Changes In Endoplasmic Reticulum Morphology Research Articles

Cannabidiol causes activated hepatic stellate cell death through a mechanism of endoplasmic reticulum stress-induced apoptosis

The major cellular event in the development and progression of liver fibrosis is the activation of hepatic stellate cells (HSCs). Activated HSCs proliferate and produce excess collagen, leading to accumulation of scar matrix and fibrotic liver. As such, the induction of activated HSC death has been proposed as a means to achieve resolution of liver fibrosis. Here we demonstrate that cannabidiol (CBD), a major non-psychoactive component of the plant Cannabis sativa, induces apoptosis in activated HSCs through a cannabinoid receptor-independent mechanism. CBD elicits an endoplasmic reticulum (ER) stress response, characterized by changes in ER morphology and the initiation of RNA-dependent protein kinase-like ER kinase-, activating transcription factor-6-, and inositol-requiring ER-to-nucleus signal kinase-1 (IRE1)-mediated signaling cascades. Furthermore, CBD induces downstream activation of the pro-apoptotic IRE1/ASK1/c-Jun N-terminal kinase pathway, leading to HSC death. Importantly, we show that this mechanism of CBD-induced ER stress-mediated apoptosis is specific to activated HSCs, as it occurs in activated human and rat HSC lines, and in primary in vivo-activated mouse HSCs, but not in quiescent HSCs or primary hepatocytes from rat. Finally, we provide evidence that the elevated basal level of ER stress in activated HSCs has a role in their susceptibility to the pro-apoptotic effect of CBD. We propose that CBD, by selectively inducing death of activated HSCs, represents a potential therapeutic agent for the treatment of liver fibrosis.

PHOSPHATIDIC ACID PHOSPHOHYDROLASE1 and 2 Regulate Phospholipid Synthesis at the Endoplasmic Reticulum in Arabidopsis

Phospholipid biosynthesis is essential for the construction of most eukaryotic cell membranes, but how this process is regulated in plants remains poorly understood. Here, we show that in Arabidopsis thaliana, two Mg(2+)-dependent phosphatidic acid phosphohydrolases called PAH1 and PAH2 act redundantly to repress phospholipid biosynthesis at the endoplasmic reticulum (ER). Leaves from pah1 pah2 double mutants contain ~1.8-fold more phospholipid than the wild type and exhibit gross changes in ER morphology, which are consistent with massive membrane overexpansion. The net rate of incorporation of [methyl-(14)C]choline into phosphatidylcholine (PC) is ~1.8-fold greater in the double mutant, and the transcript abundance of several key genes that encode enzymes involved in phospholipid synthesis is increased. In particular, we show that PHOSPHORYLETHANOLAMINE N-METHYLTRANSFERASE1 (PEAMT1) is upregulated at the level of transcription in pah1 pah2 leaves. PEAMT catalyzes the first committed step of choline synthesis in Arabidopsis and defines a variant pathway for PC synthesis not found in yeasts or mammals. Our data suggest that PAH1/2 play a regulatory role in phospholipid synthesis that is analogous to that described in Saccharomyces cerevisiae. However, the target enzymes differ, and key components of the signal transduction pathway do not appear to be conserved.

Protective effects of galantamine against Aβ-induced PC12 cell apoptosis by preventing mitochondrial dysfunction and endoplasmic reticulum stress

Amyloid β (Aβ) is considered to be responsible for the pathogenesis of Alzheimer's disease (AD). Mitochondrial and ER apoptotic pathways are considered to be involved in this process. Galantamine is an acetylcholinesterase (AChE) inhibitor widely used for patients with AD. In this study, we investigated the neuroprotective effects of galantamine on Aβ25–35-induced apoptosis in PC12 cells and the underlying mechanisms. Exposure of PC12 cells to 20μM Aβ25–35 caused significant cell viability loss and apoptosis, Aβ aggregation, mitochondrial and ER morphological changes, as well as mitochondrial membrane potential dissipation, reactive oxygen species (ROS) production, intracellular calcium elevation, and cytochrome c release from mitochondria. Pretreatment with 10μM galantamine for 24h prior to Aβ25–35 exposure significantly reduced Aβ25–35-induced apoptosis not only by preventing Aβ aggregation, mitochondrial and ER morphological changes, mitochondrial membrane potential dissipation, ROS production, intracellular calcium elevation, and cytochrome c release, but also via reversing Bcl-2/Bax ratio and suppressing the activity of GADD153, Grp78/94, caspase-9, caspase-12, and caspase-3. All these data indicate that galantamine protects PC12 cells against Aβ25–35-induced apoptosis by preventing mitochondrial dysfunction and endoplasmic reticulum (ER) stress.

Effect of heat stress on actin cytoskeleton and endoplasmic reticulum of tobacco BY-2 cultured cells and its inhibition by Co2+

Temperature stress such as heat, cold, or freezing is a principal cause for yield reduction in crops. In particular, heat stress is very common and dangerous for plants since this stress can impact several plant and cellular functions. In spite of their role in sensing local stress and in controlling fundamental processes including PCD, the responses of cellular structures and organelles to heat stress are poorly investigated. In this work, we investigated the possible changes induced by mild heat stress, medium heat stress, and heat shock (HS; 5 min at 35 degrees C, 45 degrees C, or 50 degrees C, respectively) on actin cytoskeleton and endoplasmic reticulum (ER) of tobacco BY-2 cultured cells. While mild and medium heat stresses are ineffective, HS induces depolymerization of actin microfilaments and changes in ER morphology accompanied by accumulation of the HSP70 binding protein (BiP). These effects of HS are prevented by the inhibitor of ethylene production Co(2+). While the analyzed cell structures do not seem to be involved in the establishment of mild and medium heat stresses at least in this experimental system, the strong modifications induced by the treatment at 50 degrees C suggest that actin cytoskeleton and ER may be involved in the responses to HS. Besides, the inhibiting effect of Co(2+) suggests a role for ethylene as a regulative molecule in the responses to HS here observed.

The N-terminal Amphipathic α-Helix of Viperin Mediates Localization to the Cytosolic Face of the Endoplasmic Reticulum and Inhibits Protein Secretion

Viperin is an evolutionarily conserved interferon-inducible protein that localizes to the endoplasmic reticulum (ER) and inhibits a number of DNA and RNA viruses. In this study, we report that viperin specifically localizes to the cytoplasmic face of the ER and that an amphipathic α-helix at its N terminus is necessary for the ER localization of viperin and sufficient to promote ER localization of a reporter protein, dsRed. Overexpression of intact viperin but not the amphipathic α-helix fused to dsRed induced crystalloid ER. Consistent with other proteins that induce crystalloid ER, viperin self-associates, and it does so independently of the amphipathic α-helix. Viperin expression also affected the transport of soluble but not membrane-associated proteins. Expression of intact viperin or an N-terminal α-helix-dsRed fusion protein significantly reduced secretion of soluble alkaline phosphatase and reduced its rate of ER-to-Golgi trafficking. Similarly, viperin expression inhibited bulk protein secretion and secretion of endogenous α1-antitrypsin and serum albumin from HepG2 cells. Converting hydrophobic residues in the N-terminal α-helix to acidic residues partially or completely restored normal transport of soluble alkaline phosphatase, suggesting that the extended amphipathic nature of the N-terminal α-helical domain is essential for inhibiting protein secretion.