Caspase-1 activator Ipaf is a p⁵³-inducible gene involved in apoptosis

Abstract

The tumor suppressor protein p⁵³ regulates transcription of many genes that mediate cell cycle arrest, apoptosis, DNA repair and other cellular responses. Here we show that Ipaf, a human CED-4 homologue and an activator of caspase-1, is induced by p⁵³. Overexpression of p⁵³ by transfection in U2OS and A549 cells increased Ipaf mRNA levels. Treatment of p53-positive cell lines U2OS and MCF-7 with the DNA damaging drug, doxorubicin, which increases p⁵³ protein level, induced expression of Ipaf mRNA but similar treatment of MCF-7-mp⁵³ (a clone of MCF-7 cells expressing mutant p⁵³) and p⁵³-negative K562 cells showed much less induction of Ipaf gene expression. Expression analysis for Ipaf mRNA in doxorubicin-treated human tumor cell lines suggests that p⁵³-dependent as well as p⁵³-independent mechanisms are involved in the regulation of Ipaf gene expression in a cell-type-specific manner. The Ipaf promoter was activated by normal p⁵³ but not by His⁵³ mutant of p⁵³. A functional p⁵³-binding site was identified in the Ipaf promoter. A dominant-negative mutant of Ipaf inhibited p⁵³-induced and doxorubicin-induced apoptosis by about 50%. Ipaf-directed small hairpin RNA downregulated p⁵³-induced Ipaf gene expression and also reduced p⁵³-induced apoptosis. Doxorubicin-induced apoptosis was also inhibited by Ipaf-directed small hairpin RNA. Our results show that p⁵³ can directly induce Ipaf gene transcription, which contributes to p⁵³-dependent apoptosis in at least some human cells.