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K.P. Langton, N. McKie, M.D. Barker; Characterization of Sorsby’s Fundus Dystrophy-TIMP-3 Proteins in Human RPE Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):5104.
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Purpose: Sorsby’s fundus dystrophy (SFD) is a fully penetrant, autosomal dominant macular degenerative disease caused by mutations in the tissue inhibitor of metalloproteinases-3 (TIMP-3) gene. While SFD is rare it closely resembles age-related macular degeneration (AMD) the most common form of blindness among the elderly in the developed world. Previously we have shown that SFD mutations promote TIMP-3 multimerization but do not affect its matrix metalloproteinase (MMP) inhibitory activity or extracellular matrix (ECM) binding. However, recently it has been suggested that SFD mutations do not cause TIMP-3 aggregation, but rather abolish its inhibitory action and actively promote proMMP-2 secretion/activation in human retinal pigment epithelial (RPE) cells. The aim of this study then, was to clarify the properties of SFD-TIMP-3 protein in human RPE cells. Methods: The human RPE cell-line, ARPE-19, was transfected with genes for TIMP-3, AlaTIMP-3 (which cannot inhibit MMPs) and the SFD-TIMP-3 mutants S181C, S156C and E139X. Matrix and conditioned media were analyzed for TIMP-3 content by gelatin reverse zymography and western blotting. Conditioned media was analyzed for pro-form and active MMP-2 levels by gelatin zymography. Results: ARPE-19 cells express TIMP-3 mostly as a 24 kDa non-glycosylated ECM bound protein. Transfection with TIMP-3 or AlaTIMP-3 genes augmented ECM bound TIMP-3 levels and lead to secretion of soluble TIMP-3. SFD-TIMP-3 mutants were not secreted into the culture media but were found in the ECM as a mixture of protein species, ranging from monomers through to large aggregates. These aggregates were removed by reduction. SFD-TIMP-3 and wild-type TIMP-3 protein were active MMP inhibitors, but AlaTIMP-3 was not. None of the proteins increased the levels of proMMP-2 secretion or activation from ARPE-19 cells. TIMP-3 and TIMP-3-S181C inhibited concanavalin A stimulated proMMP-2 activation but AlaTIMP-3, TIMP-3-S156C and -E139X did not. Conclusions: We have defined the common properties of a range of SFD proteins in RPE cells and shown that all are functional MMP inhibitors that are prone to aberrant intermolecular interactions. However, they do not promote MMP secretion or activation and not all are impaired with regard to their ability to prevent MMP activation. Thus dysregulation of MMP activity is not likely to be a common factor in SFD.
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