Coordinate Regulation of Cell Growth and Differentiation by TGF-B Superfamily and Runx Proteins

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.

Oncogene TGF-b signaling and Runx K Miyazono et al 4234 Both Smad binding sites and Runx binding sites are R-Smads interact with the induced Runx2 protein and essential for efficient activation of the Ig Ca promoter. further induce differentiation of cells into osteoblastic Mutations of either of these binding sites severely impair cells (Figure 1).Although Runx2 is induced in transcriptional activation by TGF-b and Runx.Smads mesenchymal cells, it may not be a direct target of interact with Runx proteins through multiple domains; BMP signaling (Lee et al., 2000). BMPs induce both the N-terminal MH1 and C-terminal MH2 expression of Dlx5, which is specifically expressed in domains in Smads are responsible for such interaction, osteogenic cells (Miyama et al., 1999; Lee et al., 2003). and the C-terminal transactivation domain as well as a Dlx5 then induces the expression of Runx2 in osteopro- region flanking the Runt domain of Runx are involved genitor cells.In contrast to BMPs, TGF- b does not in their interaction (Hanai et al., 1999; Pardali et al., induce expression of Dlx5. 2000).Runx–Smad complexes appear to be formed in BMPs prevent differentiation of mesenchymal cells the cytoplasm without ligand stimulation, but are into myoblasts and Runx2 appears to play a pivotal role detected in the nucleus only after TGF-b stimulation. in this process.Runx2 prevents differentiation of C2C12 In addition to Smad and Runx binding sites, an Ets cells into myoblasts by inducing matrix gene products, binding site and an ATF/CRE site are located in the suppressing the expression of MyoD and inhibiting vicinity of TbRE, and transcription factors of the Ets myotube formation.However, Runx2 alone is insuffi- family (Xie et al., 1999) and CREB (Zhang and cient for induction of osteoblastic differentiation; for Derynck, 2000) cooperatively regulate the transcription this process, Runx2 needs to interact with BMP-specific of Ig Ca together with Runx and Smads. R-Smads.It is currently not known how BMP-specific R-Smads regulate osteoblastic differentiation together with Runx2.It should be noted that osteoblast differentiation is not induced by TGF-b-specific Synergy between BMP and Runx in osteoblastic R-Smads.Since BMP-specific R-Smads bind to DNA differentiation sequences different from the typical Smad-binding sequences (Ishida et al., 2000), they may bind to such Runx2 is an essential transcription factor required for sequences in promoters of bone-specific genes together bone formation.CCD is characterized by patent with Runx2.Another important possibility is that fontanelles and sutures, hypoplastic clavicles, and Dlx5 induced by BMPs may support the action of certain other skeletal abnormalities, and is induced by BMP-specific R-Smads and Runx2. heterozygous mutation of the Runx2 gene (Mundlos In contrast to BMPs, TGF-b negatively regulates et al., 1997). Targeted disruption of Runx2 in mice bone formation in most instances, although the effects results in the absence of mature osteoblasts and lack of of TGF-b on osteoblast differentiation depend on the bone formation (Komori et al., 1997; Otto et al., 1997). status of differentiation of cells and the extracellular In fact, cooperation between Runx and Smads has been milieu.Although there are some controversial data (Lee most clearly documented in bone formation induced by et al., 1999, 2000), TGF-b has been reported to inhibit BMPs. the expression of Runx2 in some osteoprogenitor cells, BMP-specific Smads, including Smadl and Smad5, and this occurs independent of new protein synthesis but physically interact with Runx2, and synergistically requires Runx2 itself (Alliston et al., 2001). Smad3 induce the osoteoblast-like phenotype in C2C12 activated by TGF-b physically interacts with, and mesenchymal progenitor cells (Lee et al., 2000; Zhang represses the transcriptional activity of, Runx2.This et al., 2000). Interaction of endogenous Smadl and results in inhibition of transcription of the Runx2 gene Runx2 was observed only upon BMP stimulation in itself and some osteoblast-specific genes, including C2C12 cells.Moreover, a truncation of the C-terminal osteocalcin.Interestingly, Smad3 does not directly bind region of Runx2 found in a CCD patient resulted in loss to the osteocalcin promoter, but binds indirectly to it of interaction with BMP-specific Smads, and dominant through Runx2.Thus, the Smad3–Runx2 complex interference of differentiation of C2C12 cells into induced by TGF-b plays a critical role in repression osteoblasts upon BMP stimulation (Zhang et al., of osteoblast differentiation in certain cells. 2000).Runx proteins have nuclear matrix-targeting signals (NMTS) in their C-terminal regions.Activated Smad proteins are recruited to specific foci in the nucleus through Runx proteins, and recruitment Degradation of Runx2 by Smurf1: a novel link between of Smad-Runx complexes to specific subnuclear foci the Runx and Smad pathways is essential for transcriptional regulation of their target genes (Zaidi et al., 2002). These findings indicate a Ubiquitin-dependent protein degradation plays pivotal critical role of cooperation of Runx2 and BMP signaling roles in various biological processes (Hershko and in bone formation. Ciechanover, 1998), including TGF-b superfamily sig- Expression levels of Runx2 in mesenchymal progeni- naling.In the ubiquitin–proteasome pathway, E3 tor cells appear to be low.BMPs induce osteoblastic ubiquitin ligases play key roles in the recognition of differentiation together with Runx2 through two steps: target proteins and their degradation by the 26S first, BMPs act on mesenchymal progenitor cells and proteasomes.Smad ubiquitin regulatory factor (Smurf) induce expression of Runx2.Second, BMP-specific 1 was originally identified as a HECT type E3 ubiquitin

Oncogene TGF-b signaling and Runx K Miyazono et al 4235 ligase.Smurf1 induces the ubiquitination and degrada- Regulation of expression of Smurf1 and Smurf2 has tion of BMP-specific R-Smads, Smadl, and Smad5, in a not been extensively studied yet; however, a recent study ligand-independent manner (Figure 2) (Zhu et al., 1999). showed that Smurf2 is expressed in patients with Smurf1 has WW domains, which specifically interact esophageal squamous carcinoma, and that high expres- with PY motif in R-Smads.Smurf2 is structurally very sion of Smurf2 correlates with poor prognosis of similar to Smurf1, and also degrades Smad1 (Zhang esophageal cancer (Fukuchi et al., 2002). It will be et al., 2001). Moreover, Smurf2 was shown to interact interesting to examine whether Smurfs can regulate with activated TGF-b-specific R-Smad, Smad2, and to degradation of the other Runx family proteins, and induce its ubiquitination and degradation (Lin et al., whether expression of Smurfs is also upregulated in 2000).In addition to their effects on R-Smads, Smurf1, other tumors in which functions of Runx proteins are and Smurf2 interact with I-Smads through their PY involved (see below). motifs in the nucleus and induce their export to the cytoplasm.Smurf-I–Smad complexes are then recruited to the type I receptors for TGF-b and BMPs, and enhance their degradation (Figure 2) (Kavsak et al., Cross-talk between STAT and Smad pathways though 2000; Ebisawa et al., 2001; Murakami et al., 2003). Runx Thus, Smurfs negatively regulate TGF-b superfamily signaling by targeting R-Smads as well as type I The Smad signaling pathway has been shown to cross- receptors bound to I-Smads for ubiquitin-dependent talk with various signaling pathways, including ERK degradation. MAP kinase pathways and Wnt-b-catenin pathways Intriguingly, Smurf1 interacts directly with Runx2, (Derynck et al., 2001). Cross-talk with cytokine signal- and induces its ubiquitin-dependent degradation ing has also been reported; interferon-g (IFN-g) induces (Figure 2) (Zhao et al., 2003). Although the domain expression of Smad7 on some cells through the responsible for interaction between Smurf1 and Runx2 Jak-STAT signaling pathway, and Smad7 then inhibits has yet to be determined, Runx2 has a PY motif in the TGF-b signaling pathways (Ulloa et al., 1999) C-terminal region, which may be important for interac- (Figure 3a).In contrast, leukemia inhibitory factor tion with Smurf1.Smurf1 reduced the alkaline phos- (LIF) and BMP pathways synergistically induce differ- phatase activity and production of osteocalcin in entiation of neuroepithelial cells into astrocytes through osteoprogenitor cells, whereas a Smurf1 mutant lacking indirect binding of STAT3 and BMP-specific Smads via the catalytic activity of E3 ligase induced osoteoblastic p300 (Nakashima et al., 1999). Thus, Jak-STAT differentiation.It is currently unknown whether signaling pathways regulate Smad signaling pathways degradation of Runx2 by Smurf1 depends on BMP both positively and negatively. signaling.However, since Smurfs interact wjth R-Smads Recently, another important interaction between directly, Runx proteins may be efficiently degraded by Smad and STAT signaling pathways has been reported Smurfs as Runx-R–Smad complexes upon BMP signal- (Kim et al., 2003). Type I IFN (IFN-a/b) transduces ing.PEBP2 b has been shown to bind to the Runt signals through STAT1, STAT2, and IRF-9, whereas domain and to prevent Runx proteins from degradation type II IFN (IFN-g) does so through STAT1 homo- (Huang et al., 2001). It will thus be interesting to dimer.Both type I and type II IFNs function as negative determine how PEBP2b regulates degradation of Runx2 regulators of osteoclastogenesis (Takayanagi et al., by Smurf1. 2000, 2002).However, STAT1-null mice exhibit

BMP

INF- TGF- BMP BMPR-II BMPR-I Smad1/5 ab X T R-II BMPR-II BMPR-I Smurf T R-I JAK X X STAT1 I-Smad Smurf STAT1 Smad7 Runx2 Smad complex Smad complex X Smad complex X

Runx2 Coactivator Target Target X Target genes Runx2 Coactivator genes genes Runx2 Runx2

Figure 2 Degradation of Smads and Runx2 by Smurf proteins. Figure 3 Cross-talk between Smads and STAT.( a) IFN-g induces Smurf proteins induce degradation of BMP-speciﬁc R-Smads. expression of Smad7, which in turn inhibits TGF-b signaling.( b) Through complex formation with I-Smads, they induce degrada- Latent STAT1 interacts with Runx2, and prevents nuclear tion of type I receptors and R-Smads.In addition, Smurf1 interacts localization of Runx2.STAT1 thus inhibits osteoblast differentia- with Runx2 and induces its degradation tion induced by BMP-speciﬁc R-Smads and Runx2

Oncogene TGF-b signaling and Runx K Miyazono et al 4236 increased bone mass despite excessive osteoclastogen- cancer, and perturbations of Runx3 function may affect esis, suggesting that STAT1 inhibits the process of bone TGF-b signaling, it will be interesting to determine formation in vivo.Kim et al.(2003) demonstrated whether these genes involved in cell cycle regulation that STAT1 physically interacts with Runx2 in the or apoptosis are regulated in gastric epithelium by cytoplasm, and inhibits nuclear localization of Runx2 cooperative activity of Smads and Runx proteins. (Figure 3b).Phosphorylation of tyrosine-701 of STAT1 is not required for the interaction with Runx2, indicat- ing that the interaction between STAT1 and Runx2 Perspectives occurs independent of IFN signaling.In STAT1 À/À cells, enhanced response of osteoblasts to BMP-2 was Smads have been shown to interact with many different observed, whereas some effects of BMP that do not transcription factors and transcriptional coactivators/ require Runx2 appeared to be unaffected.STAT1 thus corepressors.However, most of these proteins preferen- functions as a cytoplasmic attenuator of Runx2.These findings suggest the interesting possibility that STAT1 tially interact with TGF-b-specific R-Smads, and only several proteins bind to BMP-specific R-Smads (Miya- regulates the function of other Runx proteins, and that zawa et al., 2002). Thus, Runx proteins are unique, since TGF-b superfamily signaling is indirectly regulated by STAT1 through the Runx proteins. they interact with both TGF-b-specific R-Smads and BMP-specific R-Smads.Cooperation between BMP- specific R-Smads and Runx2 has recently been intensively studied, and some regulatory molecules, Runx3 and TGF-b/Smad signaling in gastric mucosa including Smurf1 and STAT1, have been identified.In contrast, cooperation between TGF-b-specific R-Smads Runx3-null mice exhibit several interesting phenotypes and Runx proteins have been studied mostly in the including defects in the development of proprioceptive induction of IgA synthesis, and relatively little in other dorsal root ganglion neurons (Inoue et al., 2002; biological responses.Since Runxl is involved in defini- Levanon et al., 2002) and of cytotoxic lineage thymo- tive hematopoiesis, and mutants of it have been reported cytes (Taniuchi et al., 2002; Woolf et al., 2003). to be involved in human acute leukemias, it will be Interestingly, hyperplasia of gastric mucosa was interesting to examine whether Runxl interacts with observed in Runx3-null mice and the gastric epithelial TGF-b-specific R-Smads and regulates growth and cells of these mice were refractory to the growth differentiation of hematopoietic cells.In addition, inhibition and apoptosis induced by TGF-b (Li et al., Runx3 has been shown to play critical roles in the 2002).Moreover, Li et al.(2002) reported that development of neuronal cells, thymocytes, and gastric approximately half of human gastric cancer cells do mucosa.It will be particulaly interesting to examine not express Runx3 due to hemizygous deletion and whether growth and apoptosis of these cells are DNA methylation of the Runx3 promoter. regulated by R-Smads and Runx3. TGF-b inhibits growth of epithelial cells through suppression of c-myc and CDC-25A as well as through induction of Cdk inhibitors p21 and p15 (Massague Acknowledgements We thank Drs Tadatsugu Taniguchi and Hiroshi Takayanagi et al., 2000). Various genes including DAP-kinase (Jang for sharing unpublished observations.We also thank Keiji et al., 2002), Daxx (Perlman et al., 2001), and SHIP Miyazawa, Hiromi Fukuda, and Masao Saitoh for the (Valderrama-Carvajal et al., 2002) have been reported to valuable discussion.This work was supported by Grants-in- be involved in induction of apoptosis by TGF-b.Since Aid for Scientific Research from the Ministry of Education, Runx3 plays a critical role in the development of gastric Science, Technology, Sports and Culture of Japan.

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