The role of the unfolded protein response pathway in bone homeostasis and potential therapeutic target in cancer-associated bone disease

Abstract

The unfolded protein response pathway is an evolutionarily conserved cytoprotective signaling cascade, essential for cell function and survival. Unfolded protein response signaling is tightly integrated with bone cell differentiation and function, and chronic unfolded protein response activation has been identified in bone disease. The unfolded protein response has been found to promote oncogenesis and drug resistance, raising the possibility that unfolded protein response modulators may have activity as anti-cancer agents. Cancer-associated bone disease remains a major cause of morbidity for patients with multiple myeloma or bone-metastatic disease. Understanding the critical role of unfolded protein response signaling in cancer development and metastasis, as well as its role in bone homeostasis, may lead to novel mechanisms by which to target cancer-associated bone disease. In this review, we summarize the current research delineating the roles of the unfolded protein response in bone biology and pathophysiology, and furthermore, review unfolded protein response modulating agents in the contexts of cancer and cancer-associated bone disease.

Fig. 1

Signaling cascades of the UPR. a Adaptive UPR signaling consists of three major signaling cascades (EIF2AK3, ERN1, ATF6) and prioritizes absolution of ER stress in order to restore normal protein processing and cell function. EIF2AK3 is activated upon HSPA5 dissociation and binding of misfolded proteins in the ER lumen, promoting receptor dimerization and autophosphorylation. Activated EIF2AK3 phosphorylates EIF2A leading to global inhibition of protein translation with the exception of selective translation of ATF4 mRNA. ATF4 then mediates transcription of UPR target genes to promote amino acid biosynthesis, the antioxidant response, and autophagy to relieve ER stress. ATF4 also promotes transcription of phosphatase PP1R15A to dephosphorylate EIF2A and restore normal protein translation. HSPA5 dissociation from ERN1 leads to receptor dimerization and autophosphorylation. Activated ERN1 RNase splices XBP1 mRNA with translation generating transcriptionally active sXBP1. sXBP1 promotes transcription of genes associated with ERAD, lipid synthesis, ER biogenesis and ER chaperone expression in attempt to alleviate ER stress. Dissociation of HSPA5 from ATF6 allows for receptor translocation to the Golgi apparatus where it is proteolytically cleaved by MBTPS1 and MBTPS2 yielding its active transcriptional form. Transcriptionally active ATF6 then translocates to the nucleus where it promotes continuation of UPR signaling via induction of XBP1, ERAD, autophagy, and ER chaperone expression. b Terminal UPR, which is activated in response to chronic, prolonged ER stress. Persistent EIF2AK3-EIF2A translation of ATF4 activates expression of pro-apoptotic protein DDIT3 which inhibits BCL2, allowing for induction of apoptosis. Prolonged ERN1 activation can promote interactions with adaptor protein TRAF2 mediating downstream MAP3K5 and MAPK8-mediated apoptosis. Prolonged phosphorylation also favors higher order assembly of ERN1 oligomers, preventing XBP1 splicing and alternatively promoting regulated ERN1-dependent decay (RIDD). (Abbreviations: EIF2AK3 eukaryotic translation initiation factor 2 alpha kinase 3, ATF6 activating transcription factor 6, ERN1 ER to nucleus signaling 1, HSP70 HSPA5 heat shock protein family A member 5, EIF2A eukaryotic translation initiation factor 2 subunit alpha, p-EIF2A phosphorylated EIF2A, ATF4 activating transcription factor 4, XBP1 X-box-binding protein-1, sXBP1 spliced XBP1, MBTPS1 membrane bound transcription factor peptidase, site 1, MBTPS2 membrane bound transcription factor peptidase, site 2, DDIT3 DNA damage inducible transcript 3, Bcl-2 B-cell lymphoma 2, TRAF2 TNF receptor associate factor 2, MAP3K5 mitogen-activated protein kinase kinase kinase 5, MAPK8 mitogen-activated protein kinase 8)

Fig. 2

Signaling cascades during osteoclast differentiation. a CSF1 binds CSF1R on monocytic precursors activating transcription factors MITF and SPI1, promoting precursor survival and proliferation, and expression of TNFSF11 receptor (TNFRSF11A) and CSF1R. Surface expression of TNFRSF11A allows for TNFSF11 binding and activation of TRAF6 mediated NF-κB and MAPK signaling. Active NF-κB translocates to the nucleus where it promotes CEBPA expression and binds the NFATC1 promoter where it cooperates with NFATC2 to induce NFATC1 expression. CEBPA promotes expression of FOS which dimerizes with JUN to form active transcriptional complex AP-1. CEBPA and AP-1 in combination with NF-κB, NFATC2, and NFATC1 promote continued expression of FOS and NFATC1. b During TNFSF11-TNFRSF11A signaling, TRAF6 interacts with CYBB and RAC1 generating ROS and promoting cytoskeletal rearrangement necessary for precursor cell fusion, migration, and adhesion. TNFRSF11A signaling also mediates phosphorylation of adaptor proteins TYROBP and FCRG which activate downstream kinases, SYK, BTK, and PLCG2, triggering ER calcium release and activation of calmodulin (CALM)-dependent phosphatase calcineurin. Active calcineurin in turn dephosphorylates cytosolic NFATC1, allowing its translocation to the nucleus where it cooperates with transcription factors SPI1, MITF, AP-1, and NF-κB to promote osteoclast-specific gene expression. Additionally, NFATC1 binds its own promoter auto-amplifying its expression. c TNFRSF11A signaling continues to promote expression of proteins necessary for osteoclast resorption via NF-κB and MAPK signaling. α_Vβ₃ integrin or TREM2/OSCAR can bind RGD sequences on ECM proteins in the bone matrix initiating the resorptive signaling cascade mediated by TYROBP, CSK, SYK, RAC1, and CDC42 to regulate actin ring formation and bone resorption. Endosomal trafficking mediates acidification of the sealing zone via transport of proton pump ATP6V0D2 and chloride channel CLCN7 and release of proteases such as CTSK, MMP9, and ACP5. Degraded bone matrix is removed via transcytosis and released into the local microenvironment. (Abbreviations: CSF1 colony stimulating factor 1, CSF1R CSF1 receptor, TNFRSF11A tumor necrosis factor receptor family member 11A, TNFSF11 tumor necrosis factor soluble factor 11, MITF melanocyte-inducing transcription factor, TRAF6 tumor necrosis factor receptor-associated factor 6, MAPK mitogen-activated protein kinase, NFATC1 nuclear factor of activated T cells 1, CEBPA CCAAT enhancer binding protein alpha, AP-1 activator protein 1, TREM2 triggering receptor expressed on myeloid cells 2, OSCAR osteoclast-associated Ig-like receptor, TYROBP transmembrane immune signaling adaptor, FCRG Fc gamma receptor, SYK spleen associated tyrosine kinase, BTK Bruton’s tyrosine kinase, PLCG2 phospholipase C gamma 2, CALM calmodulin, CYBB cytochrome b-245 beta chain, ROS reactive oxygen species, DCSTAMP dendrocyte expressed seven transmembrane protein, OCSTAMP osteoclast stimulatory transmembrane protein, ATP6V0D2 ATPase H+ transporter V0 subunit d2, SH3PXD2A SH3 and PX domains 2A, ITGB3 integrin subunit beta 3, CLCN7 chloride voltage-gated channel 7, OSTM1 osteoclastogenesis associated transmembrane protein1, CTSK cathepsin K, ACP5 tartrate-resistant acid phosphatase 5, VAV3 vav guanine nucleotide exchange factor 3, CA2 carbonic anhydrase 2, MMP9 matrix metallopeptidase 9

Fig. 3

Signaling cascades during osteoblast differentiation. a Stepwise changes in gene expression throughout osteoblast lineage commitment and differentiation. b Multiple signaling cascades mediate the continued differentiation of immature osteoblast cells. TGFβ binds TGFβR and mediates RUNX2 and SP7 expression via induction of SMAD2/3 and SMAD4 transcriptional activation, and MAPK-mediated induction of DLX5. BMPs mediate precursor survival and proliferation via MAPK activation of AP-1 and promote osteoblast-specific gene expression via SMAD1/5/8 interactions with SMAD4. Canonical WNT signaling is activated by WNT3A or WNT10B binding FZD receptor allowing for binding to co-receptor LRP5/6. Binding inhibits GSK3B kinase activity, allowing for β-catenin to translocate to the nucleus where it interacts with TCF/LEF transcription factors to promote RUNX2 expression. Non-canonical WNT signaling is mediated by WNT5A binding FZD and interacting with co-receptor ROR2/RYK. Adaptor protein disheveled activates DAAM1 and RHOA mediating precursor migration, adhesion and ER expansion while also promoting DLX5-mediated induction of SP7 via MAPK signaling. PTH binding to PTH1R promotes production of cAMP, activating PRKACA, inducing calcium oscillation and activating transcription factor CREB to promote osteoblast-specific gene expression. Binding of growth factors IGF1 and FGF to cognate receptors activate PIK3CA signaling to induce PLCG2, further promoting calcium signaling and CREB mediated osteoblast gene expression. c Mature osteoblasts continue to promote osteoblast-specific gene expression via BMP, TGFβ, WNT, PTH, and growth factor signaling. RUNX2 and EIF2AK3 activation mediate induction of ATF4 which promotes expression of BGLAP. Mature osteoblasts begin to secrete matrix proteins such as BGLAP, SP1, IBSP, and COL1A1 to facilitate new bone formation, and secrete vesicles containing HAP to promote matrix maturation. (Abbreviations: SSC skeletal stem cell, SPC skeletal progenitor cell, RUNX2 runt-related transcription factor-2, SP7 Sp7 transcription factor, DMP1 dentin matrix acidic phosphoprotein 1, SOST sclerostin, GJA1 gap junction protein alpha 1, MMP13 matrix metallopeptidase 13, ICAM1 intracellular adhesion molecule 1, TGFβ transforming growth factor β, TGFβR TGFβ receptor, MAPK mitogen-activated protein kinase, DLX5 distal-less homeobox 5, BMP bone morphogenic protein, BMPR BMP receptor, AP-1 activator protein 1, LRP5/6 low-density lipoprotein receptor-related protein 5 or 6, FZD frizzled, TCF/LEF T cell factor/lymphoid enhancer factor, GSK3B glycogen synthase kinase 3 beta, FGF fibroblast growth factor, FGFR FGF receptor, IGF1 insulin-like growth factor 1, IGF1R IGF1 receptor, PIK3CA phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha, PLCG phospholipase C gamma, PTH parathyroid hormone, PTH1R PTH1 receptor, PRKACA protein kinase cAMP-activated catalytic subunit alpha, CREB cAMP-response element binding protein, CALM calmodulin, ROR2/RYK receptor tyrosine kinase of the ROR-2 and Ryk families, DAAM1 disheveled associated activator of morphogenesis 1, EIF2AK3 eukaryotic initiation factor 2 alpha kinase 3, ATF4 activating transcription factor 4, BGLAP bone gamma-carboxyglutamate protein, SP1 secreted phosphoprotein 1, IBSP integrin binding sialoprotein, COL1A1 collagen type 1 alpha 1 chain, HAP hydroxyapatite)

Fig. 4

Integration of UPR signaling cascades in bone cell differentiation. a BMP-2 SMAD1/5/8 signaling and non-canonical WNT signaling promote ER stress and UPR pathway activation. EIF2AK3 activation induces ATF4 which promotes expression of SP7 and interacts with RPS6KA3 to post-transcriptionally regulate COLα1(1) processing. ERN1 splices XBP1 with sXBP1 promoting expression of SP7 and PTH1R. Activated ATF6 and CREB3L1 translocate to the Golgi where they are proteolytically processed to produce their active transcriptional form. In the nucleus, ATF6 promotes BGLAP expression and CREB3L1 promotes COL1A1 expression. CREB3L3 is also activated upon ER stress with its active transcriptional form negatively regulating osteoblast signaling. b TNFRSF11A-TNFSF11 signaling stimulates calcium signaling and ROS production which induce ER stress and UPR activation. ERN1 and EIF2AK3 activation mediates ATF4 and sXBP1 promotion of NFATC1 and other osteoclast-specific genes. ATF4 also mediates CSF1 induced surface expression of TNFRSF11A. Activated CREB3, CREB3L3, and CRELD2 translocate to the Golgi where they are proteolytically processed to produce their active transcriptional form. In the nucleus, CREB3L3 promotes NFATC1 expression and CREB3 promotes expression of other osteoclast-specific genes. Alternatively, CRELD2 negatively regulates osteoclast differentiation via inhibition of ER calcium release. (Abbreviations: EIF2AK3 eukaryotic initiation factor 2 alpha kinase 3, ERN1 ER to nucleus signaling 1, XBP1 X-box-binding protein-1, sXBP1 spliced XBP1, MBTPS1 membrane bound transcription factor peptidase, site 1, MBTPS2 membrane bound transcription factor peptidase, site 2, RUNX2 runt-related transcription factor-2, SP7 Sp7 transcription factor, TGFβ transforming growth factor β, TGFβR TGFβ receptor, MAPK mitogen-activated protein kinase, DLX5 distal-less homeobox 5, BMP bone morphogenic protein, BMPR BMP receptor, AP-1 activator protein 1, LRP5/6 low-density lipoprotein receptor-related protein 5 or 6, FZD frizzled, TCF/LEF T cell factor/lymphoid enhancer factor, GSK3B glycogen synthase kinase 3 beta, FGF fibroblast growth factor, FGFR FGF receptor, IGF1 insulin-like growth factor 1, IGF1R IGF1 receptor, PIK3CA phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha, PLCG phospholipase C gamma, PTH parathyroid hormone, PTH1R PTH1 receptor, PRKACA protein kinase cAMP-activated catalytic subunit alpha, CREB cAMP-response element binding protein, CALM calmodulin, ROR2/RYK receptor tyrosine kinase of the ROR-2 and Ryk families, DAAM1 disheveled associated activator of morphogenesis 1, ATF4 activating transcription factor 4, BGLAP bone gamma-carboxyglutamate protein, SP1 secreted phosphoprotein 1, IBSP integrin binding sialoprotein, COL1A1 collagen type 1 alpha 1 chain, RPS6KA3 ribosomal S6 kinase A3, CREB3L1 cAMP responsive element binding protein 3 like 1, CREB3L3 cAMP responsive element binding protein 3 like 3, CSF1 colony stimulating factor 1, CSF1R CSF1 receptor, TNFRSF11A tumor necrosis factor receptor family member 11A, TNFSF11 tumor necrosis factor soluble factor 11, MITF melanocyte-induced transcription factor, TRAF6 tumor necrosis factor receptor-associated factor 6, MAPK mitogen-activated protein kinase, NFATC1 nuclear factor of activated T cells 1, CEBPA CCAAT enhancer binding protein alpha, AP-1 activator protein 1, TREM2 triggering receptor expressed on myeloid cells 2, OSCAR osteoclast-associated Ig-like receptor, TYROBP transmembrane immune signaling adaptor, FCRG Fc gamma receptor, SYK spleen associated tyrosine kinase, BTK Bruton’s tyrosine kinase, PLCG2 phospholipase C gamma 2, CALM calmodulin, CYBB cytochrome b-245 beta chain, ROS reactive oxygen species, DCSTAMP dendrocyte expressed seven transmembrane protein, OCSTAMP osteoclast stimulatory transmembrane protein, ATP6V0D2 ATPase H+ transporter V0 subunit d2, SH3PXD2A SH3 and PX domains 2A, ITGB3 integrin subunit beta 3, CLCN7 chloride voltage-gated channel 7, OSTM1 osteoclastogenesis associated transmembrane protein1, CTSK cathepsin K, ACP5 tartrate-resistant acid phosphatase 5, VAV3 vav guanine nucleotide exchange factor 3, CA2 carbonic anhydrase 2, MMP9 matrix metallopeptidase 9, CREB3 cAMP responsive element binding protein 3, CRELD2 cystine rich with EGF like domains 2

Fig. 5

Potential effects of UPR modulating agents on bone cell differentiation. a Inhibition of UPR signaling with ERN1 inhibitors, EIF2AK3 inhibitors, or ER stress modulators disrupts osteoclast-specific gene expression mediated by ATF4, sXBP1, CREB3, and CREB3L3. Alternatively, ER stress inducers may promote osteoclast differentiation through induction of the UPR and induction of osteoclast genes. Proteasome inhibitors disrupt proteasomal degradation preventing NF-κB-mediated signaling and gene expression. b Inhibition of UPR signaling with ERN1 inhibitors, EIF2AK3 inhibitors, or ER stress modulators disrupts osteoblast-specific gene expression mediated by ATF4, sXBP1, ATF6, and CREB3L1. ER stress inducers may promote osteoblast differentiation through induction of the UPR and induction of osteoblast genes. Proteasome inhibitors disrupt proteasomal degradation inducing the UPR and promoting osteoblast-specific gene expression by ATF4, sXBP1, ATF6, and CREB3L1 and ATF4 mediated COLα1(1) processing. (Abbreviations: EIF2AK3 eukaryotic initiation factor 2 alpha kinase 3, ERN1 ER to nucleus signaling 1, XBP1 X-box-binding protein-1, sXBP1 spliced XBP1, MBTPS1 membrane bound transcription factor peptidase, site 1, MBTPS2 membrane bound transcription factor peptidase, site 2, CSF1 colony stimulating factor 1, CSF1R CSF1 receptor, TNFRSF11A tumor necrosis factor receptor family member 11A, TNFSF11 tumor necrosis factor soluble factor 11, MITF melanocyte-induced transcription factor, TRAF6 tumor necrosis factor receptor-associated factor 6, MAPK mitogen-activated protein kinase, NFATC1 nuclear factor of activated T cells 1, AP-1 activator protein 1, TREM2 triggering receptor expressed on myeloid cells 2, OSCAR osteoclast-associated Ig-like receptor, TYROBP transmembrane immune signaling adaptor, FCRG Fc gamma receptor, SYK spleen associated tyrosine kinase, BTK Bruton’s tyrosine kinase, PLCG2 phospholipase C gamma 2, ACP5 tartrate resistant acid phosphatase 5, CREB3 cAMP responsive element binding protein 3, CRELD2 cystine rich with EGF like domains 2, DCSTAMP dendrocyte expressed seven transmembrane protein, OCSTAMP osteoclast stimulatory transmembrane protein, ATP6V0D2 ATPase H+ transporter V0 subunit d2, SH3PXD2A SH3 and PX domains 2A, ITGB3 integrin subunit beta 3, ACP5 tartrate-resistant acid phosphatase 5, RUNX2 runt-related transcription factor-2, SP7 Sp7 transcription factor, TGFβ transforming growth factor β, TGFβR TGFβ receptor, MAPK mitogen-activated protein kinase, DLX5 distal-less homeobox 5, BMP bone morphogenic protein, BMPR BMP receptor, AP-1 activator protein 1, LRP5/6 low-density lipoprotein receptor-related protein 5 or 6, FZD frizzled, TCF/LEF T cell factor/lymphoid enhancer factor, GSK3B glycogen synthase kinase 3 beta, FGF fibroblast growth factor, FGFR FGF receptor, IGF1 insulin-like growth factor 1, IGF1R IGF1 receptor, PLCG phospholipase C gamma, PTH parathyroid hormone, PTH1R PTH1 receptor, CREB cAMP-response element binding protein, ROR2/RYK receptor tyrosine kinase of the ROR-2 and Ryk families, DAAM1 disheveled associated activator of morphogenesis 1, ATF4 eukaryotic activating transcription factor 4, BGLAP bone gamma-carboxyglutamate protein, SP1 secreted phosphoprotein 1, IBSP integrin binding sialoprotein, COL1A1 collagen type 1 alpha 1 chain, RPS6KA3 ribosomal S6 kinase A3, CREB3L1 cAMP responsive element binding protein 3 like 1, CREB3L3 cAMP responsive element binding protein 3 like 3)