UDP-glycosyltransferases act as key determinants of host plant range in generalist and specialist Spodoptera species.

Phytophagous insects have evolved sophisticated detoxification systems to overcome the antiherbivore chemical defenses produced by many plants. However, how these biotransformation systems differ in generalist and specialist insect species and their role in determining insect host plant range remains an open question. Here, we show that UDP-glucosyltransferases (UGTs) play a key role in determining the host range of insect species within the Spodoptera genus. Comparative genomic analyses of Spodoptera species that differ in host plant breadth identified a relatively conserved number of UGT genes in generalist species but high levels of UGT gene pseudogenization in the specialist Spodoptera picta. CRISPR-Cas9 knockouts of the three main UGT gene clusters of Spodoptera frugiperda revealed that UGT33 genes play an important role in allowing this species to utilize the poaceous plants maize, wheat, and rice, while UGT40 genes facilitate utilization of cotton. Further functional analyses in vivo and in vitro identified the UGT SfUGT33F32 as the key mechanism that allows generalist S. frugiperda to detoxify the benzoxazinoid DIMBOA (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one), a potent insecticidal phytotoxin produced by poaceous plants. However, while this detoxification capacity is conserved in several generalist Spodoptera species, Spodoptera picta, which specializes on Crinum plants, is unable to detoxify DIMBOA due to a nonfunctionalizing mutation in SpUGT33F34. Collectively, these findings provide insight into the role of insect UGTs in host plant adaptation, the mechanistic basis of evolutionary transitions between generalism and specialism and offer molecular targets for controlling a group of notorious insect pests.

Juvenile hormone promotes paracellular transport of yolk proteins via remodeling zonula adherens at tricellular junctions in the follicular epithelium.

Juvenile hormone (JH) acts as a gonadotrophic hormone stimulating insect vitellogenesis and oogenesis. Paracellular transport of yolk proteins through intercellular channels (patency) in the follicular epithelium is a developmentally regulated and evolutionarily conserved process during vitellogenesis. However, the mechanisms underlying patency opening are poorly understood. Using the migratory locust Locusta migratoria as a model system, we report here that JH-regulated remodeling of zonula adherens (ZA), the belt-like adherens junction maintaining physical linking between follicle cells controlled the opening of patency. JH triggered phosphorylation of Partitioning defective protein 3 (Par3) via a signaling cascade including G protein-coupled receptor (GPCR), small GTPase Cell division cycle 42 (Cdc42) and atypical Protein kinase C (aPKC). Par3 phosphorylation resulted in its disassociation from ß-Catenin, the cytoplasmic partner of ZA core component E-Cadherin. Release of Par3 from the ß-Catenin/E-Cadherin complex caused ZA disassembly at tricellular contacts, consequently leading to patency enlargement. This study provides new insight into how JH stimulates insect vitellogenesis and egg production via inducing the opening of paracellular route for vitellogenin transport crossing the follicular epithelium barrier.

The vitellogenin receptor functionality of the migratory locust depends on its phosphorylation by juvenile hormone.

Vitellogenin receptor (VgR) plays a pivotal role in ovarian vitellogenin (Vg) uptake and vertical transmission of pathogenic microbes and Wolbachia symbionts. However, the regulatory mechanisms of VgR action as an endocytic receptor and translocation from oocyte cytoplasm to the membrane remain poorly understood. Here, by using the migratory locust Locusta migratoria as a model system, we report that juvenile hormone (JH) promotes VgR phosphorylation at Ser1361 in the second EGF-precursor homology domain. A signaling cascade including GPCR, PLC, extracellular calcium, and PKC-ι is involved in JH-stimulated VgR phosphorylation. This posttranslational regulation is a prerequisite for VgR binding to Vg on the external surface of the oocyte membrane and subsequent VgR/Vg endocytosis. Acidification, a condition in endosomes, induces VgR dephosphorylation along with the dissociation of Vg from VgR. Phosphorylation modification is also required for VgR recycling from oocyte cytoplasm to the membrane. Additionally, VgR phosphorylation and its requirement for Vg uptake and VgR recycling are evolutionarily conserved in other representative insects including the cockroach Periplaneta americana and the cotton bollworm Helicoverpa armigera This study fills an important knowledge gap of low-density lipoprotein receptors in posttranslational regulation, endocytosis, and intracellular recycling.

Juvenile hormone signaling promotes ovulation and maintains egg shape by inducing expression of extracellular matrix genes.

It is well documented that the juvenile hormone (JH) can function as a gonadotropic hormone that stimulates vitellogenesis by activating the production and uptake of vitellogenin in insects. Here, we describe a phenotype associated with mutations in the Drosophila JH receptor genes, Met and Gce: the accumulation of mature eggs with reduced egg length in the ovary. JH signaling is mainly activated in ovarian muscle cells and induces laminin gene expression in these cells. Meanwhile, JH signaling induces collagen IV gene expression in the adult fat body, from which collagen IV is secreted and deposited onto the ovarian muscles. Laminin locally and collagen IV remotely contribute to the assembly of ovarian muscle extracellular matrix (ECM); moreover, the ECM components are indispensable for ovarian muscle contraction. Furthermore, ovarian muscle contraction externally generates a mechanical force to promote ovulation and maintain egg shape. This work reveals an important mechanism for JH-regulated insect reproduction.

Trade-offs between the accumulation of cuticular wax and jasmonic acid-mediated herbivory resistance in maize.

Plants have evolved complex physical and chemical defense systems that allow them to withstand herbivory infestation. Composed of a complex mixture of very-long-chain fatty acids (VLCFAs) and their derivatives, cuticular wax constitutes the first physical line of defense against herbivores. Here, we report the function of Glossy 8 (ZmGL8), which encodes a 3-ketoacyl reductase belonging to the fatty acid elongase complex, in orchestrating wax production and jasmonic acid (JA)-mediated defenses against herbivores in maize (Zea mays). The mutation of GL8 enhanced chemical defenses by activating the JA-dependent pathway. We observed a trade-off between wax accumulation and JA levels across maize glossy mutants and 24 globally collected maize inbred lines. In addition, we demonstrated that mutants defective in cuticular wax biosynthesis in Arabidopsis thaliana and maize exhibit enhanced chemical defenses. Comprehensive transcriptomic and lipidomic analyses indicated that the gl8 mutant confers chemical resistance to herbivores by remodeling VLCFA-related lipid metabolism and subsequent JA biosynthesis and signaling. These results suggest that VLCFA-related lipid metabolism has a critical role in regulating the trade-offs between cuticular wax and JA-mediated chemical defenses.

Convergent Adaptation of Ootheca Formation as a Reproductive Strategy in Polyneoptera.

Insects have evolved numerous adaptations and colonized diverse terrestrial environments. Several polyneopterans, including dictyopterans (cockroaches and mantids) and locusts, have developed oothecae, but little is known about the molecular mechanism, physiological function, and evolutionary significance of ootheca formation. Here, we demonstrate that the cockroach asymmetric colleterial glands produce vitellogenins, proline-rich protein, and glycine-rich protein as major ootheca structural proteins (OSPs) that undergo sclerotization and melanization for ootheca formation through the cooperative protocatechuic acid pathway and dopachrome and dopaminechrome subpathway. Functionally, OSP sclerotization and melanization prevent eggs from losing water at warm and dry conditions, and thus effectively maintain embryo viability. Dictyopterans and locusts convergently evolved vitellogenins, apolipoprotein D, and laminins as OSPs, whereas within Dictyoptera, cockroaches and mantids independently developed glycine-rich protein and fibroins as OSPs. Highlighting the ecological-evolutionary importance, convergent ootheca formation represents a successful reproductive strategy in Polyneoptera that promoted the radiation and establishment of cockroaches, mantids, and locusts.

Juvenile hormone acts through FoxO to promote Cdc2 and Orc5 transcription for polyploidy-dependent vitellogenesis.

Vitellogenin (Vg) is a prerequisite for egg production and embryonic development after ovipositioning in oviparous animals. In many insects, juvenile hormone (JH) promotes fat body cell polyploidization for the massive Vg synthesis required for the maturation of multiple oocytes, but the underlying mechanisms remain poorly understood. Using the migratory locust Locusta migratoria as a model system, we report here that JH induces the dephosphorylation of Forkhead box O transcription factor (FoxO) through a signaling cascade including leucine carboxyl methyltransferase 1 (LCMT1) and protein phosphatase 2A (PP2A). JH promotes PP2A activity via LCMT1-mediated methylation, consequently triggering FoxO dephosphorylation. Dephosphorylated FoxO binds to the upstream region of two endocycle-related genes, cell-division-cycle 2 (Cdc2) and origin-recognition-complex subunit 5 (Orc5), and activates their transcription. Depletion of FoxO, Cdc2 or Orc5 results in blocked polyploidization of fat body cells, accompanied by markedly reduced Vg expression, impaired oocyte maturation and arrested ovarian development. The results suggest that JH acts via LCMT1-PP2A-FoxO to regulate Cdc2 and Orc5 expression, and to enhance ploidy of fat body cells in preparation for the large-scale Vg synthesis required for synchronous maturation of multiple eggs.

Insulin/IGF signaling and TORC1 promote vitellogenesis via inducing juvenile hormone biosynthesis in the American cockroach.

Vitellogenesis, including vitellogenin (Vg) production in the fat body and Vg uptake by maturing oocytes, is of great importance for the successful reproduction of adult females. The endocrinal and nutritional regulation of vitellogenesis differs distinctly in insects. Here, the complex crosstalk between juvenile hormone (JH) and the two nutrient sensors insulin/IGF signaling (IIS) and target of rapamycin complex1 (TORC1), was investigated to elucidate the molecular mechanisms of vitellogenesis regulation in the American cockroach, Periplaneta americana Our data showed that a block of JH biosynthesis or JH action arrested vitellogenesis, in part by inhibiting the expression of doublesex (Dsx), a key transcription factor gene involved in the sex determination cascade. Depletion of IIS or TORC1 blocked both JH biosynthesis and vitellogenesis. Importantly, the JH analog methoprene, but not bovine insulin (to restore IIS) and amino acids (to restore TORC1 activity), restored vitellogenesis in the neck-ligated (IIS-, TORC1- and JH-deficient) and rapamycin-treated (TORC1- and JH-deficient) cockroaches. Combining classic physiology with modern molecular techniques, we have demonstrated that IIS and TORC1 promote vitellogenesis, mainly via inducing JH biosynthesis in the American cockroach.

Krüppel-homolog 1 exerts anti-metamorphic and vitellogenic functions in insects via phosphorylation-mediated recruitment of specific cofactors.

BACKGROUND: The zinc-finger transcription factor Krüppel-homolog 1 (Kr-h1) exerts a dual regulatory role during insect development by preventing precocious larval/nymphal metamorphosis and in stimulating aspects of adult reproduction such as vitellogenesis. However, how Kr-h1 functions both as a transcriptional repressor in juvenile metamorphosis and an activator in adult reproduction remains elusive. Here, we use the insect Locusta migratoria to dissect the molecular mechanism by which Kr-h1 functions as activator and repressor at these distinct developmental stages. RESULTS: We report that the kinase PKCα triggers Kr-h1 phosphorylation at the amino acid residue Ser154, a step essential for its dual functions. During juvenile stage, phosphorylated Kr-h1 recruits a corepressor, C-terminal binding protein (CtBP). The complex of phosphorylated Kr-h1 and CtBP represses the transcription of Ecdysone induced protein 93F (E93) and consequently prevents the juvenile-to-adult transition. In adult insects, phosphorylated Kr-h1 recruits a coactivator, CREB-binding protein (CBP), and promotes vitellogenesis by inducing the expression of Ribosomal protein L36. Furthermore, Kr-h1 phosphorylation with the concomitant inhibition of E93 transcription is evolutionarily conserved across insect orders. CONCLUSION: Our results suggest that Kr-h1 phosphorylation is indispensable for the recruitment of transcriptional cofactors, and for its anti-metamorphic and vitellogenic actions in insects. Our data shed new light on the understanding of Kr-h1 regulation and function in JH-regulated insect metamorphosis and reproduction.

The microRNAs let-7 and miR-278 regulate insect metamorphosis and oogenesis by targeting the juvenile hormone early-response gene Krüppel-homolog 1.

Krüppel-homolog 1 (Kr-h1), a zinc-finger transcription factor, inhibits larval metamorphosis and promotes adult reproduction by transducing juvenile hormone (JH). Although the transcriptional regulation of Kr-h1 has been extensively studied, little is known about its regulation at the post-transcriptional level. Using the migratory locust Locusta migratoria as a model system, we report here that the microRNAs let-7 and miR-278 bound to the Kr-h1 coding sequence and downregulated its expression. Application of let-7 and miR-278 mimics (agomiRs) significantly reduced the level of Kr-h1 transcripts, resulting in partially precocious metamorphosis in nymphs as well as markedly decreased yolk protein precursors, arrested ovarian development and blocked oocyte maturation in adults. Moreover, the expression of let-7 and miR-278 was repressed by JH, constituting a regulatory loop of JH signaling. This study thus reveals a previously unknown regulatory mechanism whereby JH suppresses the expression of let-7 and miR-278, which, together with JH induction of Kr-h1 transcription, prevents the precocious metamorphosis of nymphs and stimulates the reproduction of adult females. These results advance our understanding of the coordination of JH and miRNA regulation in insect development.

Post-transcriptional regulation of insect metamorphosis and oogenesis.

Metamorphic transformation from larvae to adults along with the high fecundity is key to insect success. Insect metamorphosis and reproduction are governed by two critical endocrines, juvenile hormone (JH), and 20-hydroxyecdysone (20E). Recent studies have established a crucial role of microRNA (miRNA) in insect metamorphosis and oogenesis. While miRNAs target genes involved in JH and 20E-signaling pathways, these two hormones reciprocally regulate miRNA expression, forming regulatory loops of miRNA with JH and 20E-signaling cascades. Insect metamorphosis and oogenesis rely on the coordination of hormones, cognate genes, and miRNAs for precise regulation. In addition, the alternative splicing of genes in JH and 20E-signaling pathways has distinct functions in insect metamorphosis and oogenesis. We, therefore, focus in this review on recent advances in post-transcriptional regulation, with the emphasis on the regulatory role of miRNA and alternative splicing, in insect metamorphosis and oogenesis. We will highlight important new findings of miRNA interactions with hormonal signaling and alternative splicing of JH receptor heterodimer gene Taiman.

Immediate Release of Gastrin-Releasing Peptide Mediates Delayed Radiation-Induced Pulmonary Fibrosis.

Radiation-induced pulmonary fibrosis (RTPF) is a progressive, serious condition in many subjects treated for thoracic malignancies or after accidental nuclear exposure. No biomarker exists for identifying the irradiated subjects most susceptible to pulmonary fibrosis (PF). Previously, we determined that gastrin-releasing peptide (GRP) was elevated within days after birth in newborns exposed to hyperoxia who later developed chronic lung disease. The goal of the current study was to test whether radiation (RT) exposure triggers GRP release in mice and whether this contributes to RTPF in vivo. We determined urine GRP levels and lung GRP immunostaining in mice 0 to 24 after post-thoracic RT (15 Gy). Urine GRP levels were significantly elevated between 24 hours post-RT; GRP-blocking monoclonal antibody 2A11, given minutes post-RT, abrogated urine GRP levels by 6 to 12 hours and also altered phosphoprotein signaling pathways at 24 hours post-RT. Strong extracellular GRP immunostaining was observed in lung at 6 hours post-RT. Mice given one dose of GRP monoclonal antibody 2A11 24 hours post-RT had significantly reduced myofibroblast accumulation and collagen deposition 15 weeks later, indicating protection against lung fibrosis. Therefore, elevation of urine GRP could be predictive of RTPF development. In addition, transient GRP blockade could mitigate PF in normal lung after therapeutic or accidental RT exposure.

Juvenile hormone-dependent Kazal-type serine protease inhibitor Greglin safeguards insect vitellogenesis and egg production.

In addition to preventing insect metamorphosis, juvenile hormone (JH) is known to stimulate aspects of insect reproduction. However, the molecular mechanisms of JH action in insect reproduction remain largely unknown. By reanalyzing the transcriptomic data from adults and other developmental stages of the migratory locust Locusta migratoria, we identified a gene coding for Kazal-type protease inhibitor, previously named Greglin. Greglin is specifically expressed in adult females and most abundant in the fat body and ovaries. Interestingly, Greglin is among the top 3 of highly expressed genes in adult female locusts, after 2 vitellogenin ( Vg) genes. Greglin is induced by JH and expressed at remarkably high levels in the vitellogenic stage. Knockdown of Greglin in adult female locusts results in accelerated degradation of serine protease substrate and significantly reduced levels of Greglin protein in hemolymph and ovaries. The consequent phenotypes include blocked oocyte maturation, arrested ovarian growth and shrunken follicular epithelium, as well as declines in egg number and hatchability. The data provide the first evidence, to our knowledge, that JH-dependent Greglin is involved in locust vitellogenesis and oocyte maturation likely by protecting vitellogenesis and other forms of yolk precursors from proteolysis. The result offers new insights into the regulation of JH and function of protease inhibitors in insect vitellogenesis, oocyte maturation and fecundity.-Guo, W., Wu, Z., Yang, L., Cai, Z., Zhao, L., Zhou, S. Juvenile hormone-dependent Kazal-type serine protease inhibitor Greglin safeguards insect vitellogenesis and egg production.

Protein kinase C mediates juvenile hormone-dependent phosphorylation of Na+/K+-ATPase to induce ovarian follicular patency for yolk protein uptake.

In oviparous animals, vitellogenesis is prerequisite to egg production and embryonic growth after oviposition. For successful insect vitellogenesis and oogenesis, vitellogenin (Vg) synthesized in the fat body (homologue to vertebrate liver and adipose tissue) must pass through the intercellular channels, a condition known as patency in the follicular epithelium, to reach the surface of oocytes. This process is controlled by juvenile hormone (JH) in many insect species, but the underlying mechanisms remain elusive. Previous work has suggested the possible involvement of Na+/K+-ATPase in patency initiation, but again, the regulatory cascade of Na+/K+-ATPase for patency initiation has been lacking. Using the migratory locust Locusta migratoria as a model system, we report here that RNAi-mediated knockdown of gene coding for Na+/K+-ATPase, inhibition of its phosphorylation, or suppression of its activity causes loss of patency, resulting in blocked Vg uptake, arrested oocyte maturation, and impaired ovarian growth. JH triggers G protein-coupled receptor (GPCR), receptor tyrosine kinase (RTK), phospholipase C (PLC), inositol trisphosphate receptor (IP3R), and protein kinase C (PKC) to phosphorylate Na+/K+-ATPase α-subunit at amino acid residue Ser8, consequently activating Na+/K+-ATPase for the induction of patency in vitellogenic follicular epithelium. Our results thus point to a previously unidentified mechanism by which JH induces the phosphorylation and activation of Na+/K+-ATPase via a signaling cascade of GPCR, RTK, PLC, IP3R, and PKC. The findings advance our understanding of JH regulation in insect vitellogenesis and oogenesis.

Calcineurin-mediated Dephosphorylation of Acetyl-coA Carboxylase is Required for Pheromone Biosynthesis Activating Neuropeptide (PBAN)-induced Sex Pheromone Biosynthesis in Helicoverpa armigera.

Chemical signaling plays a critical role in the behavior and physiology of many animals. Female insects, as many other animals, release sex pheromones to attract males for mating. The evolutionary and ecological success of insects therefore hinges on their ability to precisely mediate (including initiation and termination) pheromone biosynthesis. Pheromone biosynthesis activating neuropeptide (PBAN) acts directly on pheromone glands to regulate sex pheromone production using Ca2+ and cyclic-AMP as secondary messengers in the majority of species. However, the molecular mechanism downstream of the secondary messengers has not yet been elucidated in heliothine species. The present study shows that calcineurin, protein kinase A (PKA) and acetyl-coA carboxylase (ACC) are key components involved in PBAN-induced sex pheromone biosynthesis in Helicoverpa armigera using PBAN-dependent phosphoproteomics in combination with transcriptomics. RNAi-mediated knockdown and inhibitor assay demonstrated that calcineurin A is required for PBAN-induced ACC activation and sex pheromone production. Calcineurin-dependent phosphoproteomics and in vitro calcineurin phosphorylation assay further revealed that calcineurin regulated ACC activity by dephosphorylating ser84 and ser92. In addition, PKA-dependent phosphoproteomics and activity analysis revealed that PKA reduces the activity of AMP-activated protein kinase (AMPK), a negative regulator of ACC by phosphorylating the conserved ser92. Taken together, our findings indicate that calcineurin acts as the downstream signal of PBAN/G-protein receptor/Ca2+ to activate ACC through dephosphorylation while inactivating AMPK via PKA to reduce ACC phosphorylation, thus facilitating calcineurin activation of ACC.

Juvenile hormone differentially regulates two Grp78 genes encoding protein chaperones required for insect fat body cell homeostasis and vitellogenesis.

Juvenile hormone (JH) has a well known role in stimulating insect vitellogenesis (i.e. yolk deposition) and oocyte maturation, but the molecular mechanisms of JH action in insect reproduction are unclear. The 78-kDa glucose-regulated protein (Grp78) is a heat shock protein 70-kDa family member and one of the most abundant chaperones in the endoplasmic reticulum (ER) where it helps fold newly synthesized peptides. Because of its prominent role in protein folding, and also ER stress, we hypothesized that Grp78 might be involved in fat body cell homeostasis and vitellogenesis and a regulatory target of JH. We report here that the migratory locust Locusta migratoria possesses two Grp78 genes that are differentially regulated by JH. We found that Grp78-1 is regulated by JH through Mcm4/7-dependent DNA replication and polyploidization, whereas Grp78-2 expression is directly activated by the JH-receptor complex comprising methoprene-tolerant and Taiman proteins. Interestingly, Grp78-2 expression in the fat body is about 10-fold higher than that of Grp78-1 Knockdown of either Grp78-1 or Grp78-2 significantly reduced levels of vitellogenin (Vg) protein, accompanied by retarded maturation of oocytes. Depletion of both Grp78-1 and Grp78-2 resulted in ER stress and apoptosis in the fat body and in severely defective Vg synthesis and oocyte maturation. These results indicate a crucial role of Grp78 in JH-dependent vitellogenesis and egg production. The presence and differential regulation of two Grp78 genes in L. migratoria likely help accelerate the production of this chaperone in the fat body to facilitate folding of massively synthesized Vg and other proteins.

Juvenile Hormone Activates the Transcription of Cell-division-cycle 6 (Cdc6) for Polyploidy-dependent Insect Vitellogenesis and Oogenesis.

Although juvenile hormone (JH) is known to prevent insect larval metamorphosis and stimulate adult reproduction, the molecular mechanisms of JH action in insect reproduction remain largely unknown. Earlier, we reported that the JH-receptor complex, composed of methoprene-tolerant and steroid receptor co-activator, acts on mini-chromosome maintenance (Mcm) genes Mcm4 and Mcm7 to promote DNA replication and polyploidy for the massive vitellogenin (Vg) synthesis required for egg production in the migratory locust (Guo, W., Wu, Z., Song, J., Jiang, F., Wang, Z., Deng, S., Walker, V. K., and Zhou, S. (2014) PLoS Genet. 10, e1004702). In this study we have investigated the involvement of cell-division-cycle 6 (Cdc6) in JH-dependent vitellogenesis and oogenesis, as Cdc6 is essential for the formation of prereplication complex. We demonstrate here that Cdc6 is expressed in response to JH and methoprene-tolerant, and Cdc6 transcription is directly regulated by the JH-receptor complex. Knockdown of Cdc6 inhibits polyploidization of fat body and follicle cells, resulting in the substantial reduction of Vg expression in the fat body as well as severely impaired oocyte maturation and ovarian growth. Our data indicate the involvement of Cdc6 in JH pathway and a pivotal role of Cdc6 in JH-mediated polyploidization, vitellogenesis, and oogenesis.

Juvenile hormone-receptor complex acts on mcm4 and mcm7 to promote polyploidy and vitellogenesis in the migratory locust.

Juvenile hormone (JH), a sesquiterpenoid produced by the corpora allata, coordinates insect growth, metamorphosis, and reproduction. While JH action for the repression of larval metamorphosis has been well studied, the molecular basis of JH in promoting adult reproduction has not been fully elucidated. Methoprene-tolerant (Met), the JH receptor, has been recently shown to mediate JH action during metamorphosis as well as in vitellogenesis, but again, the precise mechanism underlying the latter has been lacking. We have now demonstrated using Met RNAi to phenocopy a JH-deprived condition in migratory locusts, that JH stimulates DNA replication and increases ploidy in preparation for vitellogenesis. Mcm4 and Mcm7, two genes in the DNA replication pathway were expressed in the presence of JH and Met. Depletion of Mcm4 or Mcm7 inhibited de novo DNA synthesis and polyploidization, and resulted in the substantial reduction of vitellogenin mRNA levels as well as severely impaired oocyte maturation and ovarian growth. By using luciferase reporter and electrophoretic mobility shift assays, we have shown that Met directly regulates the transcription of Mcm4 and Mcm7 by binding to upstream consensus sequences with E-box or E-box-like motifs. Our work suggests that the JH-receptor complex acts on Mcm4 and Mcm7 to regulate DNA replication and polyploidy for vitellogenesis and oocyte maturation.

Radiation-induced lung injury is mitigated by blockade of gastrin-releasing peptide.

Gastrin-releasing peptide (GRP), secreted by pulmonary neuroendocrine cells, mediates oxidant-induced lung injury in animal models. Considering that GRP blockade abrogates pulmonary inflammation and fibrosis in hyperoxic baboons, we hypothesized that ionizing radiation triggers GRP secretion, contributing to inflammatory and fibrotic phases of radiation-induced lung injury (RiLI). Using C57BL/6 mouse model of pulmonary fibrosis developing ≥20 weeks after high-dose thoracic radiation (15 Gy), we injected small molecule 77427 i.p. approximately 1 hour after radiation then twice weekly for up to 20 weeks. Sham controls were anesthetized and placed in the irradiator without radiation. Lung paraffin sections were immunostained and quantitative image analyses performed. Mice exposed to radiation plus PBS had increased interstitial CD68(+) macrophages 4 weeks after radiation and pulmonary neuroendocrine cells hyperplasia 6 weeks after radiation. Ten weeks later radiation plus PBS controls had significantly increased pSmad2/3(+) nuclei/cm(2). GRP blockade with 77427 treatment diminished CD68(+), GRP(+), and pSmad2/3(+) cells. Finally, interstitial fibrosis was evident 20 weeks after radiation by immunostaining for α-smooth muscle actin and collagen deposition. Treatment with 77427 abrogated interstitial α-smooth muscle actin and collagen. Sham mice given 77427 did not differ significantly from PBS controls. Our data are the first to show that GRP blockade decreases inflammatory and fibrotic responses to radiation in mice. GRP blockade is a novel radiation fibrosis mitigating agent that could be clinically useful in humans exposed to radiation therapeutically or unintentionally.

Gastrin-releasing peptide blockade as a broad-spectrum anti-inflammatory therapy for asthma.

Gastrin-releasing peptide (GRP) is synthesized by pulmonary neuroendocrine cells in inflammatory lung diseases, such as bronchopulmonary dysplasia (BPD). Many BPD infants develop asthma, a serious disorder of intermittent airway obstruction. Despite extensive research, early mechanisms of asthma remain controversial. The incidence of asthma is growing, now affecting >300 million people worldwide. To test the hypothesis that GRP mediates asthma, we used two murine models: ozone exposure for air pollution-induced airway hyperreactivity (AHR), and ovalbumin (OVA)-induced allergic airway disease. BALB/c mice were given small molecule GRP blocking agent 77427, or GRP blocking antibody 2A11, before exposure to ozone or OVA challenge. In both models, GRP blockade abrogated AHR and bronchoalveolar lavage (BAL) macrophages and granulocytes, and decreased BAL cytokines implicated in asthma, including those typically derived from Th1 (e.g., IL-2, TNFα), Th2 (e.g., IL-5, IL-13), Th17 (IL-17), macrophages (e.g., MCP-1, IL-1), and neutrophils (KC = IL-8). Dexamethasone generally had smaller effects on all parameters. Macrophages, T cells, and neutrophils express GRP receptor (GRPR). GRP blockade diminished serine phosphorylation of GRPR with ozone or OVA. Thus, GRP mediates AHR and airway inflammation in mice, suggesting that GRP blockade is promising as a broad-spectrum therapeutic approach to treat and/or prevent asthma in humans.