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Endocrinology Vol. 145, No. 9 4103-4112
Copyright © 2004 by The Endocrine Society

Antiapoptotic Effects of Leptin in Human Neuroblastoma Cells

V. C. Russo, S. Metaxas, K. Kobayashi, M. Harris and G. A. Werther

Centre for Hormone Research, Murdoch Childrens Research Institute, Department of Paediatrics University of Melbourne, Royal Children’s Hospital, Parkville 3052, Victoria, Australia

Address all correspondence and requests for reprints to: Vincenzo C. Russo, Ph.D., Centre for Hormone Research, Murdoch Childrens Research Institute, Parkville 3052, Victoria, Australia. E-mail: vince.russo{at}mcri.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Many factors regulate nervous system development, including complex cross-talk between local neuroendocrine systems. The adipocyte-secreted hormone leptin, mainly known for its key roles in nutrition and reproductive balance, may also be involved in neuroanatomical organization, myelination processes, and neuronal/glia maturation. SK-N-SH-SY5Y neuroblastoma cells were employed as an in vitro model of human neuronal cells to determine whether leptin exerts neuroprotective activities. We show that SH-SY5Y cells express leptin, the long and short isoforms of the leptin receptor (ObRl, ObRs). In SH-SY5Y cells, leptin induced signal transducer and activator of transcription (STAT)-3 phosphorylation and suppressor of cytokine signaling-3 mRNA expression. Leptin dose-dependently increased cell number (up to 200% at 1 µM by 48 h, P < 0.01), and at 24–48 h, leptin at 100 nM increased SH-SY5Y cell number by 30–50%, respectively. SH-SY5Y cell viability was reduced in serum-free conditions at 24 h, and addition of leptin at 100 nM significantly reduced apoptosis by approximately 20% (P < 0.001). Leptin’s antiapoptotic activity required Janus kinase/STAT, MAPK, and phosphatidylinositol-3-kinase activation because the antiapoptotic effects of leptin were abolished, and caspase-3 immunoreactivity increased in the presence of the specific blockers AG490, U0126, or LY294002. Gene array demonstrated that leptin inhibits apoptosis via potent down-regulation of caspase-10 and TNF-related apoptosis-inducing ligand. Our data thus demonstrate, for the first time, that leptin stimulates, in a time- and dose-dependent manner, neuroblastoma cell proliferation and that the underlying mechanisms involve suppression of apoptosis via the Janus kinase-STAT, phosphatidylinositol-3 kinase, and MAPK pathways that culminate altogether in the down-regulation of the apoptotic factors caspase-10 and TNF-related apoptosis-inducing ligand.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PROCESSES INVOLVED in early brain development and postnatal maturation of the nervous system are poorly understood. However, a number of growth factors and hormones are known to be involved in the modulation of neurotrophic, neurogenic, and neuroprotective events that take place within the brain (1, 2, 3, 4, 5, 6, 7).

Much interest has recently been devoted to the role of leptin in the brain (8, 9, 10). Leptin is an adipocyte-secreted hormone that regulates weight centrally. Leptin deficiency causes the obese (ob/ob) mouse phenotype, characterized by obesity hyperphagia, hyperglycemia, hyperinsulinemia, insulin resistance, hypothermia, and infertility (11, 12, 13, 14). Although it is mainly known for its key role in nutrition, energy balance and reproductive regulation, leptin appears to also exert its action in a number of peripheral tissues (15, 16, 17, 18) and specialized cells (19, 20, 21, 22), in which it regulates metabolic, hematopoietic, and immune functions, suggesting that leptin action is not restricted to the hypothalamic nuclei.

In the developing central nervous system (CNS), leptin and both the long and the short isoforms of the leptin receptor are widely expressed in neuron-rich and plastic regions such as the hypothalamus, hippocampus, and cerebellum, suggesting that leptin may be involved in neurodevelopmental processes (23, 24, 25, 26, 27, 28, 29, 30). This hypothesis is supported by the observation that the obese mouse (ob/ob) shows a specific brain phenotype including reduced brain size, defective neuroanatomical organization, altered myelination processes, and reduced neuronal/glia maturation, coupled with increased apoptosis (31, 32, 33). Furthermore, studies by Ahima et al. (33) demonstrated that leptin treatment in ob/ob mice normalize the brain phenotype by reversing most of the observed CNS abnormalities. Although these findings strongly suggest a fundamental role for leptin in early development of the nervous system, the mechanisms involved remain unclear.

The Ob-Rb receptor, widely expressed in the human brain, including regions not directly associated with energy homeostasis (28), mediates leptin action by signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway (34, 35, 36, 37) including the downstream suppressor of cytokine signaling (SOCS)-3 feedback inhibitor (8, 38, 39) and/or other common pathways such as the p38 MAPK, insulin receptor substrate (IRS)-1, and phosphatidylinositol 3-kinase (PI3K) (15, 21, 27, 38, 40, 41). A short form of the leptin receptor (ObRs) and its limited signaling activity have also been reported (41, 42), although its biological significance remains unclear.

Whether leptin exerts exclusively metabolic functions or might also have trophic/survival activities is not known, and we therefore aimed to investigate these potential functions in a well-characterized in vitro model of human neuronal cells, the neuroblastoma cell line SK-N-SH-SY5Y (43, 44, 45).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Human leptin was purchased from Sigma Chemical Co. (St. Louis, MO). Leukemia inhibitory factor (LIF) was generously supplied by Prof. Perry Bartlett (Walter and Eliza Hall Institute, Parkville, Victoria, Australia). Recombinant human IGF-I was kindly supplied by Kabi Pharmacia (Kabi Pharmacia Upjohn, Peptide Hormones, Uppsala, Sweden). The random primed DNA labeling kits were from Roche (North Ryde, New South Wales, Australia). Total cellular RNA was extracted from cells using RNAzol (Biotecx Laboratories Inc., Houston, TX). Tissue culture flasks and plates were purchased from Nunc (Glastrup, Denmark). Chemical reagents (analar grade) were purchased from BDH-Merck Pty. Ltd. (Kilsyth, Victoria, Australia). PhosphorImager screens were from Molecular Dynamics (Sunnyvale, CA). X-Omat AR and BioMax films were from Eastman Kodak Co. (Rochester, NY). Intracellular pathway inhibitors AG490 (JAK2 inhibitor), SB203580 (p38 MAPK inhibitor), and U0126 [MAPK kinase (MEK)1/2 inhibitor] were obtained from Calbiochem-Novabiochem Corp. (San Diego, CA), and the MAPK inhibitor (PD98059) and PI3K inhibitor (LY294002) were purchased from Cell Signaling Technology Inc. (Beverly, MA).

Cell culture
The human neuroblastoma cell line, SK-N-SH-SY5Y, generously supplied by Prof. Eva Feldman (Department of Neurology, University of Michigan, Ann Arbor, MI), was used in this study. Cells were cultured in DMEM (Trace Biosciences, Castle Hill, New South Wales, Australia) supplemented with 10% fetal calf serum (CSL Ltd., Parkville, Victoria, Australia), penicillin (100 U/ml), and streptomycin (100 µg/ml) in a humidified atmosphere containing 5% CO2 and 95% air at 37 C.

Treatment
When cells reached the required confluence, they were placed into serum-free medium (SFM) for 24 h. They were then incubated, for up to an additional 72 h or as indicated, in the presence or absence of leptin (100 nM), with IGF-I (3 nM) being used as mitogenic or antiapoptotic control in each experiment as indicated. Leptin concentrations were initially based on those previously published and commonly used for a variety of leptin-responsive cell cultures (19, 21, 22, 46, 47) including hypothalamic neurons (48). Leptin was also used at supraphysiological concentrations of 1 µM; however, the optimal dose of 100 nM (see Results, Fig. 3Go) was used for our experiments. Cells were cultured up to 72 h with media being replaced every 24 h. Cells were then assayed at 24, 48, or 72 h as indicated. Inhibitors were used in some experiments, AG490 at 10 µM, SB203580 at 10 µM, U0126 at 25 µM, PD98059 at 100 µM, and LY294002 at 100 µM. Concentrations of the above pathway inhibitors, were consistent with those previously shown to be effective in SH-SY5Y cells (49, 50).



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  		FIG. 3. Leptin enhances cell number in a dose-dependent manner. SH-SY5Y cells were cultured in serum-free DMEM for 24–48 h in the presence or absence of leptin (0–1 µM) and cell number determined by the colorimetric NBB assay as described in Materials and Methods. The dose of 100 nM was then selected as optimal concentration for subsequent studies.

 
Cell number (NBB assay)
Cells were cultured in 24-well plates and on reaching 70% confluence were put in SFM for 24 h. Cells were then treated as above for 72 h. Cell number was determined by a colorimetric Naphtalene Blue Black (NBB) (Sigma, St. Louis, MO) assay protocol previously described by Janet et al. (51). Assays were performed three or five times and samples were run in six replicates.

Cell number (NBB) assays (inhibitors)
Cells were cultured in 24-well plates and on reaching 70% confluence were put in SFM for 24 h. They were then pretreated for 4 h with SFM containing the various inhibitors in the absence of leptin. Leptin (100 nM) was added to the cells, followed 6 h later by another addition of inhibitors. Cells were treated overnight before being harvested. Cell number was determined by a colorimetric NBB assay protocol previously described by Janet et al. (51). Assays were performed three to five times, as indicated, and samples were run in quadruplicate.

Cell death-DNA fragmentation assay (ELISA)
Cells were cultured in six-well plates and on reaching 90% confluence were placed in SFM for 24 h. They were then incubated in SFM in the presence or absence of leptin (100 nM) for 24 h. Cells were then harvested and treated according to the manufacturer’s instructions for the cell death detection ELISAPLUS kit (Roche). Assays were performed five times with samples being run in triplicate.

Cell death ELISAPLUS assay (inhibitors)
Cells were cultured in six-well plates and on reaching 90% confluence were placed in SFM for 24 h. They were then pretreated for 4 h with SFM containing the various inhibitors in the absence of leptin. Leptin (100 nM) was added to the cells, followed 6 h later by another addition of inhibitors. Cells were treated overnight before being harvested according to the manufacturer’s instructions for the cell death detection ELISAPLUS kit (Roche). Assays were performed three times with samples being run in triplicate.

Caspase-3 activity assay (inhibitors)
Cells were cultured in 96-well plates (20,000 cells/well, white plate, no. 3610, Costar, Cambridge, MA) and on reaching 80% confluence were placed in SFM for 24 h. Cells were then treated in SFM with or without the various inhibitors and in the presence or absence of leptin at 100 nM as described above. Caspase-3 activity was detected in cell lysate by the Apo-ONE kit (no. G7790, Promega, Madison, WI). Assay conditions were as per manufacturer specification. Fluorometric activity (cleavage of Z-DEVD-R110 substrate, expressed in relative fluorescent units) was measured by fluorometer (FLUOSTAR-OPTIMA, BMG Labtechnologies GmbH, Offenburg, Germany) using the excitation at 485 nm and emission at 520 nm. Assays were performed two times with samples being run in triplicate.

Preparation of RNA for RT-PCR, Northern analysis, and GEArray
Cells were plated out in T80-cm2 flasks and on reaching 70% confluence were put in SFM for 24 h. The cells were then treated with SFM in the absence and presence of leptin (100 nM) and IGF-I (3 nM), here used as potent antiapoptotic agent, for a further 48 h with media being replaced after 24 h. Cells were harvested by trypsin, and total RNA was isolated using RNAzol B (Geneworks, Adelaide, Australia) for the GEArray or RNeasy minikit (Qiagen, Santa Clarita, CA) for RT-PCR.

RT-PCR and DNA sequencing
cDNA was synthesized from 1–2 µg total RNA using oligo(dT) primer and murine leukemia virus (MuLV)-reverse transcription (RT) (Roche, PerkinElmer, Norwalk, CT) and first-stand cDNA synthesis kit (PerkinElmer), followed by PCR using the Taq polymerase. Omission of total RNA (water control) or MuLV-RT (genomic DNA control) in the RT reaction was used to assess specific amplification for each gene (not shown).

Primers used for leptin (fragment of 260 bp) were 5'-CGTCAGTCTCCTCCAAACA-3' (forward) and 5'-CAGGCTGTCCAAGGTCTC-3' (reverse) (52). Primers used for the long form of the leptin receptor (ObRl, fragment of 168 bp) were 5'-TTTCACCACACCTCACATTCTC-3' (forward) and 3'-GGCACGATACCC-TTGACTTG-5' (reverse) (52). Primers used for the ObRs (fragment of 189 bp) were 5'-TAAAAGGAAGCCCGAAGTTGT-3' (forward) and 3'-CAGAGGTCTTGGATGGTTCAGT-5' (reverse) (52), PCR conditions for leptin, the long leptin receptor isoform (ObRl), and ObRs were as previously described (52).

Primers used for SOCS3 (fragment of 224 bp) were 5'-GTCACCCACAGCAAGTTT-3' (forward) and 3'-CTGAGCGTGAAGAAGTGG-5' (reverse) (53). PCR conditions for SOCS-3 were as previously described (53). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH, fragment of 404 bp) was amplified as a control for RNA loading using the following primers: 5'-CCATGGCACCGTCAAGGCTGA-3' (forward) and 3'-GGGCCATCCACAGTCT-TCTGG-5' (reverse) (53).

To confirm the identity of the amplified genes, PCR products were then sequenced (DNA-ABI Big Dye Terminator kit, Applied Biosystems, Foster City, CA) and BLAST analysis performed (data not shown).

Northern blotting analysis
Cells were cultured in T80-cm2 flasks and treated as above in the presence or absence of leptin. Total RNA was extracted as described above. Fractionated RNA was transferred to Zeta Probe membrane and hybridized at 60 C with {alpha}32P-dCTP random primed-labeled SOCS3, leptin, ObRl, and ObRs probes. To independently confirm the expression of SOCS3, ObRl, and ObRs by Northern hybridization, a new set of probes was generated. For the detection of SOCS3, we use 681 bp mouse-SOCS3 cDNA fragment, kindly provided by Dr. Tim E. Adams (CSIRO Health Science and Nutrition, Parkville, Australia) (54). The human ObRl probe was obtained by RT-PCR (5'-TTGTGCCAGTAAT-TATTTCCTCTT-3', forward; 5'-CTGATCAGCGTGGCGTATTT-3') as recently described by Koshiba et al. (55), who reported an ObRl mRNA of about 4.5–5 kb. The ObRs probe was generated by RT-PCR (5'-AAAAGGAAGCCC-GAAGTTGT-3', forward; 5'-TGAGCAACGCAGCATAATTC-3'), this probe extends the 3' end of the original ObRs’ PCR product shown in Fig. 1Go. Filters were then stripped before reprobing with an in-house 18S probe (56) as above. Experiments were performed two times; samples were run in duplicate.



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  		FIG. 1. SH-SY5Y cells: an in vitro model to study leptin action. SH-SY5Y cells were cultured in serum-free DMEM for 24 h in the presence or absence of leptin (100 nM) and total RNA extracted as described in Materials and Methods. RT-PCR (A) was used to determine expression of leptin, ObRl, ObRs, and SOCS-3 mRNA, and Northern analysis (B) was performed to confirm gene expression. Data shown are representative of three (RT-PCR) or two (Northern analysis) individual experiments each in duplicate.

 
STAT-3 phosphorylation
SH-SY5Y cells were grown as above (six-well plates, approximately 80% confluency); serum starved overnight to deplete basal STAT-3 phosphorylation; and then induced with leptin (100 mM) or LIF (1 ng/ml), potent inducer of STAT3 and here used as positive control (reference for LIF), for 15 min. Leptin and LIF, both trigger activation of STAT3 protein. After the cytokine pulse, cells were washed with ice-cold PBS, scraped, centrifuged, and cell pellet extracted with radioimmunoprecipitation assay buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, 1% Triton X-100, and 1 mM sodium orthovanadate] containing complete protease inhibitor cocktails (Roche, Mannheim, Germany). Protein concentration, in cellular extract, was measured with BCA protein assay reagent (Pierce, Rockford, IL). Seventy micrograms of total protein were then fractionated onto 12% SDS-PAGE under reducing conditions and then transferred to nitrocellulose membranes (Protran, Dassel, Germany). STAT-3 phosphorylation was detected by the Phospho-Plus Stat3 (Try705) antibody kit (Cell Signaling) according to the manufacturer’s specifications. Phospho-STAT-3 bands were then analyzed by densitometry as above. Experiments were performed twice with samples run in triplicates

GEArray (human apoptosis genes filter-array)
This method (56, 57) was used to determine whether apoptosis-related genes are induced or suppressed in response to the experimental conditions. Twenty-three apoptosis-related genes (hGEA990510, Super Array, Inc., Bethesda, MD), including genes in the signaling of the Bcl-2 gene family, Fas, TNF-related apoptosis-inducing ligand (TRAIL), p53, and nuclear factor-kB, and two housekeeping genes, ß-actin and GAPDH, were thus simultaneously analyzed.

SH-SY5Y cells were cultured as above for 24 h, and total RNA, extracted as described earlier, was used as a template for RT using the MuLV-RT (GeneAmp, RNA PCR core kit, PerkinElmer, Roche). 32PdCTP-cDNA probes were generated by MuLV-RT using specific primers (hGEA990510, Super Array) for each of the 23 apoptosis-related genes and control genes as per manufacturer specification (Super Array). Filters were then exposed to PhosphorImager screen and data analyzed by ImageQuant software as above. Signal intensity of each gene was normalized against the housekeeping gene GAPDH control levels.

Of the 23 genes analyzed for each treatment, only those that showed statistical significant increase or decrease in gene expression was further analyzed. Data shown (see Fig. 9Go, top panels) are representative of four experiments, whereas densitometric analysis in Fig. 9Go (lower bar graph) is the combined data from four independent experiments (4 x 32PcDNAs, four sets of filters with each gene spotted in duplicate).



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  		FIG. 9. Leptin inhibits apoptosis via down-regulation of caspase-10 and TRAIL. SH-SY5Y cells were cultured in serum-free DMEM for 24 h in the presence or absence of leptin (100 nM). IGF-I (3 nM) was used as a potent antiapoptotic agent. Low-density gene array was performed as described in Materials and Methods. TRAIL and caspase-10 (top) were strongly expressed after growth factor withdrawal (SF, top left). Addition of leptin (top middle) potently down-regulated both genes. This potent effect of leptin was comparable with that seen in the IGF-I-treated cells (top right). Caspase-3 expression as seen in SF was not affected by leptin or IGF-I (bottom right panel). Data shown in the top panel (dot-blot array) are representative of four experiments. Densitometric analysis shown in the bottom panel (bar graph) is the combined data of four experiments.

 
Statistical analysis
GraphPad PRISM (Graph Pad Inc., San Diego, CA) was used to perform one way ANOVA and Bonferroni post hoc analysis on all data. Experiments were performed at least three times, or as indicated, with samples run in duplicate-quadruplicate for each indicated time and data plotted as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
An in vitro model to study leptin action in human neuronal type cells
The human neuroblastoma cell line SK-N-SH-SY5Y is widely used as an in vitro model system for human neuronal cells. This is based on its well-characterized neuronal phenotype, demonstrated morphologically (58, 59), via differentiation and expression of a wide variety of neuron-specific genes (60). We therefore aimed to establish whether SH-SY5Y cells were a suitable in vitro model to study leptin action in neuronal cells by determining the presence of a leptin system, including leptin intracellular signaling.

The expression of mRNA for the ObRl and ObRs of the leptin receptor was verified by RT-PCR in both SFM- and leptin-treated cells as shown in Fig. 1AGo. Leptin administration (100 nM) induced STAT3 phosphorylation (Fig. 2Go) and up-regulation of SOCS-3 mRNA (Fig. 1AGo), thus suggesting that in SH-SY5Y cells, leptin action is mediated via the ObRl isoform, activating the JAK-2/STAT-3 and SOCS-3 cascade. The expression of mRNA for leptin was also determined by RT-PCR (Fig. 1AGo). The expression of these genes was validated by Northern analysis (Fig. 1BGo) as described in Materials and Methods, and the identity of the amplified genes/PCR products was confirmed by DNA sequencing (data not shown). Despite some limitations in the interpretations that can be made using a stable cell line as opposed to primary neurons, the neuronal characteristics of the SH-SY5Y cells mentioned above and our findings (Figs. 1Go and 2Go) suggested that the SH-SY5Y cell line was an appropriate model system to study leptin action in neuronal cells.



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  		FIG. 2. Leptin induces STAT-3 phosphorylation in SH-SY5Y cells. SH-SY5Y cells were cultured in serum-free DMEM for 16 h followed by exposure (15 min) to leptin (100 nM). LIF (1 ng/ml) is a potent inducer of STAT-3 and here used as positive control. Cellular extract (70 µg) was fractionated onto 12% SDS-PAGE and Western immunoblotting performed with anti-phosphoSTAT-3Tyr705 (top panel) as described in Materials and Methods. Experiments were performed twice with samples run in triplicate. Densitometry is shown in the lower panel.

 
Leptin overcomes the effects of serum deprivation by increasing cell number via inhibition of apoptosis
Addition of leptin doses of 0.1–1 µM markedly increased cell number at 24 h by 30–70%, respectively, when compared with the cell number in serum-free cultures (P < 0.01, Fig. 3Go, left panel). The cell number was further increased up to 200% with 1 µM and approximately 80% with 100 nM by 48 h (P < 0.01, Fig. 3Go, right panel) over that of the serum-free cultures. The dose of 100 nM was then selected as optimal concentration for subsequent studies, consistent with published data (19, 21, 22, 46, 47).

As shown in Fig. 4Go, after exposure to SFM, SH-SY5Y cell number was decreased over the 72 h, and addition of leptin at 100 nM markedly increased cell number (~30% at 24 h, P < 0.05), with a maximal effect observed at 48–72 h (P < 0.01, Fig. 4Go, leptin). Similar to leptin, IGF-I (3 nM), here used as potent positive mitogenic control, also increased cell number; however, the magnitude of response was more dramatic (200–400%, P < 0.001, Fig. 4Go, IGF-I). SH-SY5Y cell viability was reduced in serum-free conditions with apoptotic DNA fragmentation readily detectable at 24 h (Fig. 5Go, SF). Addition of leptin (100 nM) significantly reduced apoptosis (~25% at 24 h, P < 0.001, Fig. 5Go, leptin) with IGF-I, also in this case, showing a more potent antiapoptotic effect (~75%, P < 0.001, Fig. 5Go, IGF-I).



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  		FIG. 4. Leptin overcomes the effects of serum deprivation by enhancement of cell number. SH-SY5Y cells were cultured in serum-free DMEM for 24–72 h in the presence or absence of leptin (100 nM). SH-SY5Y cells number was decreased over the 72 h, and addition of leptin at 100 nM markedly increased cell number with a maximal effect observed at 48–72 h (~40%, P < 0.01, leptin). IGF-I (3 nM), here used as a potent positive mitogenic control, increased cell number (200–400%, P < 0,001, IGF-I). Cell number determined by the colorimetric NBB assay as described in Materials and Methods.

 


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  		FIG. 5. Leptin inhibits apoptosis. SH-SY5Y cells were cultured in serum-free DMEM for 24 h in the presence or absence of leptin (100 nM). SH-SY5Y cell viability was reduced in serum-free conditions with apoptotic DNA fragmentation readily detectable at 24 h (SF). Addition of leptin (100 nM) significantly reduced apoptosis (~20%, P < 0.001, leptin). IGF-I (3 nM), here used as a potent antiapoptotic agent (~80% reduction, P < 0.001, Fig. 5Go, IGF-I).

 
Transduction of leptin enhancement of cell number requires activation of the PI3K pathway
As indicated above, leptin action is mediated by the ObRb, which is a member of the class I cytokine receptor superfamily, which signals via the JAKs (61). Another intracellular pathway recruited by leptin receptors is the MAPK cascade (38, 39). Several other pathways are triggered by leptin including IRS protein and the downstream PI3K (27). To determine which intracellular signaling pathways mediate leptin enhancement of cell number, specific signaling pathway biochemical blockers, as described in Materials and Methods, were employed.

Leptin alone (Fig. 6LGo), as shown in Figs. 3Go and 4Go, increased cell number (30% at 24 h). When leptin was administrated in the presence of the JAK/STAT inhibitor AG490 (Fig. 6Go, AG+L), the p38MAPK inhibitor SB203580 (Fig. 6Go, SB+L), the MEK inhibitor PD98059 (Fig. 6Go, PD+L), or the MAPK inhibitor U0126 (Fig. 6Go, U+L), cell number was not affected, with cell number significantly higher (P < 0.05–0.001) in the leptin-treated cells than in their paired serum-free controls (Fig. 6Go, AG, SB, PD, U, respectively). However, when leptin was administered in the presence of the PI3K inhibitor LY294002 (Fig. 6Go, LY+L), leptin-induced cell number enhancement was blunted.



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  		FIG. 6. Transduction of leptin-induced enhancement of cell number requires activation of the PI3K pathway. SH-SY5Y cells were cultured in serum-free DMEM for 24 h with or without leptin (100 nM), which was administered in the presence or absence of the JAK/STAT inhibitor AG490 (AG+L), p38MAPK inhibitor SB203580 (SB+L), MEK inhibitor PD98059 (PD+L), MAPK inhibitor U0126 (U+L), or the PI3K inhibitor LY294002 (LY+L) as described in Materials and Methods. Cell number was determined by the colorimetric NBB assay as described in Materials and Methods.

 
Leptin antiapoptotic response requires JAK/STAT, MAPK, and PI3K activation
Because inhibition of apoptosis appeared to be a major mechanism accounting for the increase in cell number, we wished to further elucidate signaling pathways specifically involved in leptin’s antiapoptotic effects in these cells. Specific signaling pathway biochemical blockers, as described in Materials and Methods, were thus employed using cell death as an end point. Leptin alone (100 nM) significantly reduced serum-free induced apoptosis (~20%, P < 0.05, Fig. 7LGo). When leptin was administered in the presence of the JAK/STAT inhibitor AG490 (Fig. 7Go, AG+L), MAPK inhibitor U0126 (Fig. 7Go, U+L), or PI3K inhibitor LY294002 (Fig. 7Go, LY+L), the antiapoptotic effects of leptin were abolished. However, when leptin was administrated in the presence the p38MAPK inhibitor SB203580 (Fig. 7Go, SB+L) or the MEK inhibitor PD98059 (Fig. 7Go, PD+L), the antiapoptotic activity of leptin was maintained and apoptosis was significantly lower (P < 0.05) than that determined in their paired serum-free controls (Fig. 7Go, SB and PD, respectively).



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  		FIG. 7. Leptin’s antiapoptotic response requires JAK/STAT, MAPK, and PI3K activation. SH-SY5Y cells were cultured in serum-free DMEM for 24 h with or without leptin (100 nM), which was administered in the presence or absence of the JAK/STAT inhibitor AG490 (AG+L), p38MAPK inhibitor SB203580 (SB+L), MEK inhibitor PD98059 (PD+L), MAPK inhibitor U0126 (U+L), or PI3K inhibitor LY294002 (LY+L) as described in Materials and Methods. Apoptotic DNA fragmentation was determined as described in Materials and Methods. Leptin alone reduced apoptosis (~20%, P < 0.05, L), whereas in the presence of AG490, U0126, or LY294002, the antiapoptotic effects of leptin were abolished.

 
Similarly, the level of caspase-3 activity (Fig. 8Go) seen in serum-free-treated cells (SF) was reduced by approximately 20% after leptin addition (leptin = P < 0.05). This effect was blunted in the presence of all the above pathway inhibitors (Fig. 8Go, leptin+AG, U, LY, P < 0.001). As expected the PI3K inhibitor blunted the antiapoptotic activity of IGF-I (Fig. 8Go, IGF-I = P < 0.001; I+LY = P < 0.01).



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  		FIG. 8. Leptin’s antiapoptotic response involves inhibition of caspase-3 activity. SH-SY5Y cells were cultured in serum-free DMEM for 24 h with or without leptin (100 nM), which was administrated in the presence or absence of the JAK/STAT inhibitor AG490 (AG+L), p38MAPK inhibitor SB203580 (SB+L), MEK inhibitor PD98059 (PD+L), MAPK inhibitor U0126 (U+L), or PI3K inhibitor LY294002 (LY+L) as described in Materials and Methods. IGF-I (3 nM) was used as a potent antiapoptotic agent. Caspase-3 activity was reduced after leptin addition (leptin, P < 0.05 to SF), and this effect was blunted by addition of the above pathway inhibitors (AG, U, LY, ***, P < 0.001 to leptin alone). The antiapoptotic activity of IGF-I was blunted by LY294002 (IGF-I, P < 0.001 to SF, I+LY; **, P < 0.01 to IGF-I alone). RFLU, Relative fluorescent unit.

 
Leptin inhibits apoptosis via down-regulation of caspase-10 and TRAIL
To determine which elements of the apoptotic cascade were modulated by leptin, low-density gene array was employed. Transcription levels of 23 key genes in the intrinsic and extrinsic apoptotic signaling pathways were determined. Two major transcripts for caspase-10 and TRAIL (Fig. 9Go, top) were strongly expressed in serum-free conditions (Fig. 9Go, SF, top left). Leptin (Fig. 9Go, top middle) strongly down-regulated both genes. This potent effect of leptin was comparable with that seen in the IGF-I-treated cells (Fig. 9Go, top right). The levels of caspase-3 expression seen in SFM (Fig. 9Go, SF, top left) were unaltered by leptin (Fig. 9Go, top middle) or IGF-I (Fig. 9Go, top right). Densitometric analysis of these data is also shown (Fig. 9Go, bottom panels, ns = not significant).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We employed the well-characterized human neuroblastoma cell line SK-N-SH-SY5Y as an in vitro model system for neuronal cells to examine the potential trophic and antiapoptotic activity of leptin. Our data show that, in neuroblastoma cells, leptin overcomes the effects of growth factor withdrawal by enhancing cell number, involving inhibition of apoptosis. Our studies further aimed to investigate which intracellular signaling pathways are activated by leptin to prevent cell loss (see model, Fig. 10Go). PI3K appeared to be a key element in the leptin-induced enhancement of cell number, a process potentially involving both enhanced cell proliferation and reduced apoptosis. Our focus was on the potential neuroprotective role of leptin, confirmed by our finding of inhibition of apoptosis, involving multiple signaling pathways in addition to PI3K, namely JAK/STAT and MAPK. We have also shown here that leptin’s antiapoptotic activity is at least in part mediated by down-regulation of two key genes of the death cascade, caspase-10 and TRAIL.



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  		FIG. 10. Leptin’s actions in neuroblastoma cells. In neuroblastoma cells, leptin overcomes the effects of growth factor withdrawal by enhancing cell number and inhibiting apoptosis. PI3K appeared to be a key element in the leptin-induced cell survival (cell number enhancement and apoptosis) with JAK/STAT, MAPK mainly involved in the leptin-induced antiapoptotic response. The antiapoptotic response of leptin in neuroblastoma cells involves down-regulation of two key genes of the death cascade (caspase-10 and TRAIL) and reduction of caspase-3 abundance.

 
Leptin (Ob protein), a protein hormone secreted by fat cells, promotes loss of body weight by acting in the brain to decrease food intake and increase sympathetic nervous system activity (8, 9, 10). In addition to body weight homeostasis, leptin is involved in various physiological events including reproduction (62), hematopoiesis (63), angiogenesis (64), wound healing (65), and insulin secretion (66).

Leptin exerts mitogenic activities in various cell types (19, 40, 67), whereas it appears to exert a dual role in apoptosis with both proapoptotic (68, 69) and antiapoptotic (22, 40, 70) activities being reported. This may represent differences in tissue or cell-specific functions. Our studies are, however, the first demonstration of such activities in neuronal cells.

Leptin action is mainly mediated by the ObRl (27). ObRs, lacking most of the intracellular domain, has also been described (27). Both ObR isoforms are expressed in the CNS (28, 42, 71, 72, 73), and we have here demonstrated their expression in SH-SY5Y cells.

The ObRl signals via a JAK2/STAT3 mechanism activating STAT-dependent transcription (i.e. SOCS3) (27). In addition, the ObRl can mediate leptin-dependent tyrosine phosphorylation of JAK2 and IRS-1 and activation of the downstream MAPK and PI3K (15, 21, 27, 34, 35, 36, 40, 73). After leptin stimulation SH-SY5Y cells showed increased phospho-STAT3 levels and increased SOCS-3 mRNA abundance, thus suggesting that the JAK/STAT pathway is functional in these cells.

The role of ObRs, which has signaling capabilities that do not involve a classic JAK/STAT transduction pathway, remains unclear. However, as for the long isoform, the ObRs is able to activate JAK2 and IRS-1, recruiting a number of downstream effectors including MAPK and the PI3K (73).

The antiapoptotic effects of leptin in SH-SY5Y cells, as demonstrated in other cell types (22, 40, 74, 75), appear to require JAK/STAT activation as well as activation of the MAPK and PI3K pathways, suggesting that, as recently demonstrated in other systems (76, 77, 78, 79), both regulation of gene transcription and pathway modulation (i.e. phosphorylation cascade), including caspase-3 modulation, are required for these responses. The effects of leptin on gene transcription were clearly shown by the dramatic down-regulation of the expression of the potent inducer of apoptosis, TRAIL, and its downstream effector, caspase-10 (extrinsic apoptotic pathway). The role of caspase-10 in apoptosis signaling by death receptor has been controversial; however, there is now clear evidence that caspase-10 is cleaved during TRAIL-induced apoptosis (80, 81).

The proapoptotic activity of TRAIL in malignant neuroectodermic cells is well documented (78, 82, 83, 84). More recently, several studies have further characterized the involvement of TRAIL in apoptosis in neuronal-type cells undergoing differentiation (56, 85) or degenerative process (86).

Very little is known about the potential neuroprotective role of leptin in both in vitro and in vivo systems. However, a number of in vivo studies in the genetically obese mouse (ob/ob) by van der Kroon et al., Bereiter et al. (31, 32), and more recently by Ahima et al. (33) show altered neuroanatomical organization in the CNS of the ob/ob mouse. These regional alterations include reduced brain weight and cortical brain volume, significantly lower amount of myelin, reduced DNA content, and overall delayed maturation of brain cells including neurons and glia (31, 32, 33). The findings of Ahima et al. (33) that treatment of these mice with leptin normalizes the brain phenotype strongly suggest a potent role in vivo for leptin in brain maturation, very likely involving inhibition of apoptosis.

Nevertheless, it is also possible that the brain phenotype as described above in the ob/ob mice (31, 32, 33) may be indirectly related to leptin deficiency as a result of other hormonal perturbations. Leptin deficiency or insensitivity is associated with a number of neuroendocrine abnormalities, which could potentially interfere with development of the immature CNS (31, 32, 33). It was precisely for this reason that we chose to study the effects of leptin using an in vitro model system. By doing so, we could assess any potential direct effect of leptin in isolation from the neuroendocrine adaptations that take place with in vivo studies.

Our data thus demonstrate the antiapoptotic activity of leptin in an in vitro model for neuronal cells. These findings might suggest that in vivo leptin, whether locally (autoparacrine) or peripherally (endocrine) derived, might also be involved in suppression of neuronal apoptosis and potentially participate in neurodevelopmental and neuroprotective processes.


   Acknowledgments
 
We thank Dr. Susie Ymer (Murdoch Childrens Research Institute, Royal Children’s Hospital) for her help with DNA sequencing of the PCR products for ObRl, ObRs, and SOCS-3. We also thank Dr. Timothy Adams (Commonwealth Scientific and Industrial Research Organization, Australia) for his generosity in providing the murine pEF-FLAG-I/mSOCS-3 construct.


   Footnotes
 
This work was supported by Grants 149219 and 209067 from the National Health and Medical Research Council of Australia.

These studies were presented in part at annual meeting of The Endocrine Society of Australia, September 2003, and the 2nd GH/IGF Symposium, April 2004.

Abbreviations: CNS, Central nervous system; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IRS, insulin receptor substrate; JAK, Janus kinase; LIF, leukemia inhibitory factor; MEK, MAPK kinase; MuLV, murine leukemia virus; ObRl, long form of the leptin receptor; ObRs, short form of the leptin receptor; PI3K, phosphatidylinositol 3- kinase; RT, reverse transcription; SFM, serum-free medium; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TRAIL, TNF-related apoptosis-inducing ligand.

Received December 31, 2003.

Accepted for publication May 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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