Nuclear localization of platelet-activating factor receptor controls retinal neovascularization.

Vikrant K Bhosle,Isabelle Lahaie,David Hamel,Samy Omri,Sylvain Chemtob,José Carlos Rivera,Tang Zhu,Areej Al Rabea,Raphael Rouget,Melanie Sanchez,Alfredo Ribeiro-Da-Silva,Xin Hou,Tianwei Zhou

doi:10.1038/celldisc.2016.17

Abstract

Platelet-activating factor (PAF) is a pleiotropic phospholipid with proinflammatory, procoagulant and angiogenic actions on the vasculature. We and others have reported the presence of PAF receptor (Ptafr) at intracellular sites such as the nucleus. However, mechanisms of localization and physiologic functions of intracellular Ptafr remain poorly understood. We hereby identify the importance of C-terminal motif of the receptor and uncover novel roles of Rab11a GTPase and importin-5 in nuclear translocation of Ptafr in primary human retinal microvascular endothelial cells. Nuclear localization of Ptafr is independent of exogenous PAF stimulation as well as intracellular PAF biosynthesis. Moreover, nuclear Ptafr is responsible for the upregulation of unique set of growth factors, including vascular endothelial growth factor, in vitro and ex vivo. We further corroborate the intracrine PAF signaling, resulting in angiogenesis in vivo, using Ptafr antagonists with distinct plasma membrane permeability. Collectively, our findings show that nuclear Ptafr translocates in an agonist-independent manner, and distinctive functions of Ptafr based on its cellular localization point to another dimension needed for pharmacologic selectivity of drugs.

Highlights

PAF, a proinflammatory phospholipid, was first discovered in 1960s for its role in platelet aggregation and release of histamine by the activated platelets [1]
The specificities of primary anti-platelet-activating factor receptor (Ptafr) and secondary nanogold antibodies were confirmed in endogenous human retinal microvascular endothelial cells (ECs) as well as in transfected human embryonic kidney 293T (HEK293T) cells by comparable staining for second antibody against c-terminal myc tag-labeled PTAFR; native HEK cells lack endogenous PTAFR (Figure 1a and Supplementary Figure S1A)
transmission electron microscopy (TEM) revealed the presence of nuclear PTAFR in human retinal microvascular ECs (hRMECs) (Figure 1a), which was confirmed on immunoreactivity of isolated nuclei by subcellular fractionation (Figure 1b), consistent with previous reports in porcine neuromicrovascular ECs [26]

Summary

Introduction

PAF (chemical name - 1-O-alkyl-2-acetyl-sn-glycerol3-phosphocholine), a proinflammatory phospholipid, was first discovered in 1960s for its role in platelet aggregation and release of histamine by the activated platelets [1]. The current evidence suggests that members of Ras superfamily of small GTPases, especially those in Rab and Arf families, are involved in the regulation of various stages of the vesicular transport as well as in the process of membrane fusion [27, 28] Another conserved eukaryotic protein family, importin (part of karyopherin superfamily), has been recently proposed to play a role in nuclear translocation of GPCRs based on the evidence from RNA interference studies [22, 23]. Importins recognize their cargo by the presence of a nuclear localization signal (NLS) [29] and many GPCRs, including Ptafr, contain a putative NLS [30]. We hypothesized that nuclear translocation of Ptafr in vascular ECs is governed by specific small GTPase and importin interaction

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Results

Conclusion