Mei-Juan Cai

Phospholipase Cγ1 Connects the Cell Membrane Pathway to the Nuclear Receptor Pathway in Insect Steroid Hormone Signaling

Background: PLCG1 plays an important role in calcium signaling. Results: PLCG1 up-regulates 20E-induced calcium signaling and regulates USP1 PKC phosphorylation in the lepidopteran insect Helicoverpa armigera. Conclusion: 20E activates PLCG1 to induce calcium influx to regulate USP1 PKC phosphorylation for gene expression. Significance: Our study establishes a link between the nongenomic pathway and genomic pathway in steroid hormone 20E signaling. In addition to the classical nuclear receptor pathway, there is a nongenomic pathway in the cell membrane that regulates gene expression in animal steroid hormone signaling; however, this mechanism is unclear. Here, we report that the insect steroid hormone 20-hydroxyecdysone (20E) regulates calcium influx via phospholipase Cγ1 (PLCG1) to modulate the protein kinase C phosphorylation of the transcription factor ultraspiracle (USP1) in the lepidopteran insect Helicoverpa armigera. The PLCG1 mRNA levels are increased during the molting and metamorphic stages. The depletion of PLCG1 by RNA interference can block 20E-enhanced pupation, cause larvae death and pupation defects, and repress 20E-induced gene expression. 20E may induce the tyrosine phosphorylation of PLCG1 at the cytosolic tyrosine kinase (Src) homology 2 domains and then determine the migration of PLCG1 toward the plasma membrane. The G-protein-coupled receptor (GPCR) inhibitor suramin, Src family kinase inhibitor PP2, and the depletions of ecdysone-responsible GPCR (ErGPCR) and Gαq restrain the 20E-induced tyrosine phosphorylation of PLCG1. PLCG1 participates in the 20E-induced Ca2+ influx. The inhibition of GPCR, PLC, inositol 1,4,5-trisphosphate receptor, and calcium channels represses the 20E-induced Ca2+ influx. Through calcium signaling, PLCG1 mediates the transcriptional activation driven by the ecdysone-response element. Through PLCG1 and calcium signaling, 20E regulates PKC phosphorylation of USP1 at Ser-21 to determine its ecdysone-response element binding activity. These results suggest that 20E activates PLCG1 via the ErGPCR and Src family kinases to regulate Ca2+ influx and PKC phosphorylation of USP1 to subsequently modulate gene transcription for metamorphosis.

The hormone-dependent function of Hsp90 in the crosstalk between 20-hydroxyecdysone and juvenile hormone signaling pathways in insects is determined by differential phosphorylation and protein interactions.

BACKGROUND Heat shock protein 90 (Hsp90) interacts with steroid hormone receptors, signaling kinases, and various transcription factors. However, the mechanism by which Hsp90 interacts with different proteins in various pathways remains unclear. METHODS Western blot was used to study Hsp90 expression profile in Helicoverpa armigera (Lepidoptera). RNA interference was performed to investigate the function of Hsp90 in 20-hydroxyecdysone (20E) and juvenile hormone (JH) signal pathways. The binding of Hsp90 to the transcription factor ultraspiracle protein (USP1) and JH candidate receptor methoprene-tolerant (Met1) was analyzed by co-immunoprecipitation. Phospho-(Ser) PKC substrate antibody was used to detect Hsp90 phosphorylation. RESULTS Hsp90 participated in 20E- or JH-induced gene expression. 20E induced the interaction between Hsp90 and USP1, whereas JH III and methoprene induced the interaction between Hsp90 and Met1, respectively. 20E and JH counteracted each other for these protein interactions. Both JH III and methoprene induced protein kinase C (PKC) phosphorylation of Hsp90. This process could be inhibited by phospholipase C (PLC) and PKC inhibitors. 20E suppressed JH III- or methoprene-induced PKC phosphorylation of Hsp90. CONCLUSION 20E maintained the non-PKC-phosphorylation status of Hsp90. Hsp90 interacted with USP1 to induce gene expression in the 20E pathway. JH regulated the PKC-phosphorylation status of Hsp90. Hsp90 also interacted with Met1 to induce gene expression in the JH pathway. GENERAL SIGNIFICANCE Our study describes a novel mechanism of Hsp90 action by altering phosphorylation and protein interaction in various hormonal signaling pathways.

G-protein-coupled receptor participates in 20-hydroxyecdysone signaling on the plasma membrane

BackgroundAnimal steroid hormones are conventionally known to initiate signaling via a genomic pathway by binding to the nuclear receptors. The mechanism by which 20E initiates signaling via a nongenomic pathway is unclear.ResultsWe illustrate that 20E triggered the nongenomic pathway through a plasma membrane G-protein-coupled receptor (named ErGPCR) in the lepidopteran insect Helicoverpa armigera. The transcript of ErGPCR was increased at the larval molting stage and metamorphic molting stage by 20E regulation. Knockdown of ErGPCR via RNA interference in vivo blocked larval–pupal transition and suppressed 20E-induced gene expression. ErGPCR overexpression in the H. armigera epidermal cell line increased the 20E-induced gene expression. Through ErGPCR, 20E modulated Calponin nuclear translocation and phosphorylation, and induced a rapid increase in cytosolic Ca2+ levels. The inhibitors of T-type voltage-gated calcium channels and canonical transient receptor potential calcium channels repressed the 20E-induced Ca2+ increase. Truncation of the N-terminal extracellular region of ErGPCR inhibited its localization on the plasma membrane and 20E-induced gene expression. ErGPCR was not detected to bind with the steroid hormone analog [3H]Pon A.ConclusionThese results suggest that ErGPCR participates in 20E signaling on the plasma membrane.

Juvenile hormone prevents 20-hydroxyecdysone-induced metamorphosis by regulating the phosphorylation of a newly identified Broad protein

Background: JH antagonizes the 20E pathway. Results: JH induces Broad protein BrZ7 phosphorylation in the lepidopteran insect Helicoverpa armigera. Conclusion: JH induces the phosphorylation of BrZ7 to inhibit 20E-mediated metamorphosis. Significance: Our study reveals a mechanism of JH antagonizing 20E-activating metamorphosis. The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5′-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7.

20-Hydroxyecdysone activates Forkhead box O to promote proteolysis during Helicoverpa armigera molting.

Insulin inhibits transcription factor Forkhead box O (FoxO) activity, and the steroid hormone 20-hydroxyecdysone (20E) activates FoxO; however, the mechanism is unclear. We hypothesized that 20E upregulates phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN) expression to activate FoxO, thereby promoting proteolysis during molting in the lepidopteran insect Helicoverpa armigera. FoxO expression is increased during molting and metamorphosis. The knockdown of FoxO in fifth instar larvae results in larval molting failure. 20E inhibits FoxO phosphorylation, resulting in FoxO nuclear translocation. Insulin, via Akt, induces FoxO phosphorylation and cytoplasmic localization. 20E represses insulin-induced Akt phosphorylation and FoxO phosphorylation. 20E, via ecdysone receptor B1 (EcRB1) and the ultraspiracle protein (USP1), upregulates PTEN expression, which represses Akt phosphorylation, thereby repressing FoxO phosphorylation. The non-phosphorylated FoxO enters the nucleus and attaches to a FoxO-binding element in the upstream region of the Broad isoform 7 (BrZ7) gene to regulate BrZ7 transcription under 20E induction. 20E upregulates FoxO expression via EcRB1 and USP1. FoxO regulation of BrZ7 expression regulates Carboxypeptidase A expression for final proteolysis during insect molting. Hence, 20E activates FoxO via upregulating PTEN expression to counteract insulin activity and promote proteolysis. Summary: Steroid hormone 20E upregulates PTEN expression to inhibit insulin-induced Akt and FoxO phosphorylation, resulting in non-phosphorylated FoxO nuclear localisation.

Small GTPase Rab4b participates in the gene transcription of 20-hydroxyecdysone and insulin pathways to regulate glycogen level and metamorphosis.

The insulin and 20-hydroxyecdysone (20E) pathways coordinately regulate insect growth and metamorphosis. However, the molecular mechanism of the interaction of these two pathways in regulating insect development is not well understood. In the present study, we found that a small GTPase Rab4b from a lepidopteran insect Helicoverpa armigera participates in gene transcription in the two pathways. The results show that RNA interference of Rab4b in larvae results in a decrease in glycogen levels, small pupae, abnormal metamorphic transition, or larval death. The molecular mechanisms are demonstrated that knockdown of Rab4b in the larvae suppresses the transcription of glycogen synthase (GS), as well as the metamorphic-initiating factor (Br) and hormone receptor 3 (HR3), but increases the transcription of Forkhead box class O (FOXO). Further studies in the cell line confirm that Rab4b is necessary for gene transcription in the insulin and 20E pathways. Rab4b locates in the cytoplasm and takes part in regulation on FOXO cytoplasmic location by insulin induction, but travels toward the cell membrane upon 20E induction without affecting the FOXO location. The transcription of Rab4b could be upregulated by insulin injection or glucose feeding to the larvae, but not by 20E or juvenile hormone analogy methoprene. Our data suggest that Rab4b takes part in metamorphosis by regulating gene transcription and glycogen level in the insulin and 20E pathways.

In a Nongenomic Action, Steroid Hormone 20-Hydroxyecdysone Induces Phosphorylation of Cyclin-Dependent Kinase 10 to Promote Gene Transcription

The insect steroid hormone 20-hydroxyecdysone (20E) regulates gene transcription via a genomic pathway by forming a transcription complex that binds to DNA with the help of the chaperone proteins, heat shock proteins (Hsps) Hsc70 and Hsp90. However, the nongenomic mechanisms by which 20E regulates gene expression remain unclear. In this study, we found that 20E regulated the phosphorylation of serine/threonine protein kinase cyclin-dependent kinase 10 (CDK10) through a nongenomic pathway to mediate gene transcription in the lepidopteran Helicoverpa armigera. The down-regulation of CDK10 by RNA interference in larvae and the epidermal cell line delayed development and suppressed 20E-induced gene transcription. CDK10 was localized to the nucleus via its KKRR motif, and this nuclear localization and the ATPase motif were necessary for the efficient expression of the 20E-inducible gene. The rapid phosphorylation of CDK10 was induced by 20E, whereas it was repressed by the inhibitors of G-protein-coupled receptors, phospholipase C, and Ca²⁺ channels. Phosphorylated CDK10 exhibited increased interactions with Hsps Hsc70 and Hsp90 and then promoted the interactions between Hsps and ecdysone receptor EcRB1 and the binding of the Hsps-EcRB1 complex to the 20E response element for the regulation of gene transcription. CDK10 depletion suppressed the formation of the Hsps-EcRB1 complex at the hormone receptor 3 promoter. These results suggest that 20E induces CDK10 phosphorylation via a nongenomic pathway to regulate gene transcription in the nucleus.

Mod(mdg4) participates in hormonally regulated midgut programmed cell death during metamorphosis

The insect midgut undergoes programmed cell death (PCD) during metamorphosis, but the molecular basis for this phenomenon has not been demonstrated. We report a mod(mdg4) protein [designated as mod(mdg4)1A] that is involved in hormonally regulated insect midgut PCD, from the lepidopteran Helicoverpa armigera. Mod(mdg4)1A is localized in the larval midgut and is highly expressed during metamorphosis. Knockdown of mod(mdg4)1a by feeding dsRNA to the larvae suppressed midgut PCD and delayed metamorphosis. The mechanism is that mod(mdg4)1a knockdown decreased the transcript levels of genes involved in PCD and metamorphosis, but increased the transcript level of inhibitor of apoptosis survivin. The transcript level of mod(mdg4)1a is independently upregulated by 20-hydroxyecdysone (20E) or juvenile hormone (JH) analog methoprene. Overlapped 20E and methoprene counteractively regulate the transcript level of mod(mdg4)1a. 20E upregulates the mod(mdg4)1a transcript level not through its nuclear receptor EcRB1. Methoprene upregulates the mod(mdg4)1a transcript level through the juvenile hormone candidate receptor Met. These findings indicate that mod(mdg4)1a participates in midgut PCD and metamorphosis by regulating the transcript levels of a network of genes via different pathways under 20E and JH regulation.

G-protein-coupled receptor controls steroid hormone signaling in cell membrane

G-protein-coupled receptors (GPCRs) are involved in animal steroid hormone signaling, but their mechanism is unclear. In this research, we report that a GPCR called ErGPCR-2 controls steroid hormone 20-hydroxyecdysone (20E) signaling in the cell membrane of the lepidopteran insect Helicoverpa armigera. ErGPCR-2 was highly expressed during molting and metamorphosis. 20E, via ErGPCR-2, regulated rapid intracellular calcium increase, protein phosphorylation, gene transcription, and insect metamorphosis. ErGPCR-2 was located in the cell surface and was internalized by 20E induction. GPCR kinase 2 participated in 20E-induced ErGPCR-2 phosphorylation and internalization. The internalized ErGPCR-2 was degraded by proteases to desensitize 20E signaling. ErGPCR-2 knockdown suppressed the entrance of 20E analog [3H] ponasterone A ([3H]Pon A) into the cells. ErGPCR-2 overexpression or blocking of ErGPCR-2 internalization increased the entrance of [3H]Pon A into the cells. However, ErGPCR-2 did not bind to [3H]Pon A. Results suggest that ErGPCR-2 transmits steroid hormone 20E signaling and controls 20E entrance into cells in the cell membrane.

Heat shock protein 90 maintains the stability and function of transcription factor Broad Z7 by interacting with its Broad-Complex-Tramtrack-Bric-a-brac domain

Heat shock protein 90 (Hsp90) is a highly conserved chaperone protein that interacts with various client proteins to mediate their folding and stability. The Broad‐Complex‐Tramtrack‐Bric‐a‐brac (BTB) domain, also known as poxvirus and zinc finger (POZ) domain, exists widely in different proteins and is highly conserved. However, the stability mechanism of BTB domain‐containing proteins has not been fully understood. Co‐immunoprecipitation and a protein pull‐down assay were performed to investigate the interaction between Hsp90 and the transcription factor Broad isoform Z7 (BrZ7) in vivo and in vitro. The middle domain of Hsp90 directly associated with the BTB domain of BrZ7. The Hsp90 inhibitor 17‐(Allylamino)‐17‐demethoxygeldanamycin (17‐AAG) interrupted the interaction between Hsp90 and BrZ7 and decreased the protein level of BrZ7 but did not affect the mRNA level of BrZ7. The addition of the proteasome inhibitor peptide aldehyde Cbz‐leu‐leu leucinal suppressed the 17‐AAG‐induced degradation of BrZ7. BTB domain deletion and 17‐AAG treatment resulted in inhibition of BrZ7 function in gene expression in the 20‐hydroxyecdysone and juvenile hormone pathways. These results reveal that the middle domain of Hsp90 associates with the BTB domain of BrZ7 to prevent BrZ7 degradation and maintain BrZ7 function in gene expression in the lepidopteran insect Helicoverpa armigera.