Attenuation of the ELAV1-like protein HuR sensitizes adenocarcinoma cells to the intrinsic apoptotic pathway by increasing the translation of caspase-2L.

Caspase-2 represents the most conserved member of the caspase family， which exhibits features of both initiator and effector caspases. Using ribonucleoprotein (RNP)-immunoprecipitation assay， we identified the proapoptotic caspase-2L encoding mRNA as a novel target of the ubiquitous RNA-binding protein HuR in DLD-1 colon carcinoma cells. Unexpectedly， crosslinking-RNP and RNA probe pull-down experiments revealed that HuR binds exclusively to the caspase-2-5' untranslated region (UTR) despite that the 3' UTR of the mRNA bears several adenylate- and uridylate-rich elements representing the prototypical HuR binding sites. By using RNAi-mediated loss-of-function approach， we observed that HuR regulates the mRNA and in turn the protein levels of caspase-2 in a negative manner. Silencing of HuR did not affect the stability of caspase-2 mRNA but resulted in an increased redistribution of caspase-2 transcripts from RNP particles to translational active polysomes implicating that HuR exerts a direct repressive effect on caspase-2 translation. Consistently， in vitro translation of a luciferase reporter gene under the control of an upstream caspase-2-5'UTR was strongly impaired after the addition of recombinant HuR， whereas translation of caspase-2 coding region without the 5'UTR is not affected by HuR confirming the functional role of the caspase-2-5'UTR. Functionally， an elevation in caspase-2 level by HuR knockdown correlated with an increased sensitivity of cells to apoptosis induced by staurosporine- and pore-forming toxins as implicated by their significant accumulation in the sub G1 phase and an increase in caspase-2， -3 and poly ADP-ribose polymerase cleavage， respectively. Importantly， HuR knockdown cells remained insensitive toward STS-induced apoptosis if cells were additionally transfected with caspase-2-specific siRNAs. Collectively， our findings support the hypothesis that HuR by acting as an endogenous inhibitor of caspase-2-driven apoptosis may essentially contribute to the antiapoptotic program of adenocarcinoma cells by HuR.