InteractiveFly: GeneBrief

Activating transcription factor-2: Biological Overview | References

BIOLOGICAL OVERVIEW

ATF-2 is a member of the ATF/CREB family of transcription factors that is activated by stress-activated protein kinases such as p38. To analyze the physiological role of Drosophila ATF-2 (dATF-2), dATF-2 knockdown flies were generated using RNA interference. Reduced dATF-2 in the fat body, the fly equivalent of the mammalian liver and adipose tissue, decreased survival under starvation conditions. This was due to smaller triglyceride reserves of dATF-2 knockdown flies than control flies. Among multiple genes that control triglyceride levels, expression of the Drosophila PEPCK (dPEPCK) gene was strikingly reduced in dATF-2 knockdown flies. PEPCK is a key enzyme for both gluconeogenesis and glyceroneogenesis, which is a pathway required for triglyceride synthesis via glycerol-3-phosphate. Although the blood sugar level in dATF-2 knockdown flies was almost same as that in control flies, the activity of glyceroneogenesis was reduced in the fat bodies of dATF-2 knockdown flies. Thus, reduced glyceroneogenesis may at least partly contribute to decreased triglyceride stores in the dATF-2 knockdown flies. Furthermore dATF-2 was shown to positively regulat dPEPCK gene transcription via several CRE half-sites in the PEPCK promoter. Thus, dATF-2 is critical for regulation of fat metabolism (Okamura, 2007).

ATF-2 (activating transcription factor-2) is a member of the ATF/CREB (CRE-binding protein) transcription factor family that has a bZIP-type DNA-binding domain. ATF-2 can form either homodimers or heterodimers with c-Jun: the dimers subsequently bind to the cyclic AMP response element (CRE: 5'-TGACGTCA-3') and positively regulate transcription. Stress-activated protein kinases (SAPKs) such as p38 and JNK (c-Jun N-terminal protein kinase) phosphorylate ATF-2 at Thr-69 and Thr-71 close to the N-terminal transcriptional activation domain containing the zinc finger domain and thereby enhance its trans-activating capacity. Activation of ATF-2 by p38/JNK is thought to play a role in apoptosis. ATF-2 is also activated by insulin, epidermal growth factor, and serum via a two-step mechanism involving two distinct Ras effector pathways. A group of ATF-2 target genes have been identified that are involved in multiple biological phenomena. The target genes of c-Jun/ATF-2 heterodimers, which are implicated in growth control, include c-jun itself and interferon-β. The platelet-derived growth factor receptor α gene, which is critical for proliferation of cytotrophoblasts, is an ATF-2 target and its expression level is decreased in the placenta of Atf-2 mutant mice (Okamura, 2007 and references therein).

ATF-2 is ubiquitously expressed and is found in liver and white adipose tissue (WAT), which are critical organs for metabolic regulation. This expression profile of ATF-2 indicates the possibility that ATF-2 plays an important role in metabolic regulation. In fact, ATF-2 activates transcription of the phosphoenolpyruvate carboxykinase-cytosolic (PEPCK-C) gene in hepatoma cells by directly binding to the CRE in its promoter via the p38 pathway (Cheong, 1998). It was also suggested that all-trans-retinoic acid activates the p38 pathway leading to phosphorylation and activation of ATF-2, thereby enhancing PEPCK gene transcription (Lee, 2002). PEPCK catalyzes the first committed step in hepatic gluconeogenesis and is a rate-limiting enzyme of gluconeogenesis. Recently, PEPCK has been also shown to play a key role in lipid homeostasis via glyceroneogenesis in WAT (Reshef, 2003). Glyceroneogenesis is defined as the de novo synthesis of glycerol-3-phosphate from pyruvate, lactate, or certain amino acids. However, it is difficult to examine in a whole animal system whether ATF-2 is critical for metabolic regulation via regulating PEPCK expression, since Atf-2 null mutant mice die immediately after birth due to a respiration defect (Maekawa, 1999). Furthermore, no tissue-specific Atf-2 knockdown mice are available at present. Thus, the role of ATF-2 in metabolic regulation still remains elusive (Okamura, 2007 and references therein).

Because Drosophila has a low degree of gene redundancy and therefore fewer related genes than mammals, it is sometimes advantageous to analyze the Drosophila homologue of a mammalian gene of interest. Furthermore, in Drosophila the expression of double-stranded RNA (dsRNA) corresponding to a part of the gene can down-regulate the gene's mRNA by RNA interference. Furthermore the Drosophila RNAi system enables the tissue-specific knockdown by using Gal4/UAS system. To elucidate the physiological role of ATF-2, the Drosophila ATF-2 homologue (dATF-2; Sano, 2005) was identified. Like mammalian ATF-2, dATF-2 has the bZIP-type DNA-binding domain and the p38 phosphorylation sites in its C- and N-terminal regions, respectively. dATF-2 binds to the cAMP response element as a homodimer or a heterodimer with Drosophila Jun and activates transcription. Drosophila p38, but not Drosophila JNK, phosphorylates dATF-2, and enhances dATF-2-dependent transcription (Okamura, 2007).

This study demonstrates that reduction of dATF-2 function in the fat body by transgenic RNAi decreases survival under dietary restriction and triglyceride as stored energy. These phenotypes are correlated with the decreased levels of dPEPCK gene transcription in the fat body, which is positively regulated by dATF-2. Thus, dATF-2 plays a critical role in regulation of fat metabolism, especially triglyceride synthesis (Okamura, 2007).

Some members of ATF/CREB family and SAPK family have been demonstrated to regulate metabolism. Using the transgenic mice expressing dominant negative CREB, CREB has been shown to control hepatic lipid metabolism and gluconeogenesis in response to a series of hormonal cues. In Drosophila cultured cells, box-B-binding factor-2 (BBF-2), a member of ATF/CREB family, binds specifically to the fat body-specific regulator element to activate transcription (Abel, 1992). Obesity increases total JNK activity and JNK1-deficient mice exhibit reduced adiposity and improved insulin sensitivity (Hirosumi, 2002). p38 mediates the free fatty acid-induced transcription of key gluconeogenic genes, including PEPCK (Collins, 2006). Furthermore, mice lacking MKP-1, which is a member of MAPK phosphatase and inactivates p38, are resistant to diet-induced obesity due to energy expenditure (Wu, 2006; Okamura, 2007 and references therein).

ATF-2 was also suggested to regulate metabolism. ATF-2 activated by p38 stimulates transcription of PTEN gene, leading to inhibition of insulin signaling (Shen, 2006). ATF-2 also activates transcription of PEPCK-C gene (Cheong, 1998). However, these data were obtained using cultured cells, and the role of ATF-2 in the regulation of metabolism remains elusive. This is partly due to a lack of appropriate model system to study the role of ATF-2 in a whole animal system. In this study, fat body-specific dATF-2 knockdown flies were generated, in which the dATF-2 mRNA level was reduced to ~40% of the wild-type level. dATF-2 knockdown flies were not lethal and exhibited a reduced triglyceride storage compared with control flies. Thus, dATF-2 knockdown flies can be thought as a good model suitable to study the role of ATF-2 in metabolism (Okamura, 2007).

Fat body and muscle are critical organs for Drosophila metabolism. Flies store energy as triglyceride in fat body, which is thought to resemble to mammalian WAT. In addition, the fat body functions like mammalian liver and has multiple enzymes that are required for glycolysis and lipid synthesis. Reduced dATF-2 activity in the fat body resulted in impaired survival under starvation condition, whereas dATF-2 down-regulation in muscle did not. The fat body-specific dATF-2 knockdown flies had a reduced reserve of triglyceride compared with control flies, suggesting that impaired survival of dATF-2 knockdown flies may be due to reduced triglyceride in the fat body. Further, dATF-2 knockdown flies did not affect either the hemolymph trehalose level or expenditure rate of triglyceride during starvation condition. Thus, dATF-2 may function in regulation of triglyceride store in the fat body by affecting the synthesis of triglyceride, but not its consumption (Okamura, 2007).

In the fat body, many enzymes that control triglyceride level are expressed. Among these enzymes, PEPCK gene expression was strikingly reduced in the fat body of dATF-2 knockdown flies. Mammalian ATF-2 was previously reported to regulate transcription of PEPCK gene in hepatoma cells (Cheong, 1998; Lee, 2002). PEPCK is a rate-limiting enzyme for gluconeogenesis in the mammalian liver, and the Drosophila fat body is an organ that has a function similar to both mammalian liver and WAT. Therefore, reduced dPEPCK activity in the fat body would be expected to decrease the blood sugar trehalose level. However, hemolymph trehalose levels in dATF-2 knockdown flies were similar to that of control flies. This may be due to the presence of multiple backup systems to keep the blood sugar levels steady because it is quite critical for the organism. In contrast to trehalose, the level of stored triglyceride in the fat bodies of dATF-2 knockdown flies was reduced compared with control flies. These data suggest that stored triglyceride may convert to trehalose to maintain blood sugar level even under normal condition. The results suggest that this was due to reduced activity of glyceronoegenesis, although a decrease in some other enzyme levels such as AcCoAS, which are involved in fatty acid synthesis, could also contribute. These results suggest that a 60% reduction of dATF-2 mRNA reduces both glyceroneogenesis and triglyceride stores (Okamura, 2007).

Although glyceroneogenesis was originally reported more than 35 years ago, it was not researched much until recently, when it has been the focus of many studies. The original discovery of glyceroneogenesis was that cytosolic PEPCK, which catalyzes the first step of hepatic and renal gluconeogenesis, is present in adipocytes. Mammalian adipose tissues do not have the gluconeogenic activity because adipocytes lack the terminal two enzymes of the pathway. This led to a finding that PEPCK converts gluconeogenic precursors such as pyruvate into glycerol-3-phosphate, the glycerol backbone of triacylglycerol, the major storage form of fat. The pathway is now described as glyceroneogenesis. Some reports described the important role of glyceroneogenesis to control the fat store. For instance, in fasting pregnant women, up to 60% of plasma glyceride-glycerol (the glycerol portion of triglyceride) was generated by glyceroneogenesis, primarily in the liver (Kalhan, 2001). Similarly, more than 80% of the glyceride-glycerol in epididymal fat of rats fed a high-protein diet (Botion, 1998) is produced by adipocyte glyceroneogenesis (Okamura, 2007).

In mammal, PEPCK is known to regulate glyceroneogenesis in addition to gluconeogenesis. The important role of PEPCK in glyceroneogenesis was demonstrated using the mice in which PEPCK was down- or up-regulated in WAT. Mice in which the PPARγ-binding site in the PEPCK-C gene promoter exhibited reduced adipose tissue size and fat content (Olswang, 2002). Conversely, transgenic mice expressing PEPCK in adipose tissue increased adipocyte size and fat mass (Franckhauser, 2002). Consistent with these reports, it was demonstrated that reduced triglyceride levels are correlated with decreased glyceroneogenesis and dPEPCK gene transcription in dATF-2 knockdown flies. Therefore, it is likely that a 60% decrease in dATF-2 mRNA reduced triglyceride levels, at least partly by affecting glyceroneogenesis via dPEPCK gene transcription (Okamura, 2007).

Glyceronegenesis is thought to occur during fasting. However, adipose tissue-specific PEPCK knockdown mice exhibited lower triglyceride levels not only under fasted condition but also under fed condition (Olswang, 2002). This observation is consistent with the current results using dATF-2 knockdown flies. The fat bodies of dATF-2 knockdown flies fed under normal condition exhibited reduced activity of glyceroneogenesis compared with the control flies. These results suggest that PEPCK contributes to glyceroneogenesis not only under fasted condition but also normal fed condition. Important role of PEPCK in glyceroneogenesis was also demonstrated by the observations that thiazolidinediones, the antidiabetic drug, reduces serum fatty acid levels by enhancing glyceroneogenesis by activating PEPCK gene transcription (Tordjman, 2003). If this result can be extended to mammals, ATF-2 may be a useful target for antidiabetic drugs like thiazolidinediones, although the role of ATF-2 in WAT has not been intensely studied (Okamura, 2007).

This study has demonstrated that dATF-2 activates dPEPCK gene transcription in part via the CRE half-sites in the dPEPCK promoter. The p38 inhibitor, SB203580, suppressed the dATF-2–dependent activation of the dPEPCK promoter, suggesting that the dp38 signal positively regulates dPEPCK transcription in the fat body. These results are consistent with the observation in mammals that ATF-2 directly binds to and activates the PEPCK promoter through the p38 pathway (Cheong, 1998). C/EBP was also reported to control PEPCK gene transcription in liver (Croniger, 1998) and can form a heterodimer with ATF-2, which binds to an asymmetric sequence composed of one consensus half-site for each monomer (Shuman, 1997). Therefore, ATF-2 could regulate PEPCK gene transcription together with other factors, such as C/EBP, which forms a heterodimer with ATF-2. If dp38 is required for dATF-2–dependent activation of dPEPCK transcription, dp38 would have to be constitutively activated in the fat body. In fact, p38 was reported to be constitutively active in the mammalian liver, which may be a result of metabolic oxidative stress (Mendelson, 1996). Retinoic acid was also shown to activate the p38 pathway leading to ATF-2–dependent activation of PEPCK gene transcription (Lee, 2002). Furthermore, dp38 mutants were shown to be more sensitive to starvation than wild-type flies (Craig, 2004). If the p38 signaling pathway is important for triglyceride stores via glyceroneogensis, this pathway may be a useful target for antidiabetic drugs, because various inhibitors for the kinases in this pathway have already been developed (Okamura, 2007).

The Drosophila MAPK p38c regulates oxidative stress and lipid homeostasis in the intestine

The p38 mitogen-activated protein (MAP) kinase signaling cassette has been implicated in stress and immunity in evolutionarily diverse species. In response to a wide variety of physical, chemical and biological stresses p38 kinases phosphorylate various substrates, transcription factors of the ATF family and other protein kinases, regulating cellular adaptation to stress. The Drosophila genome encodes three p38 kinases named p38a, p38b and p38c. This study analyzed the role of p38c in the Drosophila intestine. The p38c gene is expressed in the midgut and upregulated upon intestinal infection. p38c mutant flies are more resistant to infection with the lethal pathogen Pseudomonas entomophila but are more susceptible to the non-pathogenic bacterium Erwinia carotovora. This phenotype was linked to a lower production of Reactive Oxygen Species (ROS) in the gut of p38c mutants, whereby the transcription of the ROS-producing enzyme Dual oxidase (Duox) is reduced in p38c mutant flies. This genetic analysis shows that p38c functions in a pathway with Mekk1 and Licorne (Mkk3) to induce the phosphorylation of Atf-2, a transcription factor that controls Duox expression. Interestingly, p38c deficient flies accumulate lipids in the intestine while expressing higher levels of antimicrobial peptide and metabolic genes. The role of p38c in lipid metabolism is mediated by the Atf3 transcription factor. This observation suggests that p38c and Atf3 function in a common pathway in the intestine to regulate lipid metabolism and immune homeostasis. Collectively, this study demonstrates that p38c plays a central role in the intestine of Drosophila. It also reveals that many roles initially attributed to p38a are in fact mediated by p38c (Chakrabarti, 2014. PubMed ID: PubMed).

Drosophila activating transcription factor-2 is involved in stress response via activation by p38, but not c-Jun NH₂-terminal kinase

Activating transcription factor (ATF)-2 is a member of the ATF/cAMP response element-binding protein family of transcription factors, and its trans-activating capacity is enhanced by stress-activated protein kinases such as c-Jun NH₂-terminal kinase (JNK) and p38. However, little is known about the in vivo roles played by ATF-2. Identified here is the Drosophila homologue of ATF-2 (dATF-2) consisting of 381 amino acids. In response to UV irradiation and osmotic stress, Drosophila p38 (dp38), but not JNK, phosphorylates dATF-2 and enhances dATF-2-dependent transcription. Consistent with this, injection of dATF-2 double-stranded RNA (dsRNA) into embryos did not induce the dorsal closure defects that are commonly observed in the Drosophila JNK mutant. Furthermore, expression of the dominant-negative dp38 enhanced the aberrant wing phenotype caused by expression of a dominant-negative dATF-2. Similar genetic interactions between dATF-2 and the dMEKK1-dp38 signaling pathway also were observed in the osmotic stress-induced lethality of embryos. Loss of dATF-2 in Drosophila S2 cells by using dsRNA abrogated the induction of 40% of the osmotic stress-induced genes, including multiple immune response-related genes. This indicates that dATF-2 is a major transcriptional factor in stress-induced transcription. Thus, dATF-2 is critical for the p38-mediated stress response (Sano, 2005).

The activating transcription factor/cAMP response element-binding protein (ATF/CREB) family of proteins bears a DNA-binding domain consisting of a cluster of basic amino acids and a leucine zipper that together form the so-called b-ZIP structure. These proteins can form homodimers or heterodimers by binding via their leucine zipper motifs, after which they can bind to the cyclical AMP response element (CRE: 5'-TGACGTCA-3') via their basic region. The two major subgroups of the ATF/CREB family proteins are CREB and ATF-2. The CREB subgroup includes CREB and cAMP response element modulator (CREM), whereas the ATF-2 subgroup contains ATF-2, ATFa (recently also called ATF-7), and CRE-BPa. When the Ser-133 residue of CREB is phosphorylated by cAMP-dependent protein kinase, CREB can bind to the transcriptional coactivator CREB-binding protein (CBP), which greatly stimulates the trans-activating capacity of CREB. The trans-activating capacity of ATF-2, on the other hand, is enhanced by the phosphorylation of its Thr-69 and Thr-71 residue by stress-activated protein kinases (SAPKs) such as c-Jun NH₂-terminal kinase (JNK) and p38 (Gupta, 1995; Livingstone, 1995; van Dam, 1995). SAPKs are activated by various extracellular stress such as UV, osmotic stress, and inflammatory cytokines. All three members of the ATF-2 subgroup bear the trans-activation domain in their N-terminal region: this domain consists of two subdomains, namely, the N-terminal subdomain containing the well known zinc finger motif and the C-terminal subdomain containing the SAPK phosphorylation sites. The latter subdomain has a highly flexible and disordered structure. Although the coactivator CBP binds to the protein surface of b-ZIP domain of ATF-2 (Sano, 1998), the cofactor that binds to the N-terminal activation domain of ATF-2 remains unknown (Sano, 2005).

The physiological roles played by ATF-2 have been analyzed by using mutant mice. Null Atf-2 mutant mice die shortly after birth and display symptoms of severe respiratory distress and have lungs filled with meconium (Maekawa, 1999). In the mutant embryos, hypoxia occurs, which may lead to strong gasping respiration with the consequent aspiration of the amniotic fluid containing meconium. This is due to the impaired development of cytotrophoblast cells in the placenta that in turn is caused by decreased levels of expression of the platelet-derived growth factor receptor alpha. In addition, another Atf-2 mutant mouse, which expresses only a fragment of ATF-2, exhibits lowered postnatal viability and growth, a defect in endochondrial ossification, and reduced numbers of cerebellar Purkinje cells (Reimold, 1996). However, the physiological roles played by the other ATF-2 family proteins remain unknown (Sano, 2005).

In Drosophila, three members of the mitogen-activated protein kinase (MAPK) protein family have been identified: Rolled (Erk homologue), dJNK (JNK homologue, also called Basket), and dp38a and dp38b (p38 homologue). Rolled mediates various receptor tyrosine kinase signals in the process of tracheal elaboration, cell proliferation, mesodermal patterning, R7 photoreceptor cell differentiation, and differentiation of terminal embryonic structures. In contrast, the pathway containing Hemipterous (Hep; MAPK kinase [MAPKK] homologue), dJNK, and Drosophila Jun (dJun) is involved in dorsal closure during embryo development. All mutants of this pathway exhibit the dorsal open phenotype and a decreased level of the expression of Decapentaplegic (Dpp), a secretory ligand belonging to the transforming growth factor (TGF)-β superfamily, in leading edge cells. With regard to the dp38s, they are phosphorylated by various stresses, including UV, lipopolysaccharide (LPS), and osmotic stress. The phenotype resulting from the ectopic expression of the dominant negative (DN) dp38b in the wing imaginal disc indicates that dp38b functions downstream of thickvein (Tkv), a type I receptor of the Dpp ligand, in wing morphogenesis (Sano, 2005).

To determine the in vivo function of ATF-2, the Drosophila ATF-2 homologue (dATF-2) has been identified and characterized. dATF-2 is directly phosphorylated by dp38b but not by dJNK. Moreover, genetic analyses indicated that dATF-2 acts in the dp38 signaling pathway. In addition, DNA array analysis demonstrated that dATF-2 is a major transcriptional activator of osmotic stress-inducible genes (Sano, 2005).

The amino acid sequences of the b-Zip domain and the region containing the p38/JNK phosphorylation sites of mammalian ATF-2 are well conserved in dATF-2. However, dATF-2 lacks the N-terminal zinc finger domain that is conserved in the three members of the mammalian ATF-2 family (ATF-2, CRE-BPa, and ATF-a). The N-terminal zinc finger motif and the adjacent region that contains the p38/JNK phosphorylation sites in the mammalian ATF-2 together act as the transcriptional activation domain (Matsuda, 1991). Therefore, the mediators that regulate the transcriptional activation of mammalian ATF-2 and dATF-2 may have different characteristics (Sano, 2005).

Extracellular stress such as UV or osmotic stress induces the dp38-induced phosphorylation of dATF-2 at Thr-59 and Thr-61 and this increases the trans-activation capacity of dATF-2. Although mammalian ATF-2 is well known to be phosphorylated not only by p38 but also by JNK, this study found that dJNK neither directly phosphorylated dATF-2 nor enhanced dATF-2-dependent transcription. Furthermore, transgenic embryos expressing DN-dATF-2 or dATF-2 dsRNA did not clearly reveal the dorsal-open phenotype that is common to the Hep, Bsk, dJun, and dFos mutants. The entire amino acid sequence of JNK1 shares 65% identity with dJNK, and the ~50-amino acid stretch within the N-terminal domain of mammalian ATF-2 that contains the phosphorylation sites is also well conserved in dATF-2 (59% identity). Therefore, it is surprising that dJNK cannot phosphorylate dATF-2, unlike what is observed for mammalian JNK and ATF-2. Furthermore, it was found that although dATF-2 is phosphorylated only by dp38, dJun is phosphorylated by both dp38 and dJun. In contrast, mammalian ATF-2 is phosphorylated by both p38 and JNK, whereas Jun is phosphorylated only by JNK. It is worth noting that ATFa is not phosphorylated by JNK (De Graeve, 1999). This may raise the possibility that a regulation mechanism of dATF-2 resembles to that of ATFa, and that an ancestral ATF-2/CRE-BPa gene were derived from a duplicated ATFa-like gene. The relationship between SAPKs and transcription factors in Drosophila and mammals may be useful in understanding how the stress-inducible gene expression system is established during evolution (Sano, 2005).

The GAL4-dATF-2 fusions containing the N-terminal 150 amino acids had a stronger activity than those containing the N-terminal 274 amino acids, indicating that the region between amino acids 150 and 274 has a negative effect on the activation domain of dATF-2. In the case of vertebrate ATF-2, the b-ZIP DBD suppresses the ATF-2 activation domain via intramolecular interaction (Li, 1996). This difference may suggest that the mechanism by which the C-terminal region suppresses the activation domain is different between vertebrate ATF-2 and dATF-2. It is interesting whether the region between amino acids 150 and 274 of dATF-2 affects the stability or conformation of dATF-2 protein. Wild-type dATF-2 stimulated the luciferase expression from the CRE-containing promoter under nonstimulated condition. Because the alanine mutants of Thr-59 and Thr-61 dramatically decreased this trans-activating capacity of dATF-2, phosphorylation of these residues seems to be essential for trans-activating capacity of dATF-2. These results suggest the possibility that the Thr-59 and Thr-61 residues are phosphorylated at low levels even under nonstimulated condition. This could be due to the low levels of TNF-α or IL-1 involved in serum. Alternatively, other kinase(s) also may phosphorylate these residues, because vertebrate ATF-2 is activated by Raf-MEK-ERK pathway (Ouwens, 2002) via phosphorylation of Thr-71 (Sano, 2005).

Using two different assay systems, this study has demonstrated at the genetic level that dATF-2 acts in the dp38 signaling pathway. First, it was shown that expression of DN-dp38b enhances the aberrant wing phenotype caused by DN-dATF-2. It has been reported previously that dp38b acts downstream of the Dpp receptor Tkv, because DN-dp38b expressed in the wing imaginal disc causes a phenotype resemble to the mutant of dpp, a Drosophila homologue of mammalian bone morphogenetic protein/TGF-β/activin superfamily. Therefore, dATF-2 may functions in the Dpp signaling pathway. This may be consistent with the finding that mammalian ATF-2 is phosphorylated by TGF-β signaling via TAK1 and p38, and it then directly binds to the Smad3/4 complex to synergistically activate transcription with Smad3/4 (Sano, 1999). This study also demonstrated that DN-dp38b coexpression enhances the sensitivity of embryos expressing DN-dATF-2 to high osmolarity. Thus, dATF-2 acts in the dp38 signaling pathway, at least in wing pattern formation and the response to osmotic stress. However, no oocyte defects were observed in the transgenic flies expressing DN-dATF-2, although the dp38 MAPK pathway is known to be required during oogenesis for asymmetric egg development. Thus, dATF-2 may function only in some specific events that are regulated by the dp38 signaling pathway (Sano, 2005).

DNA array analysis indicated that ~40% of the genes that are induced by osmotic stress are also regulated by dATF-2, indicating that dATF-2 is a major inducer of osmotic stress-inducible gene expression. These genes encode cell surface and cuticle proteins, transporters, and receptors, and various endopeptidases. It is not surprising that osmotic stress may increase the production of cell surface proteins, including some receptors. In addition, the endopeptidases may be produced because high osmolarity may increase the denaturation of proteins, which must then be degraded by the cell. The dATF-2 target genes also include seven immune response genes, namely, several encoding antimicrobial peptides and one encoding a peptidoglycan recognition protein, which binds to the peptidoglycans of bacterial cell walls and triggers immune responses. LPS has been shown to increase the kinase activity of dp38. Consequently, dp38-phosphorylated dATF-2 may directly induce these immune response-related genes. However, it also has been shown that overexpression of dp38 inhibits the expression of immune response genes. This could be explained by the possibility that dp38 overexpression may inhibit the p38 signaling pathway by activating negative feedback regulatory mechanisms, such as the p38α-induced decrease of MKK6 mRNA stability in mammalian cells. In Drosophila, Gram positive bacteria and fungi predominantly induce the Toll signaling pathway to activate genes such as Drosomycin, whereas Gram negative bacteria activate the Imd pathway to activate genes such as Diptericin. DNA array analysis indicated that both Drosomycin and Diptericin are regulated by dATF-2, which suggests that dATF-2 may be a component of both the Toll and Imd pathways. Further analyses of dATF-2 will most likely enhance understanding of the molecular mechanisms involved in the Drosophila immune system (Sano, 2005).

Paternal restraint stress affects offspring metabolism via ATF-2 dependent mechanisms in Drosophila melanogaster germ cells

Paternal environmental factors can epigenetically influence gene expressions in offspring. This study demonstrates that restraint stress, an experimental model for strong psychological stress, to fathers affects the epigenome, transcriptome, and metabolome of offspring in a MEKK1-dATF2 pathway-dependent manner in Drosophila melanogaster. Genes involved in amino acid metabolism are upregulated by paternal restraint stress, while genes involved in glycolysis and the tricarboxylic acid (TCA) cycle are downregulated. The effects of paternal restraint stress are also confirmed by metabolome analysis. dATF-2 is highly expressed in testicular germ cells, and restraint stress also induces p38 activation in the testes. Restraint stress induces Unpaired 3 (Upd3), a Drosophila homolog of Interleukin 6 (IL-6). Moreover, paternal overexpression of upd3 in somatic cells disrupts heterochromatin in offspring but not in offspring from dATF-2 mutant fathers. These results indicate that paternal restraint stress affects metabolism in offspring via inheritance of dATF-2-dependent epigenetic changes (Seong, 2020).

This study indicates that paternal restraint stress induces epigenetic status of offsprings via a p38-MEKK1-dATF-2 pathway and restraint stress-induced upd3 might have a role in transmission of the stress information from somatic to germline cells. Notably, it was observed that paternal restraint stress reduces energy metabolism activity in offspring and sensitizes to rotenone toxicity (Seong, 2020).

Restraint stress information, received by the central nervous system (CNS) and/or other sensing tissues, promotes expression of upd3 in peripheral somatic tissues. In germ cells, accumulated humoral upd3 activates the JAK/STAT pathway, which subsequently activates the p38-dATF-2 pathway. Phosphorylated dATF-2 may be released from the promotor regions of its target genes, resulting in decreased H3K9me2 levels. Epigenetic marks may be retained in mature sperm and inherited by offspring. After fertilization, histone marks may act as inheritable epigenetic memory and regulate gene expression. It was observed that genes involved in the one-carbon metabolic pathway were upregulated in offspring from the paternal restraint stress-exposed fathers, while genes involved in the respiratory metabolic pathway were downregulated. It is assumed that paternal restraint stress-induced- and dATF-2-mediated upregulation of the one-carbon cycle induces downregulation of respiratory metabolism due to a trade-off relationship between these two metabolic processes (Seong, 2020).

The stress response, orchestrated by the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system via stress hormones (adrenaline, noradrenaline, and cortisol), plays a key role in physiology and in several diseases including depression. Accumulated evidence has also implicated inflammatory cytokines in depression. A peripheral infection can induce sickness behaviors, including depression-like behavior, which is caused by the immune-to-brain communication via proinflammatory cytokines produced from innate immune cells. Previous meta-analytic reports indicate that depressed patients possess significantly higher concentrations of IL-6. It was also reported that restraint stress activates the HPA axis and induces higher levels of IL-6 in a rodent model. Furthermore, IL-6 knockout mice exhibit resistance to stress-induced depression-like behaviors, suggesting that IL-6 plays a key role in eliciting depression. The present results suggest that upd3, the Drosophila homolog of IL-6, is also induced by restraint stress. Studies on the inflammatory status of the human HMC-1 cell line showed that adrenaline enhances IL-6, IL-8, and IL-13 production, mediated in part by the p38 signaling pathway. The Drosophila model of myeloproliferative neoplasm indicates that p38 pathway contributes to feed-forward loop in JAK pathway by regulating upd3 gene expression. In Drosophila, restraint stress-dependent upd-3 induction in peripheral tissue may also be mediated by the p38 pathway in response to stress hormones such as adrenaline, as occurs in mammalian systems (Seong, 2020).

The present study showed that upd3 has a role in transmitting restraint stress information to germ cells. It was observed that overexpression of upd3 in neuronal somatic tissues affected the heterochromatic status of offspring in a dATF-2 dependent manner. These results indicate that humoral Upd3, secreted from somatic cells, may be able to affect the epigenetic status of germ cells via the dATF-2 pathway. Moreover, p38 can be activated in response to several extracellular factors, such as TNF, IL-1, growth factors, insulin, salt concentration, and reactive oxygen species (ROS), either directly or indirectly. Thus, humoral components can act as key players to regulate the epigenetic status of germline cells. Upd3, a JAK ligand, is expressed in somatic gonads and activates the JAK/STAT pathway in male germ cells suggesting that testicular germ cells are responsive to the restraint stress-induced increase in Upd3. The activation of p38 by JAK signaling has been demonstrated in the Drosophila immune system, as well as in various vertebrate systems59. Somatic tissue-specific overexpression of upd3 clearly alters the heterochromatin status in the next generation. dATF-2 is highly expressed in germ cells in testes, and expression of TotA, a known upd3 signal target gene, is regulated by the MEKK1-dATF-2 pathway. Therefore, restraint stress-induced humoral upd3 in somatic tissues may control the epigenetic status of germ cells via the p38-dATF-2 signaling pathway in Drosophila. Interestingly, restraint stress-dependent activation of p38 in testes was mainly induced during the latter stages of restraint stress treatment for 10 h. A relatively long time may therefore be necessary to accumulate sufficient humoral upd3 for activation of p38 in germ cells (Seong, 2020).

In restraint stress experiments, animals may also receive mechanical stress, and it may be difficult to completely differentiate this from restraint stress. The relationship between restraint stress and mechanical stress is poorly understood. Mechanical stress induces different sets of cytokines in different types of tissues and IL-6 is one of the major components induced by mechanical stress in mammals. In Drosophila, a recent report showed that forceps squeezing of larvae induces upd3 gene expression. IL-6 induction by either restraint stress or mechanical stress may therefore reflect a common mechanism for sensing and communicating stress information between restraint stress in the nervous system and mechanical stress in the peripheral tissues (Seong, 2020).

In Drosophila, the transit time from spermatagonial stem cells to mature sperm available for insemination at 2 °C is ~260 h. During spermatogenesis, transcription mainly occurs in spermatagonial stem cells and spermatocytes. Herein, when fathers were exposed to restraint stress only once, only the first brood, but not successive broods, exhibited paternal restraint stress-induced heterochromatin disruption. However, exposure to restraint stress three times induced heterochromatin disruption not only in the first brood but also in successive broods. These results suggest that single restraint stress exposure induces heterochromatin disruption only in spermatocytes, whereas repeated restraint stress exposure also causes heterochromatin disruption in spermatagonial stem cells. Interestingly, it has been indicated that long-term memory is required for the maintained effect of the maternal transmission of ethanol preference after 24 h of wasp exposure. There is a possibility that the establishment of neuronal memory by restraint stress may be important for the stable transmission of the epigenetic status in germ cells. Further analysis is needed to elucidate the mechanistic aspect of maintenance of epigenetic change induced by restraint stress in germ cells (Seong, 2020).

Increasing evidence indicates that the paternal environment affects the transcriptome and epigenetic status of their offsprings in several animal models. Although the mechanism of paternal inheritance of epigenetic status remains unclear, some evidence suggests that histone modification is an important player. A recent study indicated that histone replacement-completed sperm retain histone H3 mainly on specific promoter regions in mice. In Drosophila, epiallelic status of H3K27me3 can be dominantly transmitted to the progeny. Drosophila paternal Cid/Cenp-A, a centromere histone protein, can be transmitted to progeny embryos. Similarly, human spermatozoa retain and transmit nucleosomes with constitutive heterochromatin containing H3K9me3 to embryos. A previous report showed that dATF-2 is required for the establishment and maintenance of heterochromatin by regulating the H3K9me2 level, and heat shock stress induces dATF-2 phosphorylation by p38 and its release from heterochromatin, and disrupts the heterochromatin status in germ cells. This causes heterochromatin disruption, which is inherited by the next generation. Based on these observations, it is likely that restraint stress disrupts heterochromatin by decreasing H3K9me2 levels in testicular germ cells, and this is transmitted to offspring. Via a similar mechanism, restraint stress may reduce H3K9me2 on dATF-2 target genes, and this may be transmitted to offspring to modulate metabolism. However, it was not possible to analyze dATF-2 target genes in testicular germ cells by ChIP because anti-dATF-2 antibodies worked only on embryo chromatin but not testis chromatin. This could be due to the presence of non-specific proteins that bind to anti-dATF-2 antibodies in testes. To detect epigenetic effect of paternal restraint stress, restraint stress-treated w1118 males were crossed with wm4 females. The level of histone H3K9me2 was observed to be slightly but significantly decreased in the w gene locus of wm4 offsprings from restraint stress-treated fathers. Although the X chromosome harboring the wm4 gene in the male offspring was derived from the unstressed female, paternal restraint stress increased w expression in male offspring, which is reminiscent of paramutation. It has been reported that paternal stress can influence epigenetic status on maternally supplied X chromosome using the wm4 strain. A previous report also indicated that quantitative content of heterochromatin on the Y chromosome affects PEV phenotypes of wm4 flies. Physical pairing between the heterochromatic regions of X chromosomes and other chromosomes, or the trans-action of some molecules, such as noncoding RNA, may induce partial disruption of the heterochromatin on the X chromosome. Small noncoding RNAs have been also reported as a factor of transgenerational transmission of paternal phenotypes in several model animals. Noncoding RNAs in sperm may also influence the transgenerational effect by paternal restraint stress. Further analysis is required to elucidate the detailed mechanism by which restraint stress affects metabolism in offspring via dATF-2 in testicular germ cells (Seong, 2020).

Consistent with a previous report on mice, the present study indicates that paternal restraint stress affects metabolism in offspring, albeit differently, possibly due to differences among species and experimental conditions. In the present study, paternal restraint stress enhanced the expression of genes involved in one-carbon metabolism, while paternal restraint stress decreased the expression of genes involved in glycolysis and the TCA cycle in the previous work on mice. Restraint stress disrupts heterochromatin possibly by decreasing the H3K9me2 level, which is thought to directly enhance the transcription of dATF-2 target genes. Therefore, downregulation of genes by paternal restraint stress, such as glycolysis-related genes, may occur indirectly via upregulation of one-carbon metabolism-associated genes. The phosphoglycerate dehydrogenase (PHGDH) gene is important in balancing glycolysis and one-carbon metabolism, and its amplification in some cancer cells causes the diversion of a relatively large amount of glycolytic carbon into serine and glycine metabolism. The upregulation of one-carbon metabolism genes, including PHGDH, enhances the levels of glutathione and taurine, which are major factors for reducing ROS during detoxification. ROS stabilizes the transcription factor hypoxia-inducible factor-1α (HIF-1α)71, which activates genes related to glycolysis. Therefore, one possibility is that indirect downregulation of glycolysis genes may be due to decreased activity of HIF-1 following the lowering of ROS levels by paternal restraint stress (Seong, 2020).

Multiple studies have shown that psychological stress affects metabolism, and metabolism conversely modulates the response to psychological stress. The incidence of both depression and diabetes increases with age, and inflammatory cytokines play a key role in eliciting both diseases. Growing evidence indicates that depression and type 2 diabetes share biological origins, particularly overactivation of a cytokine-mediated inflammatory response, potentially through dysregulation of the HPA axis. Thus, psychological stress and metabolism are tightly connected, and this study indicates that psychological stress modulates metabolism in offspring through intergenerational inheritance via sperm. Some previous evidence showed that diet composition may attenuate stress-induced symptoms, and the present results, including precise gene expression changes induced by paternal restraint stress, may contribute to the development of useful foods or supplements with therapeutic benefits (Seong, 2020).

Search PubMed for articles about Drosophila Atf-2

Abel, T., Bhatt, R. and Maniatis, T. (1992). A Drosophila CREB/ATF transcriptional activator binds to both fat body- and liver-specific regulatory elements. Genes Dev 6: 466-480. PubMed ID: 1532159

Botion, L. M., Brito, M. N., Brito, N. A., Brito, S. R., Kettelhut, I. C. and Migliorini, R. H. (1998). Glucose contribution to in vivo synthesis of glyceride-glycerol and fatty acids in rats adapted to a high-protein, carbohydrate-free diet. Metabolism 47: 1217-1221. PubMed ID: 9781624

Chakrabarti, S., Poidevin, M. and Lemaitre, B. (2014). The Drosophila MAPK p38c regulates oxidative stress and lipid homeostasis in the intestine. PLoS Genet 10: e1004659. PubMed ID: 25254641

Cheong, J., Coligan, J. E. and Shuman, J. D. (1998). Activating transcription factor-2 regulates phosphoenolpyruvate carboxykinase transcription through a stress-inducible mitogen-activated protein kinase pathway. J. Biol. Chem 273: 22714-22718. PubMed ID: 9712902

Collins, Q. F., Xiong, Y., Lupo, E. G. Jr., Liu, H. Y. and Cao, W. (2006). p38 Mitogen-activated protein kinase mediates free fatty acid-induced gluconeogenesis in hepatocytes. J. Biol. Chem 281: 24336-24344. PubMed ID: 16803882

Craig, C. R., Fink, J. L., Yagi, Y., Ip, Y. T. and Cagan, R. L. (2004). A Drosophila p38 orthologue is required for environmental stress responses. EMBO Rep 5: 1058-1063. PubMed ID: 15514678

Croniger, C., Leahy, P., Reshef, L. and Hanson, R. W. (1998). C/EBP and the control of phospho-enolpyruvate carboxykinase gene transcription in the liver. J. Biol. Chem. 273: 31629-31632. PubMed ID: 9822619

De Graeve, F., Bahr, A., Sabapathy, K. T., Hauss, C., Wagner, E. F., Kedinger, C., and Chatton, B. (1999). Role of the ATFa/JNK2 complex in Jun activation. Oncogene 18: 3491-3500. PubMed ID: 10376527

Franckhauser, S., Munoz, S., Pujol, A., Casellas, A., Riu, E., Otaegui, P., Su, B. and Bosch, F. (2002). Increased fatty acid re-esterification by PEPCK overexpression in adipose tissue leads to obesity without insulin resistance. Diabetes 51: 624-630. PubMed ID: 11872659

Gupta, S., Campbell, D., Derijard, B., and Davis, R. J. (1995). Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 267: 389-393. PubMed ID: 7824938

Hirosumi, J., Tuncman, G., Chang, L., Gorgun, C. Z., Uysal, K. T., Maeda, K., Karin, M. and Hotamisligil, G. S. (2002). A central role for JNK in obesity and insulin resistance. Nature 420: 333-336. PubMed ID: 12447443

Kalhan, S. C., Mahajan, S., Burkett, E., Reshef, L. and Hanson, R. W. (2001). Glyceroneogenesis and the source of glycerol for hepatic triacylglycerol synthesis in humans. J. Biol. Chem 276: 12928-12931. PubMed ID: 11278297

Lee, M. Y., Jung, C. H., Lee, K., Choi., Y. H., Hong, S. and Cheong, J. (2002). Activating transcription factor-2 mediates transcriptional regulation of gluconeogenic gene PEPCK by retinoic acid. Diabetes 51: 3400-3407. PubMed ID: 12453892

Li, X. Y., and Green, M. R. (1996). Intramolecular inhibition of activating transcription factor-2 function by its DNA-binding domain. Genes Dev. 10: 517-527. PubMed ID: 8598283

Livingstone, C., Patel, G., and Jones, N. (1995). ATF-2 contains a phosphorylation-dependent transcriptional activation domain. EMBO J. 14: 1785-1797. PubMed ID: 7737129

Maekawa, T., et al. (1999). Mouse ATF-2 null mutants display features of a severe type of meconium aspiration syndrome. J. Biol. Chem 274: 17813-17819. PubMed ID: 10364225

Matsuda, S., Maekawa, T., and Ishii, S. (1991). Identification of the functional domains of the transcriptional regulator CRE-BP1. J. Biol. Chem. 266: 18188-18193. PubMed ID: 1833393

Mendelson, K. G., Contois, L. R., Tevosian, S. G., Davis, R. J. and Paulson, K. E. (1996). Independent regulation of JNK/p38 mitogen-activated protein kinases by metabolic oxidative stress in the liver. Proc. Natl. Acad. Sci. 93: 12908-12913. PubMed ID: 8917518

Okamura, T., et al. (2007). ATF-2 regulates fat metabolism in Drosophila. Molec. Biol. Cell 18: 1519-1529. PubMed ID: 17314398

Olswang, Y., Cohen, H., Papo, O., Cassuto, H., Croniger, C. M., Hakimi, P., Tilghman, S. M., Hanson, R. W. and Reshef, L. (2002). A mutation in the peroxisome proliferator-activated receptor gamma-binding site in the gene for the cytosolic form of phosphoenolpyruvate carboxykinase reduces adipose tissue size and fat content in mice. Proc. Natl. Acad. Sci. 99: 625-630. PubMed ID: 11792850

Ouwens, D. M., de Ruiter, N. D., van der Zon, G. C., Carter, A. P., Schouten, J., van der Burgt, C., Kooistra, K., Bos, J. L., Maassen, J. A. and van Dam, H. (2002). Growth factors can activate ATF2 via a two-step mechanism: phosphorylation of Thr71 through the Ras-MEK-ERK pathway and of Thr69 through RalGDS-Src-p38. EMBO J. 21: 3782-3793. PubMed ID: 12110590

Reimold, A. M., et al. (1996). Chondrodysplasia and neurological abnormalities in ATF-2-deficient mice. Nature 379: 262-265. PubMed ID: 8538792

Reshef, L., Olswang, Y., Cassuto, H., Blum, B., Croniger, C. M., Kalhan, S. C., Tilghman, S. M. and Hanson, R. W. (2003). Glyceroneogenesis and the triglyceride/fatty acid cycle. J. Biol. Chem 278: 30413-30416. PubMed ID: 12788931

Sano, Y., Tokitou, F., Dai, P., Maekawa, T., Yamamoto, T., and Ishii, S. (1998). CBP alleviates the intramolecular inhibition of ATF-2 function. J. Biol. Chem. 273: 29098-29105. PubMed ID: 9786917

Sano, Y., Harada, J., Tashiro, S., Gotoh-Mandeville, R., Maekawa, T., and Ishii, S. (1999). ATF-2 is a common nuclear target of Smad and TAK1 pathways in transforming growth factor-β signaling. J. Biol. Chem. 274: 8949-8957. PubMed ID: 10085140

Sano, Y., Akimaru, H., Okamura, T., Nagao, T., Okada, M. and Ishii, S. (2005). Drosophila activating transcription factor-2 is involved in stress response via activation by p38, but not c-Jun NH(2)-terminal kinase. Mol. Biol. Cell 16: 2934-2946. PubMed ID: 15788564

Shen, Y. H., Zhang, L., Gan, Y., Wang, X., Wang, J., LeMaire, S. A., Coselli, J. S. and Wang, X. L. (2006). Up-regulation of PTEN (phosphatase and tensin homolog deleted on chromosome ten) mediates p38 MAPK stress signal-induced inhibition of insulin signaling. A cross-talk between stress signaling and insulin signaling in resistin-treated human endothelial cells. J. Biol. Chem 281: 7727-7736. PubMed ID: 16418168

Shuman, J. D., Cheong, J. and Coligan, J. E. (1997). ATF-2 and C/EBPalpha can form a heterodimeric DNA binding complex in vitro. Functional implications for transcriptional regulation. J. Biol. Chem 272: 12793-12800. PubMed ID: 9139739

Seong, K. H., Ly, N. H., Katou, Y., Yokota, N., Nakato, R., Murakami, S., Hirayama, A., Fukuda, S., Kang, S., Soga, T., Shirahige, K. and Ishii, S. (2020). Paternal restraint stress affects offspring metabolism via ATF-2 dependent mechanisms in Drosophila melanogaster germ cells. Commun Biol 3(1): 208. PubMed ID: 32367035

Tordjman, J., Chauvet, G., Quette, J., Beale, E. G., Forest, C. and Antoine, B. (2003). Thiazolidinediones block fatty acid release by inducing glyceroneogenesis in fat cells. J. Biol. Chem. 278: 18785-18790

van Dam, H., Wilhelm, D., Herr, I., Steffen, A., Herrlich, P., and Angel, P.(1995). ATF-2 is preferentially activated by stress-activated protein kinases to mediate c-jun induction in response to genotoxic agents. EMBO J. 14: 1798-1811. PubMed ID: 7737130

Wu, J. J., Roth, R. J., Anderson, E. J., Hong, E. G., Lee, M. K., Choi, C. S., Neufer, P. D., Shulman, G. I., Kim, J. K. and Bennett, A. M. (2006). Mice lacking MAP kinase phosphatase-1 have enhanced MAP kinase activity and resistance to diet-induced obesity. Cell Metab 4: 61-73. PubMed ID: 16814733

date revised: S5 August 2023

Home page: The Interactive Fly © 2008 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.