Lamprey IGF-Binding Protein-3 Has IGF-Dependent and -Independent Actions

Figures

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  Figure 1. Structural, phylogenetic, and synteny analysis of lamprey IGFBP-3. (A) The genomic structure of lamprey igfbp-3. The two PolyA signals are indicated by vertical lines. (B) Comparison of lamprey IGFBP-3 (IGFBP-3 Pm) amino acid (aa) sequence with that of human IGFBP-3 (IGFBP-3 Hs), mouse IGFBP-3 (IGFBP-3 Mm), chicken IGFBP-3 (IGFBP-3 Gg), and zebrafish Igfbp-3 (Igfbp-3 Dr). Hyphens were inserted for optimal alignment. The identical aas are dark shaded, and similar ones are gray shaded. Signal peptide, N-domain, L-domain, and C-domain are separated by vertical lines. The IGF-binding domain (IBD) and nuclear localization signal (NLS) are indicated by open boxes. The insulin-like growth factor binding protein (IGFBP) and thyroglobulin type I motifs are underlined. The aas known to be critical for transactivation activity in human IGFBPs are indicated by asterisks. (C) Phylogenetic analysis. Maximum likelihood (ML) was performed on a matrix composed of full-length aa sequences of major IGFBPs. ML bootstrap values shown are percentage of times that the two clades branched as sisters. Amphioxus IGFBP (IGFBP Bb) was used as the outgroup. (D) Synteny analysis. The IGFBP-3/igfbp3 genes are…

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  Figure 2. Lamprey IGFBP-3 contains a functional IBD, nuclear localization signal (NLS), and transactivation domain (TAD). (A) Ligand blotting (upper panel) and immunoblotting (lower panel) analyses of lamprey IGFBP-3, its IBD, NLS, and TAD mutants. DIG-labeled human IGF-I and anti-GFP antibodies were used, respectively. (B) Subcellular localization of lamprey IGFBP-3, its IBD, NLS, and TAD mutants in HeLa cells. The EGFP signal was visualized 24 h after transfection (left panel). Corresponding DAPI staining is shown in the middle column and merged views in the right column. Scale bar = 50 µm. (C) Lamprey IGFBP-3 N-domain has strong transactivation (TA) activity. Lamprey IGFBP-3 N-domain (IGFBP-3-N), its TAD mutants (TAD-N), and full-length lamprey IGFPB-3 were fused to pBIND and transfected into HEK293T cells together with a Gal4 reporter plasmid. The TA activity is expressed as fold increase over the pBind control group. Values are expressed as means ± SD (n = 3). Groups with different letters are significantly different from each other (P < 0.05).

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  Figure 3. Lamprey IGFBP-3 has insulin-like growth factor (IGF)-dependent action in vitro. (A) Lamprey IGFBP-3, but not its IBD mutant, inhibits IGF-I-stimulated cell proliferation. Confluent HEK293T cells were treated with IGF-I together with conditioned media prepared from cells transfected with the empty vector, lamprey IGFBP-3, its IBD mutant, and human IGFBP-3, respectively. The values are expressed as means ± SD (n = 3). Groups with different letters are significantly different from each other at P < 0.05. (B) Lamprey IGFBP-3, but not its IBD mutant, inhibits IGF-I-induced pAkt signaling. HEK293T cells were treated as described in panel (A). Cells were harvested and analyzed by western immunoblotting using indicated antibodies. Similar results were obtained in two additional and independent experiments.

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  Figure 4. Lamprey IGFBP-3 has insulin-like growth factor-dependent action in vivo. (A) Representative images of 24 hpf embryos injected with mRNA encoding the indicated proteins. Wild-type (WT) embryos were used as controls. Scale bar = 0.5 mm. Body length (B) and somite number (C) of the abovementioned groups were quantified and shown. Values are represented as means ± SD (n = 3). The total number of embryos in each group is shown on the top of each column. Groups with different letters are significantly different from each other (P < 0.05).

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  Figure 5. Lamprey IGFBP-3 antagonizes BMP signaling independently of its insulin-like growth factor-binding function. (A) IGFBP-3 inhibits BMP-2 signaling in vitro. Human U2 osteosarcoma cells were treated with 10 ng/ml of human BMP2 plus the indicated conditioned media. Three hours later, the cells were harvested and analyzed using the indicated antibodies. A representative image of western immunoblotting is shown on the top and quantitative results in the lower panel. pSmad1/5/8 level was normalized by total Smad1/5/8 level. Values are expressed as means ± SD (n = 3). Groups with different letters are significantly different from each other (P < 0.05). (B) IGFBP-3 inhibits BMP-2 signaling in vivo. Zebrafish embryos were injected with the indicated capped mRNA. Seven hours after injection, embryos were pooled. RNA was extracted and analyzed by quantitative RT-PCR. Wild-type (WT) embryos were used as control. Values are expressed as means ± SD (n = 3). Groups with different letters are significantly different from each other at P < 0.05.