Oncogene (2004) 23, 4232–4237 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc Coordinate regulation of cell growth and differentiation by TGF-b superfamily and Runx proteins Kohei Miyazono*,1,2, Shingo Maeda2 and Takeshi Imamura2 1Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; 2Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research (JFCR), 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan Runx proteins regulate various biological processes, of cells, including epithelial cells, endothelial cells, including growth and differentiation of hematopoietic hematopoietic cells, and lymphocytes.Perturbations cells, lymphocytes, osteoblasts, and gastric epithelial cells. of TGF-b signaling therefore result in progression of Some of the biological activities of Runx proteins are some tumors (Derynck et al., 2001; Wakefield and reminiscent of those of transforming growth factor Roberts, 2002).However, TGF- b stimulates growth of (TGF)-b superfamily cytokines. Consistent with this certain types of cells, including mesenchymal cells, notion, receptor-regulated Smads (R-Smads), signal and induces epithelial-to-mesenchymal transdifferen- mediators of the TGF-b superfamily cytokines, and Runx tiation (EMT) of some epithelial cells.Moreover, proteins have been shown to physically interact with each TGF-b stimulates extracellular matrix accumulation, other. R-Smads activated by TGF-b and Runx proteins angiogenesis, immunosuppression, and secretion of cooperatively induce synthesis of IgA in B lymphocytes, parathyroid hormone-related peptide (PTHrP).TGF- b and those activated by bone morphogenetic proteins and may thus function as a pro-oncogenic cytokine Runx2 induce osteoblastic differentiation. Moreover, the under certain conditions. R-Smad–Runx signaling pathways are regulated by an E3 Runx proteins are a family of transcription factors ubiquitin ligase Smurf1, as well as a signal transducer of having a Runt domain responsible for DNA binding interferons, STAT1. Since Runxl and Runx3 are involved (Ito, 1999).Runx was originally given three different in the development of some cancers including acute names, that is, a subunits of polyomavirus enhancer leukemia and gastric cancer, it will be of interest to binding proteins 2 (PEBP2a), acute myeloid leukemia examine in detail whether TGF-b-specific R-Smads and (AML), and core binding factor a (Cbfa).Three Runx Runx proteins coordinately regulate growth and differ- proteins are present in mammals, termed Runxl entiation of hematopoietic cells and gastric epithelial cells. (PEBP2aB/AMLl/Cbfa2), Runx2 (PEBP2aA/AML3/ Oncogene (2004) 23, 4232–4237. doi:10.1038/sj.onc.1207131 Cbfal), and Runx3 (PEBP2aC/AML2/Cbfa3).The b subunit of PEBP2 PEBP2b, binds to Runx proteins Keywords: TGF-b; BMP; Smad; Runx; leukemia; gas- through the Runt domains, and stabilizes Runx proteins tric cancer by preventing ubiquitin-dependent degradation.Runxl plays an important role in definitive hematopoiesis during development, and mutations in Runxl have been found in B30% of human acute leukemias.Runx2 plays a pivotal role in osteogenesis, and haploinsufficiency of Introduction Runx2 results in the autosomal dominant bone disease cleidocranial dysplasia (CCD).Runx3 is important for Transforming growth factor-b (TGF-b) is a family of thymogenesis and neurogenesis; and a recent study multifunctional cytokines that regulate the growth, indicated that alterations of expression of the Runx3 differentiation, apoptosis, and matrix accumulation gene result in progression of gastric cancer. of wide varieties of cells (Blobe et al., 2000). TGF-b Interestingly, some of the functions of the TGF-b belongs to a large superfamily of structurally related superfamily cytokines are similar to those of Runx proteins termed the TGF-b superfamily, which proteins.TGF- b acts on B lymphocytes and induces includes activins, Nodal, myostatin, bone morphoge- synthesis of IgA, and Runx3 exhibits a similar effect. netic proteins (BMPs), anti-Mu¨ llerian hormone BMPs induce bone formation, similar to the function of (AMH), and growth-differentiation factors (GDFs). Runx2.Runx3 has been shown to be involved in growth TGF-b acts as a potent growth inhibitor of most types and apoptosis of gastric epithelial cells, which are also regulated by TGF-b.We have recently reviewed functional cooperation between Runx and TGF-b *Correspondence: K Miyazono, Department of Molecular Pathology, superfamily signaling, focusing on their roles in Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; progression of cancer (Ito and Miyazono, 2003).In this E-mail: [email protected] review, we present the mechanisms of cooperation TGF-b signaling and Runx K Miyazono et al 4233 between TGF-b superfamily signaling and Runx, and signals.Smad4 is the only Co-Smad in mammals, and is some intriguing evidence that this cooperation is shared by the TGF-b/activin and BMP pathways. regulated through multiple mechanisms. Smad7 functions as an I-Smad for TGF-b/activin and BMP signaling, whereas Smad6 is an I-Smad preferen- tially inhibiting BMP signaling. R-Smads are anchored to the plasma membrane TGF-b signaling pathways through certain molecules, including Smad anchor for receptor activation (SARA) (Qin et al., 2002). The Members of the TGF-b superfamily bind to two distinct activated type I receptor kinases phosphorylate the last transmembrane serine/threonine kinase receptors, two serine residues at the C-terminal Ser-Ser-X- Ser termed types II and I (Heldin et al., 1997; Shi and motif of R-Smads.R-Smads then form hetero-oligo- Massague, 2003).Upon ligand binding to corresponding meric complexes with Co-Smad, and translocate into the type II and type I receptors, type II receptor kinases nucleus (Figure 1).Although the structures of the transphosphorylate the juxtamembrane GS domains of R-Smad–Co-Smad complexes have not yet been fully type I receptors, leading to activation of type I receptor determined, hetero-trimers composed of two molecules kinases.The type II and type I receptors form hetero- of R-Smads and one molecule of Co-Smad, and hetero- tetramers composed of two molecules each of type II dimers composed of one molecule each of R-Smad and and type I receptors (Figure 1).Based on the intra- Co-Smad, may be formed by them. cellular signals activated by type I receptors, the TGF-b In the nucleus, the R-Smad–Co-Smad hetero- superfamily cytokines are sorted into two subgroups: oligomers interact with various transcription factors those activating TGF-b/activin-like signals and those and transcriptional coactivators/corepressors, leading to activating BMP-like signals.In addition to TGF- b and transcriptional regulation of target genes.In addition to activin, Nodal and myostatin belong to the former these transcription factors, R-Smads and Co-Smads group, whereas most BMP family proteins and AMH directly bind to specific DNA sequences.A wide range belong to the latter (Miyazawa et al., 2002). However, in of biological activities of TGF-b may result from the endothelial cells, TGF-b binds to two distinct type I ability of Smads to interact with various transcription receptors, and activates not only TGF-b/activin-like factors.Thus, more than 30 transcription factors, signals but also BMP-like signals. including FAST1, c-Jun, Spl, TFE3, Mixer, vitamin D Type I receptors activate various intracellular sub- receptor, and GATA-3, have been reported to interact strates, among which Smad proteins play central roles in with Smads (Miyazawa et al., 2002). By selecting these exhibition of biological activities of the TGF-b super- transcription factors as their interaction partners, Smads family cytokines.Smad proteins are classified into three may exhibit specific biological effects on target cells. subtypes, that is, receptor-regulated Smads (R-Smads), common-partner Smads (Co-Smads), and inhibitory Smads (I-Smads).Smad2 and Smad3 serve as R-Smads transducing TGF-b/activin-like signals, whereas Smads Cooperative induction of IgA by Runx and TGF-b-specific 1, 5, and 8 act as R-Smads transducing BMP-like R-Smads Cooperation between TGF-b superfamily signaling and Osteoprogenitor cells Osteoblasts Runx proteins was first discovered in the regulation of BMP BMP IgA synthesis in B lymphocytes.TGF- b inhibits growth and function of B cells, T cells, and natural killer cells. R-II R-I R-II R-I However, TGF-b has been shown to act on B cells and R-Smad R-Smad to direct class switching to IgA (Coffman et al., 1989; Co-Smad Phosho-R-Smad Co-Smad Phosho-R-Smad Sonoda et al., 1989), and a TGF-b responsive element (TbRE) was identified in the promoter regions of the Smad complex Smad complex germline immunoglobulin constant a region (Ig Ca)in mouse and human (Lin and Stavnezer, 1992; Nilsson ? Runx2 and Sideras, 1993).Interestingly, the T bRE in mouse Target Target Coactivator Coactivator gene genes contains two Smad binding sites (CAGAC motif; TF Dlx5 Runx2 Dennler et al., 1998) and three Runx binding sites (Melnikova et al., 1993; Bae et al., 1994) in close vicinity.Runx proteins physically interact with Smad2/3 Inhibits differentiation Induces differentiation acting on the TGF-b signaling pathway; thus, they into myocytes into osteoblasts cooperatively stimulate the synthesis of IgA through Figure 1 Induction of osteoblastic differentiation by BMPs directly inducing the transcription of Ig Ca (Hanai et al., through Runx proteins.BMPs induce expression of Runx2 through 1999; Pardali et al., 2000). Although all three Runx Dlx5 in osteoprogenitor cells, and this process is sufficient to proteins interact with Smads, mRNA for Runx3 is inhibit their differentiation into myocytes.However, Runx2 alone is not sufficient to induce osteoblastic differentiation; BMP-specific predominantly induced by TGF-b1 in B lymphocytes R-Smads interact with Runx2 and possibly other proteins, and (Shi and Stavnezer, 1998), suggesting that Runx3 plays a induce osteoblastic differentiation.TF, transcription factors central role in IgA synthesis.