Mild Endoplasmic Reticulum Stress Research Articles

Unfolded protein response during cardiovascular disorders: a tilt towards pro-survival and cellular homeostasis.

The endoplasmic reticulum (ER) is an organelle that orchestrates the production and proper assembly of an extensive types of secretory and membrane proteins. Endoplasmic reticulum stress is conventionally related to prolonged disruption in the protein folding machinery resulting in the accumulation of unfolded proteins in the ER. This disruption is often manifested due to oxidative stress, Ca2+ leakage, iron imbalance, disease conditions which in turn hampers the cellular homeostasis and induces cellular apoptosis. A mild ER stress is often reverted back to normal. However, cells retaliate to acute ER stress by activating the unfolded protein response (UPR) which comprises three signaling pathways, Activating transcription factor 6 (ATF6), inositol requiring enzyme 1 alpha (IRE1α), and protein kinase RNA-activated-like ER kinase (PERK). The UPR response participates in both protective and pro-apoptotic responses and not much is known about the mechanistic aspects of the switch from pro-survival to pro-apoptosis. When ER stress outpaces UPR response then cell apoptosis prevails which often leads to the development of various diseases including cardiomyopathies. Therefore, it is important to identify molecules that modulate the UPR that may serve as promising tools towards effective treatment of cardiovascular diseases. In this review, we elucidated the latest advances in construing the contribution imparted by the three arms of UPR to combat the adverse environment in the ER to restore cellular homeostasis during cardiomyopathies. We also summarized the various therapeutic agents that plays crucial role in tilting the UPR response towards pro-survival.

EIF2α-ATF4 Pathway Activated by a Change in the Calcium Environment Participates in BCP-Mediated Bone Regeneration.

Biphasic calcium phosphate (BCP) ceramic is a classic bone void filler and a common basis of new materials for bone defect repair. However, the specific mechanism of BCP in osteogenesis has not been fully elucidated. Endoplasmic reticulum stress (ERs) and the subsequent PERK-eIF2α-ATF4 pathway can be activated by various factors, including trauma and intracellular calcium changes, and therefore worth exploring as a potential mechanism in BCP-mediated bone repair. Herein, a rat lateral femoral epicondyle defect model in vivo and a simulated BCP-mediated calcium environment in vitro were constructed for the analysis of BCP-related osteogenesis and the activation of ERs and the eIF2α-ATF4 pathway. An inhibitor of eIF2α dephosphorylation (salubrinal) was also used to explore the effect of the eIF2α-ATF4 pathway on BCP-mediated bone regeneration. The results showed that the ERs and eIF2α-ATF4 pathway activation were observed during 4 weeks of bone repair, with a rapid but brief increase immediately after artificial defect surgery and a re-increase after 4 weeks with the resorption of BCP materials. Mild ERs and the activated eIF2α induced by the calcium changes mediated by BCP regulated the expression of osteogenic-related proteins and had an important role during the defect repair. In conclusion, the eIF2α-ATF4 pathway activated by a change in the calcium environment participates in BCP-mediated bone regeneration. eIF2α-ATF4 and ERs could provide new directions for further studies on new materials in bone tissue engineering.

Regeneration in starved planarians depends on TRiC/CCT subunits modulating the unfolded proteinresponse.

Planarians are able to stand long periods of starvation by maintaining adult stem cell pools and regenerative capacity. The molecular pathways that are needed for the maintenance of regeneration during starvation are not known. Here, we show that down‐regulation of chaperonin TRiC/CCT subunits abrogates the regeneration capacity of planarians during starvation, but TRiC/CCT subunits are dispensable for regeneration in fed planarians. Under starvation, they are required to maintain mitotic fidelity and for blastema formation. We show that TRiC subunits modulate the unfolded protein response (UPR) and are required to maintain ATP levels in starved planarians. Regenerative defects in starved CCT‐depleted planarians can be rescued by either chemical induction of mild endoplasmic reticulum stress, which leads to induction of the UPR, or by the supplementation of fatty acids. Together, these results indicate that CCT‐dependent UPR induction promotes regeneration of planarians under food restriction.

Stimulation of myogenesis by ascorbic acid and capsaicin

Myogenesis is a complex process regulated by several factors. This study evaluated the functional interaction between vitamin C and a high dose of capsaicin (a potential endoplasmic reticulum (ER) stress inducer) on myogenesis. After the induction of differentiation, treatment with ascorbic acid or ascorbic acid phosphate (AsAp) alone had minimal effects on myogenesis in C2C12 cells. However, treatment with capsaicin (300 μM) in undifferentiated C2C12 cells increased the expression levels of genes related to ER stress as well as oxidative stress. Myogenesis was effectively enhanced in C2C12 cells treated with a combination of capsaicin (300 μM) for one day before differentiation stimulation and AsAp for four days post-differentiation; subsequently, thick and long myotubes formed, and the expression levels of myosin heavy chain (MYH) 1/2 and Myh1, Myh4, and Myh7 increased. Considering that mild ER stress stimulates myogenesis, AsAp may elicit myogenesis through the alleviation of oxidative stress-induced negative effects in capsaicin-pretreated cells. The enhanced expression of Myh1 and Myh4 coincided with the expression of Col1a1, a type I collagen, suggesting that the fine-tuning of the myogenic cell microenvironment is responsible for efficient myogenesis. Our results indicate that vitamin C is a potential stimulator of myogenesis in cells, depending on the cell context.

Downregulation of ceramide synthase 1 promotes oral cancer through endoplasmic reticulum stress

C18 ceramide plays an important role in the occurrence and development of oral squamous cell carcinoma. However, the function of ceramide synthase 1, a key enzyme in C18 ceramide synthesis, in oral squamous cell carcinoma is still unclear. The aim of our study was to investigate the relationship between ceramide synthase 1 and oral cancer. In this study, we found that the expression of ceramide synthase 1 was downregulated in oral cancer tissues and cell lines. In a mouse oral squamous cell carcinoma model induced by 4-nitroquinolin-1-oxide, ceramide synthase 1 knockout was associated with the severity of oral malignant transformation. Immunohistochemical studies showed significant upregulation of PCNA, MMP2, MMP9, and BCL2 expression and downregulation of BAX expression in the pathological hyperplastic area. In addition, ceramide synthase 1 knockdown promoted cell proliferation, migration, and invasion in vitro. Overexpression of CERS1 obtained the opposite effect. Ceramide synthase 1 knockdown caused endoplasmic reticulum stress and induced the VEGFA upregulation. Activating transcription factor 4 is responsible for ceramide synthase 1 knockdown caused VEGFA transcriptional upregulation. In addition, mild endoplasmic reticulum stress caused by ceramide synthase 1 knockdown could induce cisplatin resistance. Taken together, our study suggests that ceramide synthase 1 is downregulated in oral cancer and promotes the aggressiveness of oral squamous cell carcinoma and chemotherapeutic drug resistance.

Loss of MANF Causes Childhood-Onset Syndromic Diabetes Due to Increased Endoplasmic Reticulum Stress.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)–resident protein that plays a crucial role in attenuating ER stress responses. Although MANF is indispensable for the survival and function of mouse β-cells, its precise role in human β-cell development and function is unknown. In this study, we show that lack of MANF in humans results in diabetes due to increased ER stress, leading to impaired β-cell function. We identified two patients from different families with childhood diabetes and a neurodevelopmental disorder associated with homozygous loss-of-function mutations in the MANF gene. To study the role of MANF in human β-cell development and function, we knocked out the MANF gene in human embryonic stem cells and differentiated them into pancreatic endocrine cells. Loss of MANF induced mild ER stress and impaired insulin-processing capacity of β-cells in vitro. Upon implantation to immunocompromised mice, the MANF knockout grafts presented elevated ER stress and functional failure, particularly in recipients with diabetes. By describing a new form of monogenic neurodevelopmental diabetes syndrome caused by disturbed ER function, we highlight the importance of adequate ER stress regulation for proper human β-cell function and demonstrate the crucial role of MANF in this process.

Protective Effects of Glucose-Related Protein 78 and 94 on Cisplatin-Mediated Ototoxicity.

Cisplatin is a widely used chemotherapeutic drug for treating various solid tumors. Ototoxicity is a major dose-limiting side effect of cisplatin, which causes progressive and irreversible sensorineural hearing loss. Here, we examined the protective effects of glucose-related protein (GRP) 78 and 94, also identified as endoplasmic reticulum (ER) chaperone proteins, on cisplatin-induced ototoxicity. Treating murine auditory cells (HEI-OC1) with 25 μM cisplatin for 24 h increased cell death resulting from excessive intracellular reactive oxygen species (ROS) accumulation and caspase-involved apoptotic signaling pathway activation with subsequent DNA fragmentation. GRP78 and GRP94 expression was increased in cells treated with 3 nM thapsigargin or 0.1 μg/mL tunicamycin for 24 h, referred to as mild ER stress condition. This condition, prior to cisplatin exposure, attenuated cisplatin-induced ototoxicity. The involvement of GRP78 and GRP94 induction was demonstrated by the knockdown of GRP78 or GRP94 expression using small interfering RNAs, which abolished the protective effect of mild ER stress condition on cisplatin-induced cytotoxicity. These results indicated that GRP78 and GRP94 induction plays a protective role in remediating cisplatin-ototoxicity.

The Impact of Sugar-Sweetened Beverage Consumption on the Liver: A Proteomics-based Analysis.

Cardiometabolic complications such as the metabolic syndrome and Type 2 Diabetes Mellitus (T2DM) are major causes of global morbidity and mortality. As sugar-sweetened beverages (SSBs) are implicated in this process, this study aimed to obtain greater mechanistic insights. Male Wistar rats (~200 g) were gavaged with a local SSB every day for a period of six months while the control group was gavaged with an iso-volumetric amount of water. Experimental dosages were calculated according to the surface area-to-volume ratio and were equivalent to 125 mL/day (in human terms). A proteomic analysis was performed on isolated liver samples and thereafter, markers of endoplasmic reticulum (ER) stress, antioxidant/oxidant capacity, calcium regulation, and mitochondrial functionality were assessed. These data show that SSB consumption resulted in (a) the induction of mild hepatic ER stress; (b) altered hepatic mitochondrial dynamics; and (c) perturbed calcium handling across mitochondria-associated ER membranes. Despite significant changes in markers of ER stress, the antioxidant response and calcium handling (proteomics data), the liver is able to initiate adaptive responses to counteract such stressors. However, the mitochondrial data showed increased fission and decreased fusion that may put the organism at risk for developing insulin resistance and T2DM in the longer term.

Fat Mass and Obesity-Associated (FTO) Stimulates Osteogenic Differentiation of C3H10T1/2 Cells by Inducing Mild Endoplasmic Reticulum Stress via a Positive Feedback Loop with p-AMPK.

Fat mass and obesity-associated (FTO) gene helps to regulate energy homeostasis in mammals by controlling energy expenditure. In addition, FTO functions in the regulation of obesity and adipogenic differentiation; however, a role in osteogenic differentiation is unknown. This study investigated the effects of FTO on osteogenic differentiation of C3H10T1/2 cells and the underlying mechanism. Expression of osteogenic and endoplasmic reticulum (ER) stress markers were characterized by reverse-transcriptase polymerase chain reaction and western blotting. Alkaline phosphatase (ALP) staining was performed to assess ALP activity. BMP2 treatment increased mRNA expression of osteogenic genes and FTO. Overexpression of FTO increased expression of the osteogenic genes distal-less homeobox5 (Dlx5) and runt-related transcription factor 2 (Runx2). Activation of adenosine monophosphate-activated protein kinase (AMPK) increased FTO expression, and there was a positive feedback loop between FTO and p-AMPK. p-AMPK and FTO induced mild ER stress; however, tunicamycin-induced severe ER stress suppressed FTO expression and AMPK activation. In summary, FTO induces osteogenic differentiation of C3H10T1/2 cells upon BMP2 treatment by inducing mild ER stress via a positive feedback loop with p-AMPK. FTO expression and AMPK activation induce mild ER stress. By contrast, severe ER stress inhibits osteogenic differentiation by suppressing FTO expression and AMPK activation.

Arabidopsis GAAP1 to GAAP3 Play Redundant Role in Cell Death Inhibition by Suppressing the Upregulation of Salicylic Acid Pathway Under Endoplasmic Reticulum Stress.

The unfolded protein response (UPR) is activated to sustain cell survival by reducing misfolded protein accumulation in the endoplasmic reticulum (ER). The UPR also promotes cell death when the ER stress is severe. However, the underlying molecular mechanisms of UPR activity regulation and cell death transition are less understood in plants. Arabidopsis GAAP1 and GAAP3 are involved in the regulation of UPR and cell death. Five GAAP gene members are found in Arabidopsis. Here, we analyzed the function of GAAP2 in addition to GAAP1 and GAAP3 in ER stress response using single, double, and triple mutants. Results showed that single or double or triple mutants reduced plant survival and enhanced cell death under ER stress. And the sensitivity increased with the number of mutation genes increase. Quantitative real-time polymerase chain reaction analysis showed that mutation in triple genes promoted UPR signaling when confronted with mild ER stress, advanced SA target genes upregulation when confronted with severe stress. Moreover, Quantitative detection by UPLC-ESI-MS/MS showed that ER stress upregulated salicylic acid (SA) content in plants. These data suggest that GAAP1 to GAAP3 played redundant roles in cell death resistance and fine tuning UPR activation. And the anti-cell death function of GAAPs might be achieved by impairing the up-regulation of the SA pathway under ER stress.

Dietary restriction improves proteostasis and increases life span through endoplasmic reticulum hormesis

Unfolded protein response (UPR) of the endoplasmic reticulum (UPRER) helps maintain proteostasis in the cell. The ability to mount an effective UPRER to external stress (iUPRER) decreases with age and is linked to the pathophysiology of multiple age-related disorders. Here, we show that a transient pharmacological ER stress, imposed early in development on Caenorhabditis elegans, enhances proteostasis, prevents iUPRER decline with age, and increases adult life span. Importantly, dietary restriction (DR), that has a conserved positive effect on life span, employs this mechanism of ER hormesis for longevity assurance. We found that only the IRE-1-XBP-1 branch of UPRER is required for the longevity effects, resulting in increased ER-associated degradation (ERAD) gene expression and degradation of ER resident proteins during DR. Further, both ER hormesis and DR protect against polyglutamine aggregation in an IRE-1-dependent manner. We show that the DR-specific FOXA transcription factor PHA-4 transcriptionally regulates the genes required for ER homeostasis and is required for ER preconditioning-induced life span extension. Finally, we show that ER hormesis improves proteostasis and viability in a mammalian cellular model of neurodegenerative disease. Together, our study identifies a mechanism by which DR offers its benefits and opens the possibility of using ER-targeted pharmacological interventions to mimic the prolongevity effects of DR.

Endoplasmic Reticulum Stress and Unfolded Protein Response Pathways Involved in the Health-Promoting Effects of Allicin on the Jejunum.

Intestinal endoplasmic reticulum stress (ERS) triggered by adverse factors disturbs the normal function of the intestine. Allicin has been reported to promote intestinal health and development. In the present study, we established in vivo (35-day-old weaned piglets, 4-week-old mice) and in vitro (IPEC-J2 cell line) ERS models to explore the possible mechanisms by which allicin may benefit intestinal health. This study revealed the following: (1) allicin supplementation improved intestinal morphological indices and ameliorated mild ERS in the jejunum of the weaned piglets; (2) allicin supplementation decreased cellular reactive oxygen species and upregulated the XBP-1s signaling pathways in IPEC-J2 cells; (3) allicin supplementation reduced the prolonged ERS-mediated apoptosis of IPEC-J2 cells and in the jejunal tissues of the KM mice; (4) allicin supplementation enhanced the intercellular junction protein levels of jejunal cells by alleviating the prolonged ERS. These novel findings suggest that eating garlic could alleviate some intestinal malfunctions associated with ERS.

Environmental stresses suppress nonsense-mediated mRNA decay (NMD) and affect cells by stabilizing NMD-targeted gene expression

Nonsense-mediated mRNA decay (NMD) is a cellular mechanism that eliminates mRNAs that harbor premature translation termination codons (PTCs). Here, we investigated the effects of environmental stresses (oxidative stress and endoplasmic reticulum (ER) stress) on NMD activity. Methylmercury (MeHg) was used to cause oxidative stress and thapsigargin to stress the ER. NMD suppression, evidenced by upregulation of NMD-sensitive mRNAs and a decrease in UPF1 phosphorylation, was observed in MeHg-treated myogenic cells, cerebral cortical neuronal cells, and astroglial cells. Mild ER stress amplified NMD suppression caused by MeHg. To elucidate the cause of stress-induced NMD suppression, the role of the phospho-eIF2α/ATF4 pathway was investigated. Knockdown and non-phosphorylatable eIF2α-transfection studies demonstrated the critical role of phospho-eIF2α-mediated repression of translation in mild ER stress-induced NMD suppression. However, NMD suppression was also observed in phospho-eIF2α-deficient cells under mild ER stress. Mechanistic target of rapamycin suppression-induced inhibition of cap-dependent translation, and downregulation of the NMD components UPF1, SMG7, and eIF4A3, were probably involved in stress-induced NMD suppression. Our results indicate that stress-induced NMD suppression has the potential to affect the condition of cells and phenotypes of PTC-related diseases under environmental stresses by stabilizing NMD-targeted gene expression.

Sera of Obese Type 2 Diabetic Patients Undergoing Metabolic Surgery Instead of Conventional Treatment Exert Beneficial Effects on Beta Cell Survival and Function: Results of a Randomized Clinical Study.

Pancreatic beta cells are highly sensitive to oxidative and endoplasmic reticulum (ER) stress, commonly occurring in type 2 diabetes (T2D) and obesity. We aimed at investigating cellular responses of human beta cells exposed to sera from obese T2D patients treated differently, namely by conventional therapy or laparoscopic sleeve gastrectomy (LSG). Serum samples from obese T2D men randomized to conventional treatment or LSG were taken at baseline and 6months later. After exposing 1.1B4 cells to study patients' sera, the following were assessed: cellular viability and proliferation (by MTT and xCELLigence assays), reactive oxygen species (ROS) production (with DCFH-DA), and expression of ER stress markers, oxidative- or autophagy-related proteins and insulin (by real-time PCR and Western blot). At 6-month follow-up, patients undergoing LSG achieved an adequate glycemic control, whereas conventionally treated patients did not. As compared to 1.1B4 cells incubated with baseline sera (control), cells exposed to sera from LSG-treated participants exhibited (i) increased viability and proliferation (p < 0.05); (ii) diminished levels of ROS and p53 (p < 0.05); (iii) enhanced protein expression of autophagy-related SIRT1 and p62/SQSTM1 (p < 0.05); (iv) significantly decreased transcript levels of ER stress markers (p < 0.05); and (v) augmented insulin expression (p < 0.05). Conversely, the 6-month conventional therapy appeared not to impact on circulating redox status. Moreover, 1.1B4 cells exposed to sera from conventionally treated patients experienced mild ER stress. Circulating factors in patients with improved diabetes after metabolic surgery exerted favorable effects on beta cell function and survival.

Mild Endoplasmic Reticulum Stress Protects Against Lipopolysaccharide-Induced Astrocytic Activation and Blood-Brain Barrier Hyperpermeability.

Recent research has revealed that uncontrolled chronic neuroinflammation is closely associated with diverse neurodegenerative diseases, by impairing blood-brain barrier (BBB) function and astrocytic reaction. Endoplasmic reticulum (ER) stress is conventionally linked to the loss of neuronal structure and function and should be widely attenuated. This notion has been questioned by recent experimental studies, which have shown that non-harmful levels of ER stress had numerous beneficial roles against neurodegeneration, including neuroprotection and inhibition of cytokine production. Here, we investigated the mild ER stress-based regulation of LPS-induced inflammatory responses in astrocytes. Primary astrocytes were exposed to tunicamycin (TM), a compound that activates ER stress, with or without the ER-stress inhibitor sodium 4-phenylbutyrate (4-PBA) before LPS treatment. Astrocytic activation, proinflammatory factor production, and the extent of ER stress were assessed. In addition, the effect of mild ER stress on astrocytes and BBB function was determined in vivo. Male Sprague-Dawley rats received intracerebroventricular injections of TM with or without intraperitoneal 4-PBA before LPS administration. The levels of astrocytic activation and BBB permeability were measured after treatment. Our results showed that lower doses of TM resulted in a mild ER-stress response without inducing cytotoxicity and tissue toxicity. Non-toxic ER-stress preconditioning ameliorated LPS-induced overactivation and inflammatory responses in astrocytes. Moreover, pre-exposure to non-lethal doses of TM improved LPS-induced BBB impairment and cognitive ability dysfunction in rats. However, 4-PBA, reversed the protective effect of TM preconditioning in vitro and in vivo. We conclude that mild ER stress (“preconditioning”) can alleviate LPS-induced astrocytic activation and BBB disruption. Our findings provide a better understanding for the regulatory role of ER stress in neuroinflammation and indicate that mild ER stress might have therapeutic value for the treatment of neurodegenerative diseases.

Commentary: Mild endoplasmic reticulum stress ameliorates lpopolysaccharide-induced neuroinflammation and cognitive impairment via regulation of microglial polarization.

Commentary: Mild endoplasmic reticulum stress ameliorates lpopolysaccharide-induced neuroinflammation and cognitive impairment via regulation of microglial polarization.

The unfolded protein response as a target for anticancer therapeutics

The endoplasmic reticulum (ER) is an essential organelle in eukaryotic cells, responsible for protein synthesis, folding, sorting, and transportation. ER stress is initiated when the unfolded or misfolded protein load exceeds the capacity of the ER to properly fold protein. Tumor microenvironmental conditions, such as nutrient deprivation, hypoxia, and oxidative stress perturb protein folding and trigger chronic ER stress. Cancer cells can tolerate mild ER stress, however, persistent and severe ER stress kills cancer cells by inducing their autophagy, apoptosis, necroptosis, or immunogenic cell death. Based on this rationale, many drugs have been developed for triggering irremediable ER stress in cancer cells by targeting various processes in the secretory pathway. This review discusses the mechanisms of protein targeting to the ER, the key signaling cassettes that are involved in the ER stress response, and their correlation with cancer formation and progression. Importantly, this review discusses current experimental and FDA approved anti-cancer drugs that induce ER stress, and emerging targets within the secretory pathway for the development of new anticancer drugs.

Arabidopsis GAAP1 and GAAP3 Modulate the Unfolded Protein Response and the Onset of Cell Death in Response to ER Stress.

The function of human Golgi antiapoptotic proteins (GAAPs) resembles that of BAX inhibitor-1, with apoptosis inhibition triggered by intrinsic and extrinsic stimuli. However, little is known about the function of GAAP-related proteins in plants. Here, we studied Arabidopsis GAAP1 and GAAP3 and found that they were localized on the cellular membrane, including the endoplasmic reticulum (ER) membrane. The function of GAAP1/GAAP3 in ER-stress response was tested, and results showed that single or double mutation in GAAP1 and GAAP3 reduced plant survival and enhanced cell death under ER stress. The expression of both genes was induced by various abiotic stress signals. Quantitative real-time polymerase chain reaction analysis showed that GAAP1/GAAP3 level affected the expression pattern of the unfolded-protein response (UPR) signaling pathway genes upon prolonged ER stress. The mutation in both GAAP1 and GAAP3 genes promoted and enhanced UPR signaling when confronted with mild ER stress. Moreover, GAAP1/GAAP3 inhibited cell death caused by ER stress and promoted plant-growth recovery by turning down inositol-requiring enzyme 1 (IRE1) signaling after ER stress had been relieved. Co-immunoprecipitation (Co-Ip) and BiFC assays showed that GAAP1/GAAP3 interacted with IRE1. These data suggested that GAAP1/GAAP3 played dual roles in the negative regulation of IRE1 activity and anti-programmed cell death.

Curcumin induces osteoblast differentiation through mild-endoplasmic reticulum stress-mediated such as BMP2 on osteoblast cells

AimsCurcumin (diferuloylmethane or [1E,6E]-1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6heptadiene-3,5-dione) is a phenolic natural product derived from the rhizomes of the turmeric plant, Curcuma longa. It is reported to have various biological actions such as anti-oxidative, anti-inflammatory, and anti-cancer effects. However, the molecular mechanism of osteoblast differentiation by curcumin has not yet been reported. Main methodsThe cytotoxicity of curcumin was identified using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Expression of osteogenic markers and endoplasmic reticulum (ER) stress markers in C3H1-T1/2 cells were measured using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Alkaline phosphatase (ALP) staining was performed to assess ALP activity in C3H10T1/2 cells. Transcriptional activity was detected using a luciferase reporter assay. Key findingsCurcumin increased the expression of genes such as distal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OC), which subsequently induced osteoblast differentiation in C3H10T1/2 cells. In addition, ALP activity and mineralization was found to be increased by curcumin treatment. Curcumin also induced mild ER stress similar to bone morphogenetic protein 2 (BMP2) function in osteoblast cells. Next, we confirmed that curcumin increased mild ER stress and osteoblast differentiation similar to BMP2 in C3H10T1/2 mesenchymal stem cells. Transient transfection studies also showed that curcumin increased ATF6-Luc activity, while decreasing the activities of CREBH-Luc and SMILE-Luc. In addition, similar to BMP2, curcumin induced the phosphorylation of Smad 1/5/9. SignificanceOverall, these results demonstrate that curcumin-induced mild ER stress increases osteoblast differentiation via ATF6 expression in C3H10T1/2 cells.

Mild endoplasmic reticulum stress ameliorates lipopolysaccharide-induced neuroinflammation and cognitive impairment via regulation of microglial polarization

BackgroundNeuroinflammation, which ultimately leads to neuronal loss, is considered to play a crucial role in numerous neurodegenerative diseases. The neuroinflammatory process is characterized by the activation of glial cells such as microglia. Endoplasmic reticulum (ER) stress is commonly associated with impairments in neuronal function and cognition, but its relationship and role in neurodegeneration is still controversial. Recently, it was confirmed that nonharmful levels of ER stress protected against experimental Parkinson’s disease. Here, we investigated mild ER stress-based regulation of lipopolysaccharide (LPS)-driven neuroinflammation in rats and in primary microglia.MethodsMale Sprague–Dawley (SD) rats received the intracerebroventricular injection of the ER stress activator tunicamycin (TM) with or without intraperitoneal injection of the ER stress stabilizer sodium 4-phenylbutyrate (4-PBA) 1 h before LPS administration. The levels of neuroinflammation and memory dysfunction were assessed 24 h after treatment. In addition, the effect of mild ER stress on microglia was determined in vitro.ResultsHere, we found that low doses of TM led to mild ER stress without cell or organism lethality. We showed that mild ER stress preconditioning reduced microglia activation and neuronal death as well as improved LPS-induced memory impairment in rats. In addition, pre-exposure to nonlethal doses of TM in microglia showed significant protection against LPS-induced proinflammatory cytokine production and M1/2b polarization. However, sodium 4-PBA, a compound that ameliorates ER stress, ablated this protective effect in vivo and in vitro.ConclusionsBased on our findings, we conclude that the mild ER stress not only limits the accumulation of misfolded proteins but also protects tissues from harmful endotoxemia insults. Therefore, ER stress preconditioning has potential therapeutic value for the treatment of neurodegenerative diseases.