thapsigargin and 4-phenylbutyric-acid

Epithelial‑to‑mesenchymal transition (EMT) of human lens epithelial cells (HLECs) serve an important role in cataract formation. The endoplasmic reticulum stress response (ER stress) has been demonstrated to regulate EMT in a number of tissues. The aim of the present study was to demonstrate the role of ER stress on EMT in HLECs. HLECs were treated with tunicamycin (TM) or thapsigargin (TG) to disturb ER homeostasis, and 4‑phenylbutyric acid (PBA) or sodium tauroursodeoxycholate (TUDCA) to restore ER homeostasis. Cell morphology was evaluated after 24 h. The long axis and aspect ratio of the cells were analyzed using ImageJ software. The results demonstrated that HLECs adopted an elongated morphology following treatment with TG, and the cellular aspect ratio increased. However, this morphological change was not observed following combination treatment with TG and PBA. Western blot analysis and immunofluorescence staining were used to measure the protein expression levels. A wound‑healing assay was performed to evaluate cell migration. Treatment with TM or TG increased the expression of the ER stress markers glucose‑regulated protein 78, phosphorylated eukaryotic initiation factor 2α, activating transcription factor (ATF)6, ATF4 and inositol‑requiring protein 1α and the EMT markers fibronectin, vimentin, α‑smooth muscle actin and neural cadherin. Furthermore, treatment with TM or TG decreased the expression of the epithelial cell marker epithelial cadherin and enhanced cell migration, which effects were inhibited following treatment with PBA or TUDCA. These results indicates that enhanced ER stress induced EMT and subsequently increased cell migration in HLECs in vitro.

Topics: Cataract; Cell Line; Endoplasmic Reticulum Stress; Epithelial Cells; Epithelial-Mesenchymal Transition; Eye Proteins; Humans; Lens, Crystalline; Phenylbutyrates; Taurochenodeoxycholic Acid; Thapsigargin; Tunicamycin

B cells orchestrate pro-survival and pro-apoptotic inputs during unfolded protein response (UPR) to translate, fold, sort, secrete and recycle immunoglobulins. In common variable immunodeficiency (CVID) patients, activated B cells are predisposed to an overload of abnormally processed, misfolded immunoglobulins. Using highly accurate transcript measurements, we show that expression of UPR genes and immunoglobulin chains differs qualitatively and quantitatively during the first 4 h of chemically induced UPR in B cells from CVID patients and a healthy subject. We tested thapsigargin or tunicamycin as stressors and 4-phenylbutyrate, dimethyl sulfoxide and tauroursodeoxycholic acid as chemical chaperones. We found an early and robust decrease of the UPR upon endoplasmic reticulum (ER) stress in CVID patient cells compared to the healthy control consistent with the disease phenotype. The chemical chaperones increased the UPR in the CVID patient cells in response to the stressors, suggesting that misfolded immunoglobulins were stabilized. We suggest that the AMP-dependent transcription factor alpha branch of the UPR is disturbed in CVID patients, underlying the observed expression behavior.

Topics: B-Lymphocytes; Cells, Cultured; Common Variable Immunodeficiency; Dimethyl Sulfoxide; Endoplasmic Reticulum Stress; Gene Expression Profiling; Gene Expression Regulation; Gene Regulatory Networks; Humans; Immunoglobulins; Phenylbutyrates; Taurochenodeoxycholic Acid; Thapsigargin; Transcription Factors; Tunicamycin; Unfolded Protein Response

The present study investigated whether melatonin influences the expression/oligomerization of amylin with endoplasmic reticulum (ER) stress in rat insulinoma INS-1E cells. No change in cell survival after exposure to thapsigargin- and tunicamycin-combined melatonin treatment or melatonin-only treatment was observed when compared with the normal control cells. With thapsigargin-only or combined tunicamycin-melatonin treatments, phosphorylation of extracellular signal-regulated kinase (ERK) was significantly increased compared with control and melatonin-only treatments. A significant increase was observed in the levels of ER stress markers, namely, phosphorylated inositol-requiring protein 1α (p-IRE1α), CCAAT enhancer binding proteins (C/EBP)-homologous protein, p-eukaryotic translation initiation factor 2α and cleaved caspase-12, in the thapsigargin-combined melatonin-treated cells as compared with the tunicamycin-combined or only melatonin treatment. The melatonin-only treatment resulted in increased levels of amylin expression/oligomerization in 15-25 kDa and insulin proteins, compared with the thapsigargin- and tunicamycin-combined melatonin treatments. Treatment with ER stress inhibitor 4-phenylbutyric acid (4-PBA) did not suppress amylin expression/oligomerization or insulin production with thapsigargin or tunicamycin treatment. Levels of cleaved caspase-12 were significantly decreased in the thapsigargin- or tunicamycin-4-PBA combination treatments. Therefore, whether melatonin regulates the amylin expression/oligomerization in thapsigargin- or tunicamycin-combined with Bafilomycin A1 (autophagy inhibitor) or MG132 (proteasome inhibitor) treatments were investigated. Amylin expression/oligomerization with melatonin treatment was significantly decreased in the thapsigargin- or tunicamycin-combined Bafilomycin A1 or MG132 treatments. Since these outcomes are involved in cell viability, they indicate that increased cell death leads to decreased amylin expression/oligomerization, however, the effects of melatonin treatment on amylin expression/oligomerization induce proliferation of pancreatic β cells and improve the cellular functions of pancreatic β cells.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cell Survival; Endoplasmic Reticulum Stress; Insulin; Insulin-Secreting Cells; Islet Amyloid Polypeptide; Melatonin; Phenylbutyrates; Phosphorylation; Protein Serine-Threonine Kinases; Rats; Signal Transduction; Thapsigargin; Tunicamycin

Apoptosis of osteoblasts triggered by high-dose glucocorticoids (GCs) has been identified as a major cause of osteoporosis. However, the molecular mechanisms underlying GC-induced osteoporosis remain elusive. This study was conducted to make clear the mechanism of GC-induced osteoblast apoptosis and to examine whether reduction of ER stress by 4-PBA inhibited osteoblast apoptosis. After treatment with dexamethasone (Dex) or hydrocortisone, cell viability was assessed using an MTT assay. Flow cytometry was performed to assess the apoptosis of MC3T3-E1 cells. The expression levels of ER stress-related proteins (CHOP, GRP78, eIF2α, and phospho-eIF2α) and apoptosis-related proteins (cleaved Caspase-3, Bcl-2, and Bax) in MC3T3-E1 cells were measured by Western blot analysis. We found that both Dex and hydrocortisone reduced cell proliferation and promoted apoptosis in MC3T3-E1 cells. In addition, the protein expression levels of cleaved Caspase-3 and Bax increased and the protein expression level of Bcl-2 decreased in MC3T3-E1 cells exposed to Dex. In addition, the Dex exposure also resulted in a release of cytochrome c (Cyt C) from mitochondria. The cellular ATP content was decreased following prolonged treatment with Dex. 4-PBA attenuated ER stress and mitochondrial dysfunction induced by Dex in MC3T3-E1 cells. Dex-mediated apoptosis of MC3T3-E1 cells is aggravated by ER stress. Moreover, Dex-induced apoptosis in MC3T3-E1 cells was inhibited by 4-PBA, suggesting that ER stress involved in Dex-induced apoptosis. In conclusion, inhibition of ER stress by 4-PBA could reduce GC-induced apoptosis in MC3T3-E1 cells.

Topics: Animals; Apoptosis; Butylamines; Caspase 3; Cell Line; Dexamethasone; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Glucocorticoids; Humans; Mice; Mitochondria; Osteoblasts; Phenylbutyrates; Thapsigargin

Glucosamine is an essential substrate for N-linked protein glycosylation. However, elevated levels of glucosamine can induce endoplasmic reticulum (ER) stress. Glucosamine-induced ER stress has been implicated in the development of diabetic complications, including atherosclerosis and hepatic steatosis. In this study, we investigate the potential relationship between the effects of glucosamine on lipid-linked oligosaccharide (LLO) biosynthesis, N-linked glycosylation, and ER homeostasis. Mouse embryonic fibroblasts (MEFs) were cultured in the presence of 0-5 mM glucosamine for up to 18 h, and LLO biosynthesis was monitored by fluorescence-assisted carbohydrate electrophoresis. ER stress was determined by quantification of unfolded protein response (UPR) gene expression. We found that exposure of MEFs to ≥1 mM glucosamine significantly impaired the biosynthesis of mature (Glc

Topics: Animals; Cell Line; Dithiothreitol; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Fibroblasts; Glucosamine; Glycosylation; Lipopolysaccharides; Mice; Phenylbutyrates; Thapsigargin; Unfolded Protein Response

Newly translated proteins must undergo proper folding to ensure their function. To enter a low energy state, misfolded proteins form aggregates, which are associated with many degenerative diseases, such as Huntington's disease and chronic kidney disease (CKD). Recent studies have shown the use of low molecular weight chemical chaperones to be an effective method of reducing protein aggregation in various cell types. This study demonstrates a novel non-biased assay to assess the molecular efficacy of these compounds at preventing protein misfolding and/or aggregation. This assay utilizes a thioflavin T fluorescent stain to provide a qualitative and quantitative measure of protein misfolding within cells. The functionality of this method was first assessed in renal proximal tubule epithelial cells treated with various endoplasmic reticulum (ER) stress inducers. Once established in the renal model system, we analyzed the ability of some known chemical chaperones to reduce ER stress. A total of five different compounds were selected: 4-phenylbutyrate (4-PBA), docosahexaenoic acid (DHA), tauroursodeoxycholic acid, trehalose, and glycerol. The dose-dependent effects of these compounds at reducing thapsigargin-induced ER stress was then analyzed, and used to determine their EC

Topics: Benzothiazoles; Cell Line; Docosahexaenoic Acids; Endoplasmic Reticulum Stress; Epithelial Cells; Glycerol; Humans; Kidney Tubules, Proximal; Molecular Weight; Phenylbutyrates; Protein Aggregates; Protein Aggregation, Pathological; Protein Folding; Staining and Labeling; Taurochenodeoxycholic Acid; Thapsigargin; Thiazoles; Trehalose; Unfolded Protein Response; Xenobiotics

Adipocyte differentiation is critical in obesity. By controlling new adipocyte recruitment, adipogenesis contrasts adipocyte hypertrophy and its adverse consequences, such as insulin resistance. Contrasting data are present in literature on the effect of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR) on adipocyte differentiation, being reported to be either necessary or inhibitory. In this study, we sought to clarify the effect of ER stress and UPR on adipocyte differentiation. We have used two different cell lines, the widely used pre-adipocyte 3T3-L1 cells and a murine multipotent mesenchymal cell line, W20-17 cells. A strong ER stress activator, thapsigargin, and a pathologically relevant inducer of ER stress, glucosamine (GlcN), induced ER stress and UPR above those occurring in the absence of perturbation and inhibited adipocyte differentiation. Very low concentrations of 4-phenyl butyric acid (PBA, a chemical chaperone) inhibited only the overactivation of ER stress and UPR elicited by GlcN, leaving unaltered the part physiologically activated during differentiation, and reversed the inhibitory effect of GlcN on differentiation. In addition, GlcN stimulated proinflammatory cytokine release and PBA prevented these effects. An inhibitor of NF-kB also reversed the effects of GlcN on cytokine release. These results indicate that while ER stress and UPR activation is "physiologically" activated during adipocyte differentiation, the "pathologic" part of ER stress activation, secondary to a glucotoxic insult, inhibits differentiation. In addition, such a metabolic insult, causes a shift of the preadipocyte/adipocyte population towards a proinflammatory phenotype.

Topics: 3T3-L1 Cells; Adipocytes; Adult; Animals; Blotting, Western; Cell Differentiation; Cell Line; Cells, Cultured; Cytokines; Endoplasmic Reticulum Stress; Gene Expression; Glucosamine; Humans; Inflammation Mediators; Mice; Middle Aged; NF-kappa B; Phenotype; Phenylbutyrates; Phenylenediamines; Reverse Transcriptase Polymerase Chain Reaction; Thapsigargin; Unfolded Protein Response

Palmitic acid (C16:0) and TLR2 ligand induce, but docosahexaenoic acid (DHA) inhibits monocyte activation. C16:0 and TLR2 or TLR4 ligand induce certain ER stress markers; thus, we determined whether ER stress induced by these agonists is sufficient to induce monocyte activation, and whether the ER stress is inhibited by DHA which is known to inhibit C16:0- or ligand-induced TLR activation. Monocyte activation and ER stress were assessed by TLR/inflammasome-induced IL-1β production, and phosphorylation of IRE-1 and eIF2 and expression of CHOP, respectively in THP-1 cells. TLR2 ligand Pam3CSK4 induced phosphorylation of eIF2, but not phosphorylation of IRE-1 and CHOP expression. LPS also induced phosphorylation of both IRE-1 and eIF2 but not CHOP expression suggesting that TLR2 or TLR4 ligand, or C16:0 induces different ER stress responses. C16:0-, Pam3CSK4-, or LPS-induced IL-1β production was inhibited by 4-phenylbutyric acid, an inhibitor of ER stress suggesting that IL-1β production induced by these agonists is partly mediated through ER stress. Among two ER stress-inducing molecules, thapsigargin but not tunicamycin led to the expression of pro-IL-1β and secretion of IL-1β. Thus, not all types of ER stress are sufficient to induce inflammasome-mediated IL-1β secretion in monocytes. Although both C16:0 and thapsigargin-induced IL-1β secretion was inhibited by DHA, only C16:0-mediated ER stress was responsive to DHA. These findings suggest that the anti-inflammatory effects of DHA are at least in part mediated through modulating ER homeostasis and that the propensity of ER stress can be differentially modulated by the types of dietary fat we consume.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Biomarkers; Cell Line; Docosahexaenoic Acids; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Immunomodulation; Inflammasomes; Interleukin-1beta; Ligands; Lipopeptides; Lipopolysaccharides; Monocytes; Palmitic Acid; Phenylbutyrates; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction; Thapsigargin; Toll-Like Receptor 2; Toll-Like Receptor 4

Adipose tissue inflammation has been linked to age-related metabolic diseases. However, the underlying mechanisms are poorly understood. Adipose tissue inflammation and insulin resistance in diet associated obesity has been correlated with aberrant endoplasmic reticulum (ER) stress. This study was undertaken to test our hypothesis that increased ER stress response contributes to age-associated adipose tissue inflammation. We found elevated ER stress response in adipose tissue of old (18-20 months) compared to young (4-6 months) mice. Elevated ER stress markers BIP (GRP78), CHOP, cleaved-ATF-6, phospho-IRE1α, and XBP-1 were observed in old compared to young adipose tissue stromal cells. Additionally, old adipose tissue stromal cells were more sensitive to an ER stress inducer, thapsigargin. Similar experiments with adipose tissue macrophages showed elevated Chop and Bip expression in old adipose tissue macrophages when induced with thapsigargin. Treatment of chemical chaperone 4-phenyle-butyric acid alleviated ER stress in adipose tissue stromal cells and adipose tissue macrophages and attenuated the production of IL-6 and MCP-1 by adipose tissue stromal cells, and TNF-α by adipose tissue macrophages from both young and old mice. Finally, old mice fed with 4-phenyle-butyric acid have reduced expression of ER stress and inflammatory cytokine genes. Our data suggests that an exaggerated ER stress response in aging adipose tissue contributes to age-associated inflammation that can be mitigated by treatment with chemical chaperones.

Topics: Activating Transcription Factor 6; Adipose Tissue; Age Factors; Animals; Cell Culture Techniques; Cytokines; DNA-Binding Proteins; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Endoribonucleases; Enzyme Inhibitors; Heat-Shock Proteins; Inflammation; Macrophages; Male; Mice; Phenylbutyrates; Protein Serine-Threonine Kinases; Regulatory Factor X Transcription Factors; RNA, Messenger; Stromal Cells; Thapsigargin; Transcription Factor CHOP; Transcription Factors; X-Box Binding Protein 1

The anthraquinone compound rhein is a natural agent in the traditional Chinese medicine rhubarb. Preclinical studies demonstrate that rhein has anticancer activity. Treatment of a variety of cancer cells with rhein may induce apoptosis. Here, we report that rhein induces atypical unfolded protein response in breast cancer MCF-7 cells and hepatoma HepG2 cells. Rhein induces CHOP expression, eIF2α phosphorylation and caspase cleavage, while it does not induce glucose-regulated protein 78 (GRP78) expression in both MCF-7 and HepG2 cells. Meanwhile, rhein inhibits thapsigargin-induced GRP78 expression and X box-binding protein 1 splicing. In addition, rhein inhibits Akt phosphorylation and stimulates FOXO transactivation activity. Rhein induces Bim expression in MCF-7 and HepG2 cells, which can be abrogated by FOXO3a knockdown. Knockdown of FOXO3a or Bim abrogates rhein-induced caspase cleavage and apoptosis. The chemical chaperone 4-phenylbutyrate acid antagonizes the induction of FOXO activation, Bim expression and caspase cleavage by rhein, indicating that protein misfolding may be involved in triggering these deleterious effects. We conclude that FOXO3a-mediated up-regulation of Bim is a key mechanism underlying rhein-induced cancer cells apoptosis.

Topics: Anthraquinones; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Caspases; Cell Line; Endoplasmic Reticulum Chaperone BiP; Epithelial Cells; Eukaryotic Initiation Factor-2; Female; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Hep G2 Cells; Humans; Mammary Glands, Human; MCF-7 Cells; Membrane Proteins; Phenylbutyrates; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Thapsigargin; Transcription Factor CHOP

We have recently shown that the histidine-rich calcium binding protein (HRC) promotes the invasion and metastasis of hepatocellular carcinoma (HCC). In the current study, we evaluated whether HRC may also affect the growth of HCC. We found that ectopic expression of HRC obviously enhanced proliferation and colony formation, while suppression of HRC exhibited inhibitory effects. Furthermore, we demonstrated that HRC promoted tumor growth in nude mice. These effects may result from the ability of HRC to upregulate cyclinD1 and cyclin-dependent kinase 2 (CDK2) expressions and promote G1/S transition. Further study showed that MEK/ERK signaling pathway was involved in HRC-induced cell proliferation. Interestingly, overexpression or depletion of HRC revealed its regulation on endoplasmic reticulum stress (ERS) and apoptosis, which was partially dependent on PERK/ATF4/CHOP signaling pathway. In addition, blocking ERS using 4-phenylbutyric acid (4-PBA) not only downregulated the expression of PERK, ATF4 and CHOP, but also significantly decreased apoptosis induced by HRC silence, whereas ERS inducer thapsigargin (TG) exerted the opposite effects. Our study thus demonstrates a role of HRC in promoting HCC growth, besides its role in inducing HCC metastasis, and highlights HRC as a promising intervention target for HCC.

Topics: Activating Transcription Factor 4; Animals; Apoptosis; Calcium-Binding Proteins; Carcinoma, Hepatocellular; Caspase 3; Cell Proliferation; eIF-2 Kinase; Endoplasmic Reticulum Stress; G1 Phase Cell Cycle Checkpoints; Humans; Liver Neoplasms; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Phenylbutyrates; RNA Interference; RNA, Small Interfering; Thapsigargin; Transcription Factor CHOP; Transplantation, Heterologous

Ampelopsin (AMP), a major bioactive constituent of Ampelopsis grossedentata, exerts a number of biological effects. In this study, we investigated its anti-cancer activity in human breast cancer cell lines, and explored the underlying mechanism of this action. Our results showed that treatment with AMP dose-dependently inhibited cell viability and induced apoptosis in MCF-7 and MDA-MB-231 breast cancer cells without cytotoxicity in human normal breast epithelial cells MCF-10A. Meanwhile, AMP dose- dependently triggered reactive oxygen species (ROS) generation in both breast cancer cells. The ROS scavenger N-acetyl-L-cysteine (NAC) strongly attenuated AMP-induced ROS production, along with cell growth inhibition and apoptosis. Furthermore, AMP was observed to activate endoplasmic reticulum (ER) stress, as evidenced by the up-regulation of ER stress-related proteins, including GRP78, p-PERK, p-elF2α, cleaved ATF6α and CHOP, while knockdown of ATF6α or PERK markedly down-regulated AMP-induced CHOP expression. Blocking ER stress using 4-phenylbutyric acid not only down-regulated AMP-induced GRP78 and CHOP expression, but also significantly decreased AMP-induced cell growth inhibition and apoptosis, whereas ER stress inducer thapsigargin played opposing effects. Additionally, NAC inhibited AMP-induced ER stress by down-regulating GRP78 and CHOP expression. Conversely, blocking ER stress using CHOP siRNA decreased AMP-induced ROS production and cell apoptosis. Taken together, these results demonstrate that AMP has anti-tumor effects against breast cancer cells through ROS generation and ER stress pathway, which therefore provide experimental evidences for developing AMP as a new therapeutic drug for breast cancer.

Topics: Acetylcysteine; Activating Transcription Factor 6; Ampelopsis; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; eIF-2 Kinase; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Phenylbutyrates; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Thapsigargin; Transcription Factor CHOP; Transcription Factors

The mechanism by which IL-1β and thapsigargin (TG)-induced endoplasmic reticulum (ER) stress modulate the receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated osteoclastogenesis remains elusive. Thus, we investigated the osteoclast-specific and ER signals in osteoclastogenesis of bone marrow-derived cells.. Bone marrow cells (BMCs) were obtained from 5-week-old male ICR mice and cultured to be differentiated into osteoclasts with M-CSF and RANKL in the presence or absence of IL-1β, TG, or 4-phenylbutyric acid (PBA), an ER stress-reducing drug. The formation of osteoclasts was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and resorption pit assay with a dentine slice. The molecular mechanism of IL-1β and ER stress in osteoclastogenesis was investigated in BMCs transfected with siRNA for GRP78, PERK and IRE1 using reverse transcription-polymerase chain reaction and immunoblotting for osteoclast-specific and ER stress signaling molecules.. IL-1β and ER stress induced by TG-augmented the formation of osteoclasts, which was significantly inhibited by PBA and was mediated with osteoclast-specific signals, including c-Fos, NFATc1, and ER stress- associated signaling pathways, such as PERK, IRE1, GRP78, and eIF2α. siRNA-mediated knockdown of ER stress signals inhibited the expression of NFATc1 and c-Fos, thus reducing IL-1β and/or TG-induced formation of osteoclasts.. Osteoclastogenesis by IL-1β and/or ER stress is mainly associated with upregulation of eIF2α, GRP78, PERK and IRE1. These results suggest that the signaling pathway of ER stress-induced osteoclast formation might be a new therapeutic target to prevent inflammatory and destructive arthritic disease such as RA and diverse osteoporosis.

Topics: Animals; Bone Marrow Cells; Cell Differentiation; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Interleukin-1beta; Macrophages; Male; Mice; Mice, Inbred ICR; Models, Animal; Osteoclasts; Phenylbutyrates; RANK Ligand; Signal Transduction; Thapsigargin

Radiotherapy, which is one of the most effective approaches to the treatment of various cancers, plays an important role in malignant cell eradication in the pelvic area and abdomen. However, it also generates some degree of intestinal injury. Apoptosis in the intestinal epithelium is the primary pathological factor that initiates radiation-induced intestinal injury, but the mechanism by which ionizing radiation (IR) induces apoptosis in the intestinal epithelium is not clearly understood. Recently, IR has been shown to induce endoplasmic reticulum (ER) stress, thereby activating the unfolded protein response (UPR) signaling pathway in intestinal epithelial cells. However, the consequences of the IR-induced activation of the UPR signaling pathway on radiosensitivity in intestinal epithelial cells remain to be determined. In this study, we investigated the role of ER stress responses in IR-induced intestinal epithelial cell death. We show that chemical ER stress inducers, such as tunicamycin or thapsigargin, enhanced IR-induced caspase 3 activation and DNA fragmentation in intestinal epithelial cells. Knockdown of Xbp1 or Atf6 with small interfering RNA inhibited IR-induced caspase 3 activation. Treatment with chemical chaperones prevented ER stress and subsequent apoptosis in IR-exposed intestinal epithelial cells. Our results suggest a pro-apoptotic role of ER stress in IR-exposed intestinal epithelial cells. Furthermore, inhibiting ER stress may be an effective strategy to prevent IR-induced intestinal injury.

Topics: Animals; Apoptosis; Caspase 3; Cell Death; Cell Line; Endoplasmic Reticulum Stress; Enzyme Activation; Epithelial Cells; Intestinal Mucosa; Phenylbutyrates; Rats; Taurochenodeoxycholic Acid; Thapsigargin; Tunicamycin; Unfolded Protein Response

Cardiac fibroblast (CF) survival is important for the maintenance of the extracellular matrix homeostasis in the heart; providing a functional support to cardiomyocytes necessary for the correct myocardial function. Endoplasmic reticulum (ER) stress causes cellular dysfunction and cell death by apoptosis; and thapsigargin is a well-known ER stress inducer. On the other hand, the chemical chaperone, 4-phenylbutyric acid (4-PBA) had showed to prevent ER stress; however, in cardiac fibroblast both the ER stress induced by thapsigargin and prevention by 4-PBA, have not been studied in detail. Neonate rat CF were treated with thapsigargin in presence or absence of 4-PBA, and cell viability was evaluated by trypan blue exclusion and apoptosis by flow cytometry; whereas CHOP, BIP, PDI, ATF4 and procollagen protein levels were assessed by western blot. In CF, thapsigargin triggered the unfolded protein response detected by early increases in ATF4, CHOP, PDI and BIP protein levels as well as, the accumulation of intracellular procollagen. Thapsigargin also stimulated CF death in a time and concentration-dependent manner. ER stress, CF death and apoptosis induced by thapsigargin were prevented by 4-PBA. Conclusion our data suggest that 4-PBA prevent ER stress, intracellular procollagen accumulation, CF death and apoptosis induced by thapsigargin.

Topics: Animals; Apoptosis; Cell Survival; Cells, Cultured; Endoplasmic Reticulum Stress; Fibroblasts; Myocytes, Cardiac; Phenylbutyrates; Procollagen; Rats; Rats, Sprague-Dawley; Thapsigargin; Unfolded Protein Response

Asbestos exposure results in pulmonary fibrosis (asbestosis) and malignancies (bronchogenic lung cancer and mesothelioma) by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) apoptosis is important in the development of pulmonary fibrosis after exposure to an array of toxins, including asbestos. An endoplasmic reticulum (ER) stress response and mitochondria-regulated (intrinsic) apoptosis occur in AECs of patients with idiopathic pulmonary fibrosis, a disease with similarities to asbestosis. Asbestos induces AEC intrinsic apoptosis, but the role of the ER is unclear. The objective of this study was to determine whether asbestos causes an AEC ER stress response that promotes apoptosis. Using human A549 and rat primary isolated alveolar type II cells, amosite asbestos fibers increased AEC mRNA and protein expression of ER stress proteins involved in the unfolded protein response, such as inositol-requiring kinase (IRE) 1 and X-box-binding protein-1, as well as ER Ca²(2+) release ,as assessed by a FURA-2 assay. Eukarion-134, a superoxide dismutase/catalase mimetic, as well as overexpression of Bcl-XL in A549 cells each attenuate asbestos-induced AEC ER stress (IRE-1 and X-box-binding protein-1 protein expression; ER Ca²(2+) release) and apoptosis. Thapsigargin, a known ER stress inducer, augments AEC apoptosis, and eukarion-134 or Bcl-XL overexpression are protective. Finally, 4-phenylbutyric acid, a chemical chaperone that attenuates ER stress, blocks asbestos- and thapsigargin-induced AEC IRE-1 protein expression, but does not reduce ER Ca²(2+) release or apoptosis. These results show that asbestos triggers an AEC ER stress response and subsequent intrinsic apoptosis that is mediated in part by ER Ca²(2+) release.

Topics: Alveolar Epithelial Cells; Animals; Antioxidants; Apoptosis; Asbestos, Amosite; bcl-X Protein; Calcium Signaling; Cell Line; Cells, Cultured; DNA-Binding Proteins; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Endoribonucleases; Heat-Shock Proteins; Humans; Membrane Proteins; Organometallic Compounds; Phenylbutyrates; Protein Serine-Threonine Kinases; Rats; Regulatory Factor X Transcription Factors; RNA, Messenger; Salicylates; Thapsigargin; Transcription Factor CHOP; Transcription Factors

Nuclear lamins form the lamina on the interior of the nuclear envelope, and are involved in the regulation of various cellular processes, including DNA replication and chromatin organization. Despite this evidence, little is known about potential alterations in nuclear metabolism, specifically lamin structure and integrity in isolated β-cells subjected to stress conditions, including chronic exposure to hyperglycemia (i.e., glucotoxicity). Herein, we investigated effects of glucotoxic conditions on the catalytic activation of caspase 3 and the associated degradation of one of its substrate proteins, namely lamin-B. We report that incubation of insulin-secreting INS-1 832/13 cells, normal rat islets or human islets under glucotoxic conditions (20 mM; 12-48 h) results in the degradation of native lamin B leading to accumulation of the degraded products in non-relevant cellular compartments, including cytosol. Moreover, the effects of high glucose on caspase 3 activation and lamin B degradation were mimicked by thapsigargin, a known inducer of endoplasmic reticulum stress (ER stress). Nifedipine, a known blocker of calcium channel activation, inhibited high glucose-induced caspase 3 activation and lamin B degradation in these cells. 4-Phenyl butyric acid, a known inhibitor of ER stress, markedly attenuated glucose-induced CHOP expression (ER stress marker), caspase 3 activation and lamin B degradation. We conclude that glucotoxic conditions promote caspase 3 activation and lamin B degradation, which may, in part, be due to increased ER stress under these conditions. We also provide further evidence to support beneficial effects of calcium channel blockers against metabolic dysfunction of the islet β-cell induced by hyperglycemic conditions.

Topics: Animals; Calcium Channel Blockers; Caspase 3; Cells, Cultured; Cytosol; Endoplasmic Reticulum Stress; Glucose; Humans; Insulin-Secreting Cells; Lamin Type B; Male; Nifedipine; Phenylbutyrates; Rats; Rats, Sprague-Dawley; Thapsigargin; Transcription Factor CHOP

The ATP-binding cassette transporter ABCB4 is a phosphatidylcholine translocator specifically expressed at the bile canalicular membrane in hepatocytes, highly homologous to the multidrug transporter ABCB1. Variations in the ABCB4 gene sequence cause progressive familial intrahepatic cholestasis type 3. We have shown previously that the I541F mutation, when reproduced either in ABCB1 or in ABCB4, led to retention in the endoplasmic reticulum (ER)/Golgi. Here, Madin-Darby canine kidney cells expressing ABCB1-GFP were used as a model to investigate this mutant. We show that ABCB1-I541F is not properly folded and is more susceptible to in situ protease degradation. It colocalizes and coprecipitates with the ER chaperone calnexin and coprecipitates with the cytosolic chaperone Hsc/Hsp70. Silencing of calnexin or overexpression of Hsp70 have no effect on maturation of the mutant. We also tested potential rescue by chemical and pharmacological chaperones. Thapsigargin and sodium 4-phenyl butyrate were inefficient. Glycerol improved maturation and exit of the mutant from the ER. Cyclosporin A, a competitive substrate for ABCB1, restored maturation, plasma membrane expression, and activity of ABCB1-I541F. Cyclosporin A also improved maturation of ABCB4-I541F in Madin-Darby canine kidney cells. In HepG(2) cells transfected with ABCB4-I541F cDNA, cyclosporin A allowed a significant amount of the mutant protein to reach the membrane of bile canaliculi. These results show that the best strategy to rescue conformation-defective ABCB4 mutants is provided by pharmacological chaperones that specifically target the protein. They identify cyclosporin A as a potential novel therapeutic tool for progressive familial intrahepatic cholestasis type 3 patients.

Topics: Amino Acid Substitution; Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Calnexin; Cholestasis; Cryoprotective Agents; Cyclosporine; Dogs; Endoplasmic Reticulum; Enzyme Inhibitors; Gene Silencing; Glycerol; Hep G2 Cells; HSC70 Heat-Shock Proteins; Humans; Mutation, Missense; Phenylbutyrates; Protein Transport; Thapsigargin

Endoplasmic reticulum (ER) stress is closely connected to autophagy. When cells are exposed to ER stress, cells exhibit enhanced protein degradation and form autophagosomes. In this study, we demonstrate that the chemical chaperone, 4-phenylbutyric acid (4-PBA), regulates ER stressinduced cell death and autophagy in human gingival fibroblasts. We found that 4-PBA protected cells against thapsigargin-induced apoptotic cell death but did not affect the reduced cell proliferation. ER stress induced by thapsigargin was alleviated by 4-PBA through the regulation of several ER stress-inducible, unfolded protein response related proteins including GRP78, GRP94, C/EBP homologous protein, phospho-eIF-2α, eIF-2α, phospho-JNK1 (p46) and phospho-JNK2/3 (p54), JNK1, IRE-1α, PERK, and sXBP-1. Compared with cells treated with thapsigargin alone, cells treated with both 4-PBA and thapsigargin showed lower levels of Beclin-1, LC-3II and autophagic vacuoles, indicating that 4-PBA also inhibited autophagy induced by ER stress. This study suggests that 4-PBA may be a potential therapeutic agent against ER stress-associated pathologic situations.

Topics: Autophagy; Biomarkers; Cell Proliferation; Cells, Cultured; Cytoprotection; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Fibroblasts; Gingiva; Heat-Shock Proteins; Humans; Phenylbutyrates; Protective Agents; RNA Interference; Signal Transduction; Thapsigargin; Time Factors; Transfection; Unfolded Protein Response

Pancreatic beta cell destruction in type 1 diabetes may be mediated by cytokines such as IL-1β, IFN-γ and TNF-α. Endoplasmic reticulum (ER) stress and nuclear factor-κB (NFκB) signalling are activated by cytokines, but their significance in beta cells remains unclear. Here, we investigated the role of cytokine-induced ER stress and NFκB signalling in beta cell destruction.. Isolated mouse islets and MIN6 beta cells were incubated with IL-1β, IFN-γ and TNF-α. The chemical chaperone 4-phenylbutyric acid (PBA) was used to inhibit ER stress. Protein production and gene expression were assessed by western blot and real-time RT-PCR.. We found in beta cells that inhibition of cytokine-induced ER stress with PBA unexpectedly potentiated cell death and NFκB-regulated gene expression. These responses were dependent on NFκB activation and were associated with a prolonged decrease in the inhibitor of κB-α (IκBα) protein, resulting from increased IκBα protein degradation. Cytokine-mediated NFκB-regulated gene expression was also potentiated after pre-induction of ER stress with thapsigargin, but not tunicamycin. Both PBA and thapsigargin treatments led to preferential upregulation of ER degradation genes over ER-resident chaperones as part of the adaptive unfolded protein response (UPR). In contrast, tunicamycin activated a balanced adaptive UPR in association with the maintenance of Xbp1 splicing.. These data suggest a novel mechanism by which cytokine-mediated ER stress interacts with NFκB signalling in beta cells, by regulating IκBα degradation. The cross-talk between the UPR and NFκB signalling pathways may be important in the regulation of cytokine-mediated beta cell death.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Survival; Cytokines; Diabetes Mellitus, Type 1; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Insulin-Secreting Cells; Interferon-gamma; Interleukin-1beta; Mice; Mice, Inbred C57BL; NF-kappa B; Pancreatic Neoplasms; Phenylbutyrates; RNA, Small Interfering; Signal Transduction; Thapsigargin; Transcription Factor CHOP; Tumor Necrosis Factor-alpha; Tunicamycin; Unfolded Protein Response

Palmitic acid (PA) upregulates oxidized LDL receptor-1 (LOX-1), a scavenger receptor responsible for uptake of oxidized LDL (oxLDL), and enhances oxLDL uptake in macrophages. However, the precise underlying mechanism remains to be elucidated. PA is known to induce endoplasmic reticulum (ER) stress in various cell types. Therefore, we investigated whether ER stress is involved in PA-induced LOX-1 upregulation. PA induced ER stress, as determined by phosphorylation of PERK, eIF2α, and JNK, as well as induction of CHOP in macrophage-like THP-1 cells. Inhibitors [4-phenylbutyric acid (PBA), sodium tauroursodeoxycholate (TUDCA), and salubrinal] and small interfering RNA (siRNA) for the ER stress response decreased PA-induced LOX-1 upregulation. Thapsigargin, an ER stress inducer, upregulated LOX-1, which was decreased by PBA and TUDCA. We next examined whether unsaturated FAs could counteract the effect of PA. Both oleic acid (OA) and linoleic acid (LA) suppressed PA-induced LOX-1. Activation of the ER stress response observed in the PA-treated cells was markedly attenuated when the cells were cotreated with OA or LA. In addition, OA and LA suppressed thapsigargin-induced LOX-1 upregulation with reduced activation of ER stress markers. Our results indicate that activation of ER stress is involved in PA-induced LOX-1 upregulation in macrophages, and that OA and LA inhibit LOX-1 induction through suppression of ER stress.

Topics: Animals; Cell Line; Endoplasmic Reticulum; Fatty Acids, Unsaturated; Humans; Palmitic Acid; Phenylbutyrates; Receptors, Oxidized LDL; RNA, Small Interfering; Stress, Physiological; Thapsigargin; Up-Regulation

Saturated fatty acids promote lipotoxic ER (endoplasmic reticulum) stress in pancreatic β-cells in association with Type 2 diabetes. To address the underlying mechanisms we employed MS in a comprehensive lipidomic screen of MIN6 β-cells treated for 48 h with palmitate. Both the overall mass and the degree of saturation of major neutral lipids and phospholipids were only modestly increased by palmitate. The mass of GlcCer (glucosylceramide) was augmented by 70% under these conditions, without any significant alteration in the amounts of either ceramide or sphingomyelin. However, flux into ceramide (measured by [3H]serine incorporation) was augmented by chronic palmitate, and inhibition of ceramide synthesis decreased both ER stress and apoptosis. ER-to-Golgi protein trafficking was also reduced by palmitate pre-treatment, but was overcome by overexpression of GlcCer synthase. This was accompanied by increased conversion of ceramide into GlcCer, and reduced ER stress and apoptosis, but no change in phospholipid desaturation. Sphingolipid alterations due to palmitate were not secondary to ER stress since they were neither reproduced by pharmacological ER stressors nor overcome using the chemical chaperone phenylbutyric acid. In conclusion, alterations in sphingolipid, rather than phospholipid, metabolism are more likely to be implicated in the defective protein trafficking and enhanced ER stress and apoptosis of lipotoxic β-cells.

Topics: Animals; Apoptosis; Biomarkers; Cell Line; Endoplasmic Reticulum; Enzyme Inhibitors; Glucosylceramides; Glucosyltransferases; Insulin-Secreting Cells; Lipid Metabolism; Metabolomics; Mice; Palmitic Acid; Phenylbutyrates; Protein Biosynthesis; Protein Transport; Serine C-Palmitoyltransferase; Sphingolipids; Stress, Physiological; Thapsigargin; Transcription Factor CHOP; Tunicamycin

Tumor cells adapt to endoplasmic reticulum (ER) stress through a set of conserved intracellular pathways, as part of a process termed the unfolded protein response (UPR). The expression of UPR genes/proteins correlates with increasing progression and poor clinical outcome of several tumor types, including prostate cancer. UPR signaling can activate NF-κB, a master regulator of transcription of pro-inflammatory, tumorigenic cytokines. Previous studies have shown that Lipocalin 2 (Lcn2) is upregulated in several epithelial cancers, including prostate cancer, and recently Lcn2 was implicated as a key mediator of breast cancer progression. Here, we hypothesize that the tumor cell UPR regulates Lcn2 production.. We interrogated Lcn2 regulation in murine and human prostate cancer cells undergoing pharmacological and physiological ER stress, and tested UPR and NF-κB dependence by using pharmacological inhibitors of these signaling pathways.. Induction of ER stress using thapsigargin (Tg), a canonical pharmacologic ER stress inducer, or via glucose deprivation, a physiologic ER stressor present in the tumor microenvironment, upregulates LCN2 production in murine and human prostate cancer cells. Inhibition of the UPR using 4-phenylbutyric acid (PBA) dramatically decreases Lcn2 transcription and translation. Inhibition of NF-κB in prostate cancer cells undergoing Tg-mediated ER stress by BAY 11-7082 abrogates Lcn2 upregulation.. We conclude that the UPR activates Lcn2 production in prostate cancer cells in an NF-κB-dependent manner. Our results imply that the observed upregulation of Lipocalin 2 in various types of cancer cells may be the direct consequence of concomitant UPR activation, and that the ER stress/Lipocalin 2 axis is a potential new target for intervention in cancer progression.

Topics: Acute-Phase Proteins; Adenocarcinoma; Animals; Cell Line, Tumor; Endoplasmic Reticulum; Gene Expression Regulation, Neoplastic; Glucose; Humans; Lipocalin-2; Lipocalins; Male; Mice; Neoplasm Proteins; NF-kappa B; Nitriles; Oncogene Proteins; Phenylbutyrates; Prostatic Neoplasms; Protein Biosynthesis; Proto-Oncogene Proteins; Sulfones; Thapsigargin; Transcription, Genetic; Tunicamycin; Unfolded Protein Response; Up-Regulation

The integral tight junction protein Claudin-16 (Cldn16) is predominantly expressed in renal epithelial cells of the thick ascending limb of Henle's loop where, together with claudin-19, it forms a cation-selective pore that allows influx of Na+ from the interstitial fluid into the lumen of the kidney tubule. This leads to an electrochemical gradient that drives the reabsorbtion of Mg2+ and Ca2+ ions from the renal filtrate. Mutations in the Cldn16 gene have been identified in patients suffering from familial hypomagnesemia with hypercalciuria and nephrocalcinosis, with excessive renal wastage of Mg2+ and Ca2+ being a hallmark of this condition. Studies into the mechanism by which mutations impair Cldn16 function have shown that although several mutations affect paracellular ion transport, many interfere with intracellular trafficking of Cldn16, ultimately compromising its localization to TJs. Here, we describe the experimental approaches that can be used to monitor intracellular localization and trafficking of Cldn16. These methods can easily be adapted to study other claudins, provided suitable antibodies are available.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Cell Line; Claudins; Dogs; Electrophoresis, Polyacrylamide Gel; Endocytosis; Humans; Loop of Henle; Magnesium Deficiency; Membrane Proteins; Microscopy, Fluorescence; Molecular Sequence Data; Multiprotein Complexes; Mutation; Nephrocalcinosis; Phenylbutyrates; Plasmids; Protein Transport; Sodium Channels; Thapsigargin; Tight Junctions

The familial and sporadic forms of Alzheimer's disease (AD) have an identical pathology with a severe disparity in the time of onset [1]. The pathological similarity suggests that epigenetic processes may phenocopy the Familial Alzheimer's disease (FAD) mutations within sporadic AD. Numerous groups have demonstrated that FAD mutations in presenilin result in 'loss of function' of gamma-secretase mediated APP cleavage [2], [3], [4], [5]. Accordingly, ER stress is prominent within the pathologically impacted brain regions in AD patients [6] and is reported to inhibit APP trafficking through the secretory pathway [7], [8]. As the maturation of APP and the cleaving secretases requires trafficking through the secretory pathway [9], [10], [11], we hypothesized that ER stress may block trafficking requisite for normal levels of APP cleavage and that the small molecular chaperone 4-phenylbutyrate (PBA) may rescue the proteolytic deficit.. The APP-Gal4VP16/Gal4-reporter screen was stably incorporated into neuroblastoma cells in order to assay gamma-secretase mediated APP proteolysis under normal and pharmacologically induced ER stress conditions. Three unrelated pharmacological agents (tunicamycin, thapsigargin and brefeldin A) all repressed APP proteolysis in parallel with activation of unfolded protein response (UPR) signaling-a biochemical marker of ER stress. Co-treatment of the gamma-secretase reporter cells with PBA blocked the repressive effects of tunicamycin and thapsigargin upon APP proteolysis, UPR activation, and apoptosis. In unstressed cells, PBA stimulated gamma-secretase mediated cleavage of APP by 8-10 fold, in the absence of any significant effects upon amyloid production, by promoting APP trafficking through the secretory pathway and the stimulation of the non-pathogenic alpha/gamma-cleavage.. ER stress represses gamma-secretase mediated APP proteolysis, which replicates some of the proteolytic deficits associated with the FAD mutations. The small molecular chaperone PBA can reverse ER stress induced effects upon APP proteolysis, trafficking and cellular viability. Pharmaceutical agents, such as PBA, that stimulate alpha/gamma-cleavage of APP by modifying intracellular trafficking should be explored as AD therapeutics.

Topics: Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Anti-Bacterial Agents; Antineoplastic Agents; Apoptosis; Blotting, Western; Brefeldin A; Cell Line, Tumor; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Green Fluorescent Proteins; Immunohistochemistry; Neurons; Phenylbutyrates; Protein Transport; Recombinant Fusion Proteins; Thapsigargin; Transfection; Tunicamycin

Adiponectin acts as an insulin-sensitizing adipokine that protects against obesity-linked metabolic disease, which is generally associated with endoplasmic reticulum (ER) stress. The physiological effects of adiponectin on energy metabolism in the liver are mediated by its receptors. We found that the hepatic expression of adiponectin receptor 2 (AdipoR2) was lower, but the expression of markers of the ER stress pathway, 78 kDa glucose-regulated protein (GRP78) and activating transcription factor 3 (ATF3), was higher in the liver of ob/ob mice compared with control mice. To investigate the regulation of AdipoR2 by ER stress, we added thapsigargin, an ER stress inducer, to a human hepatocyte cell line, HepG2. Addition of the ER stress inducer increased the levels of GRP78 and ATF3, and decreased that of AdipoR2, whereas addition of a chemical chaperone, 4-phenyl butyric acid (PBA), could reverse them. Up- or down-regulation of ATF3 modulated the AdipoR2 protein levels and AdipoR2 promoter activities. Reporter gene assays using a series of 5'-deleted AdipoR2 promoter constructs revealed the location of the repressor element responding to ER stress and ATF3. In addition, using electrophoretic mobility shift and chromatin immunoprecipitation assays, we identified a region between nucleotides -94 and -86 of the AdipoR2 promoter that functions as a putative ATF3-binding site in vitro and in vivo. Thus, our findings suggest that the ER stress-induced decrease in both protein and RNA of AdipoR2 results from a concomitant increase in expression of ATF3, which may play a role in the development of obesity-induced insulin resistance and related ER stress in hepatocytes.

Topics: Activating Transcription Factor 3; Animals; Chromatin Immunoprecipitation; DNA; Electrophoretic Mobility Shift Assay; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Gene Deletion; Gene Expression; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Hep G2 Cells; Humans; Liver; Mice; Mice, Inbred C57BL; Mice, Obese; Obesity; Phenylbutyrates; Promoter Regions, Genetic; Protein Binding; Receptors, Adiponectin; Regulatory Elements, Transcriptional; RNA, Small Interfering; Stress, Physiological; Thapsigargin; Transfection

Adiponectin receptors play a key role in steatosis and inflammation; however, very little is known about regulation of adiponectin receptors in liver. Here, we examined the effects of palmitate loading, endoplasmic reticulum (ER) stress, and the hypolipidemic agent fenofibrate on adiponectin receptor R2 (AdipoR2) levels and AMP-activated protein kinase (AMPK) in human hepatoma Huh7 cells and in Huh.8 cells, a model of hepatitis C-induced steatosis. Palmitate treatment reduced AdipoR2 protein and basal AMPK phosphorylation in Huh7 cells. Fenofibrate treatment preserved AdipoR2 and phosphorylated AMPK (pAMPK) levels in palmitate-treated cells accompanied by reduced triglyceride (TG) accumulation and less activation of ER stress markers CCAAT/enhancer binding (C/EBPbeta) and eukaryotic translation initiation factor 2 alpha. ER stress agents thapsigargin and tunicamycin suppressed AdipoR2 and pAMPK levels in Huh7 cells, while fenofibrate and the chemical chaperone 4-phenylbutyrate (PBA) prevented these changes. AdipoR2 levels were lower in Huh.8 cells and fenofibrate treatment increased AdipoR2 while reducing activation of c-Jun N-terminal kinase and C/EBPbeta expression without changing TG levels. Taken together, these results suggest that fatty acids and ER stress reduce AdipoR2 protein and pAMPK levels, while fenofibrate and PBA might be important therapeutic agents to correct lipid- and ER stress-mediated loss of AdipoR2 and pAMPK associated with nonalcoholic steatohepatitis.

Topics: AMP-Activated Protein Kinases; Blotting, Western; Carcinoma, Hepatocellular; CCAAT-Enhancer-Binding Proteins; Cell Line, Tumor; Endoplasmic Reticulum; Enzyme Activation; Fatty Acids; Fatty Liver; Fenofibrate; Hepacivirus; Humans; Hypolipidemic Agents; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Phenylbutyrates; Phosphorylation; Receptors, Adiponectin; Thapsigargin; Triglycerides; Tunicamycin

Heterozygous germline mutations in the gene encoding the bone morphogenetic protein type II receptor cause familial pulmonary arterial hypertension (PAH). We previously demonstrated that the substitution of cysteine residues in the ligand-binding domain of this receptor prevents receptor trafficking to the cell membrane. Here we demonstrate the potential for chemical chaperones to rescue cell-surface expression of mutant BMPR-II and restore function. HeLa cells were transiently transfected with BMPR-II wild type or mutant (C118W) receptor constructs. Immunolocalization studies confirmed the retention of the cysteine mutant receptor mainly in the endoplasmic reticulum. Co-immunoprecipitation studies of Myc-tagged BMPR-II confirmed that the cysteine-substituted ligand-binding domain mutation, C118W, is able to associate with BMP type I receptors. Furthermore, following treatment with a panel of chemical chaperones (thapsigargin, glycerol or sodium 4-phenylbutyrate), we demonstrated a marked increase in cell-surface expression of mutant C118W BMPR-II by FACS analysis and confocal microscopy. These agents also enhanced the trafficking of wild-type BMPR-II, though to a lesser extent. Increased cell-surface expression of mutant C118W BMPR-II was associated with enhanced Smad1/5 phosphorylation in response to BMPs. These findings demonstrate the potential for rescue of mutant BMPR-II function from the endoplasmic reticulum. For the C118W mutation in the ligand-binding domain of BMPR-II, cell-surface rescue leads to at least partial restoration of BMP signalling. We conclude that enhancement of cell-surface trafficking of mutant and wild-type BMPR-II may have therapeutic potential in familial PAH.

Topics: Amino Acid Substitution; Biological Transport, Active; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Protein Receptors, Type II; Cell Membrane; Endoplasmic Reticulum; Germ-Line Mutation; Glycerol; HeLa Cells; Humans; Hypertension, Pulmonary; Models, Biological; Phenylbutyrates; Recombinant Proteins; Signal Transduction; Smad Proteins, Receptor-Regulated; Thapsigargin; Transfection

Leptin is an important circulating signal for inhibiting food intake and body weight gain. In recent years, "leptin resistance" has been considered to be one of the main causes of obesity. However, the detailed mechanisms of leptin resistance are poorly understood. Increasing evidence has suggested that stress signals, which impair endoplasmic reticulum (ER) function, lead to an accumulation of unfolded proteins, which results in ER stress. In the present study, we hypothesized that ER stress is involved in leptin resistance. Tunicamycin, thapsigargin, or brefeldin A was used to induce ER stress. The activation status of leptin signals was measured by Western blotting analysis using a phospho-(Tyr705) signal transducer and activator of transcription 3 (STAT3) antibody. We observed that ER stress markedly inhibited leptin-induced STAT3 phosphorylation. In contrast, ER stress did not affect leptin-induced c-Jun NH(2)-terminal kinase activation. These results suggest that ER stress induces leptin resistance. ER stress-induced leptin resistance was mediated through protein tyrosine phosphatase 1B but not through suppressors of cytokine signaling 3. It is noteworthy that a chemical chaperone, which could improve the protein-folding capacity, reversed ER stress-induced leptin resistance. Moreover, homocysteine, which induces ER stress, caused leptin resistance both in vitro and in vivo. Together, these findings suggest that the pathological mechanism of leptin resistance is derived from ER stress.

Topics: Animals; Brefeldin A; Cell Line; Endoplasmic Reticulum; Homocysteine; Humans; Leptin; Mice; Mice, Inbred C57BL; Phenylbutyrates; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Receptors, Leptin; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Thapsigargin; Transfection; Tunicamycin