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Keywords:

  • Embryonic stem cells;
  • Neural stem cells;
  • Hematopoietic stem cells;
  • Mesenchymal stem cells;
  • Ubiquitin;
  • Proteasome

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Embryonic Stem Cells
  5. Neural Stem Cells
  6. Hematopoietic Stem Cells
  7. Mesenchymal Stem Cells
  8. Conclusion
  9. Disclosure of Potential Conflicts of Interest
  10. Acknowledgements
  11. References

Highly ordered degradation of cell proteins by the ubiquitin-proteasome system, a sophisticated cellular proteolytic machinery, has been identified as a key regulatory mechanism in many eukaryotic cells. Accumulating evidence reveals that the ubiquitin-proteasome system is involved in the regulation of fundamental processes in mammalian stem and progenitor cells of embryonic, neural, hematopoietic, and mesenchymal origin. Such processes, including development, survival, differentiation, lineage commitment, migration, and homing, are directly controlled by the ubiquitin-proteasome system, either via proteolytic degradation of key regulatory proteins of signaling and gene expression pathways or via nonproteolytic mechanisms involving the proteasome itself or posttranslational modifications of target proteins by ubiquitin or other ubiquitin-like modifiers. Future characterization of the precise roles and functions of the ubiquitin-proteasome system in mammalian stem and early progenitor cells will improve our understanding of stem cell biology and may provide an experimental basis for the development of novel therapeutic strategies in regenerative medicine.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Embryonic Stem Cells
  5. Neural Stem Cells
  6. Hematopoietic Stem Cells
  7. Mesenchymal Stem Cells
  8. Conclusion
  9. Disclosure of Potential Conflicts of Interest
  10. Acknowledgements
  11. References

The ubiquitin-proteasome system (UPS) has been identified as the cell's major tool for extralysosomal cytosolic and nuclear protein degradation, and the characterization of its multiple biological functions has pointed out a new conceptional framework for understanding the regulation of basic cellular processes by controlled and limited proteolysis of cell proteins [1, [2], [3], [4]5]. The 26S proteasome, a large multicatalytic multisubunit protease complex, constitutes the central proteolytic machinery of the UPS and is responsible for the degradation and proteolytic processing of cell proteins essential for the regulation of development, differentiation, proliferation, cell cycling, apoptosis, gene transcription, signal transduction, senescence, antigen presentation, inflammation, and stress response, thereby governing basic cellular processes [6, [7], [8], [9]10].

Cell proteins destined to undergo processing by the UPS must be targeted for recognition and subsequent degradation by the 26S proteasome by covalent attachments of multiple monomers of the 76 amino acid polypeptide ubiquitin [1, 11]. This process, termed ubiquitylation, takes place in a multistep reaction and requires three classes of enzymes (Fig. 1A): ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin-protein ligases (E3). E1 activates ubiquitin by forming a high-energy thiol ester bond between an E1 active site-located cysteine residue and the C-terminal glycine residue of ubiquitin in a reaction that requires the hydrolysis of ATP. This activated ubiquitin moiety is then transferred to one of the cell's approximately 30 E2s via the formation of an additional thiol ester bond and finally is transferred to one of hundreds of distinct E3s, which catalyze the covalent attachment of ubiquitin to the target protein by the formation of isopeptide bonds. A single E2 may function with multiple E3s to provide specificity in a combinatorial fashion. There are several mechanistically distinct classes of E3 enzymes, including the HECT (homologous to E6-associated protein carboxyl terminus) domain family, RING (really interesting new gene) finger proteins, and a U-box protein family [2, 3]. E3s are the key determinants of substrate specificity and are capable of recognizing a few or multiple substrates through specific degradation signals. Multiple cycles of ubiquitylation finally result in the synthesis and attachment of polyubiquitin chains that serve as a recognition signal for the degradation of the target protein by the 26S proteasome [1, 2].

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Figure Figure 1.. The ubiquitin-proteasome system. (A): Attachment of ubiquitin to the target protein requires three enzymatic steps. Ubiquitin-activating enzymes activate ubiquitin by forming a high-energy thiol ester bond between an E1 active site-located cystine residue and the C-terminal glycine residue of ubiquitin. This reaction requires energy provided by the hydrolysis of ATP and forms an activated ubiquitin moiety that is transferred and bound by an additional thiol ester bond to ubiquitin-conjugating enzymes that serve as carrier proteins. Ubiquitin-protein ligases catalyze the covalent attachment of ubiquitin to the target protein by the formation of isopeptide bonds. Multiple cycles of ubiquitylation finally result in the synthesis and attachment of polyubiquitin chains that serve as a recognition signal for the degradation of the target protein by the 26S proteasome. (B): The 26S proteasome consists of the 20S catalytic core complex and two 19S regulatory complexes capping the 20S complex at both ends. The 20S complex is composed of four axially stacked rings. Each outer ring consists of seven nonproteolytic α subunits (light brown). Each of the two inner rings is formed by seven proteolytic β subunits (dark blue), and only three of them, β1, β2, and β5, are proteolytically active and harbor proteolytic sites that face the central cavity of the 20S complex. The 19S complex consists of the base and lid subcomplex. The base subcomplex contains six nonredundant ATPases of the AAA superfamily (light blue). The lid subcomplex (yellow) contains at least eight subunits including deubiquitylating enzymes and receptors for ubiquitylated proteins. The polyubiquitylated target protein enters the 19S regulatory complex and is recognized, deubiquitylated, unfolded, and translocated into the central cavity of the 20S catalytic core complex, where it is degraded by different hydrolytic activities. Ubiquitin is recycled by the UCH. Peptides as a product of degradation are released from the 26S proteasome by diffusion and further degraded to single amino acids by cytosolic peptidases or are used for major histocompatibility class I antigen presentation. Abbreviations: E1, ubiquitin-activating enzymes; E2, ubiquitin-conjugating enzymes; E3, ubiquitin-protein ligases; Ub, ubiquitin; UCH, ubiquitin carboxy terminal hydrolase.

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The proteolytic activities of the 26S proteasome occur in a barrel-shaped 20S catalytic core complex composed of four axially stacked rings (Fig. 1B). Each outer ring consists of seven different nonproteolytic α-subunits that allow conformational flexibility and substrate translocation into the central cavity of the 20S complex; the two inner rings are formed by seven different but related β-subunits, giving the complex the general stoichiometry of α1-7β1-7β1-7α1-7 [4, 5, 12]. Only β1, β2, and β5 subunits are proteolytically active and harbor proteolytic sites formed by N-terminal threonine residues that face the central cavity of the 20S complex [4, 5]. However, 20S complexes are incapable of degrading ubiquitin-conjugated and folded substrate proteins and require for this task 19S regulatory complexes bound to the α-rings and capping the 20S complex at both ends, leading to the assembly of 26S proteasome holoparticles (Fig. 1B) [4, 5]. Similar to 20S complexes, 19S regulatory complexes exhibit sophisticated multisubunit assemblies, and their functions encompass recognition, deubiquitylation, unfolding, and translocation of substrate proteins destined to be proteolytically degraded within the 20S complex [13].

Proteolytic processing of cell proteins by the UPS is a highly ordered, evolutionarily conserved, and ubiquitous process that directly impacts the development and physiology of various eukaryotic cells, tissues, organs, and even entire organisms [4, 14, [15], [16]17]. In view of this complexity, it is not surprising that the UPS can undergo substantial deregulation that contributes to the pathogenesis of various human diseases, such as cancer and neurodegenerative, autoimmune, genetic, and metabolic disorders [18, 19]. Importantly, the finding that inhibition of proteasome activity induces apoptosis selectively in cancer cells and interferes with essential functions of immune cells [20, [21]22] has led to the exploitation of the UPS as a molecular target for cancer therapy and immune modulation [23, 24]. From all these aspects, it is not surprising that the UPS also plays an important role in the biology of mammalian stem and progenitor cells, as reviewed herein.

Embryonic Stem Cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Embryonic Stem Cells
  5. Neural Stem Cells
  6. Hematopoietic Stem Cells
  7. Mesenchymal Stem Cells
  8. Conclusion
  9. Disclosure of Potential Conflicts of Interest
  10. Acknowledgements
  11. References

First murine [25, 26] and human [27] embryonic stem cell (ESC) lines were established from the inner cell mass of a preimplantation blastocyst on the foundations of earlier research on teratocarcinomas and embryonal carcinoma stem cells [28]. Since then, the ESCs have been instrumental in studying early embryonic development and in clarifying the function of many genes utilizing transgenic or knockout animals. Human and murine ESCs share the two fundamental properties of stem cells: self-renewal and pluripotency. ESCs can be maintained in culture indefinitely in pluripotent and undifferentiated states without undergoing replicative senescence, arresting the cell cycle, or being subject to contact inhibition. Nevertheless, under appropriate culture conditions, these cells can be induced to differentiate into mature cell types originating from all three germ layers [29].

A number of processes and molecular components in ESCs are directly regulated by the UPS. Significant effort has been made in recent years to clarify the molecular mechanisms regulating expression of the homeodomain transcription factor Oct4 (also known as Oct3/4), which belongs to a group of master regulators responsible for the maintenance of ESC pluripotency and self-renewal [29]. Oct4 derives its name from its ability to activate the transcription of genes containing the octameric consensus-binding motif ATGCAAAT within their promoter or enhancer regions. The expression of Oct4 is principally controlled at the transcriptional level. However, Xu and coworkers proposed recently that the abundance of Oct4 in ESCs may be finely tuned by its degradation in the UPS [30]. In addition, they provided circumstantial evidence that the activity of Oct4 may be regulated by a second mechanism involving monoubiquitylation, which would decrease the transcriptional activity of Oct4 without promoting its degradation. The ubiquitylation of Oct4 was shown to be mediated by a novel murine E3 ubiquitin ligase, Wwp2 (Table 1). This protein belongs to the neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4) family of E3 ligases characterized by the presence of a HECT domain, which provides the E3 ligase activity, and by the presence of two to four tryptophan-based WW domains that bind proteins containing proline-rich motifs and mediate specific protein-protein interactions. Although these data indicate that Wwp2 might be involved in the maintenance of Oct4 activity within a narrow range, authors failed to directly demonstrate that endogenous Oct4 is mono- or polyubiquitylated by Wwp2 in ESCs. In addition to direct ubiquitylation of Oct4 protein, the transcriptional activity of Oct4 gene also seems to be regulated by post-translational modification involving the attachment of a small ubiquitin-like modifier, SUMO-1, to orphan nuclear testicular receptor 2 (TR2), which binds to the regulatory region of Oct4 gene [31]. The activity of TR2 can be controlled by its association with other proteins in the transcriptional complex, such as coactivator PCAF (p300/CBP-associated factor, where CBP stands for “CREB-binding protein” and CREB for “cAMP response element binding”) or corepressor Rip140 (receptor-interacting protein 140). In an unSUMOylated form, TR2 is associated with the PCAF and acts as an activator of Oct4 gene expression. However, SUMOylation of TR2 induces the replacement of its coactivator PCAF with the corepressor Rip140, which suppresses the Oct4 gene expression. Thus, depending on its post-translational modification, TR2 can play a dual role in fine-tuning Oct4 expression in ESCs.

Table Table 1.. Functions of the ubiquitin-proteasome system in mammalian stem and progenitor cells
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Besides the potential role for the UPS in controlling Oct4 activity, recent genome-wide analysis of transcriptional changes induced by Oct4 knockdown revealed that Oct4 might directly or indirectly regulate the expression of many proteasome subunits [32]. In this study, the expression of four genes encoding the proteasome subunits was repressed, whereas the expression of 14 other proteasomal genes was positively affected by Oct4 in human ESC line H1. Using proteomic approaches, Baharvand and coworkers reported that, among the most abundant protein spots in three different human ESC lines, there were also the components of the UPS [33]. In a recent report, Hernebring and coworkers examined the levels of proteins damaged by carbonylation or formation of advanced glycation end products during differentiation of murine ESCs [34]. Authors found that undifferentiated ESCs contain high levels of damaged proteins but, surprisingly, these proteins were efficiently cleared when ESCs underwent differentiation. The decrease of damaged protein levels was accompanied with a threefold increase in chymotrypsin-like activity of the 20S proteasome in differentiated ESCs as compared with their undifferentiated counterparts, suggesting that this might be a mechanism of how the offspring of mammals get rid of damaged proteins during early development.

It is well-established today that the UPS is one of the most important regulators of transcriptional activity in cells [35]. This regulation is achieved either by nonproteolytic conjugation of monoubiquitin to target proteins (e.g., histone H2A) or by proteolytic removal of polyubiquitylated components of the transcriptional machinery. According to recent reports, the chromatin in lineage-specific gene loci in pluripotent ESCs is highly accessible for transcription factors and is marked by so-called bivalent domains containing at the same time both the repressive and activating histone modifications [36, 37]. However, despite this open chromatin conformation, the expression of developmental genes in ESCs is repressed as long as they remain pluripotent. Nevertheless, when differentiation is triggered, the transcription of these genes is quickly activated. In an effort to understand this phenomenon in more detail, Szutorisz and coworkers revealed that the proteasome plays a key role in maintaining open chromatin state and in preventing the incorrect transcriptional initiation at specific sites [38]. Authors showed that blocking proteasome activity leads to increased transcription at certain genomic regions, which are normally active only during early stages of differentiation but not in ESCs. In the absence of the proteasome activity, the transcription preinitiation complex was found associated with these specific genetic loci. In addition, several proteasome subunits were also bound to the same regulatory elements. The authors suggested that the proteasome maintains permissive transcription in ESCs by removing transcription initiation factors from regulatory sequences of developmental genes. By acting in this way, the proteasome contributes to the maintenance of pluripotency and keeps the chromatin in permissive state.

Gene silencing in ESCs is mediated by Polycomb group proteins, which function in two distinct multiprotein complexes, termed Polycomb repressive complexes PRC1 and PRC2. These proteins act in part by modifying histones and play essential roles in concert with master transcriptional regulators of pluripotency Oct4, Sox2, and Nanog in repressing gene expression during embryogenesis and in preventing premature differentiation of ESCs [39, 40]. PRC2 methylates histone H3 on lysine residue 27 (H3-K27), and this modification recruits PRC1. The PRC1 complex in turn exhibits the ubiquitin ligase activity capable of monoubiquitylating histone H2A at lysine position 119 (H2A-K119). The catalytic E3 ligase activity responsible for monoubiquitylation of histone H2A resides in human Ring2 protein, which is part of the PRC1 complex and is equivalent to mouse Ring1B protein [41, 42]. The RING finger proteins B lymphoma Moloney leukemia insertion region 1 (Bmi1) and Ring1A, which are also components of PRC1, greatly stimulate the E3 ligase activity of Ring1B but display no detectable E3 ligase activity themselves [43]. Both Ring1B and Bmi1 are degraded by the UPS, and their ubiquitylation is mediated by an exogenous E3, which is not a component of the PRC1. Bmi1 has been shown to play an important role in cell proliferation, adult stem cell renewal, and cancer [44, 45]. The Ring1B E3 ligase activity is activated by self-ubiquitylation during which atypical mixed K6-, K26-, and K48-based polyubiquitin chains are generated [46]. This noncanonical self-ubiquitylation of Ring1B is required for its ability to monoubiquitylate H2A but does not target this protein for proteasomal degradation. Therefore, ubiquitylation and proteasome-mediated degradation influence complex interactions between various transcriptional regulators and histones to control gene expression and help maintain pluripotency in ESCs.

Other studies have also implicated ubiquitin as a factor contributing to pluripotent phenotype of murine ES cells. In a recent functional genetic screen with the random RNA interference library, ubiquitin has been identified as a gene involved in self-renewal and differentiation of murine ESCs [47]. The ES cell line in which ubiquitin was knocked down generated 4–6 times more undifferentiated colonies in the absence of leukemia inhibitory factor (LIF) as compared with parental ES cells, suggesting a role for the ubiquitin in controlling ES cell differentiation.

Several signaling pathways are known to be involved in regulation of self-renewal and pluripotency, the most crucial being the leukemia inhibitory factor/Janus kinase/signal transducer and activator of transcription 3 (LIF/JAK/STAT3) pathway in murine ESCs [48] and the Nodal/Activin [49], Wnt/β-catenin [50], and fibroblast growth factor 2 [51] pathways in human ESCs. It is well-established that signaling through STAT3 [52, 53], Wnt [54], Notch [55], Nodal/transforming growth factor β (TGFβ)/Activin, and bone morphogenetic protein (BMP) [56, 57] and the transcriptional activity of c-Myc [58] are regulated at the level of their turnover by the UPS or by post-translational modification through ubiquitylation. For example, ubiquitylation and proteasomal degradation of STAT3 are mediated by the protein TMF/ARA160 (TATA element modulatory factor/androgen receptor coactivator 160 kDa) in myogenic C2C12 cells [53]. TMF/ARA160 was shown to directly associate with STAT3 and apparently serves as an adaptor protein to recruit a STAT3 to ubiquitylation by an E3 complex. The c-Myc protein, the downstream target of the LIF/JAK/STAT3 pathway, is normally degraded very rapidly in a glycogen synthase kinase-3β (GSK-3β)-dependent manner by the UPS with a half-life of 20–30 minutes in most cells [58], and the signaling through the canonical Wnt pathway is regulated also in a GSK-3β-dependent manner by modulating the stability of β-catenin [59]. However, it is unclear to what extent these signal transduction pathways are regulated in ESCs by the UPS.

Neural Stem Cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Embryonic Stem Cells
  5. Neural Stem Cells
  6. Hematopoietic Stem Cells
  7. Mesenchymal Stem Cells
  8. Conclusion
  9. Disclosure of Potential Conflicts of Interest
  10. Acknowledgements
  11. References

Adult neural stem cells (aNSC) are multipotent, self-renewing cells located in anatomically defined germinal zones of the adult mammalian brain: the subventricular zone along the lateral wall of the anterior lateral ventricle [60, [61], [62], [63], [64], [65], [66]67], the subgranular zone of the hippocampal dentate gyrus [66, 68, [69], [70], [71], [72], [73], [74]75], the subcallosal zone corresponding to the posterior horn of the lateral ventricle [76], and a germinal zone corresponding to the tegmentum [77]. Although the exact developmental origin of aNSC is not known, recent evidence suggests that aNSC gradually transform from embryonic neuroepithelial cells to radial glial cells to astrocyte-like aNSC [78, [79]80]. Since aNSC can give rise to neurons, astrocytes, and oligodendrocytes in vivo and ex vivo, they have been classified as multipotent cells [61, 81, [82]83]. Moreover, in vivo and ex vivo, aNSC can give rise to cells that normally derive from germ layers other than the neuroectoderm [84, [85], [86], [87], [88]89]. Embryonic neural stem cells (eNSC) can be isolated from the embryonic mammalian mesencephalon, telencephalon, and cerebral cortex [90, [91], [92], [93]94] and from the vertebrate neural crest, a migratory cell population located between the dorsal ectoderm and the neural tube that arises during early embryonal development [95, [96], [97], [98]99]. Similar to their adult counterparts, eNSC isolated from embryonic mammalian brain can give rise to neurons, astrocytes, and oligodendrocytes and are therefore classified as being multipotent [91, [92], [93]94]. Neural crest stem cells, however, exhibit a unique ability to generate different classes of neurons and glia as well as cells of all germ layers, thus demonstrating their pluripotency [96, 97, 99].

Increasing evidence suggests that the UPS plays an important role in neural stem and progenitor cell differentiation and neurogenesis. In particular, different E3 ubiquitin ligases have recently been shown to be essential for embryonic neurogenesis and neural progenitor cell differentiation and survival in mice. Mice harboring a targeted mutation of Mind bomb1 (MIB1), an E3 ubiquitin ligase of the RING finger type essential for activation of Notch signaling, which is required for proper neural development [100, 101], display dramatic neuroepithelial and cerebral defects, such as a compromised structural integrity of the embryonic neuroepithelium, a lack of neuroepithelial cells, neural tube and forebrain malformations, a lack of forebrain neural stem cells, and a precocious neurogenesis accompanied by the induction of apoptosis in premature neurons [102, 103]. This aberrant neurogenesis associated with early embryonic lethality appears as a direct consequence of disrupted Notch1 signaling due to the absence of MIB1 that finally impairs proper neural patterning and embryonal neurogenesis [102, 103]. Similar to MIB1, the E3 ubiquitin ligases UBR1 and UBR2 of the N-end rule pathway, which constitutes one regulatory proteolytic pathway of the UPS [104, 105], have been proven essential for proper differentiation, proliferation, and survival of mouse embryonic neural stem and progenitor cells [106]. Mice lacking both UBR1 and UBR2 exhibit impaired proliferation and increased apoptosis of neural progenitor cells of the neural tube and the ventricular zone of the embryonic forebrain, leading to the occurrence of thin neuroepithelial layers, a deformed neural tube, and a distorted forebrain morphology. Moreover, mitotically active neural precursor cells of UBR1−/−UBR2−/− mice embryos prematurely migrate from the ventricular zone to the subventricular zone, apparently as a result of their precocious and deregulated differentiation. As in the case of MIB1 mutant mice, aberrant neurogenesis in UBR1−/−UBR2−/− mice is associated with suppression of the Notch1 pathway and the occurrence of early embryonic lethality, revealing an essential role of UBR1 and UBR2 in mammalian neurogenesis and development [106]. Furthermore, DDB1, a component of the cullin4A-based E3 ubiquitin ligase complex that controls nuclear excision repair, DNA replication, and cell cycle progression [107, [108]109], plays an essential role in maintaining viability and genomic integrity of dividing neural progenitor cells during embryonic brain development. As recently shown, conditional inactivation of the DDB1 gene in mouse brain results in a nearly complete loss of neural stem and progenitor cells in the ventricular and subventricular zone and in the cerebellar external granular layer of newborn mice [110]. However, only proliferating, but not postmitotic, neural progenitor cells of these germinal layers undergo DNA damage and massive p53-dependent apoptosis during embryonic brain development of mice bearing a conditional deletion of DDB1, indicating a crucial role for DDB1 in maintaining embryonic neurogenesis [110]. Another component of the UPS, ubiquitin C-terminal hydrolase L1 (UCH-L1), has been shown to promote differentiation of mouse embryonic neural progenitor cells in vivo and in vitro [111]. Although UCH-L1 belongs to the large family of deubiquitinating enzymes that hydrolyze ubiquitin-protein bonds, a ubiquitin ligase activity of the enzyme has been demonstrated [112, 113]. This ubiquitin ligase activity of UCH-L1 regulates the differentiation of mouse embryonic neural progenitor cells [111], suggesting that, at least in the regulation of neurogenesis, a functional homology exists between UCH-L1 and the ubiquitin ligases discussed above.

Finally, indirect evidence for a pivotal involvement of the UPS in neural stem cell physiology and neurogenesis comes from studies showing that proteins critical for the regulation of neurogenesis are ubiquitylated and proteolytically processed by the UPS. Such proteins include the Notch receptor [114, [115], [116]117], the Notch ligands Delta and Serrate [118, [119]120], the Notch antagonist Numb [121, 122], the neuronal adaptor protein Disabled-1 [123, 124], the neuronal-specific activator of Cdk5, p35 [125, 126], and the neural stem cell-specific intermediate filament protein Nestin [127].

Hematopoietic Stem Cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Embryonic Stem Cells
  5. Neural Stem Cells
  6. Hematopoietic Stem Cells
  7. Mesenchymal Stem Cells
  8. Conclusion
  9. Disclosure of Potential Conflicts of Interest
  10. Acknowledgements
  11. References

Hematopoietic stem cells (HSC) originate during mammalian embryogenesis from temporally and anatomically restricted sites [128, 129]. Although the precise anatomical origin of definitive HSC is not fully resolved, it is commonly accepted that HSC appear first in the yolk sac blood islands [130, [131]132], closely followed by the appearance in the aorta-gonad-mesonephros region [133, [134], [135]136] in the placenta [137, [138]139] and shortly thereafter in fetal liver and spleen [137, 140, [141], [142]143], from where HSC colonize the bone marrow shortly before birth [144]. In the bone marrow, HSC reside in the hematopoietic stem cell niche, a specialized microenvironment closely associated with the endosteum, the sinusoidal endothelium, or the vasculature [145, [146]147]. Different cell types present in the hematopoietic stem cell niche provide essential signals to regulate maintenance, self-renewal, differentiation, and lineage commitment of HSC, thereby sustaining hematopoiesis throughout adult life [148, 149]. Adult human HSC uniformly express CD34 antigen and can be isolated from bone marrow, umbilical cord blood, and, after mobilization with growth factors such as granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor, from peripheral blood. Cells collected in this manner have the capacity to fully reconstitute the hematopoietic and immune system of a lethally irradiated host. This has been exploited for decades in the curative therapy of malignant and immune diseases [150]. HSC have been defined as clonogenic, self-renewing cells that can give rise to all cell types of the hematopoietic and immune systems [151, [152], [153], [154], [155]156].

Early studies showed that ubiquitin is involved in the regulation of some basic functions of mouse and human HSC and hematopoietic progenitor cells. In this regard, it has been demonstrated that ubiquitin mediates the binding of mouse hematopoietic progenitor cells to bone marrow stroma as well as their homing into the spleen [157]. Mechanistically, this effect is mediated by the cell surface expression of ubiquitin on hematopoietic stroma cells and the presence of specific binding sites for ubiquitin on hematopoietic progenitor cells [157]. The subsequent finding that extracellular ubiquitin inhibits hematopoietic lineage commitment of hematopoietic progenitor cells [158] supports the hypothesis that ubiquitin maintains hematopoietic progenitors in an undifferentiated state, a prerequisite for their migration and homing [157, 159, 160].

A series of more recent studies revealed that the central proteolytic machinery of the UPS, the 26S proteasome, regulates essential processes in HSC and hematopoietic progenitor cells. These studies have used specific and selective proteasome inhibitors of natural or synthetic origin, such as lactacystin, MG-132, MG-262, and PSI (proteasome inhibitor), for the investigation of the role and function of the 26S proteasome in survival, proliferation, differentiation, and lineage commitment of human CD34+ hematopoietic progenitor cells. In particular, inhibition of the proteolytic activities of the 26S proteasome by lactacystin, MG-132, MG-262 or PSI has been shown to induce significant apoptosis in CD34+ stem cells isolated from G-CSF-mobilized peripheral blood stem cell harvests [161, 162]. Moreover, primary human leukemic stem cells expressing CD34 and interleukin 3 receptor α-chain/CD123 undergo massive apoptosis in response to proteasome inhibition by MG-132, most likely due to their constitutive and increased activation of transcription factor nuclear factor κB (NF-κB), which is known to be proteasome-dependent, antiapoptotic, and oncogenic [163, [164], [165], [166]167]. Unlike CD34+/CD123+ leukemic stem cells, normal CD34+ stem cells lack constitutive activation of NF-κB [163] but require cytokine- and growth factor-induced activation of NF-κB for survival and hematopoietic differentiation [168, 169]. Since activation of NF-κB strictly depends on proteasome activity [165] and NF-κB activation can be blocked by proteasome inhibitors [170], it is obvious that the 26S proteasome is essentially involved in the regulation of survival and hematopoietic differentiation of CD34+ stem cells. This is supported by a study showing that proteasome inhibitors induce either apoptosis in CD34+ cells or suppress hematopoietic colony formation of CD34+ cells surviving exposure to proteasome inhibitors [161].

Additional evidence for a critical role of the UPS in regulating hematopoietic progenitor cell function and hematopoiesis came from a study showing that the transcription factors c-Rel and RelA, whose activity is tightly regulated by the UPS [171, [172]173], are required for differentiation and lineage commitment of hematopoietic progenitor cells in mice [174]. Fetal liver hematopoietic progenitor cells obtained from mice lacking both c-Rel and RelA failed to reconstitute hematopoiesis in lethally irradiated normal recipients. This failure was associated with several hematopoietic defects, including a reduction of spleen colony-forming unit progenitors, impaired erythropoiesis, and a deregulated expansion of granulocytes [174]. These findings provide indirect evidence for the involvement of the UPS in the regulation of hematopoiesis by governing the activation of c-Rel and RelA.

The recent demonstration that the activity of the zinc-finger transcription factors GATA-1 and GATA-2 (termed GATA on the basis of their ability to recognize the consensus sequence (A/T)GATA(A/G)) is tightly regulated by ubiquitin-proteasome-mediated degradation of GATA-1 and GATA-2 [175, [176]177] highlights a pivotal role of the UPS in regulating embryonic and adult hematopoiesis. GATA-1 has been shown to positively regulate the differentiation of embryonic and adult HSC toward erythroid and megakaryocytic precursors [178, [179], [180]181], whereas GATA-2 promotes survival, quiescence, expansion, and proliferation of embryonic and adult HSC [181, [182], [183]184]. In addition, expression of GATA-2 in HSC is positively regulated by the Notch signaling pathway, which constitutes a key regulating system in embryonic and adult hematopoiesis and whose components, in turn, are also regulated by ubiquitin-proteasome-mediated degradation [116, [117]118, 185, [186], [187]188]. These findings, in conjunction with the results discussed above, strongly suggest that the UPS constitutes a key regulator of HSC fate and hematopoiesis.

Mesenchymal Stem Cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Embryonic Stem Cells
  5. Neural Stem Cells
  6. Hematopoietic Stem Cells
  7. Mesenchymal Stem Cells
  8. Conclusion
  9. Disclosure of Potential Conflicts of Interest
  10. Acknowledgements
  11. References

Mesenchymal stem cells, also referred to as marrow stromal cells, represent a nonhematopoietic, adherent, fibroblast-like cell population originally isolated by their plastic-adherence properties from aspirates of bone marrow [189]. In addition to bone marrow, MSCs reside in the connective tissue of almost all adult organs, including adipose tissue, muscle, spleen, thymus, peripheral blood, and dental pulp [190]. Furthermore, MSCs are also present in fetal tissues, predominantly in blood, bone marrow, and liver of first-trimester fetuses, but also in lung, kidney, pancreas, amniotic fluid, cord blood, and placenta (approximately 1% of all cells present in placenta are MSCs) [191]. Fetal MSCs appear to be more primitive and possess greater multipotentiality than their adult counterparts [192]. Adult MSCs are characterized by expression of cell surface markers stem cell antigen-1 [193], bone marrow stromal cell glycoprotein STRO-1 [194], β1-integrin/fibronectin receptor β-subunit/CD29, endoglin/CD105 [195], hyaluronan receptor/CD44, ecto-5′-nucleotidase/CD73 [196], and stage-specific embryonic antigen-4 [197] and by the absence of lineage-specific and hematopoietic stem cell markers [196]. In culture, MSCs self-renew for approximately 50 population doublings and can be induced to differentiate primarily into mesoderm-derived tissues: bone, cartilage, and fat [198]. However, under appropriate conditions in vitro and upon transplantation in vivo, MSCs isolated from different sources may also give rise to cardiomyocytes [199], neurons [200, 201], astrocytes [202], hepatocytes [203], epidermal-like cells [204], insulin-, somatostatin-, and glucagon-expressing cells [205], and even inner ear sensory hair cells [206].

Similarly to other types of stem cells, MSCs are also characterized by their self-renewing and broad developmental capacities. Although little is known about factors responsible for maintenance of these properties in MSCs, regulation of chondro- and osteogenesis from MSCs has been explored in much more detail. Wnt/β-catenin, BMP, and Notch pathways including transcription factors NFκB, Sp7/Osterix, and runt-related transcription factor 2 (Runx2) have been implicated in osteoblast differentiation, bone formation, and chondrogenesis [207, 208], and the UPS has been recognized as an important regulator of these pathways. The UPS pathway interacts with these processes by regulating the stability of key signaling molecules and transcription factors in the cell. For example, β-catenin levels and its transcriptional activity are controlled by the UPS [209]. The physiological consequence of proteasome inhibition is the accumulation of β-catenin in the cell followed by the dedifferentiation of chondrocytes [210]. The stability of β-catenin is regulated by diverse extracellular Wnt ligands. In the absence of Wnt, intracellular β-catenin is constitutively phosphorylated and maintained at low levels by degradation in the UPS. Binding of Wnt prevents GSK-3β-mediated phosphorylation of β-catenin and results in accumulation of this factor in the cytosol, which than translocates into the nucleus and serves as an essential cofactor for the Tcf/LEF (T-cell factor/lymphoid enhancing factor) family of transcription factors [54]. Activation of peroxisome proliferative activated receptor γ (PPARγ) induces the degradation of β-catenin during preadipocyte differentiation by mechanisms that require GSK-3β and the proteasome. PPARγ2 is a key element for the differentiation of MSCs into adipocytes and is also subject to regulation by the UPS [211]. Several members of the BMP family, and particularly the BMP6, have osteoinductive properties and can induce the differentiation of MSCs into osteoblasts [212, 213] and are also subject to regulation by the UPS [214]. BMPs bind their cell surface receptors and initiate downstream signaling via DNA-binding transcriptional modulators called Smads (Caenorhabditis elegans “SMA/small”- and Drosophila melanogaster “mothers against decapentaplegic”-related proteins) to regulate gene expression. The signaling through Smads is very complex, involves the receptor-regulated Smad (R-Smad) family members Smad1, Smad5, Smad8, and the Co-Smad Smad4, and is subject to multiple layers of control, including the UPS system. Several E3 ubiquitin ligases of the Smad ubiquitin regulatory factor (Smurf) family, such as Smurf1 and Smurf2, ubiquitylate specific Smad molecules and target them to the proteasome for degradation [215, 216]. Smurf1 also recognizes bone-specific transcription factor Runx2 and mediates Runx2 degradation [217]. The turnover of some of these transcriptional modulators is regulated by the LIM mineralization protein 1 (LMP-1), which is capable of inducing bone formation. LMP-1 interacts with Smurf1 and prevents ubiquitylation of Smads, thereby contributing to their stabilization and increased responsiveness to exogenous BMPs [218].

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Embryonic Stem Cells
  5. Neural Stem Cells
  6. Hematopoietic Stem Cells
  7. Mesenchymal Stem Cells
  8. Conclusion
  9. Disclosure of Potential Conflicts of Interest
  10. Acknowledgements
  11. References

Proteolytic degradation of eukaryotic cell proteins by the UPS is a highly complex and tightly regulated process that is central to the regulation of basic cellular features, such as development, differentiation, proliferation, cell cycling, gene expression, signal transduction, and apoptosis. In addition, the UPS also functions as a quality control mechanism that selectively removes abnormal and damaged proteins, which would otherwise form toxic intracellular inclusions as seen in various neurodegenerative diseases. Nonproteolytic post-translational modifications of proteins by covalent attachment of ubiquitin or ubiquitin-like molecules represent another layer of regulation of cellular homeostasis. As delineated in this review, both of these processes play a pivotal role in controlling levels and/or activities of diverse regulatory proteins in mammalian stem and progenitor cells, especially those participating in signal transduction and transcriptional regulation. Rapid modulation of stability of these factors permits stem and progenitor cells to respond to clues from their environment by either maintaining their stem cell properties or embarking on a differentiation program toward specific cell lineages. Although the involvement of the UPS in maintaining pluripotency of ESCs at the transcriptional level and determining developmental potency of various adult stem cells have been clearly demonstrated, the evidence for the role of this system in signaling through LIF/JAK/STAT3, Nodal/TGFβ/Activin, Wnt/β-catenin, Notch, and BMP pathways, as well as in regulating activities of transcription factors such as those from Rel and GATA families in different stem and progenitor cell types, is largely circumstantial and requires direct experimental confirmation. In addition, recent assumption that the proteasome plays a role in clearing damaged proteins from differentiating ESCs in vitro as well as during normal embryonic development is based only on indirect observation, and unequivocal evidence for its involvement in this process still remains to be provided and mechanisms clarified. Proteasome inhibitors represent a powerful tool for dissecting the role of the UPS in cellular physiology and also have practical applications in cancer therapy. They have already played an essential role in advancing our understanding of the importance of the proteasome in biology of stem and progenitor cells covered in this review. In the future, they will certainly be applied in studies with different types of stem and progenitor cells and will help clarify mechanisms governing their essential biological properties. Since the proteasome acts in ESCs as a transcriptional silencer at tissue-specific gene loci, it would be interesting to test whether proteasome inhibition would modulate the differentiation kinetics and developmental potential of ESCs. Assuming that different cell populations comprising embryoid bodies will react differently to proteasome inhibition (e.g., by apoptosis), as it is the case with leukemic stem cells, proteasome inhibitors may support differentiation of ES cells into particular lineages. Whether this can be achieved and can aid in the development of novel strategies in stem cell therapy remains to be awaited.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Embryonic Stem Cells
  5. Neural Stem Cells
  6. Hematopoietic Stem Cells
  7. Mesenchymal Stem Cells
  8. Conclusion
  9. Disclosure of Potential Conflicts of Interest
  10. Acknowledgements
  11. References

The work in Tomo Šarić's laboratory is supported by grants from the German Research Foundation (DFG), Köln Fortune Program, and Stem Cell Network North Rhine Westphalia.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Embryonic Stem Cells
  5. Neural Stem Cells
  6. Hematopoietic Stem Cells
  7. Mesenchymal Stem Cells
  8. Conclusion
  9. Disclosure of Potential Conflicts of Interest
  10. Acknowledgements
  11. References