Microfibril-associated glycoprotein-1 and fibrillin-2 are associated with tropoelastin deposition in vitro

The International Journal of Biochemistry & Cell Biology 37 (2005) 120–129

Microfibril-associated glycoprotein-1 and fibrillin-2 are associated with tropoelastin deposition in vitro Eichi Tsuruga∗ , Toshihiko Yajima, Kazuharu Irie Department of Oral Anatomy, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan Received 20 April 2004; accepted 1 June 2004

Abstract Elastic system fibers consist of microfibrils and tropoelastin. During development, microfibrils act as a template on which tropoelastin is deposited. Microfibril-associated glycoprotein-1 (MAGP-1) and fibrillin-2, the major components of microfibrils, provide the likely template for tropoelastin deposition. In this study, we used the RNA interference (RNAi) technique to establish MAGP-1 and fibrillin-2 gene-specific knock-downs individually in elastin-producing cells (human gingival fibroblasts). We then examined the extracellular deposition of tropoelastin by western blotting. These two genes were specifically suppressed to <30% of the control level, and this was responsible for the diminution of tropoelastin deposition. An immunofluorescence study also confirmed that RNAi-mediated down-regulation of MAGP-1 or fibrillin-2 led to the loss of tropoelastin immunoreactivity. These results suggest that MAGP-1 and fibrillin-2 are, directly or indirectly, associated with the extracellular deposition of tropoelastin during elastic fiber formation in human gingival fibroblasts in vitro. © 2004 Elsevier Ltd. All rights reserved. Keywords: Elastin deposition; Elastogenesis; Fibrillin; MAGP; Tropoelastin

1. Introduction Elastic system fibers are an integral part of the extracellular matrix, which is distributed in blood vessels, lung, periodontal tissue and skin (Rosenbloom, Abrams, & Mecham, 1993). These fibers comprise two distinct components: microfibrils and tropoelastin, which is a soluble precursor of cross-linked elastin. Mutations in microfibril components cause Abbreviations: GAPDH, glyceroaldehyde-3-phosphate dehydrogenase; HGF, human gingival fibroblasts; MAGP, microfibrilassociated glycoprotein; MEM, minimum essential medium; siRNA, small interference RNA ∗ Corresponding author. Tel.: +81 133 23 1904; fax: +81 133 23 1218. E-mail address: [email protected] (E. Tsuruga).

severe heritable connective tissue diseases such as Marfan syndrome (Dietz, Ramirez, & Sakai, 1994) and congenital contractural arachnodactyly (Putnam, Zhang, Ramirez, & Milewicz, 1995). During elastic fiber formation, microfibrils act as a structural template on which tropoelastin is deposited (Kielty & Shuttleworth, 1995; Mecham & Davis, 1994). Among the microfibrillar molecules, fibrillins and microfibril-associated glycoprotein (MAGP) are the best characterized (Gibson et al., 1996; Henderson, Polewski, Fanning, & Gibson, 1996; Sakai, Keene, & Engvall, 1986; Zhang et al., 1994). Recent in vitro binding assays have revealed microfibrillar interaction with tropoelastin (Kielty, Sherratt, & Shuttleworth, 2002). Two isoforms of fibrillin – fibrillin-1 and fibrillin-2 – bind to tropoelastin (Trask et al., 2000).

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E. Tsuruga et al. / The International Journal of Biochemistry & Cell Biology 37 (2005) 120–129

Fibrillin-2 is found preferentially in elastic tissues such as elastic cartilage, the aorta and lung (Zhang et al., 1994), whereas fibrillin-1 is expressed in load-bearing tissues such as the aortic adventitia, the ciliary zonules and the skin (Zhang, Hu, & Ramirez, 1995). Recently, Hill, Mecham, and Starcher (2002) demonstrated in an immunohistochemical study that loss of fibrillin-2 is correlated with abnormal elastic fiber formation in chick aorta. Therefore, it is thought that fibrillin-2 may play an important role in the deposition of tropoelastin. On the other hand, MAGP-1, whose binding site is a tyrosine-rich sequence, can bind to tropoelastin at the N-terminal half (Brown-Augsburger et al., 1994). MAGP-1 is localized to the bead surface of individual microfibrils (Henderson et al., 1996), suggesting that MAGP-1 may participate in the binding of tropoelastin to the microfibril and its alignment during elastogenesis. However, it still remains unclear which microfibrillar molecule is the most important template for tropoelastin deposition. We have biochemically investigated the metabolism of elastic system fibers by comparing elastin-producing cells and elastin-non-producing cells (Tsuruga, Irie, Sakakura, & Yajima, 2002a, 2002b; Tsuruga, Irie, & Yajima, 2002; Tsuruga, Yajima, & Irie, 2004). Previously, it has been reported that human fibroblasts express fibrillin-1 and form normal microfibrils, as determined by rotary shadowing electron microscopy (Kettle, Card, Hutchinson, Sykes, & Handford, 2000). We have shown that gene expression and secretion of fibrillin-1 and fibrillin-2 in elastin-producing cells far exceeds those in elastin-non-producing cells. We have also demonstrated that gene expression of fibrillin-2 is correlated with that of tropoelastin (Tsuruga et al., 2002) and with the ultrastructural features of microfibrils bearing deposited elastin (Kunimoto, Fujii, Irie, Sakakura, & Yajima, 1999). However, the mechanism involved in tropoelastin deposition is not fully understood. To gain a better understanding of the role of microfibrils, we generated MAGP-1 and fibrillin-2 knock-down cells individually using the RNA interference (RNAi) technique to carry out biochemical analyses of tropoelastin deposition in cultures of human gingival fibroblasts (HGF). We found that siRNA-mediated gene silencing of human MAGP-1 and fibrillin-2 reduces extracellular deposition of tropoelastin.

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2. Materials and methods 2.1. Cell culture The study protocol had been reviewed and approved by the Health Sciences University of Hokkaido Research Ethics Committee. Informed consent was obtained from the tissue donors. HGF were isolated and cultured as described previously (Mochizuki, Yamaguchi, & Abiko, 1999; Palmon et al., 2000). HGF express significant amounts of microfibrils and tropoelastin (Tsuruga et al., 2002a, 2002). The cells were then cultured at 37 ◦ C in humidified air containing 5% CO2 in minimum essential medium (MEM; ICN Biomedicals Inc., Aurora, OH, USA) supplemented with non-essential amino acids (ICN Biomedicals Inc.) and 10% newborn calf serum (NCS; Life Technologies, Grand Island, NY, USA). Prior to use, the cells were trypsinized and seeded in 60 mm culture dishes at a density of 1 × 106 cells/dish (Nulge Nunc International, Roskilde, Denmark). The culture medium consisted of MEM supplemented with non-essential amino acids, 10% NCS, 100 U/ml penicillin and 100 ␮g/ml streptomycin. Cells were sampled from the third to the fifth passage of culture. All intrasubject comparisons yielded similar results for each experiment. 2.2. Small interference RNA (siRNA) design and transient transfection siRNAs for human MAGP-1 and fibrillin-2 were designed with 3 overhanging thymidine dimers, as described previously (Elbashir et al., 2001). The Gene SilencerTM siRNA preparation kit (Ambion Inc., Austin, TX, USA) was used to synthesize specific MAGP-1 and fibrillin-2 siRNAs using DNA primers. Using this kit, siRNAs are in vitro-transcribed from a 29-mer DNA primer including the specific 21-nucleotide sequence and an 8-nucleotide leader sequence used to facilitate T7 polymerase binding and transcription. The sense and antisense siRNA sequences used are summarized in Table 1. The synthesized siRNAs corresponded to the 438–456 coding region of MAGP-1 (accession no. U19718) and the 9138–9156 region of fibrillin-2 (accession no. MN 001999). BLAST searches of the database indicated that these siRNAs are specific for MAGP-1

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Table 1 Sequences of siRNAs MAGP-1

mRNA Sense Antisense

5 -AACAAGGAGAUCUGUGUUCGU-3 5 -CAAGGAGAUCUGUGUUCGUdTdT-3 5 -ACGAACACAGAUCUCCUUGdTdT-3

Fibrillin-2

mRNA Sense Antisense

5 -AAUGCAGUAAUAUAUGGAGAA-3 5 -UGCAGUAAUAUAUGGAGAAdTdT-3 5 -UUCUCCAUAUAUUACUGCAdTdT-3

and fibrillin-2, and have no homology with other proteins. The siRNA was transfected into HGF using the siPORT Amine Transfection Agent (Ambion). Briefly, 12–16 h before the first siRNA transfection, HGF were trypsinized and seeded at 1 × 106 cells per 60 mm culture dish with MEM/10% NCS without antibiotics (1.5 ml/dish). Transient transfection of siRNAs was performed using the siPORT Amine Transfection Agent (Ambion). First, 36 ␮l of OptiMEM medium/dish (Invitrogen, Gland Island, NY, USA) and 9 ␮l of the siPORT Amine Transfection Agent were preincubated for 10 min at room temperature. During this time, 150 ␮l of OptiMEM medium was mixed with 9 ␮l of 20 ␮M siRNA. The two mixtures were then combined and incubated for 20 min at room temperature to allow complex formation. After addition of 96 ␮l of OptiMEM medium to the mixture, the entire mixture was added to the cells in one dish, resulting a final concentration of 100 nM for the siRNAs. Mock transfections of cultures with the siPORT Amine Transfection Agent alone were used as a control. HGF were transfected twice with the siRNA duplex (100 nM), with a 72 h interval between, using the siPORT Amine Transfection Agent and cultured for a further 72 h. 2.3. RNA preparation and cDNA synthesis Total RNA was extracted from the cell layers at 72 h after the first transfection with an RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Total RNA treated with RNase-free DNase (Qiagen) was reverse-transcribed into cDNA using an oligo(dT)12–18 primer (Invitrogen, Carlsbad, CA, USA). One microgram of the total RNA was reverse-transcribed with the oligo(dT)12–18 primer at 1 ␮M using the Omniscript reverse transcription system (Qiagen). Briefly, cDNA was synthesized

from 1 ␮g of the total RNA for 1 h at 37 ◦ C in a final volume of 20 ␮l buffer (50 mM Tris–HCl, pH 8.3, 75 mM KCl, and 3 mM MgCl2 ) supplemented with 0.5 mM deoxy-NPTs, 2.5 mM DTT, 10 U of RNase inhibitor (Promega, Madison, WI, USA), and 4 U of reverse transcriptase. After incubation, the cDNAs were heated to 93 ◦ C and stored at −20 ◦ C until used for amplification by polymerase chain reaction (PCR). 2.4. PCR analysis For PCR amplification, specific oligonucleotide primers for human MAGP-1, fibrillin-2, fibrillin-1, tropoelastin and glyceroaldehyde-3-phosphate dehydrogenase (GAPDH) sequences were designed on the basis of sequences in GeneBank (Table 2). The reaction was performed using the Taq PCR Master Mix Kit (Qiagen) as follows: 1 ␮l of cDNA was used as the template in a 20 ␮l amplification mixture containing of 1 U of Taq DNA polymerase, 0.5 mM each of the 5 and 3 primers, and distilled water. Experiments were performed to determine the optimal number of cycles that would yield a linear phase of amplification. PCR was performed in a Gene AMP® PCR System 9700 (Applied Biosystems Japan Ltd., Tokyo, Japan) with an initial denaturation at 94 ◦ C for 2 min, followed by a cycle of 20 s at 94 ◦ C, 30 s at the temperature indicated in Table 2, extension at 72 ◦ C for 90 s, and an additional extension step at 72 ◦ C for 10 min. Amplification products were resolved by electrophoresis on a 2% (w/v) agarose gel. Ethidium bromide-stained agarose gels with separated PCR products were photographed under UV light and converted into TIFF images using Photoshop (Adobe, San Jose, CA). The signal intensity of DNA bands for MAGP-1, fibrillin-2, fibrillin-1 and tropoelastin was quantified using the NIH Image program (National Institutes of Health, Bethesda, MD, USA).

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Table 2 Primer sequences and amplification condition for PCR Tm (◦ C)

Cycle

MAGP-1

5 -CCCAAGCTTGTGAGGAACAGTACCCGT-3

(F) (R) 5 -CGGAATTCGATACTCCCCCAACCCGA-3

60

26

498

Fibrillin-2

(F) 5 -CCCAAGCTTTCAGCCTAGAGAGTGTCGAC-3 (R) 5 -GGAATTCAATACAGTAACCACGGTTGC-3

56

26

583

Fibrillin-1

(F) 5 -CCCAAGCTTGGAGAAGCACAAACCAAACT-3 (R) 5 -GGAATTCCCCCAATGGAAATACACGTC-3

56

26

698

Tropoelastin

(F) 5 -CGGAATTCACCTCTTAAGCCAGTTCCCG-3 (R) 5 -CCCAAGCTTCGGGAACACCTCCGACACTA-3

60

27

1100

GAPDH

(F) 5 -GCGGATCCCTCTGCTCCTCCTGTTCGAC-3 (R) 5 -GGAATTCTGACAAAGTGGTCGTTGAGG-3

60

20

998

Target gene

Primer sequence

All products were corrected for the level of GAPDH mRNA. 2.5. Western blot analysis At 72 h after the second transfection, cell layers were washed twice in phosphate-buffered saline (PBS), collected by scraping, transferred to a 1.5 ml microcentrifuge tube, and subjected to centrifugation to remove as much solution as possible. The pellets were suspended in a lysis buffer (50 mM Tris–HCl, pH 7.5, 1% Tween-20 in the presence of protein inhibitors: 5 mM ethylenediaminetetraacetic acid, 50 ␮M N-ethylmaleimide and 50 ␮M phenylmethylsulfonyl fluoride) and disrupted by sonication for 1 min. The supernatant after centrifugation was assayed for protein concentration using the BCA protein assay system (Pierce Chemical Co., Rockford, IL, USA), and designated the cell layer extract for Western analysis. For analysis of the medium, the transfection reagents were removed at 24 h after the second transfection, and the cells were exposed serum-free MEM for 48 h and collected medium were concentrated using VIVAPORE concentrators (VIVASCIENCE, Gloucestershire, UK). Proteins of cell lysates (5 ␮g) or medium (2 ␮g) were subjected to electrophoresis on 10% polyacrylamide gel. The electrophoresed proteins were transferred to an Immobilon-P membrane (Millipore, Bedford, MA, USA), placed in a tank blotter containing 25 mM Tris/192 mM glycine, pH 8.3, and electrophoresed at 15 V for 2 h. The blots were blocked

Product size (bp)

for 2 h with Block Ace (Dainippon Pharmaceuticals, Osaka, Japan) and then incubated for 2 h with the appropriate primary antibody (tropoelastin, 1:5000 dilution; actin, 1:200 dilution) and 10% Block Ace. Antibodies included BA4 — a monoclonal antibody against tropoelastin — and a polyclonal rabbit antibody against actin, both purchased from Sigma (Saint Louis, MO, USA). The blot was then incubated for 1 h with horseradish peroxidase-conjugated sheep anti-mouse or rabbit Ig(F(ab )2 ) at 1:5000 dilution with 10% Block Ace. Visualization of immunoreactive bands was carried out using either a SuperSignal West Pico SubstrateTM (Pierce) or an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Little Chalfont, UK). The blots were exposed to X-ray film so that the proteins could be visualized. To ensure equal loading from medium samples in each lane, the gels were silver-stained using a Silver Stain Kit (Wako Pure Chemical Industries, Osaka, Japan). Pre-stained SDS-polyacrylamide gel electrophoresis molecular mass markers (Bio-Rad Laboratories, Hercules, CA, USA) were run with each blot. 2.6. Immunofluorescence At 72 h after the second transfection, HGF were fixed in ice-cold 1% paraformaldehyde in PBS for 15 min. The culture dishes were rinsed with PBS, and treated with 6 M guanidine-HCl, 20 mM Tris, and 50 mM dithiothreitol (pH 8.0) for 15 min. Non-specific immunoreactivity was blocked with 1% bovine serum

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albumin in PBS for 1 h at room temperature. Then the cell layers were incubated for 2 h at room temperature with the appropriate primary antibody (tropoelastin, 1:50 dilution; MAGP-1, fibrillin-1, fibrillin-2, 1:80 dilution). Antibodies used for immunofluorescence included BA4 and a polyclonal rabbit antibody against MAGP-1, fibrillin-1 and fibrillin-2 (Elastin Products Co., Owensville, MO, USA). We had already confirmed the specificity of the fibrillin-1 and fibrillin-2 antibodies as reported previously (Tsuruga et al., 2002a). In this previous study immunoprecipitation was performed with each of the two antibodies from the HGF culture medium. The immunoprecipitated materials were analyzed by western blotting to confirm that the distinct proteins could be differentiated using the two antibodies. After rinsing in PBS, the cells were incubated in Alexa Fluo® 488-labeled goat anti-mouse IgG1 antibody or Alexa Fluo® 488-labeled goat anti-rabbit Ig (H + L) antibody (Molecular Probes, Eugene, OR, USA), diluted 1:100 with blocking buffer, for 1 h at room temperature. Following rinsing in PBS, immunoreactivity was observed using a fluorescence microscope (V13-6000, Keyence Co., Osaka, Japan).

3. Results 3.1. Specific RNA interference by siRNAs The semi-quantitative PCR analysis revealed that transfection of the siRNA duplex specific for MAGP-1 significantly inhibited MAGP-1 gene expression 72 h after the first transfection in HGF (69% depletion; Fig. 1). On the other hand, the expressions of fibrillin-1, fibrillin-2 and tropoelastin were not significantly affected by the MAGP-1 siRNA transfection, Fig. 1. Specific suppression of the MAGP-1 or fibrillin-2 gene in HGF by siRNAs. HGF were mock-transfected (lane 1) or transfected with the siRNA duplex for MAGP-1 (lane 2) and fibrillin-2 (lane 3). Equal amounts of cDNA were amplified with specific primers for MAGP-1, fibrillin-2, fibrillin-1, tropoelastin, and GAPDH. Each PCR cycle number was determined in the linear phase of amplification. The intensity of each band was normalized relative to that of GAPDH, and quantified by the NIH Image program, the intensity obtained for mock-transfection being set as 100%. Data represent the mean ± S.D. (standard deviation) of four separate experiments (* P < 0.05 vs. lane 1).

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indicating that inhibition of the MAGP-1 siRNA duplexes was specific. Specific inhibition was also evident with fibrillin-2 siRNA transfection (92% depletion). The same suppressions were observed 72 h after the second transfection (data not shown). 3.2. Deposition of tropoelastin on MAGP-1 siRNAor fibrillin-2 siRNA-transfected cell layers

Fig. 2. Effect of MAGP-1 or fibrillin-2 siRNA on deposition of tropoelastin in HGF. HGF were transfected by mock transfection (lane 1) or MAGP-1 (lane 2) or fibrillin-2 (lane 3) siRNA. Concentrated serum-free medium (2 ␮g) was subjected to Western blotting using anti-tropoelastin (left panel in (A)). Profiles for silver staining are shown to indicate the equal amount of protein electrophoresed (right panel in (A)). At 72 h after the second transfection, whole cell layer lysates (5 ␮g) were also detected using tropoelastin antibody (left panel in (B)). The blotting membranes used to detect tropoelastin were reprobed with anti-actin antibody as a loading control (right panel in (B)). The intensity of the tropoelastin band was normalized to actin, and showed that the density of the MAGP-1- or fibrillin-2 siRNA-transfected sample decreased significantly with mock-transfection (C). The intensity was obtained for mock-transfection set as 100%. The positions of the prestained molecular weight markers are indicated at the left of each gel. Data represent the mean ± S.D. (standard deviation) of four separate experiments (* P < 0.05 vs. lane 1).

The effect of MAGP-1 or fibrillin-2 gene suppression on tropoelastin deposition in HGF cell layers was investigated by western blotting. First, in order to verify that the level of tropoelastin secretion in the medium was correlated with that of the gene expression, we assayed tropoelastin in medium from the mock-, MAGP-1 siRNA- and fibrillin-2 siRNA-transfected cells. It is clear from Fig. 2A that the amount of tropoelastin secreted into the medium did not differ among the three samples, indicating that the siRNA transfections did not affect either the gene expression or the secretion of tropoelastin. Fig. 2B shows tropoelastin deposition in the cell layers, and Fig. 2C shows the intensity of the tropoelastin band normalized to actin. The intensity obtained with MAGP-1 siRNA transfection was decreased to <30% compared with mock-transfected samples; and that with fibrillin-2 siRNA transfection was decreased to <70%. The results of the immunohistochemical assay also supported the data from western blotting. Tropoelastin staining in the MAGP-1 siRNA- and fibrillin-2 siRNA-transfected cells was diminished in comparison with that in the mock-transfected cells (Fig. 3A–C). In addition, MAGP-1 staining was affected only by MAGP-1 siRNA transfection (Fig. 3D–F). Similarly, fibrillin-2 staining was affected only by fibrillin-2 siRNA transfection (Fig. 3G–I). Fibrillin-1 staining was observed in the mock-transfected cells and in the MAGP-1 siRNAand fibrillin-2 siRNA-transfected cells (Fig. 3J–L). These results confirmed that MAGP-1 and fibrillin-2 may be associated with tropoelastin deposition.

4. Discussion In the present study using the RNA interference technique, we have demonstrated for the first time

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Fig. 3. Blocking of tropoelastin deposition with MAGP-1 or fibrillin-2 siRNA. HGF were transiently mock-transfected (A, D, G, J), or transiently transfected with siRNAs for MAGP-1 (B, E, H, K) and fibrillin-2 (C, F, I, L). Immunofluorescence staining was performed with a tropoelastin antibody (A–C), anti-MAGP-1 antibody (D–F), anti-fibrillin-2 antibody (G–I) and anti-fibrillin-1 antibody (J–L). Tropoelastin staining was blocked by transfection with MAGP-1 or fibrillin-2 siRNA. Bars indicate 10 ␮m.

that MAGP-1 and fibrillin-2 have an effect on the extracellular deposition of tropoelastin. Recently, siRNA duplexes have become a new tool to specifically suppress the expression of endogenous genes (Elbashir et al., 2001). The gene knock-down by

MAGP-1 siRNA transfection was specific, since the transfection did not change the gene expressions of fibrillin-2 and tropoelastin, as shown in Fig. 1. Similarly, a specific effect of fibrillin-2 siRNA transfection was also observed. To our knowledge, this is the first

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study to have successfully utilized the siRNA gene silencing technique to achieve specific suppression of the MAGP-1 and fibrillin-2 genes. Fibrillin-2 has previously been demonstrated to have a close association with elastic fibers (Ramirez & Pereira, 1999; Zhang et al., 1994). Moreover, researchers have suggested that expression of fibrillin-2 participates in the assembly of elastic fibers during early morphogenesis (Ramirez & Pereira, 1999; Zhang et al., 1995). The tropoelastin-binding site of fibrillin-2 has been identified at the N-terminal domain (Trask et al., 2000). These previous reports suggest the possible participation of fibrillin-2 in elastic fiber formation. We have previously demonstrated a correlation between gene expression and matrix deposition of fibrillin-2 and tropoelastin in elastin-producing cells (Tsuruga et al., 2002). However, no cellular deposition assay has been carried out to determine whether fibrillin-2 is one of the factors determining the deposition of tropoelastin. Therefore, our present findings appear to be important, and confirm the association of fibrillin-2 with tropoelastin deposition. In fibrillin-2 mutant mice, Arteaga-Solis et al. (2001) have observed disorganized microfibrils labeled by fibrillin-1. They discussed that fibrillin-2 is required for proper microfibrillar assembly. In our in vitro study, a fibrillin-1 network was similarly observed even with fibrillin-2 knock-down. On the other hand, researchers using a fibrillin-2 null mice model demonstrated that normal microfibrils are visible and functional in elastic-rich tissues (Chaudhry et al., 2001). They suggested that loss of fibrillin-2 might be compensated for by increased fibrillin-1 expression, which could act as an alternative template for tropoelastin deposition. However, our results showed that fibrillin-1 gene expression did not increase by fibrillin-2 RNA interference. Culturing for longer periods of time may be necessary to compensate for the loss of tropoelastin deposition, because of the different compensation mechanisms in vivo and in vitro. At any rate, we have demonstrated that deposited tropoelastin decreased to <70% with fibrillin-2 gene knock-down, suggesting that fibrillin-2 may, directly or indirectly, be associated with some part of the process from the secretion of tropoelastin to its deposition. An immunoelectron microscopy study has demonstrated that MAGP-1 localizes on microfibrils (Henderson et al., 1996). Brown-Augsburger et al.

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(1994) showed that native MAGP-1 can bind to tropoelastin. In addition, it has been reported that deposition of tropoelastin in fetal bovine auricular chondroblasts can be inhibited by external addition of an antibody against MAGP-1 (Brown-Augsburger, Broekelmann, Rosenbloom, & Mecham, 1996). Therefore, MAGP-1 has been considered a candidate for tropoelastin deposition. By suppressing endogenous MAGP-1 gene expression, we indicated that MAGP-1 has a direct contribution to tropoelastin deposition, in agreement with the findings obtained by Brown-Augsburger et al. (1996). Our study is the first to employ MAGP-1 gene knock-down, at the cellular level, to analyze tropoelastin deposition. It is assumed that MAGP-1 residing on fibrillincontaining microfibrils can bind to tropoelastin to effect proper tropoelastin deposition (Henderson et al., 1996). MAGP-1 has a matrix-binding domain at its C-terminal that acts as a sufficient target for the extracellular matrix (Segade, Trask, Broekelmann, Pierce, & Mecham, 2002). MAGP-1 also interacts with type IV collagen (Finnis & Gibson, 1997). These findings further support the possibility that MAGP-1 is a bi-functional protein, with a microfibril-dependent role and a regulatory function through interacting with extracellular proteins other than microfibrils. Despite an abundant deposition of MAGP-1 in Fig. 3F, the level of tropoelastin deposition only decreased to <70%. It is unlikely that all of the deposited MAGP-1 is involved in a template of tropoelastin in this culture system. However, it is at least certain that suppression of the fibrillin-2 gene led to a decrease in tropoelastin deposition. Until now, three fibrillins had been identified and their developmental expressions examined. The developmental expression of fibrillin-1, the major fibrillin in adult tissue, has been examined in the embryo and postnatally (Quondamatteo et al., 2002; Zhang et al., 1995). Fibrillin-2 shows an overlapping distribution pattern with fibrillin-1, but is generally expressed earlier than fibrillin-1 (Zhang et al., 1995). Fibrillin-3 is mainly expressed in fetal tissues, and localizes in microfibrils such as the skin and lung (Corson, Charbonneau, Keene, & Sakai, 2004). Therefore, fibrillin-3, as well as two other fibrillins, is thought to play a role in tropoelastin deposition. Analysis of the expression of fibrillin-3 in periodontal tissues is currently under way.

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In most cell culture systems, tropoelastin is little incorporated into the cell layer, and most of it is secreted into the culture medium (Mecham, 1987). Fig. 2 shows that there were no differences in tropoelastin concentration in concentrated serum-free media in spite of the decreased tropoelastin deposition in siRNA-transfected cells. This may have been due to the small amount of tropoelastin deposition relative to the absolute rate of synthesis. In conclusion, we have obtained direct evidence supporting the involvement of both MAGP-1 and fibrillin-2 in the extracellular deposition of tropoelastin in human gingival fibroblasts cell cultures. However, in this study, it was difficult to compare the effects of MAGP-1 and fibrillin-2 on tropoelastin deposition quantitatively, because there were differences in the absolute amounts of MAGP-1 and fibrillin-2 synthesized, and the degree of gene suppression resulting from transfection with either MAGP-1 or fibrillin-2 siRNA.

Acknowledgements This work was partly supported by Grants-in-Aid for Scientific Research (nos. 14771227 and 15591942) from the Ministry of Education, Science, Sports and Culture of Japan, and for research projects from the Research Institute of Personalized Health Sciences in the Health Sciences University of Hokkaido.

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