The transcriptional activation by SRY-type high mobility group box 9 (SOX9) and the transforming growth factor ␤ (TGF-␤) signals are necessary for chondrogenic differentiation. We have previously shown that CREB-binding protein (CBP/p300) act as an important SOX9 co-activator during chondrogenesis. In the present study, we investigated the relationship between TGF-␤-dependent Smad2/3 signaling pathways and the SOX9-CBP/p300 transcriptional complex at the early stage of chondrogenesis. Overexpressed Smad3 strongly induced the primary chondrogenesis of human mesenchymal stem cells. In addition, Smad3 enhanced the transcriptional activity of SOX9 and the expression of ␣1(II) collagen gene (COL2A1), and small interference RNA against Smad3 (si-Smad3) inhibited them. We observed that Smad2/3 associated with Sox9 in a TGF-␤-dependent manner and formed the transcriptional complexes with SOX9 on the enhancer region of COL2A1. Interestingly, the association between Sox9 and CBP/p300 was increased by Smad3 overexpression and was suppressed by si-Smad3. Our findings indicate that Smad3 has a stronger potential to stimulate the SOX9-dependent transcriptional activity by modulating the interaction between SOX9 and CBP/ p300, rather than Smad2. This study suggests that the Smad3 pathway presents a key role for the SOX9-dependent transcriptional activation in primary chondrogenesis.

Smad ubiquitin regulatory factor 1 (Smurf1), a HECT type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces translocation of Smad7 to the cytoplasm. Smurf1 then associates with the transforming growth factor (TGF)-␤ type I receptor, T␤R-I, enhancing turnover. However, the mechanism of nuclear export of Smad7 by Smurf1 has not been elucidated. Here we identified a functional nuclear export signal (NES) in a Cterminal region of Smurf1. In transfected cells, the Smurf1-Smad7 complex was accumulated in the cytoplasm by the nuclear export receptor, CRM1; this action was prevented by treatment with leptomycin B, a specific inactivator of CRM1 function. A green fluorescence protein fusion protein containing the C-terminal NES motif of Smurf1, located in the cytoplasm, accumulated in the nucleus following treatment with leptomycin B. Moreover, Smurf1 was shown to bind physically to CRM1 through NES, and nuclear export of the Smurf1-Smad7 complex was prevented by mutations of Smurf1 within the NES. Finally, the Smurf1 NES mutant reduced inhibition by Smad7 of the transcriptional activation induced by TGF-␤. These results thus suggest that CRM1-dependent nuclear export of Smurf1 is essential for the negative regulation of TGF-␤ signaling by Smad7.

We have developed a high-level expression system for human blood coagulation factor VIII (FVIII) consisting of a 90 kDa heavy (H-)chain and an 80 kDa light (L-)chain. Two expression plasmids were prepared, one expressing the H-chain and the other expressing the L-chain. These recombinant plasmids were designed to produce each chain linked to short additional amino acid residues derived from the FVIII precursor sequence. Furthermore, Kozak's translation initiation consensus sequence was introduced into the start codon for the H-chain. These modifications have dramatically increased the levels of expression of these chains. Chinese hamster ovary (CHO) cells co-transfected with these two recombinant plasmids were subjected to gene amplification and cloning. The final cell line, designated CTC-CF8, secretes 15 IU/day/10(6) cells of active FVIII which is indistinguishable from plasma-derived FVIII in its structure and biochemical properties. This system is suitable for large-scale production of pathogen-free recombinant human FVIII which can be used for the treatment of haemophilia A patients.