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Impaired Caveolae Function and Upregulation of Alternative Endocytic Pathways Induced by Experimental Modulation of Intersectin-1s Expression in Mouse Lung Endothelium

DOI: 10.1155/2012/672705

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Intersectin-1s (ITSN-1s), a protein containing five SH3 (A-E) domains, regulates via the SH3A the function of dynamin-2 (dyn2) at the endocytic site. ITSN-1s expression was modulated in mouse lung endothelium by liposome delivery of either a plasmid cDNA encoding myc-SH3A or a specific siRNA targeting ITSN-1 gene. The lung vasculature of SH3A-transduced and ITSN-1s- deficient mice was perfused with gold albumin (Au-BSA) to analyze by electron microscopy the morphological intermediates and pathways involved in transendothelial transport or with dinitrophenylated (DNP)-BSA to quantify by ELISA its transport. Acute modulation of ITSN-1s expression decreased the number of caveolae, impaired their transport, and opened the interendothelial junctions, while upregulating compensatory nonconventional endocytic/transcytotic structures. Chronic inhibition of ITSN-1s further increased the occurrence of nonconventional intermediates and partially restored the junctional integrity. These findings indicate that ITSN-1s expression is required for caveolae function and efficient transendothelial transport. Moreover, our results demonstrate that ECs are highly adapted to perform their transport function while maintaining lung homeostasis. 1. Introduction ITSN-1s is a multimodular protein, evolutionary conserved and widely expressed [1, 2]; it consists of two NH2-terminal EH domains, a central coiled-coil domain, and five consecutive COOH-terminal SH3 domains, SH3A-E, [3, 4]. Similarly to Dyn, ITSN-1s localizes to endocytic clathrin-coated pits and caveolae at the plasma membrane and associates preferentially with the neck region of caveolae [5, 6]. The simultaneous presence of multiple SH3 and EH domains, best known for their function in endocytosis, as well as the subcellular localization of ITSN-1s, led to the early assumption that ITSN-1s may function as an adaptor/scaffold of the general endocytic machinery [3, 5]. Subsequent studies have shown that ITSN-1s is capable of binding essential endocytic proteins, Epsin1/2, Eps15 [7], both the neuronal and ubiquitously expressed Dyn isoforms, [3, 5, 6, 8], stonin 2 [9, 10], and the signaling proteins RaIBP-associated Eps15-homology domain protein [11], mSos [12, 13], and 5-phosphatase SHIP2 [14]. ITSN-1s binds several Dyn molecules simultaneously and clusters them at the endocytic sites, creating a high concentration of Dyn required for collar formation around the necks of endocytic vesicles [6, 8]. This is a crucial endocytic event since the GTPase activity of Dyn is allosterically dependent on Dyn protein concentration


[1]  M. Guipponi, H. S. Scott, H. Chen, A. Schebesta, C. Rossier, and S. E. Antonarakis, “Two isoforms of a human intersectin (ITSN) protein are produced by brain-specific alternative splicing in a stop codon,” Genomics, vol. 53, no. 3, pp. 369–376, 1998.
[2]  C. Pucharcos, C. Casas, M. Nadal, X. Estivill, and S. de la Luna, “The human intersectin genes and their spliced variants are differentially expressed,” Biochimica et Biophysica Acta, vol. 1521, no. 1–3, pp. 1–11, 2001.
[3]  M. Okamoto, S. Schoch, and T. C. Südhof, “EHSH1/intersectin, a protein that contains EH and SH3 domains and binds to dynamin and SNAP-25. A protein connection between exocytosis and endocytosis?” The Journal of Biological Chemistry, vol. 274, no. 26, pp. 18446–18454, 1999.
[4]  M. Yamabhai, N. G. Hoffman, N. L. Hardison et al., “Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains,” The Journal of Biological Chemistry, vol. 273, no. 47, pp. 31401–31407, 1998.
[5]  N. K. Hussain, M. Yamabhai, A. R. Ramjaun et al., “Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells,” The Journal of Biological Chemistry, vol. 274, no. 22, pp. 15671–15677, 1999.
[6]  S. A. Predescu, D. N. Predescu, B. K. Timblin, R. V. Stan, and A. B. Malik, “Intersectin regulates fission and internalization of caveolae in endothelial cells,” Molecular Biology of the Cell, vol. 14, no. 12, pp. 4997–5010, 2003.
[7]  A. S. Sengar, W. Wang, J. Bishay, S. Cohen, and S. E. Egan, “The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15,” EMBO Journal, vol. 18, no. 5, pp. 1159–1171, 1999.
[8]  I. Knezevic, D. Predescu, C. Bardita et al., “Regulation of dynamin-2 assembly-disassembly and function through the SH3A domain of intersectin-1s,” Journal of Cellular and Molecular Medicine, vol. 15, no. 11, pp. 2364–2376, 2011.
[9]  J. A. Martina, C. J. Bonangelino, R. C. Aguilar, and J. S. Bonifacino, “Stonin 2: an adaptor-like protein that interacts with components of the endocytic machinery,” Journal of Cell Biology, vol. 153, no. 5, pp. 1111–1120, 2001.
[10]  L. E. Kelly and A. M. Phillips, “Molecular and genetic characterization of the interactions between the Drosophila stoned-B protein and DAP-160 (intersectin),” Biochemical Journal, vol. 388, no. 1, pp. 196–204, 2005.
[11]  O. Dergai, O. Novokhatska, M. Dergai et al., “Intersectin 1 forms complexes with SGIP1 and Reps1 in clathrin-coated pits,” Biochemical and Biophysical Research Communications, vol. 402, no. 2, pp. 408–413, 2010.
[12]  X. K. Tong, N. K. Hussain, A. G. Adams, J. P. O'Bryan, and P. S. McPherson, “Intersectin can regulate the Ras/MAP kinase pathway independent of its role in endocytosis,” The Journal of Biological Chemistry, vol. 275, no. 38, pp. 29894–29899, 2000.
[13]  X. K. Tong, N. K. Hussain, E. de Heuvel et al., “The endocytic protein intersectin is a major binding partner for the Ras exchange factor mSos1 in rat brain,” EMBO Journal, vol. 19, no. 6, pp. 1263–1271, 2000.
[14]  F. Nakatsu, R. M. Perera, L. Lucast et al., “The inositol 5-phosphatase SHIP2 regulates endocytic clathrin-coated pit dynamics,” Journal of Cell Biology, vol. 190, no. 3, pp. 307–315, 2010.
[15]  M. H. Stowell, B. Marks, P. Wigge, and H. T. McMahon, “Nucleotide-dependent conformational changes in dynamin: evidence for a mechanochemical molecular spring,” Nature Cell Biology, vol. 1, no. 1, pp. 27–32, 1999.
[16]  B. Marie, S. T. Sweeney, K. E. Poskanzer, J. Roos, R. B. Kelly, and G. W. Davis, “Dap160/Intersectin scaffolds the periactive zone to achieve high-fidelity endocytosis and normal synaptic growth,” Neuron, vol. 43, no. 2, pp. 207–219, 2004.
[17]  P. Oh, D. P. McIntosh, and J. E. Schnitzer, “Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven fission from the plasma membrane of endothelium,” Journal of Cell Biology, vol. 141, no. 1, pp. 101–114, 1998.
[18]  J. E. Hinshaw and S. L. Schmid, “Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding,” Nature, vol. 374, no. 6518, pp. 190–192, 1995.
[19]  S. A. Predescu, D. N. Predescu, I. Knezevic, I. K. Klein, and A. B. Malik, “Intersectin-1s regulates the mitochondrial apoptotic pathway in endothelial cells,” The Journal of Biological Chemistry, vol. 282, no. 23, pp. 17166–17178, 2007.
[20]  Y. Yu, P. Y. Chu, D. N. Bowser et al., “Mice deficient for the chromosome 21 ortholog Itsn1 exhibit vesicle-trafficking abnormalities,” Human Molecular Genetics, vol. 17, no. 21, pp. 3281–3290, 2008.
[21]  E. Evergren, H. Gad, K. Walther, A. Sundborger, N. Tomilin, and O. Shupliakov, “Intersectin is a negative regulator of dynamin recruitment to the synaptic endocytic zone in the central synapse,” Journal of Neuroscience, vol. 27, no. 2, pp. 379–390, 2007.
[22]  S. Rose, M. G. Malabarba, C. Krag et al., “Caenorhabditis elegans intersectin: a synaptic protein regulating neurotransmission,” Molecular Biology of the Cell, vol. 18, no. 12, pp. 5091–5099, 2007.
[23]  T. W. Koh, P. Verstreken, and H. J. Bellen, “Dap160/intersectin acts as a stabilizing scaffold required for synaptic development and vesicle endocytosis,” Neuron, vol. 43, no. 2, pp. 193–205, 2004.
[24]  W. M. Henne, E. Boucrot, M. Meinecke et al., “FCHo proteins are nucleators of clathrin-mediated endocytosis,” Science, vol. 328, no. 5983, pp. 1281–1284, 2010.
[25]  E. Scappini, T. W. Koh, N. P. Martin, and J. P. O'Bryan, “Intersectin enhances huntingtin aggregation and neurodegeneration through activation of c-Jun-NH2-terminal kinase,” Human Molecular Genetics, vol. 16, no. 15, pp. 1862–1871, 2007.
[26]  S. A. Predescu, D. N. Predescu, and G. E. Palade, “Plasmalemmal vesicles function as transcytotic carriers for small proteins in the continuous endothelium,” American Journal of Physiology, vol. 272, no. 2, pp. H937–H949, 1997.
[27]  D. Predescu, R. Horvat, S. Predescu, and G. E. Palade, “Transcytosis in the continuous endothelium of the myocardial microvasculature is inhibited by N-ethylmaleimide,” Proceedings of the National Academy of Sciences of the United States of America, vol. 91, no. 8, pp. 3014–3018, 1994.
[28]  S. A. Predescu, D. N. Predescu, and G. E. Palade, “Endothelial transcytotic machinery involves supramolecular protein-lipid complexes,” Molecular Biology of the Cell, vol. 12, no. 4, pp. 1019–1033, 2001.
[29]  J. W. McLean, E. A. Fox, P. Baluk et al., “Organ-specific endothelial cell uptake of cationic liposome-DNA complexes in mice,” American Journal of Physiology, vol. 273, no. 1, pp. H387–H404, 1997.
[30]  K. Miyawaki-Shimizu, D. Predescu, J. Shimizu, M. Broman, S. Predescu, and A. B. Malik, “siRNA-induced caveolin-1 knockdown in mice increases lung vascular permeability via the junctional pathway,” American Journal of Physiology, vol. 290, no. 2, pp. L405–L413, 2006.
[31]  D. Predescu, S. Predescu, J. Shimizu, K. Miyawaki-Shimizu, and A. B. Malik, “Constitutive eNOS-derived nitric oxide is a determinant of endothelial junctional integrity,” American Journal of Physiology, vol. 289, no. 3, pp. L371–L381, 2005.
[32]  K. G. Rothberg, J. E. Heuser, W. C. Donzell, Y. S. Ying, J. R. Glenney, and R. G. W. Anderson, “Caveolin, a protein component of caveolae membrane coats,” Cell, vol. 68, no. 4, pp. 673–682, 1992.
[33]  K. Ihida, D. Predescu, R. P. Czekay, and G. E. Palade, “Platelet activating factor receptor (PAF-R) is found in a large endosomal compartment in human umbilical vein endothelial cells,” Journal of Cell Science, vol. 112, no. 3, pp. 285–295, 1999.
[34]  S. M. Ferguson, A. Raimondi, S. Paradise et al., “Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits,” Developmental Cell, vol. 17, no. 6, pp. 811–822, 2009.
[35]  M. Simionescu, A. Gafencu, and F. Antohe, “Transcytosis of plasma macromolecules in endothelial cells: a cell biological survey,” Microscopy Research and Technique, vol. 57, no. 5, pp. 269–288, 2002.
[36]  W. R?mer, L. Berland, V. Chambon et al., “Shiga toxin induces tubular membrane invaginations for its uptake into cells,” Nature, vol. 450, no. 7170, pp. 670–675, 2007.
[37]  M. Marsh and A. Helenius, “Virus entry: open sesame,” Cell, vol. 124, no. 4, pp. 729–740, 2006.
[38]  K. Sandvig and B. van Deurs, “Membrane traffic exploited by protein toxins,” Annual Review of Cell and Developmental Biology, vol. 18, pp. 1–24, 2002.
[39]  M. Kirkham, A. Fujita, R. Chadda et al., “Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles,” Journal of Cell Biology, vol. 168, no. 3, pp. 465–476, 2005.
[40]  D. Predescu, K. Ihida, S. Predescu, and G. E. Palade, “The vascular distribution of the platelet-activating factor receptor,” European Journal of Cell Biology, vol. 69, no. 1, pp. 86–98, 1996.
[41]  D. R. Glodowski, C. C. Chen, H. Schaefer, B. D. Grant, and C. Rongo, “RAB-10 regulates glutamate receptor recycling in a cholesterol-dependent endocytosis pathway,” Molecular Biology of the Cell, vol. 18, no. 11, pp. 4387–4396, 2007.
[42]  G. E. Palade, M. Simionescu, and N. Simionescu, “Structural aspects of the permeability of the microvascular endothelium,” Acta physiologica Scandinavica. Supplementum, vol. 463, pp. 11–32, 1979.
[43]  N. Simionescu and M. Simionescu, “Cellular interactions of lipoproteins with the vascular endothelium: endocytosis and transcytosis,” Targeted Diagnosis and Therapy, vol. 5, pp. 45–95, 1991.
[44]  S. A. Predescu, D. N. Predescu, and A. B. Malik, “Molecular determinants of endothelial transcytosis and their role in endothelial permeability,” American Journal of Physiology, vol. 293, no. 4, pp. L823–L842, 2007.
[45]  M. Drab, P. Verkade, M. Elger et al., “Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice,” Science, vol. 293, no. 5539, pp. 2449–2452, 2001.
[46]  C. R. Hopkins, “Intracellular routing of transferrin and transferrin receptors in epidermoid carcinoma A431 cells,” Cell, vol. 35, no. 1, pp. 321–330, 1983.
[47]  R. Horvat and G. E. Palade, “Thrombomodulin and thrombin localization on the vascular endothelium; their internalization and transcytosis by plasmalemmal vesicles,” European Journal of Cell Biology, vol. 61, no. 2, pp. 299–313, 1993.
[48]  R. Horvat and G. E. Palade, “The functional thrombin receptor is associated with the plasmalemma and a large endosomal network in cultured human umbilical vein endothelial cells,” Journal of Cell Science, vol. 108, no. 3, pp. 1155–1164, 1995.
[49]  S. Ahmed, W. Bu, R. T. Chuen Lee, S. Maurer-Stroh, and W. Ing Goh, “F-BAR domain proteins: families and function,” Communitative & Integrative Biology, vol. 3, no. 2, pp. 116–121, 2010.
[50]  I. K. Klein, D. N. Predescu, T. Sharma, I. Knezevic, A. B. Malik, and S. Predescu, “Intersectin-2L regulates caveola endocytosis secondary to Cdc42-mediated actin polymerization,” The Journal of Biological Chemistry, vol. 284, no. 38, pp. 25953–25961, 2009.
[51]  N. K. Hussain, S. Jenna, M. Glogauer et al., “Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP,” Nature Cell Biology, vol. 3, no. 10, pp. 927–932, 2001.
[52]  T. Takenawa and S. Suetsugu, “The WASP-WAVE protein network: connecting the membrane to the cytoskeleton,” Nature Reviews Molecular Cell Biology, vol. 8, no. 1, pp. 37–48, 2007.
[53]  M. T. Howes, S. Mayor, and R. G. Parton, “Molecules, mechanisms, and cellular roles of clathrin-independent endocytosis,” Current Opinion in Cell Biology, vol. 22, no. 4, pp. 519–527, 2010.
[54]  J. H. Chidlow Jr. and W. C. Sessa, “Caveolae, caveolins, and cavins: complex control of cellular signalling and inflammation,” Cardiovascular Research, vol. 86, no. 2, pp. 219–225, 2010.
[55]  Y. Jin, S.-J. Lee, R. D. Minshall, and A. M.K. Choi, “Caveolin-1: a critical regulator of lung injury,” American Journal of Physiology, vol. 300, no. 2, pp. L151–L160, 2011.
[56]  L. M. Popescu and M. S. Faussone-Pellegrini, “TELOCYTES—a case of serendipity: the winding way from Interstitial Cells of Cajal (ICC), via Interstitial Cajal-Like Cells (ICLC) to TELOCYTES,” Journal of Cellular and Molecular Medicine, vol. 14, no. 4, pp. 729–740, 2010.
[57]  X. M. Wang, Y. Zhang, P. K. Hong et al., “Caveolin-1: a critical regulator of lung fibrosis in idiopathic pulmonary fibrosis,” Journal of Experimental Medicine, vol. 203, no. 13, pp. 2895–2906, 2006.
[58]  R. Mathew, “Cell-specific dual role of caveolin-1 in pulmonary hypertension,” Pulmonary Medicine, vol. 2011, Article ID 573432, 12 pages, 2011.
[59]  J. Y. Park, K. S. Kim, S. B. Lee et al., “On the mechanism of internalization of α-synuclein into microglia: roles of ganglioside GM1 and lipid raft,” Journal of Neurochemistry, vol. 110, no. 1, pp. 400–411, 2009.
[60]  M. Li, H. Chen, L. Diao, Y. Zhang, C. Xia, and F. Yang, “Caveolin-1 and VEGF-C promote lymph node metastasis in the absence of intratumoral lymphangiogenesis in non-small cell lung cancer,” Tumori, vol. 96, no. 5, pp. 734–743, 2010.
[61]  H. L. Chen, L. F. Fan, J. Gao, J. P. Ouyang, and Y. X. Zhang, “Differential expression and function of the caveolin-1 gene in non-small cell lung carcinoma,” Oncology Reports, vol. 25, no. 2, pp. 359–366, 2011.
[62]  S. Singla, D. Predescu, C. Bardita et al., “Pro-inflammatory endothelial cell dysfunction is associated with intersectin-1s down-regulation,” Respiratory Research, vol. 12, article 46, 2011.
[63]  D. J. Keating, C. Chen, and M. A. Pritchard, “Alzheimer's disease and endocytic dysfunction: clues from the Down syndrome-related proteins, DSCR1 and ITSN1,” Ageing Research Reviews, vol. 5, no. 4, pp. 388–401, 2006.
[64]  J. B. Wang, W. J. Wu, and R. A. Cerione, “Cdc42 and Ras cooperate to mediate cellular transformation by intersectin-L,” The Journal of Biological Chemistry, vol. 280, no. 24, pp. 22883–22891, 2005.
[65]  D. N. Predescu, M. Wang, M. Patel, C. Bardita, and S. Predescu, “Intersectin-1s deficiency—an early molecular pathogenetic event of lung cancer,” in Proceedings of the American Thoracic Society International Meeting, 2011, abstract.


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