Tetraspanin CD9 modulates human lymphoma cellular proliferation via histone deacetylase activity.

Non-Hodgkin Lymphoma (NHL) is a type of hematological malignancy that affects two percent of the overall population in the United States. Tetraspanin CD9 is a cell surface protein that has been thoroughly demonstrated to be a molecular facilitator of cellular phenotype. CD9 expression varies in two human lymphoma cell lines, Raji and BJAB. In this report, we investigated the functional relationship between CD9 and cell proliferation regulated by histone deacetylase (HDAC) activity in these two cell lines. Introduction of CD9 expression in Raji cells resulted in significantly increased cell proliferation and HDAC activity compared to Mock transfected Raji cells. The increase in CD9-Raji cell proliferation was significantly inhibited by HDAC inhibitor (HDACi) treatment. Pretreatment of BJAB cells with HDAC inhibitors resulted in a significant decrease in endogenous CD9 mRNA and cell surface expression. BJAB cells also displayed decreased cell proliferation after HDACi treatment. These results suggest a significant relationship between CD9 expression and cell proliferation in human lymphoma cells that may be modulated by HDAC activity.

Functional relevance of tetraspanin CD9 in vascular smooth muscle cell injury phenotypes: a novel target for the prevention of neointimal hyperplasia.

Vascular smooth muscle cell (VSMC) migration and proliferation are critical events in the development of neointima following vascular injury. In this study, we found that CD9 is constitutively expressed in the VSMC of the neointima of injured carotid arteries. The in vitro migration and proliferation of human coronary artery smooth muscle (hCASM) cells were reduced by 40% and 63%, respectively, by treatment with a CD9 specific monoclonal antibody mAb7 when compared to control antibody treatment. In a mouse carotid ligation injury model, a single application of a neutralizing anti-mouse CD9 antibody resulted in a 31% (day 14, n=8, p<0.05), and 32% (day 28, n=5, p<0.01) reduction in neointima formation. In support of these findings, exogenous expression of human CD9 by CD9-adenoviral transduction led to 43% increases in neointima (p<0.05, n=6). Upon investigation of the mechanisms underlying CD9 mediated VSMC phenotypic events we found that integrin alpha5beta1 was a constitutive partner of CD9 and that CD9 significantly augmented PI-3 kinase dependent Akt phosphorylation. Furthermore, enhanced Akt phosphorylation was attenuated by mAb7 binding. Cumulatively, a functional link between CD9, alpha5beta1, PI3-K/Akt activity and enhanced VSMC migratory and proliferative phenotypes has been demonstrated. These studies suggest that agents that modulate CD9 mediated VSMC phenotypes may emerge as novel strategies for the treatment of abnormal vascular injury response.

Identification of CD9 extracellular domains important in regulation of CHO cell adhesion to fibronectin and fibronectin pericellular matrix assembly.

CD9, a 24-kDa member of the tetraspanin family, influences cellular growth and development, activation, adhesion, and motility. Our investigation focuses on the hypothesis that the CD9 second extracellular loop (EC2) is important in modulating cell adhesive events. Using a Chinese hamster ovary (CHO) cell expression system, we previously reported that CD9 expression inhibited cell adhesion to fibronectin and fibronectin matrix assembly. For the first time, a functional epitope on CD9 EC2 that regulates these processes is described. Binding of mAb7, an EC2-specific anti-CD9 monoclonal antibody, reversed the CD9 inhibitory activity on CHO cell adhesion and fibronectin matrix assembly. This reversal of cell phenotype also was observed in CHO cells expressing CD9 EC2 truncations. Furthermore, our data showed that the EC2 sequence (173)LETFTVKSCPDAIKEVFDNK(192) was largely responsible for the CD9-mediated CHO cell phenotype. Two peptides, (135)K-V(172) (peptide 5b) and (168)P-I(185) (peptide 6a), selectively blocked mAb7 binding to soluble CD9 and to CD9 on intact cells. These active peptides reversed the influence of CD9 expression on CHO cell adhesion to fibronectin. In addition, confocal microscopy revealed that CD9 colocalized with the integrin alpha(5)beta(1) and cytoskeletal F-actin in punctate clusters on the cell surface, particularly at the cell margins. Immunoprecipitation studies confirmed CD9 association with beta(1) integrin. The cellular distribution and colocalization of focal adhesion kinase and alpha-actinin with cytoskeletal actin was also influenced by CD9 expression. Thus, CD9 may exhibit its effect by modulating the composition of adhesive complexes important in facilitating cell adhesion and matrix assembly.

Chinese hamster ovary cell motility to fibronectin is modulated by the second extracellular loop of CD9. Identification of a putative fibronectin binding site.

CD9, a member of the tetraspanin family of proteins, is characterized by four transmembrane domains and two extracellular loops. Surface expression of CD9 on Chinese hamster ovary (CHO) cells dramatically enhances spreading and motility on fibronectin. To elucidate the mechanistic basis of CD9-fibronectin interaction, binding to fibronectin was investigated using purified and recombinant forms of CD9. The affinity of fibronectin for CD9 in enzyme-linked immunosorbent assay was 81 +/- 25 nm. The binding of fibronectin to immobilized CD9 was enhanced by Ca(2+) ions. Protein binding and peptide competition studies demonstrated that peptide 6 derived from CD9 extracellular loop 2 (amino acids 168-192) contained part of the fibronectin-binding domain. Additionally, enhanced adhesion of CD9-CHO-B2 cells to fibronectin was significantly reduced by peptide 6. CD9-CHO cells had a 5-fold increase in motility to fibronectin as compared with mock-transfected controls, an effect that correlated with CD9 cell surface density. Truncation of CD9 extracellular loop 2 and peptide 6 caused inhibition of CD9-CHO cell motility to fibronectin. Deletion of CD9 extracellular loop 1 had no significant effect on CHO cell motility. These findings demonstrate a critical role for CD9 extracellular loop 2 in cell motility to fibronectin and clarify the mechanism by which CD9-fibronectin interaction modulates cell adhesion and motility.