Kaposi’s sarcoma-associated herpesvirus G-protein coupled receptor activates the canonical Wnt/β-catenin signaling pathway
© Angelova et al.; licensee BioMed Central. 2014
Received: 9 June 2014
Accepted: 27 November 2014
Published: 17 December 2014
KSHV is a tumorigenic γ-herpesvirus that has been identified as the etiologic agent of Kaposi’s sarcoma (KS), a multifocal highly vascularized neoplasm that is the most common malignancy associated with acquired immunodeficiency syndrome (AIDS). The virus encodes a constitutively active chemokine receptor homologue, vGPCR that possesses potent angiogenic and tumorigenic properties, and is critical for KSHV pathobiology. To date, a number of signaling pathways have been identified as key in mediating vGPCR oncogenic potential.
In this study, we identify a novel pathway, the Wnt/β-catenin pathway, which is dysregulated by vGPCR expression in endothelial cells. Expression of vGPCR in endothelial cells enhances the nuclear accumulation of β-catenin, that correlates with an increase in β-catenin transcriptional activity. Activation of β-catenin signaling by vGPCR is dependent on the PI3K/Akt pathway, as treatment of vGPCR-expressing cells with a pharmacological inhibitor of PI3K, leads to a decreased activation of a β-catenin-driven reporter, a significant decrease in expression of β-catenin target genes, and reduced endothelial tube formation.
Given the critical role of Wnt/β-catenin signaling in angiogenesis and tumorigenesis, the findings from this study suggest a novel mechanism in KSHV-induced malignancies.
KeywordsKaposi’s sarcoma-associated herpesvirus Wnt/β-catenin signaling Angiogenesis
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a tumorigenic gammaherpesvirus that is the etiologic agent of Kaposi’s sarcoma (KS). KS is a multifocal, highly vascular tumor that develops on the lower extremities, mucous membranes or internal organs. Co-infection with HIV markedly increases the risk of KS development . Although highly active anti-retroviral therapy (HAART) has decreased the incidence of AIDS-related KS, it remains an incurable tumor for which there is no established treatment . KS tumors are characterized by highly proliferating, elongated, spindle shaped cells of endothelial origin. The majority of cells in the tumor are latently infected with KSHV, with less than 3% of the cells expressing viral lytic proteins . It has been proposed that dysregulation of the viral gene program leads to nonlytic expression of a virally-encoded G-protein coupled receptor (vGPCR) homologue . vGPCR has potent angiogenic and tumorigenic properties and its transgenic expression in vivo is sufficient to induce the development of angioproliferative lesions characterized by prominent inflammation, dysregulated angiogenesis and the presence of endothelial cells with a spindle morphology . Consequently, vGPCR has become a potential therapeutic target for KS.
The exact mechanisms involved in vGPCR-induced angiogenesis and tumorigenesis are still being elucidated. vGPCR signaling induces the secretion of potent pro-angiogenic and pro-inflammatory chemokines and growth factors, such as VEGF, IL-8 and Gro-α that can promote inflammation, cell transformation, and angiogenesis through autocrine and paracrine mechanisms ,. The large number of growth-promoting and survival pathways dysregulated by vGPCR could explain its potent transforming and oncogenic properties ,.
We previously reported that vGPCR activates expression of COX-2 that drives synthesis of PGE2, a proinflammatory molecule linked to tumor angiogenesis and upregulated in KS lesions . Recent evidence indicates that PGE2 via signaling through its E2 receptor can activate β-catenin . β-catenin is a dual function protein involved in the coordination of cell–cell adhesion and regulation of gene transcription. Numerous studies have identified aberrant activation of β-catenin signaling as a hallmark of many human cancers, which often result from stabilizing mutation in the genes encoding Wnt/β-catenin pathway components . However, many cancers do not contain such mutations ,, suggesting that there are other factors that contribute to β-catenin activation. Indeed, Wnt/β-catenin signaling is commonly manipulated by oncogenic viruses. Hepatitis C (HCV) virus activates Wnt/β-catenin signaling, which appears to be key for the malignant transformation of hepatocytes seen in hepatocellular carcinoma . Epstein Barr virus (EBV)-encoded protein, latent membrane protein 2A (LMP2A) causes β-catenin stabilization through activation of PI3K/Akt and inactivation of GSK-3β .
Here we explore the role of Wnt/β-catenin signaling in response to vGPCR, a lytic phase viral protein expressed in KS lesions. We show that expression of vGPCR in endothelial cells activates Wnt/β-catenin signaling and induces the expression of growth factors and cell-cycle regulators. The results from this study identify a novel molecular mechanism by which vGPCR modulates host signaling, which is essential to further understand the pathobiology of KS and identify novel therapeutic targets.
Results and discussion
β-catenin acts as an intracellular transducer in canonical Wnt signaling. β-catenin is normally localized in the cytoplasm in an inactive state through its interaction with a multiprotein complex comprised of axin, adenomatous polyposis coli (APC) and two serine/threonine kinases, glycogen synthase kinase-3β (GSK-3β), and casein kinase 1 (CK1). Phosphorylation of β-catenin on serine-45 by CK1 followed by phosphorylation on serine-33 and −37 by GSK-3β marks β-catenin for polyubiquitination and proteasomal degradation. Wnt ligand binding to receptors on the surface of target cells initiates a cascade of events leading to disruption of the axin/APC/GSK-3β complex thus preventing phosphorylation and degradation of β-catenin. Accumulation of stable, hypophosphorylated β-catenin in the cytoplasm promotes its translocation to the nucleus (reviewed in ) where it binds T cell-specific factor(TCF)/lymphoid enhancer-binding factor-1 (LEF-1) DNA-binding factors to activate transcription of over fifty target genes involved in cell maintenance, proliferation and survival, such as cyclin D1, c-myc, VEGFA, MMP-2 and MMP-9. Thus, the subcellular distribution of β-catenin and its transcriptional activation upon vGPCR expression was next investigated. Levels of β-catenin were found to be higher in the nucleus of vGPCR-expressing cells than in the control cells, as determined by a Western blot analysis of nuclear and cytoplasmic fractions (Figure 2B). These observations were confirmed by immunofluorescence analysis, demonstrating that more β-catenin accumulates in the nucleus of vGPCR-expressing cells (Figure 2C).
Cell lines and retrovirus infection
Primary human umbilical vein endothelial cells (HUVECs) (Lonza, Allendale, NJ) were grown in M-199 media supplemented with 20% fetal bovine serum (FBS), 1% Penicillin/Streptomycin and 50 μg/mL ECGS (BD Biosciences, San Jose, CA) on vessels coated with 2% gelatin. Recombinant retroviral vectors expressing either GFP alone (BABE) or GFP and KSHV ORF74 (BABE-vGPCR)  were used to infect HUVECs at 40-60% confluency in complete media supplemented with 8 μg/mL polybrene (hexamethidrine bromide) (Sigma). GFP fluorescence was used to determine transduction efficiency between 48–72 hr post-infection. For all experiments, transduction efficiency was >80%.
Quantitative real-time RT-PCR (qRT-PCR)
Total RNA was harvested from cells using the Qiagen RNeasy Kit (Qiagen) according to the manufacturer’s instructions. Total RNA (500 ng) was reverse transcribed using iScript cDNA Synthesis kit (Bio-Rad) and used in a qPCR using SYBR Green supermix (Bio-Rad). Oligonucleotide primers (Integrated DNA Technologies) used are as follows: cyclin D1 forward (5′-CGCCCTCGGTGTCCTACTTC-3′), cyclin D1 reverse (5′-GACCTCCTCC-TCGCACTTCTG-3′); VEGFA forward (5′- GGCAGAAGGAGGAGGGACAGAATC -3′), VEGFA reverse (5′- CATTTACACGTCTGCGGATCTTGT -3′); MMP-9 forward (5′-AGACGGGTATCCCTTCGACG-3′), MMP-9 reverse (5′-AAACCGAGTTGGAA-CCACGAC-3′); DKK1 forward (5′-TTCCAACGCTATCAACCTGC-3′), DKK1 reverse (5′- CAAGGTGGTTCTTCTGGAATACC-3′); c-myc forward (5′- GCCACGTCTCCA-CACATCAG-3), c-myc reverse (5′-TCTTGGCAGGAGGATAGTCCTT-3′); 36B4 forward (5′-TGGAGACGGATTACACCTTC-3′), 36B4 reverse (5′-CTTCCTTGGCTT-CAACCTTAG-3′); Relative quantitation was determined using the comparative CT method with data normalized to 36B4 and calibrated to the average ΔCT of BABE control at the specific time points.
Cells were lysed in RIPA buffer (25 mM TrisHCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors. Protein samples were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked in 5% non-fat dry milk and incubated in primary antibody overnight at 4°C, followed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Invitrogen). Antibody-protein complexes were detected with ECL Plus (Amersham Biosciences) and exposed to chemiluminescent film. Primary antibodies: mouse anti-β-catenin (Santa Cruz Biotechnology; 1:500 dilution), mouse anti-β-actin (Abcam; 1:5,000), mouse anti-γ-tubulin (Abcam; 1:1000), anti-Histone 3 (Cell Signaling; 1:1000). Densitometric analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD).
HUVECs were transiently transfected with the TCF/LEF-1 reporter plasmid TOPflash, which contains multimeric TCF/LEF-1 sequences upstream of a firefly luciferase reporter gene. FOPflash plasmid containing mutated TCF/LEF-1 binding sites was used as a specificity control for TOPflash. Plasmids were purchased from Millipore (Bedford, MA). A Renilla luciferase-expressing plasmid pRL-TK (Promega) was co-transfected as an internal control. Cells were transfected with 1 μg of either TOPflash or FOPflash DNA and 0.1 μg of Renilla plasmid using the Neon transfection system (Invitrogen) according to the manufacturer’s instructions. Twenty-four hr after transfection, luciferase activity was assayed with a dual-luciferase reporter assay kit (Promega) and measured in a luminometer (Berthold Technologies). For each sample, the firefly luminescence signal was normalized to the corresponding Renilla signal.
Small interfering RNA (siRNA) transfection
Cells were transfected with 100 nM siRNA to β-catenin (Santa Cruz) or the same concentration of non-silencing siRNA (Santa Cruz) using the NEON transfection system (Invitrogen). Twenty-four hours later cells were serum starved for 24 hr and harvested for subsequent assays.
Isolation of cytoplasmic and nuclear fractions
Cytoplasmic and nuclear fractions were isolated from cells using a nuclear extract kit (Active Motif, Carlsad, CA) according to the manufacturer’s protocol. Resulting fractions were separated on SDS-PAGE and immunoblotted for β-catenin, H3, and γ-tubulin as described above.
Cells were seeded onto glass coverslips coated with 0.2% gelatin, and infected with BABE or BABE-vGPCR as described above. At indicated time points after infection, cells were fixed in 2% paraformaldehyde (Ted Pella) and permeablized in 0.1% Triton X-100. The cells were blocked in 5% BSA for 1 hr at room temperature and incubated with primary antibodies for 1 hr at room temperature followed by incubation with AlexaFluor-conjugated secondary antibodies and DAPI. Coverslips were mounted with ProLong gold antifade (Invitrogen) and imaged at 40X using a Zeiss Axioplan II microscope (Carl Zeiss).
Cells were loaded into 8 μm FluoroBlok 24-well multiwell insert system (BD Biosciences) at 2x104 cells per insert, in 200 μl of M-199 supplemented with 0.5% FBS. The bottom chamber of the insert was loaded with 800 μl of complete M-199. The cells were incubated at 37°C in 5% CO2 and allowed to migrate for 12 hr. The inserts were then labeled with calcein AM (Molecular Probes) and visualized by fluorescent microscopy. Three random fields from each insert were captured with a Nikon TE200 inverted fluorescent microscope (Nikon Instruments) and the average fluorescence intensity was determined by ImageJ software.
Endothelial tube formation assay
Cells were trypsinized and resuspended in 100 μl of low serum (0.5%) M-199 medium without ECGS. Cells were seeded in a 96-well plate coated with 100 μl/well of growth factor reduced Matrigel (BD Bioscience) at a density of 1x104 cells/well and incubated at 37°C for 6 hr. Tube formation was observed using a phase-contrast microscope. The number of tubes was quantified by counting the number of tube branch points.
In vivo Matrigel plug angiogenesis assay
Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Tulane University. NIH3T3 cells were stably transduced with BABE-vGPCR or BABE as control. For each cell population, 2 × 106 cells were harvested in 200 μl of phenol-free, serum-free media containing 10 units/ml of heparin, and mixed with 200 μl of high concentration, growth-factor-reduced Matrigel (BD Bioscience). 400 μl of the Matrigel-cell suspension was injected subcutaneously into the left and right flanks (2 sites per mouse) of anaesthetized, 6–8 weeks old nu/nu athymic mice. Plugs were retrieved 7 days after implantation and fixed in 10% formalin overnight. After fixation, plugs were rinsed in PBS, paraffin embedded sectioned and stained with hematoxylin-eosin.
Significant differences between experimental groups were determined by Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc t test using GraphPad Prism 4 software. Data are presented as the means ± standard error of the means (SEM).
The PI3K inhibitor LY294002 was obtained from Cell Signaling; the selective COX-2 inhibitor NS-398 was purchased from Cayman Chemicals. The concentration for each inhibitor is specified in the figure legends.
This work was supported in part by National Institutes of Health grants R21HD076283 (DES), R01HD51998 (CAM) and P20GM103424 (HEM).
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