Epithin was originally identified as a mouse type II membrane serine protease. Its human orthologue membrane type-serine protease 1 (MT-SP1)/matriptase has been reported to be localized on the plasma membrane. In addition, soluble forms of matriptase were isolated from human breast milk and breast cancer cell-conditioned medium. In this paper, we report a processing mechanism that appears to be required for the release of epithin. CHO-K1 or COS7 cells transfected with single full-length epithin cDNA generated two different-sized proteins in cell lysates, 110 and 92 kDa. The 92-kDa epithin was found to be an N-terminally truncated form of the 110-kDa epithin, and it was the only form detected in the culture medium. The 92-kDa epithin was also found on the cell surface, where it was anchored by the N-terminal fragment. The results of in vivo cell labeling experiments indicate that the 110-kDa epithin is rapidly processed to the 92-kDa epithin. Using site-directed mutagenesis experiments, we identified Gly149 of the GSVIA sequence in epithin as required for the processing and release of the protein. These results suggest that N-terminal processing of epithin at Gly149 is a necessary prerequisite step for release of the protein.

Annals of the New York Academy of SciencesVolume 928, Issue 1 p. 376-376 p38 Mitogen-activated Protein Kinase Is Involved in H2O2-induced Phospholipase D Activation in Vascular Smooth Muscle Cells Eung-Gook Kim, Eung-Gook Kim Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorEun-Young Shin, Eun-Young Shin Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorDo Sik Min, Do Sik Min Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorByoung-Hee Park, Byoung-Hee Park Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorKyung-Sun Shin, Kyung-Sun Shin Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorMin-Soo Hyun, Min-Soo Hyun Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorJi-Cheol Shin, Ji-Cheol Shin Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorHee-Yul Ahn, Hee-Yul Ahn Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorRoger J. Davis, Roger J. Davis Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorSeung-Ryul Kim, Seung-Ryul Kim Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this author Eung-Gook Kim, Eung-Gook Kim Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorEun-Young Shin, Eun-Young Shin Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorDo Sik Min, Do Sik Min Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorByoung-Hee Park, Byoung-Hee Park Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorKyung-Sun Shin, Kyung-Sun Shin Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorMin-Soo Hyun, Min-Soo Hyun Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorJi-Cheol Shin, Ji-Cheol Shin Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorHee-Yul Ahn, Hee-Yul Ahn Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorRoger J. Davis, Roger J. Davis Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this authorSeung-Ryul Kim, Seung-Ryul Kim Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, KoreaSearch for more papers by this author First published: 25 January 2006 https://doi.org/10.1111/j.1749-6632.2001.tb05694.xRead the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume928, Issue1HEALTHY AGING FOR FUNCTIONAL LONGEVITY: MOLECULAR AND CELLULAR INTERACTIONS IN SENESCENCEApril 2001Pages 376-376 RelatedInformation

ECV304, a spontaneously transformed cell line derived from the human umbilical vein endothelial cell (HUVEC) (Takahashi et al., 1990), has been developed as an in vitro angiogenesis model. In the present study, we further characterized the angiogenic properties of this cell line. Compared to HUVEC, ECV304 cells showed distinct features including a higher activity of cellular adhesion, slower but reproducible progression of angiogenesis on Matrigel, and resistance to apoptosis. Thus, the expression of integrin and activation of extracellular-signal regulated kinase 1/2 (Erk1/2), a downstream effector of the integrin pathway, were examined. Flow cytometry revealed that alpha3beta1 integrin was markedly upregulated in ECV304 cells, while alpha(v)beta1 and alpha5beta1 integrins were slightly downregulated. Consistent with this, the binding activity to collagen type IV and laminin, major extracellular matrices of Matrigel, was increased 1.4- and 1.9-fold in ECV304 cells, respectively. This tight binding may retard the initial stage of sprouting and migration in the angiogenesis of ECV304 cells. It has been further demonstrated that Erk1/2 is constitutively active in ECV304 cells, rendering them resistent to the inhibitory effect of PD98059 on proliferation. However, migration of both HUVEC and ECV304 cells was inhibited to a similar extent by PD98059 in a dose-dependent manner. Up to 50 microM of PD98059, no significant changes in cell binding and tubulogenesis on Matrigel was observed in ECV304 cells. In contrast, the tubulogenesis of HUVEC was severely impaired by PD98059. Elevated Erk1/2 activity in ECV304 cells was suppressed by dominant negative H-Ras, but not by cytochalasin D. These results suggest that the overexpression of alpha3beta1 integrin and the constitutive activation of Erk1/2 play a key role in the alteration of the angiogenic properties of ECV304 cells.

We explored a biological role of SET as it relates to cell proliferation and differentiation. Immunohistochemical staining demonstrated that the expression of SET was ubiquitous and diffuse over the whole embryo on gestational day 15. At a later stage of development, SET was expressed at relatively lower levels and localized to specific tissues and cells. On embryonic day 19, specific SET immunoreactivity was found in the epithelium of skin, respiratory tract, intestine, and retina as well as in muscle and cartilage. In these cells SET was stained mostly in the nucleus, which was supported indirectly by nuclear transport of enhanced green fluorescence protein-SET fusion proteins in ECV304 endothelial cells. Set mRNA expression was further confirmed in various cultured cells, including NIH 3T3 cells, L6 myoblast cells, human umbilical vein endothelial cells, and ECV304 cells. Using F9 teratocarcinoma cell lines, which were stimulated to differentiate into the two different cell lineages of parietal and visceral endoderm, we have further examined the role of SET. The expression of set mRNA and SET protein was diminished about three-fold in both differentiated endoderm cells compared to the undifferentiated F9 cells. However, when F9 cells were subjected to serum starvation, reduction of set mRNA abundance also took place at a similar level to that observed in response to differentiation. Consistent with this, quiescent L6 myoblast showed a marked downregulation of set mRNA compared to proliferating cells. These results suggest that SET is involved mainly in the regulation of cell proliferation rather than differentiation during embryonic development.

Phospholipase D has been recognized as playing an important role in signal transduction in many types of cells. We investigated the expression of phospholipase D during the differentiation of F9 embryonal teratocarcinoma cells. The ADP ribosylation factor-dependent phospholipase D activity, as measured by an in vitro assay, and H2O2-induced phospholipase D activity and phospholipase D protein content in whole cells were decreased during the differentiation of F9 cells induced by a combination of dibutyryl cyclic AMP and all-trans retinoic acid. In contrast, these changes were not observed when cells were induced by retinoic acid. These results suggest that down-regulation of phospholipase D protein is associated with differentiation of F9 cells to a parietal endoderm lineage.

Fibroblast growth factor (FGF), a member of heparin-binding growth factor, is a family of nine structurally related polypeptides showing multiple biological activities: angiogenesis, wound healing and development. FGF is known to exert its biological effects by interacting with both low-affinity receptor, heparan sulfate proteoglycan which is often substituted by heparin, and high-affinity receptor, receptor tyrosine kinase. Formation of ternary complex, heparin, FGF, and the high-affinity receptor seems to be essential for the subsequent intracellular events. Therefore to interrupt an interaction between these components is a good strategy for development of a new drug. Basic FGF (bFGF) is well known for its potent angiogenic effects. To examine a feasibility of heparin as a modulator of angiogenesis, the effect of heparin on the binding of bFGF to FGF receptor (FGFR) in the human endothelial cells was evaluated. As a preliminary step, we determined the subtype of FGFRs expressed in the endothelial cells from bovine glomerulus and aorta, respectively. To date, four different FGFRs have been cloned and analyzed. In the endothelial cells FGFR-1 was found to be expressed predominantly. In human endothelial cells and NIH 3T3 cells heparin inhibited the binding of bFGF to low-affinity receptors as well as to high-affinity receptors in a dose-dependent manner. Tyrosine phosphorylation of She and other cellular proteins by bFGF were accordingly reduced in the presence of heparin (20 mu g/ml). In addition, c-fos induction was blocked by the same concentration of heparin. Collectively, these data indicate that high concentration of heparin is inhibitory to the action of bFGF during a short period of time in vitro.