CD44 isoforms containing exon V3 are responsible for the presentation of heparin-binding growth factor.

Abstract

Abstract. Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CIM4 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS-modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS-modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 3~SO4 -2-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS-modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is not likely to be the proteoglycan principally involved in presenting HS-binding growth factors to leukocytes on the vascular cell wall. C o44 represents a very heterogeneous class of molecules all encoded by a single gene. Genomic cloning of CD44 has revealed that there are 19 exons (47), 12 of which can be alternatively spliced. At least 18 different CD44 transcripts have been identified and the potential for many more exists (for review see 35). The diversity of CD44 is further magnified by the differential use of numerous N-linked and O-linked glycosylation sites as well as glycosaminoglycan (GAG) ~ attachment sites. We

A novel antiapoptotic mechanism based on interference of Fas signaling by CD44 variant isoforms

“…This suggests that the resistance to apoptosis observed in the variant positive cells is not due to the proliferation stimulus caused by growth factors binding to the heparan sulfate chains located at the variant 3 region. 26 Moreover, Jurkat cells expressing CD44v6-10 (or CD44v2-10, not shown), but lacking the cytoplasmic domain were also strongly protected from cell death. This observation clearly points out the significance of the extracellular domain of CD44v for apoptosis resistance (Figure 2d).…”

Inhibition of tumor growth in vivo by hyaluronan oligomers

“…CD44 is a broadly expressed, polymorphic, integral membrane glycoprotein whose diverse structure is determined by variable usage of at least 10 exons encoding a segment of the membrane proximal domain and by cell type-specific glycosylation and glycosaminoglycan substitution (Lesley et al, 1993;Sherman et al, 1994;Bennett et al, 1995;Bartolazzi et al, 1996). Interactions between CD44 and hyaluronan have been shown to mediate or promote macrophage aggregation (Green et al, 1988), cell migration (Thomas et al, 1992), chondrocyte pericellular matrix assembly (Knudson, 1993) and leukocyte activation (Arch et al, 1992).…”

“…The ability of CD44 to bind hyaluronan is tightly regulated, at least in part by insertion of variant exon products and by glycosylation (Lesley et al, 1993;Bennett et al, 1995;Bartolazzi et al, 1996). Thus, mere expression of CD44 by tumor cells does not imply enhanced binding of hyaluronan or enhanced tumor malignancy.…”

Recombinant CD44-HABD is a novel and potent direct angiogenesis inhibitor enforcing endothelial cell-specific growth inhibition independently of hyaluronic acid binding

“…A number of CD44-binding proteins other than HA have been described, including HGF, bFGF, fibronectin, osteopontin, selectins, erbB2 and erbB3 (Jalkanen and Jalkanen, 1992;Bennett et al, 1995;Weber et al, 1996;van der Voort et al, 1999;Sherman et al, 2000;Dimitroff et al, 2001). However, the binding of these proteins is dependent on various post-translational modifications of CD44 at alternative exons that are not present in our recombinant fusion proteins.…”