Does ERS contribute to oral diseases and oral fibrosis pathogenesis?

A systematic search of relevant literature published through July 2023 was conducted in the MEDLINE database (via PubMed) using the following controlled-vocabulary terms combined with Boolean operators: (endoplasmic reticulum stress) OR (unfolded protein response) AND (disease), (endoplasmic reticulum stress) OR (unfolded protein response) AND (fibrosis). To investigate the role of ERS in the pathogenesis of disease affecting the oral maxillofacial region, the following MeSH, Emtree, DeCS/MeSH, and other free terms combined with Boolean operators were used: (endoplasmic reticulum stress) OR (unfolded protein response) AND (oral disease).

The articles were selected based on titles and abstracts. Inclusion criteria included in vitro, in vivo, and clinical studies on ERS in oral disease. Literature reviews, irrelevant studies, non-English articles, and articles published in non-peer review journals were excluded. A manual search was also performed using the reference list of selected articles.

During periodontal and gingival inflammation, various stressors such as microbial pathogens and inflammatory mediators can induce ERS and UPR. In a study by Kim et al.7,8 of thapsigargin (TG)-induced ERS in human gingival fibroblast cells, p38 mitogen-activated protein kinase was shown to play a significant role in cell death and autophagy, with increased expression of beclin-1 and microtubule-associated protein 1 light chain 3 II (LC3II) as well as the formation of autophagic vesicles. Furthermore, selenoprotein S (SEPS) and heat shock protein 60 were highly expressed in the periodontitis, and the presence of Porphyromonas gingivalis independently aggravated alveolar bone resorption and significantly correlated with increased glucose-regulated protein (GRP) 78, X-box binding protein 1 spliced form (XBP1s), triiodothyronine receptor auxiliary protein, and cathepsin K expression9,10. Elevated expression of activating transcription factor (ATF) 6β was evident during tunicamycin-induced ERS in periodontal mice, with increased expression of the receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) in periodontal ligament cells. These biomarkers were associated with increased resorption of periodontal bone, and treatment using microRNA (miR)-1260b and ATF6β small interfering RNA (siRNA) with polyethylenimine nanoparticles significantly inhibited periodontal bone resorption11. In drug-induced gingival hyperplasia, cyclosporine can increase the expression of CCAAT/enhancer-binding protein homologous protein (CHOP), GRP78, XBP1s, vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), B-cell lymphoma 2 (Bcl-2), and cyclophilin D while decreasing cytochrome c, and shift gingival metabolism equilibrium toward an anabolic state, resulting in overgrowth and fibrosis12,13. In addition, treatment using 4-phenylbutyric acid, melatonin, and simvastatin were shown to downregulate the expression of ERS markers, mitochondrial apoptosis markers, and autophagy8,12.

In a study by Katsiougiannis et al.14, TG-induced ERS led to autophagy, as evidenced by the expression of LC3II and apoptosis in human salivary gland cells. In quiescent cells, Ro/SSA and La/SSB autoantigens are localized predominantly to the cytoplasm. However, in apoptotic cells, these autoantigens underwent relocalization and were found on the cell membrane and blebs in the apoptotic membrane. Kaira et al.15 reported the involvement of ERS in adenoid cystic carcinoma (ACC) through the identification of positive GRP78/BiP expression in ACC specimens. This is indicative of an unfavorable prognostic biomarker, demonstrating a significant association with increased cellular proliferation and angiogenesis.

In OSCC cells, heightened protein synthesis and changes in the tumor microenvironment (TME) can trigger ERS. These changes initiate an UPR, promote cell survival and facilitate tumor growth. However, persistent or unresolved ERS can also contribute to the advancement of OSCC by stimulating tumor-cell invasion, migration, and resistance to therapeutic interventions16.

Sufficient ERS can induce signal transducers and activators of transcription 3 (STAT3), NF-κB signaling, and interleukin (IL)-6. Under tunicamycin-induced ERS, normal oral keratinocytes showed significant upregulation of genes for STAT3 and leukemia inhibitory factor receptor (LIF-R), IL-6 receptor (IL-6R), and IL-18R1. Conversely, in OSCC tissue, significantly more cyclic AMP-responsive element-binding protein 3-like 3 (CREB3L3), STAT3, IL-6R, and sterol regulatory element-binding protein (SREBP) 1 were expressed17,18. The activation of STAT3 and NF-κB were activated in tunicamycin-induced ERS in OSCC cells through the upregulation of IL-5, IL-22, and LIF-R17. LIF-R is expressed by cancer-associated fibroblasts (CAFs) and plays an important role in the activation of STAT3 in the TME of OSCCs, promoting the invasion of cancer cells19. In addition, ERS can change gene-expression profiles from regulators of lipid metabolism to controllers of apoptosis18.

Cancer-associated fibroblasts are a heterogeneous population of activated fibroblasts originating from mesenchymal cell lines within the TME. Due to the absence of distinctive biomarkers, determining the source of CAFs remains challenging. The prevailing view suggests that regular fibroblasts arise from primitive mesenchymal cells, and CAFs emerge from activated fibroblasts in nearby tissues, bone marrow-derived mesenchymal precursor cells, and endothelial and epithelial cells, some of which are stimulated by cytokines such as TGFβ20,21.

Several inflammatory mediators may stimulate activation of CAFs. For example, IL-1 triggers CAF activation via the NF-κB pathway, and IL-6 primarily acts on signal transducers and STAT. Positive feedback loops and crosstalk involving Janus kinase (JAK)-STAT signaling, changes in the contractile cytoskeleton, and modifications in histone acetylation further contribute to CAF activation. Physical alterations in the extracellular matrix (ECM) can also serve as stimuli for CAF activation22.

The reverse Warburg effect is considered a hallmark of CAF regulation. Metabolite transfer from CAFs to cancer cells is important element of cancer cell growth and survival. In addition to CAF regulation, prolonged hypoxia can cause glycolysis-related genes in fibroblasts to undergo hypomethylation, indicating that metabolic interactions with epigenetic processes promote the buildup of pro-glycolytic CAFs23. Because hypoxia is associated with a UPR, a strong relationship likely exists between the ERS and CAF, and future studies are warranted.

Due to the complexity of OSCC pathogenesis, numerous studies have excluded the potential contribution of proteins and genes. Dentin sialophosphoprotein (DSPP), which facilitates dentin and bone matrix mineralization, is reportedly involved in cancer pathogenesis. DSPP is present in non-mineralizing metabolically active ductal epithelial cells of various glands, such as salivary glands, nephrons, and eccrine sweat glands, and helps repair pericellular matrix and ECM proteins damaged by free radicals generated during intense metabolic activity24. In OSCC cells transiently transfected and not exposed to puromycin, silencing of DSPP resulted in significant downregulation of GRP78, sarco/ER Ca+2-ATPase 2b (SERCA2b), protein kinase R (PKR)-like ER kinase (PERK), inositol-requiring enzyme type 1 (IRE1), ATF6, and matrix metalloproteinase (MMP)-20. Silencing DSPP can lead to reduced cell viability and migration accompanied by increased apoptosis in OSCC cells. In addition, DSPP knockdown resulted in decreased levels of proliferating cell nuclear antigen, an indicator of cell proliferation, and Bcl-2, an anti-apoptotic protein. Conversely, levels of Bax, a pro-apoptotic protein, and cytochrome c were elevated in DSPP-silenced OSCC cells25.

1) Unfolded protein response

• IRE1: inositol requiring enzyme 1

• PERK: protein kinase RNA-activated (PKR-like) ER kinase

• ATF6: activating transcription factor 6

• GRP78: glucose-regulated protein 78

• XBP1: X-box-binding protein 1

• PDIA5: protein Disulphide Isomerase 5

• PDIA6: protein Disulphide Isomerase 6

2) Apoptosis

• CHOP/GADD153: CCAAT-enhancer-binding protein homologous protein/Growth arrest-and DNA damage-inducible gene 153

• PUMA: p53-up regulated modulator of apoptosis

• Noxa: pro-apoptotic member of the Bcl-2 protein family

• BIM: Bcl-2 interacting mediator of cell death

3) Necroptosis

• RIPK1: receptor-interacting protein kinase 1

• RIPK3: receptor-interacting protein kinase 3

• MLKL: mixed-lineage kinase domain-like protein

4) Autophagy

• TRAF2: TNF receptor-associated factor 2

• JNK: c-Jun N-terminal kinases

• ASK1: apoptosis signal-regulating kinase 1

• LC3: microtubule-associated protein 1 light chain 3

5) Angiogenesis

• VEGF: vascular endothelial growth factor

• IL-8: interleukin 8

• MMP2: matrix metalloproteinase 2

• MMP9: matrix metalloproteinase 9

• ANG-2: angiopoietin 2

• HIF-1: hypoxia-inducible factor

• FGF2: fibroblast growth factor 2

6) Inflammatory signaling

• NF-kB: nuclear factor kappa light-chain-enhanced activated B-cells

• AP-1: activator protein 1

• NLRP3 inflammasome: nucleotide-binding domain (NOD)-, leucine-rich repeat (LRR)-, and pyrin domain-containing protein 3

• IL1β: interleukin 1 beta

• IL18: interleukin 18

7) Epithelial-mesenchymal transition

• TGFβ: transforming growth factor beta

• αSMA: α-smooth muscle actin

• E-cadherin: epithelial cadherin

• N-cadherin: neural cadherin

• Vimentin

• Fibronectin

• miR-150: microRNA 150

The oral mucosa consists of a stratifying squamous epithelium that is largely covered by a keratinized epithelium over a dense connective tissue or lamina propria rich in type I collagen. This structural arrangement serves as a protective barrier against external microorganisms, pathogens, and mechanical stress, particularly in the gingiva and hard palate26. For flexibility in deglutition, mastication, and speech, the floor of the mouth and buccal mucosa are composed of a nonkeratinizing epithelium. In addition, the dorsum of the tongue is covered by a specialized mosaic of keratinized and nonkeratinized epithelium that attaches tightly to the tongue muscle27.

Keratins are the main structural proteins in the keratinizing stratified epithelial cytoskeleton. Basal cells, which form the actively dividing layer of the epithelium, connect to the lamina densa of the lamina basalis via hemidesmosomes. The spinous cell layers play a crucial role in facilitating cell-cell connections through numerous desmosomes and gap junctions. The stratum granulosum contains membrane-coating granules, keratohyalin granules, and abundant tonofibrils, leading to an abrupt transition to the stratum corneum. The flattened cornified squames form a relatively thick protective layer. To maintain their function, the stratified epithelia of the oral mucosa use tight cell-cell adhesion in viable cells combined with intercellular tight and adherens junctions that connect to the actin cytoskeleton28,29.

During the healing process in response to oral mucosa injury following hemostasis, the formation of fibrin-rich granulation tissue initiates the inflammatory phase. Next, the wound-healing process enters a proliferative phase characterized by the migration of keratinocytes and fibroblasts to the wound bed. Fibroblasts actively secrete ECM components, which contribute to remodeling of granulation tissue, and keratinocytes play a crucial role in re-establishing the protective barrier function of the tissue. Unlike skin, oral mucosa heals better and typically results in rapid and scarless healing. Distinct variations in wound healing occur within the oral environment contingent on the specific location. Wounds in the non-keratinized mucosa may lead to scarring, and wounds in the tongue or gingiva and hard palate heal uneventfully without scarring. These dissimilarities can be ascribed to differences in the characteristics of resident fibroblasts26,30.

Nikoloudaki et al.26 suggested periostin may influence cell behavior during the proliferative phase of healing by identifying intracellular proximal localization between periostin and fibronectin in the ER of fibroblast cells. As a response to injury, the periostin enhances secretion of fibronectin from the ER into the ECM, leading to fibrosis and scarring. Periostin has also been identified in several pathologic conditions, including fibrous dysplasia31, odontogenic tumors of mixed epithelial/mesenchymal origin32, and OSCC33. This strengthens the hypothesis that intracellular ERS, such as in oral fibroblasts, can influence the secretion and expression of several proteins into the ECM, resulting in the development of oral disease and fibrosis.

During ERS, three transmembrane proteins in the ER membrane (IRE1, PERK, and ATF6) are activated to initiate a UPR34. Typically, the ER resident chaperone HSP A5 keeps the three ERS sensors in an inactive state. This chaperone is also referred to as GRP78 and binds to immunoglobulin protein (BiP). However, an accumulation of misfolded proteins in the ER lumen leads to engagement of BiP, releasing the three sensors to initiate the UPR2.

NF-κB plays a pivotal role in inflammation response and is typically confined within the cytoplasm by the inhibitor of NF-κB (IκB). During inflammation, various stimuli, including pro-inflammatory cytokines, activate IκB kinases, leading to phosphorylation and degradation of IκB41. Under ERS, a UPR induces the degradation of IκB through the IRE1α pathway and tumor necrosis factor receptor-associated factor 2 (TRAF2). In addition, activation of JNK by IRE1α induces the expression of activator protein (AP-1)42. The primary function of AP-1 is to bind to specific DNA sequences in the promoter regions of target genes and regulate their transcription, particularly during inflammation and apoptosis. ERS can also modify the phenotype of macrophages by activating the NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasomes5. When activated, NLRP3 can induce activation of caspase-1, which then processes pro-inflammatory cytokines, including IL-1β and IL-18, into their active forms43. In addition, ERS through IRE1α is required for the maturation and differentiation of plasma and dendritic cells as well as polarization of Th17 cells5.

Apoptosis involves initiation, signaling, and execution. Key players in the apoptotic pathway include members of the Bcl-2 proteins, which are divided into pro-apoptotic (e.g., Bax, Bak) and anti-apoptotic (e.g., Bcl-2, Bcl-XL) proteins44. The BH-3-only members of the Bcl-2 family, including p53-upregulated modulator of apoptosis (PUMA), NOXA, and Bcl-2 interacting mediator of cell death BIM, are significant in ERS-induced apoptosis45. Pro-apoptotic proteins promote the release of cytochrome c from the mitochondria into the cytoplasm, where it forms a complex with apoptotic protease-activating factor 1 (Apaf-1) and procaspase-9, ultimately producing apoptosomes46. This complex activates caspase-9, which in turn activates downstream effector caspases (e.g., caspase-3 and caspase-7), leading to the cleavage of various cellular substrates and ultimately, cell death47. In addition to direct ER-mitochondrial interactions, CHOP/GADD153 is involved in the regulation of apoptosis as the apoptosis inducer by activating BIM and PUMA as well as repressing several anti-apoptotic members of the Bcl-2 family2.

Necroptosis is a programmed form of necrosis that represents an alternative cell-death pathway when apoptotic pathways are inhibited or unavailable. Necroptosis is characterized by the rapid and uncontrolled breakdown of cellular membranes, leading to the release of cellular contents and inflammation. The molecular machinery involved in necroptosis includes RIPKs, and RIPK1 and RIPK3 in particular. These kinases form a complex, the necrosome, when apoptosis is blocked, resulting in phosphorylation of MLKL48. Phosphorylated MLKL translocates to the plasma membrane and disrupts its integrity, resulting in cell swelling, membrane rupture, and the release of intracellular contents49. In a study using a mouse fibroblast cell line, TNFR1 mediated ERS-induced necroptosis by activating caspase. The MLKL served as an effector of ERS and repression of RIPK1 shifted the ERS-induced necroptosis to apoptosis50.

In addition to ERAD, lysosome-mediated protein degradation by autophagy is another protein degradation pathway during ERS42. Bernales et al.51 reported that ERS induces abundant cell-accumulated autophagosome-like structures enclosed by two membranes and containing densely packed, closely arranged membrane cisternae. Autophagy is induced by ERS by at least two UPR pathways, PERK-elF2α and IRE1α. The IRE1α pathway involves TRAF2, ASK1, and JNK. The PERK-elF2α pathway induces autophagy through ATF4-driven transcriptional regulation by inducing vesicle elongation. Ca2+ release from the ER lumen through the inositol 1,4,5-trisphosphate receptor (IP³R) can activate calcium/calmodulin-dependent protein kinase and relieves mammalian target of rapamycin inhibition on the Unc-51-like kinase 1 complex2,52.

The interplay between ERS and angiogenesis is complex and involves various signaling pathways. Hypoxia-inducible factor 1 plays a key role in cellular responses to changes in oxygen levels, specifically hypoxia, and plays a role in angiogenesis. Under ERS conditions, the activation of PERK and IRE1 branches of the UPR can lead to increased HIF-1α stabilization and transcriptional activity. Subsequently, HIF-1α promotes the expression of pro-angiogenic factors, including VEGF, erythropoietin (EPO), angiopoietin 2 (ANG-2), and MMPs, to stimulate angiogenesis53. In a study by Ghosh et al.54, VEGF expression during ERS was promoted by the activation of ATF4, XBP1s, and cleaved ATF6, independent from HIF-1. Under ERS, IL-8 is also upregulated through the PERK pathway by ATF455. As a pro-angiogenic cytokine, IL-8 activity further induces the expression of MMP-2 and MMP-9, which are critical players in angiogenesis, by mediating ECM remodeling and promoting endothelial cell migration and proliferation while decreasing endothelial cell apoptosis56. In addition to VEGF and IL-8, FGF 2 and angiogenin, potent pro-angiogenic factors that contribute to endothelial cell proliferation and tube formation, are upregulated57.

During epithelial-mesenchymal transition, epithelial cells lose their cell-cell adhesion characteristics and acquire a migratory and invasive phenotype characteristic of mesenchymal cells. This process is regulated by various complex signaling pathways and transcription factors. In addition to the loss of adherens junctions, EMT also includes downregulation of cytokeratin and E-cadherin and increases of fibronectin, N-cadherin, and vimentin58. UPR activation can alter the function of fibroblasts by promoting TGFβ-mediated myofibroblast differentiation5; one such mechanism is through degradation of miR-150 by activated IRE1α that consecutively regulates αSMA expression37. The IRE1-XBP1 pathway can also negatively regulate E-cadherin and upregulate N-cadherin2. On PERK activation, the epithelial cells are subjected to EMT, and the process of invasion and metastasis depend on eIF2α phosphorylation59. Additional pathways have also have been suggested to induce EMT, such as autophagy and the activation of c-SRC kinase in tubular epithelial cells as well as the compensatory activation of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 (NRF-2/HO-1) antioxidative stress response pathway in colorectal adenocarcinoma cell lines60,61.