TM-induced Apoptosis Research Articles

SIRT1 inhibits apoptosis of human lens epithelial cells through suppressing endoplasmic reticulum stress in vitro and in vivo.

To explore the effect of silent information regulator factor 2-related enzyme 1 (SIRT1) on modulating apoptosis of human lens epithelial cells (HLECs) and alleviating lens opacification of rats through suppressing endoplasmic reticulum (ER) stress. HLECs (SRA01/04) were treated with varying concentrations of tunicamycin (TM) for 24h, and the expression of SIRT1 and C/EBP homologous protein (CHOP) was assessed using real-time quantitative polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Cell morphology and proliferation was evaluated using an inverted microscope and cell counting kit-8 (CCK-8) assay, respectively. In the SRA01/04 cell apoptosis model, which underwent siRNA transfection for SIRT1 knockdown and SRT1720 treatment for its activation, the expression levels of SIRT1, CHOP, glucose regulated protein 78 (GRP78), and activating transcription factor 4 (ATF4) were examined. The potential reversal of SIRT1 knockdown effects by 4-phenyl butyric acid (4-PBA; an ER stress inhibitor) was investigated. In vivo, age-related cataract (ARC) rat models were induced by sodium selenite injection, and the protective role of SIRT1, activated by SRT1720 intraperitoneal injections, was evaluated through morphology observation, hematoxylin and eosin (H&E) staining, Western blotting, and RT-PCR. SIRT1 expression was downregulated in TM-induced SRA01/04 cells. Besides, in SRA01/04 cells, both cell apoptosis and CHOP expression increased with the rising doses of TM. ER stress was stimulated by TM, as evidenced by the increased GRP78 and ATF4 in the SRA01/04 cell apoptosis model. Inhibition of SIRT1 by siRNA knockdown increased ER stress activation, whereas SRT1720 treatment had opposite results. 4-PBA partly reverse the adverse effect of SIRT1 knockdown on apoptosis. In vivo, SRT1720 attenuated the lens opacification and weakened the ER stress activation in ARC rat models. SIRT1 plays a protective role against TM-induced apoptosis in HLECs and slows the progression of cataract in rats by inhibiting ER stress. These findings suggest a novel strategy for cataract treatment focused on targeting ER stress, highlighting the therapeutic potential of SIRT1 modulation in ARC development.

ER stress increases expression of intracellular calcium channel RyR1 to modify Ca2+ homeostasis in pancreatic beta cells

Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in plasma glucose. Pulsatile insulin secretion occurs as a result of glucose-induced oscillations in beta-cell cytosolic Ca2+. The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ reduction, beta-cell dysfunction, and an increased risk of type 2 diabetes. However, the mechanistic effects of ER stress on individual calcium channels are not well understood. To determine the effects oftunicamycin-induced ER stress on ER inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, we treated INS-1 832/13cells and primary mouse islets with ER stress inducer tunicamycin (TM). We showed TM treatment increased RyR1 mRNA without affecting RyR2 mRNA and decreased both IP3R1 and IP3R3 mRNA. Furthermore, we found stress reduced ER Ca2+ levels, triggered oscillations in cytosolic Ca2+ under subthreshold glucose conditions, and increased apoptosis and that these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In addition, we demonstrated silencing RyR1-suppressed TM-induced subthreshold cytosolic Ca2+ oscillations, but silencing RyR2 did not affect these oscillations. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the TM-induced cytosolic Ca2+ oscillations and did not protect beta cells from TM-induced apoptosis although xestospongin-C inclusion did prevent ER Ca2+ reduction. Taken together, these resultsshowchanges in RyR1 play a critical role in ER stress-induced Ca2+ dysfunction and beta-cell apoptosis.

Overexpression of lncRNA Dancr inhibits apoptosis and enhances autophagy to protect cardiomyocytes from endoplasmic reticulum stress injury via sponging microRNA-6324.

Endoplasmic reticulum stress (ERS) contributes to the pathogenesis of myocardial ischemia/reperfusion injury and myocardial infarction (MI). Long non-coding RNAs (lncRNAs) serve an important role in cardiovascular diseases, and lncRNA discrimination antagonizing non-protein coding RNA (Dancr) alleviates cardiomyocyte damage. microRNA (miR)-6324 was upregulated in MI model rats and was predicted to bind to Dancr. The present study aimed to investigate the role of Dancr in ERS-induced cardiomyocytes and the potential underlying mechanisms. Tunicamycin (Tm) was used to induce ERS. Cell viability, apoptosis and levels of associated proteins, ERS and autophagy in Dancr-overexpression H9C2 cells and miR-6234 mimic-transfected H9C2 cells were assessed using Cell Counting Kit-8, TUNEL staining and western blot assay, respectively. The results suggested that Dancr expression levels and cell viability were downregulated by Tm in a concentration-dependent manner compared with the control group. Tm induced apoptosis, ERS and autophagy, as indicated by an increased ratio of apoptotic cells, increased expression levels of Bax, cleaved (c)-caspase-3/9, glucose-regulated protein 78 kDa (GRP78), phosphorylated (p)-inositol-requiring enzyme-1α (IRE1α), spliced X-box-binding protein 1 (Xbp1s), IRE1α, activating transcription factor (ATF)6, ATF4, Beclin 1 and microtubule associated protein 1 light chain 3α (LC3)II/I, and decreased expression levels of Bcl-2, unspliced Xbp1 (Xbp1u) and p62 in the Tm group compared with the control group. Moreover, the results indicated that compared with the Tm + overexpression (Oe)-negative control (NC) group, the Tm + Oe-Dancr group displayed decreased apoptosis, but enhanced ERS and autophagy to restore cellular homeostasis. Compared with the Tm + Oe-NC group, the Tm + Oe-Dancr group decreased the ratio of apoptotic cells, decreased expression levels of Bax, c-caspase-3/9 and Xbp1u, and increased expression levels of Bcl-2, p-IRE1α, Xbp1s, Beclin 1 and LC3II/I. Dancr overexpression also significantly downregulated miR-6324 expression compared with Oe-NC. The Dual-luciferase reporter assay further indicated an interaction between Dancr and miR-6324. In addition, miR-6324 mimic partially reversed the effects of Dancr overexpression on Tm-induced apoptosis, ERS and autophagy. In conclusion, lncRNA Dancr overexpression protected cardiomyocytes against ERS injury via sponging miR-6324, thus inhibiting apoptosis, enhancing autophagy and restoring ER homeostasis.

Hyperglycemia Induces Endoplasmic Reticulum Stress in Atrial Cardiomyocytes, and Mitofusin-2 Downregulation Prevents Mitochondrial Dysfunction and Subsequent Cell Death.

Mitochondrial oxidative stress and dysfunction play an important role of atrial remodeling and atrial fibrillation (AF) in diabetes mellitus. Endoplasmic reticulum (ER) stress has been linked to both physiological and pathological states including diabetes. The aim of this project is to explore the roles of ER stress in hyperglycemia-induced mitochondrial dysfunction and cell death of atrial cardiomyocytes. High glucose upregulated ER stress, mitochondrial oxidative stress, and mitochondria-associated ER membrane (MAM)- enriched proteins (such as glucose-regulated protein 75 (GRP75) and mitofusin-2 (Mfn2)) of primary cardiomyocytes in vitro. Sodium phenylbutyrate (4-PBA) prevented the above changes. Silencing of Mfn2 in HL-1 cells decreased the Ca2+ transfer from ER to mitochondria under ER stress conditions, which were induced by the ER stress agonist, tunicamycin (TM). Electron microscopy data suggested that Mfn2 siRNA significantly disrupted ER-mitochondria tethering in ER stress-injured HL-1 cells. Mfn2 silencing attenuated mitochondrial oxidative stress and Ca2+ overload, increased mitochondrial membrane potential and mitochondrial oxygen consumption, and protected cells from TM-induced apoptosis. In summary, Mfn2 plays an important role in high glucose-induced ER stress in atrial cardiomyocytes, and Mfn2 silencing prevents mitochondrial Ca2+ overload-mediated mitochondrial dysfunction, thereby decreasing ER stress-mediated cardiomyocyte cell death.

Upregulating miR-637 aggravates endoplasmic reticulum stress-induced apoptosis in gastric cancer cells by suppressing Calreticulin

ABSTRACT Gastric cancer is a leading cause of cancer death worldwide. Endoplasmic reticulum (ER) stress-induced apoptosis has been confirmed to be important in the treatment of gastric cancer. MiR-637 has recently been found to exert inhibitory effects on gastric cancer, and this study aimed to investigate whether miR-637 could regulate apoptosis through ER stress. The results showed that tunicamycin (TM) induced downregulation of miR-637 in gastric cancer cells (AGS) and increase of apoptosis and ER stress. Overexpression of miR-637 promoted TM-induced apoptosis and expression of ER stress associated proteins (GRP78 and CHOP), but inhibited expression of Calreticulin. MiR-637 could bind with the 3ʹ-UTR of CALR, and negatively regulated the expression of CALR. The co-transfection of miR-637 and CALR in AGS cells show that, CALR overexpression could reverse the pro-apoptosis effects of miR-637 in TM-treated cells. In conclusion, the present study suggests that miR-637 participates in ER stress-induced apoptosis in gastric cancer cells by suppressing CALR expression. miR-637 or CALR may be a future potential target for gastric cancer treatment.

NF-κB contributes to Smac mimetic-conferred protection from tunicamycin-induced apoptosis.

Smac mimetics that deplete cellular inhibitor of apoptosis (cIAP) proteins have been shown to activate Nuclear Factor-kappa B (NF-κB). Here, we report that Smac mimetic-mediated activation of NF-κB contributes to the rescue of cancer cells from tunicamycin (TM)-triggered apoptosis. The prototypic Smac mimetic BV6 activates non-canonical and canonical NF-κB pathways, while TM has little effect on NF-κB signaling. Importantly, ectopic expression of dominant-negative IκBα superrepressor (IκBα-SR), which inhibits canonical and non-canonical NF-κB activation, significantly reversed this BV6-imposed protection against TM. Similarly, transient or stable knockdown of NF-κB-inducing kinase, which accumulated upon exposure to BV6 alone and in combination with TM, significantly counteracted BV6-mediated inhibition of TM-induced apoptosis. Interestingly, while cIAP2 was initially degraded upon BV6 treatment, it was subsequently upregulated in an NF-κB-dependent manner, as this restoration of cIAP2 expression was abolished in IκBα-SR-overexpressing cells. Interestingly, upon exposure to TM/BV6 apoptosis was significantly increased in cIAP2 knockdown cells. Furthermore, NF-κB inhibition partially prevented BV6-stimulated expression of Mcl-1 upon TM treatment. Consistently, Mcl-1 silencing significantly inhibited BV6-mediated protection from TM-induced apoptosis. Thus, NF-κB activation by Smac mimetic contributes to Smac mimetic-mediated protection against TM-induced apoptosis.

Humanin Protects RPE Cells from Endoplasmic Reticulum Stress-Induced Apoptosis by Upregulation of Mitochondrial Glutathione.

Humanin (HN) is a small mitochondrial-encoded peptide with neuroprotective properties. We have recently shown protection of retinal pigmented epithelium (RPE) cells by HN in oxidative stress; however, the effect of HN on endoplasmic reticulum (ER) stress has not been evaluated in any cell type. Our aim here was to study the effect of HN on ER stress-induced apoptosis in RPE cells with a specific focus on ER-mitochondrial cross-talk. Dose dependent effects of ER stressors (tunicamycin (TM), brefeldin A, and thapsigargin) were studied after 12 hr of treatment in confluent primary human RPE cells with or without 12 hr of HN pretreatment (1–20 μg/mL). All three ER stressors induced RPE cell apoptosis in a dose dependent manner. HN pretreatment significantly decreased the number of apoptotic cells with all three ER stressors in a dose dependent manner. HN pretreatment similarly protected U-251 glioma cells from TM-induced apoptosis in a dose dependent manner. HN pretreatment significantly attenuated activation of caspase 3 and ER stress-specific caspase 4 induced by TM. TM treatment increased mitochondrial superoxide production, and HN co-treatment resulted in a decrease in mitochondrial superoxide compared to TM treatment alone. We further showed that depleted mitochondrial glutathione (GSH) levels induced by TM were restored with HN co-treatment. No significant changes were found for the expression of several antioxidant enzymes between TM and TM plus HN groups except for the expression of glutamylcysteine ligase catalytic subunit (GCLC), the rate limiting enzyme required for GSH biosynthesis, which is upregulated with TM and TM+HN treatment. These results demonstrate that ER stress promotes mitochondrial alterations in RPE that lead to apoptosis. We further show that HN has a protective effect against ER stress-induced apoptosis by restoring mitochondrial GSH. Thus, HN should be further evaluated for its therapeutic potential in disorders linked to ER stress.

FAM3A attenuates ER stress-induced mitochondrial dysfunction and apoptosis via CHOP-Wnt pathway

Endoplasmic reticulum (ER) stress is linked to several neurological disorders, and neuronal injury cascades initiated by excessive ER stress are mediated, in part, via mitochondrial dysfunction. In the present study, we identified FAM3A as an important regulator of ER stress-induced cell death in neuronal HT22 cells. The ER stress inductor tunicamycin (TM) significantly decreased the expression of FAM3A at both mRNA and protein levels, which was shown to be dependent on the induction of reactive oxygen species (ROS). Overexpression of FAM3A attenuated TM-induced apoptosis and activation of ER stress factors, but had no effect on ER calcium metabolism in HT22 cells. We also found decreased mitochondrial ROS generation, inhibited cytochrome c release and preserved mitochondrial membrane potential (MMP) in FAM3A overexpressed cells. In addition, the experiments using isolated mitochondria showed that overexpression of FAM3A attenuated mitochondrial swelling and loss of mitochondrial Ca2+ buffering capacity after TM exposure. By using specific targeted small interfering RNA (siRNA) to knockdown the expression of the C/EBP homologous protein (CHOP), we found that FAM3A-induced protection and inhibition of ER stress was mediated by inverting TM-induced decrease of Wnt through the CHOP pathway. Our study demonstrates a pivotal role of FAM3A in protecting against TM-induced cytotoxicity via regulating CHOP-Wnt pathway, and suggests the therapeutic values of FAM3A overexpression against ER stress-associated neuronal injury.

Inhibition of mitogen-activated protein kinase signaling pathway sensitizes breast cancer cells to endoplasmic reticulum stress-induced apoptosis.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) induces ER stress which is observed in many human diseases, including breast cancer. Cellular adaptation to ER stress is mediated by the unfolded protein response (UPR), which aims at restoring ER homeostasis. Higher levels of GRP78 expression indicates constitutive activation of the UPR in breast cancer leading to breast cancer cells that are relatively resistant to ER stress-induced apoptosis. Tunicamycin (TM), an ER stress inducer, constitutively activates the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK), and (MEK)/ERK pathway which plays a role in upregulation of GRP78 by ER stress in that inhibition of MEK by U0126 reduces the levels of GRP78 and blocks its upregulation by TM. Inhibition of the MEK/ERK pathway by U0126 sensitizes breast cancer cells to TM-induced apoptosis. Inhibition of GRP78 by siRNA knockdown enhances TM- and U0126-induced apoptosis in breast cancer cells. This sensitization of breast cancer cells to TM-induced apoptosis by inhibition of MEK/ERK and GRP78 is caspase-dependent, at least in part, by activation of caspase-4. These results seem to indicate that GRP78 has potential as a chemotherapeutical target and have important implications for new treatment strategies in breast cancer by combination with agents that induce ER stress with inhibitors of the MEK/ERK pathway.

Knockdown of 15-kDa selenoprotein (Sep15) increases hLE cells' susceptibility to tunicamycin-induced apoptosis.

In this work, we investigated the effect of Sep15 gene knockdown on apoptosis in human lens epithelial (hLE) cells, trying to understand the relevance of Sep15 to cataract formation in the Sep15 knockout (KO) mice. The results showed that sole knockdown of Sep15 by RNA interference did not result in apoptosis; however, reduction of Sep15 expression aggravated tunicamycin (Tm)-induced cell apoptosis and caspases activation. Furthermore, Tm-induced mitochondrial dysfunction was also exacerbated under the Sep15 knockdown condition by measurement of mitochondrial membrane potential decrease and human cytochrome c release into cytosol. Interestingly, the knockdown of Sep15 exacerbated Tm-induced oxidative stress while endoplasmic reticulum (ER) stress was not correspondingly elevated. These results suggest that the protective role of Sep15 against Tm-induced apoptosis in hLE cells is operated via inhibiting oxidative stress rather than regulating Tm-induced ER stress, and the protective role becomes dependent on Sep15 only in acute stress condition.

HTLV-1 infected CD4+T cells of HAM/TSP patients are susceptible to tunicamycin-induced endoplasmic reticulum stress

Lectin array analysis for membrane proteins from CD4+Tcells of four respective cases of HAM, healthy carriers (HC), and negative controls (NC) showed significant high signal for Urtica dioica agglutinin (UDA) and Solanum tuberosum (Potato) (STL) lectins, that specifically recognize N-glycan, N-acetyllactosamine (LacNAc) which consist of repeats of the disaccharide βGal(1-4)βGlcNAc(1-3). We verified that LacNAc is significantly expressed in CD4+T cells in HAM by lectin ELISA for each ten cases of HAM, HC, and NC, followed by qRT-PCR of β1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) gene which was reported as main responsible enzyme in LacNAc biosynthesis. Using tunicamycin(TM), known as a N-glycan inhibitor and an apoptosis inducing agent through endoplasmic reticulum (ER) stress, we investigated the effect of LacNAc suppression on CD4+T cells. In NC, both of LacNAc+ and LacNAc-cells showed similar survival regardless of treatment with TM of 2μM for 24h or without TM, however, both cells in HAM showed worse survival than in NC and LacNAc+cells showed better survival when treated with TM. Moreover in HAM, Tax-cells showed better survival regardless of treatment with TM and LacNAc+cells when with treatment with TM survived significantly better than when without TM. Tax+cells showed worse survival than Tax-cells and tended to decrease when treated with TM. Collectively, we hypothesized that LacNAc is a resistant factor to TM-induced apoptosis in HTLV-1 uninfected cells but HTLV-1 infected cells may be susceptible to TM even if LacNAc are expressed. To confirm this hypothesis, we performed qRT-PCR of main effector genes in ER stress pathway: x-box binding protein 1, spliced variant (XBP1s), heat shock 70kDa protein 5(GRP78), and heat shock protein 90kDa beta, member 1(GRP94). Only GRP94 was significantly suppressed in HAM and HC. We conclude that HTLV-1 infected cells may be under ER stress and be susceptible to TM, and that LacNAc positive cells are resultant survivors.

Supplementation of tauroursodeoxycholic acid during IVC did not enhance in vitro development and quality of buffalo IVF embryos but combated endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress, a dysfunction in protein-folding capacity of ER, is involved in many pathologic and physiological responses including embryonic development. This study investigated the effect of supplementation of IVC medium with an ER stress inducer, tunicamycin (TM), and an inhibitor, tauroursodeoxycholic acid (TUDCA), on the developmental competence, apoptosis, and gene expression in buffalo embryos produced by IVF. Treatment of presumed zygotes with TM resulted in a significant (P < 0.01) decrease in the blastocyst rate, whereas TUDCA supplementation did not improve the blastocyst development rate. Further, presence of TUDCA could not ameliorate the adverse effects of TM in terms of the blastocyst rate in combined (TM + TUDCA) treatment. Tunicamycin treatment increased (P < 0.01) the apoptotic index and reduced the total cell number, whereas TUDCA did not affect them significantly. However, TUDCA reduced the extent of TM-mediated apoptosis during combined (TM + TUDCA) treatment. Tunicamycin treatment increased (P < 0.01) and TUDCA treatment decreased (P < 0.01) the expression level of ER chaperones, GRP78 and GRP94. In the combined TM + TUDCA treatment, TUDCA decreased their expression level compared to that in the controls. A similar pattern was observed in the case of proapoptotic gene BAX. We did not find any significant difference in the expression level of BCl-XL, BID, P53, and CASPASE 3 after TM and TUDCA supplementation. In conclusion, our study reported that TM induces ER stress in buffalo embryos produced in vitro resulting in a decrease in the blastocyst rate and an increase in the level of apoptosis and that these actions are mediated by modulating the expression of apoptosis-related genes and ER chaperones. Tauroursodeoxycholic acid did not improve the developmental potential of buffalo embryos; however, it attenuated the TM-induced apoptosis by downregulating BAX and ER chaperones.

Inhibition of autophagy promotes caspase-mediated apoptosis by tunicamycin in HepG2 cells

Tunicamycin (TM) causes accumulation of unfolded protein in endoplasmic reticulum (ER) lumen and introduces from elsewhere ER stress. This study was to assess the apoptosis and autophagy effect induced by TM on HepG2 cells and the role of autophagy in the system. The viability of HepG2 cells was significantly inhibited by TM in a dose-dependent manner detected by MTT assay. Then, the apoptotic morphology change, increasing apoptotic cell rate suggested that apoptosis was induced by TM in a time- and dose-dependent manner. To further determine the involvement of caspase-dependent pathway in TM-induced apoptosis, we discover that the activity of caspase-3/7, 8, 9 and cleavage of PARP markedly increased after TM treatment and the apoptosis was effectively attenuated by using caspase-9 and pan caspase inhibitor. Moreover, provided the rising stained acidic vacuoles and an increased level of LC3II and activation of Beclin1, we concluded that autophagy could be triggered by TM in a time- and dose-dependent manner. In addition, the inhibition of autophagy efficiently promoted TM-induced cell death identified by MTT assay. Meanwhile, the apoptotic cell rate and caspase-3 activation increased significantly after autophagy blockage. In conclusion, we found that TM initiated apoptosis and autophagy both in a time- and dose-dependent manner in HepG2 cells; and inhibition of autophagy may promote TM-induced cell death through enhancing apoptosis.

Histone H3K4 methyltransferase Mll1 regulates protein glycosylation and tunicamycin-induced apoptosis through transcriptional regulation

Disrupting protein glycosylation induces ER (endoplasmic reticulum) stress, resulting in the activation of UPR (unfolded protein response) pathways. A key function of the UPR is to restore ER homeostasis, but prolonged or unsolved ER stress can lead to apoptosis. MLL1 (Mixed Lineage Leukemia 1, also named ALL-1 or HRX), a histone H3K4 methyltransferase in mammals, plays important roles in leukemogenesis, transcriptional regulation, cell cycle and development. Here, we find that Mll1 deficiency enhances UPR and apoptosis induced by the glycosylation inhibitor TM (tunicamycin). The abnormal regulation of the UPR appears to be caused by a defect in protein glycosylation. Furthermore, Mll1 directly binds to the promoters of H6pd, Galnt12 and Ugp2, which regulates H3K4 trimethylation and the subsequent expression of these genes. The knockdown of H6pd, Galnt12 or Ugp2 enhances TM-induced apoptosis in Mll1+/+MEF cells, whereas the ectopic expression of these proteins inhibits TM-induced apoptosis in Mll1−/− MEF cells. Together, our data suggest that the maturation of glycoproteins in the ER is subject to regulation at the epigenetic level by a histone methyltransferase whose abnormality can lead to cancer and developmental defects.

Overexpression of CREB protein protects from tunicamycin-induced apoptosis in various rat cell types.

Endoplasmic reticulum (ER) stress plays an essential role in unfolded protein response induced apoptosis contributing to several pathological conditions. Glycogen synthase kinase-3β (GSK-3β) plays a central role in several apoptotic signaling, including ER stress, as the active form of GSK-3β induces apoptosis. The phosphorylation of cAMP responsive element (CRE) binding protein (CREB) Ser-133 (S133) residue is the end-point of various signaling pathways, like growth factor signaling, while the Ser-129 (S129) residue is phosphorylated by GSK-3β. The significance of the ubiquitously expressed transcription factor CREB is demonstrated in prolonged, tunicamycin (TM)-induced ER stress in this study. In the experiments wild-type (wt) CREB, S129Ala, S133Ala or S129Ala-S133Ala mutant CREB expressing PC12 rat pheochromocytoma cell lines showed increased survival under TM-evoked prolonged ER stress compared to wtPC12 cells. After TM treatment ER stress was activated in all PC12 cell types. Lithium and SB-216763, the selective, well-known inhibitors of GSK-3β, decreased TM-induced apoptosis and promoted cell survival. The proapoptotic BH3-only Bcl-2 family member Bcl-2-interacting mediator of cell death (Bim) level was decreased in the different CREB overexpressing PC12 cells as a result of TM treatment. CREB overexpression also inhibited the sequestration of Bim protein from tubulin molecules, as it was demonstrated in wtPC12 cells. Transient expression of wtCREB diminished TM-induced apoptosis in wtPC12, Rat-1 and primary rat vascular smooth muscle cells. These findings demonstrate a novel role of CREB in different cell types as a potent protector against ER stress.

The Batten disease gene CLN3 confers resistance to endoplasmic reticulum stress induced by tunicamycin

Mutations in CLN3 gene cause juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), an early-onset neurodegenerative disorder that is characterized by the accumulation of ceroid lipofuscin within lysosomes. The function of the CLN3 protein remains unclear and is presumed to be related to Endoplasmic reticulum (ER) stress. To investigate the function of CLN3 in the ER stress signaling pathway, we measured proliferation and apoptosis in cells transfected with normal and mutant CLN3 after treatment with the ER stress inducer tunicamycin (TM). We found that overexpression of CLN3 was sufficient in conferring increased resistance to ER stress. Wild-type CLN3 protected cells from TM-induced apoptosis and increased cell proliferation. Overexpression of wild-type CLN3 enhanced expression of the ER chaperone protein, glucose-regulated protein 78 (GRP78), and reduced expression of the proapoptotic protein CCAAT/-enhancer-binding protein homologous protein (CHOP). In contrast, overexpression of mutant CLN3 or siRNA knockdown of CLN3 produced the opposite effect. Together, our data suggest that the lack of CLN3 function in cells leads to a failure of management in the response to ER stress and this may be the key deficit in JNCL that causes neuronal degeneration.

3 β-Hydroxysteroid-Δ 24 Reductase (DHCR24) Protects Neuronal Cells from Apoptotic Cell Death Induced by Endoplasmic Reticulum (ER) Stress

3β-Hydroxysteroid-Δ24 reductase (DHCR24) is an endoplasmic reticulum (ER)-localized multifunctional enzyme that possesses anti-apoptotic and cholesterol-synthesizing activities. Accumulating evidence suggests that ER stress is involved in the pathogenesis of neurodegenerative disease. In this study, we investigated whether DHCR24 may function as a neuroprotective protein under ER stress. Neuroblastoma N2A cells were infected with adenovirus expressing myc-tagged DHCR24 (Ad-DHCR24) or lacZ (Ad-lacZ, serving as a control) and subjected to ER-stress, induced with Tunicamycin (TM). Cells infected with Ad-DHCR24-myc were resistant to TM-induced apoptosis, and showed weaker level of caspase-12 activity. These cells also exhibited lower levels of Bip and CHOP proteins than Ad-LacZ-infected cells. Moreover, a stronger and rapid activation of PERK, and a prolonged activation of JNK and p38 were observed in Ad-LacZ–infected cells. The generation of intracellular reactive oxygen species from ER stress was also diminished by the overexpression of DHCR24. Additionally, intracellular cholesterol level was also elevated in the Ad-DHCR24-infected cells, accompanied by a well-organized formation of caveolae (cholesterol-rich microdomain) on the plasma membrane, and improved colocalization of caveolin-1 and insulin-like growth factor 1 receptor. These results demonstrated for the first time that DHCR24 could protect neuronal cells from apoptosis induced by ER stress.

Suppression of tunicamycin-induced CD44v6 ectodomain shedding and apoptosis is correlated with temporal expression patterns of active ADAM10, MMP-9 and MMP-13 proteins in Caki-2 renal carcinoma cells

CD44v6 has been shown to coordinate the activation of anti-apoptotic molecules as well as resistance to apoptosis. Here, we investigated CD44v6 ectodomain shedding in Caki-2 human renal carcinoma cells as well as its underlying mechanisms. Exposure of cells to tunicamycin (TM)-induced apoptosis was accompanied by cleavage of caspase-3, PARP-1 and CD44v6 ectodomain. TM-induced apoptosis was also closely associated with endoplasmic reticulum (ER) stress, as shown by increased expression of GRP-78 and CHOP proteins. Furthermore, induction of matrix metallo-proteinase (MMP)-13, MMP-9 and ADAM10 expression was highly stimulated by tunicamycin in a time- and dose-dependent manner. TM-induced PARP-1 cleavage was significantly inhibited by treatment with GM6001 (a broad spectrum MMP inhibitor), MMP-9/-13 inhibitor and GI254023X (specific ADAM10 inhibitor). In addition, inhibition of all examined MMPs resulted in reversal of TM-induced apoptosis as well as increased cell viability. When considering the functional implications of MMP-9 and ADAM10, it is likely that active MMP-9 and ADAM10 help regulate the cellular levels of CD44v6 through cleavage of CD44v6 ectodomain during TM-induced apoptosis of Caki-2 cells. Collectively, these findings suggest that multiple TM-induced MMPs may cooperate to induce apoptosis.

Tauroursodeoxycholic Acid Enhances the Pre‐Implantation Embryo Development by Reducing Apoptosis in Pigs

Apoptosis is an important determinant of the normal development of pre-implantation embryos in vitro. Recently, endoplasmic reticulum (ER) stress-mediated apoptosis has been extensively investigated in a wide variety of diseases. Efficient functioning of the ER is essential for most cellular activities and survival. Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, has been reported to attenuate ER stress-mediated cell death by interrupting the classic pathways of apoptosis. Therefore, in this study, the anti-apoptotic effect of TUDCA on ER stress-induced apoptosis was examined in pre-implantation pig embryos. Also, tunicamycin was used to investigate the effects of ER stress on pig embryo development. After in vitro maturation and fertilization, presumptive pig embryos were cultured in NCSU-23 medium supplemented with TUDCA or TM for 6 days at 39 °C, 5% CO(2) in air. All data were analysed using one-way anova and Duncan's multiple range test in the statistical analysis system (SAS). In addition, we also determined the optimal TM and TUDCA concentrations. Samples were treated with TM at concentrations of 0, 1, 2 or 5 μm and with TUDCA at concentrations of 0, 100, 200 or 300 μm. When TM was used during in vitro culture, only 8.2% (8/97) of the embryos developed to the blastocyst stage when the treatment concentration was 1 μm compared with 27.4% (28/102) of the embryos in the control group (p < 0.05). In contrast, the frequency of blastocyst formation and the number of cells were higher when treated with 200 μm TUDCA compared with the control group (32.8% and 39.5 vs 22.2% and 35.6, p < 0.05). Moreover, the developmental rate to the blastocyst stage embryo in the group treated with TM and TUDCA was not significantly different from that of the control group (17.8%, 26/142 vs 24.9%, 36/145). Furthermore, the blastocyst cell number was enhanced (31.9 vs 36.9) and apoptosis reduced (TUNEL-positive nuclei number, 6.0 vs 3.2) by TUDCA treatment in pig embryos. In the real-time quantitative RT-PCR analysis, the expression of anti-apoptotic Bcl-XL gene was shown to be increased in the blastocyst stage because of TUDCA treatment, whereas expression of pro-apoptotic Bax was decreased. In addition, we also found that TUDCA decreased the rate of TM-induced apoptosis in the pre-implantation stage. Taken together, our results indicate that TUDCA improves the developmental competence of pig embryos by modulating ER stress-induced apoptosis during the pre-implantation stage.

DIDS exerts a cardioprotective effect on tunicamycin-induced apoptosis via reduction of endoplasmic reticulum stress

Aims4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), a non-selective chloride channel blocker, has been shown to prevent apoptosis-induced by pathways of death receptor and mitochondria. However, the relationship between DIDS and apoptosis-induced by endoplasmic...