CD99, a cell surface glycoprotein with a molecular mass of 32 kDa, was originally described as a human thymus leukemia antigen (1), a Ewing sarcoma-specific membrane marker molecule (2, 3), and a putative adhesion molecule (termed E2) involved in spontaneous rosette formation of T cells with erythrocytes (4 -7). CD99 is broadly distributed on many cell types, with particularly strong expression on human cortical thymocytes, Ewing sarcoma cells, and peripheral primitive neuroectodermal tumors (3,8). The functional role of CD99 is not fully understood, and most information about its functions is derived from triggering CD99-mediated signaling events with agonistic CD99 monoclonal antibodies (mAbs) 2 in hematopoietic cells. In normal cells, CD99 has been functionally implicated in cell adhesion, migration, apoptosis, differentiation, activation, and proliferation of lymphocytes and monocyte extravasation and transport of several transmembrane proteins (9 -19). In particular, the role of CD99 in the modulation of cell adhesion has been demonstrated by the ability of anti-CD99 mAbs to induce homotypic aggregation of CD4 ϩ CD8 ϩ thymocytes, whereas other anti-CD99 antibodies block spontaneous T cell-erythrocyte rosette formation (4,6,11,20). It was also shown that antibody ligation of CD99 molecules up-regulated the expression of LFA-1 (␣ L ␤ 2 integrin), and CD99-induced cell aggregation was blocked by the addition of mAbs to LFA-1 or intracellular adhesion molecule 1 (ICAM-1) in a B cell line (20). These results suggest that signal transduction via CD99 modulates cell adhesion of lymphocytes by regulating the expression level of the cell adhesion molecule, integrin LFA-1. Several signal transducing molecules, including MAPKs and protein kinase C, have been found to mediate CD99-dependent cell adhesion of T cells (21,22). In addition, it was found that CD99 on one cell binds to another neighboring CD99 on the next, indicating that CD99 is a homophilic cell surface interacting protein (18). However, the molecular mechanisms of CD99-mediated signal transduction remain largely unknown.

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Heat shock protein 70 (Hsp70) release and its effects on pro-inflammatory cytokine production have been controversial. In this study, we investigated whether Hsp70 could be released from monocytes and activates matrix metalloproteinase-9 (MMP-9) gene expression. Hsp70 overexpression in human monocytic cell line U937 was found to increase PMAinduced MMP-9 expression and enhance cell motility. Hsp70 cDNA transfectants released Hsp70 protein into culture supernatants, and a part of released Hsp70 subsequently was bound to the surface of U937 cells. Addition of culture medium containing the extracelluar Hsp70 led to an increase not only in proMMP-9 secretion, but also the invasiveness of U937 cells through Matrigel or human umbilical vascular endothelial cells (HUVEC) in vitro. Immunodepletion of Hsp70 abolished its effect on MMP-9 expression. The released Hsp70 activated nuclear factor kappa B (NF-κB) and activating protein-1 (AP-1), which led to the activation of MMP-9 transcription. Taken together, these results suggest that extracellular Hsp70 induces the expression of MMP-9 gene through activation of NF-κB and AP-1.

To investigate the functional role of KAI1/CD82, a metastasis suppressor for human prostate cancer, in the regulation of homotypic cell adhesion, we transfected KAI1 cDNA into DU 145 human prostate cancer cells and established stable transfectant clones with high KAI1/CD82 expression. The KAI1 transfectant cells exhibited significantly increased homotypic cell aggregation in comparison with the control transfectant cells. This aggregation of the KAI1 transfectants was further enhanced upon exposure to anti-CD82 antibody, suggesting that KAI1/CD82 may be involved in the intracellular signaling for the cell adhesion. Among several signal pathway inhibitors tested, PP1, an inhibitor of Src family kinases, significantly suppressed homotypic aggregation of the KAI1 transfectant cells. Ligation of KAI1/CD82 with anti-CD82 antibody increased endogenous Src kinase activity of the KAI1 transfectant cells. When different types of src expression constructs were retransfected into the KAI1-transfected DU 145 cells, kinase-negative mutant src transfectant cells exhibited much lower homotypic aggregation than the mock cells transfected with an empty vector. Moreover, homotypic aggregation of the mutant src transfectant cells was not enhanced by KAI1/CD82 ligation with anti- CD82 antibody. These results suggest that Src mediates the intracellular signaling pathway of KAI1/CD82 for the induction of homotypic adhesion of human prostate cancer cells.

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