Matrix Metalloproteinase-2 and -9 Secreted by Leukemic Cells Increase the Permeability of Blood-Brain Barrier by Disrupting Tight Junction Proteins Saran Feng1,3, Jiannong Cen1, Yihong Huang 2 , Hongjie Shen1, Li Yao1, Yuanyuan Wang1, Zixing Chen1* 1 Leukemia Research Unit, Jiangsu Institute of Hematology, 1st Affiliated Hospital, Soochow University, Key Laboratory of Thrombosis and Hemostasis Ministry of Health, Suzhou, China, 2 Department of Hematology, Affiliated Hospital, Xuzhou Medical College, Xuzhou, China, 3 Department of Hematology, Xuzhou Central Hospital, Xuzhou, China Abstract Central nervous system (CNS) involvement remains an important cause of morbidity and mortality in acute leukemia, the mechanisms of leukemic cell infiltration into the CNS have not yet been elucidated. The blood-brain barrier (BBB) makes CNS become a refugee to leukemic cells and serves as a resource of cells that seed extraneural sites. How can the leukemic cells disrupt this barrier and invasive the CNS, even if many of the currently available chemotherapies can not cross the BBB? Tight junction in endothelial cells occupies a central role in the function of the BBB. Except the well known role of degrading extracellular matrix in metastasis of cancer cells, here we show matrix metalloproteinase (MMP)-2 and -9, secreted by leukemic cells, mediate the BBB opening by disrupting tight junction proteins in the CNS leukemia. We demonstrated that leukemic cells impaired tight junction proteins ZO-1, claudin-5 and occludin resulting in increased permeability of the BBB. However, these alterations reduced when MMP-2 and -9 activities were inhibited by RNA interference strategy or by MMP inhibitor GM6001 in an in vitro BBB model. We also found that the disruption of the BBB in company with the down- regulation of ZO-1, claudin-5 and occludin and the up-regulation of MMP-2 and -9 in mouse brain tissues with leukemic cell infiltration by confocal imaging and the assay of in situ gelatin zymography. Besides, GM6001 protected all mice against CNS leukemia. Our findings suggest that the degradation of tight junction proteins ZO-1, claudin-5 and occludin by MMP-2 and -9 secreted by leukemic cells constitutes an important mechanism in the BBB breakdown which contributes to the invasion of leukemic cells to the CNS in acute leukemia. Citation: Feng S, Cen J, Huang Y, Shen H, Yao L, et al. (2011) Matrix Metalloproteinase-2 and -9 Secreted by Leukemic Cells Increase the Permeability of Blood- Brain Barrier by Disrupting Tight Junction Proteins. PLoS ONE 6(8): e20599. doi:10.1371/journal.pone.0020599 Editor: Hava Karsenty Avraham, Beth Israel Deaconess Medical Center, United States of America Received January 10, 2011; Accepted May 5, 2011; Published August 17, 2011 Copyright: 2011 Feng 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 has been supported by a grant (30670905) from The National Natural Scientific Foundation of China, http://www.nsfc.gov.cn/Portal0/ default106.htm. 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: szchenzx@263.net Introduction The central nervous system (CNS) is one of the most frequent extramedullary locations in acute leukemia (AL). The prognosis of patients with isolated or mixed CNS relapse is particularly poor. Despite of all kinds of morden therapies (systemic chemotherapy and cranial irradiation) it has become a major obstacle to curing acute leukemia [1–5]. Despite its clinical importance, how leukemic cells enter the CNS is poorly understood. The characteristic structure of the blood-brain barrier (BBB) causes the CNS to serve as a resource of cells that seed extraneural sites. But much needs to be learned about how can the leukemic cells disrupt this barrier and invasive the CNS, even if many of the currently available chemotherapies cannot cross the BBB. Moreover, little is known about cellular response of cerebral endothelial cells during invasion of leukemic cell to the CNS. It is known that the BBB breakdown is associated with brain tumors and diseases of the central nervous system. Although the disruption of the extracellular matrix (ECM) can lead to increased permeability of the BBB in pathological states [6], it is primarily the tight junction (TJ) between adjacent brain microvascular endothelial cells (BMVECs) that confers low paracellular perme- ability and high electrical resistance, making the barrier to function 50,100 times tighter than peripheral microvessels [7– 9]. TJs are composed of transmembrane molecules (claudin, occludin and junctional adhesion molecules) linked to the actin cytoskeleton through cytoplasmic accessory proteins, including zonula occludens (ZO)-1, ZO-2 and ZO-3 [10,11]. Claudin-5 and occludin were identified as key components of BBB integrity which are localized at the leading edge of BMVECs. ZO-1 acts as a crucial central regulator of the structural organization of the TJ at the plasma membrane [11]. While the barrier and fence functions of TJ have been well understood, it is only relatively recently that the association between TJ and metastasis of cancer cells has been recognized. It is becoming increasingly clear that the TJs have a vital role in maintaining cell to cell integrity and that the loss of cohesion of the structure can lead to invasion and thus metastasis of cancer cells [12,13]. Some members of the matrix metalloproteinase (MMP) family like MMP-2 and -9 are known to be associated with tumor growth and metastasis because of their capacity to degrade collagen IV, the major ECM component [14]. Earlier reports show an association of MMP-2/-9 expression with invasive behaviour of leukemic cells in acute lymphoblastic leukemia (ALL) and acute PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e20599