CD44 is a major cell surface adhesion molecule for hyaluronan, a component of the extracellular matrix, and is implicated in tumor metastasis and invasion. We reported previously that hyaluronan oligosaccharides induce CD44 cleavage from tumor cells. Here we show that engagement of CD44 promotes CD44 cleavage and tumor cell migration, both of which were suppressed by a metalloproteinase inhibitor KB-R7785 and tissue inhibitor of metalloproteinases-1 (TIMP-1) but not by TIMP-2. We also present evidence that blockade of metalloproteinase-disintegrin ADAM10 (a disintegrin and metalloproteinase 10) by RNA interference suppresses CD44 cleavage induced by its ligation. Engagement of CD44 concurrently induced activation of the small GTPase Rac1 and led to drastic changes in cell morphology and actin cytoskeleton with redistribution of CD44 to newly generated membrane ruffling areas. A fluorescence resonance energy transfer approach to visualize GTP-bound Rac1 in living cells revealed the localization of the active Rac1 in the leading edge of the membrane ruffling areas upon ligation of CD44. Taken together, our results indicate that the cleavage of CD44 catalyzed by ADAM10 is augmented by the intracellular signaling elicited by engagement of CD44, through Rac-mediated cytoskeletal rearrangement, and suggest that CD44 cleavage contributes to the migration and invasion of tumor cells.CD44 is a cell surface receptor for several extracellular matrix components including hyaluronan (HA) 1 (1) and is implicated in a wide variety of biological processes including lymphocyte homing (2), cell migration (3), and metastasis (4). It has been demonstrated that CD44 expressed in tumor cells is proteolytically cleaved at the extracellular domain by metalloproteinases and that CD44 cleavage plays a critical role in CD44-mediated tumor cell migration (5, 6). CD44 cleavage on the tumor cell surface is enhanced by phorbol 12-myristate 13-acetate (PMA) or ionomycin treatment, indicating that the cleavage is under the regulation of the activation of protein kinase C (PKC) or the extracellular calcium influx (7). CD44 cleavage is also induced by introducing dominant active mutant of Ha-Ras small GTPase (8). In addition to these CD44 cleavage inducers acting on intracellular signaling molecules, we have shown recently (9) that HA fragments also enhance CD44 cleavage from tumor cells. HA fragments probably bind and cross-link CD44 and then induce certain intracellular signals to cause CD44 cleavage. However, almost no information is available regarding the intracellular signals transmitted upon stimulation of CD44 to the induction of CD44 cleavage. The relationship between CD44 cleavage and the enhanced cell migration also remains largely unknown.Tumor cells migrate by regulating adhesion to and detachment from extracellular matrix, dynamically changing their cytoskeleton (10). Rho family small GTPases are the key regulators of actin organization in cytoskeleton, and the changes of their activation state influence cell morphology (11). The ma...
Invasive tumour cells, such as gliomas, frequently express EGF (epidermal growth factor) receptor at a high level and they exhibit enhanced cell migration in response to EGF. We reported previously that tumour cell migration is associated with ectodomain cleavage of CD44, the major adhesion molecule that is implicated in tumour invasion and metastasis, and that the cleavage is enhanced by ligation of CD44. In the present study, we show that EGF promotes CD44 cleavage and CD44-dependent cell migration. Introduction of a dominant-negative mutant of the small GTPase Rac1 or depletion of Rac1 by RNAi (RNA interference) abrogated CD44 cleavage induced by EGF. Treatment with PD98059, an inhibitor for MEK (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase), also suppressed the CD44 cleavage. Furthermore, RNAi studies showed that EGF induced ADAM10 (a disintegrin and metalloproteinase 10)-dependent CD44 cleavage and cell migration. These results indicate that EGF induces ADAM10-mediated CD44 cleavage through Rac1 and mitogen-activated protein kinase activation, and thereby promotes tumour cell migration and invasion.
Background: Th1/Th2 cell balance is thought to be shifted toward a Th2-type immune response not only by malignancy but also by surgical stress. The aim of this study was to estimate perioperative immune responses with respect to the Th1/Th2 balance in patients with gastrointestinal cancer.
a b s t r a c tAquaporin-7 (AQP7) is expressed in adipose tissue, permeated by water and glycerol, and is involved in lipid metabolism. AQP7-null mice develop obesity, insulin resistance, and adipocyte hypertrophy. Here, we show that AQP7 is expressed in adipocyte plasma membranes, and is re-localized to intracellular membranes in response to catecholamine in mouse white adipose tissue. We found that internalization of AQP7 was induced by PKA activation and comparative gene identification 58 (CGI-58). This relocation was confirmed by functional studies in 3T3-L1 adipocytes. Collectively, these results suggest that AQP7 makes several contributions to adipocyte metabolism, in both cortical and intracellular membranes.
Neuromyelitis optica (NMO), an autoimmune disease of the central nervous system, is characterized by an autoantibody called NMO-IgG that recognizes the extracellular domains (ECDs) of aquaporin-4 (AQP4). In this study, monoclonal antibodies (mAbs) against the ECDs of mouse AQP4 were established by a baculovirus display method. Two types of mAb were obtained: one (E5415A) recognized both M1 and M23 isoforms, and the other (E5415B) almost exclusively recognized the square-array-formable M23 isoform. While E5415A enhanced endocytosis of both M1 and M23, followed by degradation in cells expressing AQP4, including astrocytes, E5415B did so to a much lesser degree, as determined by live imaging using fluorescence-labeled antibodies and by Western blotting of lysate of cells treated with these mAbs. E5415A promoted cluster formation of AQP4 on the cell surface prior to endocytosis as determined by immunofluorescent microscopic observation of bound mAbs to astrocytes as well as by Blue native PAGE analysis of AQP4 in the cells treated with the mAbs. These observations clearly indicate that an anti-AQP4-ECDs antibody possessing an ability to form a large cluster of AQP4 by cross-linking two or more tetramers outside the AQP4 arrays enhances endocytosis and the subsequent lysosomal degradation of AQP4.
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