Soluble CD44 Interacts with Intermediate Filament Protein Vimentin on Endothelial Cell Surface

CD44 is a cell surface glycoprotein that functions as hyaluronan receptor. Mouse and human serum contain substantial amounts of soluble CD44, generated either by shedding or alternative splicing. During inflammation and in cancer patients serum levels of soluble CD44 are significantly increased. Experimentally, soluble CD44 overexpression blocks cancer cell adhesion to HA. We have previously found that recombinant CD44 hyaluronan binding domain (CD44HABD) and its non-HA-binding mutant inhibited tumor xenograft growth, angiogenesis, and endothelial cell proliferation. These data suggested an additional target other than HA for CD44HABD. By using non-HA-binding CD44HABD Arg41Ala, Arg78Ser, and Tyr79Ser-triple mutant (CD443MUT) we have identified intermediate filament protein vimentin as a novel interaction partner of CD44. We found that vimentin is expressed on the cell surface of human umbilical vein endothelial cells (HUVEC). Endogenous CD44 and vimentin coprecipitate from HUVECs, and when overexpressed in vimentin-negative MCF-7 cells. By using deletion mutants, we found that CD44HABD and CD443MUT bind vimentin N-terminal head domain. CD443MUT binds vimentin in solution with a Kd in range of 12–37 nM, and immobilised vimentin with Kd of 74 nM. CD443MUT binds to HUVEC and recombinant vimentin displaces CD443MUT from its binding sites. CD44HABD and CD443MUT were internalized by wild-type endothelial cells, but not by lung endothelial cells isolated from vimentin knock-out mice. Together, these data suggest that vimentin provides a specific binding site for soluble CD44 on endothelial cells

Lovastatin Enhances Ecto-5′-Nucleotidase Activity and Cell Surface Expression in Endothelial Cells

Extracellular adenosine production by the GPI-anchored Ecto-5′-Nucleotidase (Ecto-5′-Nu) plays an important role in the cardiovascular system, notably in defense against hypoxia. It has been previously suggested that HMG-CoA reductase inhibitors (HRIs) could potentiate the hypoxic stimulation of Ecto-5′Nu in myocardial ischemia. In order to elucidate the mechanism of Ecto-5′-Nu stimulation by HRIs, Ecto-5′-Nu activity and expression were determined in an aortic endothelial cell line (SVAREC) incubated with lovastatin. Lovastatin enhanced Ecto-5′-Nu activity in a dose-dependent manner. This increase was not supported by de novo synthesis of the enzyme because neither the mRNA content nor the total amount of the protein were modified by lovastatin. By contrast, lovastatin enhanced cell surface expression of Ecto-5′-Nu and decreased endocytosis of Ecto-5′-Nu, as evidenced by immunostaining. This effect appeared unrelated to modifications of cholesterol content or Ecto-5′-Nu association with detergent-resistant membranes. The effect of lovastatin was reversed by mevalonate, the substrate of HMG-CoA reductase, by its isoprenoid derivative, geranyl-geranyl pyrophosphate, and by cytotoxic necrotizing factor, an activator of Rho-GTPases. Stimulation of Ecto-5′-Nu by lovastatin enhanced the inhibition of platelet aggregation induced by endothelial cells. In conclusion, lovastatin enhances Ecto-5′-Nu activity and membrane expression in endothelial cells. This effect seems independent of lowering cholesterol content but could be supported by an inhibition of Ecto-5′-Nu endocytosis through a decrease of Rho-GTPases isoprenylation.</jats:p