Yingru Zheng

Upregulation of scavenger receptor class B type I expression by activation of FXR in hepatocyte

OBJECTIVE The farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, has been proposed to play an important role in the pathogenesis of cardiovascular diseases by regulating the metabolism and transport of cholesterol and triglyceride. Scavenger receptor class B type I (SR-BI), a high-density lipoprotein receptor, plays an important role in decreasing lipid metabolism-associated cardiovascular diseases by regulating reverse cholesterol transport. Recent studies have shown that SR-BI expression is upregulated by several nuclear receptors. However, the role of FXR in the regulation of SR-BI expression is not well known. In the present study, we investigate the regulation of SR-BI by FXR in hepatocyte and the corresponding mechanism. METHODS AND RESULTS Treatment of human hepatoma cell line HepG2 with FXR ligands resulted in upregulation of SR-BI at the levels of both mRNA and protein. Reporter assays showed that activation of FXR significantly enhanced the SR-BI promoter activity. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that FXR induced SR-BI expression by binding to a novel FXR element (FXRE), a directed repeat DNA motif, DR8 (-703 AGGCCAcgttctagAGCTCA -684). The in vivo experiment demonstrated that gavaging mice with a natural ligand of FXR increased SR-BI expression in liver tissues. CONCLUSIONS FXR can directly upregulate SR-BI expression in hepatocyte, and DR8 is a likely novel FXRE that is involved in SR-BI regulation. FXR may serve as a novel molecular target for manipulating SR-BI expression in hepatocyte.

Differential expression of Oct4 in HPV-positive and HPV-negative cervical cancer cells is not regulated by DNA methyltransferase 3A

The colony-forming ability of cervical cancer is affected by many factors. Oct4, an important transcription factor, is highly expressed in several tumors and promotes the colony-forming ability of cancer cells. Thus, it is considered a potential target for the treatment of cancer. However, we know little about the expression level of Oct4 and its epigenetic regulatory mechanism in cervical cancer cells. In this study, we are the first to observe that human papillomavirus (HPV)-positive cervical cancer cell lines (HeLa, Caski) have a stronger colony-forming ability than HPV-negative cervical cancer cell lines (C-33A). Moreover, the expression level of Oct4 in both HeLa and Caski cells was also higher than that in C-33A cells. We then confirmed that there was a negative correlation between the expression of Oct4 and DNMT3A in these three types of cervical cancer cells, whereas DNA methyltransferase 1 and 3B had no differences among the cell lines. However, after DNA methylation in both key regulatory regions of the Oct4 gene and the genomic levels were analyzed, we found that DNA methyltransferase 3A could neither regulate the expression of Oct4 nor affect the whole level of genomic DNA methylation. These results suggest three points: (1) Oct4 might be treated as a new target for the treatment of cervical cancer, (2) we could not inhibit the expression of Oct4 by DNA demethylation, and (3) HPV virus might initiate cervical carcinogenesis by upregulation of Oct4 expression.

Peroxisome proliferator-activated receptor γ agonist troglitazone inhibits high mobility group box 1 expression in endothelial cells via suppressing transcriptional activity of nuclear factor κB and activator protein 1.

High mobility group box 1 (HMGB1), a delayed mediator of proinflammatory cytokines, could initiate and amplify inflammatory responses to infection, injury, and other inflammatory stimuli, and it has emerged as a potential therapeutic target for inflammatory diseases. The overexpression of HMGB1 in endothelial cells has been proved to contribute to the development of these diseases. Because many proinflammatory cytokines expression were suppressed by thiazolidinediones (TZDs), agonists for nuclear receptor peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;), whether TZDs can inhibit HMGB1 expression and function is of great interest, however, it remains unknown. Herein, we provide evidence that PPAR&ggr; agonist troglitazone, a member of the TZD class, modulates HMGB1 expression in the endothelial cell line EA.hy926 and propose a potential mechanism for that. Results from polymerase chain reaction experiments revealed that PPAR&ggr; is expressed in EA.hy926 cells, and it can be activated by troglitazone. Troglitazone inhibited the basal and LPS-stimulated HMGB1 expression at the mRNA level and protein level. A luciferase reporter assay showed that troglitazone inhibited not only the transcriptional activation of the HMGB1 promoter but also activities of heterologous promoters driven by nuclear factor &kgr;B (NF-&kgr;B) or activator protein 1 (AP-1) response elements. Altogether, these data suggest that NF-&kgr;B and AP-1 may participate in the inhibitory effect on HMGB1 transcription induced by troglitazone. Activation of PPAR&ggr; by troglitazone is effective for HMGB1 inhibition via suppressing NF-&kgr;B and AP-1 transcriptional activity in endothelial cells, which provides a new potential strategy to suppress excessive HMGB1 in inflammatory diseases.

Tumor targeting of vincristine by mBAFF-modified PEG liposomes in B lymphoma cells

Malignant hematologic diseases are highly malignant and refractory to conventional therapies. Ligand-mediated targeting of liposomal anticancer drugs to surface receptors expressed on malignant B cells can be an effective strategy for treating B-cell malignancies. BAFF plays an important role in the maintenance of normal B-cell development and homeostasis and the expression of its receptors is significantly increased in numerous B-cell malignancies. mBAFF (a soluble BAFF mutant with amino acid 217-224 being replaced by two glycine residues) may be used as a competitive inhibitor for BAFF to treat relevant malignant hematologic diseases. It may also hold promise as a novel ligand for targeted anticancer therapy. In this study, we show that liposomes that are sterically stabilized by PEG and surface decorated with mBAFF exhibited strong affinity and specificity to cultured human Raji B lymphoma cells. Vincristine formulated in the targeted liposomes showed significantly higher levels of cytotoxicity towards Raji cells than the nontargeted liposomal drug. Therapeutic experiments in SCID mice implanted with Raji cells showed significantly prolonged survival time with targeted liposomal vincristine compared to either free VCR or vincristine formulated in nontargeted liposomes. These studies suggest the potential of the mBAFF-modified liposomal drugs in targeted therapy of B-cell malignancies.

The Anti-Inflammatory Activity of HMGB1 A Box Is Enhanced When Fused with C-Terminal Acidic Tail

HMGB1, composed of the A box, B box, and C tail domains, is a critical proinflammatory cytokine involved in diverse inflammatory diseases. The B box mediates proinflammatory activity, while the A box alone acts as a specific antagonist of HMGB1. The C tail contributes to the spatial structure of A box and regulates HMGB1 DNA binding specificity. It is unknown whether the C tail can enhance the anti-inflammatory effect of A box. In this study, we generated fusion proteins consisting of the A box and C tail, in which the B box was deleted and the A box and C tail were linked either directly or by the flexible linker sequence (Gly4Ser)3. In vitro and in vivo experiments showed that the two fusion proteins had a higher anti-inflammatory activity compared to the A box alone. This suggests that the fused C tail enhances the anti-inflammatory effect of the A box.

sBAFF mutants induce neutralizing antibodies against BAFF

B cell activating factor belonging to the TNF family (BAFF) is a novel member of the tumor necrosis factor (TNF) ligand family and plays an important role in B lymphocyte maturation and survival. Overexpression of BAFF is closely involved in the pathogenesis and progression of many kinds of autoimmune disorders; therefore, BAFF has been considered as an ideal therapeutic target for these conditions. In this study, we generated several candidate immune inhibitors of human BAFF by conjugating foreign immunodominant T‐helper cell (Th) epitopes to the N‐ or C‐terminus of five BAFF mutants. The recombined proteins were successfully expressed in Escherichia coli (E. coli) and purified by Ni‐NTA chromatography. BALB/c mice immunized with the recombinant proteins produced high levels of anti‐BAFF antibodies, and their sera inhibited the lymphocyte proliferation‐inducing activity of recombinant soluble BAFF and natural soluble BAFF. Moreover, antibodies cross‐reactive with BAFF were detected in sera from hu‐SCID mice immunized with the recombinant proteins. These results indicated that the recombinant BAFF mutants modified with Th epitopes could induce neutralizing antibodies against BAFF in vivo. This study may provide a valuable strategy for treating BAFF‐associated autoimmune diseases.

HDAC1/DNMT3A-containing complex is associated with suppression of Oct4 in cervical cancer cells

Octamer-binding transcription factor 4 (Oct4), an important embryonic transcriptional factor, is highly expressed in several tumors and is considered as a hallmark of cancer stem cells. Knowledge about the expression and regulatory mechanisms of Oct4 can contribute to the treatment of cancers. As for cervical cancer, however, details remain obscure about Oct4 expression and its regulatory mechanism. In this study, we found that the level of Oct4 in human papillomavirus 16 (HPV16)-positive cervical cancer cells (CaSki cells) was higher than that in HPV-negative cervical cancer cells (C-33A cells), whereas both the level of histone deacetylase 1 (HDAC1) and DNA methyltransferase 3A (DNMT3A) were lower in CaSki cells than those in C-33A cells. Treatment with valproic acid, an HDAC inhibitor, could significantly increase the expression of Oct4 in C-33A cells, but only slightly increased Oct4 in CaSki cells. Co-immunoprecipitation assays showed that HDAC1 and DNMT3A existed in a common complex. The co-immunoprecipitated DNMT3A or HDAC1 was dose-dependently decreased with valproic acid treatment. These results indicated that HDAC1/DNMT3A-containing complex is associated with the suppression of Oct4 in cervical cancer cells, and the activity of HDAC1 is required in the repression of Oct4.

PinX1, a novel target gene of p53, is suppressed by HPV16 E6 in cervical cancer cells.

The aberrant activation of telomerase is critical for the initiation and development of human cervical cancer, which is dependent on the activation of human telomerase reverse transcriptase (hTERT). Recently, Pin2/TRF1-interacting protein X1 (PinX1) has been identified as a suppressor of hTERT. It has been found that the telomerase is activated while the level of PinX1 is decreased in cervical cancer. However, the regulatory mechanism of PinX1 in cervical cancer cells remains unclear. In the present study, we demonstrated that the level of PinX1 is regulated by p53, and p53 functions as a transcriptional factor to directly activate the expression of PinX1 in cervical cancer cells. Moreover, we found that HPV16 E6 suppresses the expression of PinX1 via inhibiting p53 transcriptional activity, resulting in the enhancement of telomerase activity. This study not only for the first time shows that PinX1 is a novel target gene of p53 but also suggests that suppression of p53/PinX1 pathway may be a novel mechanism by which HPV16 E6 enhances the telomerase activity in cervical cancer cells.

Upregulation of thrombomodulin expression by activation of farnesoid X receptor in vascular endothelial cells

Thrombomodulin (TM) serves as a vasoprotective molecule on the surface of vascular endothelial cells (VECs) to maintain the endothelial microenvironment by suppressing cellular proliferation, adhesion and inflammatory responses. Farnesoid X receptor (FXR), a nuclear receptor (NR) and originally considered as a bile acid-activated transcriptional factor, not only regulates metabolism homeostasis, but also influences cholesterol transport, vascular tension, and inflammation. Recent studies have shown that TM expression is upregulated by several NRs. However, it is unknown whether there is a link between FXR and TM. Our studies demonstrated that TM expression and activity were up-regulated by FXR activation in VECs. Reporter assays showed that FXR activation significantly enhanced the transcriptional activity of human TM gene promoter. Elecrophoretic mobility-shift and chromatin immunoprecipitation assays indicated that FXR induced TM expression by binding to a novel FXR-responsive element (FXRE), an inverted repeat DNA motif, IR8 (-503 AGGTCCtcccaaagTGCCCT-484) in the promoter region of TM gene. These results suggest that FXR may serve as a novel molecular target for manipulating TM expression and activity in VECs, which may be helpful for designing the therapeutic strategies to the treatment of associated diseases by targeting FXR/TM pathway.

Activation of liver X receptor attenuates endothelin-1 expression in vascular endothelial cells.

Endothelin-1 (ET-1), predominantly produced by vascular endothelial cells (VECs), plays an important role in the pathogenesis of inflammatory diseases. Liver X receptor (LXR), a typical nuclear receptor, is known for inhibiting expression of inflammatory molecules. However, it remains unclear whether LXR suppresses ET-1 expression. In the present study, we showed that pretreatment with GW3965, a specific ligand of LXR, significantly attenuated lipopolysaccharide (LPS)-induced ET-1 in mice plasma. The in vitro experiments showed that both LXRα and β were expressed in human VECs, and they are functional as demonstrated by induction of the target gene ABCA1 after treatment with GW3965. Moreover, activation of LXR with GW3965 in human VECs dramatically attenuated the basal and LPS-stimulated ET-1 production at both transcriptional and translational levels. Luciferase reporter assays indicated that LXR activation suppressed the transcriptional activity of the human ET-1 gene promoter, and repressed the activity of a heterologous promoter driven by the response elements of activator-1 (AP-1) or nuclear factor-κB (NF-κB). Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that activation of LXR reduced the binding of the transcriptional factors AP-1 and NF-κB to the ET-1 gene promoter region. In conclusion, activation of LXR represses ET-1 expression in vivo and in vitro, which may be involved in the negatively interfering with AP-1/NF-κB signaling. These results suggest that LXRs may serve as a novel molecular target for modulating ET-1 expression in VECs, and even for the treatment of ET-1-associated inflammatory diseases.