Inhibitory Smads (I-Smads) repress signaling by cytokines of the transforming growth factor-β (TGF-β) superfamily. I-Smads have conserved carboxy-terminal Mad homology 2 (MH2) domains, whereas the amino acid sequences of their amino-terminal regions (N domains) are highly divergent from those of other Smads. Of the two different I-Smads in mammals, Smad7 inhibited signaling by both TGF-β and bone morphogenetic proteins (BMPs), whereas Smad6 was less effective in inhibiting TGF-β signaling. Analyses using deletion mutants and chimeras of Smad6 and Smad7 revealed that the MH2 domains were responsible for the inhibition of both TGF-β and BMP signaling by I-Smads, but the isolated MH2 domains of Smad6 and Smad7 were less potent than the full-length Smad7 in inhibiting TGF-β signaling. The N domains of I-Smads determined the subcellular localization of these molecules. Chimeras containing the N domain of Smad7 interacted with the TGF-β type I receptor (TβR-I) more efficiently, and were more potent in repressing TGF-β signaling, than those containing the N domain of Smad6. The isolated N domain of Smad7 physically interacted with the MH2 domain of Smad7, and enhanced the inhibitory activity of the latter through facilitating interaction with TGF-β receptors. The N domain of Smad7 thus plays an important role in the specific inhibition of TGF-β signaling.

Smad ubiquitin regulatory factor 1 (Smurf1), a HECTtype E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces cytoplasmic localization of Smad7. Smurf1 then associates with transforming growth factor-␤ type I receptor (T␤R-I) and enhances the turnover of this receptor. However, the mechanisms of the nuclear export and plasma membrane localization of the Smurf1⅐Smad7 complex have not been elucidated. We show here that Smurf1 targets Smad7 to the plasma membrane through its N-terminal conserved 2 (C2) domain. Both wild-type Smurf1 (Smurf1(WT)) and Smurf1 lacking the C2 domain (Smurf1(⌬C2)) bound to Smad7 and translocated nuclear Smad7 to the cytoplasm. However, unlike Smurf1(WT), Smurf1(⌬C2) did not move to the plasma membrane and failed to recruit Smad7 to the cell surface T␤R-II⅐T␤R-I complex. Moreover, although Smurf1(⌬C2) induced ubiquitination of Smad7, it failed to induce the ubiquitination and degradation of T␤R-I and did not enhance the inhibitory activity of Smad7. Thus, these results suggest that the plasma membrane localization of Smad7 by Smurf1 requires the C2 domain of Smurf1 and is essential for the inhibitory effect of Smad7 in the transforming growth factor-␤ signaling pathway.

Basic transcription element binding protein (BTEB) is a transcription factor with a characteristic zinc finger motif and is most remarkably enhanced by thyroid hormone T 3 treatment (R. J. Denver et al., J. Biol. Chem. 272:8179-8188, 1997). To investigate the function of BTEB per se and to touch on the effects of T 3 (3,5,3-triiodothyronine) on mouse development, we generated BTEB-deficient mice by gene knockout technology. Homologous BTEB؊/؊ mutant mice were bred according to apparently normal Mendelian genetics, matured normally, and were fertile. Mutant mice could survive for at least 2 years without evident pathological defects. From the expression of lacZ, which was inserted into the reading frame of the BTEB gene, BTEB showed a characteristic tissue-specific expression profile during the developmental process of brain and bone. Dramatically increased expression of BTEB was observed in Purkinje cells of the cerebellum and pyramidal cell layers of the hippocampus at P7 when synapses start to form in the brain. Although general behavioral activities such as locomotion, rearing, and speed of movement were not so much affected in the BTEB ؊/؊ mutant mice, they showed clearly reduced activity levels in rotorod and contextual fear-conditioning tests; this finding was probably due to defective functions of the cerebellum, hippocampus, and amygdala.

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The basic structure of adventitial collagen fiber was maintained in abdominal aortas regardless of location or age. Because the molecular structure at the subfibril level changed at abdominal aorta and enhanced in aortas with AAA, alterations in the molecular structure of adventitial collagen might be associated with aneurysmal formation.