Epidermal-specific deletion of CD44 reveals a function in keratinocytes in response to mechanical stress.

CD44, a large family of transmembrane glycoproteins, plays decisive roles in physiological and pathological conditions. CD44 isoforms are involved in several signaling pathways essential for life such as growth factor-induced signaling by EGF, HGF or VEGF. CD44 is also the main hyaluronan (HA) receptor and as such is involved in HA-dependent processes. To allow a genetic dissection of CD44 functions in homeostasis and disease, we generated a Cd44 floxed allele allowing tissue- and time-specific inactivation of all CD44 isoforms in vivo. As a proof of principle, we inactivated Cd44 in the skin epidermis using the K14Cre allele. Although the skin of such Cd44Δker mutants appeared morphologically normal, epidermal stiffness was reduced, wound healing delayed and TPA induced epidermal thickening decreased. These phenotypes might be caused by cell autonomous defects in differentiation and HA production as well as impaired adhesion and migration on HA by Cd44Δker keratinocytes. These findings support the usefulness of the conditional Cd44 allele in unraveling essential physiological and pathological functions of CD44 isoforms.

Perspectives of CD44 targeting therapies.

CD44 is a family of single-span transmembrane glycoproteins. Members of this family differ in the extracellular domain where ten variant exons are either excluded or included in various combinations. CD44 isoforms participate in many physiological processes including hematopoiesis, regeneration, lymphocyte homing and inflammation. Most importantly, they are involved in pathological processes and in particular in cancer. In several types of tumors, CD44 together with other antigens specifies for cancer stem cell populations. Mechanistically, CD44 proteins act as receptors for hyaluronan, co-receptor for receptor tyrosine kinases (RTKs) or G-protein-coupled receptors or provide a platform for metalloproteinases. For all these reasons, targeting CD44 may be a successful approach in cancer therapy. In this review, we discuss the various possibilities of targeting CD44. Among these are the production of CD44 ectodomains, antibodies, peptides or aptamers. Also inhibition of CD44 expression has been proposed. Finally, the function of CD44 as a hyaluronan receptor was also taken advantage of. We are convinced that the success of these therapies will depend on an increased understanding of the molecular functions of specific CD44 isoforms in particular in cancer stem cells.

Regulation of alternative pre-mRNA splicing by the ERK MAP-kinase pathway.

Differential gene expression through alternative pre-mRNA splicing is crucial to various physiological and pathological conditions. Upon activation of B and T lymphocytes during an immune response, variant isoforms of the cell surface molecule CD44 are generated by alternative pre-mRNA splicing. We show here that in primary mouse T cells as well as in the murine LB-17 T-cell line upregulation of variant CD44 mRNA species upon T-cell activation requires activation of the MEK-ERK pathway. By employing mutant signaling molecules and a novel luciferase-based splice reporter system we demonstrate that the Ras-Raf-MEK-ERK signaling cascade, but not the p38 MAP-kinase pathway, activates a mechanism that retains variant CD44 exon v5 sequence in mature mRNA. The findings demonstrate that a highly conserved pleiotropic signaling pathway links extracellular cues to splice regulation, providing an avenue for tissue-specific, developmental or pathology-associated splicing decisions.

Soluble CD44 inhibits melanoma tumor growth by blocking cell surface CD44 binding to hyaluronic acid.

Proteolytic cleavage of the extracellular domain of CD44 from the surface of cells has been observed recently in different cell types. In cell culture supernatants of human melanoma cell lines a 70 kDa soluble CD44 protein (solCD44) was detected at concentrations of 250-300 ng/ml. Protease inhibitor studies revealed that serine proteases and metalloproteases are involved in the cleavage of CD44 from the surface of melanoma cells. To analyse a possible function of soluble CD44 a human malignant melanoma cell line was stably transfected with cDNAs encoding either wild type soluble CD44s or mutated forms with defective HA binding properties (CD44sR41A and CD44sR150A/R154A). Soluble CD44s almost completely inhibited hyaluronic acid binding by melanoma cells, whereas soluble CD44 mutated in the HA binding domain had no effect. When cultivated on hyaluronic acid, melanoma cell proliferation was induced by 30% for both the parental and the control transfected cells. This increase in proliferation was blocked completely in solCD44s-secreting transfectants, whereas solCD44sR41A and solCD44sR150A/R154A-secreting cells again showed hyaluronic acid-induced cell proliferation. These cell lines were subcutaneously injected into MF1 nu/nu mice to compare their growth as tumors in vivo. Compared to tumors derived from parental and control transfected cells, we observed a dramatic reduction of primary tumor growth with solCD44s expressing MM cells. Transfectants expressing solCD44s mutated in the HA binding domain in contrast developed fast-growing primary tumors. These results provide strong evidence that direct solCD44 interactions with hyaluronic acid interfere competitively with processes induced by hyaluronic acid binding to surface CD44. Autocrine, or drug-induced secretion of solCD44 by human melanoma cells may thus exert potent antitumoral effects in vivo.

The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44.

The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein that functions as a tumor suppressor. We found that merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypo-phosphorylated and inhibits cell growth in response to hyaluronate (HA), a mucopolysaccharide that surrounds cells. Merlin's growth-inhibitory activity depends on specific interaction with the cytoplasmic tail of CD44, a transmembrane HA receptor. At low cell density, merlin is phosphorylated, growth permissive, and exists in a complex with ezrin, moesin, and CD44. These data indicate that merlin and CD44 form a molecular switch that specifies cell growth arrest or proliferation.

Heterogeneous ribonucleoprotein A1 is part of an exon-specific splice-silencing complex controlled by oncogenic signaling pathways.

Regulation of alternative pre-mRNA splicing, recognized as increasingly important in causing human disease, was studied using the CD44 gene, whose splice variants have been implicated in tumor progression. We identified heterogeneous ribonucleoprotein (hnRNP) A1 as a protein interacting in vitro and in vivo with regulatory splice elements in CD44 variant exon v5. Transient overexpression of hnRNP A1 prevented v5 exon inclusion, dependent on the exonic elements. HnRNP A1-dependent repression was exon-specific and could be relieved by coexpression of oncogenic forms of Ras and Cdc42. The results define hnRNP A1 as a decisive part of an oncogene-regulated splice-silencing complex, which can select between multiple alternatively spliced exons.

CD44 acts both as a growth- and invasiveness-promoting molecule and as a tumor-suppressing cofactor.

High-molecular-weight splice variants of the CD44 transmembrane protein family have been implicated in tumorigenesis and metastasis formation. By contrast, in certain tumors--for example, Burkitt's lymphoma, neuroblastomas, and prostate cancer--loss of CD44 expression seems to accompany transformation. Here we describe two modes of action of CD44 proteins. They can bind growth factors and present them to their authentic high-affinity receptors, and thus promote proliferation and invasiveness of cells. Under these conditions the CD44 proteins recruit ERM proteins--for example, ezrin or moesin--to their cytoplasmic tails, thereby producing links to the cytoskeleton. This mode of action could account for the tumor-promoting action of CD44 proteins. The second mode of action of CD44 proteins comes into play when cells reach confluent growth conditions. Under specific conditions, binding of another ligand, the ECM component hyaluronate, leads to the activation and binding to the CD44 cytoplasmic tail of the tumor suppressor protein merlin. The activation of merlin confers growth arrest, so-called contact inhibition. This function of CD44 proteins defines them as tumor suppressors. The type of action of CD44 on a given cell will depend on the isoform pattern of CD44 expressed, on the cellular equipment with ERM protein members, on the nature of the ECM, and on yet-unknown conditions.

CD44 expression and regulation during mammary gland development and function.

The CD44v6 epitope has been widely reported to be expressed in human mammary carcinomas, yet its prognostic significance is controversial and its function in mammary tumors and mammary glands is unknown. To begin to resolve these issues, we analysed in detail the normal postnatal expression patterns and regulation of the CD44v6 epitope in murine mammary glands. We demonstrate that significant CD44v6 epitope expression is first seen during puberty, and that after puberty CD44v6 epitope expression follows the estrous cycle. CD44v6 epitope expression is observed in the myoepithelium and also less widely in luminal epithelial cells. During lactation, CD44v6 epitope expression is turned off and reappears during involution. The CD44 variant isoform bearing the v6 epitope is CD44v1-v10. Using HC11, a mammary epithelial cell line with stem cell characteristics, and facilitated by the cloning of the murine CD44 promoter, we show that growth factors and hormones which regulate ductal growth and differentiation modulate CD44 transcription. Together our data suggest that the CD44v6 epitope is expressed in mammary epithelial stems cells and in lineages derived from these cells, and that CD44v6 expression is regulated in part by hormones and growth factors such as IGF-1 and EGF which regulate the growth and differentiation of the mammary epithelium. The function of these same growth factors and hormones is often perturbed in mammary carcinomas, and we suggest that CD44v6 expression in tumors reflects this perturbation. We conclude that the expression of the CD44v6 epitope observed in some mammary tumors reflects the stem cell origin of breast tumors, and that whether or not the CD44v6 epitope is expressed in a mammary tumor is determined by the differentiation status of the tumor cells.

Inhibition of MT-450 rat mammary tumour growth by antibodies recognising subtypes of blood group antigen B.

Using subtractive immunization to identify cell surface epitopes expressed in a metastasis-specific fashion on cells of the rat MT-W9 mammary carcinoma model, we generated a monoclonal antibody called M-N#1. This antibody binds specifically to metastasizing cells of the MT-W9 series and also to certain other metastasizing rat mammary carcinoma cell lines. We demonstrate that the M-N#1 antibody recognizes a fucosylated N-glycosyl sugar modification, and furthermore show that the epitope specificity of the M-N#1 antibody is for blood group antigen B subtypes 2, 3 and 4 with slight cross-reactivity with blood group antigen A subtype 2. The expression of these carbohydrate epitopes on MT-450 cells is functionally important, because the M-N#1 antibody efficiently inhibits MT-450 tumour growth in spontaneous metastasis assays. These results suggest that expression of the subtypes of blood group antigen B recognized by the M-N#1 antibody does not directly participate in the metastatic cascade but rather confers a growth or survival advantage on the tumour cells.

Hyaluronate-enhanced hematopoiesis: two different receptors trigger the release of interleukin-1beta and interleukin-6 from bone marrow macrophages.

The glycosaminoglycan hyaluronate (HA) is part of the extracellular environment in bone marrow. We show here that HA activates signal transduction cascades important for hemopoiesis. In myeloid and lymphoid long-term bone marrow cultures (LTBMC), treatment with hyaluronidase (HA'ase) results in reduced production of both progenitor and mature cells. Exogeneous HA added to LTBMC had the opposite effect: it enhanced hematopoiesis. The effect of HA is mediated through two different HA receptors on bone marrow macrophage-like cells, one of which is CD44 while the other is unknown. HA induces bone marrow macrophages to secrete IL-1beta (CD44-dependent) and IL-6 (CD44-independent). The two receptors address different signal transduction pathways: CD44 links to a pathway activating p38 protein kinase while the other yet unknown receptor induces Erk activity. There was no difference of the effect of HA and HA'ase on hematopoiesis in LTBMC and on cytokine production by macrophages in CD44-deficient mice compared with wild-type mice, indicating that the CD44 hyaluronate receptor and its signal transduction can be compensated for. Our data suggest a regulatory role for the extracellular matrix component HA in hematopoiesis and show the induction of signal transduction by HA receptors.

Prognostic value of CD44 variant expression in primary breast cancer.

CD44 is a family of cell surface transmembrane glycoproteins members which differ in the extracellular part by sequences derived by alternative splicing of 10 variant exons (v1-v10). CD44 proteins containing such variant sequences have been implicated in tumor metastasis formation. Here, we have evaluated the expression of CD44 variants by immuno-histochemistry in primary breast cancer samples of 237 node-negative and 230 node-positive patients. For the analysis of samples derived from node-negative patients, the exon-specific antibodies used were DIII, vff7 and vff18 (v6), vff17 (v7/v8), fw11.24 (v9) and vff16 (v10). With the different antibodies which recognize v6 epitopes, the majority of tumors were positively stained (> or = 65% of the tumors) with varying intensities. Thirty-nine percent of the tumors were positively stained with the antibody vff16, and approximately half of the tumors with the antibodies vff17 and fw11.24. The expression of CD44 v6 epitopes in tumors from node-negative patients was associated with a favorable prognosis, both upon univariate and multivariate analysis. The expression of CD44 v7/8, v9 or v10 epitopes was not significantly related with relapse-free survival. Samples from node-positive patients were only examined with the antibodies vff7, vff17 and vff18. The staining with none of these antibodies was correlated with the length of relapse-free survival of the patients. Our data suggest that, generally, the usefulness of knowledge of CD44 variant expression is of limited value for assessing the risk of relapse in patients with primary breast cancer. However, the expression of exon v6 of CD44 may be a marker to identify patients with a relatively favorable prognosis in node-negative patients.

CD44 splice variants in draining lymph nodes precede allograft rejection of endocrine cells.

Upon allogeneic transplantation (Tx) of pancreatic islets under the kidney capsule of diabetic rats, cells from draining lymph nodes and, to a minor degree, bone marrow transiently upregulate CD44 splice variants as detected by RT-PCR using CD44 variant exon specific primers. Maximal expression was on day 5 post Tx in lymph nodes and thus precedes islet rejection sufficiently (in this model by 5 days) to still permit establishing rescue by immunosuppressive therapy. CD44 variant exon sequence could therefore serve as early markers of allograft rejection.

Characterisation of the murine gene encoding the intracellular hyaluronan receptor IHABP (RHAMM).

We have recently shown that the published cDNA sequence encoding the murine cell surface receptor for hyaluronan-mediated motility (RHAMM) in fact represents a partial sequence of the cDNA encoding a new intracellular hyaluronic acid binding protein (IHABP). Here we publish the genomic organisation, including 700bp sequences of the promoter region, of the IHABP gene. The IHABP gene consists of 18 exons and spans more than 25kb. Part of the IHABP gene is identical with the published data on RHAMM. The IHABP gene apparently possesses one promoter region with one major transcriptional start point. IHABP is ubiquitously expressed at the mRNA and the protein level in all murine tissues, suggesting that the function of this intracellular hyaluronan binding protein is not restricted to migrating cells.

How tumor cells make use of CD44.

A variant of CD44 containing exon v3 sequences is expressed in the apical ectodermal ridge of the limb bud during embryogenesis. This variant is modified by heparan sulfate moieties and acts as low affinity receptor for FGFs. These FGFs are presented by CD44 to mesenchymal cells which induces their proliferation and limb outgrowth. We suggest that a similar growthfactor presentation mechanism accounts for the function of CD44 variants on metastasizing tumor cells.

CD44 variant isoforms are essential for the function of epidermal Langerhans cells and dendritic cells.

Upon antigen encounter epidermal Langerhans cells (LC) and dendritic cells (DC) emigrate from peripheral organs and invade lymph nodes through the afferent lymphatic vessels and then assemble in the paracortical T cell zone and present antigen to T lymphocytes. Part of this process is mimicked by metastasizing tumor cells. Since splice variants of CD44 promote metastasis to lymph nodes we explored the expression of CD44 proteins on migrating LC and DC. We show that following antigen contact, LC and DC upregulate pan CD44 epitopes and epitopes encoded by variant exons v4, v5, v6 and v9. Antibodies against CD44 epitopes arrest LC in the epidermis, prevent the binding of activated LC and DC to the T cell zones of lymph nodes, and severely inhibit their capacity to induce a delayed type hypersensitivity reaction to a skin hapten in vivo. Our results demonstrate that CD44 splice variant expression is obligatory for the migration and function of LC and DC.

Two different functions for CD44 proteins in human myelopoiesis.

CD44 is important during myelopoiesis, although the contributions of variant CD44 proteins are unclear. We show here that in human long-term bone marrow culture antibodies recognizing a CD44 NH2-terminal epitope (mab 25-32) or a CD44v6 epitope (mab VFF18) inhibit myelopoiesis. However, mab 25-32 but not mab VFF18 affects myeloid colony formation. These data suggest that an early precursor cell compartment is the target for the 25-32 antibody, whereas the mab VFF18 targets later stages in myelopoiesis. Since the bulk of hemopoietic precursor cells are negative for the v6 epitope and only a minor subset of myeloid cells express the v6 epitope, we have used several human myeloid progenitor cell lines to unravel the function of different CD44 proteins. These cell lines produce variant CD44 proteins, predominantly a new variant CD44v4-v10, when stimulated towards myeloid differentiation. Features that can be acquired by the expression of CD44v4-v10 are an increased hyaluronate (HA) and a de novo chondroitin sulphate A (CS-A) binding. Although, the expression of CD44v4-v10 per se is necessary for HA and CS-A binding, the protein backbone seems to require appropriate glycosylation. HA binding results in CD44-mediated cellular self-aggregation and adhesion to the stromal cell line MS-5. In summary, our data suggest that different CD44 proteins are important for at least two different steps in myelopoiesis.

Activation-dependent modulation of hyaluronate-receptor expression and of hyaluronate-avidity by human monocytes.

During inflammation, activated monocytes (Mo) migrate into tissues where they interact with extracellular matrix components such as hyaluronate (HA), produced in high amounts at inflammatory sites. We determined whether Mo that had invaded sites of cutaneous inflammation bind HA and express the putative HA receptors CD44 isoforms, ICAM-1, or receptor for hyaluronate-mediated motility (RHAMM). In cutaneous inflammation, activated infiltrating Mo displayed high HA avidity and expressed epitopes encoded by CD44s, CD44 variant exons v3, v4, v5, v6, v7, and v9, and ICAM-1, but not RHAMM. We further investigated how activation affects the avidity of Mo for HA and which receptors were responsible for such binding. Mo freshly purified from human peripheral blood bound little HA and expressed CD44s but no epitopes encoded by CD44v exons, ICAM-1, or RHAMM. During short-term tissue culture, Mo upregulated their HA avidity and expression of ICAM-1, CD44s, and epitopes encoded by CD44v, all of which were further augmented by IFN-gamma or lipopolysaccharide, whereas RHAMM was not detectable. Thus in vitro activated Mo resembled Mo that had migrated to inflammatory sites in vivo. Lipolysaccharide or IFN-gamma-induced HA binding was inhibited by more than 90% with monoclonal antibodies directed against N-terminal HA binding domains of CD44s, but not by monoclonal antibodies against CD44v epitopes or ICAM-1. In conclusion, we show that upon in vitro or in vivo activation, Mo enhance their capacity to bind HA. This is critically dependent upon the expression ofCD44s epitopes. Regulated CD44-HA interactions may be important for the ability of Mo to migrate into and within sites of inflammation and for Mo effector functions.

CD44 variant exon v5 encodes a tyrosine that is sulphated.

Functional differences between members of the CD44 family of cell surface glycoproteins is mediated in part by differential post-translational modification of these proteins and by alternative splicing. Tyrosine sulphation is a secondary modification of the primary amino acid structure of a number of secreted, transmembrane and lysosomal proteins, which is associated with promotion of protein-protein interactions. Here we identify a cannonical tyrosine-sulphation motif within rat and mouse CD44 exon v5. We show that inorganic sulphate is incorporated into the metastasis-associated rat CD44v4-v7 splice variant. The sulphate is not incorporated into sulphated glycosaminoglycan or other sugar modifications of CD44v4-v7. A point mutation of the exon v5 tyrosine to phenylalanine destroys inorganic sulphate incorporation into CD44v4-v7. These results demonstrate that the tyrosine-sulphation motif within rat CD44 exon v5 is used in vivo, and suggest that exon v5 may be involved in mediating CD44 ligand binding-activity by means of its sulphated tyrosine.