Abstract-The endoplasmic reticulum (ER) stress response (ERSR) is activated when folding of nascent proteins in the ERlumen is impeded. Myocardial ischemia was recently shown to activate the ERSR; however, the role of this complex signaling system in the heart is not well understood. ER stress activates the transcription factor ATF6, which induces expression of proteins targeted to the ER, where they restore protein folding, thus fostering cytoprotection. We previously developed a transgenic mouse line that expresses a conditionally activated form of ATF6 in the heart. In this mouse line, ATF6 activation decreased ischemic damage in an ex vivo model of myocardial ischemia/reperfusion and induced numerous genes, including mesencephalic astrocyte-derived neurotrophic factor (MANF). In the present study, MANF expression was shown to be induced in cardiac myocytes and in other cell types in the hearts of mice subjected to in vivo myocardial infarction. Additionally, simulated ischemia induced MANF in an ATF6-dependent manner in neonatal rat ventricular myocyte cultures. In contrast to many other ER-resident ERSR proteins, MANF lacks a canonical ER-retention sequence, consistent with our finding that MANF was readily secreted from cultured cardiac myocytes. Knockdown of endogenous MANF with micro-RNA increased cell death upon simulated ischemia/ reperfusion, whereas addition of recombinant MANF to media protected cultured cardiac myocytes from simulated ischemia/reperfusion-mediated death. Thus, a possible function of the ERSR in the heart is the ischemia-mediated induction of secreted proteins, such as MANF, that can function in an autocrine/paracrine manner to modulate myocardial damage from ER stresses, including ischemia. Key Words: adenovirus Ⅲ cardiac myocytes Ⅲ endoplasmic reticulum stress Ⅲ hypoxia Ⅲ ischemia Ⅲ myocardial infarction Ⅲ unfolded protein response N umerous proteins that are critical to cellular function are synthesized on endoplasmic reticulum (ER) ribosomes and then folded and further posttranslationally modified in the ER lumen. Stresses that impede ER protein folding trigger the ER stress response (ERSR), 1-4 a signaling system that has not been studied extensively in the heart. ER stress activates the transcription factors X-box binding protein (XBP1) and activator of transcription-6 (ATF6), which induce numerous ERSR proteins designed to restore efficient ER protein folding, which contributes to resisting the stress. 5,6 Many ERSR genes are induced by ATF6 or XBP1, whereas others exhibit a requirement for only 1 of the 2 factors. 7 The folding of proteins in the ER lumen requires molecular oxygen, 8 suggesting that that hypoxia-mediated induction of ERSR proteins during myocardial ischemia may enhance ER protein folding and survival of cardiac myocytes and limit ischemic damage. Consistent with this hypothesis are recent studies showing that simulated ischemia (sI) activates the ERSR in cultured rat and mouse ventricular myocytes and that ER stress is activated in the surviving myocytes adj...

Stresses that perturb the folding of nascent endoplasmic reticulum (ER) proteins activate the ER stress response. Upon ER stress, ER-associated ATF6 is cleaved; the resulting active cytosolic fragment of ATF6 translocates to the nucleus, binds to ER stress response elements (ERSEs), and induces genes, including the ER-targeted chaperone, GRP78. Recent studies showed that nutrient and oxygen starvation during tissue ischemia induce certain ER stress response genes, including GRP78; however, the role of ATF6 in mediating this induction has not been examined. In the current study, simulating ischemia (sI) in a primary cardiac myocyte model system caused a reduction in the level of ER-associated ATF6 with a coordinate increase of ATF6 in nuclear fractions. An ERSE in the GRP78 gene not previously shown to be required for induction by other ER stresses was found to bind ATF6 and to be critical for maximal ischemiamediated GRP78 promoter induction. Activation of ATF6 and the GRP78 promoter, as well as grp78 mRNA accumulation during sI, were reversed upon simulated reperfusion (sI/R). Moreover, dominant-negative ATF6, or ATF6-targeted miRNA blocked sI-mediated grp78 induction, and the latter increased cardiac myocyte death upon simulated reperfusion, demonstrating critical roles for endogenous ATF6 in ischemia-mediated ER stress activation and cell survival. This is the first study to show that ATF6 is activated by ischemia but inactivated upon reperfusion, suggesting that it may play a role in the induction of ER stress response genes during ischemia that could have a preconditioning effect on cell survival during reperfusion.

The endoplasmic reticulum (ER)-transmembrane proteins, ATF6␣ and ATF6␤, are cleaved during the ER stress response (ERSR). The resulting N-terminal fragments (N-ATF6␣ and N-ATF6␤) have conserved DNA-binding domains and divergent transcriptional activation domains. N-ATF6␣ and N-ATF6␤ translocate to the nucleus, bind to specific regulatory elements, and influence expression of ERSR genes, such as glucose-regulated protein 78 (GRP78), that contribute to resolving the ERSR, thus, enhancing cell viability. We previously showed that N-ATF6␣ is a rapidly degraded, strong transcriptional activator, whereas ␤ is a slowly degraded, weak activator. In this study we explored the molecular basis and functional impact of these isoform-specific characteristics in HeLa cells. Mutants in the transcriptional activation domain or DNA-binding domain of N-ATF6␣ exhibited loss of function and increased expression, the latter of which suggested decreased rates of degradation. Fusing N-ATF6␣ to the mutant estrogen receptor generated N-ATF6␣-MER, which, without tamoxifen exhibited loss-of-function and high expression, but in the presence of tamoxifen N-ATF6␣-MER exhibited gain-of-function and low expression. N-ATF6␤ conferred loss-of-function and high expression to N-ATF6␣, suggesting that ATF6␤ is an endogenous inhibitor of ATF6␣. In vitro DNA binding experiments showed that recombinant N-ATF6␤ inhibited the binding of recombinant N-ATF6␣ to an ERSR element from the GRP78 promoter. Moreover, siRNA-mediated knock-down of endogenous ATF6␤ increased GRP78 promoter activity and GRP78 gene expression, as well as augmenting cell viability. Thus, the relative levels of ATF6␣ and -␤, may contribute to regulating the strength and duration of ATF6-dependent ERSR gene induction and cell viability.

Exposing cells to conditions that modulate growth can impair endoplasmic reticulum (ER) protein folding, leading to ER stress and activation of the transcription factor, ATF6. ATF6 binds to ER stress response elements in target genes, inducing expression of proteins that enhance the ER protein folding capacity, which helps overcome the stress and foster survival. To examine the mechanism of ATF6-mediated survival in vivo, we developed a transgenic mouse model that expresses a novel conditionally activated form of ATF6. We previously showed that activating ATF6 protected the hearts of ATF6 transgenic mice from ER stresses. In the present study, transcript profiling identified modulatory calcineurin interacting protein-1 (MCIP1), also known as regulator of calcineurin 1 (RCAN1), as a novel ATF6-inducible gene that encodes a known regulator of calcineurin/nuclear factor of activated T cells (NFAT)-mediated growth and development in many tissues. The ability of ATF6 to induce RCAN1 in vivo was replicated in cultured cardiac myocytes, where adenoviral (AdV)-mediated overexpression of activated ATF6 induced the RCAN1 promoter, up-regulated RCAN1 mRNA, inhibited calcineurin phosphatase activity, and exerted a striking growth modulating effect that was inhibited by RCAN1-targeted small interfering RNA. These results demonstrate that RCAN1 is a novel ATF6 target gene that may coordinate growth and ER stress signaling pathways. By modulating growth, RCAN1 may reduce the need for ER protein folding, thus helping to overcome the stress and enhance survival. Moreover, these results suggest that RCAN1 may also be a novel integrator of growth and ER stress signaling in many other tissues that depend on calcineurin/NFAT signaling for optimal growth and development.About 35% of all proteins, including secreted, cell surface and most organelle-targeted proteins are synthesized in the ER (1).Conditions that perturb ER calcium, protein glycosylation or redox status, impair nascent protein folding, triggering the ER stress response (ERSR), 4 also known as the unfolded protein response (2, 3). The ERSR has central roles in all metazoan cells, and has been shown to be important in the differentiation and function of secretory cells, including B lymphocytes, ␤-cells of the pancreas and the liver. Moreover, ER stress has been associated with neurodegenerative diseases (4), as well as pathologies that are precipitated upon impaired oxygen and nutrient delivery (i.e. ischemia) resulting from cardiovascular disease. Ischemia has been shown to activate the ERSR in brain, kidney, liver, and heart, as well as in tumor tissue in vivo (5-9).Initially, ER stress leads to remodeling of the ER protein biosynthetic machinery in attempts to overcome the stress. This initial phase of remodeling includes the transcriptional induction of genes that encode ER-targeted chaperones and folding enzymes that help proteins fold in the ER lumen. In addition, ER stress up-regulates genes encoding components of an ERassociated degradation system, which is respon...

Proper folding of secreted and transmembrane proteins made in the rough endoplasmic reticulum (ER) requires oxygen for disulfide bond formation. Accordingly, ischemia can impair ER protein folding and initiate the ER stress response, which we previously showed is activated in the ischemic heart and in culture cardiac myocytes subjected to simulated ischemia. ER stress and ischemia activate the transcription factor, activating transcription factor 6 (ATF6), which induces numerous genes, many of which have not been identified, or examined in the heart. Using an ATF6 transgenic mouse model, we previously showed that ATF6 protected the heart from ischemic damage; however, the mechanism of this protection remains to be determined. In this study, we showed that, in the mouse heart, and in cultured cardiac myocytes, ATF6 induced the protein disulfide isomerase associated 6 (PDIA6) gene, which encodes an ER enzyme that catalyzes protein disulfide bond formation. Moreover, in cultured cardiac myocytes, ER stress-mediated PDIA6 promoter activation was ATF6-dependent, and required an ER stress response element (ERSE) and a nearby CCAAT box element. Electromobility shift assays and chromatin immunoprecipitation showed that ATF6 bound to the ERSE in the PDIA6 promoter, in vitro, and in the mouse heart, in vivo. Gain- and loss-of-function studies showed that PDIA6 protected cardiac myocytes against simulated ischemia/reperfusion-induced death in a manner that was dependent on the catalytic activity of PDIA6. Thus, by facilitating disulfide bond formation, and enhanced ER protein folding, PDIA6 may contribute to the protective effects of ATF6 in the ischemic mouse heart.

Rationale Stresses, such as ischemia, impair folding of nascent proteins in the rough endoplasmic reticulum (ER), activating the unfolded protein response (UPR), which restores efficient ER protein folding, thus leading to protection from stress. In part, the UPR alleviates ER stress and cell death by increasing the degradation of terminally mis-folded ER proteins via ER-associated degradation (ERAD). ERAD is increased by the ER stress modulator, activating transcription factor 6 (ATF6), which can induce genes that encode components of the ERAD machinery. Objective Recently, it was shown that the mouse heart is protected from ischemic damage by ATF6; however, ERAD has not been studied in the cardiac context. A recent microarray study showed that the Derlin-3 (Derl3) gene, which encodes an important component of the ERAD machinery, is robustly induced by ATF6 in the mouse heart. Methods and Results In the present study, activated ATF6 induced Derl3 in cultured cardiomyocytes, and in the heart, in vivo. Simulated ischemia (sI), which activates ER stress, induced Derl3 in cultured myocytes, and in an in vivo mouse model of myocardial infarction, Derl3 was also induced. Derl3 overexpression enhanced ERAD and protected cardiomyocytes from sI-induced cell death, while dominant-negative Derl3 decreased ERAD and increased sI-induced cardiomyocyte death. Conclusions This study describes a potentially protective role for Derl3 in the heart, and is the first to investigate the functional consequences of enhancing ERAD in the cardiac context.

A nodal regulator of endoplasmic reticulum stress is the transcription factor, ATF6, which is activated by ischemia and protects the heart from ischemic damage, in vivo. To explore mechanisms of ATF6-mediated protection in the heart, a whole-genome microRNA (miRNA) array analysis of RNA from the hearts of ATF6 transgenic (TG) mice was performed. The array identified 13 ATF6-regulated miRNAs, eight of which were downregulated, suggesting that they could contribute to increasing levels of their mRNAs. The down-regulated miRNAs, including miR-455, were predicted to target 45 mRNAs that we had previously shown by microarray analysis to be up-regulated by ATF6 in the heart. One of the miR-455 targets was calreticulin (Calr), which is up-regulated in the pathologic heart, where it modulates hypertrophic growth, potentially reducing the impact of the pathology. To validate the effects of miR-455, we showed that Calr protein was increased by ATF6 in mouse hearts, in vivo. In cultured cardiac myocytes, treatment with the ER stressor, tunicamycin, or with adenovirus encoding activated ATF6 decreased miR-455 and increased Calr levels, consistent with the effects of ATF6 on miR-455 and Calr, in vivo. Moreover, transfection of cultured cardiac myocytes with a synthetic precursor, premiR-455, decreased Calr levels, while transfection with an antisense, antimiR-455, increased Calr levels. The results of this study suggest that ER stress can regulate gene expression via ATF6-mediated changes in micro-RNA levels. Moreover, these findings support the hypothesis that ATF6-mediated down-regulation of miR-455 augments Calr expression, which may contribute to the protective effects of ATF6 in the heart.

Purpose Effective therapies for KRAS mutant colorectal cancer (CRC) are a critical unmet clinical need. Previously, we described GEMMs for sporadic Kras mutant and non-mutant CRC suitable for preclinical evaluation of experimental therapeutics. To accelerate drug discovery and validation, we sought to derive low-passage cell lines from GEMM Kras mutant and wild-type tumors for in vitro screening and transplantation into the native colonic environment of immunocompetent mice for in vivo validation. Experimental Design Cell lines were derived from Kras mutant and non-mutant GEMM tumors under defined media conditions. Growth kinetics, phosphoproteomes, transcriptomes, drug sensitivity, and metabolism were examined. Cell lines were implanted in mice and monitored for in vivo tumor analysis. Results Kras mutant cell lines displayed increased proliferation, MAPK signaling, and PI3K signaling. Microarray analysis identified significant overlap with human CRC-related gene signatures, including KRAS mutant and metastatic CRC. Further analyses revealed enrichment for numerous disease-relevant biological pathways, including glucose metabolism. Functional assessment in vitro and in vivo validated this finding and highlighted the dependence of Kras mutant CRC on oncogenic signaling and on aerobic glycolysis. Conclusions We have successfully characterized a novel GEMM-derived orthotopic transplant model of human KRAS mutant CRC. This approach combines in vitro screening capability using low-passage cell lines that recapitulate human CRC and potential for rapid in vivo validation using cell line-derived tumors that develop in the colonic microenvironment of immunocompetent animals. Taken together, this platform is a clear advancement in preclinical CRC models for comprehensive drug discovery and validation efforts.