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Transforming Growth Factor-ß Signaling in Stem Cells and Cancer

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Science  07 Oct 2005:
Vol. 310, Issue 5745, pp. 68-71
DOI: 10.1126/science.1118389


Transforming growth factor–β (TGF-β) and TGF-β–related proteins, such as the bone morphogenetic proteins, have emerged as key regulators of stem cell renewal and differentiation. These proteins have disparate roles in regulating the biology of embryonic stem cells and tumor suppression, and they help define the selection of cell fate and the progression of differentiation along a lineage. Here we illustrate their roles in embryonic stem cells and in the differentiation of neural, hematopoietic, mesenchymal, and gastrointestinal epithelial stem cells.

Stem cells are characterized by their undifferentiated state, their ability to give rise to fully differentiated cells, and their capacity for self-renewal. Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of a blastocyst. They contribute to all three germ layers: ectoderm, mesoderm, and endoderm. A defining feature of ES cells is their ability to undergo prolonged symmetrical cell division in culture to produce identical pluripotent progeny. ES cells have evoked great interest, given their expected capacity to self-renew—thus resulting in an expansion of the cell population—and to differentiate into desired cell types—thus representing new sources for cell replacement therapy. Stem cells share some characteristics of cancer cells, including their ability to proliferate by a similar self-renewal process and the loss of contact inhibition. Furthermore, tumor growth and cancer progression are, at least in some cases, thought to be driven by a cancer stem cell population. Thus, studies of stem cells may provide guidance for our understanding of cancer development, and vice versa.

Multiple signaling networks orchestrate the development and differentiation of ES and somatic stem cells into functional neuronal, hematopoietic, mesenchymal, and epithelial lineages. Among these, the signaling mechanisms activated by TGF-β–family proteins have emerged as key players in the self-renewal and maintenance of stem cells in their undifferentiated state, the selection of a differentiation lineage, and the progression of differentiation along an individual lineage. Through gene knockout experiments and observations with ES cells, TGF-β–family proteins have emerged as bi-functional regulators of the maturation of cells in each of the lineages mentioned above and as suppressors of carcinogenesis (1). Gradients of signaling, activated by the TGF-β–related bone morphogenetic proteins (BMPs), often lead to the selection of a defined differentiation pathway. TGF-β–family signaling then further drives differentiation along the lineage and maintains the differentiated phenotype of epithelial, mesenchymal, and other cell types, thereby interacting with other growth factors that help promote expansion of the cell population. When TGF-β signaling is disrupted, the imbalance can result in an undifferentiated phenotype, and cancer may ensue (2).

The TGF-β family contains about 30 structurally related growth and differentiation factors that include TGF-βs, activins, nodal and BMPs (13). TGF-β–family signals are conveyed through two types (type I and type II) of transmembrane receptor serine-threonine kinases, which form a complex at the cell surface (1, 3). Ligand binding to this complex induces a conformational change that induces phosphorylation and activation of type I receptors by type II receptors. Activation of Smad transcription factors ensues and results in their nuclear translocation and activation or repression of gene expression. The Smad activation and activity are modulated by various receptor- or Smad-interacting proteins that include ubiquitin and SUMO (small ubiquitin-related modifier) ligases, as well as multiple proteins in the transcription complexes (4). Depending on the differentiation stage of the target cell, the local environment, and the identity and dosage of the ligand, TGF-β proteins promote or inhibit cell proliferation, apoptosis, and differentiation. The diverse and often seemingly contradictory TGF-β functions can be understood by gene dosage; cross-talk of TGF-β–family signaling through Smads with other signaling pathways, such as Wnt and Hedgehog signaling and receptor tyrosine kinase signaling; and interactions of Smads with a multitude of DNA binding transcription factors, which themselves are targeted by signaling pathways (15).

TGF-β–Family Signaling in ES Cells

Although human and mouse ES cells show substantial differences in their requirements for factors in the growth medium, TGF-β–family proteins play a role in both the maintenance of the cells in their undifferentiated state and in the initiation of differentiation. Nodal and activin, two TGF-β–related proteins that share the same receptors and Smads, are thought to have a role in human ES cell maintenance; accordingly, their receptors are expressed and their Smads are activated in undifferentiated ES cells (6). The activated Smad pathway downstream from these ligands is thus likely to cooperate with Wnt signaling in keeping the ES cells undifferentiated and pluripotent. BMP signals, in cooperation with leukemia-inhibiting factor (LIF), a member of the interleukin-6 (IL-6) cytokine family, are required to maintain mouse ES cells in an undifferentiated state. Apparently, the BMPs act through activation of BMP Smads, which in turn activate Id protein expression (6) transcription.

TGF-β–family signals mediate key decisions that specify germ layer differentiation. Thus, activin induces ventral or dorsal mesoderm and endoderm in Xenopus explants, depending on the dosage. In mammals, this signaling pathway is presumably activated by nodal or related factors. Conversely, inhibition of activin or TGF-β, as well as BMP signaling, gives rise to neuroectoderm formation in Xenopus, whereas the absence of these factors also allows neuroectoderm formation from mouse ES cells in culture. Activin or TGF-β also induces mesoderm differentiation, whereas BMP signals confer ectodermal and mesodermal differentiation, of human ES cells (7). Activin signaling also leads ES cells to differentiate into endoderm. Thus, the presence or absence of TGF-β–family signals is a determinant of both maintenance and initial specification of ES cells and of the primary cell fate decision in early embryogenesis that will give rise to multiple cell lineages and cell fates.

Roles of TGF-β Family Members in Neural Stem Cells

Neural differentiation from uncommitted ES cells is thought to occur in the absence of exogenous TGF-β–family factors, yet it is also regulated by other inhibitory factors and cell adhesion proteins (8). Furthermore, BMPs inhibit neural differentiation and promote epidermal differentiation in Xenopus embryo explants; however, a gradient of BMP signaling does define the dorsoventral patterning of the neural tube (6). BMPs inhibit proliferation of ventricular zone progenitor cells at embryonic day 13 but enhance astroglial and neural crest cell differentiation at day 16; at higher doses, inducing apoptosis (9). Intriguingly, BMP-2 suppresses Sonic Hedgehog (Shh)–induced proliferation of medulloblastoma granule precursor cells, displaying a tumor-suppressive role. Additionally, the loss of Cripto, a functional Nodal receptor complex, also enhances neurogenesis (10), whereas the addition of Nodal suppresses neural differentiation.

Later in development, TGF-β promotes differentiation and lineage expansion of established progenitors; for example, by inducing autonomic gangliogenesis or olfactory neuron proliferation (11). There is a decrease of cerebellar Purkinje cells in Smad4–/– mice and a proliferation of precursor cells in the developing cortex of mice lacking the Smad adaptor protein ELF (12, 13). Once a precursor lineage is established, TGF-β signaling appears to accelerate the differentiation and lineage commitment of precursor cells. Once cells are fully differentiated, TGF-β inhibits the growth of normal glial cells, setting the stage for malignant transformation into gliomas, if the growth-inhibitory and tumor-suppressor role of TGF-β is inactivated. At later stages of tumorigenesis, in gliomas, when the growth-inhibitory function of TGF-β is lost, TGF-β stimulates tumor progression and invasiveness, concomitantly with the angiogenic and immunomodulating activities of increased TGF-β expression by these cells (12, 14) (Fig. 1).

Fig. 1.

Regulation of neural and neuronal differentiation by TGF-β–family signaling. The neural crest originates from the dorsal neural tube during the early stages of embryogenesis in vertebrates. Neural crest cells migrate to give rise to diverse cell types, including neurons and glia of the peripheral nervous system, smooth muscle cells, osteoblasts, and melanocytes. CNS, central nervous system; PNS, peripheral nervous system.

Roles of TGF-β Signaling in Hematopoietic Stem Cells

TGF-β–family proteins and their downstream signaling effectors, the Smads, also have key roles in hematopoietic differentiation (15, 16). TGF-β itself inhibits the proliferation of early multipotent hematopoietic stem cells but not that of later progenitors. The effects of TGF-β on more mature progenitor cells are complex and depend on the presence of other growth factors (17). In contrast to TGF-β, BMPs, in combination with cytokines, promote hematopoietic specification, differentiation, and proliferation of human ES cells (18). Although TGF-β acts as a negative regulator of hematopoietic progenitor and stem cells in vitro, impaired TGF-β signaling in vivo does not affect hematopoietic lineage selection (17). Indeed, the absence of a functional type I TGF-β receptor allows for normal development of hematopoietic progenitors and functional hematopoiesis in mouse embryos (19). The absence of Smad5, an effector of BMP and TGF-β signaling, enhances the efficiency of hematopoietic progenitor cell generation in embryoid bodies derived from ES cells (20), supporting the notion that TGF-β accelerates the differentiation and proliferation of committed precursors (Fig. 2). Signaling by TGF-β–family proteins through Smads may also regulate cell fate commitment decisions of myeloid versus lymphoid precursors. Enhanced myeloid differentiation at the expense of lymphoid commitment is observed when Smad7, which inhibits Smad activation, is overexpressed (21). To add to the complexity, the activation of feedback loops by TGF-β–family signaling further defines the regulation of hematopoietic stem cell differentiation. For example, BMPs activate the expression of the homeobox transcription factor Dlx1, which in turn blocks activin-induced differentiation of a hematopoietic cell line by interacting with Smad4 through its homeodomain (22).

Fig. 2.

Regulation of endodermal stem cells and differentiation into hepatocytes by TGF-β–family signaling. Ventral foregut endoderm cells develop into bipotential hepatoblasts that are committed to fetal hepatocytes. In hepatocarcinogenesis, human hepatic progenitor cells most likely give rise to hepatocellular carcinoma as well as cholangiocarcinomas. The lower part of the figure shows the role of TGF-β–family signaling in mesoderm and hematopoietic precursor differentiation.

Thus, TGF-β–family signaling through Smads exerts multiple effects that are dependent on the nature of the activating ligand and activated Smad pathway, as well as the nature and differentiation state of the targeted cell. Plenty of cross-talk of TGF-β-family signaling with other signaling pathways also occurs. Again, deregulation of TGF-β–family signaling may lead to malignant transformation and cancer progression. For example, expression of an AML1/EVI-1 chimeric gene that results from fusion of a segment of the protooncogene Evi-1 to the N-terminal half of AML1, blocks the antiproliferative activity of TGF-β (23). This is achieved by the association of EVI-1 with Smad3, resulting in inactivation of the transcription function of TGF-β–activated Smad3.

TGF-β in Mesenchymal Stem Cells

Although mesenchymal stem cells are found predominantly in bone marrow, they are also interspersed in muscle, adipose, and connective tissues, which are all of mesenchymal origin. The differentiation of mesenchymal stem cells into adipose, muscle, bone, or cartilage cells is defined by select TGF-β family members, often in cooperation with other signaling pathways. Although it is stimulatory for embryonic myoblasts, TGF-β itself inhibits the progression of differentiation and maturation of myoblasts, osteoblasts, and adipocytes. Combined with its stimulatory effect on mesenchymal cell proliferation, TGF-β signaling thus allows for an expansion of the mesenchymal stem cell population and of the progenitors of these different mesenchymal lineages (24). Because TGF-β expression and signaling are activated in response to injury, this response may be at the basis of efficient wound repair in mesenchymal tissues. Myostatin, a TGF-β–related factor that acts through the same type I receptor as TGF-β, is also emerging as a key regulator of myogenesis and possibly of fat differentiation. Inactivation of the myostatin gene confers a phenotype with excessive muscle mass (25). Conversely, BMPs drive mesenchymal cells into the osteoblast lineage yet can also stimulate fat cell differentiation under some culture conditions while antagonizing adipose maturation under others. BMP signaling may also allow mesenchymal cells to switch from an immature phenotype in one lineage to differentiation into another lineage. Indeed, BMPs activate early steps of osteoblast differentiation in myoblasts and pre-adipocytes, whereas BMP signaling in combination with retinoic acid signaling allows pre-adipocytes to differentiate into mature osteoblasts (26). The potent effects of BMPs on the differentiation of mesenchymal cells are at the basis of BMPs' ability to induce ectopic osteoblast differentiation and bone deposition after administration into muscle or connective tissue. At such sites, BMPs mobilize mesenchymal cells to differentiate into bone-depositing osteoblasts.

TGF-β Signals in Gastrointestinal Tissues and Cancers

TGF-β–family signaling is most prominent at the interface between development and cancer in gut epithelial cells. Several TGF-β signaling components are bona fide tumor suppressors, with the ability to constrain cell growth and inhibit cancer development at its early stages. Inactivation of at least one of these components (such as the TGF-β receptors, Smad2, or Smad4) occurs in almost all gastrointestinal tumors (2, 27). Also, Smad4+/– mice develop gastric tumors, and intercrossing of the Smad4+/– genotype into mice with a mutation in the adenomatous polyposis coli tumor suppressor APCΔ716 results in the development of larger and more invasive colorectal tumors than those observed in the presence of the two Smad4 alleles (28). These findings are consistent with the role of Smad4 in normal gut endoderm development. Support from genetic studies in the mouse has been crucial for identifying biologically significant proteins regulated by TGF-β in cancer suppression. Thus, genetic data demonstrate that only ELF, of the many adaptor proteins, is a TGF-β stem cell/differentiating factor, without which cancer ensues. Defects in gastrointestinal epithelial cell shape and polarity are also seen in Smad2+/–/Smad3+/– double heterozygous and elf–/– homozygous mice, further arguing for a role of TGF-β signaling in normal gastrointestinal epithelial development (29).

BMP signaling may also play an active role in the stem cell compartments of the colon, presumably by suppressing the effects of Wnt signaling and consequently limiting stem cell renewal. Mutations in the BMP receptor BMPR1A and in Smad4 contribute to juvenile intestinal polyposis and Cowden disease, respectively. Furthermore, inactivation of the gene for one of the type I BMP receptors in mice allows for an expansion of the stem and progenitor cell populations, eventually leading to intestinal polyposis resembling the human juvenile polyposis syndrome (2, 3).

Finally, TGF-β signaling also appears to be important for the transition of stem cells to a progenitor and fully differentiated phenotype in the liver and biliary system. Accordingly, Smad2+/–/Smad3+/– double heterozygous and elf–/– homozygous mice all show defective liver development, with elf+/– mice developing hepatocellular carcinoma (27, 30). Moreover, the TGF-β– and BMP-regulated protein PRAJA is expressed in hepatoblasts and modulates ELF and Smad3 (3, 31). The absence of this drive to normal epithelial differentiation may thus favor the formation of human hepatocellular carcinoma (Fig. 2).

As illustrated in this overview, TGF-β–family proteins and their signaling pathways play key roles in the self-renewal and maintenance of stem cells in their undifferentiated state, whereas changes in TGF-β–family signals drive the selection of defined differentiation pathways and their progression of differentiation. When deregulated, changes in TGF-β–family signaling may contribute to impaired differentiation and allow for the development of cancers, thus linking the differentiation of stem cells with the suppression of carcinogenesis.

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