Regulation of Vascular Endothelial Cell Polarization and Migration by Hsp70/Hsp90-Organizing Protein Jingyu Li1., Xiaodong Sun1., Zaizhu Wang1, Li Chen1, Dengwen Li1, Jun Zhou 1 , Min Liu2* 1 State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, China, 2 Key Laboratory of Immune Microenvironment and Disease of the Ministry of Education, Basic Medical College, Tianjin Medical University, Tianjin, China Abstract Hsp70/Hsp90-organizing protein (HOP) is a member of the co-chaperone family, which directly binds to chaperones to regulate their activities. The participation of HOP in cell motility and endothelial cell functions remains largely unknown. In this study, we demonstrate that HOP is critically involved in endothelial cell migration and angiogenesis. Tube formation and capillary sprouting experiments reveal that depletion of HOP expression significantly inhibits vessel formation from endothelial cells. Wound healing and transwell migration assays show that HOP is important for endothelial cell migration. By examination of centrosome reorientation and membrane ruffle dynamics, we find that HOP plays a crucial role in the establishment of cell polarity in response to migratory stimulus. Furthermore, our data show that HOP interacts with tubulin and colocalizes with microtubules in endothelial cells. These findings indicate HOP as a novel regulator of angiogenesis that functions through promoting vascular endothelial cell polarization and migration. Citation: Li J, Sun X, Wang Z, Chen L, Li D, et al. (2012) Regulation of Vascular Endothelial Cell Polarization and Migration by Hsp70/Hsp90-Organizing Protein. PLoS ONE 7(4): e36389. doi:10.1371/journal.pone.0036389 Editor: Paolo Madeddu, Bristol Heart Institute, University of Bristol, United Kingdom Received January 4, 2012; Accepted March 31, 2012; Published April 30, 2012 Copyright: 2012 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Basic Research Program of China (2012CB945002 and 2010CB912204) and the National Natural Science Foundation of China (30825022). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: minliu@tijmu.edu.cn . These authors contributed equally to this work. Introduction Hsp70/Hsp90-organizing protein (HOP) is a co-chaperone protein that could directly bind to the major chaperones, Hsp70 and Hsp90, and modulate their ATPase activities [1–4]. HOP also belongs to the stress-inducible protein 1 (STI1) family, which contains various vertebrate and invertebrate homologs of HOP [5]. Human HOP has three tetratricopeptide repeat (TPR) domains, including TPR1, TPR2a, and TPR2b [6]. TPR domains are responsible for the interaction of HOP with Hsp70/Hsp90, with TPR1 for HOP binding to Hsp70 and TPR2a for HOP binding to Hsp90 [7,8]. However, no specific ligands for the TPR2b domain have been found. HOP also has non-TPR domains, including a DP repeat region and two nuclear localization signals, which render HOP to dynamically translocate between the cytoplasm and the nucleus [9,10]. In addition, HOP has been found in a number of Hsp90-independent complexes, including prion protein complex and nuclear transcription complex [11–14]. Hsp70 and Hsp90 have been shown to be overexpressed in several tumor types, and inhibition of Hsp70/Hsp90 has been reported to induce tumor cell-specific apoptosis [15]. However, whether HOP is involved in cancer development remains unclear. In addition, it is unknown whether HOP plays a role in angiogenesis,a process that refers to the generation of new blood vessels from pre-existing ones and relies largely on vascular endothelial cell proliferation and migration [16]. The angiogenic process is also critical for tumor growth, progression, and metastasis [17,18], and anti-angiogenesis strategy has been proven useful for cancer treatment [19–22]. Given that HOP harbors multiple structural motifs and interacts with various proteins, we hypothesized that HOP might participate in cell motility and endothelial cell functions. This study was designed to test this hypothesis directly, and our data demonstrate that HOP is important for angiogenesis through modulating endothelial cell polarization and migration. Materials and Methods Materials Antibodies against b-actin, a-tubulin, c-tubulin, and GST were purchased from Sigma-Aldrich. Antibodies against GFP, RhoA, Cdc42, and Rac1 were from Cell Signaling. Antibodies against HOP, Akt, and HDAC6 were from Santa Cruz Biotechnology. Anti-GFP antibody-conjugated agarose beads (MBL Internation- al), glutathione-conjugated agarose beads (Sigma-Aldrich), horse- radish peroxidase-conjugated secondary antibodies (Amersham Biosciences), and fluorescein- or rhodamine-conjugated secondary antibodies (Jackson Immuno Research Laboratories) were ob- tained from the indicated sources. Matrigel and mouse collagen IV were from BD Biosciences. Plasmids Mammalian expression plasmids of GFP-HOP and GFP-tagged mutant forms of HOP were constructed using the pEGFPC1 vector. Bacterial expression plasmid of GST-HOP was cloned using the pGEX6P3 vector, and GST-HOP fusion protein was PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e36389