XBP1 negatively regulates CENPF expression via recruiting ATF6α to the promoter during ER stress

Background Centromere protein F (CENPF) is a key component of the kinetochore complex involved in mitosis, cell differentiation and cellular response to stresses. However, the alteration of CENPF in response to endoplasmic reticulum (ER) stress has not been well described. In the present study, we investigate CENPF regulation in response to ER stress. Methods Quantitative real-time polymerase chain reaction and western blotting were used to determine CENPF expression under ER stress. Luciferase activity analysis was performed to investigate the promoter regions contributing to CENPF transcription in response to TG. Chromatin immunoprecipitation (ChIP) and ChIP Re-IP assays were used to determine if X-box binding protein 1 (XBP1) and/or activating transcription factor 6α (ATF6α) bind in the CENPF promoter region. Cell apoptosis and proliferation were analyzed using TUNEL, cell growth and clonogenic assays. Results CENPF expression is dramatically reduced under ER stress induced by thapsigargin (TG), brefeldin A (BFA), or tunicamycin (TM) and this downregulation of CENPF expression was dependent on XBP1 and ATF6α. Luciferase activity analysis of the truncated CENPF promoter indicates that regions from bases − 679 to − 488 and from − 241 to − 78 in the CENPF promoter were sensitive to TG treatment. Additionally, ChIP and ChIP Re-IP assays reveal that XBP1 and ATF6α were assembled on the same regions of CENPF promoter. Notably, we identify two XBP1 binding sequences at positions − 567 and − 192, to which XBP1 binding was enhanced by TG. Finally, CENPF overexpression inhibits cell apoptosis and promotes cell proliferation in response to ER stress. Conclusion In summary, these results demonstrate that ER stress plays a crucial role in CENPF expression, and XBP1 may up-regulate DNA-binding affinities after TG treatment to the promoter of CENPF. These findings may contribute to the understanding of the molecular mechanism of CENPF regulation.

Designing Novel Therapies to Mend Broken Hearts: ATF6 and Cardiac Proteostasis

The heart exhibits incredible plasticity in response to both environmental and genetic alterations that affect workload. Over the course of development, or in response to physiological or pathological stimuli, the heart responds to fluctuations in workload by hypertrophic growth primarily by individual cardiac myocytes growing in size. Cardiac hypertrophy is associated with an increase in protein synthesis, which must coordinate with protein folding and degradation to allow for homeostatic growth without affecting the functional integrity of cardiac myocytes (i.e., proteostasis). This increase in the protein folding demand in the growing cardiac myocyte activates the transcription factor, ATF6 (activating transcription factor 6α, an inducer of genes that restore proteostasis. Previously, ATF6 has been shown to induce ER-targeted proteins functioning primarily to enhance ER protein folding and degradation. More recent studies, however, have illuminated adaptive roles for ATF6 functioning outside of the ER by inducing non-canonical targets in a stimulus-specific manner. This unique ability of ATF6 to act as an initial adaptive responder has bolstered an enthusiasm for identifying small molecule activators of ATF6 and similar proteostasis-based therapeutics.

Sledgehammer to Scalpel: Broad Challenges to the Heart and Other Tissues Yield Specific Cellular Responses via Transcriptional Regulation of the ER-Stress Master Regulator ATF6α

There are more than 2000 transcription factors in eukaryotes, many of which are subject to complex mechanisms fine-tuning their activity and their transcriptional programs to meet the vast array of conditions under which cells must adapt to thrive and survive. For example, conditions that impair protein folding in the endoplasmic reticulum (ER), sometimes called ER stress, elicit the relocation of the ER-transmembrane protein, activating transcription factor 6α (ATF6α), to the Golgi, where it is proteolytically cleaved. This generates a fragment of ATF6α that translocates to the nucleus, where it regulates numerous genes that restore ER protein-folding capacity but is degraded soon after. Thus, upon ER stress, ATF6α is converted from a stable, transmembrane protein, to a rapidly degraded, nuclear protein that is a potent transcription factor. This review focuses on the molecular mechanisms governing ATF6α location, activity, and stability, as well as the transcriptional programs ATF6α regulates, whether canonical genes that restore ER protein-folding or unexpected, non-canonical genes affecting cellular functions beyond the ER. Moreover, we will review fascinating roles for an ATF6α isoform, ATF6β, which has a similar mode of activation but, unlike ATF6α, is a long-lived, weak transcription factor that may moderate the genetic effects of ATF6α.

ER stress signaling has an activating transcription factor 6α (ATF6)-dependent “off-switch”

In response to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen, three ER transmembrane signaling proteins, inositol-requiring enzyme 1 (IRE1), PRKR-like ER kinase (PERK), and activating transcription factor 6α (ATF6α), are activated. These proteins initiate a signaling and transcriptional network termed the unfolded protein response (UPR), which re-establishes cellular proteostasis. When this restoration fails, however, cells undergo apoptosis. To investigate cross-talk between these different UPR enzymes, here we developed a high-content live cell screening platform to image fluorescent UPR-reporter cell lines derived from human SH-SY5Y neuroblastoma cells in which different ER stress signaling proteins were silenced through lentivirus-delivered shRNA constructs. We observed that loss of ATF6 expression results in uncontrolled IRE1-reporter activity and increases X box–binding protein 1 (XBP1) splicing. Transient increases in both IRE1 mRNA and IRE1 protein levels were observed in response to ER stress, suggesting that IRE1 up-regulation is a general feature of ER stress signaling and was further increased in cells lacking ATF6 expression. Moreover, overexpression of the transcriptionally active N-terminal domain of ATF6 reversed the increases in IRE1 levels. Furthermore, inhibition of IRE1 kinase activity or of downstream JNK activity prevented an increase in IRE1 levels during ER stress, suggesting that IRE1 transcription is regulated through a positive feed-forward loop. Collectively, our results indicate that from the moment of activation, IRE1 signaling during ER stress has an ATF6-dependent “off-switch.”

Dissection of thrombospondin-4 domains involved in intracellular adaptive ER Stress responsive signaling

Thrombospondins are a family of stress-inducible secreted glycoproteins that underlie tissue remodeling. We recently reported that thrombospondin-4 (Thbs4) has a critical intracellular function where it regulates the adaptive endoplasmic reticulum (ER) stress pathway through activating transcription factor 6α (Atf6α). Here, we dissect the domains of Thbs4 that mediate interactions with ER proteins such as BiP (Grp78) and Atf6α, and the domains mediating activation of the ER stress response. Functionally, Thbs4 localized to the ER and post-ER vesicles and was actively secreted in cardiomyocytes, as were the T3R and TSP-C domains, while the LamG domain localized to the Golgi apparatus. We also mutated the major calcium-binding motifs within the T3R domain of full-length Thbs4, causing ER retention and secretion blockade. The T3R and TSP-C domains as well as wildtype Thbs4 and the calcium-binding mutant, interacted with Atf6α, induced an adaptive ER stress response, and caused expansion of intracellular vesicles. In contrast, overexpression of a related secreted oligomeric glycoprotein, Nell2, which lacks only the T3R and TSP-C domains, did not cause these effects. Finally, deletion of Atf6α abrogated Thbs4-induced vesicular expansion. Taken together, these data identify the critical intracellular functional domains of Thbs4, which was formerly thought to only have extracellular functions.

Activating Transcription Factor 6α Is Required for the Vasopressin Neuron System to Maintain Water Balance Under Dehydration in Male Mice

Activating transcription factor 6α (ATF6α) is a sensor of endoplasmic reticulum (ER) stress and increases the expression of ER chaperones and molecules related to the ER-associated degradation of unfolded/misfolded proteins. In this study, we used ATF6α knockout (ATF6α−/−) mice to clarify the role of ATF6α in the arginine vasopressin (AVP) neuron system. Although urine volumes were not different between ATF6α−/− and wild-type (ATF6α+/+) mice with access to water ad libitum, they were increased in ATF6α−/− mice compared with those in ATF6α+/+ mice under intermittent water deprivation (WD) and accompanied by less urine AVP in ATF6α−/− mice. The mRNA expression of immunoglobulin heavy chain binding protein, an ER chaperone, was significantly increased in the supraoptic nucleus in ATF6α+/+ but not ATF6α−/− mice after WD. Electron microscopic analyses demonstrated that the ER lumen of AVP neurons was more dilated in ATF6α−/− mice than in ATF6α+/+ mice after WD. ATF6α−/− mice that were mated with mice possessing a mutation causing familial neurohypophysial diabetes insipidus (FNDI), which is characterized by progressive polyuria and AVP neuronal loss due to the accumulation of mutant AVP precursor in the ER, manifested increased urine volume under intermittent WD. The aggregate formation in the ER of AVP neurons was further impaired in FNDI/ATF6α−/− mice compared with that in FNDI mice, and AVP neuronal loss was accelerated in FNDI/ATF6α−/− mice under WD. These data suggest that ATF6α is required for the AVP neuron system to maintain water balance under dehydration.