CD44 Shedding Research Articles

CD44--a sticky target for asthma.

CD44--a sticky target for asthma.

CD44 directs membrane-type 1 matrix metalloproteinase to lamellipodia by associating with its hemopexin-like domain.

Membrane-type 1 matrix metalloproteinase (MT1- MMP) localizes at the front of migrating cells and degrades the extracellular matrix barrier during cancer invasion. However, it is poorly understood how the polarized distribution of MT1-MMP at the migration front is regulated. Here, we demonstrate that MT1-MMP forms a complex with CD44H via the hemopexin-like (PEX) domain. A mutant MT1-MMP lacking the PEX domain failed to bind CD44H and did not localize at the lamellipodia. The cytoplasmic tail of CD44H, which comprises interfaces that associate with the actin cytoskeleton, was important for its localization at lamellipodia. Overexpression of a CD44H mutant lacking the cytoplasmic tail also prevented MT1-MMP from localizing at the lamellipodia. Modulation of F-actin with cytochalasin D revealed that both CD44H and MT1-MMP co-localize closely with the actin cytoskeleton, dependent on the cytoplasmic tail of CD44H. Thus, CD44H appears to act as a linker that connects MT1-MMP to the actin cytoskeleton and to play a role in directing MT1-MMP to the migration front. The PEX domain of MT1-MMP was indispensable in promoting cell migration and CD44H shedding.

Breaking Free of the ECM

CD44 is a transmembrane cell adhesion receptor that binds many components of the extracellular matrix (ECM). Kajita et al. show that when the transmembrane matrix metalloproteinases TM1-MMP or TM3-MMP are coexpressed (by transfection) with the H isoform of CD44, a soluble form of CD44 is shed into the culture medium. Such cells have a different morphology and enhanced motility relative to cells expressing CD44 alone. If the activity of the TM1-MMP was inhibited, then CD44 did not appear in the culture medium, and motility was decreased. In cells transfected to express only CD44 or when the TM1-MMP activity was inhibited, the cells formed small and large protrusions, which suggests that such cells had more substrate attachment sites than did cells in which CD44 was cleaved. The physiological importance of the cleavage of CD44 was investigated in a pancreatic tumor cell line that spontaneously sheds soluble CD44 in a manner dependent on the activity of TM1-MMP. Inhibition of CD44 cleavage and shedding decreased cell motility, as did expression of a mutant form of CD44 in which the cleavage site was eliminated. Thus, breaking the bonds that tether cells to the ECM (by disrupting the CD44 and ECM interaction) may represent one mechanism by which cell motility can be enhanced.M. Kajita, T. Itoh, T. Chiba, H. Mori, A. Okada, H. Kinoh, M. Seiki, Membrane-type 1 matrix metalloproteinase cleaves CD44 and promotes cell migration. J. Cell Biol. 153, 893-904 (2001). [Abstract] [Full Text]

Breaking Free of the ECM

CD44 is a transmembrane cell adhesion receptor that binds many components of the extracellular matrix (ECM). Kajita et al. show that when the transmembrane matrix metalloproteinases TM1-MMP or TM3-MMP are coexpressed (by transfection) with the H isoform of CD44, a soluble form of CD44 is shed into the culture medium. Such cells have a different morphology and enhanced motility relative to cells expressing CD44 alone. If the activity of the TM1-MMP was inhibited, then CD44 did not appear in the culture medium, and motility was decreased. In cells transfected to express only CD44 or when the TM1-MMP activity was inhibited, the cells formed small and large protrusions, which suggests that such cells had more substrate attachment sites than did cells in which CD44 was cleaved. The physiological importance of the cleavage of CD44 was investigated in a pancreatic tumor cell line that spontaneously sheds soluble CD44 in a manner dependent on the activity of TM1-MMP. Inhibition of CD44 cleavage and shedding decreased cell motility, as did expression of a mutant form of CD44 in which the cleavage site was eliminated. Thus, breaking the bonds that tether cells to the ECM (by disrupting the CD44 and ECM interaction) may represent one mechanism by which cell motility can be enhanced. M. Kajita, T. Itoh, T. Chiba, H. Mori, A. Okada, H. Kinoh, M. Seiki, Membrane-type 1 matrix metalloproteinase cleaves CD44 and promotes cell migration. J. Cell Biol. 153 , 893-904 (2001). [Abstract] [Full Text]

Myoepithelial-Specific CD44 Shedding Is Mediated by a Putative Chymotrypsin-like Sheddase

Our previous studies have demonstrated that myoepithelial cells, which surround incipient carcinomas in situ of the breast and other organs, exert antiinvasive and antiangiogenic effects in vitro through the elaboration of a number of different suppressor molecules among which include the shed membrane CD44. The present study addresses the mechanism of this myoepithelial CD44 shedding. This CD44 shedding is enhanced by PMA pretreatment, is specific for myoepithelial CD44, and inhibited by chymotrypsin-like inhibitors (chymostatin, α1-antichymotrypsin, TPCK, and SCCA-2) but not by trypsin-like inhibitors (TLCK), nor papain-like inhibitors (SCCA-1) nor hydroxamate-based or general metalloproteinase inhibitors (BB2516 (marimastat), 1,10-phenanthroline, and TIMP-1). The effect of PMA can be mimicked by exogenous chymotrypsin but not by other proteases. The CD44 shedding activity cannot be transferred by conditioned media, cell–cell contact, peripheral membrane, or integral membrane fractions. However, cell-free purified integral plasma membrane fractions obtained from myoepithelial cells pretreated with PMA also exhibit CD44 shedding which is inhibited by chymotrypsin-like inhibitors. These findings support the presence and activation of a putative chymotrypsin-like sheddase as the mechanism of CD44 shedding in myoepithelial cells.

Myoepithelial-Specific CD44 Shedding Contributes to the Anti-invasive and Antiangiogenic Phenotype of Myoepithelial Cells

Myoepithelial cells surround incipient ductal carcinomas of the breast and exert anti-invasive and antiangiogenic effects in vitro through the elaboration of suppressor molecules. This study examines one putative molecule, solubilized CD44 produced by myoepithelial shedding of membrane CD44. Studies with different human myoepithelial cell lines demonstrate that myoepithelial cells express and shed both the 85-kDa standard (CD44s) and the 130-kDa epithelial (CD44v8-10) isoforms, findings further confirmed by the use of isoform-specific antibodies. PMA pretreatment enhances CD44 shedding detected by two different methods at different time points: a reduction in surface CD44 at 2 h by flow cytometry and a marked decrease in both total cellular CD44 and plasma membrane CD44 at 12 h by Western blot. This shedding is both specific for CD44 and specific for myoepithelial cells. This shedding is inhibited by the chymotrypsin inhibitors chymostatin and α1-antichymotrypsin but not by general metallo-, cysteine, or other serine proteinase inhibitors. Myoepithelial-cell-conditioned medium and affinity-purified solubilized CD44 from this conditioned medium block hyaluronan adhesion and migration of both human carcinoma cell lines and human umbilical vein endothelial cells.

CD44 is involved in selective leucocyte extravasation during inflammatory central nervous system disease.

Clinical signs of experimental autoimmune encephalomyelitis (EAE) are associated with the selective recruitment of CD4+ memory (CD45RBlow CD44high) T cells into the central nervous system (CNS). However, we have found that many of these recently recruited memory cells are CD44low, suggesting that the CD44 antigen may be involved in, and transiently lost during, the extravasation process. Indeed, administration of a CD44-specific antibody (IM7.8.1) induced leucocyte CD44 shedding and both prevented the development and ameliorated the severity of established EAE by inhibiting mononuclear cell infiltration into the CNS. Trafficking of cells into lymph nodes, however, a property mainly of naïve cells, was essentially unaffected. In contrast, treatment with antibody to very late activation antigen-4 (VLA-4) prevented homing to both the CNS and to lymph nodes. This study contests previous reports that dismissed a role for CD44 in inflammation of the CNS and, coupled with observations in murine dermatitis and arthritis, suggests that CD44 is involved in the homing of primed lymphocytes to sites of inflammation. CD44 should therefore be considered a target for immunotherapy of T-cell-mediated inflammatory diseases, such as multiple sclerosis.

Modulation of hyaluronan receptor (CD44) function in vivo in a murine model of rheumatoid arthritis.

To determine how in vivo modulation of CD44 function by antibodies influences disease severity in a murine model of rheumatoid arthritis. Mice with proteoglycan (PG)-induced arthritis were subjected to systemic treatment with 3 different monoclonal antibodies against CD44. Joint swelling and serum levels of hyaluronan (HA) and soluble CD44 (sCD44) were monitored. Inflammatory leukocyte infiltration in the joints, cell surface CD44 expression, and leukocyte adhesion to HA were compared. The effects of anti-CD44 treatment on the immune status of arthritic animals were also determined. Antibody IRAWB14, which enhances HA binding, aggravated the inflammatory symptoms, while KM201, which blocks ligand binding, reduced the severity of arthritis. The most effective suppression of inflammation was noted upon treatment with antibody IM7, whose epitope lies outside the HA binding domain of CD44. Serum levels of sCD44 increased, and HA levels decreased, in response to IM7. KM201 and IM7 treatment reduced, but IRAWB14 treatment enhanced, the adhesion of leukocytes to HA. However, these antibodies had little effect on PG-specific immune responses. Each antibody acted in vivo by virtue of its combined effects on CD44-HA binding and CD44 shedding. The dramatic reduction in arthritis severity effected by IM7 treatment was associated with extensive shedding of cell surface CD44 molecules. Loss of CD44 appears to be a major factor in preventing CD44- and HA-dependent cell-matrix interactions at the inflammatory site. Our study indicates a critical role for CD44 in the pathology of joint inflammation and reveals a unique mechanism of receptor down-regulation, which can be used therapeutically.

Shedding of CD44 from PMA-Differentiated U-937 Cells Is Enhanced by Treatment with Mineral Particles

In this report, we show that enhanced shedding of CD44 might contribute to the down-regulation of this receptor observed after phagocytosis of MnO2particles by PMA-differentiated U-937. The apparent Mr of the soluble CD44 detected in culture supernatants was slightly lower than that of the membrane form suggesting that shedding resulted from proteolytic cleavage. Increased shedding of CD44 was also noted with other mineral particles (chrysotile and DQ12) but to a lower extent whereas some (TiO2and amosite) had no effect on this process. These results indicate that shedding enhancement was particle-specific rather than a general consequence of phagocytosis. The ability of the particles to enhance CD44 shedding was not directly dependent on their cytotoxic potency. Different patterns of reactivity were noted with CD11b, suggesting that the underlying mechanisms are specific.

Characterization of CD44-mediated hyaluronan binding by renal tubular epithelial cells.

CD44 is the main receptor for the extracellular polysaccharide hyaluronan (HA). We have recently shown that CD44 is strongly induced on renal tubular epithelial cells (TEC) in autoimmune renal injury and that HA accumulates in the renal interstitium (Kidney Int 1996; 50: 156-163 and Nephrol Dial Transplant 1997; 12: 1344-1353). The functional significance of enhanced tubular CD44 expression and its interaction with HA are not known. The purpose of the present study was to characterize renal tubular CD44 expression and CD44-mediated HA binding in vitro and to investigate the growth modulating effects in response to HA binding by TEC. RT-PCR analysis, flow cytometry, confocal microscopy and Western blotting were used to examine cell surface and soluble CD44 expression by cultured TEC, using SV40-transformed mouse cortical tubular (MCT) cells. HA binding characteristics were examined by flow cytometry and effects of HA on TEC cell growth by [3H]thymidine incorporation. By RT-PCR analysis MCT cells expressed predominantly the standard form of CD44 mRNA, whereas the expression of variant forms was very weak. Confocal microscopy showed that CD44 was expressed basolaterally and apically on MCT cells with strong staining on microvilli. Shedding of CD44 from MCT cells could be induced with crosslinking of anti-CD44 mAbs or with PMA stimulation. MCT cells constitutively bound HA and this binding could be modulated with anti-CD44 mAbs. Soluble and plate-bound HA markedly inhibited MCT cell growth. CD44 is a regulated HA receptor on MCT cells which can be shed into the cellular environment. Upon binding of HA, CD44 functions as a growth inhibitory cell surface protein in MCT cells. We speculate that the interaction of CD44 with HA may have important regulatory effects on cell proliferation in tubulointerstitial renal diseases.

Early detachment of colon carcinoma cells during CD95(APO-1/Fas)-mediated apoptosis. I. De-adhesion from hyaluronate by shedding of CD44

Early detachment of colon carcinoma cells during CD95(APO-1/Fas)-mediated apoptosis. I. De-adhesion from hyaluronate by shedding of CD44

Expression and shedding of CD44 variant isoforms in patients with gynecologic malignancies

The presence of CD44 isoforms was evaluated in ascitic fluid and serum samples of patients with gynecologic malignancies. Previously, the shedding of tumor-associated cell surface antigens has been demonstrated in the blood and malignant effusions of gynecologic cancer patients. Thus, the shedding of CD44 was also studied in ascitic fluids and sera of these patients, to address variant isoform expression as a biomarker of gynecologic cancer. The expression of CD44 isoforms by ovarian tumor cells was examined by flow cytometry using variant-specific monoclonal antibodies. The release of these isoforms into the peripheral circulation and ascites was assayed by Western immunoblot analysis. Flow cytometric analysis of ovarian tumor cell lines revealed a strong expression of CD44 with significant levels of v4/5 and v6 isoforms. The presence of circulating CD44 isoforms was detectable in the sera of six of eight cancer patients, as well as in 12 of 16 ascitic fluids. Of the CD44-positive specimens, all six positive sera expressed detectable levels of variant CD44. The CD44v6 was present in all of the positive sera samples tested. In the ascites, the "shed" CD44 appeared to be associated predominantly with shed particles (vesicles) of plasma membranes (membrane fragments). Of ten CD44-positive ascites samples, all expressed significant levels of variant CD44. In addition to mediating metastasis, the differential expression and shedding of CD44 isoforms into the circulation may represent important determinants in the escape of tumors from immune surveillance, and their detection may be a diagnostic or prognostic marker.

Early detachment of colon carcinoma cells during CD95(APO-1/Fas)-mediated apoptosis. I. De-adhesion from hyaluronate by shedding of CD44.

Ligation of CD95 (APO-1/Fas) cell surface receptors induces death in apoptosis-sensitive cells. Induction of apoptosis in adherent gamma interferon-stimulated HT-29 and COLO 205 colon carcinoma cells by cross-linking CD95 with anti-APO-1 monoclonal antibody resulted in detachment of the cells from hyaluronate starting about 1 h after antibody exposure. Loss of adhesion was paralleled by a substantial reduction of the multifunctional cell surface adhesion molecule CD44. As evidenced by cycloheximide treatment, this effect was not caused by impaired protein synthesis. Depletion of surface CD44 was also not due to membrane blebbing, since cytochalasin B failed to inhibit ascension from hyaluronate. Instead, ELISA and time kinetics showed increasing amounts of soluble CD44 in the supernatant of CD95-triggered cells. SDS-PAGE revealed that soluble CD44 had an apparent molecular mass of about 20 kD less than CD44 immunoprecipitated from intact cells. Thus, CD95-triggering induced shedding of CD44. Shedding is a novel mechanism operative in early steps of CD95-mediated apoptosis. Shedding surface molecules like CD44 might contribute to the active disintegration of dying epithelial cells in vivo.