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.

p38 MAPK regulates group IIa phospholipase A2 expression in interleukin-1beta -stimulated rat neonatal cardiomyocytes.

Group IIa phospholipase A(2) (GIIa PLA(2)) is released by some cells in response to interleukin-1beta. The purpose of this study was to determine whether interleukin-1beta would stimulate the synthesis and release of GIIa PLA(2) from cardiomyocytes, and to define the role of p38 MAPK and cytosolic PLA(2) in the regulation of this process. Whereas GIIa PLA(2) mRNA was not identified in untreated cells, exposure to interleukin-1beta resulted in the sustained expression of GIIa PLA(2) mRNA. Interleukin-1beta also stimulated a progressive increase in cellular and extracellular GIIa PLA(2) protein levels and increased extracellular PLA(2) activity 70-fold. In addition, interleukin-1beta stimulated the p38 MAPK-dependent activation of the downstream MAPK-activated protein kinase, MAPKAP-K2. Treatment with the p38 MAPK inhibitor, SB202190, decreased interleukin-1beta stimulated MAPKAP-K2 activity, GIIa PLA(2) mRNA expression, GIIa PLA(2) protein synthesis, and the release of extracellular PLA(2) activity. Infection with an adenovirus encoding a constitutively active form of MKK6, MKK6(Glu), which selectively phosphorylates p38 MAPK, induced cellular GIIa PLA(2) protein synthesis and the release of GIIa PLA(2) and increased extracellular PLA(2) activity 3-fold. In contrast, infection with an adenovirus encoding a phosphorylation-resistant MKK6, MKK6(A), did not result in GIIa PLA(2) protein synthesis or release by unstimulated cardiomyocytes. In addition, infection with an adenovirus encoding MKK6(A) abrogated GIIa PLA(2) protein synthesis and release by interleukin-1beta-stimulated cells. These results provide direct evidence that p38 MAPK activation was necessary for interleukin-1beta-induced synthesis and release of GIIa PLA(2) by cardiomyocytes.

alpha B-crystallin gene induction and phosphorylation by MKK6-activated p38. A potential role for alpha B-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, alphaB-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 alphaB-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 alphaB-crystallin on serine-59. Initially, exposure of cells to the hyperosmotic stressor, sorbitol, stimulated MKK6, p38, and MAPKAP-K2 and increased phosphorylation of alphaB-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 alphaB-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 alphaB-crystallin on serine 59 and to the induction of alphaB-crystallin gene expression.

p38 MAPK and NF-kappa B 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-kappaB, and protects cells from apoptosis. In the present study in primary neonatal rat cardiac myocytes, constitutively active MKK6, MKK6(Glu), bound to IkappaB kinase (IKK)-beta and stimulated its abilities to phosphorylate IkappaB and to activate NF-kappaB. MKK6(Glu) induced NF-kappaB-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-alpha, which activates both NF-kappaB and p38, also induced p38-dependent IL-6 expression and release and protected myocytes from apoptotis. While TNF-alpha 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-alpha-mediated IL-6 induction was inhibited by a kinase-inactive form of the MAPKKK, TGF-beta activated protein kinase (Tak1), which is known to activate p38 and NF-kappaB in other cell types. Thus, by stimulating both p38 and NF-kappaB, Tak1-activating cytokines, like TNF-alpha, 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.

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.

A role for the p38 mitogen-activated protein kinase pathway in myocardial cell growth, sarcomeric organization, and cardiac-specific gene expression.

Three hallmark features of the cardiac hypertrophic growth program are increases in cell size, sarcomeric organization, and the induction of certain cardiac-specific genes. All three features of hypertrophy are induced in cultured myocardial cells by alpha1- adrenergic receptor agonists, such as phenylephrine (PE) and other growth factors that activate mitogen- activated protein kinases (MAPKs). In this study the MAPK family members extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 were activated by transfecting cultured cardiac myocytes with constructs encoding the appropriate kinases possessing gain-of-function mutations. Transfected cells were then analyzed for changes in cell size, sarcomeric organization, and induction of the genes for the A- and B-type natriuretic peptides (NPs), as well as the alpha-skeletal actin (alpha-SkA) gene. While activation of JNK and/or ERK with MEKK1COOH or Raf-1 BXB, respectively, augmented cell size and effected relatively modest increases in NP and alpha-SkA promoter activities, neither upstream kinase conferred sarcomeric organization. However, transfection with MKK6 (Glu), which specifically activated p38, augmented cell size, induced NP and alpha-Ska promoter activities by up to 130-fold, and elicited sarcomeric organization in a manner similar to PE. Moreover, all three growth features induced by MKK6 (Glu) or PE were blocked with the p38-specific inhibitor, SB 203580. These results demonstrate novel and potentially central roles for MKK6 and p38 in the regulation of myocardial cell hypertrophy.

Differential effects of protein kinase C, Ras, and Raf-1 kinase on the induction of the cardiac B-type natriuretic peptide gene through a critical promoter-proximal M-CAT element.

The cardiac genes for the A- and B-type natriuretic peptides (ANP and BNP) are coordinately induced by growth promoters, such as alpha1-adrenergic receptor agonists (e.g. phenylephrine (PE)). Although inducible elements in the ANP gene have been identified, responsible elements in the BNP gene are unknown. In this study, reporter constructs transfected into neonatal rat ventricular myocytes showed that in the context of 2.5 kilobase pairs of native BNP 5'-flanking sequences, a 2-base pair mutation in a promoter-proximal M-CAT site (CATTCT) disrupted basal and PE-inducible transcription by more than 98%. Expression of constitutively active forms of Ras, Raf-1 kinase, and protein kinase C, all of which are activated by PE in cardiac myocytes, strongly stimulated BNP reporter expression. Isolated M-CAT elements conferred PE, protein kinase C, and Ras inducibility to a minimal BNP promoter, however, they did not confer Raf-1 inducibility. These results show that M-CAT elements can serve as targets for Ras-dependent, Raf-1-independent pathways, implying the involvement of c-Jun N-terminal kinase and/or p38 mitogen-activated protein kinases, but not extracellular signal-regulated protein kinase/mitogen-activated protein kinase. Moreover, the essential M-CAT element distinguishes the BNP gene from the ANP gene, which utilizes serum response elements and an Sp1-like sequence.

Dissociation of p44 and p42 mitogen-activated protein kinase activation from receptor-induced hypertrophy in neonatal rat ventricular myocytes.

In response to hormones and mechanical stretch, neonatal rat ventricular myocytes exhibit a hypertrophic response that is characterized by induction of cardiac-specific genes and increased myocardial cell size. Hypertrophic stimuli also activate mitogen-activated protein kinase (MAPK), an enzyme thought to play a central role in the regulation of cell growth and differentiation. To determine if MAPK activation is sufficient for acquisition of the molecular and morphological features of cardiac hypertrophy we compared four agonists that stimulate G protein-coupled receptors. Whereas phenylephrine and endothelin transactivate cardiac-specific promoter/luciferase reporter genes, increase atrial natriuretic factor (ANF) expression, and promote myofilament organization, neither carbachol nor ATP induces these responses. Interestingly, all four agonists activate both the p42 and the p44 isoforms of MAPK. Furthermore, the kinetics of MAPK activation are not different for the hypertrophic agonist phenylephrine and the nonhypertrophic agonist carbachol. Transient transfection of myocytes with dominant-interfering mutants of p42 and p44 MAPK failed to block phenylephrine-induced ANF expression, although Ras-induced gene expression was inhibited by expression of the mutant MAPK constructs. Moreover, PD 098059, an inhibitor of MAPK kinase, blocked phenylephrine-stimulated MAPK activity but not ANF reporter gene expression. Thus, MAPK activation is not sufficient for G protein receptor-mediated induction of cardiac cell growth and gene expression and is apparently not required for transcriptional activation of the ANF gene.

Brain natriuretic peptide is induced by alpha 1-adrenergic agonists as a primary response gene in cultured rat cardiac myocytes.

To better understand the molecular basis for increased atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) expression during overload-induced cardiac hypertrophy, we studied the induction of the genes in primary myocardial cells by the alpha 1-adrenergic agonist, phenylephrine (PE), a potent hypertrophic agent. PE augmented the transcription of both genes to similar extents, although the time course of mRNA accumulation differed. Increases in ANF mRNA were evident only after 6-8 h of PE exposure, when transcript levels were 2-4-fold over control. However, similar increases in BNP mRNA were observed as soon as 1 h of PE exposure. Moreover, while ANF mRNA levels continued to increase through 24 h of PE treatment, maximal levels of BNP mRNA (8-10-fold over control) were observed at 4 h, after which transcript levels declined to about 3-fold over control. The early induction of the BNP mRNA by PE was independent of protein synthesis, whereas the late induction of both genes required protein synthesis. Interestingly, the early BNP induction was only partially blocked by the transcription inhibitor, actinomycin D, indicating that, in part, the inductive effects of PE might be the result of transcript stabilization. Indeed, the BNP transcript, which was shown to possess a half-life of less than 1 h in control cells, was stabilized by the addition of PE, while the ANF transcript possessed a half-life of at least 24 h under all conditions. These data indicate that the induction of BNP by alpha 1-adrenergic agonists has characteristics of both a primary and secondary response gene, while ANF is a typical secondary response gene. Moreover, alpha 1-adrenergic stimulation enhances BNP expression through both transcriptional activation and transcript stabilization, while ANF expression is enhanced primarily transcriptionally.

Regulation of rat brain natriuretic peptide transcription. A potential role for GATA-related transcription factors in myocardial cell gene expression.

GATA-binding proteins are transcription factors that regulate the stage- and tissue-specific expression of globin genes in cells of the erythroid lineage. Recently, a cardiac GATA-binding protein was found to be the earliest gene expressed during cardiogenesis; however, the target genes of this transcription factor in the heart are unknown. Since brain natriuretic peptide (BNP) is activated early in cardiac growth and development, we evaluated whether it could serve as a target gene for GATA-binding protein-mediated induction. Upon isolating and sequencing 2.5 kilobases of the rat BNP 5'-flanking sequence (FS), a variety of putative transcriptional enhancer sites were identified, including several GATA consensus sequences (WGATAR), one of which apparently serves as the major promoter site. Primary myocardial cells were transfected with BNP/luciferase fusion genes; reporter expression was strongly induced by typical growth factors such as phorbol esters, serum, or alpha 1-adrenergic agonists, as well as by GATA-4 overexpression. Truncation analyses showed that inducibility mapped primarily to the proximal -116 base pair of the rat BNP 5'-FS, where there are two consensus GATA sites in addition to the GATA sequence at the TATA box. Point mutation analyses showed that at least one of the GATA sites was required to confer full GATA-4-inducible transcription. These results demonstrate that a proximal region of the rat BNP 5'-FS is required for growth factor- and GATA-inducible transcription, supporting the view that the BNP gene could serve as a target for GATA-binding proteins during early cardiac development.

The unusual sexual preferences of a chlamydomonas mutant may provide insight into mating-type evolution.

Chlamydomonas monoica undergoes intraclonal mating-type differentiation (homothallism). Although the species differs in this regard from the more commonly studied heterothallic C. reinhardtii, cell-cell interactions and progression of the sexual cycle are similar for many homothallic and heterothallic species of the genus. Regulation of chloroplast gene transmission by the nuclear mating-type alleles (mt+ and mt-) is another common denominator for Chlamydomonas species studied thus far. We have previously reported the use of chloroplast inheritance patterns to identify mutants of C. monoica that have lost the potential to function as the mt+ mating-type. A similar screening procedure led to the isolation of an unusual mutant, mtl-3 whose phenotype is less readily explained. Chloroplast gene transmission patterns in crosses involving mtl-3 suggest that the mtl-3 strain mates preferentially as mt+. However, normal mating efficiencies and high zygospore viability are observed in clonal culture, indicating the unbiased production of functional opposite mating-types. By construction of appropriately marked strains we have been able to show that mtl-3 mt- gametes prefer the mt+ gametes of their own strain. A model is presented which invokes unequal crossing over between highly homologous flagellar agglutinin genes to account for the unusual properties of the mtl-3 strain and for the evolution of mating barriers within the genus.

Sequence and fine specificity analysis of primary 511 anti-phosphorylcholine antibodies.

The primary antibody response of mice to phosphorylcholine (PC) is dominated by antibodies using the T15 L chain. Anti-PC antibodies using the 511 L chain are prominent only in secondary responses to PC coupled to proteins, are somatically mutated, and all have an extra amino acid at the Vh-D junction, compared with T15 antibodies. The aim of the experiments reported here was to determine if the extra junctional amino acid alone was sufficient to generate a 511 PC-binding antibody, or if somatic mutation or other junctional changes were also necessary. We also wished to determine if unmutated 511 antibodies had sufficient affinity for PC to appear in the primary response. To increase the frequency of primary 511 antibodies, we generated a series of hybridomas from M167 L chain transgenic mice immunized 4 days earlier with either Streptococcus pneumonia R36a or PC-keyhole limpet hemocyanin (KLH). We determined the relative affinity of the antibodies, and sequenced their H chain V regions. The results showed that: 1) somatic mutations are not required for 511 antibodies to bind PC; 2) primary 511 antibodies all had lower relative affinities for PC than T15 while having similar affinities to T15 for TNP-aminophenyl PC, and higher affinities for the PC analogs nitrophenyl PC and choline; 3) all antibodies had the 511-specific insertion of an extra amino acid, usually Ala, at the VhD junction, compared with T15; 4) immunization with R36a, but not PC-hemocyanin, elicited antibodies with a specific Tyr----Asp substitution in the D region, indicating Ag selection based on fine specificity differences; 5) the total length of CDR3 was conserved in most anti-PC-hemocyanin antibodies, whereas the anti-R36a antibodies predominantly had longer CDR3 sequences; and 6) there were unique substitutions in most antibodies, including significant sequence heterogeneity in the D-Jh junction. We conclude that Ag selection on the basis of affinity for PC biases the primary anti-PC response in favor of T15, and that 511 precursors with their alternative fine specificities contribute the precursors that are expanded in the secondary anti-PC-KLH responses.