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AKAP12 Regulates Human Blood–Retinal Barrier Formation by Downregulation of Hypoxia-Inducible Factor-1Α

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AKAP12 Regulates Human Blood–Retinal Barrier Formation by Downregulation of Hypoxia-Inducible Factor-1Α 4472 • The Journal of Neuroscience, April 18, 2007 • 27(16):4472–4481 Cellular/Molecular AKAP12 Regulates Human Blood–Retinal Barrier Formation by Downregulation of Hypoxia-Inducible Factor-1␣ Yoon Kyung Choi,1* Jeong Hun Kim,2* Woo Jean Kim,4* Hae Young Lee,1 Jeong Ae Park,5 Sae-Won Lee,3 Dae-Kwan Yoon,1 Hyun Ho Kim,1 Hum Chung,2 Young Suk Yu,2 and Kyu-Won Kim1 1NeuroVascular Coordination Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Korea, 2Department of Ophthalmology, Seoul National University College of Medicine and Seoul Artificial Eye Center, 3Clinical Research Institute, Seoul National University Hospital, Seoul 110-744, Korea, 4Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02115, and 5Department of Marine Biotechnology, College of Liberal Arts and Sciences, Anyang University, Incheon 417-833, Korea Many diseases of the eye such as retinoblastoma, diabetic retinopathy, and retinopathy of prematurity are associated with blood–retinal barrier (BRB) dysfunction. Identifying the factors that contribute to BRB formation during human eye development and maintenance could provide insights into such diseases. Here we show that A-kinase anchor protein 12 (AKAP12) induces BRB formation by increasing angiopoietin-1 and decreasing vascular endothelial growth factor (VEGF) levels in astrocytes. We reveal that AKAP12 downregulates the level of hypoxia-inducible factor-1␣ (HIF-1␣) protein by enhancing the interaction of HIF-1␣ with pVHL (von Hippel-Lindau tumor suppressor protein) and PHD2 (prolyl hydroxylase 2). Conditioned media from AKAP12-overexpressing astrocytes induced barriergen- esis by upregulating the expression of tight junction proteins in human retina microvascular endothelial cells (HRMECs). Compared with the retina during BRB maturation, AKAP12 expression in retinoblastoma patient tissue was markedly reduced whereas that of VEGF was increased. These findings suggest that AKAP12 may induce BRB formation through antiangiogenesis and barriergenesis in the develop- ing human eye and that defects in this mechanism can lead to a loss of tight junction proteins and contribute to the development of retinal pathologies such as retinoblastoma. Key words: A-kinase anchor protein 12 (AKAP12); barriergenesis; blood–retinal barrier (BRB); hypoxia-inducible factor-1 ␣; retinoblas- toma; retina Introduction intermediate filaments (Kivela et al., 1986), leaky vessels, and The blood–retinal barrier (BRB) is a selective barrier of the eye high levels of vascular endothelial growth factor (VEGF). The composed of well differentiated microvessels. It plays an essential expression of VEGF is mainly regulated by hypoxia-inducible role in protecting neural tissues from toxic materials and in main- factor-1␣ (HIF-1␣) (Ikeda et al., 1995; Ema et al., 1997; Maxwell taining vision and neural function in the retina. The inner mature et al., 1997), which is the most important transcriptional factor in BRB is a complex system composed of highly specialized micro- hypoxia-induced angiogenesis (Semenza, 2003). vascular endothelial cells embedded in a basement membrane HIF-1 is composed of two subunits, an oxygen-sensitive and surrounded by astrocytic end feet. In many in vitro models, HIF-1␣ subunit and oxygen-insensitive HIF-1␤ subunit (Wang astrocytes can induce barrier-like features when cocultured with et al., 1995). HIF-1␣ is stable under hypoxia, whereas it is unsta- cerebral brain endothelial cells (Stanness et al., 1996; Lee et al., ble under normoxia. In normoxic conditions, the HIF-1␣ sub- 2003). A typical BRB-defect eye disease, retinoblastoma, is the unit is rapidly degraded via the von Hippel-Lindau tumor sup- most common intraocular malignant tumor in children. Retino- pressor protein (pVHL)-mediated ubiquitin-proteasome blastoma arises in the retina and is characterized by a lack of pathway (Salceda and Caro, 1997; Maxwell et al., 1999). The association of pVHL and HIF-1␣ is triggered by prolyl hydroxy- Received Dec. 13, 2006; revised March 6, 2007; accepted March 12, 2007. lation within a polypeptide segment known as the oxygen- This work was supported by the Creative Research Initiatives (NeuroVascular Coordination Research Center) of dependent degradation domain through the function of specific the Ministry of Science and Technology. S.-W.L. is the recipient of a grant from the Korea Health 21 R&D Project, HIF-prolyl hydroxylases (PHDs) (Ivan et al., 2001; Jaakkola et al., Ministry of Health and Welfare (A062260). We thank Dr. G. Y. Koh (Korea Advanced Institute of Science and Tech- 2001). In mammalian cells, three isoforms, PHD1, PHD2, and nology,Daejeon,Korea)forprovidingCOMP-Ang1.WealsothankDr.N.JoanAbbott(WolfsonCentreforAgeRelated Diseases, King’s College, London, UK) for critical reading of this manuscript. We declare that we have no competing PHD3, have been identified and shown to hydroxylate in vitro the financial interests. key proline residues of HIF-1␣ (Epstein et al., 2001). *Y.K.C., J.H.K., and W.J.K. contributed equally to this work. A-kinase anchor protein 12 (AKAP12) is a putative tumor CorrespondenceshouldbeaddressedtoDr.Kyu-WonKim,NeuroVascularCoordinationResearchCenter,College suppressor that associates with protein kinase A (PKA) and pro- of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.5368-06.2007 tein kinase C (PKC) and serves as a scaffolding protein in signal Copyright © 2007 Society for Neuroscience 0270-6474/07/274472-10$15.00/0 transduction (Gelman et al., 2000; Gelman, 2002). AKAP12 ex- Choi et al. • Barrier Formation by AKAP12 J. Neurosci., April 18, 2007 • 27(16):4472–4481 • 4473 agnosed with retinoblastoma were obtained from the Seoul National University Children’s Hospital. After nucleation, the eyeballs were fixed by immersion in Carnoy’s solution for 2 h at room temperature. Each specimen was then dehydrated through a series of graded ethanol solutions and embedded in paraffin using stan- dard techniques. Paraffin-embedded eyes were sectioned into 4 ␮m sections, and sections were mounted on slides coated with 0.5% Elmer’s glue for immunohistochemistry. Immunohis- tochemistry was performed using an Inno- Genex (San Ramon, CA) immunohistochemis- try kit. For quantitative analysis of immunostaining, Image-Pro Plus (Media Cybernetics, Bethesda, MD) was used. Immunofluorescence staining. Cryosections were incubated with the indicated primary an- tibodies, followed by FITC-conjugated IgG and tetramethylrhodamine isothiocyanate- conjugated IgG (Jackson ImmunoResearch, West Grove, PA) as secondary antibodies. Flu- orescence immunostaining was evaluated us- ing a confocal microscope (Bio-Rad, Hercules, CA). Human astrocyte cells were incubated overnight at 4°C with the indicated primary antibodies, followed by incubation with Alexa Fluor antibodies. Nuclei were stained using propidium iodide (Invitrogen, San Diego, CA). Images were obtained with an Axiovert M200 microscope (Zeiss, Oberkochen, Germany) and analyzed using Image-Pro Plus (Media Cybernetics). Cell culture. Primary human brain astrocyte cells and dissociated normal human brain cor- tex tissue were purchased from the Applied Cell Figure 1. Immunohistochemical analysis of AKAP12, vWF, GFAP, Ang1, VEGF, and tight junction proteins in the developing Biology Research Institute (Kirkland, WA). human retina. Human fetal retinal sections were immunostained with antibodies to AKAP12, vWF, GFAP, Ang1, claudin-1, occlu- The CTX-TNA2 cell line, an established astro- din, and VEGF (red). Nuclei were counterstained with hematoxylin and are seen in blue. The negative control was immunostained cytic cell line from the neonatal Sprague Daw- with normal rabbit IgG. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 50 ␮m. ley rat cerebral cortex, was purchased from the American Type Culture Collection (Manassas, VA). Primary human brain astrocyte cells, pression is strongly suppressed in a variety of cancers (Xia et al., CTX-TNA2 cells, 786-O renal clear cell carcinoma cells, and HT1080 2001; Liu et al., 2006). Src-suppressed C-kinase substrate cells were cultured in DMEM supplemented with 10% FBS (Invitrogen) (SSeCKS), the rodent ortholog of human AKAP12, is important and antibiotics. Human retina microvascular endothelial cells for mouse brain homeostasis and regulating the formation of the (HRMECs) were purchased from the Applied Cell Biology Research In- blood–brain barrier (Lee et al., 2003). A recent report showed stitute and grown on attachment factor-coated plates in complete me- that SSeCKS/AKAP12 attenuates expression of angiogenic genes dium (Cell Systems, Kirkland, WA) or in M199 medium supplemented with 20% FBS, 3 ng/ml basic FGF (Millipore, Bedford, MA), and 10 U/ml by suppressing Src-induced oncogenesis (Liu et al., 2006). How- ␣ ever, it is not clear whether AKAP12 participates in antiangiogen- heparin (Sigma, St. Louis, MO). For the analysis of HIF-1 stability in 786-O renal clear cell carcinoma cells, cells were transfected with a plas- esis and barriergenesis in the human retina. Moreover, the mech- mid encoding myc-pVHL and selected using G418 (Invitrogen; or BRL, anism of barrier failure in the human eye is not well understood. Bethesda, MD). For hypoxia experiments, astrocytes were incubated in a In this study, we identified AKAP12
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