Stress-induced phosphorylation of eIF2α inhibits global protein synthesis to conserve energy for repair of stress-induced damage. Stress-induced translational arrest is observed in cells expressing a nonphosphorylatable eIF2α mutant (S51A), which indicates the existence of an alternative pathway of translational control. In this paper, we show that arsenite, heat shock, or ultraviolet irradiation promotes transfer RNA (tRNA) cleavage and accumulation of tRNA-derived, stress-induced small RNAs (tiRNAs). We show that angiogenin, a secreted ribonuclease, is required for stress-induced production of tiRNAs. Knockdown of angiogenin, but not related ribonucleases, inhibits arsenite-induced tiRNA production and translational arrest. In contrast, knockdown of the angiogenin inhibitor RNH1 enhances tiRNA production and promotes arsenite-induced translational arrest. Moreover, recombinant angiogenin, but not RNase 4 or RNase A, induces tiRNA production and inhibits protein synthesis in the absence of exogenous stress. Finally, transfection of angiogenin-induced tiRNAs promotes phospho-eIF2α–independent translational arrest. Our results introduce angiogenin and tiRNAs as components of a phospho-eIF2α–independent stress response program.

Copper ions stimulate proliferation of human umbilical artery and vein endothelial cells but not human dermal fibroblasts or arterial smooth muscle cells. Incubation of human umbilical vein endothelial cells for 48 h with 500 microM CuSO4 in a serum-free medium in the absence of exogenous growth factors results in a twofold increase in cell number, similar to the cell number increase induced by 20 ng/ml of basic fibroblast growth factor under the same conditions. Copper-induced proliferation of endothelial cells is not inhibited by 10% fetal bovine serum or by the presence of antibodies against a variety of angiogenic, growth, and chemotactic factors including angiogenin, fibroblast growth factors, epidermal growth factor, platelet-derived growth factor, tumor necrosis factor-alpha, transforming growth factor-beta, macrophage/monocyte chemotactic and activating factor, and macrophage inflammatory protein-1alpha. Moreover, despite the previous observations that copper increased total specific binding of 125I-angiogenin to endothelial cells, binding to the 170 kDa receptor is not changed; hence, the mitogenic activity of angiogenin is not altered by copper. Copper-induced proliferation, along with early reports that copper induces migration of endothelial cells, may suggest a possible mechanism for the involvement of copper in the process of angiogenesis.

SUMMARY Regulation of stem and progenitor cell populations is critical in the development, maintenance and regeneration of tissues. Here, we define a novel mechanism by which a niche-secreted ribonuclease, angiogenin (ANG), distinctively alters the functional characteristics of primitive hematopoietic stem/progenitor cells (HSPC) compared with lineage-committed myeloid-restricted progenitors (MyePro). Specifically, ANG reduces the proliferative capacity of HSPC while simultaneously increasing proliferation of MyePro. Mechanistically, ANG induces cell type-specific RNA processing events: tRNA-derived stress-induced small RNA (tiRNA) generation in HSPC and ribosomal RNA (rRNA) induction in MyePro, leading to respective reduction and increase in protein synthesis. Recombinant ANG protein improves survival of irradiated animals and enhances hematopoietic regeneration of mouse and human HSPC in transplantation. Thus, ANG plays a non-cell autonomous role in regulation of hematopoiesis by simultaneously preserving HSPC stemness and promoting MyePro proliferation. These cell type-specific functions of ANG suggest considerable therapeutic potential.

Angiogenin (ANG), also known as ribonuclease (RNASE) 5, is a member of the vertebrate-specific, secreted RNASE superfamily. ANG was originally identified as a tumor angiogenic factor, but its biological activity has been extended from inducing angiogenesis to stimulating cell proliferation and more recently, to promoting cell survival. Under growth conditions, ANG is translocated to nucleus where it accumulates in nucleolus and stimulates ribosomal RNA (rRNA) transcription, thus facilitating cell growth and proliferation. Under stress conditions, ANG is accumulated in cytoplasmic compartments and modulates the production of tiRNA, a novel class of small RNA that is derived from tRNA and is induced by stress. tiRNA suppress global protein translation by inhibiting both cap-dependent and -independent translation including that mediated by weak IRESes. However, strong IRES-mediated translation, a mechanism often used by genes involved in pro-survival and anti-apoptosis, is not affected. Thus, ANG-mediated tiRNA reprogram protein translation, save anabolic energy, and promote cell survival. This recently uncovered function of ANG presents a novel mechanism of action in regulating cell growth and survival.

Cyclin-dependent kinase 6 (CDK6) promotes cell cycle progression and is overexpressed in human lymphoid malignancies. To determine the role of CDK6 in development and tumorigenesis, we generated and analyzed knockout mice. Cdk6-deficient mice show pronounced thymic atrophy due to reduced proliferative fractions and concomitant transitional blocks in the double-negative stages. Using the OP9-DL1 system to deliver temporally controlled Notch receptordependent signaling, we show that CDK6 is required for Notch-dependent survival, proliferation, and differentiation. Furthermore, CDK6-deficient mice were resistant to lymphomagenesis induced by active Akt, a downstream target of Notch signaling. These results show a critical requirement for CDK6 in Notch/Akt-dependent T-cell development and tumorigenesis and strongly support CDK6 as a specific therapeutic target in human lymphoid malignancies. [Cancer Res 2009; 69(3):810-8]

The 42-kDa angiogenin binding protein isolated previously has been purified to electrophoretic homogeneity. It has been identified as a member of the actin family by peptide mapping and partial amino acid sequencing. The interaction of bovine muscle actin with angiogenin is similar to that of the angiogenin binding protein. Angiogenin induces the polymerization of actin below the critical concentration for spontaneous polymerization. The interaction occurs both in solution and on a poly(vinylidene difluoride) membrane. It is inhibited by excess unlabeled angiogenin and also by platelet factor 4 and protamine, which are known inhibitors of angiogenesis. Two other angiogenic molecules, basic fibroblast growth factor and tumor necrosis factor a, bind to 125I-labeled actin and can be crosslinked by a water-soluble carbodilmide. Both actin and an anti-actin antibody inhibit the angiogenic activity of angiogenin in the chicken embryo chorioallantoic membrane assay. The results indicate that the angiogenin binding protein is a cell surface actin and suggest that the reaction between angiogenin and this actin is an essential step in the angiogenesis process induced by angiogenin.Angiogenin is a 14-kDa protein purified initially from serumfree supernatants of an established human adenocarcinoma cell line, HT-29 (1). It was the first human tumor-derived angiogenic protein to be isolated based on its in vivo activity. It stimulates endothelial cells to produce diacylglycerol (2) and secrete prostacyclin (3) by phospholipase activation. It supports endothelial and fibroblast cell adhesion (4) and modulates a mitogenic effect in certain cells (5). An angiogenin binding protein (AngBP), which has properties consistent with its being a component of a cellular receptor for angiogenin, has been identified and isolated from a transformed endothelial cell line, GM7373 (6). It is a cell-surface protein with an apparent molecular mass of 42 kDa and is released from endothelial cells by exposure to heparnn, heparan sulfate, or angiogenin itself.We report here the further purification and characterization of AngBP. Tryptic peptide mapping and amino acid sequence analysis indicate that AngBP is a member of the actin family. The binding of angiogenin to bovine muscle actin and the inhibitory effect of actin and anti-actin antibodies on angiogenin-induced neovascularization on the chicken embryo chorioallantoic membrane (CAM) are also reported. The mechanism by which AngBP is released from the cell surface and the physiological significance of its presence and displacement remain to be elucidated.

Angiogenin stimulates both [ 3 H]thymidine incorporation and proliferation of human endothelial cells in sparse cultures. Under these conditions, a 170-kDa cell surface protein can be detected that binds angiogenin specifically. Angiogenin-stimulated cell growth is concentrationdependent and is completely inhibited by an anti-angiogenin monoclonal antibody, but not by a nonimmune control antibody. It is not affected by the nonangiogenic homolog, RNase A, nor by other angiogenic proteins, such as basic fibroblast growth factor and its antibody. Results suggest that under specific conditions, endothelial cells express an angiogenin receptor that may mediate angiogenin-stimulated DNA synthesis and proliferation and play an important role in angiogenin-induced angiogenesis.

Angiogenin, a potent inducer of neovascularization in the chicken chorioallantoic membrane and rabbit cornea, promotes endothelial cell invasion of Matrigel basement membrane. A transformed bovine aortic endothelial cell line, GM 7373, Is 5 times more invasive when cultured in the presence of 1 pg of bovine anglogenin per ml than in its absence. A polyclonal anti-angiogenin antibody and a2-antipsmin neutralize the effect of angiogenin, but an angiogenin-bng protein (actin) does not. Further, this concentration of angoeni induces a 14-fold increase in the cellassociated proteolytic activity of cultured endothelial cells, determined with a tissue-type plasminogen activator-specific peptide as the substrate. In addition, cells cultured on a three-dimensional fibrin gel in the presence of angiogenin are 3 times more capable of dissolving the gel and forming focal defects in the underlying matrix. The results indicate that angiogenin can enhance the ability of endothelial cells to digest extracellular matrix components and degrade basement membrane, thereby facilitating cell invasion and migration. Binding of angioenin to its cell-surface binding protein (actin) followed by dissociation of the angiogenin-actin complex from the cell surface and subsequent activation of tissue-type plasminogen activator/plasmin are likely steps involved in the processes of endothelial cell invasion and angiogenesis.