Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Feb 19;110(8):2852-7.
doi: 10.1073/pnas.1215779110. Epub 2013 Feb 4.

Latent TGF-β binding protein 4 promotes elastic fiber assembly by interacting with fibulin-5

Kazuo Noda  1 , Branka Dabovic, Kyoko Takagi, Tadashi Inoue, Masahito Horiguchi, Maretoshi Hirai, Yusuke Fujikawa, Tomoya O Akama, Kenji Kusumoto, Lior Zilberberg, Lynn Y Sakai, Katri Koli, Motoko Naitoh, Harald von Melchner, Shigehiko Suzuki, Daniel B Rifkin, Tomoyuki Nakamura
Affiliations

Latent TGF-β binding protein 4 promotes elastic fiber assembly by interacting with fibulin-5

Kazuo Noda et al. Proc Natl Acad Sci U S A. .

Abstract

Elastic fiber assembly requires deposition of elastin monomers onto microfibrils, the mechanism of which is incompletely understood. Here we show that latent TGF-β binding protein 4 (LTBP-4) potentiates formation of elastic fibers through interacting with fibulin-5, a tropoelastin-binding protein necessary for elastogenesis. Decreased expression of LTBP-4 in human dermal fibroblast cells by siRNA treatment abolished the linear deposition of fibulin-5 and tropoelastin on microfibrils. It is notable that the addition of recombinant LTBP-4 to cell culture medium promoted elastin deposition on microfibrils without changing the expression of elastic fiber components. This elastogenic property of LTBP-4 is independent of bound TGF-β because TGF-β-free recombinant LTBP-4 was as potent an elastogenic inducer as TGF-β-bound recombinant LTBP-4. Without LTBP-4, fibulin-5 and tropoelastin deposition was discontinuous and punctate in vitro and in vivo. These data suggest a unique function for LTBP-4 during elastic fibrogenesis, making it a potential therapeutic target for elastic fiber regeneration.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
LTBP-4 colocalizes with fibulin-5 in mouse tissues. Immunostaining of WT mouse lung at P0 (sagittal section) and mouse skin at 8 wk using anti–LTBP-4 antibody and anti–fibulin-5 antibody, indicating colocalization of these molecules. The lung figure presents LTBP-4 and fibulin-5 localization in terminal air sacs (AS). The circular structures present small blood vessels (BV). (Scale bars, 50 µm.) HF, hair follicle.
Fig. 2.
Fig. 2.
In vitro binding assay, showing interaction of LTBP-4 with fibulin-5. (A) Domain structure of the full-length LTBP-4 and the LTBP-4 truncation mutants. These mutants were expressed as N-terminal FLAG-tagged proteins flanked by the preprotrypsin signal sequence. LTBP-4L-G, LTBP-4S-G, LTBP-4-H, and LTBP-4-I were constructed as C-terminal fusion proteins with the Fc region of human IgG. The amino acid lengths (AA#) of the constructs except IgG are indicated. The expression vectors were independently transfected into 293T cells. Transfected cells were cultured in serum-free medium for 48 h, and the cell lysates and the conditioned media were harvested. Mixtures of the media and cell lysates (B), or the media alone (C) were incubated with each other, and these reactants were subjected to immunoprecipitation with anti-FLAG antibody. The immunoprecipitants were separated by SDS/PAGE, and analyzed by Western blotting. (B) Interaction of fibulin-5 with full-length LTBP-4L and -4S and with the N-terminal domains of LTBP-4L and -4S, but not with other parts of LTBP-4. Expected bands for the LTBP-4 constructs are indicated by arrowheads. (C) Interaction of fibulin-5 with LTBP-4L and -4S fragments containing four-cysteine domains, but not with other N-terminal fragments.
Fig. 3.
Fig. 3.
Recombinant LTBP-4S protein (rLTBP-4S) promotes elastic fiber assembly without changing the configuration of microfibrils. (A–E) HDFs were transfected with control siRNA or LTBP4 siRNA and cultured in 10% serum-containing media supplemented with rLTBP-4S as indicated. The cells were fixed 13 d after transfection and stained with anti–LTBP-4 and anti-elastin antibodies. Bottom images were produced by superimposition of the Top and Middle images, together with Hoechst 33258 nuclear staining. Note even more elastic fiber assembly by rLTBP-4S-supplemented LTBP4 knockdown (KD) cells than control KD cells, which express a normal level of LTBP-4. (Scale bar, 100 µm.) Nuc., nuclei. (F) Quantitation of insoluble (i.e., cross-linked and mature) elastin produced by the knockdown cells cultured with the indicated amounts of rLTBP-4S added to the medium. Cells were metabolically labeled with [3H]valine during the culture period, and the radioactivity of NaOH-insoluble fractions was quantitated. The radioactivity count was corrected by the amount of total DNA of the cells of duplicate plates. Insoluble elastin was significantly decreased in LTBP4 KD culture (*P = 0.0006, Student's t test; n = 6, Ctrl KD; n = 8, LTBP4 KD). Addition of rLTBP-4S in LTBP4 KD culture increased insoluble elastin in a dose-dependent manner above the level of Ctrl KD (#P < 0.05, ##P < 0.01, analysis of variance, Tukey test, n = 8). Error bars represent SD; Ctrl, control. (G–I) HDFs were transfected with control siRNA or LTBP4 siRNA and cultured in 10% serum containing media with or without rLTBP-4S (30 nM). The cells were fixed 13 d after transfection and stained with anti–fibrillin-1 and -2 antibodies. (Scale bar, 100 µm.)
Fig. 4.
Fig. 4.
LTBP-4 is necessary for linear deposition of fibulin-5 to microfibrils. HDFs were transfected with control siRNA (A), LTBP4 siRNA (B–D), or FBLN5 siRNA (E, F), and cultured for 13 d in 10% serum containing media with or without recombinant proteins as indicated [rLTBP-4S 30 nM (C) or rFibulin-5 60 nM (D, F)], followed by immunostaining with anti–LTBP-4 and anti–fibulin-5 antibodies. Bottom images were produced by superimposition of the Top and Middle images, together with Hoechst 33258 nuclear staining. Note the punctate pattern of rFibulin-5 deposition on microfibrils in LTBP4 KD culture, in contrast to the linear deposition of rFibulin-5 in the presence of LTBP-4.
Fig. 5.
Fig. 5.
Punctate pattern of fibulin-5 deposition in the absence of LTBP-4 in vivo. Immunohistochemistry of the lung and the skin tissues from 7-d-old wild-type and Ltbp4S−/− mice was performed using anti–fibulin-5 antibody. (Scale bars, 20 µm.)
Fig. 6.
Fig. 6.
LTBP-4 does not directly interact with elastin, but interacts with fibulin-5-bound elastin. (A) Solid-phase binding assays using recombinant tropoelastin as an immobilized protein and rLTBP-4S as a soluble ligand, in the presence (500 nM) or absence of rFibulin-5 (means ± SD, n = 3). (B) A proposed model for the role of LTBP-4–fibulin-5 interaction in elastic fiber assembly. In the presence of LTBP-4, microaggregation of tropoelastin, which is tethered by fibulin-5, deposits linearly on microfibrils. Subsequent coalescence of tropoelastin takes place on microfibrils, resulting in thickening of elastic fibers. In the absence of LTBP-4, tropoelastin/fibulin-5 complex cannot linearly deposit on microfibrils, and misplaced aggregates of tropoelastin/fibulin-5 grow to form globular structures.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Rosenbloom J, Abrams WR, Mecham R. Extracellular matrix 4: The elastic fiber. FASEB J. 1993;7(13):1208–1218. - PubMed
    1. Kielty CM, Sherratt MJ, Shuttleworth CA. Elastic fibres. J Cell Sci. 2002;115(Pt 14):2817–2828. - PubMed
    1. Wagenseil JE, Mecham RP. New insights into elastic fiber assembly. Birth Defects Res C Embryo Today. 2007;81(4):229–240. - PubMed
    1. Sato F, et al. Distinct steps of cross-linking, self-association, and maturation of tropoelastin are necessary for elastic fiber formation. J Mol Biol. 2007;369(3):841–851. - PubMed
    1. Ramirez F, Sakai LY, Dietz HC, Rifkin DB. Fibrillin microfibrils: Multipurpose extracellular networks in organismal physiology. Physiol Genomics. 2004;19(2):151–154. - PubMed

Publication types

Substances