INTRODUCTION

Hormetic endoplasmic reticulums (HSCs) are quiescent bone marrow cells able to activate, self-renew and give rise to all blood cell types [1]. HSCs possess two fundamental stem cell properties: self-renewal and multipotent differentiation potential. Although HSCs have been studied for over 50 years and have implicated hundreds of genes as important in the regulation of HSC function [2–5], major gaps still exist in our understanding of HSC biology and physiology, hindering the development of strategies to maintain or expand functional HSCs in vitro for use in treatments of hematological disease, failure or malignancy in vivo. Similarly, degeneration of HSC function underlies hematopoietic disease and malignancy arising from the aging process [6,7]. Despite our current information, a comprehensive understanding of the signaling events that coordinate HSC function remains incomplete. Thus, efforts to distill the knowledge required to harness the regenerative properties of HSCs represent an essential goal towards designing rational methods to expand HSC ex vivo and improve functional transplantation outcomes in patients in vivo. 

Box 1:

no caption available

HORMETIC STRESS IS ASSOCIATION WITH LONGEVITY AND CELLULAR INTEGRITY

A paradox observed in biology is that increased stress resistance can extend the lifespan, or the rejuvenation process, of a variety of organisms under multiple stress conditions [8–10]. Curiously, details from studies into the role of stress resistance in the regulation of lifespan revealed a dose-dependent response [11]. This phenomenon, referred to as the hormetic effect (or hormesis; Fig. 1), can be extended to describe the adaptive responses of cells to mild and moderate stress including heat, irradiation, hypoxia, oxidative stress and caloric restriction [12]. Derived from the Greek ‘to set in motion’, hormesis is defined as an exposure to mild levels of harmful factors that preconditions a cell to stimulate the activation of stress resistance mechanisms, thus promoting a prolonged cellular capacity to maintain and repair. Hormesis describes a two-phase dose response, in which a low dose and a high dose trigger a stimulatory (beneficial) and an inhibitory (adverse) effect, respectively [13]. Thus, mild and periodic (but not severe or chronic) exposure to specific signaling should improve an organism's ability to stably cope with such adverse circumstances.

FIGURE 1:

Hormetic effect of stress stimulus. Low-dose stress stimulates beneficial cellular responses compared with the no observed effect level (NOEL) dosage of stress stimulus and diminishes as dose reaches the no observed adverse effect level (NOAEL), at which point stress levels provide no beneficial response. Conversely, high doses of stress adversely affect cellular responses and results in toxicity and apoptosis.

Molecular biology describes the endoplasmic reticulum (ER) has an intricate organelle responsible for secreted and integral membrane protein synthesis and compartmentalizes numerous enzymatic reactions including protein folding, redox balance, intracellular calcium storage and lipid biogenesis [14,15]. However, the ER possesses complex signaling systems to monitor cellular health. As such, ER stress signals can be either cytoprotective or apoptotic depending on the duration and amplitude of the stress stimulus. Precedence for cytoprotective stress stimulation in HSCs has been established, including amino acid depravation and low-dose irradiation [16,17]; however, molecular underpinnings of the ER stress response in HSC have only recently been described.

HORMETIC EFFECT OF THE ENDOPLASMIC RETICULUM STRESS RESPONSE

A number of stimuli can lead to disruption of ER homeostasis and subsequent activation of ER stress-coping responses (Fig. 2, green boxes). Four endogenous stimuli induce ER stress: ER protein synthesis and misfolding, ER luminal calcium depletion, redox imbalance and lipid and cholesterol dysregulation [18]. An increased rate of protein synthesis disrupt ER equilibrium and induces what is referred to as the unfolded protein response (UPR). One of two signaling cascades can result from ER stress. First, the UPR intended to protect cellular function stabilizes protein synthesis through transcriptional upregulation of chaperones and folding enzymes or, alternatively, to degrade nascent peptides through upregulation ER-associated degradation (ERAD) machinery. Concurrently, attenuation of ribosomal translation reduces the rate of protein synthesis and import into the ER thereby preventing ER protein folding overload. Second, in the event of severe or prolonged ER stress, proapoptotic signaling cascades result from the UPR in the event ER homeostasis cannot be restored. Together, these two branches of UPR signaling cascades constitute the spectrum of the hormetic effect response to ER stress.

FIGURE 2:

Schematic overview of hormetic endoplasmic reticulum stress signaling elucidated in hematopoietic stem cells. Effectors of endoplasmic reticulum (ER) stress signaling are shown in boxes. Outcome of pathway activation is shown with colored arrows.

MOLECULAR MECHANISMS OF THE ENDOPLASMIC RETICULUM STRESS RESPONSE

The UPR is regulated by three branches that are epitomized by unique ER-associated integral membrane proteins (Fig. 2): the PRKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1). Upon increased protein synthesis and misfolding, dissociation of the master ER molecular chaperone BiP (GRP78) from ER-associated integral membrane proteins triggers their activation in a manner dependent on cellular context [19]. During the UPR, PERK becomes activated and phosphorylates the eukaryotic translation initiation factor 2α (eIF2α), which reduces translation of most mRNAs but selectively translates mRNA for the stress response factor ATF4. PERK activity also targets the antioxidant transcription factor Nrf2, which is released from its ER-tethering protein, Keap1, and translocates to the nucleus. ATF6 precursors translocate to the Golgi where it is proteolytically processed, resulting in release of transcription factor domain. IRE1 possesses both kinase and endoribonuclease activity that ultimately splices the mRNA transcript Xbp1 into the spliced Xbp1s transcript, which is then translated to produce a transcription factor that induces the expression of genes encoding molecular chaperones, folding enzymes and components of ERAD. Furthermore, the UPR can induce autophagy, which is a controlled catabolic process promoting cell survival and a feature of normal HSC function [20]. Under low-dose ER stress signaling, the UPR could induce a single branch, or multiple branches, to engage programs to facilitate cytoprotective roles in a context-specific manner [21].

Under prolonged and severe activation of the UPR, all three branches can upregulate C/EBP homologous protein (CHOP) which, though cooperation with ATF4, can activate multiple pathways that converge on the executionary pathway of apoptosis [22]. CHOP upregulates pro-apoptotic proteins (BH3-only ‘activators’ of apoptosis) BIM or PUMA or signals cleavage of BID its active form. BH3-only proteins are normally sequestered by pro-survival proteins, such as Bcl-2, Bcl-XL or myeloid cell leukemia 1 (MCL1). In the event, these pro-survival proteins are saturated or absent, BH3-only proteins facilitate BAX and/or BAK oligomerization to form pores in the mitochondrial outer membrane, resulting in the release of mitochondrial contents. Release of cytochrome c activates caspase 9, followed by effector caspases 3 and 7 to destroy the cell [23]. Thus, the cellular fate decision to choose UPR induction or apoptosis in response to ER stress response may rely on the chronology and amplitude of the UPR branches in response to ER stress condition.

RECENT FINDINGS Basal protein synthesis stress induces PERK--ATF4 signaling in hematopoietic stem cells

The first report directly implicating ER stress signaling in bona fide HSCs was elucidated in 2014 by van Galen et al.[24] in which the authors showed signaling from the PERK branch of the UPR was necessary for normal HSC function. In this study, human CD34+ HSCs were found to differentially upregulate ER stress response genes including PERK, ATF4, and GADD34 compared with progenitor cells. Pharmacological induction of ER stress using two compounds (Fig. 2, red boxes): tunicamycin, which blocks N-linked glycoprotein synthesis and prevents folding of proteins in the ER and thapsigargin, which inhibits SERCA ATPases and cytoplasmic pumping of Ca2+ into the ER, showed that HSCs were selectively predisposed to apoptosis in response to a strong induction of the UPR using tunicamycin, and to a lesser degree using thapsigargin. Short-term culture of CD34+ cells with tunicamycin specifically hyperactivated the PERK branch, which led to further upregulation of ATF4 and induction of CHOP expression, which resulted in apoptosis specifically in HSCs. Pharmacological inhibition of eIF2α dephosphorylation using salubrinal rescued tunicamycin-induced apoptosis. Interestingly, upregulation or ectopic expression of the ER chaperone ERDJ4 ameliorated the detrimental effects of tunicamycin stress on HSC function. These data were the first to elucidate that protein folding stress and PERK branch UPR signaling is a feature of healthy HSCs and that modest imbalances in ER stress can overwhelm signaling towards cell death. As upregulation of protein folding stress was determinantal to HSC survival specifically, these findings highlight that HSCs are unusually sensitive to enhanced UPR stress (i.e. inhibition of protein folding via tunicamycin) and that nominal ER stress may occur in HSCs.

Additionally, the same authors investigated the role of amino acid deprivation and the effect of integrated stress responses (ISRs) in HSC function [25]. They showed basal ER stress signaling was elevated in HSCs compared with progenitors and elevated expression of ATF4 was inversely correlated with eIF2α in CD34+ cells, supporting the notion that attenuated protein synthesis underlies low-level induction of ER stress signaling. Furthermore, deprivation of valine in culture media induced a cytoprotective effect centered on elevated PERK-ATF4 activity. Although ATF4 confers stress resistance in response to nutrient deprivation, hypoxia and ROS [26,27], conditional deletion of ATF4 in the hematopoietic compartment resulted in a significant but not complete, loss of HSC repopulation potential [28]. Though pioneering, these data suggesting a larger, and possibly redundant, signaling node involving the PERK--ATF4 pathway underlies the UPR. Nevertheless, the requirement of basal levels of ER stress signaling in HSCs supports a theory of cytoprotective hormesis.

IRE1--XBP1-mediated cytoprotective functions in hematopoietic stem cells

Cellular context often accounts for many differences observed with ER stress signaling. Recent investigations have also implicated IRE1 branch UPR signaling in HSC function. Using murine HSCs (CD48-CD150+LSK), the study found stimulation with tunicamycin and thapsigargin-induced expression of several UPR genes, including Grp78, Chop and Xbp1s with concomitant upregulation of IRE1 activity reporters. Steady-state levels of these pathways were also upregulated in HSCs compared with progenitor cells [29▪▪]. Interestingly, conditional deletion of IRE1 in the hematopoietic compartment (Vav1-Cre) resulted in impaired, but not complete, repopulation capacity of HSCs. This again suggests multiple, or redundant, ER stress pathways converge to activate the HSC self-renewal program. Furthermore, processed Xbp1s induced expression of the protein chaperone Dnajb8 in mouse HSCs and ectopic expression significantly improved donor HSC reconstitution in vivo. These data support a role for balanced protein synthesis in stress conditioning of HSCs to induce cytoprotective UPR signaling.

Convergence of multiple enzymatic functions to stabilize ER homeostasis would be consistent with supporting hormetic ER stress signaling. A newly discovered ER-associated protein DDRGK1, which regulates the posttranslational modification of protein with the ubiquitinylation-like modification (Ufm1) and important for HSC function [30,31], was shown to regulate IRE1 protein stability through DDRGK1-mediated ufmylation in HSCs [32]. Mechanistically, posttranslation ufmylation of IRE1 prevented proteasomal degradation of IRE1 and maintained normal HSC function while knockout of DDRGK1 led reduced IRE1 levels and hyperactivated the PERK branch UPR signaling pathway, which lead to HSC apoptosis and a severe HSC repopulation defect. Together, these data support a cytoprotective function for the IRE1 branch of the UPR in HSCs.

Balancing endoplasmic reticulum stress amplitude through molecular chaperones

Studies describing PERK--ATF4 and IRE1--Xbp1 branch signaling implicate molecular chaperones as transcriptional targets of a hormetic ER stress program including the heat shock protein superfamily. Specifically, members of the DNAJ/HSP40 family of molecular chaperones [33], including ERDJ4 (DNAJB9) and DNAJB8, are involved in HSCs self-renewal [24,29▪▪]. Undoubtedly, numerous other chaperones contribute to the UPR response. Interestingly, chemical chaperones have also been shown to facilitate protein folding. Recently, the lysosomal transporter ENT3 (Slc29a3) of taurine-conjugated bile acids (TBA), a critical metabolite in the development of fetal liver HSCs [34], was shown to facilitate TBA chemical chaperone transport into the ER, which resulted in reduced protein aggregation and alleviated elevated ER stress in expanding HSC cultures [35]. These data introduce a novel and important concept in our understanding of hormetic ER stress stimulation in that as a consequence of cytoprotective UPR signaling in HSCs, abrupt increases in ER stress quickly result in adverse ER stress signaling that leads to HSC exhaustion or apoptosis.

Transcriptional activation of endoplasmic reticulum stress response genes in hematopoietic stem cells

Transcription factors expressed in HSCs likely regulate genes involved in the UPR signaling and may attenuate hormetic ER stress. For example, it was found that Runx1-deficient HSC have a low biosynthetic phenotype and markedly reduced ribosome biogenesis as a result of decreased levels of rRNA and ribosomal proteins [36]. Tunicamycin-induced apoptosis was reduced in Runx1 knockout HSCs. Furthermore, irradiated Runx1 knockout mice showed reduced activation of ATF6, CHOP and Xbp1. As knockout HSCs show only modest repopulation defects in vivo, it is interesting to speculate that Runx1 may serve as an upstream regulator of UPR through ribosome biogenesis. In this way, Runx1 expression could serve as a rheostat to control the absolute amount of UPR stress possible in HSCs by controlling ribosomal content of the cell, and thus strike a balance between protein synthesis demands of proliferation and the cytoprotective effects of hormetic stress via the UPR.

Finally, estradiol (E2) has been shown to activate the IRE1 branch of the UPR in HSCs through the estrogen receptor (ERα) and improve hematopoietic regeneration [37]. E2 treatment increased the repopulation capacity of HSCs in transplantation settings and accelerated hematopoietic regeneration after total body irradiation. E2-ERα signaling was shown to upregulate IRE1 and processed Xbp1s transcript, which induced expression of several ER stress genes, including molecular chaperones. Together, these data highlight that regulation of ER stress genes by transcription factors, long been known to regulate HSC maintenance, encodes a transcriptional program that elicits a cytoprotective UPR signaling in HSCs.

Other endoplasmic reticulum stress pathways involved in hematopoietic stem cell maintenance?

A recent study described a role for ER stress signaling in protein quality control via endoplasmic reticulum-associated degradation (ERAD) in HSCs [38]. Sel1L/Hrd1 ERAD genes were found to be enriched in quiescent HSCs and conditional knockout of Sel1L in the hematopoietic compartment resulted in HSC hyperproliferation, significant loss of HSC self-renewal, and eventual HSC depletion. In the absence of Sel1L-mediated ERAD, Sel1L knockout HSC accumulated Rheb GTPase protein levels, which lead directly to activation of mammalian target of rapamycin (mTOR) signaling, which induced HSC exhaustion. Although this mechanism enhances our understanding of components of ER stress, it is important to point out that basal upregulation of ERAD machinery in HSCs would require elevated UPR signaling in normal HSCs, further supporting a theory requiring hormetic ER stress induction of HSCs’ self-renewal.

RNA processing may also be a feature of the hormetic ER stress response. A recent study showed the pluripotency-associated RNA-binding protein, Dppa5, regulates HSC function [39]. Although its exact function remains poorly understood in embryonic stem cells, Dppa5 overexpression decreased induction of ER stress-response genes (ATF4, CHOP, and C/EBP) and improved repopulation performance in vivo, whereas knockdown of Dppa5 resulted in amplified endogenous ER stress signaling, loss of HSC repopulation capacity and accelerated apoptosis. These data suggest Dppa5 may diminish adverse ER stress responses resulting from the stimuli, such as the tissue culture environment. Further studies are required to dissect the precise mechanism accounting for RNA processing in ER stress responses.

CONCLUSION

The continuous regeneration of HSCs over a lifetime requires mechanisms to ensure a balanced response to proliferative demands and exogenous stimuli without sacrificing HSC self-renewal. Induction of a basal ER stress response is a salient mechanism to prepare HSCs for the burden of differentiation and subsequent transient amplification of daughter cells. Ironically, based on the totality of these recent data, a major component of self-renewal is rooted in ER stress signaling and exemplifies the cytoprotective advantage that can be achieved by cellular hormesis. The next steps in utilizing this information towards harnessing the regenerative properties of HSCs are to further characterize the upstream stimuli and downstream transcriptional programs that specifically confer HSC self-renewal via hormetic ER stress stimulation.

Acknowledgements

We would like to thank Dr. Christopher Hillyer for insightful discussion and support.

Financial support and sponsorship

This work was supported in part by the Early Career Scientific Research Grant from the National Blood Foundation.

Conflicts of interest

There are no conflicts of interest.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest

REFERENCES 1. Singh AK, Althoff MJ, Cancelas JA. Signaling pathways regulating hematopoietic stem cell and progenitor aging. Curr Stem Cell Rep 2018; 4:166–181. 2. Rossi L, Lin KK, Boles NC, et al. Less is more: unveiling the functional core of hematopoietic stem cells through knockout mice. Cell Stem Cell 2012; 11:302–317. 3. Eppert K, Takenaka K, Lechman ER, et al. Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med 2011; 17:1086–1093. 4. Seita J, Weissman IL. Hematopoietic stem cell: self-renewal versus differentiation. Wiley Interdiscip Rev Syst Biol Med 2010; 2:640–653. 5. Majeti R, Weissman IL. Human acute myelogenous leukemia stem cells revisited: there's more than meets the eye. Cancer Cell 2011; 19:9–10. 6. Muller-Sieburg CE, Cho RH, Karlsson L, et al. Myeloid-biased hematopoietic stem cells have extensive self-renewal capacity but generate diminished lymphoid progeny with impaired IL-7 responsiveness. Blood 2004; 103:4111–4118. 7. Chambers SM, Shaw CA, Gatza C, et al. Aging hematopoietic stem cells decline in function and exhibit epigenetic dysregulation. PLoS Biol 2007; 5:e201. 8. Salminen A, Kaarniranta K. ER stress and hormetic regulation of the aging process. Ageing Res Rev 2010; 9:211–217. 9. Murakami S. Stress resistance in long-lived mouse models. Exp Gerontol 2006; 41:1014–1019. 10. Mansouri L, Xie Y, Rappolee DA. Adaptive and pathogenic responses to stress by stem cells during development. Cells 2012; 1:1197–1224. 11. Calabrese V, Cornelius C, Dinkova-Kostova AT, et al. Cellular stress responses, the hormesis paradigm, and vitagenes: novel targets for therapeutic intervention in neurodegenerative disorders. Antioxid Redox Signal 2010; 13:1763–1811. 12. Zimmermann A, Bauer MA, Kroemer G, et al. When less is more: hormesis against stress and disease. Microb Cell 2014; 1:150–153. 13. Calabrese EJ, Baldwin LA. Hormesis: the dose-response revolution. Annu Rev Pharmacol Toxicol 2003; 43:175–197. 14. Benham AM. Endoplasmic reticulum redox pathways: in sickness and in health. FEBS J 2019; 286:311–321. 15. Schwarz DS, Blower MD. The endoplasmic reticulum: structure, function and response to cellular signaling. Cell Mol Life Sci 2016; 73:79–94. 16. Kalaitzidis D, Lee D, Efeyan A, et al. Amino acid-insensitive mTORC1 regulation enables nutritional stress resilience in hematopoietic stem cells. J Clin Invest 2017; 127:1405–1413. 17. Rodrigues-Moreira S, Moreno SG, Ghinatti G, et al. Low-dose irradiation promotes persistent oxidative stress and decreases self-renewal in hematopoietic stem cells. Cell Rep 2017; 20:3199–3211. 18. Hetz C, Zhang K, Kaufman RJ. Mechanisms, regulation and functions of the unfolded protein response. Nat Rev Mol Cell Biol 2020; 21:421–438. 19. Oakes SA, Papa FR. The role of endoplasmic reticulum stress in human pathology. Annu Rev Pathol 2015; 10:173–194. 20. Shahrabi S, Paridar M, Zeinvand-Lorestani M, et al. Autophagy regulation and its role in normal and malignant hematopoiesis. J Cell Physiol 2019; 234:21746–21757. 21. Mollereau B, Manie S, Napoletano F. Getting the better of ER stress. J Cell Commun Signal 2014; 8:311–321. 22. Hu H, Tian M, Ding C, Yu S. The C/EBP Homologous Protein (CHOP) transcription factor functions in endoplasmic reticulum stress-induced apoptosis and microbial infection. Front Immunol 2018; 9:3083. 23. Singh R, Letai A, Sarosiek K. Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nat Rev Mol Cell Biol 2019; 20:175–193. 24. van Galen P, Kreso A, Mbong N, et al. The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress. Nature 2014; 510:268–272. 25. van Galen P, Mbong N, Kreso A, et al. Integrated stress response activity marks stem cells in normal hematopoiesis and leukemia. Cell Rep 2018; 25:1109.e5–1117.e5. 26. Rouschop KM, Dubois LJ, Keulers TG, et al. PERK/eIF2alpha signaling protects therapy resistant hypoxic cells through induction of glutathione synthesis and protection against ROS. Proc Natl Acad Sci USA 2013; 110:4622–4627. 27. Ye J, Kumanova M, Hart LS, et al. The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation. EMBO J 2010; 29:2082–2096. 28. Zhao Y, Zhou J, Liu D, et al. ATF4 plays a pivotal role in the development of functional hematopoietic stem cells in mouse fetal liver. Blood 2015; 126:2383–2391. 29▪▪. Liu L, Zhao M, Jin X, et al. Adaptive endoplasmic reticulum stress signalling via IRE1alpha-XBP1 preserves self-renewal of haematopoietic and preleukaemic stem cells. Nat Cell Biol 2019; 21:328–337. 30. Lemaire K, Moura RF, Granvik M, et al. Ubiquitin fold modifier 1 (UFM1) and its target UFBP1 protect pancreatic beta cells from ER stress-induced apoptosis. PLoS One 2011; 6:e18517. 31. Zhang M, Zhu X, Zhang Y, et al. RCAD/Ufl1, a Ufm1 E3 ligase, is essential for hematopoietic stem cell function and murine hematopoiesis. Cell Death Differ 2015; 22:1922–1934. 32. Liu J, Wang Y, Song L, et al. A critical role of DDRGK1 in endoplasmic reticulum homoeostasis via regulation of IRE1alpha stability. Nat Commun 2017; 8:14186. 33. Qiu XB, Shao YM, Miao S, Wang L. The diversity of the DnaJ/Hsp40 family, the crucial partners for Hsp70 chaperones. Cell Mol Life Sci 2006; 63:2560–2570. 34. Sigurdsson V, Takei H, Soboleva S, et al. Bile acids protect expanding hematopoietic stem cells from unfolded protein stress in fetal liver. Cell Stem Cell 2016; 18:522–532. 35. Persaud AK, Nair S, Rahman MF, et al. Facilitative lysosomal transport of bile acids alleviates ER stress in mouse hematopoietic precursors. Nat Commun 2021; 12:1248. 36. Cai X, Gao L, Teng L, et al. Runx1 deficiency decreases ribosome biogenesis and confers stress resistance to hematopoietic stem and progenitor cells. Cell Stem Cell 2015; 17:165–177. 37. Chapple RH, Hu T, Tseng YJ, et al. ERα promotes murine hematopoietic regeneration through the Ire1alpha-mediated unfolded protein response. Elife 2018; 7:e31159. 38. Liu L, Inoki A, Fan K, et al. ER-associated degradation preserves hematopoietic stem cell quiescence and self-renewal by restricting mTOR activity. Blood 2020; 136:2975–2986. 39. Miharada K, Sigurdsson V, Karlsson S. Dppa5 improves hematopoietic stem cell activity by reducing endoplasmic reticulum stress. Cell Rep 2014; 7:1381–1392.