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...

Because of the probable causal relationship between constitutive p210bcr/abl protein tyrosine kinase activity and manifestations of chronic-phase chronic myelogenous leukemia (CML; myeloid expansion), a key goal is to identify relevant p210 substrates in primary chronic-phase CML hematopoietic progenitor cells. We describe here the purification and mass spectrometric identification of a 155-kD tyrosine phosphorylated protein associated with src homologous and collagen gene (SHC) from p210bcr/abl-expressing hematopoietic cells as SHIP2, a recently reported, unique SH2-domain–containing protein closely related to phosphatidylinositol polyphosphate 5-phosphatase SHIP. In addition to an N-terminal SH2 domain and a central catalytic region, SHIP2 (like SHIP1) possesses both potential PTB(NPXY) and SH3 domain (PXXP) binding motifs. Thus, two unique 5-ptases with striking structural homology are coexpressed in hematopoietic progenitor cells. Stimulation of human hematopoietic growth factor responsive cell lines with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrate the rapid tyrosine phosphorylation of SHIP2 and its resulting association with SHC. This finding suggests that SHIP2, like that reported for SHIP1 previously, is linked to downstream signaling events after activation of hematopoietic growth factor receptors. However, using antibodies specific to these two proteins, we demonstrate that, whereas SHIP1 and SHIP2 selectively hydrolyze PtdIns(3,4,5)P3 in vitro, only SHIP1 hydrolyzes soluble Ins(1,3,4,5)P4. Such an enzymatic difference raises the possibility that SHIP1 and SHIP2 may serve different functions. Preliminary binding studies using lysates from p210bcr/abl-expressing cells indicate that both Ptyr SHIP2 and Ptyr SHIP1 bind to the PTB domain of SHC but not to its SH2 domain. Interestingly, SHIP2 was found to selectively bind to the SH3 domain of ABL, whereas SHIP1 selectively binds to the SH3 domain of Src. Furthermore, in contrast to SHIP1, SHIP2 did not bind to either the N-terminal or C-terminal SH3 domains of GRB2. These observations suggest (1) that SHIP1 and SHIP2 may have a different hierarchy of binding SH3 containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and (2) that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. Because recent evidence has clearly implicated both PI(3,4,5)P3 and PI(3,4)P2 in growth factor-mediated signaling, our finding that both SHIP1 and SHIP2 are constitutively tyrosine phosphorylated in CML primary hematopoietic progenitor cells may thus have important implications in p210bcr/abl-mediated myeloid expansion.

Because of the probable causal relationship between constitutive p210bcr/abl protein tyrosine kinase activity and manifestations of chronic-phase chronic myelogenous leukemia (CML; myeloid expansion), a key goal is to identify relevant p210 substrates in primary chronic-phase CML hematopoietic progenitor cells. We describe here the purification and mass spectrometric identification of a 155-kD tyrosine phosphorylated protein associated with src homologous and collagen gene (SHC) from p210bcr/abl-expressing hematopoietic cells as SHIP2, a recently reported, unique SH2-domain–containing protein closely related to phosphatidylinositol polyphosphate 5-phosphatase SHIP. In addition to an N-terminal SH2 domain and a central catalytic region, SHIP2 (like SHIP1) possesses both potential PTB(NPXY) and SH3 domain (PXXP) binding motifs. Thus, two unique 5-ptases with striking structural homology are coexpressed in hematopoietic progenitor cells. Stimulation of human hematopoietic growth factor responsive cell lines with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrate the rapid tyrosine phosphorylation of SHIP2 and its resulting association with SHC. This finding suggests that SHIP2, like that reported for SHIP1 previously, is linked to downstream signaling events after activation of hematopoietic growth factor receptors. However, using antibodies specific to these two proteins, we demonstrate that, whereas SHIP1 and SHIP2 selectively hydrolyze PtdIns(3,4,5)P3 in vitro, only SHIP1 hydrolyzes soluble Ins(1,3,4,5)P4. Such an enzymatic difference raises the possibility that SHIP1 and SHIP2 may serve different functions. Preliminary binding studies using lysates from p210bcr/abl-expressing cells indicate that both Ptyr SHIP2 and Ptyr SHIP1 bind to the PTB domain of SHC but not to its SH2 domain. Interestingly, SHIP2 was found to selectively bind to the SH3 domain of ABL, whereas SHIP1 selectively binds to the SH3 domain of Src. Furthermore, in contrast to SHIP1, SHIP2 did not bind to either the N-terminal or C-terminal SH3 domains of GRB2. These observations suggest (1) that SHIP1 and SHIP2 may have a different hierarchy of binding SH3 containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and (2) that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. Because recent evidence has clearly implicated both PI(3,4,5)P3 and PI(3,4)P2 in growth factor-mediated signaling, our finding that both SHIP1 and SHIP2 are constitutively tyrosine phosphorylated in CML primary hematopoietic progenitor cells may thus have important implications in p210bcr/abl-mediated myeloid expansion.