Ⅰ. INTRODUCTION

Endoplasmic Reticulum (ER) is an important organelle in eukaryotic cells, involved in protein synthesis, folding, modification, lipid synthesis and calcium ion (Ca²⁺) homeostasis ^[1]. When ER homeostasis is disrupted (such as though Ca²⁺ homeostasis imbalance, viral infection or dysregulated redox homeostasis), an imbalance between the ER's protein- folding capacity and its functional requirements, resulting in the accumulation of unfolded or misfolded proteins. This condition can triggers an intracellular Stress response called Endoplasmic Reticulum Stress (ER Stress). Under ER Stress, cells activate the unfolded protein response (UPR) as an adaptive response^{[2, 3]}. Normally folded proteins require specific conditions to remain stable in the ER lumen, and newly synthesized proteins first bind to Ca²⁺ dependent companion proteins such as glucose regulatory protein 78 (GRP78, also known as BiP). However, severe ER Stress promotes the expression of proapoptotic proteins such as CHOP (CIEBP homologous protein) and caspase-12, ultimately leading to apoptosis^[4]. Disruption of endoplasmic reticulum homeostasis affects Ca²⁺ signaling, protein folding and modification, lipid synthesis, and other signaling pathways. Therefore, ER stress is closely associated with the occurrence and development of many diseases, including neurodegenerative diseases, metabolic diseases, cancers, and oral diseases.

Previous studies have had found that in response to ER Stress, cells have evolved the UPR, in which cells can detect unfolded or misfolded proteins in the ER, transmit this information to the nucleus through a series of signal cascades, and take corresponding protective measures to restore the protein homeostatic state of the ER^[5]. When ER stress occurs, the cell initiates the UPR program to slow down the protein translation process, thereby reducing the burden of ER protein synthesis. Additionally, UPR promotes the clearance of misfolded proteins by up-regulating the expression of chaperone proteins in the ER, thereby improving protein folding capacity helping cells resist stress and promoting cell survival^{[6, 7]}. However, when ER stress cannot be reversed, cell function will be impaired, often resulting in cell death^[8]. Currently, three UPR receptors are known to exist in higher mammalian cells, all of which are single transmembrane proteins in the endoplasmic reticulum: inositol-requiring enzyme 1 (IRE1), protein kinase R-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6(ATF6). There is growing evidence that ER stress-induced cell dysfunction and cell death are major triggers for a variety of diseases, making ER stress-related proteins potential therapeutic targets for these diseases. Moreover, cytoplasmic Ca²⁺ levels have been suggested to play a key role in the regulation of UPR, and calcineurin (Ca²⁺-dependent proteins) have been found to promote the survival of human and mouse astrocytes and African claw frog- oocytes during the acute phase of UPR^[9]. Rapid elevation of calcineurin interact with the cytoplasmic structure of PERK to reduce protein translation, Which in turn affects Ca²⁺ levels. Additionally, changes in Ca²⁺ levels in the ER also induce ER stress.

Ca²⁺ ions are important second messengers in cells. Ca²⁺ signaling is widely involved in many biological processes, such as proliferation, development, learning, and memory. At the cellular and molecular level, Ca²⁺ signal is involved in the regulation of cell proliferation, differentiation, apoptosis and other physiological processes^[10]. Therefore, the maintenance of Ca²⁺ homeostasis is essential for ER function. Proper storage of Ca²⁺ in the ER is essential for normal Ca²⁺ signaling and critical cellular functions such as protein synthesis, folding, and maturation. The disruption of Ca²⁺ homeostasis in ER may cause ER stress. Imbalance of Ca²⁺ homeostasis in ER is associated with many diseases, such as heart disease, neurological diseases and oral diseases^[11]. At present, both Ca²⁺ depletion and Ca²⁺ overload in the ER are known to cause ER stress, which activates UPR. ER Ca²⁺ homeostasis involves of Ca²⁺ binding proteins (such as calreticulin, BIP, and GRP94), ER-resident Ca²⁺ transporters, and Ca²⁺ channel regulators, including inositol 1,4,5 -triphosphate receptor (IP3R), ryanodine receptors (RyRs), Transient receptor potential cation channels(TRP), Transmembrane and Coiled-Coil Domains 1（TMCO1）, Sarcoplasmic/ Endoplasmic reticulum Ca²⁺ ATPase (SERCA) and Store-operated Ca²⁺ entry （SOCE）of STIM1/Orai l pathway^[12]. There is a complex bidirectional regulatory relationship between ER stress and Ca²⁺. The changes of Ca²⁺ can not only induce ER stress, but also play a key role in the process of cellular stress response ^[13]. The maintenance of ER Ca²⁺ homeostasis is essential for the physiological function of cells, and imbalances in Ca²⁺ may lead to the development of ER dysfunction and related diseases. Thus, studying the relationship between ER stress and Ca²⁺ is of great significance for understanding cellular stress mechanism response and developing new therapeutic strategies.

Disturbed Ca²⁺ homeostasis in the ER is also associated with many oral diseases. For example, bacterial infection in periodontitis can lead to ER stress in periodontal ligament cells, resulting in apoptosis and ultimately periodontal tissue destruction^[14]. ER stress has been implicated in adenoid cystic carcinoma (ACC) and is significantly associated with increased cell proliferation and angiogenesis. One study showed that exogenous ceramides induce ACC cancer cell death through disruption of ER Ca²⁺ homeostasis, triggering severe ER stress^[15]. Similarly Osteoarthritis is a common chronic degenerative joint disease characterized by progressive cartilage erosion, has been linked to ER stress. Decreased Derlin-3 expression is related to the apoptosis of mandibular condyle chondrocytes mediated through biomechanically induced ER stress pathway, suggesting it as a therapeutic target ^[16]. Moreover, ER Ca²⁺ homeostasis imbalance is closely associated with oral squamous cell carcinoma development^[16]. Therefore, in-depth study of ER Ca²⁺ homeostasis and its mechanism in oral diseases is of great significance to reveal the pathogenesis and discover novel therapeutic targets.

In this review, we focus on the mechanism between ER Ca²⁺ homeostasis and ER stress, and discuss their relevance to oral disease.

1. ER Stress and UPR

Various pathological conditions, including hypoxia, gene mutation, inhibition of glycosylation, nutrient deficiency, oxidative stress and Ca²⁺ homeostasis imbalance can disrupt protein homeostasis, resulting in accumulation of misfolded proteins in the ER lumen, commonly referred to as ER stress. To restore protein folding capacity and meet protein synthesis, cells activate the UPR, cellular stress response mechanism. UPR is a complex and highly regulated network designed to restore normal protein homeostasis by enhancing ER folding capacity and removing misfolded proteins.

UPR activation can buffers ER stress by restoring protein folding homeostasis, and UPR enhances ER protein folding capacity by increasing the expression of molecular chaperons (e.g. GRP78 or BiP) and folding enzymes. This assist in repairing misfolded proteins, ensuring proper protein folding and efficient quality control within the ER^[17]. UPR also promotes the degradation of unfolded proteins by activating the endoplasmic reticulum associated protein degradation (ERAD) pathway. ERAD identifies misfolded proteins and directs them to the proteasome for degradation, thus preventing intracellular accumulating^{[6, 18]}. To further reduce ER stress, the UPR inhibits translation via the PERK pathway, reducing synthesis of new proteins and providing time for the cells to restore ER function. By regulating gene expression, the UPR enables cellular adaptation to ER stress^[19]. For example, ATF6 and XBP1s enhances the expression of genes relevant to antioxidant and metabolic adaptation, facilitating cells survival under stress^{[20, 21]}. Furthermore, The UPR detects ER protein folding level and adjusts the folding capacity according balancing protein homeostasis. Depending on the severity and duration of ER stress, the UPR can shift from an adaptive mechanism to a triggering apoptosis implicated in the development of various diseases.

UPR consist of two type; adaptive UPR and terminal UPR. When cells face ER stress, IRE1α, PERK, and ATF6 pathway are activated to relieve ER stress and promote cell survival. However, if the adaptive UPR is fail due to severe or prolonged ER stress, terminal UPR is activated, leading to apoptosis ^[22].

IRE1α is the most conserved UPR receptor. Under normal physiological conditions, BIP maintains IRE1αthe monomer inactivation state by binding its lumen domain^[23]. During the ER stress, BIP from IRE1α, which subsequently dimerize or oligomerize, activating its Ribonuclease (RNase) domain ^[24]. Activated IRE1αexcises, producing spliced XBP1 (X-box binding protein 1) mRNA, translated into active XBP1s (XBP1 splicing) transcription factors^{[25, 26]}. XBP1s promote expression of URP target gene, facilitating protein transport, folding, quality control, and ERAD to reduce ER stress^[27]. Overactivation of IRE1αcan also degrade ER-localized mRNAs via regulated IRE1α-dependent decay (RIDD), reducing the burden of protein folding ^{[28, 29]}. PERK is also a type I single transmembrane protein on the ER, and is activated on ER Stress when BIP dissociates from it, leading to phosphorylation of eukaryotic translation initiation factor 2α (eIF2α)^[30,31]. Phosphorylated eIF2α reduces the translation initiation rate and inhibits the translation process by preventing the formation of ribosome initiation complexes^{[32, 33]}. In addition, under ER stress, activated PERK promotes NRF2 phosphorylation, resulting in NRF2 dissociation from KEAP1 and activation. ATF4 also induces DNA-damage-inducible 34(GADD34), which, through interaction with the catalytic subunit of type 1 protein serine/ threonine phosphatase (PP1), enables the dephosphorylation of eI2Fαand restoration of protein synthesis^[34]. ATF6 is a type II transmembrane protein on the ER. NRF2 is then transferred to the nucleus to further activate the transcription of proteins with antioxidant activity and counteract the ROS level in ER stress cells, thus playing its role in anti- oxidative stress^[35]. Under normal physiological conditions, the interaction between BIP and ATF6’s ER domain masks the inactivation of ATF6’s Golgi localization sequences (GLS). Under ER stress, Bip dissociates from ATF6, exposing the Golgi localization sequence GLS, which is then exported from ER to the Golgi complex via coat protein Ⅱ (CopII) vesicles. Subsequently in the Golgi apparatus, ATF6 is cleaved by proteases S1P and S2P, releasing its cytoplasmic domain (releasing the n-terminal transcriptional activation domain (TAD) fragment into the nucleus), which acts as a transcription factor (ATF6f). ATF6f induces the expression of genes involved in protein folding, secretion, and ER-re lated degradation^{[36, 37]}.

Each of the three receptors of UPR has a set of pro-apoptotic signaling pathways. When ER stress is over-activated, phosphorylated IRE1α is transformed into polymers or oligomers. In the case of continuous oligomerization, the Ribonuclease (RNase) domain of IRE1-α cuts hundreds of ER localized mRNA containing N-terminal signal sequences for degradation through the IRE1-dependent decay (RIDD) pathway, resulting in the consumption of protein folding components, further exacerbating ER stress^{[26, 28]}. Overactivation of IRE1α was found to raise thioredoxin-interacting protein （TXNIP）by lowering the levels of microRNA miR-17 ( TXNIP was a linchpin of the terminal UPR). Elevated TXNIP activates the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome, leading to the cleavage of procaspase-1 and the secretion of interleukin- 1β (IL-1β), ultimately leading to cell death^[39]. Additionally, oligomerization of IRE1αleads to activation of pro-inflammatory and pro-apoptotic proteins^{[38, 39]}. The IRE1 α-TXNIP pathway is therefore thought to be involved in terminal UPR, promoting aseptic inflammation and programmed cell death, which may be targeted to develop effective therapies for cell degenerative diseases. Under severe ER stress, IRE1α can activate the downstream target JNK, and phosphorylation of JNK leads to apoptosis^[40]. Several studies have found that ER stress-induced apoptosis is closely related to the pathogenesis of a range of cancers. Asiatic acid induces ER stress and activates the Grp78/IRE1α /JNK and calpain pathways, inhibiting tongue cancer growth^[42]. Activation of apoptosis-signal regulating kinase 1 (ASK1) and JUN N-terminal kinase (JNK) in the downstream apoptosis signaling pathway, as well as nuclear factor kappa- B (NF-KB) activation in downstream inflammatory signaling pathway, are involved, promoting programmed cell apoptosis^[41]. For PERK, under prolonged stress conditions, PERK mediated sustained phosphorylation of eIF2α and increased ATF4 expression lead to CHOP-induced apoptotic cell death through various signaling pathways. ATF4 and CHOP directly induce genes involved in protein synthesis and URP, leading to ATP depletion, Oxidative stress and apoptosis^{[43, 44]}. For ATF6, ATF6 translocates to the Golgi, where it is cleaved. Additionally, studies have shown that PERK activation phosphorylates eIF2α during ER stress, which can induce the activation of nuclear factor-KappaB (NF-kB) signaling pathway^[45]. The subsequent ATF6 fragment (pATF6(N)) translocates to the nucleus and initiates the expression of its target genes, such as chaperones, ERAD-associated genes, and the pro-apoptotic genes CHOP and pXBP1(S)^[25]. Multiple pro-apoptotic signals from UPR sensors eventually converge on the mitochondrial apoptotic pathway, and then mitochondrial proteins such as cytochrome C and Smac/Diablo are released into the cytoplasm, this pathway is triggered, leading to a cascade of downstream Caspase-related proteins, ultimately resulting in apoptosis. It has been found that siRNA-mediated silencing of ATF6 leads to a decrease in acute-phase CHOP induction but results in prolonged CHOP induction, suggesting that ATF6 is an important regulator of cell fate decisions under chronic ER stress^[46]. The mitochondrial apoptosis pathway is mainly regulated by the BCL-2 protein family, including BCL-2-associated X protein (BAX) protein and BCL-2 homologous antagonist/killer (BAK) proteins. The BCL-2 family consists of pro-death and anti-death proteins that control the apoptotic pathway of ER stress by regulating the integrity of the mitochondrial outer membrane^[47].

In addition, UPR also affects the composition of mitochondria. ER stress can be transferred mitochondria by altering metabolite (Ca²⁺) translocation or through the UPR signaling pathway. ATF4, for example, has been shown to control the expression of the ubiquitin ligase Parkin, a key regulator of mitochondrial function and dynamics^[48]. In turn, Parkin's ability to enhance UPR signal transduction by activating the sXBP1 pathway illustrates mutual activation^[49]. Thus, UPR affects mitochondrial function at multiple levels: it induces mitochondrial autophagy to clear stress-damaged mitochondria, regulates mitochondrial bioenergetics by influencing Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM), and promotes the loss of mitochondrial membrane potential^[51]. In addition, ATF6 is associated with the activity of peroxisome proliferator-activated receptor-γ -coactivator 1α (PCG1α), a major regulator of mitochondrial biogenesis, thus linking UPR to metabolic gene programs^[50].

Ⅱ. Ca²⁺ and ER stress

The ER is the largest reservoir of calcium ions in the cell, and there are a large number of chaperone proteins with an affinity for Ca²⁺ ions in the ER lumen. Changes in Ca²⁺ concentration in the ER will affect protein folding in the ER, leading to ER stress^[52]. At physiological rest, the concentration of Ca²⁺ in the ER ranges from 500μmol/L to 1mmol/L, and Ca²⁺ is generally released into the cytoplasm through the ER Ca²⁺ channel RyRs or IP3R. ER Stress caused by Ca²⁺ is closely related to the pathogenesis of many diseases, such as salivary gland dysfunction, enamel development, and oral squamous cell carcinoma. Under the stimulation of physiological activities, the precise regulation of Ca²⁺ concentration can generate accurate calcium signals to fine-regulate cell functions. Both extracellular Ca²⁺ flow and intracellular Ca²⁺ release can lead to increased cytoplasmic Ca²⁺ concentration and calcium signaling^{[53, 54]}. The generation of accurate calcium signals requires that the Ca²⁺ concentration in the ER and cytoplasm be maintained within a stable range, which is called Ca²⁺ homeostasis. Ca²⁺ release channels and transporters of Ca²⁺ on the ER include IP3R, RyRS, TMCO1, SERCA, TRP and SOCE of STIM1/Orai l pathway^[55]. Therefore, the regulation of intracellular Ca²⁺ concentration is important for achieving normal cell function.

IP3R, RyRs, TMCO1, and SERCA are located on the ER membrane. When Ca²⁺ in the ER is released into the cytoplasm, signaling first begins with signals generated at the plasma membrane. G-protein coupled receptors (GPCRs) and receptor tyrosine kinases (PTKs) activate phospholipase C(PLC), which then hydrolyzes phosphatidylinositol 4, 5-diphosphate (PIP2) in the plasma membrane to produce inositol 1, 4, 5-triphosphate (IP3), which then diffuses to bind to IP3 receptors (IP3R) on the ER, causing them to open. If IP3 is abscent, IP3R remains inactive. This eventually results in the release of Ca²⁺ from the ER into the cytoplasm ^[56]. On stimulation, IP3 binds to the IP3R receptor and promotes the opening of IP3R channels to release Ca²⁺. Cytoplasmic Ca²⁺ also binds to the activation site of these channels to promote the release of Ca²⁺ from the ER. Upon Ca²⁺ depletion from the ER via IP3R, the Ca²⁺-sensitive the sigma-1 receptor (Sig-1R) dissociates from BiP and associates with IP3R, thereby protecting the otherwise unstable IP3R from Endoplasmic-reticulum-associated protein degradation (ERAD) and prolonging Ca²⁺ signaling to the mitochondria. This process is called Ca²⁺-induced Ca²⁺ release (CICR)^[57]. When the concentration of cytoplasmic free Ca²⁺ is 100-200 nmol/L, RyRs remain a closed ^[58]. When the concentration of cytoplasmic Ca²⁺ exceeds above a certain threshold, it acts on RyRs, triggering the opening of homotetrameric channels, ultimately releasing ER-stored Ca²⁺ into the cytoplasm.

TMCO1 is a transmembrane protein located on the ER, and its amino acid sequences is highly conserved, indicating that the physiological function of TMCO1 is evolutionally conserved^[60]. This activation of RyR is activated by Ca²⁺ to release Ca²⁺ from the ER is also referred to CICR^{[ 59]}. TMCO1 detects ER Ca²⁺ overload and actively regulates ER Ca²⁺ content. TMCO1 is able to sense the ER Ca²⁺ overload state in the ER, which lead to TMCO1 dimerization and the formation of tetrameric Ca²⁺ channels assemblies, facilitating the release of excess Ca²⁺ from the ER. When ER Ca²⁺ returns to resting levels, the TMCO1 tetramer depolymerizes into a dimeric state and monomeric state, terminating Ca²⁺ channel activity. this channel is also referred to as the Ca²⁺ load-activated Ca²⁺ channel (CLAC)^[61].

SERCA is a Ca²⁺ ATPase. Its main function is to transport excess cytoplasmic Ca²⁺ back into the ER for storage. A 1000-fold Ca²⁺ concentration gradient between the ER and cytoplasm is maintained, Which is critical for a variety of ER-dependent cellular functions, including protein synthesis, folding and export, nucleoplasmic transport, stress response, regulation of IP3, and SERCA expression. There is a strong link between ER stress and SERCA. Therefore, SERCA is essential in maintaining ER Ca²⁺ homeostasis. SERCA inhibitor induces ER stress and prevent the reuptake of Ca²⁺ into the ER, which enhances Ca²⁺ flux from the ER to mitochondria and promotes apoptosis. Additionally, a redox- active form of Selenoprotein N (SEPN1) interacts with SERCA at mitochondria-associated endoplasmic reticulum membranes (MAMS) to regulate ER Ca²⁺ levels^[62]. Meanwhile, SEPN1 antagonizes oxidative damage that occurs during ER stress, which triggers the UPR. After UPR induction, the amount of reactive oxygen species (ROS) increases, and oxidized derivatives inactivate SERCA^[62].

TRP and SOCE exist not only in ER membrane, but also in cytoplasmic membrane. TRP is a non-selective cation permeable channel that maintains calcium balance in the cellular environment. The TRP superfamily has seven major subfamilies: TRPC, TRPV, RRPA, TRPM, TRPP, TRPML, and TRPN. In addition, some findings suggest that TRP activity is also associated with the regulation of UPR pathway. There are six types of channels in TRPV family. Among them, TRPV1, TRPV2, TRPV3 and TRPV4 are found in ER, whereas TRPV5 and TRPV6 are highly selective Ca²⁺ channels. In addition, TRPV channels interact with three ER stress receptors to induce an UPR, thereby reducing ER stress and protecting cells^[64]. In most tissues, TRPV acts as a sensor for different stimuli (eg, heat, pressure, and PH) and contribute to electrolyte homeostasis, barrier function maintenance, and cell development^[63]. In human embryonic stem cell-derived cardiomyocytes, inducing of ER stress using thapsigargin (TG) and tunicamycin(TM) resulted in increased expression of TRPV6 via the ATF6α signaling branch. Additionally the signaling pathway downstream of TRPV6 is the MAPK-JNK pathway. Activation of Store-operated Ca²⁺ entry (SOCE) rapidly triggers the activation of Ca²⁺ channels in the cytoplasmic membrane when the ER is depleted of Ca²⁺, allowing extracellular Ca²⁺ to enter. Overexpression of TRPV6 attenuates apoptosis induced by ER stress in HESC-CMs cells^[65]. The electrophysiological characterization of SOCE channels identifies them as Ca²⁺ release- activated channels (CRAC)^[66]. CRAC is activative is believed to follow an ordered process. Participants in the SOCE process are stromal interacting molecule (STIM) and Orai. Orai, an essential component of the CRAC channel, is a cytoplasmic membrane protein with four transmembrane structural domains that from the channel pore. STIM1 is a single transmembrane protein located on the ER membrane with its N-terminus in the ER lumen that regulates CRAC channels by sensing changes in ER Ca²⁺ concentration. First, the concentration of Ca²⁺ in the ER lumen decreases. Then the Ca²⁺ sensor STIMl, through binding and dissociation with Ca²⁺ in the ER lumen, undergo conformational changes and migrates to the contact site between the ER and the cytoplasmic membrane. STIMl must first coupl to the C terminal the Orai l to act as a bridge. It then interacts with the N terminal of the Orai l and finally activates the Orai l Ca²⁺ channel, allowing Ca²⁺ influx. It has been reported that the UPR receptor IRE1α interacts with STIM1, promotes ER-plasma membrane contact sites, and enhances SOCE, ultimately leading to decreased T cell activation. Under ER stress, the IRE1α-STIM1 axis maintains immune cell function by promoting SOCE, and this study also reveals a potential pathway for cancer immunotherapy ^[68]. SOCE maintains adequate Ca²⁺ levels in the ER lumen during Ca²⁺ signaling, and disruption of ER Ca²⁺ homeostasis activates the UPR to restore protein homeostasis ^[67].

The imbalance in ER Ca²⁺ homeostasis consists of two aspects: Ca²⁺ depletion and Ca²⁺ overload. Ca²⁺ uptake from the cytoplasm into the ER is mediated by SERCA. TG, a SERCA inhibitor, causes Ca²⁺ leakage from the ER, leading to ER dysfunction and activation of the UPR^[69]. IP3R is activated through GPCR-mediated activation of PLC, which produces IP3. Sustained Ca²⁺ leakage from the ER is mediated by a range of proteins, such as Sec61 or Bcl-2. Upon depletion of ER Ca²⁺ stores, dissociation of Ca²⁺ from STIM proteins leads to STIM oligomerization and interaction with the plasma membrane Ca²⁺ channel Orai, increasing Ca²⁺ influx from the extracellular space. In addition to inducing SOCE, prolonged ER Ca²⁺ depletion is also a potent inducer of ER stress and will lead to destabilization of the ER, misfolding of proteins, and activation of the three branches of the UPR. SERCA is localized in the ER- PM Ca²⁺ microregion, allowing the ER Ca²⁺ store to be replenished repopulate^{[70, 71]}. Within the physiological range, when the ER is depleted of Ca²⁺, the occupancy of Ca²⁺-binding sites decrease, which may, in turn, affect protein folding^[72]. Fine-tuning the UPR response determines whether cells survive or apoptosis under ER stress^[70]. Under ER Stress, unfolded proteins displace UPR sensor-bound BIPs, leading to activation of the UPR pathway. During ER Ca²⁺ depletion, the relative acceleration of ADT to ATP exchange promotes substrate release, leading to BIP dissociation from the UPR sensor. Bip, a particularly important Ca²⁺-binding protein, is involved in sensing the accumulation of misfolded proteins in the ER and regulates the UPR together with three ER transmembrane proteins; ATF6, IREI and PERK. BIP also plays an important role as a Ca²⁺ buffering protein in the ER. In addition, It is now generally accepted that ER Ca²⁺ depletion leads to ER stress. At high Ca²⁺ concentrations, on the other hand, BIP stabilizes its interaction with nascent polypeptides^{[72, 73]}. However ER Ca²⁺ overload is also an crucial factor. TMCO1 acts as a Ca²⁺ overload-activated ion channel in the ER. Defects in TMCO1 lead to Ca²⁺ overload, providing a useful cellular model to study the effect of ER Ca²⁺ overload on ER stress. ER Ca²⁺ overload occurs in various diseases, including Alzheimer's disease, ischemic stroke, abnormal bone development and oral disease^[74]. It has been found that in mouse granulosa cells, deletion of TMCO1 protein leads to ER Ca²⁺ overload, which ER stress^[75]. Further studies showed that overloaded ER Ca²⁺ in TMCO1-deficient cells lead to the BIP dissociation from IRE1α and promote IRE1α dimerization , thereby enhancing promoting the IRE1 αactivation^[74].

ER mitochondrial contact sites (MAMs) and Ca²⁺ homeostasis

In addition, there are dynamic membrane contact sites (ER-Mitochondrial contact sites) between the ER and mitochondria, which function as a dynamic platform called MAMs. Through the MAM structure, the ER is able to transmit appropriate Ca²⁺ signals to the mitochondria. At MAMs, SERCA transports Ca2+ from the cytoplasm to the ER, and IP3Rs and RyRs release Ca2+ from the ER, leading to a rapid increase in intracellular Ca2+. The mitochondria interpret these signals and use them as a specific input to regulate basic cellular functions. MAMs are key regions for Ca²⁺ transport. Glucose regulatory protein 75(GRP75) links IP3R, a channel responsible for calcium efflux from the ER, to the mitochondrial voltage-dependent ion channel VDAC1 to regulate mitochondrial Ca²⁺ uptake. ER-mitochondrial interactions are physiologically important in the regulation of Ca²⁺ homeostasis. Ca²⁺ released from the ER passes through the outer mitochondrial membrane via the VDAC1 channel, reaches the intermembrane space, and is transferred to the mitochondrial matrix via the mitochondrial calcium uniporter (MCU) complex. The study of ER-mitochondrial interactions and Ca²⁺ homeostasis will be highly significant for understanding the pathogenesis of related diseases and developing effective prevention and treatment strategies^[76]. Imbalance of Ca²⁺ homeostasis between these organelles contribute to various physiological and pathological processes, Many cancer-associated factors are linked to Ca²⁺ regulated proteins localized in MAMs.

Ⅲ. ER stress and Ca²⁺ regulation in oral diseases

1. Salivary gland diseases

Salivary glands secrete saliva in the mouth, which aids in taste perception, food chewing, and digestion of starches and lipids. For optimal oral health, a basal flow of saliva must be maintained to protect the mouth from bacterial infections, regulate the pH level, maintain the integrity of the oral mucosa, and keep the mouth clean. The key event in fluid secretion is the increase in intracellular Ca²⁺ concentration ([Ca²⁺]i) caused by inositol 1,4,5-trisphosphate (IP3) via IP3 receptor (IP3R)-induced Ca²⁺ release from the ER. However, abnormal Ca²⁺ signaling in the salivary glands can lead to oral dryness, loss of taste, salivation disorders, and other oral diseases^[77]. IP3Rs determine the site and mode of initiation of intracellular [Ca²⁺]i signaling. However, Ca²⁺ entry into the cell is essential to sustain [Ca²⁺]i elevation and fluid secretion, which is mediated by the SOCE mechanism. Orai1, TRPC1, TRPC3 and STIM1 are identified as key components of SOCE in these cells. It has been found that SERCA activity is inhibited in the submandibular gland of streptozotocin-induced diabetic rats. Cells finely regulate the generation and amplification of [Ca²⁺]i signaling to maintain cellular function. As a result, ER Ca²⁺ and IP3-induced increases in cytoplasimic Ca²⁺ are reduced^[78]. A reduction in ER Ca²⁺ leads to improper post-translational processing, protein folding and ER proteins export^[79]. Unregulated [Ca2+]i signaling can directly lead to cellular damage, dysfunction, and disease. This could explain the reduced salivary protein content and amylase activity. Abnormal [Ca²⁺]i signaling can exacerbate and accelerate cellular damage. These defects are associated with altered function or expression of key Ca²⁺ signaling components, such as STIM proteins and TRP channels. This Ca²⁺ signaling defect has been described in the salivary glands in association with radiation-induced salivary gland dysfunction and autoimmune ectodermal gland disease, Sjögren's syndrome. These studies provide new avenues for investigating the underlying mechanisms of disease while aiding in the developing new clinical targets and therapeutic strategies^[78].

2. Gingival and periodontal disease

Chronic inflammatory periodontal disease manifests clinically as two distinct forms；gingivitis and periodontitis. Gingivitis is caused by a bacterial biofilm that accumulates on the teeth near the gums. As the mildest form of periodontal disease, gingivitis does not cause attachment loss and can be reversed with proper treatment. However, periodontitis leads to loss of connective tissue and bone support, and the pathogenesis of periodontal disease is thought to be infection-induced inflammatory tissue destruction. At the site of tissue destruction, proinflammatory and anti-inflammatory cytokines, as well as inflammatory mediators, are elevated. In addition, various enzymes (and their inhibitors) involved in matrix degradation and alveolar bone resorption are increased^[80]. In patients with periodontitis, antibody synthesis is significantly upregulated in gingival lesions. Due to accelerated protein synthesis, the transcriptional upregulation of these molecules may induce ER stress via the UPR or ER activation. The UPR enables cells to reduce burden unfolded proteins in the ER through translational attenuation, transcriptional induction of ER chaperones, and the degradation of misfolded proteins. In Gram-positive bacteria, peptidoglycan (PGN) makes up 90% of the bacterial cell wall, and PGN is an important bacterial component in periodontal disease. Cells that are unable to cope with ER stress undergo apoptosis^[69]. PGN-induced Ca²⁺ signaling triggers the secretion of pro-inflammatory cytokines such as interleukin-8 (IL-8). It has been found that PGN induces an increase in intracellular Ca²⁺ levels through the PLC/IP3 pathway, which in turn enhances IL-8 mRNA expression. Furthermore, IL-8 mRNA expression is dependent on intracellular Ca²⁺ levels in human gingival epithelial cells. The development and application of intracellular Ca²⁺ inhibitors may provide novel therapeutic for approaches of periodontal disease^[81].

3. Oral squamous cell carcinoma

Oral cancer is the sixth most common type of cancer worldwide, With Oral squamous cell carcinoma (OSCC) accounting for approximately 90% of cases. The tongue is the most common site of OSCC, followed by the lips. OSCC is highly aggressive, with high morbidity and mortality rates. Currently, surgery alone or in combination with radiotherapy or chemotherapy is the primary treatment option for OSCC patients^[82]. However, these treatments can lead to facial disfigurement and impair affect eating and speech function. Despite treatment the prognosis for OSCC remains poor, as evidenced by high rates of recurrence, metastasis, and drug resistance^[83]. Some mechanisms, such as ER stress, can regulate apoptotic signaling. Thus, the search for novel therapeutic strategies for OSCC is crucial. Under pathological conditions or drug-induced Ca²⁺ metabolism disorders, ER stress and UPR may be triggered due to the Ca²⁺-dependent function of ER molecular chaperones. ER stress is strongly associated with tumor progression. It has been found that Asiatic acid (AA) treatment significantly increases intracellular Ca²⁺ levels and calpain expression in tongue cancer cells^[42]. Studies have found that ER stress induces apoptosis in various cancer cells, including malignant melanoma, hepatocellular carcinoma, cervical cancer and squamous cells carcinoma^[84]. Capsazepine (CPZ) also sensitizes OSCC cells and other cancer types to apoptosis^[85]. AA treatment upregulated Grp78 expression and enhance IRE1α and JNK phosphorylation in tongue cancer cells, suggesting that AA induces apoptosis through activation of the ER Grp78/IRE1α/JNK signaling pathway. One study^[86] using capsazepine (CIDD-99) not only reduced Ca²⁺ entry, but also lowered ER Ca²⁺ levels, inducing ER stress and leading to apoptosis in oral cancer cells. Additionally, mitochondrial function was also found to be inhibited by CIDD-99, which may be due to loss of calcium homeostasis. Recent studies have shown that ER-mitochondrial crosstalk play a crucial role in determining cell fate by regulating mitochondrial Ca²⁺ homeostasis and metabolism^[87].

4. DSSP and ER stress in OSCC

Dentin salivary phosphoprotein (DSPP) is up regulated in various human cancers, including head and neck squamous cell carcinoma. Cancer cells are typically experience chronic ER stress due to genetic mutations and stressful microenvironments leading to increase levels of misfolded proteins. It has been suggested that DSPP silencing may disrupt of ER Ca²⁺ homeostasis in OSCC cells. DSPP silencing resulted in decreased expression of Bcl2 and PCNA, while increasing in Bax and cytochrome C levels. Downregulation of DSPP has been shown to decrease cell proliferation and increase apoptosis in OSCC cancer cells. In addition, ER stress enhances Bax translocation and insertion into the ER membrane, while Bax or Bak upregulation induces ER Ca²⁺ efflux and cytochrome c release from mitochondria. In conclusion, a detailed understanding of how cancer cells survive under ER Stress may aid in development of better therapeutic strategies for OSCC. Down-regulation of DSPP in OSCC cells affects major ER stress-related proteins (GRP78, SERCA2b, and UPR sensor proteins), leading to UPR disfunction and systemic failure^[88].

5. Odontodysplasia

Enamel formation is a complex process that is tightly regulated by enamel-forming cells. Traditionally, Amelogenesis imperfecta (AI) has been studied from the perspective that mutations in genes encoding secreted enamel matrix proteins affect proteins function and behavior within the extracellular matrix. The correct spatial pattern of enamel matrix protein (EMP) expression is essential for coordinated enamel crystal formation, which depends on a strong calcium ions supply^[89]. It has been demonstrated that stimulation of LS8 cells or mouse primary enamel organoids with TG to activate SOCE resulted in increased expression of Amelx, Ambn, Enam, and Mmp20. However, recent finding suggest that AI may also be associated with intracellular phenomena, particularly ER stress and UPR activation in enamel- forming cells under certain conditions^[90]. LS8 cells express the CRAC channel components STIM and ORAI1. Activation of SOCE by ER Ca²⁺ depletion cause a conformational change of Ca²⁺ sensors in ER STIM1 to aggregate in specific regions of the ER (called puncta). This effect is reversed when cells are treated with CRAC channel inhibitors. Ca²⁺ endocytosis is reported to be virtually eliminated in LS8 cells pretreated with pharmacological CRAC channel inhibitors, suggesting that SOCE in LS8 cells is mediated by CRAC channels. This suggests that Ca²⁺ influx is mediated by CRAC channels in LS8 cells and enamel organoids, and that Ca²⁺ signaling enhances the EMPs expression^[89]. These findings are consistent with the observation of severely hypocalcified enamel in patients with mutations in the CRAC channel genes STIM1 and ORAI1. Ca²⁺ plays an essential role not only in enamel mineralization but also in regulating the EMPs expression. Excessive fluoride consumption can lead to dental fluorosis. Studies have shown that in enamel cells, fluoride exposure affects the function of the ER-localized Ca²⁺ channel IP3R and the activity of the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump during Ca²⁺ refilling ^[91].

6. Temporomandibular joint disorders

Temporomandibular Joint disease (TMD) is an osteoarthritis (OA)-like condition characterized by cartilage thinning, followed by subchondral bone erosion, synovitis, and chronic pain. Therefore, cartilage thinning is considered a key event in the onset and progression of TMD. Studies using in vivo and in vitro models have found that mechanical stress can induce ER stress in mandibular chondrocytes. ER stress as well as ER stress-mediated apoptosis can be mitigated by inhibiting IP3R and RyR, suggesting that Ca²⁺ signaling plays a critical role in mechanical stress-mediated chondrocyte apoptosis and cartilage thinning^[92]. Recent studies have found that the phosphatidylinositol 3-kinase/ Akt pathway plays an important role in the regulation of chondrocyte apoptosis and survival and may help prevent OA. Therefore, novel drug targets are being investigated for the treatment of TMD. In addition, hypoxia-inducible factor-2α(HIF2α) activatesβ-catenin and nuclear factor- KB（NF-kB） pathways to promote chondrocyte apoptosis. However, the crosstalk between these pathways remains unclear. Further studies are required to explore Ca²⁺ independent mechanisms^[93].

Ⅳ. TREATMENT

1. Photobiomodulation

Photobiomodulation (PBM), also known as low-level laser therapy, involves the interaction of specific wavelengths of natural or artificial light with living cells to trigger various biological Responses. It is non-invasive and widely used. In recent years, red or near-infrared (NIR) applications have been among the most widely used light sources, with wavelengths ranging from 620nm to 670 nm. PBM is utilized for irradiation therapy to relieve pain, reduce inflammation, and accelerate wound healing. It is absorbed by cytochrome C oxidase (CCO), the terminal enzyme of the mitochondrial respiratory chain, which produces reactive oxygen species (ROS) and adenosine triphosphate (ATP)^[94]. Low-intensity NIR light irradiation has been reported to reduce intracellular Ca²⁺ overload and decrease ER stress. Treatment with low-level laser therapy (LLLT) at 850 nm reduces high glucose-induced intracellular Ca²⁺ overload and ER stress via downregulation of Caspase-12 and CHOP. In addition, ROS production, which activates Ca²⁺ release from the ER, is considered a potential light-dependent signaling pathway^[95-97]. Further comparisons using 650nm light irradiation revealed that low-intensity light at 650nm rapidly increased intracellular Ca²⁺ in neuronal cells by 88% within the first 1-2 minutes, followed by decrease. Zhang et al used the human mast cell line HMC-1 to demonstrate that 640 nm RED light irradiation increase intracellular Ca²⁺ concentration and activate TRPV2 leading to Ca²⁺ channel opening^[98].

2. Calcium Targeted Therapy

Calcium Targeted Therapy considered a novel approach in tumor therapy ^[99]. This strategy involves the use of exogenous Ca²⁺ and multimodal endogenous Ca²⁺ regulation through two main mechanisms. Exogenous Ca²⁺ sources include calcium carbonate (CaCO3), calcium phosphate (Ca₃(Po₄)₂), and calcium peroxide (Cao2). These nanomaterials exhibit high transport capacity and can be modified for targeted delivery to tumor site. Endogenous Ca²⁺ regulation involves modulating Ca²⁺ channels, membrane integrity, and ER stress. However, the ability of tumor cells to buffer intracellular Ca²⁺ concentrations via Ca²⁺ pumps and ER Ca²⁺ stores significantly limits the effectiveness of Calcium targeted therapy.

Ⅴ. CONCLUSION and EXPECTATION

ER stress and Ca²⁺ homeostasis are intricately connected. Ca²⁺ not only play a key role in signaling for ER stress but also influences cellular physiological responses by regulating the ER Ca²⁺ balance. When the ER is subjected to stress, Ca²⁺ release increase, from the ER, which process not only activates the ER stress pathway, but also regulate apoptosis and survival decisions through Ca²⁺-dependent signaling mechanisms. As research progresses, growing evidence suggest that the ER stress and Ca²⁺ homeostasis play an crucial role in the onset and progression various oral diseases. For example, abnormal release of Ca²⁺ may promote the malignant growth in oral cancer cells or intensity localized inflammatory responses in periodontal disease. In oral diseases, ER stress exacerbates tissue damage through Ca²⁺ imbalance. ER stress and Ca²⁺ dysregulation significantly impact on the development and progression of oral diseases. Therefore, modulation of ER stress and Ca²⁺ homeostasis may serve as an effective strategy for oral diseases.

In the future, Targeting ER stress and Ca²⁺ imbalance may provide new breakthroughs in the prevention and treatment of oral diseases. By working together to regulate cellular function and immune responses, they influence pathophysiology of condition such as oral cancer, periodontal disease, and dental caries. Further in-depth study on their mechanism will provide new theoretical basis and therapeutic targets for clinical application.