Acta Biochim Biophys Sin, 2016, 48(5), 399–410 doi: 10.1093/abbs/gmv131 Advance Access Publication Date: 23 December 2015 Review

Review

Three decades of research on angiogenin: a review and perspective Jinghao Sheng1,2,3 and Zhengping Xu1,2,3,* 1

Institute of Environmental Health, Zhejiang University School of Public Health, Hangzhou 310058, China, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou 310003, China, and 3Program in Molecular Cell Biology, Zhejiang University School of Medicine, Hangzhou 310058, China 2

*Correspondence address. Tel: +86-571-88208008; Fax: +86-571-88208164; E-mail: [email protected] Received 28 October 2015; Accepted 23 November 2015

Abstract As a member of the vertebrate-specific secreted ribonucleases, angiogenin (ANG) was first isolated and identified solely by its ability to induce new blood vessel formation, and now, it has been recognized to play important roles in various physiological and pathological processes through regulating cell proliferation, survival, migration, invasion, and/or differentiation. ANG exhibits very weak ribonucleolytic activity that is critical for its biological functions, and exerts its functions through activating different signaling transduction pathways in different target cells. A series of recent studies have indicated that ANG contributes to cellular nucleic acid metabolism. Here, we comprehensively review the results of studies regarding the structure, mechanism, and function of ANG over the past three decades. Moreover, current problems and future research directions of ANG are discussed. The understanding of the function and mechanism of ANG in a wide context will help to better delineate its roles in diseases, especially in cancer and neurodegenerative diseases. Key words: angiogenin, angiogenesis, tumorigenesis, neurodegeneration disease, RNA metabolism

Introduction Angiogenin (ANG) was first characterized by Vallee and colleagues at Harvard in 1985. It is the first human tumor-derived protein that was found to stimulate the growth of blood vessels, and it provides the first direct supporting evidence for Folkman’s hypothesis that tumor growth is angiogenesis dependent [1–3]. ANG is potently angiogenic in comparison with most other angiogenic factors, as it can induce new blood vessel formation in chicken chorioallantoic membrane, rabbit cornea, and meniscus at femtomolar doses [2,4]. Indeed, ANG has been proposed as a permissive factor for angiogenesis induced by other angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, acidic fibroblast growth factor, and epidermal growth factor [5]. The mechanisms underlying the induction of tumor angiogenesis by ANG have become more and more clear over the past 30 years. ANG is a secreted growth factor present in normal human tissues and fluids, such as plasma [6], amniotic fluid [7], tumor microenvironment

[8], and cerebrospinal fluid (CSF) [9]. The widespread expression pattern of ANG suggests that its physiological and pathological functions are not restricted to neovascularization. Indeed, ANG has been shown to be involved in many other pathophysiological processes, including tumorigenesis [10], neuroprotection [11], inflammation [12], innate immunity [13], reproduction [14], and regeneration of damaged tissues [4]. Loss-of-function mutations of the human ANG gene have been identified in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) [15–17]. Most of the mutations that segregate with ALS do not significantly alter the secondary structure or stability of ANG, but rather disrupt its ribonucleolytic activity or subcellular distribution [16,18]. This has stimulated interest in understanding the roles of ANG in the central nervous system, especially in neurodegenerative conditions, which may provide novel diagnostic methods and therapeutic targets. Here, we review the structure, mechanism, and functions of ANG reported over the past three decades. In addition, current questions and future directions regarding ANG research will also be discussed.

© The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 399

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By understanding ANG in a wide context, it may be possible to better delineate its roles in diseases.

Basic Characteristics of ANG ANG gene arrangement The gene encoding ANG has been identified in nearly all vertebrates including fishes, reptiles, birds, and mammals, but not in invertebrates [19–22] (Table 1). Analyses of complete or nearly complete mammalian genomes indicated the absence of ANG in six mammalian species (gibbon, naked mole rat, guinea pig, dog, giant panda, and African elephant) [23]. Notably, evolutionary and structural feature analyses showed that the ANG family evolves and duplicates from early ANGlike genes in fish [20]. The operation of positive selection on the ANG

family results in divergent functions of the paralogous ANG genes [20]. To date, the mouse has been shown to possess the largest ANG family, including five mouse ANG genes (Ang1, Ang2, Ang4, Ang5, and Ang6) and three ANG pseudogenes (Ang-ps1-3) [24]. However, only Ang1 shows angiogenic activity and has the same structure as the human ANG gene [25,26]. Similar to all the other known ribonucleases (RNases), ANG is encoded by a single exon and is usually located in the middle of the RNase A superfamily gene cluster. Particularly, the human and mouse ANG loci have a unique gene arrangement, characterized by shared promoters and 5′-untranslated regions (5′-UTR) directing two distinct exons encoding ANG and Ribonuclease 4 (RNASE4, the fourth member of the RNase A superfamily), respectively [24,26,27] (Fig. 1). In this unique gene arrangement, the transcription of ANG and RNASE4 is

Table 1. Numbers of ANG genes and pseudogenes identified from complete or nearly complete genome sequences of vertebrates Species

ANG ( pseudogenes)

Classification

Reference

Human (Homo sapiens) Mouse (Mus musculus) Rat (Rattus norvegicus) Dog (Canis lupus familiaris) Cattle (Bos taurus) Opossum (Malus domestica) Pig (Sus scrofa) Rabbit (Oryctolagus cuniculus) Turtle (Chrysemys picta) Zebrafish (Danio rerio)

1 5 (3) 2 (1) 3 1 2 1 2 5

Primates Rodentia Rodentia Carnivora Artiodactyla Marsupial Artiodactyla Lagomorpha Testudines Cypriniformes

[19] [19] [19] [21] [21] [21] This study This study This study [22]

Figure 1. Gene structure of angiogenin (ANG) and the ribonuclease 4 (RNASE4) locus in mammalian whole-genome sequences The locus usually contains four exons, three introns, and two promoters. The exons containing the coding regions of ANG and RNASE4 are indicated by gray arrows. The chromatin and nucleotide numbering of the locus are indicated below the chromatin line.

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Functions and mechanisms of angiogenin controlled by a universal promoter or a liver-specific promoter [26]. As the promoter is generally involved in regulation of mRNA transcription and the 5′-UTR affects the translation efficiency, the expression levels of ANG and RNASE4 are expected to be similar. Indeed, the tissue distribution and cellular localization or secretion of ANG and RNASE4 are almost identical or highly similar in human, mouse, and cattle [26–30]. Results from our laboratory indicated that the transcription of these two genes is regulated by RNA polymerase III elements and a CCCTC binding factor (CTCF)-dependent intragenic chromatin loop [31]. This unique gene arrangement of ANG and RNASE4 was proposed to ensure the coexpression of these two proteins, which regulates important biological events. The promoter sharing and conservation of the 5′-UTR sequence between ANG and RNASE4 are also observed in other mammalian species for which the whole-genome sequence is available (Fig. 1). Conservation of this genomic structure through evolution probably reflects a selection constraint to maintain coregulation of the two genes, which in turn suggests a functional relationship between these genes. Unlike ANG that has very low ribonucleolytic activity, RNASE4 is a very active endonuclease with high evolution conservation and unique uridine specificity [32]. However, the biological roles of RNASE4 are still largely unclear. Recent studies have shown that, similar to ANG, RNASE4 is able to protect against neuron degeneration through promotion of angiogenesis, neurogenesis, and neuronal survival under conditions of stress [33]. Our unpublished data indicated that RNASE4 possesses tumorigenic activity. These data suggested that the two genes may be functionally associated. It will be interesting to experimentally determine the biological functions and mechanisms of action of RNASE4, alone or in combination with ANG, in physiological and pathological processes.

Structure of ANG protein ANG is a homolog of bovine pancreatic RNase A (EC 3.1.27.5), albeit with marked differences in both its specificity and level of activity [34]. This 14-kDa basic single-chain protein has 33% sequence identity and 65% homology with bovine pancreatic RNase A as well as the same general catalytic residues [34]. It has all three main catalytic residues (His-13, Lys-40, and His-114) that form the catalytic center (P1) at which phosphodiester bond cleavage occurs [34,35]. The pyrimidine base-binding center (B1) and purine base-binding center (B2) are also present in ANG. However, the B1 center in ANG is occluded by the side chain of Gln-117 due to the formation of two hydrogen bonds with Thr-44, when compared with the structure of RNase A. Ile1-19 and Phe-120 form intramolecular hydrophobic interactions, thereby helping to keep Gln-117 in its obstructive position [36]. This blocking of the B1 center is one of the reasons for the reduced ribonucleolytic activity of ANG [36]. In addition, ANG lacks the fourth disulfide bond, which results in a loop region (from Lys-60 to Lys-68) to interact with a yet to be defined cell-surface receptor [37,38]. It also has a nuclear localization sequence (NLS) of 30 Met-Arg-Arg-Arg-Gly35 [39,40]. These special structures of ANG not only explain its unique ribonucleolytic activity but also confer diverse biological functions (Fig. 2).

Ribonucleolytic activity of ANG Although ANG cleaves the 3′-side of pyrimidine nucleotides by a transphosphorylation-hydrolysis mechanism as a general endonuclease [41], the ribonucleolytic activity of ANG is 10−5- to 10−6-fold less than that of RNase A because of its special structure, suggesting that ANG may have specific substrates and functions distinct from those of other

Figure 2. Schematic representation of the crystal structure of human ANG at a resolution of 1.8 Å The file with PDB ID IB1I (human ANG) [160] was retrieved from protein data bank and visualized with Cn3D. ANG consists of 123 amino acid residues with two α-helices (green tubular arrow), seven β-sheets (yellow sheet arrow), and three disulfide bonds (red line). The protein contains three main catalytic residues (His-13, Lys-40, and His-114) that compose the catalytic center (P1) at which phosphodiester bond cleavage occurs. NLS and receptor-binding site are colored by yellow. Further details can be found in the text.

RNases [42,43]. The results of in vitro studies have established some features of ANG cleavage in natural polyribonucleotides. The base cleavage specificity toward RNA has also been determined with Saccharomyces cerevisiae and Escherichia coli 5S RNA [44]. Unlike RNase A, which has no base specificity, ANG usually cleaves the 3′-side of cytidylic or uridylic acid residues when the pyrimidine is followed by adenine, but not restrict to all the potential cleavage sites [42,44]. ANG targets RNA sequences in the order of CpA > CpG > UpA > UpG [42,45]. In addition, ANG shows preferential cleavage of single-stranded RNA as the substrate and the cleavage specificity depends on its secondary structure [44]. Although ANG has been shown to bind DNA in vivo, it does not cleave DNA [46].

ANG-interacting proteins Protein–protein interactions are involved in a wide range of biological processes and have become one of the major topics of protein research. The first identified ANG-binding protein is human placental ribonuclease inhibitor (RNH1), a 50-kDa leucine-rich repeat protein [47]. ANG binds human RNH1 with an extremely low Ki value of 0.7 fM, which is the tightest protein–protein binding recorded to date [48]. The ANG- and RNH1-binding interface is a large contact surface from both proteins; however, the important contacts only involve the C-terminal segment 434–460 of RNH1 and the notable catalytic residue Lys-40 of ANG [49]. Binding of RNH1 inhibits both the enzymatic and angiogenic activities of ANG [47]. Cellular ANG is bound to RNH1, which prevents random cleavage of cellular RNA. It has also been reported that RNH1 regulates the subcellular localization of ANG to control growth and survival [50]. To date, a total of 19 unique ANG-interacting proteins have been reported with experimental evidence (Table 2). Based on the available data in the literature, we have classified the ANG interactions into several function groups: (i) cell migration, adhesion, and invasion, mainly including plasminogen activation system

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Table 2. ANG-interacting proteins No.

Interactor (gene symbol)

Experimental evidence

Throughput

Reference

1 2 3 4 5 6 7

RNH1 TP53 TDGF1 PTEN ATP6AP1 MDM2 ACTN2

Low Low High High High Low High

[49] [51] [161] [161] [161] [51] [162]

8

FST

High

[52]

9 10 11 12

MYH9 ACTN4 ACTB FHL3

High High High High

[53] [53] [53] [54]

13 14 15

ANXA2 FBLN1 ACTA1

High High Low

[55] [163] [56]

16

HIST3H3

High

[65]

17

PLSCR1

High

[57]

18 19

HSF1 SDC4

Co-crystal structure Co-IP Two-hybrid Two-hybrid Two-hybrid Co-IP Two-hybrid, GTS pull-down, FRET Two-hybrid, GTS pull-down, FRET Co-IP Co-IP Co-IP Two-hybrid, GTS pull-down, Co-IP Co-IP Pull-down, FRET Affinity Capture Western Affinity Capture Western Two-hybrid, GTS pull-down, FRET Pull-down In situ proximity ligation assay

Low Low

[58] [59]

Co-IP, co-immunoprecipitation; FRET, fluorescence resonance energy transfer; GTS, glutathione S transferase.

proteins [60,61] and cytoskeletal proteins [56,62,63]; (ii) cell proliferation, including follistatin [52,64], phospholipid scramblase 1 (PLSCR1) [57], histone H3 [65], RNH1 [50], four and a half LIM domains 3 (FHL3) [54,66], and ANG receptor that is a 170-kDa cellsurface protein expressed in human endothelial cells [67] or syndecan 4 in astrocyte cells [59]; and (iii) cell apoptosis, including p53 [51], MDM2 [51], heat shock factor 1 (HSF1) [58], and RNH1 [50]. A full identification of ANG-interacting proteins may help to draw the ANG interaction maps and to better understand its roles and mechanisms.

Mechanisms of Action of ANG After three decades of research, a more comprehensive understanding of the mechanisms of action of ANG is emerging. Based on our review published in 2008 [68], here, we incorporate novel discoveries over the past 7 years and summarize the main mechanisms of action of ANG (Fig. 3).

Cellular ANG regulates nucleic acid metabolism Secreted ANG undergoes receptor-mediated endocytosis from the cell surface to the nucleus and accumulates in the nucleoli. A series of studies have highlighted that cellular ANG contributes to the metabolism of nucleic acids. It is now clear that nuclear ANG can promote 47S pre-rRNA transcription by binding the ABE (Angiogenin Binding Element) and UCE (Upstream Core Element) region on the promoter of ribosomal DNA (rDNA), where ANG increases the number of actively transcribing rDNAs and promotes the assembly of the initiation complex by epigenetic activation through promoter methylation and histone modification [46,65,69–71]. Subsequently, ANG may enhance rRNA processing by acting as an endonuclease to cleave the first

cleavage site (A0) of the 47S pre-rRNA under growth conditions [72]. In addition, ANG can degrade 28S and 18S rRNAs to the major products from 100 to 500 nucleotides in length in vitro [34]. Therefore, ANG not only regulates rRNA transcription, it may also be involved in processing and degradation of this RNA as well. Further in vivo studies of the latter two functions are needed. On the other hand, several lines of evidence have also indicated that surplus ANG in the nucleus may be related to mRNA transcription. ANG binds the first exon region of estrogen receptor-related receptor gamma (ERRγ) and inhibits ERRγ expression [73]. To screen and identify mRNAs regulated by ANG at a genome-wide level, chromatin immunoprecipitation-chip assay was carried out to identify a total of 699 genes that may be regulated by ANG. These genes were shown to be significantly enriched in tumorigenesis, Wnt, and TGF-β pathways on KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis (data not shown). Based on the observation that ANG is able to remodel the histone modification through direct binding to the histone protein, it is likely that ANG acts as a chromatin remodeling activator to regulate mRNA transcription. As there is no evidence that ANG has methyltransferase or acetyltransferase activities, it is possible that ANG serves as a structural or adaptor protein to recruit other modifying enzymes to the DNA. Thus, a great deal of research remains to be done to fully determine the mechanisms of action of ANG in gene transcription. ANG also plays an important role in tRNA metabolism that occurs in the cytoplasm. Interestingly, tRNA was first used for quantitative enzymatic assay of ANG [43]. Recent studies have revealed that ANG cleaves the conserved single-stranded 3′-CCA termini of tRNA or the tRNA anticodon loop to form tiRNA (tRNA-derived, stress induced small RNA) in response to stress (e.g. oxidative, hypoxia, and starvation stress) [74–77]. Moreover, ANG-mediated tiRNA production is precisely regulated. For example, the patterns of tiRNA production are stress specific or dependent on the intensity of stress [75]. Modification of the selected tRNA (ValAAC, GlyGCC, and AspGTC) anticodon loop protects the tRNA from ANG-induced tRNA cleavage [78]. RNH1 can also regulate the stress-induced subcellular localization of ANG to control tiRNA production under stress conditions [50]. Respiratory syncytial virus (RSV) infection leads to abundant production of tiRNAGlu, which is also mediated by ANG and can target mRNA in the cytoplasm and promote RSV replication [79]. In addition, sex hormones and their receptors can promote ANG-mediated tiRNA production, which plays a significant functional role in cell proliferation [80]. ANG-induced tiRNAs reprogram protein translation, thereby promoting damage repair and cell survival [73]. In the two tRNA halves produced by ANG, only the 5′ tiRNAs (but not 3′-tiRNAs) can inhibit translation in vitro. The different functions of these two tRNA fragments might be the consequence of the fragment size and stability, but need to be further investigated [76]. For example, only 5′-tiRNAAla and 5′-tiRNACys are active. The common feature of these two tiRNAs is the presence of a 5′-oligo-G motif (four to five consecutive guanine residues) at the 5′-end. The 5′-end of tiRNA interacts with the translational silencer YB-1 to inhibit translational initiation by recruiting eIF4E/G/A from capped mRNAs or eIF4G/A from uncapped mRNAs. These tiRNAs also cooperate with the assembly of stress granules under conditions of stress. Through this mechanism, the eukaryotic cell dynamically represses translation at low metabolic cost under stress conditions. It should be noted that precise molecular roles for the majority of tiRNAs as well as their localization, relative abundance, and stability have yet to be determined. ANG may participate in the metabolism of other RNAs. Recent data also showed that stressed motor neurons secrete ANG, and

Functions and mechanisms of angiogenin

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Figure 3. Schematic representation of the mechanisms of action of ANG Secreted ANG undergoes receptor-mediated endocytosis from the cell surface to the inside of the cell where it accumulates in the nucleoli under growth conditions or in the cytoplasm under stress conditions. First, the extracellular ANG activates signal transduction pathways including SAPK/JNK, ERK1/2, and PI3K/AKT pathways, in different cells and cell conditions, although full pathway investigations have yet to be performed. The interaction between ANG and cell-surface complex could lead to ECM degradation and matrix metalloproteinase activation that promote cell migration and invasion. Second, the cytoplasmic ANG cleaves the tRNA to form tiRNA in response to stress, while the 5′-tiRNA interacts with the translational silencer YB-1 to inhibit translational initiation by recruiting eIF4G/A from uncapped mRNAs. ANG also inhibits the serine-15 phosphorylation of p53 and the subsequent binding of Mdm2, resulting in ubiquitination of p53. In addition, cytoplasmic ANG can optimize stress fiber assembly and focal adhesion formation to accommodate cell migration. Third, the nuclear ANG contributes to the rRNA transcription/procession and mRNA transcription under growth conditions. Further details can be found in the text. SAPK/JNK, stress-associated protein kinase/c-Jun N-terminal kinase; ERK1/2, extracellular signal-related kinase 1/2; YB-1, Y box binding protein 1; eIF4, eukaryotic initiation factor-4; Mdm2, MDM2 protooncogene, E3 ubiquitin protein ligase; RNH1, human placental ribonuclease inhibitor; ECM, extracellular matrix; LR, lipid raft; uPAR, urokinase plasminogen activator receptor; uPA, urokinase plasminogen activator; A2, annexin A2; S100, calpactin S100-A10; VT, vitronectin; PLSG, plasminogen; PLN, plasmin; PLSCR1, phospholipid scramblase 1.

then astrocytes endocytose ANG to cleave an unknown group of RNAs [59]. Our laboratory’s preliminary data indicated that ANG can play an important role in microRNA degradation. However, further research is needed to determine whether ANG participates in other RNA cleavage, identify the special subgroups of the target RNAs, and elucidate the function and mechanism of that cleavage.

Extracellular ANG activates signal transduction pathways In the extracellular environment, secreted ANG can trigger a series of signaling responses by binding to the receptor, a 170-kDa

transmembrane protein that has yet to be fully characterized [67]. It has been reported that ANG can activate extracellular signal-related kinase 1/2 (ERK1/2), protein kinase B/Akt, and/or stress-associated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in different target cells [81–85]. Activation of these key signaling mediators by ANG is considered to produce more ribosomal proteins to enhance cell growth. ANG can also induce synthesis of nitric oxide, an important biological regulator involved in vascular physiology, in endothelial cells through the PI3K/Akt kinase signaling transduction cascade [86]. Moreover, the RNH1 protein blocks ANG-mediated PI3K/ AKT/mTOR signaling pathway to regulate the biological functions of ANG in cancer cells [87–89]. Finally, ANG could activate the

404 NF-κB-mediated cell survival pathway and Bcl-2-mediated antiapoptotic pathway [51,66,90]. Taken together, ANG may activate several signaling pathways in different cells and cell conditions. Currently, however, no information is available regarding the relations among these pathways.

ANG stimulates basement membrane degradation In the tumor microenvironment, extracellular ANG can reach the cell surface of endothelial cells, where it binds to actin and dissociates as a complex called AngBP [37,56]. This complex stimulates tissue-type plasminogen activator-catalyzed generation of plasmin from plasminogen [60,91]. The interaction between ANG and cell-surface actin also induces changes in the cell cytoskeleton, such as inhibition of G-actin polymerization and changes in the physical properties of F-actin [62,63]. The interaction between ANG and cell-surface actin could also lead to the activation of several protease cascades, including the plasminogen activator/plasmin serine protease system and the matrix metalloproteinase system [92]. Recent data indicated that ANG interacts with uPAR, A2, and S100-A10 proteins at the junction of lipid raft and nonlipid raft regions of cell membranes, where ANG acts as a bridging molecule to facilitate indirect interactions between uPAR and the plasminogen receptor, A2, S100-A10 complex that is necessary for plasmin formation and cell migration [61,91,93]. Further studies identified a peptide (ANI-E) by phage-display technology that could specifically inhibit the interaction between ANG and actin and then ANG-induced angiogenesis [55,94]. Moreover, ANG promotes fibrinolytic activity in GM7373 cells grown on fibrin gels, and also enhances endothelial cell migration and invasion [92,95]. ANG also supports the adhesion of different target cells when it is coated at concentrations ≥100 ng/cm2, whereas RNase A does not [96,97]. Thus, ANG may promote degradation of the basement membrane and extracellular matrix, thus allowing endothelial cells to penetrate and migrate. In addition, our laboratory has demonstrated that cytoplasmic ANG optimizes stress fiber assembly and focal adhesion formation to accommodate cell migration by interacting with β-actin, α-actinin 4, and nonmuscle myosin heavy chain 9 [98].

Functions of ANG At the cell level, ANG regulates cell proliferation, migration, invasion, adhesion, and/or differentiation in various experimental cell models [33,68]. In zebrafish, an ANG homolog gene has been reported to regulate angiogenesis and yolk extension [53]. Although many reports have focused on the functions of ANG, and ANG has been detected in human organs, such as the heart, spleen, lung, liver, colon, prostate, breast, brain, retina, melanocytes, and foreskin [99–102], there is a lack of direct in vivo data to fully determine the roles of ANG in normal physiological and pathophysiological processes. Overall, ANG is thought to play roles in pregnancy, innate immunity, tumorigenesis, neurodegeneration, and other pathological conditions.

Functions and mechanisms of angiogenin confluent cell membrane [24]. However, ANG is involved in wound neovascularization. When injured clots disrupt endothelial cell confluence, high concentration of ANG in blood vessels could facilitate rapid blood vessel growth and tissue repair, suggesting that ANG may promote wound healing while losing vascular integrity [104]. In addition, the hemodynamic forces are associated with cell cycle activity of endothelial cells, and endothelial cells turn over very slowly in adult tissues [105]. Therefore, plasma ANG may control vascular homeostasis through maintenance of endothelial cell self-renewal.

ANG is involved in fetal vascularization and immune tolerance Pregnancy is a physiological state that triggers angiogenesis and vascular remodeling within normal tissues [106]. The serum level of ANG increases in uncomplicated pregnancies, suggesting that ANG may play an important role in normal vascular development during the perinatal period [107,108]. Indeed, ANG is expressed during early human placental development [109]. It has been reported that cytotrophoblasts may release ANG as a paracrine signal to stimulate endothelial cell proliferation and differentiation [109]. ANG is also expressed in decidual cells from first-trimester placenta, and is present in placental stromal and epithelial cells, with increased expression in the endometrium in the mid- and late-secretory phases and early gestation [109,110]. The decidual environment is known to be immunosuppressive [111]. It is possible that ANG, an immunomodulator as discussed below, could participate in maternal immune tolerance toward the semiallogeneic fetus [112,113].

ANG participates in immune responses As mentioned above, ANG is involved in the innate immune system and is related to diverse inflammatory activation. In innate host defense, ANG is a component of tears and protects the ocular surface as an antimicrobial peptide [114,115]. ANG is also abundantly induced in Paneth cells and has been demonstrated to act as a factor for innate antimicrobial defense of the intestine [13]. In addition to antibacterial activities, ANG also has antiviral activities. As one of the two RNases produced by primary T cells, ANG can suppress the replication of the X4 HIV strains [116]. ANG-mediated tiRNAs are also increased and abundant in chronic hepatitis B and C infection, suggesting that ANG may play roles in viral infection [117]. Moreover, ANG may have antiinflammatory activity because its protein concentrations in serum are increased during the inflammatory response or tumour necrosis factor alpha and interleukin -1β treatment [102,118,119]. The mechanisms by which ANG suppresses the inflammatory response may involve the inhibition of TBK1-mediated NF-κB nuclear translocation [120]. Additional data are required to support this suggestion. In addition, ANG can inhibit the degranulation of polymorphonuclear leukocytes, suggesting that ANG may participate in endogenous inhibitory mechanisms to counterbalance plasma-derived molecules released during inflammatory responses [112].

ANG may maintain blood vessel homeostasis ANG circulates in human plasma at a concentration of 200–400 ng/ml [103]. Although ANG induces new blood vessel formation in the chicken embryo chorioallantoic membrane at a concentration of as little as 0.5 ng, it does not exert the same function under such high concentrations in human adults [6]. This appears to be a paradox. In fact, ANG does not trigger ribosome biogenesis in the confluent endothelial cells that constitute the blood vessels [10]. ANG receptor is present only on the sparsely cultured endothelial cell membrane, but not on

Roles of ANG in Diseases ANG and tumorigenesis Tumorigenesis is a multistep process involving not only genetic and epigenetic changes in tumor cells but also selective supporting conditions of the tumor microenvironment. Available data indicated that ANG influences nearly all steps of tumorigenesis including protecting tumor cells from poor survival conditions, promoting

405

Functions and mechanisms of angiogenin tumor cell proliferation, enhancing tumor cell migration and invasion, and inducing angiogenesis. In general, solid tumors frequently contain regions of suboptimal growth conditions, and cells in these regions show reprogramming of gene expression to adapt to these conditions, and then survive and grow. ANG is one of the stress-responsive proteins. For example, ANG secretion is significantly increased in human malignant melanoma and cervical cancer cell lines in hypoxic environments [102,121]. ANG is also crucial for maintaining the survival of body cavity-based lymphoma cells infected by the tumor virus, such as Kaposi sarcoma-associated herpesvirus [122,123]. Hypoxia-inducible factor-1, a transcription factor that regulates the expression of hypoxia-responsive genes, is necessary and sufficient to activate ANG expression in cells exposed to hypoxia [124]. Mechanistically, ANG has been shown to inactivate p53, including inhibition of its serine-15 phosphorylation and subsequent binding of Mdm2, resulting in ubiquitination of p53 [51]. ANG initiates the mammalian stress response by cleaving tRNA, resulting in reprogramming of the protein translation, thereby promoting damage repair and cell survival [125]. However, the mechanism underlying the regulation of cell survival and apoptosis by ANG is still largely unknown. In the tumor microenvironment, ANG is secreted by tumor cells and promotes tumor development. ANG can directly promote tumor cell proliferation. This protein can constantly translocate into the nuclei of tumor cells in a cell density-independent manner and promote cell proliferation [10,126]. As one of the major angiogenic components in the microvesicles (MVs), ANG is released by glioblastoma and stimulates tubule formation of endothelial cells [8]. Hepatocellular carcinoma cells also secrete ANG to induce hepatic stellate cells and extracellular matrix composition remodeling [127]. In prostate cancer, microenvironmental ANG can stimulate the invasion of normal prostate fibroblasts and is sufficient for invasion of prostate cancerassociated fibroblasts [128]. Therefore, ANG may promote degradation of the basement membrane and extracellular matrix to allow endothelial cells to penetrate and migrate into the tumor. The mast cell, a unique immune cell that accumulates in the stroma surrounding certain tumors, can also release ANG to induce neovascularization [129]. Moreover, MVs derived from mesenchymal stem cells under conditions of hypoxia or serum deprivation contain ANG, which mainly contributes to the angiogenesis-promoting functions of the MVs [130]. ANG can also induce vasculogenic mimicry of

fibrosarcoma HT1080 cells [131]. These studies suggested that ANG could facilitate tumor angiogenesis or/and vasculogenic mimicry to permit tumor cell metastases through the blood vessels. Another aspect of ANG functions in tumor microenvironment may involve suppression of the immune system, as ANG has an immunosuppression action. The ANG level in serum may be useful for the evaluation of tumor growth and progression. Various types of cancer have been investigated concerning the serum ANG level during cancer progress (reviewed in [132]). Indeed, increased serum levels of ANG have been found in solid tumors, and patients with higher levels of serum ANG were found to have poor prognosis [133–135]. For example, ANG is significantly and progressively upregulated in prostatic epithelial cells as they evolve from a benign to an invasive phenotype in the same patients [133,136]. However, serum ANG levels vary between cancer types and study cohorts. There is a decrease of ANG level in tumors after treatment, and in some cases, an increase with tumor recurrence [137,138]. It has been suggested that ANG may be clinically useful in monitoring the responses to tumor therapy and for detection of tumor recurrence.

ANG and neurodegenerative diseases The first study suggesting a link between ANG in ALS, one of the most common neuromuscular diseases worldwide, was published in 2004 [139]. Analysis of the ANG gene in an Irish population indicated a significant allelic association with the rs11701 single nucleotide polymorphism and identified a novel mutation in two individuals with sporadic ALS that potentially inhibits ANG functions [139]. To date, a total of 29 unique, nonsynonymous variants of ANG gene have been identified in 6471 ALS patients (0.46%) and 3146 PD patients (0.45%) compared with 7668 control subjects [17] (Fig. 4). Several mutations have been shown to impair the ribonucleolytic activity, nuclear translocation capacity, and angiogenic activity of this protein [18,140]. In fact, ANG is the first ‘loss-of-function’ gene identified in ALS and PD patients to date [16]. ANG is also the second angiogenic factor associated with the pathogenesis of ALS. Mice with a homozygous deletion in the hypoxia-responsive element of the VEGF gene showed an ALS-like phenotype [141]. Further studies indicated that VEGF has neuroprotective functions on motor neurons not only by increasing neurovascular perfusion but also via direct effects on the neuronal cells

Figure 4. Schematic representation of ANG mutations identified to date in ALS and PD patients All the reported human ANG mutations are shown. Newly synthesized ANG has 147 amino acids and contains several conserved domains: signal peptide, receptor-binding site, RNase A canonical domain, catalytic site, and NLS. Numbers under the protein line indicate the boundaries of each domain. Mutations identified in ALS (black and blue) and PD (orange) patients are shown above the protein line.

406 themselves [142]. As ANG-mediated rRNA transcription is essential for VEGF to stimulate angiogenesis [5], it is possible that a deficiency in ANG function may also impair the physiological actions of VEGF toward motor neurons. On the other hand, there are also reports showing that ANG concentration is abnormally elevated in the plasma of some ALS patients, suggesting that ANG might act as a stressresponding protein to protect neuron damage. However, so far no significant correlation has been found between CSF ANG level and clinical state of ALS patients [9,143,144]. Due to the limited sample size, further investigation is required to assess the utility of serum or CSF ANG level as a biomarker for ALS and as a predictor of disease progression. ANG has a survival-promoting effect on neural progenitors and different postmitotic neurons of the CNS and peripheral nervous system [145,146]. Mouse Ang1 is enriched in motor neurons of the spinal cord and dorsal root ganglia, and ANG could protect motor neurons against excitotoxic insult or serum starvation in vitro [11,124]. Recombinant ANG is neuroprotective for cultured motor neurons, and administration of ANG to SOD1G93A mice, a standard laboratory model of ALS, significantly promotes both lifespan and motor function. Mechanistically, formation of tiRNA may contribute to motor neuron survival via its ability to inhibit apoptosis or promote the formation of stress granules. ANG-induced 5′ tiRNAs, which form a G-quadruplex structure, interact with stress granule proteins (TDP43, eIF4G, and eIF4E) and are capped by a 5′ monophosphate, which is necessary for optimal stress granule assembly [73,76,147,148]. Taken together, these observations suggest that pathological changes in ALS may result in the inability of mutant ANG to inhibit this novel stress pathway. Future studies addressing the interplay between stress-activated ANG, stress granules, and the effects of ALS-associated ANG mutation in neuron cells would significantly advance our understanding of the role of ANG in neurodegeneration.

ANG and other diseases ANG also plays a role in a variety of nonmalignant pathological conditions, such as endometriosis [149], peripheral vascular disease [150], inflammatory bowel disease (IBD) [12], rheumatoid arthritis [151], and diabetes [152,153]. However, the majority of the related studies focused on the serum level of ANG in nonmalignant diseases. For example, peripheral arterial occlusive disease patients with more severe disease was found to have higher ANG levels compared with those with mild or moderate disease severity and controls [150]. Hypertension, a major risk factor for thrombotic events, has been characterized by low serum levels of ANG, which promotes the onset of the disease [154,155]. ANG levels are significantly higher in IBD patients than in healthy controls [118]. Moreover, ANG levels are also increased in children and adolescent patients with type 1 diabetes [156]. Therefore, change in serum ANG level may potentially be an indicator of these diseases.

Perspectives After three decades of research, >1,000 reports related to ANG have been published, and ANG is known to play roles in cell growth and/or apoptosis, differentiation, migration, and/or invasion in vitro, and angiogenesis, tumorigenesis, and neurodegeneration in vivo. However, the precise functions of ANG in the body remain unclear. Meanwhile, ANG is known to activate several enzymolysis and signaling pathways, but no full pathway information is available. ANG has been shown to enhance rRNA transcription and processing, mediate

Functions and mechanisms of angiogenin tiRNA production, and regulate transcription of certain mRNAs, but in vivo supporting data and mechanistic explanations are still lacking. The crystal structure of ANG has been determined, but nothing is known about the structures of ANG in complex with its partners. There will likely be significant breakthroughs in a number of areas related to ANG, as outlined below. With the wide application of modern genetic manipulation tools in animal models, it is likely that the in vivo roles of ANG will be determined in the near future. Although we recently reported that depletion of zebrafish RNase-like 5, a homolog of ANG, results in defects of intersegmental vessel sprouting [53], it is still not clear whether this is its physiological function, given that there are five homologs in fish. Mice have the largest number of ANG homologs, and only Ang1 shares the same gene structure as human ANG. However, Ang1-knockout mice do not show any significant phenotypic changes (unpublished data), suggesting that there may be functional complementation or redundancy among these genes. The CRISPR/CAS9 system has been adapted as a powerful technology for multiplexed genome editing [157]. Using this system to generate mice carrying deletions of different combinations of ANG homologs will facilitate studies of the in vivo functions as well as the interactions among these homologs. Although we have partially elucidated the mechanism of ANG-enhanced rRNA transcription, and identified a group of ANG-interacting proteins, ANG-binding genomic sequences, ANG-activated signal molecules, ANG-related miRNAs, and ANG-mediated tiRNAs, the current understanding of its mechanism of action is fragmentary. First, it will be necessary to determine the precise genomic organization and transcriptional regulation of the ANG gene, including its epigenetic regulation and alternative splicing. Second, it would be beneficial to determine whether ANG, as a secreted protein, functions in a paracrine or autocrine mode, and to clarify the process and regulation of its subcellular translocation. Finally, it would be helpful to carry out a high-throughput full screen for DNA sequences to which ANG binds, and for RNA substrates targeted by ANG, as well as different proteins that interact with ANG under physiological and pathological conditions. The potential clinical applications of ANG have been highlighted due to its important roles in angiogenesis, tumorigenesis, and neuroprotection. In cancer therapies, the dual roles of ANG in tumor growth, of stimulating both tumor angiogenesis and cancer cell proliferation, suggested that ANG may be an excellent cancer therapeutic target. ANG inhibitors (including the antihuman monoclonal antibody, 26-2F, small chemical compounds, neomycin and neamine, siRNA, antisense, soluble binding proteins, and enzymatic inhibitors) have been shown to inhibit tumor growth in various animal models [5,10,94,133,158,159]. However, the high level of ANG in human serum makes it very difficult to neutralize by inhibitors. Alternatively, ANG receptor plays prominent roles in mediating tumor progression, and therefore, antagonists that disrupt binding of ANG to its receptor would represent novel therapeutic targets for the treatment of cancer. ANG is also associated with a range of inflammatory diseases and innate immunity, suggesting that it may be a useful drug target in tumor immunotherapy. In neuroprotective therapies, recombinant ANG was shown to effectively postpone the death of ALS mice. Therefore, attention should be paid to the results of clinical trials of ANG in neurodegenerative diseases. The changes in serum and urine ANG levels in various diseases suggested that ANG is a strong candidate as a diagnostic marker for cancer, neurodegeneration diseases, and other diseases. However, there is a paucity of clinical trial data, and further studies on its potential medical applications are needed.

Functions and mechanisms of angiogenin In summary, ANG serves as a multiple-function protein with distinct and varied mechanisms of action, and could be an ideal clinical target and diagnostic marker. However, studies on ANG are approaching a bottleneck regarding its biological functions and underlying mechanisms of action. It is likely that additional functions and molecular mechanisms of action of ANG will be uncovered in the near future with the emergence of new technologies.

Funding This work was supported by the grants from the National Natural Science Foundation of China (Nos. 81372303 and 31170721 to Z.X. and No. 31400648 to J.S.).

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Three decades of research on angiogenin: a review and perspective.

As a member of the vertebrate-specific secreted ribonucleases, angiogenin (ANG) was first isolated and identified solely by its ability to induce new ...
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