CD44 variant isoforms associate with tetraspanins and EpCAM

The metastasizing subline of the rat pancreatic adenocarcinoma BSp73 expresses a set of membrane molecules, the combination of which has not been detected on non-metastasizing tumor lines. Hence, it became of interest whether these molecules function independently or may associate and exert specialized functions as membrane complexes. Separation of CD44v4-v7 containing membrane complexes in mild detergent revealed an association with the alpha3 integrin, annexin I, EpCAM, and the tetraspanins D6.1A and CD9. EpCAM and the tetraspanins associate selectively with CD44 variant (CD44v), but not with the CD44 standard (CD44s) isoform. The complexes are found in glycolipid-enriched membrane (GEM) microdomains, which are dissolved by stringent detergents, but the complexes are not destroyed by methyl-beta-cyclodextrin (MbetaCD) treatment, which implies that complex formation does not depend on a lipid-rich microenvironment. However, a complex-associated impact on cell-matrix and cell-cell adhesion as well as on resistance towards apoptosis essentially depended on the location in GEMs. Thus, CD44v-specific functions may well be brought about by complex formation of CD44v with EpCAM, the tetraspanins, and the alpha3 integrin. Because CD44v4-v7-EpCAM complex-specific functions strictly depended on the GEM localization, linker or signal-transducing molecules associating with the complex are likely located in GEMs.     

CD44 variant,but not standard CD44 isoforms,mediate disassembly of endothelial VE-cadherin junction on metastatic melanoma cells

Loss of endothelial adherens junctions is involved in tumor metastasis. Here, we demonstrate that, in the metastatic Lu1205 melanoma cells, expression of the CD44 variant CD44v8-v10 induced junction disassembly and vascular endothelial (VE)-cadherin phosphorylation at Y658 and Y731. Short interfering RNA (siRNA)-mediated CD44 knockdown or sialic acid cleavage reversed these effects. Moreover, microspheres coated with recombinant CD44v8-v10 promoted endothelial junction disruption. Overexpression of CD44v8-v10 but not of standard CD44 (CD44s) promoted gap formation in the non-metastatic WM35 melanoma cells, whereas CD44 knockdown or neuraminidase treatment dramatically diminished melanoma transendothelial migration. Endothelial cells transfected with the phosphomimetic VE-cadherin mutant Y658E supported transmigration of CD44-silenced Lu1205 cells. Our findings imply that CD44 variant isoform (CD44v) but not CD44s regulates endothelial junction loss, promoting melanoma extravasation.     

Expression of CD44 isoforms during bleomycin-or radiation-induced pulmonary fibrosis in rats and mini-pigs

The distribution of CD44s and CD44v molecules in normal and injured lung tissue of rats and minipigs was studied by examining the immunohistochemical binding of monoclonal antibodies against CD44 isoforms. We showed that the expression of CD44v and CD44s varies greatly among different pulmonary fibrosis samples and that some tissues express either enhanced expression of CD44s, particularly in the interstitium and on alveolar macrophages, or very low levels of CD44v in the alveolar epithelium. Normal type II pneumocytes expressed the CD44s and CD44v molecules at the basolateral aspect of the cell. Such localisation favours a role for CD44 in epithelial cell-fibroblast interaction during lung development and repair.     

CD44 isoforms during differentiation and development

During mouse early development cell adhesion molecules are indispensable for the embryo organisation. A family of molecules probably involved in development is the transmembrane glycoprotein CD44 family, which exists in multiple isoforms. These are generated by alternative splicing of the pre-mRNA, resulting in the enlargement of the extracellular part of the molecule. The standard form of CD44 is widely expressed in adult tissues and in embryos from day 9.5 post coitum onwards, while the numerous variant isoforms exhibit highly specialised patterns of expression that are already in the egg cylinder at day 6.5 of development. In lymphohemopoiesis, specific variant isoforms also emerge at decisive differentiation stages. Although specific ligands for the variant region still await isolation, the highly organised expression of CD44 variant isoforms suggests they have a pivotal role in cellular interactions during early development, pattern formation and hemopoiesis.     

CD44 variant isoforms are preferentially expressed in basal epithelia of non-malignant human fetal and adult tissues

CD44 is a transmembrane glycoprotein, which can exist in a multitude of isoforms due to alternative splicing of the pre-mRNA. We have generated monoclonal antibodies to several of these variant regions, which are encoded by 10 additional exons in the extracellular part of the molecule. CD44 variant isoforms have been reported to be involved in the malignant progression of rat and human tumours. The precise localization of CD44 variant isoforms in normal developmental and morphogenetic processes is essential for diagnostic studies of human tumorigenesis. Therefore, we have analysed a large number of different human tissues by immunohistochemistry for the expression of CD44 isoforms containing either exons 4v, 6v or 9v. Expression of exon 9v-isoforms was detected in almost all epithelia analysed, with a few exceptions. Exon 6v isoforms are expressed only in squamous and glandular epithelia, e.g. skin epidermis, sweat and sebaceous glands, oesophagus, ducts of the mammary gland, salivary and prostate glands. Detection of exon 4v-encoded isoforms was restricted to the epidermis and the oesophagus. Similar tissue distributions of CD44 variant isoforms were observed in 10-week-old fetal tissues. Since one of the ligands of CD44 is hyaluronic acid (HA), we also analysed the tissue distribution of HA synthetase. HA synthetase was detected in all tissues analysed, showing good correlation with the expression of the standard form of CD44, CD44s.     

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

There is growing evidence that one of the central common characteristics of tumor and inflammatory cells is their resistance to programmed cell death. This feature results in the accumulation of harmful cells, which are mostly refractory to Fas (FAS, APO-1)-mediated apoptosis. A molecule found on these cells is the transmembrane receptor CD44 with its variant isoforms (CD44v). The establishment of transfectants expressing different CD44v isoforms allowed us to demonstrate that the CD44v6 and CD44v9 isoforms exhibit an antiapoptotic effect and can block Fas-mediated apoptosis. Moreover, we observed that CD44v6 and CD44v9 colocalize and interact with Fas. Importantly, an anti-CD44v6 antibody can abolish the antiapoptotic effect of CD44v6. These results are the first to show that CD44v isoforms interfere with Fas signaling. Our findings improve the understanding of the pathogenesis of cancer and autoimmunity and open new strategies to treat such disorders.     

Expression of CD44 isoforms in normal salivary gland tissue: an immunohistochemical and ultrastructural study

We studied the expression of CD44 isoforms immunoreactivity in normal human salivary gland tissue, aiming at its full characterisation in normal epithelial and myoepithelial cell types. Optical immunohistochemistry techniques using monoclonal antibodies anti-CD44v3, CD44v4/5 and, for CD44v6, together with immunoelectron microscopy, were performed in serous, seromucinous and mucinous glands. Normal human breast and a case of lactating breast adenoma were used for comparative purposes and as controls. CD44v3 was positive in acinar and myoepithelial cells and was absent in mucin-producing cells from the different gland types. CD44v4/5 was consistently negative in all types of salivary tissue. CD44v6 was constantly positive in serous acinar cells, focally positive in basal cells of ducts, and myoepithelial cells consistently expressed it. At the ultrastructural level, CD44v6 was localised to the interdigitating processes of acinar cells, whenever they were not covered by basal lamina and to the cell membrane facing myoepithelial cells. In myoepithelial cells, immunolabelling was found at the membranes facing the acinar cells and in caveolae present at this interface. No labelling was found at cell membranes of both acinar and myoepithelial cells in contact with basal lamina or at the luminal aspect of the former. The finding of CD44v3 and v6 in myoepithelium of normal salivary glands may argue in favour of the role of these molecules in the regulation of growth and renewal of normal tissues and, potentially, on the morphogenesis of salivary gland neoplasms.     

A complex of EpCAM, claudin-7, CD44 variant isoforms, and tetraspanins promotes colorectal cancer progression

High expression of EpCAM and the tetraspanin CO-029 has been associated with colorectal cancer progression. However, opposing results have been reported on CD44 variant isoform v6 (CD44v6) expression. We recently noted in rat gastrointestinal tumors that EpCAM, claudin-7, CO-029, and CD44v6 were frequently coexpressed and could form a complex. This finding suggested the possibly that the complex, rather than the individual molecules, could support tumor progression. The expression of EpCAM, claudin-7, CO-029, and CD44v6 expression was evaluated in colorectal cancer (n = 104), liver metastasis (n = 66), and tumor-free colon and liver tissue. Coexpression and complex formation of the molecules was correlated with clinical variables and apoptosis resistance. EpCAM, claudin-7, CO-029, and CD44v6 expression was up-regulated in colon cancer and liver metastasis. Expression of the four molecules did not correlate with tumor staging and grading. However, coexpression inversely correlated with disease-free survival. Coexpression was accompanied by complex formation and recruitment into tetraspanin-enriched membrane microdomains (TEM). Claudin-7 contributes to complex formation inasmuch as in the absence of claudin-7, EpCAM hardly associates with CO-029 and CD44v6 and is not recruited into TEMs. Notably, colorectal cancer lines that expressed the EpCAM/claudin-7/CO-029/CD44v6 complex displayed a higher degree of apoptosis resistance than lines devoid of any one of the four molecules. Expression of EpCAM, claudin-7, CO-029, and CD44v6 by themselves cannot be considered as prognostic markers in colorectal cancer. However, claudin-7-associated EpCAM is recruited into TEM and forms a complex with CO-029 and CD44v6 that facilitates metastasis formation.     

Carcinoembryonic antigen and CD44 variant isoforms cooperate to mediate colon carcinoma cell adhesion to E- and L-selectin in shear flow

Selectin-mediated adhesion of tumor cells to platelets, leukocytes, and endothelial cells may regulate their hematogenous dissemination in the microvasculature. We recently identified CD44 variant isoforms (CD44v) as functional P-, but not E- or L-, selectin ligands on colon carcinoma cells. Moreover, an approximately 180-kDa sialofucosylated glycoprotein(s) mediated selectin binding in CD44-knockdown cells. Using immunoaffinity chromatography and tandem mass spectrometry, we identify this glycoprotein as the carcinoembryonic antigen (CEA). Blot rolling assays and flow-based adhesion assays using microbeads coated with CEA immunopurified from LS174T colon carcinoma cells and selectins as substrate reveal that CEA possesses E- and L-, but not P-, selectin ligand activity. CEA on CD44-knockdown LS174T cells exhibits higher HECA-452 immunoreactivity than CEA on wild-type cells, suggesting that CEA functions as an alternative acceptor for selectin-binding glycans. The enhanced expression of HECA-452 reactive epitopes on CEA from CD44-knockdown cells correlates with the increased CEA avidity for E- but not L-selectin. Through the generation of stable knockdown cell lines, we demonstrate that CEA serves as an auxiliary L-selectin ligand, which stabilizes L-selectin-dependent cell rolling against fluid shear. Moreover, CEA and CD44v cooperate to mediate colon carcinoma cell adhesion to E- and L-selectin at elevated shear stresses. The novel finding that CEA is an E- and L-selectin ligand may explain the enhanced metastatic potential associated with tumor cell CEA overexpression and the supportive role of selectins in metastasis.     

Hyaluronidases and CD44 undergo differential modulation during chondrogenesis

Hyaluronan, a high-molecular-weight glycosaminoglycan of cartilage, is deposited directly into the extracellular space by hyaluronan synthases, while hyaluronan catabolism is mediated by the hyaluronidases. An in vitro cell culture system has been established in which human dermal fibroblasts are induced to undergo chondrogenesis. Here, we describe the differential modulation of the hyaluronidases and the up-regulation of the hyaluronan receptor, CD44, during such chondrogenesis. Dermal fibroblasts, plated in micromass cultures in the presence of lactic acid and staurosporine for 24 h, were then placed in serum-free, chemically defined medium. At 3 days, RNA was extracted and RT-PCR performed using primers for the hyaluronidase genes. Marked increase in HYAL1 expression was observed, with only moderate increases occurring in HYAL2 and HYAL3. No expression of HYAL4 and PH-20, the sperm-associated hyaluronidase, was detected. RNA levels correlated well with changes in hyaluronidase enzyme activity. Finally, greater expression and staining for the hyaluronan receptor, CD44s, the standard form, were detected. Differential expression of the somatic hyaluronidases and CD44-mediated hyaluronan turnover play a critical role in cartilage development from mesenchymal precursors.     

结肠癌组织中CD44S和CD44V6基因蛋白的表达及意义

摘要：目的 研究CD44S和CD44V6的表达与结肠癌临床病理特征之间的关系,以明确二者在结肠癌发展和预后中的作用。方法 选取98例结肠癌组织做为实验组,采用免疫组织化学方法,检测组织中CD44S和CD44V6基因蛋白产物表达,与组织学分型、肠壁浸润深度、脉管有无浸润、淋巴结有无转移、预后等相关因素进行实验研究。结果 CD44S和CD44V6的表达与结肠癌组织类型无关,与癌组织浸润肠壁深度、脉管浸润、淋巴结转移、远处转移等密切相关。结论 CD44S和CD44V6异常表达可能参与了结肠癌的发展并可能影响患者的预后。     

Alternatively spliced CD44 isoforms containing exon v10 promote cellular adhesion through the recognition of chondroitin sulfate-modified CD44

Correlations have been noted between the expression of certain alternatively spliced CD44 isoforms and the metastatic propensity of various histologically distinct tumor cell types. The precise mechanism by which particular CD44 isoforms contribute to the metastatic process is, however, unclear. In the present study we demonstrate that CD44R2, a CD44 isoform highly expressed on activated and transformed hemopoietic cells, can recognize and bind a common determinant present on CD44H and CD44R1. Importantly, CD44H lacked this activity. Pretreatment of TIL1 cells expressing CD44H or CD44R1 with chondroitinase ABC inhibited adhesion to CD44R2, suggesting that the unique inserted region present within the CD44R2 molecule, encoded by exon v10, mediates cell adhesion by potentiating the recognition of chondroitin sulfate moieties presented in association with other CD44 molecules. These data help explain the differential involvement of v10-containing CD44 isoforms in tumor metastasis.     

Coexpression of CD44 variant (v10/ex14) and CD44S in human mammary epithelial cells promotes tumorigenesis

CD44 is the major hyaluronan cell surface receptor and functions as an adhesion molecule in many different cell types, including human breast epithelial cells. The coexpression of certain CD44 variants (CD44v), such as CD44v (v10/ex14), with CD44s (standard form) appears to be closely associated with human breast tumor metastasis. In this study we have established a stable transfection of CD44v (v10/ex14) cDNA into nontumorigenic human breast epithelial cells (HBL100) which contain endogenous CD44s. Our results indicate that coexpression of both CD44v (v10/ex14) and CD44s alters the following important biological properties of these cells: 1) there is a significant reduction in hyaluronic acid (HA)-mediated cell adhesion; 2) there is an increased migration capability in collagen-matrix gel; and 3) these cells constitutively produce certain angiogenic factors and effectively promote tumorigenesis in athymic nude mice. These findings suggest that coexpression of CD44v (v10/ex14) and CD44s may trigger the onset of cell transformation required for breast cancer development. J. Cell. Physiol. 171:152–160, 1997. © 1997 Wiley-Liss, Inc.