CyclinD2 at the edge: splitting up cell fate

EMBO J 31 8, 1879–1892 March062012Asymmetric cell division and cell cycle length are two fundamental mechanisms that influence the fate of neural stem cells during mammalian brain development. In this issue of The EMBO Journal, the team of Noriko Osumi proposes an elegant link between the two by showing that the mRNA of cyclinD2, a positive regulator of G1 progression, is confined to the basal end-foot of radial glial cells and is asymmetrically distributed upon mitosis to the two resulting daughter cells (Tsunekawa et al, 2012). According to this model, the daughter cell inheriting cyclinD2 mRNA maintains its self-renewal capability, while lengthening of G1 and differentiation would occur in the sibling cell.All neurons and glia of the mammalian central nervous system derive from highly elongated radial glial cells spanning the apical (ventricular) to basal (pial) axis of the neural tube. As development proceeds, an increasing proportion of radial glial cells switches from divisions that generate two additional radial glial cells (proliferative divisions) to divisions that generate one radial glial plus one differentiated cell (differentiative divisions) (Gotz and Huttner, 2005). The observation that radial glia retain their basal process while undergoing apical mitosis (Miyata et al, 2001; Noctor et al, 2001) has made it evident that these cells are intrinsically prone to divide asymmetrically, since only one daughter would inherit the process upon cytokinesis. Reinforcing the view that asymmetric inheritance of cellular components may underlie asymmetric cell fate change, this observation has added a new level of complexity to asymmetric division of neural progenitors that was previously thought to involve mainly apical, as opposed to basal, components (reviewed by Kosodo and Huttner, 2009). De facto, formal demonstration that asymmetric inheritance of the basal process is a prerequisite for differentiative divisions is still missing and it is even debated whether the daughter inheriting the process is the radial glial cell, her differentiated sibling, or if in some instance the process itself could be split in two (Kosodo and Huttner, 2009). Nevertheless, it is well recognized that the peculiar morphology or radial glial cells can provide the means to control asymmetric cell division and fate and many laboratories are investigating the mechanisms underlying the inheritance of the basal process and its role in neurogenesis.Similar to asymmetric cell division, cell cycle length has long been recognized as an important parameter for neural differentiation, since lengthening of G1 in radial glial cells was shown to correlate with neurogenesis at the temporal, spatial, and cellular level (reviewed by Salomoni and Calegari, 2010). Interestingly, manipulations that lengthened G1 induced differentiative divisions and neurogenesis while, conversely, shortening G1 promoted proliferative divisions and progenitors expansion supporting the notion (the cell cycle length hypothesis) that longer cell cycles are more likely to allow the accumulation of factors necessary for cell fate change to occur (Salomoni and Calegari, 2010). It is still unclear whether cell cycle regulators influence differentiation solely through a change in G1 length or, indirectly, through still unknown additional functions. Nevertheless, an overwhelming number of reports have pointed out a strong correlation between the activity of G1-specific cdk/cyclin complexes, G1 length, and cell fate change in various paradigms of stem cell differentiation including embryonic, neural, and hematopoietic stem cells (Lange and Calegari, 2010).It now comes as an interesting surprise that the mRNA of cyclinD2, a positive regulator of G1 progression mediating the transition from radial glial cells to intermediate/basal progenitors (Glickstein et al, 2009), is almost exclusively localized to the basal end-foot of radial glial cells (Tsunekawa et al, 2012). Tsunekawa and Osumi must have thought that this cannot be a mere coincidence and embarked on a detailed characterization of the subcellular distribution of cyclinD2 mRNA and protein, its inheritance upon mitosis, and the effects of manipulating the levels of cyclinD2 on neurogenesis. By using in vivo electroporation either in whole embryo culture or in utero, two techniques that the Osumi group has pioneered (Osumi and Inoue, 2001), Tsunekawa et al now show that a 50 bp cis-acting transport element is present in the 3′UTR of cyclinD2 that is responsible for its localization at the basal end-foot of radial glial cells (Figure 1). From fixed samples, the authors concluded that the majority of basally localized siblings resulting for a radial glial division appeared to be the ones inheriting the process and having higher levels of cyclinD2. Moreover, disrupting the asymmetry in cyclinD2 by forcing its overexpression or downregulation resulted in decreased or increased neurogenesis, respectively. From these observations, the authors concluded that the active transport of cyclinD2 mRNA to the basal end-foot of radial glial cells can ensure that only one daughter would inherit high levels of this positive regulator of G1 progression. As a result, and consistent with the cell cycle length hypothesis, the sibling cell that did not inherit the cyclinD2 mRNA-containing basal process would lengthen G1 in the subsequent cell cycle leading to a change in her fate and consequent differentiation (Tsunekawa et al, 2012).Open in a separate windowFigure 1CyclinD2 crowns stem cell identity. Loss of stemness represents a revolutionary event allowing the generation of differentiated cells. According to the model by Tsunekawa et al, radial glial cells during mammalian cortical development ensure that daughter cells without basal process would lengthen G1 upon cytokinesis thereby triggering asymmetric cell fate change and neurogenesis. This model elegantly links the subcellular localization of a cell cycle regulator with asymmetric cell division and fate. Note that the deposed cell must not necessarily need to be a neuron but could also be an intermediate/basal progenitor. Bystanders watch aghast while undergoing interkinetic nuclear migration waiting for their turn to divide.Importantly, this model implies that the basally localized daughter cell is the one inheriting the process and remaining as a proliferative radial glial cell while her apical sibling would undergo differentiation. Whether this is consistent with other studies is difficult to say because inheritance of the basal process is a controversial issue with opposite views proposed by different groups (Kosodo and Huttner, 2009). Nevertheless, in support to their model Tsunekawa et al show that the apically localized sibling, the one with lower levels of cyclinD2, tends to express higher levels of the neurogenic factor Ngn2. Interestingly, the group of Anna Philpott has recently shown that G1-cdk/cyclins can destabilize Ngn2 by direct phosphorylation (perhaps an ‘additional function'' for these cell cycle regulators?) thereby inhibiting differentiation concomitantly to shortening G1 (Ali et al, 2011). Reinforcing each other, these two recent observations (Ali et al, 2011; Tsunekawa et al, 2012) depict an elegant interplay between cell cycle regulators and cell fate determinants that mechanistically link asymmetric cell division, G1 length, and neurogenesis at the molecular and cellular levels.Several points still need to be addressed to fully validate the model proposed by the team of Noriko Osumi. In fact, it is fair to say that the use of fixed samples to infer about differentiation is suboptimal and that time-lapse microscopy becomes necessary when asymmetric cell division and cell fate change are correlated at the single cell level. Moreover, while the analysis of cell cycle parameters performed by Tsunekawa et al revealed to be entirely consistent with the model proposed, no direct measurement of G1 length has been provided neither upon overexpression nor downregulation experiments. Finally, active transport of cyclinD2 mRNA, as opposed to other possibilities including passive diffusion or selective degradation, was only deduced. Yet, no step in science is typically complete per se and Tsunekawa et al can certainly be granted the rare merit of providing solid evidence for the proposal of an original, elegant, and simple model that will likely stimulate and inspire the field in the years to come.     

Split decisions: oesophageal progenitor cell behaviour

Science advance online publication July192012; doi:10.1126/science.1218835The maintenance and regeneration of continually shedding epithelial tissues that make up the linings and barriers of our bodies requires rapid and continual input of proliferative progenitor cells for tissue homeostasis. The mechanisms by which epithelial progenitors cells maintain tissues remain controversial. In a recent Science paper, Doupé et al (2012) demonstrate that a population of equivalent progenitor cells support tissue homeostasis of the oesophagus without the need for slow cycling cells as described in other rapidly dividing epithelia.In tissues such as blood and skin in which differentiated cells constantly turnover, proliferative progenitor populations are required to continually produce lost differentiated cells. Several models have been proposed to explain mechanisms by which progenitor cells contribute to tissue maintenance (Figure 1). A hierarchical model has been suggested in which longer lived stem cells, which may also cycle slowly, produce highly proliferative cells with less self-renewal potential that differentiate into a restricted number of cells. Following proliferative cells in pulse-chase experiments and genetic lineage tracing has supported a hierarchical model in the blood, epidermis and intestine (Fuchs, 2009). Alternatively, an equivalency model has been proposed in which all proliferative progenitor cells are equally able to produce proliferative and differentiated progeny in a stochastic manner. Analysis of labelled clones has supported an equivalency model for progenitors in the interfollicular epidermis and intestine (Clayton et al, 2007; Doupé et al, 2010; Snippert et al, 2010).Open in a separate windowFigure 1Two types of models have been put forward to describe the pattern of progenitor behaviour within mammalian tissues. In the hierarchical model, a stem cell can produce proliferative progenitors with less self-renewal potential that differentiate into lineage-specific cells. Alternatively, an equivalency model has been proposed that assumes equal behaviour of progenitor cells to maintain tissue homeostasis.An elevated interest in understanding the dynamics of oesophageal epithelium has resulted, in part, from the rapid increase in the incidence of oesophageal adenocarcinoma (Devesa et al, 1998). The oesophagus is a stratified epithelium that lacks any appendages or glands, and thus consists of a basal layer of proliferative keratinocytes and several suprabasal layers of differentiated cells, which are continually shed. Previously, labelling of proliferative cells with DNA analogues has demonstrated that proliferation is restricted to the basal cells, which all proliferate in 5 days seemingly stochastically, supporting an equivalency model (Marques-Periera and Leblond, 1965). In contrast, studies using chimeric mice have suggested that proliferation of labelled progenitor cells occurs in a hierarchical manner (Thomas et al, 1988; Croagh et al, 2008).To address this controversy, a recent study in Science uses several genetic mouse models to define the contribution of proliferative basal cells to oesophageal homeostasis (Doupé et al, 2012). In one mouse model, the authors utilized a genetic pulse-chase system based on the tetracycline-regulated expression of the histone H2B-GFP (Tumbar et al, 2004). They find that the rapidly dividing epithelial cells of the oesophagus lose H2B-GFP expression after 4 weeks. These data suggest that either H2B-GFP is degraded (Waghmare et al, 2008) or oesophageal progenitor cells proliferate faster than their counterparts in skin epithelial appendages or blood lineages, which retain H2B-GFP after 4 weeks (Tumbar et al, 2004; Foudi et al, 2009).To analyse the properties of oesophageal progenitor cells in more detail, the authors label single cells using an inducible cre-lox genetic system and followed clones for a year. Similar to their results with this system in the tail and ear epidermis (Clayton et al, 2007; Doupé et al, 2010), the authors find that the size of the persistent clones is linear with time. Statistical analysis of the clone size data supports the ability of the cells to contribute to proliferative and non-proliferative (i.e., differentiated) progeny with equal probability. Thus, these data support a model in which all of the labelled cells are equivalent.In addition to homeostasis, the authors explore how proliferative progenitors contribute to alterations in tissue homeostasis. After inflicting wounds by biopsy, marked clones span both proliferative and non-proliferative zones of the healing oesophageal epithelium, suggesting that they maintain a progenitor fate with distinct phenotypes. With atRA treatment, the authors show that suprabasal cell formation increases, which is consistent with the known effect of atRA on the oesophagus (Lasnitzki, 1963). Statistical analysis reveals that the probability of forming basal and suprabasal cells was not altered with atRA administration. However, since proliferative cells exist in suprabasal layers during epithelial hyperplasia, additional analyses of cell state are required to determine if atRA maintains stochastic fate decisions of progenitor cells. Furthermore, the progenitor response to atRA treatment might be limited by niche space along the basement membrane like in intestinal crypt progenitor cells (Snippert et al, 2010).In summary, this study together with the authors'' previous work provides additional support for the existence of equivalent progenitor cells within stratified epithelium in several tissues. Additional studies revealing how epithelial progenitor cells behave when proliferation and differentiation are altered in the oesophagus could shed light on mechanisms for the pathogenesis of oesophageal tumours or diseases such as Barrett''s oesophagus.     

Lrig1: a new master regulator of epithelial stem cells

Nat Cell Biol 14 4, 401–408 March042012The intestine represents the most vigorously renewing, adult epithelial tissue that makes maintenance of its homeostasis a delicate balance between proliferation, cell cycle arrest, migration, differentiation, and cell death. These processes are precisely controlled by a network of developmental signalling cascades, which include Wnt, Notch, BMP/TGFβ, and Hedgehog pathways. A new, elegant study by Wong et al (2012) now adds Lrig1 as a key player in the control of intestinal homeostasis. As for epidermal stem cells, Lrig1 limits the size of the intestinal progenitor compartment by dampening EGF/ErbB-triggered stem cell expansion.The epithelium of the small intestine is separated into two distinct compartments: a proliferative crypt, containing tissue-specific stem cells, and a villus with differentiated, short-lived cells, which are replenished by a constant stream of cell migration from the underlying crypt (Scoville et al, 2008). In particular, the canonical Wnt pathway in combination with Notch signals control stem cell maintenance and proliferation in the crypt. In addition, both pathways direct differentiation into the Paneth and the absorptive cell lineage, respectively. Intensive cross-talk between the epithelium and the underlying mesenchyme helps to define the crypt–villus boundary. This relies on epithelial-derived Hedgehog and Wnt ligands that trigger stromal BMP production, which in turn signals back to the epithelium to restrict proliferation to the crypt. A gradient of BMP antagonists produced by mesenchymal cells at the bottom of the crypts supports compartmentalization. In addition, a Wnt gradient in the crypt defines EphB expression and establishes repulsion-mediated separation into Paneth cell, proliferative, and differentiation zones along the crypt–villus axis (Figure 1A).Open in a separate windowFigure 1(A) The epithelium of the small intestine contains two populations of multipotent stem cells that reside at the bottom of the crypts. These give rise to transit-amplifying progenitors, which rapidly divide while migrating upwards. Cell cycle arrest and functional differentiation occur when these cells pass from the upper part of the crypt into the villus where they continue their upward movement until they finally undergo apoptosis. Only long-living Paneth cells follow a different path as they migrate downwards to populate the base of the crypt. Control of proliferation and lineage specification of all intestinal epithelial cells is directed in a self-organizing, dynamically regulated process based on cell–cell and cell–environment interactions. Among them, Wnt and Notch signalling have been defined as major determinants for stem cell maintenance, for proliferation of stem cells in the crypt and lineage specification. Epithelial-derived Hedgehog ligands and reciprocal stromal BMP ligands establish a connection between the epithelium and the stroma that regulates the crypt–villus boundary. In addition, repulsive interactions mediated by the Eph/ephrin family allow establishment of stable compartments. Importantly, ErbB signalling, which is partially suppressed by Lrig1 at the base of the crypt, is now shown to be a new key player in the control of stem and progenitor cell expansion. (B) Cross-talk of signalling pathways in intestinal homeostasis with an emphasis on ErbB signalling. A negative feedback loop via Lrig1 helps to fine-tune population size and proliferative activity of intestinal progenitor cells. Lrig1 has been identified as a direct target of Myc and is known to repress ErbB signalling. Myc itself is a main target of the ErbB and Wnt pathways implicated in intestinal stem and progenitor cell expansion. Moreover, Lrig1 has been found to promote BMP signalling, which interferes with intestinal proliferation by restricting AKT activation via PTEN.In the small intestine, two stem cell (SC) populations coexist: Lgr5⁺crypt base columnar cells (CBCs) that cycle every 24 h and are interspersed between Paneth cells, and slower dividing SCs concentrated above (around position +4 relative to the crypt bottom) the Lgr5⁺position (Takeda et al, 2011). The localization of these Hopx⁺mTert⁺slowly cycling SCs partly overlaps with that of quiescent cells, which show long-term label retention upon irradiation damage and pulse labelling with BrdU. Lgr5⁺CBCs are, however, dispensable (Tian et al, 2008) and can be replaced by the second stem cell population, which also shows greater activity during damage repair. The relationship between these two stem cell populations, which can reciprocally generate each other, and the mechanisms that govern quiescence are being elucidated. Importantly, leucine-rich repeats and Ig-like domains 1 (Lrig1), a transmembrane protein that interacts with ErbBs and promotes its degradation, has now been found to be enriched at the crypt base and in the progenitor compartment of the small intestine and colon (Wong et al, 2012). Lrig1 is highly expressed in Lgr5⁺, Musashi1⁺, Ascl2⁺, and Olfm4⁺CBCs, and shows an inverse relation to the pattern of activated, phosphorylated EGFR above the crypt base (Figure 1A). In line with these patterns, deletion of Lrig1 in the mouse causes a dramatic crypt expansion and increased numbers of CBCs, transit-amplifying and Paneth cells. Whether the increase of Paneth cells, which actually do not express Lrig1, is a secondary effect due to the progenitor expansion remains open. Importantly, reduction of EGFR signalling by pharmacological (Gefitinib) and genetic modulation (Egfr^wa-2 mice) is able to partially normalize all Lrig1 phenotypes. These data establish EGF/ErbB signalling, as an important regulator of the crypt compartment, and suggest Lrig1 as a central control that dampens the expansion of stem cells during normal intestinal homeostasis.Lrig1 was initially identified in the skin and proposed to maintain epidermal stem cells in a quiescent state (Watt and Jensen, 2009). Lrig1 marks human interfollicular epidermal stem cells, which can give rise to all epithelial lineages including hair follicle cells in skin reconstitution assays. However, during normal homeostasis, these cells are only bipotent, contributing to the sebaceous gland and the interfollicular epidermis. In contrast to quiescent Lrig1⁺SCs in the skin, Lrig1⁺ intestinal SCs are rapidly dividing and Lrig1 appears to only reduce their proliferative capacity. However, similar to the situation in the skin, Lrig1 and EGF signalling may play an important role during damage repair. Earlier experiments analysed the phenotype of mice lacking major EGF family members (Egger et al, 1997; Troyer et al, 2001). While these mice display some duodenal lesions during normal homeostasis, further experiments established EGF signalling as a key protective component that ameliorates mucosal damage. It remains to be seen whether activation of intestinal SCs during damage repair involves mitigation of Lrig1 dampening.Lrig1 is known to repress ErbB signalling by mediating ubiquitinylation and degradation of activated receptors, thereby limiting the amplitude of EGF signalling (Watt and Jensen, 2009). Consequently, Lrig1 deletion in the intestine induced upregulation of EGFR, ErbB2, and ErbB3, promoting downstream activation of c-Myc within intestinal stem and progenitor cells (Wong et al, 2012). Importantly, Lrig1 is a direct Myc target gene, and thereby part of a negative feedback loop that helps to fine-tune the population size and proliferative activity of intestinal progenitor cells (Figure 1B).Since the rescue of the Lrig1^−/− phenotype by EGFR deficiency was only partial (Wong et al, 2012), other mechanisms may contribute. Intriguingly, Lrig1 has been shown to promote BMP signalling by direct binding to Type I (ALK6) and Type II (ALK1, ALK2, ALK3, and ActRIB) BMP receptors (Gumienny et al, 2010). BMPR1A inactivation, deficiency of its downstream effector PTEN, and transgenic overexpression of the BMP inhibitor Noggin display crypt expansion and increased SC numbers. Inhibition of BMP signalling in these genetic models enhanced AKT activation and increased Wnt signalling, promoting proliferation and adenoma formation (Figure 1B; Scoville et al, 2008). Future work will reveal a potential involvement of BMP and Wnt signalling in the Lrig1 knockout phenotype.The ErbB pathway has been linked to inflammatory bowel disease, and progression and metastatic potential of colorectal cancer. EGFR inhibition blocks adenoma formation in preclinical models, and ErbB pathway inhibition is currently being evaluated in clinical trials with colorectal cancer patients, where promising results have been reported (Cunningham et al, 2004). In contrast, Lrig1 is expressed at low levels in several cancer types but is overexpressed in some prostate and colorectal tumours (Hedman and Henriksson, 2007). Given this heterogeneity, the Lrig1 function in tumours appears to be cell- and context-dependent. Due to early postnatal lethality of Lrig1 knockout mice, the exciting possibility that Lrig1 may act as an intestinal tumour suppressor could not be answered by the current study but clearly deserves further attention.     

Enlightening the impact of immunogenic cell death in photodynamic cancer therapy

EMBO J 31 5, 1062–1079 (2012); published online January172012In this issue of The EMBO Journal, Garg et al (2012) delineate a signalling pathway that leads to calreticulin (CRT) exposure and ATP release by cancer cells that succumb to photodynamic therapy (PTD), thereby providing fresh insights into the molecular regulation of immunogenic cell death (ICD).The textbook notion that apoptosis would always take place unrecognized by the immune system has recently been invalidated (Zitvogel et al, 2010; Galluzzi et al, 2012). Thus, in specific circumstances (in particular in response to anthracyclines, oxaliplatin, and γ irradiation), cancer cells can enter a lethal stress pathway linked to the emission of a spatiotemporally defined combination of signals that is decoded by the immune system to activate tumour-specific immune responses (Zitvogel et al, 2010). These signals include the pre-apoptotic exposure of intracellular proteins such as the endoplasmic reticulum (ER) chaperon CRT and the heat-shock protein HSP90 at the cell surface, the pre-apoptotic secretion of ATP, and the post-apoptotic release of the nuclear protein HMGB1 (Zitvogel et al, 2010). Together, these processes (and perhaps others) constitute the molecular determinants of ICD.In this issue of The EMBO Journal, Garg et al (2012) add hypericin-based PTD (Hyp-PTD) to the list of bona fide ICD inducers and convincingly link Hyp-PTD-elicited ICD to the functional activation of the immune system. Moreover, Garg et al (2012) demonstrate that Hyp-PDT stimulates ICD via signalling pathways that overlap with—but are not identical to—those elicited by anthracyclines, which constitute the first ICD inducers to be characterized (Casares et al, 2005; Zappasodi et al, 2010; Fucikova et al, 2011).Intrigued by the fact that the ER stress response is required for anthracycline-induced ICD (Panaretakis et al, 2009), Garg et al (2012) decided to investigate the immunogenicity of Hyp-PDT (which selectively targets the ER). Hyp-PDT potently stimulated CRT exposure and ATP release in human bladder carcinoma T24 cells. As a result, T24 cells exposed to Hyp-PDT (but not untreated cells) were engulfed by Mf4/4 macrophages and human dendritic cells (DCs), the most important antigen-presenting cells in antitumour immunity. Similarly, murine colon carcinoma CT26 cells succumbing to Hyp-PDT (but not cells dying in response to the unspecific ER stressor tunicamycin) were preferentially phagocytosed by murine JAWSII DCs, and efficiently immunized syngenic BALB/c mice against a subsequent challenge with living cells of the same type. Of note, contrarily to T24 cells treated with lipopolysaccharide (LPS) or dying from accidental necrosis, T24 cells exposed to Hyp-PDT activated DCs while eliciting a peculiar functional profile, featuring high levels of NO production and absent secretion of immunosuppressive interleukin-10 (IL-10) (Garg et al, 2012). Moreover upon co-culture with Hyp-PDT-treated T24 cells, human DCs were found to secrete high levels of IL-1β, a cytokine that is required for the adequate polarization of interferon γ (IFNγ)-producing antineoplastic CD8⁺ T cells (Aymeric et al, 2010). Taken together, these data demonstrate that Hyp-PDT induces bona fide ICD, eliciting an antitumour immune response.By combining pharmacological and genetic approaches, Garg et al (2012) then investigated the molecular cascades that are required for Hyp-PDT-induced CRT exposure and ATP release. They found that CRT exposure triggered by Hyp-PDT requires reactive oxygen species (as demonstrated with the ¹O₂ quencher L-histidine), class I phosphoinositide-3-kinase (PI3K) activity (as shown with the chemical inhibitor wortmannin and the RNAi-mediated depletion of the catalytic PI3K subunit p110), the actin cytoskeleton (as proven with the actin inhibitor latrunculin B), the ER-to-Golgi anterograde transport (as shown using brefeldin A), the ER stress-associated kinase PERK, the pro-apoptotic molecules BAX and BAK as well as the CRT cell surface receptor CD91 (as demonstrated by their knockout or RNAi-mediated depletion). However, there were differences in the signalling pathways leading to CRT exposure in response to anthracyclines (Panaretakis et al, 2009) and Hyp-PDT (Garg et al, 2012). In contrast to the former, the latter was not accompanied by the exposure of the ER chaperon ERp57, and did not require eIF2α phosphorylation (as shown with non-phosphorylatable eIF2α mutants), caspase-8 activity (as shown with the pan-caspase blocker Z-VAD.fmk, upon overexpression of the viral caspase inhibitor CrmA and following the RNAi-mediated depletion of caspase-8), and increased cytosolic Ca²⁺ concentrations (as proven with cytosolic Ca²⁺ chelators and overexpression of the ER Ca²⁺ pump SERCA). Moreover, Hyp-PDT induced the translocation of CRT at the cell surface irrespective of retrograde transport (as demonstrated with the microtubular poison nocodazole) and lipid rafts (as demonstrated with the cholesterol-depleting agent methyl-β-cyclodextrine). Of note, ATP secretion in response to Hyp-PDT depended on the ER-to-Golgi anterograde transport, PI3K and PERK activity (presumably due to their role in the regulation of secretory pathways), but did not require BAX and BAK (Garg et al, 2012). Since PERK can stimulate autophagy in the context of ER stress (Kroemer et al, 2010), it is tempting to speculate that autophagy is involved in Hyp-PDT-elicited ATP secretion, as this appears to be to the case during anthracycline-induced ICD (Michaud et al, 2011).Altogether, the intriguing report by Garg et al (2012) demonstrates that the stress signalling pathways leading to ICD depend—at least in part—on the initiating stimulus (Figure 1). Speculatively, this points to the coexistence of a ‘core'' ICD signalling pathway (which would be common to several, if not all, ICD inducers) with ‘private'' molecular cascades (which would be activated in a stimulus-dependent fashion). Irrespective of these details, the work by Garg et al (2012) further underscores the importance of anticancer immune responses elicited by established and experimental therapies.Open in a separate windowFigure 1Molecular mechanisms of immunogenic cell death (ICD). At least three processes underlie the immunogenicity of cell death: the pre-apoptotic exposure of calreticulin (CRT) at the cell surface, the secretion of ATP, and the post-apoptotic release of HMGB1. ICD can be triggered by multiple stimuli, including photodynamic therapy, anthracycline-based chemotherapy, and some types of radiotherapy. The signalling pathways elicited by distinct ICD inducers overlap, but are not identical. In red are indicated molecules and processes that—according to current knowledge—may be required for CRT exposure and ATP secretion in response to most, if not all, ICD inducers. The molecular determinants of the immunogenic release of HMGB1 remain poorly understood. ER, endoplasmic reticulum; P-eIF2α, phosphorylated eIF2α; PI3K, class I phosphoinositide-3-kinase; ROS, reactive oxygen species.     

A Janus Tale of Two Active High Mobility Group Box 1 (HMGB1) Redox States

High mobility group box 1 (HMGB1), the prototypic damage–associated molecular pattern molecule, is released at sites of inflammation and/or tissue damage. There, it promotes cytokine production and chemokine production/cell migration. New work shows that the redox status of HMGB1 distinguishes its cytokine-inducing and chemokine activity. Reduced all-thiol-HMGB1 has sole chemokine activity, whereas disulfide-HMGB1 has only cytokine activity, and oxidized, denatured HMGB1 has neither. Autophagy (programmed cell survival) and apoptosis (programmed cell death) have been implicated in controlling both innate and adaptive immune functions. Reduced HMGB1 protein promotes autophagy, whereas oxidized HMGB1 promotes apoptosis. Thus, the differential activity of HMGB1 in immunity, inflammation and cell death depends on the cellular redox status within tissues.High mobility group box 1 (HMGB1), a nonhistone nuclear factor, acts extracellularly as a damage-associated molecular pattern (DAMP) molecule to modulate inflammation, promoting autophagy and innate immune responses (1–5). HMGB1 has compartment-specific functions: nuclear, intracellular (but extranuclear) and extracellular. Its extracellular functions can now be divided further into cytokine-like or cytokine-inducing, chemokinelike and proangiogenic. Signaling pathways that induce variations on the posttranslational modification, such as phosphorylation and acetylation, have been implicated in the regulation of HMGB1 release. Importantly, HMGB1 contains three cysteines, each of which is susceptible to redox modification (6,7). The redox state of these cysteines is important for the proinflammatory cytokine-stimulating and proautophagic activity of HMGB1 (8–10). Autophagy (literally “self-eating”), a lysosome-mediated catabolic process, contributes to maintenance of intracellular homeostasis and promotes cell survival in response to environmental stress (11–13).Treatment with reduced but not oxidized HMGB1 protein increases autophagy in cancer cells (9). In contrast, oxidized HMGB1 protein activates the caspase-dependent apoptotic cell death pathway (9). Venereau et al.(14) described a new role for redox control of both the cytokine-inducing and chemokine activity of HMGB1 in the setting of sterile inflammation, regulating leukocyte recruitment and their ability to secrete inflammatory cytokines (Figure 1).Open in a separate windowFigure 1Redox control of HMGB1 activity. To act as a DAMP/danger signal and inflammatory mediator, HMGB1 is transported extracellularly by two principal means: active secretion from living inflammatory cells (for example, macrophages) or passive release from necrotic cells. The activities of extracellular HMGB1 are redox dependent. All-thiol-HMGB1 promotes chemokine production and leukocyte recruitment. Disulfide-HMGB1, originating from infiltrating leukocytes, promotes release of proinflammatory cytokines and thus participates in the inflammatory response. Reactive oxygen species produced by leukocytes induces the terminal oxidation of HMGB1, which is inactivated during resolution of inflammation.Structurally, HMGB1 is composed of three domains: two positively charged proximal DNA-binding domains (A box and B box) and a negatively charged carboxyl terminus. Three cysteines are encoded within the molecule: two vicinal cysteines in box A (C23 and C45) and a single one in box B (C106). C23 and C45 can form an intermolecular disulfide bond, whereas C106 is unpaired. Therefore, three different redox forms HMGB1 (all-thiol-HMGB1, disulfide-HMGB1 and oxidized HMGB1) were derived from bacterial expression systems (14). In addition, by using tryptic digests and liquid chromatography tandem mass spectrometric analysis, Venereau et al. observed that recombinant HMGB1 can be reversibly oxidized and reduced in the presence of electron donors (for example, dithiothreitol) or acceptors (oxygen) (14).Next, Venereau et al. assessed whether individual redox forms of HMGB1 have a differential role in cytokine-stimulating and chemoattractant activities (14). They found that disulfide-HMGB1 induced activation of the nuclear factor (NF)-κB pathway and production of proinflammatory cytokines (for example, tumor necrosis factors-α, interleukin [IL]-6 and IL-8) in fibroblasts and macrophages. Interestingly, all-thiol-HMGB1 failed to induce a proinflammatory response. In contrast, all-thiol-HMGB1, but not disulfide-HMGB1, had chemoattractant activity in fibroblasts. These findings prompted them to determine whether HMGB1 inhibitors, such as box A and monoclonal antibody PDH1.1, block the chemoattractant and/or cytokine-inducing activities of HMGB1. Unexpectedly, these inhibitors prevented cell migration but not cytokine production, although they are widely used as HMGB1-targeting agents in experimental inflammatory diseases.Reactive oxygen species oxidize the HMGB1 released from dying cells, thereby neutralizing its stimulatory activity and promoting tolerance in immune cells (15,16). In addition, oxidation of C106 or lack of a disulfide bridge between C23 and C45 then causes HMGB1 to lose its proinflammatory effects in macrophages (8). Venereau et al. found that terminal oxidation by hydrogen peroxide results in the loss of both the cytokine-stimulating and chemoattractant activities of HMBG1. Moreover, the authors found that the three HMGB1 cysteine residues were required for the cytokine-stimulating activity but not for the chemoattractant activity of HMGB1. Cysteine mutant HMGB1 promotes fibroblast migration, but not cytokine expression in macrophages (14). Collectively, these findings establish a crucial role for redox in the regulation of HMGB1 activity in inflammation and migration.What is the redox state of HMGB1 in the pathogenesis of individual diseases? The redox state of HMGB1 from the human acute monocytic leukemia cell line THP-1 was measured in the presence or absence of lipopolysaccharide (LPS) and necrotic medium in vitro. Intracellular HMGB1 was all-thiol-HMGB1, whereas secreted HMGB1 contained both all-thiol- and disulfide-HMGB1 (14). Furthermore, disulfide-HMGB1 was present later and time-dependently increased in cardiotoxin-injured muscles in vivo, confirming that the redox state of HMGB1 is altered during tissue damage and inflammation. HMGB1 protein with all three cysteines mutated to serine are resistant to oxidation and induce leukocyte recruitment without inducing cytokine production (14). The activities of HMGB1 are thus redox-dependent and can be modified within the injured tissues after HMGB1 release. Therefore, release of dynamic redox-regulated HMGB1 contributes to the orderly orchestrated recruitment of leukocytes, activation of cytokine release and subsequent resolution of inflammation.Several issues remain unresolved regarding the redox control of HMGB1 activity. First, HMGB1 is specifically recognized by several cell surface receptors (2), including Toll-like receptor (TLR)-4 and the receptor for advanced glycation end products (RAGE), but most recently was joined by T-cell immunoglobulin and mucin domain 3 (TIM-3) (17). Initial studies suggest that reduced C106 is necessary for the binding of HMGB1 to one of its receptors, TLR4, to stimulate cytokine release (8). HMGB1-induced recruitment of inflammatory cells depends on forming a complex with CXCL12 and signaling via CXCR4 (18). Moreover, RAGE is required for reduced HMGB1-mediated autophagy, but not oxidized HMGB1-induced apoptosis (9). All-thiol-HMGB1, but not disulfide-HMGB1, binds CXCL12 (14). The influence of HMGB1 receptors (for example, RAGE, TLR4, TLR2, CD24, TIM-3 and triggering receptor expressed on myeloid cells 1 [TREM1]) on biological activities of individual redox forms of HMGB1 remains to be carefully investigated. Second, HMGB1 forms highly inflammatory complexes with DNA, lipoteichoic acid, LPS, IL-1β, chemokine (C-X-C motif) ligand 12 (CXCL12)/ stromal cell–derived factor-1 (SDF-1) and nucleosomes (19). There is great interest in determining whether the individual redox forms of HMGB1 have varying affinity profiles active in inflammation and immunity. Third, HMGB1 has multiple intracellular and extracellular functions in health and disease, including cancer (1,2,6,20). Additional studies will be needed to determine whether redox is required for other functions of HMGB1, such as regeneration and cellular differentiation as well as the complex interactions between autophagy and immunity (5). One additional unanswered question is where and how the formation of the disulfide takes place and whether there is an enzyme specific for regulating this. This is important, knowing that the nuclear form is mostly all thiol. Finally, the development and performance of a simple, sensitive method for the detection of individual HMGB1 redox state isoforms in clinical specimens remains to be accomplished.     

Niche science: The aging stem cell

Studies on stem cell aging are uncovering molecular mechanisms of regenerative decline, providing new insight into potential rejuvenating therapies.Studies on stem cell aging are uncovering molecular mechanisms of regenerative decline, providing new insight into potential rejuvenating therapies. Most human tissues retain an amazing ability to regenerate well into adulthood. Somatic stem cells are central to this ability, replacing damaged cells and thus keeping the body in a highly functional state. Yet this process does not continue unabated forever, as aging is accompanied by a loss of this regenerative capacity. Presently, studies in invertebrate and vertebrate model systems are advancing our understanding of regenerative decline and are identifying strategies for ‘rejuvenating’ therapies that have the potential to extend human health- and lifespan.The feasibility of rejuvenating interventions was demonstrated by classic studies in which exposure to a young systemic environment restored regenerative capacity of muscle stem cells in old mice.¹ Similar rejuvenation has now been demonstrated for the central nervous system, suggesting that such interventions have systemic potential² and raising the question of whether the lifespan of the organism could be extended by restoring the regenerative capacity of adult stem cells. This has already been demonstrated in flies, where improved intestinal stem cell function leads to enhanced longevity.³Such studies have inspired the burgeoning field of “stem cell aging.”^4,5 A recent symposium at the Buck Institute for Research on Aging in Novato, CA showcased the field, bringing together researchers interested in the biology of aging and experts in stem cell biology, and covering topics ranging from basic research in stem cell aging to the use of stem cells in clinical applications. Clear from the meeting is that new molecular insight into stem cell aging is emerging at a rapid pace, revealing both the promises and challenges of deploying stem cell therapies for age-related diseases. The key questions are starting to be answered.