The prosaposin gene downregulates oxidative stress–induced apoptosis in human retinal pigment epithelial cells: Hammerhead ribozymes and proximal promoter studies

Abstract

Abstract: : Purpose: The prosaposin (PSAP) gene encodes a multifunctional glycoprotein that includes neuroprotective saposin C. In our search for endogenous neuroprotective signaling in the RPE we used ARPE–19 cells and hammerhead ribozyme–mediated cleavage of PSAP mRNA for the functional knockout of PSAP. To test the significance of saposin C, we transfected ARPE–19 cells with expression vectors of PSAP hammerhead ribozyme constructs and then assessed oxidative stress–induced apoptosis. We also studied LAU 0901, a neuroprotective platelet–activating factor antagonist, in the induction of promoter constructs of PSAP. Methods: ARPE–19 cells were transfected with PSAP promoter–luciferase constructs, its deletion mutants, or hammerhead ribozymes. Four hours later cells were fed normal medium and incubated 12 hours. Then cells were serum–starved before the addition of inducers. Oxidative stress was induced by TNFα /H₂O₂ for 8 hours. Results: LAU 0901, but not PAF, activated PSAP transcription in RPE cells. This up–regulation of the PSAP promoter was not detected when deletion mutants lacking the first 1100 bp of the promoter sequences were used. In fact, the 813–bp and 1100–bp proximal PSAP promoter construct was the most responsive to LAU–0901. Expression of RZ–D (which degrades intracellular PSAP mRNA) substantially protected against oxidative stress–induced apoptosis. Conclusions: The deletion–mutant analysis of PSAP promoters indicated that the first 1100 bp of PSAP promoter contains LAU–0901–response elements. Up–regulation of PSAP promoter by LAU 0901 suggests that this experimental drug's neuroprotective properties may be mediated at least in part by this effect. Moreover, the use of PSAP hammerhead ribozymes that targeted the saposin C region of the PSAP mRNA counteracted apoptosis induced by oxidative stress further suggests that PSAP and more specifically saposin C may be an endogenous mediator of RPE survival. (Supported by R01EY05121 and P20RR016816–COBRE; LAU–0901 patent assignee LSUHSC.)

Keywords: cell death/apoptosis • gene/expression • signal transduction