Microfibril-associated MAGP-2 Stimulates Elastic Fiber Assembly
2006; Elsevier BV; Volume: 282; Issue: 1 Linguagem: Inglês
10.1074/jbc.m609692200
ISSN1083-351X
AutoresRaphaël Lemaire, J Bayle, Robert P. Mecham, Robert Lafyatis,
Tópico(s)Elasticity and Material Modeling
ResumoElastic fibers are complex structures composed of a tropoelastin inner core and microfibril outer mantle guiding tropoelastin deposition. Microfibrillar proteins mainly include fibrillins and microfibril-associated glycoproteins (MAGPs). MAGP-2 exhibits developmental expression peaking at elastic fiber onset, suggesting that MAGP-2 mediates elastic fiber assembly. To determine whether MAGP-2 regulates elastic fiber assembly, we used an in vitro model featuring doxycycline-regulated cells conditionally overexpressing exogenous MAGP-2 and constitutively expressing enhanced green fluorescent protein-tagged tropoelastin. Analysis by immunofluorescent staining showed that MAGP-2 overexpression dramatically increased elastic fibers levels, independently of extracellular levels of soluble tropoelastin, indicating that MAGP-2 stimulates elastic fiber assembly. This was associated with increased levels of matrix-associated MAGP-2. Electron microscopy showed that MAGP-2 specifically associates with microfibrils and that elastin globules primarily colocalize with MAGP-2-associated microfibrils, suggesting that microfibril-associated MAGP-2 facilitates elastic fiber assembly. MAGP-2 overexpression did not change levels of matrix-associated fibrillin-1, MAGP-1, fibulin-2, fibulin-5, or emilin-1, suggesting that microfibrils and other elastic fiberassociated proteins known to regulate elastogenesis do not mediate MAGP-2-induced elastic fiber assembly. Moreover, mutation analysis showed that MAGP-2 does not stimulate elastic fiber assembly through its RGD motif, suggesting that integrin receptor binding does not mediate MAGP-2-induced elastic fiber assembly. Because MAGP-2 interacts with Jagged-1 that controls cell-matrix interaction and cell motility, two key factors in elastic fiber macroassembly, microfibril-associated MAGP-2 may stimulate elastic fiber macroassembly by targeting the release of elastin globules from the cell membrane onto developing elastic fibers. Elastic fibers are complex structures composed of a tropoelastin inner core and microfibril outer mantle guiding tropoelastin deposition. Microfibrillar proteins mainly include fibrillins and microfibril-associated glycoproteins (MAGPs). MAGP-2 exhibits developmental expression peaking at elastic fiber onset, suggesting that MAGP-2 mediates elastic fiber assembly. To determine whether MAGP-2 regulates elastic fiber assembly, we used an in vitro model featuring doxycycline-regulated cells conditionally overexpressing exogenous MAGP-2 and constitutively expressing enhanced green fluorescent protein-tagged tropoelastin. Analysis by immunofluorescent staining showed that MAGP-2 overexpression dramatically increased elastic fibers levels, independently of extracellular levels of soluble tropoelastin, indicating that MAGP-2 stimulates elastic fiber assembly. This was associated with increased levels of matrix-associated MAGP-2. Electron microscopy showed that MAGP-2 specifically associates with microfibrils and that elastin globules primarily colocalize with MAGP-2-associated microfibrils, suggesting that microfibril-associated MAGP-2 facilitates elastic fiber assembly. MAGP-2 overexpression did not change levels of matrix-associated fibrillin-1, MAGP-1, fibulin-2, fibulin-5, or emilin-1, suggesting that microfibrils and other elastic fiberassociated proteins known to regulate elastogenesis do not mediate MAGP-2-induced elastic fiber assembly. Moreover, mutation analysis showed that MAGP-2 does not stimulate elastic fiber assembly through its RGD motif, suggesting that integrin receptor binding does not mediate MAGP-2-induced elastic fiber assembly. Because MAGP-2 interacts with Jagged-1 that controls cell-matrix interaction and cell motility, two key factors in elastic fiber macroassembly, microfibril-associated MAGP-2 may stimulate elastic fiber macroassembly by targeting the release of elastin globules from the cell membrane onto developing elastic fibers. Elastic fibers are major insoluble extracellular matrix structures that provide connective tissues with flexibility and extensibility. These properties are critical to the functions of arteries, lung, skin, and all other dynamic tissues and have been essential requirements in the evolution of multicellular organisms. The importance of elastic fibers is highlighted by severe heritable connective tissue diseases caused by mutations in components of elastic fibers (1Milewicz D.M. Urban Z. Boyd C. Matrix Biol. 2000; 19: 471-480Crossref PubMed Scopus (150) Google Scholar). Elastic fibers are complex structures composed of two major components as follows: an inner core of amorphous cross-linked tropoelastin and an outer microfibril mantle composed of multiple proteins. Various other components of the elastic fiber localize at the elastin-microfibril or cell-elastic fiber interface (for review see Ref. 2Kielty C.M. Sherratt M.J. Shuttleworth C.A. J. Cell Sci. 2002; 115: 2817-2828Crossref PubMed Google Scholar), including fibulin-2, fibulin-5, and emilin-1. These elastic fiber-associated molecules are important mediators of elastic fiber assembly as deletion of these genes induces elastic fiber defects in mice (3Chu M. Communication: 2nd National Meeting of the American Society for Matrix Biology, San Diego, CA, November, 10-13, 2004, Abstr. 28.American Society for Matrix Biology. 2004; Google Scholar, 4Yanagisawa H. Davis E.C. Starcher B.C. Ouchi T. Yanagisawa M. Richardson J.A. Olson E.N. Nature. 2002; 415: 168-171Crossref PubMed Scopus (487) Google Scholar, 5Zanetti M. Braghetta P. Sabatelli P. Mura I. Doliana R. Colombatti A. Volpin D. Bonaldo P. Bressan G.M. Mol. Cell. Biol. 2004; 24: 638-650Crossref PubMed Scopus (142) Google Scholar). Microfibrils play an essential role in elastic fiber assembly by orchestrating deposition of tropoelastin onto the developing elastic fiber. Recent data have shown that a microfibril scaffold is required for deposition of tropoelastin into the extracellular matrix (6Kozel B.A. Ciliberto C.H. Mecham R.P. Matrix Biol. 2004; 23: 23-34Crossref PubMed Scopus (65) Google Scholar). This confirms previous ultrastructural studies showing that microfibrillar proteins are generally expressed prior to tropoelastin during development (7Mariencheck M.C. Davis E.C. Zhang H. Ramirez F. Rosenbloom J. Gibson M.A. Parks W.C. Mecham R.P. Connect. Tissue Res. 1995; 31: 87-97Crossref PubMed Scopus (75) Google Scholar) and that elastin in early stages of assembly is always associated with microfibrils in developing elastic tissues (8Cleary E.G. Gibson M.A. Int. Rev. Connect. Tissue Res. 1983; 10: 97-209Crossref PubMed Google Scholar, 9Greenlee Jr., T.K. Ross R. Hartman J.L. J. Cell Biol. 1966; 30: 59-71Crossref PubMed Scopus (297) Google Scholar). However, because of the structural and functional heterogeneity of microfibrils, the molecular basis of tropoelastin alignment by microfibrils remains unclear. In this respect, each microfibrillar component may selectively determine the structure of microfibrils and/or their function in tropoelastin alignment. The biological role of these microfibrillar molecules is still under investigation. Fibrillin-1 and -2 are the principal structural components of microfibrils in elastic fibers (10Sakai L.Y. Keene D.R. Engvall E. J. Cell Biol. 1986; 103: 2499-2509Crossref PubMed Scopus (889) Google Scholar, 11Zhang H. Apfelroth S.D. Hu W. Davis E.C. Sanguineti C. Bonadio J. Mecham R.P. Ramirez F. J. Cell Biol. 1994; 124: 855-863Crossref PubMed Scopus (318) Google Scholar). Mutation of fibrillin-1 in the Tight-skin (Tsk) mice induces major alterations in microfibril structures (12Gayraud B. Keene D.R. Sakai L.Y. Ramirez F. J. Cell Biol. 2000; 150: 667-680Crossref PubMed Scopus (80) Google Scholar, 13Lemaire R. Farina G. Kissin E. Shipley J.M. Bona C. Korn J.H. Lafyatis R. Arthritis Rheum. 2004; 50: 915-926Crossref PubMed Scopus (42) Google Scholar). They also have a function in tropoelastin deposition as they regulate tropoelastin coacervation (14Clarke A.W. Wise S.G. Cain S.A. Kielty C.M. Weiss A.S. Biochemistry. 2005; 44: 10271-10281Crossref PubMed Scopus (56) Google Scholar). They are large glycoproteins that have distinct but overlapping pattern of expression (15Zhang H. Hu W. Ramirez F. J. Cell Biol. 1995; 129: 1165-1176Crossref PubMed Scopus (255) Google Scholar). Fibrillin-2 is generally expressed earlier in development than fibrillin-1 and may be particularly important in elastic fiber formation (16Pereira L. Andrikopoulos K. Tian J. Lee S.Y. Keene D.R. Ono R. Reinhardt D.P. Sakai L.Y. Biery N.J. Bunton T. Dietz H.C. Ramirez F. Nat. Genet. 1997; 17: 218-222Crossref PubMed Scopus (309) Google Scholar). Apart from the fibrillins, MAGP-1 (microfibril-associated glycoprotein 1) is possibly the best candidate for an integral microfibril molecule important for structural integrity. It is associated with virtually all microfibrils and is widely expressed in mesenchymal and connective tissue cells throughout the development. MAGP-1 binds to amino-terminal exons 1-10 of fibrillin-1 (17Jensen S.A. Reinhardt D.P. Gibson M.A. Weiss A.S. J. Biol. Chem. 2001; 276: 39661-39666Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) through its carboxyl-terminal region containing seven common cysteine residues (18Penner A.S. Rock M.J. Kielty C.M. Shipley J.M. J. Biol. Chem. 2002; 277: 35044-35049Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). MAGP-1 forms ternary complexes with fibrillin-1 and decorin (19Trask B.C. Trask T.M. Broekelmann T. Mecham R.P. Mol. Biol. Cell. 2000; 11: 1499-1507Crossref PubMed Scopus (118) Google Scholar), as well as with biglycan and tropoelastin (20Reinboth B. Hanssen E. Cleary E.G. Gibson M.A. J. Biol. Chem. 2002; 277: 3950-3957Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). MAGP-1 also binds to the α3 chain of type VI collagen (21Finnis M.L. Gibson M.A. J. Biol. Chem. 1997; 272: 22817-22823Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). MAGP-2, the other member of this small microfibrillar protein family, is a 170-173 residue protein structurally related to MAGP-1 mainly in its central region. MAGP-2 contains an RGD motif through which it specifically binds to the αvβ3 integrin (22Gibson M.A. Leavesley D.I. Ashman L.K. J. Biol. Chem. 1999; 274: 13060-13065Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and interacts with Jagged-1, an activating ligand for Notch receptor signaling (23Nehring L.C. Miyamoto A. Hein P.W. Weinmaster G. Shipley J.M. J. Biol. Chem. 2005; 280: 20349-20355Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). MAGP-2 is specifically associated with fibrillin-containing microfibrils. It binds to a carboxyl-terminal 7 tandem cbEGF-like repeat-containing region of fibrillin-1 through its carboxyl-terminal region (18Penner A.S. Rock M.J. Kielty C.M. Shipley J.M. J. Biol. Chem. 2002; 277: 35044-35049Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), as well as to amino-terminal and central regions of fibrillin-1 (24Hansen H.E. Hew F.H. Moore G. Gibson M.A. J. Biol. Chem. 2004; 279: 29185-29194Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The role of MAGP-2 in microfibril structure remains unclear because it exhibits restricted patterns of tissue localization and developmental expression. However, it may have a specialized function in matrix homeostasis. We have recently shown that fibrosis in skin of scleroderma patients and Tsk mice is associated with increased MAGP-2 levels (13Lemaire R. Farina G. Kissin E. Shipley J.M. Bona C. Korn J.H. Lafyatis R. Arthritis Rheum. 2004; 50: 915-926Crossref PubMed Scopus (42) Google Scholar) and that MAGP-2 stimulates expression of type I procollagen (25Lemaire R. Korn J.H. Shipley J.M. Lafyatis R. Arthritis Rheum. 2005; 52: 1812-1823Crossref PubMed Scopus (28) Google Scholar). Other evidence suggests that MAGP-2 may also modulate microfibril function in elastogenesis, as MAGP-2 is associated with elastin-associated microfibrils in developing nuchal ligaments (26Gibson M.A. Hatzinikolas G. Kumaratilake J.S. Sandberg L.B. Nicholl J.K. Sutherland G.R. Cleary E.G. J. Biol. Chem. 1996; 271: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar); MAGP-2 mRNA expression peaks at the period of onset of elastogenesis in this tissue (27Gibson M.A. Finnis M.L. Kumaratilake J.S. Cleary E.G. J. Histochem. Cytochem. 1998; 46: 871-886Crossref PubMed Scopus (78) Google Scholar), and increased matrix-associated MAGP-2 in the hypodermis of Tsk mice is associated with increased elastic fiber assembly. Consistent with MAGP-2 function in elastogenesis, several fibrillin-1 mutations giving rise to Marfan syndrome have been mapped to the 7 cbEGF repeats-containing region that binds MAGP-2 (28Handford P.A. Downing A.K. Reinhardt D.P. Sakai L.Y. Matrix Biol. 2000; 19: 457-470Crossref PubMed Scopus (114) Google Scholar). In this study, using cell lines conditionally overexpressing MAGP-2 and constitutively expressing EGFP 2The abbreviations used are: EGFP, enhanced green fluorescent protein; MEF, mouse embryonic fibroblast; TGF-β, transforming growth factor-β; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin; dox, doxycycline; TRE, tet-responsive element; tet, tetracycline; CMV, cytomegalovirus; GFP, green fluorescent protein. 2The abbreviations used are: EGFP, enhanced green fluorescent protein; MEF, mouse embryonic fibroblast; TGF-β, transforming growth factor-β; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin; dox, doxycycline; TRE, tet-responsive element; tet, tetracycline; CMV, cytomegalovirus; GFP, green fluorescent protein.-tagged tropoelastin, we show that microfibril-associated MAGP-2 stimulates elastic fiber assembly independently of its RGD motif and without affecting microfibril assembly or matrix-associated levels of elastic fiber associated molecules, fibulin-2, fibulin-5, and emilin-1. Cells and Plasmids—All cells used in this study were developed from a mouse embryonic fibroblast line (MEF 3T3; Clontech) that harbors the pTet-Off regulator, a plasmid expressing a tetracycline (tet)-controlled transactivator protein that binds to promoter tet-responsive element (CMVmin + TRE). MEF-TE cells constitutively overexpressing tropoelastin were created by transfecting MEF cells with pEGFP-bTE-FL, a vector supporting CMV-driven expression of bovine tropoelastin tagged with EGFP (B. Kozel). MG-TE cells conditionally overexpressing mouse MAGP-2 and constitutively tropoelastin were developed by transfecting MEF-TE cells with pTRE-MAGP-2, a vector supporting tet-regulated TRE-driven expression of MAGP-2 (25Lemaire R. Korn J.H. Shipley J.M. Lafyatis R. Arthritis Rheum. 2005; 52: 1812-1823Crossref PubMed Scopus (28) Google Scholar). MGV5-TE and MGΔRGD-TE cells conditionally overexpressing V5-tagged MAGP-2 and ΔRGD MAGP-2, respectively, were developed by transfecting MEF-TE cells with pTRE2-MAGP2-V5 and pTRE2-MAGP2ΔRGD (25Lemaire R. Korn J.H. Shipley J.M. Lafyatis R. Arthritis Rheum. 2005; 52: 1812-1823Crossref PubMed Scopus (28) Google Scholar). MEF-ΔcFbn cells conditionally overexpressing carboxyl-terminal truncated fibrillin-1 mutant were developed by transfecting MEF cells with pTRE-ΔcFbn-EGFP, vector supporting tet-regulated TRE-driven expression of the truncated fibrillin-1 mutant. pTRE-ΔcFbn-EGFP was constructed, first by creating pTRE-Fbn-1, a vector supporting conditional expression of wild-type fibrillin-1. pTRE-Fbn-1 was constructed by deleting the 3.0-kb Sph-1 fragment from pTRE2-Tsk fibrillin (13Lemaire R. Farina G. Kissin E. Shipley J.M. Bona C. Korn J.H. Lafyatis R. Arthritis Rheum. 2004; 50: 915-926Crossref PubMed Scopus (42) Google Scholar), corresponding to the duplicated region within Tsk fibrillin-1, followed by religation. Second, a 792-bp EGFP cDNA-containing fragment from pEGFP-N1 (Clontech) was inserted in-frame into pTRE-Fbn-1 in place of a 300-bp SacI/XbaI fragment, corresponding to the 91 carboxyl-terminal amino acid of fibrillin-1. Cell Transfection—Cell lines were transfected using Lipofectamine plus reagent (Invitrogen). After transfection, cells were selected for 12-14 days using G418 (100 μg/ml) and hygromycin (60 μg/ml). 40-50 selected cell colonies were then ring-cloned, expanded, and analyzed for the transfected gene by Northern blot and Western blot. SDS-PAGE and Immunoblotting—Secreted proteins were precipitated from culture media by trichloroacetic acid. Proteins from cell and matrix layers were directly lysed in SDS-PAGE buffer, 2% β-mercaptoethanol. Proteins were resolved on an 8% SDS-polyacrylamide gel, transferred to nitrocellulose, and then incubated with primary polyclonal rabbit antisera directed against mouse MAGP-2 (1/1000 dilution) (13Lemaire R. Farina G. Kissin E. Shipley J.M. Bona C. Korn J.H. Lafyatis R. Arthritis Rheum. 2004; 50: 915-926Crossref PubMed Scopus (42) Google Scholar), mouse fibrillin-1 (Ab9543, gift from L. Sakai), mouse fibulin-5 (1/2000, gift from B. Schiemann), bovine fibronectin (1/2000; Calbiochem), EGFP (1/1000; Abcam, Cambridge, MA), or monoclonal mouse antisera to bovine α-tropoelastin (1/400, clone BA4; RDI, Flanders, NJ). The secondary antibody consisted of a secondary horseradish peroxidase-conjugated donkey antimouse IgG antibody (1/10,000). Signal was detected using Supersignal West Pico or Femto chemiluminescent reagent (Pierce) followed by autoradiography. RNA Analysis—Total RNA was prepared using RNeasy Total RNA kit (Qiagen, Valencia, CA), separated by electrophoresis through a 1 m formaldehyde, 1% agarose gel and blotted onto a nylon membrane (Hybond-H; Amersham Biosciences). Blotted RNAs were then hybridized to random primed 32P-labeled cDNA probes for either mouse MAGP-2, tropoelastin, or 18S ribosome, as described previously (29Kissin E.Y. Lemaire R. Korn J.H. Lafyatis R. Arthritis Rheum. 2002; 46: 3000-3009Crossref PubMed Scopus (53) Google Scholar). Immunofluorescence—Cells were grown in Lab-Tek 8-chamber culture slides (Nalge Nunc International, Naperville, IL), fixed for 10 min in paraformaldehyde at room temperature, blocked for 30 min with 3% BSA in TBS, and then incubated for 2 h with polyclonal rabbit antisera directed against either mouse MAGP-2 (gift from J. M. Shipley), mouse fibrillin-1 (Ab9543), mouse MAGP-1 (R. Mecham), mouse fibulin-2 (gift from R. Timpl), mouse emilin-1 (gift form A. Colombatti), mouse tropoelastin (EPC, Owensville, MO) (30Lemaire R. Korn J.H. Shipley J.M. Lafyatis R. J. Investig. Dermatol. 2004; 123: 1063-1069Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), or monoclonal mouse antisera to bovine α-tropoelastin (clone BA4) or V5 epitope (Invitrogen) at a 1/200 dilution in 3% BSA/TBS. Cells were washed three times with TBS for 5 min and incubated for 1 h with a rhodamine-conjugated donkey anti-rabbit IgG or fluorescein isothiocyanate-conjugated donkey anti-mouse IgG antibody (Jackson ImmunoResearch, West Grove, PA) at a 1/200 dilution. Specific fluorescence signals were then visualized by an Olympus BH-2 microscope under fluorescent light associated with high resolution Olympus DP70 camera. Exposure times were set manually and were identical between dox + and dox - conditions. Immunoelectron Microscopy of Elastic Fibers—MGV5-TE cells overexpressing MAGP-2 were plated 1/3 in 35-mm tissue culture dishes and cultured without dox in the presence of TGF-β. After 2 weeks, cells were fixed in 2% paraformaldehyde for 30 min, washed three times for 5 min with PBS, and aldehydes quenched with 0.1 m ammonium chloride for 10 min. Cells were then blocked for 30 min with 1% BSA/PBS, incubated with primary antibodies directed to MAGP-2-V5 and/or elastin-EGFP in 1% BSA/PBS for 90 min, washed three times for 5 min with PBS, and incubated with secondary gold-labeled anti-IgG antibodies in 1% BSA/PBS for 60 min. Immunolocalization of MAGP-2-V5 was performed using a mouse anti-V5 antibody in combination with a 6-nm gold-labeled anti-mouse IgG antibody (1/30 dilution; Jackson ImmunoResearch). Coimmunolocalizations of MAGP-2-V5 and elastin-EGFP were performed using two antibody combinations. The first combination used mouse antibody to V5 (1/100) and rabbit antibody to GFP (1/300, Ab290; Abcam, Cambridge, MA) associated with 6-nm gold-labeled anti-mouse and 18-nm gold-labeled anti-rabbit IgG antibodies. The second combination used rabbit antibody to MAGP-2 (1/200) and mouse antibody to bovine elastin (1/200, clone BA4) associated with 18-nm gold-labeled anti-rabbit and 6-nm gold-labeled anti-mouse IgG antibodies. Following antibody incubation, cells were washed three times for 5 min with PBS, prior to final fixation in 1% glutaraldehyde in 0.1 m sodium cacodylate buffer for 15 min on ice. Cells were then stained with 1.25% osmium tetroxide, 0.1 m sodium cacodylate for 30 min, washed three times for 5 min with 0.1 m sodium cacodylate buffer, incubated with 2% tannic acid in 0.1 m cacodylate for 30 min, washed three times with cacodylate buffer, rinsed twice for 5 min with 15% ethanol, followed by a 30-min incubation in 4% aqueous uranyl acetate before sequential dehydration with ethanol. The samples were then embedded in PolyBed812-filled gelatin capsules and baked at 60 °C for 18 h. 60-nm thin sections were counterstained with uranyl acetate and lead citrate and examined on a Zeiss 902 electron microscope. MAGP-2 Overexpression Stimulates Elastic Fiber Assembly in MEF Cells—As a tool to investigate the molecular mechanisms underlying elastic fiber assembly, we used cultured MEF cells, a 3T3-derived mouse embryonic fibroblast cell line that assembles fibrillin microfibrils but not elastic fibers because tropoelastin is not expressed (Fig. 1A). Because these cells produce all components required to form the scaffolding of elastic fibers, but not tropoelastin itself, they provide an excellent in vitro system for studying elastic fiber assembly in association with constitutive expression of an exogenous tropoelastin molecule. To generate MEF cells constitutively expressing tropoelastin (MEF-TE cells), MEF cells were transfected with a vector supporting CMV-driven expression of bovine tropoelastin tagged with EGFP. Their ability to deposit exogenous tropoelastin onto microfibrils was then tested by EGFP fluorescence and immunofluorescence. Cells were cultured in the presence of TGF-β in order to maximize the assembly of fibrillin-1-containing microfibrils in the matrix (29Kissin E.Y. Lemaire R. Korn J.H. Lafyatis R. Arthritis Rheum. 2002; 46: 3000-3009Crossref PubMed Scopus (53) Google Scholar). At 1-week post-confluence, MEF cells constitutively overexpressing tropoelastin (MEF-TE cells) showed a sparse and restricted network of thin developing elastic fibers that stretched between a discontinuous network of dense elastin globules (Fig. 1B). Importantly, MEF-TE cells cultured for longer times, up to a month, eventually showed a much more robust elastic fiber network as elastic fibers built up in diameter and length (data not shown). To investigate the role of MAGP-2 in elastic fiber assembly, we developed MEF-TE cells conditionally overexpressing exogenous mouse MAGP-2 (MG-TE cells) under the control of tet via the Tet-Off system (31Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4214) Google Scholar). ME-TE cells, originally designed to constitutively express a tet-controlled transactivator, were stably transfected with a plasmid supporting mouse MAGP-2 expression under the control of the tet-responsive element. In the absence of dox, a tet analog, MG-TE cells showed high levels of MAGP-2 in the culture medium (Fig. 1C, dox -). In the presence of dox, MG-TE cells showed only low levels of endogenous MAGP-2 in the medium as expression of exogenous MAGP-2 is repressed (Fig. 1C, dox +). Overexpression of MAGP-2 did not change mRNA expression or medium levels of tropoelastin (Fig. 1, D and E, dox + versus dox -). Of note, TGF-β greatly enhanced levels of exogenous MAGP-2 present in the medium (Fig. 1C, dox -). This is associated with a similar increase in levels of exogenous MAGP-2 mRNA (data not shown). Because expression of exogenous MAGP-2 mRNA is driven by a CMV minimum promoter (see "Experimental Procedures"), this suggests that TGF-β stabilizes the MAGP-2 mRNA. To look at the effect of MAGP-2 overexpression on elastic fiber assembly, hyper-confluent MG-TE cells were cultured for 1 week with or without dox in the presence of TGF-β, and then elastic fibers were analyzed by EGFP fluorescence. Consistent with parental MEF cells (Fig. 1B), MG-TE cells cultured with dox, and therefore expressing only low levels of endogenous MAGP-2, showed an extremely poor and limited network of elastic fibers associated with numerous and prominent elastin globules (Fig. 2A, dox +). Thin, sparse developing elastic fibers stretched between these large elastin globules that were arranged in a discontinuous, loose network (Fig. 2B, panels d and j). In contrast, MEG-TE cells cultured with dox, and therefore overexpressing exogenous MAGP-2, showed a strong and extended network of elastic fibers (Fig. 2A, dox -). Elastic fibers were thick and dense and seemed to be generated from the aggregation of the elastin globules (Fig. 2B, panels c and i). Of note, both control transfection with the empty pTRE2 and control dox treatment of parental MEF-TE cells failed to alter elastic fibers assembly, indicating that the effect of MAGP-2 on elastic fibers was not because of either the plasmid pTRE-2 or dox (data not shown). These results suggest that MAGP-2 overexpression stimulates assembly of tropoelastin onto microfibrils, independent of extracellular levels of soluble tropoelastin available to the matrix (Fig. 1E). MAGP-2-induced Elastic Fibers Are Associated with Increased Matrix-associated Levels of MAGP-2—To investigate the molecular basis of the stimulation of elastic fiber assembly by MAGP-2, MG-TE cells were also stained in parallel for MAGP-2 by immunofluorescence. MG-TE overexpressing exogenous MAGP-2 showed a dramatic increase in matrix-associated MAGP-2 compared with the same cells expressing only endogenous MAGP-2 (Fig. 2B, panels a versus b and g versus h). Remarkably, increases in elastic fibers and matrix-associated MAGP-2 upon expression of exogenous MAGP-2 were closely associated as they largely colocalized (Fig. 2B, panels e and k). Similarly, in the absence of exogenous MAGP-2, the sparse network of elastic fibers also colocalized with basal endogenous matrix-associated MAGP-2 (Fig. 2B, panels f and l). MAGP-2-induced Matrix-associated Elastin Primarily Colocalizes with MAGP-2-associated Microfibrils and Forms Elastic Fibers—Ultrastructural analysis of elastic fibers assembled by MGV5-TE cells overexpressing V5-tagged MAGP-2 and EFGP-tagged tropoelastin was performed by EM associated with immunolocalization of MAGP-2 alone (Fig. 3, A and B) or in combination with elastin (Fig. 3, C and D). Gold-labeled anti-V5 and MAGP-2 antibodies targeting MAGP-2-V5 specifically localized to 10-12 nm fibrillin microfibrils within the matrix (Fig. 3, A-C, 6-nm gold labeling, and D, 18-nm gold labeling). This is consistent with previous immunoelectron microscopy studies performed on various animal tissues showing specific localization of MAGP-2 to fibrillin microfibrils (26Gibson M.A. Hatzinikolas G. Kumaratilake J.S. Sandberg L.B. Nicholl J.K. Sutherland G.R. Cleary E.G. J. Biol. Chem. 1996; 271: 1096-1103Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Dark, electron dense, globular structures typical of elastin were found exclusively associated with bundles of fibrillin microfibrils (Fig. 3, A-D), providing molecular evidence for the presence of elastic fibers in our in vitro system. Although a substantial amount of MAGP-2-associated microfibrils was elastin-free, elastin globules primarily localized to MAGP-2-associated microfibrils (Fig. 3, A-D), not to MAGP-2-free microfibrils (Fig. 3, A-C, arrowheads). These globules were gold-labeled by antibody to GFP targeting elastin-EGFP (Fig. 3C, 18 nm-gold particles) and antibody to bovine tropoelastin (Fig. 3D, 6 nm-gold particles), confirming their elastin nature. The lack of uniform labeling of these globules by the antibody to GFP is likely to represent a technical artifact inherent to the complexity of immuno-EM technique and/or lack of accessibility of EGFP epitopes within highly cross-linked elastin globules. Of note, control experiments using secondary 6- or 18-nm gold-labeled secondary antibodies alone showed no immunogold labeling (data not shown). The close association of elastin globules with MAGP-2 molecules within the matrix suggests that MAGP-2 facilitates elastic fiber assembly from its matrix-associated form, as opposed to its soluble non-bound form. To selectively assess the effect of matrix-associated and soluble MAGP-2 on elastic fiber assembly, we took advantage of the inability of soluble MAGP-2 provided as an external source from conditioned medium to assemble within the matrix of ME-TE cells (data not shown). ME-TE cells were cultured in high content MAGP-2-containing media conditioned from dox-regulated MG-TE overexpressing MAGP-2 and then elastic fibers analyzed by EGFP fluorescence. Although conditioned media were renewed every other day for 2 weeks, ME-TE cells cultured in MAGP-2-containing medium (dox -) showed no increase in elastic fibers compared with cells cultured in MAGP-2-free media (dox +) (data not shown). Because soluble MAGP-2 failed to stimulate elastic fiber assembly in matrix-associated MAGP-2-free conditions, this result supports the electron microscopy data, indicating that matrix-associated MAGP-2 mediates MAGP-2-induced elastic fiber assembly. Mat
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