G-protein-coupled receptor (GPCR) agonists are well-known inducers of cardiac hypertrophy. We found that the shedding of heparin-binding epidermal growth factor (HB-EGF) resulting from metalloproteinase activation and subsequent transactivation of the epidermal growth factor receptor occurred when cardiomyocytes were stimulated by GPCR agonists, leading to cardiac hypertrophy. A new inhibitor of HB-EGF shedding, KB-R7785, blocked this signaling. We cloned a disintegrin and metalloprotease 12 (ADAM12) as a specific enzyme to shed HB-EGF in the heart and found that dominant-negative expression of ADAM12 abrogated this signaling. KB-R7785 bound directly to ADAM12, suggesting that inhibition of ADAM12 blocked the shedding of HB-EGF. In mice with cardiac hypertrophy, KB-R7785 inhibited the shedding of HB-EGF and attenuated hypertrophic changes. These data suggest that shedding of HB-EGF by ADAM12 plays an important role in cardiac hypertrophy, and that inhibition of HB-EGF shedding could be a potent therapeutic strategy for cardiac hypertrophy.
Recently we reported that N-glycans on the -propeller domain of the integrin ␣5 subunit (S-3,4,5) are essential for ␣51 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type ␣5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the ␣5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type ␣5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the ␣5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.Glycosylation is a crucial post-translational modification of most secreted and cell surface proteins (1). Glycosylation is involved in a variety of physiological and pathological events, including cell growth, migration, differentiation, and tumor invasion. It is well known that glycans play important roles in cell-cell communication, intracellular signal transduction, protein folding, and stability (2, 3).Integrins comprise a family of receptors that are important for cell adhesion. The major function of integrins is to connect cells to the extracellular matrix, activate intracellular signaling pathways, and regulate cytoskeletal formation (4). Integrin ␣51 is well known as a fibronectin (FN) 3 receptor. The interaction between integrin ␣5 and FN is essential for cell migration, cell survival, and development (5-8). In addition, integrins are N-glycan carrier proteins. For example, ␣51 integrin contains 14 and 12 putative N-glycosylation sites on the ␣5 and 1 subunits, respectively. Several studies suggest that N-glycosylation is essential for functional integrin ␣51. When human fibroblasts were cultured in the presence of 1-deoxymannojirimycin, which prevents N-linked oligosaccharide processing, immature ␣51 integrin appeared on the cell surface, an...
1 We investigated the ability of a newly synthesized sugar derivative, OJ-R9188, {N-(2-tetradecylhexadecanoyl)-O-(L-alpha-fucofuranosyl)-D-seryl}-L-glutamic acid 1-methylamide 5-L-arginine salt, to block binding of selectins to their ligands in vitro and inhibit the in®ltration of leukocytes in vivo. 2 OJ-R9188 prevented the binding of human E-, P-and L-selectin-IgG fusion proteins to immobilized sialyl Lewis x (sLe x )-pentasaccharide glycolipid, with IC 50 values of 4.3, 1.3, and 1.2 mM, respectively.3 In a mouse model of thioglycollate-induced peritonitis, OJ-R9188 at 10 mg kg 71 , i.v. inhibited neutrophil accumulation in the peritoneal cavity. In the IgE-mediated skin reaction, OJ-R9188 at 3 and 10 mg kg 71 , i.v. signi®cantly inhibited extravasation of neutrophils and eosinophils into the in¯ammatory sites and at 10 mg kg 71 , i.v. also inhibited in®ltration caused by picryl chlorideinduced delayed-type hypersensitivity in mice. These results suggest that OJ-R9188 may be a useful selectin blocker, with activity against human and mouse E-, P-and L-selectins in vitro and in vivo, and that blocking selectin-sLe x binding is a promising strategy for the treatment of allergic skin diseases.
1 Selectins play an important role on leukocytes in®ltration into in¯ammatory tissues. To understand the role of selectins, we investigated the eects of selectin-IgG chimeras and anti selectin antibodies on the murine IgE-mediated skin in¯ammation model. 2 Biphasic skin reactions were induced by intradermal challenge with ovalbumin (OA) to ears of actively sensitized mice. This reaction was characterized by immediate and late phase responses observed as which were induced via a rapid increase in capillary permeability and leukocyte in®ltration, respectively. The expression of E-selectin mRNA was signi®cantly increased to reach its highest level at 2 h after OA challenge. 3 E-, P-, and L-selectin-IgG chimeras inhibited the late phase responses, i.e. ear swelling, neutrophil in®ltration and eosinophil in®ltration at 24 h after OA challenge in a dose-dependent manner at dose range of 0.1 ± 10 mg kg 71 , i.v. Antiselectin antibodies did not inhibit the increase of ear swelling. But anti E-and P-selectin antibodies signi®cantly inhibited neutrophil in®ltration and eosinophil in®ltration. 4 These results indicate that selectins play an important role on the late phase response of the murine IgE-mediated skin in¯ammation model by mediating in¯ammatory cell adhesion to endothelium.
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