Donna J. Thuerauf

p38 MAPK and NF-κB Collaborate to Induce Interleukin-6 Gene Expression and Release EVIDENCE FOR A CYTOPROTECTIVE AUTOCRINE SIGNALING PATHWAY IN A CARDIAC MYOCYTE MODEL SYSTEM

In cardiac myocytes, the stimulation of p38 MAPK by the MAPKK, MKK6, activates the transcription factor, NF-κB, and protects cells from apoptosis. In the present study in primary neonatal rat cardiac myocytes, constitutively active MKK6, MKK6(Glu), bound to IκB kinase (IKK)-β and stimulated its abilities to phosphorylate IκB and to activate NF-κB. MKK6(Glu) induced NF-κB-dependent interleukin (IL)-6 transcription and IL-6 release in a p38-dependent manner. IL-6 protected myocardial cells against apoptosis. Like IL-6, TNF-α, which activates both NF-κB and p38, also induced p38-dependent IL-6 expression and release and protected myocytes from apoptotis. While TNF-α was relatively ineffective, IL-6 activated myocardial cell STAT3 by about 8-fold, indicating a probable role for this transcription factor in IL-6-mediated protection from apoptosis. TNF-α-mediated IL-6 induction was inhibited by a kinase-inactive form of the MAPKKK, TGF-β activated protein kinase (Tak1), which is known to activate p38 and NF-κB in other cell types. Thus, by stimulating both p38 and NF-κB, Tak1-activating cytokines, like TNF-α, can induce IL-6 expression and release. Moreover, the myocyte-derived IL-6 may then function in an autocrine and/or paracrine fashion to augment myocardial cell survival during stresses that activate p38.

Activation of the Unfolded Protein Response in Infarcted Mouse Heart and Hypoxic Cultured Cardiac Myocytes

Endoplasmic reticulum (ER) stresses that reduce ER protein folding activate the unfolded protein response (UPR). One effector of the UPR is the transcription factor X-box binding protein-1 (XBP1), which is expressed on ER stress–mediated splicing of the XBP1 mRNA. XBP1 induces certain ER-targeted proteins, eg, glucose-regulated protein 78 (GRP78), that help resolve the ER stress and foster cell survival. In this study, we determined whether hypoxia can activate the UPR in the cardiac context. Neonatal rat ventricular myocyte cultures subjected to hypoxia (16 hours) exhibited increased XBP1 mRNA splicing, XBP1 protein expression, GRP78 promoter activation, and GRP78 protein levels; however, the levels of these UPR markers declined during reoxygenation, suggesting that the UPR is activated during hypoxia but not during reoxygenation. When cells were infected with a recombinant adenovirus (AdV) encoding dominant-negative XBP1 (AdV-XBP1dn), UPR markers were reduced; however, hypoxia/reoxygenation-induced apoptosis increased. Confocal immunocytofluorescence demonstrated that hypoxia induced GRP78 in neonatal rat and isolated adult mouse ventricular myocytes. Moreover, mouse hearts subjected to in vivo myocardial infarction exhibited increased GRP78 expression in cardiac myocytes near the infarct, but not in healthy cells distal to the infarct. These results indicate that hypoxia activates the UPR in cardiac myocytes and that XBP1-inducible proteins may contribute to protecting the myocardium during hypoxic stress.

Endoplasmic Reticulum Stress Gene Induction and Protection From Ischemia/Reperfusion Injury in the Hearts of Transgenic Mice With a Tamoxifen-Regulated Form of ATF6

Ischemia/reperfusion (I/R) affects the integrity of the endoplasmic reticulum (ER), the site of synthesis and folding of numerous proteins. Therefore, I/R may activate the unfolded protein response (UPR), resulting in the induction of a collection of ER stress proteins, many of which are protective and function to resolve the ER stress. In this study, we showed that when mouse hearts were subjected to ex vivo I/R, the levels of 2 ER stress-inducible markers of the UPR, the ER-targeted cytoprotective chaperones glucose-regulated proteins 78 and 94 (GRP78 and GRP94), were increased, consistent with I/R-mediated UPR activation in the heart. The UPR-mediated activation of ATF6 (Activation of Transcription Factor 6) induces cytoprotective ER stress proteins, including GRP78 and GRP94. To examine whether ATF6 protects the myocardium from I/R injury in the heart, we generated transgenic (TG) mice featuring cardiac-restricted expression of a novel tamoxifen-activated form of ATF6, ATF6-MER. When NTG and ATF6-MER TG mice were treated with or without tamoxifen for 5 days, only the hearts from the tamoxifen-treated TG mice exhibited increased levels of many ER stress–inducible mRNAs and proteins; for example, GRP78 and GRP94 transcript levels were increased by 8- and 15-fold, respectively. The tamoxifen-treated TG mouse hearts also exhibited better functional recovery from ex vivo I/R, as well as significantly reduced necrosis and apoptosis. These results suggest that the UPR is activated in the heart during I/R and that, as a result, the ATF6 branch of the UPR may induce expression of proteins that can function to reduce I/R injury.

Mimicking Phosphorylation of αB-Crystallin on Serine-59 Is Necessary and Sufficient to Provide Maximal Protection of Cardiac Myocytes From Apoptosis

Abstract— &agr;B-Crystallin (&agr;BC), a small heat shock protein expressed in high levels in the heart, is phosphorylated on Ser-19, 45, and 59 after stress. However, it is not known whether &agr;BC phosphorylation directly affects cell survival. In the present study, constructs were prepared that encode forms of &agr;BC harboring Ser to Ala (blocks phosphorylation) or Ser to Glu (mimics phosphorylation) mutations at positions 19, 45, and 59. The effects of each form on apoptosis of cultured cardiac myocytes after hyperosmotic or hypoxic stress were assessed. Compared with controls, cells that expressed &agr;BC with Ser to Ala substitutions at all three positions, &agr;BC(AAA), exhibited more stress-induced apoptosis. Cells expressing either &agr;BC(AAE) or (EEE) exhibited 3-fold less apoptosis than cells expressing &agr;BC(AAA), indicating that phosphorylation of Ser-59 confers protection. &agr;BC is known to bind to procaspase-3 and to decrease caspase-3 activation. Compared with cells expressing &agr;BC(AAA), the activation of caspase-3 was decreased by 3-fold in cells expressing &agr;BC(AAE). These results demonstrate that mimicking the phosphorylation of &agr;BC on Ser-59 is necessary and sufficient to confer caspase-3 inhibition and protection of cardiac myocytes against hyperosmotic or hypoxic stress. These findings provide direct evidence that &agr;BC(S59P) contributes to the cardioprotection observed after physiologically relevant stresses, such as transient hypoxia. Identifying the targets of &agr;BC(S59P) will reveal important details about the mechanism underlying the cytoprotective effects of this small heat shock protein.

αB-crystallin Gene Induction and Phosphorylation by MKK6-activated p38 A POTENTIAL ROLE FOR αB-CRYSTALLIN AS A TARGET OF THE p38 BRANCH OF THE CARDIAC STRESS RESPONSE

The MAPK kinase MKK6 selectively stimulates p38 MAPK and confers protection against stress-induced apoptosis in cardiac myocytes. However, the events lying downstream of p38 that mediate this protection are unknown. The small heat shock protein, αB-crystallin, which is expressed in only a few cell types, including cardiac myocytes, may participate in MKK6-mediated cytoprotection. In the present study, we showed that, in cultured cardiac myocytes, expression of MKK6(Glu), an active form of MKK6, led to p38-dependent increases in αB-crystallin mRNA, protein, and transcription. MKK6(Glu) also induced p38-dependent activation of the downstream MAPK-activated protein kinase, MAPKAP-K2, and the phosphorylation of αB-crystallin on serine-59. Initially, exposure of cells to the hyperosmotic stressor, sorbitol, stimulated MKK6, p38, and MAPKAP-K2 and increased phosphorylation of αB-crystallin on serine 59. However, after longer times of exposure to sorbitol, the cells began to undergo apoptosis. This sorbitol-induced apoptosis was increased when p38 was inhibited in a manner that would block αB-crystallin induction and phosphorylation. Thus, under these conditions, the activation of MKK6, p38, and MAPKAP-K2 by sorbitol can provide a degree of protection against stress-induced apoptosis. Supporting this view was the finding that sorbitol-induced apoptosis was nearly completely blocked in cells expressing MKK6(Glu). Therefore, the cytoprotective effects of MKK6 in cardiac myocytes are due, in part, to phosphorylation of αB-crystallin on serine 59 and to the induction of αB-crystallin gene expression.

Ischemia activates the ATF6 branch of the endoplasmic reticulum stress response.

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 ischemia-mediated 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.

p38 Mitogen-activated Protein Kinase Mediates the Transcriptional Induction of the Atrial Natriuretic Factor Gene through a Serum Response Element A POTENTIAL ROLE FOR THE TRANSCRIPTION FACTOR ATF6

In various cell types certain stresses can stimulate p38 mitogen-activated protein kinase (p38 MAPK), leading to the transcriptional activation of genes that contribute to appropriate compensatory responses. In this report the mechanism of p38-activated transcription was studied in cardiac myocytes where this MAPK is a key regulator of the cell growth and the cardiac-specific gene induction that occurs in response to potentially stressful stimuli. In the cardiac atrial natriuretic factor (ANF) gene, a promoter-proximal serum response element (SRE), which binds serum response factor (SRF), was shown to be critical for ANF induction in primary cardiac myocytes transfected with the selective p38 MAPK activator, MKK6 (Glu). This ANF SRE does not possess sequences typically required for the binding of the Ets-related ternary complex factors (TCFs), such as Elk-1, indicating that p38-mediated induction through this element may take place independently of such TCFs. Although p38 did not phosphorylate SRF in vitro, it efficiently phosphorylated ATF6, a newly discovered SRF-binding protein that is believed to serve as a co-activator of SRF-inducible transcription at SREs. Expression of an ATF6 antisense RNA blocked p38-mediated ANF induction through the ANF SRE. Moreover, when fused to the Gal4 DNA-binding domain, an N-terminal 273-amino acid fragment of ATF6 was sufficient to support trans-activation of Gal4/luciferase expression in response to p38 but not the other stress kinase, N-terminal Jun kinase (JNK); p38-activating cardiac growth promoters also stimulated ATF6 trans-activation. These results indicate that through ATF6, p38 can augment SRE-mediated transcription independently of Ets-related TCFs, representing a novel mechanism of SRF-dependent transcription by MAP kinases.

Mesencephalic Astrocyte-Derived Neurotrophic Factor Is an Ischemia-Inducible Secreted Endoplasmic Reticulum Stress Response Protein in the Heart

The endoplasmic reticulum (ER) stress response (ERSR) is activated when folding of nascent proteins in the ER lumen 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.

Effects of the Isoform-specific Characteristics of ATF6α and ATF6β on Endoplasmic Reticulum Stress Response Gene Expression and Cell Viability

The endoplasmic reticulum (ER)-transmembrane proteins, ATF6α and ATF6β, are cleaved during the ER stress response (ERSR). The resulting N-terminal fragments (N-ATF6α and N-ATF6β) have conserved DNA-binding domains and divergent transcriptional activation domains. N-ATF6α and N-ATF6β 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-ATF6α is a rapidly degraded, strong transcriptional activator, whereas β 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-ATF6α exhibited loss of function and increased expression, the latter of which suggested decreased rates of degradation. Fusing N-ATF6α to the mutant estrogen receptor generated N-ATF6α-MER, which, without tamoxifen exhibited loss-of-function and high expression, but in the presence of tamoxifen N-ATF6α-MER exhibited gain-of-function and low expression. N-ATF6β conferred loss-of-function and high expression to N-ATF6α, suggesting that ATF6β is an endogenous inhibitor of ATF6α. In vitro DNA binding experiments showed that recombinant N-ATF6β inhibited the binding of recombinant N-ATF6α to an ERSR element from the GRP78 promoter. Moreover, siRNA-mediated knock-down of endogenous ATF6β increased GRP78 promoter activity and GRP78 gene expression, as well as augmenting cell viability. Thus, the relative levels of ATF6α and -β, may contribute to regulating the strength and duration of ATF6-dependent ERSR gene induction and cell viability.

Sarco/endoplasmic Reticulum Calcium ATPase-2 Expression Is Regulated by ATF6 during the Endoplasmic Reticulum Stress Response INTRACELLULAR SIGNALING OF CALCIUM STRESS IN A CARDIAC MYOCYTE MODEL SYSTEM

The recently described transcription factor, ATF6, mediates the expression of proteins that compensate for potentially stressful changes in the endoplasmic reticulum (ER), such as reduced ER calcium. In cardiac myocytes the maintenance of optimal calcium levels in the sarcoplasmic reticulum (SR), a specialized form of the ER, is required for proper contractility. The present study investigated the hypothesis that ATF6 serves as a regulator of the expression of sarco/endoplasmic reticulum calcium ATPase-2 (SERCA2), a protein that transports calcium into the SR from the cytoplasm. Depletion of SR calcium in cultured cardiac myocytes fostered the translocation of ATF6 from the ER to the nucleus, activated the promoter for rat SERCA2, and led to increased levels of SERCA2 protein. SERCA2 promoter induction by calcium depletion was partially blocked by dominant-negative ATF6, whereas constitutively activated ATF6 led to SERCA2 promoter activation. Mutation analyses identified a promoter-proximal ER stress-response element in the rat SERCA2 gene that was required for maximal induction by ATF6 and calcium depletion. Although this element was shown to be responsible for all of the effects of ATF6 on SERCA2 promoter activation, it was responsible for only a portion of the effects of calcium depletion. Thus, SERCA2 induction in response to calcium depletion appears to be a potentially physiologically important compensatory response to this stress that involves intracellular signaling pathways that are both dependent and independent of ATF6.