CD44v6 promotes proliferation by persisting activation of MAP kinases

Abstract

CD44v6 is transiently expressed during T cell activation, and constitutively CD44v4-v7 expressing transgenic T cells show accelerated responses towards nominal antigens. The underlying mechanism is unknown.

The mouse thymoma EL4 was transfected with CD44 standard isoform (CD44s) or CD44v6 cDNA (EL4-s, EL4-v6). Only EL4-v6 cells proliferated at an over 10-fold higher rate than untransfected cells, displayed up-regulated expression of CD69, CD25, and IL-2, and were protected from apoptosis by CD44v6 cross-linking. In the absence of any stimulus, ERK1/2 was partly phosphorylated, and phosphorylation was significantly increased by CD44v6 cross-linking. The same accounted for JNK, c-jun, and IκBα. Moreover, NF-κB was partly translocated into the nucleus. Instead, CD44s cross-linking induced ERK1/2, JNK, c-jun, and IκBα phosphorylation only in the context of TCR engagement. No selectively CD44v6 associated transmembrane proteins were uncovered in EL4 cells. However, CD44v6, as opposed to CD44s, did not colocalise with the TCR/CD3 complex after CD3 cross-linking. Furthermore, a CD44-associated 85-kDa protein became hypophosphorylated only after CD44v6 cross-linking. Threonine hypophosphorylation of this protein coincided with the activation of MAP and SAP kinases, which was prohibited in the presence of a phosphatase inhibitor.

Thus, CD44v6, distinct to CD44s, stimulates autonomously growth and IL-2 secretion of a thymoma line and rescues cells from apoptosis.

Introduction

CD44 glycoproteins, encoded by a single gene, vary by glycosylation and the insertion of variant exon products (CD44v) in the extracellular domain [1]. CD44 molecules display a multitude of functions involving a large array of signal transduction pathways (rev. in Refs. [2], [3], [4], [5]). It is supposed that the manifold functions rely on the existence of CD44v isoforms [6], [7], [8] and concerted activities with associating molecules.

CD44-mediated signal transduction can be initiated by CD44–ligand binding, hyaluronic acid being one of the prominent ligands [9] or by associating transmembrane kinases or phosphatases that become activated via the association with CD44, e.g., ERBB receptor tyrosine kinases [10], [11], [12]. The interactions can be CD44 isoform-specific as shown for scatter factor binding which essentially requires CD44v6 expression.

The cytoplasmic tail, too, is most important for CD44-mediated signal transduction. First, transmembrane molecules can associate with the CD44 cytoplasmic tail, like TGFβ-RI, which associates via a disulfide bridge that leads to activation and nuclear localisation of Smad2 and Smad3 [13]. Second, the CD44 cytoplasmic tail is constitutively associated with lck, lyn, and fyn [14], [15], [16]. In T cells, CD44-associated lck supports phosphorylation of ZAP-70 and downstream signal transduction by apposition of lck in the proximity of the TCR [16]. CD44-associated PTKs are engaged also in the phosphorylation and activation of Vav1, an important upstream regulator of Rac1 [17], [18] which colocalizes with CD44 [19]. These complexes supposedly account for CD44 triggered F-actin polymerization and cell spreading [19], [20], [21], [22], [23]. Third, CD44 can interact with the actin cytoskeleton via binding of ankyrin [24] or ERM (ezrin/radixin/moesin) proteins [25], [26]. Binding of ERM proteins depends on the activity state of CD44, which is triggered by PKC [27]. Rho-kinase may also be involved in CD44-mediated signal transduction [28], [29], [30].

In view of the multiple signaling pathways involving CD44, it may not be surprising that the outcome of CD44–ligand binding revealed partly opposing results (rev. in Refs. [5], [8], [31]) as exemplified for the involvement of CD44 in apoptosis. In CD44 knockout mice, apoptosis is impaired independent of Fas and FasL expression [32], [33]. A CD44 blockade can also be accompanied by a striking decrease in apoptosis due to bcl-2 up-regulation [34], [35]. In T lymphoma and thymocytes CD44 ligation induces apoptosis by up-regulation of Fas and FasL [16] or Bax and down-regulation of Bcl-Xl [36]. On the other hand, CD44 can enhance survival by suppressing apoptosis induction [37] via Fas down-regulation [38] or up-regulation of Akt and BAD phosphorylation and/or activation and up-regulation of anti-apoptotic genes as Bcl-Xl and Bcl-2 [22], [39], [40]. The latter requires CD44v6 expression and involves the PI3K/Akt pathway [41]. We showed that CD44v7 knockout mice are resistant towards experimental colitis induction due to CD44v7-supported survival of activated T cells by BAD phosphorylation and Bcl-2 and Bcl-Xl up-regulation [42], [43]. Also, the anti-apoptotic effect of osteopontin, accompanied by up-regulation of Bcl-Xl [44], likely depends on CD44v expression [45]. Thus, opposing results with respect to CD44 preventing vs. supporting apoptosis may at least partly depend on the involved CD44v isoforms.

Considering in particular CD44v6, we described that anti-CD44v6 inhibits T cell proliferation [46], [47]. Induction of immune responses in transgenic mice overexpressing ratCD44v4-v7 on T lymphocytes is accelerated [48]. CD44v6 knockout mice have a disadvantage in T cell recovery after myeloablation and bone marrow cell reconstitution [49]. As we also noted a CD44v7-selective activation of anti-apoptotic proteins [43], it became important to define strictly CD44v6-mediated functions. Here, we used EL4 T lymphoma cells transfected with CD44 standard isoform (CD44s) or CD44v6 cDNA. CD44v6 overexpression provides a very strong proliferative stimulus, which is TCR/CD3 complex independent and is accompanied by IL-2 and CD25 up-regulation and persisting activation of MAP and SAP kinases.