Latent TGF-β-binding protein 2 binds to DANCE/fibulin-5 and regulates elastic fiber assembly
2007; Springer Nature; Volume: 26; Issue: 14 Linguagem: Inglês
10.1038/sj.emboj.7601768
ISSN1460-2075
AutoresMaretoshi Hirai, Masahito Horiguchi, Tetsuya Ohbayashi, Toru Kita, Kenneth R. Chien, Tomoyuki Nakamura,
Tópico(s)Cell Adhesion Molecules Research
ResumoArticle21 June 2007free access Latent TGF-β-binding protein 2 binds to DANCE/fibulin-5 and regulates elastic fiber assembly Maretoshi Hirai Maretoshi Hirai Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto, Japan Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Masahito Horiguchi Masahito Horiguchi Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto, Japan Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Tetsuya Ohbayashi Tetsuya Ohbayashi Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Toru Kita Toru Kita Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Kenneth R Chien Kenneth R Chien Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Tomoyuki Nakamura Corresponding Author Tomoyuki Nakamura Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto, Japan Department of Pharmacology, Kansai Medical University, Osaka, Japan Search for more papers by this author Maretoshi Hirai Maretoshi Hirai Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto, Japan Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Masahito Horiguchi Masahito Horiguchi Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto, Japan Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Tetsuya Ohbayashi Tetsuya Ohbayashi Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Toru Kita Toru Kita Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Kenneth R Chien Kenneth R Chien Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA Search for more papers by this author Tomoyuki Nakamura Corresponding Author Tomoyuki Nakamura Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto, Japan Department of Pharmacology, Kansai Medical University, Osaka, Japan Search for more papers by this author Author Information Maretoshi Hirai1,2,‡, Masahito Horiguchi1,2,‡, Tetsuya Ohbayashi1, Toru Kita2, Kenneth R Chien3 and Tomoyuki Nakamura 1,4 1Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto, Japan 2Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan 3Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA 4Department of Pharmacology, Kansai Medical University, Osaka, Japan ‡These authors contributed equally to this work *Corresponding author. Department of Pharmacology, Kansai Medical University, 10-15, Fumizono-cho, Moriguchi, Osaka, 570-8506, Japan. Tel.: +81 6 6993 9427; Fax: +81 6 6993 9428; E-mail: [email protected] The EMBO Journal (2007)26:3283-3295https://doi.org/10.1038/sj.emboj.7601768 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Elastic fibers play the principal roles in providing elasticity and integrity to various types of human organs, such as the arteries, lung, and skin. However, the molecular mechanism of elastic fiber assembly that leads to deposition and crosslinking of elastin along microfibrils remains largely unknown. We have previously shown that developing arteries and neural crest EGF-like protein (DANCE) (also designated fibulin-5) is essential for elastogenesis by studying DANCE-deficient mice. Here, we report the identification of latent transforming growth factor-β-binding protein 2 (LTBP-2), an elastic fiber-associating protein whose function in elastogenesis is not clear, as a DANCE-binding protein. Elastogenesis assays using human skin fibroblasts reveal that fibrillar deposition of DANCE and elastin is largely dependent on fibrillin-1 microfibrils. However, downregulation of LTBP-2 induces fibrillin-1-independent fibrillar deposition of DANCE and elastin. Moreover, recombinant LTBP-2 promotes deposition of DANCE onto fibrillin-1 microfibrils. These results suggest a novel regulatory mechanism of elastic fiber assembly in which LTBP-2 regulates targeting of DANCE on suitable microfibrils to form elastic fibers. Introduction Elastic matrices impart resilience and structural integrity to various types of human organs, such as the large arteries, lungs, and skin (Rosenbloom et al, 1993). They play especially vital roles in the functions of the arteries and lungs, which undergo repeated cycles of extension and recoil to maintain the blood pressure or breathing process. Injury and degeneration of elastic matrices are profoundly associated with age-related symptoms and diseases, such as arteriosclerosis, lung emphysema, and wrinkled skin (Pasquali-Ronchetti and Baccarani-Contri, 1997; Bailey, 2001). Elucidating the mechanisms of elastogenesis might lead to new treatments for these age-related symptoms. However, little is known about the molecular mechanism of elastic fiber assembly. We and others reported that mice deficient in a newly identified protein, developing arteries and neural crest EGF-like (DANCE) (also designated fibulin-5 or EVEC), exhibit a severely disorganized elastic fiber system throughout the body (Nakamura et al, 2002; Yanagisawa et al, 2002). DANCE is a secreted 66-kDa molecule, which colocalizes with elastic fibers and is abundantly expressed in developing arteries (Nakamura et al, 1999). We have recently demonstrated that DANCE has an elastogenic organizer activity, by showing that recombinant DANCE can induce elastogenesis in cell culture (Hirai et al, 2007). DANCE deposits on microfibrils, then promotes coacervation and crosslinking of tropoelastin molecules along microfibrils. Thus, DANCE is not only necessary for elastogenesis, but also able to promote elastic fiber organization. Therefore, DANCE may provide important clues to the molecular basis of elastogenesis. Elastic fibers are known to consist of two morphologically distinct components: microfibrils and polymerized elastin (Rosenbloom et al, 1993). Microfibrils are 10–12 nm filaments in the extracellular matrices, and mainly consist of large extended glycoproteins, fibrillin-1 and -2 (Sakai et al, 1986; Zhang et al, 1994), together with several kinds of proteins that are associated with them, such as latent transforming growth factor β-binding proteins (LTBPs) (Gibson et al, 1995; Taipale et al, 1995) and microfibril-associated glycoproteins (MAGPs) (Gibson et al, 1986, 1996). Microfibrils are considered to provide a scaffold for the orientated deposition of tropoelastin monomers and to play a central role in elastogenesis, although they can also form macroaggregates devoid of elastin (Ramirez et al, 2004). The expression of fibrillin-1 and -2 is differentially controlled, but overlaps in the development of elastic tissues (Mariencheck et al, 1995). Although fibrillin-1-mutated mice develop normal elastic matrices (Pereira et al, 1997) and fibrillin-2-mutated mice show syndactyly but normal elastogenesis (Arteaga-Solis et al, 2001; Chaudhry et al, 2001), mice deficient in both fibrillin-1 and -2 were recently reported to exhibit impaired elastogenesis (Carta et al, 2006). However, why only a part of microfibrils deposit elastin to form elastic fibers and how fibrillin-1 and -2 microfibrils are differentially utilized in elastogenesis remains unclear. These considerations prompted us to investigate whether there might be any microfibril molecules that regulate elastic fiber assembly. For example, LTBP proteins share a high degree of homology with fibrillins. There are four isoforms in the LTBP family (LTBP-1, -2, -3, and -4) (Kanzaki et al, 1990; Moren et al, 1994; Yin et al, 1995; Giltay et al, 1997; Saharinen et al, 1998; Hyytiainen et al, 2004). This family is named based on its ability to bind the latent form of transforming growth factor-β (TGF-β) and to modulate TGF-β bioactivity, but the family members also serve as structural components of extracellular matrices. Notably, LTBP-2 has the distinctive characteristic that it cannot interact with TGF-β, whereas LTBP-1, -3, and -4 can interact with TGF-β (Saharinen and Keski-Oja, 2000). In addition, LTBP-2 is reported to be specifically localized to the elastin-associated microfibrils (Gibson et al, 1995). These findings suggest that LTBP-2 may play an important role in elastogenesis unrelated to the regulatory function of TGF-β activity. To elucidate the molecular mechanisms of elastic fiber assembly and the roles of DANCE in elastogenesis, we attempted here to identify DANCE-binding proteins. Several groups have previously identified some DANCE-binding proteins, such as elastin (Yanagisawa et al, 2002; Freeman et al, 2005), lysyl oxidase-like 1 (Liu et al, 2004), EMILIN (Zanetti et al, 2004), apolipoprotein(a) (Kapetanopoulos et al, 2002), and extracellular superoxide dismutase (Nguyen et al, 2004). However, it is still not possible to fully understand the role that DANCE plays in elastic fiber development. In the present study, we demonstrate the interaction of DANCE and LTBP-2. We show that the middle calcium-binding epidermal growth factor-like domain (cbEGF-like domain) of DANCE specifically interacts with the amino- (N-) terminal domain of LTBP-2. Knockdown experiments using small interfering RNA (siRNA) reveal that deposition of DANCE and elastin is dependent on fibrillin-1, not on either fibrillin-2 or LTBP-2. Intriguingly, downregulation of LTBP-2 rescues fibrillar deposition of DANCE and elastin onto fibrillin-2 or other potential microfibrils, even in the absence of fibrillin-1. Moreover, recombinant LTBP-2 protein promotes fibrillar deposition of DANCE onto fibrillin-1 microfibrils. These results imply that LTBP-2 may function not only as a structural protein, but also as a regulatory protein that determines which microfibrils DANCE should deposit on for subsequent assembly of elastic fiber components. Results Latent TGF-β-binding protein-2 as a DANCE-binding protein To clarify the molecular role of DANCE in elastogenesis, we attempted to identify DANCE-binding proteins. For this purpose, we performed immunoprecipitation/SDS–PAGE. Bovine aortic smooth muscle cells were metabolically labeled with 35S-cysteine/methionine overnight, and the conditioned medium was harvested. An aliquot of the conditioned medium was combined with a purified FLAG-tagged DANCE recombinant protein or a mock control, followed by immunoprecipitation with a monoclonal anti-FLAG antibody. Other aliquots were subjected to immunoprecipitation with available antibodies against elastic fiber components, including elastin, fibrillin-1, fibrillin-2, and latent TGF-β-binding protein-2 (LTBP-2), to see whether there are these proteins in the conditioned medium. The immunoprecipitated complexes were separated by SDS–PAGE, and three major bands were detected as candidates for DANCE-binding proteins compared with the mock control (Figure 1, compare lanes 1 and 2, asterisks). The molecular weights of two of these bands are higher than 182 kDa, and that of the other is approximately 60 kDa (lane 2). We assumed that the larger molecules might be LTBP-2, because the molecular weight seems to be very similar to that of the protein immunoprecipitated with a monoclonal anti-LTBP-2 antibody (Figure 1, compare lanes 2 and 8). Fibrillin-1 and -2 were not detected, suggesting that these antibodies might not react with bovine fibrillin-1 and -2. In an analogous experiment using neonatal skin fibroblasts from wild-type mice, we found similar bands precipitated with the recombinant DANCE (data not shown). However, the 60-kDa band was not detected in the DANCE−/− neonatal fibroblast culture medium (data not shown); therefore, we infer that the 60-kDa molecule may be the DANCE itself, which is abundantly expressed in aorta (lane 2). To see whether these larger molecules are LTBP-2, we carried out double immunostaining of human skin fibroblasts with monoclonal antibodies against DANCE and LTBP-2. The result reveals that these two molecules colocalize, which suggests that they interact each other (Supplementary Figure S1). Figure 1.Latent TGF-β-binding protein-2 (LTBP-2) as a candidate DANCE-binding protein. Bovine smooth muscle cells were metabolically labeled with 35S-cysteine/methionine, and conditioned media were subjected to immunoprecipitation. Several DANCE-binding proteins are detected (lane 2, asterisks). The sizes of the bands larger than 182 kDa are similar to those of proteins precipitated with anti-LTBP-2 antibody (compare lanes 2 and 8). Another 60-kDa band is inferred to be DANCE, which is abundantly expressed in aortic smooth muscle cells (lane 2). Download figure Download PowerPoint LTBP-2 specifically interacts with the sixth calcium-binding EGF-like domain of DANCE To examine whether DANCE actually interacts with LTBP-2, we performed in vitro binding assays. Myc-tagged LTBP-2 proteins overexpressed by 293T cells were incubated with FLAG-tagged DANCE or a series of FLAG-tagged DANCE deletion mutant proteins (Figure 2A) also overexpressed by 293T cells. Each mixture was subjected to immunoprecipitation with anti-FLAG antibody, and then Myc-tagged proteins associated with the FLAG-tagged proteins were detected by Western blotting. As shown in Figure 2B, DANCE interacts with LTBP-2 (compare lane 1 with 11). Among the deletion mutant proteins, ΔM5-DANCE does not interact with LTBP-2 at all (Figure 2B, upper panel, lane 8). This result indicates that DANCE directly interacts with LTBP-2 through the sixth cbEGF-like domain. The carboxy- (C-) terminal domain of DANCE may also be involved in the interaction, as the binding of ΔC1- and ΔC2-DANCE with LTBP-2 was weaker than that of full-length DANCE with LTBP-2. Figure 2.Schematic representation of the DANCE deletion mutants, and mapping of the binding domain of DANCE with LTBP-2. (A) Domain structure of the full-length DANCE and the DANCE deletion mutants used for the in vitro binding assay. These mutants were expressed as C-terminally FLAG-tagged proteins. We prepared expression vectors encoding FLAG-tagged DANCE (FL-DANCE-FLAG) or DANCE deletion mutants, including N-terminal domain deletion mutants (ΔN1- and ΔN2-DANCE-FLAG), central calcium-binding EGF (cbEGF)-like repeat domain deletion mutants (ΔM1-, ΔM2-, ΔM3-, ΔM4-, and ΔM5-DANCE-FLAG), and C-terminal domain deletion mutants (ΔC1- and ΔC2-DANCE-FLAG). The asterisk indicates the sixth cbEGF-like domain of DANCE, where LTBP-2 binds. (B) The sixth cbEGF-like domain of DANCE interacts with LTBP-2. 293T cells were transiently transfected with the vectors shown in A or mock vector. Expression vectors for Myc-tagged full-length LTBP-2 (LTBP-2-Myc) were also independently transfected into 293T cells. Transfected cells were cultured in serum-free medium for 48 h, and then the cell lysates and the conditioned media were harvested and mixed. After incubation of LTBP-2-Myc with a set of FLAG-tagged DANCE deletion mutants, each mixture was subjected to immunoprecipitation with anti-FLAG antibody. These immunoprecipitates were then separated by SDS–PAGE, and analyzed by Western blotting with a monoclonal anti-Myc antibody. Download figure Download PowerPoint To see whether DANCE deletion mutants maintain their structural integrity, we performed a solid phase binding assay on recombinant tropoelastin. The result shows that ΔM5-DANCE binds tropoelastin as strongly as full-length DANCE (Supplementary Figure S2). We also carried out a deglycosylation assay using N-glycosidase F. Incubating ΔM5-DANCE with glycosidase did not change its molecular size, whereas the same glycosidase treatment caused size reduction of ΔM4-DANCE to a similar size of ΔM5-DANCE (Supplementary Figure S3). Taking into account that the sixth cbEGF-like domain contains putative N-glycosilation site (Nakamura et al, 1999) and that both mutants miss one cbEGF-like domain, this result indicates that not only ΔM4-DANCE, but also other DANCE mutants are properly processed after translation as well as the full-length DANCE. DANCE interacts with the N-terminal domain of LTBP-2 To identify the DANCE-binding domain in the LTBP-2 molecule, we constructed expression vectors encoding five types of LTBP-2 truncation mutants with FLAG-tag (LTBP-2-A-FLAG through LTBP-2-E-FLAG, Figure 3A). FLAG-tagged LTBP-2 truncation mutants and Myc-tagged full-length DANCE recombinant proteins were transiently expressed in 293T cells, and were subjected to in vitro binding assays. As shown in Figure 3B (left panel), only LTBP-2-A fragment can interact with DANCE (lane 2). This result demonstrates that DANCE specifically interacts with the N-terminal domain of LTBP-2. Figure 3.Schematic representation of the LTBP-2 truncation mutants, and mapping of the binding domain of LTBP-2 with DANCE. (A) Domain structure of the full-length LTBP-2 and the LTBP-2 truncation mutants used for the in vitro binding assay. These mutants were expressed as N-terminally FLAG-tagged proteins flanked by the preprotrypsin signal sequence, except for the full-length LTBP-2. The LTBP-2-H fragment is prone to degradation, so we constructed C-terminal fusion proteins with the constant region of human IgG to prevent the degradation (LTBP-2-G-Ig, LTBP-2-H-Ig, and LTBP-2-I-Ig). The characteristics of each domain are described below the figure. The asterisk indicates the second four-cystein domain of LTBP-2, where DANCE binds. (B) Left panel, DANCE interacts with the N-terminal domain of LTBP-2 (LTBP-2-A). Right panel, DANCE interacts with the second four-cysteine domain of LTBP-2 (LTBP-2-I). The expression vector of each LTBP-2 truncation mutant was transfected into 293T cells. Mixtures of the media and cell lysates of FLAG-tagged LTBP-2 truncation mutants were incubated with DANCE-Myc, and then these reactants were subjected to immunoprecipitation with anti-FLAG antibody. The immunoprecipitates were separated by SDS–PAGE, and analyzed by Western blotting with anti-Myc antibody. (C) Calcium dependency of the LTBP-2–DANCE interaction. The expression vector for Myc-tagged full-length LTBP-2 was transfected into 293T cells. Mixtures of the conditioned media and cell lysates of Myc-LTBP-2 were incubated with the conditioned media of DANCE-FLAG in the presence of EDTA (0, 1, 2, 5, or 10 mM). These cocktails were subjected to immunoprecipitation with anti-FLAG antibody. The immunoprecipitates were separated by SDS–PAGE, and analyzed by Western blotting with anti-Myc antibody. Download figure Download PowerPoint To more precisely identify the domain of LTBP-2 involved in this interaction, we constructed two expression vectors encoding FLAG-tagged LTBP-2-F and -G fragments by dividing the LTBP-2-A fragment (Figure 3A). As shown in Figure 3B (right panel), in vitro binding assays reveal that DANCE specifically interacts with LTBP-2-G fragment (lane 10), but not with LTBP-2-F fragment (lane 9). We further constructed expression vectors encoding FLAG-tagged LTBP-2-H and -I fragments by dividing the LTBP-2-G fragment (Figure 3A). Because the LTBP-2-H fragment is easily degraded, we constructed immunoglobulin fusion proteins to prevent degradation (LTBP-2-G-Ig, LTBP-2-H-Ig, and LTBP-2-I-Ig). DANCE interacted with the immunoglobulin-fused LTBP-2-G-Ig, as well as the intact LTBP-2-G fragment, indicating that the fused immunoglobulin does not affect the interaction with DANCE (Figure 3B, compare lanes 10 and 11). As shown in Figure 3B, in vitro binding assays revealed that DANCE specifically interacts with the LTBP-2-I-Ig, but not with the LTBP-2-H-Ig (Figure 3B, lanes 12 and 13). These results demonstrate that DANCE specifically interacts with the second four-cysteine repeat of LTBP-2. Next, we examined the Ca2+ dependency of the interaction between DANCE and LTBP-2. Myc-tagged LTBP-2 and FLAG-tagged DANCE recombinant proteins were expressed in 293T cells, and subjected to in vitro binding assays in the presence or absence of EDTA (ethylenediaminetetraacetic acid). As shown in Figure 3C, the interaction between DANCE and LTBP-2 was markedly diminished in the presence of 1 mM EDTA, and almost abolished in the presence of 2 mM EDTA (Figure 3C, upper panel, lanes 1–3). These results indicate that the DANCE–LTBP-2 interaction is likely to be Ca2+ dependent, which is consistent with our finding that LTBP-2 interacts with the Ca2+-binding EGF (cbEGF)-like domain of DANCE, whereas the N-terminal domain of LTBP-2 does not contain any cbEGF domain. DANCE interacts with LTBP-2 in vivo To investigate whether DANCE colocalizes with LTBP-2 at the tissue level, we performed in situ hybridization using neonatal mice. As shown in Figure 4A and B, both the DANCE transcript and the LTBP-2 transcript are strongly detected in the cardiac outflow tract, cardiac valves, aorta, and lung. The expression patterns of these two transcripts are strikingly similar, except for the minor difference that DANCE is more strongly expressed in arteries than in lung distal airspace walls, whereas the expression of LTBP-2 is similar in these tissues. These expression patterns suggest that DANCE is localized in close proximity to LTBP-2 in vivo. Figure 4.Expression patterns of DANCE (A) and LTBP-2 (B) transcripts in neonatal mice as shown by in situ hybridization with dark field (A, B) and bright field (C, D) views. Ao, aorta; PA, pulmonary artery; OT, cardiac outflow tract; MV, mitral valve; TV, tricupsid valve, LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium; Rt.Lu, right lung; Lt.Lu, left lung. Scale bar; 500 μm. Download figure Download PowerPoint To examine the interaction of DANCE and LTBP-2 in vivo, we performed co-immunoprecipitation analysis using lung extracts from wild-type and DANCE−/− mice. Immunoreactive DANCE protein was precipitated from lung extracts with anti-DANCE antibody followed by Western blotting using an anti-LTBP-2 antibody. As shown in Figure 5, the interaction of DANCE and LTBP-2 was detected (upper panel, compare lanes 1, 2 and lanes 3, 4). This result indicates that endogenous DANCE interacts with endogenous LTBP-2 in vivo. Figure 5.DANCE interacts with LTBP-2 in vivo. Whole lungs were dissected from two lines each of wild-type and DANCE-deficient mice. Proteins were extracted using a homogenizer on ice. Immunoreactive DANCE protein was precipitated from lung extracts with anti-DANCE antibody, followed by Western blotting with a polyclonal anti-LTBP-2 antibody. Download figure Download PowerPoint BIAcore analysis of the interaction between DANCE and LTBP-2 To investigate the affinity of the binding of DANCE to LTBP-2, we performed kinetic analyses using surface plasmon resonance with a Biacore X instrument. The proteins used for the assay were purified from 293T cell lines stably overexpressing C-terminal histidine-tagged full-length DANCE and LTBP-2. Analysis by SDS–PAGE followed by Coomassie blue staining revealed that the purities of these proteins were more than 80% (Figure 6A). The purified DANCE recombinant protein was immobilized on a CM5 sensor chip, and LTBP-2 was used as the analyte. Kinetic analyses were performed at a range of concentrations of 15–360 μg/ml (75–1800 nM) of LTBP-2 on the DANCE-immobilized chip, and the dissociation constant (KD) of the binding of LTBP-2 to DANCE was determined to be 265 nM, which indicates that this interaction is direct and specific (Figure 6B). Figure 6.Quantification of the affinity of DANCE binding to LTBP-2 by surface plasmon resonance. (A) Recombinant full-length DANCE and full-length LTBP-2 proteins were purified by chelating chromatography, separated by SDS–PAGE, and stained with Coomassie Blue. (B) LTBP-2 was injected over a DANCE-immobilized sensor chip surface. Each sensorgram shows seven different analyte concentrations of 15, 30, 60, 90, 180, 240, and 360 μg/ml. Response difference; the difference between experimental and control flow cells in response units (RU). Time is shown in seconds (s). Download figure Download PowerPoint Deposition of DANCE is dependent on fibrillin-1, not on either fibrillin-2 or LTBP-2 To investigate the physiological significance of the interaction of DANCE and LTBP-2, we used RNA interference (RNAi) to knock down target genes. For this purpose, we developed an in vitro culture system of human skin fibroblasts to assess the effects of gene knockdown on elastic fiber assembly. We reverse transfected each siRNA duplex into human skin fibroblasts, cultured the transfected cells in 10% serum-containing medium for more than 10 days, and subjected them to immunostaining with anti-elastin and anti-DANCE antibody. As controls, we used siRNA with a scrambled sequence of protein phosphatase PP2C gamma, a gene irrelevant to the extracellular matrix. Because LTBP-2 is a constituent of elastic microfibrils, we also examined the role of fibrillin-1 and -2, major constituents of elastic microfibrils, in addition to LTBP-2. We confirmed that treatment with siRNA results in more than a 90% decrease in the respective transcripts even in double or triple knockdown cells as detected by quantitative polymerase chain reaction (qPCR) (Figure 7A). No off-target knockdown was observed at least in mRNAs of fibrillin-1 and -2, LTBP-2, DANCE and elastin (data not shown). Fibrillin-1 and -2 knockdown specifically abolished each meshwork of fibrillin-1 and -2 microfibrils (Figure 7B–O). As shown in Figure 8B, fibrillin-1-knockdown cells develops only a faint meshwork of elastic fibers, whereas fibrillin-2-knockdown cells and LTBP-2-knockdown cells each develop abundant meshworks of elastic fibers like those in the control cells (Figure 8A, C and D). Moreover, DANCE is barely deposited on fibrillin-1-knockdown cells (Figure 8J), whereas DANCE is abundantly deposited and colocalizes with elastin on fibrillin-2- or LTBP-2-knockdown cells (Figure 8I, K and L). These results indicate that DANCE and elastin are deposited mainly on fibrillin-1 microfibrils in human skin fibroblast culture. Figure 7.Quantitative PCR analysis of gene knockdown in skin fibroblasts (A), and fibrillin-1 and -2 knockdown specifically abolishes the meshwork of fibrillin-1 and -2 microfibrils, respectively (B–O). (A) Total RNA from siRNA-transfected skin fibroblasts was extracted 9 days after transfection. Complementary DNA was synthesized and was subjected to quantitative real-time PCR for the expression of fibrillin-1, fibrillin-2, LTBP-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts. A graphic presentation of the results obtained by qPCR is shown. Levels of GAPDH transcript are used to normalize the cDNA levels. The relative amount of the PCR product amplified from control siRNA-treated fibroblasts is set at 100. Data are presented as the means±s.e. of three independent experiments, each performed in duplicate. (B–O) HSFs were transfected with each RNAi oligo as indicated and cultured in 10% serum containing media. Cells were stained with anti-fibrillin-1 (B–H) or anti-fibrillin-2 antibody (I–O). Lower panels are superimpositions of upper panels with DAPI (4,6-diamidino-2-phenylindole) nuclear staining. KD, knockdown; FBN-1, fibrillin-1; FBN-2, fibrillin-2. Scale bars; 60 μm. Download figure Download PowerPoint Figure 8.DANCE and elastin deposition is dependent on fibrillin-1 microfibril, but LTBP-2 knockdown induces fibrillin-1-independent deposition of DANCE and elastin. HSFs were transfected with each RNAi oligo as indicated, cultured in 10% serum containing media for 14 days and fixed. Cells were stained with anti-elastin (A–H) and anti-DANCE (I–P) antibodies. In (F) and (N), recombinant LTBP-2 (rLTBP2) was added to the culture medium to cancel the knockdown effect of LTBP-2. (Q–X) Superimpositions of (A–H) and (I–P) with DAPI nuclear staining, showing that DANCE colocalizes with elastic fibers. Scale bars; 60 μm. Download figure Download PowerPoint LTBP-2 inhibits fibrillin-1-independent deposition of DANCE and elastin Next, we investigated the role of LTBP-2 in the deposition of DANCE in the absence of fibrillin-1. For this purpose, we doubly knocked down LTBP-2 in addition to fibrillin-1. As shown in Figure 8E and M, the nearly abolished deposition of DANCE and elastin resulting from fibrillin-1 knockdown is unexpectedly rescued by additional LTBP-2 knockdown. The rescue effect of LTBP-2 knockdown is similar when each of three different siRNAs to LTBP-2 is independently transfected with fibrillin-1 siRNA (data not shown). To rule out off-target effects of LTBP-2 RNAi, we added recombinant LTBP-2 protein to these fibrillin-1–LTBP-2 double knockdown cells. As shown in Figure 8F and N, addition of recombinant LTBP-2 greatly reduces the deposition of DANCE and elastin to a similar level as o
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