Lack of Bcr and Abr Promotes Hypoxia-Induced Pulmonary Hypertension in Mice Min Yu1.¤, Dapeng Gong1., Min Lim2, Anna Arutyunyan1, John Groffen1,2, Nora Heisterkamp1,2* 1 Section of Molecular Carcinogenesis, Division of Hematology/Oncology, and The Saban Research Institute of Children’s Hospital, Los Angeles, California, United States of America, 2 Departments of Pediatrics and Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America Abstract Background: Bcr and Abr are GTPase activating proteins that specifically downregulate activity of the small GTPase Rac in restricted cell types in vivo. Rac1 is expressed in smooth muscle cells, a critical cell type involved in the pathogenesis of pulmonary hypertension. The molecular mechanisms that underlie hypoxia-associated pulmonary hypertension are not well-defined. Methodology/Principal Findings: Bcr and abr mutant mice were compared to wild type controls for the development of pulmonary hypertension after exposure to hypoxia. Also, pulmonary arterial smooth muscle cells from those mice were cultured in hypoxia and examined for proliferation, p38 activation and IL-6 production. Mice lacking Bcr or Abr exposed to hypoxia developed increased right ventricular pressure, hypertrophy and pulmonary vascular remodeling. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxia bcr2/2 and abr2/2 macrophages generated more reactive oxygen species. Consistent with a contribution of inflammation and oxidative stress in pulmonary hypertension- associated vascular damage, Bcr and Abr-deficient animals showed elevated endothelial leakage after hypoxia exposure. Hypoxia-treated pulmonary arterial smooth muscle cells from Bcr- or Abr-deficient mice also proliferated faster than those of wild type mice. Moreover, activated Rac1, phosphorylated p38 and interleukin 6 were increased in these cells in the absence of Bcr or Abr. Inhibition of Rac1 activation with Z62954982, a novel Rac inhibitor, decreased proliferation, p38 phosphorylation and IL-6 levels in pulmonary arterial smooth muscle cells exposed to hypoxia. Conclusions: Bcr and Abr play a critical role in down-regulating hypoxia-induced pulmonary hypertension by deactivating Rac1 and, through this, reducing both oxidative stress generated by leukocytes as well as p38 phosphorylation, IL-6 production and proliferation of pulmonary arterial smooth muscle cells. Citation: Yu M, Gong D, Lim M, Arutyunyan A, Groffen J, et al. (2012) Lack of Bcr and Abr Promotes Hypoxia-Induced Pulmonary Hypertension in Mice. PLoS ONE 7(11): e49756. doi:10.1371/journal.pone.0049756 Editor: James West, Vanderbilt University Medical Center, United States of America Received December 19, 2011; Accepted October 16, 2012; Published November 12, 2012 Copyright: 2012 Yu 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 a Children’s Hospital Los Angeles Research Career Development award to MY and the National Institutes of Health (HL071945) to JG. 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: heisterk@hsc.usc.edu . These authors contributed equally to this work. ¤ Current address: Department of Anesthesiology, Jiangsu Province Hospital, Nanjing Medical University, Nanjing, China Introduction BCR (breakpoint cluster region) was originally identified as a gene involved in the development of Ph-chromosome-positive leukemias [1]. The normal Bcr protein of around 160-kDa contains multiple domains. One of these has an enzymatic function that is shared with Abr, a related protein encoded by a separate gene. The function of this domain has been relatively well characterized in vivo. It interacts with the small GTPase Rac when Rac is in its active GTP-bound conformation [2,3]. The interaction of Bcr and Abr with RacGTP results in the conversion of bound GTP to GDP and the inactivation of Rac. Thus Bcr and Abr belong to the class of GTPase activating proteins (GAPs), of which around 70 have been identified for Rho family members [4]. Although many of these GAPs are expressed in a cell-specific or developmentally-restricted fashion, both Abr and Bcr are expressed in many cell types in an overlapping pattern. There are only three Rac proteins, that regulate a diversity of biological functions. The expression of Rac2 and Rac3 is relatively restricted, whereas that of Rac1 is ubiquitous. Specificity of Rac function is regulated through a tightly controlled cycle of activation and deactivation that is mediated by upstream activators, the guanine nucleotide exchange factors (GEFs), and through deactivation by the GAPs [5,6]. Mice lacking Bcr or Abr are phenotypically normal and fertile. However, when they are examined in more detail under conditions that generate pathology, significant differences with control wild type (WT) animals can be measured in specific functions. We found that ablation of Bcr and Abr results in abnormal reactivity of the innate immune system, and mice without both Abr and Bcr function also exhibit neuronal defects and inner ear developmental abnormalities [7–11]. Rac1 is also expressed in smooth muscle cells, which contribute to the pathology of pulmonary hypertension (PH), a life- PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49756