Hyaluronan oligosaccharides induce CD44 cleavage and promote cell migration in CD44-expressing tumor cells

CD44 is an adhesion molecule that serves as a cell surface receptor for several extracellular matrix components, including hyaluronan (HA). The proteolytic cleavage of CD44 from the cell surface plays a critical role in the migration of tumor cells. Although this cleavage can be induced by certain stimuli such as phorbol ester and anti-CD44 antibodies in vitro, the physiological inducer of CD44 cleavage in vivo is unknown. Here, we demonstrate that HA oligosaccharides of a specific size range induce CD44 cleavage from tumor cells. Fragmented HA containing 6-mers to 14-mers enhanced CD44 cleavage dose-dependently by interacting with CD44, whereas a large polymer HA failed to enhance CD44 cleavage, although it bound to CD44. Examination using uniformly sized HA oligosaccharides revealed that HAs smaller than 36 kDa significantly enhanced CD44 cleavage. In particular, the 6.9-kDa HA (36-mers) not only enhanced CD44 cleavage but also promoted tumor cell motility, which was completely inhibited by an anti-CD44 monoclonal antibody. These results raise the possibility that small HA oligosaccharides, which are known to occur in various tumor tissues, promote tumor invasion by enhancing the tumor cell motility that may be driven by CD44 cleavage.     

Hyaluronan binding by cell surface CD44

CD44 is the primary cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan. Here we determined the relative avidities of unlabeled hyaluronan preparations for cell surface CD44 by their ability to block the binding of fluorescein-conjugated hyaluronan to a variety of cells. We show that hyaluronan binding at the cell surface is a complex interplay of multivalent binding events affected by the size of the multivalent hyaluronan ligand, the quantity and density of cell surface CD44, and the activation state of CD44 as determined by cell-specific factors and/or treatment with CD44-specific monoclonal antibody (mAb). Using low M(r) hyaluronan oligomers of defined sizes, we observed monovalent binding between 6 and 18 sugars. At approximately 20 to approximately 38 sugars, there was an increase in avidity (approximately 3x), suggesting that divalent binding was occurring. In the presence of the inducing mAb IRAWB14, monovalent binding avidity was similar to that of noninduced CD44, but beginning at approximately 20 residues, there was a dramatic and progressive increase in avidity with increasing oligomer size ( approximately 22 < 26 < 30 < 34 < 38 sugars). Kinetic studies of binding and dissociation of fluorescein-conjugated hyaluronan indicated that inducing mAb treatment had little effect on the binding kinetics, but dissociation from the cell surface was greatly delayed by inducing mAb.     

Hyaluronan substratum induces multidrug resistance in human mesenchymal stem cells via CD44 signaling

Little information is available concerning multidrug resistance (MDR) in mesenchymal stem cells, although several studies have reported that MDR is associated with hyaluronan in neoplastic cells. We have evaluated whether a hyaluronan-coated surface modulates MDR in placenta-derived human mesenchymal stem cells (PDMSCs). We have found that PDMSCs cultured on a tissue-culture polystyrene surface coated with 30 μg/cm² hyaluronan are more resistant than control PDMSCs to doxorubicin. Inhibiting PI3K/Akt signaling has shown that the PI3K/Akt pathway modulates both P-glycoprotein activity and doxorubicin resistance. In addition, 10 μM verapamil dramatically suppresses the doxorubicin resistance induced by the hyaluronan-coated surface, indicating that P-glycoprotein activity is necessary for MDR. We have further found that PDMSCs treated with CD44 small interfering RNA (siRNA) and grown on a polystyrene surface coated with 30 μg/cm² hyaluronan have fewer P-glycoprotein⁺ cells and lower CD44 expression levels (less than 60% in both cases) than PDMSCs not treated with CD44 siRNA and grown on the hyaluronan-coated surface. Moreover, treatment with CD44 siRNA suppresses the hyaluronan-substratum-induced doxorubicin resistance. We conclude that a hyaluronan substratum induces MDR in PDMSCs through CD44 signaling. This work was supported by research grant NSC95–2745-B-006–003-MY2 from the National Science Council, Taiwan, and by Landmark Project Grant A25, funded by the Taiwan Ministry of Education, from National Cheng Kung University.     

Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism

CD147 is a multifunctional transmembrane protein and promotes cancer progression. We found that the anti-human CD147 mouse monoclonal antibody MEM-M6/1 strongly induces necrosis-like cell death in LoVo, HT-29, WiDr, and SW620 colon cancer cells and A2058 melanoma cells, but not in WI-38 and TIG-113 normal fibroblasts. Silencing or overexpression of CD147 in LoVo cells enhanced or decreased the MEM-M6/1 induced cell death, respectively. CD147 is known to form complex with proton-linked monocarboxylate transporters (MCTs), which is critical for lactate transport and intracellular pH (pHi) homeostasis. In LoVo cells, CD147 and MCT-1 co-localized on the cell surface, and MEM-M6/1 inhibited the association of these molecules. MEM-M6/1 inhibited lactate uptake, lactate release, and reduced pHi. Further, the induction of acidification was parallel to the decrease of the glycolytic flux and intracellular ATP levels. These effects were not found in the normal fibroblasts. As cancer cells depend on glycolysis for their energy production, CD147 inhibition might induce cell death specific to cancer cells.     

T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta + T cells.

mAb directed against the TCR/CD3 complex activate resting T cells. However, TCR/CD3 signaling induces death by apoptosis in immature (CD4+CD8+) murine thymocytes and certain transformed leukemic T cell lines. Here we show that anti-TCR and anti-CD3 mAb induce growth arrest of cloned TCR-gamma delta + T cells in the presence of IL-2. In the absence of exogenous IL-2, however, the very same anti-TCR/CD3 mAb stimulated gamma delta (+)-clones to proliferation and IL-2 production. In the presence of exogenous IL-2, anti-TCR/CD3 mAb induced the degradation of DNA into oligosomal bands of approximately 200 bp length in cloned gamma delta + T cells. This pattern of DNA fragmentation is characteristic for the programmed cell death termed apoptosis. These results demonstrate that TCR/CD3 signaling can induce cell death in cloned gamma delta + T cells. In addition, this report is the first to show that apoptosis triggered by TCR/CD3 signaling is not restricted to CD4+CD8+ immature thymocytes and transformed leukemic T cell lines but can be also observed with IL-2-dependent normal (i.e., TCR-gamma delta +) T cells.     

Engagement of CD44 up-regulates Fas Ligand expression on T cells leading to activation-induced cell death

Activation-induced cell death (AICD) plays a pivotal role in self-tolerance by deleting autoreactive T cells, but a defect of AICD results in expansion of autoreactive T cells and is deeply involved in the pathogenesis of rheumatoid arthritis. Although the process of AICD is mainly mediated by Fas Ligand (FasL)/Fas signaling, it remains unclear what induces FasL expression on T cells. In the present study, we found that CD44 was the most potent stimulator of FasL expression on human peripheral T cells. CD44 cross-linking rapidly up-regulated FasL expression on the T cell surface by delivery from the cytoplasm without new FasL protein synthesis. This up-regulation of FasL was mediated by activation of a tyrosine kinase, IP3 receptor-dependent Ca²⁺ mobilization and actin cytoskeletal rearrangements. Furthermore, AICD induced by CD3 restimulation was inhibited by hyaluronidase as well as by soluble Fas, indicating an interaction between membrane-bound hyaluronan and the cell surface CD44 was involved in the up-regulation of FasL expression on T cells and subsequent AICD. We therefore propose that the engagement of CD44 on T cells can eliminate autoreactive T cells by expression of FasL and FasL-mediated AICD. Grant support: Scientific Research by the Ministry of Health, Labor and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan and University of Occupational and Environmental Health, Japan.     

Semaphorin CD100 from activated T lymphocytes induces process extension collapse in oligodendrocytes and death of immature neural cells

An inappropriate cross talk between activated T lymphocytes infiltrating the CNS and neural cells can sustain the onset and progression of demyelination and axonal degeneration in neuroinflammatory diseases. To mimic this deleterious cross talk, we designed an experimental paradigm consisting of transient cocultures of T lymphocytes chronically activated by retrovirus infection (not virus productive) with human multipotent neural precursors or primary oligodendrocytes from rat brain. We showed that activated T lymphocytes induced apoptotic death of multipotent neural progenitors and immature oligodendrocytes after a progressive collapse of their process extensions. These effects were reminiscent of those induced by brain semaphorin on neural cells. Blockade by specific Abs of soluble CD100 (sCD100)/semaphorin 4D released by activated T cells, or treatment with rsCD100, demonstrated that this immune semaphorin has the ability to collapse oligodendrocyte process extensions and to trigger neural cell apoptosis, most likely through receptors of the plexin family. The specific presence of sCD100 in the cerebrospinal fluid and of CD100-expressing T lymphocytes in the spinal cord of patients suffering with neuroinflammatory demyelination pointed to the potential pathological effect of sCD100 in the CNS. Thus, our results show that CD100 is a new important element in the deleterious T cell-neural cell cross talk during neuroinflammation and suggest its role in demyelination or absence of remyelination in neuroinflammatory diseases including multiple sclerosis and human T lymphotropic virus type 1-associated myelopathy.     

Hyaluronan and CD44 antagonize mitogen-dependent cyclin D1 expression in mesenchymal cells

High molecular weight (HMW) hyaluronan (HA) is widely distributed in the extracellular matrix, but its biological activities remain incompletely understood. We previously reported that HMW-HA binding to CD44 antagonizes mitogen-induced S-phase entry in vascular smooth muscle cells (SMCs; Cuff, C.A., D. Kothapalli, I. Azonobi, S. Chun, Y. Zhang, R. Belkin, C. Yeh, A. Secreto, R.K. Assoian, D.J. Rader, and E. Puré. 2001. J. Clin. Invest. 108:1031-1040); we now characterize the underlying molecular mechanism and document its relevance in vivo. HMW-HA inhibits the mitogen-dependent induction of cyclin D1 and down-regulation of p27(kip1) in vascular SMCs. p27(kip1) messenger RNA levels were unaffected by HMW-HA, but the expression of Skp2, the rate-limiting component of the SCF complex that degrades p27(kip1), was reduced. Rescue experiments identified cyclin D1 as the primary target of HMW-HA. Similar results were observed in fibroblasts, and these antimitogenic effects were not detected in CD44-null cells. Analysis of arteries from wild-type and CD44-null mice showed that the effects of HMW-HA/CD44 on cyclin D1 and Skp2 gene expression are detected in vivo and are associated with altered SMC proliferation after vascular injury.     

Achacin induces cell death in HeLa cells through two different mechanisms

Achacin, which belongs to the L-amino acid oxidase group, oxidizes free amino acids and produces hydrogen peroxide in cell culture systems. Morphological changes in cells incubated with achacin were similar to those of cells incubated with H(2)O(2). In both cases, the end result was cell death. To examine the mechanism of achacin-associated cytotoxicity, the H(2)O(2) scavenger catalase was added to culture media. Features typical of apoptosis, including morphological changes, DNA fragmentation, and PARP cleavage, were observed when cells were incubated with achacin in the presence of catalase. Moreover, apoptosis was inhibited by Z-VAD-fmk, a broad-spectrum caspase inhibitor. Herein, we present evidence that two pathways are involved in achacin-induced cell death. One is direct generation of H(2)O(2) through the L-amino acid oxidase activity of achacin. The other is the caspase-mediated apoptotic pathway that is induced by depletion of L-amino acids by achacin.     

CD44 contributes to T cell activation

We demonstrate here that the CD44 molecule, which mediates lymphocyte adhesion to high endothelial venules (HEV), is also involved in the delivery of an activation signal to the T cell. We have produced a CD44 mAb (H90) which is able to block the binding of lymphocytes to high endothelial venules. H90 had no effect on [3H]TdR incorporation of whole PBL stimulated by lectins, allogeneic cells, or CD3 mAb in the soluble phase; in contrast, it strongly increased [3H]TdR incorporation of PBL stimulated by CD2 pairs of mAb or by CD3 mAb linked to the plastic culture plates, when purified T cells were used, H90 mAb could efficiently induce them to proliferate after a primary signal of activation delivered via cross-linked CD3 or via CD2, an effect mediated by Il-2 synthesis and Il-2R expression. Thus, the effect of H90 mAb resembles the mitogenic effect of CD28 "9.3" mAb. However, several results show that CD28 and CD44 mediate different signals to the T cells: i) in contrast to CD28 mAb, CD44 mAb cannot complement the signal delivered by a soluble CD3 mAb, lectins, or PMA; ii) CD44 mAb, at the difference of CD28 mAb, cannot induce CD3+ thymocytes to proliferate in conjunction with a first signal provided via cross-linked CD3 or via CD2; iii) F(ab) fragments of H90 were efficient, whereas divalent fragments of CD29 9.3 mAb are required to produce activation signals; and iv) CD44 and CD28 mAb produce a very strong synergistic effect on T cell proliferation. These results fit with previous ones showing that endothelial cells can play the role of accessory cell in T cell activation and that a hierarchy of signaling can be delivered to T cells via CD3 and CD2.     

Adoptive transfer of CD3+ T cells and CD4+CD44high memory T cells induces autoimmune pancreatitis in MRL/MpJ mice

The immunopathogenesis of autoimmune pancreatitis (AIP) is poorly understood. Here, we have used MRL/MpJ mice, a model of spontaneous AIP, to address the role of cellular autoimmune processes in the initiation and progression of the disease. Therefore, different T cell subpopulations were adoptively transferred from sick to still healthy (but susceptible) MRL/MpJ mice. Unpurified splenocytes and CD3⁺ T cells both efficiently induced AIP, while CD4⁺ and CD8⁺ T cells alone, as well as splenocytes from healthy mice, were insufficient to trigger the disease. Strikingly, CD4⁺CD44^high memory T cells, although transferred at lower numbers than other T cells, also induced AIP in recipient mice. Employing a modified experimental design, we also evaluated the effects of regulatory T cells (T_regs) on the progression of AIP in already diseased mice. Under the given experimental conditions, there was no significant suppressive effect of adoptively transferred T_regs on pancreatic histopathology. The results of our studies suggest a key role of T cell‐mediated processes in murine AIP. The effects of CD4⁺CD44^high memory T cells are in accordance with genetic studies of our group, which had previously implicated this cell type into the pathogenesis of AIP. In follow‐up studies, we will focus on the interplay of cellular and humoral autoimmunity in the context of AIP.     

Curcumin induces cell death in esophageal cancer cells through modulating Notch signaling

Background

Curcumin inhibits the growth of esophageal cancer cell lines; however, the mechanism of action is not well understood. It is becoming increasingly clear that aberrant activation of Notch signaling has been associated with the development of esophageal cancer. Here, we have determined that curcumin inhibits esophageal cancer growth via a mechanism mediated through the Notch signaling pathway.

Methodology/Principal Findings

In this study, we show that curcumin treatment resulted in a dose and time dependent inhibition of proliferation and colony formation in esophageal cancer cell lines. Furthermore, curcumin treatment induced apoptosis through caspase 3 activation, confirmed by an increase in the ratio of Bax to Bcl2. Cell cycle analysis demonstrated that curcumin treatment induced cell death and down regulated cyclin D1 levels. Curcumin treatment also resulted in reduced number and size of esophagospheres. Furthermore, curcumin treatment led to reduced Notch-1 activation, expression of Jagged-1 and its downstream target Hes-1. This reduction in Notch-1 activation was determined to be due to the down-regulation of critical components of the γ-secretase complex proteins such as Presenilin 1 and Nicastrin. The combination of a known γ-secretase inhibitor DAPT and curcumin further decreased proliferation and induced apoptosis in esophageal cancer cells. Finally, curcumin treatment down-regulate the expressions of Notch-1 specific microRNAs miR-21 and miR-34a, and upregulated tumor suppressor let-7a miRNA.

Conclusion/Significance

Curcumin is a potent inhibitor of esophageal cancer growth that targets the Notch-1 activating γ-secretase complex proteins. These data suggest that Notch signaling inhibition is a novel mechanism of action for curcumin during therapeutic intervention in esophageal cancers.     

Internalization of the Hyaluronan Receptor CD44 by Chondrocytes

Chondrocytes express CD44 as a primary receptor for the matrix macromolecule hyaluronan. Hyaluronan is responsible for the retention and organization of proteoglycan within cartilage, and hyaluronan-chondrocyte interactions are important for the assembly and maintenance of the cartilage matrix. Bovine articular chondrocytes were used to study the endocytosis and turnover of CD44 and the effects of receptor occupancy on this turnover. Matrix-intact chondrocytes exhibit approximately a 6% internalization of cell surface CD44 by 4 h. Treatment with Streptomyces hyaluronidase to remove endogenous pericellular matrix increased internalization to approximately 20% of cell surface CD44 at 4 h. This turnover could be partially inhibited by the addition of exogenous hyaluronan to these matrix-depleted chondrocytes. Cell surface biotin-labeled CD44 was internalized by chondrocytes and this internalization was decreased in the presence of hyaluronan. Colocalization of internalized CD44 and fluorescein-labeled hyaluronan in intracellular vesicles correlates with the previous results of receptor-mediated endocytosis pathway for the degradation of hyaluronan by acid hydrolases. Taken together, our results indicate that CD44 is internalized by chondrocytes and that CD44 turnover is modulated by occupancy with hyaluronan.     

CD44 mediated Hyaluronan adhesion of Toxoplasma gondii-infected leukocytes

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects humans and animals. Ingested parasites cross the intestinal epithelium, invade leukocytes and are then disseminated to peripheral organs. However, the mechanism of extravasation of the infected leukocytes remains poorly understood. In this study, we demonstrate that T. gondii-invaded human and mouse leukocytes express higher level of CD44, a ligand of hyaluronan (HA), and its expression on myeloid and non-myeloid leukocytes causes T. gondii-invaded human and mouse leukocyte to adhere to HA more effectively than non-invaded leukocytes. The specific adherence of parasite-invaded leukocytes was inhibited by anti CD44 antibody. Leukocytes of CD44 knockout mice did not show parasite-invaded leukocyte specific adhesion. Our results indicate that parasite-invaded leukocytes, regardless of whether myeloid or not, gain higher ability to adhere to HA than non-invaded leukocytes, via upregulation of CD44 expression and/or selective invasion to CD44 highly expressing cells. The difference in ability to adhere to HA between parasite-invaded cells and non-invaded neighboring cells might facilitate effective delivery of parasite-invaded leukocytes to the HA-producing endothelial cell surface and/or HA-rich extra cellular matrix.     

A novel protein, MUDENG, induces cell death in cytotoxic T cells

A screening system comprised of a randomized hybrid-ribozyme library has previously been used to identify pro-death genes in Fas-mediated apoptosis, and short sequence information of candidate genes from this system was previously reported by Kawasaki and Taira [H. Kawasaki, K. Taira, A functional gene discovery in the Fas-mediated pathway to apoptosis by analysis of transiently expressed randomized hybrid-ribozyme libraries, Nucleic Acids Res. 30 (2002) 3609-3614]. In this study, we have cloned the full-length of the candidate’s open reading frames and found that one of the candidates, referred to as MUDENG (Mu-2 related death-inducing gene), which is composed of 490 amino acids that contain the adaptin domain found in the μ2 subunit of APs related to clathrin-mediated endocytosis, is able to induce cell death by itself. Ectopic expression of MUDENG induced cell death in Jurkat T cells and HeLa cells. In addition, when MUDENG expression was evaluated by immnuohistochemical staining, it was found in most tissues, including the intestine and testis. Furthermore, MUDENG appears to be evolutionary conserved from mammals to amphibians, suggesting that it may have a common role in cell death. Taken together, these results suggest that MUDENG is likely to play an important role in cell death in various tissues.     

T cells bearing the CD44hi "memory" phenotype display characteristics of activated cells in G1 stage of cell cycle.

T cells capable of anamnestic proliferative responses to antigen in vitro (i.e., "memory" cells) have been shown to display the CD44hi CD45RBlo surface phenotype. To assess the state of activation of these T cells, CD4+ T cells expressing the CD44hi or CD45RBlo phenotype were compared to CD4+ T cells expressing the CD44lo or CD45RBhi phenotype in the context of expression of the "activated" (asialo-GM1hi) vs "resting" (asialo-GM1lo) phenotype and in the context of cell size, total protein content, and total RNA content. Dual fluorescence analysis demonstrated that all CD4+ T cells expressing the CD44hi phenotype also expressed the asialo-GM1hi phenotype associated with cell activation. In vitro proliferative assays confirmed that the CD4+ asialo-GM1hi, the CD4+ CD45RBlo, and the CD4+ CD44hi FACS-sorted populations displayed stronger in vitro responsiveness to stimulation with immobilized anti-CD3 mAB than the CD4+ asialo-GM1lo, CD45RBhi, or CD44lo populations. Acridine orange analysis of sorted CD44hi/lo fractions revealed that the diploid (G1) population of the CD44hi T cells displayed a higher mean RNA content than the CD44lo T cells. Similarly, the CD44hi T cells displayed a higher mean cell size and a higher mean total protein content than the CD44lo CD4+ T cells. Similar results were obtained with asialo-GM1 and CD45RB subsets of CD4+ T cells. The basal rate of protein synthesis, as determined by [3H]leucine incorporation, was approximately 50% higher in the CD44hi small CD4+ T cells than in the CD44lo CD4+ T cells. Based on the knowledge that cell size, total protein and RNA content, and responsiveness to signals inducing proliferation are lowest in G0 stage of cycle and increase through G1 stage of cycle, it appears that the CD44hi CD45RBlo T cells exist in a higher activation state than CD44lo CD45RBhi T cells. The previously demonstrated association of CD44hi CD45RBlo phenotype with memory T cells suggests that the CD44hi memory T cells are maintained in G1 (not necessarily cycling) rather than resting "out of cycle" in G0.