InteractiveFly: GeneBrief

Growth-blocking peptide 1: Biological Overview | References

Antimicrobial peptides (AMPs), major innate immune effectors, are induced to protect hosts against invading microorganisms. AMPs are also induced under non-infectious stress; however, the signaling pathways of non-infectious stress-induced AMP expression are yet unclear. This study demonstrates that growth-blocking peptide (GBP) is a potent cytokine that regulates stressor-induced AMP expression in insects. GBP overexpression in Drosophila elevates expression of AMPs. GBP-induced AMP expression does not require Toll and immune deficiency (Imd) pathway-related genes, but imd and basket are essential, indicating that GBP signaling in Drosophila does not use the orthodox Toll or Imd pathway but uses the JNK pathway after association with the adaptor protein Imd. The enhancement of AMP expression by non-infectious physical or environmental stressors is apparent in controls but not in GBP-knockdown larvae. These results indicate that the Drosophila GBP signaling pathway mediates acute innate immune reactions under various stresses, regardless of whether they are infectious or non-infectious (Tsuzuki, 2012).

The innate immune system of animals provides the first and most primitive line of defense against invading microorganisms. Antimicrobial peptides (AMPs) are produced as immune effector molecules to fight pathogenic infection, and the induction of AMPs is regulated through activation of the Toll and immune deficiency (Imd) pathways in Drosophila. Although the activation of both signaling pathways in response to infection has been extensively investigated, it is also known that changes in innate immune activities are sometimes unrelated to microbial infection. Various physical and physiological factors such as temperature, starvation, and diapause also elevate AMP expression levels. It is also known that AMP expression is highly sensitive to developmental stage in mammals as well as insects. Further, it has been recently reported that AMP expression in starved Drosophila is enhanced in response to the transcription factor FOXO, a key regulator of stress resistance, metabolism, and ageing, independently of the immunoregulatory pathways (Becker, 2010). Insulin signaling is currently the only known pathway for the induction of AMP expression by non-infectious stress. However, it is unlikely that animals cope with various non-infectious stressors by using the same signaling pathway that manages the regulation of innate immunity (Tsuzuki, 2012).

To investigate extracellular signaling in the innate immune regulation under non-infectious stresses, focus was placed on insect cytokines because cytokines in general regulate many physiological events including stress resistance through transmission of signals from outside the cell to the inside. While a large number of cytokines have been identified and their roles in mammals studied extensively, the number of known insect cytokines is quite limited. In Drosophila, spätzle is known as the cytokine that activates Toll signaling after microbial infection, which leads to expression of the target AMPs. In lepidopteran insects, structurally similar bioactive peptides have been reported in the last 20 years and are now recognized as the insect cytokine family referred to as the ENF peptides on the basis of their common N-terminal sequence, Glu-Asn-Phe- (Strand, 2000). These peptides are typically 23-25 amino acids long, and growth-blocking peptide (GBP) was the first member of this peptide family discovered (Hayakawa, 1990). Other known insect-specific cytokines include Unpaired-3, which was first identified in Drosophila, and hemocyte chemotactic peptide (HCP), identified in the armyworm Pseudaletia separata. Stress-responsive peptide (SRP) has also recently been identified in the common cutworm Spodoptera litura. Drosophila eda-like cell death trigger (eiger) HAS been reported to modify AMP expression levels. Among these insect cytokines, this study focused on characterizing the functional role of GBP in innate immunity because GBP was initially identified as the factor responsible for the reduced growth exhibited by armyworm P. separata larvae under stress conditions such as parasitization by the parasitoid wasp Cotesia kariyai and exposure to low temperature (Ohnishi, 1995). NMR analysis of GBP showed that it consists of flexible N- and C-termini, and a structured core stabilized by a disulfide bridge and a short antiparallel β-sheet (β-hairpin) (Aizawa, 1999). Structural comparisons indicated that the core β-hairpin region adopts the C-terminal subdomain structure of human epidermal growth factor. Consistent with this structural similarity, GBP at concentrations of 10^-1 to 10² pmol/ml induced proliferation of human keratinocytes as well as insect Sf 9 cells (Hayakawa, 1998; Tsuzuki, 2012 and references therein).

At least 16 members of the insect ENF cytokine family have been identified. They have diverse functions such as growth retardation, paralysis induction, cardioacceleration, embryogenic morphogenesis, and immune cell stimulation. Characterization of some of these peptide cDNAs demonstrated that the ENF peptides are synthesized as a precursor form in which the active peptide is located at the C-terminal region. Because it has been demonstrated that ENF family peptides stimulate insect immune cells like plasmatocytes to spread on foreign surfaces, this study first examined whether GBP affects humoral immune activity in a lepidopteran insect, the silkworm Bombyx mori. As expected, injection of B. mori GBP into B. mori larvae elevated the expression of AMPs. Further, GBP-induced elevation of AMP expression was demonstrated in silkworm larvae exposed to heat stress. Although this result demonstrated GBP-dependent induction of AMP expression in non-infected stressed silkworm larvae, elucidating in detail the pathway of GBP signaling in the immune system required analysis in Drosophila because little is known about the signaling pathways that activate AMP gene expression in non-Drosophila insects like B. mori. However, none of the ENF family cytokines have been identified in insect orders outside the Lepidoptera, making it necessary to identify the Drosophila GBP homolog. Database searches did not reveal any obvious homologs in the fly genome, which suggested either that the Diptera lack ENF genes or that members of this gene family might have diverged too much to be identified on a sequence level in Diptera. Therefore, a peptidergic factor with GBP-like activity was purified from the bluebottle fly Lucilia cuprina. Using the sequence of the bluebottle fly GBP homolog for motif and FASTA searches, five Drosophila melanogaster homologs were identified, among which CG15917 was most similar to lepidopteran GBPs in terms of the primary structure of its ORF. Overexpression and RNAi knockdown of GBP in the Drosophila larvae indicated that GBP regulates the expression of AMPs through a novel pathway associated with Imd and JNK. The Drosophila GBP signaling pathway stimulated AMP expression in Drosophila larvae in response to external stressors, whether they were infectious or non-infectious (Tsuzuki, 2012).

This study has revealed the innate immune activity of GBP in insects by demonstrating that it elevates expression levels of some AMPs in Drosophila larvae as well as in Bombyx larvae. In order to clarify the role and mechanism of GBP in inducing AMP expression in insects, its signaling pathway was analyzed in Drosophila because no detailed signaling pathway of an innate immune system had been recognized in non-Drosophila species. To perform the analyses, a GBP-like cytokine was isolated from bluebottle fly larvae, and then 46 Drosophila genes were identified encoding its homologous peptide by (a cysteine-based motif search) using the C-(X except for C)_6-8-C-(X except for C)_1-4 pattern. Among these genes, CG14069 and CG17244 were selected as candidate Drosophila GBP genes whose ORFs consist of 100-200 amino acid residues. Subsequent FASTA searches for the C-terminal 25-amino acid peptide sequence of CG14069 identified CG11395, CG15917, and CG12517. These five genes were found to encode GBP-like peptide sequences at the C-terminal ends. Among the five genes, the peptide gene most homologous to the bluebottle fly GBP and lepidopteran GBP genes were further selected based on the characteristics common to those GBP genes: the size (100-200 amino acids) of the ORF and the presence of an arginine residue at an appropriate position (7-9 residues upstream of the first cysteine). Further, after confirming the gene expression in the larval fat body, CG15917 remained as the most plausible candidate. The functional GBP peptide located in the C-terminal region of the CG15917 ORF stimulated aggregation of hemocytes from Drosophila y w larvae. Further, overexpression of CG15917 retarded larval development, creating a phenotype similar to that seen following injection of GBP into lepidopteran larvae. These results were interpreted as an indication that the CG15917 product has a physiological role identical to that of lepidopteran GBPs (Tsuzuki, 2012).

Overexpression of proGBP as well as GBP significantly elevated the expression of the antimicrobial peptides Mtk and Dpt with the aid of the adaptor protein Imd in Drosophila larvae. The results are partially consistent with previously published data on the Drosophila gene CG15917: the gene was detected, along with many other genes, by microarray analysis as being transcriptionally activated three hours after septic injury of Drosophila adults. It was further revealed that the GBP-dependent induction of Mtk expression was abolished in larvae whose bsk expression was reduced by RNAi, indicating that the GBP pathway stimulated AMP expression through JNK signaling after recruitment of Imd to the activated GBP receptor (GBPR). This interpretation was partially confirmed by showing that GBP induced Mtk expression independently of PGRP-LC, PGRP-LE, and Relish. The biological importance of GBP was demonstrated by the finding that RNAi targeting of GBP significantly repressed AMP expression during the initial phase of bacterial infection and consequently made the larvae more susceptible to the bacteria than control larvae. Moreover, GBP RNAi repression of AMP expression was found to occur when test larvae were subcutaneously damaged without bleeding by pinching. The GBP RNAi-induced repression of AMP expression was also observed in Drosophila larvae exposed to temperature stresses: AMP expression was significantly elevated in control larvae by transferring them from 4^oC to 25^oC or exposing to 4^oC, but it was not seen in GBP RNAi larvae. The results clearly show that induction of GBP-dependent AMP expression does not require a pathogen-associated molecular pattern, and that such non-infectious or non-injurious stimuli-dependent AMP expression is probably mediated by the GBP signaling pathway (Tsuzuki, 2012).

In lepidopteran larvae, GBP is abundantly present in a precursor form (proGBP) in hemolymph (Kamimura, 2001; Oda, 2010). The present experiments showed that this is also true for Drosophila. Non-injurious stimuli instantly trigger activation of GBP-processing enzyme(s) in hemolymph, by which the proGBP is proteolytically activated to GBP. The active GBP that results from the proteolytic processing under various stresses should trigger GBP-dependent AMP expression. It is reasonable to expect that such processing of proGBP enables the production of AMPs for the swift supply of active GBP under stress conditions. This interpretation is consistent with the fact that AMP expression was enhanced more by overexpression of active GBP than that of proGBP. Therefore, it is reasonable to propose that GBP serves as a key cytokine in the enhancement of AMP gene expression through the Imd/JNK pathway in Drosophila larvae exposed to various stressors, regardless of whether they are infectious or non-infectious (Tsuzuki, 2012).

Although there have been reports of the cross-regulation or cross-modification of two signaling pathways, Toll and Imd or JNK (or JAK/STAT) and NF-kappaB, the current knowledge is not still adequate to fully understand all mechanisms controlling the regulatory system. Further, recent studies suggest the existence of an evolutionarily conserved mechanism of cross-regulation of metabolism and innate immunity. It might be worth emphasizing that GBP was initially identified as a growth inhibitory factor in armyworm larvae. Although this study confirmed that GBP overexpression retards normal larval development in Drosophila, it was confirmed that this GBP-induced growth retardation did not affect AMP expression levels in test larvae under the present experimental conditions. Further, GBP-induced AMP expression was observed in Drosophila adults. Therefore, it is reasonable to propose that the insect cytokine GBP contributes to regulation of both growth and innate immunity. It may be possible to solve important problems concerning the adaptation of organismal defense to environmental stresses if the contributions of the novel signaling pathway through GBP-JNK are taken into account there (Tsuzuki, 2012).

Activation of PLC by an endogenous cytokine (GBP) in Drosophila S3 cells and its application as a model for studying inositol phosphate signalling through ITPK1

Using immortalized [³H]inositol-labelled S3 cells, this study has demonstrated that various elements of the inositol phosphate signalling cascade are recruited by a Drosophila homologue from a cytokine family of so-called GBPs (growth-blocking peptides) (see The pathways of inositol phosphate metabolism). HPLC analysis revealed that dGBP (Drosophila GBP) elevates Ins(1,4,5)P₃ levels 9-fold. By using fluorescent Ca²⁺ probes, it was determined that dGBP initially mobilizes Ca²⁺ from intracellular pools; the ensuing depletion of intracellular Ca²⁺ stores by dGBP subsequently activates a Ca²⁺ entry pathway. The addition of dsRNA (double-stranded RNA) to knock down expression of the Drosophila Ins(1,4,5)P₃ receptor almost completely eliminates mobilization of intracellular Ca²⁺ stores by dGBP. Taken together, the results of the present study describe a classical activation of PLC (phospholipase C) by dGBP. The peptide also promotes increases in the levels of other inositol phosphates with signalling credentials: Ins(1,3,4,5)P₄, Ins(1,4,5,6)P₄ and Ins(1,3,4,5,6)P₅. These results greatly expand the regulatory repertoire of the dGBP family, and also characterize S3 cells as a model for studying the regulation of inositol phosphate metabolism and signalling by endogenous cell-surface receptors. Therefore a cell-line (S3^ITPK1) was created in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. It was found that dGBP-stimulated S3^ITPK1 cells did not synthesize Ins(3,4,5,6)P₄, contradicting a hypothesis that the PLC-coupled phosphotransferase activity of ITPK1 [Ins(1,3,4,5,6)P₅+Ins(1,3,4)P₃→Ins(3,4,5,6)P₄+Ins(1,3,4,6)P₄] is driven solely by the laws of mass action. This conclusion represents a fundamental breach in understanding of ITPK1 signalling (Zhou, 2012).

Stimulus-dependent activation of PLC (phospholipase C) hydrolyses PtdIns(4,5)P₂ to generate two intracellular messengers: diacylglycerol which activates protein kinase C, and Ins(1,4,5)P₃, which binds to specific receptors {IP3R [Ins(1,4,5)P₃ receptor]; itpr in Drosophila} that gate intracellular Ca²⁺ stores. This is a ubiquitous signalling response that regulates diverse aspects of cellular biology in almost all animal cell types. As such, it is an intensively studied area of cell biology. Additionally, research into the metabolism of Ins(1,4,5)P₃ has spurred the development of a largely separate signalling industry: the study of an elaborate network of interconnected metabolites, many of which have multiple biological functions. Thus the discovery that PLC is activated by an extracellular agonist endows that agent with a large number of new signalling activities (Zhou, 2012).

Drosophila melanogaster is a genetically-tractable eukaryotic model that has proved useful in unraveling the complexities of many aspects of Ca²⁺ signalling. For example, experiments with Drosophila mutants have demonstrated that the fly's single itpr gene is important for a range of physiological response. Cultured Drosophila cells have also been used to study the receptor-dependent activation of Ins(1,4,5)P₃ production and Ca²⁺ mobilization. However, the range of opportunities offered by this model organism have not yet been fully exploited for studies into the function and agonist-regulated metabolism of the other inositol phosphate signals. Such studies, which typically rely on analysing cells that have been radiolabelled with [³H]inositol during several days of proliferation, are best suited to immortalized cell lines. Such cells have been derived from Drosophila. However, there is limited insight into the nature of peptides that act through endogenous receptors to activate PLC in these immortalized cells. The application of these cells to inositol phosphate research has therefore been restricted. To bypass this problem, some groups have heterologously expressed exogenous receptors into immortalized Drosophila cells. A more physiologically relevant model is described that utilizes endogenous receptors: the S3 imaginal-disc cell line, in which this study shows that PLC is activated by a recently discovered insect cytokine, dGBP [Drosophila GBP (growth-blocking peptide)](Zhou, 2012).

dGBP is a member of a large family of insect cytokines. These peptides range from 19-30 amino acid residues in length, and are produced upon proteolytic cleavage of longer prepropeptides. The first GBP to be identified was the factor responsible for the reduction in the rate of growth of lepidopteran larvae following their colonization by the larvae of a parasitic wasp, Costesia kariyai. The endocrinological perturbation that up-regulates GBP synthesis in the host stops it from forming the sclerotized pupal cuticle that would otherwise prevent the parasitic larvae from emerging. The GBP family also regulate morphogenesis, cell proliferation and innate immune responses, for example by stimulating plasmatocyte adhesion and spreading However, to date little progress has been made in understanding the molecular mechanisms of action of these cytokines. Recently, the discovery of dGBP has revealed that this peptide acts through c-Jun N-terminal kinase to regulate gene expression. However, in view of the multi-functionality of the GBP family, it has remained probable that these peptides recruit additional signalling pathways. The present study adds substantially to dGBP's signalling repertoire by demonstrating its ability to activate multiple facets of the PLC-dependent inositol phosphate/Ca²⁺ cascade (Zhou, 2012).

An increased insight into the molecular actions of insect cytokines such as dGBP is of interest in itself, but the importance of this field of research goes beyond the goal of expanding understanding of insect physiology. Knowledge of the roles of GBPs in immune responses in pest insects that impair human health or reduce crop yield may lead to the development of improved control programs. There is also considerable evolutionary conservation of innate immunity genes, pathways and effector mechanisms. Research into these defense mechanisms in Drosophila may ultimately improve understanding of human immune responses. Indeed, the identification of the human homologue of the Toll receptor was initially prompted by the discovery that this protein mediates antifungal immune responses in Drosophila (Zhou, 2012).

Another goal of the present study was to determine whether the discovery that dGBP activates PLC in S3 cells could be exploited to explore the signalling activities of inositol phosphate metabolizing enzymes from higher animals. In pursuit of this idea, it is noted that the Drosophila genome does not encode a homologue of the mammalian ITPK1 (inositol tetrakisphosphate 1-kinase). Therefore S3 cells offer a rare example of a model for characterizing any gain-of-function that might arise from the heterologous expression of human ITPK1. For example, the phosphorylation of Ins(1,3,4)P₃ by ITPK1 in mammals has been proposed to be the rate-limiting step in the synthesis of Ins(1,3,4,5,6)P₅ and InsP₆. However, there continue to be differences of opinion as to the relative importance of this pathway compared with the alternative ITPK1-independent route to Ins(1,3,4,5,6)P₅ and InsP₆. This study aimed to address this debate by studying the impact of ITPK1 upon Ins(1,3,4,5,6)P₅ and InsP₆ synthesis in basal and PLC-activated S3 cells. Furthermore, ITPK1 has additional significance to mammalian cell signalling because this enzyme controls the synthesis of Ins(3,4,5,6)P₄, which regulates the conductance of the Cl⁻ channel/transporter ClC-3 (chloride channel, voltage-sensitive 3). In this way, ITPK1 helps regulate a number of physiological processes, such as salt and fluid secretion, insulin secretion, and neurotransmission. However, it has proved difficult to characterize the mechanism by which Ins(3,4,5,6)P₄ synthesis is accelerated following PLC activation. To explain this phenomenon, previous studies put forward a hypothesis that is based on the laws of mass action. This study proposes that an elevated rate of phosphorylation of Ins(1,3,4)P₃ to Ins(1,3,4,6)P₄ by ITPK1 is coupled through an unusual phosphotransferase reaction to an increased rate of dephosphorylation of Ins(1,3,4,5,6)P₅ to Ins(3,4,5,6)P₄. The present study has tested this hypothesis by determining whether PLC-dependent Ins(3,4,5,6)P₄ synthesis could be heterologously introduced into S3 cells by the expression of ITPK1 (Zhou, 2012).

A central conclusion in the present study is that PLC is activated by dGBP, a member of a multi-functional family of insect cytokines. The Drosophila homologue of the GBP family was identified only quite recently, and it has been shown to stimulate transcription of antimicrobial peptides by activation of c-Jun NH₂-terminal kinase. However, the GBP family is known to regulate other diverse aspects of insect cell physiology, including larval growth, morphogenesis and cell proliferation. Therefore it was thw goal of this study to search for additional signalling pathways that dGBP might recruit, in order to gain a more complete understanding of the mechanisms that underlie the various actions of these cytokines. The demonstration that dGBP activates PLC in S3 cells opens up a number of directions for further research into insect physiology. PLC-mediated hydrolysis of PtdIns(4,5)P₂ yields diacylglycerol, which activates protein kinase C, and Ins(1,4,5)P₃, which binds itpr and gates intracellular Ca²⁺ stores. Furthermore dGBP also promotes increases in a number of other inositol phosphates, several of which have their own distinct cellular functions (Zhou, 2012).

This study used several experimental approaches to ascertain that, in S3 cells, dGBP mobilizes Ca²⁺ from intracellular Ins(1,4,5)P₃-releasable stores as a consequence of PLC activation. First, HPLC and chemical analyses were used to determine that dGBP promotes the increases in Ins(1,4,5)P₃ and related metabolites that are typical of agonist-dependent stimulation of PLC activity in mammalian cells. Secondly, fluorescent Ca²⁺ probes were used to demonstrate that dGBP promotes the biphasic Ca²⁺ response that is typically observed following the activation of PLC-coupled receptors: depletion of intracellular Ca²⁺ stores, which then drives enhanced Ca²⁺ entry. Thirdly, dsRNA was used to knock down itpr expression, whereupon the ability of dGBP to mobilize intracellular Ca²⁺ stores was almost completely eliminated (Zhou, 2012).

The various elements of the inositol phosphate cascade that are activated by dGBP have independent functions, and so may contribute to the peptide's multi-functionality. The Ins(1,4,5)P₃-mediated mobilization of Ca²⁺ probably mediates several physiological responses. The dGBP-mediated increases in Ins(1,3,4,5)P₄ levels may also have some signalling significance. Changes in Ins(1,4,5,6)P₄ levels also promise to be important, in the light of evidence that Ins(1,4,5,6)P₄ regulates gene transcription. Ins(1,3,4,5,6)P₅ levels in wild-type S3 cells were also elevated by 70% within 2 min of dGBP addition. Such a sizable Ins(1,3,4,5,6)P₅ response to PLC activation is not usually observed in mammalian cells. However, in the murine F9 teratocarcinoma cell line, PLC activation has been associated with large increases in Ins(1,3,4,5,6)P₅ levels, which regulate key aspects of the β-catenin signalling pathway. In F9 cells, Ins(1,3,4,5,6)P₅ synthesis occurs primarily through direct phosphorylation of Ins(1,4,5)P₃ by IPMK (inositol polyphosphate multikinase). This is also the case in Drosophila. Thus S3 cells may be of value for further exploring these signalling functions for Ins(1,3,4,5,6)P₅ and their contribution to the physiology of dGBP (Zhou, 2012).

The genetic tractability of Drosophila S3 cells is also an advantage when using them to explore the mechanisms of agonist-dependent regulation of the metabolism and functions of the inositol phosphate family. The present study exploited the observation (Seeds, 2004) that the Drosophila genome is itpk1-null, and so human ITPK1 was heterologously expressed in S3 cells in order to gain insight into the signalling functions of that particular kinase. These experiments yielded three significant new conclusions with regards to the actions of ITPK1 (Zhou, 2012).

First, data was obtained that are relevant to the ongoing debate concerning the degree to which ITPK1 contributes to Ins(1,3,4,5,6)P₅ synthesis in mammalian cells. Specifically, it was found that the expression of ITPK1 in S3 cells yielded several-fold increases in the steady-state levels of Ins(1,3,4,5,6)P₅ and one of its metabolites, Ins(1,4,5,6)P₄. Nevertheless, in both wild-type S3 and S3^ITPK1 cells, the ratio of Ins(1,3,4,5,6)P₅ to InsP₆ was relatively low compared with that in mammalian cells. These results suggest that factors other than ITPK1 expression itself contribute to the synthesis of the relatively large quantities of Ins(1,3,4,5,6)P₅ that are typically present in mammalian cells. For example, there is evidence that much of the mammalian cell's Ins(1,3,4,5,6)P₅ resides in a metabolically-resistant pool. The mechanism by which Ins(1,3,4,5,6)P₅ transfers into that pool in mammalian cells may be key to the accumulation of that inositol phosphate at relatively high levels (Zhou, 2012).

Secondly, the sizable accumulation of Ins(1,2,3,4,6)P₅ in PLC-activated S3^ITPK1 cells is also of interest. That result is attributed to the relatively high levels of Ins(1,3,4,6)P₄ successfully out-competing the lower levels of Ins(1,3,4,5,6)P₅ for phosphorylation by IP5K; such competition has previously been shown in vitro. Such a phenomenon could help explain a previously puzzling observation that the InsP₆ pool in rat-1 cells is largely unaffected when Ins(1,3,4,5,6)P₅ synthesis was compromised by knockdown of IPMK expression. An ITPK1-dependent alternative pathway of InsP₆ synthesis in rat-1 in cells could solve that problem: phosphorylation of Ins(1,3,4)P₃ into Ins(1,3,4,6)P₄, then into Ins(1,2,3,4,6)P₅ which then can be converted into InsP₆ (Zhou, 2012).

The third, and arguably the most important, observation to emerge from these studies with ITPK1, was the discovery that the phosphotransferase activity of ITPK1 is not sufficient by itself to recapitulate PLC-dependent synthesis of Ins(3,4,5,6)P₄, an intracellular signal that serves multiple biological roles through its regulation of Cl⁻ channel activity. Thus the results of the present study counter a previous hypothesis that Ins(3,4,5,6)P₄ levels are solely regulated by the mass-action effects of substrate supply that are exerted upon ITPK1. This outcome was unexpected, because previous structural and metabolic data had indicated that the phosphotransferase activity of human ITPK1 is inherently well-adapted to regulation by mass-action effects in vitro. Perhaps the absence of Ins(3,4,5,6)P₄ in PLC-activated S3^ITPK1 cells is because metabolic compartmentalization prevents ITPK1 from accessing Ins(1,3,4,5,6)P₅. It is also possible that there is a yet to be discovered mechanism, not active in S3 cells, by which the phosphotransferase activity of ITPK1 might be activated relative to its kinase activity. A potential mechanism might involve covalent modification of ITPK1, such as by its phosphorylation or acetylation. In any case, the present study indicates that the regulation of the Ins(3,4,5,6)P₄ signalling cascade is more complex than hitherto was appreciated. As the number of physiological responses that are regulated by Ins(3,4,5,6)P₄ increases, so the need to unravel these mechanisms becomes more urgent (Zhou, 2012).

In previous work, the heterologous expression in yeast of inositol phosphate kinases of animal and plant origin uncovered new aspects of inositol phosphate metabolism and signalling. However, the use of immortalized Drosophila cells as a host offers several advantages over the yeast model, including their more direct relevance to mammalian systems: unlike yeasts, flies have an Ins(1,4,5)P₃-releasable Ca²⁺ pool. Genetic manipulation by dsRNA is also cheap and effective in cultured fly cells. Furthermore, the demonstration that dGBP stimulates PLC adds value to S3 cells as a resource for further studies into receptor-dependent regulation of inositol phosphate function and metabolism. This work with ITPK1 offers an example of how that model can usefully be exploited. Finally, this study adds substantially to the repertoire of the GBP family by demonstrating its ability to activate multiple facets of the PLC-dependent inositol phosphate signalling cascade (Zhou, 2012).

Proteolytic activation of Growth-blocking peptides triggers calcium responses through the GPCR Mthl10 during epithelial wound detection

The presence of a wound triggers surrounding cells to initiate repair mechanisms, but it is not clear how cells initially detect wounds. In epithelial cells, the earliest known wound response, occurring within seconds, is a dramatic increase in cytosolic calcium. This study shows that wounds in the Drosophila notum trigger cytoplasmic calcium increase by activating extracellular cytokines, Growth-blocking peptides (Gbps; see Gbp1), which initiate signaling in surrounding epithelial cells through the G-protein-coupled receptor Methuselah-like 10 (Mthl10). Latent Gbps are present in unwounded tissue and are activated by proteolytic cleavage. Using wing discs, this study showed that multiple protease families can activate Gbps, suggesting that they act as a generalized protease-detector system. Experimental and computational evidence is presented that proteases released during wound-induced cell damage and lysis serve as the instructive signal: these proteases liberate Gbp ligands, which bind to Mthl10 receptors on surrounding epithelial cells, and activate downstream release of calcium (O'Connor, 2021).

When a tissue is wounded, the cells surrounding the wound rapidly respond to repair the damage. Despite the non-specific nature of cellular damage, there is remarkable specificity to the earliest cellular response: cells around the wound increase cytosolic calcium, and this damage response is conserved across the animal kingdom. The calcium response is not limited to cells at the wound margin but extends even to distal cells. Multiple molecular mechanisms have been identified that regulate wound-induced gene expression or cell behavior downstream of calcium, but the upstream signals remain unclear. How exactly do cells detect wounds? Thia study investigate the molecular mechanisms by which a wound initiates cytosolic calcium signals (O'Connor, 2021).

The immediate increase in cellular calcium in turn initiates repair or defense responses. Calcium has been well established as a versatile and universal intracellular signal that plays a role in the modulation of numerous intracellular processes. Several calcium-regulated processes are required for proper wound repair, including actomyosin dynamics, JNK pathway activation and plasma membrane repair. Unsurprisingly, an increase in cytosolic calcium is necessary for wound repair. Nonetheless, there is less clarity on the mechanisms that trigger increased cytosolic calcium in cells near to and distant from the wound. In some cases, wound-induced cytoplasmic calcium enters from the extracellular environment, either directly through plasma membrane damage. In others, calcium is released from the endoplasmic reticulum (ER) through the IP3 Receptor and initiated by an unknown G-protein-coupled receptor (GPCR) or receptor tyrosine kinase (RTK). Further, calcium responses can be initiated by mechanical stimuli alone. Elucidating the mechanisms by which calcium signaling is triggered in vivo is critical to understanding how wound information is transmitted through a tissue in order to change cellular behavior and properly repair the wound (O'Connor, 2021).

By live imaging laser wounds in Drosophila pupae, previously work showed that damaged cells around wounds become flooded within milliseconds by extracellular calcium entering through microtears in the plasma membrane. Although this calcium influx expands one or two cell diameters through gap junctions, it does not extend to more distal cells. Strikingly, after a delay of 45-75 s, a second independent calcium response expands outward to reach a larger number of distal cells. This study identified the relevant signal transduction pathway and receptor, the GPCR Mthl10. Downstream, signals are relayed through Gαq and PLCβ to release calcium from the ER. Upstream, Mthl10 is activated around wounds by the cytokine ligands Growth-blocking peptides (Gbps). Further, experimental and computational evidence is provided that the initiating event for the distal calcium response in vivo is a wound-induced release of proteases that activate the latent Gbp cytokines, cleaving them from inactive/pro-forms into active signaling molecules (O'Connor, 2021).

It was already known that Gbps are synthesized in an inactive pro-form, requiring proteolytic cleavage for activation, and that they are secreted by the fat body. Although Gbps are present in unwounded tissues, they activate Mthl10 only in the presence of a wound. Interestingly, Gbps have cleavage consensus sequences for multiple protease families. Further, the addition of cell lysate or the addition of the unrelated proteases trypsin or clostripain to unwounded tissue is sufficient to generate a calcium signal in wing discs through Mthl10/Gbp signaling. These results lead to a model in which the lysis of cells inherent in wounding releases non-specific cellular proteases into the extracellular environment. These proteases cleave and activate extracellular Gbps, which in turn activate the Mthl10 GPCR on cells around the wound, initiating wound-induced calcium signaling. Such cell lysis and protease release should be a general feature of cell destruction, whether caused by trauma, pathogen-induced lysis, or a lytic form of cell death such as pyroptosis or necroptosis (immunologically silent apoptosis may well be an exception). A variety of epithelial damage mechanisms may thus converge through the Gbps to signal via the GPCR Mthl10 and alert surrounding cells to the presence of a nearby wound. This molecular mechanism is supported by a computational model that accurately describes the pattern and timing of wound-induced calcium, predicted its dependence on wound size and initial levels of Gbps, and led to the observation that cell lysis is not immediate but rather takes place over tens of seconds. Thus, this study offers a model for how surrounding cells detect the damage of cell lysis, utilizing a Gbp-based protease-detector system (O'Connor, 2021).

Two superimposed mechanisms increase cytoplasmic calcium levels around wounds Laser wounds generate complex yet reproducible patterns of increased cytoplasmic calcium, and the complexity of this pattern has undoubtedly made it difficult to unravel its underlying mechanisms. Within the first ~90 s after wounding, two mechanisms drive the increase of calcium, and the complexity is generated by the temporal and spatial superimposition of these two mechanisms. Previously, it was reported that a different type of cellular damage initiates a different mechanism for increasing cytoplasmic calcium. In that report, wound-induced microtears were identified in the plasma membranes of surviving cells, and these microtears provided an entry for extracellular calcium to flood into the cytoplasm and then flow out to neighboring undamaged cells via gap junctions. This direct entry of calcium through damaged plasma membranes is evident within milliseconds after wounding. In this report we describe a second mechanism that extends to more distal cells, initiated by cell lysis at wounding. The dynamics of protease release from lysed cells, along with the gradual accumulation of active Gbp and its rapid diffusion, all contribute to the appearance of this distal calcium response 45-75 s after wounding. The earliest and closest cells to be activated by Mthl10/Gbp signaling cannot be identified visually because the initial flood of calcium through microtears takes time to subside (O'Connor, 2021).

Three tools allowed deciphering od these superimposed mechanisms. The first tool was the laser itself, which generates a highly stereotyped pattern of damage within a circular wound bed. Although cell lysis and plasma membrane damage are features of nearly every wound, their reproducible pattern in a laser wound allowed distinguishing the signaling mechanisms each type of damage potentiated. The second tool was a spatiotemporal analytical framework to measure radius over time, which clearly identified two peaks, the first induced by microtears and the second induced by cell lysis. The third tool was experimental, using RNAi-knockdown of genes in a limited region and comparing it with an internal control. The ability to identify asymmetry between the control and experimental sides of wounds allowed bypassing of concerns about variable wound sizes, which otherwise would have made it difficult to recognize patterns and interpret data. Complex overlapping patterns may have obscured the mechanisms upstream of wound-induced calcium in other systems as well as the current one (O'Connor, 2021).

Previous studies identified other molecules and phenomena upstream of wound-induced calcium. Studies in cell culture found that wound-generated cell lysis releases ATP, which diffuses extracellularly to bind to purinergic receptors and activate calcium release from intracellular stores. Although reproducible in many types of cultured cells, there has been little evidence to support ATP signaling from lytic cells in vivo, likely because extracellular ATP is rapidly hydrolyzed by nucleotidases in vivo. Interestingly, ATP does appear to signal damage and promote motility in response to injuries associated with cell swelling in zebrafish, animals that inhabit a hypotonic aqueous environment; however, even in this wounding paradigm, ATP does not signal from lytic cells at an appreciable level. No evidence was found for ATP signaling upstream of calcium in the wounding experiments, as knockdown of the only fly adenosine receptor did not alter the calcium pattern around wounds (O'Connor, 2021).

Some in vivo studies have implicated a TrpM ion channel upstream of calcium release. This role of TrpM was first identified in laser-wounding studies of the C. elegans hypodermis, a giant syncytial cell where great overlap in the spatial extent of microtear-initiated calcium, which would diffuse quickly throughout a syncytium, and receptor-mediated calcium released from the ER would be expected. In the hypodermis, loss of TrpM reduced by half the intensity of wound-induced calcium signaling, but without spatial and temporal analysis, the exact contribution of TrpM is not known. In the Drosophila notum, a previous study identified TrpM as a regulator of wound-induced actin remodeling, and a slight reduction in wound-induced calcium intensity over time was noted in TrpM knockdowns. In contrast, this study did not observe any change in the spatial or temporal aspects of the calcium response in TrpM knockdown cells compared with the internal control, and given wound-to-wound variability, it would have been hard to identify a small effect without an internal control. A study in the fly embryo determined that wound-induced calcium originates from both the external environment and internal stores, suggesting to that two superimposed calcium response mechanisms may have been at play in these experiments. That study found when TrpM was knocked down, calcium intensity was reduced by half, but again without spatial and temporal analysis or an internal control, it is difficult to know what pathway TrpM regulates. Tissue mechanics are upstream of increases in cytoplasmic calcium in a non-wounding context. Several labs have reported calcium flashes and waves in unwounded wing discs, dissected from larvae and cultured ex vivo. Cell and organ culture requires serum to support metabolism outside the organism, and in fly culture, this 'serum' is generated by grinding whole adult flies and collecting the supernatant. Because such serum would undoubtedly contain secreted signals from wounded cells, calcium signaling in wing discs ex vivo is probably a wound response; indeed, it was founs to be transduced by the same mechanism as wound signals, requiring protease, Gbps, and Mthl10. One aspect of calcium signaling in wing discs that we have not tested in our wounding model, however, is the role of mechanical tension. In carefully controlled mechanical experiments, fly serum was found to induce calcium flashes in wing discs specifically on the release of mechanical compression, indicating that tension is a requirement for calcium signaling in these wing discs. It is interesting to consider the TrpM results in light of these mechanical studies, as some TrpM channels can be mechanosensitive. Together, these data suggest that there may be a role for mechanical tension in wound-induced calcium responses (O'Connor, 2021).

Two independent mechanisms were founs that increase cytoplasmic calcium, and in the cells at the wound margin these mechanisms would appear to act redundantly. Such redundancy indicates that the role of calcium in these cells is very important for wound healing. One biological pathway that may be downstream of calcium in these cells is recruitment of actin and myosin to the wound margin to form an actomyosin purse string that cinches the wound closed. What about calcium in the distal cells, regulated by Gbp/Mthl10? There are many possible functions, but currently, all of them are speculative. One possibility is that the cytosolic calcium response initiates distal epithelial cells to modify their cellular behavior from a stationary/non-proliferative state to a migratory and/or proliferative state necessary to repair the wound. Alternatively, an increase in cytosolic calcium may act to modulate an inflammatory response through DUOX leading to the formation of hydrogen peroxide to recruit inflammatory cells to the wound or through the calcium-dependent activation of cytoplasmic Phospholipase A2 leading to the rapid recruitment of immune cells to tissue damage. This possibility is intriguing because Gbp is known to activate an immune response leading to the upregulation of antimicrobial peptides and to increased activity of phagocytic plasmatocytes in a calcium-dependent manner. Interestingly, loss of Methuselah-like (Mthl) GPCRs results in increased lifespans, and Gbps are nutrition-sensitive peptides whose expression is reduced under starvation conditions. Increased lifespan, caloric restriction and decreased inflammation have all been linked, and Gbp/Mthl10 activation at wounds may be part of this link (O'Connor, 2021).

Although the cytokine and GPCR families are widely conserved, Gbp and Mthl10 do not have direct orthologs in chordates. Nonetheless, similarities exist between the Gbp/Mthl10 mechanism and wound responses in other organisms. Damage- or pathogen-induced activation of proteins by proteolytic cleavage has been well documented in the cases of Spatzle in the Toll pathway, thrombin and fibrin in the blood coagulation pathway, and IL-1β and IL-18 in the pyroptosis pathway. Additionally, wound-defense signaling in plants relies on an immunomodulatory plant elicitor peptide that is cleaved into its active form by cysteine proteases upon damage-induced cytosolic calcium, and the plant defense hormone systemin is cleaved into its active form by phytaspases in response to damage or predation. Because the basic circuitry is similar across kingdoms, the current study suggests an ancient strategy for wound detection based on proprotein cleavage, activated by proteases released via cell lysis. As these examples make clear, proteases are already known to play critical roles in blood clotting and immune signaling, and this study finds that proteases are also instructive signals in epithelial wound detection (O'Connor, 2021).

As noted above, the Gbp ligands and Mthl10 receptor are not present in mammals, so the extent of mechanistic conservation is unclear. Further, this study did not experimentally tested this wound-detection mechanism in other developmental stages of Drosophila. For the computational model, several simplifications were made: the use of one variable for all proteases and one variable for all Gbps, rather than having separate Gbp1 and Gbp2; the use of simplified receptor/ligand dynamics that do not include uptake or recycling; and the use of a ligand-receptor-binding threshold rather than inclusion of the signal transduction cascade between receptor-binding and calcium release. Finally, this study does not describe or address the mechanism behind the calcium flares that continue for at least one houe after wounding (O'Connor, 2021).

Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress

This study used Drosophila to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca(2+) signaling pathway mediates innate immune responses to GBP, this study conducted a dsRNA library screen for genes that modulate Ca(2+) mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. The metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This was established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. It was further reported that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these homeostatic attributes of GBP: Elevated GBP expression reduces lifespan. Conversely, Mthl10 knockdown extended lifespan (Sung, 2017).

The most important development to emerge from this study is the deorphanization of Mthl10, through the placement of this GPCR at the epicenter of a molecular pathway that pits stress responses against lifespan. Various immunological and metabolic properties of a single cytokine, GBP, are integrated through its interactions with Mthl10. In particular, it was shown how the operation of the GBP/Mthl10 axis usefully matches nutrient supply to the degree of a metabolically expensive inflammatory response; this is an important topic in immunology. The model for GBP/Mthl10 functionality also shows how it has the potential to exacerbate metabolic inflammation; this may be one of the reasons that nutrient excess in Drosophila can model human metabolic syndrome. Furthermore, these homeostatic functions for Mthl10 were linked to its strong influence upon longevity. This provides a molecular foundation for a theory of aging, namely, that a shortened lifespan can be the ultimate price that a young organism pays to successfully combat short-term environmental stresses (Sung, 2017).

These findings were considered in relation to previous work that provides a detailed analysis of the expression pattern of Mthl10 in Drosophila embryos and larvae. For example, due to extensive expression of Mthl10 in imaginal discs, it has been proposed this gene may influence organogenesis. It is therefore relevant that cytokines-including the Mthl10 ligand, GBP-are well-known to regulate tissue remodeling and development. Additionally, the determination that Mthl10 regulates GBP-mediated innate immune responses seems pertinent to earlier observations that Mthl10 is expressed in hematopoietic tissue (which has immunological functions) and also crystal cells, which encapsulate foreign material. Nevertheless, the possibility cannot be excluded that other ligands for Mthl10 remain to be identified, perhaps as a consequence of the expression of alternate Mthl10 transcripts (Sung, 2017).

The significance of Mthl10 to longevity and metabolism is shared by Mth. In fact, it was the first gene duplication within the Mth superclade that is believed to have given rise to Mthl10, which did not then undergo any further expansion in Drosophila. In contrast, five further rounds of gene duplication apparently occurred before Mth emerged. Thus, it is concluded that the connection between lifespan and metabolic homeostasis that was observed for Mthl10 is an ancestral trait rather than adaptive specifically to Mth (Sung, 2017).

It is not unusual for gene regulatory networks to be widely conserved, even when certain components might undergo evolutionary turnover. Indeed, recent work has shown that although selection pressure has caused GPCR ectodomains and their ligands to codiversify, there has nevertheless been considerable conservation of the receptor's intracellular interactions with G proteins; as a result, flies and mammals share many of the same downstream signaling cascades. Indeed, GBP exhibits some sequence similarity with the human defensin BD2; both are small, cationic cytokines produced by protease action upon larger, precursor proteins. Furthermore, human BD2 acts through an uncharacterized GPCR to stimulate PLC/Ca2+ signaling to initiate inflammatory responses; the current study demonstrates that GBP is also a GPCR ligand that initiates PLC/Ca2+ signaling. Thus, it is proposed that there is general applicability to the concepts that emerge from our integration of immunological, metabolic, and lifespan functions for the GBP/Mthl10 axis (Sung, 2017).

Search PubMed for articles about Drosophila Gbp

Aizawa, T., Fujitani, N., Hayakawa, Y., Ohnishi, A., Ohkubo, T., Kumaki, Y., Kawano, K., Hikichi, K. and Nitta, K. (1999). Solution structure of an insect growth factor, growth-blocking peptide. J Biol Chem 274: 1887-1890. PubMed ID: 9890941

Becker, T., Loch, G., Beyer, M., Zinke, I., Aschenbrenner, A. C., Carrera, P., Inhester, T., Schultze, J. L. and Hoch, M. (2010). FOXO-dependent regulation of innate immune homeostasis. Nature 463: 369-373. PubMed ID: 20090753

Hayakawa, Y. (1990). Juvenile hormone esterase activity repressive factor in the plasma of parasitized insect larvae. J Biol Chem 265: 10813-10816. PubMed ID: 2358440

Hayakawa, Y. and Ohnishi, A. (1998). Cell growth activity of growth-blocking peptide. Biochem Biophys Res Commun 250: 194-199. PubMed ID: 9753606

Kamimura, M., Nakahara, Y., Kanamori, Y., Tsuzuki, S., Hayakawa, Y. and Kiuchi, M. (2001). Molecular cloning of silkworm paralytic peptide and its developmental regulation. Biochem Biophys Res Commun 286: 67-73. PubMed ID: 11485309

O'Connor, J. T., Stevens, A. C., Shannon, E. K., Akbar, F. B., LaFever, K. S., Narayanan, N. P., Gailey, C. D., Hutson, M. S. and Page-McCaw, A. (2021). Proteolytic activation of Growth-blocking peptides triggers calcium responses through the GPCR Mthl10 during epithelial wound detection. Dev Cell. PubMed ID: 34273275

Oda, Y., Matsumoto, H., Kurakake, M., Ochiai, M., Ohnishi, A. and Hayakawa, Y. (2010). Adaptor protein is essential for insect cytokine signaling in hemocytes. Proc Natl Acad Sci U S A 107: 15862-15867. PubMed ID: 20798052

Ohnishi, A., Hayakawa, Y., Matsuda, Y., Kwon, K. W., Takahashi, T. A. and Sekiguchi, S. (1995). Growth-blocking peptide titer during larval development of parasitized and cold-stressed armyworm. Insect Biochem Mol Biol 25: 1121-1127. PubMed ID: 8580912

Seeds, A. M., Sandquist, J. C., Spana, E. P. and York, J. D. (2004). A molecular basis for inositol polyphosphate synthesis in Drosophila melanogaster. J Biol Chem 279: 47222-47232. PubMed ID: 15322119

Strand, M. R., Hayakawa, Y. and Clark, K. D. (2000). Plasmatocyte spreading peptide (PSP1) and growth blocking peptide (GBP) are multifunctional homologs. J Insect Physiol 46: 817-824. PubMed ID: 10742531

Sung, E. J., et al. (2017). Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress. Proc Natl Acad Sci U S A 114(52): 13786-13791. PubMed ID: 29229844

Tsuzuki, S., Ochiai, M., Matsumoto, H., Kurata, S., Ohnishi, A. and Hayakawa, Y. (2012). Drosophila growth-blocking peptide-like factor mediates acute immune reactions during infectious and non-infectious stress. Sci Rep 2: 210. PubMed ID: 22355724

Zhou, Y., Wu, S., Wang, H., Hayakawa, Y., Bird, G. S. and Shears, S. B. (2012). Activation of PLC by an endogenous cytokine (GBP) in Drosophila S3 cells and its application as a model for studying inositol phosphate signalling through ITPK1. Biochem J 448: 273-283. PubMed ID: 22928859

date revised: 26 December 2023

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.

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