Changes in Human Trabecular Meshwork Cells Induced by Overexpression of SPARC

Abstract

Abstract: : Purpose:Overexpression of proteins by recombinant adenovirus transduction is a method currently used in our laboratory. We wished to study the effect of this overexpression on the proteasome degradation system of primary cultures of human trabecular meshwork cells (HTM). Methods:We used the pd1EGFP-N1 vector from Clontech to expresses a short-live form of GFP which in normal conditions is rapidly turned over by the proteasome system in mammalian cells. HTM primary cultures were transfected with pd1EGFP-N1 vector and infected with recombinant adenovirus expressing secreted proteins at different multiplicity of infection (m.o.i). Accumulation of GFP protein resulting from a failure in the proteasome system was analyzed by fluorescence microscopy. Results:HTM cells infected with recombinant adenovirus expressing secreted proteins show higher fluorescence when compare to non-infected controls. The fluorescence seems to be proportional to the m.o.i. Conclusion:The method allows us to detect saturation of proteasome function by analyzing the accumulation of a short-lived form of GFP protein in HTM cells. Overexpression of large amounts of secreted protein seems to saturate the degradation capacity of the proteasome, resulting in intracellular accumulation of proteins. When studying the effect of overexpression of proteins by an adenovirus system in HTM cells it may be important to determine the range of protein production so that the desired effect can be studied without potential interference caused by saturation of the proteasome degradation system of the cell.

Keywords: 307 adenovirus • 601 trabecular meshwork • 592 stress response