Leukemia (2011) 25, 1379–1388 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu SPOTLIGHT REVIEW The role of Smad signaling in hematopoiesis and translational hematology U Blank and S Karlsson Division of Molecular Medicine and Gene Therapy, Laboratory Medicine, Lund Stem Cell Center, Lund University Hospital, Lund, Sweden Hematopoietic stem cells (HSCs) reside in the bone marrow ligands are reflected in vitro, often leading to opposing findings (BM) of adult individuals and function to produce and between in vitro and in vivo systems. regenerate the entire blood and immune system over the In this review, we will discuss the role of TGF-b and Smad course of an individual’s lifetime. Historically, HSCs are among the most thoroughly characterized tissue-specific stem cells. signaling in normal hematopoiesis, featuring aspects of transla- Despite this, the regulation of fate options, such as self-renewal tional hematology, particularly the role of Smad signaling in the and differentiation, has remained elusive, partly because of the development of hematologic malignancies and how the Smad expansive plethora of factors and signaling cues that govern circuitry may be exploited for the purpose of stem cell HSC behavior in vivo. In the BM, HSCs are housed in expansion. It should be emphasized that although some of the specialized niches that dovetail the behavior of HSCs with the findings discussed here come from studies of human and need of the organism. The Smad-signaling pathway, which operates downstream of the transforming growth factor-b patients’ cells, the majority of the data derive from studies in (TGF-b) superfamily of ligands, regulates a diverse set of bio- well-defined mouse models. logical processes, including proliferation, differentiation and apoptosis, in many different organ systems. Much of the function of Smad signaling in hematopoiesis has remained Smad signaling nebulous due to early embryonic lethality of most knockout mouse models. However, recently new data have been The Smad-signaling circuitry embodies an evolutionary con- uncovered, suggesting that the Smad-signaling circuitry is served signaling module, which functions to convert biological intimately linked to HSC regulation. In this review, we bring the information from activated TGF-b receptor complexes at the cell Smad-signaling pathway into focus, chronicling key concepts and recent advances with respect to TGF-b-superfamily signal- surface to concrete transcriptional regulation in the nucleus. ing in normal and leukemic hematopoiesis. TGF-b ligands transmit signals through two types of serine/ Leukemia (2011) 25, 1379–1388; doi:10.1038/leu.2011.95; threonine kinase receptors known as type I and type II published online 13 May 2011 receptors.1 In vertebrates, seven different type I receptors Keywords: hematopoietic stem cell; smad signaling; TGF-b (ALK1–7) and five distinct type II receptors have been identified.1 Although some promiscuity occurs, each ligand generally signals through a specific combination of receptors (Figure 1). Following ligand binding, the type I receptor Introduction becomes activated through phosphorylation by the type II receptor. Activated type I receptors subsequently phosphorylate Transforming growth factor-b (TGF-b) is the founding member of the Smad proteins at residues in the C-terminus, leading to a large superfamily of secreted polypeptide growth factors, propagation of the signal intracellularly. The Smad family of which additionally includes activins, nodal, bone morpho- intracellular mediators is comprised of eight members in genetic proteins (BMPs), growth and differentiation factors and 1 mammals, Smad1–8, which can be further subdivided into others. From early development and continuously throughout three distinct classes based on structural properties and the adult life, TGF-b members carry out pivotal functions by specific functions they carry out.7 Receptor-regulated Smads (R- regulating biological events ranging from gastrulation and organ Smads), Smad1, 2, 3, 5 and 8, are the only Smads directly morphogenesis to homeostatic tissue turnover. Alterations in phosphorylated and activated by the kinase domain of type I components of the TGF-b-superfamily pathway lead to severe receptors. Phosphorylation of R-Smads results in a conforma- SPOTLIGHT developmental abnormalities and have been shown to underlie tional change, allowing complex formation with the common- a range of human diseases, including autoimmune and 2,3 Smad, Smad4. Activated complexes subsequently accumulate in cardiovascular disorders as well as cancer. A fundamental the nucleus, where they cooperate with other transcriptional co- feature of the TGF-b superfamily is its highly pleiotropic nature, regulators to modify target gene transcription. The third class of a phenomenon well illustrated within the hematopoietic system; Smads includes the inhibitory Smads, Smad6 and Smad7, which depending on the differentiation stage and environmental function in a negative feedback loop to inhibit TGF-b-super- context of the target cell, these factors can affect proliferation, 4–6 family signaling. TGF-b/activin/nodal and BMP/growth and differentiation and apoptosis either positively or negatively. differentiation factor employ different subsets of R-Smads. Part of the molecular basis for this is thought to stem from the R-Smad2 and 3 specifically relay signals from TGF-b and activin unique repertoire of transcriptional co-factors expressed by each receptors, whereas R-Smad1, 5 and 8 primarily operate specific cellular target. The context-dependent actions of TGF-b downstream of BMP receptors.1,8 Correspondence: Dr U Blank, Division of Molecular Medicine and Gene Therapy, Laboratory Medicine, Lund Stem Cell Center, Lund TGF-b in hematopoiesis University Hospital, BMC A12, Lund 221 84, Sweden. E-mail: [email protected] Received 13 January 2011; revised 25 March 2011; accepted 6 April TGF-b is categorized as one of the most potent inhibitors of 2011; published online 13 May 2011 hematopoietic stem cell (HSC) growth in vitro and a large body Smad signaling in hematopoiesis U Blank and S Karlsson 1380 Ligand Activin/Nodal TGF-β BMP Receptor ActR-IIA BMPR-II ActR-IIB ActR-IIA β TGF- RII ActR-IIB ALK4 ALK1 ALK2 ALK7 ALK5 P ALK3 P ALK6 P P I-Smads Smad1 Smad6 Smad2 Smad5 R-Smads R-Smads Smad7 Smad3 Smad8 SPOTLIGHT Smad4 Co-Smad Cytoplasm Nucleus DNA-binding partner P R-Smad P Smad4 R-Smad Smad4 Target gene Figure 1 The TGF-b superfamily. Schematic representation of the various components of the TGF-b-superfamily pathway, including ligands, receptors and Smads. Following ligand binding to type I and type II receptors, R-Smads become phosphorylated by activated type I receptors. Phosphorylation of R-Smads results in a conformational change allowing for complex formation with Smad4. The R-Smad/Smad4 complex subsequently translocates to the nucleus, where target gene transcription is modified in cooperation with other DNA-binding factors. P indicates phosphorylation. R-Smad denotes receptor-activated Smad; Co-Smad, common-Smad and I-Smad indicates inhibitory Smad. 4,9–11 of work from a variety of culture systems supports this notion. Vasculature Owing to the naturally quiescent state of HSCs, TGF-b has been hypothesized to be a cardinal regulator of HSC quiescence, maintaining a slow-cycling state of HSCs in vivo (Figure 2). In keeping with this, neutralization of TGF-b in vitro was shown to release early hematopoietic progenitor cells from quiescence.12–14 Several molecular mechanisms have been proposed to account HSC for TGF-b-mediated growth inhibition, including alterations in TGFβ cytokine receptor expression and up-regulation of cyclin- Stroma cell G0 dependent kinase inhibitors, such as p15, p21 and p27.13,15–21 Osteoblast BMP Smad4 However, it has been shown that TGF-b can exert growth Smad7 inhibitory actions independently of p21 and p27.22 Addition- ally, neutralization of TGF-b coupled with antisense knockdown ALK3 of p27 was shown to result in synergistically increased retroviral gene transfer efficiency in human CD34 þ bone marrow (BM) cells, implying that TGF-b and p27 work in separate pathways.16 þ b In human CD34 cells, TGF- -mediated cell-cycle arrest has Bone been suggested to occur through up-regulation of p57, another 23 member of the cyclin-dependent kinase inhibitor family. This Figure 2 The BM niche. Quiescent HSCs reside in the BM endosteal finding was further corroborated by the observation that p57 region in close proximity to osteoblastic cells and other cellular and was highly enriched in mouse CD34ÀKit þ lineageÀSca1 þ structural components with supportive and regulatory functions. BMPs (CD34ÀKLS) cells as opposed to the more mature CD34 þ KLS signal via ALK3 on osteoblastic cells, regulating the size of the fraction.24 Interestingly, a high level of p57 was shown to osteoblastic niche and consequently the size of the HSC pool. Autocrine and/or paracrine TGF-b is thought to induce quiescence of correlate with the activation status of Smad2 and Smad3, HSCs, contributing to maintenance of HSCs. Loss of Smad4 in HSCs which were reported to be uniquely phosphorylated in freshly results in impaired self-renewal capacity, whereas overexpression of isolated CD34ÀKLS cells but not in CD34 þ KLS progenitors.25 Smad7 leads to increased self-renewal of HSCs. Leukemia Smad signaling in hematopoiesis U Blank and S Karlsson 1381 In addition, TGF-b was shown to up-regulate p57 in CD34ÀKLS of redundant mechanisms in vivo, its role as a critical regulator cells in vitro.25 These findings point to a mechanism, where of HSC quiescence in vivo remains to be fully proven, despite TGF-b functions to induce p57 within the most primitive HSC intense research and increased knowledge of TGF-b signaling. compartment, thus maintaining their quiescent state in vivo. Most work regarding TGF-b in hematopoiesis has been b b carried out using TGF- 1. However, TGF- exists in three TGF-b signaling diversified: a role for transcriptional b b isoforms: TGF- 1–3.