Xu RH et al. (1999), Functional analysis of human Smad1: role of the...

XB-ART-13025

Biochem Biophys Res Commun 1999 May 10;2582:366-73.

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Functional analysis of human Smad1: role of the amino-terminal domain.

Xu RH , Lechleider RJ , Shih HM , Hao CF , Sredni D , Roberts AB , Kung H .

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The signals originating from transforming growth factor beta/activin/bone morphogenetic proteins (BMPs) are transduced by a set of evolutionarily conserved family of Smad proteins which, upon activation, directly translocate to the nucleus where they may activate transcription. Smad proteins of different species contain conserved amino- (N) and carboxy- (C) terminal domains separated by a proline-rich linker. Human, Drosophila, and Xenopus Smad1 all have been shown to mediate the biological effects of BMP-4 in Xenopus embryos. We have investigated the functional domains of human Smad1 (hSmad1) using the Xenopus embryo system. Dorsal injection of hSmad1 RNA into the 4-cell-stage embryos results in embryonic ventralization. Since the C-terminus of Smads has been shown to mediate the transcriptional activity, whereas this activity is masked by the presence of the N-terminus, we tested the effect of a hSmad1 construct lacking the C-terminal domain [hSmad1(N)] in the Xenopus embryo system. Surprisingly, we found that hSmad1(N) not only synergizes with hSmad1 in embryonic ventralization, but induces ventralization by itself. Ectopic expression of a dominant negative BMP receptor (DN-BR) as well as neural inducers noggin and chordin induce neurogenesis in the animal cap, which is inhibited by co-expression of either hSmad1 or hSmad1(N). Ventral expression of DN-BR induces formation of a second body axis at tailbud stage, which is also prevented by hSmad1 and hSmad1(N). It has recently been reported that calmodulin interacts with the N-terminal domain of Smad proteins. We demonstrate that the ventralizing activity of hSmad1 and hSmad1(N) is markedly inhibited by calmodulin. Thus, calmodulin acts as a Smad1 inhibitor. A model is proposed to accomodate these findings.

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Species referenced: Xenopus laevis
Genes referenced: bmp4 chrd krt12.4 mad1l1 ncam1 nog smad1 uqcc6
???displayArticle.antibodies??? Hba3 Ab1 Ncam1 Ab1 Somite Ab3

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	FIG. 1. Whole-mount immunostaining of embryos ventralized by hSmad1 and hSmad1(N). The 4-cell-stage embryos were injected with 2 ng RNA encoding b-gal (A and D), hSmad1 (B and E), or hSmad1(N) (C and F). The embryos were allowed to develop to tailbud stage when they were fixed and bleached for immunostaining. The first antibodies used for the staining were as follows: 12/101 (for muscle actin), and 4d (for NCAM). Muscle actin (A-C) was stained purple by using secondary antibodies conjugated with alkaline phosphotase, while NCAM (D-F) was stained brown by using secondary antibodies conjugated with horseradish peroxidase.
	FIG. 2. Inhibition of neurogenesis in Xenopus ectoderm by hSmad1 and hSmad1(N). RNAs were injected into the two animal blastomeres of 2-cell-stage embryos. Animal caps were dissected from the embryos at stages 8.5 to 9, cultured until tailbud stage, and harvested for RT-PCR analysis of NCAM expression. EF-1a expression was detected as a RNA loading control. b-gal RNA was used as an injection control. Co-injection of hSmad1 RNA (2 ng) with RNA encoding noggin (0.1 ng), chordin (1.5 ng), or DN-BR (1 ng) led to inhibition of NCAM expression induced by each of the neural inducing agents (A). Co-injection of hSmad1/hSmad1 (N) with noggin RNA inhibited NCAM expression and rescued expression of the epidermal marker gene XK81 (B).
	FIG. 3. Proposed model for the mechanism of action of hSmad1(N).