Expression of Mutant and Wild Type TIMP3 in Primary Gingival Fibroblasts from Sorsby’s Fundus Dystrophy Patients

Abstract

Abstract: : Purpose:To establish primary gingival fibroblast cell lines from Sorsby’s fundus dystrophy (SFD) patients carrying the S181C TIMP3 and the E139X TIMP3 mutations in order to study expression of wild type and mutant tissue inhibitor of metalloproteinase 3 (TIMP3), and matrix metalloproteinases –2 and –9 (MMP2 and MMP9) from a normal diploid cell type. Methods: Cell lines were established by gingival explant culture. Expression of wild type and mutant TIMP3 mRNA was studied using restriction fragment length polymorphism (RFLP) analysis. Expression of TIMP3 protein was studied by immunoblotting. MMP2 inhibitory activity was studied using reverse zymography. To facilitate studies of the E139X TIMP3 protein the allele was expressed using HighFive insect cells. Results: Patient cells were found to co-express wild type and mutant TIMP3 mRNA and protein. S181C TIMP3 from these cells was found to dimerize and retain MMP2 inhibitory activity. In HighFive insect cells E139X TIMP3 was synthesized as a mixture of monomer and dimer. Both monomeric and dimeric E139X TIMP3 protein retained MMP2 inhibitory activity in reverse zymography. Expression of mutant E139X or S181C TIMP3 protein had no effect on either MMP2 or MMP9 expression or activation when transcribed from their normal promoter context. Conclusions: This study confirms that mutant TIMP3 genes are transcribed and translated to protein in fibroblast cells derived from patients with SFD. This supports the general hypothesis that mutant TIMP3 proteins cause SFD by a gain of function mechanism, possibly related to oligomerization of the mutant TIMP3 protein. Furthermore, due to the retention of MMP2 inhibitory activity by the mutated TIMP3 proteins this pathological gain of function is likely to be unrelated to MMP inhibition. Our results with primary fibroblast cell lines derived from two different SFD pedigrees do not support the view that increased synthesis and activation of MMP2 and/or MMP9 is a general facet of the possession of an SFD TIMP3 allele.

Keywords: retinal degenerations: hereditary • retina • extracellular matrix