title: Attenuation of the ELAV1-like protein HuR sensitizes adenocarcinoma cells to the intrinsic apoptotic pathway by increasing the translation of caspase-2L.
abstract: 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.
authors.affiliation: pharmazentrum frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Winkler
authors.affiliation: pharmazentrum frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Doller
authors.affiliation: Institut für Biochemie II, Universitätsklinikum Frankfurt, Frankfurt am Main, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Imre
authors.affiliation: pharmazentrum frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Badawi
authors.affiliation: Institut für Biochemie I, Universitätsklinikum Frankfurt, Frankfurt am Main, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Schmid
authors.affiliation: pharmazentrum frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Schulz
authors.affiliation: pharmazentrum frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Steinmeyer
authors.affiliation: pharmazentrum frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Pfeilschifter
authors.affiliation: Institut für Biochemie II, Universitätsklinikum Frankfurt, Frankfurt am Main, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Rajalingam
authors.affiliation: pharmazentrum frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Eberhardt
mesh_terms: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000230:Adenocarcinoma / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D017209:Apoptosis; D053143:Caspase 2 / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D051959:ELAV Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D015854:Up-Regulation
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D051959:ELAV Proteins; C490360:CASP2 protein, human; D053143:Caspase 2; D003546:Cysteine Endopeptidases