Mechanisms of VE-cadherin Processing and Degradation in Microvascular Endothelial Cells
2003; Elsevier BV; Volume: 278; Issue: 21 Linguagem: Inglês
10.1074/jbc.m211746200
ISSN1083-351X
AutoresKanyan Xiao, David F. Allison, Margaret D. Kottke, Susan Summers, George P. Sorescu, Victor Faúndez, Andrew P. Kowalczyk,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoVE-cadherin is an endothelial-specific cadherin that plays important roles in vascular morphogenesis and growth control. To investigate the mechanisms by which endothelial cells regulate cadherin cell surface levels, a VE-cadherin mutant containing the non-adhesive interleukin-2 (IL-2) receptor extracellular domain and the VE-cadherin cytoplasmic tail (IL-2R-VE-cadcyto) was expressed in microvascular endothelial cells. Expression of the IL-2R-VE-cadcyto mutant resulted in the internalization of endogenous VE-cadherin and in a dramatic decrease in endogenous VE-cadherin levels. The internalized VE-cadherin co-localized with early endosomes, and the lysosomal inhibitor chloroquine dramatically inhibited the down-regulation of VE-cadherin in cells expressing the IL-2R-VE-cadcyto mutant. Chloroquine treatment also resulted in the accumulation of a VE-cadherin fragment lacking the β-catenin binding domain of the VE-cadherin cytoplasmic tail. The formation of the VE-cadherin fragment could be prevented by treating endothelial cells with proteasome inhibitors. Furthermore, inhibition of the proteasome prevented VE-cadherin internalization and inhibited the disruption of endothelial intercellular junctions by the IL-2RVE-cadcyto mutant. These results provide new insights into the mechanisms of VE-cadherin processing and degradation in microvascular endothelial cells. VE-cadherin is an endothelial-specific cadherin that plays important roles in vascular morphogenesis and growth control. To investigate the mechanisms by which endothelial cells regulate cadherin cell surface levels, a VE-cadherin mutant containing the non-adhesive interleukin-2 (IL-2) receptor extracellular domain and the VE-cadherin cytoplasmic tail (IL-2R-VE-cadcyto) was expressed in microvascular endothelial cells. Expression of the IL-2R-VE-cadcyto mutant resulted in the internalization of endogenous VE-cadherin and in a dramatic decrease in endogenous VE-cadherin levels. The internalized VE-cadherin co-localized with early endosomes, and the lysosomal inhibitor chloroquine dramatically inhibited the down-regulation of VE-cadherin in cells expressing the IL-2R-VE-cadcyto mutant. Chloroquine treatment also resulted in the accumulation of a VE-cadherin fragment lacking the β-catenin binding domain of the VE-cadherin cytoplasmic tail. The formation of the VE-cadherin fragment could be prevented by treating endothelial cells with proteasome inhibitors. Furthermore, inhibition of the proteasome prevented VE-cadherin internalization and inhibited the disruption of endothelial intercellular junctions by the IL-2RVE-cadcyto mutant. These results provide new insights into the mechanisms of VE-cadherin processing and degradation in microvascular endothelial cells. Endothelial adherens junctions are adhesive intercellular contacts that are crucial for the maintenance and regulation of normal microvascular function (1Stevens T. Garcia J.G. Shasby D.M. Bhattacharya J. Malik A.B. Am. J. Physiol. 2000; 279: L419-L422Crossref PubMed Google Scholar, 2Dejana E. Spagnuolo R. Bazzoni G. Thromb. Haemost. 2001; 86: 308-315Crossref PubMed Scopus (192) Google Scholar, 3Bazzoni G. Dejana E. Microcirculation. 2001; 8: 143-152Crossref PubMed Google Scholar). Alterations in adherens junction assembly influence endothelial cell motility, vascular morphogenesis, and permeability. Moreover, recent studies indicate that components of adherens junctions also function in intracellular signaling, leading to the current view that these complexes are plasma membrane domains that integrate chemical and mechanical signaling information (4Gottardi C.J. Gumbiner B.M. Curr. Biol. 2001; 11: R792-R794Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The major cell-cell adhesion molecule at endothelial adherens junctions is VE-cadherin, a cadherin family member that is specifically expressed in endothelial cells (5Breviario F. Caveda L. Corada M. Martin-Padura I. Navarro P. Golay J. Introna M. Gulino D. Lampugnani M.G. Dejana E. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1229-1239Crossref PubMed Scopus (241) Google Scholar). The cytoplasmic tail of the classic cadherins, including VE-cadherin, comprises two well-characterized domains. The juxtamembrane domain (JMD) 1The abbreviations used are: JMD, juxtamembrane domain; CBD, catenin binding domain; IL-2, interleukin-2; IL-2R, IL-2 receptor; MECs, microvascular endothelial cells; CMV, cytomegalovirus; EEA-1, early endosome antigen-1; Ab, antibody; PBS, phosphate-buffered saline; ECD, extracellular domain; ICD, intracellular domain; E3, ubiquitin-protein isopeptide ligase; PECAM-1, platelet endothelial cell adhesion molecule-1. binds to the catenin p120, an armadillo family protein that is thought to regulate cadherin adhesive interactions by modulating the activity of Rho family GTPases (6Anastasiadis P.Z. Moon S.Y. Thoreson M.A. Mariner D.J. Crawford H.C. Zheng Y. Reynolds A.B. Nat. Cell Biol. 2000; 2: 637-644Crossref PubMed Scopus (380) Google Scholar, 7Thoreson M.A. Anastasiadis P.Z. Daniel J.M. Ireton R.C. Wheelock M.J. Johnson K.R. Hummingbird D.K. Reynolds A.B. J. Cell Biol. 2000; 148: 189-202Crossref PubMed Scopus (389) Google Scholar, 8Noren N.K. Liu B.P. Burridge K. Kreft B. J. Cell Biol. 2000; 150: 567-580Crossref PubMed Scopus (470) Google Scholar). At the carboxyl-terminal region of the cadherin cytoplasmic tail, a domain termed the catenin binding domain (CBD) interacts with β-catenin or plakoglobin (9Angst B.D. Marcozzi C. Magee A.I. J. Cell Sci. 2001; 114: 629-641Crossref PubMed Google Scholar). β-Catenin and plakoglobin both interact with α-catenin, which links cadherins to the actin cytoskeleton and to other actin-binding proteins such as α-actinin (10Aberle H. Butz S. Stappert J. Weissig H. Kemler R. Hoschuetzky H. J. Cell Sci. 1994; 107: 3655-3663Crossref PubMed Google Scholar, 11Knudsen K.A. Wheelock M.J. J. Cell Biol. 1992; 118: 671-679Crossref PubMed Scopus (229) Google Scholar, 12Knudsen K.A. Soler A.P. Johnson K.R. Wheelock M.J. J. Cell Biol. 1995; 130: 67-77Crossref PubMed Scopus (564) Google Scholar, 13Jou T.-S. Stewart D.B. Stappert J. Nelson W.J. Marrs J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5067-5071Crossref PubMed Scopus (305) Google Scholar). VE-cadherin also associates with the vimentin cytoskeletal network in endothelial cells through interactions with plakoglobin and the intermediate filament-binding protein desmoplakin (14Kowalczyk A.P. Navarro P. Dejana E. Bornslaeger E.A. Green K.J. Kopp D.S. Borgwardt J.E. J. Cell Sci. 1998; 111: 3045-3057Crossref PubMed Google Scholar). These unique intercellular junctions, containing both actin and vimentin-binding proteins, have been referred to as complexus adhaerentes (15Schmelz M. Franke W.W. Eur. J. Cell Biol. 1993; 61: 274-289PubMed Google Scholar, 16Schmelz M. Moll R. Kuhn C. Franke W.W. Differentiation. 1994; 57: 97-117Crossref PubMed Scopus (101) Google Scholar, 17Valiron O. Chevier V. Usson Y. Breviario F. Job D. Dejana E. J. Cell Sci. 1996; 109: 2141-2149Crossref PubMed Google Scholar). Although our understanding of the mechanisms of junction assembly has advanced significantly, much less is known about how cellular levels of cadherins are regulated. The loss of cadherin expression in epithelial cells is associated with a variety of pathologies, including tumor metastasis (18Christofori G. Semb H. Trends Biochem. Sci. 1999; 24: 73-76Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar), and changes in cadherin expression are associated with epithelial-mesenchymal transitions during development (19Miller J.R. McClay D.R. Dev. Biol. 1997; 192: 323-339Crossref PubMed Scopus (126) Google Scholar). Interestingly, initiation of endothelial-mesenchymal transdifferentiation was found to correlate with the disruption of cell-cell contacts and has been marked by a loss of VE-cadherin expression in endothelial monolayers (20Frid M.G. Kale V.A. Stenmark K.R. Circ. Res. 2002; 90: 1189-1196Crossref PubMed Scopus (359) Google Scholar). A recent study (21Bobryshev Y.V. Cherian S.M. Inder S.J. Lord R.S. Cardiovasc. Res. 1999; 43: 1003-1017Crossref PubMed Scopus (68) Google Scholar) found that down-regulation of VE-cadherin in intimal neovessels was closely related to intimal inflammation. These data suggest that regulated changes in VE-cadherin levels have important consequences on endothelial function and pathophysiology. However, the cellular mechanisms that control cadherin expression and presentation at the cell surface are poorly characterized. Some insights into these issues have come from studies in which dominant negative mutants of cadherins were expressed in epithelial cells (22Amagai M. Fujimori T. Masunaga T. Shimizu H. Nishikawa T. Shimizu N. Takeichi M. Hashimoto T. J. Invest. Dermatol. 1995; 104: 27-32Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 23Fujimori T. Takeichi M. Mol. Biol. Cell. 1993; 4: 37-47Crossref PubMed Scopus (181) Google Scholar). In a variety of model systems, cadherin mutants with a non-adhesive extracellular domain disrupt cell-cell adhesion (24Kintner C. Cell. 1992; 69: 225-236Abstract Full Text PDF PubMed Scopus (332) Google Scholar) and decrease cell proliferation (25Zhu A.J. Watt F.M. J. Cell Sci. 1996; 109: 3013-3023Crossref PubMed Google Scholar). The mechanism by which these mutants disrupt adhesion is not fully understood; however, several studies (26Norvell S.M. Green K.J. J. Cell Sci. 1998; 111: 1305-1318Crossref PubMed Google Scholar, 27Nieman M.T. Kim J.B. Johnson K.R. Wheelock M.J. J. Cell Sci. 1999; 112: 1621-1632Crossref PubMed Google Scholar, 28Troxell M.L. Chen Y.T. Cobb N. Nelson W.J. Marrs J.A. Am. J. Physiol. 1999; 276: C404-C418Crossref PubMed Google Scholar) have indicated that dominant negative cadherin mutants cause the down-regulation of endogenous cadherins. Using a mutant VE-cadherin in which the extracellular domain of VE-cadherin was replaced with the non-adhesive IL-2 receptor, we have observed similar effects in an endothelial model system (29Venkiteswaran K. Xiao K. Summers S. Calkins C.C. Vincent P.A. Pumiglia K. Kowalczyk A.P. Am. J. Physiol. 2002; 283: C811-C821Crossref PubMed Scopus (105) Google Scholar). These observations suggest that cellular mechanisms common to a wide range of epithelial cell types control cadherin expression, and that cadherin mutants can be used to expose the molecular nature of these regulatory pathways. Recent studies indicate that some pools of cadherin on the cell surface are endocytosed and recycled back to the plasma membrane (30Le T.L. Joseph S.R. Yap A.S. Stow J.L. Am. J. Physiol. 2002; 283: C489-C499Crossref PubMed Scopus (100) Google Scholar, 31Le T.L. Yap A.S. Stow J.L. J. Cell Biol. 1999; 146: 219-232Crossref PubMed Scopus (484) Google Scholar). These observations suggest that cadherin endocytosis is an important regulatory mechanism that controls cadherin cell surface levels and perhaps regulates overall levels of cadherin expression. Because endogenous cadherins are down-regulated by mutant cadherins, we hypothesized that these mutants trigger the endocytosis and degradation of endogenous cadherins. This model system might therefore be used to reveal the cellular machinery involved in regulating cadherin levels in vascular endothelial cells. In the present study, we utilized an adenoviral system to introduce the dominant negative IL-2R-VE-cadcyto mutant into primary cultures of microvascular endothelial cells (MECs). The results of this study indicate that expression of the IL-2R-VE-cadcyto mutant caused a rapid and dramatic down-regulation of endogenous VE-cadherin. Furthermore, the mutant triggered the internalization of endogenous VE-cadherin, which was subsequently processed through the endosomal-lysosomal pathway. Lysosomal inhibitors prevented VE-cadherin degradation and revealed the formation of a VE-cadherin fragment that is generated during endocytic processing. The internalization and fragmentation of VE-cadherin could be blocked using proteasome inhibitors, suggesting a role for the proteasome in cadherin processing. Together, these observations provide new insights into the mechanisms by which cell surface levels of cadherins are regulated in microvascular endothelial cells. Cell Culture—Primary cultures of dermal microvascular endothelial cells (MECs) from human neonatal foreskin were purchased from the Emory Skin Diseases Research Center (Core B) and cultured in MCDB131 medium (Invitrogen, Carlsbad, CA). The culture medium was supplemented with 10% fetal bovine serum (HyClone, Logan, UT), l-glutamine (Herndon, VA), cAMP (Sigma, St. Louis, MO), hydrocortisone (Sigma), epidermal growth factor (Intergen, Purchase, NY), and antibiotic/antimycotic (Invitrogen). Cells were typically cultured 24–48 h and grown to 80% confluency for most experiments. For some experiments, MECs were seeded onto Matrigel to induce migration and branching as previously described (29Venkiteswaran K. Xiao K. Summers S. Calkins C.C. Vincent P.A. Pumiglia K. Kowalczyk A.P. Am. J. Physiol. 2002; 283: C811-C821Crossref PubMed Scopus (105) Google Scholar). Monkey kidney cell line COS-7 and human embryonic kidney cell line QBI-293A (Qbiogene, Carlsbad, CA) were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin/amphotericin. For transient transfection experiments, a subclone of COS-7 cells (COS-7-20) was transfected by calcium phosphate precipitation. Chloroquine was purchased from Sigma and used at 100 μm, and the proteasome inhibitor MG132 was obtained from Calbiochem (San Diego, CA) and used at 24 μg/ml. cDNA Constructs—A cDNA clone encoding full-length human VE-cadherin was generously provided by Dr. E. Dejana (32Navarro P. Caveda L. Breviario F. Mandoteanu I. Lampugnani M.G. Dejana E. J. Biol. Chem. 1995; 52: 30965-30972Abstract Full Text Full Text PDF Scopus (193) Google Scholar), and an expression construct encoding the extracellular domain of the IL-2 receptor (IL-2R) was provided by Dr. S. LaFlamme (33LaFlamme S.E. Thomas L.A. Yamada S.S. Yamada K.M. J. Cell Biol. 1994; 126: 1287-1298Crossref PubMed Scopus (207) Google Scholar). This IL-2R construct was used to construct a chimeric cDNA with the IL-2R extracellular domain, the entire VE-cadherin cytoplasmic domain, and a carboxyl-terminal c-myc epitope tag, as described previously (29Venkiteswaran K. Xiao K. Summers S. Calkins C.C. Vincent P.A. Pumiglia K. Kowalczyk A.P. Am. J. Physiol. 2002; 283: C811-C821Crossref PubMed Scopus (105) Google Scholar). The IL-2R-α5 integrin cytoplasmic tail mutant was generously provided by R. Keller (34Pham C.G. Harpf A.E. Keller R.S. Vu H.T. Shai S.Y. Loftus J.C. Ross R.S. Am. J. Physiol. 2000; 279: H2916-H2926Crossref PubMed Google Scholar). A deletion mutant of the VE-cadherin cytoplasmic tail lacking the catenin binding domain of VE-cadherin was generously provided by Dr. E. Dejana (32Navarro P. Caveda L. Breviario F. Mandoteanu I. Lampugnani M.G. Dejana E. J. Biol. Chem. 1995; 52: 30965-30972Abstract Full Text Full Text PDF Scopus (193) Google Scholar). A VE-cadherin entire cytoplasmic domain construct encoding the VE-cadherin extracellular and transmembrane domains with a cytoplasmic myc epitope tag was generated by PCR using Vent polymerase and the following primers: 5′-primer: 5′-ACGGGATCCGGGAAGATGCAGAGGCTCATGATGCTCCTC and the 3′-primer: 5′-GCAGTCTCGAGCTACAAGTCCTCTTCAGAAATGAGCTTTTGCTCCACCTTGCCGTGCGCGCGGGCCTGCTT. This 3′-primer includes an in-frame c-myc epitope tag followed by a stop codon. The resulting PCR product was ligated into PKS and subcloned into a CMV expression vector. All constructs were characterized fully by DNA sequence analysis, Western blot, and immunofluorescence analysis. Adenovirus Production—The pAdeasy adenovirus-packaging system, including pAdTrack-CMV and pAdeasy-1, was kindly provided by Dr. B. Vogelstein. Escherichia coli BJ5183 electroporation-competent cells were purchased from Stratagene (La Jolla, CA). Packaging and production of recombinant adenoviruses carrying the VE-cadherin constructs was achieved using the pAdeasy system according to published protocols (35He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3256) Google Scholar). Recombinant adenoviruses were amplified using QBI-293A cells and purified using VirakitTM Adeno4 kit (Viropur, LLC, Carlsbad, CA). Infection rates in MECs were monitored using green fluorescent protein, which is expressed in tandem with the construct of interest. In all experiments infection rates of ∼80% for MEC cultures were used for all constructs. Immunofluorescence—MECs cultured on gelatin-coated glass cover-slips were rinsed in PBS+ (PBS containing Ca2+ and Mg2+) and fixed in methanol at –20 °C for 3 min. Alternatively, cells were fixed in 3.7% paraformaldehyde in PBS followed by extraction in 0.5% Triton X-100 in PBS+. Endogenous VE-cadherin was detected using mouse monoclonal antibody Cad-5 (Transduction Laboratories, Lexington, KY); VE-cadherin mutants were followed using a rabbit antibody directed against the c-myc epitope tag (Bethyl Laboratories, Inc., Montgomery, TX). The localization of early endosomes was monitored using a mouse monoclonal EEA-1 antibody (Transduction Laboratories) or a rabbit polyclonal EEA-1 antibody (Affinity Bioreagents). In some experiments, the IL-2 receptor was detected using a mouse monoclonal antibody directed against the human IL-2 receptor (BioSource, Camarillo, CA). Appropriate species cross-absorbed secondary antibodies conjugated to various Alexa Fluors (Molecular Probes, Eugene, OR) were used for dual-label immunofluorescence. Control experiments were carried out routinely to verify that fluorescence was not due to secondary antibody cross-reactivity. Microscopy was carried out using a Leica DMR-E fluorescence microscope equipped with narrow band pass filters and a Hammamatsu Orca camera. Images were captured, pseudo-colored, and processed using Open Lab software (Improvision Inc., Lexington, MA). Western Blot Analysis—MECs were harvested in Laemmli gel sample buffer (Bio-Rad Laboratories, Hercules, CA) and analyzed by SDS-PAGE and immunoblot using antibodies directed against the extracellular domain of VE-cadherin (cad-5, ECD Ab, Transduction Laboratories), the intracellular domain of VE-cadherin (ICD Ab, Santa Cruz Biotechnology, Santa Cruz, CA), the myc epitope tag (Bethyl Laboratories, Montgomery, TX), PECAM-1 (Santa Cruz Biotechnology), or vimentin (V9, Sigma). The IL-2R extracellular domain was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology). Horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) were used at 1: 3000 dilution and detected using ECL (Amersham Biosciences, England). Trypsinization Experiments—To distinguish cell surface and intracellular pools of VE-cadherin, MECs were rinsed and incubated in trypsin/EDTA at 37 °C to proteolytically remove cell surface VE-cadherin. Trypsin was subsequently inactivated using normal growth medium with serum, and cells were recovered by centrifugation. Cell pellets were dissolved in SDS-PAGE sample buffer for Western blot analysis as described above. For controls, parallel cultures were harvested in SDS-PAGE sample buffer without trypsinization. VE-cadherin Mutants Induce Internalization and Down-regulation of Endogenous VE-cadherin—To investigate the early events associated with the disassembly of endothelial junctions, recombinant adenoviruses were used to express a VE-cadherin mutant in primary dermal microvascular endothelial cells (MECs). Using this adenoviral system, the IL-2R-VE-cadcyto mutant cadherin was expressed in MECs, and the levels of endogenous VE-cadherin were evaluated over the course of 24 h after infection (Fig. 1). Expression of the IL-2R-VE-cadcyto mutant was evident within 8 h of infection, and endogenous VE-cadherin was dramatically decreased by 12 h in MECs expressing the IL-2R-VE-cadcyto mutant. In contrast to the IL2-R-VE-cadcyto mutant, a similar mutant with the α5 integrin cytoplasmic tail (34Pham C.G. Harpf A.E. Keller R.S. Vu H.T. Shai S.Y. Loftus J.C. Ross R.S. Am. J. Physiol. 2000; 279: H2916-H2926Crossref PubMed Google Scholar) did not alter VE-cadherin levels (Fig. 1C). To determine the localization of endogenous VE-cadherin in MECs expressing IL-2R-VE-cadcyto mutant, immunofluorescence analysis was performed at 12 h after infection with adenovirus encoding the empty vector or the IL-2R-VE-cadcyto mutant (Fig. 2). An antibody directed against the VE-cadherin extracellular domain (cad-5) was used to specifically identify endogenous VE-cadherin and not the mutant, which lacks the VE-cadherin extracellular domain. The mutant cadherin was detected using antibodies directed against the myc epitope tag present at the carboxyl terminus. In uninfected MECs (not shown) and in control cells expressing empty adenoviral vector, extensive VE-cadherin staining was observed at MEC cell borders (Fig. 2A). In striking contrast, in MEC cultures expressing the IL-2R-VE-cadcyto mutant, endogenous VE-cadherin was distributed in a punctate cytoplasmic distribution (Fig. 2B). This punctate distribution was observed in virtually every cell expressing the IL-2R-VE-cadcyto mutant. Expression of the IL-2R-α5cyto integrin mutant had no effect on VE-cadherin distribution (Fig. 2, C and F). To determine if the cadherin mutant was specifically altering VE-cadherin levels, the distribution and expression levels of PECAM-1 were monitored in MECs expressing the IL-2R-VE-cadcyto mutant (Fig. 3). In MECs infected with empty virus, PECAM-1 was present at the cell surface and accumulated at intercellular junctions (Fig. 3, A and B). In MECs expressing the IL-2R-VE-cadcyto mutant, PECAM-1 appeared more diffuse, and junctional accumulation of the protein was decreased (Fig. 3, C and D). However, the IL-2R-VE-cadcyto mutant had no effect on PECAM-1 protein levels (Fig. 3E). Together, these data indicate that the expression of VE-cadherin was modulated specifically by the IL-2RVE-cadcyto mutant cadherin.Fig. 2The IL-2R-VE-Cadcyto mutant disrupts MECs intercellular junctions. MECs were infected with empty adenovirus (A and D), the IL-2R-VE-Cadcyto mutant (B and E), or the IL2-R-α5cyto chimeric integrin mutant (C and F) for 18 h. The cells were then fixed in methanol and processed for dual label immunofluorescence using antibodies directed against endogenous VE-cadherin (A–C), the myc epitope tag (D and E), or the IL-2 receptor extracellular domain (F). Bar, 50 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Expression of the IL-2R-VE-Cadcyto mutant disrupts PECAM-1 localization but does not down-regulate PECAM-1 levels. MECs were infected by empty adenoviral vector (A and B) or the IL-2R-VE-Cadcyto (C and D) mutant for 10 h. The cells were then fixed in paraformaldehyde plus 0.5% Triton X-100 and processed for dual-label immunofluorescence using antibodies directed against PECAM-1 (B and D) and the myc epitope tag (A and C). Bar, 50 μm. E, MECs were infected with empty virus or the IL-2R-VE-Cadcyto mutant for 10 h. Western blot analysis was carried out using antibodies directed against VE-cadherin, PECAM-1, the myc epitope tag, and vimentin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) VE-cadherin Is Internalized and Processed through the Endosomal/Lysosomal Pathway—To determine if the punctate staining for VE-cadherin in cells expressing the IL-2R-VE-cadcyto mutant represented an internalized pool of protein, MECs were fixed in paraformaldehyde to detect cell surface VE-cadherin (Fig. 4A) or paraformaldehyde followed by Triton X-100 extraction to detect internalized VE-cadherin (Fig. 4B). No punctate staining for VE-cadherin was observed in cells fixed in paraformaldehyde, but a loss of cell surface cadherin staining was observed in cells infected with adenovirus expressing the IL-2R-VE-cadcyto mutant (Fig. 4A, asterisk). In contrast, in cells fixed in paraformaldehyde followed by permeabilization with Triton X-100, obvious VE-cadherin punctate staining was observed, indicating that the punctate VE-cadherin staining represents an internalized pool rather than cell surface clusters (Fig. 4B). These results indicate that dominant negative cadherin mutants induce internalization of endogenous VE-cadherin in primary human microvascular endothelial cells. To determine if internalized VE-cadherin is destined for the endosome-lysosome pathway, co-localization of endogenous VE-cadherin with EEA-1, a marker for early endosomes, was carried out using dual-label immunofluorescence analysis (Fig. 4, C–E). As shown in Fig. 4E, the endogenous VE-cadherin localized to cytoplasmic vesicles containing early endosome antigen-1 (EEA-1), indicating that the internalized VE-cadherin was processed through the endosomal pathway. To determine if the internalized VE-cadherin was degraded via a lysosomal pathway, uninfected MECs and MECs expressing IL-2R-VE-cadcyto were treated with the lysosomal inhibitor chloroquine. Interestingly, a dramatic increase in vesicular VE-cadherin staining was observed in uninfected MECs after treatment with chloroquine for 8 h (Fig. 5B), indicating that lysosomal inhibitors increase vesicular VE-cadherin accumulation even in quiescent MECs. In MECs expressing the IL-2RVE-cadcyto mutant, ∼90% of the cells expressing this mutant exhibited vesicular VE-cadherin staining even in the absence of chloroquine (Fig. 5C). Treatment of MECs expressing the mutant cadherin with chloroquine resulted in a slight increase in vesicular cadherin, and the vesicular structures were often larger in chloroquine treated cells (Fig. 5D). Together, these results suggest that, in quiescent MECs, VE-cadherin is normally internalized and degraded via a lysosomal pathway and that the expression of cadherin mutants dramatically accelerates this process. Lysosome Inhibitors Prevent VE-cadherin Degradation and Reveal the Formation of a VE-cadherin Fragment—The above results demonstrate that endogenous VE-cadherin is internalized and that the expression level of endogenous VE-cadherin is decreased upon the introduction of dominant negative cadherin mutants. Furthermore, the internalized VE-cadherin is processed through the endosome-lysosome pathway. To determine if lysosomal inhibitors could prevent the decrease in VE-cadherin levels in cells expressing the mutant cadherin, MECs that were infected with empty virus or IL-2R-VE-cadcyto were treated with chloroquine, and Western blot analysis was performed to evaluate the expression level of endogenous VE-cadherin. As shown in Fig. 6A, chloroquine treatment prevented the down-regulation of VE-cadherin in MECs expressing the IL-2R-VE-cadcyto mutant. The regulation of VE-cadherin expression was explored further by seeding MECs onto Matrigel, which induces rapid endothelial cell migration and the formation of branching networks (29Venkiteswaran K. Xiao K. Summers S. Calkins C.C. Vincent P.A. Pumiglia K. Kowalczyk A.P. Am. J. Physiol. 2002; 283: C811-C821Crossref PubMed Scopus (105) Google Scholar, 36Benelli R. Albini A. Int. J Biol. Markers. 1999; 14: 243-246Crossref PubMed Scopus (60) Google Scholar). As shown in Fig. 6B, VE-cadherin levels were dramatically decreased in MECs seeded onto Matrigel for 6 h. To determine if the down-regulation of VE-cadherin in endothelial cells induced to migrate on Matrigel could be inhibited with chloroquine, MECs were allowed to adhere to Matrigel or plastic for 1 h, and then treated with chloroquine for 6 h. Similar to MECs expressing the IL-2R-VE-cadcyto mutant, chloroquine treatment attenuated the down-regulation of VE-cadherin in MECs seeded onto Matrigel. These data suggest that lysosomal degradation of VE-cadherin is used to control VE-cadherin expression in migrating endothelial cells. Surprisingly, treatment of MECs with chloroquine also resulted in the appearance of a 95-kDa VE-cadherin fragment in both control MECs, and in MECs expressing the mutant cadherin (Fig. 6A). The amount of the VE-cadherin fragment generated in chloroquine-treated cells was also increased in MECs seeded onto Matrigel compared with control cells seeded onto plastic (Fig. 6B). This VE-cadherin fragment likely represents an intermediary in the processing of VE-cadherin during endocytosis and degradation. To determine the kinetics of the appearance of the VE-cadherin fragment in chloroquine-treated cells, time course experiments were conducted on uninfected MECs. MECs were treated with chloroquine for 0–9 h, and the appearance of the fragment was monitored by Western blot using an antibody directed against the VE-cadherin extracellular domain. As shown in Fig. 6C, a steady increase in the accumulation of the VE-cadherin fragment is observed over this time frame. To determine how rapidly the fragmented VE-cadherin is subsequently processed for complete degradation, MECs were treated with chloroquine for 6 h and then transferred to normal growth medium without chloroquine for various amount of time. Three hours after the return to normal medium, the fragment was almost completely absent, indicating that the chloroquine treatment is reversible (Fig. 6D). The results of these experiments suggest that chloroquine treatment halts VE-cadherin degradation in the lysosome subsequent to an earlier cleavage of the VE-cadherin tail. Removal of chloroquine then allows the process to continue, resulting in c
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