Inhibition of Secretion of Interleukin (IL)-12/IL-23 Family Cytokines by 4-Trifluoromethyl-celecoxib Is Coupled to Degradation via the Endoplasmic Reticulum Stress Protein HERP

Interleukin-12 (IL-12), p80, and IL-23 are structurally related cytokines sharing a p40 subunit. We have recently demonstrated that celecoxib and its COX-2-independent analogue 4-trifluoromethyl-celecoxib (TFM-C) inhibit secretion but not transcription of IL-12 (p35/p40) and p80 (p40/p40). This is associated with a mechanism involving altered cytokine-chaperone interaction in the endoplasmic reticulum (ER). In the present study, we found that celecoxib and TFM-C also block secretion of IL-23 (p40/p19 heterodimers). Given the putative ER-centric mode of these compounds, we performed a comprehensive RTPCR analysis of 23 ER-resident chaperones/foldases and associated co-factors. This revealed that TFM-C induced 1.5-3-fold transcriptional up-regulation of calreticulin, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29. However, more significantly, a 7-fold up-regulation of homocysteine-inducible ER protein (HERP) was observed. HERP is part of a high molecular mass protein complex involved in ER-associated protein degradation (ERAD). Using co-immunoprecipitation assays, we show that TFM-C induces protein interaction of p80 and IL-23 with HERP. Both HERP siRNA knockdown and HERP overexpression coupled to cycloheximide chase assays revealed that HERP is necessary for degradation of intracellularly retained p80 by TFM-C. Thus, our data suggest that targeting cytokine folding in the ER by small molecule drugs could be therapeutically exploited to alleviate in appropriate inflammation in autoimmune conditions.

Overexpression of endoplasmic reticulum protein 29 regulates mesenchymal-epithelial transition and suppresses xenograft tumor growth of invasive breast cancer cells

Endoplasmic reticulum protein 29 (ERp29) is a novel endoplasmic reticulum ( ER) secretion factor that facilitates the transport of secretory proteins in the early secretory pathway. Recently, it was found to be overexpressed in several cancers; however, little is known regarding its function in breast cancer progression. In this study, we show that the expression of ERp29 was reduced with tumor progression in clinical specimens of breast cancer, and that overexpression of ERp29 resulted in G(0)/G(1) arrest and inhibited cell proliferation in MDA-MB-231 cells. Importantly, overexpression of ERp29 in MDA-MB-231 cells led to a phenotypic change and mesenchymal-epithelial transition (MET) characterized by cytoskeletal reorganization with loss of stress fibers, reduction of fibronectin (FN), reactivation of epithelial cell marker E-cadherin and loss of mesenchymal cell marker vimentin. Knockdown of ERp29 by shRNA in MCF-7 cells reduced E-cadherin, but increased vimentin expression. Furthermore, ERp29 overexpression in MDA-MB-231 and SKBr3 cells decreased cell migration/invasion and reduced cell transformation, whereas silencing of ERp29 in MCF-7 cells enhanced cell aggressive behavior. Significantly, expression of ERp29 in MDA-MB-231 cells suppressed tumor formation in nude mice by repressing the cell proliferative index (Ki-67 positivity). Transcriptional profiling analysis showed that ERp29 acts as a central regulator by upregulating a group of genes with tumor suppressive function, for example, E-cadherin (CDH1), cyclin-dependent kinase inhibitor (CDKN2B) and spleen tyrosine kinase (SYK), and by downregulating a group of genes that regulate cell proliferation (eg, FN, epidermal growth factor receptor ( EGFR) and plasminogen activator receptor ( uPAR)). It is noteworthy that ERp29 significantly attenuated the overall ERK cascade, whereas the ratio of p-ERK1 to p-ERK2 was highly increased. Taken together, our results showed that ERp29 is a novel regulator leading to cell growth arrest and cell transition from a proliferative to a quiescent state, and reprogramming molecular portraits to suppress the tumor growth of MDA-MB-231 breast cancer cells. Laboratory Investigation (2009) 89, 1229-1242; doi: 10.1038/labinvest.2009.87; published online 21 September 2009

Oxidative and endoplasmic reticulum stresses mediate apoptosis induced by modified LDL in human retinal Müller cells

We previously showed that extravasated, modified LDL is implicated in pericyte loss in diabetic retinopathy (DR). Here, we investigate whether modified LDL induces apoptosis in retinal Müller glial cells.

Activation of AMP-activated protein kinase inhibits oxidized LDL-triggered endoplasmic reticulum stress in vivo

The oxidation of LDLs is considered a key step in the development of atherosclerosis. How LDL oxidation contributes to atherosclerosis remains poorly defined. Here we report that oxidized and glycated LDL (HOG-LDL) causes aberrant endoplasmic reticulum (ER) stress and that the AMP-activated protein kinase (AMPK) suppressed HOG-LDL-triggered ER stress in vivo.

Antitumor alkyl-lysophospholipid analog edelfosine induces apoptosis in pancreatic cancer by targeting endoplasmic reticulum

Pancreatic cancer remains as one of the most deadly cancers, and responds poorly to current therapies. The prognosis is extremely poor, with a 5-year survival of less than 5%. Therefore, search for new effective therapeutic drugs is of pivotal need and urgency to improve treatment of this incurable malignancy. Synthetic alkyl-lysophospholipid analogs (ALPs) constitute a heterogeneous group of unnatural lipids that promote apoptosis in a wide variety of tumor cells. In this study, we found that the anticancer drug edelfosine was the most potent ALP in killing human pancreatic cancer cells, targeting endoplasmic reticulum (ER). Edelfosine was taken up in significant amounts by pancreatic cancer cells and induced caspase-and mitochondrial-mediated apoptosis. Pancreatic cancer cells show a prominent ER and edelfosine accumulated in this subcellular structure, inducing a potent ER stress response, with caspase-4, BAP31 and c-Jun NH 2-terminal kinase (JNK) activation, CHOP/GADD153 upregulation and phosphorylation of eukaryotic translation initiation factor 2 a-subunit that eventually led to cell death. Oral administration of edelfosine in xenograft mouse models of pancreatic cancer induced a significant regression in tumor growth and an increase in apoptotic index, as assessed by TUNEL assay and caspase-3 activation in the tumor sections. The ER stress-associated marker CHOP/GADD153 was visualized in the pancreatic tumor isolated from edelfosine-treated mice, indicating a strong in vivo ER stress response. These results suggest that edelfosine exerts its pro-apoptotic action in pancreatic cancer cells, both in vitro and in vivo, through its accumulation in the ER, which leads to ER stress and apoptosis. Thus, we propose that the ER could be a key target in pancreatic cancer, and edelfosine may constitute a prototype for the development of a new class of antitumor drugs targeting the ER. © 2012 Macmillan Publishers Limited All rights reserved.

Endoplasmic reticulum targeting in Ewing’s sarcoma by the alkylphospholipid analog edelfosine

Ewing's sarcoma (ES) is the second most common bone cancer in children and young people. Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) is the prototype of a family of synthetic antitumor compounds, collectively known as alkylphospholipid analogs (APLs). We have found that APLs ranked edelfosine>perifosine>erucylphosphocholine>miltefosine for their capacity to promote apoptosis in ES cells. Edelfosine accumulated in the endoplasmic reticulum (ER) and triggered an ER stress response that eventually led to caspase-dependent apoptosis in ES cells. This apoptotic response involved mitochondrial-mediated processes, with cytochrome c release, caspase-9 activation and generation of reactive oxygen species. Edelfosine-induced apoptosis was also dependent on sustained c-Jun NH2-terminal kinase activation. Oral administration of edelfosine showed a potent in vivo antitumor activity in an ES xenograft animal model. Histochemical staining gave evidence for ER stress response and apoptosis in the ES tumors isolated from edelfosine-treated mice. Edelfosine showed a preferential action on ES tumor cells as compared to non-transformed osteoblasts, and appeared to be well suited for combination therapy regimens. These results demonstrate in vitro and in vivo antitumor activity of edelfosine against ES cells that is mediated by caspase activation and ER stress, and provide the proof of concept for a putative edelfosine-and ER stress-mediated approach for ES treatment.

the liver endoplasmic reticulum in malaria liver infection

Malaria, a disease caused by Plasmodium, represents a major health problem with a still disconcertingly high mortality rate (655 000 malaria deaths were estimated by the World Health Organization in 2012), mainly in Africa [1]. After a bite by an infected Anopheles mosquito occurs, Plasmodium sporozoites reach their target organ, the liver, within minutes. After traversing several hepatocytes, the parasite invades a final one and establishes a parasitophorous vacuole, where it replicates exponentially generating thousands of infective merozoites, the red blood cell infectious forms that are released in the blood stream. The liver stage is the first obligatory phase of malaria infection and, although no symptoms are associated with it, it is absolutely crucial to the establishment of a successful infection.(...)

Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum.

BACKGROUND: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD). Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub)-proteasome degradative system through an interaction with beta-TrCP, a component of the SCFbeta-TrCP E3 Ub ligase complex. RESULTS: Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFbeta-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation. CONCLUSION: Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFbeta-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.

Comparison of proteins of the endoplasmic reticulum from control rat liver with proteins of the endoplasmic reticulum from dissected liver tumor nodules

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