Proteomic Analysis of Vascular Endothelial Growth Factor-induced Endothelial Cell Differentiation Reveals a Role for Chloride Intracellular Channel 4 (CLIC4) in Tubular Morphogenesis
2005; Elsevier BV; Volume: 280; Issue: 51 Linguagem: Inglês
10.1074/jbc.m506724200
ISSN1083-351X
AutoresSvante Bohman, Tarô Matsumoto, Kwang S. Suh, Anna Dimberg, Lars Jakobsson, Stuart H. Yuspa, Lena Claesson‐Welsh,
Tópico(s)Cancer Cells and Metastasis
ResumoFormation of new vessels from pre-existing capillaries demands extensive reprogramming of endothelial cells through transcriptional and post-transcriptional events. We show that 120 protein spots in a two-dimensional isoelectric focusing/electrophoretic analysis were affected during vascular endothelial growth factor-A-induced endothelial cell tubular morphogenesis in vitro, as a result of changes in charge or expression level of the corresponding proteins. For about 22% of the spots, the protein products could be identified, of which several previously have been implicated in cytoskeletal reorganization and angiogenesis. One such protein was heat shock protein 27, a chaperone involved in β-actin rearrangement that was identified as regulated in degree of serine phosphorylation. We also identified regulation of chloride intracellular channel 4 (CLIC4), the expression of which decreased during tubular morphogenesis. CLIC4 was expressed at high levels in resting vessels, whereas expression was modulated during pathological angiogenesis such as in tumor vessels. The subcellular localization of CLIC4 in endothelial cells was dependent on whether cells were engaged in proliferation or tube formation. Antisense- and small interfering RNA-mediated suppression of CLIC4 expression led to arrest in tubular morphogenesis. Our data implicate CLIC4 in formation of a vessel lumen. Formation of new vessels from pre-existing capillaries demands extensive reprogramming of endothelial cells through transcriptional and post-transcriptional events. We show that 120 protein spots in a two-dimensional isoelectric focusing/electrophoretic analysis were affected during vascular endothelial growth factor-A-induced endothelial cell tubular morphogenesis in vitro, as a result of changes in charge or expression level of the corresponding proteins. For about 22% of the spots, the protein products could be identified, of which several previously have been implicated in cytoskeletal reorganization and angiogenesis. One such protein was heat shock protein 27, a chaperone involved in β-actin rearrangement that was identified as regulated in degree of serine phosphorylation. We also identified regulation of chloride intracellular channel 4 (CLIC4), the expression of which decreased during tubular morphogenesis. CLIC4 was expressed at high levels in resting vessels, whereas expression was modulated during pathological angiogenesis such as in tumor vessels. The subcellular localization of CLIC4 in endothelial cells was dependent on whether cells were engaged in proliferation or tube formation. Antisense- and small interfering RNA-mediated suppression of CLIC4 expression led to arrest in tubular morphogenesis. Our data implicate CLIC4 in formation of a vessel lumen. Angiogenesis, the formation of new blood vessels from the preexisting vasculature (1Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4752) Google Scholar, 2Folkman J. Semin. Oncol. 2001; 28: 536-542Crossref PubMed Scopus (283) Google Scholar), arises as a result of endothelial cell activation by increased production of angiogenic growth factors such as vascular endothelial growth factor (VEGF-A) 5The abbreviations used are: VEGFvascular endothelial growth factorsiRNAsmall interfering RNACLIC4chloride intracellular channel 4PBSphosphate-buffered salineCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidTIMEtelomerase-immortalized dermal microvascular endothelialMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightMSmass spectrometry.5The abbreviations used are: VEGFvascular endothelial growth factorsiRNAsmall interfering RNACLIC4chloride intracellular channel 4PBSphosphate-buffered salineCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidTIMEtelomerase-immortalized dermal microvascular endothelialMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightMSmass spectrometry. or decreased production of angiogenesis inhibitors (3Folkman J. 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Mechanistically, the formation of new vessels from preexisting ones can be divided in at least two categories, capillary sprouting and intussusception (splitting of vessels). In capillary sprouting, the endothelial cells respond to VEGF-A by producing proteases that degrade the basement membrane. The detached endothelial cells migrate to form cord structures headed by a nondividing tip cell (8Gerhardt H. Golding M. Fruttiger M. Ruhrberg C. Lundkvist A. Abramsson A. Jeltsch M. Mitchell C. Alitalo K. Shima D. Betsholtz C. J. Cell Biol. 2003; 161: 1163-1177Crossref PubMed Scopus (2031) Google Scholar). This is followed by proliferation of cells in the stalk of the cord and later differentiation to form a lumen-containing vessel. Circulating endothelial cell precursors may also contribute to the formation of new vessels (9Ribatti D. Vacca A. Nico B. Roncali L. Dammacco F. Mech. Dev. 2001; 100: 157-163Crossref PubMed Scopus (101) Google Scholar). vascular endothelial growth factor small interfering RNA chloride intracellular channel 4 phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid telomerase-immortalized dermal microvascular endothelial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. vascular endothelial growth factor small interfering RNA chloride intracellular channel 4 phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid telomerase-immortalized dermal microvascular endothelial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A number of different in vitro assays of angiogenesis have been shown to model specific steps (proliferation, migration, cord formation, and lumen creation) of the capillary sprouting process (10Auerbach R. Lewis R. Shinners B. Kubai L. Akhtar N. Clin. Chem. 2003; 49: 32-40Crossref PubMed Scopus (587) Google Scholar). In the three-dimensional collagen gel assay, endothelial cells are induced to migrate and fuse to form a cord of cells. This is followed by rudimentary lumen formation in a manner closely mimicking the pattern of vessel formation in a collagenous matrix in vivo (11Montesano R. Orci L. Vassalli P. J. Cell Biol. 1983; 97: 1648-1652Crossref PubMed Scopus (496) Google Scholar) making it a suitable model for analysis of early regulation of angiogenesis. Indeed, the vessel structures assembled in a collagen matrix have the ability to anastomose with the pre-existing vasculature and to form functional vessels in vivo (12Koike N. Fukumura D. Gralla O. Au P. Schechner J.S. Jain R.K. Nature. 2004; 428: 138-139Crossref PubMed Scopus (566) Google Scholar). The three-dimensional collagen assay has been employed in analyses of gene regulation during the sprouting process (13Kahn J. Mehraban F. Ingle G. Xin X. Bryant J.E. Vehar G. Schoenfeld J. Grimaldi C.J. Peale F. Draksharapu A. Lewin D.A. Gerritsen M.E. Am. J. Pathol. 2000; 156: 1887-1900Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 14Bell S.E. Mavila A. Salazar R. Bayless K.J. Kanagala S. Maxwell S.A. Davis G.E. J. Cell Sci. 2001; 114: 2755-2773Crossref PubMed Google Scholar), but until now, post-transcriptional events critical in capillary formation have not been identified. We have therefore undertaken a proteomic approach to identify key regulatory events at the protein level, in tubular morphogenesis. One of the identified VEGF-A-regulated endothelial cell proteins was chloride intracellular channel four (CLIC4). The CLIC proteins have the unusual capacity to translocate from the cytoplasm to various cellular membranes (15Ashley R.H. Mol. Membr. Biol. 2003; 20: 1-11Crossref PubMed Scopus (104) Google Scholar, 16Fernandez-Salas E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. J. Biol. Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Overexpression of CLIC4 promotes plasma membrane localization where it is associated with anion channel activity (17Proutski I. Karoulias N. Ashley R.H. Biochem. Biophys. Res. Commun. 2002; 297: 317-322Crossref PubMed Scopus (26) Google Scholar). CLIC4 has also been shown to engage in complex formation with cytoskeletal components such as β-actin, tubulin, and dynamin I (18Suginta W. Karoulias N. Aitken A. Ashley R.H. Biochem. J. 2001; 359: 55-64Crossref PubMed Scopus (90) Google Scholar). The expression of CLIC4 is regulated in a p53-dependent manner (16Fernandez-Salas E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. J. Biol. Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), and it contributes to p53-regulated apoptosis (19Fernandez-Salas E. Suh K.S. Speransky V.V. Bowers W.L. Levy J.M. Adams T. Pathak K.R. Edwards L.E. Hayes D.D. Cheng C. Steven A.C. Weinberg W.C. Yuspa S.H. Mol. Cell. Biol. 2002; 22: 3610-3620Crossref PubMed Scopus (141) Google Scholar). CLIC4 has also been described as involved in the differentiation of fibroblasts into myofibroblasts (20Ronnov-Jessen L. Villadsen R. Edwards J.C. Petersen O.W. Am. J. Pathol. 2002; 161: 471-480Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In Caenorhabditis elegans, CLIC-like proteins have been implicated in formation of the excretory tube (21Berry K.L. Bulow H.E. Hall D.H. Hobert O. Science. 2003; 302: 2134-2137Crossref PubMed Scopus (123) Google Scholar). In this study, we demonstrate a role for CLIC4 in endothelial cell tubular morphogenesis. Tube Formation Assay—Human telomerase-immortalized dermal microvascular endothelial cells (TIME) (22Venetsanakos E. Mirza A. Fanton C. Romanov S.R. Tlsty T. McMahon M. Exp. Cell Res. 2002; 273: 21-33Crossref PubMed Scopus (95) Google Scholar) were maintained on gelatinized dishes in endothelial microvascular EBM MV2 growth medium with the following supplements: 5 ng/ml endothelial growth factor, 0.2 μg/ml hydrocortisone, 0.5 ng/ml VEGF-A, 10 ng/ml basic fibroblast growth factor, 20 ng/ml insulin-like growth factor-1 (Promocell). The cells were starved overnight in 1% serum in EBM MV2 without growth supplements. Collagen gels were prepared by mixing 10× Ham's F-12 medium, 0.1 m NaOH, collagen type 1 (1:1:8), supplemented with bicarbonate solution (Invitrogen) to 0.117% and Glutamax-I (Invitrogen) to 1%. The gels were allowed to set at 37 °C overnight. To allow cell proliferation, tissue culture plastic was coated overnight with fibronectin at 20 μg/ml (Sigma). Cells were seeded on collagen or fibronectin matrices at 6.3 × 104 cells/cm2. The cells were allowed to set for 2 h at 37 °C on the collagen gel before a second layer of collagen was added on top. After top gel polymerization (1 h), the cells were kept in medium containing serum and VEGF-A to final concentrations of 1% and 50 ng/ml, respectively. Two-dimensional Gel Electrophoresis and Protein Identification—At the indicated time points, cells in collagen gels were harvested by disrupting the gel with 2.5 mg/ml collagenase SC2674 (Sigma) for 13 min in the presence of 1 mm proteasome inhibitor MG-132 (Calbiochem), 10 μg/ml cycloheximide, and 50 μm Na3VO4, to prevent protein degradation, new protein synthesis, and dephosphorylation, respectively. The cells were collected by centrifugation and washed with ice-cold PBS to remove collagen monomers. The cell pellet was lysed in a modified RIPA buffer (1% Triton X-100, 40 mm Tris-HCl, pH 8 (Amersham Biosciences), 0.1% SDS, and 1× Complete protease inhibitor mixture (Roche Applied Science)). Cells on fibronectin were washed once with PBS before lysis in RIPA buffer. Whole cell protein extracts were prepared for two-dimensional gel analysis by using a two-dimensional clean-up kit following the manufacturer's instructions (Amersham Biosciences). Immediately thereafter, proteins were precipitated and washed before being dissolved in isoelectric focusing compatible buffer containing 8 m urea, 2% CHAPS, and 40 mm dithiothreitol (Amersham Biosciences). Protein concentration was determined using the two-dimensional Quant kit (Amersham Biosciences) to ensure equal gel loading. Two-dimensional gel electrophoresis and protein identification by mass spectrometry were carried out by the WCN Expression Proteomics Facility (Department of Medical Biochemistry and Microbiology, Uppsala University). Carrier ampholytes (0.5%) and bromphenol blue (0.002%) were added, and the sample was loaded on an isoelectrofocusing strip (Amersham Biosciences) by “in-gel rehydration loading” and run essentially according to the manufacturer's instructions (Amersham Biosciences). Protein samples were separated on analytical gels (220-μg sample) or on preparative gels (500-μg or 1-mg samples). Prior to the second dimension separation, proteins in the immobilized pH gradient strips were reduced and alkylated using dithiothreitol and iodoacetamide, respectively. The second dimension SDS-PAGE was carried out using the Ettan DALTsix vertical gel electrophoresis system (Amersham Biosciences) with 12.5% acrylamide gels. Proteins were subsequently stained with SYPRO ruby (Molecular Probes) essentially according to the manufacturer's instructions. The gel image was recorded by a Typhoon 9400 scanner (Amersham Biosciences). Regulated proteins were identified and extracted by in gel digestion essentially as described (23Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7735) Google Scholar). The generated peptides were analyzed by MALDI-MS using α-cyano-4-hydroxy-trans-cinnamic acid as matrix and MS spectra recorded on an Ultraflex TOF/TOF mass spectrometer (Bruker Daltonics). Spectra were internally calibrated with trypsin autolysis products (m/z = 842.51, m/z = 1045.56, and m/z = 2211.10). Finally, proteins were identified by peptide mass fingerprinting using the Mascot search engine (Matrix Science). Confirmation of Regulated Proteins on Protein Blots—Whole cell protein samples from collagen and fibronectin matrices were subjected to SDS-PAGE in 10% acrylamide gels, followed by transfer onto Hybond-C filters (Amersham Biosciences). The protein expression was detected using rabbit anti-HSP27 and phospho-HSP27 (Upstate) and rabbit anti-CLIC4 (24Suh K.S. Mutoh M. Nagashima K. Fernandez-Salas E. Edwards L.E. Hayes D.D. Crutchley J.M. Marin K.G. Dumont R.A. Levy J.M. Cheng C. Garfield S. Yuspa S.H. J. Biol. Chem. 2004; 279: 4632-4641Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Primary antibody reactivity was visualized by secondary horseradish peroxidase-conjugated anti-rabbit IgG antibody, followed by enhanced chemiluminescence (Amersham Biosciences). Real Time PCR Analysis—Total RNA was prepared from cells cultured in collagen or on fibronectin using the RNeasy mini kit (Qiagen). The cell lysis buffer containing guanidine isothiocyanate completely dissolved the collagen matrix, allowing the RNA to be purified directly from the cells. Contaminating genomic DNA was removed with DNase I (Amersham Biosciences), and 0.5 μg of DNase-treated RNA was used to prepare cDNA using oligo(dT) primers and the Advantage RT-for-PCR kit (Clontech). PCR was performed by mixing the cDNA with primers and SYBR Green PCR master mix (Applied Biosystems). CLIC4 primer sequences are as follows: 5′-GGTGATTCTGAACCTTGCCTCA-3′ and 5′-TCCTCTTGTTAGCCCTCCACCT-3′ (Invitrogen). The PCR was run in an ABI Prism 7700 instrument (Applied Biosystems). Immunofluorescence—Induction of angiogenesis in mouse embryoid bodies was performed as described previously (25Magnusson P. Rolny C. Jakobsson L. Wikner C. Wu Y. Hicklin D.J. Claesson-Welsh L. J. Cell Sci. 2004; 117: 1513-1523Crossref PubMed Scopus (45) Google Scholar). Collagen gels, with tube-forming TIME cells and embryoid bodies, were fixed overnight at 4 °C in zinc fix (0.1 m Tris-HCl, pH 7.5, 3 mm calcium acetate, 23 mm zinc acetate, 37 mm zinc chloride), permeabilized in Triton X-100, and blocked using 3% bovine serum albumin in Tris-buffered saline for 1 h at room temperature. Frozen T241 tumor and normal C57Bl/6 kidney sections were fixed in ice-cold methanol and blocked in 3% bovine serum albumin in PBS. Collagen gels, embryoid bodies, and tumor sections were incubated with the primary antibodies rabbit anti-CLIC4 and rat anti-CD31 (BD) as indicated and then with appropriate secondary antibodies (anti-rabbit-Fab2-Alexa-488 and anti-rat IgG-Alexa-555; Invitrogen). Collagen gels were transferred to microscope slides, and coverslips were mounted using Fluoromount-G (Southern Biotechnology Associates) and examined using a Nikon Eclipse E1000 microscope or using an LSM 510 META confocal microscope (Carl Zeiss). CLIC4 Antisense Transfections—CLIC4 antisense construct (24Suh K.S. Mutoh M. Nagashima K. Fernandez-Salas E. Edwards L.E. Hayes D.D. Crutchley J.M. Marin K.G. Dumont R.A. Levy J.M. Cheng C. Garfield S. Yuspa S.H. J. Biol. Chem. 2004; 279: 4632-4641Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) was transfected into TIME cells by electroporation using the human umbilical vein endothelial cell nucleofector kit (Amaxa Biosystems) following the manufacturer's instructions. In brief, 1 × 106 cells per condition were mixed with electroporation solution and 8 μg of plasmid and electroporated to transfer the DNA into the nucleus. The cells were resuspended in EBM MV2 medium and seeded on collagen gels or on fibronectin-coated plastics. Cells in three-dimensional collagen were fixed after 24 h with Zn-fix and incubated with 0.8 units/ml Texas Redphalloidin (Molecular Probes) and 1 μg/ml Hoechst 33342. Tube structures were defined as structures containing four or more connected cells and were counted in nine fields per well and in three gels per condition. Statistical analyses were performed using the Statistica® software and an independent t test. Identification of Proteins Regulated in Angiogenesis—Primary endothelial cells are known to undergo tubular morphogenesis and form capillary-like structures in three-dimensional collagen I gels in response to treatment with VEGF-A (26Qi J.H. Matsumoto T. Huang K. Olausson K. Christofferson R. Claesson-Welsh L. Angiogenesis. 1999; 3: 371-380Crossref PubMed Scopus (36) Google Scholar, 27Yang S. Xin X. Zlot C. Ingle G. Fuh G. Li B. Moffat B. de Vos A.M. Gerritsen M.E. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1934-1940Crossref PubMed Scopus (50) Google Scholar). We have studied tubular morphogenesis of TIME cells, which retain the characteristics of the primary human dermal endothelial cells from which they were derived (22Venetsanakos E. Mirza A. Fanton C. Romanov S.R. Tlsty T. McMahon M. Exp. Cell Res. 2002; 273: 21-33Crossref PubMed Scopus (95) Google Scholar). TIME cells express several markers for vascular endothelial cells and lack lymphatic endothelial cell markers (22Venetsanakos E. Mirza A. Fanton C. Romanov S.R. Tlsty T. McMahon M. Exp. Cell Res. 2002; 273: 21-33Crossref PubMed Scopus (95) Google Scholar). 6R. Rennel, S. Mellberg, A. Dimberg, A. Ameur, Westholm J. Orzechowski, J. Komorowski, P. Lassalle, M. J. Cross, and P. Gerwins, manuscript in preparation. The cells continue to divide for at least 200 population doublings, whereas parental primary cells will become senescent on average within 35-45 doublings. TIME cells between passage 21 and 25 were seeded between two layers of collagen I and induced with VEGF-A to form capillary structures (Fig. 1A). Gradually, cells would send out fine branches and make contact with neighboring cells, followed by swelling and fusion of cells into tubes. By 24 h, mature vessel-like structures had formed in which the actin cytoskeleton was lined up in parallel bundles seemingly expanding across cell borders (Fig. 1). These structures have been shown previously to contain rudimentary lumens and be growth-arrested (28Folkman J. Haudenschild C. Nature. 1980; 288: 551-556Crossref PubMed Scopus (848) Google Scholar, 29Matsumoto T. Turesson I. Book M. Gerwins P. Claesson-Welsh L. J. Cell Biol. 2002; 156: 149-160Crossref PubMed Scopus (174) Google Scholar). In contrast, cells seeded on a fibronectin matrix proliferated and failed to form vessel-like structures (Fig. 1B). To detect proteins regulated during different steps of vessel formation, cells were harvested and proteins isolated at specific time points corresponding to the discrete morphological steps in vessel formation. Whole cell protein lysates were generated from cells spreading on collagen 2 h after seeding and at 1, 3, 8, 16, and 24 h after onset of VEGF-A treatment. The relative expression patterns of total cellular proteins were analyzed by two-dimensional gel electrophoresis and compared between different time points on collagen, and also between differentiating cells on collagen and proliferating cells on fibronectin. Three gels for each time point were analyzed to identify consistent changes in expression pattern. Initially, two-dimensional gels with a broad isoelectric focusing range (pH 3-10) were used to visualize as many proteins as possible (Fig. 2A). Because most proteins have an isoelectric point between 4 and 8, rare proteins may be masked by more abundant ones. Therefore, we also separated the protein extracts on two-dimensional gels with a more narrow isoelectric focusing range (pH 4-7). This strategy allows better separation and loading of more protein. Guided by the analytical gels, we selected certain time points for preparative two-dimensional gels and spot identification as follows: 2 h after seeding but before addition of VEGF-A (time point 0), and after 1 and 16 h in the presence of VEGF-A. Selected spots were subjected to in-gel trypsin digestion, and the resulting peptide mix was analyzed by MALDI-TOF MS. The resulting mass spectra were run against the Mascot search engine (Matrix Science) for protein identification. Over 120 protein spots were identified as differentially regulated on the two-dimensional gels, but reliable sequence identification could only be generated for 27 spots (TABLE ONE). Twelve of the identified proteins have been implicated in regulation of the cytoskeleton, reflecting the dramatic cytoskeletal rearrangements required to form new vascular tubes. Five of the proteins were classified as involved in metabolism and three proteins as involved in protein turnover, which indicates the need for reprogramming in conjunction with transition from proliferation to differentiation. Some of the proteins identified belong to protein families already associated with a role in angiogenesis, such as HSP27 (30Rousseau S. Houle F. Landry J. Huot J. Oncogene. 1997; 15: 2169-2177Crossref PubMed Scopus (711) Google Scholar, 31Keezer S.M. Ivie S.E. Krutzsch H.C. Tandle A. Libutti S.K. Roberts D.D. Cancer Res. 2003; 63: 6405-6412PubMed Google Scholar) and annexins (32Oh P. Li Y. Yu J. Durr E. Krasinska K.M. Carver L.A. Testa J.E. Schnitzer J.E. Nature. 2004; 429: 629-635Crossref PubMed Scopus (438) Google Scholar). The family of chloride intracellular channels has been shown to have an important role in tubular morphogenesis in C. elegans (21Berry K.L. Bulow H.E. Hall D.H. Hobert O. Science. 2003; 302: 2134-2137Crossref PubMed Scopus (123) Google Scholar), which in many aspects resembles lumen formation in vascular sprouts.TABLE ONEIdentification of proteins subject to up- or down-regulation in tube forming and proliferating endothelial cellsProtein designationAccession no.Change in tube forming ECChange in proliferating EC0+1 h+16 hCytoskeleton-associated proteinsβ-Tubulin9112276+a+ and − indicate the presence of the protein on the two-dimensional gels at the indicated time point.+−−F-acting capping protein β subunit4826659+++−Chloride intracellular channel 47330335++−+Vimentin25074+++−Actin γ1ATHUG−+−−HSP27 spot 1980237++−−HSP27 spot 2bHSP27 spot 2 indicates that the protein changed in position of the gel indicative of a change in charge (pI).980237−−++Heat shock 60-kDa protein306890−−+−Heat shock 70-kDa protein24234688−−+−Chaperonin containing TCP124307939−++−Tropomyosin 1 α chain339956++−−Myosin regulatory light chain5453740−−+−Filamin 196C61−−+−MetabolicGlyceraldehyde-3-phosphate dehydrogenase31645+++−N-Acetylneuramic acid phosphate synthase17939512++−−Purine-nucleoside phosphorylasePHHUPN+++−Transitional endoplasmic reticulum ATPaseAAH20946−+−−Protein turnoverProteosome S26 subunit p457110703+−−−Proteasome S26 subunit 67435741+−−−Elongation factor 2αAAA50308−−+−VarioushD54 + ins2 isoformsAAC98478++−−Orofacial clefting Chr breakpoint region 1 proteinQ8IUL6−+−−Unnamed protein product21752016−−+−Annexin V999926−−+−Annexin V1-like gene (splice variant)BAC85290−−+−Laminin-binding protein/ribosomal protein 40CAA43469−−+−Similar to mosaic serine proteaseQ96C21−−+−a + and − indicate the presence of the protein on the two-dimensional gels at the indicated time point.b HSP27 spot 2 indicates that the protein changed in position of the gel indicative of a change in charge (pI). Open table in a new tab Verification of Identified Protein Regulation—The changes in expression pattern of individual proteins identified in the two-dimensional gels were validated by immunoblotting for selected proteins (Fig. 2B). We focused on CLIC4 and HSP27 because of their implication in angiogenesis and because they displayed different patterns of regulation during tube formation in collagen. CLIC4 was down-regulated at the later stage of tube formation (16 h); in contrast, its expression did not change during culture on fibronectin. HSP27 on the other hand was identified in two different spots when two-dimensional gel analyses of the 0- and the 16-h time points were compared. Horizontal shifts in spot position on a two-dimensional gel are often indicative of a change in protein charge, e.g. because of phosphorylation. In accordance with this, the immunoblotting analyses showed that the total HSP27 levels decreased with time on collagen, but the relative level of phosphorylated HSP27 increased 2.6 times at 16 h (in relation to β-actin expression levels). Because both HSP27 (33Lavoie J.N. Gingras-Breton G. Tanguay R.M. Landry J. J. Biol. Chem. 1993; 268: 3420-3429Abstract Full Text PDF PubMed Google Scholar) and CLIC4 (18Suginta W. Karoulias N. Aitken A. Ashley R.H. Biochem. J. 2001; 359: 55-64Crossref PubMed Scopus (90) Google Scholar) are known to be associated with the cytoskeleton, cells were lysed with a harsher SDS-containing lysis buffer to identify potential changes in protein subcellular localization, to a more insoluble compartment, with time. Similar levels of CLIC4 protein were generated independent of the lysis technique, indicating that the apparent decrease in CLIC4 expression level at 16 h was not because of trapping of protein. The decrease in HSP27 expression at the 16-h collagen gel time point also persisted under harsher lysis conditions, as did the relative increase in serine phosphorylation. On fibronectin cultures, increased levels of HSP27 protein were detected after lysis in SDS buffer compared with RIPA buffer indicating relocalization of HSP27. CLIC4 Down-regulation through Post-translational Mechanisms—To determine whether the observed decrease in CLIC4 expression in cells kept in three-dimensional collagen gels for 16 h was dependent on transcriptional regulation, total mRNA was prepared from the indicated time points, and the levels of CLIC4 transcripts were analyzed by real time PCR (Fig. 2C). No changes in transcript levels could be observed at different time points, suggesting that the decrease in CLIC4 protein involved post-translational mechanisms. CLIC4 Is Expressed by Vessels in Vivo—Expression of CLIC4 in endothelial cells has not yet been studied despite the intriguing link to lumen formation for the CLIC homologue EXC-4 in C. elegans. In other cell types, CLIC4 has been linked to differentiation (myofibroblasts) (20Ronnov-Jessen L. Villadsen R. Edwards J.C. Petersen O.W. Am. J. Pathol. 2002; 161: 471-480Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), and apoptosis (keratinocytes and tumor cells) (19Fernandez-Salas E. Suh K.S. Speransky V.V. Bowers W.L. Levy J.M. Adams T. Pathak K.R. Edwards L.E. Hayes D.D. Cheng C. Steven A.C. Weinberg W.C. Yuspa S.H. Mol. Cell. Biol. 2002; 22: 3610-3620Crossref PubMed Scopus (141) Google Scholar). To gain insight into the role of CLIC4 in vessels of different angiogenic status in vivo, we examined CLIC4 immunoreactivity in normal mouse kidney vessels, in developing murine embryonic vessels, and in T241 fibrosarcoma tumor vessels (Fig. 3). In normal mouse kidney, in which CLIC4 is known to be abundantly expressed in a constitutive manner (16Fernandez-Salas E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. J. Biol. Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), CLIC4 was expressed in all vessels, and relative to the surrounding stroma, the expression seemed high. CLIC4 was also expressed at high
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