Larvae of an F1 racial hybrid, Guofu × Nongfong, of B. mori were reared on fresh mulberry leaves at 25°C under a 12-h light: 12-h dark photoperiod. Newly-ecdysed last instar larvae were collected and used for each experiment.

SP600125, N-acetylcysteine (NAC), and diphenylene iodonium (DPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Grace's insect cell culture medium was purchased from Invitrogen (Carlsbad, CA, USA). A MAPK/ERK kinase (MEK) inhibitor (U0126), a PI3K inhibitor (LY294002), A23187, and thapsigargin were purchased from Calbiochem (San Diego, CA, USA). All other reagents used were of analytical grade. [23, 24-³H] Ecdysone was obtained from New England Nuclear (Boston, MA, USA). Recombinant B. mori PTTH (PTTH) was kindly provided by Dr. David R. O'Reilly; it was produced by infection of Spodoptera frugiperda-SF21 cells with the vWTPTTHM baculovirus as described previously (O'Reilly et al., 1995). The same PTTH as that previously reported (O'Reilly et al., 1995; Gu et al., 2010) was used in the present study. In the present study, extracellular fluid from cells infected with vWTPTTHM was used as the PTTH source, and it was diluted 500 times with Grace's medium. Each incubation (50 μl) contained about 0.15 ng of PTTH.

In vitro incubation of PGs and the RIA of ecdysteroids followed protocols described in a previous study (Lin and Gu, 2007). To study the in vivo activation of JNK phosphorylation of PGs by the PTTH, day-6 last instar larvae were injected with 10 μl saline containing 0.3 μl of the original PTTH solution. Larvae injected with 10 μl saline only were used as controls.

Anti-phospho-SAPK/JNK (Thr183/Tyr185) (#9251), anti-phospho-ERK (#9101), anti-phospho-4E-BP (#9459), anti-total ERK (#9102), and anti-α-tubulin (#2144) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-JNK1/3 antibody (sc-474) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-labeled anti-rabbit immunoglobulin G (IgG) was purchased from PerkinElmer Life Sciences (Boston, MA, USA).

Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed as described previously (Lin and Gu, 2007; Gu et al., 2010). Briefly, PGs from day-6 last instar larvae were homogenized in lysis buffer (10 mM Tris and 0.1% Triton x100) at 4°C, and then boiled in an equal volume of SDS sample buffer for 4 min followed by centrifugation at 15,800 g for 3 min to remove any particulate matter. One percent protease inhibitor cocktail (Sigma-Aldrich, cat. no. P8340) and 1% phosphatase inhibitor cocktail (Calbiochem, cat. no. 524629) were added at the time of homogenization. Aliquots of the supernatants were loaded onto SDS gels. Following electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes using an Owl (Portsmouth, NH, USA) Bandit™ Tank Electroblotting System, and then washed with Tris-buffered saline (TBS) for 5 min at room temperature. Blots were blocked at room temperature for 1 h in TBS containing 0.1% Tween 20 (TBST) and 5% (w/v) nonfat powdered dry milk, followed by washing three times for 5 min each with TBST. Blots were incubated overnight at 4°C with primary antibodies against phospho-SAPK/JNK (1:1,000), phospho-ERK (1:10,000), phospho-4E-BP (1:3,000), JNK (1:1,000), ERK (1:1,000), or α-tubulin (1:10,000) in TBST with 5% bovine serum albumin (BSA). Blots were then washed three times in TBST for 10 min each and further incubated with an HRP-linked second antibody in TBST with 1% BSA. Following three additional washes, immunoreactivity was visualized by chemiluminescence using Western Lightning Chemiluminescence Reagent Plus from PerkinElmer Life Sciences. Films exposed to the chemiluminescent reaction were scanned and quantified using an AlphaImager Imaging System and AlphaEaseFC software (Alpha Innotech, San Leandro, CA, USA).

To measure JNK activity, control or treated PGs (each of a pair of glands for control and treatment from 10 larvae) were lysed in JNK lysis buffer (20 mM Tris at pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na₃VO₄, and 1 μg/ml leupeptin). Subsequently, 200 μl of gland lysate was incubated with 20 μl of c-Jun fusion protein beads overnight at 4°C, and then a kinase assay was performed following the manufacturer's instructions (Cell Signaling Technology). The reaction was carried out for 30 min at 30°C and was stopped by boiling the samples in 3x SDS sample buffer. Levels of phospho-c-Jun were measured by Western blotting as described in the section “Western blot analysis”.

PG lysates were treated with lambda protein phosphatase (New England Biolabs, Beverly, MA, USA), which removes phosphates from serine, threonine, and tyrosine residues (Zhuo et al., 1993). The lysates were incubated for 30 min at 30°C with 1000 units of lambda protein phosphatase in 50 μl of reaction buffer according to the manufacturer's specifications. At the end of the incubation period, the sample was boiled for 10 min and stored at −70°C until the PAGE analysis.

Total RNA from PGs was extracted using the TRI Reagent (New England Biolabs, Beverly, MA, USA) according to the manufacturer's protocol. Purification of total RNA, reverse transcription and real-time qRT-PCR were performed as described previously (Young et al., 2012). Transcript levels were normalized to the Bombyx ribosomal protein 49 (rp49, GenBank accession: NM_001098282.1). The real-time qRT-PCR was performed using the following primers: JNK forward, 5′-ACCAAGACTGATATTGTGATAC-3′ and JNK reverse, 5′-AAGTTGTCCGTGTCCATA-3′; Jun forward, 5′-ATGACGAGCAATATCCTC-3′ and Jun reverse, 5′-GATCCGAGCTTCAACATT-3′; and rp49 forward, 5′-CAGGCGGTTCAAGGGTCAATAC-3′ and rp49 reverse, 5′-TGCTGGGCTCTTTCCACGA-3′.

Data are shown as the mean ± standard error of the mean (SEM). Statistical comparisons between different groups were done using either Student's t-test for comparison of two groups or a one-way analysis of variance (ANOVA) followed by Tukey's test for more than two groups. A p < 0.05 was considered significant.

Although there are 10 different splicing isoforms of the three JNK genes (JNK 1, 2, and 3) in mammals, only one JNK (DJNK) exists in Drosophila. B. mori JNK (BmJNK) was cloned as previously reported by Katsuma et al. (2007). BmJNK was respectively reported to have 79.8 and 74.5% amino acid sequence identities with Drosophila DJNK (Basket) and human JNK1 (Katsuma et al., 2007). In the kinase domain, Bombyx JNK contains all conserved residues of protein kinases, and the TPY motif between kinase subdomains VII and VIII, which is phosphorylated on threonine and tyrosine residues to activate JNKs (Riesgo-Escovar et al., 1996; Sluss et al., 1996; Davis, 2000). High similarities of B. mori JNK with those of human and Drosophila suggest that commercial antibodies against mammalian JNK can likely be used in B. mori.

Previous studies showed that PTTH stimulates ERK phosphorylation in both Manduca and Bombyx PGs (Rybczynski et al., 2001; Lin and Gu, 2007). In the present study, we further examined whether PTTH stimulates JNK phosphorylation. Figure 1A shows whole blots of lysates of PGs. Using an anti-phospho-JNK (Thr183/Tyr185) antibody, one immunoreactive protein with a molecular weight (MW) of about 46 kDa was detected in the lysate of a control PG from day-6 last instar larvae. The PTTH greatly increased the phosphorylation level of this protein. In addition, the phosphorylation level of another immunoreactive protein with an MW of ~38 kDa was also strongly increased upon PTTH stimulation, although it remained low in the lysate of the control PG. Between these two bands, there was another small immunoreactive protein with an MW of ~40 kDa. Currently, it is not clear whether these two bands (of 38 and 40 kDa) correspond to minor splice variants of Bombyx JNK or were due to non-specificity of the antibody. Considering the difference in MW between Bombyx JNK and these two bands, we only focused on the 46 kDa Bombyx JNK in subsequent experiments.

Studies were conducted with an antibody that recognizes total JNK. Results (Figure 1A) showed that the JNK level did not change after PTTH treatment, thus confirming that PTTH activates phosphorylation levels of this kinase. Lysates of PTTH-stimulated PGs were incubated with lambda protein phosphatase prior to electrophoresis, and the immunoreactivity detected by the antibody directed against human anti-phospho-JNK decreased (Figure 1B); this indicates that the above antibody, raised against mammalian sequences, indeed recognized phosphorylated epitopes in B. mori PGs.

The above results clearly showed that the PTTH activated JNK phosphorylation of B. mori PGs in vitro. In subsequent experiments, we examined in vivo activation of JNK phosphorylation of PGs by PTTH. PTTH was injected into day-6 last instar larvae. After 30 min, the PGs were quickly dissected out, and JNK phosphorylation was examined and compared to that of control larvae. We chose 30 min after the in vivo PTTH injection because in vitro time-dependent effects of the PTTH revealed that the highest stimulation was detected 30 min after the PTTH injection (see below). Results showed that the PTTH injection greatly increased the JNK phosphorylation level, compared to that of the controls, verifying the in vitro stimulation of JNK phosphorylation (Figure 1C).

In a subsequent experiment, we studied the in vitro JNK phosphorylation by the PTTH in greater detail. Figure 2A shows that a time-dependent increase in JNK phosphorylation in vitro was detected when PGs from day-6 last instar larvae were treated with the PTTH. Activation of JNK by the PTTH was detected after 10 min and reached a peak at 30 min after treatment, and activation persisted for 120 min (Figure 2A). Figure 2B shows the dose-dependent effects of the PTTH on JNK phosphorylation. To examine whether or not PTTH specifically exerts its action on B. mori PGs, PGs, subesophageal ganglions, wing disks, salivary glands, and a small piece of fat body were incubated with or without the PTTH. Results showed that only PGs, but not other tissues, exhibited activation of JNK phosphorylation after stimulation by the PTTH (Figure 2C), indicating the specificity of PTTH's action on PGs.

In addition, when PGs were treated with extracellular fluid from cells only or cells infected with WT AcMNPV, no activation of JNK phosphorylation was observed (O'Reilly et al., 1995); this indicates the specificity of the recombinant PTTH (Figure 2D). Treatment with the PTTH, but not with extracellular fluid from cells only or cells infected with the WT AcMNPV, stimulated ERK and 4E-BP phosphorylation, two well-documented signaling pathways downstream of PTTH stimulation (Gu et al., 2010, 2017), further confirming the specificity of the recombinant PTTH in the present study.

U73122, a selective pharmacological inhibitor of phosphoinositide-specific PLC (Smith et al., 1990), was used to demonstrate the involvement of PLC in PTTH-stimulated JNK phosphorylation. As shown in Figure 3A, pretreatment of PGs with U73122 (10 μM) blocked PTTH-stimulated JNK phosphorylation, thus indicating PLC's involvement.

To further examine whether PTTH-stimulated JNK phosphorylation is dependent on Ca²⁺, we used A23187, a Ca²⁺ ionophore. Figure 3B shows that treatment with A23187 greatly increased JNK phosphorylation. Similar to A23187, thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺-ATPase, also increased JNK phosphorylation. Gu et al. (2010) previously reported that A23187 and thapsigargin increase ERK phosphorylation and ecdysteroid secretion in Bombyx PGs.

We used U0126, a specific MEK inhibitor, to determine whether PTTH-stimulated JNK phosphorylation is linked to ERK phosphorylation. As shown in Figure 4A, U0126 did not block PTTH-stimulated JNK phosphorylation. We further examined the effect of LY294002, a PI3K inhibitor, on PTTH-stimulated JNK phosphorylation. PGs pretreated with LY294002 were subsequently challenged with the PTTH. Figure 4B shows that LY294002 did not affect PTTH-stimulated JNK phosphorylation, which indicates that PI3K signaling is not related in PTTH-stimulated JNK phosphorylation.

In our previous study, we found that PTTH-stimulated phosphorylation of ERK is dependent on the redox status (Hsieh et al., 2014). To determine whether the JNK signaling pathway is redox-sensitive, PGs were pretreated with NAC, a superoxide scavenger, to block PTTH-stimulated ROS production (Hsieh et al., 2013), and then were stimulated with the PTTH. As shown in Figure 5A, blocking the PTTH-stimulated production of ROS by NAC completely blocked PTTH-stimulated JNK phosphorylation compared to PTTH treatment only. Treatment with a mitochondrial oxidative phosphorylation inhibitor (DPI) also greatly decreased PTTH-stimulated JNK phosphorylation (Figure 5B). In addition, treatment with NAC also reduced the basal JNK phosphorylation level compared to the controls, possibly due to inhibition of ROS production. Figure 5C shows that treatment with 1 mM of H₂O₂ greatly stimulated JNK phosphorylation. This result demonstrated that ROS alone can regulate JNK phosphorylation, thus confirming that ROS play a critical role in PTTH-stimulated JNK signaling. These findings suggest that JNK phosphorylation might be a critical component of the redox-sensitive signaling pathway activated by the PTTH in B. mori PGs.

The above data clearly show that the PTTH stimulates JNK phosphorylation. We further confirmed the role of JNK signaling in regulating PTTH-stimulated ecdysteroid synthesis with SP600125, a specific JNK kinase inhibitor. After PGs were pretreated with SP600125, they were challenged with the PTTH. Phosphorylated JNK and ERK levels in PGs were examined, and ecdysteroid secretion was determined. Results (Figure 6) showed that SP600125 treatment decreased PTTH-stimulated JNK phosphorylation levels, but did not affect PTTH-stimulated ERK phosphorylation levels. The basal ERK phosphorylation level was inhibited by treatment with SP600125, indicating that basal ERK phosphorylation is sensitive to this inhibitor. Moreover, although SP600125 treatment did not completely block PTTH-stimulated ecdysteroid secretion, it greatly inhibited it. These results confirm the involvement of JNK signaling in PTTH-stimulated ecdysteroid secretion.

We further examined the effect of the PTTH on the kinase activity of JNK. Lysates from PGs were incubated with 20 μl of c-Jun fusion protein beads overnight, and then a kinase assay was performed per the manufacturer's instructions. Results (Figure 7) show that the kinase activity of JNK from PTTH-stimulated PG lysates greatly increased compared to that from control gland lysates, and that SP600125 inhibited the PTTH-stimulated kinase activity of JNK. In addition, SP600125 also inhibited the basal kinase activity of JNK. These results confirmed that the PTTH-activated phosphorylation of JNK is indeed related to its kinase activity.

We further conducted an experiment to determine changes in JNK (GenBank accession: NP_001103396.1) and Jun (http://silkworm.genomics.org.cn/: BGIBMGA004164) mRNA expression levels upon PTTH treatment. As shown in Figure 8, no significant differences were detected in JNK or Jun transcription levels between control glands and those treated with the PTTH during the 1- or 2-h incubation periods after in vitro treatment with PTTH.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.