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. 1997 Jan 13;136(1):193-204.
doi: 10.1083/jcb.136.1.193.

Extracellular fibrillar structure of latent TGF beta binding protein-1: role in TGF beta-dependent endothelial-mesenchymal transformation during endocardial cushion tissue formation in mouse embryonic heart

Y Nakajima  1 , K Miyazono, M Kato, M Takase, T Yamagishi, H Nakamura
Affiliations

Extracellular fibrillar structure of latent TGF beta binding protein-1: role in TGF beta-dependent endothelial-mesenchymal transformation during endocardial cushion tissue formation in mouse embryonic heart

Y Nakajima et al. J Cell Biol. .

Abstract

Transforming growth factor-beta (TGF beta) is a dimeric peptide growth factor which regulates cellular differentiation and proliferation during development. Most cells secrete TGF beta as a large latent TGF beta complex containing mature TGF beta, latency associated peptide, and latent TGF beta-binding protein (LTBP)-1. The biological role of LTBP-1 in development remains unclear. Using a polyclonal antiserum specific for LTBP-1 (Ab39) and three-dimensional collagen gel culture assay of embryonic heart, we examined the tissue distribution of LTBP-1 and its functional role during the formation of endocardial cushion tissue in the mouse embryonic heart. Mature TGF beta protein was required at the onset of the endothelial-mesenchymal transformation to initiate endocardial cushion tissue formation. Double antibody staining showed that LTBP-1 colocalized with TGF beta 1 as an extracellular fibrillar structure surrounding the endocardial cushion mesenchymal cells. Immunogold electronmicroscopy showed that LTBP-1 localized to 40-100 nm extracellular fibrillar structure and 5-10-nm microfibrils. The anti-LTBP-1 antiserum (Ab39) inhibited the endothelial-mesenchymal transformation in atrio-ventricular endocardial cells cocultured with associated myocardium on a three-dimensional collagen gel lattice. This inhibitory effect was reversed by administration of mature TGF beta proteins in culture. These results suggest that LTBP-1 exists as an extracellular fibrillar structure and plays a role in the storage of TGF beta as a large latent TGF beta complex.

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Figures

Figure 1
Figure 1
Specificity of anti–LTBP-1 antiserum (Ab39). COS-1 cells were transfected with human LTBP-1 (BP-1) or LTBP-2 (BP-2) cDNA. The cells were metabolically labeled, and resulting conditioned medium was immunoprecipitated with Ab39 or anti– LTBP-2 antiserum (Ab178) and analyzed by SDS-PAGE (5– 20% linear gradient gel) in the presence of dithiothreitol.
Figure 2
Figure 2
Immunoblot of plasmin digest of a deoxycholate insoluble fraction obtained from homogenizing 9.5- or 14.5-d whole embryo probed with anti–LTBP-1 antiserum (Ab39). Deoxycholate insoluble fraction of whole homogenate of the embryos was digested with plasmin, and the resulting supernatant containing 50 μg of protein was electrophoresed (6% SDS-PAGE) under nonreducing conditions. Separated proteins were transferred to Immobilon-P and stained with Ab39. Ab39 recognizes a 190-kD band both in 9.5- and in 14.5-d embryo (arrowhead).
Figure 3
Figure 3
Immunohistochemical staining for LTBP-1 in fresh frozen sections of endocardial cushion tissue. (a and b) At the onset of endothelial-mesenchymal transformation (9.5 d), fibrillar LTBP-1–like molecules are distributed beneath the hypertrophied transforming cell (arrowhead) and surrounding mesenchymal cells (arrows). There is no LTBP-1 beneath the stationary endothelial cells (E). (c and d) Fibrillar LTBP-1–like molecules are observed surrounding the invaded mesenchymal cells in 10.5 d AV cushion tissue. (e and f) Fresh frozen section treated with plasmin (PL) and stained with Ab39. Ab39-immunoreactivity disappeared after plasmin digestion. (g and h) The Ab39-immunostaining was blocked by a pre-incubation with purified human LTBP-1. E, endocardium; M, myocardium; arrows, mesenchymal cells. b, d, f, and h are light microscopic images of a, c, e, and g. Bar, 200 μm.
Figure 4
Figure 4
Immunogold electron microscopic detection of LTBP-1 in endocardial cushion tissue. (a) A 40–100 nm extracellular fibrillar network surrounding the invading mesenchymal cell (Me) is decorated with an Ab39-gold-particles complex (arrowheads). (b) Extracellular microfibril is stained with Ab39 (pre-embedding method). (c) The immunoreactivity of Ab39 was blocked by preincubation of an antibody with purified human LTBP-1. Bar, 0.5 μm.
Figure 5
Figure 5
Immunohistochemical colocalization of TGFβ1 and LTBP-1 in 9.75-d embryonic heart and cultured atrioventricular (AV) explant. Double antibody staining for anti-TGFβ1 IgY (a, c, e, and g) and anti–LTBP-1 antiserum (Ab39; b, d, f, and h) was performed. Extracellular fibrillar structures containing TGFβ1 (arrowheads in a and e) coincides with LTBP-1 (arrowheads in b and f) surrounding the AV cushion tissue mesenchymal cells. The immunoreactivities of anti-TGFβ1 IgY and AB39 were blocked by preincubation of antibody mixture with recombinant human TGFβ1 and purified human LTBP-1 (c and d and g and h). Note that an explant (EX in e and f) containing myocardium, mesenchyme, and endocardium shows immunoreactivity. Bar, 20 μm.
Figure 6
Figure 6
Three-dimensional collagen gel culture of the AV region. AV explants were prepared from 9.5-d mouse embryonic heart and cultured on the gel lattice. After 12 h incubation, explants were subjected to various culture conditions as described in Materials and Methods. (a and b) After an additional 48 h in culture with CM199, an endothelial outgrowth (E in a) and mesenchymal cell invasion into the gel lattice (arrows in b) were both present. (c and d) When explants were cultured with Ab39 (1 μl/ml), endothelial outgrowth (E in c) and mesenchymal cell invasion (d) into the gel lattice were both inhibited. (e and f) Rescue experiment: AV explant cultured in the presence of both Ab39 (1 μl/ml) and mature TGFβ1 (2 ng/ml) in CM199 showed definite endothelial outgrowth (E in e) and mesenchymal cell invasion (arrows in f) in the gel lattice. E, endothelial outgrowth; EX, explant; arrows, mesenchymal cell; a, c, and e, gel surface; b, d, and f, under the gel surface observed by optical sectioning, Bar, 200 μm.
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