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Shoujun Chen, Mei Sun, David E. Birk; Intracellular Degradation/Retention of Mutant C-terminal Truncated Decorin In Human Congenital Stromal Corneal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3643.
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© ARVO (1962-2015); The Authors (2016-present)
We established a transgenic mouse model that allowed partial definition of mechanisms involved in the pathophysiology of Human Congenital Stromal Corneal Dystrophy (HCSCD) caused by a mutation resulting in a C-terminal truncated decorin. Using this model combined with an in vitro system, we characterized the cellular/molecular mechanisms whereby C-terminal truncated decorin leads to HCSCD.
In vivo, the mouse model was analyzed using immune-chemical and morphological approaches. In vitro, HEK293 cell lines transfected with wild type decorin or 952delT mutant decorin were analyzed by molecular and biochemical approaches.
The expression of 952delT mutant decorin was demonstrated in vivo; however, the extracellular content of mutant decorin was significantly lower than wild type decorin. Histological analysis of mutant corneas showed altered keratocyte morphology, with mutant keratocytes exhibiting enlarged cell bodys and an increased number of vesicles, suggesting that alteration of intracellular processing of mutant decorin may affect keratocyte function. Mutant decorin, which migrated 3-4 KDa faster than wild type decorin in both the cell layer and cell medium, also was demonstrated in transfected HEK293 cell lines. Immuno-blots showed that the intracellular expression of mutant decorin was comparable with that of wild type decorin; this was confirmed by immunofluorescence microscopy. However, mutant decorin expression in the cell medium was significantly lower than the expression of wild type decorin, an observation that was consistent with our in vivo study. In a 952delT mutant decorin transfected cell line, we also observed an anti-decorin reactivity against a 30KDa fragment on immuno-blots, suggesting a degradation of mutant decorin. Using cycloheximide to inhibit newly synthesized decorin, we demonstrated that the intracellular mutant decorin was significantly decreased after 12 hours compared to wild type decorin, indicating increased intracellular degradation. However, the proteosome inihibitor MG132 did not increase mutant decorin expression, which suggests that the increased intracellular degradation may be through an autophagy-lysosome pathway.
The C-terminal "ear-repeat" of decorin is important for maintaining its structure and conformation. Truncation of the C-terminus may decrease decorin stability, increase its degradation/retention and reduce its secretion, all of which affect intracellular homeostasis, and in turn may affect the expression of extracellular matrix components, as we observed in our transgenic mouse model of HCSCD.
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