Epidermal growth factor induction of c-jun expression requires ATF1 and MEF2 sites in the c-jun promoter. We find that activation of the c-jun promoter through the ATF1 site requires phosphorylation of ATF1 at serine 63. A serine 63 to alanine mutation of ATF1 acts to block epidermal growth factor (EGF) induction of a transfected c-jun gene. ATF1 can be phosphorylated by mitogen- and stress-activated protein kinase 1 (MSK1), which is activated by EGF and ERK1/2. Kinase-dead MSK1 mutants blocked EGF induction of a transfected c-jun gene suggesting that MSK1 or a similar family member is required for induced c-jun expression. Use of the MEK1 inhibitor U0126 and dominant negative MEK1 further showed that MSK1 activation and c-jun induction require the ERK pathway. In contrast, a JNK inhibitor blocked EGF induction of c-jun expression but not ATF1 phosphorylation. These results show that the two MAPK pathways, ERK and JNK, are required for EGF-induced c-jun expression and that the ERK pathway acts through downstream phosphorylation of ATF1.

FRA-1, a member of the FOS family of transcription factors, is overexpressed in a variety of human tumors, and contributes to tumor progression. In addition to mitogens, various toxicants and carcinogens persistently induce FRA-1 expression in vitro and in vivo. Although the mitogen induced expression of c-FOS is relatively well understood, it is poorly defined in the case of FRA-1. Our recent analysis of the FRA-1 promoter has shown a critical role for a TRE located at -318 in mediating the TPA-induced expression. The -379 to -283 bp promoter segment containing a critical TRE (-318), however, is insufficient for the induction of FRA-1 promoter. Here, we show that a 40-bp (-276/-237) segment, comprising a TCF binding site and the CArG box (collectively known as serum response element, SRE), and an ATF site, is also necessary for the FRA-1 induction by TPA and EGF. Interestingly, the -283 to +32 bp FRA-1 promoter fragment containing an SRE and an ATF site alone was also insufficient to confer TPA sensitivity to a reporter gene. However, in association with the -318 TRE, the SRE and ATF sites imparted a strong TPA-inducibility to the reporter. Similarly, EGF also required these motifs for the full induction of this gene. Using ChIP assays we show that, in contrast to c-Jun, SRF, Elk1, ATF1 and CREB proteins bind to SRE and ATF sites of the FRA-1 promoter, constitutively. RNAi-mediated knockdown of endogenous SRF, ELK1 and c-JUN protein expression significantly reduced TPA-stimulated FRA-1 promoter activity. Thus, a bipartite enhancer formed by an upstream TRE and the downstream SRE and ATF sites and the cognate factors is necessary and sufficient for the regulation of FRA-1 in response to mitogens.

In Schizosaccharomyces pombe, the Sty1 mitogen-activated protein kinase and the Atf1 transcription factor control transcriptional induction in response to elevated salt concentrations. Herein, we demonstrate that two repressors, Tup11 and Tup12, and the Prr1 transcription factor also function in the response to salt shock. We find that deletion of both tup genes together results in hypersensitivity to elevated cation concentrations (K(+) and Ca(2+)) and we identify cta3(+), which encodes an intracellular cation transporter, as a novel stress gene whose expression is positively controlled by the Sty1 pathway and negatively regulated by Tup repressors. The expression of cta3(+) is maintained at low levels by the Tup repressors, and relief from repression requires the Sty1, Atf1, and Prr1. Prr1 is also required for KCl-mediated induction of several other Sty1-dependent genes such as gpx1(+) and ctt1(+). Surprisingly, the KCl-mediated induction of cta3(+) expression occurs independently of Sty1 in a tup11Delta tup12Delta mutant and so the Tup repressors link induction to the Sty1 pathway. We also report that in contrast to a number of other Sty1- and Atf1-dependent genes, the expression of cta3(+) is induced only by high salt concentrations. However, in the absence of the Tup repressors this specificity is lost and a range of stresses induces cta3(+) expression.

Carbon dioxide (CO(2)) sensing and metabolism via carbonic anhydrases (CAs) play pivotal roles in survival and proliferation of pathogenic fungi infecting human hosts from natural environments due to the drastic difference in CO(2) levels. In Cryptococcus neoformans, which causes fatal fungal meningoencephalitis, the Can2 CA plays essential roles during both cellular growth in air and sexual differentiation of the pathogen. However the signaling networks downstream of Can2 are largely unknown. To address this question, the present study employed comparative transcriptome DNA microarray analysis of a C. neoformans strain in which CAN2 expression is artificially controlled by the CTR4 (copper transporter) promoter. The P(CTR4)CAN2 strain showed growth defects in a CO(2)-dependent manner when CAN2 was repressed but resumed normal growth when CAN2 was overexpressed. The Can2-dependent genes identified by the transcriptome analysis include FAS1 (fatty acid synthase 1) and GPB1 (G-protein beta subunit), supporting the roles of Can2 in fatty acid biosynthesis and sexual differentiation. Cas3, a capsular structure designer protein, was also discovered to be Can2-dependent and yet was not involved in CO(2)-mediated capsule induction. Most notably, a majority of Can2-dependent genes were environmental stress-regulated (ESR) genes. Supporting this, the CAN2 overexpression strain was hypersensitive to oxidative and genotoxic stress as well as antifungal drugs, such as polyene and azole drugs, potentially due to defective membrane integrity. Finally, an oxidative stress-responsive Atf1 transcription factor was also found to be Can2-dependent. Atf1 not only plays an important role in diverse stress responses, including thermotolerance and antifungal drug resistance, but also represses melanin and capsule production in C. neoformans. In conclusion, this study provides insights into the comprehensive signaling networks orchestrated by CA/CO(2)-sensing pathways in pathogenic fungi.

Dendritic cells (DCs) are critical for the initiation of immune responses including activation of CD8 T cells. Intracellular reactive oxygen species (ROS) levels influence DC maturation and function. Intracellular heme, a product of catabolism of heme-containing metalloproteins, is a key inducer of ROS. Intracellular heme levels are regulated by heme oxygenase-1 (HO-1), which catalyzes the degradation of heme. Heme oxygenase-1 has been implicated in regulating DC maturation; however, its role in other DC functions is unclear. Furthermore, the signaling pathways modulated by HO-1 in DCs are unknown. In this study, we demonstrate that inhibition of HO-1 activity in murine bone marrow-derived immature DCs (iDCs) resulted in DCs with raised intracellular ROS levels, a mature phenotype, impaired phagocytic and endocytic function, and increased capacity to stimulate antigen-specific CD8 T cells. Interestingly, our results reveal that the increased ROS levels following HO-1 inhibition did not underlie the changes in phenotype and functions observed in these iDCs. Importantly, we show that the p38 mitogen-activated protein kinase (p38 MAPK), cAMP-responsive element binding protein (CREB), and activating transcription factor 1 (ATF1) pathway is involved in the mediation of the phenotypic and functional changes arising from HO-1 inhibition. Furthermore, up-regulation of HO-1 activity rendered iDCs refractory to lipopolysaccharide-induced activation of p38 MAPK-CREB/ATF1 pathway and DC maturation. Finally, we demonstrate that treatment of iDC with the HO-1 substrate, heme, recapitulates the effects that result from HO-1 inhibition. Based on these results, we conclude that HO-1 regulates DC maturation and function by modulating the p38 MAPK-CREB/ATF1 signaling axis.

Soft tissue myoepithelial tumors, a recently defined entity, include benign and malignant lesions showing a considerable morphological and immunohistochemical heterogeneity. EWSR1 rearrangements are well recognized in this tumor type, and some of the partner genes have been identified. Herein we describe a soft tissue myoepithelioma arising in the pelvis with an EWSR1-ATF1 fusion, therefore extending the spectrum of partner genes of EWSR1. In addition, this case indicates that there are overlapping genetic features of myoepithelial tumors, clear cell sarcoma, angiomatoid fibrous histiocytoma, and hyalinizing clear-cell carcinoma of the salivary gland.

The specific induction of genes in response to distinct environmental stress is vital for all eukaryotes. To study the mechanisms that result in selective gene responses, we examined the role of the fission yeast Tup1 family repressors in chromatin regulation. We found that chromatin structure around a cAMP-responsive element (CRE)-like sequence in ade6-M26 that is bound by Atf1.Pcr1 transcriptional activation was altered in response to osmotic stress but not to heat and oxidative stresses. Such chromatin structure alteration occurred later than the Atf1 phosphorylation but correlated well with stress-induced transcriptional activation at ade6-M26. This chromatin structure alteration required components for the stress-activated protein kinase (SAPK) cascade and both subunits of the M26-binding CREB/ATF-type protein Atf1.Pcr1. Cation stress and glucose starvation selectively caused chromatin structure alteration around CRE-like sequences in cta3(+) and fbp1(+) promoters, respectively, in correlation with transcriptional activation. However, the tup11Delta tup12Delta double deletion mutants lost the selectivity of stress responses of chromatin structure and transcriptional regulation of cta3(+) and fbp1(+). These data indicate that the Tup1-like repressors regulate the chromatin structure to ensure the specificity of gene activation in response to particular stresses. Such a role for these proteins may serve as a paradigm for the regulation of stress response in higher eukaryotes.

Chromatin remodeling plays crucial roles in the regulation of gene expression and recombination. Transcription of the fission yeast fbp1(+) gene and recombination at the meiotic recombination hotspot ade6-M26 (M26) are both regulated by cAMP responsive element (CRE)-like sequences and the CREB/ATF-type transcription factor Atf1*Pcr1. The Tup11 and Tup12 proteins, the fission yeast counterparts of the Saccharomyces cerevisiae Tup1 corepressor, are involved in glucose repression of the fbp1(+) transcription. We have analyzed roles of the Tup1-like corepressors in chromatin regulation around the fbp1(+) promoter and the M26 hotspot. We found that the chromatin structure around two regulatory elements for fbp1(+) was remodeled under derepressed conditions in concert with the robust activation of fbp1(+) transcription. Strains with tup11delta tup12delta double deletions grown in repressed conditions exhibited the chromatin state associated with wild-type cells grown in derepressed conditions. Interestingly, deletion of rst2(+), encoding a transcription factor controlled by the cAMP-dependent kinase, alleviated the tup11delta tup12delta defects in chromatin regulation but not in transcription repression. The chromatin at the M26 site in mitotic cultures of a tup11delta tup12delta mutant resembled that of wild-type meiotic cells. These observations suggest that these fission yeast Tup1-like corepressors repress chromatin remodeling at CRE-related sequences and that Rst2 antagonizes this function.

Schizosaccharomyces pombe expresses a putative transcription factor, named Prr1, which is intriguing in the sense that it contains a bacterial type of phospho-accepting receiver domain, preceded by a mammalian heat shock factor (HSF2)-like DNA-binding domain. The receiver domain is most probably involved in an as yet unidentified histidine-to-aspartate (His-to-Asp) phosphorelay pathway in S. pombe. In this study, the structure, function, and cellular localization of Prr1 were assessed in the context of oxidative stress and His-to-Asp phosphorelay. As the most intriguing result of this study, we found that Prr1 is essential not only for the expression of genes induced by oxidative stress (e.g., ctt1+ and trr1+), but also for the expression of ste11+, which in turn is responsible for the expression of a variety of genes required for sexual development. Accordingly, Prr1-deficient cells are not only hypersensitive to oxidative stress, but also severely defective in conjugation and/or spore formation. These results suggested that the transcription factor Prr1 plays a pivotal role in an as yet unknown signal transduction pathway that is implicated in sexual differentiation. These findings are discussed with special reference to the well-characterized transcription factors Pap1 and Atf1 of S. pombe.

Cardiac-directed expression of adenylyl cyclase type VI (AC(VI)) increases stimulated cAMP production, improves heart function, and increases survival in cardiomyopathy. In contrast, pharmacological agents that increase intracellular levels of cAMP have detrimental effects on cardiac function and survival. We wondered whether effects that are independent of cAMP might be responsible for these salutary outcomes associated with AC(VI) expression. We therefore conducted a series of experiments focused on how gene transcription is influenced by AC(VI) in cultured neonatal rat cardiac myocytes, with a particular focus on genes that might influence cardiac function. We found that overexpression of AC(VI) down-regulated mRNA and protein expression of phospholamban, an inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase. We determined that the cAMP-responsive-like element in the phospholamban (PLB) promoter was critical for down-regulation by AC(VI). Overexpression of AC(VI) did not alter the expression of CREB, CREM, ATF1, ATF2, or ATF4 proteins. In contrast, overexpression of AC(VI) increased expression of ATF3 protein, a suppressor of transcription. Following AC(VI) gene transfer, when cardiac myocytes were stimulated with isoproterenol or NKH477, a water-soluble forskolin analog that directly stimulates AC, expression of ATF3 protein was increased even more, which correlated with reduced expression of PLB. We then showed that AC(VI)-induced ATF3 protein binds to the cAMP-responsive-like element on the PLB promoter and that overexpression of ATF3 in cardiac myocytes inhibits PLB promoter activity. These findings indicate that AC(VI) has effects on gene transcription that are not directly dependent on cAMP generation.

In fission yeast, knockout of the calcineurin gene resulted in hypersensitivity to Cl(-), and the overexpression of pmp1(+) encoding a dual-specificity phosphatase for Pmk1 mitogen-activated protein kinase (MAPK) or the knockout of the components of the Pmk1 pathway complemented the Cl(-) hypersensitivity of calcineurin deletion. Here, we showed that the overexpression of ptc1(+) and ptc3(+), both encoding type 2C protein phosphatase (PP2C), previously known to inactivate the Wis1-Spc1-Atf1 stress-activated MAPK signaling pathway, suppressed the Cl(-) hypersensitivity of calcineurin deletion. We also demonstrated that the mRNA levels of these two PP2Cs and pyp2(+), another negative regulator of Spc1, are dependent on Pmk1. Notably, the deletion of Atf1, but not that of Spc1, displayed hypersensitivity to the cell wall-damaging agents and also suppressed the Cl(-) hypersensitivity of calcineurin deletion, both of which are characteristic phenotypes shared by the mutation of the components of the Pmk1 MAPK pathway. Moreover, micafungin treatment induced Pmk1 hyperactivation that resulted in Atf1 hyperphosphorylation. Together, our results suggest that PP2C is involved in a negative feedback loop of the Pmk1 signaling, and results also demonstrate that Atf1 is a key component of the cell integrity signaling downstream of Pmk1 MAPK.

The ade6-M26 meiotic recombination hot spot of fission yeast is defined by a cyclic AMP-responsive element (CRE)-like heptanucleotide sequence, 5'-ATGACGT-3', which acts as a binding site for the Atf1/Pcr1 heterodimeric transcription factor required for hot spot activation. We previously demonstrated that the local chromatin around the M26 sequence motif alters to exhibit higher sensitivity to micrococcal nuclease before the initiation of meiotic recombination. In this study, we have examined whether or not such alterations in chromatin occur at natural meiotic DNA double-strand break (DSB) sites in Schizosaccharomyces pombe. At one of the most prominent DSB sites, mbs1 (meiotic break site 1), the chromatin structure has a constitutively accessible configuration at or near the DSB sites. The establishment of the open chromatin state and DSB formation are independent of the CRE-binding transcription factor, Atf1. Analysis of the chromatin configuration at CRE-dependent DSB sites revealed both differences from and similarities to mbs1. For example, the tdh1+ locus, which harbors a CRE consensus sequence near the DSB site, shows a meiotically induced open chromatin configuration, similar to ade6-M26. In contrast, the cds1+ locus is similar to mbs1 in that it exhibits a constitutive open configuration. Importantly, Atf1 is required for the open chromatin formation in both tdh1+ and cds1+. These results suggest that CRE-dependent meiotic chromatin changes are intrinsic processes related to DSB formation in fission yeast meiosis. In addition, the results suggest that the chromatin configuration in natural meiotic recombination hot spots can be classified into at least three distinct categories: (i) an Atf1-CRE-independent constitutively open chromatin configuration, (ii) an Atf1-CRE-dependent meiotically induced open chromatin configuration, and (iii) an Atf1-CRE-dependent constitutively open chromatin configuration.

Clear cell sarcoma (CCS) of tendons and aponeuroses is a deadly soft-tissue malignancy resembling melanoma, with a predilection for young adults. EWS-ATF1, the fusion product of a balanced chromosomal translocation between chromosomes 22 and 12, is considered the definitional feature of the tumor. Conditional expression of the EWS-ATF1 human cDNA in the mouse generates CCS-like tumors with 100% penetrance. Tumors, developed through varied means of initiating expression of the fusion oncogene, model human CCS morphologically, immunohistochemically, and by genome-wide expression profiling. We also demonstrate that although fusion oncogene expression in later stages of differentiation can transform mesenchymal progenitor cells and generate tumors resembling CCS generally, expression in cells retaining stem cell markers permits the full melanoma-related phenotype.

Multiple Aspergillus fumigatus isolates from a patient with two aspergillomas complicating chronic pulmonary aspergillosis were pan-azole resistant. Microsatellite typing was identical for all isolates despite major phenotypic and some growth rate differences. Three different cyp51A mutations were found (G138C, Y431C, and G434C), of which the first two were demonstrated by heterologous expression in a hypersusceptible Saccharomyces cerevisiae strain to be at least partly responsible for elevated MICs. cyp51A and cyp51B gene duplication was excluded, but increased expression of cyp51A was demonstrated in three isolates selected for additional study (7-to 13-fold increases). In the isolate with the greatest cyp51A expression, an Aft1 transposon was found inserted 370 bp upstream of the start codon of the cyp51A gene, an integration location never previously demonstrated in Aspergillus. Two transcription start sites were identified at 49 and 136 bp upstream of the start codon. The role of the Aft1 transposon, if any, in modulating cyp51A expression remains to be established. Increased mRNA expression of the transporters AfuMDR1 and AfuMDR4 also was demonstrated in some isolates, which could contribute to azole resistance or simply represent a stress response. The diversity of confirmed and possible azole resistance mechanisms demonstrated in a single series of isogenic isolates is remarkable, indicating the ability of A. fumigatus to adapt in the clinical setting.

Clear cell sarcoma is an aggressive malignancy occurring most commonly in the distal extremities of young adults, characterized by t(12;22)(q13;q12) creating the chimeric fusion oncoprotein EWS-ATF1. We assessed growth inhibition and differentiation effects of histone deacetylase inhibitors MS-275 and romidepsin (depsipeptide, FK228) on clear cell sarcoma cells and evaluated drug sensitivity among related translocation-associated sarcomas and other cell models. Three clear cell sarcoma cell lines, seven other sarcomas, six nonsarcoma malignant cell lines, and two nonneoplastic mesenchymal cell models were treated with MS-275 or romidepsin. Growth inhibition was assayed by monolayer 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Induction of cell cycle arrest and apoptosis were assessed by propidium iodide/Annexin V flow cytometry in monolayer and spheroid cultures and by immunoblotting analysis. Expression levels of key genes involved in mesenchymal differentiation and of EWS-ATF1 were measured by quantitative real-time PCR in clear cell sarcoma cells treated with histone deacetylase inhibitors. MS-275 and romidepsin inhibited growth in clear cell sarcoma cells by inducing cell cycle arrest and apoptosis in a time- and dose-dependent manner. Sarcomas showed greater sensitivity than other tumor types, with clear cell sarcomas most sensitive of all, whereas nonmalignant mesenchymal cells were highly resistant. MS-275 at 1 micromol/L and romidepsin at 1 nmol/L induced histone H3 acetylation, cell cycle arrest, apoptosis, and differentiation in clear cell sarcoma cells within 24 hours. Histone deacetylase inhibitors increased expression of SOX9, MYOD1, and PPARG and decreased EWS-ATF1 expression in clear cell sarcoma cells. Histone deacetylase inhibitors show promising preclinical activity in multiple clear cell sarcoma models.

Clear cell sarcoma of soft tissue (CCSST), also known as malignant melanoma of soft parts, represents a rare lesion of the musculoskeletal system usually affecting adolescents and young adults. CCSST is typified by a chromosomal t(12;22)(q13;q12) translocation resulting in a fusion between the Ewing sarcoma gene (EWSR1) and activating transcription factor 1 (ATF1), of which the activity in nontransformed cells is regulated by cyclic AMP. Our aim was to identify critical differentially expressed genes in CCSST tumor cells in comparison with other solid tumors affecting children and young adults to better understand signaling pathways regulating specific features of the development and progression of this tumor entity. We applied Affymetrix Human Genome U95Av2 oligonucleotide microarrays representing approximately 12,000 genes to generate the expression profiles of the CCSST cell lines GG-62, DTC-1, KAO, MST2, MST3, and Su-CC-S1 in comparison with 8 neuroblastoma, 7 Ewing tumor, and 6 osteosarcoma cell lines. Subsequent hierarchical clustering of microarray data clearly separated all four of the tumor types from each other and identified differentially expressed transcripts, which are characteristically up-regulated in CCSST. Statistical analysis revealed a group of 331 probe sets, representing approximately 300 significant (P < 0.001) differentially regulated genes, which clearly discriminated between the CCSST and other tumor samples. Besides genes that were already known to be highly expressed in CCSST, like S100A11 (S100 protein) or MITF (microphthalmia-associated transcription factor), this group shows an obvious portion of genes that are involved in cyclic AMP response or regulation, in pigmentation processes, or in neuronal structure and signaling. Comparison with other expression profile analyses on neuroectodermal childhood tumors confirms the high robustness of this strategy to characterize tumor entities based on their gene expression. We found the avian erythroblastic leukemia viral oncogene homologue 3 (ERBB3) to be one of the most dramatically up-regulated genes in CCSST. Quantitative real-time PCR and Northern blot analysis verified the mRNA abundance and confirmed the absence of the inhibitory transcript variant of this gene. The protein product of the member of the epidermal growth factor receptor family ERBB3 could be shown to be highly present in all of the CCSST cell lines investigated, as well as in 18 of 20 primary tumor biopsies. In conclusion, our data demonstrate new aspects of the phenotype and the biological behavior of CCSST and reveal ERBB3 to be a useful diagnostic marker.

Yeast Sin1 binds to the Sty1 kinase, a member of the stress-activated kinases (SAPKs), and is required for stress-induced phosphorylation and activation of the transcription factor Atf1, a homolog of the vertebrate-activating transcription factor-2 (ATF-2). Here we report that mammalian Sin1 plays an important role in the SAPK signaling pathway by binding to both ATF-2 and p38. In response to stress, ATF-2, a member of the ATF/cAMP response element-binding protein family, is phosphorylated by p38/Jun NH2-terminal protein kinase and activates the transcription of apoptosis-related genes. In contrast, in response to serum stimulation, ATF-2 is phosphorylated via the Ras effector pathway and leads to the induction of growth-related genes. We found that Sin1 binds directly to both ATF-2 and p38. Sin1 over-expression enhanced osmotic stress-induced phosphorylation of ATF-2 and ATF-2-mediated transcription, whereas knockdown of Sin1 expression by siRNA suppressed these responses. Moreover, a reduction in Sin1 expression suppressed osmotic stress-induced apoptosis and the expression of Gadd45beta, one of the ATF-2 target genes that is correlated with apoptosis. Decreased Sin1 expression, however, did not affect the serum stimulation-induced phosphorylation of ATF-2. Sin1 may contribute to ATF-2 signaling specificity by acting as a nuclear scaffold.

The AP-1 and ATF/CREB families of eukaryotic transcription factors are dimeric DNA-binding proteins that contain the bZIP structural motif. The AP-1 and ATF/CREB proteins are structurally related and recognize identical half-sites (TGAC), but they differ in their requirements for half-site spacing. AP-1 proteins such as yeast GCN4 preferentially bind to sequences with overlapping half-sites, whereas ATF/CREB proteins bind exclusively to sequences with adjacent half-sites. Here we investigate the distinctions between AP-1 and ATF/CREB proteins by determining the DNA-binding properties of mutant and hybrid proteins. First, analysis of GCN4-ATF1 hybrid proteins indicates that a short surface spanning the basic and fork regions of the bZIP domain is the major determinant of half-site spacing. Replacement of two GCN4 residues on this surface (Ala244 and Leu247) by their ATF1 counterparts largely converts GCN4 into a protein with ATF/CREB specificity. Secondly, analysis of a Fos derivative containing the GCN4 leucine zipper indicates that Fos represents a novel intermediate between AP-1 and ATF/CREB proteins. Thirdly, we examine the effects of mutations in the invariant arginine residue of GCN4 (Arg243) that contacts the central base pair(s) of the target sites. While most mutations abolish DNA binding, substitution of a histidine residue results in a GCN4 derivative with ATF/CREB binding specificity. These results suggest that the AP-1 and ATF/CREB proteins differ in positioning a short surface that includes the invariant arginine and that AP-1 proteins may represent a subclass (and perhaps evolutionary offshoot) of ATF/CREB proteins that can tolerate overlapping half-sites.

int-6 is one of the frequent integration sites for mouse mammary tumor viruses. Although its product is the e-subunit of translation initiation factor eIF3, other evidence indicates that it interacts with proteasomes or other proteins to regulate protein stability. Here we report that the fission yeast int6(+) is required for overcoming stress imposed by histidine starvation, using the drug 3-aminotriazole (3AT). Microarray and complementary Northern studies using wild-type, int6Delta or gcn2Delta mutants indicate that 3AT-treated wild-type yeast induces core environmental stress response (CESR) genes in addition to typical general amino acid control (GAAC) genes whose transcription depends on the eIF2 kinase, Gcn2. In agreement with this, Sty1 MAPK and its target transcription factor Atf1, which signal the CESR, are required for overcoming 3AT-induced starvation. We find that Int6 is required for maintaining the basal level of Atf1 and for rapid transcriptional activation of the CESR on 3AT-insult. Pulse labeling experiments indicate that int6Delta significantly slows down de novo protein synthesis. Moreover, Atf1 protein half-life was reduced in int6Delta cells. These effects would account for the compromised Atf1 activity on 3AT-induced stress. Thus, the robust protein synthesis promoted by intact eIF3 appears to be a part of the requisites for sound Sty1 MAPK-dependent signaling governed by the activity of the Atf1 transcription factor.

Chromosomal translocations that fuse the N-terminal region of the Ewings sarcoma oncogene (EWS) to the C-terminal region (including the DNA-binding domain) of the cellular transcription factor ATF1 are associated with a tumour type termed malignant melanoma of soft parts (MMSP). It is envisioned that transformation by the EWS/ATF1 fusion protein results from aberrant transcriptional regulation of genes that are normally regulated by ATF1. To examine this hypothesis we have expressed exogenous EWS-ATF1 in JEG3 cells and tested its ability to activate several promoters that contain binding sites for ATF1. We show that EWS-ATF1 is a strong constitutive activator of some promoters tested but represses others. Significantly, the ability of particular promoters to be activated by EWS/ATF1 in JEG3 cells correlates with promoter activity in two MMSP-derived cell lines (SU-CCS-1 and DTC1). Our results therefore provide evidence that endogenous EWS/ATF1 can de-regulate transcription and that this capacity may contribute to transformation in MMSP.

Certain HMB-45-positive epithelioid cell tumors have recently been categorized under a unified concept: perivascular epithelioid cell tumor (PEComa). In this report, we describe ajejunal PEComa arising in a 32-year-old woman without other tumors or stigmata of tuberous sclerosis. The tumor consisted of nests of epithelioid cells with clear to granular eosinophilic cytoplasm. The nests were separated by thin fibrovascular septa. The tumor cells were positive for HMB-45 and progesterone receptor, and negative for cytokeratin, epithelial membrane antigen, vimentin, desmin, alpha-smooth muscle actin and CD34. RT-PCR analysis failed to reveal fusion transcript ETW/ATF1, which is characteristic of clear cell sarcoma of the soft parts. She developed a recurrent tumor at the pelvic wall and the left ovary at 13 and 25 months after the first operation, respectively. Each tumor was resected surgically, and no additional therapy was performed. We think the tumor of this case is a malignant form of PEComa because of the clinical history of multiple recurrences and the size of the primary tumor. Our case underscores that to make a correct diagnosis, clinical information and immunohistochemical examination are essential.

The receptor of activated C kinase (RACK1) is a protein highly conserved among eukaryotes. In mammalian cells, RACK1 functions as an adaptor to favor protein kinase C (PKC)-mediated phosphorylation and subsequent activation of c-Jun NH(2)-terminal kinase mitogen-activated protein kinase. Cpc2, the RACK1 orthologue in the fission yeast Schizosaccharomyces pombe, is involved in the control of G2/M transition and interacts with Pck2, a PKC-type protein member of the cell integrity Pmk1 mitogen-activated protein kinase (MAPK) pathway. Both RACK1 and Cpc2 are structural components of the 40S ribosomal subunit, and recent data suggest that they might be involved in the control of translation. In this work, we present data supporting that Cpc2 negatively regulates the cell integrity transduction pathway by favoring translation of the tyrosine-phosphatases Pyp1 and Pyp2 that deactivate Pmk1. In addition, Cpc2 positively regulates the synthesis of the stress-responsive transcription factor Atf1 and the cytoplasmic catalase, a detoxificant enzyme induced by treatment with hydrogen peroxide. These results provide for the first time strong evidence that the RACK1-type Cpc2 protein controls from the ribosome the extent of the activation of MAPK cascades, the cellular defense against oxidative stress, and the progression of the cell cycle by regulating positively the translation of specific gene products involved in key biological processes.

Proinsulin C-peptide has been reported to have some biological activities and to be possibly involved in the development of diabetic microangiopathy. In the present study, we examined the effects of C-peptide on the mitogen-activated protein kinase pathway in LEII mouse lung capillary endothelial cells. Stimulation of the cells with C-peptide increased both p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK1/2) activities and activity-related site-specific phosphorylation of the respective kinases in a concentration-dependent manner, but failed to activate c-Jun N-terminal kinase. Stimulation of the cells with C-peptide also induced site-specific phosphorylation of cAMP response element (CRE)-binding protein (CREB)/activating transcription factor 1 (ATF1), and thereby binding of these transcription factors to CRE. Among three CREB kinases tested, phosphorylation of mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2) was induced after stimulation with C-peptide. The phosphorylation of CREB, ATF1 and MAPKAP-K2 were inhibited by SB203580, a p38MAPK inhibitor, but not by PD98059, an ERK kinase inhibitor. These results indicate that C-peptide activates p38MAPK followed by MAPKAP-K2 to enhance DNA-CREB/ATF1 interactions.

Stress-induced alterations in neuronal plasticity and in hippocampal functions have been suggested to be involved in the development of mood disorders. In this context, we investigated in the hippocampus the activation of intracellular signaling cascades, the expression of epigenetic markers and plasticity-related genes in a mouse model of stress-induced hyperactivity and of mixed affective disorders. We also determined whether the antidepressant drug agomelatine, a MT1/MT2 melatonergic receptor agonist/5-HT2C receptor antagonist, could prevent some neurobiological and behavioral alterations produced by stress. C57BL/6J mice, exposed for 3 weeks to daily unpredictable socio-environmental stressors of mild intensity, were treated during the whole procedure with agomelatine (50 mg kg(-1) per day, intraperitoneal). Stressed mice displayed robust increases in emotional arousal, vigilance and motor activity, together with a reward deficit and a reduction in anxiety-like behavior. Neurobiological investigations showed an increased phosphorylation of intracellular signaling proteins, including Atf1, Creb and p38, in the hippocampus of stressed mice. Decreased hippocampal level of the repressive epigenetic marks HDAC2 and H3K9me2, as well as increased level of the permissive mark H3K9/14ac suggested that chronic mild stress was associated with increased gene transcription, and clear-cut evidence was further indicated by changes in neuroplasticity-related genes, including Arc, Bcl2, Bdnf, Gdnf, Igf1 and Neurod1. Together with other findings, the present data suggest that chronic ultra-mild stress can model the hyperactivity or psychomotor agitation, as well as the mixed affective behaviors often observed during the manic state of bipolar disorder patients. Interestingly, agomelatine could normalize both the behavioral and the molecular alterations induced by stress, providing further insights into the mechanism of action of this new generation antidepressant drug.