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Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by type-II alveolar epithelial cell (AECII) injury and fibroblast hyperproliferation. Severe AECII endoplasmic reticulum (ER) stress is thought to underlie IPF, but is yet incompletely understood. We studied the regulation of C/EBP homologous protein (CHOP), a proapoptotic ER-stress-related transcription factor (TF) in AECII-like cells. Interestingly, single or combined overexpression of the active ER stress transducers activating transcription factor-4 (Atf4) and activating transcription factor-6 (p50Atf6) or spliced x-box-binding protein-1 (sXbp1) in MLE12 cells did not result in a substantial Chop induction, as compared to the ER stress inducer thapsigargin. Employing reporter gene assays of distinct CHOP promoter fragments, we could identify that, next to the conventional amino acid (AARE) and ER stress response elements (ERSE) within the CHOP promoter, activator protein-1 (AP-1) and c-Ets-1 TF binding sites are necessary for CHOP induction. Serial deletion and mutation analyses revealed that both AP-1 and c-Ets-1 motifs act in concert to induce CHOP expression. In agreement, CHOP promoter activity was greatly enhanced upon combined versus single overexpression of AP-1 and c-Ets-1. Moreover, combined overexpression of AP-1 and c-Ets-1 in MLE12 cells alone in the absence of any other ER stress inducer was sufficient to induce Chop protein expression. Further, AP-1 and c-Ets-1 were upregulated in AECII under ER stress conditions and in human IPF. Finally, Chop overexpression in vitro resulted in AECII apoptosis, lung fibroblast proliferation, and collagen-I production. We propose that CHOP activation by AP-1 and c-Ets-1 plays a key role in AECII maladaptive ER stress responses and consecutive fibrosis, offering new therapeutic prospects in IPF.Key messagesOverexpression of active ER stress sensors Atf4, Atf6, and Xbp1 does not induce Chop.AP-1 and c-Ets-1 TFs are necessary for induction of the ER stress factor Chop.AP-1 and c-Ets-1 alone induce Chop expression in the absence of any ER stress inducers.AP-1 and c-Ets-1 are induced in AECII under ER stress conditions and in human IPF.Chop expression alone triggers AECII apoptosis and consecutive profibrotic responses.

Background The development of potent non-toxic chemotherapeutic drugs against castration resistant prostate cancer (CRPC) remains a major challenge. Corosolic acid (CA), a natural triterpenoid, has anti-cancer activity with limited side effects. However, CA anti-prostate cancer activities and mechanisms, particularly in CRPC, are not clearly understood. In this study, we investigated CA anti-tumor ability against human CRPC and its mechanism of action. Methods The cell apoptosis and proliferation effects were evaluated via MTT detection, colony formation assay and flow cytometry. Western blot, gene transfection and immunofluorescence assay were applied to investigate related protein expression of Endoplasmic reticulum stress. A xenograft tumor model was established to investigate the inhibitory effect of CA on castration resistant prostate cancer in vivo. Results The results showed that CA inhibited cell growth and induced apoptosis in human prostate cancer cell (PCa) line PC-3 and DU145, as well as retarded tumor growth in a xenograft model, exerting a limited toxicity to normal cells and tissues. Importantly, CA activated endoplasmic reticulum (ER) stress-associated two pro-apoptotic signaling pathways, as evidenced by increased protein levels of typical ER stress markers including IRE-1/ASK1/JNK and PERK/eIF2α/ATF4/CHOP. IRE-1, PERK or CHOP knockdown partially attenuated CA cytotoxicity against PCa cells. Meanwhile, CHOP induced expression increased Tribbles 3 (TRIB3) level, which lead to AKT inactivation and PCa cell death. CHOP silencing resulted in PCa cells sensitive to CA-induced apoptosis. Conclusion Our data demonstrated, for the first time, that CA might represent a novel drug candidate for the development of an anti-CRPC therapy.

Ischemic stroke is the leading cause of human disability and mortality in the world. The main problem in stroke therapy is the search of efficient neuroprotector capable to rescue neurons in the potentially salvageable transition zone (penumbra), which is expanding after brain damage. The data on molecular mechanisms of penumbra formation and expression of diverse signaling proteins in the penumbra during first 24 h after ischemic stroke are discussed. Two basic features of cell death regulation in the ischemic penumbra were observed: (1) both apoptotic and anti-apoptotic proteins are simultaneously over-expressed in the penumbra, so that the fate of individual cells is determined by the balance between these opposite tendencies. (2) Similtaneous and concerted up-regulation in the ischemic penumbra of proteins that execute apoptosis (caspases 3, 6, 7; Bcl-10, SMAC/DIABLO, AIF, PSR), signaling proteins that regulate different apoptosis pathways (p38, JNK, DYRK1A, neurotrophin receptor p75); transcription factors that control expression of various apoptosis regulation proteins (E2F1, p53, c-Myc, GADD153); and proteins, which are normally involved in diverse cellular functions, but stimulate apoptosis in specific situations (NMDAR2a, Par4, GAD65/67, caspase 11). Hence, diverse apoptosis initiation and regulation pathways are induced simultaneously in penumbra from very different initial positions. Similarly, various anti-apoptotic proteins (Bcl-x, p21/WAF-1, MDM2, p63, PKBα, ERK1, RAF1, ERK5, MAKAPK2, protein phosphatases 1α and MKP-1, estrogen and EGF receptors, calmodulin, CaMKII, CaMKIV) are upregulated. These data provide an integral view of neurodegeneration and neuroprotection in penumbra. Some discussed proteins may serve as potential targets for anti-stroke therapy.

Diabetes mellitus (DM) is associated with the increased risk of the central nervous system complications as cerebrovascular disease, impaired cognition, dementia and neurodegeneration. Curcumin is a polyphenol with anti-oxidant, anti-inflammatory, anti-hyperlipidemic, and anti-cancer effects. Therefore, the present study was aimed to focus on the mechanistic insights of diabetes-induced hippocampal neurodegeneration in addition to shedding the light on the modulatory effect of curcumin. Twenty-eight male Wistar rats were randomly divided into four groups. Type I DM was induced by a single intra-peritoneal injection of streptozotocin (STZ) (65 mg/kg b.w.). Curcumin (100 mg/kg b.w.) was given to the diabetic group after the induction and for eight weeks. Hippocampal glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF-4), Bcl2 and choline acetyl transferase (ChAT) genes expression were assessed. Heat shock protein 70 (HSP70), Bcl-2-Associated X protein (Bax), Interferon-γ (INF-γ) and CCAAT-enhancer-binding protein homologous protein (CHOP) levels in the hippocampus were immunoassayed, in addition to the assessment of glycemic and redox status. Curcumin significantly improved blood glucose level, redox status, cellular stress, and decreased INF-γ and Bax levels, down-regulated GRP78 and ATF-4 expression, meanwhile, up-regulated Bcl2 and ChAT expression in hippocampus. Histological findings proved the biochemical and molecular findings. Our results support curcumin as a potential neuro-protective agent against diabetes induced hippocampal neurodegeneration.

Neuronal apoptosis is an important factor accounting for the poor outcomes of intracerebral hemorrhage (ICH). This study first showed that inhibition of activating transcription factor 6 (ATF6) could alleviate secondary brain injury through anti-apoptosis after ICH in rats. Melatonin, ATF6 and CCAAT/enhancer-binding protein homologous protein (CHOP) siRNAs were applied in this study. Brain edema, neurological functions, blood-brain barrier (BBB) integrity were evaluated at 24 h after ICH. Western blot analysis was used to evaluate the protein level of target proteins (ATF6, CHOP, Bip, Bcl-2, Bax, and cleaved caspase-3). Reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the mRNA level of ATF6, CHOP and cleaved caspase-3. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase-3 immunofluorescence staining were applied to evaluate the neuronal cell death. The results suggested that the levels of ATF6 and its downstream protein, CHOP, were upregulated and reached the peak at 24 h after ICH. ATF6 was highly expressed in neurons. The administration of melatonin significantly decreased the mRNA and protein levels of ATF6, and its downstream targets, CHOP and cleaved caspase-3, but increased the Bcl-2/Bax ratio, which ameliorated the neurological functions. The CHOP siRNA significantly reversed the pro-apoptotic effect induced by the increased ATF6 level after ICH. Melatonin could protect against neuronal apoptosis via suppression of ATF6/CHOP arm of ER-stress-response pathway.

Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in various neurodegenerative diseases including Parkinson Disease, Alzheimer Disease and Huntington Disease. PUMA (p53 upregulated modulator of apoptosis) and BIM (BCL2 interacting mediator of cell death), pro-apoptotic BH3 domain-only, BCL2 family members, have previously been shown to regulate ER stress-induced cell death, but the upstream signaling pathways that regulate this response in neuronal cells are incompletely defined. Consistent with previous studies, we show that both PUMA and BIM are induced in response to ER stress in neuronal cells and that transcriptional induction of PUMA regulates ER stress-induced cell death, independent of p53. CHOP (C/EBP homologous protein also known as GADD153; gene name Ddit3), a critical initiator of ER stress-induced apoptosis, was found to regulate both PUMA and BIM expression in response to ER stress. We further show that CHOP knockdown prevents perturbations in the AKT (protein kinase B)/FOXO3a (forkhead box, class O, 3a) pathway in response to ER stress. CHOP co-immunoprecipitated with FOXO3a in tunicamycin treated cells, suggesting that CHOP may also regulate other pro-apoptotic signaling cascades culminating in PUMA and BIM activation and cell death. In summary, CHOP regulates the expression of multiple pro-apoptotic BH3-only molecules through multiple mechanisms, making CHOP an important therapeutic target relevant to a number of neurodegenerative conditions.

New and more effective therapies for canine cancer patients are urgently required and this necessitates advanced experimental research. Dogs are good models for studies in comparative oncology; however, canine cancer cell biology research is currently limited by low availability of validated antibody reagents and techniques. This study characterises the expression of key components of the unfolded protein response (UPR) in a panel of haematopoietic canine cancer cell lines using commercially available antibodies, and validates the methods used to study this pathway.The CLBL-1 canine lymphoma cell line and the GL-1 canine leukaemia cell line sourced externally and two counterparts established in house (CNK-89 and CLB70) were used as models of different lymphoma and leukaemia canine cell lines for the study. The human U2OS cell line served as the control. Antibodies were selected for identifying UPR proteins according to known canine cell reactivity and canine–murine and canine–human homology. Endoplasmic reticulum stress was induced with thapsigargin and MG132 in the cell lines. Etoposide was used to induce DNA damage in the cells. The techniques used for this validation analysis were RNA sequencing to observe the expression of UPR components in canine cell lines, Western blot to observe changes of protein expression levels after inducing ER stress in the cells, and flow cytometry in order to study cell death.Substantial variations in both the basic expression and agonist-induced activation of the UPR pathway were observed in canine cancer cell lines, although the biological significance of these differences requires further investigation.These findings will be a starting point for future studies on cancer biology in dogs. They will also contribute to developing novel anticancer therapies for canine patients and may provide new insights into human oncology.

C/EBP homologous protein (CHOP), known also as DNA damage-inducible transcript 3 and as growth arrest and DNA damage-inducible protein 153 (GADD153), is induced in response to certain stressors. CHOP is universally acknowledged as a main conduit to endoplasmic reticulum stress-induced apoptosis. Ongoing research established the existence of CHOP-mediated apoptosis signaling networks, for which novel downstream targets are still being determined. However, there are studies that contradict this notion and assert that apoptosis is not the only mechanism by which CHOP plays in the development of pathologies. In this review, insights into the roles of CHOP in pathophysiology are summarized at the molecular and cellular levels. We further focus on the newest advances that implicate CHOP in human diseases including cancer, diabetes, neurodegenerative disorders, and notably, fibrosis.

Anticancer drugs induce apoptotic and non-apoptotic cell death in various cancer types. The signaling pathways for anticancer drug-induced apoptotic cell death have been shown to differ between drug-sensitive and drug-resistant cells. In atypical multidrug-resistant leukemia cells, the c-Jun/activator protein 1 (AP-1)/p53 signaling pathway leading to apoptotic death is altered. Cancer cells treated with anticancer drugs undergo c-Jun/AP-1–mediated apoptotic death and are involved in c-Jun N-terminal kinase activation and growth arrest- and DNA damage-inducible gene 153 (Gadd153)/CCAAT/enhancer-binding protein homologous protein pathway induction, regardless of the p53 genotype. Gadd153 induction is associated with mitochondrial membrane permeabilization after anticancer drug treatment and involves a coupled endoplasmic reticulum stress response. The induction of apoptosis by anticancer drugs is mediated by the intrinsic pathway (cytochrome c, Cyt c) and subsequent activation of the caspase cascade via proapoptotic genes (e.g., Bax and Bcl-xS) and their interactions. Anticancer drug-induced apoptosis involves caspase-dependent and caspase-independent pathways and occurs via intrinsic and extrinsic pathways. The targeting of antiapoptotic genes such as Bcl-2 enhances anticancer drug efficacy. The modulation of apoptotic signaling by Bcl-xS transduction increases the sensitivity of multidrug resistance-related protein-overexpressing epidermoid carcinoma cells to anticancer drugs. The significance of autophagy in cancer therapy remains to be elucidated. In this review, we summarize current knowledge of cancer cell death-related signaling pathways and their alterations during anticancer drug treatment and discuss potential strategies to enhance treatment efficacy.

Endoplasmic reticulum of all eukaryotic cells is a membrane-bound organelle. Under electron microscope it appears as parallel arrays of “rough membranes” and a maze of “smooth vesicles” respectively. It performs various functions in cell, i.e., synthesis of proteins to degradation of xenobiotics. Bioaccumulation of drugs/chemicals/xenobiotics in the cytosol can trigger ER stress. It is recognized by the accumulation of unfolded or misfolded proteins in the lumen of ER. Present review summarizes the present status of knowledge on ER stress caused by toxic elements, viz arsenic, cadmium, lead, mercury, copper, chromium, and nickel. While inorganic arsenic may induce various glucose-related proteins, i.e., GRP78, GRP94 and CHOP, XBP1, and calpains, cadmium upregulates GRP78. Antioxidants like ascorbic acid, NAC, and Se inhibit the expression of UPR. Exposure to lead also changes ER stress related genes, i.e., GRP 78, GRP 94, ATF4, and ATF6. Mercury too upregulates these genes. Nickel, a carcinogenic element upregulates the expression of Bak, cytochrome C, caspase-3, caspase-9, caspase-12, and GADD 153. Much is not known on ER stress caused by nanoparticles. The review describes inter-organelle association between mitochondria and ER. It also discusses the interdependence between oxidative stress and ER stress. A cross talk amongst different cellular components appears essential to disturb pathways leading to cell death. However, these molecular switches within the signaling network used by toxic elements need to be identified. Nevertheless, ER stress especially caused by toxic elements still remains to be an engaging issue.