Endoplasmic reticulum stress-mediated pathways to both apoptosis and autophagy: Significance for melanoma treatment

Melanoma is the most aggressive form of skin cancer.Disrupted intracellular signaling pathways are responsible for melanoma's extraordinary resistance to current chemotherapeutic modalities. The pathophysiologic basis for resistance to both chemo- and radiation therapy is rooted in altered genetic and epigenetic mechanisms that, in turn, result in the impairing of cell death machinery and/or excessive activation of cell growth and survival-dependent pathways. Although most current melanoma therapies target mitochondrial dysregulation,there is increasing evidence that endoplasmic reticulum(ER) stress-associated pathways play a role in the potentiation,initiation and maintenance of cell death machinery and autophagy. This review focuses on the reliability of ER-associated pathways as therapeutic targets for melanoma treatment.

Roles of TGF-β Signaling Pathway in Endoplasmic Reticulum Stress in Endothelial Cells Stimulated with Cigarette Smoke Extract

To investigate the role of signaling pathway in the effect of endoplasmic reticulum stress（ER stress） in endothelial cells stimulated with cigarette smoke extract（CSE）. Human umbilical vein endothelial cells（HUVECs） were cultured and divided into 3 groups： CSE-stimulated group, CSE-stimulated with 4-PBA group, and negative control group. HUVECs were cultured and stimulated with CSE at concentrations of 5%, 10% and 20%, respectively, mR NA of CXCL-8 and GRP78 was detected by real-time PCR. ELISA was performed to test the expression of CXCL-8 protein, and neutrophils migration was detected by Transwell board test. The NF-κB, ERK, p38 MAPK and transforming growth factor beta（TGF-β） were detected by flow cytometry. The mRNA of CXCL-8 and GRP78 increased in CSE-stimulated HUVECs（P〈0.05）. Furthermore, it was concentration-dependent. 4-PBA significantly reduced the expression of CXCL-8 protein（P〈0.05） and neutrophil migration（P〈0.05）. The TGF-β, rather than the NF-κB, ERK and P38 MAPK pathway might be involved in ER stress stimulated by CSE. CSE induced neutrophils migration by increasing the expression of CXCL-8 in endothelial cells. ER stress might play a role in the effect of neutrophils migration stimulated with CSE, and TGF-β pathway may contribute to the ER stress in HUVECs.

Effects of Shenling Baizhu powder on endoplasmic reticulum stress related signaling pathway in liver tissues of nonalcoholic fatty liver disease rats

Background:According to our previous studies,Shenling Baizhu powder has an excellent preventive and therapeutic effect on nonalcoholic fatty liver disease,but the prevention mechanism is still not clear.In this study,we intended to explore the effects of Shenling Baizhu powder on the endoplasmic reticulum stress related signaling pathway in nonalcoholic fatty liver disease rats’liver tissues.Methods:After 16 weeks,the levels of serum total cholesterol,triglyceride,high-density lipoprotein cholesterol,low-density lipoprotein cholesterol,alanine transaminase and aspartate transaminase were evaluated by an automatic biochemical analyzer,and the levels of serum free fatty acid and hepatic total cholesterol and triglyceride were evaluated by commercial kits.Then,histological changes of the liver were observed by hematoxylin and eosin and oil red-O staining.Protein expression related to the liver unfolded protein response signalling pathway was assessed using Western blot analysis.Results:The results showed that Shenling Baizhu powder supplementation reduced serum total cholesterol,triglyceride,free fatty acid,alanine transaminase,and aspartate transaminase(P<0.05 or P<0.01),as well as the levels of hepatic total cholesterol and triglyceride(P<0.01).Pathological examination showed that Shenling Baizhu powder improved hepatic steatosis and lipid accumulation.The results of biochemical parameters and histological changes indicated that Shenling Baizhu powder administration exerted protective effects against nonalcoholic fatty liver disease.In addition,Shenling Baizhu powder decreased the protein expression levels of binding immunoglobulin protein,activating transcription factor 6,phosphorylation of eukaryotic initiation factor 2 alpha,protein kinase RNA-like endoplasmic reticulum kinase and X-box binding protein 1s in the liver(P<0.05 or P<0.01).Conclusion:Shenling Baizhu powder can ameliorate high-fat diet-induced liver lipid metabolism disorder in nonalcoholic fatty liver disease rats.The mechanisms may be related to the inhibition of the expression of proteins related to unfolded protein response signaling pathways in endoplasmic reticulum stress.

The emerging role of nitric oxide in the synaptic dysfunction of vascular dementia

With an increase in global aging,the number of people affected by cerebrovascular diseases is also increasing,and the incidence of vascular dementia-closely related to cerebrovascular risk-is increasing at an epidemic rate.However,few therapeutic options exist that can markedly improve the cognitive impairment and prognosis of vascular dementia patients.Similarly in Alzheimer’s disease and other neurological disorders,synaptic dysfunction is recognized as the main reason for cognitive decline.Nitric oxide is one of the ubiquitous gaseous cellular messengers involved in multiple physiological and pathological processes of the central nervous system.Recently,nitric oxide has been implicated in regulating synaptic plasticity and plays an important role in the pathogenesis of vascular dementia.This review introduces in detail the emerging role of nitric oxide in physiological and pathological states of vascular dementia and summarizes the diverse effects of nitric oxide on different aspects of synaptic dysfunction,neuroinflammation,oxidative stress,and blood-brain barrier dysfunction that underlie the progress of vascular dementia.Additionally,we propose that targeting the nitric oxide-sGC-cGMP pathway using certain specific approaches may provide a novel therapeutic strategy for vascular dementia.

DHA induces adipocyte lipolysis through endoplasmic reticulum stress and the cAMP/PKA signaling pathway in grass carp(Ctenopharyngodon idella)

Docosahexaenoic acid(DHA)is a biologically active fatty acid that reduces the accumulation of lipids.However,the molecular mechanism underlying this process,particularly in fish,is not well understood.Recent studies show that endoplasmic reticulum(ER)stress triggers the activation of the unfolded protein response,which has been revealed to play an essential role in lipid metabolism.In this study,we explored the effect of DHA on ER stress and investigated the potential molecular mechanisms underlying DHA-induced adipocyte lipolysis in grass carp(Ctenopharyngodon idella)both in vivo and in vitro.We found that DHA remarkably reduced the triglyceride content,increased the secretion of glycerol,pro-moted lipolysis in adipocytes and evoked ER stress,whereas inhibiting ER stress using 4-phenyl butyric acid(4-PBA)inhibited the effects of DHA(P<0.05).These results implied that ER stress potentially participates in DHA-induced adipocyte lipolysis.Additionally,STF-083010,a specific inositol-requiring enzyme 1a(IRE1a)-inhibitor,attenuated the effects of DHA on lipolysis,demonstrating that IRE1a and X-box binding protein 1 potentially participate in DHA-induced lipolysis.DHA also activated the cyclic adenosine monophosphate(cAMP)-dependent protein kinase A(PKA)pathway by increasing the level of cAMP and activating the PKA enzyme(P<0.05).Nevertheless,H89,a PKA inhibitor,weakened DHA-induced lipolysis by inhibiting the cAMP/PKA signaling pathway.Furthermore,inhibiting ER stress us-ing 4-PBA also inhibited lipolysis and alleviated DHA-induced activation of the cAMP/PKA signaling pathway,suggesting that ER stress may participate in DHA-induced lipolysis through the activation of the cAMP/PKA signaling pathway.Our data illustrate that DHA supplementation can be a promising nutritional strategy for ameliorating lipid accumulation in grass carp.The present study elucidated the molecular mechanism for DHA-induced lipolysis in grass carp adipocytes and emphasized the impor-tance of ER stress and the cAMP/PKA pathway in DHA-induced lipolysis.These results deepen our un-derstanding of ameliorating lipids deposition in freshwater fish by targeting DHA.

Pollen Typhae Total Flavone Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis in Human Aortic-Vascular Smooth Muscle Cells through Down-Regulating PERK-eIF2α-ATF4-CHOP Pathway

Objective: To test the hypothesis that the inhibition of endoplasmic reticulum(ER) stress-induced apoptosis in oxidized low-density lipoproteins(ox-LDL)-induced human aortic-vascular smooth muscle cells(HA-VSMCs) was associated with suppression of the protein kinase RNA-like ER kinase(PERK)-eukaryotic translation initiation factor 2α(e IF2α)-activating transcription factor 4(ATF4)-CCAAT/enhancer binding protein homologous protein(CHOP) signaling pathway by Pollen Typhae total flavone(PTF). Methods: Primary HA-VSMCs were cultured and identified. The cultured HA-VSMCs were randomized into 5 groups, including a normal control group, an ox-LDL group(70 μg/m L high ox-LDL), an HPTF group(70 μg/m L high ox-LDL+500 μg/m L PTF), an MPTF group(70 μg/m L high ox-LDL+250 μg/m L PTF), and a LPTF group(70 μg/m L high ox-LDL+100 μg/m L PTF) in the first part;and a normal control group, an ox-LDL group(70 μg/mL high ox-LDL), an MPTF group(70 μg/m L high ox-LDL+250 μg/m L PTF), a sh RNA group(transducted with PERK shRNA lentiviral particles), a scramble shRNA group(transducted with control shRNA lentiviral particles), an MPTF+ox-LDL+shRNA group(250 μg/mL PTF+70 μg/mL high ox-LDL+PERK shRNA lentiviral particles) and an ox-LDL+shRNA group(70 μg/mL high ox-LDL+PERK shRNA lentiviral particles) in the second part. The protein expression levels of ER-associated apoptosis proteins were detected by Western blot, and their m RNA expression levels were detected by quantitative real-time reverse transcription-polymerase chain reaction. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay was applied to test cel viability, and the level of apoptosis was monitored by flow cytometry. Results: The MTT assay and flow cytometry showed that the ox-LDL group had a significant increase in apoptosis, which was attenuated in PTF treatment groups and sh RNA groups. Moreover, the ox-LDL group had increased protein and m RNA levels of binding immunoglobulin protein and ER-associated apoptosis proteins, such as PERK, e IF2α, ATF4 and CHOP, which were attenuated in PTF treatment groups and sh RNA groups. Conclusions: The apoptosis induced by ox-LDL had a strong relation to ER stress. The protective effect of PTF on ER stressinduced apoptosis was associated with inhibition of the PERK-eIF2α-ATF4-CHOP pathway, which might be a potential therapeutic strategy for enhancing the stability of atherosclerotic plaques.

Endoplasmic reticulum stress and proteasome pathway involvement in human podocyte injury with a truncated COL4A3 mutation

Background:Collagen type Ⅳ(COL4)-related nephropathy includes a variety of kidney diseases that occur with or without extra-renal manifestations caused by COL4A3-5 mutations.Previous studies revealed several novel mutations,including three COL4A3 missense mutations (G619R,G801R,and C1616Y) and the COL4A3 chr:228172489delA c.4317delA p.Thr1440ProfsX87 frameshift mutation that resulted in a truncated NC1 domain (hereafter named COL4A3 c.4317delA);however,the mutation mechanisms that lead to podocyte injury remain unclear.This study aimed to further explore the mutation mechanisms that lead to podocyte injury.Methods:Wild-type (WT) and four mutant COL4A3 segments were constructed into a lentiviral plasmid,then stably transfected into human podocytes.Real-time polymerase chain reaction and Western blotting were applied to detect endoplasmic reticulum stress (ERS)-and apoptosis-related mRNA and protein levels.Then,human podocytes were treated with MG132 (a proteasome inhibitor) and brefeldin A (a transport protein inhibitor).The human podocyte findings were verified by the establishment of a mus-Col4a3 knockout mouse monoclonal podocyte using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Casg) technology.Results:Our data showed that COL4A3 mRNA was significantly overexpressed in the lentivirus stably transfected podocytes.Moreover,the COL4A3 protein level was significantly increased in all groups except the COL4A3 c.4317delA group.Compared to the other test groups,the COL4A3 c.4317delA group showed excessive ERS and apoptosis.Podocytes treated with MG 132 showed remarkably increased intra-cellular expression of the COL4A3 c.4317delA mutation.MG132 intervention improved higher ERS and apoptosis levels in the COL4A3 c.4317delA group.Mouse monoclonal podocytes with COL4A3 chr:82717932insA c.4852insA p.Arg1618ThrfsX4 were successfully acquired;this NC1-truncated mutation suggested a higher level of ERS and relatively remarkable level of apoptosis compared to that of the WT group.Conclusions:We demonstrated that excessive ERS and ERS-induced apoptosis were involved in the podocyte injury caused by the NC1-truncated COL4A3 mutation.Furthermore,proteasome pathway intervention might become a potential treatment for collagen type Ⅳ-related nephropathy caused by a severely truncated COL4A3 mutation.

Endoplasmic Reticulum Stress Induces the Early Appearance of Pro-apoptotic and Anti-apoptotic Proteins in Neurons of Five Familial Alzheimer＇s Disease Mice

Background： Amyloid β （Aβ） deposits and the endoplasmic reticulum stress （ERS） are both well established in the development and progression of Alzheimer＇s disease （AD）. However, the mechanism and role of Aβ-induced ERS in AD-associated pathological progression remain to be elucidated. Methods： The five familial AD （5×FAD） mice and wild-type （WT） mice aged 2, 7, and 12 months were used in the present study. Monis water maze test was used to evaluate their cognitive performance, lmmunofluorescence and Western blot analyses were used to examine the dynamic changes of pro-apoptotic （CCAAT/enhancer-binding protein homologous protein [CHOP] and cleaved caspase-12） and anti-apoptotic factors （chaperone glucose-regulated protein [GRP] 78 and endoplasmic reticulum-associated protein degradation-associated ubiquitin ligase synovial apoptosis inhibitor 1 [SYVN 1]） in the ERS-associated unfolded protein response （UPR） pathway. Results： Compared with age-matched WT mice, 5 xFAD mice showed higher cleaved caspase-3, lower neuron-positive staining at the age of 12 months, but earlier cognitive deficit at the age of 7 months （all P 〈 0.05）. Interestingly, for 2-month-old 5×FAD mice, the related proteins involved in the ERS-associated UPR pathway, including CHOP, cleaved caspase-12, GRP 78, and SYVN 1, were significantly increased when compared with those in age-matched WT mice （all P 〈 0.05）. Moreover, ERS occurred mainly in neurons, not in astrocytes. Conclusions： These findings suggest that compared with those of age-matched WT mice, ERS-associated pro-apoptotic and anti-apoptotic proteins are upregulated in 2-month-old 5×FAD mice, consistent with intracellular Aβ aggregation in neurons.

Investigating the cellular functions of β-Glucosidases for synthesis of lignocellulose-degrading enzymes in Trichoderma reesei

β-glucosidases play an important role in the synthesis of cellulase in fungi,but their molecular functions and mechanisms remain unknown.We found that the 10 putativeβ-glucosidases investigated in Trichoderma ree-sei facilitate cellulase production,with cel3j being the most crucial.Transcriptional analysis revealed that the most affected biological processes in△cel3j strain were cellulase synthesis,ribosome biogenesis,and RNA poly-merases.Moreover,CEL3J was unconventionally transported through the endoplasmic reticulum,bypassing the Golgi apparatus,whereas cel3j overexpression altered cellulase secretion from conventional to unconventional,likely owing to the activated unconventional protein secretion pathway(UPS),as indicated by the upregulation of genes related to UPS.The mTORC1-GRASP55 signaling axis may modulate the unconventional secretion of CEL3J and cellulase.The transcriptional levels of genes associated with DNA replication,the cell cycle,and meiosis were noticeably affected by overexpressing cel3j.These data give new clues for exploring the roles ofβ-glucosidases and the molecular mechanisms of their unconventional secretion in fungi.

Common variants in PERK, JNK, BIP and XBP1 genes are associated with the risk of prediabetes or diabetes-related phenotypes in a Chinese population

Background Prediabetes is an early stage of β-cell dysfunction presenting as insulin resistance.Evidences suggest that endoplasmic reticulum （ER） stress is involved in the pathogenesis of type 2 diabetes mellitus and prediabetes.In a Chinese population with prediabetes,we investigated single nucleotide polymorphisms （SNPs） in the genes of PERK,JNK,XBP1,BIP and CHOP which encode molecular proteins involved in ER stress pathways.Methods Nine SNPs at the PERK,JNK,XBP1,BIP and CHOP loci were genotyped by mass spectrometry in 1 448 unrelated individuals.By using a 75 g oral glucose tolerance test （OGTT）,828 subjects were diagnosed as prediabetes and 620 subjects aged 55 years and over as normal controls based on WHO diagnostic criteria （1999） for diabetes mellitus.Results The allele C of SNP rs867529 at PERK locus was a risk factor for prediabetes,with the carriers of C allele genotype at a higher risk of prediabetes compared to non-carriers （OR=1.279,95％ CI：1.013-1.614,P=0.039,after adjustment for age,sex and body mass index （BMI）.The SNPs rs6750998 at PERK locus was associated with homeostasis model assessments of insulin resistance （HOMA-IR） （P=0.019）,and rs17037621 with BMI （P=0.044）.The allele G of SNP rs10986663 in BIP gene was associated with a decreased risk of prediabetes （OR=0.699,95％ CI：0.539-0.907,P=0.007）.The SNP rs2076431 in JNK gene was associated with fasting plasma glucose levels （P=0.006） and waist-hip ratios （P=0.019）.The SNP rs2239815 in XBP1 gene was associated with 2-hour plasma glucose levels after 75 g oral glucose load （P=0.048） in the observed population.Conclusion Common variants at PERK and BIP loci contributed to the risk of prediabetes,and the genetic variations in JNK and XBP1 genes are associated with diabetes-related clinical parameters in this Chinese population.