InteractiveFly: GeneBrief

Peroxinectin-like: Biological Overview | References

BIOLOGICAL OVERVIEW

Prostaglandins (PGs)-lipid signals produced downstream of cyclooxygenase (COX) enzymes-regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. This study defines a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton-temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin remodeling, including actin filaments and aggregates, within the posterior nurse cells of S9 follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts-RC) and Villin (Quail), an actin bundler, localize to all early actin structures, whereas Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to a model in which PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, whereas at S10B, it promotes Ena-dependent actin remodeling (Spracklen, 2014).

Development and adult tissue homeostasis require dramatic movements and reorganization of both cells and whole tissues. Underlying all of these processes is the actin cytoskeleton, which serves as a dynamic scaffold to facilitate cell migration, cell division, and cell shape. Tight regulation of actin cytoskeletal dynamics is mediated by the concerted activity of over one hundred known actin binding proteins. While much is known about how the activity of individual actin binding proteins are regulated, very little is known about the mechanisms by which the activity of multiple actin binding proteins is coordinated to mediate developmental processes and tissue homeostasis (Spracklen, 2014).

One possible mechanism by which such coordination may occur is through prostaglandin (PG) signaling. PGs are small, bioactive lipids that act as paracrine and autocrine signaling molecules to regulate numerous physiological processes including pain, inflammation, fertility, and cardiovascular function. PGs are synthesized downstream of cyclooxygenase enzymes (COX1 and COX2), which convert free arachidonic acid into the precursor PGH2, and are the pharmacologic targets of non-steroidal anti-inflammatory drugs. PGH2 is then processed into biologically active prostanoids (including PGD₂, PGE₂, PGF_2α, PGI₂, and TXA₂) downstream of COX enzymes through the activity of specific synthases. Following their synthesis, PGs most commonly serve as ligands for specific G protein-coupled receptors (GPCRs), which elicit their downstream effects through activation of Gα and, in some cases, Gβγ. Additionally, PGs may induce MAPK signaling pathways, activate Rho GTPases, or serve as PPARγ nuclear hormone receptor ligands (Spracklen, 2014).

In vitro studies have provided evidence that PG signaling can regulate the actin cytoskeleton in both a cell-type and PG-type dependent manner. For example, TXA₂ and PGF2_α stimulate actomyosin-based contractility, whereas PGE2 and PGI2 promote relaxation in hepatic stellate cells. Subsequently, PGs were found to have opposing effects on cytoplasmic actin filaments (i.e., actin stress fibers) in multiple cell types. While PGE₂ promotes actin stress fiber assembly in rat IMCD cells and stability in IEC-6 cells, it induces actin stress fiber disassembly in A431 cells, HeLa cells, rat-1 fibroblasts and human aortic smooth muscle cells. Similarly, both PGE2 and PGI2 promote actin stress fiber disassembly in human pulmonary artery endothelial cells. PGF_2α promotes filopodia retraction and actin stress fiber assembly in 293-EBNA cells. In human umbilical vein endothelial cells, TXA₂ slows α_vβ₃-dependent cell adhesion and inhibits cell spreading, while PGE2 accelerates cell adhesion and promotes cell spreading. Interestingly, cytoskeletal inputs (i.e., mechanical stretching) have been shown to induce COX2- dependent production of PGE₂, which subsequently leads to disassembly of actin stress fibers in murine podocytes (Spracklen, 2014 and references therein).

PG signaling is also known to directly regulate platelet activation and aggregation, which requires actin cytoskeletal remodeling, including the rapid generation of filopodia that mediate protrusion and adhesion. TXA₂, the major prostanoid produced in human platelets, is a potent activator of platelet aggregation, while PGI₂, PGE₁, and PGD₂ inhibit platelet aggregation. Additionally, PGE₂ has been shown to both potentiate and inhibit platelet aggregation (Spracklen, 2014 and references therein).

The above-mentioned studies have provided some insight into the mechanisms by which PGs regulate cytoplasmic actin filament remodeling. Multiple in vitro studies demonstrate that the PG-dependent morphological changes in cytoplasmic actin bundles occur via cAMPdependent mechanisms, albeit through different downstream events including: cAMP-dependent kinase (PKA) and nucleotide exchange proteins directly activated by cAMP (Epac1)/Ras-related protein 1 (Rap1)-dependent activation of Rac (Birukova, 2007), PKA-dependent Rac activation and Rac-independent activities (Dormond, 2002), or PKA-dependent decreases in focal adhesion kinase (FAK) phosphorylation (Bulin, 2005). Other in vitro studies implicate Rho activation downstream of PGs in driving the changes in actin stress fiber assembly. Furthermore, PGI₂ and PGE1 block platelet activation through cAMP/cGMPdependent phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of actin elongation factors. Thus, while these studies have provided some insight into the mechanisms by which PG signaling regulates cytoplasmic actin filament assembly/disassembly, much remains to be determined including how multiple PG signals are integrated to coordinate actin remodeling and the mechanisms through which particular PG signals regulate actin dynamics (Spracklen, 2014 and references therein).

Drosophila oogenesis is a well-established model system for studying actin cytoskeletal remodeling and regulation. It consists of 14 well-characterized, morphological stages of follicle development. At stage 9 (S9) of follicle development, the follicle consists of 16 germline-derived cells (15 support or nurse cells and a single oocyte), which are surrounded by ~1000 somatically-derived epithelial cells. Multiple processes occur during S9 that are critical for female fertility. A small group (6-8) of cells, termed border cells, delaminate from the anterior of the follicle and migrate between the nurse cells toward the dorsal-anterior of the oocyte, while the remaining follicle cells migrate posteriorly over the nurse cells and oocyte to form an anterior-posterior gradient of follicle cell thickness. During S9, the oocyte actively takes up yolk granules from the hemolymph, and microtubule-dependent, slow cytoplasmic streaming establishes oocyte polarity. Aside from cortical actin deposits, the cytoplasm of the nurse cells is largely devoid of actin filament bundles through the end of stage 10A (S10A). During stage 10B (S10B), the actin cytoskeleton within the nurse cells undergoes rapid remodeling resulting in increased cortical actin deposition and the formation of a cage-like network of parallel actin filament bundles extending from the nurse cell membranes inward, toward the nurse cell nuclei. This dramatic actin remodeling is required to provide the contractile force necessary for the rapid transfer of nurse cell cytoplasm (nurse cell dumping) into the growing oocyte at S11, while preventing the nurse cell nuclei from obstructing the ring canals-specialized cytoplasmic bridges-that the cytoplasm must flow through (Spracklen, 2014).

Previous studies have identified critical roles for PG signaling in regulating actin bundle formation during S10B (Tootle, 2008) and gene expression (Tootle, 2011) during Drosophila oogenesis. Using this same model, Fascin, an actin bundling protein, has been established as a novel downstream target of PG signaling during PG-dependent actin remodeling during S10B (Groen, 2012). Thus, Drosophila oogenesis is an attractive model for identifying the likely conserved mechanisms by which PG signaling coordinates actin cytoskeletal remodeling. This study shows that PG signaling temporally regulates the onset of actin remodeling during Drosophila oogenesis. While prior studies have largely focused on the cytoskeletal events occurring during S10B, this study primarily focused on the previously undescribed role of PGs in preventing actin filament formation during S9. Wild-type S9 follicles exhibit a low level of early actin structures in the posterior nurse cells, whereas loss of Pxt, the Drosophila COX-like enzyme, results in the highly penetrant presence of extensive actin filament and aggregate formation in the posterior nurse cells at S9. This study found that two actin binding proteins, Hts-RC (Adducin) and Quail (Villin), localize to early actin structures in wild type and pxt mutant S9 follicles, while Enabled (Ena), the sole Ena/VASP family member found in Drosophila, localizes preferentially to those early actin structures found in pxt mutants. Furthermore, genetic reduction of Ena in pxt mutants suppresses this early actin remodeling. Additionally, this study found that Ena localization to the sites of parallel actin filament bundle formation at S10B is reduced in pxt mutants. Together, these data are consistent with the model that PG signaling cascades regulate Ena localization/activity to temporally regulate actin filament formation during Drosophila oogenesis, at least in part, by restricting Ena localization/activity earlier in oogenesis (S9) and promoting appropriate Ena localization/activity later in oogenesis (S10B). Further understanding the mechanisms by which PGs exert opposing effects on Ena localization/activity during Drosophila oogenesis is likely to shed light on conserved mechanisms by which PGs may generally regulate the Ena/VASP family of proteins (Spracklen, 2014).

This study provides strong evidence that PGs temporally regulate cytoplasmic F-actin rtical actin, suggest that Fascin may generate microspikes or short filopodia that are requ penetrant, early induction of filamentous and aggregated actin structures in the posterior nurse cells of S9 follicles. Importantly, overexpression of Pxt suppresses early actin remodeling, and similar structures are observed at a low frequency in wild-type follicles. Previously, it has been shown that Pxt is also required for cortical actin integrity and bundle formation during S10B (Tootle, 2008; Groen, 2012). Together these data lead to the model that, during S9, Pxt-dependent PG production initiates a signaling cascade that prevents or restricts early actin remodeling, while during S10B, Pxt-dependent PG signaling induces actin remodeling events necessary for nurse cell dumping. These opposing activities could be achieved through different PGs at S9 and S10B, or the same PG may be produced at both stages, but elicit distinct signaling cascades. The first possibility is favored as exogenous PGE₂ inhibits, while PGF2α promotes in vitro nurse cell dumping and restores dumping in the presence of COX inhibitor treatment or genetic loss of Pxt (Tootle, 2008; Groen, 2012; Spracklen, 2014).

While early actin remodeling in the posterior nurse cells is observed in response to certain stresses and in a few mutant backgrounds, current understanding of how these structures form and the consequences of such formation is severely limited. Overexpression of death-inducing factors in the follicle cells, or starvation, results in follicle death at S8/9 and the accumulation of actin filaments and aggregates. Interestingly, these actin structures colocalize with Hts-RC, similar to what was observed in both wild-type and pxt mutants. Additionally, expression of active Dcp-1 disrupts the actin cytoskeleton within the nurse cells at S10B, suggesting that limiting caspase activation may prevent the destruction of the nurse cell cytoskeleton. Furthermore, loss of Midway, a diacylglycerol acyltransferase, causes S8 checkpoint death and the dying follicles accumulate extensive actin filaments in the posterior nurse cells (Buszczak, 2002). Thus, early actin structures may either cause or be caused by the induction of follicle death (Spracklen, 2014).

If premature actin remodeling in pxt mutants is either driving or caused by induction of follicle death, then the prevalence of the actin structures should be similar to the levels of death. This is not what was observed, as 34% of pxt^f and 74% of pxt^pxtEY S9 follicles exhibit early actin structures, while there is a much higher level of follicle death in pxt^f (54% death), the allele with a lower frequency of early actin structures, than in the pxt^EY allele (22% death). These data suggest that early actin structures can form and not result in follicle death, and that follicles can die without forming such structures. Thus, the function of these early actin structures remains unclear (Spracklen, 2014).

These early actin structures may function to assess whether the nurse cells are capable of the dramatic S10B remodeling events. In this case, small structures form and are rapidly depolymerized, as few wild-type S9 follicles exhibit visible, early actin structures. Supporting this idea, it was found that strong germline expression of actin labeling tools, which likely stabilize actin structures, result in increased frequency and size of these structures. Alternatively, the early actin structures may regulate nuclear position. Indeed, Hts (one of the factors found to be associated with the early actin structures) localizes to a perinuclear actin meshwork that maintains nuclear position during nurse cell dumping. Defining the function of these early actin structures will require further analyses of their structure, dynamics, and regulation (Spracklen, 2014).

The data are consistent with the model that both the early actin remodeling during S9 and the inhibition of canonical actin remodeling during S10B observed in pxt mutants are due, at least in part, to misregulation of Ena, the sole Drosophila Ena/VASP family member. Supporting this model, this study found that while the actin regulators Hts and Villin localize the early actin structures in both wild-type and pxt mutant follicles, Ena preferentially localizes to the early actin structures in pxt mutants. Furthermore, a reduction in Ena level suppresses the early actin remodeling observed in pxt mutant S9 follicles, but has no effect on the prevalence of those structures in a wild-type background. Ena has been previously shown to promote actin remodeling during S10B (Gates, 2009). Interestingly, Ena localization to the sites of canonical F-actin elongation is reduced in pxt mutants during S10B. The alterations in Ena localization in pxt mutants during both S9 and S10B are not due to changes in mRNA (Tootle, 2011) or protein expression. These data lead to the hypothesis that Pxt-dependent production of PGs results in the activation of signaling cascades that either directly or indirectly lead to altered Ena localization/activity. Ena may be regulated by protein-protein interactions, its antagonist Capping protein, or phosphorylation. Interestingly, loss of kinases known to regulate Ena/VASP proteins, PKA and Abl also result in early actin remodeling (Spracklen, 2014).

While PG signaling is known to regulate VASP, the extent to which the other homologs, Mena and Evl, are regulated by PG signaling is unclear. As Ena exhibits a higher level of homology to Mena and Evl than to VASP, PG signaling is likely to regulate all three mammalian forms. Uncovering the means by which PG signaling regulates, either directly or indirectly, Drosophila Ena to temporally regulate actin remodeling during oogenesis is expected to reveal conserved mechanisms through which PG signaling modulates the activity of this family of actin regulators. Such mechanisms are likely to play critical roles, not only during development, but also in human diseases including heart disease and cancer (Spracklen, 2014).

Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling

Although prostaglandins (PGs)-lipid signals produced downstream of cyclooxygenase (COX) enzymes-regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. Drosophila oogenesis, in particular nurse cell dumping, serves as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. This study provides the first link between Fascin (Drosophila Singed, Sn), an actin-bundling protein, and PGs. Loss of either pxt or fascin results in similar actin defects. Fascin interacts, both pharmacologically and genetically, with PGs, as reduced Fascin levels enhance the effects of COX inhibition and synergize with reduced Pxt levels to cause both parallel bundle and cortical actin defects. Conversely, overexpression of Fascin in the germline suppresses the effects of COX inhibition and genetic loss of Pxt. These data lead to the conclusion that PGs regulate Fascin to control actin remodeling. This novel interaction has implications beyond Drosophila, as both PGs and Fascin-1, in mammalian systems, contribute to cancer cell migration and invasion (Groen, 2012).

PGs regulate actin remodeling during Drosophila nurse cell dumping (Tootle, 2008). Dumping has been widely used to identify and characterize the conserved functions of actin-binding proteins, making it an ideal system with which to determine the mechanisms by which PGs regulate actin dynamics. This study provides the first evidence that Drosophila Singed is a downstream target of PGs and is required for PG-dependent formation of parallel actin filament bundles and maintenance of cortical actin integrity (Groen, 2012).

Mutations in fascin and pxt exhibit similar phenotypes, including the lack of or a reduction in bundles, breakdown of cortical actin, inhibition of dumping, and, ultimately, female sterility. Reduced Fascin levels enhance the effects of COX inhibitor treatment and synergize with reduced Pxt levels to cause actin-remodeling defects and a block in dumping. This supports the model that Drosophila Fascin is a downstream target of PGs. Additional evidence for this model comes from overexpression experiments in which increased Fascin levels suppress the defects due to the loss of PG synthesis (Groen, 2012).

Although there are many similarities in the actin defects in pxt and fascin mutants, there are also differences. The bundles in pxt-mutant follicles are variable in length and are not evenly distributed along membranes, whereas those in fascin mutant follicles exhibit uniform length and distribution but fail to elongate. These differences are intrepeted as evidence that whereas Fascin is a downstream target of PGs, PGs regulate additional factors to control actin remodeling. Indeed, a number of other putative PG effectors have been identified in a pharmaco-interaction screen (Groen, 2012).

The integrity of the cortical actin in the nurse cells is not maintained in either pxt or fascin mutants. The breakdown of the cortical actin occurs starting in S8 in pxt mutants (Tootle, 2008). This breakdown is suppressed by overexpression of Fascin. These data indicate that Fascin plays an important, and previously undescribed, role in cortical actin. Supporting this idea, Fascin levels appear enriched in the subcortical region and green fluorescent protein-Fascin localizes to the cortical actin. At first glance, it seems surprising that Fascin regulates the cortical actin, given that it is believed to be a branched network of actin filaments. Supporting that this is indeed the structure of the cortical actin in the nurse cells are the findings that cortical actin integrity requires Arp2/3 subunits and Wash (Liu, 2009), a regulator of Arp2/3 activity. Other factors regulating nurse cell cortical actin include Profilin, an actin monomer-binding protein, Enabled (Gates, 2009), an actin filament elongation factor, and Capping (Gates, 2009), a barbed end-binding protein and antagonist of Enabled. All of these actin regulators are implicated in the formation of branched actin networks. Fascin, however, has not been widely implicated in the formation of such a network because Fascin is generally found in parallel bundles. Of interest, a few mammalian cell culture studies have revealed a role of Fascin-1 in lamellipodia, which are composed of branched actin networks. Specifically, Rac and Cdc42 trigger the localization of Fascin-1 to the lamellopodia, where it contributes to the formation of microspikes necessary for cell motility. In addition, in fish fibroblasts it has been shown that Fascin 1-dependent bundles are folded into the lamella network. These previous findings, along with the current evidence that Fascin regulates nurse cell cortical actin, suggest that Fascin may generate microspikes or short filopodia that are required to strengthen the likely branched network of this cortical actin. Furthermore, there is evidence that, in vitro, Arp2/3 and Fascin-1 activity must be balanced to regulate the type and extent of actin polymerization. Such an Arp2/3-dependent network can lead to Fascin-1 recruitment and bundle formation. These data make it tempting to speculate that Arp2/3 and Fascin activity must be balanced in order to generate properly structured nurse cell cortical actin. If there is too much Arp2/3 or too little Fascin activity, then the cortical actin structure is altered such that integrity is lost. Thus, in pxt mutants, where Fascin activity is likely reduced, cortical actin breaksdowns; this is rescued by overexpression of Fascin. It will be interesting to determine whether reduced Arp2/3 activity also suppresses the cortical actin defects in pxt mutants. Another possibility for the role of Fascin in cortical actin is that bundle elongation is required to maintain the cortical actin. Therefore the bundle defects in pxt and fascin mutants would cause the breakdown of the cortical actin, and because overexpression of Fascin in pxt mutants restores bundle formation/elongation, the cortical actin defects are also suppressed. This is thought unlikely because cortical actin defects are apparent in S8 pxt-mutant follicles, well before the onset of bundle formation (Tootle, 2008). Further characterization of the structure of the nurse cell cortical actin and the interplay between the factors required for its integrity is required to determine the role of Fascin (Groen, 2012).

Although this study shows that Fascin is a downstream target of PG signaling during nurse cell dumping, Villin (Quail), another actin-bundling protein required for nurse cell dumping, does not interact with PGs. Previous work indicates that Villin mediates initial bundle formation during nurse cell dumping. Fascin then bundles the filaments more tightly, increasing bundle strength. It was found that heterozygosity for villin fails to enhance the dumping defects due to reduced PG synthesis. These results may seem surprising, given that overexpression of Villin partially rescues fascin-mutant phenotypes. However, such bundles are structurally different, and overexpression may not completely reflect endogenous function. Therefore the current findings are interpreted to mean that Villin is not likely to be regulated by PGs. Supporting this model, Villin expression and localization are grossly normal in pxt mutants. An alternative interpretation is that Villin levels may not have been reduced enough to detect an interaction by these assays (Groen, 2012).

Prostaglandins could regulate Fascin activity in a number of ways. In human cells, protein kinase C (PKC) phosphorylates Fascin-1, blocking filamentous actin (F-actin) binding. In addition, human Fascin-1 competes with caldesmon and tropomyosin for F-actin. Calmodulin, and thus Ca²⁺/cAMP signaling, negatively regulates these two proteins, promoting Fascin-1's bundling activity. Rac, a Rho-type GTPase, also positively regulates human Fascin-1. A recent study revealed that Fascin-1 is also regulated by Rho via LIM kinase 1. Of note, PGs are known to signal through all of these mechanisms. Given that Drosophila Fascin PKC-site phosphomutants (S52A/E) restore nurse cell dumping in fascin mutants, it is unlikely that PGs regulate Fascin in this manner during this process. However, an additional phosphorylation site (S289), associated with a bundling-independent function, has recently been identified in Drosophila; perhaps this role of Fascin contributes to cortical actin integrity. Because both cAMP and Rho GTPase regulate nurse cell dumping, it will be important to determine whether PGs signal via these pathways to regulate Fascin. It remains possible that PGs regulate Fascin by a previously unidentified means (Groen, 2012).

One alternative mechanism by which PGs could regulate Fascin is through direct modification. 15-Deoxy-prostaglandin J₂, produced by nonenzymatic processing of PGF_2α, modifies (prostanylates) cysteine residues on proteins in mammalian cells. It is intriguing that actin and cytoskeletal regulatory proteins, including tropomyosin, have been shown to be prostanylated. Therefore PGs could directly modify Fascin or a protein that regulates Fascin (Groen, 2012).

This work is the first evidence linking PGs to Fascin. Of interest, high levels of both PGs and Fascin-1 independently correlate with highly aggressive cancers in patients. In addition, both are critical for cancer cell invasion in human cell culture and mouse models. Specifically, Fascin-1 bundles actin filaments within filopodia and invadopodia, structures required for cancer metastasis. These parallel actin bundles are nearly structurally identical to those found in the nurse cells during dumping. This leads to the speculation that PGs control Fascin-1 within human cancer cells to tightly regulate the formation of invasive cytoskeletal structures and thus cancer metastasis. It will be critical to determine the detailed mechanisms and signaling cascade by which PGs regulate Fascin and whether this regulation is conserved from Drosophila follicle development to human cancer progression (Groen, 2012).

Drosophila eggshell production: identification of new genes and coordination by Pxt

Drosophila ovarian follicles complete development using a spatially and temporally controlled maturation process in which they resume meiosis and secrete a multi-layered, protective eggshell before undergoing arrest and/or ovulation. Microarray analysis revealed more than 150 genes that are expressed in a stage-specific manner during the last 24 hours of follicle development. These include all 30 previously known eggshell genes, as well as 19 new candidate chorion genes and 100 other genes likely to participate in maturation. Mutations in pxt, encoding a putative Drosophila cyclooxygenase, cause many transcripts to begin expression prematurely, and are associated with eggshell defects. Somatic activity of Pxt is required, as RNAi knockdown of pxt in the follicle cells recapitulates both the temporal expression and eggshell defects. One of the temporally regulated genes, cyp18a1, which encodes a cytochromome P450 protein mediating ecdysone turnover, is downregulated in pxt mutant follicles, and cyp18a1 mutation itself alters eggshell gene expression. These studies further define the molecular program of Drosophila follicle maturation and support the idea that it is coordinated by lipid and steroid hormonal signals (Tootle, 2011).

These studies show that a large fraction of the genes involved in eggshell production can be identified by simply scoring for stage-specific changes in transcript levels during late oogenesis. Not only were virtually all of the previously known structural genes involved in producing yolk, the vitelline membrane and the chorion identified, but at least 19 new candidate eggshell proteins were discovered. Despite the simplicity of the protocol, the expression profiles revealed by these experiments agreed closely with previous studies. Many of the genes identified as eggshell genes are included among 81 genes reported as candidate targets of Egfr signaling. Additionally, the expression of many genes involved in follicle maturation are spatially regulated within the folliclar epithelium, and the method described in this study could serve as an efficient pre-screen before undertaking such studies (Tootle, 2011).

The nature of the peroxidase that crosslinks the eggshell has been controversial. Various proteins have been suggested to function as the crosslinking peroxidase, including Pxd and Pxt. This study found pxt, the COX-like enzyme, is expressed and pxd is absent throughout all stages of egg maturation. In contrast, the eggshell protein and putative peroxidase CG4009 is very highly expressed during S12. We propose that CG4009 is the peroxidase that crosslinks the eggshell (Tootle, 2011).

Pxt mutations partially uncouple morphological development and gene expression. Yolk protein genes turn off normally in pxt mutant follicles, but vitelline membrane genes continue to be expressed longer than normal. Some chorion genes turn on earlier than normal, while the expression of others is delayed or prolonged. Many possible mechanisms may underlie these changes. However, the possibility is particularly interesting that Pxt coordinates the production of PGs that interact with other mechanisms to precisely control egg maturation (Tootle, 2011).

In all sexually reproducing organisms the growth and development of the somatic and germ cells are mutually dependent and must be coordinated. Such coordination requires bi-directional communication. Historically, somatic cells were thought to regulate follicle development, including maintaining meiotic arrest, promoting meiotic resumption, and suppressing oocyte transcription prior to nuclear maturation. It has more recently been shown that the oocyte also signals to the soma. Oocyte signaling is necessary for follicular formation, and regulating the proliferation and differentiation of the somatic cells. It is generally thought that the oocyte has a greater influence on the soma early in follicular development and this is reversed during the later stages (Tootle, 2011).

There is emerging evidence that PG signaling coordinates germline and somatic development within mammalian follicles. While both oocyte and somatic maturation are delayed in COX2 knockout mice, it has been shown that the PGs are required in the soma for fertility (Takahashi, 2006). Specifically, COX2 is required in the somatic cells for cumulus (somatic) cell expansion and survival. However, meiotic resumption is not controlled by PGs from the soma. These germline and somatic events must be coordinated for the follicle to be competent for fertilization. This study has found that PG signaling is required for both germline and somatic development during Drosophila follicle development. Fertility requires both of these signals. Specifically, PG signaling within the germline is necessary for mediating nurse cell dumping, the contractile process by which the oocyte is supplied with materials required for embryonic development, while PG signaling within the follicle cells is needed to regulate the timing of eggshell gene expression and subsequent eggshell structure. Thus PG signals, from insects to mammals, maintain the synchronized development of the germline and somatic cells within the individual follicle (Tootle, 2011).

Female reproduction is regulated by a complement of hormones that are cyclically produced and secreted. One such hormone that interacts with PG signaling in mammals is oxytocin. Oxytocin plays critical roles in regulating the function of the corpus luteum, a transient endocrine organ that secretes hormones to regulate the menstrual cycle and the early stages of pregnancy. In the absence of pregnancy, PGF_2α stimulates the release of oxytocin to mediate luteolysis or the regression of the corpus luteum. During parturition, oxytocin and PGF_2α also play critical roles. Oxytocin initiates labor, inducing PGF_2α, which maintains labor and dilates the cervix (Tootle, 2011 and references therein).

PGs and estrogen co-regulate each other in multiple cells types, including breast cancer cells. Breast tissue is the largest producer of estrogen in post-menopausal women; aromatase, Cyp19, leads to the production of estradiol. There is a high correlation between aromatase and COX2 expression in human breast cancer samples. Specifically, PGE₂ signals via cAMP and PKA to stimulate a promoter upstream of cyp19, leading to increased aromatase expression. Autocrine and paracrine feedback loops via estradiol subsequently increase PGE₂ secretion. Therefore, in breast cancer cells, PG and estrogen signaling are intimately linked (Tootle, 2011 and references therein).

PGs and estrogen also interact in endometriotic tissue. Both PGE₂ and PGF_2α are excessively produced in uterine and endometriotic tissues of women with endometriosis. In the endometriotic stromal cells, PGE₂ stimulates the expression of all the steroidogenic genes needed to synthesis estradiol from cholesterol. This occurs via PGE₂ activation of cAMP/PKA signaling which upregulates of the expression of steroidogenic acute regulatory gene (StAR) and cyp19. The expression of these steroidogenic genes is regulated by Steroidogenic Factor 1 (SF1), a nuclear hormone receptor. PGE₂ signaling leads to SF1 out competing other transcription factors, Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) and Wilms' tumor-1 (WT-1), for binding to steroidogenic gene promoters. Thus, PG signaling coordinates the expression of all steroidogenic genes (Tootle, 2011).

The current results encourage future efforts to further establish the roles for PG signaling during Drosophila egg maturation and specifically, to learn how PGs are connected to steroid hormones. The Drosophila hormone ecdysone plays several critical roles during oogenesis. The loss of ecdysone signaling arrests follicle development at stage 8. Additionally, ecdysone signaling is needed to control the onset of chorion gene amplification, and to activate eggshell gene expression via transcriptional regulation. Temporally programmed changes in ecdysone levels may contribute to the timed control of eggshell gene expression. These studies reportedprovide a foundation for further dissecting the roles of Pxt and ecdysone-mediated signaling during late follicle development. If important aspects of these interactions have been conserved during evolution, the Drosophila ovary may emerge as a model for understanding the cellular and molecular changes underlying mammalian follicular maturation, endometriosis and infertility (Tootle, 2011).

Drosophila Pxt: a cyclooxygenase-like facilitator of follicle maturation

Prostaglandins are local transient hormones that mediate a wide variety of biological events, including reproduction. The study of prostaglandin biology in a genetically tractable invertebrate model organism has been limited by the lack of clearly identified prostaglandin-mediated biological processes and prostaglandin metabolic genes, particularly analogs of cyclooxygenase (COX), the rate-limiting step in vertebrate prostaglandin synthesis. This study presents pharmacological data that Drosophila ovarian follicle maturation requires COX-like activity. Genetic evidence is presented that this activity is supplied in vivo by the Drosophila peroxidase Pxt. pxt mutant females are sterile, and maturing follicles show defects in actin filament formation, nurse cell membrane stability and border cell migration. Maturation of pxt follicles in vitro is stimulated by prostaglandin treatment and fertility is restored in vivo to pxt mutants by expressing mammalian Cox1 protein. These experiments suggest that prostaglandins promote Drosophila follicle maturation, in part by modulating the actin cytoskeleton, and establish Drosophila oogenesis as a model for understanding these critical biological regulators (Tootle, 2008).

PGs play important and diverse roles in insects and other invertebrates, such as regulating immune responses and reproduction, including oogenesis (Stanley, 2006; Stanley-Samuelson, 1996). Biochemical studies suggest that invertebrate PGs are produced in a similar manner to those in vertebrates , and mammalian COX inhibitors have been shown to block insect PG synthetic activity in tissue extracts (Buyukguzel, 2002; Stanley-Samuelson, 1994). However, the genes encoding the proteins that mediate invertebrate PG production have not been previously identified. This has slowed attempts to understand the detailed functions of PGs in Drosophila and precluded the use of genetics to address many important issues about PG signaling. This study has found that Drosophila S10B-14 follicle development requires a Cox1-like activity that can be satisfied by exogenous PGs, including PGF_2α. Thus, follicle maturation provides a phenotypic focus for further genetics studies of PG action in Drosophila (Tootle, 2008).

Using pharmacology, sequence homology and developmental genetics, this study has identified Pxt as a candidate COX enzyme. Pxt is a heme peroxidase, and like mammalian COX enzymes, is predicted to be glycosylated and membrane bound. Despite only moderate homology to known COX enzymes, genetic loss of pxt resembles the effects of COX inhibitors on follicle development in vitro. Exogenous PGs can overcome the block in development caused by COX inhibitors and by pxt mutation. Therefore, PGs act downstream or in parallel to Pxt, supporting the idea that Pxt is responsible for the synthesis of PGs. Furthermore, this study found that expression of mouse Cox1 in pxt ^f01000 mutant adults fully rescues female fertility and follicle development. The fact that mouse Cox1 can functionally replace Pxt during Drosophila oogenesis strongly argues that Pxt is a Drosophila COX enzyme (Tootle, 2008).

Although Drosophila COX and mammalian COX enzymes appear to have diverged over time, they are likely to have the same basic three-dimensional structure within the conserved domain. The critical residues (Gln203, His207, His388) for heme coordination necessary for the peroxidase activity are conserved in Pxt (Gln396, His402, His590). The conservation is too weak to predict the existence of the hydrophobic substrate-binding channel within the cyclooxygenase active site and the substrate binding residues Arg120 and Tyr355 are not clearly conserved. However, Pxt does contain a candidate Tyr385 (Pxt Tyr564), the residue at the active site that acquires an activating electron from the peroxidase part of the enzyme and is required for the cyclooxygenase activity. This suggests that there may be substantial differences in the structure of Pxt and Cox1, despite the ability of Cox1 to substitute functionally for Pxt (Tootle, 2008).

The limited previous studies of Pxt structure and expression have suggested alternative functions for the protein. Distant homologies exist with peroxinectin (17-31% identity), a crayfish cell-adhesion peroxidase. Pxt has also been suggested, by homology to a putative Aedes aegypti chorion peroxidase, to be the peroxidase responsible for eggshell hardening, a process caused by the formation of dityrosine crosslinks. Although the current detailed expression data and the phenotypes of pxt mutants indicate that Pxt functions as a COX enzyme and is required during oogenesis prior to eggshell formation, additional functions for the protein cannot be ruled out. Studies of purified Pxt will be required to address its full range of biochemical and enzymatic properties (Tootle, 2008).

Both pharmacological and genetic studies show that PGs are required for mammalian follicles to mature normally and undergo ovulation. Cox2 knockout mice are defective in both follicle maturation and ovulation (Loftin, 2002). In rats, inhibition of PG synthesis results in mistargeted and incomplete follicle rupture. The small percentage of follicles that do ovulate fail to be fertilized, most probably owing to impaired oocyte maturation. COX inhibitors, such as NSAIDs, cause reversible female infertility and may act in a similar manner. Like many mammals, Drosophila females also ovulate only one mature oocyte at a time, most probably by inducing contraction of the muscles surrounding just one of the more than 30 ovarioles that make up the two ovaries. In addition to their defects in follicle maturation, pxt mutants ovulate only rarely. Thus, the roles of PGs during follicle development and ovulation may be conserved between Drosophila and mammals (Tootle, 2008).

Each cycle, the single mammalian follicle that will be ovulated, the dominant follicle, accumulates high levels of smooth muscle myosin and actin which contributes to the contractile force needed for ovulation. These changes in actin organization are hypothesized to be downstream of PG signaling. PGF_2α, the PG that had the most effect on Drosophila in vitro egg maturation (IVEM), is intimately associated with muscle contractions in mammals (Funk, 2001; Langenbach, 1999). Consequently, the finding that Pxt and PGs affect some oogenic processes that are modulated by actin and myosin, including border cell migration and nurse cell dumping, represents a potential parallel between the role of PGs in Drosophila and mammalian oogenesis (Tootle, 2008).

Even the induction of PG-dependent maturation may occur in a similar manner. In mammals, PG synthesis is hormonally upregulated during the 10 hours preceding ovulation, and thereafter mediates the terminal differentiation of follicles. Similarly, it was found that pxt levels are upregulated during stages 9-10, and that COX activity, Pxt and PGs are subsequently required to complete the last 10-15 hours of Drosophila follicular development. Interestingly, the steroid hormone ecdysone is known to regulate the S8 oogenic checkpoint, which commits egg chambers to finish developing into mature follicles. Drosophila pxt expression and PG synthesis may be upregulated by ecdysone at the onset of S8 as part of the maturation program (Tootle, 2008).

Many PGs serve as short-range hormones that act as ligands for G protein-coupled receptors (GPCRs). In mammals there are eight receptors with distinct specificities for the active PGs, and each receptor favors the initiation of a specific signaling cascade. Such GPCR signaling can secondarily modulate additional signaling pathways. This study found that PGF_2α mediates Drosophila egg maturation in vitro. Mammalian PGF_2α acts as a ligand not only for the F receptor (FP), but also for two E receptors (EP₁ and EP₃), thereby increasing the possible signaling outcomes. One known downstream target is protein kinase A (PKA), which can then activate multiple MAPKs (Bos, 2004). It should be possible in the future to discern which effects of Pxt are exerted autonomously, and which are downstream of intercellular signals (Tootle, 2008).

Despite a possible plethora of mechanisms, PGs have been frequently found ultimately to affect muscle contraction and ovulation, or to modulate the actin cytoskeleton in mammals. Within many cultured mammalian cell types, PGs cause changes in the organization, stability and polymerization of actin that influence membrane permeability and cell motility (Banan, 2000; Sheng, 2001). PG action appears to be rapid, suggesting that sophisticated feedback circuits may be involved. For example, reduction in the level of actin microfilaments has been reported to stimulate PG synthesis and release (Wang, 2006). Thus, PGs may facilitate fine-tuning of the actin cytoskeleton in a rapid time frame (Tootle, 2008).

These studies show that Pxt and PGs can affect the actin cytoskeleton in Drosophila. Actin filaments are greatly reduced in pxt mutant stage 10B follicles, which phenotypically resemble follicles lacking major components of the actin cytoskeleton. PG-mediated regulation of actin dynamics is currently not well understood, particularly when intercellular interactions are involved. Drosophila oogenesis provides an attractive system to elucidate how PGs modulate actin in the context of developing tissues (Tootle, 2008).

These data suggests that disruption of PG signaling has a graded effect within the developing follicle. The actin-related defects in pxt mutants are more prevalent at the anterior of the egg chamber, including nurse cell membrane loss and accumulation of actin puncta. Conversely, actin bundles are more likely to form in a normal fashion in the nurse cells closest to the oocyte. A PG signaling gradient might be relevant to many incompletely understood biological differences that exist along the AP follicle axis, such as the gradient of nurse cell ploidy, the temporal gradient of vitelline membrane formation within the oocyte, the directional migration signal for border cells and the programmed reorganization of the oocyte cytoskeleton in mid-oogenesis that underlies patterning. Ultimately, by using Drosophila genetics, it may be possible to better understand how PGs act, providing new insights and therapeutic targets for the widely conserved biological processes they influence (Tootle, 2008).

Search PubMed for articles about Drosophila Pxt

Banan, A., Smith, G. S., Kokoska, E. R. and Miller, T. A. (2000). Role of actin cytoskeleton in prostaglandin-induced protection against ethanol in an intestinal epithelial cell line. J Surg Res 88: 104-113. PubMed ID: 10644474

Birukova, A. A., Zagranichnaya, T., Fu, P., Alekseeva, E., Chen, W., Jacobson, J. R. and Birukov, K. G. (2007). Prostaglandins PGE(2) and PGI(2) promote endothelial barrier enhancement via PKA- and Epac1/Rap1-dependent Rac activation. Exp Cell Res 313: 2504-2520. PubMed ID: 17493609

Bos, C. L., Richel, D. J., Ritsema, T., Peppelenbosch, M. P. and Versteeg, H. H. (2004). Prostanoids and prostanoid receptors in signal transduction. Int J Biochem Cell Biol 36: 1187-1205. PubMed ID: 15109566

Bulin, C., Albrecht, U., Bode, J. G., Weber, A. A., Schror, K., Levkau, B. and Fischer, J. W. (2005). Differential effects of vasodilatory prostaglandins on focal adhesions, cytoskeletal architecture, and migration in human aortic smooth muscle cells. Arterioscler Thromb Vasc Biol 25: 84-89. PubMed ID: 15458982

Buszczak, M., Lu, X., Segraves, W. A., Chang, T. Y. and Cooley, L. (2002). Mutations in the midway gene disrupt a Drosophila acyl coenzyme A: diacylglycerol acyltransferase. Genetics 160: 1511-1518. PubMed ID: 11973306

Dormond, O., Bezzi, M., Mariotti, A. and Ruegg, C. (2002). Prostaglandin E2 promotes integrin alpha Vbeta 3-dependent endothelial cell adhesion, rac-activation, and spreading through cAMP/PKA-dependent signaling. J Biol Chem 277: 45838-45846. PubMed ID: 12237321

Funk, C. D. (2001). Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294: 1871-1875. PubMed ID: 11729303

Gates, J., Nowotarski, S. H., Yin, H., Mahaffey, J. P., Bridges, T., Herrera, C., Homem, C. C., Janody, F., Montell, D. J. and Peifer, M. (2009). Enabled and Capping protein play important roles in shaping cell behavior during Drosophila oogenesis. Dev Biol 333: 90-107. PubMed ID: 19576200

Groen, C. M., Spracklen, A. J., Fagan, T. N. and Tootle, T. L. (2012). Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling. Mol Biol Cell 23: 4567-4578. PubMed ID: 23051736

Langenbach, R., Loftin, C., Lee, C. and Tiano, H. (1999). Cyclooxygenase knockout mice: models for elucidating isoform-specific functions. Biochem Pharmacol 58: 1237-1246. PubMed ID: 10487525

Liu, R., Abreu-Blanco, M. T., Barry, K. C., Linardopoulou, E. V., Osborn, G. E. and Parkhurst, S. M. (2009). Wash functions downstream of Rho and links linear and branched actin nucleation factors. Development 136: 2849-2860. PubMed ID: 19633175

Loftin, C. D., Tiano, H. F. and Langenbach, R. (2002). Phenotypes of the COX-deficient mice indicate physiological and pathophysiological roles for COX-1 and COX-2. Prostaglandins Other Lipid Mediat 68-69: 177-185. PubMed ID: 12432917

Sheng, H., Shao, J., Washington, M. K. and DuBois, R. N. (2001). Prostaglandin E2 increases growth and motility of colorectal carcinoma cells. J Biol Chem 276: 18075-18081. PubMed ID: 11278548

Spracklen, A. J., Kelpsch, D. J., Chen, X., Spracklen, C. N. and Tootle, T. L. (2014). Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis. Mol Biol Cell 25: 397-411. PubMed ID: 24284900

Stanley-Samuelson, D. W. and Ogg, C. L. (1994). Prostaglandin biosynthesis by fat body from the tobacco hornworm, Manduca sexta. Insect Biochem Mol Biol 24: 481-491. PubMed ID: 8205144

Stanley-Samuelson, D. W. and Pedibhotla, V. K. (1996). What can we learn from prostaglandins and related eicosanoids in insects? Insect Biochem Mol Biol 26: 223-234. PubMed ID: 8900594

Stanley, D. (2006). Prostaglandins and other eicosanoids in insects: biological significance. Annu Rev Entomol 51: 25-44. PubMed ID: 16332202

Takahashi, T., Morrow, J. D., Wang, H. and Dey, S. K. (2006). Cyclooxygenase-2-derived prostaglandin E(2) directs oocyte maturation by differentially influencing multiple signaling pathways. J Biol Chem 281: 37117-37129. PubMed ID: 17023426

Tootle, T. L. and Spradling, A. C. (2008). Drosophila Pxt: a cyclooxygenase-like facilitator of follicle maturation. Development 135: 839-847. PubMed ID: 18216169

Tootle, T. L., Williams, D., Hubb, A., Frederick, R. and Spradling, A. (2011). Drosophila eggshell production: identification of new genes and coordination by Pxt. PLoS One 6: e19943. PubMed ID: 21637834

Wang, Y. F. and Hatton, G. I. (2006). Mechanisms underlying oxytocin-induced excitation of supraoptic neurons: prostaglandin mediation of actin polymerization. J Neurophysiol 95: 3933-3947. PubMed ID: 16554501

date revised: 2 March 2014

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