Huifen Zhu

Hepatitis B Virus Protein X-induced Expression of the CXC Chemokine IP-10 Is Mediated through Activation of NF-κB and Increases Migration of Leukocytes

Interferon-γ inducible protein 10 (IP-10) involves inflammatory cell recruitment and cellular immune damage during virus infection. Although an increase of the peripheral IP-10 level is known in HBV-infected patients, the molecular basis of HBV infection inducing IP-10 expression has remained elusive. In the present study, we demonstrate that hepatitis B virus protein X (HBx) increases IP-10 expression in a dose-dependent manner. Transfection of the HBx-expressing vector into HepG2 cells results in nuclear translocation of NF-κB, which directly binds the promoter of IP-10 at positions from −122 to −113, thus facilitating transcription. The addition of the NF-κB inhibitor blocks the effect of HBx on IP-10 induction. In parallel, increase of NF-κB subunits p65 and p50 in HepG2 cells also augments IP-10 expression. Furthermore, we show that HBx induces activation of NF-κB through the TRAF2/TAK1 signaling pathway, leading to up-regulation of IP-10 expression. As a consequence, up-regulation of IP-10 may mediate the migration of peripheral blood leukocytes in a NF-κB-dependent manner. In conclusion, we report a novel molecular mechanism of HBV infection inducing IP-10 expression, which involves viral protein HBx affecting NF-κB pathway, leading to transactivation of the IP-10 promoter. Our study provides insight into the migration of leukocytes in response to HBV infection, thus causing immune pathological injury of liver.

Transcriptional upregulation of HSP70-2 by HIF-1 in cancer cells in response to hypoxia.

Heat shock protein 70‐2 (HSP70‐2) can be expressed by cancer cells and act as an important regulator of cancer cell growth and survival. Here, we show the molecular mechanisms by which hypoxia regulate HSP70‐2 expression in cancer cells. When cells were subjected to hypoxia (1% O2), the expression of HSP70‐2 had a significant increase in cancer cells. Such increase was due to the direct binding of hypoxia‐inducible factor to hypoxia‐responsive elements (HREs) in the HSP70‐2 promoter. By luciferase assays, we demonstrated that the HRE1 at position −446 was essential for transcriptional activation of HSP70‐2 promoter under hypoxic conditions. We also demonstrated that HIF‐1α binds to the HSP70‐2 promoter and the binding is specific, as revealed by HIF binding/competition and chromatin immunoprecipitation assays. Consequently, the upregulation of HSP70‐2 enhanced the resistance of tumor cells to hypoxia‐induced apoptosis. These findings provide a new insight into how tumor cells overcome hypoxic stress and survive, and also disclose a new regulatory mechanism of HSP70‐2 expression in tumor cells.

Transforming growth factor-β1 upregulates the expression of CXC chemokine receptor 4 (CXCR4) in human breast cancer MCF-7 cells

AbstractAim:To investigate whether rhTGF-β1 or a recombinant vector encoding a fusion protein comprising an extracellular domain of TGF-β receptor II and an IgG Fc fragment) affects the regulation of CXC chemokine receptor 4 (CXCR4) expression in MCF-7 human breast cancer cells.Methods:MCF-7 breast cancer cells were treated with rhTGF-β1 or transfected with a recombinant vector, pIRES2-EGFP-TβRII-Fc. Expression of CXCR4 in these cells was then analyzed at the mRNA and protein levels by quantitative RT-PCR and flow cytometry assay, respectively. A transwell assay was used to measure the chemotactic response of these cells to SDF-1α.Results:CXCR4 mRNA and protein expression were upregulated in TGF-β1-treated MCF-7 cells. These cells also demonstrated an enhanced chemotactic response to SDF-1α. In MCF-7 cells transiently transfected with pIRES2-EGFP-TβRII-Fc, a fusion protein named TβRII-Fc (approximately 41 kDa) was produced and secreted. In these transfected cells, there was a reduction in CXCR4 expression and in the SDF-1α-mediated chemotactic response.Conclusion:TGF-β1 upregulated CXCR4 expression in MCF-7 cells, which subsequently enhanced the SDF-1α-induced chemotactic response. The results suggest a link between TGF-β1 and CXCR4 expression in MCF-7 human breast cancer cells, which may be one of the mechanisms of TGF-β1-mediated enhancement of metastatic potential in breast cancer cells.

Interleukin-32 expression induced by hepatitis B virus protein X is mediated through activation of NF-κB.

HBV replicates noncytopathically in hepatocytes, but HBV or proteins encoded by HBV genome could induce cytokines, chemokines expression by hepatocytes. Moreover, liver damage in patients with HBV infection is immune-mediated and cytokines play important roles in immune-mediated liver damage after HBV infection. Interleukin-32 (IL-32) is a proinflammatory cytokine and plays a critical role in inflammation. However, the role of HBV in IL-32 expression remains unclear. In the present study, we demonstrate that hepatitis B virus protein X (HBx) increases IL-32 expression through the promoter of IL-32 at positions from -746 to +25 and in a dose-dependent manner. Furthermore, we demonstrate that increase of NF-κB subunits p65 and p50 in Huh7 cells also augments IL-32 expression, and the NF-κB inhibitor blocks the effect of HBx on IL-32 induction. These results indicate that NF-κB activation is required for HBx-induced IL-32 expression. In conclusion, IL-32 is induced by HBx in Huh7 cells. Our results suggest that IL-32 might play an important role in inflammatory response after HBV infection.

The antiproliferative effects of somatostatin receptor subtype 2 in breast cancer cells.

AbstractAim:Somatostatin receptor subtype 2 (SSTR2) is the principal mediator of somatostatins (SST) antiproliferative effects on normal and cancer cells. Therefore, we investigated whether the enhanced expression of SSTR2 could inhibit the proliferation of tumor cells, and, if so, the mechanisms that might be involved.Methods:SSTR2 expression levels were determined by qRT-PCR in several tumor cell lines. Then, a plasmid pIRES2-EGFP-SSTR2 (pSIG) was constructed and stably transfected into MCF-7 cells (MCF-7/pSIG). After SSTR2 overexpression was identified by qRT-PCR, immunofluorescence staining and a receptor binding assay, the MCF-7/pSIG cells were analyzed by PI staining for apoptosis and cell cycle arrest was tested by flow cytometry for epidermal growth factor receptor (EGFR) expression. The EGF-stimulated proliferation of MCF-7 cells was assayed by MTT.Results:The human breast cancer cell line MCF-7 expresses a lower level of SSTR2, thereby partly accounting for the decreased response to SST. The overexpression of SSTR2 in MCF-7 cells resulted in apoptosis, cytostasis and G1/S cell cycle arrest. Furthermore, the expression of EGFR, together with EGF-stimulated proliferation, was markedly decreased in the MCF-7/pSIG cells.Conclusion:Enhanced SSTR2 expression played an antiproliferative role in MCF-7 cells through inducing apoptosis and G1/S cell cycle arrest, and also by decreasing EGFR expression, thereby counteracting the growth-stimulating effect of EGF. Our data seem to indicate that developing a new therapeutic agent capable of upregulating SSTR expression could potentially be a way to block tumor progression.

Epidermal growth factor (EGF) induces apoptosis in a transfected cell line expressing EGF receptor on its membrane.

Interaction between epidermal growth factor (EGF) and EGF receptor (EGFR) promotes cell growth in most cell lines, but in a number of cell lines, EGF paradoxically inhibits proliferation. In the present study, we established a cell line expressing full‐length human EGFR on membrane with a GFP fluorescence reporter at the C‐terminal and studied the effects of EGF on cell proliferation in the transfected cell line. Our results suggested that low concentrations of EGF promoted proliferation, while high concentrations of EGF induced loss of adhesion, cell cycle arrest, apoptosis, and inhibition of proliferation. The effects of EGF on cell proliferation correlated well with the expression levels of EGFR. High concentrations of EGF induced both EGFR expression and apoptosis in a dose‐dependent manner. Our study reported, for the first time, a relationship between the effects of EGF on cell proliferation and levels of EGFR expression in one cell line expressing different levels of EGFR caused by different concentrations of EGF treatment. The study should provide considerable insight into the effects of EGF on cell proliferation and tumor cell metastasis.

Programmed death-1 signaling is essential for the skin allograft protection by alternatively activated dendritic cell infusion in mice.

Background. Alternatively activated dendritic cell (aaDC) can prolong allograft survival in the mouse model. However, the molecular mechanism(s) by which these DCs function to regulate alloreactive T-cell responses remains to be clearly defined. Methods. Bone marrow-derived DCs were incubated in the presence of interleukin (IL)-10 (immature DC), stimulated with lipopolysaccharide only (mature DC), or pretreated with IL-10 and then activated with lipopolysaccharide (aaDC). These cells were compared for their phenotypes and regulatory capacities both in vitro and in vivo. In addition, programmed death-1 (PD-1)/PD-L pathway was blocked to test its contribution to the regulatory function of aaDC. Results. The expression of surface major histocompatibility complex class II, CD80, and CD86 on aaDC was lower than that on mDC, whereas aaDC had a higher expression of PD-L1 and PD-L2 compared with immature DC or untreated DC. In vitro co-culture of aaDC with allogeneic T cells led to a significant decrease in the T-cell response as well as a reduction of interferon-&ggr; secretion and an enhanced IL-10 production while CD4+ CD25+ Foxp3+ T cells were expanded. Interestingly, these regulatory effects of aaDC were partially abolished when PD-1/PD-L pathway was blocked using anti-PD-1 blocking antibody. Infusion of BALB/c donor-derived aaDC into naive C57BL/6 recipients resulted in a significantly prolonged skin allograft survival, which was, at least in part, PD-1/PD-L pathway dependent. Conclusion. Our data indicate that the PD-1/PD-L pathway plays an important role in aaDC-mediated prolongation of skin allograft survival.

CTLA4-Ig-modified dendritic cells inhibit lymphocyte-mediated alloimmune responses and prolong the islet graft survival in mice

The induction of antigen specific tolerance is critical for prevention and treatment of allograft rejection. In this study, we transfected CTLA4-Ig gene into dendritic cells (DCs), and investigated their effect on inhibition of lymphocyte activity in vitro and induction of immune tolerance on pancreatic islet allograft in mice. An IDDM C57BL/6 murine model induced by streptozotocin is as model mouse. The model mice were transplanted of the islet cells isolated from the BALB/c mice to their kidney capsules, and injected of CTLA4-Ig modified DCs (mDCs). The results showed that mDCs could significantly inhibit T lymphocyte proliferation and induce its apoptosis; whereas, unmodified DCs (umDCs) promoted the murine lymphocyte proliferation. Compared with injection of umDCs and IgG1 modified DCs, the injection of mDCs prolonged IDDM mices allograft survival, and normalized their plasma glucose (PG) levels within 3 days and maintained over 2 weeks. The level of IFN-gamma was lower and the level of IL-4 was higher in mDCs treated recipient mice than that in control mice, it indicated that mDCs led to Th1/Th2 deviation. After 7 days of islet transplantation, HE stain of the renal specimens showed that the islets and kidneys were intact in structure, and islet cells numbers are increased in mDCs treated mice. Our studies suggest that DCs expressing CTLA4-Ig fusion protein can induce the immune tolerance to islet graft and prolong the allograft survival through the inhibition of T cell proliferation in allogeneic mice.

IL-10 Gene Modified Dendritic Cells Inhibit T Helper Type 1-Mediated Alloimmune Responses and Promote Immunological Tolerance in Diabetes

Dendritic cells (DCs) have the potency to regulate the outcome of autoimmunity through the modulation of immune responses. The induction of antigen specific tolerance is critical for prevention and treatment of allograft rejection. In the present study, we transfected IL-10 gene into DCs and investigated their effect on inhibition of lymphocyte activity in vitro and induction of immune tolerance on islet allograft in mice. An IDDM C57BL/6 mouse model was induced by streptozotocin. The islet cells isolated from the BALB/c mice were transplanted into the kidney capules of the model mice followed by injection of IL-10 modified DCs (mDCs). The results showed that mDCs could significantly inhibit T lymphocyte proliferation mediated by allotype cells and induce its apoptosis, whereas, unmodified DCs (umDCs) could promote the murine lymphocyte proliferation markedly. The injection of mDCs could prolong the survival of allotype islet transplanted IDDM mice. The average plasma glucose (PG) level in mDCs treated mice returned to normal within 3 days and lasted for about 2 weeks. The rejection response in control mice occurred for 5 days after transplantation. The level of IFN-γ was lower while IL-4 was higher in mDCs treated mice than that in umDCs treated mice, which indicated that Th1/Th2 deviation occurred. Our studies suggest that IL-10 gene modified DCs can induce the immune tolerance to islet graft and prolong survival of the recipients by the inhibiting of T cell proliferation in allotype mice.

Tumor transfected with CCL21 enhanced reactivity and apoptosis resistance of human monocyte-derived dendritic cells

Chemokine CCL21 can effectively attract CCR7(+) dendritic cells (DCs), however its role in this event is poorly understood. In this report, we investigated the effect of exogenous CCL21 expressed in breast cancer MCF-7 on human monocyte-derived DCs. CCL21-transfected MCF-7 stimulation prompted DC functions: migration, antigen-uptake and presentation. The stimulated DCs facilitated Th1 type cytokines production, perforin-forming CD8(+) T cell transformation and final T cell-associated clearance of MCF-7. Moreover, the MCF-7-resourced CCL21 protected DCs from apoptosis significantly, involving up-regulations of Bcl-2 expression and NF-kappaB activity, and reduction of caspase-3. This study provides evidence that tumor-derived CCL21 increases the presentation and apoptosis resistance of DCs, suggesting such a mechanism may be useful for the improvement of tumor cell immunogenicity and anti-tumor response.