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RESEARCH PAPER Key role of heat shock protein 90 in leptin-induced STAT3 activation and feeding regulation Correspondence Koichiro Ozawa or Toru Hosoi, Department of Pharmacotherapy, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. E-mail: [email protected]; [email protected]
Received 6 February 2015; Revised 16 May 2016; Accepted 17 May 2016
Toru Hosoi1, Toshiko Kohda1, Syu Matsuzaki1, Mizuho Ishiguchi1, Ayaka Kuwamura1, Tomoyuki Akita2, Junko Tanaka2 and Koichiro Ozawa1 1
Department of Pharmacotherapy, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan, and 2Department of
Epidemiology, Infectious Disease Control and Prevention, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
BACKGROUND AND PURPOSE Leptin, an important regulator of the energy balance, acts on the brain to inhibit feeding. However, the mechanisms involved in leptin signalling have not yet been fully elucidated. Heat shock protein 90 (HSP90) is a molecular chaperone that is involved in regulating cellular homeostasis. In the present study, we investigated the possible involvement of HSP90 in leptin signal transduction.
EXPERIMENTAL APPROACH HEK293 and SH-SY5Y cell lines stably transfected with the Ob-Rb leptin receptor (HEK293 Ob-Rb, SH-SY5Y Ob-Rb) were used in the present study. Phosphorylation of JAK2 and STAT3 was analysed by western blotting. An HSP90 inhibitor was administered i.c.v. into rats and their food intake was analysed.
KEY RESULTS The knock-down of HSP90 in the HEK293 Ob-Rb cell line attenuated leptin-induced JAK2 and STAT3 signalling. Moreover, leptininduced JAK2/STAT3 phosphorylation was markedly attenuated by the HSP90 inhibitors geldanamycin, radicicol and novobiocin. However, these effects were not mediated through previously known factors, which are known to be involved in the development of leptin resistance, such as suppressor of cytokine signalling 3 or endoplasmic reticulum stress. The infusion of an HSP90 inhibitor into the CNS blunted the anorexigenic actions of leptin in rats (male Wister rat).
CONCLUSIONS AND IMPLICATIONS HSP90 may be a novel factor involved in leptin-mediated signalling that is linked to anorexia.
Abbreviations ER stress, endoplasmic reticulum stress; HSP90, heat shock protein 90; POMC, proopiomelanocortin; PTP1B, protein tyrosine phosphatase-1B; SOCS3, suppressor of cytokine signalling 3
DOI:10.1111/bph.13520
© 2016 The British Pharmacological Society
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Tables of Links TARGETS GPCRs
LIGANDS
a
Enzymes
GPR78 Catalytic receptors
c
JAK2
HSP90α1
Leptin
HSP90β
POMC (ACTH)
b
Leptin receptor These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016) and are permanently archived in the Concise Guide a,b,c Alexander et al., 2015a,b,c). to PHARMACOLOGY 2015/16 (
Introduction Obesity is a complex condition associated with an increased risk of diseases, such as diabetes, cardiovascular disease and hypertension. Therefore, the underlying mechanisms involved in the development of obesity need to be elucidated in more detail. Leptin is an anti-obesity hormone that was identified by Friedman’s group in 1994 (Zhang et al., 1994). It is mostly secreted from adipose tissue, circulates in the blood, and acts on the brain. Leptin activates the Ob-Rb leptin receptor, which is expressed on neurons within the hypothalamus, and induces the JAK2/STAT3 signalling pathway (Vaisse et al., 1996; Hosoi et al., 2002). By activating the JAK2/STAT3 pathway, leptin inhibits food intake and body weight gain (Campfield et al., 1995). The activation of STAT3 in proopiomelanocortin (POMC) neurons has been shown to increase the production of α-melanocyte-stimulating hormone (αMSH), which, in turn, activates melanocortin receptors. The activation of melanocortin receptors activates catabolic pathways by inhibiting food intake. Furthermore, leptin has been shown to inhibit anabolic pathways by inhibiting neuropeptide Y/agouti-related neuropeptide (NPY/AgRP) neurons (Schwartz et al., 2000). Although leptin has anti-obesity effects and was initially expected to be useful as an anti-obesity drug, most obese patients were found to be unresponsive to leptin, which indicated that leptin resistance was involved in the development of obesity (Münzberg and Myers, 2005). Therefore, elucidation of the underlying mechanisms involved in the development of leptin resistance is of importance (Friedman, 2003). Until now, suppressors of cytokine signalling 3 (SOCS3) (Endo et al., 1997; Naka et al., 1997; Starr et al., 1997; Bjørbaek et al., 1998), protein tyrosine phosphatase-1B (PTP1B) (Cheng et al., 2002; Zabolotny et al., 2002) and endoplasmic reticulum (ER) stress (Hosoi et al., 2008; Zhang et al., 2008; Ozcan et al., 2009; Hosoi et al., 2014) were the only pathways thought to be involved in the development of leptin resistance (Hosoi and Ozawa, 2010). SOCS3 is up-regulated in the arcuate nucleus of the hypothalamus from diet-induced obese mice (Münzberg et al., 2004) and inhibits leptin signalling by binding to Ob-Rb leptin receptors (Bjørbaek et al., 1998, Bjorbak et al., 2000). PTP1B-deficient mice are resistant to high-fat diet-induced obesity (Elchebly et al., 1999; Klaman et al., 2000), and the activation of PTP1B inhibits leptin signalling by dephosphorylating the leptin receptorassociated kinase, JAK2 in the hypothalamus (Zabolotny et al., 2002, Cheng et al., 2002). Endoplasmic reticulum stress was found to be activated in the hypothalamus of obese mice
(Zhang et al., 2008; Ozcan et al., 2009), and leptin resistance developed under ER-stressed conditions (Hosoi et al., 2008; Ozcan et al., 2009). Taken together, these findings indicate that several mechanisms are involved in the development of leptin resistance. However, the mechanisms underlying leptin resistance are still unclear and need to be examined in more detail. HSP90 is a molecular chaperone that is involved in regulating cellular homeostasis and stress responses. Several studies have demonstrated its important role in regulating CNS function. HSP90 was previously shown to promote tau solubility and binding to microtubules in the CNS, thereby preventing tau aggregation in Alzheimer’s disease (AD) (Dou et al., 2003). HSP90 was also shown to inhibit amyloid β-(1–42) aggregation, which has been implicated in the progression of AD (Evans et al., 2006). These findings suggested the critical involvement of HSP90 in neuronal function. In addition to its function as a molecular chaperone, HSP90 has been reported to regulate signal transduction. Previous studies suggested that HSP90 interacts with STAT3 and regulates its activation in IL-6 stimulated cells (Shah et al., 2002; Sato et al., 2003). However, the detailed relationships between the two molecular functions of HSP90: as a chaperone and as a mediator of STAT3 signalling had not yet been elucidated. HSP90 is expressed in the hypothalamus (Olazábal et al., 1992), and immunohistochemical analyses revealed that its expression is primarily neuronal (Itoh et al., 1993; Gass et al., 1994). The actions of leptin on the regulation of feeding behavior are known to be mediated through neuronal cells; therefore, we hypothesized that HSP90 may regulate leptin-induced signal transduction, activating leptin-induced STAT3, which has been linked to anorexia. In the present study, we demonstrated that HSP90 plays a key role in regulating the leptin-induced activation of the JAK2/STAT3 signalling. Furthermore, we showed that HSP90 inhibitors attenuate leptininduced anorexia, suggesting the important role of HSP90 in regulating the actions of leptin.
Methods Generation of Ob-Rb leptin receptor-transfected cells The Ob-Rb leptin receptor is a long isoform of the leptin receptor, which plays an important role in activating leptin-induced JAK2-STAT3 signalling (Ghilardi et al., 1996). The human ObRb leptin receptor construct, a gift from Genetech Inc., was British Journal of Pharmacology (2016) 173 2434–2445
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transfected into SH-SY5Y and HEK293 cell lines using LipofectAMINE PLUS Reagent (Life Technologies Inc.) according to the manufacturer’s instructions. Stable transfectants (SH-SY5Y-Ob-Rb and HEK293-Ob-Rb cells) were obtained by selection with the antibiotic G418 (Hosoi et al., 2006).
Transient transfection of Ob-Rb plasmid into GT1–7 cell line The human Ob-Rb leptin receptor construct was transfected into GT1–7 cells using Lipofectamine 2000 Reagent (Life Technologies Inc.) according to the manufacturer’s instructions. Forty-eight hours after the transfection, the cells were stimulated with leptin.
nitrocellulose membranes. The membranes were incubated with anti-KDEL (StressGen; diluted to 1:1000), anti-CHOP (Santa Cruz; diluted to 1:500), anti-HSP90 (Sigma or Santa Cruz; diluted to 1:1000), anti-JAK2 (Santa Cruz; diluted to 1:500), anti-Phospho (Tyr1007/1008)-JAK2 (Cell Signaling or upstate; diluted to 1:1000), anti-Phospho (Tyr705)-STAT3 (Cell Signaling; diluted to 1:1000), anti-STAT3 (Cell Signaling; diluted to 1:1000), anti-Phospho-Tyr (upstate; diluted to 1:2000), and anti-GAPDH (Chemicon; diluted to 1:2000) antibodies followed by anti-horseradish peroxidase-linked antibody. Peroxidase was detected by chemiluminescence using an enhanced chemiluminescence system.
Immunoprecipitation Experimental design The analyses were not performed blind. However, we made an effort to be close to the conditions of blinded assays. All the samples were obtained using the same procedures, and the samples were treated in the same way. Each of the samples was numbered, and the identity of each sample was not obvious during the experiment. The analysis was not performed with randomization. However, the experiments were carried out using the same conditions (the room temperature was same for all of the experiments), and we made an effort to keep the conditions close to those of a randomization assay.
RNAi experiment Lipofectamine RNAiMAX (Life Technologies) was used to transfect siRNA at a final concentration of 25 nM (at Santa Cruz siRNAs) or 10 nM (at Life Technologies siRNAs) according to the manufacturer’s directions. An Opti-MEM1 medium was used for the transfection. HSP90 α/β siRNA (h) (SantaCruz, sc-35608) and control siRNA-A (Santa Cruz, sc-37007) were used for the siRNA transfection. HSP 90α/β siRNA (h) (sc-35608) is a pool of four different siRNA duplexes. Sequence of these siRNAs are as follows: sc-35608A: sense; CGUGAGAUGUUGCAACAAAtt, antisense; UUUGUUGCAACAUCUCACGtt, sc-35608B: sense; CCUGUUCAGUACUCUACAAtt, antisense; UUGUAGAGUACUGAACAGGtt, sc-35608C: sense; GAAGACAAGGAGAAUUACAtt, antisense; UGUAAUUCUCCUUGUCUUCtt, sc-35608D: sense; GCAAGGCAAAGUUUGAGAAtt, antisense; UUCUCAAACUUUGCCUUGCtt. Strands A and B are specific to HSP 90α. Strands C and D are specific to HSP 90β. We also used HSP90 siRNA and Silencer® select negative control siRNA #1 from Life technologies (Supporting Information Fig. S1). The sequence of the siRNA is as follows: sense: CUAUGGGUCGUGGAACAAAtt, antisense: UUUGUUCCACGACCCAUAGgt. Cells were harvested 72 h after the transfection.
Western blotting Western blotting was performed as described previously (Hosoi et al., 2012). Cells were washed with ice-cold PBS and lysed in a buffer containing 10 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 10 μg·mL 1 aprotinin, 10 μg·mL 1 leupeptin, 1 mM PMSF and 1% NP-40 for 20 min. The lysates were centrifuged at 20630 × g for 20 min at 4°C, and the supernatants were collected. The samples were boiled with Laemmli buffer for 3 min, fractionated by SDS-PAGE and transferred at 4°C to 2436
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Cells were lysed in lysis buffer (10 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 10 μg·mL 1 aprotinin, 10 μg·mL 1 leupeptin, 1 mM PMSF and 0.1% NP-40), and samples were homogenized using a 21G needle. The lysates were centrifuged at 20630 × g for 20 min at 4°C, and the supernatants were collected. An antibody was added to the lysate and rotated at 4°C. Dynabeads Protein G (Invitrogen) was then added and rotated at 4°C for 20 min. Immunoprecipitates were washed three times with lysis buffer. The immunoprecipitates from cell lysates were resolved on SDS-PAGE and transferred to a nitrocellulose transfer membrane. The filters were then immunoblotted with each antibody. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system. Immunohistochemistry Cells were fixed with methanol for 10 min at 20°C. After being washed with PBS, the cells were incubated with 5% normal bovine serum at 37°C for 1 h and allowed to react with anti-HSP90 (Santa Cruz; diluted to 1:200) and antiphospho-STAT3 (Cell Signaling; diluted to 1:50) antibodies at 4°C overnight. The cells were then incubated with antimouse Alexa 488 (1:2000) and anti-rabbit Alexa 488 (1:2000) at 37°C for 1 h. The cells were visualized using confocal laser scanning microscopy. The confocal laser scanning microscopy was carried out at the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University.
I.c.v. injections and measurement of food intake in rats To install a stainless-steel guide cannula for the i.c.v. injection of leptin and geldanamycin, the male Wister rat was anaesthetized with sodium pentobarbital (50 mg·kg 1, i.p.) and placed in a stereotaxic apparatus. A 24G stainless-steel guide cannula was inserted into the brain (1.0 mm posterior to the bregma, 1.5 mm to the right lateral side and 3.7 mm below the surface). The guide cannula was secured with dental cement anchored by two stainless steel screws fixed on the dorsal surface of the skull. After surgery, a dummy cannula (30G) was inserted into the guide cannula. Animals were allowed to recover for at least 10 days after this operation. Four days before the leptin and geldanamycin injection, the dummy cannula was replaced by a microinjection cannula, all rats were injected with saline and their food intake was measured. Food was removed at 17:30, and saline was injected at 18:00. Food was placed back in the cage and at
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19:30 and their food intake was measured after 4, 14 and 24 h. On the day of the experiment, the dummy cannula was replaced by a microinjection cannula. The food was removed at 17:30. Geldanamycin (100 nmol·3 μL 1) and leptin 5 μg·3 μL 1 was injected at 18:00. As the geldanamycin was dissolved in DMSO, we injected DMSO (3 μL) for the control experiments. Food was placed back in the cage at 19:30, and their food intake was measured after 4, 14 and 24 h. The placement of the cannula was verified at the end of the experiment by injecting 10 μL dye (5 μg·mL 1 Evans Blue). All animal experiments were carried out in accordance with the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals. Considering welfare and ethics, the present experiments were approved by the animal care and use committee at Hiroshima University. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilly, 2015). The sex and ages of the mice and rats were the same and the room temperature was the same for all of the experiments.
I.c.v. injections and measurement of STAT3 phosphorylation in mice Food-deprived (16 h) male C57BL/6 mice were anaesthetized with sodium pentobarbital (50 mg·kg 1, i.p.). Novobiocin (0.5 μmol, 1 μL) or saline was injected i.c.v into the skull 0.3 mm caudal from the bregma, 0.9 mm right lateral side from the midline and 2.0 mm below the dura matter. Five minutes after the injection, leptin (2.25 μg, 2 μL) or saline was injected i.c.v. Thirty minutes after this injection, the animal was killed by decapitation and the hypothalamus was snap-frozen in liquid nitrogen and stored in 80°C until use. Samples were homogenized using Precellys 24 (Bertin Technologies, Orleans, France) in a buffer containing 10 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 1 mM EGTA, 21 mM Na3VO4, 10 mM NaF, 10 μg·mL 1 aprotinin, 10 μg·mL 1 leupeptin, 1 mM PMSF and 1% Nonidet P-40. Samples were then centrifuged at 20630 × g for 45 min at 4°C, and the supernatants were collected for western blot analysis.
Data and statistical analysis The present studies comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). Results are expressed as the means ± SEM. Statistical analysis was performed using Student’s t-test, paired t-test or Dunnett’s test. ANOVA analysis and post hoc multiple comparison tests (Holm’s test) were applied to compare groups. Before applying ANOVA, we checked homoscedasticity by Bartlett test. Statistical significance was assumed when the P value was 0.05). From the result of ANOVA and Holm’s test, there are significant difference among groups (ANOVA: P < 0.05) and significant difference between pairs of 0–14 h and 0–24 h were confirmed by Holm’s test (all P < 0.05). *P < 0.05 n = 10–13 per group of five independent experiments. In each experiment, we used the following rat numbers: control group: 1–4 rats, leptin treatment group: 1–5 rats and GA + leptin treatment group: 1–5 rats. In total, control group 10 rats, leptin treatment group 12 rats and GA+ leptin treatment group 13 rats were used for the overall experiments. (B) HSP90 inhibitor (novobiocin, NB: 0.5 μmol) and leptin (2.25 μg) were administered through an intracerebroventricular route to male mice. Animals were pretreated with novobiocin (5 min) prior to leptin injection. Thirty minutes after the treatment, the hypothalamus was removed, and the phosphorylation level of STAT3 was analysed by western blotting. Student’s ttest, *P < 0.05 versus control mice treated with leptin. n = 8 per group of three independent days of experiments. In each experiment, we used the following mouse numbers: control group two mice, leptin treatment group four mice, NB treatment group two mice and NB + leptin treatment group four mice for two independent experiments. Control group of four mice and NB treatment group of four mice were used for another experimental group. In total, control group eight mice, leptin treatment group eight mice, NB treatment group eight mice and NB + leptin treatment group eight mice were used for the overall experiments. Each set of data was expressed as fold increase over control rodents.
This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organizations engaged with supporting research.
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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: http://dx.doi.org/10.1111/bph.13520 Figure S1 HSP90 knockdown attenuated leptin-induced STAT3 phosphorylation. Western blotting analysis of leptininduced STAT3 activation in the HSP90-knocked down HEK293-Ob-Rb cell line. Cells were stimulated with leptin (0.5 μg/mL) for 15 min and Western blotting was then performed. (A) Expression level of HSP90 was attenuated by HSP90 siRNA. (B) The leptin-induced phosphorylation of STAT3 was inhibited in HSP90-knocked down cells. Typical blot among three independent experiments was shown. Figure S2 A HSP90 inhibitors inhibited leptin-induced activation of STAT3 in neuronal cells. SH-SY5Y-Ob-Rb cells were treated with the HSP90 inhibitor, radicicol (Rad: 10 μM) and novobiocin (NB: 1000 μM), and leptininduced STAT3 phosphorylation was analyzed. *P < 0.05 v.s. control cells treated with leptin. Dunnett’s test. n = 4. © 2016 The British Pharmacological Society.
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RESEARCH PAPER Key role of heat shock protein 90 in leptin-induced STAT3 activation and feeding regulation Correspondence Koichiro Ozawa or Toru Hosoi, Department of Pharmacotherapy, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. E-mail: [email protected]; [email protected]
Received 6 February 2015; Revised 16 May 2016; Accepted 17 May 2016
Toru Hosoi1, Toshiko Kohda1, Syu Matsuzaki1, Mizuho Ishiguchi1, Ayaka Kuwamura1, Tomoyuki Akita2, Junko Tanaka2 and Koichiro Ozawa1 1
Department of Pharmacotherapy, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan, and 2Department of
Epidemiology, Infectious Disease Control and Prevention, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
BACKGROUND AND PURPOSE Leptin, an important regulator of the energy balance, acts on the brain to inhibit feeding. However, the mechanisms involved in leptin signalling have not yet been fully elucidated. Heat shock protein 90 (HSP90) is a molecular chaperone that is involved in regulating cellular homeostasis. In the present study, we investigated the possible involvement of HSP90 in leptin signal transduction.
EXPERIMENTAL APPROACH HEK293 and SH-SY5Y cell lines stably transfected with the Ob-Rb leptin receptor (HEK293 Ob-Rb, SH-SY5Y Ob-Rb) were used in the present study. Phosphorylation of JAK2 and STAT3 was analysed by western blotting. An HSP90 inhibitor was administered i.c.v. into rats and their food intake was analysed.
KEY RESULTS The knock-down of HSP90 in the HEK293 Ob-Rb cell line attenuated leptin-induced JAK2 and STAT3 signalling. Moreover, leptininduced JAK2/STAT3 phosphorylation was markedly attenuated by the HSP90 inhibitors geldanamycin, radicicol and novobiocin. However, these effects were not mediated through previously known factors, which are known to be involved in the development of leptin resistance, such as suppressor of cytokine signalling 3 or endoplasmic reticulum stress. The infusion of an HSP90 inhibitor into the CNS blunted the anorexigenic actions of leptin in rats (male Wister rat).
CONCLUSIONS AND IMPLICATIONS HSP90 may be a novel factor involved in leptin-mediated signalling that is linked to anorexia.
Abbreviations ER stress, endoplasmic reticulum stress; HSP90, heat shock protein 90; POMC, proopiomelanocortin; PTP1B, protein tyrosine phosphatase-1B; SOCS3, suppressor of cytokine signalling 3
DOI:10.1111/bph.13520
© 2016 The British Pharmacological Society
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Tables of Links TARGETS GPCRs
LIGANDS
a
Enzymes
GPR78 Catalytic receptors
c
JAK2
HSP90α1
Leptin
HSP90β
POMC (ACTH)
b
Leptin receptor These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016) and are permanently archived in the Concise Guide a,b,c Alexander et al., 2015a,b,c). to PHARMACOLOGY 2015/16 (
Introduction Obesity is a complex condition associated with an increased risk of diseases, such as diabetes, cardiovascular disease and hypertension. Therefore, the underlying mechanisms involved in the development of obesity need to be elucidated in more detail. Leptin is an anti-obesity hormone that was identified by Friedman’s group in 1994 (Zhang et al., 1994). It is mostly secreted from adipose tissue, circulates in the blood, and acts on the brain. Leptin activates the Ob-Rb leptin receptor, which is expressed on neurons within the hypothalamus, and induces the JAK2/STAT3 signalling pathway (Vaisse et al., 1996; Hosoi et al., 2002). By activating the JAK2/STAT3 pathway, leptin inhibits food intake and body weight gain (Campfield et al., 1995). The activation of STAT3 in proopiomelanocortin (POMC) neurons has been shown to increase the production of α-melanocyte-stimulating hormone (αMSH), which, in turn, activates melanocortin receptors. The activation of melanocortin receptors activates catabolic pathways by inhibiting food intake. Furthermore, leptin has been shown to inhibit anabolic pathways by inhibiting neuropeptide Y/agouti-related neuropeptide (NPY/AgRP) neurons (Schwartz et al., 2000). Although leptin has anti-obesity effects and was initially expected to be useful as an anti-obesity drug, most obese patients were found to be unresponsive to leptin, which indicated that leptin resistance was involved in the development of obesity (Münzberg and Myers, 2005). Therefore, elucidation of the underlying mechanisms involved in the development of leptin resistance is of importance (Friedman, 2003). Until now, suppressors of cytokine signalling 3 (SOCS3) (Endo et al., 1997; Naka et al., 1997; Starr et al., 1997; Bjørbaek et al., 1998), protein tyrosine phosphatase-1B (PTP1B) (Cheng et al., 2002; Zabolotny et al., 2002) and endoplasmic reticulum (ER) stress (Hosoi et al., 2008; Zhang et al., 2008; Ozcan et al., 2009; Hosoi et al., 2014) were the only pathways thought to be involved in the development of leptin resistance (Hosoi and Ozawa, 2010). SOCS3 is up-regulated in the arcuate nucleus of the hypothalamus from diet-induced obese mice (Münzberg et al., 2004) and inhibits leptin signalling by binding to Ob-Rb leptin receptors (Bjørbaek et al., 1998, Bjorbak et al., 2000). PTP1B-deficient mice are resistant to high-fat diet-induced obesity (Elchebly et al., 1999; Klaman et al., 2000), and the activation of PTP1B inhibits leptin signalling by dephosphorylating the leptin receptorassociated kinase, JAK2 in the hypothalamus (Zabolotny et al., 2002, Cheng et al., 2002). Endoplasmic reticulum stress was found to be activated in the hypothalamus of obese mice
(Zhang et al., 2008; Ozcan et al., 2009), and leptin resistance developed under ER-stressed conditions (Hosoi et al., 2008; Ozcan et al., 2009). Taken together, these findings indicate that several mechanisms are involved in the development of leptin resistance. However, the mechanisms underlying leptin resistance are still unclear and need to be examined in more detail. HSP90 is a molecular chaperone that is involved in regulating cellular homeostasis and stress responses. Several studies have demonstrated its important role in regulating CNS function. HSP90 was previously shown to promote tau solubility and binding to microtubules in the CNS, thereby preventing tau aggregation in Alzheimer’s disease (AD) (Dou et al., 2003). HSP90 was also shown to inhibit amyloid β-(1–42) aggregation, which has been implicated in the progression of AD (Evans et al., 2006). These findings suggested the critical involvement of HSP90 in neuronal function. In addition to its function as a molecular chaperone, HSP90 has been reported to regulate signal transduction. Previous studies suggested that HSP90 interacts with STAT3 and regulates its activation in IL-6 stimulated cells (Shah et al., 2002; Sato et al., 2003). However, the detailed relationships between the two molecular functions of HSP90: as a chaperone and as a mediator of STAT3 signalling had not yet been elucidated. HSP90 is expressed in the hypothalamus (Olazábal et al., 1992), and immunohistochemical analyses revealed that its expression is primarily neuronal (Itoh et al., 1993; Gass et al., 1994). The actions of leptin on the regulation of feeding behavior are known to be mediated through neuronal cells; therefore, we hypothesized that HSP90 may regulate leptin-induced signal transduction, activating leptin-induced STAT3, which has been linked to anorexia. In the present study, we demonstrated that HSP90 plays a key role in regulating the leptin-induced activation of the JAK2/STAT3 signalling. Furthermore, we showed that HSP90 inhibitors attenuate leptininduced anorexia, suggesting the important role of HSP90 in regulating the actions of leptin.
Methods Generation of Ob-Rb leptin receptor-transfected cells The Ob-Rb leptin receptor is a long isoform of the leptin receptor, which plays an important role in activating leptin-induced JAK2-STAT3 signalling (Ghilardi et al., 1996). The human ObRb leptin receptor construct, a gift from Genetech Inc., was British Journal of Pharmacology (2016) 173 2434–2445
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transfected into SH-SY5Y and HEK293 cell lines using LipofectAMINE PLUS Reagent (Life Technologies Inc.) according to the manufacturer’s instructions. Stable transfectants (SH-SY5Y-Ob-Rb and HEK293-Ob-Rb cells) were obtained by selection with the antibiotic G418 (Hosoi et al., 2006).
Transient transfection of Ob-Rb plasmid into GT1–7 cell line The human Ob-Rb leptin receptor construct was transfected into GT1–7 cells using Lipofectamine 2000 Reagent (Life Technologies Inc.) according to the manufacturer’s instructions. Forty-eight hours after the transfection, the cells were stimulated with leptin.
nitrocellulose membranes. The membranes were incubated with anti-KDEL (StressGen; diluted to 1:1000), anti-CHOP (Santa Cruz; diluted to 1:500), anti-HSP90 (Sigma or Santa Cruz; diluted to 1:1000), anti-JAK2 (Santa Cruz; diluted to 1:500), anti-Phospho (Tyr1007/1008)-JAK2 (Cell Signaling or upstate; diluted to 1:1000), anti-Phospho (Tyr705)-STAT3 (Cell Signaling; diluted to 1:1000), anti-STAT3 (Cell Signaling; diluted to 1:1000), anti-Phospho-Tyr (upstate; diluted to 1:2000), and anti-GAPDH (Chemicon; diluted to 1:2000) antibodies followed by anti-horseradish peroxidase-linked antibody. Peroxidase was detected by chemiluminescence using an enhanced chemiluminescence system.
Immunoprecipitation Experimental design The analyses were not performed blind. However, we made an effort to be close to the conditions of blinded assays. All the samples were obtained using the same procedures, and the samples were treated in the same way. Each of the samples was numbered, and the identity of each sample was not obvious during the experiment. The analysis was not performed with randomization. However, the experiments were carried out using the same conditions (the room temperature was same for all of the experiments), and we made an effort to keep the conditions close to those of a randomization assay.
RNAi experiment Lipofectamine RNAiMAX (Life Technologies) was used to transfect siRNA at a final concentration of 25 nM (at Santa Cruz siRNAs) or 10 nM (at Life Technologies siRNAs) according to the manufacturer’s directions. An Opti-MEM1 medium was used for the transfection. HSP90 α/β siRNA (h) (SantaCruz, sc-35608) and control siRNA-A (Santa Cruz, sc-37007) were used for the siRNA transfection. HSP 90α/β siRNA (h) (sc-35608) is a pool of four different siRNA duplexes. Sequence of these siRNAs are as follows: sc-35608A: sense; CGUGAGAUGUUGCAACAAAtt, antisense; UUUGUUGCAACAUCUCACGtt, sc-35608B: sense; CCUGUUCAGUACUCUACAAtt, antisense; UUGUAGAGUACUGAACAGGtt, sc-35608C: sense; GAAGACAAGGAGAAUUACAtt, antisense; UGUAAUUCUCCUUGUCUUCtt, sc-35608D: sense; GCAAGGCAAAGUUUGAGAAtt, antisense; UUCUCAAACUUUGCCUUGCtt. Strands A and B are specific to HSP 90α. Strands C and D are specific to HSP 90β. We also used HSP90 siRNA and Silencer® select negative control siRNA #1 from Life technologies (Supporting Information Fig. S1). The sequence of the siRNA is as follows: sense: CUAUGGGUCGUGGAACAAAtt, antisense: UUUGUUCCACGACCCAUAGgt. Cells were harvested 72 h after the transfection.
Western blotting Western blotting was performed as described previously (Hosoi et al., 2012). Cells were washed with ice-cold PBS and lysed in a buffer containing 10 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 10 μg·mL 1 aprotinin, 10 μg·mL 1 leupeptin, 1 mM PMSF and 1% NP-40 for 20 min. The lysates were centrifuged at 20630 × g for 20 min at 4°C, and the supernatants were collected. The samples were boiled with Laemmli buffer for 3 min, fractionated by SDS-PAGE and transferred at 4°C to 2436
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Cells were lysed in lysis buffer (10 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 10 μg·mL 1 aprotinin, 10 μg·mL 1 leupeptin, 1 mM PMSF and 0.1% NP-40), and samples were homogenized using a 21G needle. The lysates were centrifuged at 20630 × g for 20 min at 4°C, and the supernatants were collected. An antibody was added to the lysate and rotated at 4°C. Dynabeads Protein G (Invitrogen) was then added and rotated at 4°C for 20 min. Immunoprecipitates were washed three times with lysis buffer. The immunoprecipitates from cell lysates were resolved on SDS-PAGE and transferred to a nitrocellulose transfer membrane. The filters were then immunoblotted with each antibody. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system. Immunohistochemistry Cells were fixed with methanol for 10 min at 20°C. After being washed with PBS, the cells were incubated with 5% normal bovine serum at 37°C for 1 h and allowed to react with anti-HSP90 (Santa Cruz; diluted to 1:200) and antiphospho-STAT3 (Cell Signaling; diluted to 1:50) antibodies at 4°C overnight. The cells were then incubated with antimouse Alexa 488 (1:2000) and anti-rabbit Alexa 488 (1:2000) at 37°C for 1 h. The cells were visualized using confocal laser scanning microscopy. The confocal laser scanning microscopy was carried out at the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University.
I.c.v. injections and measurement of food intake in rats To install a stainless-steel guide cannula for the i.c.v. injection of leptin and geldanamycin, the male Wister rat was anaesthetized with sodium pentobarbital (50 mg·kg 1, i.p.) and placed in a stereotaxic apparatus. A 24G stainless-steel guide cannula was inserted into the brain (1.0 mm posterior to the bregma, 1.5 mm to the right lateral side and 3.7 mm below the surface). The guide cannula was secured with dental cement anchored by two stainless steel screws fixed on the dorsal surface of the skull. After surgery, a dummy cannula (30G) was inserted into the guide cannula. Animals were allowed to recover for at least 10 days after this operation. Four days before the leptin and geldanamycin injection, the dummy cannula was replaced by a microinjection cannula, all rats were injected with saline and their food intake was measured. Food was removed at 17:30, and saline was injected at 18:00. Food was placed back in the cage and at
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19:30 and their food intake was measured after 4, 14 and 24 h. On the day of the experiment, the dummy cannula was replaced by a microinjection cannula. The food was removed at 17:30. Geldanamycin (100 nmol·3 μL 1) and leptin 5 μg·3 μL 1 was injected at 18:00. As the geldanamycin was dissolved in DMSO, we injected DMSO (3 μL) for the control experiments. Food was placed back in the cage at 19:30, and their food intake was measured after 4, 14 and 24 h. The placement of the cannula was verified at the end of the experiment by injecting 10 μL dye (5 μg·mL 1 Evans Blue). All animal experiments were carried out in accordance with the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals. Considering welfare and ethics, the present experiments were approved by the animal care and use committee at Hiroshima University. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilly, 2015). The sex and ages of the mice and rats were the same and the room temperature was the same for all of the experiments.
I.c.v. injections and measurement of STAT3 phosphorylation in mice Food-deprived (16 h) male C57BL/6 mice were anaesthetized with sodium pentobarbital (50 mg·kg 1, i.p.). Novobiocin (0.5 μmol, 1 μL) or saline was injected i.c.v into the skull 0.3 mm caudal from the bregma, 0.9 mm right lateral side from the midline and 2.0 mm below the dura matter. Five minutes after the injection, leptin (2.25 μg, 2 μL) or saline was injected i.c.v. Thirty minutes after this injection, the animal was killed by decapitation and the hypothalamus was snap-frozen in liquid nitrogen and stored in 80°C until use. Samples were homogenized using Precellys 24 (Bertin Technologies, Orleans, France) in a buffer containing 10 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 1 mM EGTA, 21 mM Na3VO4, 10 mM NaF, 10 μg·mL 1 aprotinin, 10 μg·mL 1 leupeptin, 1 mM PMSF and 1% Nonidet P-40. Samples were then centrifuged at 20630 × g for 45 min at 4°C, and the supernatants were collected for western blot analysis.
Data and statistical analysis The present studies comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). Results are expressed as the means ± SEM. Statistical analysis was performed using Student’s t-test, paired t-test or Dunnett’s test. ANOVA analysis and post hoc multiple comparison tests (Holm’s test) were applied to compare groups. Before applying ANOVA, we checked homoscedasticity by Bartlett test. Statistical significance was assumed when the P value was 0.05). From the result of ANOVA and Holm’s test, there are significant difference among groups (ANOVA: P < 0.05) and significant difference between pairs of 0–14 h and 0–24 h were confirmed by Holm’s test (all P < 0.05). *P < 0.05 n = 10–13 per group of five independent experiments. In each experiment, we used the following rat numbers: control group: 1–4 rats, leptin treatment group: 1–5 rats and GA + leptin treatment group: 1–5 rats. In total, control group 10 rats, leptin treatment group 12 rats and GA+ leptin treatment group 13 rats were used for the overall experiments. (B) HSP90 inhibitor (novobiocin, NB: 0.5 μmol) and leptin (2.25 μg) were administered through an intracerebroventricular route to male mice. Animals were pretreated with novobiocin (5 min) prior to leptin injection. Thirty minutes after the treatment, the hypothalamus was removed, and the phosphorylation level of STAT3 was analysed by western blotting. Student’s ttest, *P < 0.05 versus control mice treated with leptin. n = 8 per group of three independent days of experiments. In each experiment, we used the following mouse numbers: control group two mice, leptin treatment group four mice, NB treatment group two mice and NB + leptin treatment group four mice for two independent experiments. Control group of four mice and NB treatment group of four mice were used for another experimental group. In total, control group eight mice, leptin treatment group eight mice, NB treatment group eight mice and NB + leptin treatment group eight mice were used for the overall experiments. Each set of data was expressed as fold increase over control rodents.
This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organizations engaged with supporting research.
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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: http://dx.doi.org/10.1111/bph.13520 Figure S1 HSP90 knockdown attenuated leptin-induced STAT3 phosphorylation. Western blotting analysis of leptininduced STAT3 activation in the HSP90-knocked down HEK293-Ob-Rb cell line. Cells were stimulated with leptin (0.5 μg/mL) for 15 min and Western blotting was then performed. (A) Expression level of HSP90 was attenuated by HSP90 siRNA. (B) The leptin-induced phosphorylation of STAT3 was inhibited in HSP90-knocked down cells. Typical blot among three independent experiments was shown. Figure S2 A HSP90 inhibitors inhibited leptin-induced activation of STAT3 in neuronal cells. SH-SY5Y-Ob-Rb cells were treated with the HSP90 inhibitor, radicicol (Rad: 10 μM) and novobiocin (NB: 1000 μM), and leptininduced STAT3 phosphorylation was analyzed. *P < 0.05 v.s. control cells treated with leptin. Dunnett’s test. n = 4. © 2016 The British Pharmacological Society.
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