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

Abstract

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.

Introduction

Vitellogenesis is one of the most emblematic processes in the reproduction of oviparous animals. During insect vitellogenesis, vitellogenin (Vg)² and other forms of yolk proteins are mostly synthesized in the fat body (analogue to the liver and adipose tissue in vertebrates), secreted into the hemolymph, and taken up by the developing oocytes (1–3). This process is stimulated by the arthropod-specific sesquiterpenoid hormone, juvenile hormone (JH), in insects belonging to diverse orders, such as the coleopteran beetle Tribolium castaneum, the dictyopteran cockroach Blattella germanica, and the orthopteram migratory locust Locusta migratoria (1–5). In the process of insect vitellogenesis, JH acts on follicle cells, initiating the intercellular channels (termed patency) to permit Vg in the hemolymph to gain access to the oocyte membrane where Vg is internalized into maturing oocytes by receptor-mediated endocytosis (6–9). JH-dependent patency in the follicular epithelium has been reported previously in many insects, from the basal species with panoistic ovaries like L. migratoria to the more advanced ones with meroistic ovaries like the fruit fly Drosophila melanogaster and the mosquito Aedes aegypti (1–3). However, the underlying machinery in the regulation of patency remains largely unknown.

Earlier studies on the kissing bug Rhodnius prolixus have shown that patency initiation is inhibited by ouabain treatment, suggesting the involvement of Na⁺/K⁺-ATPase in this process (6, 10). Thereafter, the possible role of Na⁺/K⁺-ATPase in patency has been reported in several other insect species including L. migratoria, the beetle Tenebrio molitor, and the moth Heliothis virescens (6, 8, 11–14). Na⁺/K⁺-ATPase is a transmembrane transporter controlling the internal and external ion concentration gradient of cells (15–17). Recent studies have illustrated the function of Na⁺/K⁺-ATPase in osmoregulation of crustaceans, neurological cancers, and therapeutic targets for diseases (18–20). Na⁺/K⁺-ATPase is also reported as an efficient target of insecticides (21, 22). Whereas extensive studies have been carried out to elucidate the function of Na⁺/K⁺-ATPase in controlling the ion transport through cell membranes, the regulatory cascade of Na⁺/K⁺-ATPase activity in JH-dependent initiation of patency has been lacking.

Na⁺/K⁺-ATPase is a heterodimer protein consisting of evolutionarily conserved α- and β-subunits present in equimolar ratios (15, 23, 24). The α-subunit, showing 10 transmembrane domains containing short extracellular loops and larger cytoplasmic regions, plays the main function in catalytic action because of its ATP binding site and phosphorylation (15, 25). Protein kinase C (PKC) mediates the phosphorylation of α-subunit at Ser¹¹, Ser¹⁶, or Ser¹⁸ to enhance or repress the catalytic action of Na⁺/K⁺-ATPase in vertebrates (26–30). Interestingly, in the rat Rattus norvegicus, PKC phosphorylation of α-subunit at Ser²³ corresponding to Ser¹⁸ of the mature cleaved form stimulates its activity, whereas phosphorylation of α-subunit at Ser⁹⁴³ by protein kinase A (PKA) inhibits its activity (31–33). The activity of Na⁺/K⁺-ATPase can be rapidly induced by hormones, including cortisol, growth hormone, insulin-like growth factor I, prolactin, thyroid hormones, and steroids (34). Regulation of Na⁺/K⁺-ATPase activity by JH in relation to patency initiation has been proposed for R. prolixus, L. migratoria, and H. virescens (6, 8, 35), but the molecular basis of JH action on Na⁺/K⁺-ATPase activity has not been determined.

JH exerts both genomic and nongenomic actions. In genomic action, JH induces the heterodimerization of methoprene-tolerant (Met) with taiman (Tai) to form an active receptor complex that regulates the transcription of JH-responsive genes (36–39). In nongenomic actions, JH is reported to trigger receptor tyrosine kinase (RTK), phospholipase C (PLC), inositol trisphosphate (IP₃), and calcium/calmodulin-dependent protein kinase II (CaMKII) to stimulate the phosphorylation of Met for enhanced transcriptional regulation activity in A. aegypti (40–42). In the bollworm Helicoverpa armigera, JH acts on phosphorylation of BrC-Z7 to inhibit 20E-mediated metamorphosis via a GPCR, PLC, and PKC signaling pathway (40). Through PLC and PKC, JH also stimulates Hsp90 phosphorylation to regulate JH-responsive gene transcription (43). Previously, pharmaceutical experiments with PKC inhibitors and activators have suggested that this enzyme is involved in the activation of Na⁺/K⁺-ATPase (1, 6). We therefore hypothesized that JH might trigger nongenomic pathways to activate PKC-mediated phosphorylation of Na⁺/K⁺-ATPase for patency in vitellogenic follicular epithelium. L. migratoria is a favorable model for studying JH-dependent patency and vitellogenesis (1). With this model system, we report in the present study that depletion of Na⁺/K⁺-ATPase or blocking its activity caused loss of patency, leading to inhibited Vg uptake, accompanied by arrested oocyte maturation and impaired ovarian growth. We demonstrated that JH triggered a GPCR, RTK, PLC, IP3R, and PKC pathway to phosphorylate the α-subunit at Ser⁸, consequently activating Na⁺/K⁺-ATPase for the induction of patency during vitellogenesis. These results shed some light on the regulatory mechanisms of JH-dependent vitellogenesis and oogenesis in insects.

Results

Na⁺/K⁺-ATPase α-subunit knockdown blocks ovarian Vg uptake and oocyte maturation

As the role of Na⁺/K⁺-ATPase in locust vitellogenesis and oogenesis had not been previously determined by gene knockdown, we initially performed Na⁺/K⁺-ATPase α-subunit (GenBank^TM number MH450018) RNAi in vitellogenic adult female locusts. qRT-PCR showed that the mRNA levels of Na⁺/K⁺-ATPase α-subunit were reduced by 81% in the ovary of adult females at 6 days post-adult eclosion (PAE) (Fig. 1A). Western blotting demonstrated that the protein levels of Na⁺/K⁺-ATPase α-subunit declined by 85% after RNAi (Fig. 1B). The migratory locust has two Vg genes, VgA (GenBank^TM number KF171066) and VgB (GenBank^TM number KX709496) (45). Depletion of Na⁺/K⁺-ATPase α-subunit caused 77 and 73% reduction of VgA and VgB protein levels, respectively, in the ovaries (Fig. 1, B and C). Consequently, the primary oocytes and ovaries of Na⁺/K⁺-ATPase α-subunit–depleted adult females remained small on day 6 (Fig. 1D). In contrast, the primary oocytes and ovaries of dsGFP controls were remarkably enlarged (Fig. 1D). We measured the length of primary oocyte as an indicator of oocyte maturation. Statistically, the average length of primary oocytes of Na⁺/K⁺-ATPase α-subunit–depleted locusts was 1.9 mm, whereas that of dsGFP controls was 5.3 mm (Fig. 1D). These results reinforce our view that Na⁺/K⁺-ATPase plays a pivotal role in ovarian Vg uptake and oocyte maturation of L. migratoria.

Figure 1.

Effects of Na⁺/K⁺-ATPase α-subunit knockdown on ovarian Vg uptake and oocyte maturation. A, knockdown efficiency of Na⁺/K⁺-ATPase α-subunit (dsNK) in the ovary measured by qRT-PCR. **, p < 0.01 compared with the dsGFP control. n = 5. B, Western blotting showing the relative protein levels of VgA, VgB, and Na⁺/K⁺-ATPase α-subunit (NK) in the ovary of adult females subjected to Na⁺/K⁺-ATPase α-subunit knockdown versus the dsGFP controls. C, quantitative analysis of band intensity by ImageJ on ovarian Vg uptake as representatively shown in B. **, p < 0.01 compared with the respective dsGFP controls. n = 3. D, representative phenotypes of ovaries and ovarioles after Na⁺/K⁺-ATPase α-subunit knockdown (dsNK) versus the dsGFP control. Scale bars, 0.5 cm (blue) and 1 mm (red). E, statistical analysis for the length of primary oocytes in Na⁺/K⁺-ATPase α-subunit–depleted locusts compared with the dsGFP controls. **, p < 0.01. n = 15. Error bars, S.E.

JH induces PKC-mediated phosphorylation and activation of Na⁺/K⁺-ATPase

Western blots were conducted to reveal the dynamics of ovarian Vg uptake during the first gonadotrophic cycle, using the proteins extracted from ovaries of adult females collected from 1 to 8 days PAE. As shown in Fig. 2A, the levels of VgA and VgB proteins in the ovary were substantially increased after day 4 and remained high at 5–7 days PAE and then decreased at the end of the first gonadotrophic cycle. Our ELISA showed that Na⁺/K⁺-ATPase activity in the ovary was significantly increased on day 4, continually elevated at 5–6 days PAE, and thereafter (7–8 days PAE) declined to levels similar to those at 0–3 days PAE (Fig. 2B). The Na⁺/K⁺-ATPase activity was lowest at 8 days PAE (Fig. 2B). These data suggest a correlation between Na⁺/K⁺-ATPase activity and ovarian Vg uptake in the vitellogenic stage. As PKC has been suggested to phosphorylate and activate Na⁺/K⁺-ATPase (1, 6), we next performed immunoprecipitation using an anti-phospho-(Ser)-PKC substrate antibody to explore the developmental profiles of PKC-mediated phosphorylation of Na⁺/K⁺-ATPase α-subunit. As shown in Fig. 2 (C and D), the levels of phosphorylated Na⁺/K⁺-ATPase α-subunit were significantly increased on day 2, reached peaks at 4–7 days PAE, and then declined on day 8. As locust hemolymph JH titer is undetectable at eclosion but elevates significantly in the previtellogenic stage and rises to a peak during vitellogenesis (46, 47), the elevation of Na⁺/K⁺-ATPase α-subunit phosphorylation appeared to correlate with its enhanced activity and the phase of increased JH titers.

Figure 2.

JH-induced and PKC-mediated phosphorylation and activation of Na⁺/K⁺-ATPase. A, Western blotting showing the developmental profiles of Vg uptake in the ovary of adult female locusts during the first gonadotrophic cycle. B, Na⁺/K⁺-ATPase activity in the ovary of adult females from 1 to 8 days PAE. Means labeled with different letters indicate significant difference at p < 0.05. n = 3. C, temporal abundance of PKC-mediated phosphorylation of Na⁺/K⁺-ATPase α-subunit in the ovary. NK, Na⁺/K⁺-ATPase α-subunit. P-NK, phosphorylated Na⁺/K⁺-ATPase α-subunit. WB, Western blotting. IP, immunoprecipitation. D, quantitative analysis of band intensity by ImageJ on PKC-phosphorylated Na⁺/K⁺-ATPase α-subunit (P-NK) as representatively shown in C. Different letters indicate significant differences at p < 0.05 (n = 3). E, effects of methoprene (Meth) treatment on the activity of Na⁺/K⁺-ATPase in locust ovary. **, p < 0.01 compared with the DMSO controls. n = 3. F, effects of methoprene treatment on PKC-mediated phosphorylation of Na⁺/K⁺-ATPase α-subunit in the ovary. G, quantitative analysis of band intensity by ImageJ on PKC-phosphorylated Na⁺/K⁺-ATPase α-subunit as representatively shown in F. **, p < 0.01. n = 3. Error bars, S.E.

To assess the responsiveness of Na⁺/K⁺-ATPase α-subunit phosphorylation and activation to JH, we performed ELISA and immunoprecipitation using proteins extracted from ovaries of 2-day-old adult females treated with methoprene for 0–60 min. The activity of Na⁺/K⁺-ATPase was significantly enhanced 30 min post-methoprene administration (Fig. 2E). With respect to PKC-mediated phosphorylation of Na⁺/K⁺-ATPase α-subunit, the levels were significantly increased 15 min after methoprene application (Fig. 2, F and G). Collectively, the above data indicate that JH stimulates PKC-mediated phosphorylation of Na⁺/K⁺-ATPase α-subunit for its activation.

Phosphorylation and activation of Na⁺/K⁺-ATPase promote ovarian Vg uptake

To determine the effect of Na⁺/K⁺-ATPase phosphorylation and activation on ovarian Vg uptake, we carried out in vivo Vg uptake assays by treating JH-deprived adult females with methoprene and the hemolymph containing Vg but without endogenous JH. As evaluated by immunoprecipitation and Western blotting, depletion of Na⁺/K⁺-ATPase α-subunit led to significant reduction of PKC-mediated phosphorylation of Na⁺/K⁺-ATPase α-subunit (Fig. 3, A and B). Application of ouabain, an inhibitor of Na⁺/K⁺-ATPase α-subunit (48), had no significant effect on its phosphorylation (Fig. 3, A and B). Either Na⁺/K⁺-ATPase α-subunit knockdown or ouabain treatment caused significant reduction of Na⁺/K⁺-ATPase activity (Fig. 3C). As shown in Fig. 3 (D–F), methoprene treatment induced Vg uptake in the ovary. Knockdown of Na⁺/K⁺-ATPase α-subunit precluded methoprene-induced ovarian Vg uptake (Fig. 3, D–F). The capacity of methoprene to induce ovarian Vg uptake was also blocked by ouabain treatment (Fig. 3, D–F). These observations indicate that activated Na⁺/K⁺-ATPase by JH and PKC is crucial to Vg uptake in the ovary.

Figure 3.

Phosphorylation-dependent activation of Na⁺/K⁺-ATPase and Vg uptake by ovaries. A, immunoprecipitation (IP) and Western blotting (Wb) showing effects of RNAi or ouabain treatment on PKC-mediated phosphorylation of Na⁺/K⁺-ATPase α-subunit in the ovary. Precocene, precocene III; Meth, methoprene; dsNK, dsRNA for Na⁺/K⁺-ATPase α-subunit. B, quantitative analysis of band intensity by ImageJ on PKC-mediated phosphorylation of Na⁺/K⁺-ATPase α-subunit (P-NK) as representatively shown in A. **, p < 0.01. C, relative activity of Na⁺/K⁺-ATPase in the ovary after RNAi (dsNK) or ouabain treatment. **, p < 0.01. n = 3. D, effects of Na⁺/K⁺-ATPase α-subunit knockdown or ouabain treatment on JH analogue–induced ovarian Vg uptake. E and F, quantitative analysis of band intensity by ImageJ on ovarian VgA (E) and VgB (F) uptake as representatively shown in D. **, p < 0.01. Error bars, S.E.

JH activates Na⁺/K⁺-ATPase via triggering its α-subunit phosphorylation at Ser⁸

It is well-known that PKC phosphorylates R. norvegicus Na⁺/K⁺-ATPase α-subunit 1 at Ser²³ (49, 50). We therefore performed N terminus amino acid sequence alignment of α-subunits between R. norvegicus and L. migratoria. As shown in Fig. 4A, Ser⁸ of locust Na⁺/K⁺-ATPase α-subunit is highly homologous to Ser²³ of R. norvegicus α-subunit 1. Intriguingly, the motif RKEPS at amino acid residues 4–8 of the locust Na⁺/K⁺-ATPase α-subunit (Fig. 4A) is a conserved sequence recognized by PKC (51, 52). These findings suggest that Ser⁸ is a potential phosphorylation site of locust Na⁺/K⁺-ATPase α-subunit. We next tested whether the phospho-Na⁺/K⁺-ATPase α1 (Ser²³) antibody against PKC-phosphorylated Na⁺/K⁺-ATPase α-subunit 1 of R. norvegicus recognizes the Na⁺/K⁺-ATPase α-subunit of L. migratoria. Clearly, JH-induced phosphorylation of locust Na⁺/K⁺-ATPase α-subunit was detectable (Fig. 4B), and the pattern resembled that observed in immunoprecipitation using an anti-phospho-(Ser)-PKC substrate antibody (Fig. 2F). To confirm this phosphorylation site, we mutated Ser⁸ to Ala⁸ by site-directed mutagenesis. The WT (pIEx-4-NK-RFP-His) or mutated (pIEx-4-NK^S8A-RFP-His) vectors were constructed and transfected into Sf9 cells for expression of recombinant proteins and further Western blotting analyses. When Sf9 cells were transfected with the WT pIEx-4-NK-RFP-His, additional methoprene treatment induced phosphorylation of Na⁺/K⁺-ATPase α-subunit, as detected by phospho-Na⁺/K⁺-ATPase α1 (Ser²³) antibody (Fig. 4C). In contrast, methoprene-induced phosphorylation of Na⁺/K⁺-ATPase α-subunit was not detectable when mutated pIEx-4-NK^S8A-RFP-His was transfected (Fig. 4C). In parallel control experiments with the empty vector pIEx-4-RFP-His, no phosphorylation band was observed with phospho-Na⁺/K⁺-ATPase α1 (Ser²³) antibody (Fig. 4D). Mutation of Ser⁸ to Ala⁸ also inhibited methoprene-induced Na⁺/K⁺-ATPase activity as measured by ELISA (Fig. 4E). Taken together, these results imply that locust Na⁺/K⁺-ATPase α-subunit is phosphorylated at Ser⁸, which mediates JH-induced activation of Na⁺/K⁺-ATPase.

Figure 4.

JH-stimulated Na⁺/K⁺-ATPase α-subunit phosphorylation at Ser⁸. A, alignment of N terminus amino acid sequences of Na⁺/K⁺-ATPase α-subunits (NK) from R. norvegicus (Rn) (GenBank^TM number NP_036636.1) and L. migratoria (Lm) (GenBank^TM number MH450018). Numbers denote amino acid residues. B, Western blotting showing JH induction on phosphorylation of Na⁺/K⁺-ATPase α-subunit in locust ovary, detected by phospho-Na⁺/K⁺-ATPase α1 (Ser²³) antibody. NK, Na⁺/K⁺-ATPase α-subunit. C, JH triggered locust Na⁺/K⁺-ATPase α-subunit phosphorylation at Ser⁸. Sf9 cells transfected with pIEx-4-NK-RFP-His (NK-RFP-His) or pIEx-4-NK^S8A-RFP-His (NKS8A-RFP-His) were treated with methoprene (Meth) or DMSO, followed by Western blotting with phospho-Na⁺/K⁺-ATPase α1 (Ser²³) antibody. D, Sf9 cells transfected with the empty vector, pIEx-4-RFP-His as the negative control. E, inhibition of JH-triggered Na⁺/K⁺-ATPase activity by mutation of locust Na⁺/K⁺-ATPase α-subunit Ser⁸ to Ala⁸. **, p < 0.01. n = 3. Error bars, S.E.

Na⁺/K⁺-ATPase α-subunit phosphorylation mediates JH-induced patency

In our in vitro assays, patency was induced by methoprene treatment (Fig. 5A). Application of either ouabain or chelerythrine chloride restrained methoprene-induced patency (Fig. 5A), suggesting that JH-induced patency depends on PKC-mediated Na⁺/K⁺-ATPase activation. We next performed Western blotting and ELISA to further characterize the role of Na⁺/K⁺-ATPase α-subunit phosphorylation at Ser⁸ in Na⁺/K⁺-ATPase activity and function. Western blotting showed that methoprene-induced phosphorylation of Na⁺/K⁺-ATPase α-subunit at Ser⁸ was alleviated by treatment of chelerythrine chloride but not ouabain (Fig. 5B), indicating that PKC mediates the phosphorylation of locust Na⁺/K⁺-ATPase α-subunit at Ser⁸. As measured by ELISA, either ouabain or chelerythrine chloride treatment resulted in significant decrease of methoprene-stimulated Na⁺/K⁺-ATPase activity (Fig. 5C). Notably, ouabain or chelerythrine chloride (CC) administration blocked ovarian Vg uptake that was induced by methoprene (Fig. 5D). Collectively, these results suggest that JH induces patency and subsequent ovarian Vg uptake via triggering PKC-mediated phosphorylation of Na⁺/K⁺-ATPase α-subunit at Ser⁸.

Figure 5.

Na⁺/K⁺-ATPase α-subunit phosphorylation and JH-induced patency. A, inhibition of JH-induced patency by ouabain and CC treatment. Meth, methoprene. White arrows, patency. Red bars, 10 μm. B, Western blotting showing the effects of ouabain and CC treatment on JH-induced phosphorylation of Na⁺/K⁺-ATPase α-subunit at Ser⁸. NK, Na⁺/K⁺-ATPase α-subunit. C, effects of ouabain and CC treatment on JH-induced Na⁺/K⁺-ATPase activity. **, p < 0.01. n = 3. D, abolishment of JH-induced ovarian Vg uptake by ouabain and CC treatment. Error bars, S.E.

JH induces phosphorylation of Na⁺/K⁺-ATPase α-subunit via a GPCR/RTK-PLC-IP3R-PKC signaling pathway

To unveil the molecules potentially involved in the regulation of JH-dependent phosphorylation of Na⁺/K⁺-ATPase α-subunit at Ser⁸, we selected a series of inhibitors to treat 5-day-old adult female locusts for ovarian protein extraction, followed by Western blotting using the phospho-Na⁺/K⁺-ATPase α1 (Ser²³) antibody. When the GPCR inhibitor suramin and the RTK inhibitor Su6668 were applied, the levels of methoprene-induced Na⁺/K⁺-ATPase α-subunit phosphorylation at Ser⁸ were significantly reduced (Fig. 6, A and B). As well, application of the PLC inhibitor U73122 and the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) led to significant decrease of methoprene-induced phosphorylation of Na⁺/K⁺-ATPase α-subunit at Ser⁸ (Fig. 6, A and B). Nevertheless, injection of the TRPC3 channel inhibitor Pyr3 or the T-type voltage-gated calcium channel inhibitor flunarizine dihydrochloride (Fl) had no significant effect on methoprene-induced phosphorylation of Na⁺/K⁺-ATPase α-subunit at Ser⁸ (Fig. 6, A and B). The results indicate the possible involvement of GPCR, RTK, PLC, and IP3R in methoprene-induced phosphorylation of locust Na⁺/K⁺-ATPase α-subunit.

Figure 6.

Involvement of GPCR, RTK, PLC, IP3R, and PKC in JH-stimulated phosphorylation and activation of Na⁺/K⁺-ATPase. A, effects of GPCR inhibitor suramin, RTK inhibitor Su6668, PLC inhibitor U73122, IP3R inhibitor 2-APB, TRPC3 channel inhibitor Pyr3, and T-type voltage-gated calcium channel inhibitor flunarizine dihydrochloride (Fl) treatment on Na⁺/K⁺-ATPase α-subunit phosphorylation at Ser⁸. Meth, methoprene. NK, Na⁺/K⁺-ATPase α-subunit. B, quantitative analysis of band intensity by ImageJ on Na⁺/K⁺-ATPase α-subunit phosphorylation at Ser⁸ as representatively shown in A. **, p < 0.01. n = 3. C, effects of PKA inhibitor H89, CaMKII inhibitor KN-93, PKC inhibitor CC, and staurosporine treatment on Na⁺/K⁺-ATPase α-subunit phosphorylation at Ser⁸. D, quantitative analysis of band intensity by ImageJ on Na⁺/K⁺-ATPase α-subunit phosphorylation at Ser⁸ as representatively shown in B. **, p < 0.01. n = 3. E, effects of inhibitor treatment on Na⁺/K⁺-ATPase activity. *, p < 0.05; **, p < 0.01. n = 3. Error bars, S.E.

We next employed the PKA inhibitor H89, the CaMKII inhibitor KN-93, the PKC inhibitor CC, and staurosporine to further determine the kinases mediating the phosphorylation of Na⁺/K⁺-ATPase α-subunit at Ser⁸. Administration of CC and staurosporine, but not H89 or KN-93 treatment, caused significant reduction of Na⁺/K⁺-ATPase α-subunit phosphorylation at Ser⁸ (Fig. 6, C and D), indicating that PKC, but not PKA or CaMKII, mediates the phosphorylation of Na⁺/K⁺-ATPase α-subunit at Ser⁸. Interestingly, methoprene-stimulated Na⁺/K⁺-ATPase activity was significantly decreased by application of suramin, Su6668, U73122, 2-APB, CC, and staurosporine (Fig. 6E), which was in accordance with the reduced phosphorylation of Na⁺/K⁺-ATPase α-subunit at Ser⁸ caused by treatment of these inhibitors (Fig. 6, A–D). These data indicate the tie of Na⁺/K⁺-ATPase activity with α-subunit phosphorylation at Ser⁸.

Discussion

Function of Na⁺/K⁺-ATPase in JH-dependent vitellogenesis and oogenesis

Na⁺/K⁺-ATPase is known to regulate the equilibrium osmotic pressure and maintain membrane potential through facilitating active transport of three Na⁺ out of the cell and two K⁺ into the cell with the expense of an ATP (53). Previous studies have focused on its role as a drug target for human diseases like cardiac failure, cancers, leukemia, and tissue fibrosis (20, 54). Na⁺/K⁺-ATPase is also investigated as a target of insecticides, including pyrethroids, organophosphorus, and carbamates (55–60). The function of Na⁺/K⁺-ATPase in patency initiation and ovarian Vg uptake is proposed by pharmaceutical experiments utilizing inhibitors (6, 12, 13, 61, 62). In the present study, we performed RNAi and demonstrated that depletion of Na⁺/K⁺-ATPase α-subunit led to substantially reduced Vg accumulation in the ovary, blocked oocyte maturation, and arrested ovarian growth. By in vivo Vg uptake assays, we documented that knocking down Na⁺/K⁺-ATPase α-subunit precluded methoprene-induced ovarian Vg uptake. As locust ovaries are panoistic, Vg is synthesized in the fat body, released into hemolymph, and transported to maturing oocytes through patency in the follicular epithelium (1, 63, 64). The reduced Vg uptake from Na⁺/K⁺-ATPase α-subunit–depleted adult female locusts might consequently result in impaired oocyte maturation and ovarian growth. Our results thus address the importance of Na⁺/K⁺-ATPase in JH-dependent vitellogenesis and oogenesis in locusts. It is likely that Na⁺/K⁺-ATPase enlarges patency for Vg transportation by reducing follicle cell volume via ion exchange between the cells and extracellular media.

JH signaling for phosphorylation and activation of Na⁺/K⁺-ATPase

The possible regulation of Na⁺/K⁺-ATPase by JH has been reported many years ago for several insect species, including R. prolixus, L. migratoria, and H. virescens (1, 2, 6, 35, 62). In this study, immunoprecipitation and ELISA with an anti-phospho-(Ser)-PKC substrate antibody demonstrated that phosphorylation of Na⁺/K⁺-ATPase peaked in the vitellogenic stage, which was in concert with its enhanced activity and Vg uptake in the ovary. Moreover, exogenous application of methoprene rapidly induced α-subunit phosphorylation and activated Na⁺/K⁺-ATPase. These observations raised the hypothesis that PKC mediates JH-stimulated phosphorylation of Na⁺/K⁺-ATPase α-subunit for patency initiation. Indeed, chelerythrine chloride treatment precluded JH-induced patency and ovarian Vg uptake. Thus, it is likely that JH activates a signaling pathway including PKC to phosphorylate the Na⁺/K⁺-ATPase α-subunit for its activation.

Interestingly, a conserved motif, RKEPS (amino acid residues 4–8), recognized by PKC was identified at the N terminus of locust Na⁺/K⁺-ATPase α-subunit, and Ser⁸ is homologous to a known PKC phosphorylation site (Ser²³) in rat Na⁺/K⁺-ATPase α-subunit 1 (51, 52). When anti-rat phospho-Na⁺/K⁺-ATPase α1 (Ser²³) antibody was used in our Western blotting, the phosphorylation patterns of locust Na⁺/K⁺-ATPase α-subunit matched those observed in immunoprecipitation with an anti-phospho-(Ser)-PKC substrate antibody, which was abolishable by chelerythrine chloride treatment. Notably, mutation of Ser⁸ to Ala⁸ abrogated JH-induced phosphorylation and activation of locust Na⁺/K⁺-ATPase α-subunit. These results together suggest that JH induces patency and subsequent ovarian Vg uptake via triggering PKC-mediated phosphorylation of Na⁺/K⁺-ATPase α-subunit at Ser⁸. Our findings, therefore, extend the view of Na⁺/K⁺-ATPase activation by JH-stimulated and PKC-mediated phosphorylation of α-subunit.

By utilizing a series of inhibitors, we unfolded the potential regulation of GPCR, RTK, PLC, IP3R, and PKC in JH-stimulated phosphorylation of Na⁺/K⁺-ATPase α-subunit at Ser⁸ and activation of Na⁺/K⁺-ATPase in locusts. The involvement of GPCR, RTK, PLC, IP₃, or PKC in JH-triggered phosphorylation of Met, BrC-Z7, and Hsp90 have been reported previously in A. aegypti and H. armigera (41, 44). Our results point to a previously unidentified mechanism by which JH triggers the phosphorylation of Na⁺/K⁺-ATPase via GPCR, RTK, PLC, IP3R, and PKC signaling pathways.

Based on our findings, we propose a model for the regulation and function of locust Na⁺/K⁺-ATPase in JH-stimulated vitellogenesis and oogenesis (Fig. 7). JH triggers GPCR, RTK, PLC, IP3R, and PKC cascades to phosphorylate Na⁺/K⁺-ATPase α-subunit at Ser⁸, leading to activation of Na⁺/K⁺-ATPase. Activated Na⁺/K⁺-ATPase is likely to change the ionic balance of follicle cells and cause cell shrinkage, which consequently initiates patency for Vg transportation to the surface of oocytes. Currently, the specific GPCR or RTK involved in this signaling pathway is unclear. Further identification and characterization of molecules downstream of JH and upstream of PKC in the regulation of Na⁺/K⁺-ATPase phosphorylation and activation should help unveil the picture of patency induction during insect vitellogenesis and oogenesis. It may also provide knowledge for developing new strategies of insect pest control.

Figure 7.

A proposed conceptual model for the regulation and function of locust Na⁺/K⁺-ATPase in JH-stimulated Vg uptake and oogenesis. The GPCR/RTK, PLC, IP3R, and PKC pathway transduces JH signaling to activate Na⁺/K⁺-ATPase via phosphorylating its α-subunit at Ser⁸. Consequently, the ionic balance of follicle cells is changed, causing cell shrinkage and patency initiation. Patency between follicle cells allows Vg to pass through the follicular epithelium and reach the surface of maturing oocyte. Fc, follicle cells. NK, Na⁺/K⁺-ATPase α subunit. P-NK, phosphorylated Na⁺/K⁺-ATPase α-subunit.

Experimental procedures

Insects and pharmaceutical treatment

The gregarious phase of migratory locusts was maintained under a light/dark photoperiod of 14 h/10 h and at 30 ± 2 °C as described previously (46). The diet included a continuous supply of wheat bran and fresh wheat seedlings provided once daily. The JH-deprived adult females were achieved by inactivation of corpora allata with intra-abdominal injection of 500 μg of precocene III (Sigma-Aldrich) per locust within 12 h PAE. For JH treatment, adult females at 2 days PAE were intra-abdominally injected with a potent JH analogue, S-(+)-methoprene (Abcam), at 150 μg/locust, and tissues were collected at 5, 15, 30, and 60 min post-methoprene treatment. In pharmaceutical experiments with inhibitors, adult females at 2 or 6 days PAE were injected with suramin (150 μg/locust), Su6668 (3 μg/locust), U73122 (10 μg/locust), 2-aminoethoxydiphenyl borate (18 μg/locust), Pyr3 (10 μg/locust), flunarizine dihydrochloride (50 μg/locust), H89 (10 μg/locust), KN-93 (10 μg/locust), chelerythrine chloride (5 μg/locust), staurosporine (10 ng/locust), and ouabain (30 μg/locust) in PBS buffer. After 30 min, methoprene or DMSO was applied for an additional 30 min.

RNA isolation and qRT-PCR

Total RNA was extracted from ovaries using TRIzol reagent (Invitrogen), and cDNA was reverse transcribed using the FastQuant RT kit (with gDNase) (Tiangen). qRT-PCR was performed using a SuperReal PreMix Plus (with SYBR Green I) kit (Tiangen) in a LightCycler 96 System (Roche Applied Science), initiated at 95 °C for 2 min, followed by 40 cycles of 95 °C for 20 s, 58 °C for 20 s, and 68 °C for 20 s. The relative expression levels were calculated using the 2^−ΔΔCt method, with β-actin as the reference control. Primers used for qRT-PCR are listed in Table 1.

Table 1.

Primers used in PCR cloning, qRT-PCR, and RNAi

Primers	Oligonucleotide sequence (5′–3′)
qRT-PCR
Na+/K+-ATPase α subunit-qRTF	5′-gcatacacactgacatccaaca-3′
Na+/K+-ATPase α subunit-qRTR	5′-ctgtccatatgccatggagat-3′
β-Actin-qRT F	5′-aattaccattggtaacgagcgatt-3′
β-Actin-qRT R	5′-tgcttccatacccaggaatga-3′
RNA interference
Na+/K+-ATPase α subunit-RNAi F	5′-gcgtaatacgactcactatagggttctgtgacttcatgctgcc-3′
Na+/K+-ATPase α subunit-RNAi R	5′-gcgtaatacgactcactatagggtcatgtcagcagcctgtttc-3′
GFP-RNAi F	5′-gcgtaatacgactcactataggtggtcccaattctcgtggaac-3′
GFP-RNAi R	5′-gcgtaatacgactcactataggcttgaagttgaccttgatgcc-3′
Expression in Sf9
Na+/K+-ATPase α subunit-OE F	5′-cggatcccaattggcagatctcgatgcctgccgataagcac-3′
Na+/K+-ATPase α subunit-OE R	5′-ggaggccatggtaccgtcgacgtagtatgtttcctgttccaa-3′
Prokaryotic expression
Na+/K+-ATPase α subunit-exp F	5′-tactcaggatccttgacacagaatcgtatgact-3′
Na+/K+-ATPase α subunit-exp R	5′-tactcactcgagctttgctacagcatcaggtac-3′
Vitellogenin A-exp F	5′-tactcagtcgacaaatggctgtagcccaggccggt-3′
Vitellogenin A-exp R	5′-tactcagcggccgcgtatggttgcttgcatgacaggtgg-3′
Vitellogenin B-exp F	5′-tactcagcggccgcatgggatacaacgtagggact-3′

Antiserum preparation

cDNA fragments of locust VgA, VgB, Na⁺/K⁺-ATPase α-subunit, and β-actin were amplified with the respective specific primers (Table 1) using Pfu DNA polymerase (Novoprotein), cloned into pET-32a (+)-His or pET-30a (+)-His, and confirmed by sequencing. The recombinant proteins (peptides) were expressed in Rosetta host cells under isopropyl-β-d-thiogalactopyranoside induction, purified by an Ni²⁺-nitrilotriacetic acid affinity column (CWbiotech), and examined by SDS-PAGE. Polyclonal antisera were raised in New Zealand White rabbits using the purified proteins (peptides) mixed with Freund's complete adjuvant (Sigma-Aldrich) to form a stable emulsion for immunization. The rabbits were injected subcutaneously at four sites and boosted once a week for a total of 4–5 times. The antiserum specificity was verified by Western blotting using proteins extracted from ovaries of adult females subjected to respective gene knockdown.

RNAi and tissue imaging

cDNA templates were amplified by PCR, cloned into pGM-T easy vector (Tiangen), and confirmed by sequencing. dsRNA was then synthesized by in vitro transcription with the T7 RiboMAX Express System (Promega) following the manufacturer's instruction. Adult female locusts within 6 h after eclosion were intraabdominally injected with 15 μg of dsRNA and boosted on day 3. Phenotypes were examined at 6 days PAE. Ovaries and ovarioles were photographed by a Canon EOS550D camera and Leica M205C stereomicroscope, respectively. The length of primary oocytes was measured with Image-Pro Plus version 6.0 software. dsRNA for GFP was used as the mock control. Primers for dsRNA synthesis are included in Table 1.

Protein extraction and Western blotting

Total proteins from ovaries were extracted by tissue homogenate method using ice-cold PBS buffer containing 137 mm NaCl, 2.7 mm KCl, 10 mm Na₂HPO₄, and 1.8 mm KH₂PO₄ at pH 7.4 plus 1 mm phenylmethylsulfonyl fluoride and a protease inhibitor mixture (Roche Applied Science). Lysates were cleared by centrifugation at 8,000 × g for 10 min at 4 °C. Extracted proteins were quantified by BCA protein assay kit (Pierce). Proteins were fractionated on 7.5% SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Millipore). Western blots were performed using VgA, VgB, Na⁺/K⁺-ATPase α-subunit, or phospho-Na⁺/K⁺-ATPase α1 (Ser²³) antibodies, the corresponding HRP-conjugated secondary antibodies (ProteinTech), and an ECL chemiluminescence detection kit (Boster). Application of β-actin antibody was used as the loading control. Bands were imaged by an Amersham Biosciences Imager 600 (GE Healthcare) and analyzed by ImageJ software.

Immunoprecipitation

Protein extracts were precleaned with Protein A resin (CWbiotech) for 1 h at 4 °C and then incubated with the antibody of Na⁺/K⁺-ATPase α-subunit at 4 °C for overnight. The immunocomplexes were captured with separate Protein A resin (CWbiotech) for 2 h at 4 °C and eluted in Laemmli sample buffer, followed by Western blotting with anti-phospho-(Ser)-PKC-substrate antibody (Cell Signaling Technology).

Na⁺/K⁺-ATPase activity measurement

ELISA for Na⁺/K⁺-ATPase activity measurement were carried out using a Micro Na⁺/K⁺-ATPase assay kit (Solarbio) according to the manufacturer's instructions. Briefly, total proteins were extracted from ovaries of adult females using lysis buffer provided in the kit. After centrifugation at 8,000 × g for 10 min at 4 °C, the supernatant was added into microlon ELISA plates and incubated for 30 min at 28 °C. The absorbance was measured at the 660-nm wavelength using a microplate reader (Molecular Devices). The amount of total proteins was used as the reference value.

In vitro patency induction

Ovaries dissected from 5-day-old adult female locusts were incubated in Grace's insect cell medium (Gibco) containing 10% FBS (Gibco) and 0.1 μm methoprene. For inhibition of Na⁺/K⁺-ATPase and PKC activities, 100 nm ouabain and 5 μm CC were separately added. DMSO was used as the control. After 2 h, CellMask Orange Plasma membrane Stain (Thermo) was added and further incubated for 30 min. The images were captured with a ZEISS LSM 710 laser confocal microscope and processed with ZEN2012 software (Carl Zeiss).

In vivo Vg uptake assay

Hemolymph was collected from adult females at 6 days PAE. Endogenous JH in the collected hemolymph was removed by dialysis with PBS for >24 h at 4 °C. Adult females previously treated with precocence III for 6 days were injected with dialytic hemolymph. After 30 min, methoprene was applied at 150 μg/locust. DMSO treatment was used as a parallel control. Proteins were then extracted from ovaries for further analysis by Western blotting.

Cell culture and gene overexpression

The protein coding sequence of Na⁺/K⁺-ATPase α-subunit was amplified by PCR, cloned into the pIEx-4-RFP-His vector (Invitrogen), and confirmed by sequencing. To obtain the mutant of phosphorylation site at Ser⁸, the sequence of 5′-GAACCATCAGCAAAG-3′ was substituted by 5′-GAACCAGCAGCAAAG-3′ using the Q5 site-directed mutagenesis kit (New England Biolabs). Sf9 cells were transfected with the recombinant vectors using Lipofectamine 3000 (Thermo). Cells were used in the subsequent experiments 24 h after vector transfection. The empty vector pIEx-4-RFP-His was used as the negative control.

Data analysis

MAFFT online software was used for multiple-sequence alignments of cDNA and proteins. Statistical analyses were performed by Student's t test or Duncan's test with SPSS version 20.0 software. Significant difference was considered at p < 0.05 and p < 0.01. Values are shown as mean ± S.E.

Author contributions

Y.-P. J. and S. Z. designed the research. Y.-P. J., H. A., S. Z., and N. W. performed the experiments and acquired the data. S. Z. and Y. J. analyzed the data and wrote the paper.