The guanine exchange factor SWAP70 mediates vGPCR-induced endothelial plasticity

Abstract

Background: The viral G protein-coupled receptor (vGPCR) is proposed to act as one of the predominant mediators of Kaposi's sarcoma (KS), a human herpes virus 8 (HHV8)-elicited disease. The actions of vGPCR manifest pathogenesis, in part, through increased permeability of endothelial cells. Endothelial cell-cell junctions have indeed emerged as an instrumental target involved in the vasculature defects observed within the tumor microenvironment. The pathway leading to adherens junction destabilization has been shown to involve the activation of the small GTPase Rac, in the context of either latent infection or the sole expression of vGPCR. However, the precise molecular mechanisms governed by vGPCR in vascular leakage require further elucidation.

Findings: Guanine exchange factors (GEFs) function as critical molecular switches that control the activation of small GTPases. We therefore screened the effects of 80 siRNAs targeting GEFs on vGPCR-driven endothelial permeability and identified switch-associated protein 70 (SWAP70) as necessary for its elevating effects. Pull-down experiments further showed that Rac activation by vGPCR was dependent on SWAP70. Examination of tissues and cells from HHV8-positive patients revealed that SWAP70 was ubiquitously expressed. Furthermore, SWAP70 was found to be crucial for vGPCR-driven endothelial tube formation and endothelial sprouting in vitro.

Conclusions: SWAP70 appears to act as a molecular intermediate between vGPCR and endothelial activation. Because of the important role of vGPCR-mediated endothelial plasticity in KS pathogenesis, inhibition of SWAP70 function could be of interest for blocking vGPCR-driven activities in HHV8-defined diseases.

Figure 1

A functional siRNA library screen identifies 6 putative GEF downstream of vGPCR. A. Schematic of GEF siRNA permeability screen. B. The fluorescence intensity was determined for each targeting sequence (2 sequences/target) and expressed as means of the ratio to control siRNA-transfected cells (4 non-silencing control sequences). Shared hits between vGPCR and IL-8 screens are highlighted in red. C. A two-step screen was performed; the first examined vGPCR-driven permeability. Hits from that screen were then tested for involvement in IL-8-driven permeability. The six GEFs that significantly decreased permeability when silenced are indicated.

Figure 2

SWAP70 modulates vGPCR- and IL-8-mediated permeability through Rac activation. A. Permeability of SVEC (parental) or SVEC stably expressing vGPCR (vGPCR) that were transfected with non-silencing siRNA (non^si) or siRNA against SWAP70 (S70^si). B. Activated Rac was detected by Rac pulldown in cells with (+) and without (−) vGPCR expression following transfection with non^si or S70^si. C. Permeability of non-stimulated SVEC (control) or those stimulated with either VEGF or IL-8 was determined after transfection S70^si or non^si. D. Activated Rac was detected by Rac pulldown in cells with (+) and without (−) IL-8 stimulation following transfection with non^si or S70^si. Data are representative of three independent experiments. ***p < 0.001; **p < 0.01; ns, non significant by analysis of variance (ANOVA).

Figure 3

SWAP70 is expressed in HHV8-infected cells and tissues. A. Semi-quantitative PCR was used to analyze expression of vGPCR and SWAP70 in patient-derived non-KS (Ctrl) and KS skin lesions (KS #1 to #4), as well as in latently infected HHV8 cell lines (BC3, BC1, BCBL1, CROAP6), and patient-isolated KS-positive (KS-B cell) and –negative (Ctrl-B cell) peripheral B cells. GAPDH mRNA levels were detected as a control for input and equal loading. B. Immunohistochemical staining of hematoxylin/eosin (H&E) coloration, LNA, and SWAP70 in tissue sections from KS-positive lesions (KS #5 and KS #6) and a KS-negative lesion (angioma).

Figure 4

SWAP70 is required to convey vGPCR pro-angiogenic actions in endothelial cells. A-D. Parental (Par) and vGPCR-expressing SVEC (−) were stably transfected with control (shC) or two shRNAs against SWAP70 (sh1 and sh3) plasmids. A. Cells were processed for semi-quantitative RT-PCR to determine SWAP70 mRNA expression. Actin mRNA levels were detected as a control for input and equal loading. B. Permeability assays were performed as described in methods. C-D. SVEC cells were also tested for tube formation capacity (C) and sprouting ability (D). Representative images are shown. Tube length and number of branch points per field of view (FOV) were quantified in C. Number of sprouting cells and mean sprout length were determined in D. ***p < 0.001, **p < 0.01 by analysis of variance (ANOVA).