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Signaling of reactive oxygen species in PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori
Journal of Insect Physiology 63 (2014) 32–39
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Signaling of reactive oxygen species in PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori Yun-Chih Hsieh, Pei-Ling Lin, Shi-Hong Gu ⇑ Department of Biology, National Museum of Natural Science, 1 Kuan-Chien Road, Taichung 404, Taiwan, ROC
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Article history: Received 28 October 2013 Received in revised form 7 February 2014 Accepted 7 February 2014 Available online 15 February 2014 Keywords: Bombyx mori Prothoracic glands Ecdysone ROS signaling Ca2+ PTTH
a b s t r a c t Our previous study demonstrated that mitochondria-derived reactive oxygen species (ROS) generation is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs). In the present study, we further investigated the mechanism of ROS production and the signaling pathway mediated by ROS. PTTH-stimulated ROS production was markedly attenuated in a Ca2+-free medium. The phospholipase C (PLC) inhibitor, U73122, greatly inhibited PTTH-stimulated ROS production, indicating the involvement of Ca2+ and PLC. When the PGs were treated with agents that directly elevate the intracellular Ca2+ concentration (either A23187, or the protein kinase C (PKC) activator, phorbol 12-myristate acetate (PMA)), a great increase in ROS production was observed. We further investigated the action mechanism of PTTH-stimulated ROS signaling. Results showed that in the presence of either an antioxidant (N-acetylcysteine, NAC), or the mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and diphenyleneiodonium (DPI)), PTTH-regulated phosphorylation of ERK, 4E-BP, and AMPK was blocked. Treatment with 1 mM of H2O2 alone activated the phosphorylation of ERK and 4E-BP, and inhibited AMPK phosphorylation. From these results, we conclude that PTTH-stimulated ROS signaling is Ca2+- and PLC-dependent and that ROS signaling appears to lie upstream of the phosphorylation of ERK, 4E-BP, and AMPK. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Insect growth, molting, and metamorphosis are initiated and regulated by the steroid hormone ecdysteroids, which are synthesized and secreted in the prothoracic glands (PGs) (De Loof, 2008; Marchal et al., 2010; Smith and Rybczynski, 2012). The mechanism regulating ecdysteroid biosynthesis appears to be a key for clear understanding of how insect metamorphosis is regulated (De Loof, 2008; Marchal et al., 2010; Smith and Rybczynski, 2012; Rewitz et al., 2013; Yamanaka et al., 2013). One of the major stimulators for ecdysteroidogenesis is the prothoracicotropic hormone (PTTH), a neuropeptide, which is produced by brain neurosecretory cells (De Loof, 2008; Marchal et al., 2010; Smith and Rybczynski, 2012; Rewitz et al., 2013; Yamanaka et al., 2013). PTTH activates ecdysteroidogenesis in PGs by binding to its receptor Torso, a receptor tyrosine kinase (Rewitz et al., 2009b, 2013; Smith and Rybczynski, 2012). Downstream of PTTH receptor activation, a complex signaling transduction network is activated (Rewitz ⇑ Corresponding author. Tel.: +886 42 3226940; fax: +886 42 3232146. E-mail address: [email protected] (S.-H. Gu). http://dx.doi.org/10.1016/j.jinsphys.2014.02.004 0022-1910/Ó 2014 Elsevier Ltd. All rights reserved.
et al., 2009a; Marchal et al., 2010; Smith and Rybczynski, 2012). This network includes rapid increase of Ca2+ (Fellner et al., 2005; Gu et al., 1998; Birkenbeil and Dedos, 2002), cAMP generation (Smith et al., 1984, 1985; Gu et al., 1996), and activation of protein kinase A (PKA), phospholipase C (PLC), protein kinase C (PKC), p70S6 kinase (S6K), ribosomal protein S6, and tyrosine kinase (Song and Gilbert, 1994, 1995, 1997; Smith et al., 2003; Rybczynski and Gilbert, 2006; Lin and Gu, 2007, 2011; Gu et al., 2010). In addition, phosphorylation of extracellular signal-regulated kinase (ERK) appears to be involved in PTTH-stimulated ecdysteroidogenesis in both Manduca sexta and Bombyx mori (Rybczynski et al., 2001; Lin and Gu, 2007). Our recent studies further indicate that phosphoinositide 3-kinase (PI3K)/adenosine 50 -monophosphateactivated protein kinase (AMPK)/the target of rapamycin (TOR) signaling is involved in PTTH-stimulated ecdysteroidogenesis in B. mori PGs (Gu et al., 2011, 2012, 2013). For many years, reactive oxygen species (ROS) were viewed as the inevitable but unwanted by-products of cellular metabolism. If the cellular production of ROS overwhelms the cell’s antioxidant capacity, damage to cellular constituents, such as lipids, protein, and DNA, may ensue (Veal et al., 2007). An excessive amount of
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ROS is harmful and is considered a causal factor for various pathological conditions including cardiac hypertrophy and insulin resistance (Freeman and Crapo, 1982). However, recent findings suggest that a low concentration of ROS is important for physiological cellular activity (Veal et al., 2007). In mammalian systems, there is growing recognition that ROS may serve as intracellular messenger following receptor activation by a variety of extracellular stimuli including growth factors, cytokines, and hormones (Thannickal and Fanburg, 2000; Veal et al., 2007; Bashan et al., 2009). The mechanism is only now emerging and includes modulation of the thiol proteome, thereby controlling the redox tone that regulates the sensitivity of redox signaling pathways to ROS-dependent activation, inactivation of phosphatases, and activation of tyrosine kinases (Finkel, 1998, 2012; Forman et al., 2010; Collins et al., 2012; Sena and Chandel, 2012; Ray et al., 2012; Liochev, 2013). In a previous study, we demonstrated that mitochondriaderived ROS signaling is involved in PTTH-stimulated ecdysteroidogenesis in B. mori PGs (Hsieh et al., 2013). We found that PTTH rapidly stimulated ROS production and that the antioxidant (N-acetylcysteine (NAC)) and mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and diphenyleneiodonium (DPI)) not only greatly inhibited ROS production, but blocked PTTH-stimulated ecdysteroidogenesis. Although these results point to the role of ROS in PTTH signaling network, how ROS are generated and what is the cellular action mechanism of ROS signaling are not clear. In the present study, we demonstrated that ROS production induced by PTTH is Ca2+-and PLC-dependent. We further investigated the modulatory function of redox state on the phosphorylation of ERK, 4E-BP, and AMPK. These results implicate ROS as key modulators in PTTH signaling transduction processes.
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2.3. In vitro incubation of PGs PGs from day-5 last instar larvae were dissected in lepidopteran saline (12 mM NaCl, 21 mM KCl, 3 mM CaCl2, 18 mM MgCl2, 9 mM KOH, 170 mM glucose, 5 mM PIPES; pH 6.6). Following dissection, the saline was replaced with fresh medium (± any inhibitors), and a 30-min preincubation period was initiated. After preincubation, glands were rapidly transferred to fresh medium (± experimental materials, such as an inhibitor or PTTH), and then incubated for the indicated times with gentle shaking. In most experiments except for the experiments of Ca2+-free incubation, Grace’s medium was used. In the experiments requiring Ca2+-free condition, dissected PGs were preincubated in Ca2+-free saline (i.e. saline without Ca2+ but containing 5 mM EGTA) and then transferred to either Ca2+-free saline or saline containing Ca2+ (control). 2.4. ROS measurements ROS generation was monitored by measuring changes in fluorescence resulting from intracellular oxidation of the dye DCFDA (Keller et al., 2004; Hsieh et al., 2010, 2011, 2013). The DCFDA probe enters a cell, is hydrolyzed by cellular esterases, where upon non-fluorescent DCFDA is trapped inside the cell. Subsequent oxidation by ROS yields DCF. Groups of PGs were pre-loaded with DCFDA (20 lM) at 37 °C for 30 min in the dark and washed before a 30-min preincubation period was initiated. After preincubation, the PGs were then incubated for the indicated times with gentle shaking. At the end of incubation the medium was removed, and the DCF fluorescence intensity in each pair of PGs was measured using a fluorescence plate reader (WallacVictor3 1420 multilabel counter, Perkin Elmer) with an excitation wavelength at 485 nm and emission wavelength of 535 nm. Results are expressed as the percent change from a pair of PGs incubated in control medium. 2.5. Western blot analysis
2. Materials and methods 2.1. Experimental animals Larvae of an F1 racial hybrid, Guofu Nongfong, were reared on fresh mulberry leaves at 25 °C under a 12-L: 12-D photoperiod. Newly ecdysed last instar larvae were collected and used for each experiment. 2.2. Reagents A23187, the PKC activator (phorbol 12-myristate acetate, PMA), NAC, rotenone, antimycin A, DPI, and FCCP were supplied by Sigma–Aldrich (St. Louis, MO, USA). Grace’s insect cell culture medium and 20 ,70 -dichlorodihydrofluorescein diacetate (DCFDA) were obtained from Molecular Probes/Invitrogen (Carlsbad, CA, USA). All other reagents used were of analytical grade. Recombinant B. mori PTTH (PTTH) was produced by infection of Spodoptera frugiperda-SF21 cells with the vWTPTTHM baculovirus as previously described (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 medium. Each incubation (50 ll) contained about 0.15 ng PTTH. Anti-phospho-AMPKa (Thr172), anti-phospho-4E-BP1 (Thr37/ 46), anti-phospho-ERK, anti-total-ERK, and anti-a-tubulin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). A horseradish peroxidase (HRP)-linked goat anti-rabbit second antibody was purchased from PerkinElmer Life Sciences (Boston, MA, USA).
SDS–PAGE and immunoblotting were performed as previously described (Lin and Gu, 2007; Gu et al., 2011, 2012, 2013). Briefly, the treated or control PGs were homogenized in lysis buffer (10 mM Tris and 0.1% Triton 100) at 4 °C, then boiled in an equal volume of SDS sample buffer for 4 min followed by centrifugation at 15,800g for 3 min to remove any particulate matter. Aliquots of the supernatants were loaded onto SDS gels. Following electrophoresis, proteins were transferred to polyvinylidenedifluoride (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 the primary antibody in TBST with 5% bovine serum albumin (BSA). Blots were then washed three times in TBST for 10 min each and further incubated with the 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). 3. Results 3.1. Involvement of Ca2+ and PLC in PTTH-stimulated ROS production In a previous study, we demonstrated that PTTH-stimulated ROS production is involved in ecdysteroidogenesis in B. mori PGs
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(Hsieh et al., 2013). In the present study, the involvement of Ca2+ in regulating PTTH-stimulated ROS production was further investigated. Fig. 1A shows that when PGs were incubated in saline containing Ca2+, treatment with PTTH greatly increased ROS production. However, ROS production induced by PTTH was completely blocked in Ca2+-free saline (with 5 mM EGTA). In addition, treatment with PLC inhibitor, U73122, also completely prevented PTTH-stimulated ROS production (Fig. 1B). These results indicate that PTTH-stimulated ROS production is Ca2+- and PLC-dependent.
Bombyx PGs (Gu et al., 2010). Fig. 2A shows that treatment with either A23187 or PMA greatly increased ROS production. A23187-stimulated ROS production appears to be time- and
3.2. Agents that directly elevate the intracellular Ca2+ concentration stimulate ROS production The existence of Ca2+-dependent ROS production stimulated by PTTH prompted us to study the effects of agents that directly increase the intracellular Ca2+ concentration on ROS production. The Ca2+ ionophore, A23187, and the PKC activator, PMA, were used, because it was previously reported that both A23187 and PMA increase ERK phosphorylation and ecdysteroidogenesis in
Fig. 1. Effect of external Ca2+ and U73122 on PTTH-stimulated ROS production. (A) Effect of external Ca2+. PGs were preincubated in Ca2+-free saline (with 5 mM EGTA) for 30 min and then transferred to either control saline (+Ca2+), saline with the PTTH (+Ca2++PTTH), Ca2+-free saline (Ca2+), or Ca2+-free saline with PTTH (Ca2++PTTH). (B) Effect of U73122. PGs were pretreated with 50 lM U73122 or vehicle alone for 30 min, then, transferred to medium containing the same dose of U73122, with or without PTTH. Incubations were maintained for 5 min. ROS production was detected by the fluorescence of DCF. Results were normalized to the controls. Each value represents the mean ± SEM of five separate assays. Different letters above the bars indicate a significant difference (ANOVA followed by a Tukey’s multiple comparisons test, p < 0.05).
Fig. 2. Effects of A23187 and PMA on ROS production. (A) Effects of A23187 and PMA. PGs were preincubated in control medium for 30 min, then, transferred to control medium containing vehicle alone (Control) or medium containing either A23187 (50 lM) or PMA (10 lM). Incubations were maintained for 5 min. (B) Timedependence of A23187. PGs were preincubated in control medium for 30 min and then transferred to control medium containing vehicle alone (Control) or medium containing A23187 (50 lM) for different periods. (C) Dose-dependent effect of A23187. PGs were preincubated in control medium for 30 min and then transferred to control medium containing vehicle alone (0) or medium containing different concentration of A23187 for 5 min. ROS production was detected by the fluorescence of DCF. Results were normalized to the controls. Each value represents the mean ± SEM of five separate assays. Different letters above the bars indicate a significant difference (ANOVA followed by a Tukey’s multiple comparisons test, p < 0.05).
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Fig. 3. Effects of NAC (A), rotenone (B), antimycin A (C), FCCP (D), and DPI (E) on PTTH-stimulated ERK phosphorylation. PGs from day-5 last instar larvae were pretreated with either NAC (20 mM), rotenone (10 lM), antimycin A (10 lM), FCCP (10 lM), DPI (10 lM), or vehicle alone for 30 min, then transferred to medium containing the same dose of the inhibitors, with or without PTTH. Incubations were maintained for 60 min. CN, PGs incubated in control medium; N, PGs incubated in medium containing NAC; P, PGs incubated in medium containing PTTH; P + N, PGs incubated in medium containing both PTTH and NAC; Ro, PGs incubated in medium containing rotenone; P + Ro, PGs incubated in medium containing both PTTH and rotenone; An, PGs incubated in medium containing antimycin A; P + An, PGs incubated in medium containing both PTTH and antimycin A; F, PGs incubated in medium containing FCCP; P + F, PGs incubated in medium containing both PTTH and FCCP; DP, PGs incubated in medium containing DPI; P + DP, PGs incubated in medium containing both PTTH and DPI. Gland lysates were prepared and subjected to immunoblot analysis with anti-phospho-ERK (P-ERK) and anti-total-ERK (T-ERK) antibodies. Results shown are representative of three independent experiments.
dose-dependent (Fig. 2B and C). A peak in ROS production was detected as early as 1 min after the glands were treated with A23187 and the high levels were maintained between 1 min and 15 min. The level then decreased 30 min after A23187 treatment. The dose-dependent effect of A23187 showed that 50 lM A23187 treatment results in the highest ROS production.
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Fig. 4. Effects of NAC (A), rotenone (B), antimycin A (C), FCCP (D), and DPI (E) on PTTH-stimulated 4E-BP phosphorylation. PGs from day-5 last instar larvae were pretreated with either NAC (20 mM), rotenone (10 lM), antimycin A (10 lM), FCCP (10 lM), DPI (10 lM), or vehicle alone for 30 min, then transferred to medium containing the same dose of the inhibitors, with or without PTTH. Incubations were maintained for 60 min. CN, PGs incubated in control medium; N, PGs incubated in medium containing NAC; P, PGs incubated in medium containing PTTH; P + N, PGs incubated in medium containing both PTTH and NAC; Ro, PGs incubated in medium containing rotenone; P + Ro, PGs incubated in medium containing both PTTH and rotenone; An, PGs incubated in medium containing antimycin A; P + An, PGs incubated in medium containing both PTTH and antimycin A; F, PGs incubated in medium containing FCCP; P + F, PGs incubated in medium containing both PTTH and FCCP; DP, PGs incubated in medium containing DPI; P + DP, PGs incubated in medium containing both PTTH and DPI. Gland lysates were prepared and subjected to an immunoblot analysis with anti-phospho-4E-BP1 (Thr37/46) (P-4E-BP) and anti-a-tubulin (a-tubulin) antibodies. Results shown are representative of three independent experiments.
PTTH-stimulated production of ROS by NAC completely blocked PTTH-stimulated ERK phosphorylation as compared with PTTH treatment only. Treatment with the mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, FCCP, and DPI) also greatly decreased PTTH-stimulated ERK phosphorylation (Fig. 3B–E). These findings suggest that ERK phosphorylation might be a critical component of the redox-sensitive signaling pathway activated by PTTH in B. mori PGs.
3.3. Activation of ERK signaling by PTTH is ROS-dependent
3.4. Activation of 4E-BP phosphorylation by PTTH is ROS-dependent
The previous studies have shown that PTTH-stimulated phosphorylation of ERK appears to be involved in ecdysteroidogenesis (Rybczynski et al., 2001; Lin and Gu, 2007; Gu et al., 2010). However, it remains unclear whether the activation of the ERK pathway is redox-dependent. To determine whether the ERK signaling pathway is redox-sensitive, PGs were pretreated with NAC, a superoxide scavenger, to block PTTH-stimulated production of ROS, then stimulated with PTTH. As shown in Fig. 3A, blocking
TOR signaling is another signaling pathway stimulated by PTTH (Gu et al., 2012). We also examined whether the phosphorylation of 4E-BP, which is generally accepted to be the marker of TOR activity, is redox-sensitive. As shown in Fig. 4, blocking PTTHstimulated production of ROS by either NAC or the mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, FCCP, and DPI) completely abolished PTTH-stimulated 4E-BP phosphorylation as compared with PTTH treatment only.
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Fig. 5. Effects of NAC (A), rotenone (B), antimycin A (C), FCCP (D), and DPI (E) on PTTH-inhibited AMPK phosphorylation. PGs from day-5 last instar larvae were pretreated with either NAC (20 mM), rotenone (10 lM), antimycin A (10 lM), FCCP (10 lM), DPI (10 lM), or vehicle alone for 30 min, then transferred to medium containing the same dose of the inhibitors, with or without PTTH. Incubations were maintained for 60 min. CN, PGs incubated in control medium; N, PGs incubated in medium containing NAC; P, PGs incubated in medium containing PTTH; P + N, PGs incubated in medium containing both PTTH and NAC; Ro, PGs incubated in medium containing rotenone; P + Ro, PGs incubated in medium containing both PTTH and rotenone; An, PGs incubated in medium containing antimycin A; P + An, PGs incubated in medium containing both PTTH and antimycin A; F, PGs incubated in medium containing FCCP; P + F, PGs incubated in medium containing both PTTH and FCCP; DP, PGs incubated in medium containing DPI; P + DP, PGs incubated in medium containing both PTTH and DPI. Gland lysates were prepared and subjected to an immunoblot analysis with anti-phospho-AMPKa (Thr172) (P-AMPK) and antia-tubulin (a-tubulin) antibodies. Results shown are representative of three independent experiments.
Fig. 6. Effects of exogenous H2O2 on phosphorylation of ERK (A), 4E-BP (B) and AMPK (C). PGs from day-5 last instar larvae were preincubated in control medium for 30 min, then treated with H2O2 (HP, 1 mM), or incubated in control medium (Con). Incubations were maintained for 30 min. Gland lysates were prepared and subjected to an immunoblot analysis with anti-phospho-ERK (P-ERK), anti-totalERK (T-ERK), anti-phospho-4E-BP1 (Thr37/46) (P-4E-BP), anti-phospho-AMPKa (Thr172) (P-AMPK), and anti-a-tubulin (a-tubulin) antibodies. Results shown are representative of three independent experiments.
that treatment with 1 mM of H2O2 greatly stimulated the phosphorylation of ERK and 4E-BP and inhibited AMPK phosphorylation. This result demonstrated that ROS alone can regulate the phosphorylation of ERK, 4E-BP, and AMPK, thus confirming that ROS play a critical role in PTTH signaling.
3.5. PTTH-inhibited AMPK phosphorylation is ROS-dependent
4. Discussion
Our previous study showed that PTTH-inhibited AMPK phosphorylation is the upstream signaling of TOR and is involved in ecdysteroidogenesis by B. mori PGs (Gu et al., 2013). In the present study, we further investigated whether or not PTTH-inhibited AMPK phosphorylation is ROS-dependent. Fig. 5 shows that PTTH markedly inhibited AMPK phosphorylation and that blocking PTTH-stimulated ROS production by either NAC or the mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, FCCP, and DPI) blocked PTTH-inhibited AMPK phosphorylation as compared with PTTH treatment only.
Our previous study demonstrated that in B. mori PGs, the mitochondria-derived ROS production stimulated by PTTH is required for PTTH-stimulated ecdysteroidogenesis (Hsieh et al., 2013). The present study further investigated the releasing mechanism of ROS upon stimulation by PTTH and the cellular action mechanism of ROS. Our results showed that PTTH-stimulated ROS production is Ca2+- and PLC-dependent and that agents that directly elevate the intracellular Ca2+ concentration also stimulate ROS production. Moreover, the present study showed that ROS play a critical role in regulating PTTH-regulated phosphorylation of ERK, 4E-BP, and AMPK. To our knowledge, this is the first publication linking from PLC/Ca2+ to ROS as well as to protein phosphorylation regulated by PTTH in an insect endocrine organ (Fig. 7). It is necessary, however, to be cautious in this conclusion, because the specificity of the inhibitors used in these experiments has not been proved in insects. PLC/Ca2+ plays a critical role in PTTH signaling transduction. It has been reported that an inhibitor of PLC blocked increase in [Ca2+]i in the PGs of Manduca, indicating that PLC-activated formation of IP3 is a crucial step in elevating [Ca2+]i in Manduca PGs (Fellner et al., 2005). The rapid activation of PLC and an increase in [Ca2+]i in turn result in the activation of Ca2+/calmodulin dependent adenylate cyclase and an increase in cAMP content. In
3.6. Effects of hydrogen peroxide (H2O2) on phosphorylation of ERK, 4E-BP, and AMPK While the effects of NAC and mitochondrial inhibitors implicated the involvement of ROS in PTTH signaling transduction, it remained unclear whether ROS could directly participate in phosphorylation of ERK, 4E-BP, and AMPK. To determine whether ROS have a direct effect on cell signaling in B. mori PGs, PGs from day-5 last instar larvae were treated with H2O2 (1 mM) and alternation in downstream protein phosphorylation (ERK, 4E-BP, and AMPK) was assessed by Western blotting. Results (Fig. 6) showed
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Fig. 7. The signaling network involved in PTTH-stimulated ecdysteroidogenesis in B. mori PGs. See text for details.
addition, several other protein kinases, such as the phosphorylation of ERK appear to be also Ca2+/calmodulin dependent (Gu et al., 2010). In the present study, we further showed that PLC/Ca2+affects PTTH-stimulated ROS production. Blocking PLC activation by U73122 greatly inhibited ROS production stimulated by PTTH. PTTH-stimulated ROS was significantly reduced in Ca2+-free saline compared with those incubated in saline containing Ca2+. In addition, we found that the agents that directly increase the intracellular Ca2+ concentration (A23187 and PMA) also increased ROS
production and that A23187-stimulated ROS production was both dose- and time-dependent. In mammalian cells, the regulation of ROS production by Ca2+ has been well documented (Camello-Almaraz et al., 2006). Ca2+ modulates ROS homeostasis by regulating ROS production and annihilation mechanisms in both the mitochondria and the cytosol. Recent evidences have underscored the notion that the Ca2+ and ROS signaling systems are intimately integrated such that Ca2+-dependent regulation of components of ROS homeostasis might influence intracellular redox balance, and vice versa (Brookes
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et al., 2004; Camello-Almaraz et al., 2006). In Drosophila, it has been documented that PLC/Ca2+ modulates dual oxidase activity to produce microbicidal ROS essential for normal host survival (Ha et al., 2009). The cooperative action between Ca2+ and ROS production has also been reported in Drosophila ovarian smooth muscle: ROS generation in response to proctolin is Ca2+-dependent, and vice versa (Ritsick et al., 2007). The exact cooperative regulation between Ca2+ and ROS production in PGs remains to be investigated. We are currently studying whether an increase in [Ca2+]i upon PTTH stimulation is dependent on ROS production. Another objective was to determine whether ROS are involved in the phosphorylation of several key signaling proteins regulated by PTTH. The previous studies showed that ERK signaling is involved in PTTH-stimulated ecdysteroidogenesis in both Manduca and Bombyx (Rybczynski et al., 2001; Lin and Gu, 2007, 2011). Our recent studies indicated that PI3K/AMPK/TOR signaling is another signaling pathway involved in PTTH action (Gu et al., 2011, 2012, 2013). In the present study, pretreatment with either NAC or the mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, FCCP, and DPI) blocked PTTH-stimulated phosphorylation of ERK and 4E-BP. PTTH-inhibited AMPK phosphorylation was blocked by pretreatment with either NAC or the mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, FCCP, and DPI). In addition, we found that ROS alone can activate the phosphorylation of ERK and 4E-BP and inhibit AMPK phosphorylation. These results clearly showed that PTTHstimulated ROS production lies upstream signaling in regulating the phosphorylation of ERK, 4E-BP, and AMPK. In mammalian systems, the most often cited mechanism by which ROS contribute to cellular signaling is by modifying the actions of proteins through the reversible oxidation of essential cysteine residues (Sena and Chandel, 2012; Ray et al., 2012; Liochev, 2013). Oxidative modification of mitogen-activated protein (MAP) kinase signaling proteins and inactivation and/or degradation of MAP kinase phosphatases (MKPs) may provide the plausible mechanism for activation of MAP kinase pathways by ROS (Finkel, 1998, 2012; Forman et al., 2010; Collins et al., 2012; Sena and Chandel, 2012; Ray et al., 2012; Liochev, 2013). Previous studies also suggested that ROS can activate the PI3K/Akt/TOR signaling pathway through the reversible inactivation of the PTEN protein (Finkel, 1998, 2012; Forman et al., 2010; Collins et al., 2012; Sena and Chandel, 2012; Ray et al., 2012; Liochev, 2013). These observations have led us to hypothesize that oxidant signals induced by PTTH may regulate protein phosphorylation by either triggering tyrosine phosphorylation or affecting phosphatase activity. In addition, the phosphorylation and activation of Spook, a possible rate-limiting enzyme in the ecdysone biosynthetic pathway (Ono et al., 2006; Rewitz et al., 2009a), and the rate-limiting Black Box in this pathway may also be modulated by PTTH-stimulated oxidant signals. Further study is needed to clarify the exact cellular mechanism by which PTTH-stimulated ROS signaling affects phosphorylation of ERK, 4E-BP, AMPK, Spook, as well as the oxidation of the rate-limiting Black Box. In summary, we have demonstrated that ROS production stimulated by PTTH is Ca2+- and PLC-dependent and that agents that directly elevate the intracellular Ca2+ concentration also stimulate ROS production. PTTH-stimulated ROS signaling appears to lie upstream signaling of the phosphorylation of ERK and AMPK/TOR signaling (Fig. 7). Acknowledgements The authors thank the National Science Council, Taipei, Taiwan for grants (NSC99-2628-B-178-002-MY3 and NSC100-2313-B-178001-MY3); and the National Museum of Natural Science, Taichung, Taiwan for their financial support.
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Y.-C. Hsieh et al. / Journal of Insect Physiology 63 (2014) 32–39 O’Reilly, D.R., Kelly, T.J., Masler, E.P., Thyagaraja, B.S., Robson, R.M., Shaw, T.C., Miller, L.K., 1995. Overexpression of Bombyx mori prothoracicotropic hormone using baculovirus vectors. Insect Biochem. Mol. Biol. 25, 475–485. Ray, P.D., Huang, B.W., Tsuji, Y., 2012. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal. 24, 981–990. Rewitz, K.F., Larsen, M.R., Lobner-Olesen, A., Rybczynski, R., O’Connor, M.B., Gilbert, L.I., 2009a. A phosphoproteomics approach to elucidate neuropeptide signal transduction controlling insect metamorphosis. Insect Biochem. Mol. Biol. 39, 475–483. Rewitz, K.F., Yamanaka, N., Gilbert, L.I., O’Connor, M.B., 2009b. The insect neuropeptide PTTH activates receptor tyrosine kinase torso to initiate metamorphosis. Science 326, 1403–1405. Rewitz, K.F., Yamanaka, N., O’Connor, M.B., 2013. Developmental checkpoints and feedback circuits time insect maturation. Curr. Top. Dev. Biol. 103, 1–33. Ritsick, D.R., Edens, W.A., Finnerty, V., Lambeth, J.D., 2007. Nox regulation of smooth muscle contraction. Free Radic. Biol. Med. 43, 31–38. Rybczynski, R., Bell, S.C., Gilbert, L.I., 2001. Activation of an extracellular signalregulated kinase (ERK) by the insect prothoracicotropic hormone. Mol. Cell. Endocrinol. 184, 1–11. Rybczynski, R., Gilbert, L.I., 2006. Protein kinase C modulates ecdysteroidogenesis in the prothoracic gland of the tobacco hornworm Manduca sexta. Mol. Cell. Endocrinol. 251, 78–87. Sena, L.A., Chandel, N.S., 2012. Physiological roles of mitochondrial reactive oxygen species. Mol. Cell 48, 158–167. Smith, W.A., Gilbert, L.I., Bollenbacher, W.E., 1984. The role of cyclic AMP in the regulation of ecdysone synthesis. Mol. Cell. Endocrinol. 37, 285–294.
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Smith, W.A., Gilbert, L.I., Bollenbacher, W.E., 1985. Calcium-cyclic AMP interactions in prothoracicotropic hormone stimulation of ecdysone synthesis. Mol. Cell. Endocrinol. 39, 71–78. Smith, W.A., Priester, J., Morais, J., 2003. PTTH-stimulated ecdysone secretion is dependent upon tyrosine phosphorylation in the prothoracic glands of Manduca sexta. Insect Biochem. Mol. Biol. 33, 1317–1325. Smith, W.A., Rybczynski, R., 2012. Prothoracicotropic hormone. In: Gilbert, L.I. (Ed.), Insect Endocrinology. Elsevier, Oxford, UK, pp. 1–62. Song, Q., Gilbert, L.I., 1994. S6 phosphorylation results from prothoracicotropic hormone stimulation of insect prothoracic glands: a role for S6 kinase. Dev. Genet. 15, 332–338. Song, Q., Gilbert, L.I., 1995. Multiple phosphorylation of ribosomal protein S6 and specific protein synthesis are required for prothoracicotropic hormonestimulated ecdysteroid biosynthesis in the prothoracic glands of Manduca sexta. Insect Biochem. Mol. Biol. 25, 591–602. Song, Q., Gilbert, L.I., 1997. Molecular cloning, developmental expression, and phosphorylation of ribosomal protein S6 in the endocrine gland responsible for insect molting. J. Biol. Chem. 272, 4429–4435. Thannickal, V.J., Fanburg, B.L., 2000. Reactive oxygen species in cell signaling. Am. J. Physiol. Lung Cell. Mol. Physiol. 279, L1005–L1028. Veal, E.A., Day, A.M., Morgan, B.A., 2007. Hydrogen peroxide sensing and signaling. Mol. Cell 26, 1–14. Yamanaka, N., Rewitz, K.F., O’Connor, M.B., 2013. Ecdysone control of developmental transitions: lessons from Drosophila research. Annu. Rev. Entomol. 58, 497–516.
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Signaling of reactive oxygen species in PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori Yun-Chih Hsieh, Pei-Ling Lin, Shi-Hong Gu ⇑ Department of Biology, National Museum of Natural Science, 1 Kuan-Chien Road, Taichung 404, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 28 October 2013 Received in revised form 7 February 2014 Accepted 7 February 2014 Available online 15 February 2014 Keywords: Bombyx mori Prothoracic glands Ecdysone ROS signaling Ca2+ PTTH
a b s t r a c t Our previous study demonstrated that mitochondria-derived reactive oxygen species (ROS) generation is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs). In the present study, we further investigated the mechanism of ROS production and the signaling pathway mediated by ROS. PTTH-stimulated ROS production was markedly attenuated in a Ca2+-free medium. The phospholipase C (PLC) inhibitor, U73122, greatly inhibited PTTH-stimulated ROS production, indicating the involvement of Ca2+ and PLC. When the PGs were treated with agents that directly elevate the intracellular Ca2+ concentration (either A23187, or the protein kinase C (PKC) activator, phorbol 12-myristate acetate (PMA)), a great increase in ROS production was observed. We further investigated the action mechanism of PTTH-stimulated ROS signaling. Results showed that in the presence of either an antioxidant (N-acetylcysteine, NAC), or the mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and diphenyleneiodonium (DPI)), PTTH-regulated phosphorylation of ERK, 4E-BP, and AMPK was blocked. Treatment with 1 mM of H2O2 alone activated the phosphorylation of ERK and 4E-BP, and inhibited AMPK phosphorylation. From these results, we conclude that PTTH-stimulated ROS signaling is Ca2+- and PLC-dependent and that ROS signaling appears to lie upstream of the phosphorylation of ERK, 4E-BP, and AMPK. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Insect growth, molting, and metamorphosis are initiated and regulated by the steroid hormone ecdysteroids, which are synthesized and secreted in the prothoracic glands (PGs) (De Loof, 2008; Marchal et al., 2010; Smith and Rybczynski, 2012). The mechanism regulating ecdysteroid biosynthesis appears to be a key for clear understanding of how insect metamorphosis is regulated (De Loof, 2008; Marchal et al., 2010; Smith and Rybczynski, 2012; Rewitz et al., 2013; Yamanaka et al., 2013). One of the major stimulators for ecdysteroidogenesis is the prothoracicotropic hormone (PTTH), a neuropeptide, which is produced by brain neurosecretory cells (De Loof, 2008; Marchal et al., 2010; Smith and Rybczynski, 2012; Rewitz et al., 2013; Yamanaka et al., 2013). PTTH activates ecdysteroidogenesis in PGs by binding to its receptor Torso, a receptor tyrosine kinase (Rewitz et al., 2009b, 2013; Smith and Rybczynski, 2012). Downstream of PTTH receptor activation, a complex signaling transduction network is activated (Rewitz ⇑ Corresponding author. Tel.: +886 42 3226940; fax: +886 42 3232146. E-mail address: [email protected] (S.-H. Gu). http://dx.doi.org/10.1016/j.jinsphys.2014.02.004 0022-1910/Ó 2014 Elsevier Ltd. All rights reserved.
et al., 2009a; Marchal et al., 2010; Smith and Rybczynski, 2012). This network includes rapid increase of Ca2+ (Fellner et al., 2005; Gu et al., 1998; Birkenbeil and Dedos, 2002), cAMP generation (Smith et al., 1984, 1985; Gu et al., 1996), and activation of protein kinase A (PKA), phospholipase C (PLC), protein kinase C (PKC), p70S6 kinase (S6K), ribosomal protein S6, and tyrosine kinase (Song and Gilbert, 1994, 1995, 1997; Smith et al., 2003; Rybczynski and Gilbert, 2006; Lin and Gu, 2007, 2011; Gu et al., 2010). In addition, phosphorylation of extracellular signal-regulated kinase (ERK) appears to be involved in PTTH-stimulated ecdysteroidogenesis in both Manduca sexta and Bombyx mori (Rybczynski et al., 2001; Lin and Gu, 2007). Our recent studies further indicate that phosphoinositide 3-kinase (PI3K)/adenosine 50 -monophosphateactivated protein kinase (AMPK)/the target of rapamycin (TOR) signaling is involved in PTTH-stimulated ecdysteroidogenesis in B. mori PGs (Gu et al., 2011, 2012, 2013). For many years, reactive oxygen species (ROS) were viewed as the inevitable but unwanted by-products of cellular metabolism. If the cellular production of ROS overwhelms the cell’s antioxidant capacity, damage to cellular constituents, such as lipids, protein, and DNA, may ensue (Veal et al., 2007). An excessive amount of
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ROS is harmful and is considered a causal factor for various pathological conditions including cardiac hypertrophy and insulin resistance (Freeman and Crapo, 1982). However, recent findings suggest that a low concentration of ROS is important for physiological cellular activity (Veal et al., 2007). In mammalian systems, there is growing recognition that ROS may serve as intracellular messenger following receptor activation by a variety of extracellular stimuli including growth factors, cytokines, and hormones (Thannickal and Fanburg, 2000; Veal et al., 2007; Bashan et al., 2009). The mechanism is only now emerging and includes modulation of the thiol proteome, thereby controlling the redox tone that regulates the sensitivity of redox signaling pathways to ROS-dependent activation, inactivation of phosphatases, and activation of tyrosine kinases (Finkel, 1998, 2012; Forman et al., 2010; Collins et al., 2012; Sena and Chandel, 2012; Ray et al., 2012; Liochev, 2013). In a previous study, we demonstrated that mitochondriaderived ROS signaling is involved in PTTH-stimulated ecdysteroidogenesis in B. mori PGs (Hsieh et al., 2013). We found that PTTH rapidly stimulated ROS production and that the antioxidant (N-acetylcysteine (NAC)) and mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and diphenyleneiodonium (DPI)) not only greatly inhibited ROS production, but blocked PTTH-stimulated ecdysteroidogenesis. Although these results point to the role of ROS in PTTH signaling network, how ROS are generated and what is the cellular action mechanism of ROS signaling are not clear. In the present study, we demonstrated that ROS production induced by PTTH is Ca2+-and PLC-dependent. We further investigated the modulatory function of redox state on the phosphorylation of ERK, 4E-BP, and AMPK. These results implicate ROS as key modulators in PTTH signaling transduction processes.
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2.3. In vitro incubation of PGs PGs from day-5 last instar larvae were dissected in lepidopteran saline (12 mM NaCl, 21 mM KCl, 3 mM CaCl2, 18 mM MgCl2, 9 mM KOH, 170 mM glucose, 5 mM PIPES; pH 6.6). Following dissection, the saline was replaced with fresh medium (± any inhibitors), and a 30-min preincubation period was initiated. After preincubation, glands were rapidly transferred to fresh medium (± experimental materials, such as an inhibitor or PTTH), and then incubated for the indicated times with gentle shaking. In most experiments except for the experiments of Ca2+-free incubation, Grace’s medium was used. In the experiments requiring Ca2+-free condition, dissected PGs were preincubated in Ca2+-free saline (i.e. saline without Ca2+ but containing 5 mM EGTA) and then transferred to either Ca2+-free saline or saline containing Ca2+ (control). 2.4. ROS measurements ROS generation was monitored by measuring changes in fluorescence resulting from intracellular oxidation of the dye DCFDA (Keller et al., 2004; Hsieh et al., 2010, 2011, 2013). The DCFDA probe enters a cell, is hydrolyzed by cellular esterases, where upon non-fluorescent DCFDA is trapped inside the cell. Subsequent oxidation by ROS yields DCF. Groups of PGs were pre-loaded with DCFDA (20 lM) at 37 °C for 30 min in the dark and washed before a 30-min preincubation period was initiated. After preincubation, the PGs were then incubated for the indicated times with gentle shaking. At the end of incubation the medium was removed, and the DCF fluorescence intensity in each pair of PGs was measured using a fluorescence plate reader (WallacVictor3 1420 multilabel counter, Perkin Elmer) with an excitation wavelength at 485 nm and emission wavelength of 535 nm. Results are expressed as the percent change from a pair of PGs incubated in control medium. 2.5. Western blot analysis
2. Materials and methods 2.1. Experimental animals Larvae of an F1 racial hybrid, Guofu Nongfong, were reared on fresh mulberry leaves at 25 °C under a 12-L: 12-D photoperiod. Newly ecdysed last instar larvae were collected and used for each experiment. 2.2. Reagents A23187, the PKC activator (phorbol 12-myristate acetate, PMA), NAC, rotenone, antimycin A, DPI, and FCCP were supplied by Sigma–Aldrich (St. Louis, MO, USA). Grace’s insect cell culture medium and 20 ,70 -dichlorodihydrofluorescein diacetate (DCFDA) were obtained from Molecular Probes/Invitrogen (Carlsbad, CA, USA). All other reagents used were of analytical grade. Recombinant B. mori PTTH (PTTH) was produced by infection of Spodoptera frugiperda-SF21 cells with the vWTPTTHM baculovirus as previously described (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 medium. Each incubation (50 ll) contained about 0.15 ng PTTH. Anti-phospho-AMPKa (Thr172), anti-phospho-4E-BP1 (Thr37/ 46), anti-phospho-ERK, anti-total-ERK, and anti-a-tubulin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). A horseradish peroxidase (HRP)-linked goat anti-rabbit second antibody was purchased from PerkinElmer Life Sciences (Boston, MA, USA).
SDS–PAGE and immunoblotting were performed as previously described (Lin and Gu, 2007; Gu et al., 2011, 2012, 2013). Briefly, the treated or control PGs were homogenized in lysis buffer (10 mM Tris and 0.1% Triton 100) at 4 °C, then boiled in an equal volume of SDS sample buffer for 4 min followed by centrifugation at 15,800g for 3 min to remove any particulate matter. Aliquots of the supernatants were loaded onto SDS gels. Following electrophoresis, proteins were transferred to polyvinylidenedifluoride (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 the primary antibody in TBST with 5% bovine serum albumin (BSA). Blots were then washed three times in TBST for 10 min each and further incubated with the 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). 3. Results 3.1. Involvement of Ca2+ and PLC in PTTH-stimulated ROS production In a previous study, we demonstrated that PTTH-stimulated ROS production is involved in ecdysteroidogenesis in B. mori PGs
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(Hsieh et al., 2013). In the present study, the involvement of Ca2+ in regulating PTTH-stimulated ROS production was further investigated. Fig. 1A shows that when PGs were incubated in saline containing Ca2+, treatment with PTTH greatly increased ROS production. However, ROS production induced by PTTH was completely blocked in Ca2+-free saline (with 5 mM EGTA). In addition, treatment with PLC inhibitor, U73122, also completely prevented PTTH-stimulated ROS production (Fig. 1B). These results indicate that PTTH-stimulated ROS production is Ca2+- and PLC-dependent.
Bombyx PGs (Gu et al., 2010). Fig. 2A shows that treatment with either A23187 or PMA greatly increased ROS production. A23187-stimulated ROS production appears to be time- and
3.2. Agents that directly elevate the intracellular Ca2+ concentration stimulate ROS production The existence of Ca2+-dependent ROS production stimulated by PTTH prompted us to study the effects of agents that directly increase the intracellular Ca2+ concentration on ROS production. The Ca2+ ionophore, A23187, and the PKC activator, PMA, were used, because it was previously reported that both A23187 and PMA increase ERK phosphorylation and ecdysteroidogenesis in
Fig. 1. Effect of external Ca2+ and U73122 on PTTH-stimulated ROS production. (A) Effect of external Ca2+. PGs were preincubated in Ca2+-free saline (with 5 mM EGTA) for 30 min and then transferred to either control saline (+Ca2+), saline with the PTTH (+Ca2++PTTH), Ca2+-free saline (Ca2+), or Ca2+-free saline with PTTH (Ca2++PTTH). (B) Effect of U73122. PGs were pretreated with 50 lM U73122 or vehicle alone for 30 min, then, transferred to medium containing the same dose of U73122, with or without PTTH. Incubations were maintained for 5 min. ROS production was detected by the fluorescence of DCF. Results were normalized to the controls. Each value represents the mean ± SEM of five separate assays. Different letters above the bars indicate a significant difference (ANOVA followed by a Tukey’s multiple comparisons test, p < 0.05).
Fig. 2. Effects of A23187 and PMA on ROS production. (A) Effects of A23187 and PMA. PGs were preincubated in control medium for 30 min, then, transferred to control medium containing vehicle alone (Control) or medium containing either A23187 (50 lM) or PMA (10 lM). Incubations were maintained for 5 min. (B) Timedependence of A23187. PGs were preincubated in control medium for 30 min and then transferred to control medium containing vehicle alone (Control) or medium containing A23187 (50 lM) for different periods. (C) Dose-dependent effect of A23187. PGs were preincubated in control medium for 30 min and then transferred to control medium containing vehicle alone (0) or medium containing different concentration of A23187 for 5 min. ROS production was detected by the fluorescence of DCF. Results were normalized to the controls. Each value represents the mean ± SEM of five separate assays. Different letters above the bars indicate a significant difference (ANOVA followed by a Tukey’s multiple comparisons test, p < 0.05).
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Fig. 3. Effects of NAC (A), rotenone (B), antimycin A (C), FCCP (D), and DPI (E) on PTTH-stimulated ERK phosphorylation. PGs from day-5 last instar larvae were pretreated with either NAC (20 mM), rotenone (10 lM), antimycin A (10 lM), FCCP (10 lM), DPI (10 lM), or vehicle alone for 30 min, then transferred to medium containing the same dose of the inhibitors, with or without PTTH. Incubations were maintained for 60 min. CN, PGs incubated in control medium; N, PGs incubated in medium containing NAC; P, PGs incubated in medium containing PTTH; P + N, PGs incubated in medium containing both PTTH and NAC; Ro, PGs incubated in medium containing rotenone; P + Ro, PGs incubated in medium containing both PTTH and rotenone; An, PGs incubated in medium containing antimycin A; P + An, PGs incubated in medium containing both PTTH and antimycin A; F, PGs incubated in medium containing FCCP; P + F, PGs incubated in medium containing both PTTH and FCCP; DP, PGs incubated in medium containing DPI; P + DP, PGs incubated in medium containing both PTTH and DPI. Gland lysates were prepared and subjected to immunoblot analysis with anti-phospho-ERK (P-ERK) and anti-total-ERK (T-ERK) antibodies. Results shown are representative of three independent experiments.
dose-dependent (Fig. 2B and C). A peak in ROS production was detected as early as 1 min after the glands were treated with A23187 and the high levels were maintained between 1 min and 15 min. The level then decreased 30 min after A23187 treatment. The dose-dependent effect of A23187 showed that 50 lM A23187 treatment results in the highest ROS production.
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Fig. 4. Effects of NAC (A), rotenone (B), antimycin A (C), FCCP (D), and DPI (E) on PTTH-stimulated 4E-BP phosphorylation. PGs from day-5 last instar larvae were pretreated with either NAC (20 mM), rotenone (10 lM), antimycin A (10 lM), FCCP (10 lM), DPI (10 lM), or vehicle alone for 30 min, then transferred to medium containing the same dose of the inhibitors, with or without PTTH. Incubations were maintained for 60 min. CN, PGs incubated in control medium; N, PGs incubated in medium containing NAC; P, PGs incubated in medium containing PTTH; P + N, PGs incubated in medium containing both PTTH and NAC; Ro, PGs incubated in medium containing rotenone; P + Ro, PGs incubated in medium containing both PTTH and rotenone; An, PGs incubated in medium containing antimycin A; P + An, PGs incubated in medium containing both PTTH and antimycin A; F, PGs incubated in medium containing FCCP; P + F, PGs incubated in medium containing both PTTH and FCCP; DP, PGs incubated in medium containing DPI; P + DP, PGs incubated in medium containing both PTTH and DPI. Gland lysates were prepared and subjected to an immunoblot analysis with anti-phospho-4E-BP1 (Thr37/46) (P-4E-BP) and anti-a-tubulin (a-tubulin) antibodies. Results shown are representative of three independent experiments.
PTTH-stimulated production of ROS by NAC completely blocked PTTH-stimulated ERK phosphorylation as compared with PTTH treatment only. Treatment with the mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, FCCP, and DPI) also greatly decreased PTTH-stimulated ERK phosphorylation (Fig. 3B–E). These findings suggest that ERK phosphorylation might be a critical component of the redox-sensitive signaling pathway activated by PTTH in B. mori PGs.
3.3. Activation of ERK signaling by PTTH is ROS-dependent
3.4. Activation of 4E-BP phosphorylation by PTTH is ROS-dependent
The previous studies have shown that PTTH-stimulated phosphorylation of ERK appears to be involved in ecdysteroidogenesis (Rybczynski et al., 2001; Lin and Gu, 2007; Gu et al., 2010). However, it remains unclear whether the activation of the ERK pathway is redox-dependent. To determine whether the ERK signaling pathway is redox-sensitive, PGs were pretreated with NAC, a superoxide scavenger, to block PTTH-stimulated production of ROS, then stimulated with PTTH. As shown in Fig. 3A, blocking
TOR signaling is another signaling pathway stimulated by PTTH (Gu et al., 2012). We also examined whether the phosphorylation of 4E-BP, which is generally accepted to be the marker of TOR activity, is redox-sensitive. As shown in Fig. 4, blocking PTTHstimulated production of ROS by either NAC or the mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, FCCP, and DPI) completely abolished PTTH-stimulated 4E-BP phosphorylation as compared with PTTH treatment only.
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Fig. 5. Effects of NAC (A), rotenone (B), antimycin A (C), FCCP (D), and DPI (E) on PTTH-inhibited AMPK phosphorylation. PGs from day-5 last instar larvae were pretreated with either NAC (20 mM), rotenone (10 lM), antimycin A (10 lM), FCCP (10 lM), DPI (10 lM), or vehicle alone for 30 min, then transferred to medium containing the same dose of the inhibitors, with or without PTTH. Incubations were maintained for 60 min. CN, PGs incubated in control medium; N, PGs incubated in medium containing NAC; P, PGs incubated in medium containing PTTH; P + N, PGs incubated in medium containing both PTTH and NAC; Ro, PGs incubated in medium containing rotenone; P + Ro, PGs incubated in medium containing both PTTH and rotenone; An, PGs incubated in medium containing antimycin A; P + An, PGs incubated in medium containing both PTTH and antimycin A; F, PGs incubated in medium containing FCCP; P + F, PGs incubated in medium containing both PTTH and FCCP; DP, PGs incubated in medium containing DPI; P + DP, PGs incubated in medium containing both PTTH and DPI. Gland lysates were prepared and subjected to an immunoblot analysis with anti-phospho-AMPKa (Thr172) (P-AMPK) and antia-tubulin (a-tubulin) antibodies. Results shown are representative of three independent experiments.
Fig. 6. Effects of exogenous H2O2 on phosphorylation of ERK (A), 4E-BP (B) and AMPK (C). PGs from day-5 last instar larvae were preincubated in control medium for 30 min, then treated with H2O2 (HP, 1 mM), or incubated in control medium (Con). Incubations were maintained for 30 min. Gland lysates were prepared and subjected to an immunoblot analysis with anti-phospho-ERK (P-ERK), anti-totalERK (T-ERK), anti-phospho-4E-BP1 (Thr37/46) (P-4E-BP), anti-phospho-AMPKa (Thr172) (P-AMPK), and anti-a-tubulin (a-tubulin) antibodies. Results shown are representative of three independent experiments.
that treatment with 1 mM of H2O2 greatly stimulated the phosphorylation of ERK and 4E-BP and inhibited AMPK phosphorylation. This result demonstrated that ROS alone can regulate the phosphorylation of ERK, 4E-BP, and AMPK, thus confirming that ROS play a critical role in PTTH signaling.
3.5. PTTH-inhibited AMPK phosphorylation is ROS-dependent
4. Discussion
Our previous study showed that PTTH-inhibited AMPK phosphorylation is the upstream signaling of TOR and is involved in ecdysteroidogenesis by B. mori PGs (Gu et al., 2013). In the present study, we further investigated whether or not PTTH-inhibited AMPK phosphorylation is ROS-dependent. Fig. 5 shows that PTTH markedly inhibited AMPK phosphorylation and that blocking PTTH-stimulated ROS production by either NAC or the mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, FCCP, and DPI) blocked PTTH-inhibited AMPK phosphorylation as compared with PTTH treatment only.
Our previous study demonstrated that in B. mori PGs, the mitochondria-derived ROS production stimulated by PTTH is required for PTTH-stimulated ecdysteroidogenesis (Hsieh et al., 2013). The present study further investigated the releasing mechanism of ROS upon stimulation by PTTH and the cellular action mechanism of ROS. Our results showed that PTTH-stimulated ROS production is Ca2+- and PLC-dependent and that agents that directly elevate the intracellular Ca2+ concentration also stimulate ROS production. Moreover, the present study showed that ROS play a critical role in regulating PTTH-regulated phosphorylation of ERK, 4E-BP, and AMPK. To our knowledge, this is the first publication linking from PLC/Ca2+ to ROS as well as to protein phosphorylation regulated by PTTH in an insect endocrine organ (Fig. 7). It is necessary, however, to be cautious in this conclusion, because the specificity of the inhibitors used in these experiments has not been proved in insects. PLC/Ca2+ plays a critical role in PTTH signaling transduction. It has been reported that an inhibitor of PLC blocked increase in [Ca2+]i in the PGs of Manduca, indicating that PLC-activated formation of IP3 is a crucial step in elevating [Ca2+]i in Manduca PGs (Fellner et al., 2005). The rapid activation of PLC and an increase in [Ca2+]i in turn result in the activation of Ca2+/calmodulin dependent adenylate cyclase and an increase in cAMP content. In
3.6. Effects of hydrogen peroxide (H2O2) on phosphorylation of ERK, 4E-BP, and AMPK While the effects of NAC and mitochondrial inhibitors implicated the involvement of ROS in PTTH signaling transduction, it remained unclear whether ROS could directly participate in phosphorylation of ERK, 4E-BP, and AMPK. To determine whether ROS have a direct effect on cell signaling in B. mori PGs, PGs from day-5 last instar larvae were treated with H2O2 (1 mM) and alternation in downstream protein phosphorylation (ERK, 4E-BP, and AMPK) was assessed by Western blotting. Results (Fig. 6) showed
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Fig. 7. The signaling network involved in PTTH-stimulated ecdysteroidogenesis in B. mori PGs. See text for details.
addition, several other protein kinases, such as the phosphorylation of ERK appear to be also Ca2+/calmodulin dependent (Gu et al., 2010). In the present study, we further showed that PLC/Ca2+affects PTTH-stimulated ROS production. Blocking PLC activation by U73122 greatly inhibited ROS production stimulated by PTTH. PTTH-stimulated ROS was significantly reduced in Ca2+-free saline compared with those incubated in saline containing Ca2+. In addition, we found that the agents that directly increase the intracellular Ca2+ concentration (A23187 and PMA) also increased ROS
production and that A23187-stimulated ROS production was both dose- and time-dependent. In mammalian cells, the regulation of ROS production by Ca2+ has been well documented (Camello-Almaraz et al., 2006). Ca2+ modulates ROS homeostasis by regulating ROS production and annihilation mechanisms in both the mitochondria and the cytosol. Recent evidences have underscored the notion that the Ca2+ and ROS signaling systems are intimately integrated such that Ca2+-dependent regulation of components of ROS homeostasis might influence intracellular redox balance, and vice versa (Brookes
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et al., 2004; Camello-Almaraz et al., 2006). In Drosophila, it has been documented that PLC/Ca2+ modulates dual oxidase activity to produce microbicidal ROS essential for normal host survival (Ha et al., 2009). The cooperative action between Ca2+ and ROS production has also been reported in Drosophila ovarian smooth muscle: ROS generation in response to proctolin is Ca2+-dependent, and vice versa (Ritsick et al., 2007). The exact cooperative regulation between Ca2+ and ROS production in PGs remains to be investigated. We are currently studying whether an increase in [Ca2+]i upon PTTH stimulation is dependent on ROS production. Another objective was to determine whether ROS are involved in the phosphorylation of several key signaling proteins regulated by PTTH. The previous studies showed that ERK signaling is involved in PTTH-stimulated ecdysteroidogenesis in both Manduca and Bombyx (Rybczynski et al., 2001; Lin and Gu, 2007, 2011). Our recent studies indicated that PI3K/AMPK/TOR signaling is another signaling pathway involved in PTTH action (Gu et al., 2011, 2012, 2013). In the present study, pretreatment with either NAC or the mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, FCCP, and DPI) blocked PTTH-stimulated phosphorylation of ERK and 4E-BP. PTTH-inhibited AMPK phosphorylation was blocked by pretreatment with either NAC or the mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, FCCP, and DPI). In addition, we found that ROS alone can activate the phosphorylation of ERK and 4E-BP and inhibit AMPK phosphorylation. These results clearly showed that PTTHstimulated ROS production lies upstream signaling in regulating the phosphorylation of ERK, 4E-BP, and AMPK. In mammalian systems, the most often cited mechanism by which ROS contribute to cellular signaling is by modifying the actions of proteins through the reversible oxidation of essential cysteine residues (Sena and Chandel, 2012; Ray et al., 2012; Liochev, 2013). Oxidative modification of mitogen-activated protein (MAP) kinase signaling proteins and inactivation and/or degradation of MAP kinase phosphatases (MKPs) may provide the plausible mechanism for activation of MAP kinase pathways by ROS (Finkel, 1998, 2012; Forman et al., 2010; Collins et al., 2012; Sena and Chandel, 2012; Ray et al., 2012; Liochev, 2013). Previous studies also suggested that ROS can activate the PI3K/Akt/TOR signaling pathway through the reversible inactivation of the PTEN protein (Finkel, 1998, 2012; Forman et al., 2010; Collins et al., 2012; Sena and Chandel, 2012; Ray et al., 2012; Liochev, 2013). These observations have led us to hypothesize that oxidant signals induced by PTTH may regulate protein phosphorylation by either triggering tyrosine phosphorylation or affecting phosphatase activity. In addition, the phosphorylation and activation of Spook, a possible rate-limiting enzyme in the ecdysone biosynthetic pathway (Ono et al., 2006; Rewitz et al., 2009a), and the rate-limiting Black Box in this pathway may also be modulated by PTTH-stimulated oxidant signals. Further study is needed to clarify the exact cellular mechanism by which PTTH-stimulated ROS signaling affects phosphorylation of ERK, 4E-BP, AMPK, Spook, as well as the oxidation of the rate-limiting Black Box. In summary, we have demonstrated that ROS production stimulated by PTTH is Ca2+- and PLC-dependent and that agents that directly elevate the intracellular Ca2+ concentration also stimulate ROS production. PTTH-stimulated ROS signaling appears to lie upstream signaling of the phosphorylation of ERK and AMPK/TOR signaling (Fig. 7). Acknowledgements The authors thank the National Science Council, Taipei, Taiwan for grants (NSC99-2628-B-178-002-MY3 and NSC100-2313-B-178001-MY3); and the National Museum of Natural Science, Taichung, Taiwan for their financial support.
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