remodeling of the extracellular matrix (ECM) of the optic nerve head and cupping of the optic disc are characteristics of glaucoma. Endothelin–1 levels are increased in aqueous and vitreous humor in glaucoma patients and animal models of glaucoma. Whether the elevated ET–1 induces ECM remodeling resulting in pathological changes in the optic nerve head is still unknown. In the present study, the regulation of MMPs and TIMPs and ECM remodeling in ET–1–activated astrocytes were determined.
Human optic nerve head astrocytes (hONAs) were exposed to ET–1 for 1 day and 4 days. Culture media and cell lysates were subjected to perform zymography and Western blot. A zymography assay was used for quantifying the activity of MMP–2, 3 and 9. Western Blot was employed to determine the expression of MMP–2, 3, 9 and TIMP–1, 2. siRNA was employed to knock down the expression of MMP–2, 3, 9 or TIMP–1, 2. Fibronectin and collagen IV were monitored by ELISA and immunofluorescent staining.
ET–1 slightly increased the activity of MMP2, which was also increased in presence of U0126 (a MEK inhibitor) and chelerythrine (a PKC inhibitor) in hONAs. The activity of MMP3 was detectable using casein zymography, but not Western Blot. In addition, blockade of ERK–MAPK by U0126 and PKC by chelerythrine increased the activity of MMP3. Whereas, the significantly increased expression of TIMP–1 and 2 induced by ET–1 was abolished by U0126 and chelerythrine in hONAs. Furthermore, there were no apparent differences in the expression profile of MMPs and TIMPs in hONA cells from normal human and POAG patients. Knock–down of MMP2 and 3 using siRNA not only decreased the activity of MMP2 and expression of TIMP–1 and 2 but also increased fibronectin concentration in cell supernatant. Fibronectin deposition was also significantly increased and formed ECM network in hONA at day 1 after cells were exposed to ET–1, but only slightly increased at day 4. Blockade of MAPK using U0126 did not alter the expression and deposition pattern of fibronectin in hONAs.
ET–1 increased the expression and activity of MMP2 and TIMP–1, 2. ERK–MAPK and PKC pathways are involved in the regulation of MMP2 and TIMP–1, 2. A balance of MMPs/TIMPs may be important but only to regulate the expression of MMPs and TIMPs but also to regulate ECM remodeling. Current data show that ECM remodeling is controlled in a temporal fashion. In future studies, the concise temporal and spatial regulation of MMPs and TIMPs induced by ET–1 will be investigated in both cell cultures and animal models.
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