Hongbin He

Ribosomal protein S27-like and S27 interplay with p53-MDM2 axis as a target, a substrate and a regulator

Several ribosomal proteins regulate p53 function by modulating MDM2. We recently found that RPS27L, a RPS27-like protein, is a direct p53-inducible target. Here we showed that RPS27 itself is a p53-repressible target. Furthermore, the N-terminal region of either RPS27L or RPS27 binds to MDM2 on the central acidic domain of MDM2. RPS27L or RPS27 forms an in vivo triplex with MDM2-p53 and competes with p53 for MDM2 binding. Similar to p53, RPS27L, but not RPS27, is a short-lived protein and a novel MDM2 substrate. Degradation of RPS27L requires the RING or acidic domain of MDM2. Ectopic expression of RPS27L or RPS27 inhibits MDM-2-mediated p53 ubiquitination and increases p53 levels by extending p53 protein half-life, whereas siRNA silencing of RPS27L decreases p53 levels by shortening p53 half-life, with a corresponding reduction in p53 transcription activity. RPS27L is mainly localized in the cytoplasm, but upon p53-activating signals, a portion of RPS27L shuttled to the nucleoplasm where it colocalizes with MDM2. Both the cytoplasmic and the nuclear p53, induced by ribosomal stress, were reduced upon RPS27L silencing. Our study reveals a multilevel interplay between RPS27L/S27 and p53-MDM2 axis, with RPS27L functioning as a p53 target, a MDM2 substrate and a p53 regulator.

SAG/ROC2/RBX2 is a HIF-1 target gene that promotes HIF-1α ubiquitination and degradation

SAG (sensitive to apoptosis gene) or ROC2/RBX2 is the second family member of ROC1/RBX1, a component of SCF (Skp1, Cullin, F-box protein) and VCB (von Hippel–Lindau (VHL), Cullin and Elongin B/C) E3 ubiquitin ligases. SAG protected cells from hypoxia-induced apoptosis when overexpressed. We report here that SAG was subjected to hypoxia induction at the levels of mRNA and protein. Hypoxia induction of SAG was largely HIF-1α dependent. A consensus HIF-1-binding site, GCGTG was identified in the first intron of the SAG gene. In response to hypoxia, HIF-1 bound to this site and transactivated SAG expression. SAG transactivation required both the intact binding site in cis and HIF-1α in trans. On the other hand, like its family member, ROC1, SAG promoted VHL-mediated HIF-1α ubiquitination and degradation, which was significantly inhibited upon small interfering RNA silencing of SAG or ROC1. Furthermore, the endogenous HIF-1α at both basal and hypoxia-induced levels was significantly increased upon SAG silencing. Finally, SAG forms in vivo complex with Cul-5 and VHL under hypoxia condition. These results suggest an HIF-1-SAG feedback loop in response to hypoxia, as follows: hypoxia induces HIF-1 to transactivate SAG. Induced SAG then promotes HIF-1α ubiquitination and degradation. This feedback loop may serve as a cellular defensive mechanism to reduce potential cytotoxic effects of prolonged HIF-1 activation under hypoxia.

Ribosomal protein S27L is a direct p53 target that regulates apoptosis.

Ribosomal proteins were recently shown to regulate p53 activity by abrogating Mdm2-induced p53 degradation (L23, L11, L5) or by enhancing p53 translation (L26). Here, we report that a novel ribosomal protein, RPS27L (S27-like protein), is a direct p53 target. RPS27L, but not its family member RPS27, was identified as a p53 inducible gene in a genome-wide chip-profiling study. Further characterization revealed a p53-dependent induction of RPS27L in multiple cancer cell models. Indeed, a consensus p53-binding site was identified in the first intron of the RPS27L gene and a direct binding of p53 to this site was demonstrated both in vitro and in vivo. Characterization of a luciferase reporter driven by the RPS27L intron fragment revealed a p53-binding site-dependent transaction by wild-type p53, but not by several transactivating-deficient p53 mutants. This transactivation was enhanced by etoposide, a DNA damaging agent that activates p53 and was completely blocked by a dominant-negative p53 mutant. Functionally, overexpression of RPS27L within the physiological inducible levels promoted, whereas siRNA silencing of RPS27L inhibited, apoptosis induced by etoposide. This is the first report, to our knowledge, that p53 directly induces the expression of a ribosomal protein, RPS27L, which in turn promotes apoptosis.

Validation of SAG/RBX2/ROC2 E3 Ubiquitin Ligase as an Anticancer and Radiosensitizing Target

Purpose: Sensitive to apoptosis gene (SAG; also known as RBX2 or ROC2) was originally cloned as a redox-inducible antioxidant protein and was later characterized as a RING component of SCF E3 ubiquitin ligases. SAG overexpression inhibits apoptosis induced by many stimuli both in vitro and in vivo. SAG mRNA was overexpressed in human lung tumor tissues with a correlation to poor patient survival. To investigate whether SAG serves as an anticancer target, we determined the effect of SAG silencing on cell proliferation, survival, and radiosensitivity. Experimental Design: SAG protein expression in human tumors was evaluated by immunohistochemical staining using tumor tissue arrays. SAG expression in cancer cells was knocked down by siRNA silencing. The anticancer effects of SAG silencing were evaluated by in vitro assays for cell growth and survival and by an in vivo orthotopic xenograft tumor model. Radiosensitization by SAG silencing of human cancer cells was determined by clonogenic survival assay. Apoptosis induction was evaluated by fluorescence-activated cell sorting analysis, caspase-3 activation assay, and Western blotting of apoptosis-associated proteins. Results: SAG was overexpressed in multiple human tumor tissues compared with their normal counterparts. SAG silencing selectively inhibited cancer cell proliferation, suppressed in vivo tumor growth, and sensitized radiation-resistant cancer cells to radiation. Mechanistically, SAG silencing induced apoptosis with accumulation of NOXA, whereas SAG overexpression reduced NOXA levels and shortened NOXA protein half-life. Conclusions: The findings showed that SAG E3 ubiquitin ligase plays an essential role in cancer cell proliferation and tumor growth and may serve as a promising anticancer and radiosensitizing target. Clin Cancer Res; 16(3); 814–24

SAG/ROC2/RBX2 E3 ligase promotes UVB-induced skin hyperplasia, but not skin tumors, by simultaneously targeting c-Jun/AP-1 and p27

Sensitive to apoptosis gene (SAG)/regulator of cullins-2/RING box protein 2 is a stress-responsive RING component of Skp-1/Cullins/F-box protein E3 ubiquitin ligase. When overexpressed, SAG inhibits apoptosis induced by reactive oxygen species or hypoxia. Here, we report that SAG overexpression inhibits ultraviolet (UV) B-induced apoptosis in mouse JB6 epidermal cells. Using a transgenic mouse model, in which SAG expression was targeted primarily to epidermis by a K14 promoter, we showed that, at the early stage of UVB skin carcinogenesis (10 weeks post-UVB exposure), c-Jun, p27, p53, c-Fos and cyclin D1 were strongly induced. While having no effect on UVB-induced p53, c-Fos and cyclin D1, SAG-transgenic expression reduced the levels of c-Jun and p27 and inhibited AP-1 activity. The net outcome of SAG-mediated inhibition of c-Jun/AP-1 (pro-tumor promotion) and of p27 (antiproliferation) increased skin hyperplasia, with no apparent effect on apoptosis, as evidenced by increased skin thickness, and increased rate of DNA synthesis, but hardly any apoptosis. Although skin hyperplasia was promoted, SAG-transgenic expression had no significant effect on tumor formation in the later stage of UVB carcinogenesis. Thus, by simultaneously targeting c-Jun and p27, SAG accelerates UVB-induced skin hyperplasia, but not carcinogenesis.

Treating Cloned Embryos, But Not Donor Cells, with 5-aza-2’-deoxycytidine Enhances the Developmental Competence of Porcine Cloned Embryos

The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression.

Identification of Short Hairpin RNA Targeting Foot-And-Mouth Disease Virus with Transgenic Bovine Fetal Epithelium Cells

Background Although it is known that RNA interference (RNAi) targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV), it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. Principal Finding Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2), VP3 (RNAi-VP3), or VP4 (RNAi-VP4) of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h. Conclusion RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.

Identification of a natural human serotype 3 parainfluenza virus.

Parainfluenza virus is an important pathogen threatening the health of animals and human, which brings human many kinds of disease, especially lower respiratory tract infection involving infants and young children. In order to control the virus, it is necessary to fully understand the molecular basis resulting in the genetic diversity of the virus. Homologous recombination is one of mechanisms for the rapid change of genetic diversity. However, as a negative-strand virus, it is unknown whether the recombination can naturally take place in human PIV. In this study, we isolated and identified a mosaic serotype 3 human PIV (HPIV3) from in China, and also provided several putative PIV mosaics from previous reports to reveal that the recombination can naturally occur in the virus. In addition, two swine PIV3 isolates transferred from cattle to pigs were found to have mosaic genomes. These results suggest that homologous recombination can promote the genetic diversity and potentially bring some novel biologic characteristics of HPIV.

New genetic mechanism, origin and population dynamic of bovine ephemeral fever virus

Bovine ephemeral fever virus (BEFV) is a typical species of the genusEphemerovirus in the family Rhabdoviridae. Today, prevailing BEFV can be divided into three phylogeographic lineages, East Asia, Mideast, and Australia. In this study, we provide evidence that the whole East Asia lineage originates from a homologous recombination (HR) between the Mideast and Australia lineages that probably occurred in the 1940s. To our knowledge, HR has not been proposed before as the genetic mechanism of BEFV. According to the HR event and Bayesian estimation, the three BEFV lineages might originate from Africa, and may have spread to Asia and Australia through the Mideast. In addition, the population of the virus may have augmented significantly in the 2000s, suggesting that the risk for outbreaks of BEFV may be high at present.

Analysis of biofilm formation and associated gene detection in Staphylococcus isolates from bovine mastitis

The objective of this study was to investigate the biofilm-forming ability and distribution of biofilm associated genes in clinically isolated Staphylococcus in bovine mastitis. Silver staining, scanning electron microscopy (SEM) and crystal violet staining were conducted for the detection of biofilmforming ability in 24-well plates. The bap, icaAD, icaBC, Staphylococcal accessory regulator (sar), accessory gene regulator (agr), sigB, clumping factor A (clfA), clfB, fibronectin-binding proteins (fnbpA) and fnbpB genes were amplified by polymerase chain reaction (PCR). Formation of biofilms was found macroscopically in 120 of the 137 strains after being stained with silver (biofilm-forming rate 87.6%). Five strains did not adhere to the surface of the silica gel after being stained with crystal violet, while the remaining 132 strains did adhere. Bap was amplified in 57 isolates, and icaAD and icaBC were isolated in 43 and 54 strains, respectively. SigB, sar and agr were amplified in 73, 49 and 38 isolates, respectively, and clfA and clfB were isolated in 76 and 50 strains, respectively. FnbpA was present in 52 strains and fnbpB in 26 isolates. Our study reveals that bap, sigB, sar, icaAD and icaBC may be crucial biofilm associated genes since these genes were present more often in biofilm-positive strains than in biofilm-negative strains. There was no obvious difference between the frequencies of agr in the biofilmpositive strains and biofilm-negative strains, which indicates that the role of agr in biofilm development is still controversial. The distribution of clfA, clfB, fnbpA and fnbpB in biofilm-positive strains were not greatly different from that in biofilm-negative strains. Key words: Bovine mastitis Staphylococcus , biofilm, silver staining, crystal violet staining.