Signaling via the epidermal growth factor receptor (EGFR), which has critical roles in development and diseases such as cancer, is regulated by proteolytic shedding of its membrane-tethered ligands. Sheddases for EGFR-ligands are therefore key signaling switches in the EGFR pathway. Here, we determined which ADAMs (a disintegrin and metalloprotease) can shed various EGFR-ligands, and we analyzed the regulation of EGFR-ligand shedding by two commonly used stimuli, phorbol esters and calcium influx. Phorbol esters predominantly activate ADAM17, thereby triggering a burst of shedding of EGFR-ligands from a late secretory pathway compartment. Calcium influx stimulates ADAM10, requiring its cytoplasmic domain. However, calcium influx-stimulated shedding of transforming growth factor ␣ and amphiregulin does not require ADAM17, even though ADAM17 is essential for phorbol ester-stimulated shedding of these EGFR-ligands. This study provides new insight into the machinery responsible for EGFR-ligand release and thus EGFR signaling and demonstrates that dysregulated EGFR-ligand shedding may be caused by increased expression of constitutively active sheddases or activation of different sheddases by distinct stimuli.

Key Words: ADAMs Ⅲ metalloproteinase-disintegrins Ⅲ TNF␣-convertase Ⅲ proliferative retinopathy Ⅲ pathological neovascularization P athological neovascularization has a critical role in diseases such as cancer, 1,2 rheumatoid arthritis 3 and proliferative retinopathies, including retinopathy of prematurity, diabetic retinopathy and the wet form of macular degeneration. 4,5 Therefore molecules with roles in pathological neovascularization are considered potential targets for treatment of these conditions. Previous studies have identified a role for the cell surface metalloproteinase ADAM17 (a disintegrin and metalloproteinase 17, also referred to as TACE [tumor necrosis factor ␣-converting enzyme]) in crosstalk between the VEGFR2 and extracellular signal-regulated kinase 1/2 in endothelial cells, and in processing several receptors with key functions in angiogenesis, including the VEGFR2 and Tie2. 6 The goal of the present study was to determine whether ADAM17 has a role in angiogenesis or pathological neovascularization in vivo by subjecting conditional knockout mice carrying floxed alleles of ADAM17 7 and a Cre-recombinase expressed either in endothelial cells (Tie2-Cre) or in smooth muscle cells and pericytes (␣-smooth muscle actin [␣sma] Cre) to mouse models of pathological neovascularization.ADAM17 was first discovered as the converting enzyme for tumor necrosis factor (TNF)␣, 8,9 a potent proinflammatory cytokine that is a causative factor in autoimmune diseases such as rheumatoid arthritis and Crohn's disease as well as in septic shock in mice. 10 Once mice lacking ADAM17 were generated, it became clear that ADAM17 is Original received August 14, 2009; revision received January 13, 2010; accepted January 19, 2010 The ability of ADAM17 to release endothelial cell membrane proteins on stimulation with VEGF-A raised questions about what role ADAM17 has during developmental angiogenesis and in pathological neovascularization in adult animals. Although mice lacking ADAM17 die perinatally, most likely as a consequence of their severe heart valve defects, 11,12 there have been no reports of defects in developmental angiogenesis in these animals. To address whether ADAM17 has a role in angiogenesis or pathological neovascularization or both, we conditionally inactivated ADAM17 in endothelial cells or in smooth muscle cells such as pericytes, and then determined how lack of ADAM17 affects two mouse models for pathological neovascularization, the oxygen induced retinopathy model for retinopathy of prematurity, and growth of heterotopically injected tumor cells. Moreover, we assessed proliferation and chord formation of endothelial cells lacking ADAM17, and evaluated the role of ADAM17 in the proteolytic release of membrane proteins with known roles in angiogenesis and pathological neovascularization. Methods Reagents and Cell LinesPorcine aortic endothelial cells expressing VEGFR2/KDR (PAE/ KDR cells) and mouse embryonic fibroblasts (mEFs) lacking ADAM17 have been described previously. 6,15 Reagents were from...

Ngkelo et al. use a mast cell–deficient mouse model to reveal a protective role of mast cells in myocardial infarction, through regulation of the cardiac contractile machinery.