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Biomedical Research Centre (O.O., G.M., I.L.P.B.), School of Medicine, Health Policy, and Practice, University of East Anglia, Norwich NR4 7TJ, United Kingdom; and Department of Gastroenterology (I.L.P.B.), Norfolk and Norwich University Hospital, Norwich NR4 7UZ, United Kingdom
Address all correspondence and requests for reprints to: Dr. Ian Beales, School of Medicine, Health Policy, and Practice, University of East Anglia, Norwich NR4 7TJ, United Kingdom. E-mail: i.beales{at}uea.ac.uk.
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Introduction |
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Leptin exerts its effects through interaction with the specific cell membrane localized receptor. There appear to be at least six different receptor variants (Ob-Ra through Ob-Rf), all generated by differential splicing of the db leptin receptor gene product. All receptors share the same extracellular ligand binding domain but differ in the intracellular portion and presumably in the ability to transduce signals. The full-length form (Ob-Rb) is believed to be predominantly responsible for active signal transduction (7), although activation of the MAPK cascade by the Ob-Ra short receptor form has been described (8, 9). The Ob-Rb isoform is extensively expressed in the hypothalamus, in which it mediates the anorectic effects of leptin, but in keeping with the pleiotropic effects of leptin, leptin receptors are widely expressed in peripheral tissues. Typically expression of the Ob-Ra predominates over the other shorter forms in peripheral tissues. The specific actions, if any, of all the isoforms remain unclear.
Recent studies have shown that leptin is a growth factor for a variety of cell types. Leptin stimulated proliferation of hemopoietic cells and breast, colon, gastric, pancreatic ß-, kidney, and lung cell lines in culture (5, 6, 10, 11, 12, 13, 14, 15), and exogenous leptin stimulated colonic proliferation in vivo (10). However, leptin has been reported to reduce the proliferation of the pancreatic PANC-1 and Mia-PaCa cell lines and T24 bladder cancer cells (11, 16).
Esophageal adenocarcinoma (EAC) is increasing in incidence rapidly in the developed world; in contrast, the incidence of squamous cell esophageal cancer remains stable. In 2004 there were more than 14,000 cases of EAC in the United States and more than 13,000 deaths (17). The incidence of EAC has increased by more than 6-fold in the United States over the last 30 yr, and even greater increases have been reported in the United Kingdom (17). The etiology of EAC remains unclear, but the two most important risk factors are obesity and gastroesophageal reflux. The increasing incidence of EAC parallels that of obesity, and detailed case-control studies have confirmed that obesity is a potent risk factor for EAC (18, 19). There is a dose-dependent relationship between body mass index (BMI) and risk of EAC: the odds ratio of developing EAC for those in the upper quartile for body weight is 7.6, compared with those in the lowest quartile and the odds ratio for the obese (BMI > 30 kg/m2) is 16.2, compared with the leanest (BMI < 22 kg/m2) (18). In contrast, there is no relationship between esophageal squamous cancer and body mass. Initial suggestions that the relationship between obesity and EAC was secondary to increased acid reflux in obesity have not been supported by evidence: obesity and acid reflux appear to be independent (18). Whereas there are undoubtedly many possible explanations for this association, it is notable that link between EAC and obesity is stronger than most other cancers, and it is possible that the increased circulating levels of leptin in obesity directly contribute to progression of EAC.
Most cases of OAC develop from metaplastic esophageal columnar epithelium (Barrett’s esophagus). The factors determining progression are not well understood, but compared with normal esophageal epithelium, the metaplastic glandular epithelium has a higher basal proliferative rate and reduced apoptotic index (19). It is believed that these changes are important in the progression to cancer, although the specific factors driving these remain to be determined, the increased proliferation and reduced apoptosis promoting the accumulation and persistence of genetic abnormalities. EAC has not typically been thought of as an endocrine-related cancer, in the manner of breast or prostate cancer, but recent studies have shown that endocrine influences might be important. For example, cholecystokinin2 receptors are expressed by premalignant and malignant esophageal epithelial cells, and gastrin is a growth factor for Barrett’s esophagus and EAC (20). Therefore, we hypothesized that leptin promotes the development of EAC by acting as a growth factor and/or inhibiting apoptosis. We examined the effects of leptin on the OE33 Barrett’s adenocarcinoma cell line. This has been used successfully as an in vitro model for Barrett’s esophagus, results being comparable with human biopsy explants in short-term culture and preliminary data from noncancerous cell lines (21, 22).
We examined the receptor expression, effects on proliferation and apoptosis and early signal transduction events stimulated by leptin in OE33 cells. Previous studies have shown that a wide variety of signaling pathways may be involved in mediating the effects of leptin (7, 23). However, the specific sequence or interactions between these pathways remains to be clarified. Increased activation of the ERK, p38 MAPK, and c-Jun NH2-terminal kinase (JNK) and protein kinase C (PKC) pathways has been described in Barrett’s esophagus, and these may regulate proliferation and apoptosis in EAC and Barrett’s esophagus (19). There is also increased expression of cyclooxygenase (COX)-2 with progression of EAC (24) and overexpression and activation of the epidermal growth factor receptor (EGFR) has been documented (25). Again there is no consensus on the interactions and significance of these signaling pathways in EAC. Therefore, we examined the role of these pathways specifically in the effects of leptin in EAC.
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Materials and Methods |
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Cell culture and chemicals
The OE33 human Barrett’s-derived esophageal adenocarcinoma cell line was obtained from the European Collection of Cell Cultures (Salisbury, UK) and cultured in DMEM containing 4500 mg/liter glucose, 100 mg/liter penicillin, 100 mg/liter streptomycin, and 2 mM [SCAP]L-glutamine and supplemented with 10% fetal bovine serum (FBS) as previously described (20). All cell culture media and supplements were from Invitrogen (Paisely, UK).
RT-PCR
Total RNA was extracted from cells using a one-step RNA extraction kit (QIAGEN, Crawley, UK) following the manufacturer’s instructions; 1.568 µg RNA was reverse transcribed using previously published custom primers (10, 27). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-actin primers were gifts from Dr. John Winpenny (University of East Anglia, Norwich, UK); prostaglandin receptor and leptin receptor primers were obtained from Invitrogen. The sequences of all primers and expected product sizes are shown in Table 1
. Reverse transcriptase comprised incubation at 50 C for 30 min and 95 C for 15 min. PCR was performed for 40 cycles comprising a 45-sec denaturation step at 94 C, a 45-sec annealing step at 58 C, and a 60-sec extension step at 72 C followed by 10 min at 72 C. Electrophoresis on a 0.8% agarose gel stained with ethidium bromide was used to separate PCR products. PCR products were then extracted from the gel using the QIAGEN gel extraction kit, and confirmatory sequencing was performed by the John Innes Centre Genome Laboratory (Norwich, UK).
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Immunocytochemistry
Cells were seeded onto coverslips in 10% FBS-containing medium. Coverslips were air dried at room temperature before washing in PBS once. Cells were then fixed in 4% formaldehyde for 10 min at room temperature and washed again once in PBS. Fixed cells were permeabilized with 0.2% Triton X-100 for 5 min at room temperature followed by three washing steps at room temperature for 5 min each in PBS. Cells were incubated in 2% rabbit serum for 45 min, washed once in PBS for 5 min and then incubated in 1:200 M-18 antileptin receptor antibody in 1% BSA in PBS overnight at 4 C. After further washing in PBS, cells were incubated with 1:400 donkey antigoat fluorescein isothiocyanate (FITC) conjugated secondary antibody (Santa Cruz) for 30 min at room temperature and then washed for 5 min in PBS at room temperature and in low-lighting conditions three times, mounted on slides, and visualized with a Axioplan 2 charge-coupled device upright fluorescent microscope equipped with AxioVision 4.3 digital imaging software (Zeiss, Göttingen, Germany). Images were acquired at x63 magnification.
MTT assay
Cells were seeded at 5 x 104 cells/well in 48-well plates in 10% FBS-containing medium for 24 h and subsequently serum starved for 24 h. Cells were treated with inhibitors and leptin and relative cell numbers after 24 h determined using the MTT colorimetric assay as previously described (28).
Bromodeoxyuridine (BrdU) incorporation assay
A total of 2 x 103 cells/well were cultured in 96-well plates in 10% FBS-containing medium until 60–70% confluent and subsequently serum starved for 24 h. Cells were then treated with 10 nM leptin and 1 µM prostaglandin E2 (PGE2) and incubated for a further 24 h. DNA synthesis and relative cell proliferation were assessed using the BrdU incorporation assay (Roche, Mannheim, Germany). Briefly, cells were incubated with BrdU labeling solution for 2 h at 37 C. After removal of the labeling solution, cells were fixed and denatured and incubated for 90 min with anti-BrdU antibody conjugate, which was subsequently removed by rinsing three times. Finally, cells were incubated in substrate solution at room temperature and proliferation assessed by colorimetric detection.
Assessment of apoptosis
Measurement of intracellular nucleosomes was used for the quantification of apoptosis as described (29, 30). Serum-starved OE33 cells were treated with inhibitors, leptin, or PGE2 for 24 h, and cells were lysed and nucleosomes quantified using the cell death ELISA kit (Roche) according to the manufacturer’s instructions.
COX-2 mRNA assay
A total of 1 x 106 cells/well were seeded into 12-well plates and cultured in complete culture medium for 48 h. After 24 h further culture in serum-free medium, cells were treated with inhibitors and stimulants. Four hours after stimulation, the media were aspirated and cells lysed with cell lysis reagent (R&D Systems). COX-2 and GAPDH mRNA levels in the cell lysates were measured as described previously using Quantikine mRNA ELISA (R&D Systems) according to the manufacturer’s instructions (31).
PGE2 release
Cells were seeded at 1 x 105 cells/well in 24-well plates and cultured in complete medium for 72 h. To test for induction of COX-2 activity, cells were exposed to kinase inhibitors and 10 nM leptin or IL-1ß (10 ng/ml) as a positive control for 18 h before assessment of PGE-2 secretion as previously described using a specific PGE2 ELISA (R&D Systems) (30).
Detection of activated phosphorylated Akt, EGFR, and MAPKs
A total of 104 cells/well were subcultured in 96-well plates for 72 h and subsequently serum starved for 24 h. Cells were then treated with inhibitors for 60 min followed by leptin or PGE2. After exposure to leptin or PGE2, cells were fixed with 4% formaldehyde. Nonphosphorylated total and active phosphorylated Akt, EGFR (tyrosine-1173 phosphorylated), ERK, JNK, and p38 MAPK were quantified using the FACE ELISA kits (Active Motif, Rixensart, Belgium) as described previously (26, 32).
Detection of activated JAK2
Total and tyrosine-phosphorylated JAK2 were quantified in stimulated and formalin-fixed cells using a specific cell-based ELISA as previously described (26).
Statistical analysis
Proliferation studies were done in triplicate or quadruplicate wells. COX-2 mRNA and bioactivity studies were performed in duplicate wells with each well assayed in duplicate. Experiments for direct detection of other signaling intermediates were performed in triplicate wells. The mean of all duplicates from one experiment were regarded as n = 1. Each experiment was repeated three to eight times. Results are expressed as mean ± SEM, and effects were compared with untreated control cells on the same plate. One-way ANOVA was used for dose-response curve and paired t tests were used to analyze the effect of inhibitors. P < 0.05 was considered significant.
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Results |
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). Correct identification of the sequences was confirmed by sequencing. The mRNA band for the long isoform was less prominent than the bands for the short and common variants. Expression of the leptin receptor protein in OE33 cells was confirmed with Western blotting and immunofluorescence microscopy. Three proteins bands of 120, 100, and 60 kDa were consistently detected in OE33 cells, the largest two corresponding to the Ob-Rb and Ob-Ra receptors, respectively (Fig. 1B). Ob-Ra expression was relatively greater than Ob-Rb. Similar results were obtained with both the H-300 antibody, which binds to an internal domain (residues 541–840) of the full-length leptin receptor and M-18 antibody, which is directed against the C-terminal region of the Ob-Ra isoform sequence. Strong cell surface immunoreactivity was detected using FITC immunofluorescence microscopy (Fig. 1C). Immunofluorescence was absent in control cells that had not been treated with anti-Ob-R antibodies (data not shown).
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). Reliable maximal effects were produced by 10 nM, and this concentration was used for further characterization. To substantiate that the increase in cell number detected with the MTT assay was due to increased proliferation, we confirmed that leptin stimulated DNA synthesis as measured by a BrdU incorporation assay. In this case 10 nM leptin stimulated DNA synthesis by 43 ± 5% (Fig. 1E). Having determined the concordance between the assays, the MTT assay was used in preference for further studies because of cost and convenience. Leptin also significantly inhibited serum starvation-induced apoptosis by 39 ± 9% (Fig. 1F).
Leptin-induced proliferation of OE33 cells is ERK, p38 MAPK, JNK, JAK2, PI3-kinase/Akt, COX-2, and EGFR dependent but mTOR independent
Leptin-induced proliferation was abolished by the selective COX-2 inhibitors NS-398 and celecoxib as well as the nonselective COX inhibitor indomethacin (Fig. 2A). Pharmacologic inhibition of ERK with PD 98059, JAK2 with AG490, and the PI3-kinase/Akt pathway with LY 294002 also abolished leptin-induced proliferation. Inhibition of p38 MAPK with SB 203580 and JNK with SP600125 inhibited leptin-induced proliferation. Pretreatment of cells with the EGFR kinase inhibitor AG1478 also significantly inhibited leptin-induced proliferation, but leptin-induced proliferation was unaffected by pretreatment of cells with rapamycin, the specific mTOR inhibitor (Fig. 2B).
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Leptin-induced COX-2 bioactivity in OE33 cells is dependent on activation of ERK, p38 MAPK, JAK2, and Akt but not mTOR, JNK, and EGFR kinase
Leptin increased secretion of PGE2 by OE33 cells by 163% (Fig. 3A). In this regard leptin was almost as effective as IL-1ß used as positive control (191% increase). The effect of leptin was blocked by the selective COX-2 inhibitor NS-398 and also by inhibition of ERK, p38 MAPK, PI3-kinase, and JAK2 (Fig. 3A). However, inhibition of JNK with SP600125 or EGFR with AG1478 did not reduce leptin-stimulated PGE2 production. Similarly rapamycin did not alter leptin-stimulated PGE2 release (Fig. 3A). To confirm the results of the PGE2 release experiments, we then assessed the effect of 4 h treatment with leptin on COX-2 mRNA expression. Leptin increased COX-2 mRNA levels by 112%. Pretreatment of cells with inhibitors of ERK, p38 MAPK, JAK2, and PI3-kinase/Akt abolished induction of COX-2 mRNA by leptin, but the inhibitors of JNK and EGFR failed to affect leptin-stimulated COX-2 mRNA levels (Fig. 3B).
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). Time-course studies show no significant increase in either JNK or EGFR phosphorylation between 2 and 30 min after leptin stimulation. The specific inhibitors of each kinase blocked leptin-stimulated phosphorylation of that kinase. The JAK2 inhibitor AG490 blocked phosphorylation of JAK2 and also abolished leptin-stimulated activation of ERK and Akt. However, AG490 did not affect leptin-stimulated p38 MAPK activation (Fig. 4). The specific inhibitors of ERK, Akt, p38 MAPK, JNK, and EGFR did not reduce the immediate phosphorylation of any other kinase studied (n = 3, data not shown). NS-398, the specific COX-2 inhibitor, did not affect the immediate activation of ERK, p38 MAPK, Akt, or JAK2 (n = 3, data not shown).
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). Expression of EP-4 receptor protein was confirmed by immunoblotting (Fig 5B). We examined the functional involvement of the EP-4 receptor using the specific pharmacological antagonist AH23848 (27). PGE2 (1 µM) itself significantly stimulated proliferation of OE33 cells by 104% above control assessed by the MTT assay and 73% when measured using BrdU incorporation (Fig. 5, C and D). PGE2 also significantly reduced OE33 cell apoptosis, reducing serum starvation-induced apoptosis by 46% (Fig. 5E). The stimulation of proliferation and inhibition of apoptosis induced by PGE2 were both blocked by AH23848 (Fig. 5, D and E). The EP-4 antagonist also effectively blocked the growth-promoting effect of leptin (Fig. 5D).
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) and JNK (Fig. 6B) were seen 6 h after initial stimulation with leptin. Inhibition of COX-2 with NS-398 and antagonism of the EP-4 receptor blocked this late kinase activation. Inhibition of EGFR kinase activity with AG1478 abolished the late leptin-induced phosphorylation of both EGFR and JNK, but the specific JNK inhibitor SP600125 did not affect EGFR phosphorylation, suggesting that JNK was activated downstream of the EGFR. Consistent with these data showing that activation of EGFR and JNK are delayed events in the signal transduction pathway, adding the EGFR kinase inhibitor 60 min after the leptin was as efficacious in blocking leptin-induced proliferation as pretreating the cells before adding leptin (Fig. 6C).
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) and JNK phosphorylation by 194% (Fig. 7B). The COX-2 inhibitor NS-398 did not affect these effects of PGE2, consistent with COX-2 being upstream of PGE2 in the sequence. The JNK inhibitor SP600125 abolished PGE2-stimulated JNK activation but did not affect EGFR phosphorylation, and the EGFR inhibitor AG1478 blocked activation of both EGFR and JNK, again consistent with JNK being activated downstream of the EGFR (Fig. 7, A and B).
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) and JNK (Fig. 7B), and similar effects were seen with the specific PKC inhibitor GF109203X and the nonspecific MMP inhibitor GM6001. Consistent with these data, PP2, GF109203X, and GM6001 all significantly inhibited both leptin and PGE2-stimulated OE33 cell proliferation (Fig. 7C). Inhibition of COX-2 with NS-398 inhibited leptin but not PGE2-stimulated proliferation. |
Discussion |
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OE33 cells express mRNA and protein for both the full-length Ob-Rb and shorter Ob-Ra isoforms. These appear to be functional as demonstrated by the biological activity of leptin in regulating proliferation, apoptosis, and kinase phosphorylation. To our knowledge this is the first demonstration of functional leptin receptors in OAC or Barrett’s esophagus. This gives support to our original hypothesis linking obesity and OAC and supporting our conclusions about signaling distal to the Ob-R. A recent preliminary study has reported increased expression of Ob-Rb detected by real-time PCR and immunohistochemistry in Barrett’s esophagus and EAC: expression increasing with malignant transformation from normal to cancer tissues (33). Whereas further studies examining the expression of leptin receptors in EAC are warranted, these data do support the idea that leptin may be a pathogenic factor in EAC. Similarly leptin has been reported to stimulate the growth of two other EAC cell lines (SEG-1 and BIC-1), although that study did not examine any dose-response relationships, receptor expression, or signaling events (34). Leptin was not found to have antiapoptotic actions in that study, but proliferation and apoptosis experiments were performed in serum-containing media, which may have blunted the proliferative and antiapoptotic effects.
The dose-response curve for leptin reported in this study corresponds to the binding affinity of the Ob-R and proliferative responses reported in other model systems (32, 35), consistent with a receptor-mediated effect. The leptin concentrations stimulating cell growth correspond to the physiological circulating concentrations seen in obesity (1). Leptin is also secreted by chief cells into gastric juice (36); it is possible, although speculative at present, that leptin in esophagogastric refluxate also contributes to growth promotion and apoptosis inhibition in vivo.
From our data it is not possible to determine whether the Ob-Rb or Ob-Ra is responsible for signaling in OE33 cells. Both receptors are expressed, although mRNA and protein expression of the shorter form was more prominent. The Ob-Rb has a full-length cytoplasmic domain containing all the motifs and tyrosine residues required for activation of the JAK/signal transducer and activator of transcription (STAT) pathways and has generally been regarded as the fully functional receptor (7). However, signaling via JAK2, ERK, PI3-kinase, and insulin receptor substrate-1 has been documented by Ob-Ra (8). Although activation of STAT proteins is important in mediating many of the biological effects of leptin and activation of STAT3 in the stomach by leptin has been reported (14), we have not specifically examined activation of STAT or other transcription factors because there are no data currently available concerning the involvement of these pathways in EAC, although in other models and disease states, STAT3 appears to be oncogenic and increases the transcription of genes such as survivin, Bcl-xl, and c-fos that may be important in carcinogenesis (37). In contrast, in vivo and in vitro studies have all suggested that the MAPKs, Akt, and COX-2 pathways are involved and important in the pathogenesis of EAC, although the interrelationship of these pathways has not been determined (19, 22, 29, 38). Although we have illustrated the sequence of early signaling events induced by leptin, it must be remembered that other as-yet-uncharacterized pathways may be involved. Because JAK2 is the predominant upstream activator of STAT3, it is possible that STAT3 is activated by leptin in EAC, and it is clear that there is considerable potential for cross-talk between leptin-activated pathways. Serine phosphorylation of STAT3 by MAPKs alters transcriptional activity and STAT3 cooperates with several transcription factors activated by the MAPK cascades (39, 40). Therefore, further studies examining the role of leptin-stimulated STAT protein activation in EAC are likely to prove fruitful.
Previous studies have shown than the proproliferative effects of leptin in HT-29 colon cancer cells and MKN28 gastric cancer cells were ERK dependent (10, 14). Similarly in the MDCK kidney cell line, leptin-induced invasion was dependent on ERK, PI3-kinase, JAK2, and mTOR, although the sequence of these effects was not determined (23). The antiapoptotic effect of leptin in HT-29 cells was ERK dependent, and pharmacological inhibitor studies in SK-N-SH-SY5Y neuroblastoma cells showed that the antiapoptotic effect of leptin involved JAK2, ERK, and PI3-kinase (32, 41).
In this study we have shown that a sequence of signaling events is initiated by leptin and so promotes growth and inhibits apoptosis. Leptin rapidly stimulated phosphorylation of JAK2, ERK, p38 MAPK, and Akt, and all of these kinases were essential to the effects of leptin. In contrast, although JNK and EGFR were essential to the actions of leptin, activation of these appeared to be a later event in signaling: no immediate phosphorylation of these kinases was seen after leptin exposure, phosphorylation was detectable 6 h after stimulation, and the inhibitor of EGFR was effective at abolishing proliferation, even if added 1 h after the leptin. We demonstrated that leptin increased COX-2 mRNA and increased PGE2 production. Inhibitor studies showed that activation of COX-2 and prostaglandin production was an essential event in signal transduction: JAK2, Akt, p38 MAPK, and ERK were upstream of COX-2 and EGFR and JNK were downstream.
Our studies also confirmed that JAK2 was required for activation of the ERK and PI3-kinase/Akt pathways but that activation of p38 MAPK appeared to be JAK2 independent. In gastric cancer cells, JAK2, tyrosine phosphorylation, and the scaffold function but not the phosphatase action of SHP-2 were required for leptin-induced ERK activation (14), and such a pathway could be responsible for ERK and PI3-kinase activation in OE33 cells. The link between Ob-R and p38 MAPK activation remains to be determined. Signaling via JAK1 by the leptin receptors has been demonstrated, and further studies confirming the kinases, small GTPase proteins, and scaffold proteins linking signal transduction pathways may determine further potential therapeutic targets.
Previous data from in vivo and in vitro studies have suggested that ERK, p38 kinase, and Akt are important proliferative and antiapoptotic signals in EAC (29, 38). The precise downstream targets remain to be elucidated: our study suggests that COX-2 is an essential downstream target for all three kinases. Although activation of mTOR and subsequently regulation of transcription via ribosomal p70 S6 kinase has been reported as an important action of Akt, the lack of effect of the specific mTOR inhibitor rapamycin suggests that this pathway is not an essential downstream target of leptin in OE33 cells. Although when activated all three MAPKs and Akt translocate to the nucleus and can phosphorylate a variety of transcription factors, our data do not allow us to determine whether the predominant activities are in increasing COX-2 transcription or increasing mRNA stability. A study has shown that the proliferative responses of acid in esophageal SEG-1 cells are mediated via ERK and p38 MAPK, leading to increased COX-2 transcription (42). However, it is quite clear that COX-2 mRNA levels and bioactivity are increased after leptin treatment. Experimental and observation data have implicated COX-2 in the progression of EAC (19). COX-2 is up-regulated in the transition from normal to premalignant to malignant EAC, Barrett’s esophageal epithelium secretes more PGE2 than normal esophageal epithelium, PGE2 stimulates proliferation of Barrett’s biopsy explants, and COX-2 inhibitors reduce EAC cell proliferation and increase apoptosis in vitro and in vivo (22, 24, 43, 44).
There are relatively few data concerning leptin and COX-2 activation. In rabbit oviduct leptin has been reported to increase PGF2
but decrease PGE2 (45), and in macrophage J774A.1 cells, leptin increased COX-2 expression and PGE2 production (46). Our data show that COX-2-derived prostaglandins are essential to the proliferative and antiapoptotic effects of leptin. Although COX-2 is a downstream mediator of the growth factor effects of several hormones, to our knowledge this is the first demonstration of the involvement of this pathway in the effects of leptin. Although COX-2 is up-regulated in Barrett’s esophagus and EAC, this does not appear to be due to the inflammatory response (47), our data suggest that leptin may be an important factor in driving COX-2 expression in vivo.
Prostaglandins have a variety of procarcinogenic actions, but there a very limited data concerning the cellular actions of prostaglandins in EAC. Our data suggest that in OE33 cells the downstream effects of PGE2 are mediated via the EP-4 receptor and involve activation of EGFR and JNK. The effects of leptin were replicated by adding exogenous PGE2, and the effects of both leptin and PGE2 were abolished by the specific EP-4 receptor antagonist. Using RT-PCR, we were able to detect expression of the EP-4 receptor type only in OE33 cells, and immunoblotting confirmed expression on EP-4 receptor protein, consistent with this receptor mediating the effects of leptin. There are very limited data on EP-receptor expression in OAC; one study in a rat model showed increased expression of EP-2, EP-3, and EP-4 by RT-PCR. Although not truly quantitative, EP-4 seemed to be increased the most (48), but clearly further studies in humans are required.
In recent years it has been reported that transactivation of EGFR can occur secondary to activation of several hepatohelical G protein-linked receptors. Such an effect is important in mediating the growth-promoting effects of gastrin via the cholecystokinin2 receptors, acetylcholine at muscarinic receptors, and prostaglandins (28, 49, 50, 51). A variety of methods seems to be involved in this effect including cleavage of membrane bound EGFR ligands leading to autocrine and paracrine activation of the EGFR as well as more direct enzymatic tyrosine phosphorylation of the intracellular portion of EGFR (49). In this study we have shown that the broad spectrum MMP inhibitor GM6001, the PKC inhibitor GF109203X, and the src inhibitor PP2 all inhibited the proliferative effects of leptin and PGE2 as well as blocked EGFR phosphorylation. This suggests that all these transduce signals between the EP-4 receptor and EGFR, although further studies are required to dissect this further. Cleavage of EGFR ligands stimulated by PKC and mediated by MMPs has been reported, and we believe that this is the likely mechanism in this case, although further studies concerning the specific ligands are needed (28). Although leptin has been shown to increase the production of various MMPs, this is the first study to implicate MMP activity in the growth-promoting and antiapoptotic effects of leptin (52). The specific MMPs responsible require determination. The predominant MMP expressed in EAC is MMP-7, but clearly this mechanism may provide further therapeutic targets (53). Recently leptin was reported to stimulate immediate tyrosine phosphorylation of the EGFR in gastric cancer cell lines; this mechanism appears to be separate from the one we have described and suggests that there are alternate possible tissue or tumor-specific routes of EGFR activation (54).
Both the EGFR and receptor ligands are increased with malignant progression in Barrett’s esophagus and EAC (25, 55), once again suggesting how leptin can be involved in driving progression in EAC. Our data show that activation of JNK is a necessary downstream target of EGFR activation. Leptin stimulated late phosphorylation of JNK in a COX-2-, EP-4-, and EGFR-dependent manner, and PGE2 stimulated early JNK phosphorylation, again dependent on EP-4 and EGFR kinase activity. In contrast, the specific JNK inhibitor did not affect EGFR phosphorylation. Activation of JNK has been reported to have both pro- and antiapoptotic effects in different models, probably dependent on which specific downstream targets are phosphorylated (26). The predominant target of JNK is phosphorylation and activation of c-Jun as part of activator protein-1 dimers, and this is probably important in the proliferative and antiapoptotic effects; however, JNK has also been reported to regulate apoptosis by regulating the function of Bax and Bcl-2 (56, 57). In SEG-1 OAC cells, acid exposure and subsequent neutralization caused JNK activation that was important in the proliferative response, although there was no indication of any involvement of prostaglandins and the EGFR in this immediate response (29). Although we have described a sequence of events secondary to leptin activating the Ob-R, leading eventually to JNK activation, our present data do not allow us to conclude than JNK is the final common pathway, although clearly this whole complex cascade is essential to the biological activities. It is that possible that continued activation of the other MAPKs or Akt secondary to either direct Ob-R or subsequent EGFR activation is also required as part of the ongoing portfolio of signaling promoting growth and inhibiting apoptosis.
The main aim of our study was to examine the early signaling events stimulated by leptin regulating proliferation and apoptosis. We have not yet examined the main downstream effectors of these leptin (and PGE2) mediated effects. Further studies will be required to determine the alterations in gene expression and protein activity regulating the cell cycle and apoptosis. Our study could be criticized for our reliance use of pharmacological inhibitors and detection of kinase phosphorylation as a marker of enzyme activity. However, we have used widely used, potent, and specific inhibitors of the respective kinases, and detection of specific phosphorylated kinases (whether by ELISA or immunoblotting) is widely used as reliable surrogate read-out of kinase activity. We have demonstrated that leptin induced biological effects (inhibition of apoptosis, stimulation of proliferation, increased COX-2 mRNA), that these were associated with increases in the active forms of the implicated kinases, and that the pharmacological inhibitors specifically abolished phosphorylation of the kinases and blocked the biological end responses. Thus, we feel our data are robust enough to support our conclusions about the involvement of the signaling pathways.
In view of the poor prognosis of EAC, new therapeutic agents are required both for prevention and as sensitizers for current therapies. The complexity of the pathways induced by leptin also suggests several new points at which chemopreventive or therapeutic agents may be targeted. The involvement of COX-2 suggests that inhibition of the pathway might be beneficial in obese patients. Natural plant products that inhibit Akt such as derivatives of quercetin and deguelin may also be potentially useful as part of a dietary approach (58, 59).
In conclusion, we have demonstrated receptor-mediated stimulation of growth and inhibition of apoptosis by leptin in esophageal adenocarcinoma cells. These effects were mediated via a complex cascade of reactions. Initial stimulation with leptin stimulates JAK2 activation and subsequent activation of ERK and Akt; leptin also stimulated rapid activation of p38 MAPK. Increased COX-2 mRNA and PGE2 production occur secondary to ERK, Akt, and p38 MAPK activation. Subsequent activation of the prostaglandin EP-4 receptor leads to transactivation of the EGFR via MMP-, src-, and PKC-dependent pathways and activation of JNK (Fig. 8). These pathways are essential to the effects of leptin and may provide a biological link between obesity and the increased proliferation and reduced apoptosis seen in Barrett’s esophagus and EAC.
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Acknowledgments |
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Footnotes |
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Part of this work was presented in abstract form at the United European Gastroenterology Week, Copenhagen, Denmark, 2005, and published in abstract form [Gut 2005;54(Suppl VII):A70].
Disclosure statement: The authors have nothing to disclose.
First Published Online June 1, 2006
Abbreviations: BrdU, Bromodeoxyuridine; COX, cyclooxygenase; BMI, body mass index; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JAK, Janus tyrosine kinase; JNK, c-Jun NH2-terminal kinase; MMP, matrix metalloproteinase; mTOR, mammalian target of rapamycin; MTT, 3-[4, 5-dimethylthiazol-2-y-l]2, 5-diphenyltetrazolium bromide; EAC, esophageal adenocarcinoma; Ob-Ra, short receptor form; Ob-Rb, full-length receptor form; PGE2, prostaglandin E2; PI3-kinase, phosphatidylinositol 3'-kinase; PKC, protein kinase C; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine; STAT, signal transducer and activator of transcription.
Received February 22, 2006.
Accepted for publication May 22, 2006.
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B J Kendall, G A Macdonald, N K Hayward, J B Prins, I Brown, N Walker, N Pandeya, A C Green, P M Webb, D C Whiteman, et al. Leptin and the risk of Barrett's oesophagus Gut, April 1, 2008; 57(4): 448 - 454. [Abstract] [Full Text] [PDF]
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D C Whiteman, S Sadeghi, N Pandeya, B M Smithers, D C Gotley, C J Bain, P M Webb, A C Green, and for the Australian Cancer Study Combined effects of obesity, acid reflux and smoking on the risk of adenocarcinomas of the oesophagus Gut, February 1, 2008; 57(2): 173 - 180. [Abstract] [Full Text] [PDF]
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F Francois, J Roper, A J Goodman, Z Pei, M Ghumman, M Mourad, A Z O. de Perez, G I Perez-Perez, C-H Tseng, and M J Blaser The association of gastric leptin with oesophageal inflammation and metaplasia Gut, January 1, 2008; 57(1): 16 - 24. [Abstract] [Full Text] [PDF]
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Abstracts Gut, April 1, 2007; 56(suppl_2): a1 - a145. [Full Text] [PDF]
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