Tài liệu Báo cáo khoa học: Bone morphogenetic protein signaling in stem cells ) one signal, many consequences pptx

MINIREVIEW Bone morphogenetic protein signaling in stem cells ) one signal, many consequences Toni U. Wagner Physiological Chemistry I, University of Wuerzburg, Germany BMP signals in stem cells Bone morphogenetic protein (BMP) signals have tre- mendous effects on all kinds of cells. Most striking and defining, however, are the reactions that stem and progenitor cells show upon exposure to BMP ligands. Various stem cell types utilize BMP signals in a multi- tude of ways in order to define their fates. The integra- tion of this pathway with a variety of other signals is still poorly understood, but recent discoveries strongly suggest that stem cell niches, areas with a certain sig- nal–molecule cocktail, are responsible for the final out- come of BMP signaling, be it by modulating the ligand itself, or the cascade transducing the signal within the cell. Regulation of BMP signaling is seen at all molecular levels: ligands, receptors, transducers, tran- scription complex composition and chromatin state. The present review focuses on data gathered on the role of BMP signaling in selected stem cell systems. Due to space limitations, numerous stem cell niches described to be influenced by BMP are not reviewed. We try to focus on publications that represent the wide variety of effects induced by BMP signals. The BMP signaling cascades The basic BMP signaling process is started by homo- or heterodimeric BMP ligands. Upon binding to type I receptors, formation of a heteromeric complex with type II receptors is induced. In this simple transduction Keywords apoptosis; BMP; differentiation; pluripotency; signaling; stem cells Correspondence T. U. Wagner, Physiological Chemistry I, University of Wuerzburg, 97070 Wuerzburg, Germany Fax: +49 931 888 4150 Tel: +49 931 888 4165 E-mail: toni.wagner@biozentrum. uni-wuerzburg.de Website: http://www.biozentrum. uni-wuerzburg.de/pc/pc1/ (Received 1 December 2006, revised 29 March 2007, accepted 19 April 2007) doi:10.1111/j.1742-4658.2007.05839.x Bone morphogenetic protein (BMP) signals play key roles throughout embryology, from the earliest patterning events, via tissue specification, through organ development and again in germ cell differentiation. While both input and the transducer molecules are rather well studied, the final outcome of a BMP signal is basically unpredictable and differs enormously between previously studied cell types. As already suggested by their name, BMPs exhibit most of their (known) functions on stem cells and precursor cells, usually driving them into various types of differentiation or death. In this minireview, some prime examples of BMP effects on several very different stem-cell types are discussed. Abbreviations BMP, bone morphogenetic protein; ES, embryonic stem; ID, inhibitors of DNA-binding; GDF3, growth and differentiation factor-3; GFAP, glial fibrillary acidic protein; LIF, leukaemia inhibitory factor; MAPK, mitogen-activated protein kinase; MPC, mesodermal progenitor cell; NSC, neural stem ⁄ progenitor cell; PGC, primordial germ cell; STAT3, signal transducer and activator of transcription-3; TGF-b, transforming growth factor b. 2968 FEBS Journal 274 (2007) 2968–2976 ª 2007 The Author Journal compilation ª 2007 FEBS version, the type II receptors then phosphorylate the type I receptors, which subsequently activate R-Smads (Smads 1, 5 and 8 for BMP ligands) by serine-threonine phosphorylation [1,2]. R-Smads are transcription fac- tors, which need activation (usually by phosphoryla- tion) and subsequent multimerization in order to become active and accumulate in the nucleus. Once activated, the R-Smad is able to bind to the Co-Smad (Smad4) and translocate to, or accumulate in the nuc- leus [3]. There, together with a wide variety of cofac- tors, target gene transcription is usually activated when R-Smads are in play. Of course, this very linear pathway is far from real- ity. BMP receptors have been shown to convey signals not only by Smad phosphorylation, but also through p38 activation [3]. Furthermore, transduction can as well occur through so-called repressor-Smads and Smads specific for transforming growth factor b (TGF-b) signals such as Nodal (Table 1) [2]. BMP signals are strongly influenced by many addi- tional parameters, such as the mode of oligomerization of receptors even prior to ligand-binding and resulting differences in downstream targets have also been clar- ified in much more detail [1]. Regulation is further fine-tuned by BMP receptor regulation through degra- dation and dephosphorylation [4], different modes of endocytosis [5], interaction with other pathways and expression of pseudo-receptors [6,7]. Further downstream, Smads are again subject to massive functional modulation by interaction with other transcriptional modifiers [8–11], nuclear import and export regulation [3], as well as dephosphorylation [12–15]. BMP signals aid to keep pluripotency in embryonic stem cell cultures Among the hot topics in current research is the genera- tion and use of stem cells. The theoretical applications of having expandable and differentiation-controllable stem cell cultures are extremely promising. However, basic knowledge of molecular changes happening in cells that are transferred from the embryonic milieu to the cell culture dish is missing. A different side of the same problem is the lack of information on factors that guide self-renewal and plu- ripotency in the embryo or adult. Since 1988, the key player in embryonic stem (ES)- cell media for the best established culture system, murine ES-cells, has been leukaemia inhibitory factor (LIF) [16]. Even though cultures have to be addition- ally supplemented by serum, feeder cells and other factors, LIF is considered to be necessary for pluripo- tency. The signaling cascade triggered by LIF is trans- duced through phosphorylation and subsequent translocation of the signal transducer and activator of transcription-3 (STAT3) to the nucleus [17,18]. Although most of the data reviewed below has been collected with mouse embryonic stem cells, it should be noted that recent studies in nonhuman primate ES-cell cultures [19] as well as in human ES-cell culture systems [20,21], have demonstrated complete independ- ence of LIF and STAT3. Back in the mouse system, feeder cells and serum can be omitted if BMP2 ⁄ 4 and LIF are present in the medium [22], resulting in a very defined two-factor sys- tem to study pluripotency. The same work demonstra- ted that the downstream target genes primarily responsible for the pluripotency maintenance effect of BMPs under these conditions are the inhibitors of DNA-binding (ID) genes. ID gene transcription was previously shown to be enhanced by a Smad1–Smad4 complex directly binding GC-rich elements in combina- tion with Smad-binding elements (SBE, sequence: GTCT) present in the ID1 promoter region [23]. ID gene expression is further enhanced by another well known pluripotency associated factor called Nanog [24] in a not yet understood way. Adding to the picture are data obtained by micro- array-based analysis [25] of murine stem cells, in which Table 1. BMP and TGF-b signal transducer molecules of the Smad family and their respective function [1,2]. Name Type Ligands Receptors (type I) Function Smad1 R-Smad AMH, BMP2 ⁄ 4 ⁄ 7 ALK1 ⁄ 2 ⁄ 3 ⁄ 6 Transcriptional regulation Smad2 R-Smad Activin, Nodal, TGF-b ALK4 ⁄ 5 ⁄ 7 Transcriptional regulation Smad3 R-Smad Activin, Nodal, TGF-b ALK4 ⁄ 5 ⁄ 7 Transcriptional regulation Smad4 Co-Smad all – Co-Smad needed for All R-Smads Smad5 R-Smad AMH, BMP2 ⁄ 4 ⁄ 7 ALK1 ⁄ 2 ⁄ 3 ⁄ 6 Transcriptional regulation Smad6 I-Smad – All Decoy Smad, inhibition of Smad interactions Smad7 I-Smad – All Decoy Smad, inhibition of Smad interactions Smad8 R-Smad AMH, BMP2 ⁄ 4 ⁄ 7 ALK1 ⁄ 2 ⁄ 3 ⁄ 6 Transcriptional regulation T. U. Wagner BMP signaling in stem cells FEBS Journal 274 (2007) 2968–2976 ª 2007 The Author Journal compilation ª 2007 FEBS 2969 several genes with STAT3 dependent expression were identified. Among the list of strongly up-regulated genes after LIF ⁄ STAT3 signaling inactivation are fac- tors generally associated with TGF-b and BMP signa- ling cascades including Lefty1, ID1 and ID2. These data already indicate negative transcriptional regula- tion between LIF ⁄ STAT3 signaling on one end, and TGF-b ⁄ BMP signaling on the other end. Strong activation of the BMP pathway was shown to lead to differentiation of embryonic stem cells into mesodermal and endodermal lineages, whereas neural differentiation is actively suppressed [22]. Low levels of BMP, followed by transduction via Smad1, already leads to up-regulation of typical mark- ers for early stages of mesodermal differentiation inclu- ding the transcription factor Brachyury. Present in only low amounts, Brachyury forms a complex with STAT3. This complex has been shown to bind to the Nanog promoter and enhance its transcription. Nanog protein in turn was proven to bind to Smad1 and sup- press formation of Smad1 transcriptional activator complexes [26]. Closing the regulatory circle, a prime target of the thus inhibited Smad1-complex mediated transcriptional activation would be Brachyury. Thus, early stages of differentiation triggered by low level BMP signaling are reversible by simultaneous presence and action of Nanog and STAT3. Consequently, sup- port of pluripotency by BMP signals is not only highly dose-dependent, but also needs to be counter-regulated (e.g. by STAT3 and Nanog). Recapitulating, the BMP-signaling pathway promotes pluripotency only indirectly by driving expression of ID genes in a Smad1-dependent manner. ID-proteins block neural differentiation of ES-cells by sequestering tran- scription factors needed to initiate commitment to this lineage. Concurrently, the differentiation induction effects of BMP are counteracted by STAT3 and Nanog, which are able to suppress activation of Smad1-target genes necessary for differentiation into mesodermal and endodermal cell fates. In other words, the essence of defined medium murine ES-cell culture appears to be the simultaneous action of STAT3 and Smad in a certain ratio. Down- stream, negative regulation of differentiation pro- grammes for mesodermal and endodermal fates (mediated by STAT3) as well as neuro-ectodermal line- ages (controlled by IDs) is initiated, resulting in the blockage of any kind of differentiation (Fig. 1). Taking these data from the culture system, it is inter- esting to look at studies on BMP signaling proteins in early embryonic development. Although Smad4 – ⁄ – mouse embryos do not successfully undergo gastrula- tion and die before embryonic day 7.5, it was possible to derive ES-cell lines from the inner cell mass of these mutants [27]. As the only Co-Smad, Smad4 is abso- lutely necessary for any Smad-linked BMP signal con- duction to the nucleus. Thus, these experiments suggest that the pluripotent stem cell state within the embryo is not depending on Smad signaling. Initially, this contra- dicts a basal role of BMP in pluripotency described before based on cell culture experiments. There is, how- ever, dependency on BMPR-IA (ALK3) because it is impossible to derive ES-cell lines from ALK3 null embryos [27]. The discrepancy of BMP receptor dependence on one hand and Smad4 independence on the other suggests a Smad-independent mode of BMP signal transduction. The only other known transduc- tion pathway of BMP receptor activation is mediated by p38, a mitogen-activated protein kinase (MAPK) family member, via a complex of adapter proteins including XIAP, Tab1 ⁄ 2 and Tak1 [28,29]. Here, it is interesting to note that BMP4 treatment of mouse ES-cell cultures results not only in up-regulation of ID genes, but also in the up-regulation of Oct4, a definitive marker of pluripotency, accompanied by a short- termed drop of p38 phosphorylation levels [27]. The mediators of these effects remain unidentified, with many candidates from MAPK phosphatase families. In a key experiment, simulation of the BMP induced de-phosphorylation effect on p38 by its inhibitor SB23580 enabled derivation of pluripotent stem cell lines from ALK3 – ⁄ – embryos. Even more striking is the finding that these cell lines were subsequently able to Fig. 1. Model of pluripotency control in cultured feeder and serum- free mouse embryonic stem cells. Parallel activity of STAT3 and Smad leads to inhibition of differentiation programs induced by the other pathway, thereby upholding the pluripotent state of the cells. The balance is easily broken upon signal increase in any direction. BMP signaling in stem cells T. U. Wagner 2970 FEBS Journal 274 (2007) 2968–2976 ª 2007 The Author Journal compilation ª 2007 FEBS tolerate lack of ALK3 in absence of the inhibitor. Although functional proof is missing, the authors found up-regulation of ALK1 and ALK2 in these cells and suggest these receptors to be able to compensate ALK3 loss. Bringing the different aspects together, alternative BMP signaling pathways all seem to be able to support pluripotency, but a complete loss of BMP signal transduction is not compatible with stemness. To truly clarify this situation, additional studies using con- ditional depletion of all combinations of the suggested transduction ways are needed. Yet another BMP signal influencing factor associ- ated with pluripotency has been identified, namely growth and differentiation factor-3 (GDF3) [30]. GDF3 is exclusively expressed in the undifferentiated state in both mouse and human ES-cell culture. GDF3 is a secreted factor, which is able to bind to and thereby inactivate BMP4. Reduction of GDF3 expres- sion in murine ES-cells lead to increased independence of LIF but, at the same time, to a lack of mesodermal and endodermal differentiation-ability in vitro. In vivo, GDF3 is expressed during early embryogenesis in mice, notably in the inner cell mass. Protein localization shows extracellular distribution throughout the blasto- cyst embryo. During gastrulation, GDF3 mRNA was detected in the node. These data substantiate the notion that fine-tuning BMP-signal strength, timing and downstream pathway choices are strongly influencing cell fate decisions both in early embryonic cells and stem cell cultures. How- ever, the molecular mechanisms underlying this non- linear behaviour are not yet identified. They likely include cross-talk with other prominent signaling path- ways such as Wnts on multiple levels of the cascades. BMP action in other stem cell niches BMP pathways are not only involved in pluripotency control, but also in various other stem cell niches, both in adults and embryos. The roles of BMP in those niches are far from uniform. BMP signaling induces differentiation of neural stem cells Another variant of signal integration between BMPs and STAT3 has been identified in the case of differen- tiation of neural stem ⁄ progenitor cells (NSCs). Here, the neurogenesis versus astrocytogenesis decision during early differentiation is based on a network of negative regulation. Co-immunoprecipitation assays showed that STAT3 and Smad1 are complexed via the transcriptional cofactor p300 [31]. This complex enhances the differentiation of fetal neuroepithelial cells into astrocytes, by binding and hyper activating the promoter of glial fibrillary acidic protein (GFAP). In parallel, BMP signals lead to enhanced expression of ID proteins, which in turn bind and sequester bHLH transcription factors such as Neurogenin1 and Mash1, both responsible for neurogenesis [32]. Addi- tionally, BMP exposure results in down-regulation of Olig2 expression [33]. Olig2 in turn inhibits formation of the GFAP superactivator complex STAT3–p300– Smad1 [34], thus clearing the path for neuronal differ- entiation for cells exposed to low amounts of BMPs. The p300–Smad1 complex is target for yet another reg- ulatory input. Neurogenin has been shown to compete with STAT3 for its recruitment. Although the GFAP promoter is hyper activated when bound by Smad1– p300–STAT3, the neuroD promoter is strongly driven by binding of Smad1–p300–Neurogenin [35], giving BMP signals a role in neurogenesis as well. Other stud- ies [36] have shown that LIF or BMP4 alone are also able to drive GFAP expression in neurosphere cul- tures. Phenotypically, the resulting GFAP + cells gener- ated by either LIF or BMP4 differ strongly: whereas LIF induces GFAP expressing NSCs to become elon- gated and stay proliferative, BMP4 application results in cell-cycle exit and a star-like cell-morphology. Fur- thermore, LIF treatment leads to an upkeep of progen- itor features, such as prolonged culture ability and the potential to undergo neural differentiation, whereas BMP4 decreases both. These results stress that BMP signaling is indeed an antiproliferative and differenti- ation inductive signal for neural stem cells, again (as shown and discussed for murine ES-cells) modulated by LIF ⁄ STAT3 in a highly dose-dependent manner. BMP signals as inhibitors of differentiation of pancreatic progenitors A prototypic example for a cell type where BMP sig- nals play an inhibitory role for differentiation are pan- creatic progenitor cells. Upon treatment of these progenitors with BMP4, increased levels of ID2 result in inhibition of NeuroD function [37]. NeuroD is a classical target for ID proteins because it is a member of the bHLH transcription factor family, which can be efficiently bound and thereby inactivated by ID pro- teins. In the rat tumour cell line AR42J, derived from the acinar pancreas, and isolated primary interferon-c- NOD epithelial duct cells, ID2 expression leads to down-regulation of the NeuroD target gene PAX6, an important factor for final differentiation of the progen- itors into endocrine cells. In parallel, BMP4 treatment increases proliferation activity of the progenitors. T. U. Wagner BMP signaling in stem cells FEBS Journal 274 (2007) 2968–2976 ª 2007 The Author Journal compilation ª 2007 FEBS 2971 These data once more demonstrate that BMP signals are able to promote stemness and block differentiation in specific contexts. BMP as a specification switch in hair-cell progenitors NeuroD also plays essential roles in hair-cell specifi- cation in the inner ear. Recently, the effects of BMP on hair-cell progenitors have been investigated. BMP4 is a well established marker for otic sensory patches in various species. Over expression of BMP4 in explanted chicken otic vesicles leads to fewer hair cells [38]. Inhibition of BMP signals by Noggin results in increasing numbers of hair-cells. Cell-cycle and apoptosis analyses of these experiments reveal that BMP4 not only suppresses expression of prosen- sory markers (itself including), but also drives the proliferative sensory precursor cells into apoptosis. In the same context, Noggin application is able to expand the sensory patches without increasing pro- genitor proliferation. This suggests that BMP4 has a double function in restriction of hair-cell number: block of differentiation and stop of progenitor prolif- eration, with both effects leading to apoptosis. The molecular mechanism at work in these progenitor cells has not yet been addressed, but might include ID protein mediated block of bHLH factors such as NeuroD, which are responsible for correct progres- sion of final differentiation steps. BMP signals lead to proliferation, apoptosis and cell-cycle arrest within the eye Studies in the chick embryo have revealed a role for BMP4 in eye development. Implantation experiments of beads soaked in BMP4 have shown that BMP is able to induce programmed cell death (apoptosis). Blocking the BMP pathway by using Noggin-leaded beads does not lead to over proliferation but, on the contrary, restricts growth and, when applied for longer periods, will result in reduced size of the optic cup [39]. Surprisingly, apoptosis is inhibited at the same time. In line with this, BMP4, even though responsible for programmed cell death in the optic cup, increases cell proliferation. The effects on lens tissue are completely different: Noggin cannot stop apoptosis there, and BMP4 ⁄ 7 application leads to over proliferation. This set of experiments clearly demonstrates how versatile the BMP pathway really is. Within a very small region, clear subdivision of pro-apoptotic, pro- proliferative and ignorant cell responses to BMP signal effects alternate. To date, the intracellular mechanisms are not well understood. In almost all cases studied, BMP signalling will directly lead to target gene up- regulation. The best studied ones with functions in stem and progenitor cells are ID1-4 and Msx1 ⁄ 2. Probably, there are many more direct as well as cell-type specific targets with yet unknown functions to be found once more in-depth molecular analyses are performed. Cell fate determination as a consequence of Nodal versus BMP signals Patterning events during early development are often guided by BMP activity gradients. In a recently des- cribed case, mesodermal progenitor cell commitment was shown to be controlled by a graded exposure to BMP and Nodal ligands. With zebrafish as a model system, Szeto and Kimmelman [40] used elegant trans- plantation experiments to demonstrate that the somites along the anterior–posterior body axis are divided into three regions: the anterior trunk, the posterior trunk and the tail. According to their experiments, the fate of mesodermal progenitor cells (MPCs) is set at gastru- lation. A BMP signal gradient originating from the posterior end of the embryo establishes a boundary between trunk and tail domains. Determination of the two trunk domains is probably due to higher levels of Nodal signaling and weakening of BMP signaling by antagonists such as Chordin or Follistatin, which are most likely genetically downstream of Nodal. The MPCs are then able to integrate and interpret the signal strengths of Nodal and BMP by entering the somite regions at different somites. Strong Nodal drives them in early, at somite 1, whereas strong BMP delays their entry until somite 16. MPCs receiving both weak Nodal and weak BMP input enter in-between, at somite 9. So far, it has not been possible to truly visualize gra- ded signal activities in living embryos. Furthermore, the cellular machinery for signal integration also remains elusive. Whether this process is strictly nuc- lear, transcriptional control of sets of target genes or happens in the cytoplasm where translocation and acti- vation of transducer molecules is modulated in is also unclear. There is a significant gap between the avail- able in vivo knowledge coming from analyses of pheno- types as a result of BMP signal strength and in vitro knowledge about intracellular processes downstream of signal triggering. There is another example of stemness decisions by Nodal versus BMP signaling emerging from human embryonic stem cell research. Testing human ES-cells BMP signaling in stem cells T. U. Wagner 2972 FEBS Journal 274 (2007) 2968–2976 ª 2007 The Author Journal compilation ª 2007 FEBS for signaling activity, James et al. [41] found constantly active TGF-b signaling as demonstrated by nuclear Smad2 staining and detectable phosphorylation of this signal transducer. The main transducer of BMPs how- ever, Smad1, resided mostly in the cytoplasm and was not phosphorylated. As expected, addition of BMP to the medium of these cultures resulted in a down-regu- lation of Oct4 and an accordant change in cell mor- phology reminiscent of differentiation. Interestingly, supplementation with activin A did not alter Oct4 lev- els and the cells retained their typical embryonic stem cell morphology. Only recently, proof for the involve- ment of TGF-b signaling in pluripotent cell cultures has been extended to the murine system. Ogawa et al. [42] used either ectopic expression of Smad7 or the chemical inhibitor SB-431542 to block activin ⁄ TGF-b signaling in murine stem cell cultures. Both experi- ments resulted in a strong growth inhibition of the stem cells but, fascinatingly, did not interfere with their pluripotency, as judged by mRNA levels of Oct4, Nanog and Sox2. Inhibition of BMP signaling by ectopic expression of Smad6 neither interfered with proliferation, nor did it lead to changes in pluripotency [42]. These results suggest that there is not only a mechanistic difference between proliferation and pluri- potency of stem cells, but also that both BMP and TGF-b signaling are dispensable for stemness, even though they are capable of supporting it under specific conditions as described before. BMP as an initiator of the germ line Cells of the germ line are unique in many aspects. Their DNA and differentiation state have to be con- trolled over generations. They are extremely mobile and on their long way from being defined to arriving in the gonad they are exposed to, and ignore, practically all extracellular cues used for building and patterning the embryo. Generally, with the appearance of primordial germ cells (PGCs), the germ line is usually the earliest cell-lineage that is determined in the embryo. In many lower ani- mals, such as flies and fish, they are defined by maternally deposited factors. Strikingly, they have even been shown to be pluripotent after in vitro expansion [43]. In the mouse embryo, PGCs are formed during embryonic day 6 at the posterior proximal epiblast through a location dependent mechanism. Using gene knockouts, the molecules responsible were identified as BMPs. The primary induction of PGCs is driven by BMP4 [44], whereas the number of PGCs is guided by BMP2, BMP4 and BMP8b in synergistic action [45]. As demonstrated by in vitro culture assays [30], inde- pendent BMP signals originating from the visceral endoderm and the extra embryonic ectoderm are necessary for proper PGC induction. The receiving receptors were found by knockout experiments, where no PGCs were present in ALK2 – ⁄ – embryos, and lower than wild-type numbers are found in ALK2 heterozy- gous animals. The same effect is true for BMP4 knock- outs and heterozygotes, which can then be rescued by ectopic expression of a constantly active variant of ALK2. How these different BMP signals are finally integrated to first guide induction and later number is yet unclear. Summary BMP signals influence various kinds of stem cells, inter- estingly, with very diverse outcomes (Table 2). This pathway is a prime example of how cells are able to integrate the very same signal into their current molecular state. How exactly this is achieved is far from being understood. There are examples of this integration on many levels of signal transduction (Table 3). In some cases, the interaction of downstream signal transducers produces different transcriptional Table 2. BMP signal outcome in described stem cell types. Stem cell type BMP signal outcome Embryonic stem cells (i) Stemness upkeep when counteracting STAT3 is balancing BMP effects [10] Embryonic stem cells (ii) Differentiation when dominating other signals (especially LIF, STAT3) [14] Hair-cell progenitors (inner ear) Apoptosis of proliferative progenitors [24] Mesodermal progenitor cells Exposure to BMP during gastrulation defines mesodermal progenitors in their identity along the anterior–posterior axis of the embryo [26] Neural progenitor cells Differentiation into astrocytes [22] Pancreatic progenitor cells Inhibition of differentiation and increased proliferation of progenitors [23] Primordial germ cells Cell lineage definition and cell number control [29] Progenitors of the eyefield Depending on the exact cell type increases proliferation, induces cell-cycle arrest or drives cells into apoptosis [25] T. U. Wagner BMP signaling in stem cells FEBS Journal 274 (2007) 2968–2976 ª 2007 The Author Journal compilation ª 2007 FEBS 2973 outcomes. This has been seen when Smad1 and STAT3 interact to guide astrocyte fates. For embryonic stem cell cultures, it is more likely an effect of negative regulation of target genes: here, STAT3 and its targets seem to block transcription of BMP signal targets and vice versa. Certainly, the signal integration process has to be extended beyond the transducer molecules. One such level of signal modulation occurs outside of the signal receiving cell, by secretion of blocking ligands such as Noggin, Chordin and Follistatin or GDF3. Another way to modulate ligand ⁄ receptor inter- action is by receptor localization within the membrane [46]. Additionally, the makeup of BMP-receptor com- plexes is crucial for the choice of the transduction route and resulting cellular response. BMP2, for example, can bind a type I receptor (e.g. BRIa) with high affinity [47] and induce subsequent BRII recruitment to the complex. This mode of complex formation leads to signal conveyance via the p38 MAPK pathway. Alter- natively, a preformed receptor complex including type I and type II receptors is able to bind BMP2, in which case transduction will occur through Smad activation [48]. There is further evidence for higher-order cross-talk between BMP signaling proteins and those of other pathways close to the membrane (e.g. with PI3-Kin- ase [49] or Dullard [4]), in cytoplasmic complexes (e.g. Smad1 and Enofin [50]) and nuclear complexes (e.g. b-catenin, Tcf4 and Smad1 [9] or Notch-IC and Smad3 [11]). Taken together, the currently available evidence strongly suggests that cells are defined in their identity by the sequence of signals they are exposed to, whereas their respective responses to a molecule cocktail (often referred to as niche) appear to be highly variable. BMP signaling cannot be attributed to define or differ- entiate a stem cell of any kind on its own. Rather, it is an integral part of stemness and also differentiation signaling depending on the the current transduction programme of receiving cell, which involves multilevel integration of a variety of signals. Acknowledgements I would like to thank Dr M. Schartl for critical read- ing and general help. Furthermore, I need to thank Dr A. Herpin for his patience and inspiring discussions. I also have to thank the reviewers for helpful comments, enhancing this article and making it more comprehen- sive. I want to thank sources of funding: Deutsche Forschungsgemeinschaft through GK1048 ‘Vertebrate Organogenesis’ and the European Community through Plurigenes. I have to apologize to a high number of scientists whom I was not able to cite due to space limitations, especially those cited indirectly via more general BMP signaling reviews. Table 3. BMP signal modulation. PIAS, protein inhibitor of activated STAT proteins. Site Mechanism Example Extracellular space Modification of ligand-receptor affinity ) usually by binding to ligand dimers Dimeric secreted proteins such as noggin, chordin, follistatin and GDF3 bind and therby inactivate BMP dimers [16] Cell membrane Predimerization of type I receptors, type I–type II receptors Receptor heterooligomers induced by ligand binding signal via Smads, preformed complexes signal via p38 [31] Receptor complex inhibiton BAMBI pseudoreceptors block receptor activation [1] Receptor–adapter junction R-Smads are kept from interacting with receptor complexes Smad7 binds to type I receptors and thus blocks R-Smads from being activated by the receptors [1] Adapters inhibit receptor activation FKBP12 inhibits type I receptor phosphorylation [1] Cytoplasm Smad expression and covalent modification Sumoylation by PIAS, ubiquitylation by Smurf1 ⁄ 2 [1] Competition for Smad4 binding Smad6 sequesters Smad4, thereby blocks Smad1 binding and nuclear accumulation [1] Nucleus Co-factor-availability, corepressors and coactivators can form a complex Ngn competes with Stat3 for Smad1–p300 complexes in glial differentiation [21] Nuclear import, export and retention MAPK phosphorylation of Smads leads to export from the nucleus [2] Dephosphorylation of Smads to end a signal Several phosphatases targeting the SXS C-terminal regions of Smad1 ⁄ 2 ⁄ 3 [12–15] BMP signaling in stem cells T. 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Wuerzburg, Germany BMP signals in stem cells Bone morphogenetic protein (BMP) signals have tre- mendous effects on all kinds of cells. Most striking and defining,