Proteostenosis and plasma cell pathophysiology

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Proteostenosis and plasma cell pathophysiology Simone Cenci, Eelco van Anken and Roberto Sitia Plasma cells differentiate from B lymphocytes to sustain antibody production. As professional secretors, they allow dissecting proteostasis in the exocytic compartment, the stresses that protein production entails and their possible roles in signaling. Most plasma cells are short-lived to limit antibody responses. After a few days of intense immunoglobulin production, they undergo apoptosis, offering a unique model of cellular senescence. Recent observations reveal that proteotoxic stresses physiologically contribute to regulate their biogenesis, function and lifespan, explaining partly the sensitivity of multiple myeloma cells to proteasome inhibitors. This essay summarizes these plasma cell lessons, and their general implications for the regulation of proteostasis, cell senescence and cancer therapeutics. Address Division of Genetics and Cell Biology, San Raffaele Scientific Institute and Universita` Vita-Salute San Raffaele, Milano, Italy Corresponding author: Sitia, Roberto ([email protected])

Current Opinion in Cell Biology 2011, 23:216–222 This review comes from a themed issue on Cell regulation Edited by Laurie Glimcher and Claudio Hetz Available online 18th December 2010 0955-0674/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2010.11.004

Introduction Cells of the B lineage have been goldmines for cell and molecular biologists, providing numerous novel paradigms of general significance [1,2!!,3]. Plasma cells, specialists in antibody (Ab) production, have yielded key mechanistic insights on the biogenesis of the endoplasmic reticulum (ER) and secretory pathway, and its astonishing efficiency and precision [4–6,7!!]. But they probably have more surprises up their sleeves (Box 1). Most plasma cells are short-lived, while some gain lifelong survival in bone marrow niches, contributing to shape immunological memory [8]. Over the past decade, a milestone observation in plasma cell pathophysiology came from the clinics, with the discovery that proteasome inhibitors (bortezomib, Velcade) are particularly effective against multiple myeloma (MM) [9!!,10,11!!], the malignant transformation of plasma cells. The sensitivity to proteasome inhibitors is a general feature of Ig-secreting plasma cells, as Ab responses to both T-dependent and Current Opinion in Cell Biology 2011, 23:216–222

independent antigens are blunted by bortezomib [12!], which offers an avenue for reversing autoimmune disease through shipwrecking autoreactive plasma cells [13!]. For many MM patients, bortezomib heralds higher chances for successful treatment. For cell biologists, it opened up novel questions and perspectives on the mechanisms that coordinate differentiation, proliferation and death of plasma cells, a powerful model for cellular senescence and lifespan control. How is their demise controlled? Is it linked to Ab synthesis and secretion, and if so how? Why are certain myelomas particularly sensitive to proteasome inhibitors? The nature of the molecular timers or counters setting apoptotic programs in normal and malignant plasma cells holds great immunological and biomedical interest [5,14]. This essay focuses on the role of proteostasis as a signaling device and therapeutic target in plasma cell pathophysiology.

How do (most) plasma cells die? As they differentiate from small B lymphocytes, plasma cells meticulously prepare for large scale synthesis, folding and secretion of Ig chains by enlarging their ER and secretory organelles [7!!]. After a few days of intense labor, in which each of them releases thousands of Ab per second, most eventually die (Figure 1). The B to plasma cell metamorphosis is orchestrated via diverse pathways. Some are lineage specific, like the transcription factor Blimp-1, whose expression is necessary and sufficient to launch the differentiation program [15]. B cell differentiation hijacks also general homeostatic pathways, amongst which a primary role is reserved for the unfolded protein response (UPR), a multidimensional signaling cascade aimed at adjusting ER capacity according to need during stress or differentiation [16,17]. Plasma cell development is blocked in the absence of the key UPR transcription factor, XBP-1 [18!!], which Box 1 The many virtues of the plasma cell model. The differentiation of B lymphocytes into plasma cells can be readily induced with mitogen or antigen, either in vitro (with inducible B lymphomas or with primary B lymphocytes obtained from different animal models, especially genetically modified mice) or in vivo, by immunization or polyclonal activation. After a proliferative burst, cell cycle arrests, differentiation allows exuberant antibody secretion and eventually massive apoptosis ensues after 3 to 6 days. The dramatic morphological and functional changes allow investigating the complex relationship between signaling via developmental and stress pathways, cell cycle, cell function, proteostasis, and lifespan control.

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Figure 1

Antibody secreting cells Differentiation Homing to bone marrow

Antibody titers

Apoptosis Ag Proliferation Affinity maturation

Long-lived Plasma cell

B cell Time Days

Months Current Opinion in Cell Biology

Terminal B to plasma cell differentiation. Upon encounter with antigen (Ag) in a suitable context, resting B cells rapidly proliferate, some undergoing affinity maturation and class switch to become memory cells. For the remainder, B cells rapidly differentiate into antibody secreting plasma cells that sustain massive antibody production, folding, assembly and secretion, causing a sharp rise in the titer of specific antibodies. After a few days of intense secretion, most antibody secreting cells undergo apoptosis, which explains the decrease in serum Ab titers. Some plasma cells return to the bone marrow, and home in niches where they survive for long periods, releasing antibodies at a level that confers immunological memory and protection.

promotes expansion of the secretory pathway [19!] and ER associated degradation [20!,21!,22!,23]. Increased expression of XBP-1 and its target genes also accompanies plasma cell differentiation from memory B cells [24]. While Blimp-1 promotes XBP-1 transcription [15], the mechanisms that in plasma cells activate the Ire1-dependent splicing of XBP-1 transcripts—an essential event for signal propagation [25,26]—are not fully understood. Ire1 is also required for early B cell development, independent of XBP-1 [27!], which is also dispensable for generation of memory B cells [28]. Intriguingly, another UPR pathway, propagated via PERK, is essential for pancreatic b cell survival [29!], but dispensable for B cell differentiation [14,27!,30,31]. Plasma cells may hence prove valuable to address how different branches of the UPR, once considered a monolithic response, are selectively activated in tissues. The propensity of plasma cells to undergo apoptosis is probably linked to their stressful life, following the adagium: ‘those who live fast, die young’. In view of their full gear secretory occupation, the UPR—which like most adaptive responses can lead to apoptosis if excessive or prolonged—is a primary suspect for sealing plasma cells’ fate. Yet, their development and lifespan are essentially normal in the absence of Chop, a UPR factor generally involved in maladaptive responses and cell death [14]. It remains to be elucidated whether other pro-apoptotic www.sciencedirect.com

branches of the UPR [16] are specifically engaged in plasma cells. Besides elements of the UPR, plasma cell differentiation also entails oxidative stress, which could play an important role especially considering that H2O2 is produced during Ero1-driven disulfide bond formation [32!,33!,34], and that a single plasma cell can form over 105 disulfides per second to sustain Ig folding and assembly. Plasma cells lacking peroxiredoxin IV (PrxIV), an efficient H2O2 scavenger localized in the secretory pathway [35], however, do not display a significantly different lifespan [36]. As surprisingly, plasma cells lacking both Ero1a and b display only some slowdown in Ig assembly [37!!]. Thus, additional pathways probably operate in the ER to maintain redox homeostasis [38]. Also Ca2+ signaling may contribute to plasma cell development and lifespan control, as the contact sites between mitochondria and ER—key hubs for Ca2+ and redox signal integration [39!,40!,41]—probably undergo profound reshaping during B to plasma cell differentiation. The lack of success in pinpointing whether any of these stress pathways specifically provokes plasma cell death may reflect a multifactorial and perhaps synergic control.

Challenged proteostasis as a signal Our knowledge of the biology of protein degradation advanced greatly in the past decade. Milestone discovCurrent Opinion in Cell Biology 2011, 23:216–222

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Figure 2

secretory machinery antibody production ERAD load ubiquitinated proteins proteasomal capacity days

quiescent phase

anticipatory phase

full-gear secretory phase

proteostenotic apoptosis phase Current Opinion in Cell Biology

Schematic representation of changes in proteostasis during B to plasma cell differentiation. As soon as B cells have been awakened from quiescence by Ag stimulation, they undergo a complete rearrangement, most notably expanding the secretory capacity, in anticipation of bulk antibody secretion. The complexity of antibody folding and assembly at massive loads then exceedingly burdens the folding-quality control systems with excess or aberrant antibody subunits being retrotranslocated to the cytosol via ER associated degradation (ERAD) to be disposed of by proteasomes. Paradoxically, the abundance of proteasomes decreases in differentiating cells. As a consequence poly-ubiquitinated proteins accumulate and antibody-secreting cells become proteostenotic. To cope with proteotoxicity, detergent insoluble proteins that accumulate in the lumen of the early secretory compartment (ESC) are often sorted in defined sub-regions, such as Russell Bodies [79]. Finally, the full-gear antibody production demands its toll and the cells perish, probably as a result of the accumulated proteotoxic waste.

eries include the following: a substantial pool of newly synthesized proteins is rapidly degraded by proteasomes in a variety of cell lines and tissues, hence the concept of rapidly degraded polypeptides (RDP) [42!!]; cells are equipped with excess idle proteasomes, probably as a functional reserve for the rapid clearance of aberrant proteins under stress conditions [43!,44,45]; proteasome biogenesis is tuned to demand via a proteasome stress response [46]. Owing to the intrinsic complexity of Ab molecules and their high rate of synthesis, plasma cells face a great demand for protein degradation. Like many substrates of ER quality control, unassembled Ig subunits are degraded by proteasomes [6]. Moreover, the reshaping of B cells into plasma cells increases RDP production [47!,48!!], even before Ig synthesis (Cenci et al., unpublished). Paradoxically, while the demand for proteasomal degradation increases exponentially in activated B cells, proteasome abundance and activity decrease [48!!]. As a consequence, the ubiquitin proteasome system (UPS) is overloaded (Figure 2): ubiquitinated proteins accumulate, free ubiquitin diminishes, and certain proteasome targets—including pro-apoptotic factors—are stabilized [12!,48!!]. The decrease of the proteasome pool following Current Opinion in Cell Biology 2011, 23:216–222

B cell activation is a challenging contradiction of the proteasome stress response [46]. An unfavorable proteasome load vs. capacity ratio correlates with the accumulation of Bax, Bim and IkBa and may hence sensitize plasma cells to spontaneous and pharmacological apoptosis [12!,48!!]. Indeed, such imbalance coincides with increased sensitivity to proteasome inhibitors, shedding light on their anti-myeloma toxicity [47!] (see below). The coup de grace may then be offered jointly or in a redundant fashion, by other imbalances experienced by plasma cells, such as prolonged UPR, oxidative stress, or altered Ca2+ homeostasis. From the perspective of cell biology, we are left wondering what could be the advantage for disabling the proteasome stress response in terminal B cell differentiation. A strict control of plasma cells lifespan is important to limit humoral immune response. The reduced proteostatic capacity ( proteostenosis) may provide an inbuilt counter for secreted molecules. By engineering a bottleneck in an otherwise efficient quality control strategy devoted to Ig folding and assembly, plasma cells may keep track of the work accomplished (Ab secretion), and limit the humoral response accordingly. A key issue that remains to be addressed is what decreases the proteasome complement during plasma cell differenwww.sciencedirect.com

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tiation, despite the demand increases. Apart from the effects of interferon-g on the three b-peptidases that hallmark immunoproteasomes [49], and of antioxidant responses involving Nrf2 and Nrf1 [50,51!], the mechanisms regulating proteasome biogenesis in metazoans are unclear. The plasma cell paradox can provide a powerful system to address this central issue in molecular cell biology.

Proteostenosis, senescence and cell death

By linking cell death to protein degradation [48!!], the plasma cell paradigm provides a unique model for cellular aging. In yeasts and bacteria, mother cells retain damaged proteins, preventing replicative senescence [52!,53]. In metazoans, asymmetric division may have been co-opted to enable cell fate diversification, lineage commitment and morphogenesis [54]. At the cellular level, aging is characterized by a progressive reduction of the capacity to adapt to stress and maintain homeostasis [55]. Protein degradation is key to enable rapid proteome adaptations and minimize protein damage [56]. Senescent cells and tissues accumulate oxidized and damaged proteins, with increased propensity to aggregate, further challenging proteostasis [57–59]. An age-associated decline in proteasomal degradation may explain why aging predisposes to many diseases [60]. A generalized collapse of the proteostatic capacity precedes tissue damage and may be causal to the aging process in C. elegans [61!]. An integral role for proteostenosis in the aging process is also suggested by the cross-talk between signaling pathways that control lifespan and proteostasis. For example, inhi-

bition of insulin growth factor (IGF) signaling has beneficial effects in animal models of Alzheimer’s disease [62,63] and prolongs lifespan in worms, flies, and mammals [64!!,65,66]. Although the mechanism is not fully understood, reduced IGF signaling may decrease the buildup of aggregation-prone proteins and the ensuing proteotoxicity by reinforcing the cellular protein folding capacity [67,68]. By offering a cellular model of progressive proteostenosis linked to accelerated senescence, plasma cells enable the dissection of the molecular mechanisms of how proteostasis, cellular senescence and lifespan control intertwine, likely to unveil novel strategies against age-related diseases, in primis cancer.

Flipping the coin: exploiting proteotoxicity against multiple myeloma and other cancers If an unbalanced proteasomal load vs. capacity ratio limits the lifespan of normal plasma cells, we can flip the coin and ask whether we can manipulate it against MM, a condition in which cells deemed to die no longer do. The wealth of clinical reports on the efficacy of proteasome inhibitors against MM [10,11!!,69,70] generates great promise, although a substantial proportion of patients fail to respond [10,71]. The findings obtained with non-malignant plasma cells have provided clues for the diverse susceptibility of MM cells to proteasome inhibitors. In accordance with the load vs. capacity model (Figure 3), bortezomib sensitivity was found to correlate with the burden on the UPS because of either increased levels of Ig synthesis [72], high RDP

Figure 3

resistant

sensitive Current Opinion in Cell Biology

Schematical representation of the load vs. capacity model as a determinant for sensitivity to proteasomal inhibitors of MM cells. Multiple myeloma cell lines as well as primary CD138+ cells from patients display different sensitivity to proteasome inhibitors. In both cell lines and patient derived cells, sensitivity was found to correlate with the proteasomal load vs. capacity ratio. In resistant cells, the proteasomal capacity (depicted in orange) exceeds the load of poly-ubiquitinated proteins (depicted in gray). Conversely, in sensitive cells, more proteins are degraded by fewer proteasomes, generating long queues of poly-ubiquitinated proteins. The free ubiquitin pool (depicted as white spheres) decreases, and many proteins that otherwise would be proteasome substrates are no longer ubiquitinated. These proteins (depicted in dark blue) are stabilized, because they are no longer ‘visible’ to the proteasome. Such stabilization of proteins may contribute to predisposing cells to apoptosis, as has been shown for Ig-H, Bim, Bax and IkBa. www.sciencedirect.com

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production, or reduced proteasome pools [47!]. As a result, the highest vulnerability was found in MM populations hallmarked by reduced proteasome capacity and accumulation of poly-ubiquitinated proteins, clear symptoms of proteostenosis. We could therefore extrapolate the stress threshold paradigm and attempt to further manipulate stress levels against resistant MM and other malignancies. While novel inhibitors are being tested that target the proteasome differently [73], overall stress levels may be further manipulated targeting a variety of homeostatic control pathways. For instance, proteostenotic MM lines fail to increase de novo proteasome expression in response to proteasome stress, while less sensitive cells respond to proteasome stress with increased proteasome biogenesis, acquiring resistance [47!]. Hence, the mechanisms that control (immuno)proteasome biogenesis could unveil important targets for MM treatment. The emerging complementarity of UPS and autophagy [74] predicts that the latter pathway may serve an important role in plasma cells, sustaining viability and secretory function. Our preliminary evidence supports this prediction both in normal (N Pengo et al., unpublished) and malignant (F Fontana et al., unpublished) plasma cells, providing valuable models to dissect the molecular circuits linking these two degradative systems. The paradoxical discrepancy between proteasomal load and capacity observed in plasma cells opens the possibility to design, test, and exploit pro-apoptotic synergies for therapeutic intervention against MM and possibly other cancers. In particular, treatments that further compromise proteolysis or increase the degradative burden, for example, by manipulating the UPR or redox stress, may help tip the balance in favor of increased susceptibility to proteasome inhibitors.

Conclusions Studies on the B-lymphocytic lineage continue to offer novel paradigms in cell biology. The metamorphosis of long-lived B cells into short-lived Ab secreting cells provides a powerful system to recapitulate and dissect mechanistically senescence and death. Indeed, the features revealed by normal and neoplastic plasma cells support the general, novel idea that cytotoxic stress can be exploited against cancer [75–78]. Most stress responses entail apoptotic pathways that can be activated if stress duration or intensity increase, turning these responses maladaptive. In cancer, these responses are generally intact, with lower apoptotic thresholds, while physiological apoptotic responses to genotoxic stress are often disabled. Owing to deregulated growth, cancer cells generally experience more cytotoxic stress than normal counterparts (e.g. hypoxia, nutrient deprivation, pH changes, oxidative stress), and adaptive responses are often activated to higher levels, providing therapeutic specificity. Thus, strategies exploiting cytotoxic stress Current Opinion in Cell Biology 2011, 23:216–222

hold therapeutic promise against cancer, and preliminary results generate some optimism.

Acknowledgements We thank Anna Mondino for critical reading of the manuscript and helpful suggestions, Niccolo` Pengo and Francesca Fontana for providing novel results and discussions, Ana Fella and Raffaella Brambati for secretarial assistance, and AIRC, Cariplo, Ministero della Salute, MIUR, Telethon, and the Armenise-Harvard Foundation for generously supporting our work.

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