Abstract
Proteolytic shedding is an important step in the functional down-regulation and turnover of most membrane proteins at the cell surface. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a multifunctional glycoprotein that has two Ig-like domains in its extracellular portion and functions in cell adhesion as an inducer of matrix metalloproteinase (MMP) expression in surrounding cells. Although the shedding of EMMPRIN is reportedly because of cleavage by metalloproteinases, the responsible proteases, cleavage sites, and stimulants are not yet known. In this study, we found that human tumor HT1080 and A431 cells shed a 22-kDa EMMPRIN fragment into the culture medium. The shedding was enhanced by phorbol 12-myristate 13-acetate and inhibited by TIMP-2 but not by TIMP-1, suggesting the involvement of membrane-type MMPs (MT-MMPs). Indeed, down-regulation of the MT1-MMP expression in A431 cells using small interfering RNA inhibited the shedding. The 22-kDa fragment was purified, and the C-terminal amino acid was determined. A synthetic peptide spanning the cutting site was cleaved by MT1-MMP in vitro. The cleavage site is located in the linker region connecting the two Ig-like domains. The N-terminal Ig-like domain is important for the MMP inducing activity of EMMPRIN and for cell-cell interactions, presumably through its ability to engage in homophilic interactions, and the 22-kDa fragment retained the ability to augment MMP-2 expression in human fibroblasts. Thus, the MT1-MMP-dependent cleavage eliminates the functional N-terminal domain of EMMPRIN from the cell surface, which is expected to down-regulate its function. At the same time, the released 22-kDa fragment may mediate the expression of MMPs in tumor tissues.
Highlights
Lating factor, basigin, and M6) is a multifunctional glycoprotein that belongs to the immunoglobulin superfamily [1,2,3,4]
Our knowledge about the physiological substrates of MT1-matrix metalloproteinase (MMP) is still fragmentary, but proteins that can be cleaved by MT1-MMP are often co-purified with it from cell lysates, and Extracellular matrix metalloproteinase inducer (EMMPRIN) is among the proteins we identified as co-purifying with MT1-MMP.3
We examined the expression of EMMPRIN in cell lysates and its shedding into the conditioned medium (CM) with Western blotting using anti-EMMPRIN pAb (Fig. 1)
Summary
Cells and Culture Conditions—The human fibrosarcoma cell line HT1080, human epidermoid carcinoma cell line A431, and human melanoma cell line A375 were obtained from the American Type Culture Collection (ATCC, Manassas, VA), and the African green monkey kidney cell line COS-7 was obtained from Health Science Research Resources Bank (Osaka, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum for 3 days in the presence or absence of 1 g/ml doxycycline (Sigma). The CM containing the FLAG-tagged EMMPRIN fragment was purified on an affinity column filled with agarose beads conjugated to an anti-FLAG M2 mouse monoclonal antibody (Sigma). Immunocytochemistry and Immunohistochemistry—A431 cells expressing FLAG-tagged MT1-MMP were cultured on glass coverslips for 16 h in the presence of 10 M MMI270 and fixed with 4% paraformaldehyde in PBS for 5 min. After a blocking step using 5% goat serum, 3% bovine serum albumin in PBS, the cells were treated with a rabbit anti-FLAG polyclonal antibody (Sigma) or a mouse anti-EMMPRIN mAb. An Alexa 488-conjugated goat anti-mouse IgG and an Alexa 568-conjugated goat anti-rabbit IgG (Invitrogen) were used as secondary antibodies. Peroxidase activity was detected by a solution of diaminobenzidine containing H2O2 in a Tris-HCl buffer, which develops a brown color
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