CD9 is an integral membrane protein associated with integrins and other membrane proteins. Mice lacking CD9 were produced by homologous recombination. Both male and female CD9-/- mice were born healthy and grew normally. However, the litter size from CD9-/- females was less than 2% of that of the wild type. In vitro fertilization experiments indicated that the cause of this infertility was due to the failure of sperm-egg fusion. When sperm were injected into oocytes with assisted microfertilization techniques, however, the fertilized eggs developed to term. These results indicate that CD9 has a crucial role in sperm-egg fusion.

Ectodomain shedding is an important mechanism to regulate the biological activities of membrane proteins. We focus here on the signaling mechanism of the ectodomain shedding of heparin-binding epidermal growth factor (EGF)-like growth factor (pro HB-EGF). Lysophosphatidic acid (LPA), a ligand for seven-transmembrane G protein-coupled receptors, stimulates the shedding of pro HB-EGF, which constitutes a G proteincoupled receptor-mediated transactivation of the EGF receptor. Experiments using a series of inhibitors and overexpression of mutant forms of signaling molecules revealed that the Ras-Raf-MEK signal is essential for the LPA-induced shedding. In addition, the small GTPase Rac is involved in the LPA-induced shedding, possibly to promote MEK activation. 12-O-Tetradecanoylphorbol-13-acetate is another potent inducer of pro HB-EGF shedding. We also demonstrate that the LPA-induced pathway is distinct from the 12-O-tetradecanoylphorbol-13-acetate-induced pathway and that these pathways constitute a dual signaling cascade that regulates the shedding of pro HB-EGF.

To examine whether cancer cell dissemination results from incisional biopsy, we tried to detect squamous cell carcinoma (SCC) cells in peripheral blood before and after incisional biopsy by means of cytokeratin 19 (CK19) reverse-transcriptase polymerase chain reaction (RT-PCR). The study population consisted of 20 patients with oral SCC; 10 were given incisional biopsies followed by radical excision (the incisional biopsy group), and the remaining 10 were treated by excisional biopsy alone (the excisional biopsy group). Ten non-oral cancer patients with benign oral lesions served as controls. Five-ml blood aspirates collected before and after incision were used for CK19 RT-PCR. Two (20.0%) of 10 patients from the incisional biopsy group were positive for CK19 transcripts in their peripheral blood drained 15 min after incision. In contrast, CK19 transcript was not detected either in the excisional biopsy group or in controls. Surgical invasiveness for oral cancer, including incisional biopsy, causes dissemination of cancer cells into circulation, resulting in increased risk of metastasis.

Because CD9 is implicated in cell growth, cell adhesion and cell motility, altered CD9 expression might be involved in cancer invasion and metastasis. We have studied the immunolocalization of CD9 in oral squamous cell carcinoma (SCC). Sections prepared from paraffin-embedded specimens from patients with SCC of the oral cavity were stained with a monoclonal anti-CD9 antibody by means of the streptoavidin biotin method. Significant reduction or complete loss of CD9 expression was observed in cancer cells at the periphery of the cancer nests in the advancing front of invading tumor. Among 78 cases of oral SCCs examined, 46 (59.0%) cases were completely negative for CD9 expression. Loss of CD9 expression in cancer tissue strongly correlated with a high incidence of cervical lymph node metastasis and poorer prognosis (P=0.001). Thus a close examination of CD9 in SCC tissue would be useful for the prognosis of patients with oral carcinoma.

ABSTRACT. CD9 and CD63 belong to a tetramembrane-spanning glycoprotein family called tetraspanin, and are involved in a wide variety of cellular processes, but the structure-function relationship of this family of proteins has yet to be clarified. CD9 associates with diphtheria toxin receptor (DTR), which is identical to the membraneanchored form of heparin-binding EGF-like growth factor (proHB-EGF). CD9 upregulates the diphtheria toxin (DT) binding activity of DTR/proHB-EGF, while CD63 does not upregulate the DT binding activity in spite of the fact that this protein also associates with DTR/proHB-EGF on the cell surface. CD9 molecules localize on the cell surface, while those of CD63 localize predominantly at lysosomes and intracellular compartments. We made CD9/ CD63 chimeric molecules and then studied their intracellular localization and upregulation activities. The C-terminal regions of CD63, which includes the lysosome sorting motif, showed a strong inhibitory effect on the expression of the chimeric proteins at the cell surface, while mutants lacking the lysosome sorting motif delivered more efficiently on the cell surface, indicating that the lysosome sorting motif contributes to the inhibitory effect of the C-terminal region. However, the N-terminal half of this family of proteins containing the 1st to 3rd transmembrane domains also seems to influence the cell surface expression. For the upregulation of DT binding activity the large extracellular loop (EC2) of CD9 was essential, while the remaining regions influenced the upregulation activity by changing the efficiency of cell surface expression. From these results we discussed the structure-function relationship of this family of proteins.