Alternative Splicing of Caspase-8 mRNA during Differentiation of Human Leukocytes Leopold Eckhart,* ,1 Michael Henry,* ,1 Antonio M. Santos-Beneit,† Ingo Schmitz,‡ Andreas Krueger,‡ Heinz Fischer,* Ju ¨ rgen Bach,* Jozef Ban,* Sabine Kirchhoff,‡ Peter H. Krammer,‡ Faustino Mollinedo,† and Erwin Tschachler* , § ,2 *Department of Dermatology, University of Vienna Medical School, A-1090 Vienna, Austria; ‡Tumorimmunology Program, German Cancer Research Center, D-69120 Heidelberg, Germany; †Centro de Investigacio ´n del Ca ´ ncer, CSIC-Universidad de Salamanca, E-37007 Salamanca, Spain; and §Centre de Recherches et d'Investigations E ´ pidermiques et Sensorielles (CERIES), 92521 Neuilly, France Received November 2, 2001 Caspase-8 is a key initiator of death receptor- induced apoptosis. Here we provide evidence that caspase-8 expression is subject to posttranscriptional regulation in human leukocytes. Resting peripheral blood lymphocytes preferentially use a distant splice donor site at the 3-end of caspase-8 exon 8 to generate mRNAs with a truncated open reading frame. When lymphocytes were activated, the expression of caspase-8 variants was shifted to caspase-8/a and b which lack the extension of exon 8. The opposite change of the splicing pattern was found in a neutro- phil differentiation model. Promyelocytic HL-60 cells mainly expressed caspase-8 mRNAs with the normal exon 8, but the splicing pattern was changed to the distant exon 8 splice site during DMSO-induced differ- entiation of HL-60 cells. In spite of the presence of these novel mRNAs, the corresponding translation products were not detectable in either cell type. Our findings suggest that leukocyte differentiation and al- ternative splicing of caspase-8 pre-mRNA are inter- dependent processes. © 2001 Elsevier Science Key Words: caspase-8; alternative splicing; leuko- cytes; neutrophils; HL-60; apoptosis; differentiation. Caspase-8 (FLICE, MACH, Mch5) (1– 4) is the most upstream caspase of the CD95 (APO-1/Fas)-induced apoptosis pathway (reviewed by Krammer (5)). In ad- dition caspase-8 was implicated in the activation of the c-Jun N-terminal kinase/stress-activated protein ki- nase (JNK) and of the NF-B signaling pathway (6, 7). Recent publications also suggest a role of caspase-8 in promotion of T cell proliferation (8, 9). Gene knock-out studies revealed that caspase-8 is essential for heart muscle development and hematopoiesis (10). Like most other caspase mRNAs, the mRNA of hu- man caspase-8 exists in various isoforms that are gen- erated by alternative pre-mRNA splicing (casp-8/a through casp-8/h) (2, 3). Since specific protein functions such as binding to adapter molecules and proteolytic activity can be ascribed to sequence elements derived from certain exons, different physiological functions have been predicted for variants with different exon composition (4, 11). The translation products of two pro-apoptotic isoforms, caspase-8/a and caspase-8/b were shown to be part of the death inducing signaling complex (DISC) whereas other protein variants were not detectable (12). Recently, we and others (our own unpublished data, GenBank Accession No. AF207672; 13) detected a novel facultative splice donor site at the 3'-end of exon 8 of the human caspase-8 gene. Since exon 8b (our name for the resulting extension of the previously described exon 8) contains an in frame stop codon, utilization of this splice site redirects expression of the caspase-8 gene from functional isoforms to trun- cated proteins. Horiuchi and colleagues showed that a splice variant named caspase-8L which contains exon 8b is the predominant caspase-8 mRNA form in human peripheral blood lymphocytes (13). Their finding that lymphocytes of systemic lupus erythematosus (SLE) patients express less caspase-8L indicated that splic- ing of exon 8b might play a role in this and possibly other disorders of the immune system. Here we report that expression of caspase-8 splice variants utilizing the novel exon 8 splice donor site is subject to differ- ential regulation in leukocytes. 1 These authors contributed equally to this paper. 2 To whom correspondence should be addressed at Department of Dermatology, University of Vienna Medical School, Wa ¨ hringer Gu ¨ r- tel 18-20, A-1090 Vienna, Austria. Fax: (43)-1-403 4922. E-mail: Erwin.Tschachler@akh-wien.ac.at. Biochemical and Biophysical Research Communications 289, 777–781 (2001) doi:10.1006/bbrc.2001.6055, available online at http://www.idealibrary.com on 777 0006-291X/01 $35.00 © 2001 Elsevier Science All rights reserved.