Yinyin Wang

Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference

The severe acute respiratory syndrome (SARS) has been one of the most epidemic diseases threatening human health all over the world. Based on clinical studies, SARS‐CoV (the SARS‐associated coronavirus), a novel coronavirus, is reported as the pathogen responsible for the disease. To date, no effective and specific therapeutic method can be used to treat patients suffering from SARS‐CoV infection. RNA interference (RNAi) is a process by which the introduced small interfering RNA (siRNA) could cause the degradation of mRNA with identical sequence specificity. The RNAi methodology has been used as a tool to silence genes in cultured cells and in animals. Recently, this technique was employed in anti‐virus infections in human immunodeficiency virus and hepatitis C/B virus. In this study, RNAi technology has been applied to explore the possibility for prevention of SARS‐CoV infection. We constructed specific siRNAs targeting the S gene in SARS‐CoV. We demonstrated that the siRNAs could effectively and specifically inhibit gene expression of Spike protein in SARS‐CoV‐infected cells. Our study provided evidence that RNAi could be a tool for inhibition of SARS‐CoV.

CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation

Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage.

IL-17RD (Sef or IL-17RLM) interacts with IL-17 receptor and mediates IL-17 signaling

Interleukin-17 (IL-17 or IL-17A) production is a hallmark of TH17 cells, a new unique lineage of CD4+ T lymphocytes contributing to the pathogenesis of multiple autoimmune and inflammatory diseases. IL-17 receptor (IL-17R or IL-17RA) is essential for IL-17 biological activity. Emerging data suggest that the formation of a heteromeric and/or homomeric receptor complex is required for IL-17 signaling. Here we show that the orphan receptor IL-17RD (Sef, similar expression to FGF genes or IL-17RLM) is associated and colocalized with IL-17R. Importantly, IL-17RD mediates IL-17 signaling, as evaluated using a luciferase reporter driven by the native promoter of 24p3, an IL-17 target gene. In addition, an IL-17RD mutant lacking the intracellular domain dominant-negatively suppresses IL-17R-mediated IL-17 signaling. Moreover, IL-17RD as well as IL-17R is associated with TRAF6, an IL-17R downstream molecule. These results indicate that IL-17RD is a part of the IL-17 receptor signaling complex, therefore providing novel evidence for IL-17 signaling through a heteromeric and/or homomeric receptor complex.

Protein tyrosine phosphatase Meg2 dephosphorylates signal transducer and activator of transcription 3 and suppresses tumor growth in breast cancer

IntroductionSignal transducer and activator of transcription 3 (STAT3) is over-activated or phosphorylated in breast cancers. The hyper-phosphorylation of STAT3 was attributed to either up-regulated phosphorylation by several tyrosine-kinases or down-regulated activity of phosphatases. Although several factors have been identified to phosphorylate STAT3, it remains unclear how STAT3 is dephosphorylated by PTPMeg2. The aim of this study was to determine the role of PTPMeg2 as a phosphatase in regulation of the activity of STAT3 in breast cancers.MethodsImmunoprecipitation assays were used to study the interaction of STAT3 with PTPMeg2. A series of biochemistry experiments were performed to evaluate the role of PTPMeg2 in the dephosphorylation of STAT3. Two breast cancer cell lines MCF7 (PTPMeg2 was depleted as it was endogenously high) and MDA-MB-231 (PTPMeg2 was overexpressed as it was endogenously low) were used to compare the level of phosphorylated STAT3 and the tumor growth ability in vitro and in vivo. Samples from breast carcinoma (n = 73) were subjected to a pair-wise Pearson correlation analysis for the correlation of levels of PTPMeg2 and phosphorylated STAT3.ResultsPTPMeg2 directly interacts with STAT3 and mediates its dephosphorylation in the cytoplasm. Over-expression of PTPMeg2 decreased tyrosine phosphorylation of STAT3 while depletion of PTPMeg2 increased its phosphorylation. The decreased tyrosine phosphorylation of STAT3 is coupled with suppression of STAT3 transcriptional activity and reduced tumor growth in vitro and in vivo. Levels of PTPMeg2 and phosphorylated STAT3 were inversely correlated in breast cancer tissues (P = 0.004).ConclusionsPTPMeg2 is an important phosphatase for the dephosphorylation of STAT3 and plays a critical role in breast cancer development.

CHIP/Stub1 functions as a tumor suppressor and represses NF-κB-mediated signaling in colorectal cancer

The carboxyl terminus of Hsc70-interacting protein (CHIP, also named Stub1), a U-box containing E3 ubiquitin ligase, is involved in degradation of certain oncogenic proteins. Recent studies indicated that CHIP suppresses tumor progression in human cancers by targeting Src-3, hypoxia inducible factor 1α, NF-κB, ErbB2 and c-Myc. Here, we report that CHIP was downregulated, predominantly, in the late stages of human colorectal cancer (CRC), and that the CHIP promoter was hypermethylated in CRC specimens. Overexpression of CHIP in HCT-116 cells resulted in impaired tumor growth in nude mice and decreased abilities of tumor cell migration and invasion. Conversely, depletion of CHIP in HCT-116 cells promoted tumor growth and increased tumor cell migration and invasion. CHIP was further found to negatively regulate NF-κB signaling in HCT-116 cells by promoting ubiquitination and degradation of p65, a subunit of the NF-κB complex. The suppressive effect of CHIP led to decreased expression of NF-κB-targeted oncogenes including Cyclin D1, c-Myc, MMP-2, VEGF and IL-8. We proposed that CHIP inhibits the malignancy of CRC cells, possibly through targeting NF-κB signaling. This study provides functional evidence for CHIP as a potential tumor suppressor in CRC, and CHIP expression may be a marker for stages of CRC.

siRNA targeting the Leader sequence of SARS-CoV inhibits virus replication

SARS-CoV (the SARS-Associated Coronavirus) was reported as a novel virus member in the coronavirus family, which was the cause of severe acute respiratory syndrome. Coronavirus replication occurs through a unique mechanism employing Leader sequence in the transcripts when initiating transcription from the genome. Therefore, we cloned the Leader sequence from SARS-CoV(BJ01), which is identical to that identified from SARS-CoV(HKU-39849), and constructed specific siRNA targeting the Leader sequence. Using EGFP and RFP reporter genes fused with the cloned SARS-CoV Leader sequence, we demonstrated that the siRNA targeting the Leader sequence decreased the mRNA abundance and protein expression levels of the reporter genes in 293T cells. By stably expressing the siRNA in Vero E6 cells, we provided data that the siRNA could effectively and specifically decrease the mRNA abundance of SARS-CoV genes as analyzed by RT-PCR and Northern blot. Our data indicated that the siRNA targeting the Leader sequence inhibited the replication of SARS-CoV in Vero E6 cells by silencing gene expression. We further demonstrated, via transient transfection experiments, that the siRNA targeting the Leader sequence had a much stronger inhibitory effect on SARS-CoV replication than the siRNAs targeting the Spike gene or the antisense oligodeoxynucleotides did. This report provides evidence that targeting Leader sequence using siRNA could be a powerful tool in inhibiting SARS-CoV replication.

Varp interacts with Rab38 and functions as its potential effector.

Varp, a novel protein containing a VPS9 domain and ankyrin repeats, can function as a guanine nucleotide exchange factor (GEF) of Rab21. We previously reported that Varp plays an important role in the regulation of endosome dynamics. To further investigate the function of Varp, a yeast two-hybrid screen was performed and Rab38 was identified as a Varp-associated protein. We demonstrate that Varp physically interacts with Rab38, and preferentially binds to the active GTP-bound form of Rab38 both in vitro and in vivo. Furthermore, Varp was shown to be recruited to Rab38-positive organelles in an ankyrin-repeat 1 (ANK1)-dependent manner. Our data demonstrate that Varp is a potential effector of Rab38. Together with our previous study, we propose Varp serves as both an effector and a GEF by interacting with different Rabs in mammalian cells.

miR‐764‐5p promotes osteoblast differentiation through inhibition of CHIP/STUB1 expression

Differentiation of committed precursor cells into the osteoblast lineage is tightly regulated by several factors, including Runx2 and BMP2. We previously reported that C terminus of Hsc70‐interacting protein/STIP1 homology and U‐Box containing protein 1 (CHIP/STUB1) negatively regulated osteoblast differentiation through promoting Runx2 protein degradation. However, how CHIP is regulated during osteoblast differentiation remains unknown. In this study, we found that miR‐764‐5p is up‐expressed during the osteoblast differentiation in calvarial and osteoblast progenitor cells, coupled with down‐expression of CHIP protein. We observed that forced expression or inhibition of miR‐764‐5p decreased or increased the CHIP protein level through affecting its translation by targeting the 3′‐UTR region. Perturbation of miR‐764‐5p resulted in altered differentiation fate of osteoblast progenitor cells and the role of miR‐764‐5p was reversed by overexpression of CHIP, whereas depletion of CHIP impaired the effect of miR‐764‐5p. Our data showed that miR‐764‐5p positively regulates osteoblast differentiation from osteoblast progenitor cells by repressing the translation of CHIP protein.

p15RS Attenuates Wnt/β-Catenin Signaling by Disrupting β-Catenin·TCF4 Interaction

The formation of a β-catenin·TCF4 complex in the nucleus of cells is well known as a prerequisite for the transcription of Wnt target genes. Although many co-factors have been identified to regulate the activity of the β-catenin·TCF4 complex, it remains unclear how the complex association is negatively regulated. In this study, we report that p15RS, a negative regulator of the cell cycle, blocks β-catenin·TCF4 complex formation and inhibits Wnt signaling. We observed that p15RS interacts with β-catenin and TCF4. Interestingly, whereas the interaction of p15RS with β-catenin is increased, its interaction with TCF4 is decreased upon Wnt1 stimulation. Moreover, overexpression of p15RS reduces the interaction of β-catenin with TCF4, whereas the depletion of p15RS enhances their interaction. We further demonstrate that overexpression of p15RS suppresses canonical Wnt signaling and results in retarded cell growth, whereas depletion of p15RS shows an enhanced effect on Wnt signaling. We analyzed that inhibition of Wnt signaling by p15RS leads to decreased expression of CYCLIN D1 and c-MYC, two Wnt targeted genes critical for cell growth. Our data suggest that p15RS inhibits Wnt signaling by interrupting β-catenin·TCF4 complex formation and that Wnt signaling initiates downstream gene expression by removing p15RS from promoters.

Caspase 3 is Activated through Caspase 8 instead of Caspase 9 during H2O2-induced Apoptosis in HeLa Cells

Oxidative stress is known to be involved in a variety of pathological processes including atherosclerosis, diabetes, and neurodegenerative diseases. Understanding how intracellular signaling pathways respond to oxidative stress will have a significant implication in the therapy of these diseases. In this study, we applied hydrogen peroxide (H2O2) to trigger apoptosis and investigated the dynamic activation of various caspases using a FRET technique. We measured the activation dynamics of caspase 3 and caspase 9 based on two reporter systems, SCAT 3 and SCAT 9. We found that caspase 3 activation was earlier than that of caspase 9 following H2O2 treatment. Caspase 3 was activated rapidly, reaching a maximum in 12±3 min, while the average duration of caspase 9 activation was 21±3 min. When cells were pretreated with Z-LEHD-fmk, a caspase 9 specific inhibitor, caspase 3 activation and apoptosis by H2O2 treatment were little affected, although the caspase 9 activation was completely inhibited. When cells were pretreated with Z-DEVD-fmk, a caspase 3 specific inhibitor, the activation of both caspase 3 and caspase 9, as well as apoptosis, were inhibited. When cells were pretreated with Z-IETD-fmk, a caspase 8 specific inhibitor, the activation of caspase 3 and caspase 9 were significantly delayed. Finally, we found that Bax did not translocate from the cytosol to the mitochondrial membrane during H2O2-induced apoptosis. Our results suggest that, during H H2O2-induced apoptosis, caspase 3 is activated directly through caspase 8 and is not through the mitochondria-dependent caspase 9 activation.