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PubMed1999

Regulated CD44 cleavage under the control of protein kinase C, calcium influx, and the Rho family of small G proteins.

CD44 is a cell surface receptor for several extracellular matrix components and is implicated in tumor cell invasion and metastasis. Our previous studies have shown that CD44 expressed in cancer cells is proteolytically cleaved at the extracellular domain through membrane-associated metalloproteases and that CD44 cleavage plays a critical role in CD44-mediated tumor cell migration (Okamoto, I., Kawano, Y., Tsuiki, H., Sasaki, J., Nakao, M., Matsumoto, M., Suga, M., Ando, M., Nakajima, M., and Saya, H. (1999) Oncogene 18, 1435-1446). In the present study, we first demonstrate rapid degradation of the membrane-tethered CD44 cleavage product through intracellular proteolytic pathways, and it occurs only after CD44 extracellular cleavage. To address the mechanisms regulating CD44 cleavage at the extracellular domain, we show that 12-O-tetradecanoylphorbol 13-acetate (TPA) and the calcium ionophore ionomycin rapidly enhance metalloprotease-mediated CD44 cleavage in U251MG cells via protein kinase C-dependent and -independent pathways, respectively, suggesting the existence of multiple distinct pathways for regulation of CD44 cleavage. Concomitant with TPA-induced CD44 cleavage, TPA treatment induces redistribution of CD44 and ERM proteins (ezrin, radixin, and moesin) to newly generated membrane ruffling areas. Treatment with lysophosphatidic acid, which is known to activate the Rho-dependent pathway, inhibits TPA-induced CD44 redistribution and CD44 cleavage. Furthermore, overexpression of Rac dominant active mutants results in the redistribution of CD44 to the Rac-induced ruffling areas and the enhancement of CD44 cleavage. These results suggest that the Rho family proteins play a role in regulation of CD44 distribution and cleavage.

Calcium↗
PubMed1992

Requirements for hyaluronic acid binding by CD44: a role for the cytoplasmic domain and activation by antibody.

The CD44-negative T lymphoma AKR1 (CD44.2 genotype) was transfected with a CD44.1 cDNA. The intact cDNA conferred on the transfected cells the ability to bind hyaluronic acid (HA) both from solution and immobilized on culture plates. It also conferred a CD44-dependent and hyaluronidase-sensitive increase in adhesion to a lymph node endothelial cell line. A mutant cDNA which codes for a CD44 molecule lacking most of the cytoplasmic domain of CD44 was also transfected into AKR1, and cell sorting was used to select transfectants expressing levels of cell surface CD44 expression comparable with the line transfected with the wild-type CD44 cDNA. The cells transfected with the mutant construct bound fluoresceinated HA from solution very poorly, but did adhere to immobilized HA, though less well than cells transfected with the wild-type construct. This result indicates that the cytoplasmic domain of CD44 is necessary for binding of HA from solution but is not required for binding to immobilized HA, although it may contribute to adhesion following ligand recognition. A monoclonal antibody (mAb), IRAWB 14, which reacts with CD44 on all CD44+ cells dramatically induced HA binding by some CD44+ cell lines that did not constitutively bind HA. The transfectant expressing a CD44 molecule with a truncated cytoplasmic domain could be induced by this antibody to bind fluoresceinated-HA from solution. Splenic T cells did not bind fluoresceinated HA constitutively. In the presence of the IRAWB 14 mAb, virtually all CD44+ splenic T cells bound HA. Induction was immediate and occurred equally well at room temperature and at 4 degrees C, indicating that the new HA-binding activity was due to preexistent CD44 molecules. These results are compatible with an antibody-induced activation of CD44 by either a conformational change in the CD44 molecule or a change in the distribution of CD44 molecules on the cell surface.

Animals↗
PubMed2000

The CD44 receptor of lymphoma cells: structure-function relationships and mechanism of activation.

Migration of some tumor cells, and their lodgment in target organs, is dependent on the activation of cell surface CD44 receptor, usually detected by its ability to bind hyaluronic acid (HA) or other ligands. In an attempt to reveal the mechanism of tumor cell CD44 activation, we compared the physical and chemical properties of CD44 in nonactivated LB cell lymphoma with those in phorbol 12-myristate 13-acetate (PMA)-activated LB cells and of an LB cell subline (designated HA9) expressing constitutively-active CD44. In contrast to nonactivated LB cells, PMA-activated LB cells and HA9 cells displayed a CD44-dependent ability to bind HA. The ability of activated cell CD44 to bind HA was not dependent on microfilament or microtubule integrity or on changes in CD44 mobility on the membrane plane, indicating that the CD44 activation status is not associated with cytoskeleton function. Aside from the increased expression of CD44 on the surface of PMA-activated LB cells and HA9 cells, qualitative differences between the CD44 of nonactivated and activated LB cells were also detected: the CD44 of the activated lymphoma was (i) larger in molecular size, (ii) displayed a broader CD44 isoform repertoire, including a CD44 variant that binds HA, and (iii) its glycoprotein contained less sialic acid. Indeed, after removal of sialic acid from their cell surface by neuraminidase, LB cells acquired the ability to bind HA. However, a reduced dose of neuraminidase did not confer HA binding on LB cells, unless they were also activated by a low concentration of PMA, which by itself was ineffective. Similarly, under suboptimal conditions, a synergistic effect was obtained with tunicamycin and PMA: each one alone was ineffective but in combination they induced the acquisition of HA binding by the lymphoma cells, while their CD44 expression was not enhanced. Unveiling of the activation mechanism of CD44, by exposing the cells to PMA stimulation or to deglycosylation, is not only academically important, but it also has practical implications, as activated CD44 may be involved in the support of tumor progression.

Animals↗
PubMed1997

Absence of functional CD44 hyaluronan receptor on human NMYC-amplified neuroblastoma cells.

CD44 represents a heterogeneous group of surface glycoproteins, involved in cell-cell and cell-matrix interactions. CD44 is the major receptor for hyaluronate (HA), a component of cell matrices, and most of CD44 known functions are attributed to its ability to recognize HA. We have recently shown that although a majority of human neuroblastomas (NBs), a childhood cancer, express high levels of CD44H, high stages and tumors with amplification of the NMYC proto-oncogene fail to express CD44. Lack of CD44 expression is strongly associated with the presence of NMYC amplification and has been further shown to represent a new feature for predicting risk of disease progression and dissemination. In the present study, we have investigated the role of CD44 expressed by NB cell lines and the possible relationship among the presence of NMYC amplification, functional expression of CD44 receptor, and tumorigenic properties of NB cells. A panel of cell lines with variable NMYC amplification and/or overexpression, as well as clonal and stable NMYC-transfected NB cells, were analyzed for CD44 expression and ability to bind HA. Our results confirmed previous observations that in NB cell lines lack of CD44 is not always related to the presence of NMYC amplification, with a number of cell lines or transfectants with both CD44 expression and NMYC amplification. However, the ability of the CD44 receptor to bind immobilized hyaluronan was restricted to CD44H+ cell lines without NMYC amplification (SH-EP and ACN). The HA-binding function was CD44 dependent and could be specifically blocked by an anti-CD44 antibody. No induction of functional HA binding was obtained with NMYC-amplified cell lines or NMYC transfectants, despite an induced increase of CD44 expression upon differentiation or after tentative activation of the receptor with phorbol esters. Inhibition of N-linked glycosylation with tunicamycin resulted in decreased HA binding of cells bearing an active CD44 receptor. We conclude that NMYC-amplified NB cell lines either do not express CD44 at all or express a nonfunctional receptor, whereas nonamplified cells constitutively express an active receptor. The lack of functional HA binding in NB cells might be partly due to incomplete N-glycosylation. The involvement of NMYC in the regulation of N-linked glycosylation can be suspected.

Antibodies, Blocking↗
PubMed2002

Trafficking of CD44-deficient murine lymphocytes under normal and inflammatory conditions.

CD44 has been implicated in hyaluronan (HA)-dependent primary adhesion between leukocytes and endothelium. We studied the trafficking of lymphocytes of CD44-deficient DBA/1 mice under normal conditions, and during chronic and transient forms of inflammation. Animals homozygous for the CD44 mutation (CD44(-/-)) showed no abnormalities in the composition of the lympho-hemopoietic system, but their leukocytes could not recognize HA as an adhesion ligand. T cells from CD44-deficient mice responded normally to immunization with type II collagen or stimulation with a bacterial superantigen. Lymphocytes harvested from naive CD44(-/-) and wild-type (WT) animals showed similar trafficking properties when injected into naive recipients. However, cells from WT and CD44-deficient mice with collagen-induced arthritis showed distinct migration kinetics upon transfer to arthritic recipients. While lymphocytes from CD44(-/-) mice preferentially homed to lymph nodes, their entry into the inflamed synovial joints was delayed as compared with WT cells. Similar differences were observed in the migration kinetics of CD44-deficient and CD44-competent (CD44(+/+)) lymphocytes in bacterial superantigen-induced peritonitis. These results suggest that CD44 plays opposite roles in the regulation of leukocyte traffic to inflammatory sites versus the lymph nodes. CD44-deficient lymphocytes from animals with chronic arthritis, but not from those with transient peritonitis, expressed markedly reduced levels of the lymph node homing receptor, L-selectin. Extreme down-modulation of L-selectin from CD44(-/-) cells in arthritic condition might be a counter-regulatory response, which, by extending lymphocyte transit time in the circulation at the expense of lymph node homing, allows CD44-deficient cells to gain entry to the site of chronic inflammation via secondary adhesion mechanisms.

Animals↗
PubMed2004

Cytokines regulate the affinity of soluble CD44 for hyaluronan.

CD44, a receptor for the extracellular matrix glycosaminoglycan hyaluronan, has been implicated in many adhesion-dependent cellular processes including tumor growth and metastasis. Soluble CD44 has been identified in the serum of normal individuals. Furthermore, tumor progression is often associated with marked increases in plasma levels of soluble CD44. Release of soluble CD44 by proteolytic cleavage (shedding) of membrane-anchored CD44 is likely to alter cellular responses to the environment due to modification of the cell surface and the potential for soluble CD44 to influence CD44-mediated hyaluronan binding to cell surfaces. Cellular activation is typically required to induce hyaluronan binding to cell surface CD44 but the affinity of endogenous soluble CD44 for hyaluronan remains unknown. In this study, we demonstrate that oncostatin M and transforming growth factor beta1 (TGF-beta1) which stimulate hyaluronan binding to HTB58 lung epithelial-derived tumor cells, also induce the release of soluble CD44. Interestingly, soluble CD44 released by oncostatin M-treated cells retained the ligand-binding properties of the membrane-anchored receptor. In contrast, soluble CD44 released from TGF-beta1-treated HTB58 cells differed in its hyaluronan-binding capacity from cell surface CD44 expressed on TGF-beta1-stimulated cells. These data indicate that the mechanisms that regulate the generation of soluble CD44 may also govern the binding of the released receptor to hyaluronan and therefore determine the impact on CD44-dependent physiologic and pathologic processes.

Cell Line, Tumor↗
PubMed1994

CD44 isoform expression mediated by alternative splicing: tissue-specific regulation in mice.

CD44 is a widely distributed cell surface glycoprotein which shows heterogeneity in molecular expression as a result of post-translational modification as well as alternative splicing of CD44 mRNA. Functional studies have indicated that CD44 plays a role as an adhesion molecule and that different CD44-expressing cells differ in their capacities for CD44-dependent ligand binding. These observations have raised the possibility that structural modifications of CD44, including those resulting from alternatively spliced mRNA isoforms, are involved in the functional heterogeneity of CD44. To assess the expression of CD44 isoforms in the mouse, we examined CD44 cDNA by reverse transcription polymerase chain reaction (RT-PCR). Southern blotting of PCR products with a CD44 cDNA probe or with internal oligonucleotides revealed the expression in mouse tumor cell lines and normal tissues of multiple CD44 mRNA products which are larger than that observed in the absence of variable exon expression. Interestingly, different mouse tissues, including lymphoid cells, showed unique patterns of alternative CD44 mRNA in Southern blotting analysis. The use of exon-specific primers allowed detection of multiple alternatively spliced mRNA species involving expression of at least seven variable exons. Cloning and sequencing of these PCR products revealed sequence identity with recently identified genomic CD44 sequences and confirmed that the PCR products correspond to mature mRNA expressing alternatively spliced CD44 exons. Taken together, these findings demonstrate that the mouse expresses multiple variably spliced CD44 isoforms and that expression is regulated in a tissue- and cell-type specific manner.

Alternative Splicing↗
PubMed1998

CD44 expression in normal adrenal tissue and adrenal tumours.

BACKGROUND: CD44 is a cell surface glycoprotein found on many normal cells, mainly lymphoid and epithelial. Normal cells usually express standard CD44 (CD44-S), whereas malignant tumours may express CD44 variant isoforms (CD44-V). CD44 expression has been described for neural crest derivatives. Characterisation of differences in CD44 expression may help in the diagnosis and differentiation of distinct adrenal tumours. AIMS: To examine CD44 expression in different layers of cortical cortex, in adrenal medulla, and in adrenal tumours. METHODS: CD44-S and CD44-V6 expression were studied in 12 cases of adrenal cortical adenoma, 3 of adrenal cortical carcinoma, 10 of pheochromocytoma, and 4 normal adrenal glands. RESULTS: CD44-V6 staining showed cytoplasmic expression in normal adrenal cortex and in cortical adenomas and carcinomas. Pheochromocytomas also showed CD44-V6 expression but in 5 of the 10 cases it was sparse, focal, and sometimes perinuclear. Strong membranous staining for CD44-S was observed in normal adrenal medulla. Analysis of CD44-S expression revealed differences between cortical adrenal tumours and pheochromocytomas. Ten of 12 cortical adenomas and 2 of 3 cortical carcinoma cells showed weak to moderate cytoplasmic staining, but all cases of pheochromocytoma had strong membranous staining. CONCLUSIONS: Membranous CD44-S staining may help to distinguish pheochromocytoma from adrenal cortical adenoma.

Adrenal Cortex↗
PubMed1998

The normal structure and function of CD44 and its role in neoplasia.

CD44 is a transmembrane glycoprotein, the variant isoforms of which are coded for by alternative splicing, with the most prolific isoform being CD44 standard. CD44 is found in a wide variety of tissues including the central nervous system, lung, epidermis, liver, and pancreas, whereas variant isoforms of CD44 (CD44v) appear to have a much more restricted distribution. Variants of CD44 are expressed in tissues during development, including embryonic epithelia. Known functions of CD44 are cellular adhesion (aggregation and migration), hyaluronate degradation, lymphocyte activation, lymph node homing, myelopoiesis and lymphopoiesis, angiogenesis, and release of cytokines. The functions of CD44 are principally dependant on cellular adhesion in one setting or another. The role of CD44 in neoplasia is less well defined, although metastatic potential can be conferred on non-metastasising cell lines by transfection with a variant of CD44 and high levels of CD44 are associated with several types of malignant tumours. The physiological functions of CD44 indicate that the molecule could be involved in the metastatic spread of tumours. Many studies have investigated the pattern of CD44 distribution in tumours and some observations suggest that certain cells do not use CD44 in tumorigenesis or in the production of metastases. However, the data are extremely conflicting, and further studies are needed to establish the prognostic value of CD44 and its variant isoforms. The precise function of CD44 in the metastatic process and the degree of involvement in human malignancies has yet to be established fully.

Antigens, Neoplasm↗
PubMed2005

The role of the hyaluronan receptor CD44 in mesenchymal stem cell migration in the extracellular matrix.

In a previous investigation, we demonstrated that mesenchymal stem cells (MSCs) actively migrated to cardiac allografts and contributed to graft fibrosis and, to a lesser extent, to myocardial regeneration. The cellular/molecular mechanism responsible for MSC migration, however, is poorly understood. This paper examines the role of CD44-hyaluronan interaction in MSC migration, using a rat MSC line Ap8c3 and mouse CD44-/- or CD44+/+ bone marrow stromal cells (BMSCs). Platelet-derived growth factor (PDGF) stimulation of MSC Ap8c3 cells significantly increased the levels of cell surface CD44 detected by flow cytometry. The CD44 standard isoform was predominantly expressed by Ap8c3 cells, accounting for 90% of the CD44 mRNA determined by quantitative real-time polymerase chain reaction. Mouse CD44-/- BMSCs bonded inefficiently to hyaluronic acid (HA), whereas CD44+/+ BMSC and MSC Ap8c3 adhered strongly to HA. Adhesions of MSC Ap8c3 cells to HA were suppressed by anti-CD44 antibody and by CD44 small interfering RNA (siRNA). HA coating of the migration chamber significantly promoted passage of CD44+/+ BMSC or Ap8c3 cells, but not CD44-/- BMSCs, through the insert membranes (p < .01). Migration of MSC Ap8c3 was significantly inhibited by anti-CD44 antibodies (p < .01) and to a lesser extent by CD44 siRNA (p = .05). The data indicate that MSC Ap8c3 cells, in response to PDGF stimulation, express high levels of CD44 standard (CD44s) isoform, which facilitates cell migration through interaction with extracellular HA. Such a migratory mechanism could be critical for recruitment of MSCs into wound sites for the proposition of tissue regeneration, as well as for migration of fibroblast progenitors to allografts in the development of graft fibrosis.

Animals↗
PubMed2001

Antibody-induced shedding of CD44 from adherent cells is linked to the assembly of the cytoskeleton.

CD44 is a widely expressed integral membrane glycoprotein that serves as a specific adhesion receptor for the extracellular matrix glycosaminoglycan hyaluronan. CD44 participates in a variety of physiological and pathological processes through its role in cell adhesion. Under appropriate conditions, the ectodomain of CD44 is proteolytically removed from the cell surface. In this study we show that excessive CD44 shedding can be induced in mouse fibroblasts and monocytes upon exposure of these cells to a CD44-specific Ab immobilized on plastic, whereas treatment with phorbol ester induces significantly enhanced CD44 release from the monocytes only. CD44 shedding proceeds normally in fibroblasts and monocytes deficient in TNF-alpha converting enzyme (TACE), a sheddase involved in the processing of several substrates. Conversely, activation of the CD44 protease has no effect on the release of TNF-alpha from TACE-expressing cells, although the same metalloprotease inhibitor effectively blocks both TACE and the CD44 sheddase. Concomitant with anti-CD44 Ab- or phorbol ester-induced CD44 shedding, dramatic changes are observed in cell morphology and the structure of the actin cytoskeleton. Disruption of actin assembly with cytochalasin reduces CD44 shedding, but not the release of TNF-alpha. Moreover, pharmacological activation of Rho family GTPases Rac1 and Cdc42, which regulate actin filament assembly into distinct cytoskeletal structures, has a profound effect on CD44 release. We conclude that the CD44 sheddase and TACE are distinct enzymes, and that Ab- and phorbol ester-enhanced cleavage of CD44 is controlled in a cell type-dependent fashion by Rho GTPases through the cytoskeleton.

ADAM Proteins↗
PubMed2005

[The role of CD44 in the proliferation, adhesiveness and invasiveness of osteosarcoma cell lines].

OBJECTIVE: To study the influence of CD44 a cell-matrix adhesion molecule on the proliferation, adhesiveness and invasiveness of osteosarcoma cell lines, in order to investigate the growth and invasion mechanism of osteosarcoma. METHODS: Three osteosarcoma cell lines MG-63, HOS and U2-OS were routinely cultured. Flow cytometry and Western blot analysis were used for detecting the positive rates and relative amount of CD44 protein in the three cell lines. RT-PCR method was also used to compare the differences in the expression of CD44 mRNA among the 3 cell lines. Then, MTT method, adhesion detection, and Microcon-migration assay were used to detect the changes of the cells' proliferation rate, adhesive and invasive abilities after blocking the role of CD44 by using a special neutralizing antibody. RESULTS: The results of flow cytometry showed that the percentage of CD44 positive cells were both over 99% in HOS and U2-OS, while that in MG-63 was only (2.10 +/- 0.46)%. The average fluorescence density of CD44 in HOS was significantly higher than in U2-OS. Western blot also showed that the relative content of CD44 protein in HOS was notably higher than that in U2-OS, while CD44 was negatively expressed in MG-63. The expression of CD44 mRNA was significantly lower in MG-63 than in both HOS and U2-OS, which were consistent with the expression of CD44 protein. The proliferation rates and adhesive abilities of MG-63 and HOS have no significant difference, but both were significantly higher than that of U2-OS. The invasive abilities of HOS was dramatically higher than MG-63 and U2-OS. After the role of CD44 was blocked by anti-CD44 neutralizing antibody, the proliferation rates of the 3 cell lines did not change significantly. While the HOS and MG-63 adhesion indices decreased dramatically (P < 0.05), the invasive abilities of HOS and U2-OS also decreased notably (P < 0.01). CONCLUSIONS: CD44 could promote the adhesiveness and invasiveness of osteosarcoma cell line HOS. CD44 may take part in promoting the process of U2-OS invasion and the adhesion of MG-63. On the other hand, CD44 could not affect the osteosarcoma cell proliferation rates.

Bone Neoplasms↗
PubMed1993

Regulation of human CD44H and CD44E isoform binding to hyaluronan by phorbol myristate acetate and anti-CD44 monoclonal and polyclonal antibodies.

CD44 molecules are comprised of multiple alternatively spliced forms and are associated with diverse functions such as mediation of carcinoma metastasis and T cell coactivation. To study the function of individual CD44 isoforms, we have transfected CD44 isoforms into CD44-negative Jurkat T cells and produced cloned Jurkat cell lines that are stably transfected with either a CD44 isoform containing no alternatively spliced insert (CD44H) or a CD44 variant (CD44E) containing an insert of 132 amino acids derived from exons 12, 13, and 14 of the CD44 gene. We found that neither CD44H- nor CD44E-transfected Jurkat T cells constitutively bound hyaluronan (HA), whereas PMA treatment induced Jurkat cells transfected with CD44H but not CD44E to bind HA. CD44 mAb against noninsert regions of the CD44 extracellular domain (A3D8, A1G3) and polyclonal antisera against the COOH-terminal extracellular glycosaminoglycan region of CD44H (anti-6A serum) both induced CD44H-transfected cells to bind HA, whereas only one CD44 mAb (A1G3) induced CD44E-transfected Jurkat T cells to bind HA. Studies of Jurkat cells transfected with CD44H forms with truncations of the CD44 cytoplasmic domain demonstrated that the cytoplasmic COOH-terminal 52 amino acids were critical for binding of HA to the CD44 extracellular domain. Thus, these data underscore the importance of the CD44 cytoplasmic domain in the function of the extracellular portion of CD44H, and demonstrate a role for ligation of human CD44 isoforms at multiple distinct sites in regulation of expression of CD44 binding to HA.

Amino Acid Sequence↗
PubMed1994

Characterization of soluble CD44 in the circulation of mice. Levels are affected by immune activity and tumor growth.

ELISA determinations revealed substantial concentrations (0.49 to 2.10 micrograms/ml) of soluble CD44 in murine serum, with some variation among normal mouse strains. At least three species of CD44 were identified by immunoprecipitation and SDS-PAGE analysis of serum. The most prominent was indistinguishable in mobility from that extracted from normal and transformed lymphocytes and was estimated in this way to be approximately 90 kDa. A similar estimate resulted from gel filtration under nondenaturing conditions, followed by ELISA. However, lymphocyte membrane-extracted and soluble CD44 had different mobilities after treatment with neuraminidase plus O-glycosidase, and the core protein of soluble CD44 might be 17 to 20 kDa smaller than that of CD44 on lymphocyte membranes. Furthermore, an Ab to cytoplasmic residues of CD44 failed to recognize soluble CD44 recovered from the circulation or in lymphoma culture supernatants. These observations would be consistent with cleavage of CD44 from cell surfaces; and protease inhibitors slowed the loss of CD44 from cultured lymphomas. Serum CD44 levels were significantly reduced in immunodeficient CD17.SCID and BALB/c.Xid mice, and elevated in tumor-bearing mice. Mild graft-vs-host (GVH) reactions also resulted in increased concentrations of CD44, as did autoimmune disease in BXSB and MRL/lpr strains of mice. Serum with high concentrations of CD44 partially blocked the binding of one ligand, hyaluronate, to CD44-bearing hybridoma cells. The degree of inhibition was positively correlated with CD44 concentration. These findings indicate that substantial quantities of CD44 can be released into the circulation by cleavage from cell surfaces and that this process is markedly influenced by immune system activity and tumor growth. The material seemed to be intact and potentially functional.

Amino Acid Sequence↗
PubMed2000

Regulation of the cell adhesion molecule CD44 after nerve transection and direct trauma to the mouse brain.

CD44 is a cell surface glycoprotein involved in cell adhesion during neurite outgrowth, leukocyte homing, and tumor metastasis. In the current study, we examined the regulation of this molecule 4 days after neural trauma in different forms of central and peripheral injury. Transection of the hypoglossal, vagus, or sciatic nerve led to the appearance of CD44-immunoreactivity (CD44-IR) on the surface of the affected motoneurons, their dendrites, and their axons. Fimbria fornix transection led to CD44-IR on a subpopulation of cholinergic neurons in the ipsi- and contralateral medial septum and diagonal band of Broca and colocalized with galanin-IR. Central projections of axotomized sensory neurons to the spinal cord (substantia gelatinosa, Clarke's column) also showed an increase in CD44-IR, which was abolished by spinal root transection. Nonneuronal CD44-IR was mainly restricted to sites of direct injury. In the crushed sciatic nerve, CD44-IR was found on the demyelinating Schwann cells and on infiltrating monocytes and granulocytes. Direct parasagittal transection of the cerebral cortex led to CD44-IR on resident astrocytes and on leukocytes entering the injured forebrain tissue. CD44-IR also increased on reactive retinal astrocytes and microglia after the optic nerve crush. Additional time points in the retina and hypoglossal nucleus (days 1, 2, and 14) and cerebral cortex (day 2) injury models also showed the same cell type pattern for the CD44-IR. Finally, polymerase chain reaction analysis confirmed the posttraumatic expression of CD44 mRNA and detected only the standard haematopoietic CD44 splice isoform both in direct and indirect brain injury models. Overall, the current study shows the widespread, graded appearance of CD44-IR on neurons and on nonneuronal cells, depending on the form of neural injury. Here, the ability of CD44 to bind to a variety of extracellular matrix and cell adhesion proteins and its common presence in different forms of brain pathology could suggest an important role for this cell surface glycoprotein in the neuronal, glial, and leukocyte response to trauma and in the repair of the damaged nervous system.

Animals↗
PubMed2004

Osteopontin modulates CD44-dependent chemotaxis of peritoneal macrophages through G-protein-coupled receptors: evidence of a role for an intracellular form of osteopontin.

Expression of osteopontin (OPN) by activated T-cells and macrophages is required for the development of cell-mediated inflammatory responses. Acting through integrin alpha(v)beta(3) and CD44 receptors, OPN can promote chemoattraction and pro-inflammatory cytokine expression by macrophages. In this study, we have used peritoneal macrophages from OPN-/, CD44-/-, and WT mice to study the relationship between OPN and CD44 in macrophage migration. Using confocal microscopy, we show that OPN co-distributes with CD44 inside macrophages at cell edges and in cell processes in a mutually dependent manner. The existence of an intracellular form of OPN is supported by pulse-chase studies in which a thrombin-sensitive, phosphorylated protein immunoprecipitated with OPN antibodies is retained inside macrophages. In OPN-/- and CD44-/- macrophages, the absence of CD44 and OPN, respectively, is associated with the formation of fewer cell processes, reduced cell fusion required to form functional multinucleated osteoclasts in the presence of CSF-1 and RANKL, and impaired chemotaxis. Whereas the chemotaxis of CD44-/- cells to various chemoattractants is almost completely abrogated, a differential effect is seen with the OPN-/- cells. Thus, OPN-/- cells migrate normally towards CSF-1 but not towards fMLP and MCP-1, which signal through G-protein coupled receptors (GPCRs). That the GPCR-mediated migration is dependent upon the level of cell-surface CD44 is indicated by the reduced cell-surface expression of CD44 in OPN-/- cells and a comparable impairment in the chemotaxis of CD44+/- cells. Although chemotaxis of OPN-/- cells could be rescued by an OPN substratum, or by addition of high levels of OPN in solution, no response is evident with physiological levels of OPN, indicating a requirement for the CD44-associated intracellular OPN in CD44 cell-surface expression. These studies indicate, therefore, that the level of cell surface CD44 is critical for GPCR-mediated chemotaxis by peritoneal macrophages and suggest that a novel intracellular form of OPN may modulate CD44 activities involved in these processes.

Actins↗
PubMed1996

O-linked glycosylation modifies CD44 adhesion to hyaluronate in colon carcinoma cells.

CD44 alternative splicing patterns differ between normal and malignant tissue, and accordingly, modulation of CD44 splicing has received the most attention in studies that have examined the role of CD44 in tumor progression. Many investigators have examined functional differences between individual CD44 alternative splice variants. However, specific CD44 isoforms function uniquely depending on the type of cell on which they are expressed, thereby suggesting that additional tissue-specific mechanisms regulate CD44 function. In the present study we have demonstrated that colon carcinoma cells modify CD44 with O-linked glycosyl groups, and blockade of this glycosylation enhances their CD44-mediated adhesion to hyaluronate. This enhancement is attributable principally to CD44H (CD44s) rather than high molecular weight CD44 variants. Use of site-directed mutant CD44H cDNA transfectants demonstrated that CD44 O-linked glycosylation modulates interaction between hyaluronate and the B loop domain of CD44. The influence of glycosylation on CD44 function in colon carcinoma cells is specific to the presence of O-linked sugars; inhibition of N-linked glycosylation had minimal influence on CD44 function. These findings indicate that O-linked glycosylation may be as important as alternative splicing in the regulation of CD44 function and the broad spectrum of biological processes attributed to it, including normal development, tumor metastases, and lymphocyte function.

Alternative Splicing↗
PubMed1995

Evaluation of CD44 prognostic value in neuroblastoma: comparison with the other prognostic factors.

CD44 gene products are potential markers of aggressiveness in different tumour models, a result which prompted us to study clinical neuroblastoma (NB) specimens. CD44 expression was determined by immunostaining of 52 tumour samples from newly diagnosed NB with a monoclonal antibody (J173) directed against an epitope common to all CD44 isoforms. CD44 immunoreactivity was detected in 37 of the tumours (71%). CD44 was expressed in all 22 NBs with favourable prognoses (stages 1, 2 or 4S), but only 50% (15/30) of advanced NB (stages 3 and 4) (P < 10(-4)), suggesting that the absence, rather than the overexpression, of CD44 is a signal of tumour aggressiveness. The cumulative progression-free survival was significantly longer in patients with CD44 positive tumours compared with patients with CD44 negative tumours (P < 10(-5)). More importantly, progression-free survival was also significantly higher in CD44 positive patients within the high-risk group (P < 0.01). In univariate analysis, we tested the prognostic value of tumour expression of CD44 in comparison with tumour stage, age, tumour histology, and presence or absence of amplification of the MYCN protooncogene. All five measures had significant prognostic value. The expression of CD44 and the absence of MYCN amplification were the most powerful predictors of a favourable outcome. In a multivariate analysis of these measures, CD44 expression and tumour stage were the only independent prognostic factors for the prediction of patient survival. NB is the first clinical model described in which tumour aggressiveness correlates with repression rather than stimulation of CD44 expression. We recommend the use of CD44 as an additional biological marker in the initial staging of NB.

Adolescent↗