Exploring The Mechanism Of Action Of Angiogenin

Angiogenin (ANG) was originally isolated from the conditioned medium of HT-29 human colon adenocarcinoma cells based solely on its angiogenic activity. It has been recognized that angiogenin is closely related with tumor growth, progression and metastasis in many types of cancers. However, the molecular mechanisms underlying the functions of angiogenin remain elusive. It is known that angiogenin binds to actin and promotes invasiveness of cultured endothelial cells by stimulation of cell-associated proteolytic activities; supports endothelial adhesion; activates ERK1/2 and PKB/Akt in human umbilical vein endothelial cells, as well as JNK/SAPK in human umbilical artery smooth muscle cells. Meanwhile, exogenously applied angiogenin can translocate into the nucleus of target cells and enhance rRNA transcription. Recently angiogenin has been reported to translocate into the nucleus of cancer cells such as HeLa cells and PC-3 cells and stimulate rRNA synthesis and cell proliferaction. However, these lines of evidence are fragmentary and their interconnections remain to be established. Since protein interactions are critical in every biological process, we reason that interactions between angiogenin and other proteins mediate a series of biological activities in angiogenin-induced angiogenesis and tumor cell growth. Unfortunately, only a few proteins have so far been identified as binding partners of angiogenin. To identify additional mediators or modulators of angiogenin activity, yeast two-hybrid technology was employed and 21 proteins were identified as angiogenin potential interacting molecules from human liver cDNA library and heart cDNA library, including follistatin (FS). Follistatin was recognized as an important mediator of cell secretion, proliferation, and apoptosis. Therefore, in the first part of my thesis, we carried out further studies on the interaction between follistatin and angiogenin.Angiogenin has been reported to translocate into the nucleus in endothelial cells, smooth muscle cells as well as cancer cells and stimulate rRNA transcription. An angiogenin-binding element (ABE) has been identified in vitro and exhibited angiogenin-dependent promoter activity in the luciferase reporter system, indicating that angiogenin may serve as a trans-acting factor. However, we do not know which part of genomic rDNA could bind to angiogenin and what is the mechanism underlining angiogenin-induced rRNA transcription. Therefore, in the second part of the thesis, we studied the binding of angiogenin to rDNA in vivo and the subsequent influences on the epigenetic characteristics of rDNA.Part I: The interaction between follistatin and angiogenin.To corroborate the interaction between follistatin and angiogenin, we first performed a pull-down experiment. Recombinant 6×His-tagged follistatin was expressed and roughly purified from the transformed bacterial supernatant by complexation with Ni²⁺-agarose, Angiogenin was added to the mixture, and the final Ni²⁺-agarose bound products were immunoblotted with polyclonal antibody against angiogenin. Data revealed that angiogenin was detected in pulled-down products only when 6×His-tagged follistatin was present, indicating follistatin interacted with angiogenin specifically in the in vitro system.To detect the localization of follistatin and angiogenin, their genes were tagged with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) sequences, respectively, and transfected into HeLa cells. The transfected cells were monitored with a confocal microscope. Data showed that each single expression of either protein appeared as even distribution of the corresponding protein in the nucleus. However, when both proteins were co-expressed, they co-localized in the nucleus and appeared as granules, suggesting angiogenin and follistatin interact at particular foci within the nucleus. Fluorescence resonance energy transfer (FRET) analysis further confirmed their interaction in the nucleus.The mature follistatin molecule is composed of an N-terminal segment followed by three homologous FS domains named FS1, FS2 and FS3 respectively. To determine the specific structural motif(s) in follistatin that mediates its binding to angiogenin, we tested the binding abilities of truncted constructs of follistatin with angiogenin using yeast two-hybrid approach. Data showed that FS domains 2 and 3 are both necessary and sufficient for angiogenin interacting.Follistatin is traditionally recognized as a secretion protein. To fully study the cellular distribution of follistatin, its genes with the signal peptide sequence was tagged with CFP and transfected into HeLa cells. The localization of the protein was monitored by a confocal microscope. The data showed that in some transfected cells, CFP-tagged protein was localized in the nucleus, while in others it was distributed in the cytoplasm and entered into a secretory pathway. The secreted protein in the medium of transfected cells could be detected by Western Blot method. It is reported that the translation of some proteins can be initiated at different Methionines, creating proteins with variable or even no signal peptides and with different cellular distribution. Follistatin also has a second Met in itssignal sequence, which raises the possibility that the use of alternative Met for follistatin translation initiation could result in a portion of translated protein being translocated into the nucleus. To test this hypothesis, we made mutants of follistatin with either the Met (AUG) at -29 or -8 amino acid changed to Ile (AUC). The M-29I mutant showed exclusive nuclear translocation and was not detectable in the medium. When M-8 was mutated into Ile, the protein could not be observed in the nucleus while it could be detected in the medium, indicating that M-8I mutant goes into the secretory pathway. These results suggested that follistatin could be translated from different AUG start codon, which resulted in different distribution of the protein. When the CFP-tagged follistatin M-29I mutant was co-expressed with ANG-YFP, the two proteins co-localized at particular foci within the nucleus. The following FRET analysis further confirmed their interaction in the formed foci.To determine the specific structural motif(s) in follistatin that mediates its nuclear translocation, we constructed a series of plasmids containing truncated follistatin cDNA fused with CFP gene. All of the constructs were transfected into HeLa cells and the localization of the truncated proteins was monitored by a confocal microscope. Deletion mutants with the 64-100 amino acid in follistatin domain 1 (FS1) all showed obvious nuclear accumulation, while deletion of this part produced dispersed distribution, indicating that aa64-100 is necessary for follistatin nuclear translocation.Based on the above data, a question was raised regarding the function of the interaction between follistatin and angiogenin in the nucleus. It is reported that angiogenin has transcription-stimulating activity in the nucleus. An angiogenin-binding DNA sequence in the non-transcribed region of rDNA was also identified and designated as angiogenin-binding element (ABE). ABE binds angiogenin specifically and exhibits angiogenin-dependent promoter activity in the luciferase reporter system. We speculated that follistatin may regulate angiogenin's transcription-stimulating activity. To test that hypothesis,a luciferase system was applied. The expression level of luciferase in HeLa cells transfected with pGL3E-ABE was 3.14 times higher than those transfected with pGL3E vector, indicating that ABE acted as a promoter. When co-transfected with angiogenin-expressing plasmid, the expression level of luciferase increased greatly (about 8.02 times higher than those transfected with pGL3E), indicating that ABE's promoter activity was ANG-dependent. Follistatin had no effect on ABE's promotor activity, as the luciferase expression level did not change much in follistatin co-transfection group (about 3.76 times higher than those transfected with pGL3E). When the cells were co-transfected with both angiogenin and follistatin, the ABE activity reduced to the basic level (about 2.69 times higher than those transfected with pGL3E), indicating that follistatin inhibited angiogenin's ABE-stimulating activity.Part II: The effect of angiogenin's binding on epigeneticcharacteristics of rDNA region.Angiogenin was reported to bind to the nucleolar DNA and enhance rRNA transcription. To determine its genomic binding sequence, chromatin immunoprecipitation (ChIP) was applied to study the binding capacity of angiogenin with different regions of rRNA gene (rDNA) in vivo. First, angiogenin was crosslinked with DNA and the ChIP experiment was performed with rabbit polyclonal antibody against angiogenin (ChIP experiment performed with no antibody as a control). Then real time PCR was carried out with primers spanning the promoter region, 18S region, 28S region, ABE region, intergenic spacer (IGS) region, respectively, using the ChIP products as the template. Data showed that when immunoprecipitated with ANG antibody, the content of rDNA at those regions was 2-3 times more than the control group, indicating that angiogenin binds to the entire rDNA region. Next we compared the binding capacity of angiogenin with rDNA in HeLa cells before and after angiogenin treatment. HeLa cells were divided into two groups, one group was treated with angiogenin and the other group was untreated. Then ChIP experiments were carried out using equal amout of ANG antibody in both groups. Real time PCR analysis showed that the binding of angiogenin with the entire rDNA in the ANG treated group was 4 times higher than the untreated group, which further confirmed the binding of angiogenin with rDNA.Many rRNA transcription factors have been reported to bind to the entire rDNA and induce the chromatin remodeling at rDNA region by altering histone acetylation and/or methylation status. Since our previous results indicate that angiogenin may bind to the entire rDNA region, we speculate that the angiogenin's binding with rDNA may influence the histone modifications at rDNA region. Therefore, chromatin immnoprecipitation experiments were carried out to study angiogenin's effects on H4 acetylation and H3K9 (lysine 9 in H3) dimethylation. First, we stably transfected HeLa cells with angiogenin antisense vector to down-regulate the angiogenin expression. The chromatin of angiogenin down-regulated group and the control group were immunoprecipitated with anti-acetyl-H4 and anti-dimethyl-H3 antibodies respectively. The rDNA content at different regions was detected by real time PCR, which reflected the extent of H4 acetylation and H3K9 dimethylation respectively. Data showed that in the angiogenin down-regulated group, the extent of H4 acetylation at the entire rDNA region reduced, which was about 0.5 times less than the control group. While the extent of H3 acetylation slightly improved, about 1.2 to 1.7 times more than the control group. H4 acetylation is related with euchromatin formation and switches on transcription, while H3 dimethylation acts in the opposite way. Our data demonstrated that angiogenin may influence the chromatin structure by enhancing H4 acetylation and reducing H3 dimethylation, thus stimulate rRNA transcription.Based on the data, the innovative conclusions in the thesis are:1. Translation from the M-8 in the signal peptide induces follistatin's nuclear translocation. Its interaction with angiogenin happens in the nucleus.2. Amino acids from 64 to 100 of FS1 domain mediate follistatin's nuclear localization while FS2 and FS3 domains are needed for angiogenin's interaction.3. Follistatin inhibits angiogenin's ABE-stimulating activity through interacting with angiogenin.4. Angiogenin binds to the entire rDNA in vivo and induces epigenetic changes at the rDNA region by enhancing H4 acetylation and slightly reducing H3K9 dimethylation.