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Keywords:

  • endoplasmic reticulum;
  • unfolded protein response;
  • mitochondrial dysfunction

Abstract

  1. Top of page
  2. Abstract
  3. ENDOPLASMIC RETICULUM UNFOLDED PROTEIN RESPONSE
  4. MITOCHONDRIAL PROTEIN HOMEOSTASIS
  5. MITOCHONDRIAL UNFOLDED PROTEIN RESPONSE
  6. MITOCHONDRIAL ADAPTATIONS
  7. ER MITOCHONDRIAL NETWORK
  8. MITOCHONDRIA AS INTEGRATORS OF CELLULAR DANGER SIGNALING
  9. ER UPR AND MITOCHONDRIAL DYSFUNCTION IN IBD
  10. CONCLUSION
  11. REFERENCES

Inflammatory bowel diseases (IBDs), like many other chronic diseases, feature multiple cellular stress responses including endoplasmic reticulum (ER) unfolded protein response (UPR). Maintaining protein homeostasis is indispensable for cell survival and, consequently, distinct signaling pathways have evolved to transmit organelle stress. While the ER UPR, aiming to restore ER homeostasis after challenges to ER function, has been extensively studied in the context of chronic diseases, only recently the related mitochondrial UPR (mtUPR), induced by disturbances of mitochondrial proteostasis, has drawn some attention. ER and mitochondria are in close contact and interact physically and functionally. Accumulating data have placed mitochondria at the center of diverse cellular functions and suggest mitochondria as integrators of signaling pathways such as autophagy and inflammation. Consequently, it is likely that mitochondrial stress and ER stress cannot be regarded separately and that mitochondrial stress, as well as ER stress, participates in the pathology of IBD. Protein homeostasis is particularly sensitive toward infections, oxidative stress, and energy deficiency. Thus, environmental disturbances impacting organelle function lead to the concerted activation of distinct UPRs. The metabolic status might therefore serve as an innate mechanism to sense the epithelial environment, including luminal-derived and host-derived factors. This review highlights mtUPR and its interrelation with ER UPR, focuses on recent studies identifying mitochondria as integrators of cellular danger signaling, and, furthermore, illustrates the importance ER UPR and mitochondrial dysfunction in IBD. (Inflamm Bowel Dis 2011;)

A cellular condition present in various chronic diseases including inflammatory bowel diseases (IBDs) is endoplasmic reticulum (ER) stress and the associated ER unfolded protein response (UPR).1–3 In eukaryotic cells, distinct cellular processes occur in specialized organelles such as the ER, mitochondria, peroxisomes, and the Golgi apparatus. As a consequence, the abundance and/or capacity of each organelle have to be tightly regulated to meet fluctuating cellular demands. For example, this is reflected by enlarged mitochondria found in exercise-conditioned muscle cells and secretory cells containing large amounts of ER.4, 5 Autoregulatory mechanisms have been proposed to control organelle abundance, whereby sensor molecules monitor organelle function and, if the demand exceeds the capacity, elicit retrograde signaling enhancing biogenesis5 (Fig. 1).

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Figure 1. The metabolic state of the epithelium as an innate sensing mechanism for the tissue environment and schematic illustration of unfolded protein responses. Accumulation of unfolded or misfolded proteins can be triggered by environmental factors like bacteria or nutrients as well as host-derived signals such as inflammatory cytokines. The insufficient protein folding capacity initiates a signaling cascade leading to the transcription of nuclear-encoded genes involved in the organelle-specific protein folding machinery and in cellular stress response. These signals result in augmentation of organelle capacity and lower the burden of unfolded proteins.

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The so-called UPRs are a paradigm for this type of signaling. Maintaining protein homeostasis is essential to all cells and is dependent on chaperones, which promote protein folding and prevent protein aggregation.6 Proteostasis is particularly sensitive to environmental challenges and triggers like infections, oxidative stress, and metabolic alterations impact protein folding in different cellular compartments7, 8 (Fig. 1). Under normal conditions, chaperones of the heat shock protein (HSP) family, HSP70 in the cytosol and glucose-regulated protein (GRP) 78 in the ER, bind to stress-sensor proteins to repress their signaling.7, 9 Upon stress-induced accumulation of unfolded proteins, these chaperones dissociate from their binding partners, preferentially interacting with unfolded proteins.

In the case of the HSP70, this leads to the release of the transcription factor HSF1, allowing its nuclear translocation and inducing genes involved in cytosolic protein folding.9 Similarly, dissociation of GRP78 activates three ER transmembrane proteins, IRE1 (inositol requiring enzyme 1), ATF6 (activation transcription factor 6), and PERK (PKR-like ER kinase), initiating sophisticated downstream signaling to increase the ER folding capacity.7

In addition to the cytosolic and the ER UPR, a third, cognate signaling pathway responding to mitochondrial stress has been described, the mitochondrial UPR (mtUPR).10–12 Although the underlying molecular mechanisms of the mtUPR are less understood, studies in Caenorhabditis elegans suggested that the initial signal might be peptides generated from unfolded proteins of the mitochondrial matrix by the protease ClpP and their efflux into the cytosol.13 Many components of mtUPR-signaling have been identified using C. elegans, but recently we found mtUPR also to be dependent on ClpP in mice.14 Furthermore, the cytosolic dsRNA-dependent protein kinase (PKR), which is also implicated in metabolic signaling,15 has been shown to be indispensable for mtUPR downstream signaling in a murine cell line.14 As a result, mtUPR responsive genes, mitochondrial proteases, and chaperones such as chaperonin (CPN) 60, are activated mainly through the transcription factor C/EBP homologous protein (CHOP).11, 16, 17 Cytosolic, ER, and mtUPR are distinct signaling pathways targeting specific stress proteins of different cellular compartments, even though ER- and mtUPR share features like phosphorylation of eukaryotic translation initiation factor (eIF) 2α as well as the transcription factors CHOP and activating protein (AP) 1.7, 11, 14, 16

At the same time, ER and mitochondria interact physically and functionally via mitochondria-associated membranes (MAM).18 Consistently, ER stress impacts mitochondrial gene expression19, 20 and vice versa mitochondria have been shown to modulate ER UPR.21–23 Various metabolically driven and inflammatory diseases feature activated ER UPR1–3, 7 and both ER- and mtUPR are associated with IBD24.14 Also, mitochondrial dysfunction and alterations in energy metabolism in general have been implicated during the onset and course of neoplasia, metabolic diseases, and inflammation.21, 23, 25 Beyond their well-established functions in cell metabolism and during apoptosis, mitochondria have gained increasing attention for their role in innate immune signaling.26–29 These data have placed mitochondria at the center of diverse cellular functions and suggest mitochondria as integrators of various signaling pathways. However, even though cellular stress responses and mitochondrial dysfunction represent key regulators of metabolically driven disorders, the role of mitochondria-related signaling in the pathogenesis of IBD is virtually unknown.

This review highlights ER- and mtUPR as well as their interrelation, gives a short overview of mitochondrial signal-integration, and illustrates the relevance of mitochondrial stress to intestinal inflammation.

ENDOPLASMIC RETICULUM UNFOLDED PROTEIN RESPONSE

  1. Top of page
  2. Abstract
  3. ENDOPLASMIC RETICULUM UNFOLDED PROTEIN RESPONSE
  4. MITOCHONDRIAL PROTEIN HOMEOSTASIS
  5. MITOCHONDRIAL UNFOLDED PROTEIN RESPONSE
  6. MITOCHONDRIAL ADAPTATIONS
  7. ER MITOCHONDRIAL NETWORK
  8. MITOCHONDRIA AS INTEGRATORS OF CELLULAR DANGER SIGNALING
  9. ER UPR AND MITOCHONDRIAL DYSFUNCTION IN IBD
  10. CONCLUSION
  11. REFERENCES

In mammalian cells, the ER is essential for cholesterol production, for calcium homeostasis, and for the transit of correctly folded proteins to the extracellular space, the plasma membrane, and the exo- and endocytic compartments. Among the conditions that challenge ER functions and elicit ER stress responses are changes in calcium homeostasis or redox status, elevated protein synthesis, accumulation of unfolded or misfolded proteins, energy deficiency and glucose deprivation, altered protein glycosylation, cholesterol depletion, and microbial infections.7 In unstressed cells, the transmembrane proteins IRE1, ATF6, and PERK are bound by the ER chaperone GRP78 (also referred to as immunoglobulin heavy chain-binding protein, BIP) in their intraluminal domains and rendered inactive. Accumulation of mis- or unfolded proteins in the ER triggers recruitment of GRP78 away from these sensors and sequentially initiates three distinct branches of the ER UPR.30

IRE1

Liberation of IRE1 and PERK results in dimerization and activation of the two kinases and engages a complex downstream signaling pathway.7 IRE1 serves as Ser/Thr protein kinase as well as endoribonuclease31 capable of splicing a 26-nucleotide intron out of the mRNA encoding the transcription factor XBP1, thereby facilitating the translation of the active protein (XBP1s).7 XBP1s induces a broad spectrum of ER UPR-related genes including chaperones, proteins involved in ER-associated degradation (ERAD), and protein quality control.32, 33 Since XBP1s also controls the expansion of secretory pathways by ER/Golgi biogenesis,34, 35 it is crucial for survival and function of secretory cells, which are particularly susceptible to ER stress.24 Furthermore, the cytosolic domain of activated IRE1 can bind to the adaptor protein TNFR-associated factor (TRAF) 2 to activate the apoptosis signal-regulating kinase (ASK) 1 and cJun-N terminal kinase (JNK).36, 37

PERK

Activation of PERK results in the inhibition of global protein synthesis by phosphorylation of eIF2α38 and downstream in the induction of ATF4 through alternative translation. Target genes of ATF4 comprise genes related to a negative feedback of eIF2α phosphorylation, genes involved in ER redox control (ER oxidoreductin [ERO] 1), and apoptosis (CHOP) as well as regulators of glucose metabolism.39, 40 PERK-dependent phosphorylation also triggers dissociation of Nrf2/Keap1 complexes, subsequent nuclear translocation of Nrf2, and transcription of genes harboring antioxidant response elements (ARE) in their promoters, a process thought to counteract oxidative stress evoked by ER stress.41, 42

ATF6

The third branch of ER UPR is mediated by ATF6 and requires ATF6-release from the ER, migration to the Golgi apparatus, and cleavage by site 1 protease (S1P) and site 2 protease (S2P) to generate an active transcription factor (nATF6).43 Subsequently, nATF6 translocates to the nucleus, binds to promoters containing ER stress elements (ERSE), UPR elements (UPRE), and cAMP response elements (CRE) and enhances gene transcription of xbp1 and many other ER UPR genes related to ERAD and protein folding.33, 44, 45 In addition, ATF6 may also modulate lipid biosynthesis and ER expansion under stress conditions.46

Together, the three branches of the ER UPR restore ER homeostasis by 1) enhancing the degradation of misfolded proteins 2) global translational attenuation, and 3) expanding the protein folding capacity of the cell through upregulation of ER chaperones like GRP78. However, if the ER stress is prolonged or excessive, ER UPR can ultimately lead to cell death via mitochondria-dependent and -independent apoptotic pathways.47, 48

MITOCHONDRIAL PROTEIN HOMEOSTASIS

  1. Top of page
  2. Abstract
  3. ENDOPLASMIC RETICULUM UNFOLDED PROTEIN RESPONSE
  4. MITOCHONDRIAL PROTEIN HOMEOSTASIS
  5. MITOCHONDRIAL UNFOLDED PROTEIN RESPONSE
  6. MITOCHONDRIAL ADAPTATIONS
  7. ER MITOCHONDRIAL NETWORK
  8. MITOCHONDRIA AS INTEGRATORS OF CELLULAR DANGER SIGNALING
  9. ER UPR AND MITOCHONDRIAL DYSFUNCTION IN IBD
  10. CONCLUSION
  11. REFERENCES

Mitochondria are enclosed by two membranes and are well known for the production of ATP from acetyl CoA in the tricarboxylic acid (TCA) cycle and the respiratory chain. Outer and inner membranes separate two compartments, the intermembrane space and the matrix, which is bound by the impermeable inner membrane that possess specific, gated channels for metabolite and protein exchange.8, 49 Furthermore, the inner membrane harbors five (in mammals) respiratory complexes involved in oxidative phosphorylation (OXPHOS) and ATP production.49 Within the matrix, several copies of circular mitochondrial DNA are contained as well as the components required for its replication, transcription, and subsequent translation of the encoded proteins. Additionally, various enzymes essential for metabolic processes such as TCA, fatty-acid oxidation, iron-sulfur cluster formation, and heme synthesis reside in the matrix.8 Enclosing matrix and inner membrane, the intermembrane space forms cristae, long tubules or folds that project into the matrix as well as a narrow intermembrane boundary between the inner and outer membranes.50 Cytochrome c, which is involved in respiration in normal cells and apoptotic induction upon its release into the cytosol and other potential apoptotic inducers, are present in this compartment.51, 52

Mitochondrial Protein Import

Due to the structure of mitochondria, mitochondrial protein homeostasis encounters unique challenges. More than 98% of the total mitochondrial protein is encoded by the nuclear genome, making it necessary to import cytosolically synthesized mitochondrial precursor proteins.8 Both in the outer and inner membrane, protein translocases enable precursor protein translocation and/or integration into membranes. The translocase of the outer mitochondrial membrane (TOM) acts as entry site and, depending on targeting sequences, precursor proteins are subsequently directed to their destination by translocase of the mitochondrial inner membrane (TIM) family members.53 Protein import into the matrix requires proteins to be unfolded, in order to transit the protein translocase channels.54, 55 Following import, signal sequences are cleaved and proteins are (re-)folded. The mitochondrial matrix contains its own set of molecular chaperones facilitating the folding of newly synthesized or imported proteins with the evolutionary conserved Hsp70 and Hsp60/10 (chaperonin60/10) as main players.8, 11 Additional chaperones and proteases essential for protein maturation and mitochondrial quality control residing in the matrix include ClpP, Lon, Yme1, DnaJ, and Hsp78.8, 56, 57

Furthermore, expression of nuclear and mitochondrial DNA-encoded proteins has to be accurately coordinated since 13 out of 89 subunits of the respiratory chain are encoded by mitochondrial DNA in humans8 and need to assemble into stoichiometric complexes with nuclear-encoded proteins.

Thus, cellular stress leading to increased mitochondrial biogenesis,10 generation of reactive oxygen species (ROS),10, 58 or metabolic alterations might impact mitochondrial protein homeostasis and evoke mtUPR.

MITOCHONDRIAL UNFOLDED PROTEIN RESPONSE

  1. Top of page
  2. Abstract
  3. ENDOPLASMIC RETICULUM UNFOLDED PROTEIN RESPONSE
  4. MITOCHONDRIAL PROTEIN HOMEOSTASIS
  5. MITOCHONDRIAL UNFOLDED PROTEIN RESPONSE
  6. MITOCHONDRIAL ADAPTATIONS
  7. ER MITOCHONDRIAL NETWORK
  8. MITOCHONDRIA AS INTEGRATORS OF CELLULAR DANGER SIGNALING
  9. ER UPR AND MITOCHONDRIAL DYSFUNCTION IN IBD
  10. CONCLUSION
  11. REFERENCES

Like ER UPR, mtUPR senses insufficient protein-handling capacity, i.e., misfolded, misassembled, or aggregated proteins and aims to restore protein homeostasis by expanding the folding capacity of the organelle and enhancing protein degradation.8, 59

mtUPR in Mammalian Cells

Experiments using cells depleted of mitochondrial DNA through ethidium bromide provided the first evidence for a mammalian mitochondrial-specific stress response.12 Probably due to the disturbance of respiratory complex assembly and accumulation of orphaned nuclear-encoded subunits in the mitochondrial matrix, an induction of Cpn60 and Cpn10 was observed.12 Consistently, overexpression of a truncated protein prone to aggregate in the mitochondrial matrix (truncated ornithine transcarbamylase, OTCΔ) increased the expression of Cpn60, Cpn10, and the mitochondrial protease ClpP in an organelle-specific manner since ER-associated chaperones were not induced.11 Subsequent studies suggested a model in which protein aggregation in the mitochondrial matrix results in the transcriptional activation of the transcription factor CHOP and its cofactor C/EBPβ via cJun-N terminal kinase (JNK) 2 and the transcription factor AP1.11, 16, 17 In turn, CHOP induces mtUPR-target genes containing a CHOP-C/EBPβ element flanked by conserved sequences, so-called mtUPR elements (MURE), in their promoters17 (Fig. 2). CHOP is engaged both by ER- and mtUPR but deletion analysis of its promoter identified two adjacent but distinct elements for transcriptional activation, providing evidence for a separate induction of the Chop gene in response to either UPR pathway.16, 17

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Figure 2. Peptides generated by ClpP seem to be the initial signal of mtUPR in both, C.elegans and mammalian cells. Additionally, several transcription factors and target genes associated with mtUPR-signaling have been identified. However, mitochondria-to-nucleus signaling is largely unknown under mtUPR, even though the kinase PKR has been recently shown to be a cytosolic mediator in murine cells.

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mtUPR in C. elegans

Genetic approaches in C. elegans further characterized the mtUPR and led to the identification of additional signaling components. Reflecting results obtained in mammalian cells,7, 12 RNAi-mediated knockdown of mitochondrial chaperones and proteases as well as factors required for mitochondrial DNA expression activated mtUPR in worms that expressed green fluorescent protein (GFP) under the control of the hsp-60 or hsp-6 (a homolog of mitochondrial Hsp70) promoter.10 A subsequent genome-wide RNAi screening identified additional nuclear genes involved in mtUPR signaling, dve-1 encoding a putative homeobox-like transcription factor, ubl-5 encoding a small ubiquitin-like protein, and clpp-1 encoding a mitochondrial matrix-localized homolog of the E. coli ClpP protease.13, 60 Furthermore, HAF-1, an ATP-binding cassette transporter similar to the yeast mitochondrial peptide transporter Mdl1p and the mammalian transporters associated with antigen presentation (TAP) as well as ZC376.7, a basic leucine zipper transcription factor, were found to be essential for signaling mtUPR.61 In summary, these results suggest a signaling cascade for mtUPR in which insufficient protein-handling capacity within the mitochondrial matrix leads to the accumulation of unfolded proteins and activation of mtUPR. The unfolded proteins are degraded by the matrix-localized ClpP protease to peptides that are transported through the mitochondrial inner membrane by HAF-1. Since small peptides like that generated by ClpP have been shown to be able to diffuse freely through the mitochondrial outer membrane into the cytoplasm62 an additional transporter out of the mitochondria might not be needed.63 Downstream, the transcription factor ZC376.7 translocates to the nucleus, UBL-5 and DVE-1 form a complex, and DVE-1 redistributes within nuclei and binds to the hsp-60 promoter (Fig. 2). It is unclear whether ZC376.7 and the DVE-1/UBL-5 complex interact, but since the mammalian homolog of DVE-1, SATB2, functions as a global chromatin organizer, it has been suggested that DVE-1/UBL-5 complex-induced chromatin rearrangements might facilitate access of ZC376.7 to promoters of mtUPR target genes.63 Ubl-5 itself is a target gene of mtUPR, probably representing an amplification circuit to enhance mtUPR signaling, and it has been speculated that ZC376.7 might fulfill similar functions to CHOP in C. elegans, as worms lack a conspicuous homolog of CHOP.63 The importance of mtUPR-signaling in protecting the mitochondrial protein homeostasis by transcriptional upregulation of mitochondrial chaperones has been demonstrated by knockdown or deletion of signaling components, all of which sensitized worms to mitochondrial stress, resulting in altered mitochondrial morphology and decreased function, slowed development, and shortened lifespan.13, 60, 61, 63

mtUPR and dsRNA-activated Protein Kinase (PKR)

Recently, we were able to confirm the importance of ClpP during mtUPR in mammalian cells and to identify an additional signaling component, the cytosolic kinase PKR.14 Using OTCΔ to induce mtUPR in a murine intestinal epithelial cell line, we demonstrated PKR to be a target gene of mtUPR and the transcriptional activation of the gene to be associated with AP1 binding to its promoter. PKR was previously described to be activated by various triggers including ER UPR, Toll-like receptors (TLRs), growth receptor signaling, cytokines, and palmitic acid.15, 64, 65 In turn, PKR is able to phosphorylate eIF2α, modulate tumor necrosis factor (TNF)-induced signaling,66 IkB kinase (IKK)64, 66 activity and can induce insulin receptor substrate (IRS) phosphorylation at serine 307, thereby blocking insulin action.15 Activation of PKR under mtUPR was dependent on ClpP activity and, moreover, inhibition or siRNA-mediated knockdown of PKR abrogated phosphorylation of eIF2α and activation of the transcription factors AP1 and CHOP, as well as the induction of PKR itself. Of note, mtUPR and ER UPR converge on the level of eIF2α phosphorylation and activation of AP1 as well as CHOP, suggesting PKR integrating mtUPR into ER UPR signaling (Fig. 3). The relevance of PKR-mediated could also be confirmed in vivo, since Pkr−/− mice were unable to activate the mtUPR target-gene CPN60 in intestinal epithelial cells (IECs) upon dextran sodium sulfate (DSS) administration and were protected from DSS-induced colitis.14

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Figure 3. Schematic illustration of mitochondrial and ER UPR integration. eIF2α phosphorylation is a feature of both mtUPR as well as ER UPR. While eIF2α is phosphorylated by PERK under ER UPR, PKR seems to mediate phosphorylation under mtUPR. In addition, PKR activates the transcription factor AP1 via MEK and JNK2/3. The transcription factor CHOP is transcriptionally activated by mtUPR and ER UPR. However, distinct compartment-specific chaperones such as GRP78 and CPN60 are induced as downstream targets, probably due to different coactivators.

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However, the mechanism leading to PKR activation remains to be identified. Several possibilities have been suggested how ClpP-generated peptides might activate downstream signaling.61, 63 Since peptides released from mitochondria in an HAF-1-dependent manner originate from a broad spectrum of mainly matrix-localized proteins and differ in amino acid composition and length,61 rather the rate of efflux than the peptides themselves might provide a specific signal. Yet the presence of a peptide-specific receptor cannot be ruled out.61 Alternatively, ClpP-mediated proteolysis might release a nonpeptide ligand that is subsequently transported by HAF-1.61 Interestingly, the related mammalian ABC transporter ABCB10 has been implicated in heme transport across the mitochondrial inner membrane.67

MITOCHONDRIAL ADAPTATIONS

  1. Top of page
  2. Abstract
  3. ENDOPLASMIC RETICULUM UNFOLDED PROTEIN RESPONSE
  4. MITOCHONDRIAL PROTEIN HOMEOSTASIS
  5. MITOCHONDRIAL UNFOLDED PROTEIN RESPONSE
  6. MITOCHONDRIAL ADAPTATIONS
  7. ER MITOCHONDRIAL NETWORK
  8. MITOCHONDRIA AS INTEGRATORS OF CELLULAR DANGER SIGNALING
  9. ER UPR AND MITOCHONDRIAL DYSFUNCTION IN IBD
  10. CONCLUSION
  11. REFERENCES

MtUPR has been suggested to participate in the tightly and dynamically regulated mitochondrial biogenesis program to adjust mitochondrial abundance to cellular energy demands.4, 68

Mitochondrial Biogenesis

Mitochondria cannot be made de novo, but divide by a process that recruits lipids and new proteins leading to organelle growth and finally fission.8 Defects in the respiratory chain and low cellular ATP levels have been shown to increase the number of mitochondria69, 70 as well as external factors such as nutrients, hormones, temperature, exercise, hypoxia, and aging impact mitochondrial biogenesis.8 However, the underlying mechanisms are not completely understood. Mitochondrial calcium release has been implicated in mitochondrial retrograde signaling,71 NO,40, 72 as well as reduced mitochondrial membrane potential (Δψ), accumulation of NADH or reduced FAD,8 and the activation of AMP activated protein kinase (AMPK) by a high AMP:ATP ratio.73 As a consequence of these initial signaling events, nuclear and mitochondrial genes encoding mitochondrial proteins as well as nonmitochondrial proteins involved in energy metabolism are transcriptionally inducted.74, 75 Several transcription factors and coactivators involved in mitochondrial biogenesis have been identified in mammals.76 These include Tfam, NRF1, NRF2, SP1, YY1, CREB, MEF2, and PGC-1α76 and through interaction with various binding partners they constitute a regulatory cascade controlling the expression target genes in a tissue- and stimulus-specific way. Recently, an additional factor regulating total mitochondrial mass was identified, mitochondrial DNA absence sensitive factor (MIDAS), a protein stimulating the synthesis of cardiolipin and total mitochondrial lipids.77

Mitochondrial Morphology

Furthermore, the morphology of mitochondria seems to be directly related to mitochondrial function and respiratory activity. This was demonstrated by overexpression or knockdown of mitochondrial fusion-relevant proteins, Mtfn2 and Optic atrophy protein (Opa) 1, resulting in altered mitochondrial structures and respiration.78, 79 Small, mobile mitochondria are needed for rapid distribution of ATP to subcellular sites with high energy demand,80, 81 whereas larger mitochondria are more efficient in producing ATP.82 In addition, mitochondrial fusion is important for maintaining mitochondrial DNA integrity.83 On the other hand, mitochondrial fission has been described to produce metabolically different daughter units, segregating dysfunctional mitochondria, and targeting them for mitophagy.84

Mitophagy

It has been suggested that serum withdrawal preferentially promotes autophagic degradation of mitochondria carrying mtDNA mutations.85 Mitophagy is a specialized form of autophagy degrading damaged, nonfunctional mitochondria, thereby ensuring the maintenance of a functional mitochondrial population. Several factors have been implicated in selectively targeting mitochondria for autophagy such as Atg32 and the cargo adaptor protein Atg11 in yeast and Nip3-like protein X (Nix, also known as Bnip3l) in mammals (reviewed86). Recently, also PINK1 has been shown to be a signal for the lack of mitochondrial fitness.87 Expression of PINK1 on individual mitochondria is regulated by voltage-dependent proteolysis to maintain low levels of PINK1 on healthy, polarized mitochondria, while facilitating the rapid accumulation of PINK1 on mitochondria that sustain damage. In turn, PINK1 provides the signal for selective recruiting of Parkin to damaged mitochondria to trigger their mitophagy.87

Remarkably, Parkin has been shown to be transcriptionally regulated by the ER UPR-associated transcription factor ATF4, strengthening the evidence for an interconnection between mitochondrial stress and ER stress.20 Damaged, dysfunctional mitochondria display toxicity to cells possibly by enhanced ROS generation. Consequences of ROS-mediated damage due to lack of active autophagy have been demonstrated in mice with a liver-specific deficiency in Atg7.88 These mice show liver dysfunction associated with accumulation of ubiquitin-positive protein aggregates as well as enhanced expression of oxidative stress-inducible proteins demonstrating autophagy to be required to reduce oxidative stress, and hence cellular damage.88, 89 Additional evidence for a protective role of autophagy under inflammatory conditions came from a study suggesting that autophagy preserves mitochondrial integrity, thereby preventing mitochondrial DNA translocation to the cytosol.29 In this model, depletion of autophagic proteins promoted the accumulation of dysfunctional mitochondria, mitochondrial ROS production, and activation of the NALP3 inflammasome leading to the activation of caspase 1 and secretion of interleukin (IL)-1β and IL-18.29 Consistently, Zhou et al26 showed that mitophagy/autophagy blockade not only lead to the accumulation of dysfunctional mitochondria and activation of the NLRP3 inflammasome, but also that NLRP3 and its adaptor ASC redistribute from ER structures to ER/mitochondria organelle clusters upon inflammasome activation. Thus, the authors suggest that the NLRP3 inflammasome senses mitochondrial dysfunction and that this might explain the frequent association of mitochondrial damage with inflammatory diseases.26

ER MITOCHONDRIAL NETWORK

  1. Top of page
  2. Abstract
  3. ENDOPLASMIC RETICULUM UNFOLDED PROTEIN RESPONSE
  4. MITOCHONDRIAL PROTEIN HOMEOSTASIS
  5. MITOCHONDRIAL UNFOLDED PROTEIN RESPONSE
  6. MITOCHONDRIAL ADAPTATIONS
  7. ER MITOCHONDRIAL NETWORK
  8. MITOCHONDRIA AS INTEGRATORS OF CELLULAR DANGER SIGNALING
  9. ER UPR AND MITOCHONDRIAL DYSFUNCTION IN IBD
  10. CONCLUSION
  11. REFERENCES

As mentioned above, mtUPR and ER UPR share several signaling molecules, yet there are many more levels of communication between mitochondria and ER. In living cells, mitochondria form a dynamic network, constantly remodeled by fission and fusion events, and should not be regarded as single organelles.8 In fact, they are in close contact with the ER via MAM.18, 90 These specialized ER domains are enriched in ER folding chaperones and oxidoreductases,91–93 and are sites of calcium and lipid exchange. Furthermore, the proximity of ER and mitochondria might ensure the constant supply of the ER with ATP.94, 95

Mitochondria-associated Membranes

On the MAM, calcium release from the ER occurs at inositol 1,4,5-triphosphate receptors (IP3R). Mitochondria can take up these local high amounts of calcium in a quasi-synaptic manner96 and upon mitochondrial calcium efflux sarcoplasmic/endoplasmic reticulum calcium ATPases (SERCA) mediate calcium reuptake into the ER.97 This calcium flux is regulated through calcium and redox-dependent interactions of IP3R and SERCA with ER chaperones such as calnexin and calreticulin and oxidoreductases such as ERp44, ERp57, and Ero1α.90, 97 ER-associated calcium signaling is not only important during apoptosis.98 ER chaperones like GRP78 depend on calcium and ATP99 and, moreover, increased mitochondrial calcium levels promote the activity of ATP synthase.100 Another link between mitochondrial and ER metabolism at the MAM is provided by Ero1α, an oxidoreductase that oxidizes protein disulfide isomerase (PDI), which in turn catalyzes the formation of disulfide bonds within newly synthesized proteins in the ER.101–103 Ero1α requires flavin adenine dinucleotide (FAD) derived from the mitochondrial metabolism as cofactor and generates ROS as a byproduct,101 probably directly impacting mitochondrial permeability transition pore (MPTP), SERCA, and IP3R.90 Interestingly, Ero1α is induced under ER UPR7 and at the same time riboflavin-deficiency, and therefore FAD metabolism impairs oxidative folding in the ER.104 An additional level of regulation of mitochondria-ER interaction is the regulation of MAM itself. Three proteins are implicated in MAM-regulation in mammalian cells, mitofusin (Mtfn) 2 and the chaperones GRP75 and phosphofurin acidic cluster sorting protein (PACS) 2.105–107 Mtfn2 is a GTPase localized to the mitochondrial outer membrane and plays a central role in mitochondrial fusion, but additionally has been shown to mediate tethering of the ER to mitochondria and to affect ER morphology as well as calcium homeostasis.107 In contrast, GRP75 bridges the mitochondrial voltage-dependent anion channel (VDAC) to the regulatory domain of IP3R, thereby enhancing MAM formation.90, 106 Of particular interest in the context of UPR is PACS2, since siRNA-mediated knockdown of this chaperone not only uncouples ER from mitochondria but also induces ER UPR.105 In summary, the MAM represents an interface linking protein folding in the ER to mitochondrial function via energy, calcium, and metabolite exchange.

Interrelated Stress Signaling

Consistently, changes in calcium homeostasis and redox status or energy deficiency trigger ER UPR and at the same time ER stress impacts mitochondrial gene expression,19, 20 and vice versa mitochondria have been shown to modulate ER UPR.21–23 Confirming the fact that this organelle interplay is not one-sided, the generation of ROS can be both cause as well as consequence of ER UPR and mitochondrial dysfunction. Under cellular stress conditions, mitochondria and ER also interact on more sophisticated levels. Recent data present evidence that dysfunctional mitochondria contain endogenous high-affinity human TLR4 ligands and induce TLR4-mediated inflammatory reactions,108 suggesting a model in which mitochondria could impact TLR signaling and associated ER UPR. Furthermore, it has been reported that nuclear genes encoding mitochondrial proteins such as the mitochondrial matrix proteases Lon, mtHsp70, and Yme1 are induced by ER stress.19 Likewise, the ER-mitochondria interconnection plays a prominent role in the caspase-mediated induction of neuronal cell death,22 and mitochondria modulate the ER UPR under glucose deprivation conditions.21 Conversely, it has been suggested that mitochondria support ER function via adenylate kinase (AK) 2109 and mitochondrial dysfunction triggers the ER stress response and aggravates hepatic insulin resistance.23

MITOCHONDRIA AS INTEGRATORS OF CELLULAR DANGER SIGNALING

  1. Top of page
  2. Abstract
  3. ENDOPLASMIC RETICULUM UNFOLDED PROTEIN RESPONSE
  4. MITOCHONDRIAL PROTEIN HOMEOSTASIS
  5. MITOCHONDRIAL UNFOLDED PROTEIN RESPONSE
  6. MITOCHONDRIAL ADAPTATIONS
  7. ER MITOCHONDRIAL NETWORK
  8. MITOCHONDRIA AS INTEGRATORS OF CELLULAR DANGER SIGNALING
  9. ER UPR AND MITOCHONDRIAL DYSFUNCTION IN IBD
  10. CONCLUSION
  11. REFERENCES

Next to the well-established functions of mitochondria in cell metabolism and during apoptosis, accumulating data have placed mitochondria at the center of diverse cellular functions and suggest mitochondria as integrators of various signaling pathways. As mentioned above, mitochondria participate in cellular calcium homeostasis and constitute a major source of cellular ROS,8, 52 thereby affecting processes such as autophagy and inflammation.110–112 Under conditions of nutrient deficiencies mitochondria have evolved mechanisms to regulate the cell cycle.113 Additionally, they have been suggested to serve as a signaling platform by providing microdomains for molecule activation in their unique membrane environment.114 Moreover, since the description of a protein located in the mitochondrial outer membrane, mitochondrial antiviral signaling (MAVS), required for the production of type I interferon in response to viral infection in 2005,27 mitochondria are thought to modulate innate immune signaling. During viral infection, RIG-I binds single- or double-stranded RNA and is recruited to the mitochondrial outer membrane to bind MAVS, subsequently leading to activation of interferon-regulatory factors (IRF) and nuclear factor kappaB (NF-κB), and downstream to the production of IFN as well as proinflammatory cytokines.27, 115 Notably, with regard to mtUPR-induced PKR activation, PKR has also been shown to act in concert with mitochondria-dependent RIG-I/MAVS-signaling to mount an efficient IFN-β-secretion in response to rotavirus infection.116

Recent work has confirmed the role of mitochondria in immune responses by linking mitochondrial ROS production and autophagy to the activation of the NLRP3 inflammasome,26, 29 a multiprotein complex involved in proteolytic maturation and release of IL-1β and IL-18.28 Notably, polymorphisms in NLRP3 have been associated with Crohn's disease (CD) in a candidate-gene study117 and a single nucleotide polymorphism (SNP) within the IL-18 receptor accessory protein gene (IL18RAP) has been identified as a risk factor for both CD as well as ulcerative colitis (UC).118 Consistently, IL-1β as well as IL-18 expression is enhanced in IBD, particularly in the epithelium.119–121 In line with these data, neutralization of IL-18 as well as genetic ablation of caspase-1, a downstream effector of the NALP3 inflammasome involved in IL-1β and IL-18 processing,122 has been shown to ameliorate DSS colitis. Moreover, administration of anti-IL-1β was effective in experimental rabbit complex colitis.123, 124 Interestingly, also ATG16L1, encoded by the CD-associated gene, has been demonstrated to regulate inflammasome activation125 (Fig. 4).

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Figure 4. Mitochondria as integrators of cellular danger signaling. Several cellular signaling pathways converge on mitochondria. Mitochondrial function is reflected by the protein folding environment in the matrix, metabolic rates, mitochondrial membrane potential (ψΔ), and generation of ROS. Whereas mitochondrial biogenesis-related pathways as well as mtUPR might serve to sustain a functional mitochondrial population, mitochondria additionally represent a signaling platform for inflammation-related signals.

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ER UPR AND MITOCHONDRIAL DYSFUNCTION IN IBD

  1. Top of page
  2. Abstract
  3. ENDOPLASMIC RETICULUM UNFOLDED PROTEIN RESPONSE
  4. MITOCHONDRIAL PROTEIN HOMEOSTASIS
  5. MITOCHONDRIAL UNFOLDED PROTEIN RESPONSE
  6. MITOCHONDRIAL ADAPTATIONS
  7. ER MITOCHONDRIAL NETWORK
  8. MITOCHONDRIA AS INTEGRATORS OF CELLULAR DANGER SIGNALING
  9. ER UPR AND MITOCHONDRIAL DYSFUNCTION IN IBD
  10. CONCLUSION
  11. REFERENCES

IBD and its two main idiopathic pathologies, UC and CD, are chronic, immunologically mediated disorders of the gastrointestinal tract and represent another aspect of chronic pathologies. UC as well as CD are multifactorial diseases and are characterized by alterations of the innate and adaptive immune system, luminal and mucosa-associated microbiota, as well as epithelial function.126 IECs are crucial for maintaining intestinal homeostasis, constituting an interface between the two major factors influencing intestinal inflammation, the gut microbiota and the immune system.127 Conversely, failure to control inflammatory processes at the IEC level may critically contribute to IBD pathogenesis.

During the last years, accumulating evidence implicated ER UPR at the epithelial cell level in promotion and perpetuation of intestinal inflammation,24, 128, 129 and at the same time, it has been repeatedly suggested that chronic intestinal inflammation represents an energy-deficiency disease involving mitochondria and featuring alterations in epithelial cell oxidative metabolism.25, 130 Inflammation and ER stress are linked at many levels, since inflammation is characterized by the production of large amounts of proteins such as cytokines or chemokines and, furthermore, ER UPR signaling can directly intersect with inflammatory pathways including NF-κB, JNK, TLR-mediated signaling and production of ROS.131–134 Moreover, the ER has been suggested to be essential in the coordination of metabolic responses through its ability to control the synthetic and catabolic pathways of various nutrients.135 Our results, demonstrating ER UPR as well as mtUPR to be activated in two murine models of chronic, immune-mediated colitis as well as human patients with IBD, further emphasizes the importance of mitochondrial signaling in intestinal inflammation.14 Moreover, identifying PKR as an mtUPR-mediator as well as an integrator of ER UPR and mtUPR signaling strengthened the hypothesis of interrelated, disease-relevant organelle signaling (Fig. 5).

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Figure 5. Mitochondria at the center of a tightly interrelated cellular stress network. Stress responses including metabolic, inflammatory, mitochondrial, and ER UPR pathways are interrelated and associated with bacterial and nutrient sensing. Polymorphisms in genes associated with these cellular processes have been identified as disease susceptibility factors and underlie several chronic diseases.

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UPR and Microbial Sensing

Recent work using mice deficient in ER UPR-mediators (IRE1β, XPB1, S1P) or mice with a mutation in the Muc2 gene links ER stress in the highly secretory subtypes of IEC, antimicrobial peptides producing Paneth cells, and mucin-producing goblet cells, with antimicrobial defense and intestinal inflammation.24, 129, 136 The commensal microbiota is one of the key drivers of intestinal inflammation in IBD and pathways crucial for sensing and controlling the composition of bacteria, like TLR signaling and autophagy, interact with ER UPR as well as mitochondrial signaling. Genome-wide association studies (GWAS) have identified multiple polymorphisms as disease susceptibility factors in CD such as the autophagy-related ATG16L1137, 138 and IRGM,139, 140 as well as bacterial sensing-related NOD2141, 142 and TLR4143 genes. In addition, a polymorphism leading to lower expression levels in the promoter of the gene encoding the mitochondrial carrier protein uncoupling protein (UCP) 2 was associated with CD as well as UC.144 Consistently with CD patients harboring the NOD2 or ATG16L1 alleles,145–147 NOD2- as well as XBP1-deficient and ATG16L1-hypomorphic mice display alterations in Paneth cell structure and function, but in addition, NOD1 and NOD2 have been demonstrated to be critical for the autophagic response to invasive bacteria by recruiting ATG16L1 to the bacterial entry site. Cells homozygous for the CD-associated frameshift mutation of NOD2 failed to recruit ATG16L1 and displayed impaired bacterial autophagy, providing a functional link between the proteins encoded by the two most prominent CD-associated genes.148

In contrast, IRGM as well as UCP2 directly impact mitochondrial function. By affecting mitochondrial fission, IRGM was shown to induce autophagy of intracellular mycobacteria but also to influence mitochondrial membrane polarization and cause Bax/Bak-dependent cell death, suggesting IRGM to be a double-edged sword in the context of intestinal inflammation.149 On the other hand, UCP2 is thought to promote a metabolic shift from glucose oxidation to fatty acid oxidation and by controlling the speed of the Krebs cycle, decrease the production of mitochondrial ROS.150–152 Macrophages stimulated with lipopolysaccharide (LPS) have been shown to quickly downregulate UCP2 in a TLR4- and MAP kinase-dependent manner to increase mitochondrial ROS production, thereby linking UCP2 and immune cell activation.153

IBD and Metabolism

It has been suggested that chronic intestinal inflammation might represent an energy-deficiency disease involving the mitochondria and featuring alterations in epithelial cell oxidative metabolism.25, 130 In particular, β-oxidation is implicated in CD pathogenesis and a polymorphism in SLC22A5,154, 155 encoding the carnitine transporter OCTN2, has been described as a risk factor in IBD. Carnitine is essential to the energy metabolism of IEC by transporting long-chain fatty acids into mitochondria for β-oxidation.156 Consequently, genetic ablation of OCTN2 as well as pharmacological inhibition of intestinal fatty acid β-oxidation also results in experimental colitis.157, 158 Sustaining β-oxidation and thereby energy supply might be particularly important in IEC metabolically challenged by alterations in the microbiota and/or in the context of energy-consuming inflammatory processes.159 Furthermore, several pathogens and their toxins specifically target mitochondria to disrupt their function160 and proinflammatory cytokine-evoked ROS generation is associated with a drop in mitochondrial membrane potential.161 Contrarily, treating epithelial cells with the oxidative phosphorylation-uncoupler dinitrophenol (DNP) to induce mitochondrial stress caused a decrease in TER and increased translocation of E. coli.162 Supporting the relevance of these data, enterocytes of IBD patients have been reported to display swollen mitochondria with irregular cristae indicative of impaired function.163, 164 In accordance, reduced ATP levels have been found in the colon of some CD patients165 and biopsies from patients with IBD can be more susceptible to uncouplers of oxidative phosphorylation.164

CONCLUSION

  1. Top of page
  2. Abstract
  3. ENDOPLASMIC RETICULUM UNFOLDED PROTEIN RESPONSE
  4. MITOCHONDRIAL PROTEIN HOMEOSTASIS
  5. MITOCHONDRIAL UNFOLDED PROTEIN RESPONSE
  6. MITOCHONDRIAL ADAPTATIONS
  7. ER MITOCHONDRIAL NETWORK
  8. MITOCHONDRIA AS INTEGRATORS OF CELLULAR DANGER SIGNALING
  9. ER UPR AND MITOCHONDRIAL DYSFUNCTION IN IBD
  10. CONCLUSION
  11. REFERENCES

The current paradigm for the pathogenesis of IBD is a dysregulated interaction between the intestinal microbiota and the mucosal immune system in genetically predisposed individuals, whereby onset and recurrence of disease are most likely triggered by unknown environmental agents.166 The loss of immune homeostasis in IBD might be due to insults generated by disease-associated alterations such as reduced epithelial barrier function, defective production of antimicrobial peptides, impaired intracellular handling of bacterial products, and an inadequate regulation of innate and adaptive immune responses.159, 166

The host can sense microorganisms by various pattern recognition receptors, such as TLR and NOD-like receptors; however, accumulating evidence indicates that the mucosal metabolic environment created by the microbiota also impacts host cellular functions and immune responses. Expressing short-chain fatty acid (SCFA) receptors such as GPR41, GPR43, and GPR109A and the butyrate transporter SLC5A8, IEC and mucosal immune cells actively sense bacterial metabolites.167–170 In particular, the SCFA butyrate, generated by bacterial fermentation of dietary fibers, is not only supposed to be the main energy source of colonocytes,171 but also to exert antiinflammatory and anticarcinogenic functions.169 Interestingly, IBDs are associated with shifts in the microbiota and a reduction in SCFA.172 These changes might be particularly important in the context of intestinal inflammation, characterized by increased energy demand and catabolism.

On the other hand, using a metabolomic approach in a murine model of Crohn's-like ileitis, we were able to demonstrate shifts in host intestinal lipid metabolism concomitant to the histological onset of inflammation. Moreover, disease progression was characterized by significantly altered metabolism of cholesterol, triglycerides, phospholipids, plasmalogens, and sphingomyelins in the inflamed tissue (ileum) and the adjacent intestinal parts (proximal colon).173

With regard to the interrelation of mitochondrial stress, ER stress, and metabolism, it is remarkable that butyrate has already been shown to impact ER UPR-signaling and mitochondrial pathways in IEC.174, 175 Considering these data and the fact that protein homeostasis is particularly sensitive to environmental changes, it is attractive to hypothesize that a concerted UPR-activation serves as an innate mechanism to sense potentially threatening changes of the metabolic environment.

Thus, manipulating the bacterial composition of the microbiota or changing food composition to alter bacterial metabolism, complemented with therapeutic approaches to improve ER function such as administration of chemical chaperones like phenyl butyric acid (PBA) or tauro-ursodeoxycholic acid (TUDCA), as well as targeting mtUPR by mitochondria-specific antioxidants such as acetyl-L-carnitine might have beneficial outcomes in multiple chronic diseases.

REFERENCES

  1. Top of page
  2. Abstract
  3. ENDOPLASMIC RETICULUM UNFOLDED PROTEIN RESPONSE
  4. MITOCHONDRIAL PROTEIN HOMEOSTASIS
  5. MITOCHONDRIAL UNFOLDED PROTEIN RESPONSE
  6. MITOCHONDRIAL ADAPTATIONS
  7. ER MITOCHONDRIAL NETWORK
  8. MITOCHONDRIA AS INTEGRATORS OF CELLULAR DANGER SIGNALING
  9. ER UPR AND MITOCHONDRIAL DYSFUNCTION IN IBD
  10. CONCLUSION
  11. REFERENCES