Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Transl Med. Dec 12, 2015; 4(3): 101-112
Published online Dec 12, 2015. doi: 10.5528/wjtm.v4.i3.101
p38 mitogen-activated protein kinase regulates type-I vs type-II phenotyping of human vascular endothelial cells
Masako Nakahara, Miwako Nishio, Koichi Saeki, Akira Yuo, Kumiko Saeki
Masako Nakahara, Miwako Nishio, Akira Yuo, Kumiko Saeki, Department of Disease Control, Research Institute, National Center for Global Health and Medicine, Tokyo 162-8655, Japan
Koichi Saeki, Section of Cell Engineering, Department of Basic Research, DNAVEC Center, ID Pharma Co., Ltd., Ibaraki 300-2611, Japan
Kumiko Saeki, PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan
Author contributions: Nakahara M and Nishio M performed the experiments and analyzed the data; Saeki K constructed viral vectors; Yuo A designed and coordinated the research; Saeki K designed the research, constructed viral vectors and wrote the paper.
Supported by A Grant-in-Aid from the Ministry of Health, Labour and Welfare of Japan, No. KHD1017; and by that from JST, PRESTO.
Institutional review board statement: The study was performed after having received an approval by the Japanese Government and the institutional review board of National Center for Global Health and Medicine.
Conflict-of-interest statement: None of the authors has any potential financial conflict of interest related to this manuscript.
Data sharing statement: Technical appendix and dataset are available from the corresponding author (saeki@ri.ncgm.go.jp).
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Kumiko Saeki, MD, PhD, Division Chief, Department of Disease Control, Research Institute, National Center for Global Health and Medicine, 1-21-1 Toyama Shinjuku-ku, Tokyo 162-8655, Tokyo 162-8655, Japan. saeki@ri.ncgm.go.jp
Telephone: +81-3-32027181 Fax: +81-3-32027364
Received: June 23, 2015
Peer-review started: June 29, 2015
First decision: August 16, 2015
Revised: September 3, 2015
Accepted: December 1, 2015
Article in press: December 2, 2015
Published online: December 12, 2015
Processing time: 175 Days and 0.7 Hours
Abstract

AIM: To identify kinases involved in phenotype regulation of vascular endothelial cells (VECs): Pro-proliferative G-protein signaling 5 (RGS5)high (type-I) vs anti-proliferative RGS5low (type-II) VECs.

METHODS: Proteomic kinase assays were performed to identify the crucial kinase involved in the phenotype regulation of human VECs using type-I VECs, which promotes the proliferation of human vascular smooth muscle cells (VSMCs), and type-II VECs, which suppress the proliferation of human VSMCs. The assays were performed using multiple pairs of type-I and type-II VECs to obtain the least number of candidates. The involvement of the candidate kinases was verified by evaluating the effects of their specific inhibitors on the phenotype regulation of human VECs as well as the expression levels of regulator of RGS5, which is the causative gene for the “type-II to type-I” phenotype conversion of human VECs.

RESULTS: p38α mitogen-activated protein kinase (p38α MAPK) was the only kinase that showed distinctive activities between type-I and type-II VECs: p38α MAPK activities were low and high in type-I and type-II VECs, respectively. We found that an enforced expression of RGS5 indeed lowered p38α MAPK activities in type-II VECs. Furthermore, treatments with a p38α MAPK inhibitor nullified the anti-proliferative potential in type-II VECs. Interestingly, MAPK inhibitor treatments enhanced the induction of RGS5 gene. Thus, there is a vicious cycle between “RGS5 induction” and “p38α MAPK inhibition”, which can explain the unidirectional process in the stress-induced “type-II to type-I” conversions of human VECs. To understand the upstream signaling of RGS5, which is known as an inhibitory molecule against the G protein-coupled receptor (GPCR)-mediated signaling, we examined the effects of RGS5 overexpression on the signaling events from sphingosine-1-phosphate (S1P) to N-cadherin, because S1P receptors belong to the GPCR family gene and N-cadherin, one of their downstream effectors, is reportedly involved in the regulation of VEC-VSMC interactions. We found that RGS5 specifically bound with S1P1. Moreover, N-cadherin localization at intercellular junctions in type-II VECs was abolished by “RGS5 overexpression” and “p38α MAPK inhibition”.

CONCLUSION: p38α MAPK plays crucial roles in “type-I vs type-II” phenotype regulations of human VECs at the downstream of RGS5.

Keywords: Vascular endothelial cells; Vascular smooth muscle cells; Proteomic kinase assay; p38α mitogen-activated protein kinase; Regulator of G-protein signaling 5; Sphingosine-1-phosphate; N-cadherin

Core tip: We previously reported that human vascular endothelial cells (VECs) are categorized into two types by their effects on the proliferation of vascular smooth muscle cells and the expressions of regulator of G-protein signaling 5 (RGS5): Pro-proliferative RGS5high (type-I) and anti-proliferative RGS5low (type-II) VECs. Performing proteomic kinase assays and inhibitor studies, we show here that p38 mitogen-activated protein kinase (p38 MAPK) is the crucial kinase that determines VEC phenotyping at the downstream of RGS5. Not only RGS5 overexpression suppressed p38 MAPK activities but also p38 MAPK inhibitions up-regulated RGS5 expression, indicating that “RGS5 induction” and “p38 MAPK inhibition” creates a vicious cycle in “type-II to type-I” conversions of human VECs.