Gene name - p38b MAP kinase
Cytological map position - 34C4--D1
Function - protein kinase
Symbol - p38b
FlyBase ID: FBgn0024846
Genetic map position -
Classification - MAP kinase
Cellular location - probably both nuclear and cytoplasmic
|Recent literature||Huang, D., Li, X., Sun, L., Huang, P., Ying, H., Wang, H., Wu, J. and Song, H. (2016). Regulation of Hippo signalling by p38 signalling. J Mol Cell Biol [Epub ahead of print]. PubMed ID: 27402810
The Hippo signalling pathway has a crucial role in growth control during development, and its dysregulation contributes to tumorigenesis. Recent studies uncover multiple upstream regulatory inputs into Hippo signalling, which affects phosphorylation of the transcriptional coactivator Yorkie/YAP/TAZ by Warts/Lats. This study identifies the p38 MAPK pathway as a new upstream branch of the Hippo pathway. In Drosophila, overexpression of MAPKK gene licorne (lic), or MAPKKK gene Mekk1, promotes Yki activity and induces Hippo target gene expression. Loss-of-function studies show that lic regulates Hippo signalling in ovary follicle cells and in the wing disc. Epistasis analysis indicates that Mekk1 and lic affect Hippo signalling via p38b and wts It was further demonstrated that the Mekk1-Lic-p38b cascade inhibits Hippo signalling by promoting F-actin accumulation and Jelly belly phosphorylation. In addition, p38 signalling modulates actin filaments and Hippo signalling in parallel to small GTPases Ras, Rac1, and Rho1. Lastly, p38 signalling was shown to regulate Hippo signalling in mammalian cell lines. The Lic homolog MKK3 promotes nuclear localization of YAP via the actin cytoskeleton. Upregulation or downregulation of the p38 pathway regulates YAP-mediated transcription. This work thus reveals a conserved crosstalk between the p38 MAPK pathway and the Hippo pathway in growth regulation.
|Eom, H. J., Liu, Y., Kwak, G. S., Heo, M., Song, K. S., Chung, Y. D., Chon, T. S. and Choi, J. (2017). Inhalation toxicity of indoor air pollutants in Drosophila melanogaster using integrated transcriptomics and computational behavior analyses. Sci Rep 7: 46473. PubMed ID: 28621308
This study conducted an inhalation toxicity test on the alternative animal model, Drosophila melanogaster, to investigate potential hazards of indoor air pollution. The inhalation toxicity of toluene and formaldehyde was investigated using comprehensive transcriptomics and computational behavior analyses. The ingenuity pathway analysis (IPA) based on microarray data suggests the involvement of pathways related to immune response, stress response, and metabolism in formaldehyde and toluene exposure based on hub molecules. A toxicity test was conducted using mutants of the representative genes in these pathways to explore the toxicological consequences of alterations of these pathways. Furthermore, extensive computational behavior analysis showed that exposure to either toluene or formaldehyde reduced most of the behavioral parameters of both wild-type and mutants. Interestingly, behavioral alteration caused by toluene or formaldehyde exposure was most severe in the p38b mutant, suggesting that the defects in the p38 pathway underlie behavioral alteration. Overall, the results indicate that exposure to toluene and formaldehyde via inhalation causes severe toxicity in Drosophila, by inducing significant alterations in gene expression and behavior, suggesting that Drosophila can be used as a potential alternative model in inhalation toxicity screening.
Mitogen-activated protein kinase (MAPK) proteins are conserved eukaryotic factors that are integral to various signal transduction pathways. Three subgroups of the MAPK superfamily have been identified: the extracellular signal-regulated kinase (ERK); c-Jun N-terminal kinase (JNK, also referred to as stress-activated protein kinase [SAPK]), and p38 (also called Mpk2). MAPKs are activated through phosphorylation by specific MAPK kinases (MAPKKs or MEKs), which are themselves phosphorylated and activated by specific MAPKK kinases (MAPKKKs), thus comprising a series of separate MAPK cascades. Both ERK and JNK, termed Rolled and Basket, respectively, have been extensively characterized in Drosophila, but Drosophila p38 has been all but neglected. This neglect has now come to an end for several reasons: (1) two p38 homologs have now been cloned; (2) two MEKs (MAPK kinases) functioning immediately upstream of p38 have also been cloned; (3) a role for one of these MEKs, called Licorne, has been identified in oogenesis; (4) potential roles for these p38s and MEKs in the immune response have also been identified, and (5) in the study described in this overview, p38b is involved in transforming growth factor beta superfamily signal transduction in Drosophila wing morphogenesis (Adachi-Yamada, 1999).
Two Drosophila homologs of p38, Mpk2 (also known as p38a or simply p38) and p38b, have been identified on the basis of their homology to mammalian p38 and to one another (S. Han, 1998 and Z. Han, 1998). p38b, the subject of this overview, is maternally expressed and is present ubiquitously during embryonic development (Z. Han, 1998). A second approach, in addition to that of sequence homology, was also taken when cloning p38s in Drosophila. p38b was cloned on the basis of its ability to complement a defect in MAPK in yeast. The yeast hog1 pathway mediates cellular responses to an increase in external osmolarity (Herskowitz, 1995). Mammalian p38 has been shown to complement the high osmolarity-sensitive growth phenotype of the hog1 MAPK mutant. To isolate cDNAs for the Drosophila homolog(s) of p38, genetic screens were used to isolate suppressors of the hog1delta mutation. A total of 16 positive clones were obtained and assigned to either of two classes. The cDNAs from one class were identical to Drosophila Basket/JNK. cDNAs from the other class were identical to p38b. Comparison of the cDNA and genomic sequences revealed that the D-p38b gene is organized into two exons, although its coding region is continuous within a single exon (Adachi-Yamada, 1999).
The chromosomal region around the p38b locus has been well characterized genetically. However, a p38b transgene was unable to rescue any of the known mutations mapping to this region. Likewise, attempts to isolate a mutant of p38b were unsuccessful. These failures are possibly due to the functional redundancy of the two p38 homologs. Various alternative methods were therefore use to interfere with endogenous p38(s) in order to investigate its function. A dominant-negative allele of p38b, designated D-p38bDN, was generated by replacing the Thr-183 of the MAPKK target site with Ala, analogous to the change in ERK2 that produces a dominant-negative allele. This recombinant mutant protein lost its ability to suppress the yeast hog1delta mutant phenotype (Adachi-Yamada, 1999).
Two lines were prepared which express D-p38bDN at different levels: D-p38bDN-S (Strong), which expresses high levels, and D-p38bDN-W (Weak), which expresses low levels. When two copies of the D-p38bDN-S transgene are expressed in the wing, a certain fraction of adult flies that escape death exhibit ectopic vein fragments around the end of the longitudinal vein L2 and a reduction in the distance between L4 and L5. Both of these features have also been observed with some mutant alleles of decapentaplegic (dpp) and thick veins (tkv), a gene encoding a type I receptor for Dpp. This wing phenotype is rescued by coexpression of the wild type p38b+ transgene. When two copies of the D-p38bDN-S transgene are weakly expressed in the wing of a dpp mutant, the vein phenotype of dpp is strongly enhanced. These phenotypes suggest the involvement of Drosophila p38(s) in Dpp function in the early and late stages of wing pattern development. Dpp is known to play a dual role during wing development, acting as a morphogen and mitogen at early stages, while activating vein differentiation at later stages (Adachi-Yamada, 1999).
To examine whether p38(s) functions in the Dpp signaling pathway, the genetic interaction was examined between p38(s) and a constitutively active mutant of Tkv (TkvCA). Two classes of tkvCA insertions, tkvCA-S (Strong) and tkvCA-W (Weak), were used. When tkvCA-S is expressed, normal wing venation is severely distorted and extensive production of fragments of vein material is observed. The abdominal-cuticle pattern also appears irregular. This wing phenotype suggests that TkvCA may influence Dpp action during vein formation. Ectopic coexpression of dpp+ and tkv+ causes similar phenotypes, indicating that these TkvCA-induced aberrations are indeed the result of an increase in Dpp signaling. It was thus expected that reducing the levels of downstream components would suppress tkvCA. In fact, reducing by one-half the gene dosage of Mothers against dpp (Mad), a well-documented Dpp-signaling factor, significantly suppresses the tkvCA wing phenotype (Adachi-Yamada, 1999).
The effect of the imidazole compound SB203580, a p38 inhibitor, was tested on the tkvCA wing phenotype. SB203580 has been reported to inhibit both p38a and p38b (Z. Han, 1998), and penetration of various imidazole compounds through the insect epidermis is well known. Exposure of growing larvae to SB203580 indeed results in suppression of the phenotype. Tests were performed to see whether endogenous p38 genes are involved in the tkvCA wing phenotype by reducing endogenous gene dosage using chromosomal hemizygosity. Interestingly, reduction of p38b suppresses the tkvCA wing phenotype, while reduction of p38a is not effective. Suppression by reduction of the p38b gene dosage is abrogated by the introduction of a transgene for p38b+. Thus, the gene within the deficiency that suppresses tkvCA is indeed p38b. These results suggest that p38b plays a major role in this morphogenetic process, and attention was focussed on this gene in further analyses (Adachi-Yamada, 1999).
Antisense RNA can often be used to mimic the effects of mutation. When antisense p38d RNA is coexpressed with TkvCA-S, the tkvCA-S phenotype is markedly suppressed. Four of five independently established p38bantisense lines showed significant suppression. Suppression affects the various pleiotropic phenotypes associated with the tkvCA allele, including wing blade morphology, abdominal-cuticle morphology, and wing posture. Similar suppression of the tkvCA phenotype is also achieved by coexpression of p38bDN-W in a dose-dependent manner. This suppression is greater when the strong dominant negative p38b (p38bDN-S) is coexpressed instead of p38bDN-W. Furthermore, this suppression is abrogated by simultaneous coexpression of wild type p38d, demonstrating that p38bDN and wild type p38b competitively sequester endogenous factors essential to signaling. These results suggest either that p38b functions downstream of Tkv or that inhibition of p38b causes a reduction in endogenous dpp activity. Since the expression pattern of dpp in the developing wing of the D-p38bDN-S producer has been found to be indistinguishable from that of the wild type, and reduction in the gene dosage of dpp is not effective in suppressing the tkvCA phenotype, it is concluded that p38b does not affect Dpp production per se but rather acts as a downstream component of the Dpp-Tkv signaling pathway, operating late in wing development. The fact that the weak phenotype of tkvCA-W is significantly enhanced by wild type p38b is also consistent with this conclusion (Adachi-Yamada, 1999).
The effect of p38b on optomotor blind transcription was examined in order to study the involvement of p38b in the Dpp signaling pathway. The omb gene encodes a T-box family transcription factor, and its expression in the wing imaginal disc is dependent on early Dpp-Tkv signaling. In the wing discs of flies ectopically expressing tkvCA-5, the omb expression domain is greatly expanded and overgrowth of the disc is evident. Expression of p38bDN or p38bantisense markedly suppresses both omb expression and disc overgrowth. Induction of omb in the tkvCA-expressing clones in regions outside those where dpp is expressed is also inhibited by coexpression of p38bDN, consistent with the possibility that p38b functions downstream of Tkv. Furthermore, while p38bDN slightly affects omb expression in a tkv+ genetic background, the wing phenotype of a hypomorphic omb allele is clearly enhanced by expression of p38bDN or p38bantisense, as observed in the wing phenotype of severe omb alleles. These results suggest that p38b is also involved in early Dpp-Tkv signaling in wing development to activate omb transcription. Evidence is presented that p38b, or possibly both p38s, are phosphorylated in vivo downstream of ectopically expressed constitutively active Tkv (Adachi-Yamada, 1999).
To investigate whether p38b is activated by Tkv signaling, a preliminary biochemical characterization of p38b was carried out. Immediately after heat treatment of flies, the amount of p38b immunoprecipitated by anti-p-Tyr antibody was found to increase considerably, demonstrating that p38b is tyrosine phosphorylated following heat shock, like mammalian p38. The site of tyrosine phosphorylation is expected to be in the 'activation loop' region recognized by MAPKK, as is the case in mammalian p38. Thus, a test was performed to see whether an anti-phospho-p38 (anti-p-p38) antibody raised against a phosphorylated peptide from the activation loop of mammalian p38 could cross-react with p38b. This anti-p-p38 antibody detects a protein with a calculated size of 42 kDa whose amount increases immediately after heat shock. This protein is also more abundant in the flies overproducing p38b regardless of heat treatment. Therefore, it has been concluded that anti-phospho-p38 can cross-react with the phosphorylated from of p38b and can be used to assay recombinant p38b phosphorylation in vitro. Treatment of p38b with recombinant human MKK6, a MAPKK that activates p38, causes a marked increase in the level of p38b, as detected with anti-phospho-Tyr and anti-p-p38 antibodies, and a drastic increase in the level of Drosophila p38-dependent phosphorylation of recombinant human activating transcription factor 2 (ATF2), a physiological substrate for mammalian p38. The correlation between the phosphorylation state and kinase activity of p38b indicates that the anti-p-p38 antibody recognizes the active form of p38b. This allowed activation of p38b by TkvCA to be examined in vivo. The amount of active p38b was found to be slightly but significantly higher in larvae carrying ectopically expressed tkvCA relative to that in wild-type Canton-S larvae. However, it has been reported that p38a protein expressed in yeast, which was presumed to have the same molecular mass as p38b, is also recognized by anti-p-p38 antibody. It is therefore possible that p38b, or both D-p38's, may be activated by Tkv signaling in vivo (Adachi-Yamada, 1999).
The integrity of the intestinal epithelium is crucial for the barrier function of the gut. Replenishment of the gut epithelium by intestinal stem cells contributes to gut homeostasis, but how the differentiated enterocytes are protected against stressors is less well understood. This study used the Drosophila larval hindgut as a model system in which damaged enterocytes are not replaced by stem cell descendants. By performing a thorough genetic analysis, it was demonstrates that a signalling complex consisting of p38b and MK2 forms a branch of SAPK signalling that is required in the larval hindgut to prevent stress-dependent damage to the enterocytes. Impaired p38b/MK2 signalling leads to apoptosis of the enterocytes and a subsequent loss of hindgut epithelial integrity, as manifested by the deterioration of the overlaying muscle layer. Damaged hindguts show increased JNK activity, and removing upstream activators of JNK suppresses the loss of hindgut homeostasis. Thus, the p38/MK2 complex ensures homeostasis of the hindgut epithelium by counteracting JNK-mediated apoptosis of the enterocytes upon chronic stress (Seisenbacher, 2011).
The p38 SAPK belongs to the MAPK family and is conserved from yeast to humans. In higher eukaryotes, p38 associates with its major target, the MAPK-activated protein kinase MK2. This complex resides in the nucleus in the resting state. Upon stress, p38 is activated by MKK3/MKK6 and phosphorylates MK2, which results in an exposure of the nuclear export signal of MK2 and a subsequent nuclear export of the complex (ter Haar, 2008). Another consequence of the p38/MK2 complex formation is the stabilisation of p38 protein. Interestingly, the kinase activity of MK2 is required neither for the nucleo-cytoplasmic shuttling nor for the p38 protein stabilisation (see ter Haar, 2008). Conversely, MK2 kinase activity is crucial to phosphorylate small heat-shock proteins, transcription factors (e.g., SRF and HSF-1), and Tristetraprolin (TTP) is the only trans-acting factor shown to be capable of regulating AU-rich element-dependent mRNA turnover at the level of the intact animal. The inhibitory phosphorylation of TTP by the p38/MK2 complex has been shown to increase the translation of AU-rich elements (ARE)-containing mRNAs including the mRNA encoding the proinflammatory cytokine TNFα. Furthermore, p38 and MK2 have been shown to act as cytoplasmic checkpoint kinases in parallel to CHK1. Due to the plethora of p38/MK2 functions, targeting the p38 SAPK branch with inhibitors might lead to harmful side effects. Thus, it is important to understand the roles of p38 signalling in a tissue-specific context. The availability of mouse models has helped to decipher some in vivo roles of p38 SAPK signalling but the complex nature of the intestinal system has hampered a detailed analysis (Seisenbacher, 2011 and references therein).
Gut homeostasis, under normal and stress conditions, is ensured by complex interactions between the intestinal epithelium, the immune system and the gut flora. Drosophila has been used as a simple model organism to address different aspects of intestinal homeostasis. Replenishment of the gut epithelium by intestinal stem cells (ISCs) clearly contributes to epithelial homeostasis but how the differentiated enterocytes (ECs) are protected against stressors has remained largely unknown. This study used the larval hindgut of Drosophila as a simple intestinal model organ to address how stress signalling in the hindgut ECs ensures intestinal homeostasis in the absence of proliferative cells. This analysis identifies the p38b/MK2 signalling module as a critical component in the protection of hindgut ECs against salt stress (Seisenbacher, 2011).
A model is proposed that puts a p38b/MK2 complex in the centre of stress-protection of the hindgut ECs . In the absence of this signalling module, cells are undergoing JNK-dependent apoptosis upon stress. The lesion in the EC monolayer results in the damage of the overlying hindgut musculature. This regional loss of the barrier function leads to systemic defects in the larvae, further weakening the larvae and impairing growth under stress conditions. As a consequence, pathogens and toxic substances might enter the body cavity, eventually resulting in the melanisation of pericardial cells and the induction of cecropin in the midgut (Seisenbacher, 2011).
Interestingly, JNK activation in MK2 mutant hindguts precedes the melanisation, and it consistently occurs in patches. Within these areas, some cells acquire highest amounts of JNK activity and eventually undergo apoptosis. The surrounding cells maintain JNK activity, forming a rim around the scar in the tissue. The number of affected ECs remains roughly constant for a given stress. Presently it is not known what determines the patches with high JNK activity within the tissue. Although the dorsal domain (hd, positive for engrailed expression) ECs of the hindgut form a homogeneous epithelium and are facing the same stressor, JNK signalling is only induced in clusters of a certain size but not in surrounding cells. Increased JNK activation was also observed in the p38α deleted intestinal epithelium of a mouse model for inflammatory bowel diseases (IBDs). Furthermore, ulcerations occur in similar patchy patterns in IBDs. Thus, the MK2 mutant phenotype may be useful to decipher how a group of cells within a tissue of genetically identical cells transforms into the weakest link in the chain upon stressful conditions (Seisenbacher, 2011).
Several lines of evidence support the notion that the p38b/MK2 signalling complex is key to EC protection against chronic salt stress. (1) p38b and MK2 mutant larvae both develop BDs upon stress conditions. (2) The severity of the p38b; MK2 double mutant phenotype upon stress is similar to the p38b single mutant phenotype, suggesting that they act in the same signalling pathway. (3) p38b but not p38a physically associates with MK2 via its C-terminal CD domain (DPTD motif). (4) The binding of p38b to MK2 stabilises MK2. (5) Upon co-expression, p38b but not p38a redirects MK2 to the cytoplasm. (6) Both the activation and the catalytic activity of p38b are required to efficiently relocalise MK2. (7) The binding of MK2 to p38 and the catalytic activities of both kinases are essential to protect the ECs of the larval hindgut upon salt stress. Taken together, stabilisation, localisation and activation of MK2 by p38b are required for a proper stress response (Seisenbacher, 2011).
The genetic analysis also revealed that p38 SAPK signalling is contributing to stress protection in different ways in addition to the pivotal role of the p38b/MK2 complex. First, p38b impacts on hindgut homeostasis in an MK2-independent manner. This is apparent from the p38b mutant larvae that, in contrast to MK2 mutant larvae, develop BDs even at normal conditions. Consistently, a p38b protein version that no longer binds MK2 partially rescues the p38b mutant phenotype. Second, p38a is also required for full stress protection. The strong phenotype of MK2; p38a double mutants underscores the importance of the p38a SAPK pathway upon severe salt stress. However, the double mutants do not display an increase in BD formation but rather a decrease in viability. Thus, p38a may be involved in general stress protection that is not specific to the hindgut tissue. Third, a common p38 SAPK branch, encompassing p38a, p38b and potentially also p38c, is essential as the p38a; p38b double mutants die. Since the p38a1 allele affects the coding sequences of p38a and p38c, the p38a1; p38bd27 double mutants may even represent p38a; p38b; p38c triple mutants. However, it is unclear to date whether p38c is a pseudogene. Recent studies have suggested an involvement of p38c function in immune gene regulation, early larval survival, and fertility. Thus, further studies will be required to clarify whether p38c contributes to p38 signalling subbranches. Finally, the slight increase in BDs seen in MK2; p38b double mutants under normal conditions suggests that MK2 also performs a p38b-independent function in the hindgut. p38b mutants always impact on MK2 signalling since the MK2 protein is not stable and probably not correctly localised if not bound to p38b. Negative feedback regulation acting from MK2 on the activation of p38b further complicates the SAPK signalling network (Seisenbacher, 2011).
What are the upstream components regulating the p38b/MK2 complex? Surprisingly, MKK3/Lic does not appear to play a role in the hindgut function of the p38b/MK2 branch. MKK3 but not MKK4 can activate p38 proteins in cell culture. On the other hand, siRNA mediated knockdown of both MKK4 and MKK3 is required to fully block the activation of p38 under certain stresses in S2 cells. Both p38b and MKK3/lic mutants show a strong reduction in p38 activation but no BDs are observed in MKK3/lic mutants. In contrast, mutants for MEKK1, which acts upstream of MKK3, do develop BDs similar to p38b and MK2 mutants. In mammalian cells, it has been shown that p38 can be activated independently of a MAP2 kinase. However, no activation of p38 occurred in fibroblasts of MKK3 MKK6 double mutant mice. Since MKK4 is a suppressor of the BD phenotype and MKK7 most likely does not activate p38 in Drosophila, a scenario of MAP2K-independent p38 activation could also apply for the p38b/MK2 signalling branch in the larval hindgut (Seisenbacher, 2011).
Interestingly, overexpression of the kinase-dead or of the non-activatable p38b protein version results in an even stronger phenotype than the deletion of p38b, probably by titrating upstream partners that would have additional functions besides activating p38b. A common p38a/p38b activator would be a strong candidate for such an upstream component. Saturating this common p38 activator with p38bKR or p38bAGF would essentially result in a p38a/p38b double mutant situation and therefore would explain the strong phenotype, especially at high salt conditions (Seisenbacher, 2011).
This analysis of the p38b/MK2 signalling module in hindgut ECs reveals how deletion of SAPK members results in increased sensitivity towards a particular stressor from the molecular level to the level of the whole organism. These findings provide a new model of how hindgut homeostasis is maintained and how different SAPK branches act together in vivo to ensure cellular survival upon stress. The p38 SAPK pathway efficiently protects hindgut ECs over a wide range of stress conditions and for an astonishingly long time period of at least five days without cell replenishment. In this light, a comparative analysis of the larval and the adult hindgut (which has to be maintained for a time period of up to forty days) will be of great interest (Seisenbacher, 2011).
How does p38 interfere with JNK activation in the hindgut epithelium? In mammalian cells, p38α binds to and phosphorylates TAB1 (and potentially TAB2). As a consequence, the activities of TAK1 and thereby JNK are reduced. TAB1 is not conserved in Drosophila but TAB2 might be involved in a similar negative feedback loop. TAB2 has been implicated in JNK activation in response to peptidoglycans and lipopolysaccharides. However, in the absence of TAB2, no change in JNK activation in response to Sorbitol or NaCl has been observed. Alternatively, p38 might induce a JNK phosphatase. p38α has been shown to impact on JNK activation by inducing DUSP1/MKP-1 in mammalian cells. Sustained JNK activation due to a loss of DUSP1/MKP-1 resulted in increased cell death in response to UV stress. Interestingly, p38 activation was also increased in DUSP1/MKP-1 mutant cells but p38 activity was not linked to cell death induction. Similarly, MK2 mutant larvae bearing BDs display an overactivation of both p38 and JNK, and blocking JNK only is sufficient to prevent the BD phenotype (Seisenbacher, 2011).
Despite the more complex nature of mammalian guts, the strong conservation of stress-signalling pathways and the similar demands of ECs make it likely that the current results will also be important in the context of various diseases of the intestinal system. A variety of different signalling pathways have been implicated in IBDs, underscoring the complex nature of these diseases. p38 and MK2 are critical regulators of TNFα production and are thereby associated with IBDs but the role of p38 SAPK in IBDs has remained controversial. This study identifies a crucial role of p38b/MK2 signalling in the first line of defence against a particular stressor in a model system devoid of an adaptive immune system. The consequences of lacking this immune system-independent protective function of a SAPK branch might parallel early steps of IBD development in intestinal epithelial cells. Indeed, a recent study has revealed tissue-specific effects of p38α in a DSS-induced mouse model for IBDs. Deleting p38α in the myeloid lineage had beneficial effects, consistent with the inflammatory nature of IBDs. In contrast, p38α deletion in the intestinal epithelium resulted in a loss of gut homeostasis, marked by increased proliferation and by a reduction in goblet cells. Thus, these studies on Drosophila p38 signalling and its role in the larval hindgut provide a basis to specifically address the role of ECs in the maintenance of an intestinal epithelium in the absence of proliferation and immune response (Seisenbacher, 2011).
The fruit fly Drosophila melanogaster is a powerful model system for the study of innate immunity in vector insects as well as mammals. For vector insects, it is particularly important to understand all aspects of their antiviral immune defenses, which could eventually be harnessed to control the transmission of human pathogenic viruses. The immune responses controlling RNA viruses in insects have been extensively studied, but the response to DNA virus infections is poorly characterized. This study reports that infection of Drosophila with the DNA virus Invertebrate iridescent Virus 6 (IIV-6) triggers JAK-STAT signaling and the robust expression of the Turandots, a gene family encoding small secreted proteins. To drive JAK-STAT signaling, IIV-6 infection more immediately induced expression of the unpaireds, a family of IL-6-related cytokine genes, via a pathway that required one of the three Drosophila p38 homologs, p38b. In fact, both Stat92E and p38b were required for the survival of IIV-6 infected flies. In addition, in vitro induction of the unpaireds required an NADPH-oxidase, and in vivo studies demonstrated Nox was required for induction of TotA. These results argue that ROS production, triggered by IIV-6 infection, leads to p38b activation and unpaired expression, and subsequent JAK-STAT signaling, which ultimately protects the fly from IIV-6 infection (West, 2018).
This study shows that infection of Drosophila with the DNA virus IIV-6 triggers a protective p38b-dependent response. While previous work has demonstrated that Drosophila p38b is critical for survival to bacterial or fungal infections and affects the tolerance to bacterial infections, this is the first time p38b has been linked to antiviral defenses. Critical targets for p38b for the protection against IIV-6 infection are the unpaireds, a family of three IL-6-like genes clustered together on Chromosome X. The genetic data presented in this study argue that the three Unpaireds function together, in a partially redundant manner, to activate the JAK-STAT pathway following IIV-6 infection, thereby driving Tot gene expression. The JAK-STAT pathway also protects against IIV-6 infection, although the role of the Tots in antiviral defense requires more study. These results also imply that p38b is activated following IIV-6 infection. While the mechanisms leading from virus infection to p38 activation are unclear, they likely involve ROS-mediated signaling as the induction of TotA expression is potently blocked by an NADPH oxidase inhibitor and require the Nox gene. This is reminiscent of the activation of p38a by ROS generated from apoptotic cells in models of tissue regeneration (West, 2018).
Interestingly, p38b has also been shown to provide tolerance to Salmonella typhimurium infections, promoting survival of the host without reducing bacterial burden. This study suggested that p38b contributes to tolerance by enabling hemocyte enlargement, and hence, engulfment of larger quantities of bacteria. In the context of IIV-6 infection, p38b could be acting to promote engulfment of infected and damaged cells, thereby providing a repair mechanism to enable the animals to better tolerate and limit virus infection. Future studies will be necessary to probe all the roles of p38b in antiviral defense (West, 2018).
Although the data presented in this study demonstrate that the JAK-STAT pathway is protective against IIV-6 infection, the protective mechanisms require further study. In the case of the RNA virus DCV, the JAK-STAT pathway is also protective, possibly through the induction of vir-1. However, the JAK-STAT pathway is not broadly antiviral and vir-1 was not induced by IIV-6. Curiously, a previous study examining the role of the JAK-STAT pathway during IIV-6 infection, using one particular hypomorphic allelic combination hopscotch (JAK), concluded that hopscotch (and by inference the JAK-STAT pathway) was not involved in protecting flies against IIV-6 infection. The current data, with multiple RNAi lines targeting stat92E, as well as the S2 cell based results with RNAi targeting domeless, hopscotch, and stat92E, demonstrate a consistent and reproducible role for this pathway in the response to and survival from IIV-6 infection. These contradictory outcomes may be due to differences in alleles used or dose delivered (West, 2018).
The Tots are intriguing candidates for JAK-STAT induced antivirals. They are rapidly evolving with evidence of positive selection, typical for immune effectors. However, the Tots have not yet been demonstrated to provide direct antimicrobial activity. To date, this study has been unable to demonstrate any antiviral activity for the Tots. In particular, over-expression of TotA resulted in reduced survival following IIV-6 infection and no change in viral titers, consistent with the previously reported general toxicity caused by over expression of this gene. Further studies, examining all six of the IIV-6 induced Tots, with both loss- and gain-of-function approaches, will be necessary to more fully examine this possibility (West, 2018).
The sensitivity of STAT knockdowns to IIV-6 infection argues that JAK-STAT signaling is an important antiviral target of p38b. However, other p38b targets are also possible. For example, an established target of p38b is the heat shock response. In the context of bacterial and fungal infections, p38b is known to regulate Heat shock factor (Hsf) expression and the induction of heat shock proteins (Hsps). In addition, another report has shown that Hsf protects flies against both RNA and DNA viral infections. Together, these results suggest that the antiviral effects of p38b could be mediated, at least in part, through Hsf and Hsps. Indeed, Hsf mutant flies display an increased rate of death after IIV-6 infection. It will be interesting to learn if the heat shock response is activated by p38b following IIV-6 infection, and how this response interacts with JAK-STAT dependent viral protection (West, 2018).
Successful host defenses detect multiple characteristics of an invading pathogen. For example, cellular damage is one common indicator of pathogenic infection that can be sensed by the innate immune system. In mammals, several danger-associated molecular patterns (DAMPs) have been characterized, including HMGB1, F-actin, and histones. Likewise, a recent report examining a Drosophila model of sterile injury demonstrated that extracellular actin activates JAK-STAT signaling. In this paradigm, detection of extracellular actin, via an unknown receptor, triggered Nox-dependent ROS generation, the activation of Src42A and Shark (Syk homolog), and induction of unpaireds and eventually Tots. This pathway is very similar to that reported in this study, although p38b was not examined in this actin-DAMP, and suggests that IIV-6 infection may cause cellular damage, rupture and the release of actin, which in turn triggers ROS production, unpaired expression, JAK-STAT signaling and the induction of Tots. Formally testing this model will be facilitated by the identification of an extracellular actin receptor (West, 2018).
In summary, this study has found a novel role for Drosophila p38b in protecting against DNA virus infection. Virus infection leads to p38b dependent responses, including the induction of the JAK-STAT activating cytokines, the Unpaireds, and the induction of downstream target genes such as the Tots. Based on the analysis of viral load, the p38b pathway appears to function primarily by increasing tolerance to IIV-6, as viral loads were not altered in the p38b strain. Whether the Tots contribute to this tolerance and, more generally, whether p38b induces a directly antiviral response, or relies entirely on the Unpaired and JAK-STAT signaling for its ability to tolerize against this viral infection will be probed in future studies (West, 2018).
A p38 DNA sequence deposited by the Drosophila Genome Project was identified by searching GenBank with the p38a (Mpk2) sequence. This genomic DNA sequence contains an open reading frame (p38b), which also does not contain an intron and encodes a putative protein of 365 amino acids. The deduced amino acid sequence is 75% identical to that of p38a (Z. Han, 1998).
date revised: 30 July 99
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