B-Myb Represses Elastin Gene Expression in Aortic Smooth Muscle Cells
2004; Elsevier BV; Volume: 280; Issue: 9 Linguagem: Inglês
10.1074/jbc.m412501200
ISSN1083-351X
AutoresClaudia S. Hofmann, Xiaobo Wang, Christopher P. Sullivan, Paul Toselli, Phillip J. Stone, Sean E. McLean, Robert P. Mecham, Barbara M. Schreiber, Gail E. Sonenshein,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoB-Myb represses collagen gene transcription in vascular smooth muscle cells (SMCs) in vitro and in vivo. Here we sought to determine whether elastin is similarly repressed by B-Myb. Levels of tropoelastin mRNA and protein were lower in aortas and isolated SMCs of adult transgenic mice expressing the human B-myb gene, driven by the basal cytomegalovirus promoter, compared with age-matched wild type (WT) animals. However, the vessel wall architecture and levels of insoluble elastin revealed no differences. Since elastin deposition occurs early in development, microarray analysis was performed using nontransgenic mice. Aortic levels of tropoelastin mRNA were low during embryonal growth and increased substantially in neonates, whereas B-myb levels varied inversely. Tropoelastin mRNA expression in aortas of 6-day-old neonatal transgenic and WT animals was comparable. Recently, we demonstrated that cyclin A-Cdk2 prevents B-Myb-mediated repression of collagen promoter activity. Cyclin A2 levels were higher in neonatal versus adult WT or transgenic mouse aortas. Ectopic cyclin A expression reversed the ability of B-Myb to repress elastin gene promoter activity in adult SMCs. These results demonstrate for the first time that B-Myb represses SMC elastin gene expression and that cyclin A plays a role in the developmental regulation of elastin gene expression in the aorta. Furthermore, the findings provide additional insight into the mechanism of B-myb-mediated resistance to femoral artery injury. B-Myb represses collagen gene transcription in vascular smooth muscle cells (SMCs) in vitro and in vivo. Here we sought to determine whether elastin is similarly repressed by B-Myb. Levels of tropoelastin mRNA and protein were lower in aortas and isolated SMCs of adult transgenic mice expressing the human B-myb gene, driven by the basal cytomegalovirus promoter, compared with age-matched wild type (WT) animals. However, the vessel wall architecture and levels of insoluble elastin revealed no differences. Since elastin deposition occurs early in development, microarray analysis was performed using nontransgenic mice. Aortic levels of tropoelastin mRNA were low during embryonal growth and increased substantially in neonates, whereas B-myb levels varied inversely. Tropoelastin mRNA expression in aortas of 6-day-old neonatal transgenic and WT animals was comparable. Recently, we demonstrated that cyclin A-Cdk2 prevents B-Myb-mediated repression of collagen promoter activity. Cyclin A2 levels were higher in neonatal versus adult WT or transgenic mouse aortas. Ectopic cyclin A expression reversed the ability of B-Myb to repress elastin gene promoter activity in adult SMCs. These results demonstrate for the first time that B-Myb represses SMC elastin gene expression and that cyclin A plays a role in the developmental regulation of elastin gene expression in the aorta. Furthermore, the findings provide additional insight into the mechanism of B-myb-mediated resistance to femoral artery injury. The B-myb gene was isolated based on its homology with c-myb in its DNA binding region (∼90% homology) (1Nomura N. Takahashi M. Matsui M. Ishii S. Date T. Sasamoto S. Ishizaki R. Nucleic Acids Res. 1988; 16: 11075-11089Crossref PubMed Scopus (200) Google Scholar). The mRNA, which is ∼3.3–3.5 kb, codes for a B-Myb protein of ∼704 amino acids that migrates at ∼93 kDa (2Arsura M. Luchetti M.M. Erba E. Golay J. Rambaldi A. Introna M. Blood. 1994; 83: 1778-1790Crossref PubMed Google Scholar, 3Marhamati D.J. Sonenshein G.E. J. Biol. Chem. 1996; 271: 3359-3365Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The consensus Myb binding site (MBS) 1The abbreviations used are: MBS, Myb binding site; SMC, smooth muscle cell, CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription; WT, wild type; CAT, chloramphenicol acetyltransferase; CREB, cAMP-response element-binding protein. is (C/T)AACNG (4Howe K.M. Watson R.J. Nucleic Acids Res. 1991; 19: 3913-3919Crossref PubMed Scopus (96) Google Scholar). B-Myb forms complexes of comparatively lower stability showing less tolerance for binding site variations compared with c-Myb or A-Myb (5Bergholtz S. Andersen T.O. Andersson K.B. Borrebaek J. Luscher B. Gabrielsen O.S. Nucleic Acids Res. 2001; 29: 3546-3556Crossref PubMed Scopus (33) Google Scholar). B-Myb was also found to be able to regulate several reporter constructs without MBS sequences, including the DNA polymerase α promoter, the fibroblast growth factor-4 promoter, and its own promoter (6Mizuguchi G. Nakagoshi H. Nagase T. Nomura N. Date T. Ueno Y. Ishii S. J. Biol. Chem. 1990; 265: 9280-9284Abstract Full Text PDF PubMed Google Scholar, 7Watson R.J. Robinson C. Lam E.W. Nucleic Acids Res. 1993; 21: 267-272Crossref PubMed Scopus (69) Google Scholar, 8Johnson L.R. Johnson T.K. Desler M. Luster T.A. Nowling T. Lewis R.E. Rizzino A. J. Biol. Chem. 2002; 277: 4088-4097Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 9Sala A. Saitta B. De Luca P. Cervellera M.N. Casella I. Lewis R.E. Watson R. Peschle C. Oncogene. 1999; 18: 1333-1339Crossref PubMed Scopus (42) Google Scholar). B-Myb expression is low in early G1 and is induced in late G1 and S phases in many cell types, including vascular smooth muscle cells (SMCs) (3Marhamati D.J. Sonenshein G.E. J. Biol. Chem. 1996; 271: 3359-3365Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 10Golay J. Capucci A. Arsura M. Castellano M. Rizzo V. Introna M. 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Saville M. Watson R. Koeffler H.P. Blood. 2001; 97: 2091-2097Crossref PubMed Scopus (47) Google Scholar). We showed that B-Myb displays a strong negative regulatory activity on MBS element-driven reporter activity and on type I and type V collagen gene promoters in vascular SMCs in culture (3Marhamati D.J. Sonenshein G.E. J. Biol. Chem. 1996; 271: 3359-3365Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 13Kypreos K.E. Marhamati D.J. Sonenshein G.E. Matrix Biol. 1999; 18: 275-285Crossref PubMed Scopus (11) Google Scholar). Furthermore, B-Myb mediates the decrease in type I collagen gene transcription induced by basic fibroblast growth factor (21Kypreos K.E. Nugent M.A. Sonenshein G.E. Cell Growth Differ. 1998; 9: 723-730PubMed Google Scholar). Interestingly, B-Myb does not induce proliferation of bovine SMCs (22Marhamati D.J. Bellas R.E. Arsura M. Kypreos K.E. Sonenshein G.E. Mol. Cell. Biol. 1997; 17: 2448-2457Crossref PubMed Scopus (27) Google Scholar), and cyclin A dramatically reduces its ability to repress collagen gene expression (23Hofmann C.S. Sullivan C.P. Jiang H.Y. Stone P.J. Toselli P. Reis E.D. Chereshnev I. Schreiber B.M. Sonenshein G.E. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1608-1613Crossref PubMed Scopus (10) Google Scholar, 24Petrovas C. Jeay S. Lewis R.E. Sonenshein G.E. Oncogene. 2003; 22: 2011-2020Crossref PubMed Scopus (7) Google Scholar). Consistent with our observations, B-Myb inhibits c-Myb-mediated transactivation of the α2(I) collagen promoter in scleroderma fibroblasts (25Luchetti M.M. Paroncini P. Majlingova P. Frampton J. Mucenski M. Baroni S.S. Sambo P. Golay J. Introna M. Gabrielli A. J. Biol. Chem. 2003; 278: 1533-1541Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) and represses the α1(I) collagen gene transcription via interaction with Sp1 and CBF factors (26Cicchillitti L. Jimenez S.A. Sala A. Saitta B. Biochem. J. 2004; 378: 609-616Crossref PubMed Google Scholar). SMCs are the major cellular constituents of the medial layer of an artery and are responsible for the synthesis and deposition of connective tissue proteins, including elastin, that maintain vascular tone in the adult blood vessel (27Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19339) Google Scholar, 28Ross R. Klebanoff S.J. J. Cell Biol. 1971; 50: 159-171Crossref PubMed Scopus (237) Google Scholar, 29Kadar A. Gardner D.L. Bush V. J. Pathol. 1971; 104: 253-260Crossref PubMed Scopus (38) Google Scholar). Elastin is one of the major structural proteins of large arteries, contributing the physical properties of extensibility and elastic recoil. Elastin is synthesized as a soluble monomeric precursor called tropoelastin, which has an apparent molecular mass of 62–75 kDa, depending on animal species and isoform (30Foster J.A. Curtiss S.W. Am. J. 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The number of lamellar units appears to be species-specific, fixed, and genetically predetermined (34Wolinsky H. Glagov S. Circ. Res. 1967; 20: 99-111Crossref PubMed Scopus (541) Google Scholar). During the development of the aorta, synthesis of elastin occurs very early, and its expression is regulated, in part, at the transcriptional level (35Perrin S. Foster J.A. Crit. Rev. Eukaryot. Gene Expr. 1997; 7: 1-10Crossref PubMed Scopus (19) Google Scholar). Once the artery has been fully formed, SMCs differentiate into a contractile phenotype in which they normally remain (36Chamley-Campbell J. Campbell G.R. Ross R. Physiol. Rev. 1979; 59: 1-61Crossref PubMed Scopus (1266) Google Scholar). In culture, vascular SMCs exhibit a synthetic phenotype, and an inverse relationship exists between cell proliferative state and matrix deposition (36Chamley-Campbell J. Campbell G.R. Ross R. Physiol. Rev. 1979; 59: 1-61Crossref PubMed Scopus (1266) Google Scholar). In exponential growth, cultured SMCs synthesize low levels of matrix proteins, and expression of matrix genes is induced when SMCs reach confluence or are deprived of serum growth factors (37Beldekas J.C. Gerstenfeld L. Sonenshein G.E. Franzblau C. J. Biol. Chem. 1982; 257: 12252-12256Abstract Full Text PDF PubMed Google Scholar, 38Chang C.J. Sonenshein G.E. Matrix. 1991; 11: 242-251Crossref PubMed Scopus (10) Google Scholar, 39Kindy M.S. Chang C.J. Sonenshein G.E. J. Biol. Chem. 1988; 263: 11426-11430Abstract Full Text PDF PubMed Google Scholar). Specifically, it has been shown that the expression of elastin varies inversely with the growth rate of SMCs (40Barone L.M. Wolfe B.L. Faris B. Franzblau C. Biochemistry. 1988; 27: 3175-3182Crossref PubMed Scopus (12) Google Scholar, 41Toselli P. Faris B. Sassoon D. Jackson B.A. Franzblau C. Matrix. 1992; 12: 321-332Crossref PubMed Scopus (15) Google Scholar, 42Tajima S. Keio J. Med. 1996; 45: 58-62Crossref PubMed Scopus (7) Google Scholar). Confluent primary chick vascular SMCs displayed a dramatic increase in elastin mRNA levels upon serum deprivation, and when serum was added back to the cultures, cell proliferation was induced, and elastin mRNA levels dropped (43Wachi H. Seyama Y. Yamashita S. Tajima S. Biochem. J. 1995; 309: 575-579Crossref PubMed Scopus (23) Google Scholar). Similarly, it was shown that types I, III, and V/XI collagen mRNA levels were relatively low when SMCs were subconfluent and growing exponentially, and levels increased as cells become confluent (37Beldekas J.C. Gerstenfeld L. Sonenshein G.E. Franzblau C. J. Biol. Chem. 1982; 257: 12252-12256Abstract Full Text PDF PubMed Google Scholar, 43Wachi H. Seyama Y. Yamashita S. Tajima S. Biochem. J. 1995; 309: 575-579Crossref PubMed Scopus (23) Google Scholar, 44Brown K.E. Lawrence R. Sonenshein G.E. J. Biol. Chem. 1991; 266: 23268-23273Abstract Full Text PDF PubMed Google Scholar, 45Stepp M.A. Kindy M.S. Franzblau C. Sonenshein G.E. J. Biol. Chem. 1986; 261: 6542-6547Abstract Full Text PDF PubMed Google Scholar) or upon serum starvation, which renders SMCs quiescent (39Kindy M.S. Chang C.J. Sonenshein G.E. J. Biol. Chem. 1988; 263: 11426-11430Abstract Full Text PDF PubMed Google Scholar, 46Kindy M.S. Sonenshein G.E. J. Biol. Chem. 1986; 261: 12865-12868Abstract Full Text PDF PubMed Google Scholar). Recently, to explore the role of B-Myb on collagen gene expression in vascular SMCs in vivo, we studied the effects of overexpression of the human B-myb gene in a transgenic mouse model. We observed a reduction in type I and type V collagen expression in adult animals and a reduction in neointima formation upon vascular injury (23Hofmann C.S. Sullivan C.P. Jiang H.Y. Stone P.J. Toselli P. Reis E.D. Chereshnev I. Schreiber B.M. Sonenshein G.E. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1608-1613Crossref PubMed Scopus (10) Google Scholar). Here, these transgenic mice are characterized with respect to elastin gene expression. A substantial reduction in tropoelastin mRNA expression is observed in aortas as well as isolated aortic SMCs from adult B-myb transgenic versus age-matched wild type (WT) animals, although measurements of insoluble elastin and numbers of elastic layers in the vessel wall revealed no differences between WT and transgenic animals. Importantly, elastin mRNA levels are not reduced in neonatal transgenic mice, apparently due to high cyclin A expression, which ablates B-Myb-mediated repression of the elastin promoter. Thus, these findings help to elucidate the mechanism of B-myb-mediated resistance to vascular injury that we reported recently (23Hofmann C.S. Sullivan C.P. Jiang H.Y. Stone P.J. Toselli P. Reis E.D. Chereshnev I. Schreiber B.M. Sonenshein G.E. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1608-1613Crossref PubMed Scopus (10) Google Scholar) and implicate cyclin A and B-Myb in developmental regulation of expression of elastin, a key structural component of the vessel wall. Isolation and Culture of SMCs—FVB mice overexpressing human B-myb (2300-bp human B-myb cDNA driven by the basal cytomegalovirus (CMV) promoter) in the aorta were generated as described previously (23Hofmann C.S. Sullivan C.P. Jiang H.Y. Stone P.J. Toselli P. Reis E.D. Chereshnev I. Schreiber B.M. Sonenshein G.E. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1608-1613Crossref PubMed Scopus (10) Google Scholar). Murine SMCs were prepared as described previously (23Hofmann C.S. Sullivan C.P. Jiang H.Y. Stone P.J. Toselli P. Reis E.D. Chereshnev I. Schreiber B.M. Sonenshein G.E. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1608-1613Crossref PubMed Scopus (10) Google Scholar). Briefly, the aortas of 3-month-old female CMV-B-myb transgenic or WT FVB mice (Taconic Farms) were removed from the aortic arch to the femural bifurcation, minced, and subjected to digestion with 0.5 μg/ml bacterial collagenase (Sigma type I) and 0.125 μg/ml porcine pancreatic elastase (Sigma type III) in Dulbecco's modified Eagle's medium (DMEM) supplemented with nonessential amino acids, 1 mmol/liter sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) (complete DMEM). SMCs were collected by centrifugation at 400 × g for 10 min at room temperature and grown in complete DMEM containing 10% fetal bovine serum. Second passage cultures were subjected to immunohistochemistry with an antibody against SMC α-actin, which demonstrated that contamination with endothelial cells and fibroblasts was negligible (data not shown). Bovine aortic SMCs, derived by explants from the aortic arches of female calves, were grown in complete DMEM plus 10% fetal bovine serum as we described previously (37Beldekas J.C. Gerstenfeld L. Sonenshein G.E. Franzblau C. J. Biol. Chem. 1982; 257: 12252-12256Abstract Full Text PDF PubMed Google Scholar). Cell culture medium was changed every 2–3 days, and SMCs were used between passages 2 and 6. RNA Isolation—For extraction of total RNA from aortas, the region from the aortic arch to the diaphragm was removed from age-matched mice, and samples (100–200 mg) were frozen and homogenized in liquid nitrogen. RNA was prepared using the Ultraspec-II RNA Isolation System (Biotecx Laboratories, Inc.) according to the manufacturer's instructions. Total RNA was isolated from cultured SMCs by guanidinium isothiocyanate extraction followed by purification on CsCl density gradients, as described (47.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1987)Google Scholar). Northern Blot Analysis—For Northern blot analysis, RNA samples (12 μg for RNA from tissues and 20 μg for RNA from cultured SMCs) were denatured and separated by electrophoresis in 1.0% formaldehyde agarose gels (48Dean M. Kent R.B. Sonenshein G.E. Nature. 1983; 305: 443-446Crossref PubMed Scopus (37) Google Scholar). RNA was stained with ethidium bromide to verify integrity and equal loading and then transferred to nylon membranes. Probes, prepared as described (21Kypreos K.E. Nugent M.A. Sonenshein G.E. Cell Growth Differ. 1998; 9: 723-730PubMed Google Scholar), included human B-myb (2300-bp cDNA), elastin (1.1-kb fragment of rat cDNA (49Rich C.B. Foster J.A. Arch. Biochem. Biophys. 1989; 268: 551-558Crossref PubMed Scopus (26) Google Scholar)), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (600-bp cDNA fragment). Quantitation by scanning densitometry was performed using KDS1D version 2.0 (Eastman Kodak Co.). RT-PCR—RNA was digested for 30 min at 37 °C with RQ1 RNase-free DNase (Promega) according to the manufacturer's directions. Reverse transcription (RT) was performed using 5 μg of RNA in the presence or absence of SUPERSCRIPT™ RNase H– reverse transcriptase and 200 ng of random primers. PCRs were performed using one-fortieth volume of the total RT reaction and Taq DNA polymerase according to the manufacturer's instructions (Invitrogen). A primer pair for the cyclin A2 gene, located at bp 789 and 1334 within the mouse cDNA sequence (5′-GAGACCCTGCATTTGGCTGTG-3′ and 5′-GGTAGGTCTGGTGAAGGTCC-3′), yielding a fragment of 550 bp, was employed. To control for RNA integrity, the following primer pair was employed, which amplified a 600-bp RT-PCR product of GAPDH: 5′-TCACCATCTTCCAGGAG-3′ and 5′-GCTTCACCACCTTCTTG-3′. Annealing of the reaction was performed at 63 °C for cyclin A2 (28 cycles) and at 53 °C for GAPDH (20 cycles). Immunoblot Analysis—For preparation of protein extracts, tissues from age-matched B-myb transgenic and WT mice were frozen in liquid nitrogen and then homogenized in radioimmune precipitation buffer (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 1% sodium sarcosyl, 0.2 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 mm dithiothreitol, 3 μg/ml aprotinin) using a Polytron homogenizer (Kinematica GmBH). Following incubation on ice for 30 min, the DNA was sheared either by sonication for 5–10 s or by passing the lysate 20 times through a 23-gauge and then a 25-gauge needle. The debris was removed by centrifugation at 13,000 rpm in an Eppendorf centrifuge for 30 min at 4 °C. Protein concentration was determined using the Bio-Rad Dc protein assay (Bio-Rad). Proteins were resolved in 10% polyacrylamide-SDS gels and subjected to immunoblotting, as we described previously (50Sovak M.A. Bellas R.E. Kim D.W. Zanieski G.J. Rogers A.E. Traish A.M. Sonenshein G.E. J. Clin. Invest. 1997; 100: 2952-2960Crossref PubMed Scopus (650) Google Scholar). A goat antibody against rat tropoelastin (RA75) was purchased from Elastin Products. The antibodies against human B-Myb (sc-724) and β-actin were purchased from Santa Cruz Biotechnology, Inc. and Sigma, respectively. The rabbit polyclonal cyclin A antibody (A5) was purchased from Lab Vision. RNA Isolation and Gene Microarray Analysis—Aortas were removed from C57BL/6 embryos at embryonic day 12, 14, 16, and 18, postnatal days 0 (representing the first 24 h of life), 4, 7, 10, 14, 21, 30, and 60, and 5.5 months of life. Total RNA was extracted from each sample of pooled aortas (10–14 aortas per time point) using a modified guanidinium/phenol extraction method. RNA quality and purity was evaluated with the use of an RNA 6000 Nano Labchip (Agilent, Palo Alto, CA) and Agilent 2100 bioanalyzer per the manufacturer's protocol (Agilent). cRNA target and gene chip hybridization were performed by the Multiplexed Gene Analysis Core Facility of the Siteman Cancer Institute at the Washington University in St. Louis School of Medicine. A duplicate of the P7 time point was taken and analyzed, which indicated that the variability was minimal. The MU74Av2 chips were scanned, and the intensities of the images were analyzed by the GeneChip Analysis Suite software (Affymetrix). All chip intensities were scaled by the software to an average of 1500 units. Probe sets were removed from the analysis if the difference between their maximum and minimum raw average difference values over the time series was less than 300. Details of the array procedures, sample selection and preparation, and data analysis can be found as described. 2S. E. McLean, B. H. Mecham, T. J. Mariani, S. Corry, C. H. Ciliberto, and R. P. Mecham, manuscript in preparation. Transient Transfection Assays—Bovine adult aortic SMCs were plated at 60 × 104 cells/well in 6-well dishes and transfected the following day using Lipofectamine (2.4–3.2 μg of DNA total in 5 μl of Lipofectamine reagent) according to the manufacturer's instructions (Invitrogen). The following vectors were employed: elastin 2.2-CAT and elastin 0.5-CAT, which contain 2.2 and 0.5 kb of the proximal region of the human elastin promoter linked to a chloramphenicol acetyltransferase (CAT) reporter (kindly provided by J. Rosenbloom, University of Pennsylvania, Philadelphia, PA) (51Rich C.B. Fontanilla M.R. Nugent M. Foster J.A. J. Biol. Chem. 1999; 274: 33433-33439Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar); pB14, bovine B-myb expression vector (3Marhamati D.J. Sonenshein G.E. J. Biol. Chem. 1996; 271: 3359-3365Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar); and pCMVcyclinA, a human cyclin A expression vector, generously provided by Jim Xiao (Boston University School of Medicine, Boston, MA). Cells were harvested 72 h after transfection, and CAT activity was analyzed as described (21Kypreos K.E. Nugent M.A. Sonenshein G.E. Cell Growth Differ. 1998; 9: 723-730PubMed Google Scholar). Verhoeff and Van Gieson Staining—Age-matched 5-week-old to 2-month-old B-myb transgenic and WT mice were perfused with 5 ml of 1× phosphate-buffered saline followed by 10 ml of 4% paraformaldehyde, as described previously (23Hofmann C.S. Sullivan C.P. Jiang H.Y. Stone P.J. Toselli P. Reis E.D. Chereshnev I. Schreiber B.M. Sonenshein G.E. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1608-1613Crossref PubMed Scopus (10) Google Scholar). The aortas were dissected out, fixed overnight in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Five-μm cross-sections were prepared and subjected to Verhoeff and Van Gieson staining with the Accustain Elastic Stain kit (catalog no. HT25-A; Sigma) according to the manufacturer's instructions. Layers of elastic lamellae were analyzed by counting the stained sections of the aortas. Measurement of Total Insoluble Aortic Elastin Protein—To measure the levels of insoluble elastin, aortas from age-matched adult (3-monthold) B-myb transgenic and WT mice were dissected out, weighed, homogenized, and incubated in 0.1 n NaOH at 95 °C for 45 min to isolate insoluble elastin. The insoluble residue was subjected to acid hydrolysis and amino acid analysis (Beckman model 6300 with System Gold software) to determine the quantity of the elastin (52Lansing A.I. Rosenthal T.B. Alex M. Dempsey E.W. Anat. Rec. 1952; 114: 555-575Crossref PubMed Scopus (334) Google Scholar, 53Stone P.J. McMahon M.P. Morris S.M. Calore J.D. Franzblau C. In Vitro Cell Dev. Biol. 1987; 23: 663-676Crossref PubMed Scopus (23) Google Scholar, 54Stone P.J. Morris S.M. Thomas K.M. Schuhwerk K. Mitchelson A. Am. J. Respir. Cell Mol. Biol. 1997; 17: 289-301Crossref PubMed Scopus (32) Google Scholar, 55Starcher B.C. Galione M.J. Anal. Biochem. 1976; 74: 441-447Crossref PubMed Scopus (165) Google Scholar). The amount of elastin was calculated as the sum of the amino acids (in nmol) multiplied by the average amino acid mass of 85 ng/nmol. Nonelastin protein in the aorta was measured by combining the supernatant from the 0.1 n NaOH incubation with an equal volume of 12 n HCl followed by hydrolysis and amino acid analysis, as described above. Protein was estimated by multiplying the sum of the amino acids (in nmol) by 100 ng/nmol. The mean value for the elastin content of the aortas normalized by total protein of the WT group of mice (n = 6) were compared with those for Line 2, 4, and 16 transgenic mice using the Bonferroni/Dunn procedure for the analysis of variance. Elastin mRNA Levels Are Reduced in the Aorta of Adult Transgenic B-myb Mice—Recently, we demonstrated that B-Myb represses type I collagen gene expression in the aorta and isolated aortic SMCs from adult transgenic mice expressing human B-myb (23Hofmann C.S. Sullivan C.P. Jiang H.Y. Stone P.J. Toselli P. Reis E.D. Chereshnev I. Schreiber B.M. Sonenshein G.E. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1608-1613Crossref PubMed Scopus (10) Google Scholar). Thus, it was of interest to evaluate whether B-Myb affects the expression of elastin, another matrix gene important for the architecture of the vessel wall. In the previous report, the characterization of three B-myb transgenic mouse lines (Lines 2, 4, and 16) was described. It was demonstrated that the level of expression of B-Myb was greatest in Line 16, followed by Line 4, with the least amount of B-Myb in Line 2. RNA was isolated from pooled aortas of the three 6–10-week-old B-myb transgenic mouse lines as well as from age-matched WT mouse aortas. As in the previous report, Northern blot analysis was performed for elastin gene expression, and the data were normalized for the levels of GAPDH. Using the average of six independent experiments with age-matched animals, Lines 2, 4, and 16 displayed 40.6 ± 23.0%, 38.4 ± 12.6%, and 21.4 ± 17.1% of WT normalized elastin mRNA levels, respectively (Fig. 1A). These data indicate that there is a statistically significant decrease in elastin mRNA expression in aortas from all of the lines versus WT mice. The relationship between the levels of expression of B-Myb protein and elastin mRNA was determined, using previously reported values for the levels of B-Myb protein in the aortas of the three transgenic mouse lines (23Hofmann C.S. Sullivan C.P. Jiang H.Y. Stone P.J. Toselli P. Reis E.D. Chereshnev I. Schreiber B.M. Sonenshein G.E. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1608-1613Crossref PubMed Scopus (10) Google Scholar), specifically 1.6-, 2.5-, and 3.2-fold overexpression of B-Myb, respectively (Fig. 1B). These data suggest that elastin is another matrix gene target of repression by B-Myb in the aorta. Soluble Elastin Levels Are Reduced in the Aorta of Adult Transgenic Mice—To evaluate the effects of B-Myb on intracellular tropoelastin levels, the two transgenic lines that expressed the highest levels of B-Myb were selected for study. Total soluble protein extracts were prepared from individual aortas from WT and transgenic Line 16 and 4 mice and subjected to immunoblot analysis for soluble tropoelastin protein, as well as for β-actin, as control to normalize for differences in loading (Fig. 2, A and B, respectively). Tropoelastin was substantially reduced in three of three aortas from transgenic Line 16 tested as compared with the fo
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