View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library Apoptosis (2007) 12:2051–2061 DOI 10.1007/s10495-007-0115-3 ORIGINAL PAPER The CD44 standard/ezrin complex regulates Fas-mediated apoptosis in Jurkat cells Ainhoa Mielgo Æ Vincent Brondani Æ Lukas Landmann Æ Andrea Glaser-Ruhm Æ Peter Erb Æ Dwayne Stupack Æ Ursula Gu¨nthert Published online: 28 August 2007 Ó Springer Science+Business Media, LLC 2007 Abstract The transmembrane receptor CD44 conveys ezrin-actin is an important modulator of Fas-mediated important signals from the extracellular microenvironment apoptosis. The results highlight a mechanism by which to the cytoplasm, a phenomena known as ‘‘outside-in’’ signals from the extracellular milieu regulate intracellular signaling. CD44 exists as several isoforms that result from signaling activities involved in programmed cell death. alternative splicing, which differ only in the extracellular domain but yet exhibit different activities. CD44 is a Keywords CD44 Á Ezrin Á Apoptosis Á binding partner for the membrane-cytoskeleton cross-linker Actin cytoskeleton Á Interaction protein ezrin. In this study, we demonstrate that only CD44 standard (CD44s) colocalizes and interacts with the actin cross-linkers ezrin and moesin using well-characterized Introduction cell lines engineered to express different CD44 isoforms. Importantly, we also show that the association CD44s- CD44 is a ubiquitously expressed cell adhesion trans- membrane receptor which exists as different isoforms. The large number of isoforms results from alternative splicing Electronic supplementary material The online version of this of the extracellular domain, while all isoforms retain a article (doi: 10.1007/s10495-007-0115-3) contains supplementary common transmembrane and intra-cytoplasmic domain. material, which is available to authorized users. Post-translational modifications (such as glycosylation) of the extracellular domain and alternative splicing are known A. Mielgo Á A. Glaser-Ruhm Á P. Erb Á U. Gu¨nthert (&) Institute of Medical Microbiology, Department of Clinical and to influence CD44 function, but nonetheless do not com- Biological Sciences, University of Basel, Petersplatz 10, 4003 pletely explain the multifunctional activity of CD44. Basel, Switzerland Many studies have reported the ability of the CD44 e-mail: [email protected] cytoplasmic domain as critical in coordinating signaling Present Address: events. Indeed, signaling via CD44 can induce the activa- A. Mielgo tion of focal adhesion kinase and mediate resistance to Moores Cancer Center, University of California San Diego, apoptosis [1]. CD44 may also associate with protein kin- La Jolla, CA 92093, USA ases, enhance T cell receptor signaling and thereby support V. Brondani proliferation, or cell death [2]. CD44 expression and liga- Genome Center and Medical Pharmacology and Toxicology, tion can promote actin cytoskeleton rearrangement during University of California, 4513 GBSF, Davis, CA 95616, USA T cell activation [3]. The carboxy terminal cytoplasmic L. Landmann domain of CD44 supports the binding of proteins with Center of Biomedicine, Department of Clinical and Biological crucial functions in cytoskeletal organization and signaling Sciences, University of Basel, 4003 Basel, Switzerland (reviewed in [4]). These proteins include ankyrin [5], annexin II [6] and members of the ERM (Ezrin, Radixin, D. Stupack Moores Cancer Center, University of California San Diego, Moesin, Merlin) family [7–10]. ERM proteins act as cross- La Jolla, CA 92093, USA linkers between actin cytoskeleton and membrane 123 2052 Apoptosis (2007) 12:2051–2061 receptors [11]. ERM proteins can become activated either observed during apoptosis induction. Here, we extend these by phospholipid binding by their N-terminal domain or by studies, investigating the interaction between ERM proteins phosphorylation of C-terminal threonine residue [9, 10]. and different CD44 isoforms, in Jurkat cells in the context Once in their ‘‘open and active’’ conformation, the ERM C- of cell death. Our results implicate CD44-ERM interaction terminal domain can bind actin cytoskeleton, while the N- as an important regulator of Fas-mediated killing. terminal domain is able to associate with a number of transmembrane receptors, including CD44 [11, 12]. These linker proteins have been shown to localize to membrane Material and methods ruffles, microvilli, filopodia and cleavage furrows [11]. In addition to their structural function, they have been Cell lines and generation of CD44 transfectants implicated in several cellular functions that involve actin cytoskeleton rearrangement, including cell polarization, Jurkat cells (human T cell leukemia line) obtained from the migration, morphology, survival and apoptosis [13–18]. American Type Culture Collection (ATCC) were co- The prototype member of the ERM protein family is ezrin. transfected by electroporation as described in [26] with a Ezrin and CD44 are ubiquitously expressed and associate via neomycin resistance plasmid and CD44s, CD44v or CD44s the binding of ezrin to the cytoplasmic domain of CD44, lacking the cytoplasmic domain (CD44sDcyt) encoding proximal to the transmembrane domain [9]. This association DNA. The constructs contain EGFP (enhanced green flu- may function to regulate its subsequent interactions with orescence protein) fused at the CD44 carboxy terminal end downstream signaling pathways (reviewed by [7]). Phos- (Fig. 1A). phorylation of serine 291 by protein kinase C (PKC), and Expression of CD44 isoforms was confirmed by flow dephosphorylation of serine 325 in the cytoplasmic tail of cytometry with fluorescently labeled antibodies for CD44 result in dissociation of ezrin, and promote cell panCD44 and CD44v isoforms. Cells with similar green migration [18]. In myeloid cells, the binding of phosphory- fluorescence intensity were sorted on a MoFlo high-speed lated ERM proteins to CD44 regulates hyaluronan binding to cell sorter (DakoCytomation, Zug, Switzerland). CD44 [19]. Supporting this idea, reports in other cell types suggest that phosphorylated ERM and CD44 play a critical role in microvilli formation, cell adhesion and cell migration Cytochalasin D treatment [16, 17]. Confocal microscopy studies on endothelial cells show colocalization of CD44, ERM proteins, actin filaments Cells were exposed to 0.5 lg/ml of cytochalasin D (CD, and PKC during wound healing [20]. Recruitment of ezrin Sigma) for 90 min before apoptosis induction for 5 h with and the actin cytoskeleton by CD44 has also been shown to FasL. The short-term treatment used in this study inhibits be required for c-met signaling and activation of Ras [21]. In actin polymerization without general cytotoxicity, as contrast, a recent study reveals that merlin, a member of the assessed by analytical cytology analyses [27]. Apoptosis ERM family suppresses tumor growth by inhibiting CD44- was evaluated by flow cytometry by measuring the sub G1 hyaluronic acid interaction [22]. population. Actin stabilization and destabilization have both been shown to be implicated in regulation of apoptosis [23–25]. Many reports also suggest that ERM proteins regulate the Induction and evaluation of apoptosis initial phase of apoptosis through their effects on the actin cytoskeleton rearrangement. Indeed, it has been demon- FasL and anti-human Fas treatment strated that formation of blebs and disruption of nuclear integrity during apoptosis requires an intact actin cyto- As previously described [26], Neuro-2a cells producing skeleton, and that actin accumulates at the basis of the recombinant mouse FasL were kindly provided by A. apoptotic bodies [25]. All these studies together suggest that Fontana (University Hospital, Zu¨rich, Switzerland). Jurkat membrane receptors, actin cross-linker proteins and cyto- transfectants (5 · 105 cells/ml) were incubated with serial skeleton remodeling are tightly regulated and implicated in dilutions of FasL or with a Fas cross-linking antibody the control of the cell’s fate. Nonetheless, the impact of (100 ng/ml) (clone 7C11, Immunotech, Marseille, France) CD44 on the actin cytoskeleton, via its effector protein for 1, 3 or 6 h at 37°C. ezrin, has not been elucidated in the context of apoptosis. Untreated and Fas triggered cells were collected, and Recently, we showed that CD44 variant (CD44v) isoforms evaluated for apoptosis with different methods. block Fas mediated apoptosis [26] whereas the shorter standard isoform (CD44s) does not. This mechanism 1. AnnexinV/PI. Cells were stained with annexinV explains the different behavior of CD44s and CD44v labeled with allophycocyanine (APC) (Alexis, Basel, 123 Apoptosis (2007) 12:2051–2061 2053 Fig. 1 Expression of CD44 isoforms in Jurkat transfectants and CD44s/ezrin colocalization. (A) Scheme of the different CD44-EGFP constructs used for transfection of Jurkat cells. Jurkat cells transfected with CD44s (B), CD44sDcyt (C) and CD44v2-10 (D). Cells were stained with anti-panCD44 antibody (Hermes-3), followed by Cy2 labeled goat anti-mouse antibody (green fluorescence) and with anti-ezrin antibody followed by TxR labeled goat anti-rabbit antibody. The merge in yellow represents colocalization between CD44s and ezrin. The 2D histograms display the voxel pattern of colocalization between CD44 and ezrin. (E) Histogram representing the percentage of colocalization between CD44 and ezrin (black bars) and ezrin and actin (gray bars). Data are means of three to four data sets and were analyzed