Endoplasmic Reticulum Expansion Research Articles

IL-6 mediates ER expansion during hyperpolarization of alternatively activated macrophages.

Although recent evidence has shown that IL‐6 is involved in enhanced alternative activation of macrophages toward a profibrotic phenotype, the mechanisms leading to their increased secretory capacity are not fully understood. Here, we investigated the effect of IL‐6 on endoplasmic reticulum (ER) expansion and alternative activation of macrophages in vitro. An essential mediator in this ER expansion process is the IRE1 pathway, which possesses a kinase and endoribonuclease domain to cleave XBP1 into a spliced bioactive molecule. To investigate the IRE1‐XBP1 expansion pathway, IL‐4/IL‐13 and IL‐4/IL‐13/IL‐6‐mediated alternative programming of murine bone marrow‐derived and human THP1 macrophages were assessed by arginase activity in cell lysates, CD206 and arginase‐1 expression by flow cytometry, and secreted CCL18 by ELISA, respectively. Ultrastructural intracellular morphology and ER biogenesis were examined by transmission electron microscopy and immunofluorescence. Transcription profiling of 128 genes were assessed by NanoString and Pharmacological inhibition of the IRE1‐XBP1 arm was achieved using STF‐083010 and was verified by RT‐PCR. The addition of IL‐6 to the conventional alternative programming cocktail IL‐4/IL‐13 resulted in increased ER and mitochondrial expansion, profibrotic profiles and unfolded protein response‐mediated induction of molecular chaperones. IRE1‐XBP1 inhibition substantially reduced the IL‐6‐mediated hyperpolarization and normalized the above effects. In conclusion, the addition of IL‐6 enhances ER expansion and the profibrotic capacity of IL‐4/IL‐13‐mediated activation of macrophages. Therapeutic strategies targeting IL‐6 or the IRE1‐XBP1 axis may be beneficial to prevent the profibrotic capacity of macrophages.

The Human Cytomegalovirus Endoplasmic Reticulum-Resident Glycoprotein UL148 Activates the Unfolded Protein Response.

Eukaryotic cells are equipped with three sensors that respond to the accumulation of misfolded proteins within the lumen of the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR), which functions to resolve proteotoxic stresses involving the secretory pathway. Here, we identify UL148, a viral ER-resident glycoprotein from human cytomegalovirus (HCMV), as an inducer of the UPR. Metabolic labeling results indicate that global mRNA translation is decreased when UL148 expression is induced in uninfected cells. Further, we find that ectopic expression of UL148 is sufficient to activate at least two UPR sensors: the inositol-requiring enzyme-1 (IRE1), as indicated by splicing of Xbp-1 mRNA, and the protein kinase R (PKR)-like ER kinase (PERK), as indicated by phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) and accumulation of activating transcription factor 4 (ATF4). During wild-type HCMV infection, increases in Xbp-1 splicing, eIF2α phosphorylation, and accumulation of ATF4 accompany UL148 expression. UL148-null infections, however, show reduced levels of these UPR indicators and decreases in XBP1s abundance and in phosphorylation of PERK and IRE1. Small interfering RNA (siRNA) depletion of PERK dampened the extent of eIF2α phosphorylation and ATF4 induction observed during wild-type infection, implicating PERK as opposed to other eIF2α kinases. A virus with UL148 disrupted showed significant 2- to 4-fold decreases during infection in the levels of transcripts canonically regulated by PERK/ATF4 and by the ATF6 pathway. Taken together, our results argue that UL148 is sufficient to activate the UPR when expressed ectopically and that UL148 is an important cause of UPR activation in the context of the HCMV-infected cell.IMPORTANCE The unfolded protein response (UPR) is an ancient cellular response to ER stress that is of broad importance to viruses. Certain consequences of the UPR, including mRNA degradation and translational shutoff, would presumably be disadvantageous to viruses, while other attributes of the UPR, such as ER expansion and upregulation of protein folding chaperones, might enhance viral replication. Although HCMV is estimated to express well over 150 different viral proteins, we show that the HCMV ER-resident glycoprotein UL148 contributes substantially to the UPR during infection and, moreover, is sufficient to activate the UPR in noninfected cells. Experimental activation of the UPR in mammalian cells is difficult to achieve without the use of toxins. Therefore, UL148 may provide a new tool to investigate fundamental aspects of the UPR. Furthermore, our findings may have implications for understanding the mechanisms underlying the effects of UL148 on HCMV cell tropism and evasion of cell-mediated immunity.

Impact of the Reticular Stress and Unfolded Protein Response on the inflammatory response in endometrial stromal cells

During decidualization, endometrial stromal cells undergo reticular stress (RS) and unfolded protein response (UPR), allowing the endoplasmic reticulum-expansion and immunomodulators production. Physiological RS generates the activation of sensing proteins, inflammasome activation and mature-IL-1β secretion, associated with pro-implantatory effects. We focus on the impact of RS and UPR on decidualized cells and whether they induce a physiological sterile inflammatory response through IL-1β production. Human endometrial stromal cell line (HESC) after decidualization treatment with MPA + dibutyryl-cAMP (Dec) increased the expression of RS-sensors (ATF6, PERK and IRE1α) and UPR markers (sXBP1 and CHOP) in comparison with Non-dec cells. Then we found increased NLRP3 expression in Dec cells compared with Non-dec cells. In fact STF-083010 (an IRE1α inhibitor) prevented this increase. Downstream, increased levels of active caspase-1 on Dec cells were detected by FAM-Flica Caspase-1 associated with an increase in IL-1β production. Moreover, the treatment with STF-083010 decreased the invasion index observed in Dec cells, evaluated by an in vitro model of implantation. In endometrial biopsies from recurrent spontaneous abortion patients an increased expression of IRE1α was found in comparison with fertile women; while recurrent implantation failure samples showed a lower expression of sXBP1, TXNIP and NLRP3 than fertile women, suggesting that RS/UPR tenors might condition endometrial receptivity.

Endoplasmic reticulum turnover: ER-phagy and other flavors in selective and non-selective ER clearance.

The endoplasmic reticulum (ER) is a highly dynamic organelle in eukaryotic cells. It is deputed to lipid and protein biosynthesis, calcium storage, and the detoxification of various exogenous and endogenous harmful compounds. ER activity and size must be adapted rapidly to environmental and developmental conditions or biosynthetic demand. This is achieved on induction of thoroughly studied transcriptional/translational programs defined as “unfolded protein responses” that increase the ER volume and the expression of ER-resident proteins regulating the numerous ER functions. Less understood are the lysosomal catabolic processes that maintain ER size at steady state, that prevent excessive ER expansion during ER stresses, or that ensure return to physiologic ER size during recovery from ER stresses. These catabolic processes may also be activated to remove ER subdomains where proteasome-resistant misfolded proteins or damaged lipids have been segregated. Insights into these catabolic mechanisms have only recently emerged with the identification of so-called ER-phagy receptors, which label specific ER subdomains for selective lysosomal delivery for clearance. Here, in eight chapters and one addendum, we comment on recent advances in ER turnover pathways induced by ER stress, nutrient deprivation, misfolded proteins, and live bacteria. We highlight the role of yeast (Atg39 and Atg40) and mammalian (FAM134B, SEC62, RTN3, and CCPG1) ER-phagy receptors and of autophagy genes in selective and non-selective catabolic processes that regulate cellular proteostasis by controlling ER size, turnover, and function.

Rab7a modulates ER stress and ER morphology

The Endoplasmic Reticulum (ER) is a membranous organelle with diverse structural and functional domains. Peripheral ER includes interconnected tubules, and dense tubular arrays called “ER matrices” together with bona fide flat cisternae. Transitions between these states are regulated by membrane-associated proteins and cytosolic factors. Recently, the small GTPases Rab10 and Rab18 were reported to control ER shape by regulating ER dynamics and fusion. Here, we present evidence that another Rab protein, Rab7a, modulates the ER morphology by controlling the ER homeostasis and ER stress. Indeed, inhibition of Rab7a expression by siRNA or expression of the dominant negative mutant Rab7aT22 N, leads to enlargement of sheet-like ER structures and spreading towards the cell periphery. Notably, such alterations are ascribable neither to a direct modulation of the ER shaping proteins Reticulon-4b and CLIMP63, nor to interactions with Protrudin, a Rab7a-binding protein known to affect the ER organization. Conversely, depletion of Rab7a leads to basal ER stress, in turn causing ER membrane expansion. Both ER enlargement and basal ER stress are reverted in rescue experiments by Rab7a re-expression, as well as by the ER chemical chaperone tauroursodeoxycholic acid (TUDCA). Collectively, these findings reveal a new role of Rab7a in ER homeostasis, and indicate that genetic and pharmacological ER stress manipulation may restore ER morphology in Rab7a silenced cells.

Brown adipose tissue whitening leads to brown adipocyte death and adipose tissue inflammation

In mammals, white adipose tissue (WAT) stores and releases lipids, whereas brown adipose tissue (BAT) oxidizes lipids to fuel thermogenesis. In obese individuals, WAT undergoes profound changes; it expands, becomes dysfunctional, and develops a low-grade inflammatory state. Importantly, BAT content and activity decline in obese subjects, mainly as a result of the conversion of brown adipocytes to white-like unilocular cells. Here, we show that BAT "whitening" is induced by multiple factors, including high ambient temperature, leptin receptor deficiency, β-adrenergic signaling impairment, and lipase deficiency, each of which is capable of inducing macrophage infiltration, brown adipocyte death, and crown-like structure (CLS) formation. Brown-to-white conversion and increased CLS formation were most marked in BAT from adipose triglyceride lipase (Atgl)-deficient mice, where, according to transmission electron microscopy, whitened brown adipocytes contained enlarged endoplasmic reticulum, cholesterol crystals, and some degenerating mitochondria, and were surrounded by an increased number of collagen fibrils. Gene expression analysis showed that BAT whitening in Atgl-deficient mice was associated to a strong inflammatory response and NLRP3 inflammasome activation. Altogether, the present findings suggest that converted enlarged brown adipocytes are highly prone to death, which, by promoting inflammation in whitened BAT, may contribute to the typical inflammatory state seen in obesity.

Floral biology and pistil receptivity of the drumstick tree (Moringa oleifera Lam.)

Drumstick ( Moringa oleifera Lam.) has a wide range of uses due to its high nutritional value and the high oil content of its seeds. Many aspects of its reproductive biology remain poorly understood. We investigated the floral morphology of drumstick, its stigma receptivity and the structural and cytochemical features of the stigma and style at different developmental stages. The inflorescences are panicles of hermaphroditic flowers, with a pistil consisting of one open-type stigma and a hollow stylar canal. Stigma receptivity was assayed based on pollen germination, pollen tube growth and fruit set following artificial pollination. Flowers at later developmental stages exhibited greater stigma receptivity, higher percentages of pollen germination and a higher fruit set than those in earlier stages. Enhanced stigma receptivity was associated with increased amounts of insoluble polysaccharides, lipids and proteins in the canal cells at later developmental stages. An ultrastructural study of the cells lining the canal indicated that they were secretory cells containing an enlarged endoplasmic reticulum, dictyosomes, mitochondria, plastids and ribosomes. Post-anthesis, these organelles exhibited degeneration at the end of the secretory phase. This study provides an important contribution to current knowledge of the anatomy and ultrastructure of the style and stigma in drumstick. https://doi.org/10.2298/ABS170205046Z Received: February 5, 2017; Revised: April 10, 2017; Accepted: May 8, 2017; Published online: November 20, 2017 How to cite this article: Zhang J, Lin M, Chen H, Zhu Q, Linh VN, Chen X. Floral biology and pistil receptivity of the drumstick tree ( Moringa oleifera Lam.). Arch Boil Sci. 2018;70(2):299-305.

Sheng-Jiang Powder Ameliorates High Fat Diet Induced Nonalcoholic Fatty Liver Disease via Inhibiting Activation of Akt/mTOR/S6 Pathway in Rats.

Background and Aims Nonalcoholic fatty liver disease (NAFLD) is an alarming public health problem that directly contributes to increased prevalence of liver cirrhosis and hepatic cell cancer, but without any specific pharmacological option. Sheng-jiang powder (SJP), an empirical Chinese medicine formula to treat NAFLD, showed great efficacy but the specific mechanisms have never been reported. Therefore, we performed this study to explore the effect of SJP on NAFLD and the potential mechanism. Methods NAFLD was induced by high fat diet (HFD) feeding. Rats were treated with SJP/normal saline daily for 10 weeks and all rats were euthanized after 12 weeks' feeding. Liver tissue samples were obtained for biochemistry test and pathological evaluation. Additionally, oleic acid induced LO2 cells were employed to simulate a cell model of NAFLD. Cells were subjected to western blotting for Akt, mTOR, S6, SREBP1-c, and FASN detection after coincubated with SJP, LY294002 (a selective PI3K inhibitor), or the combination for 24h. Results HFD significantly induced hepatic steatosis. Plenty of lipid droplets were observed under transmission electron microscope. The ultrastructure of liver cells showed distinct changes with obvious endoplasmic reticulum expansion, mitochondrial contraction, and cell matrix solidification. Although no difference was detected in serum hepatic enzymes and tissue proinflammatory cytokines, the tissue level of SOD and GSH-px was much lower and the pathologic injuries were much severe in HFD feeding rats. However, SJP treatment significantly attenuated the ultrastructure changes and protected rat liver against inflammatory injury. Abundant of lipid droplets and high expression of pAkt, pmTOR, pS6, and FASN were observed in oleic acid treated LO2 cells, while these changes were restored by SJP treatment. Conclusions SJP is efficient in attenuating HFD induced NAFLD in rats and this effect might be partly related to the inhibition of Akt/mTOR/S6 pathway.

Mutations in SRP54 Gene Cause Severe Primary Neutropenia As Well As Shwachman-Diamond-like Syndrome

Context. Congenital neutropenia (CN) is a heterogeneous group of diseases characterized by low neutrophil count, severe bacterial infections, increased risk of leukemic transformation and various extra-hematopoietic organ dysfunctions. Even if 24 different genes have been linked to the etiology of CN, in many patients the genetic causes of CN remain unknown.Methods. Whole-exome sequencing (WES) was performed on a trio-based approach in 8 sporadic cases and 6 multiplex families. Sanger sequencing was performed in a second phase on 66 additional patients from the French CN registry. Structural and functional studies were performed using primary cells from patients including fibroblasts and hematopoietic cells. In vitro granulocytic differentiation was conducted by culturing CD34+ in serum-free medium with SCF, IL-3 and G-CSF for 21 days.Results. WES analysis identified a heterozygous mutation in the SRP54 gene, encoding the signal recognition particle (SRP) 54 GTPase protein, in 3 sporadic cases and an autosomal dominant family. Considering these results we directly sequenced SRP54 in the French CN cohort and identified 13 additional sporadic cases and 2 multiplex families, thus reaching a total of 23 cases carrying a SRP54 mutation (19 families, Figure 1A). The Thr117del in-frame deletion was found in 12 probands. In all patients, neutropenia was profound (mean neutrophil absolute count 0.23x109/L), diagnosed in the neonate period or during childhood (mean age 4.2 months), and required long-term G-CSF therapy (mean dose 9 µg/kg/day). Bone marrow examination showed a maturation arrest at the promyelocytic stage. In contrast to CN associated with ELANE mutations, SRP54-mutated patients presented an important degree of dysgranulopoiesis (abnormal localization and number of mature granules) and enlarged endoplasmic reticulum (ER) in promyelocytes as well as dystrophic neutrophils (Figure 1B-1C). No evolution into acute myeloid leukemia was observed in this cohort after a median follow-up period of 14.8 years despite high doses of G-CSF. Six out of the 23 patients had extra-hematopoietic manifestations comprising severe neurodevelopmental delay (n=5) and/or exocrine pancreatic insufficiency (n=3), and/or bone abnormalities (n=2) (Figure 1A).The SRP54 protein is a key component of the ribonucleoprotein complex SRP that mediates the co-translational targeting and the insertion of secretory and membrane proteins to the ER. We identified 7 distinct mutations that affect highly conserved residues within or interacting with G motifs involved in the GTPase activity of SRP54. Of note, 17 out of 18 patients with mutations located within the G1 element and predicted to affect the structure and/or stability of the NG domain presented only a severe neutropenia. In contrast, the other 5 patients with mutations affecting either the magnesium-binding or the guanine-binding site and/or implied in the heterodimeric association with the receptor of SRP54, were associated with Shwachman-Diamond-like syndrome features. During the in vitro granulocytic differentiation of both normal and mutated hematopoietic progenitors, we found a strong increase in SRP54 mRNA expression levels associated with a slight decrease in the protein level in patients compared to controls. Moreover, SRP54 mutations induced a major deleterious effect on proliferation (Figure 1D) and a delay in the differentiation associated with increased apoptosis. Using both in vitro-derived granulocytic cells and primary fibroblasts, we also observed that SRP54 mutations led to ER stress (increased eIF2a phosphorylation, XBP1 splicing, ATF4 and CHOP expression) and enhanced autophagy (higher levels of LC3-II and ULK1 expression).Conclusions. This study thus identifies a novel pathological pathway implicating the co-translational process of protein targeting. This new genetic subtype which represents the second cause of CN in the French registry (prevalence 6.9%), is characterized by a promyelocytic maturation arrest with dysgranulopoiesis leading to a profound neutropenia, with a poor response to G-CSF, and in few patients, is associated with severe neurodevelopmental delay and exocrine pancreatic insufficiency. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Autophagy mediated by endoplasmic reticulum stress enhances the caffeine-induced apoptosis of hepatic stellate cells.

Caffeine has been identified to have beneficial effects against chronic liver diseases, particularly liver fibrosis. Many studies have reported that caffeine ameliorates liver fibrosis by directly inducing hepatic stellate cell (HSC) apoptosis; however, the molecular mechanisms involved in this process remain unclear. The presents study aimed to detect the underlying mechanisms by which caffeine mediates HSC apoptosis and eliminates activated HSCs. For this purpose, the LX-2 cell line was applied in this study and the cells were exposed to various concentrations of caffeine for the indicated times. The effects of caffeine on cell viability and apoptosis were assessed by Cell Counting Kit-8 assay and flow cytometry, respectively. Autophagy and endoplasmic reticulum (ER) stress were explored by morphological assessment and western blotting. In the present study, caffeine was found to inhibit the viability and increase the apoptosis of the LX-2 cells in dose- and time-dependent manners. ER stress was stimulated by caffeine as demonstrated by increased levels of GRP78/BiP, CHOP and inositol-requiring enzyme 1 (IRE1)-α as well as many enlarged ERs detected by electron microscopy. Caffeine induced autophagy as shown by increased p62 and LC3II accumulation and the number of GFP/RFP-LC3 puncta and autophagosomes/autolysosomes. Moreover, IRE1-α knockdown decreased the level of autophagic flux, and inhibition of autophagy protected LX-2 cells from apoptotic death. In conclusion, our study showed that the caffeine-enhanced autophagic flux in HSCs was stimulated by ER stress via the IRE1-α signaling pathway, which further weakened HSC viability via the induction of apoptosis. These findings provide new insight into the mechanism of caffeine's anti-fibrotic effects.

Role of Endoplasmic Reticulum Stress in Silica-induced Apoptosis in RAW264.7 Cells

ObjectiveWe investigated the role of endoplasmic reticulum stress (ERS) in silica-induced apoptosis in alveolar macrophages in vitro. MethodsRAW264.7 cells were incubated with 200 μg/mL silica for different time periods. Cell viability was assayed by the MTT assay. Cell apoptosis was evaluated by DAPI staining, flow cytometry analysis, and Western blot analysis of caspase-3. Morphological changes in the endoplasmic reticulum were observed by transmission electron microscopy. The expression of ERS markers binding protein (BiP) and CCAAT-enhancer-binding protein homologous protein (CHOP) was examined by Western blotting and real-time PCR. As an inhibitor of ERS, 4-phenylbutyric acid (4-PBA) was used in the experiments. ResultsSilica exposure induced nuclear condensation and caspase-3 expression in RAW264.7 cells. The number of apoptotic cells increased after silica exposure in a time-dependent manner. Silica treatment induced expansion of the endoplasmic reticulum. In addition, the expression of BiP and CHOP increased in silica-stimulated cells. Furthermore, 4-PBA treatment inhibited silica-induced endoplasmic reticulum expansion and the expression of BiP and CHOP. Moreover, 4-PBA treatment attenuated nuclear condensation, reduced apoptotic cells, and downregulated caspase-3 expression in silica-stimulated cells. ConclusionSilica-induced ERS is involved in the apoptosis of alveolar macrophages.

Atherosusceptible Shear Stress Activates Endoplasmic Reticulum Stress to Promote Endothelial Inflammation

Atherosclerosis impacts arteries where disturbed blood flow renders the endothelium susceptible to inflammation. Cytokine activation of endothelial cells (EC) upregulates VCAM-1 receptors that target monocyte recruitment to atherosusceptible regions. Endoplasmic reticulum (ER) stress elicits EC dysregulation in metabolic syndrome. We hypothesized that ER plays a central role in mechanosensing of atherosusceptible shear stress (SS) by signaling enhanced inflammation. Aortic EC were stimulated with low-dose TNFα (0.3 ng/ml) in a microfluidic channel that produced a linear SS gradient over a 20mm field ranging from 0–16 dynes/cm2. High-resolution imaging of immunofluorescence along the monolayer provided a continuous spatial metric of EC orientation, markers of ER stress, VCAM-1 and ICAM-1 expression, and monocyte recruitment. VCAM-1 peaked at 2 dynes/cm2 and decreased to below static TNFα-stimulated levels at atheroprotective-SS of 12 dynes/cm2, whereas ICAM-1 rose to a maximum in parallel with SS. ER expansion and activation of the unfolded protein response also peaked at 2 dynes/cm2, where IRF-1-regulated VCAM-1 expression and monocyte recruitment also rose to a maximum. Silencing of PECAM-1 or key ER stress genes abrogated SS regulation of VCAM-1 transcription and monocyte recruitment. We report a novel role for ER stress in mechanoregulation at arterial regions of atherosusceptible-SS inflamed by low-dose TNFα.

Effect of endoplasmic reticulum stress in trophocytes on the pathogenesis of intrahepatic cholestasis of pregnancy

Objective: To evaluate the effect of endoplasmic reticulum stress in trophocytes, in patients with intrahepatic cholestasis of pregnancy (ICP). Methods: Sixty-one pregnant women who were hospitalized in Women's Hospital, School of Medicine, Zhejiang University from January to December 2015 were recruited. Thirty-one women who were diagnosed as ICP were defined as the ICP group and 30 healthy pregnant women were defined as the control group. The localization and expression intensity of glucose regulated protein 78 (GRP-78) in placental tissues were detected by immunohistochemistry technique. Electronic microscope was used to observe ultra-microstructure change of the endoplasmic reticulum in trophocytes and cell line Swan71. Reverse transcription (RT)-PCR and western blot were used to investigate the expression of GRP-78 mRNA and protein in Swan 71 cell. Results: (1) GRP-78 protein was mainly expressed in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts. The protein expression of GRP-78 in placentas of the ICP group (13.2±2.4) was significantly higher than that in the control group (7.8±1.3, P<0.01). (2) The volume of endoplasmie reticulum did not increase and the microvilli developed well, with no swelling and no expansion of endoplasmic reticulum in the control group.In the ICP group, microvilli injury, endoplasmic reticulum edema were found; the volume of endoplasmic reticulum increased, with dilation, vacuolation and significant degranulation. After treated with 100 μmol/L cholyglycine for 24 hours, universal dilatation of the endoplasmic reticulum were seen in the Swan71 cells. (3) In Swan71 cells, cholylglycine displayed a concentration-dependent up-regulation on the expression of GRP-78. The expressions of GRP-78 mRNA in 0, 25, 50, 100 μmol/L cholylglycine experimental group were 1.01±0.17, 2.17±0.16, 5.47±0.36, 5.65±0.82, respectively. The expression of GRP-78 protein in 0, 25, 50, 100 μmol/L cholylglycine experimental group were 1.01±0.04, 1.17±0.15, 1.33±0.13, 1.73±0.13, respectively. The expression of GRP-78 mRNA and protein in 100 and 50 μmol/L cholylglycine experimental group were significantly higher than 0 μmol/L (all P<0.01). Conclusion: The obvious expansion of endoplasmic reticulum and the increased expression of GRP-78 in trophocytes indicated that endoplasmic reticulum stress of trophocytes may be involved in the pathogenesis of ICP.

In vitro additive antitumor effects of dimethoxycurcumin and 5-fluorouracil in colon cancer cells.

Dimethoxycurcumin (DMC) is a lipophilic analog of curcumin, an effective treatment for colon cancer, which has greater chemical and metabolic stability. Chemotherapy treatments, such as 5‐fluorouracil (5‐Fu), play a key role in the current management of colon cancer. In this study, we investigated the antitumor efficacy of DMC in combination with 5‐Fu in SW480 and SW620 colon cancer cells. CCK‐8 assay was used to evaluate the inhibitory effect of DMC and 5‐Fu on cancer cells proliferation, and the combination index was calculated. The influence of DMC and 5‐Fu on cell cycle, apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential in SW480 and SW620 cells was determined using flow cytometry, and the related signaling pathways were detected by western blot. Transmission electron microscopy was used to observe endoplasmic reticulum expansion. DMC‐ and/or 5‐Fu‐induced apoptosis, stimulated G0/G1 phase arrest, increased ROS levels, decreased mitochondrial membrane potential, and enhanced endoplasmic reticulum expansion. The induction of apoptosis is involved in the increasing of Bax and cytochrome c and decreasing of Bcl2 expressions. Increased production of ROS was accompanied by upregulation of CHOP and Noxa. Combination therapy of DMC and 5‐Fu had increased efficacy on the above pathways compared with either drug alone. Based on the calculated IC 50, combination treatment with DMC and 5‐Fu had an additive antitumor effect in both cell lines. Combined treatment with DMC and 5‐Fu led to an additive antitumor effect in colon cancer cells that was related to apoptosis induction, G0/G1 phase arrest, increased ROS production, decreased mitochondrial membrane potential, and enhanced endoplasmic reticulum expansion.

The amyloidogenic light chain is a stressor that sensitizes plasma cells to proteasome inhibitor toxicity

AbstractSystemic light chain (AL) amyloidosis is caused by the clonal production of an unstable immunoglobulin light chain (LC), which affects organ function systemically. Although pathogenic LCs have been characterized biochemically, little is known about the biology of amyloidogenic plasma cells (PCs). Intrigued by the unique response rates of AL amyloidosis patients to the first-in-class proteasome inhibitor (PI) bortezomib, we purified and investigated patient-derived AL PCs, in comparison with primary multiple myeloma (MM) PCs, the prototypical PI-responsive cells. Functional, biochemical, and morphological characterization revealed an unprecedented intrinsic sensitivity of AL PCs to PIs, even higher than that of MM PCs, associated with distinctive organellar features and expression patterns indicative of cellular stress. These consisted of expanded endoplasmic reticulum (ER), perinuclear mitochondria, and a higher abundance of stress-related transcripts, and were consistent with reduced autophagic control of organelle homeostasis. To test whether PI sensitivity stems from AL LC production, we engineered PC lines that can be induced to express amyloidogenic and nonamyloidogenic LCs, and found that AL LC expression alters cell growth and proteostasis and confers PI sensitivity. Our study discloses amyloidogenic LC production as an intrinsic PC stressor, and identifies stress-responsive pathways as novel potential therapeutic targets. Moreover, we contribute a cellular disease model to dissect the biology of AL PCs.

The Chemical Chaperone, PBA, Reduces ER Stress and Autophagy and Increases Collagen IV α5 Expression in Cultured Fibroblasts From Men With X-Linked Alport Syndrome and Missense Mutations

IntroductionX-linked Alport syndrome (OMIM 301050) is caused by COL4A5 missense variants in 40% of families. This study examined the effects of chemical chaperone treatment (sodium 4-phenylbutyrate) on fibroblast cell lines derived from men with missense mutations.MethodsDermal fibroblast cultures were established from 2 affected men and 3 normals. Proliferation rates were examined, the collagen IV α5 chain localized with immunostaining, and levels of the intra- and extracellular chains quantitated with an in-house enzyme-linked immunosorbent assay. COL4A5 mRNA was measured using quantitative reverse transcriptase polymerase chain reaction. Endoplasmic reticulum (ER) size was measured on electron micrographs and after HSP47 immunostaining. Markers of ER stress (ATF6, HSPA5, DDIT3), autophagy (ATG5, BECN1, ATG7), and apoptosis (CASP3, BAD, BCL2) were also quantitated by quantitative reverse transcriptase polymerase chain reaction. Measurements were repeated after 48 hours of incubation with 10 mM sodium 4-phenylbutyrate acid.ResultsBoth COL4A5 missense variants were associated with reduced proliferation rates on day 6 (P = 0.01 and P = 0.03), ER enlargement, and increased mRNA for ER stress and autophagy (all P values < 0.05) when compared with normal. Sodium 4-phenylbutyrate treatment increased COL4A5 transcript levels (P < 0.01), and reduced ER size (P < 0.01 by EM and P < 0.001 by immunostaining), ER stress (p HSPA5 and DDIT3, all P values < 0.01) and autophagy (ATG7, P < 0.01). Extracellular collagen IV α5 chain was increased in the M1 line only (P = 0.06).DiscussionSodium 4-phenylbutyrate increases collagen IV α5 mRNA levels, reduces ER stress and autophagy, and possibly facilitates collagen IV α5 extracellular transport. Whether these actions delay end-stage renal failure in men with X-linked Alport syndrome and missense mutations will only be determined with clinical trials.

Does Cimicifuga racemosa have the effects like estrogen on the sublingual gland in ovariectomized rats?

BackgroundCimicifuga racemosa is one of the herbs used for the treatment of climacteric syndrome, and it has been cited as an alternative therapy to estrogen. Apart from hectic fevers, dyspareunia and so on, dry mouth also increase significantly after menopause. It has not yet been reported whether C. racemosa has any impact on the sublingual gland, which may relate to dry mouth. In an attempt to determine this, we have compared the effects of estrogen and C. racemosa on the sublingual gland of ovariectomized rats.ResultsHE staining showed that the acinar cell area had contracted and that the intercellular spaces were broadened in the OVX (ovariectomized rats) group, while treatment with estradiol (E2) and iCR (isopropanolic extract of C. racemosa) improved these lesions. Transmission electron microscopy showed that rough endoplasmic reticulum expansion in mucous and serous acinar epithelial cells and apoptotic cells was more commonly seen in the OVX group than in the SHAM (sham-operated rats) group. Mitochondria and plasma membrane infolding lesions in the striated ducts were also observed. These lesions were alleviated by both treatments. It is of note that, in the OVX + iCR group, the volume of mitochondria in the striated duct was larger than in other groups. Immunohistochemical staining showed that the ratio of caspase-3 positive cells was significantly increased in the acinar cells of the OVX group compared with the SHAM group (p < 0.05); and the MA (mean absorbance) of caspase-3 in the striated ducts also increased (p < 0.05). Estradiol decreased the ratio of caspase-3 positive cells and the MA of caspase-3 in striated ducts significantly (p < 0.05). ICR also reduced the ratio of caspase-3 positive cells and the MA in the striated ducts (p < 0.05), but the reduction of the MA in striated ducts was inferior to that of the OVX + E2 group (p < 0.05).ConclusionBoth estradiol and iCR can inhibit subcellular structural damage, and down-regulate the expression of caspase-3 caused by ovariectomy, but their effects were not identical, suggesting that both drugs confer a protective effect on the sublingual gland of ovariectomized rats, but that the specific location and mechanism of action producing these effects were different.

A novel form of necrosis, TRIAD, occurs in human Huntington\u2019s disease

We previously reported transcriptional repression-induced atypical cell death of neuron (TRIAD), a new type of necrosis that is mainly regulated by Hippo pathway signaling and distinct from necroptosis regulated by RIP1/3 pathway. Here, we examined the ultrastructural and biochemical features of neuronal cell death in the brains of human HD patients in parallel with the similar analyses using mutant Htt-knock-in (Htt-KI) mice. LATS1 kinase, the critical regulator and marker of TRIAD, is actually activated in cortical neurons of postmortem human HD and of Htt-KI mouse brains, while apoptosis promoter kinase Plk1 was inactivated in human HD brains. Expression levels of YAP/YAPdeltaC were decreased in cortical neurons of human HD brains. Ultra-structural analyses revealed extreme enlargement of endoplasmic reticulum (ER), which characterizes TRIAD, in cortical neurons of human HD and those of Htt-KI mice. These biochemical and morphological results support that TRIAD occurs in human and mouse neurons under the HD pathology.

Roles of Endoplasmic Reticulum Stress in NECA-Induced Cardioprotection against Ischemia/Reperfusion Injury.

Objective This study aimed to investigate whether the nonselective A2 adenosine receptor agonist NECA induces cardioprotection against myocardial ischemia/reperfusion (I/R) injury via glycogen synthase kinase 3β (GSK-3β) and the mitochondrial permeability transition pore (mPTP) through inhibition of endoplasmic reticulum stress (ERS). Methods and Results H9c2 cells were exposed to H2O2 for 20 minutes. NECA significantly prevented H2O2-induced TMRE fluorescence reduction, indicating that NECA inhibited the mPTP opening. NECA blocked H2O2-induced GSK-3β phosphorylation and GRP94 expression. NECA increased GSK-3β phosphorylation and decreased GRP94 expression, which were prevented by both ERS inductor 2-DG and PKG inhibitor KT5823, suggesting that NECA may induce cardioprotection through GSK-3β and cGMP/PKG via ERS. In isolated rat hearts, both NECA and the ERS inhibitor TUDCA decreased myocardial infarction, increased GSK-3β phosphorylation, and reversed GRP94 expression at reperfusion, suggesting that NECA protected the heart by inhibiting GSK-3β and ERS. Transmission electron microscopy showed that NECA and TUDCA reduced mitochondrial swelling and endoplasmic reticulum expansion, further supporting that NECA protected the heart by preventing the mPTP opening and ERS. Conclusion These data suggest that NECA prevents the mPTP opening through inactivation of GSK-3β via ERS inhibition. The cGMP/PKG signaling pathway is responsible for GSK-3β inactivation by NECA.

Role of endoplasmic reticulum stress in disuse osteoporosis

Osteoporosis is a major skeletal disease with low bone mineral density, which leads to an increased risk of bone fracture. Salubrinal is a synthetic chemical that inhibits dephosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) in response to endoplasmic reticulum (ER) stress. To understand possible linkage of osteoporosis to ER stress, we employed an unloading mouse model and examined the effects of salubrinal in the pathogenesis of disuse osteoporosis. The results presented several lines of evidence that osteoclastogenesis in the development of osteoporosis was associated with ER stress, and salubrinal suppressed unloading-induced bone loss. Compared to the age-matched control, unloaded mice reduced the trabecular bone area/total area (B.Ar/T.Ar) as well as the number of osteoblasts, and they increased the osteoclasts number on the trabecular bone surface in a time-dependent way. Unloading-induced disuse osteoporosis significantly increased the expression of Bip, p-eIF2α and ATF4 in short-term within 6h of tail suspension, but time-dependent decreased in HU2d to HU14d. Furthermore, a significant correlation of ER stress with the differentiation of osteoblasts and osteoclasts was observed. Administration of salubrinal suppressed the unloading-induced decrease in bone mineral density, B.Ar/T.Ar and mature osteoclast formation. Salubrinal also increased the colony-forming unit-fibroblasts and colony-forming unit-osteoblasts. It reduced the formation of mature osteoclasts, suppressed their migration and adhesion, and increased the expression of Bip, p-eIF2α and ATF4. Electron microscopy showed that rough endoplasmic reticulum expansion and a decreased number of ribosomes on ER membrane were observed in osteoblast of unloading mice, and the abnormal ER expansion was significantly improved by salubrinal treatment. A TUNEL assay together with CCAAT/enhancer binding protein homologous protein (CHOP) expression indicated that ER stress-induced osteoblast apoptosis was rescued by salubrinal. Collectively, the results support the notion that ER stress plays a key role in the pathogenesis of disuse osteoporosis, and salubrinal attenuates unloading-induced bone loss by altering proliferation and differentiation of osteoblasts and osteoclasts via eIF2α signaling.