Perspective

Id: A Target of BMP Signaling

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Science's STKE  24 Sep 2002:
Vol. 2002, Issue 151, pp. pe40
DOI: 10.1126/stke.2002.151.pe40

Abstract

Cytokines of the transforming growth factor-β (TGF-β) superfamily transduce their signals by activating receptor-regulated Smads (R-Smads). Distinct R-Smads or combinations of R-Smads are activated by TGF-β, activin, or bone morphogenetic proteins (BMPs). R-Smads activated by BMPs induce expression of Id proteins, which act as inhibitors of differentiation and stimulators of cell growth by inhibiting the function of basic helix-loop-helix transcription factors. In endothelial cells, TGF-β binds to two distinct type I receptor serine-threonine kinases, ALK-5 and ALK-1; the latter activates the same R-Smads that are activated by BMP and induces synthesis of Id (inhibitor of differentation or inhibitor of DNA binding) proteins. Growing evidence suggests that Id proteins may play crucial roles in angiogenesis, neurogenesis, and osteogenesis and act as key molecules in regulating biological responses induced by BMPs and TGF-β.

In the animal body, various cytokines bind to their receptors and transmit diverse arrays of intracellular signals. These cytokines and intracellular signals may synergize with or antagonize each other and, as a consequence, determine the fate of target cells. To produce certain biological effects, cytokines may regulate multiple signaling pathways. For example, in order to arrest cell growth, transforming growth factor-β (TGF-β) induces synthesis of cyclin-dependent kinase (CDK) inhibitors p15 and p21 or represses expression of c-Myc and tyrosine phosphatase Cdc25A, depending on the cell type (1). Sometimes, however, a single component acts as a "conductor" in signaling pathways and exhibits wide varieties of biological effects in multiple tissues. Expression of Id (inhibitor of differentiation or inhibitor of DNA binding) proteins, which are negative regulators of basic-helix-loop-helix (bHLH) transcription factors, is induced by bone morphogenetic proteins (BMPs), which are members of the TGF-β superfamily, in many cell types (2, 3). TGF-β also causes increased expression of Id proteins in certain types of cells (4). Recent findings suggest that Id proteins act as conductors in controlling multiple biological responses induced by BMPs and TGF-β.

Members of the TGF-β superfamily bind to two distinct cell surface receptors that contain serine-threonine kinase domains in their intracellular portions, known as type II and type I receptors. Upon ligand binding, these two receptors form heteromeric complexes in which type II receptors phosporylate type I receptors. Type I receptors then activate receptor-regulated Smads (R-Smads), which form oligomeric complexes with common partner (also called common mediator) Smad (Co-Smad), Smad4 (5) (Fig. 1A). Type I receptors thus act as downstream components of type II receptors in signaling pathways, and the former therefore determine the specificity of intracellular signals. The R-Smad and Co-Smad complexes then translocate into the nucleus and recruit various transcription factors and transcriptional coactivators or corepressors to regulate transcription of target genes. Although more than 30 proteins have been identified as members of the TGF-β superfamily, they can be sorted into two major subclasses on the basis of the R-Smads that they activate.

Fig. 1.

Two distinct pathways activated by the TGF-β superfamily. (A) Members of the TGF-β superfamily bind to type II and type I receptors. They form heteromeric complexes upon ligand binding, in which type II receptors phosporylate type I receptors. Type I receptors then activate R-Smads (Smad1 and Smad5, or Smad2 and Smad3), which associate with Co-Smad (Smad4). The R-Smad and Co-Smad complexes then move into the nucleus and regulate the expression of target genes. In endothelial cells, TGF-β binds to ALK-1, as well as to ALK-5; the former activates Smad1 and Smad5 and transmits BMP-like signals. Synthesis of Id proteins is increased in response to the Smad1-Smad5 pathway. (B) The Id proteins act as dominant negative antagonists of bHLH transcription factors. Tissue-specific bHLH transcription factors form heteromers with ubiquitous bHLH factors and activate the transcription of E-box-containing genes. Id proteins sequester ubiquitous bHLH factors and inhibit the action of bHLH transcription factors.

TGF-βs bind to TGF-β type I receptor (TβR-I), also known as activin receptor-like kinase-5 (ALK-5). Activins bind to ALK-4, and the cytokine Nodal binds to ALK-4 and ALK-7. ALKs 5, 4, and 7 are structurally similar to each other and activate two structurally related R-Smads: Smad 2 and Smad3. Thus, these cytokines transmit similar, although not identical, intracellular signals. In contrast, BMPs, including BMP-2 and BMP-4, bind to BMP-specific type I receptors, ALK-3 and ALK-6. BMP-6 and BMP-7 also bind to another type I receptor, ALK-2. Although ALK-2 is not highly similar to ALK-3 and ALK-6, these three receptors activate Smad1 and Smad5, but not Smad2 or Smad3. Anti-Müllerian hormone (AMH) also binds to ALK-2 and ALK-6 and activates Smads 1 and 5. Thus, members of the TGF-β superfamily can be classified as activators of Smad2 and Smad3 or of Smad1 and Smad5 (6).

A large number of target genes activated by TGF-β-specific Smad2 and Smad3 have been identified, including those encoding plasminogen activator-1 (PAI-1); type I collagen; cell cycle regulators p15 and p21; the junB transcription factor; and Smad7. Transcription of the c-myc gene is inhibited by TGF-β-specific Smads. To regulate these target genes, TGF-β-specific R-Smads physically interact with various transcription factors, including TFE3, the AP-1 complex, Sp1, FoxH3 (also called FAST1), and Runx. In contrast, BMP-specific Smads 1 and 5 interact with only a limited number of transcription factors, and most of them, including Runx transcription factors, are shared with TGF-β-specific R-Smads (7). Only a few proteins (for example, Vent-2 and Smad6) have been reported as direct targets of BMPs. However, several recent studies have revealed that Id HLH proteins are one of the most crucial targets of BMPs and that they may be responsible for exhibition of the biological activities of BMPs.

Id proteins were identified as negative regulators of bHLH transcription factors. Subsequently, retinoblastoma (Rb) and Ets family members were also shown to be interacting partners of Id proteins (8, 9). Four Id proteins, Id1 through Id4, have been identified in mammals. Mammalian Id proteins partially overlap in their profiles of expression and display some functional redundancy in vivo. The extramacrochaetae (emc) gene product is a Drosophila ortholog of mammalian Id proteins (10).

Generally, Id proteins act as positive regulators of cell proliferation and negative regulators of cell differentiation. Tissue-specific bHLH transcription factors form heteromers with ubiquitously expressed bHLH proteins, including HEB, E2-2, and E2A gene products, and activate the transcription of genes containing an E-box in their promoters. Id proteins lack a basic DNA binding region but possess an HLH dimerization motif (Fig. 1B) (8). Id proteins physically interact with ubiquitous bHLH transcription factors through their HLH regions, but these heterodimers containing Id proteins are unable to bind DNA. Id proteins thus sequester ubiquitous bHLH factors and act as dominant negative antagonists of bHLH transcription factors. Id proteins behave as inhibitors of differentiation, because many bHLH transcription factors positively regulate cell differentiation in various tissues and cells. For example, Id1 proteins in myoblasts associate with E2A protein and inhibit the formation of functional E2A-MyoD heterodimer, thus maintaining an undifferentiated phenotype of myoblasts.

Id proteins also positively regulate cell cycle progression. Id2 physically interacts with the active, hypophosphorylated form of Rb family proteins and inhibits antiproliferative functions of those proteins. Rb family proteins interact with E2F transcription factors through the pocket domain of the Rb family; because the HLH region of Id interacts with the pocket domain, E2F factors are released from Rb family proteins when the Rb proteins interact with Id2 (11). Although Id family proteins have similar biological activities in most cases, the ability to interact with Rb family proteins can be observed only in Id2 and possibly Id4. However, other Id proteins can also regulate cell cycle progression; in addition to their effects on Rb family proteins, Id proteins can regulate expression of CDK inhibitors, including p21, which occurs through suppression of bHLH transcription factors (9).

What then are the in vivo roles of Id proteins? During embryonic development as well as regeneration, the balance of cell proliferation and differentiation must be well coordinated. Id proteins are likely to be involved in such coordination. In fact, an important role for the Id proteins in cell proliferation and differentiation has been demonstrated in Id1-Id3 double knockout mice (12). Id1-, Id2-, or Id3-null mice are all viable. However, Id1 and Id3 double knockout mice die at day 13.5 of embryonic development, exhibiting small brain size with premature withdrawal of neuroblasts from the cell cycle and expression of neural-specific differentiation markers, indicating that Id proteins control the timing of switching from cell proliferation to differentiation. In addition, hemorrhage in the forebrain has been observed in Id1-Id3-null mice. Vasculogenesis is normal in these mice; however, dilated vessels without sprouting or branching were observed, indicating that the process of angiogenesis is impaired. The migration of endothelial cells appears to be abnormal in the mutant mice, although the molecular mechanism resulting in this phenotype remains to be determined. An important question thus arises: Are these functional roles of Id proteins related to the activity of BMPs?

Although the expression of Id proteins is increased by various stimuli, including some growth factors, BMPs are one of the most important signals that increase synthesis of Id1, Id2, and Id3 (2, 13). Analysis of the Id1 promoter revealed that the promoter activity was increased in cells treated with BMP but not in response to TGF-β, and that the promoter activation by BMPs occurred in a manner that required Smad1or Smad5, and Smad4 (14, 15).

Because Id proteins negatively regulate the action of myogenic bHLH factors, including MyoD, the Id proteins may be involved in the determination of the cell fate of mesenchymal cells induced by BMPs. Id proteins also act as important targets of BMPs in neuronal cells. During early embryogenesis in Xenopus, BMP antagonists induce formation of neural tissues, suggesting that BMPs inhibit neurogenesis in vivo. BMPs alter the developmental fate of fetal mouse brain cells and inhibit neurogenesis, but they are not sufficient to promote gliogenesis (3). In the nervous system, neurogenic bHLH transcription factors such as Mash1, neurogenin, and NeuroD induce neurogenesis. BMP-2 induces expression of Id1 and Id3 in neuroepithelial cells, which occurs in a Smad-dependent manner. Both Id1 and Id3 repress the promoter activation induced by bHLH heteordimers containing neurogenic bHLH factors, Id1 and Id3 also inhibit neurogenesis. Thus, these findings agree with the findings that Id1-Id3 double knockout mice exhibit premature neuronal differentiation in the brain, and they further support the notion that BMPs exert their antineurogenic effects by promoting the accumulation of Id proteins.

BMPs also induce the expression of Id1 in endothelial cells (16). Mutations in the human BMPR-II gene have been found in patients with primary pulmonary hypertension (PPH), a disorder of the pulmonary arteries characterized by formation of plexiform lesions and obliteration of small pulmonary arteries (17). These findings suggest important roles for Id proteins in the development and homeostasis of vascular tissues. Consistent with this observation, Smad1- or Smad5-null mice exhibit enlarged and dilated blood vessels (18-20), which are reminiscent of the vascular abnormalities observed in Id1-Id3 double knockout mice (12), indicating a functional link between BMP-Smad pathways and Id proteins in endothelial cells.

An important question is whether expression of Id proteins is induced only by BMPs or by TGF-β signals as well. TGF-β can bind not only to ALK5 (TβR-I) but also to ALK-1 in endothelial cells, although the affinity of TGF-β for ALK-1 is weaker than that for ALK-5 (21). ALK-1 is structurally quite similar to ALK-2 and is preferentially expressed in endothelial cells. ALK-1 thus induces expression of Id1 in endothelial cells upon stimulation by TGF-β. Mutations in ALK-1 genes are responsible for a vascular disorder, hereditary hemorrhagic telangiectasia (HHT) type II, characterized by multisystemic vascular dysplasia and recurrent bleeding (22).

Once TGF-β binds to the TGF-β type II receptor, ALK-5 and ALK-1 can be recruited to the receptor complexes in endothelial cells (Fig. 1A). ALK-5 induces the phosphorylation of Smad2 and Smad3 and promotes the expression of PAI-1 and other genes, and thus plays crucial roles in inhibiting the growth and migration of endothelial cells. ALK-1 specifically activates Smad1 and Smad5; in contrast to the phosphorylation of Smad2 and Smad3 by ALK-5, this phosphorylation of Smad1 and Smad5 by ALK-1 occurs only transiently (4). Smad1 and Smad5 induce expression of Id1 and possibly other Id proteins, which stimulate the migration of endothelial cells. Thus, two type I receptors activated by TGF-β have apparently opposing effects in endothelial cells. Balance between these two types of receptor signaling thus appears to be crucial for determining the state of activation of endothelial cells.

BMPs and TGF-β exert diverse biological effects. BMPs regulate growth, differentiation, and apoptosis of various types of cells, including mesenchymal progenitor cells, vascular cells, neuronal cells, and lymphocytes. In vivo, BMPs induce bone and cartilage formation when they are implanted in subcutaneous tissues, but they also regulate morphogenesis in nonskeletal tissues. Important questions that remain to be answered include which of these BMP-regulated biological effects are propagated by Id proteins. Id proteins may regulate neurogenesis, angiogenesis, and bone formation through inhibition of bHLH proteins. Id proteins could influence other effects of BMPs as well. ALK-1 activation by TGF-β also increases expression of Id proteins. Id proteins regulate apoptosis, cell senescence, and oncogenesis. It remains to be determined whether these effects of Id proteins are regulated by BMPs or TGF-β and, if so, what the effector proteins regulated by Id proteins might be. Identification of Id proteins as targets of BMP-specific R-Smads enables understanding of the mechanisms by which BMPs and other TGF-β superfamily proteins induce a wide variety of biological activities, but it also raises several important questions that must be answered to understand the functional roles of these cytokines in the animal body.

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