Hypoxia-Induced Matrix Metalloproteinase-13 Expression in Astrocytes Enhances Permeability of Brain Endothelial Cells DAH-YUU LU, 1,2 WEI-HSUAN YU, 3 WEI-LAN YEH, 2 CHIH-HSIN TANG, 4 YUK-MAN LEUNG, 1,5 KAR-LOK WONG, 6 YUH-FUNG CHEN, 4,7 CHIH-HO LAI, 8 AND WEN-MEI FU 2 * 1 Graduate Institute of Neural and Cognitive Sciences, China Medical University, Taichung, Taiwan 2 Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan 3 Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan 4 Department of Pharmacology, China Medical University, Taichung, Taiwan 5 Department of Physiology, China Medical University, Taichung, Taiwan 6 Department of Anesthesiology, China Medical University and Hospital, Taichung, Taiwan 7 Graduate Institute of Chinese Pharmaceutical Sciences, College of Pharmacy, China Medical University, Taichung, Taiwan 8 Department of Microbiology, College of Medicine, China Medical University, Taichung, Taiwan Matrix metalloproteinase-13 (MMP-13) is involved in the degradation of extracellular matrix in many kinds of tissues. Here we found that hypoxia increased MMP-13 protein and mRNA levels in primary rat astrocyte cultures. Hypoxia stimulation also increased the secretion of MMP-13 from astrocytes, as shown by zymographic analysis. In addition, exposure to hypoxia up-regulated the expression of c-Fos and c-Jun time-dependently. Hypoxia-induced MMP-13 overexpression was antagonized by transfection with antisense oligodeoxynucleotides (AS-ODN) of c-Fos or c-Jun. Furthermore, hypoxic-conditioned medium (Hx-CM) collected from astrocytes exposed to hypoxia increased paracellular permeability of adult rat brain endothelial cells (ARBECs). Administration of MMP-13 neutralizing antibody antagonized Hx-CM-induced paracellular permeability of ARBECs. Furthermore, pre-transfection of astrocytes with AS-ODN of c-Fos, c-Jun or MMP-13-shRNA significantly decreased hyperpermeability of ARBECs induced by Hx-CM. The arrangement of tight junction protein (TJP) zonular occludens-1 (ZO-1) of ARBECs disorganized in response to Hx-CM. Administration of Hx-CM to ARBECs also resulted in the production of proteolytic fragments of ZO-1, which was antagonized by transfection of MMP-13-shRNA in primary astrocytes. Administration of MMP-13 recombinant protein to ARBECs led to the disorganization and fragmentation of ZO-1 protein and also increased paracellular permeability. These results suggest that hypoxia-induced MMP-13 expression in astrocytes is regulated by c-Fos and c-Jun. MMP-13 is an important factor leading to the disorganization of ZO-1 and hyperpermeablility of blood–brain barrier in response to hypoxia. J. Cell. Physiol. 220: 163–173, 2009. ß 2009 Wiley-Liss, Inc. Blood–brain barrier (BBB) integrity protects the neuronal microenvironment (Cserr and Bundgaard, 1986). When this integrity is lost, inflammatory cells and fluid penetrate the brain, causing edema and cell death (Fishman, 1975). Tight junction proteins in endothelial cells are a major structural component of BBB formed by components of the neurovascular unit (Huber et al., 2001). MMPs are involved in the remodeling of the extracellular matrix (ECM) in a variety of physiological and pathological conditions. Encountered with ischemia/hypoxia, TJPs in endothelial cell cultures or in situ models of BBB are disrupted. Proteases of the serine and MMP gene families have been shown to degrade extracellular matrix molecules and proteins of the basal lamina around the cerebral blood vessels (Wagner et al., 1997; Heo et al., 1999). Among MMPs, both MMP-2 and MMP-9, also known as type IV collagenases, are able to digest the endothelial basal lamina and lead to the opening of the BBB (Rosenberg et al., 1992, 1996a,b, 1998). Expression of MMP-2 and MMP-9 has also been shown to be significantly increased during stroke in human (Anthony et al., 1997; Romanic et al., 1998; Montaner et al., 2001) and in rat models of focal ischemia (Rosenberg et al., 1996a, 1998; Romanic et al., 1998). Abbreviations: AP-1, activating-protein-1; ARBECs, adult rat brain endothelial cell culture; AS-ODN, antisense oligodeoxynucleotides; BBB, blood–brain barrier; CNS, central nervous system; DMEM, Dulbecco’s modified Eagle’s medium; DWI, diffusion- weighted image; ECM, extracellular matrix; FBS, fetal bovine serum; Hx-CM, hypoxia-conditioned medium; LF2000, Lipofectamine 2000; MMP-13, matrix metalloproteinase-13; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; rMMP-13, MMP-13 recombinant protein; TJP, tight junction protein; ZO-1, zonular occludens-1. Additional supporting information may be found in the online version of this article. Contract grant sponsor: National Science Council of Taiwan; Contract grant number: 97-2314-B-039-039. Contract grant sponsor: China Medical University; Contract grant number: CMU97-229. *Correspondence to: Wen-Mei Fu, Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan. E-mail: wenmei@ntu.edu.tw Received 11 September 2008; Accepted 27 January 2009 Published online in Wiley InterScience (www.interscience.wiley.com.), 24 February 2009. DOI: 10.1002/jcp.21746 ORIGINAL ARTICLE 163 Journal of Journal of Cellular Physiology Cellular Physiology ß 2009 WILEY-LISS, INC.