BMP Receptor

BMP Receptor A. Hari Reddi* Department of Orthopaedic Surgery, Center for Tissue Regeneration and Repair, University of California, Davis, 4635 Second Avenue, Room 2000, Sacramento, CA 95817, USA * corresponding author tel: 916-734-5749, fax: 916-734-5750, e-mail: [email protected] DOI: 10.1006/rwcy.2000.18006.

SUMMARY Bone morphogenetic proteins (BMPs) are osteotrophic and osteoinductive cytokines. BMPs are pleiotropic cytokines with actions not only on bone and cartilage but also on brain, teeth, skin, heart, lung, kidney, and a host of other tissues. They act on chemotaxis, mitosis, differentiation, cell survival and cell death. The biological actions of BMPs are mediated by binding to specific BMP receptors types I and II. There is a collaboration between the receptors and downstream substrates such as receptor-regulated R-Smads 1, 5, and 8. The phosphorylated R-Smads partner with Co-Smad, Smad4, and enter the nucleus to initiate the transcription of BMP-responsive genes. Smad6 inhibits the activity of type I BMP receptor protein kinase. Thus, the BMP receptors provide a finely regulated homeostatic system with checks and balances.

BACKGROUND It is axiomatic in biology that critical cytokines, growth factors, and morphogens bind to specific receptors and act in collaboration to initiate signal transduction to activate effector genes, culminating in biological action. In certain instances the biological actions may translate into therapeutic actions with clinical applications. Certainly, this is the case with members of the bone morphogenetic protein family (BMPs) and their cognate receptors and downstream effectors. The BMP family is a burgeoning area of signaling molecules with wide-ranging biological actions. However, although there are over 15 members of the family in mammals, they convergence on a paradoxically small number of receptors. In addition to the BMPs found in mammals, there are a number of homologs found in nematodes,

Drosophila, sea urchins, and in nonmammalian vertebrates. BMPs are distantly related to the TGF family, and it is customary to discuss them together. However, in this article the focus is exclusively on BMP receptor signaling in mammals, with emphasis on the conserved pathways between mammals and Drosophila.

Discovery The early experiments demonstrated the binding of radio-iodinated BMP-4 to cellular proteins (Paralkar et al., 1991). Specific interactions between BMP-4 and BMP-7 and BMP receptors were discovered by ten Dijke et al. (1994). Members of the BMP family bind to BMP type I and II receptors (Heldin et al., 1997; Kawabata et al., 1998; Reddi, 1998). The BMPs play a pivotal role in heteroligomerization of the BMP type IA and B receptors with BMP type II receptor. Type IA BMP receptor (earlier known as ALK-3) and type IB BMP receptor (earlier known as ALK-6) are phosphorylated by type II receptor (Kawabata et al., 1998). During the course of work on TGF type I receptor, seven different activin receptor-like kinases (ALKs) were cloned and called ALK-1 through ALK-7 (ten Dijke et al., 1994; Kang and Reddi, 1996). Certain type I receptors for TGF and BMPs interact with an immunophilin called FKBP-12 (Wang et al., 1994).

Main activities and pathophysiological roles BMPs are multifunctional morphogens involved in pattern formation, mesoderm induction, differentiation, regeneration, and the inevitable cell death and

1886 A. Hari Reddi related apoptosis (Reddi, 1998; Bhatia et al., 1999). Unlike TGF , which causes fibrosis of diverse tissues, BMPs are critical for embryonic development and recapitulation of developmental stages during regeneration and tissue repair (Reddi, 1998). In amphibians such as Xenopus, BMPs induce ventral mesoderm derivatives, while activin induces dorsal mesoderm. Disruption of the BMP signals may induce neural lineages by default. Thus BMP signaling mechanisms play a key morphogenetic role. The general scheme of the BMP signaling pathway includes interaction and binding to BMP type I and II receptors that function as serine/threonine kinases (Heldin et al., 1997; Kawabata et al., 1998; Reddi, 1998). The resulting phosphorylated acceptor proteins are called Smad1, Smad5, and Smad8. These receptor-regulated Smads (R-Smads) interact intimately with plasma membrane proteins, including SARA (Smad anchor for receptor activation). The phosphorylation of R-Smads enables them to seek their common functional partner Smads, the so-called Co-Smads. Smads are trimeric molecules forming oligomeric cytoplasmic complexes that are translocated into the nucleus. In the nucleus, Smads interact with specific DNA sequences directly or indirectly via Smad-interacting proteins (SIPs). The biological functions of the R-Smad/Co-Smad partnership is regulated by inhibitory Smads (I-Smads), such as Smad6 and Smad7. The inhibitory Smads are generally resident in the nucleus and BMPs appear to translocate them into cytoplasm to competitively inhibit receptormediated phosphorylation of R-Smads.

GENE Little is known at present.

PROTEIN Little is known at present.

receptor phosphorylates the BMP receptor-regulated R-Smads called Smad1, Smad5, and Smad8. It is noteworthy to recall that TGF and activin receptors phosphorylate Smad2 and Smad3. The receptorregulated Smads are in this respect pathwayrestricted. However, irrespective of whether the BMP or TGF /activin pathway is involved, the various R-Smads interact with a common Smad4 (Co-Smad) and form oligomeric complexes which translocate into the nucleus, possibly due to a conformational change (Heldin et al., 1997; Attisano and Wrana, 1998; Reddi, 1998). Smads are an evolutionarily conserved family of molecules from nematodes (Savage et al., 1996). They have a molecular mass between 40 and 65 kDa. The three functional classes of Smads are: (1) The pathway-restricted receptor-regulated Smads (RSmads), of which Smad1, Smad5, and Smad8 are specific for the BMP signaling pathway (Tamaki et al., 1998; Nishimura et al., 1998); (2) the Co-Smads, of which the primary member is Smad4, also known as dpc4 (deleted in pancreatic carcinoma 4), a tumor suppressor gene (Hahn et al., 1996; Zawel et al., 1998); (3) the inhibitory Smads (I-Smads, also called anti-Smads), as exemplified by Smad6 and Smad7. Smad1 is also activated by BMP-7 type I receptor via ALK-2, an activin type I receptor (Yamamoto et al., 1997; Macias-Silva et al., 1998). Smads have conserved N- and C-terminal domains called MAD (Mothers against dpp in Drosophila) homology domain (MH) 1 and 2, respectively. A linker domain with a proline-rich sequence is found between the MH1 and MH2 domains. The `C'terminal SSXS motif is the critical phosphorylation site in R-Smads (Souchelnytskyi et al., 1998). The MH2 domain of Smad is the effector domain involved in oligomerization and DNA interaction. The MH1 domain consists of about 130 amino acids and is implicated in DNA binding. The prolinerich linker region contains a PY motif with potential to interact with the WW domain. Emerging data from several groups have pointed to Smads as the key signal mediators of the BMP family. Smad1 and Smad5 are the signaling substrates for BMP type I receptor kinase.

SIGNAL TRANSDUCTION

Associated or intrinsic kinases

Cytoplasmic signaling cascades

The interaction of the BMP family ligand with the type I and II BMP receptor serine/threonine kinases results in activation of type I receptor kinase. The type I and II receptors for BMPs appear to interact with the ligand concurrently. The activated type I BMP

Although considerable evidence has accumulated in favor of Smad pathways, it is possible that BMPs may act via other signaling cascades, including Jun and MAP kinases (Adachi-Yamada et al., 1999; Ulloa et al., 1999; Liberati et al., 1999).

BMP Receptor 1887

DOWNSTREAM GENE ACTIVATION

Transcription factors activated The precise nature of the BMP-induced promoter sequences are not clear. Smad1 interacts with DNAbinding proteins (Shi et al., 1999). BMPs induce a homeobox gene related to human Hox 11 gene and called Tlx-2 (Tang et al., 1998). R-Smads are phosphorylated by type I BMP receptors. The potential role of the plasma membrane protein called Smad anchor for receptor activation (SARA) in BMP signaling is not clear, although it has been suggested that it might play a role in recruitment of Smad2 and Smad3 to TGF receptors. Upon translocation into the nucleus, Smad1 and Smad5 in partnership with Smad4 activate downstream genes. Although BMP-response genes are known to be activated by BMPs few studies have implicated BMPs in the activation of specific transcription factors such as AP-1, c-bfa1, and c-fos. Although mice devoid of core-binding factor A1 (CBFA1) lack bone, the role of BMP-4 or BMP-7 in the regulation of CBFA1 is not clear. Equal attention should also be paid to transcriptional repressors. The recent work of Verschueren et al. (2000) has implicated Smadinteracting protein 1 (SIP-1), a zinc finger-containing homeodomain protein that binds to R-Smads. SIP-1 interacts with the brachyury gene in Xenopus. It is probable that SIP-1 functions as a transcriptional repressor and R-Smads may overcome this repression by interaction with SIP-1 (Verschueren et al., 2000). Both Smad6 and Smad7 are induced by BMPs and are translocated from nucleus to cytoplasm. In the cytoplasm Smad6 and Smad7 may inhibit type I receptor kinase-mediated phosphorylation of Smad1, Smad5, and Smad8 (Imamura et al., 1997; Nakao et al., 1997; Kawabata et al., 1998; Bhushan et al., 1998).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY The most important action of the BMP family of ligands is new bone formation in vivo. Despite the advances of the past two years there is a gap in our knowledge of the processes between the entry of the oligomeric R-Smads ± Smad1, Smad5, and Smad8 ± and Co-Smad4 into the nucleus and the attendant

gene expression and the lineage of bone differentiation. What cascade of genes is induced and/or repressed to set into motion the train of events leading to the developmental cascade of bone and cartilage formation? Although a flash of excitement was heralded by the discovery of CBFA1 in bone formation, the connection between the Smad pathway and BMP signaling is still shrouded in mystery. Perhaps BMP-binding proteins such as noggin and chordin, which interact with BMP-4 with the same affinity as the type I and II serine/threonine kinase BMP receptors, may hold some secrets. However, the nuclear gene activation needs considerable scrutiny and is the next big step in the BMP receptor pathway.

THERAPEUTIC UTILITY There is much expectation and hope that BMPs can be used with BMP receptors and the signaling substrate Smads in preclinical and clinical situations. In this regard it is noteworthy that upon transfection into mesenchymal cells, adenovirus vectors with Smad1 and Smad5 confer an osteoblastic phenotype as assessed by alkaline phosphatase activity (Fujii and Miyazono, personal communication). This raises the possibility that Smad1 and Smad5 may initiate the lower threshold of the biological response and that additional stimulation by BMP ligand might complete the progression of the cascade. However, additional experiments in vivo are needed to further extend the conceptual framework for the action of R-Smads. The critical role of R-Smad5 and Co-Smad4 is illustrated by embryonic lethality and other developmental defects. Smad4 knockout mice die by embryonic day 7.5 in utero due to gastrulation defects (Sirard et al., 1998; Yang et al., 1998). Smad5 knockout results in impaired angiogenesis with decreased smooth muscle cells and attendant vascular pathology (Yang et al., 1999). It is noteworthy that angiogenesis and vascular invasion into hypertrophic cartilage is a prerequisite for optimal bone formation and modeling by osteoclasts.

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