Yutao Yang

Highly pathogenic avian influenza A virus H5N1 NS1 protein induces caspase-dependent apoptosis in human alveolar basal epithelial cells.

BackgroundIt is widely considered that the multifunctional NS1 protein of influenza A viruses contributes significantly disease pathogenesis by modulating a number of virus and host-cell processes, but it is highly controversial whether this non-structural protein is a proapoptotic or antiapoptotic factor in infected cells.ResultsNS1 protein of influenza A/chicken/Jilin/2003 virus, a highly pathogenic H5N1 strain, could induce apoptosis in the carcinomic human alveolar basal epithelial cells (A549) by electron microscopic and flow cytometric analyses. NS1 protein-triggered apoptosis in A549 cells is via caspase-dependent pathway.ConclusionsInfluenza A virus NS1 protein serves as a strong inducer of apoptosis in infected human respiratory epithelial cells and plays a critical role in disease pathogenesis.

FBI-1 functions as a novel AR co-repressor in prostate cancer cells.

The pro-oncogene FBI-1, encoded by Zbtb7a, is a transcriptional repressor that belongs to the POK (POZ/BTB and Krüppel) protein family. In this study, we investigated a potential interaction between androgen receptor (AR) signaling and FBI-1 and demonstrated that overexpression of FBI-1 inhibited ligand-dependent AR activation. A protein–protein interaction was identified between FBI-1 and AR in a ligand-dependent manner. Furthermore, FBI-1, AR and SMRT formed a ternary complex and FBI-1 enhanced the recruitment of NCoR and SMRT to endogenous PSA upstream sequences. Our data also indicated that the FBI-1-mediated inhibition of AR transcriptional activity is partially dependent on HDAC. Interestingly, FBI-1 plays distinct roles in regulating LNCaP (androgen-dependent) and PC-3 cell (androgen-independent) proliferation.

LINE-1 ORF-1p functions as a novel androgen receptor co-activator and promotes the growth of human prostatic carcinoma cells

Widespread interest in the mechanism of transcriptional regulation by the androgen receptor (AR) has been stimulated by the finding that AR signaling is critically important in the progression of human prostate cancers. Co-factors, the co-repressors, or the co-activators are responsible for the regulation of AR activation. The pro-oncogene human Long Interspersed Nucleotide acid Element-1 (LINE-1) encodes LINE-1 ORF-1p and plays important roles in the development and progression of several human carcinomas. In this study, the results showed that LINE-1 ORF-1p increased the AR transcriptional activity and in turn enhanced the expression of prostate specific antigen (PSA) in the presence of R1881. A physical protein-protein interaction between the AR signaling and the LINE-1 ORF-1p was identified by the immunoprecipitation assays and GST pull-down assays. Furthermore, LINE-1 ORF-1p would function as a novel AR positive co-regulator through modulating its cytoplasm/nucleus translocation and the recruitment to the androgen response element in the PSA gene promoter. Our date also showed that the LINE-1 ORF-1p promoted the proliferation and anchor-independent growth of LNCaP (ligand dependent) and PC-3 (ligand independent) human prostatic carcinoma cells. By investigating a novel role of the LINE-1 ORF-1p in the androgen/androgen receptor signaling pathway regulation, our study identifies that LINE-1 ORF-1p may be a novel AR co-regulator and molecular target for human prostate carcinoma therapy.

The NS1 protein of influenza a virus interacts with heat shock protein Hsp90 in human alveolar basal epithelial cells: Implication for virus-induced apoptosis

BackgroundOur previous study showed that the NS1 protein of highly pathogenic avian influenza A virus H5N1 induced caspase-dependent apoptosis in human alveolar basal epithelial cells (A549), supporting its function as a proapoptotic factor during viral infection, but the mechanism is still unknown.ResultsTo characterize the mechanism of NS1-induced apoptosis, we used a two-hybrid system to isolate the potential NS1-interacting partners in A549 cells. We found that heat shock protein 90 (Hsp90) was able to interact with the NS1 proteins derived from both H5N1 and H3N2 viruses, which was verified by co-immunoprecitation assays. Significantly, the NS1 expression in the A549 cells dramatically weakened the interaction between Apaf-1 and Hsp90 but enhanced its interaction with cytochrome c (Cyt c), suggesting that the competitive binding of NS1 to Hsp90 might promote the Apaf-1 to associate with Cyt c and thus facilitate the activation of caspase 9 and caspase 3.ConclusionsThe present results demonstrate that NS1 protein of Influenza A Virus interacts with heat hock protein Hsp90 and meidates the apoptosis induced by influenza A virus through the caspase cascade.

Long interspersed nuclear element ORF-1 protein promotes proliferation and resistance to chemotherapy in hepatocellular carcinoma.

AIM To clarify the specific roles and mechanisms of long interspersed nuclear element-1 ORF-1 protein [human long interspersed nuclear element-1 (LINE-1), ORF-1p] in chemotherapeutic drug resistance and cell proliferation regulation in hepatocellular carcinoma (HCC) cells. METHODS MTT assays were performed to identify the effect of the chemotherapeutic drug toxicity on HepG2 cells. Cell proliferation inhibition and the IC50 were calculated by the Origin 8.0 software. Western blotting assays were performed to investigate whether LINE-1 ORF-1p modulates the expression of some important genes, including p53, p27, p15, Bcl-2, mdr, and p-gp. To corroborate the proliferation and anchor-independent growth results, the HepG2 cells were analyzed by flow cytometry to investigate the effect of LINE-1 ORF-1p on the apoptosis regulation. RESULTS LINE-1 ORF-1p contributed to the resistance to several chemotherapeutic drugs (cisplatin and epirubicin) in HepG2 cells. The IC50 of the epirubicin and cisplatin increased from 36.04 nmol/L to 59.11 nmol/L or from 37.94 nmol/L to 119.32 nmol/L. Repression of LINE-1 ORF-1p expression by the siRNA could markedly enhance the response of HepG2 cells to the epirubicin and cisplatin. The IC50 correspondingly decreased from 28.06 nmol/L to 3.83 nmol/L or from 32.04 nmol/L to 2.89 nmol/L. Interestingly, down-regulation of LINE-1 ORF-1p level by siRNA could promote the response of HepG2 cells to the paclitaxel. The IC50 decreased from 35.90 nmol/L to 7.36 nmol/L. However, overexpression of LINE-1 ORF-1p did not modulate the paclitaxel toxicity in HepG2 cells. Further Western blotting revealed that LINE-1 ORF-1p enhanced mdr and p-gp gene expression. As a protein arrested in the nucleus, LINE-1 ORF-1p may function through modulating transcriptional activity of some important transcription factors. Indeed, LINE-1 ORF-1p promoted HepG2 cell proliferation, anchor-independent growth and protected the cells against apoptosis through modulating the expression of p15, p21, p53, and Bcl-2 genes. CONCLUSION LINE-1 ORF-1p promotes HepG2 cell proliferation and plays an important role in the resistance of chemotherapeutic drugs. By establishing novel roles and defining the mechanisms of LINE-1 ORF-1p in HCC chemotherapeutic drug resistance and cell proliferation regulation, this study indicates that LINE-1 ORF-1p is a potential target for overcoming HCC chemotherapeutic resistance.

The interaction between the PARP10 protein and the NS1 protein of H5N1 AIV and its effect on virus replication.

BackgroundDuring the process that AIV infect hosts, the NS1 protein can act on hosts, change corresponding signal pathways, promote the translation of virus proteins and result in virus replication.ResultsIn our study, we found that PARP domain and Glu-rich region of PARP10 interacted with NS1, and the presence of NS1 could induce PARP10 migrate from cytoplasm to nucleus. NS1 high expression could reduce the endogenous PARP10 expression. Cell cycle analysis showed that with inhibited PARP10 expression, NS1 could induce cell arrest in G2-M stage, and the percentage of cells in G2-M stage rise from the previous 10%-45%, consistent with the cell proliferation result. Plague forming unit measurement showed that inhibited PARP10 expression could help virus replication.ConclusionsIn a word, our results showed that NS1 acts on host cells and PARP10 plays a regulating role in virus replication.

MEIS1 functions as a potential AR negative regulator

The androgen receptor (AR) plays critical roles in human prostate carcinoma progression and transformation. However, the activation of AR is regulated by co-regulators. MEIS1 protein, the homeodomain transcription factor, exhibited a decreased level in poor-prognosis prostate tumors. In this study, we investigated a potential interaction between MEIS1 and AR. We found that overexpression of MEIS1 inhibited the AR transcriptional activity and reduced the expression of AR target gene. A potential protein-protein interaction between AR and MEIS1 was identified by the immunoprecipitation and GST pull-down assays. Furthermore, MEIS1 modulated AR cytoplasm/nucleus translocation and the recruitment to androgen response element in prostate specific antigen (PSA) gene promoter sequences. In addition, MEIS1 promoted the recruitment of NCoR and SMRT in the presence of R1881. Finally, MEIS1 inhibited the proliferation and anchor-independent growth of LNCaP cells. Taken together, our data suggests that MEIS1 functions as a novel AR co-repressor.

Cloning and functional analysis of 5′‐upstream region of the Pokemon gene

Pokemon, the POK erythroid myeloid ontogenic factor, not only regulates the expression of many genes, but also plays an important role in cell tumorigenesis. To investigate the molecular mechanism regulating expression of the Pokemon gene in humans, its 5′‐upstream region was cloned and analyzed. Transient analysis revealed that the Pokemon promoter is constitutive. Deletion analysis and a DNA decoy assay indicated that the NEG‐U and NEG‐D elements were involved in negative regulation of the Pokemon promoter, whereas the POS‐D element was mainly responsible for its strong activity. Electrophoretic mobility shift assays suggested that the NEG‐U, NEG‐D and POS‐D elements were specifically bound by the nuclear extract from A549 cells in vitro. Mutation analysis demonstrated that cooperation of the NEG‐U and NEG‐D elements led to negative regulation of the Pokemon promoter. Moreover, the NEG‐U and NEG‐D elements needed to be an appropriate distance apart in the Pokemon promoter in order to cooperate. Taken together, our results elucidate the mechanism underlying the regulation of Pokemon gene transcription, and also define a novel regulatory sequence that may be used to decrease expression of the Pokemon gene in cancer gene therapy.

FBI-1 enhances ETS-1 signaling activity and promotes proliferation of human colorectal carcinoma cells.

In this study, we investigated a potential regulatory role of FBI-1 in transcription factor activity of ETS-1. The protein interaction was identified between ETS-1 and FBI-1 in lovo cells. The accumulating data showed that FBI-1 promoted the recruitment of ETS-1 to endogenous promoter of its target genes and increase ETS-1 accumulation in the nuclear. Our work also indicated that the FBI-1 enhances ETS-1 transcription factor activity via down-regulating p53-mediated inhibition on ETS-1. Further, FBI-1 plays a role in regulation of colorectal carcinoma cells proliferation. These findings supported that FBI-1 might be a potential molecule target for treating colorectal carcinoma.

Characterization of the Rat GAL2R Promoter: Positive Role of ETS-1 in Regulation of the Rat GAL2R Gene in PC12 Cells

Galanin receptor 2 (GAL2R) is a G protein-coupled receptor for the neuropeptide galanin that regulates many important physiological functions and pathological processes. To investigate the molecular mechanism governing GAL2R gene transcription, the rat GAL2R promoter was isolated and analyzed. We found that the region from −320 to −300 of the GAL2R promoter contains two putative ETS-1 elements and plays an important role in regulating GAL2R promoter activity. We also showed that transcription factor ETS-1 bound to this region in vitro and in vivo. Overexpression of ETS-1 significantly increased GAL2R promoter activity and transcription of the GAL2R gene, whereas knockdown of ETS-1 produced the opposite effects. In addition, we showed that ETS-1 recruited co-activator p300 to the GAL2R promoter. These data indicate a role for ETS-1 in the control of the GAL2R gene expression and provide a basis for understanding the transcriptional regulation of the GAL2R gene.