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. 2012;7(11):e48935.
doi: 10.1371/journal.pone.0048935. Epub 2012 Nov 8.

Constitutive β-catenin signaling by the viral chemokine receptor US28

Ellen V Langemeijer  1 , Erik Slinger, Sabrina de Munnik, Andreas Schreiber, David Maussang, Henry Vischer, Folkert Verkaar, Rob Leurs, Marco Siderius, Martine J Smit
Affiliations

Constitutive β-catenin signaling by the viral chemokine receptor US28

Ellen V Langemeijer et al. PLoS One. 2012.

Abstract

Chronic activation of Wnt/β-catenin signaling is found in a variety of human malignancies including melanoma, colorectal and hepatocellular carcinomas. Interestingly, expression of the HCMV-encoded chemokine receptor US28 in intestinal epithelial cells promotes intestinal neoplasia in transgenic mice, which is associated with increased nuclear accumulation of β-catenin. In this study we show that this viral receptor constitutively activates β-catenin and enhances β-catenin-dependent transcription. Our data illustrate that this viral receptor does not activate β-catenin via the classical Wnt/Frizzled signaling pathway. Analysis of US28 mediated signaling indicates the involvement of the Rho-Rho kinase (ROCK) pathway in the activation of β-catenin. Moreover, cells infected with HCMV show significant increases in β-catenin stabilization and signaling, which is mediated to a large extent by expression of US28. The modulation of the β-catenin signal transduction pathway by a viral chemokine receptor provides alternative regulation of this pathway, with potential relevance for the development of colon cancer and virus-associated diseases.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. US28 induces constitutively activates ß-catenin signaling.
US28 is expressed and functional in NIH-3T3 cells. A, Whole cell binding of [125I]-CCL5 on NIH-3T3 cells expressing wild-type (WT), mutant R129A or HA-tagged US28 is displaced by fractalkine (CX3CL1). B, US28-WT constitutively stimulates inositol phosphate (IPx) accumulation in NIH-3T3, while the non-G protein-coupling mutant US28-R129A shows no activation. C, Total cell extracts of NIH-3T3 cells stably expressing US28, the non G-protein-coupling mutant and empty plasmid control (mock) were analysed on Western blot with antibodies recognizing the non-phospho (active) ß-catenin, total ß-catenin and actin. D, NIH-3T3 cells stably expressing US28 and the non G-protein coupling US28 mutant R129A were transfected with the Tcf-Lef reportergene construct. Luciferase activity was measured 24 h after transfection. E, US28 dose-dependently induces Tcf-Lef transcriptional activation in HEK293T cells. The non-G protein-coupling mutant US28 R129A does not display activation of the reportergene at 25 ng DNA transfected (dark grey bar). Treatment of HEK293T cells transfected with 25 ng US28 DNA with inverse agonist VUF 6064 (10 µM) prevents activation of Tcf-Lef reortergene (light grey bar). F, HEK293T cells transfected with the human chemokine receptor CCR1 and the Tcf-Lef reportergene construct do not show Tcf-Lef activation nor after exposure to 100 nM CCL5 (RANTES). US28 expressing HEK293T cells display constitutive signaling to the Tcf-Lef reportergene, which is significantly enhanced by exposure to 100 nM CCL5 (RANTES).
Figure 2
Figure 2. Classical Wnt/Frizzled/ß-catenin
signaling is not involved in US28-mediated Tcf-Lef activation. A, Western blot analysis of total cell extracts of NIH-3T3 cells, stably expressing US28 or an empty plasmid (mock) which were treated with Wnt3a- (overnight, 200 ng/ml) and vehicle-treated mock cells. The blot was probed with antibodies recognizing the non-phosphorylated (active ß-catenin, total ß-catenin and actin. A representative blot is shown and normalized quantifications of (active) ß-catenin of independent experiments are shown below the blot. B, Western blot analysis of total cell extracts of NIH-3T3 cells stably expressing US28, the non G-protein coupling US28 mutant R129A or an empty plasmid (mock) and Wnt3a-treated mock cells. The blot was probed with antibodies recognizing Lrp6-phospho-ser1490 and actin. A representative blot is shown and normalized quantifications of Lrp6-phospho-ser1490 of independent experiments are shown below the blot. C, HEK293T cells co-transfected with the Tcf-Lef reporter gene construct and either US28-expressing or an empty control plasmid (mock) exposed to Wnt3a (overnight, 200 ng/ml). Luciferase activity was measured 24 hr after transfection and is displayed here as the percentage of the non-treated mock control that is set at 100%. D, HEK293T cells co- transfected with the Tcf-Lef reportergene and an US28-expressing construct or empty plasmid control (mock) were exposed to various concentrations (ON, 10–25 µM) of the COX2 inhibitor celecoxib (Cxb). Tcf-Lef reporter gene activation was measured 24 hr after transfection and is displayed here as the percentage of the mock control that is set at 100%.
Figure 3
Figure 3. G protein involvement in US28-mediated Tcf-Lef activation.
A, HEK293T cells were co-transfected with the Tcf-Lef reporter gene construct, a US28-expressing construct or empty plasmid control (mock) and various constructs expressing Gα-proteins as indicated, Gαq-11 shRNA construct or a construct expressing regulator of G protein signaling 2 (RGS2), known to specifically interfere with Gαq signaling. Tcf-Lef reporter gene activation was measured 24 hr after transfection and is displayed here as the percentage of the mock control that is set at 100%. B, HEK293T cells were co-transfected with the Tcf-Lef reporter gene construct, US28-expressing construct or empty plasmid control (mock) and an shRNA construct to decrease protein levels of Gαq. Total cell extracts were analysed on Western blot using antibodies recognizing Gαq or actin (insert). Bars represent level of Gαq protein level compared to the actin levels, with the ratio in non-treated mock cells set at 100%. C, HEK293T cells were co-transfected with the Tcf-Lef reporter gene construct, a US28-expressing construct or empty plasmid control (mock) and various constructs expressing Gα13, a constitutive active (CA) Gα13 or Lsc-RGS, encoding the RGS domain of the Rho GTPase guanine nucleotide exchange factor (Rho-GEF) Lsc, known to specifically interfere with transmembrane signaling mediated by activated Gα12/13. Tcf-Lef reporter gene activation was measured 24 hr after transfection and is displayed here as the percentage of the mock control that is set at 100%. D, HEK293T cells co-transfected with the Tcf-Lef reporter gene construct, a US28-expressing construct or empty plasmid control (mock) were treated (overnight) with various concentrations of the ROCK inhibitor Y27632 as indicated.
Figure 4
Figure 4. HCMV-infected cells stimulate activation of ß-catenin in a US28 dependent manner.
A. HFF cells were infected with HCMV-WT or HCMV-ΔUS28 with a M.O.I of 1 on IBIDI slides. Cells were fixed 24 hours post-infection (hpi) and stained with antibodies recognizing the HCMV immediate early antigen (IEA) and activated ß-catenin respectively. B. U373-MG cells transfected with Tcf-Lef reporter gene were either infected with HCMV-WT or HCMV-ΔUS28 with a M.O.I. of 2, or left uninfected (mock). Luciferase activity was measured 48 h post-infection.
Figure 5
Figure 5. Schematic representation of the classic Wnt signaling pathway and model of US28-mediated activation of ß-catenin signaling pathway.
The left side of the model indicates components of the classic Wnt/Frizzled mediated activation of ß-catenin. In this pathway the disruption complex (Axin, APC) that enables GSK3ß- and Caseine kinase 1 (CK1)-mediated phosphorylation of ß-catenin leading to its degradation, is disrupted in a Dishevelled-mediated way upon Wnt binding to Frizzled/LRP. US28 activates ß-catenin signaling in a ligand-dependent and independent manner, involving respectively Gα12/13 and Gαq proteins and respective RhoGEFs, converging at RhoA/ROCK, resulting in increased Tcf-Lef transcriptional activation.
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Grants and funding

This work was supported by The Netherlands Organization for Scientific Research (to E.L., S.d.M., F.V. and M.J.S.), The Royal Netherlands Academy of Arts and Sciences (to M.J.S.) and Echo grant (to E.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.