Proteolytic cleavage of CD44, a cell surface protein that acts as a receptor for hyaluronan and other components of the extracellular matrix, has been implicated in tumor cell migration. CD44 is present in cholesterol-rich plasma membrane microdomains known as lipid rafts [structures implicated in the proteolytic cleavage and release (shedding) of various membrane proteins], leading Murai et al. to investigate the effects of decreasing cell cholesterol content (a manipulation that disrupts lipid rafts) on CD44 location, shedding, and function. After confirming that methyl-β-cyclodextrin (MβCD) treatment decreased the cellular and membrane cholesterol content of U-251 MG human glioblastoma cells, the authors determined that it increased CD44 shedding, whereas a cholesterol-MβCD complex did not. Moreover, filipin, which binds to cholesterol and thereby disrupts lipid rafts, also stimulated CD44 shedding. MβCD-dependent CD44 shedding was blocked by the metalloproteinase inhibitor TAP, as well as by tissue inhibitor of metalloproteinase (TIMP)–1, and siRNA knockdown of members of the ADAM (a disintegrin and metalloproteinase) family of metalloproteinases implicated ADAM10 in this process. Analyses of CD44 and ADAM10 location in Triton-X-100–insoluble (lipid raft) and Triton-X-100–soluble (nonlipid raft) cell fractions revealed that CD44 was present in both Triton-X-100–insoluble and Triton-X-100–soluble fractions of untreated cells, whereas ADAM10 was largely in the Triton-X-100–soluble fraction. Treatment with MβCD or filipin, however, led to loss of CD44 from the Triton-X-100–insoluble fraction. Moreover, atmospheric scanning electron microscopic analysis revealed that MβCD led to the dispersal of CD44 clusters on the plasma membrane. Sustained exposure to simvastatin, which is used to treat hypercholesterolemia, decreased cholesterol content and increased CD44 shedding in U-251 MG cells. Intriguingly, simvastatin also blocked stimulation of U-251 MG cell migration on a hyaluronan-coated substrate by hyaluronan oligosaccharides or epidermal growth factor (treatments that promote CD44 shedding), as did a blocking antibody directed against CD44. The authors thus propose that manipulation of CD44 shedding by reducing cholesterol might provide an approach for preventing tumor cell migration.
T. Murai, Y. Maruyama, K. Mio, H. Nishiyama, M. Suga, C. Sato, Low cholesterol triggers membrane microdomain-dependent CD44 shedding and suppresses tumor cell migration. J. Biol. Chem. 286, 1999–2007 (2011). [Abstract] [Full Text]