TGF-β signaling by Smad proteins
Introduction
Members of the transforming growth factor-β (TGF-β) superfamily are multifunctional cytokines, which include TGF-βs, activins, and bone morphogenetic proteins (BMPs). These cytokines bind to two different types of serine/threonine kinase receptors (type I and type II), and activate intracellular substrates, e.g. Smad proteins [1], [2], [3]. The type II receptor kinases are constitutively active, and transactivate the type I receptor kinases, which then transmit signals inside cells. Thus, the type I receptors act downstream of the type II receptors, and determine the specificity of intracellular signals.
Smads are subdivided into three subclasses based on their structure and function. Receptor-regulated Smads (R-Smads) have a C-terminal Ser-Ser-X-Ser motif, and are directly phosphorylated by the type I receptor kinases. R-Smads then form complexes with common-partner Smads (Co-Smads) and translocate into the nucleus, where they regulate the transcription of target genes. Inhibitory Smads (I-Smads) are induced by ligand stimulation and interfere with the receptor activation or complex formation of R-Smads. In this review, I will focus on the biological activities of Smads determined using adenoviral expression vectors, and the function of Smads in the nucleus.
Section snippets
Bioactivity of Smads: adenoviral expression vector-based approach
C2C12 cells are undifferentiated mouse mesenchymal cells which differentiate into mature myocytes after serum deprivation in culture. If they are treated with BMPs, e.g. BMP-2, BMP-6 or BMP-7/OP-1 (osteogenic protein-1), they differentiate into osteoblast-like cells, and synthesize alkaline phosphatase [4], [5], [6]. Using adenoviral expression vectors, we examined the biological activities of Smads and compared them with ligands or constitutively active forms of type I receptors.
Function of Smads in the nucleus
After translocation into the nucleus, Smads regulate the transcription of target genes by directly binding to consensus DNA sequences, interacting with other DNA binding proteins, and recruiting transcriptional co-activators or co-repressors (Fig. 4).
Conclusion
Here I have described the biological activity of Smads determined using the adenoviral expression vector system. The adenoviral vector system will be useful for study of the biological activity of Smads in vivo, as shown for the lung fibrosis induced by bleomycin in mice [15]. The adenovirus system may also be helpful in studying the in vitro bioactivity of these proteins in cells which are difficult to transfect genes. In fact, more than 80% of C2C12 cells could be transfected using the
Acknowledgements
I would like to thank the colleagues in my laboratory and collaborators in other laboratories. This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan, Research for the Future Program of the Japan Society for the Promotion of Science, and the Creative BioMolecules Fund.
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