To more fully understand the role of ENSA expression in MCs with regard to ECM formation, a stably overexpressing a-endosulfine cell line was produced by retroviral transduction. The resulting cell line reliably upregulated ENSA by eightfold. In cells, exposed to a high-glucose environment (25 mM) for 2 wk, there was increased accumulation of collagen type I in the conditioned medium, compared with control cells transduced by a nonvirus-containing empty vector. A similar finding was noted for type-4 collagen. Contrastingly, ENSA downregulation by RNA interference during transient transfection experiments with three different siRNAs led to decrements of types I and IV collagen accumulation in the conditioned media of cells incubated for 2 wk in 5 mM glucose. We conclude that, although modifications of ECM gene expression via ENSA may not be translated directly to changes in ECM protein expression, these results imply that a-endosulfine enhances ECM accumulation, an effect that is particularly invoked by a high-glucose environment. In addition, in other preliminary data, these effects occur downstream of the perinuclear and nuclear translocation of a-endo-sulfine in MCs following their cAMP-mediated PKA phosphorylation.
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