Aberrant Proliferation in CXCR7+ Endothelial Cells via Degradation of the Retinoblastoma Protein

Angiogenesis is a critical factor in the growth and dissemination of solid tumors. Indeed, tumor vasculature is abnormal and contributes to the development and spread of malignancies by creating a hostile microenvironment. The alternative SDF-1/CXCL12 receptor, CXCR7, is frequently and specifically expressed in tumor-associated vessels. In this study, we examine the role of endothelium-expressed CXCR7 in tumor vascular dysfunction by specifically examining the contribution of CXCR7 to endothelial cell (EC) proliferation. We demonstrate that CXCR7 expression is sufficient to drive post-confluent growth in EC cultures. Further, we provide a novel mechanism for CXCR7-mediated proliferation via proteasomal degradation of the tumor suppressor protein Rb. These findings identify a heretofore unappreciated role for CXCR7 in vascular dysfunction and confirm this receptor as a plausible target for anti-tumor therapy

KSHV infection of endothelial cells manipulates CXCR7-mediated signaling: implications for Kaposi’s Sarcoma progression and intervention

CXCR7 was recently characterized as an alternative receptor for the chemokine CXCL12/SDF-1, previously thought to bind and signal exclusively through CXCR4.We recently identified CXCR7 as a key cellular factor in the endothelial cell (EC) dysfunction associated with KSHV infection. CXCL12 signaling is critically associated with tumor growth, angiogenesis and metastasis in several diverse tumors and is one of the most studied chemokine/chemokine receptor interactions in cancer systems. The tumorigenic activity of the CXCL12 signaling axis offers an attractive target for therapeutic intervention in multiple cancers including Kaposi’s Sarcoma (KS). However, most of the research to date was based on the assumption that CXCR4 was the sole CXCL12 receptor, and thus focused on the development of CXCR4-targeted treatments

IRF-3, IRF-5, and IRF-7 coordinately regulate the type I IFN response in myeloid dendritic cells downstream of MAVS signaling

Although the transcription factors IRF-3 and IRF-7 are considered master regulators of type I interferon (IFN) induction and IFN stimulated gene (ISG) expression, Irf3(-/-)×Irf7(-/-) double knockout (DKO) myeloid dendritic cells (mDC) produce relatively normal levels of IFN-β after viral infection. We generated Irf3(-/-)×Irf5(-/-)×Irf7(-/-) triple knockout (TKO) mice to test whether IRF-5 was the source of the residual induction of IFN-β and ISGs in mDCs. In pathogenesis studies with two unrelated positive-sense RNA viruses (West Nile virus (WNV) and murine norovirus), TKO mice succumbed at rates greater than DKO mice and equal to or approaching those of mice lacking the type I IFN receptor (Ifnar(-/-)). In ex vivo studies, after WNV infection or exposure to Toll-like receptor agonists, TKO mDCs failed to produce IFN-β or express ISGs. In contrast, this response was sustained in TKO macrophages following WNV infection. To define IRF-regulated gene signatures, we performed microarray analysis on WNV-infected mDC from wild type (WT), DKO, TKO, or Ifnar(-/-) mice, as well as from mice lacking the RIG-I like receptor adaptor protein MAVS. Whereas the gene induction pattern in DKO mDC was similar to WT cells, remarkably, almost no ISG induction was detected in TKO or Mavs(-/-) mDC. The relative equivalence of TKO and Mavs(-/-) responses suggested that MAVS dominantly regulates ISG induction in mDC. Moreover, we showed that MAVS-dependent induction of ISGs can occur through an IRF-5-dependent yet IRF-3 and IRF-7-independent pathway. Our results establish IRF-3, -5, and -7 as the key transcription factors responsible for mediating the type I IFN and ISG response in mDC during WNV infection and suggest a novel signaling link between MAVS and IRF-5

CXCR7+ EC are not contact inhibited.

<p>(A) pLEC were infected with Trans only or Trans+CXCR7. At 20 hours post-infection cells were trypsinized, counted and replated onto chamber slides at confluence and allowed to attach for a further 20 hours. Resulting monolayers were fixed and stained for DAPI (blue), CD31 (green), and CXCR7 (red). (B) Nuclei were counted in 10 random fields from 2 independent wells from 2 independent experiments (n = 40, <i>P</i><0.0001).</p

A small molecule inhibitor of CXCR7 prevents overconfluent growth and restores Rb expression.

<p>(A) Confluent pLEC cultures were infected with Trans only or Trans+CXCR7. At 6 hours post-infection, media was replaced with media containing DMSO vehicle control or CCX733 at 10 nM, 50 nM or 100 nM. At 20 hours post-infection, cells were lysed in the presence of Cyquant. Flourescence values are normalized to Trans+DMSO control. n>10 from two independent experiments. **<i>P</i><0.01 (B) pLEC cultures were infected and treated as described for (A). At 20 hours post-infection, cells were lysed and analyzed by western blot for total Rb levels. Densitometry analysis was performed and fold change values were calculated from GAPDH-normalized optical density ratios as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069828#pone-0069828-g003" target="_blank">Figure 3</a>.</p

Supernatant concentrations of selected cytokines in Trans or CXCR7-transduced EC.

<p>Quantities are displayed in pg/ml.</p><p>LOD: Limit of Detection; MAX: maximum concentration of standard curve.</p><p>ANG: angiopoietin; EGF: epidermal growth factor; HGF/SF: hepatocyte growth factor/scatter factor; VEGF: vascular endothelial growth factor.</p

Selected proliferation-related proteins displaying modified expression or phosphorylation in CXCR7+ EC.

<p>Selected proliferation-related proteins displaying modified expression or phosphorylation in CXCR7+ EC.</p

Fold changes in mRNA expression in CXCR7-transduced EC compared to uninfected controls.

<p>Average ± standard error of two experimental replicates.</p