Endoplasmic reticulum membrane reorganization is regulated by ionic homeostasis.

Recently we described a new, evolutionarily conserved cellular stress response characterized by a reversible reorganization of endoplasmic reticulum (ER) membranes that is distinct from canonical ER stress and the unfolded protein response (UPR). Apogossypol, a putative broad spectrum BCL-2 family antagonist, was the prototype compound used to induce this ER membrane reorganization. Following microarray analysis of cells treated with apogossypol, we used connectivity mapping to identify a wide range of structurally diverse chemicals from different pharmacological classes and established their ability to induce ER membrane reorganization. Such structural diversity suggests that the mechanisms initiating ER membrane reorganization are also diverse and a major objective of the present study was to identify potentially common features of these mechanisms. In order to explore this, we used hierarchical clustering of transcription profiles for a number of chemicals that induce membrane reorganization and discovered two distinct clusters. One cluster contained chemicals with known effects on Ca(2+) homeostasis. Support for this was provided by the findings that ER membrane reorganization was induced by agents that either deplete ER Ca(2+) (thapsigargin) or cause an alteration in cellular Ca(2+) handling (calmodulin antagonists). Furthermore, overexpression of the ER luminal Ca(2+) sensor, STIM1, also evoked ER membrane reorganization. Although perturbation of Ca(2+) homeostasis was clearly one mechanism by which some agents induced ER membrane reorganization, influx of extracellular Na(+) but not Ca(2+) was required for ER membrane reorganization induced by apogossypol and the related BCL-2 family antagonist, TW37, in both human and yeast cells. Not only is this novel, non-canonical ER stress response evolutionary conserved but so also are aspects of the mechanism of formation of ER membrane aggregates. Thus perturbation of ionic homeostasis is important in the regulation of ER membrane reorganization

Perturbation of Ca²⁺ homeostasis by compounds clustered with apogossypol.

<p>Listing of the eleven compounds that clustered with apogossypol in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056603#pone-0056603-g001" target="_blank">Figure 1</a> and their pharmacological activities. HeLa cells after 4 h exposure to ivermectin (20 µM), clotrimazole (50 µM), niclosamide (50 µM), pyrvinium (20 µM), dequalinium (10 µM), gossypol (10 µM), valinomycin (20 µM), thapsigargin (10 µM), chlorpromazine (20 µM), trifluoperazine (20 µM) or NDGA (50 µM) exhibited varying levels of ER membrane reorganization, from minimal (<b>+</b>) to extensive reorganization (<b>+++</b>). Ten of the eleven compounds have been described to perturb cellular Ca²⁺ homeostasis by different mechanisms.</p

Enhanced Ca²⁺ influx is not critical for apogossypol-mediated ER membrane reorganization.

<p>(A) Down-regulation of STIM1, ORAI1 or ORAI3, using RNA interference for 72 h, had no significant inhibitory effects on apogossypol-induced Ca²⁺ entry, as measured by the maximal ratio change in fura-2 fluorescence in Ca²⁺-free conditions followed by addition of extracellular Ca²⁺. In contrast, THG-mediated SOCE was dramatically reduced following STIM1 and ORAI1 siRNA but not with ORAI3 siRNA. The graph for THG represents the mean from 3 independent experiments with 20 cells recorded in each experiment. Following apogossypol, the effects were not uniform (the cells behaving inconsistently, often exhibiting multiple peaks) and the graph represents data from a single cell in one of three independent experiments. (B) HeLa cells, transfected for 72 h with siRNA against STIM1, ORAI1 or ORAI3, were exposed to apogossypol (10 µM) for 4 h and immunostained with BAP31 antibody to assess ER membrane reorganization (scale bar, 20 µm). (C) HeLa cells, exposed to apogossypol (1 µM) or THG (5 µM) for 1 h, in serum-free normal DMEM or in serum- and Ca²⁺-free DMEM, were immunostained with BAP31 antibody to assess ER membrane reorganization (scale bar, 20 µm). (D) <i>S. pombe</i>, harboring <i>rtn1-GFP</i> at its endogenous <i>rtn1</i> locus to visualize the ER membranes, were grown for 2 h in minimal media or Ca²⁺-free media (supplemented with 20 mM EDTA), in the presence or absence of apogossypol (10 µM) (scale bar, 10 µm).</p

ER membrane reorganization may be due to enhanced Ca²⁺ entry.

<p>(A) Fura-2-loaded HeLa cells were exposed to either apogossypol (10 µM) or THG (10 µM) in Ca²⁺-free media. This was followed by addition of extracellular Ca²⁺. Changes in fura-2 fluorescence were measured throughout as an index of changes in [Ca²⁺]_cyt. The graph represents the mean from 3 independent experiments with 20 cells recorded in each experiment. (B) Extensive ER membrane reorganization, assessed by BAP31 staining, was observed in HeLa cells exposed for 4 h to the calmodulin inhibitors, calmidazolium (10 µM) and A-7 (20 µM) (scale bar, 20 µm). (C) Apogossypol-mediated ER membrane reorganization was abolished when HeLa cells were pretreated for 1 h with 2-APB (10 µM) (scale bar, 20 µm). (D) Transient overexpression of YFP, STIM1-YFP, ORAI1-YFP, ORAI2-Myc or ORAI3-Myc and immunostaining with BAP31 antibody, revealed that STIM1-YFP caused extensive ER membrane reorganization, similar to that observed following apogossypol (scale bar, 10 µm). (E) Ultrastructure of STIM1-YFP expressing HeLa cells showing STIM1-mediated reorganization of ER membranes (lower two panels). The bottom panel (scale bar, 5 µm) shows a detail of one of two regions of pronounced ER membrane organizations (indicated by black arrows in the middle panel). It also shows a clear continuity of membranes with the rest of the ER (white arrows). The upper panel shows a comparable area of control transfected HeLa cell (the scale bar in the top 2 panels is 2 µm).</p

Apogossypol-induced ER membrane reorganization is not due to depletion of intracellular Ca²⁺ stores.

<p>(A) Permeabilized HeLa cells, exposed to DMSO (control), apogossypol (10 µM), or THG (10 µM), were labelled with ⁴⁵Ca²⁺ and samples collected at the indicated times to assess ⁴⁵Ca²⁺ uptake. (B) Permeabilized HeLa cells labelled with ⁴⁵Ca²⁺ for 20 minutes, were exposed to DMSO (control), apogossypol (10 µM), THG (10 µM), IP₃ (3 µM) or ionomycin (1 µM) for the indicated times and samples collected and assessed for the extent of ⁴⁵Ca²⁺ release. (C) Experiments were carried out as in (B) except using DT40 cells, that either lack all 3 isoforms of IP₃ receptors (DT40 KO) or were reconstituted with IP₃R1 (DT40-IP₃R1). (D) Extensive ER membrane reorganization, as assessed by electron microscopy (scale bar, 1 µm) was observed in DT40- IP₃R1 and DT40 KO cells, exposed to apogossypol (10 µM) for 4 h. Error bars shown in A–C represent the standard error of the mean.</p

Hierarchical clustering reveals a possible role for Ca²⁺ homeostasis in ER membrane reorganization.

<p>Heat map analysis comparing the top 50 differentially expressed genes following the indicated drug treatments as available in the connectivity map reference database. Yellow and blue indicate up or down regulation, respectively. Hierarchical clustering was used to analyze microarray data for chemicals known to induce ER membrane reorganization. The microarray data were normalized, compared with the equivalent control samples, and filtered by mean channel intensity (<250) and mean log₂ fold changes across all the data sets (<1.8). Trends in the resulting gene expression matrix were analyzed, using a Pearson correlation metric and average link clustering. Based on transcriptional trends, two distinct groups of chemicals were identified. Apogossypol clustered with a number of chemicals that are known to disrupt Ca²⁺ homeostasis (shaded in blue box).</p

Apogossypol induces ER membrane reorganization <i>via</i> an enhanced influx of Na⁺.

<p>(A) <i>S. pombe</i> cells, harbouring <i>rtn1-GFP</i>, exhibited extensive ER membrane reorganization following exposure for 2 h to apogossypol (10 µM), when grown in minimal media, or minimal media lacking K⁺. Apogossypol-mediated ER membrane reorganization was abolished when cells were suspended in Na⁺-free media or exposed to either LOE-908 (10 µM) or benzamil (100 µM) 30 minutes before exposure to apogossypol (scale bar, 10 µm). (B) Influx of Na⁺ is required for ER membrane reorganization induced by some but not all agents in HeLa cells. Thus, LOE-908 (10 µM) or benzamil (100 µM) abolished apogossypol- (10 µM) and TW37- (50 µM)-induced membrane aggregates but not those induced by THG (10 µM) (scale bar, 20 µm). (C) Scheme summarizing the formation, regulation and consequences of ER membrane reorganization. Exposure to stress conditions, such as inhibition of the BCL-2 family (apogossypol, TW37) or SERCA (THG) induced ER membrane reorganization (right hand panel). Multiple mechanisms, including an influx of Na⁺, through LOE-908- and benzamil-sensitive Na⁺ channels, and ER Ca²⁺ depletion or altered Ca²⁺ handling regulate ER membrane reorganization. These membrane aggregates are reversible and occur independent of canonical ER stress, as tunicamycin (Tunic) and brefeldin A (BFA), two inducers of canonical ER stress, do not result in ER membrane reorganization <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056603#pone.0056603-Varadarajan1" target="_blank">[14]</a>. However, at later times, the extensive ER membrane reorganization may eventually lead to canonical ER stress and the UPR, thus placing ER membrane reorganization upstream and/or independent of the UPR.</p