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The Journal of Biological Chemistry logoLink to The Journal of Biological Chemistry
. 2012 Mar 13;287(18):15066–15075. doi: 10.1074/jbc.M111.327262

Neurotensin-induced Proinflammatory Signaling in Human Colonocytes Is Regulated by β-Arrestins and Endothelin-converting Enzyme-1-dependent Endocytosis and Resensitization of Neurotensin Receptor 1*

Ivy Ka Man Law ‡, Jane E Murphy §, Kyriaki Bakirtzi ‡, Nigel W Bunnett §,, Charalabos Pothoulakis ‡,1
PMCID: PMC3340238  PMID: 22416137

Background: Neurotensin induces proinflammatory responses in human colonic epithelial cells.

Results: β-Arrestins and endothelin-converting enzyme 1 regulate neurotensin receptor 1-mediated inflammatory signaling in human colonocytes.

Conclusion: Neurotensin-induced proinflammatory responses depend on β-arrestins and are regulated by receptor recycling.

Significance: This is a previously unrecognized pathway for regulating neurotensin-induced colonic inflammatory responses.

Keywords: Arrestin, Cell Signaling, Inflammation, Receptor Recycling, Receptors, Neurotensin, Neurotensin Receptor 1

Abstract

The neuropeptide/hormone neurotensin (NT) mediates intestinal inflammation and cell proliferation by binding of its high affinity receptor, neurotensin receptor-1 (NTR1). NT stimulates IL-8 expression in NCM460 human colonic epithelial cells by both MAP kinase- and NF-κB-dependent pathways. Although the mechanism of NTR1 endocytosis has been studied, the relationship between NTR1 intracellular trafficking and inflammatory signaling remains to be elucidated. In the present study, we show that in NCM460 cells exposed to NT, β-arrestin-1 (βARR1), and β-arrestin-2 (βARR2) translocate to early endosomes together with NTR1. Endothelin-converting enzyme-1 (ECE-1) degrades NT in acidic conditions, and its activity is crucial for NTR1 recycling. Pretreatment of NCM460 cells with the ECE-1 inhibitor SM19712 or gene silencing of βARR1 or βARR2 inhibits NT-stimulated ERK1/2 and JNK phosphorylation, NF-κB p65 nuclear translocation and phosphorylation, and IL-8 secretion. Furthermore, NT-induced cell proliferation, but not IL-8 transcription, is attenuated by the JNK inhibitor, JNK(AII). Thus, NTR1 internalization and recycling in human colonic epithelial cells involves βARRs and ECE-1, respectively. Our results also indicate that βARRs and ECE-1-dependent recycling regulate MAP kinase and NF-κB signaling as well as cell proliferation in human colonocytes in response to NT.

Introduction

Neurotensin (NT)2 is a 13-amino acid neuropeptide/hormone secreted by cells in the ileum (1) and colon (2). Three NT receptors have been identified. Neurotensin receptor 1 (NTR1), which has a higher affinity for NT (3), and NTR2 (4), both of which are coupled to G proteins, whereas the third receptor, sortilin 1 (SORT1), is a non-G protein-coupled receptor (GPCR) (5). NTR1 mediates most of the intestinal responses to NT (6, 7). In the intestine, NT/NTR1 interactions promote both proliferation and inflammation (2, 8, 9). Moreover, NTR1 is overexpressed in cancer cell lines (10) and intestinal tumors (11), and NTR1 coupling induces activation of MAP kinases (9, 12, 13). NTR1 expression is up-regulated during acute and chronic colitis of diverse etiologies in both animals and humans (2, 8), whereas in human colonocytes overexpressing NTR1, NT stimulates NF-κB-dependent IL-8 expression that involves insulin-like growth factor-1 receptor and Rho GTPases (14–16).

The trafficking of GPCRs between plasma and endosomal membranes is a critically important determinant of cellular responsiveness to extracellular agonists. Agonist-stimulated receptor endocytosis depletes GPCRs from plasma membranes to prevent overstimulation (17), whereas cell resensitization is achieved by receptor dephosphorylation (18) and receptor recycling following receptor internalization (19). Many activated GPCRs are phosphorylated by G protein-coupled receptor kinases, which promotes receptor interaction with β-arrestins (βARRs), key mediators of receptor desensitization, endocytosis, and intracellular signaling (18, 20). The NTR1 receptor is a “class B” receptor (21, 22) that exhibits sustained, high affinity interactions with both βARR1 and βARR2 during receptor endocytosis (21, 22) and remains associated with βARRs until they translocate to endosomes for dissociation (23). In all cell types investigated for NTR1 endocytosis, βARRs are important for internalization of NTR1 (21–24). However, whether NTR1 is recycled back to the surface remains controversial. Although NTR1 recycles in human neuroblastoma CHP212 cells (25) and in Cos7 cells (26), NTR1 is internalized and degraded in lysosomes (26–29).

GPCR trafficking also participates in signal transduction. GPCRs at the plasma membrane can activate MAP kinases by G protein-dependent PKA or PKC and by G protein-mediated transactivation of the EGF receptor. However, G protein-mediated signaling at the plasma membrane is rapidly attenuated by βARR-mediated desensitization mechanisms. βARRs are also scaffold proteins that recruit certain GPCRs and MAP kinases to endosomes and thereby mediate a second wave of βARR-dependent and G protein-independent signaling from endosomes (30). βARRs interact with c-Src, c-Raf-1, MEK1, ERK2, and JNK3 (30–34) and recruit MAP kinases to form “signalosomes” facilitating GPCR signaling (35–38). When compared with G protein-dependent ERK activation, βARR-associated ERK activation is more persistent, depends on βARRs expression, and takes place in the cytosol (20, 39, 40).

Endothelin-converting enzyme (ECE-1) plays an important role in the endosomal trafficking and signaling of neuropeptide/hormone GPCRs (41–44). ECE-1 exists in four isoforms (isoforms a–d), all of which share a common catalytic domain (45). All four isoforms are present in early endosomes, although ECE-1a and ECE-1c are mainly localized at the plasma membrane (45, 46), whereas ECE-1b and ECE-1d are predominantly in endosomes (46, 47). ECE-1 degrades certain neuropeptides, such as substance P and calcitonin gene-related peptide (42), in the acidified endosomal environment and thereby destabilizes the peptide/receptor/βARR signalosome (48). This destabilization has a dual effect on signal transduction. In the case of the substance P neurokinin-1 receptor and the calcitonin gene-related peptide receptor, endosomal ECE-1 allows the receptors, freed from βARRs, to recycle, which mediates resensitization of G protein-mediated signaling at the plasma membrane. However, endosomal ECE-1 disrupts the substance P/neurokinin 1 receptor/βARR signalosome and thereby terminates βARR-dependent ERK2 signaling from endosomes (48).

Sustained proinflammatory signaling responses represent a major component of NTR1 function that could be attenuated by disrupting receptor resensitization or intracellular signaling. Therefore, in this study, using nontransformed colonic epithelial cells as an in vitro model, we investigated the role of βARRs and ECE-1, the key mediators in NTR1 trafficking for NT-stimulated proinflammatory signaling and proliferation. Our results suggest that both proinflammatory and proliferative responses in colonic epithelial cells are regulated by βARRs and ECE-1-dependent recycling of NTR1.

EXPERIMENTAL PROCEDURES

Reagents

NT was purchased from Bachem Americas, Inc (Torrance, CA). Cell culture medium M3:D was purchased from INCEL Corp. (San Antonio, TX). Antibodies against phosphorylated ERK1/2, JNK, and p65 were from Cell Signaling (Danvers, MA). Antibodies against pan βARRs were from Abcam (Cambridge, MA); βARR-1 and early endosomal antigen 1 (EEA1) were from BD Transduction Laboratories (San Jose, CA); ECE-1 was from Zymed Laboratories Inc. (South San Francisco, CA); NTR1 was from Santa Cruz Biotechnology (Santa Cruz, CA); and β-tubulin was from Cell Signaling (Beverly, MA). Inhibitors for JNK (JNK(AII)), ERK1/2 (PD98059), and NF-κB (caffeic acid phenethyl ester) were from Cell Signaling. SM19712, an inhibitor for ECE-1, was from Sigma-Aldrich. Bafilomycin A1, cycloheximide, and siRNAs specific for βARR1 and βARR2 were obtained from Santa Cruz Biotechnology. Lipofectamine 2000 and LipofectamineTM RNAiMAX were from Invitrogen. Plasmids overexpressing green fluorescent-tagged βARR1 and βARR2 were previously described (49, 50).

Transduction of NCM460 Cells with NTR1-GFP (NCM460-NTR1-GFP)

The full-length human NTR1 gene was isolated from the original plasmid backbone pCR2.1 (9) with EcoRV. The enzyme-digested NTR1 construct (1.2 kb) was extracted from agarose gels and inserted into the lentiviral backbone CMV-IRES-GFP-PGK-Puro at EcoRV site just 5′ of the IRES site (CMV-IRES-GFP-PGK-Puro-NTR1). The GFP-tagged NTR1 construct was generated by ligating PCR-amplified GFP and NTR1 constructs and inserted into the lentiviral backbone CMV-IRES-Puro at XbaI and EcoRI sites just 5′ of the IRES site (CMV-IRES-Puro-NTR1-GFP). Lentiviral particles expressing both constructs were generated by transient co-transfection of 293T cells with three other plasmids. Generation of lentil virus particles using the third generation of lentil virus is as previously described (51). Briefly, nonconfluent 293T cells were co-transfected with pMDLg/pRRE, pMDG (encoding the VSV-G envelope), pRSV-REV, and either CMV-IRES-GFP-PGK-Puro-NTR1 or CMV-IRES-Puro-NTR1-GFP, by the CaPi-DNA co-precipitation method (52–54). Viral titer was determined by assessing viral p24 antigen concentration by ELISA (Coulter Immunetech, Miami, FL) and hereafter expressed as μg of p24 equivalent units/ml.

For transfection, NCM460 were seeded at 2 × 105 cells/well in 6-well plates (Corning) and incubated (37 °C for 24 h) in 1 ml of M3:D, including serial dilutions of lentiviral vector supernatants. Infected cells were then incubated in medium supplemented with 10% (v/v) FBS and 2 μg/ml puromycin for 6 days. Positively selected cells, designated as NCM460-NTR1-GFP were pooled and used for this study.

NT Degradation by ECE-1

NT (250 μm) was incubated with 300 nm recombinant human ECE-1 (rhECE-1) (R & D Systems, Minneapolis, MN) in 50 mm MES-KOH, pH 5.5, or 50 mm Tris-HCl, pH 7.4, for 0–8 h at 37 °C. The products were separated by reversed phase HPLC and identified by MALDI-TOF/TOF mass spectrometry. Mass spectrometry data were provided by the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF (A. L. Burlingame, Director) supported by the Biomedical Research Technology Program of the National Institutes of Health National Center for Research Resources (National Institutes of Health Grants P41RR001614 and 1S10RR014606).

Localization of NTR1, βARRs, ECE-1, and EEA1

NCM460 stably expressing NTR1-GFP were incubated with 100 nm NT (0–30 min, 37 °C) before fixation. To follow NTR1 and βARRs trafficking, NCM460 cells stably expressing untagged NTR1 (NCM460-NTR1) (55) were transiently transfected with βARR1 or 2-GFP using Lipofectamine 2000, according to the manufacturer's instructions. After 48–72 h, the cells were incubated with 100 nm NT (0–30 min, 37 °C) prior to fixation. The cells were fixed in PBS containing 4% (w/v) paraformaldehyde, (pH 7.4, 20 min, 4 °C) and blocked in PBS containing 0.1% (w/v) saponin and either 1% (v/v) normal goat serum or 2% (v/v) normal donkey serum. The cells were incubated with the following antibodies overnight at 4 °C: mouse anti-EEA1 (1:200), rabbit anti-ECE-1 (1:400), or goat anti-NTR1 (1:200). The cells were washed and incubated with fluorescent secondary antibodies (1:400 for 2 h at room temperature), and slides were mounted on Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). The cells were imaged using a LSM 510 Meta confocal microscope (Carl Zeiss, Inc.) with a Plan Apo 63× (NA 1.4) objective.

Western Blot Analysis

After appropriate treatment, NCM460-NTR1 were washed by ice-cold PBS twice and then incubated with radiolabeled immunoprecipitation assay buffer containing protease inhibitors and supplemented with PMSF and sodium orthovanadate (Santa Cruz Biotechnology) for 5 min. The cell lysates were centrifuged (12,000 rpm for 15 min at 4 °C), and supernatants were analyzed by Western blot analysis. Proteins from equal amounts of cell extracts were separated in 10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad) at 70 V for 2 h at 4 °C. Membranes were blocked by 5% nonfat dried milk in TBS (Boston Bioproducts, Ashland, MA) supplemented with 0.05% Tween 20 for 1 h. Appropriate antibodies were incubated with the membranes overnight at 4 °C, washed with TBS/Tween 20, and incubated with secondary antibodies conjugated with horseradish peroxidase. Signals from target proteins were detected with SuperSignal chemiluminescent substrate (Pierce). Western blot bands were quantified by densitometry using Multi Gauge V3.1 (Fuji).

NF-κB ELISA

Following appropriate treatment, nuclei-enriched portions of NCM460-NTR1 were isolated with Nuclei EZ prep nuclei isolation kit (Sigma-Aldrich) according to the manufacturer's instructions. The cells were washed with ice-cold PBS twice and lysed with Nuclei EZ lysis buffer. The nuclei were centrifuged (500 × g for 5 min at 4 °C) and resuspended in Nuclei EZ storage buffer. The supernatant (cytosolic portion) was stored at −80 °C for further analysis. Translocation of p65 into the nucleus was detected by TransAM NF-κB (Active Motif, Carlsbad, CA) as described by the manufacturer. In brief, 5 μg of nuclei-enriched cell extracts were allowed to bind to the oligonucleotide containing the NF-κB consensus site coated in 96-well plates. The p65 in nuclear extracts was detected by primary antibodies against p65 and secondary antibodies conjugated with horseradish peroxidase. The signals were detected at A405 nm using a 96-well plate reader.

IL-8 ELISA

NCM460-NTR1 cells were exposed to NT for 6 h, and IL-8 in cell supernatants was measured by Duo-Set® ELISA for IL-8 (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions.

Luciferase Activity

The NF-κB-driven luciferase reporter construct was purchased from Clontech and was used as previously described (15). A reporter construct with the IL-8 promoter (nucleotides −1481 to +40) was used to measure the transcription activity of IL-8 as described (15). Either one of the reporter constructs, a control luciferase construct (pRL-TK; Promega, Madison, WI) and siRNAs (control and βARRs) were transfected into NCM460-NTR1 using Lipofectamine 2000. NCM460-NTR1 were seeded at 1 × 105 cells/well in 24-well plates 24 h prior to transfection. After 48 h, transfected cells were serum-fasted (16 h) and pretreated with pharmacological inhibitors where appropriate, followed by stimulation with NT for 1.5 h. Firefly and Renilla luciferase cell activities were detected using dual luciferase reporter assay system (Promega). The relative luciferase activities were calculated by normalizing the firefly luciferase activity with that from Renilla luciferase. The results were presented as the relative luciferase activity (means ± S.E.) from at least three independent sets of experiments, each with five replicated measurements.

Cell Proliferation Assay

NCM460-NTR1 were seeded at 1 × 104 cells/well in 96-well culture plates 24 h prior to incubation with 10 or 100 nm NT for 24 h in M3:D medium supplemented with 1% FBS and antibiotics. Cell proliferation assay was performed with CellTiter 96® AQueous nonradioactive cell proliferation assay (Promega) and cell proliferation ELISA, BrdU (colorimetric) (Roche Applied Science) according to the manufacturer's instructions.

Statistical Analysis

All of the results were derived from at least three sets of repeated experiments. The results are expressed as the means ± S.D. The data were analyzed with Student's t tests. In all statistical comparisons, p < 0.05 was used to indicate significant differences.

RESULTS

β-Arrestins Are Internalized with NTR1 to Endosomes

Previous studies identified NTR1 as class B GPCR that forms stable interactions with βARR1 and βARR2 in endosomes (21, 22). βARR1 and βARR2 initiate internalization in NTR1 endocytosis by docking with the phosphorylated NTR1 (22). To determine whether βARR1 and βARR2 traffic with NTR1 to endosomes, we localized βARR1, βARR2, and NTR1 by confocal microscopy. Plasmids expressing either βARR1 or βARR2 tagged with GFP were transfected into NCM460-NTR1 cells 48 h prior to NT exposure, and NTR1 was localized with a specific antibody. As shown in Fig. 1, the majority of NTR1 before stimulation was localized at the plasma membrane, whereas βARR1 and βARR2 were distributed evenly throughout the cytoplasm. Following 30 min of NT stimulation, NTR1 was co-localized with βARR1 and βARR2 in endosomes (Fig. 1, A and B, arrows). Thus, βARR1 and βARR2 were translocated with NTR1 to endosomes following stimulation with NT.

FIGURE 1.

FIGURE 1.

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β-Arrestins co-localize with NTR1 in endosomes following stimulation with NT. NCM460-NTR1 cells transiently transfected with βARR1-GFP (A) or βARR2-GFP (B) were incubated with 100 nm NT for 0 or 30 min at 37 °C. In nonstimulated cells, βARR1-GFP and βARR2-GFP are distributed throughout the cytosol, and NTR1 is present at the plasma membrane. Following stimulation with NT, βARR1-GFP and βARR2-GFP co-localized with NTR1 in endosomes (arrows). Scale bars, 10 μm.

NTR1 Translocates to ECE-1 Containing Early Endosomes upon NT Stimulation

We next examined whether endogenous ECE-1, the metalloendopeptidase capable of degrading NT (56), was present in early endosomes in NCM460-NTR1 cells. ECE-1 was localized using an antibody to the ECE-1b and ECE-1d isoforms (42). In unstimulated cells, ECE-1 immunoreactivity co-localized with EEA1, a specific marker for early endosomes (Fig. 2A), confirming the presence of endogenous ECE-1 in early endosomes, as previously shown in rat kidney epithelial cells (42). We next exposed NCM460-NTR1-GFP to NT for 30 min and localized NTR1-GFP and ECE-1 immunoreactivity by confocal microscopy. In unstimulated conditions, NTR1 was localized mainly at the plasma membrane, whereas ECE-1 was localized in endosomes (Fig. 2B). However, upon NT stimulation, NTR1 co-localized with ECE-1 (Fig. 2B, arrows), suggesting that this receptor translocates to early endosomes containing ECE-1 following NT exposure.

FIGURE 2.

FIGURE 2.

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NTR1-GFP traffics to early endosomes containing ECE-1. A, endogenous ECE-1 immunoreactivity, localized using an antibody specific for ECE-1b and ECE-1d isoforms, was present in EEA1-positive early endosomes (arrows). B, NCM460-NTR1-GFP cells were incubated with 100 nm NT for 0 or 30 min at 37 °C. NTR1-GFP trafficked from the plasma membrane (arrowheads) to endosomes containing ECE-1 (arrows). Scale bars, 10 μm.

ECE-1 Degrades NT at Acidic pH

NT is an ECE-1 substrate (56). By analogy with previous studies of the role of ECE-1 in regulating endosomal trafficking and signaling of certain class B GPCRs (41–44), we hypothesized a similar role for ECE-1 in regulating NTR1. To assess whether ECE-1 could degrade NT within endosomes or at the plasma membrane, we incubated NT with recombinant human ECE-1 in buffer with physiological pH of early endosomes (pH 5.5) or of extracellular fluid (pH 7.4) and assessed degradation by HPLC. As shown in representative HPLC chromatograms, NT degradation products eluted at ∼20 min and were detected only after incubation at endosomal pH (Fig. 3B) and not at extracellular fluid pH (Fig. 3, A–C). Time course experiments confirmed that ECE-1 degraded NT only in acidic, endosomal conditions (Fig. 3D). Analysis by mass spectrometry revealed that ECE-1 degraded NT at three sites, which would be expected to inactivate this neuropeptide/hormone (Fig. 3E). Thus, ECE-1 can degrade NT at the acidic pH of endosomes but not at the neutral pH of extracellular fluid.

FIGURE 3.

FIGURE 3.

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Degradation of neurotensin by rhECE-1 at endosomal pH. A–C, the HPLC profiles of neurotensin (250 μm) incubated with rhECE-1 (300 nm) at pH 5.5 for 0 h (A) or 8 h (B) or at pH 7.4 for 8 h (C). D, time course of degradation of neurotensin by rhECE-1. Neurotensin was degraded by rhECE-1 at pH 5.5 but not at pH 7.4. E, the sequence of neurotensin with ECE-1 cleavage sites indicated by arrows.

ECE-1 Mediates Receptor Recycling

In the case of the substance P and calcitonin gene-related peptide receptors, ECE-1 inhibition or knockdown causes retention of the receptors and βARRs in early endosomes and thereby prevents receptor recycling and resensitization of plasma membrane signaling (41, 42). Bafilomycin A1, a vacuolar H+ATPase inhibitor similarly prevents recycling because it impedes ECE-1 activity, which requires endosomal acidification. To examine whether the translocation of NTR1 from early endosomes also requires endogenous ECE-1 activity and endosomal acidification, NCM460-NTR1 cells were treated with the protein synthesis inhibitor cycloheximide (100 nm), ECE-1 inhibitor SM19712 (10 μm), and the endosomal acidification inhibitor bafilomycin A1 (100 nm) 30 min prior to NT exposure (100 nm, 1 h) as previously described (20). The cells were then washed twice with ice-cold HBSS and allowed to recover in NT-free medium. The majority of NTR1 was recovered from early endosomes at 6 h after NT removal (Fig. 4A). Our results show that cycloheximide did not affect receptor endocytosis or NTR1 translocation to plasma membrane (Fig. 4B). However, inhibition of ECE-1 activity by 10 μm SM19712 and inhibition of endosomal acidification by 100 nm bafilomycin A1 reduced translocation of NTR1 to plasma membrane, as shown by the retention of NTR1 in endosomes 6 h after recovery (Fig. 4, C and D). Our results suggest that recovery of NTR1 from early endosomes depends on endogenous ECE-1 activity in acidified endosomal vesicles.

FIGURE 4.

FIGURE 4.

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ECE-1 activity and endosomal acidification mediate NTR1 receptor recycling. NCM460-NTR1 cells were incubated in medium containing vehicle (A), 100 nm cycloheximide (B), 10 μm SM19712 (C), or 100 nm bafilomycin A1 (D). The cells were then stimulated with 100 nm NT for 1 h at 37 °C. After washing with HBSS, the cells were allowed to recover in NT-free medium for 6 h. In unstimulated cells, NTR1 were distributed at the plasma membrane in all groups. NT-exposed NCM460-NTR1 treated with SM19712 and bafilomycin A1 show reduced NTR1 on the cell surface after recovery. Scale bars, 10 μm.

ECE-1 Inhibition and βARR1 and βARR2 Gene Silencing Attenuate NTR1-dependent MAP Kinase Activation

The effectiveness of cell desensitization depends on the rates of receptor endocytosis and recycling. Although we have shown that NTR1 is recycled from ECE-1-containing acidified early endosomes in NCM460-NTR1, agonist-stimulated endocytosis of the NTR1 is mediated by βARRs (21–23). NT stimulates MAP kinase signaling, especially ERK-dependent signaling (9, 13), and activates NF-κB (14, 15). Both these pathways are involved in NT-mediated proinflammatory and cell proliferative responses (15, 57), and βARRs are also implicated in MAP kinase signaling of endocytosed GPCR (38). Therefore, we investigated whether ECE-1 and βARRs contribute to NT-stimulated MAP kinase signaling in colonocytes. NCM460-NTR1 cells were treated with siRNA to knockdown expression of βARR1 or βARR2. Fig. 5 (A and B) shows that the transcription levels of βARR1 and βARR2 in transfected cells are significantly reduced compared with cells transfected with scrambled siRNA. To examine whether ECE-1-mediated recycling contributes to ERK1/2 signaling, we treated cells with the ECE-1 inhibitor SM-19712. All the experiments were repeated 5 times and densitometric analysis was performed. Inhibition of ECE-1 activity also reduced ERK1/2 phosphorylation upon NT stimulation (Fig. 5C). On the other hand, although previous studies suggested that both βARR1 and βARR2 participate in GPCR internalization (22, 23), our Western blot analysis showed that only βARR1, but not βARR2 silencing significantly reduced ERK1/2 phosphorylation upon 100 nm NT stimulation, whereas the basal phosphorylation levels were similar (Fig. 5C). Thus, NT-induced ERK1/2 signaling can be regulated by βARR1 expression and ECE-1-dependent NTR1 receptor recycling and resensitization.

FIGURE 5.

FIGURE 5.

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Inhibition of NTR1 endocytosis and ECE-1-dependent resensitization attenuated MAP kinase signaling. A and B, NCM460-NTR1 were transfected with scrambled siRNA (si-Control) and siRNA specific for βARR1 (si-βARR1) or siRNA specific for βARR2 (si-βARR2) 48 h prior to NT treatment. Transcription levels of βARR1 and βARR2 were compared against the control group and examined by quantitative PCR. C, NCM460-NTR1 cells were preincubated with SM19712 (10 μm) or transfected with siRNA 48 h and subsequently treated with NT (100 nm) for 5 min. Equal amounts of proteins were subjected to Western blot analysis using anti-phospho-ERK1/2 antibody (p-ERK1/2) or anti-β-tubulin to ensure equal loading. D, NCM460-NTR1 cells were preincubated with SM19712 (10 μm) or transfected with siRNA 48 h and subsequently treated with NT (100 nm) for 40 min. Equal amounts of proteins were subjected to Western blot analysis using anti-phospho-JNK antibody (p-JNK) or anti-β-tubulin to ensure equal loading. E, NCM460-NTR1 cells were pretreated with or without JNK(AII) (10 μm) before incubated with NT for 24 h. Cell proliferation was assessed by microculture tetrazolium assay according to manufacturer's instructions (n = 5). F, NCM460-NTR1 cells were transfected with plasmids expressing with IL-8 promoter-driven luciferase and plasmids expressing control luciferase construct 48 h before NT (100 nm) treatment for 1.5 h in the presence or absence of JNK(AII) and caffeic acid phenethyl ester (CAPE). Luciferase activity of each group was measured with luminometer. *, p < 0.05 when compared with intergroup NT-treated control. #, p < 0.05 when compared with intragroup NT-treated control.

Next, we tested whether βARR expression and ECE-1 activity influence the activity of JNK. We found that NT-induced JNK activation was attenuated by inhibition of NTR1 recycling with SM-19712 and by βARR1 and βARR2 silencing (Fig. 5D). Because the ability of NT to activate JNK has not previously reported, we examined the pathophysiologic consequences of this response in human colonocytes. NT enhanced cell proliferation (58, 59), whereas increased JNK phosphorylation is linked to cell proliferation (60). NCM460-NTR1 cells were incubated in the presence or absence of NT for 24 h, and cell proliferation was assessed by the assay. As shown in Fig. 5E, NT (10 nm) increased cell proliferation, and co-incubation with the JNK inhibitor JNK(AII) reversed the effect. To confirm this response, we also used BrdU incorporation colorimetric assay. We found that NT-associated (10 nm, 24 h) increased BrdU incorporation was inhibited by JNK(AII) (by ∼20%, n = 5, p < 0.05). Because JNK phosphorylation is associated with inflammatory responses, we determined its involvement in NT-induced IL-8 secretion. NCM460-NTR1 cells were transfected with a plasmid encoding IL-8 promoter-driven luciferase or a control luciferase plasmid 48 h prior to NT stimulation in the presence or absence of JNK(AII). We found no significant difference in IL-8 promotor-driven luciferase activity between JNK(II)-treated and control cells (Fig. 5F). The reduction in luciferase activity in cells pretreated with the NF-κB inhibitor caffeic acid phenethyl ester prior to NT exposure (Fig. 5F), confirmed that IL-8 transcription in NCM460-NTR1 cells involved NF-κB activation (15). These results indicate that, at least in colonocytes, NTR1-associated JNK activation is linked to cell proliferation, but not to NF-κB-driven IL-8 transcription.

ECE-1 Inhibition and βARR1 and βARR2 Gene Silencing Attenuate NT-induced NF-κB Activation

We next investigated whether ECE-1 and βARRs regulated NT-induced NF-κB activation and subsequent NF-κB-dependent IL-8 transcription. Western blot analysis of NCM460-NTR1 cells stimulated with 100 nm NT showed that p65 phosphorylation was attenuated by gene silencing of both βARRs (Fig. 6A), and IκB-α phosphorylation was attenuated by gene silencing of βARR2 (Fig. 6B). Transcription of NF-κB-dependent proinflammatory cytokines involves translocation of p65 to the nucleus and binding of the corresponding p65 promoter sequences to cytokine genes. We next isolated nuclear extracts from cells transfected with βARR1 and βARR2 siRNAs, before or after treatment with the ECE-1 inhibitor SM19712 and measured p65 by ELISA. Our results showed a significant reduction in NT-induced p65 levels in SM19712-treated cells as well as in βARR2-silenced cells, whereas in contrast, βARR1 silencing slightly increased p65 nuclear translocation (Fig. 6C). Interestingly, SM19712 reduced p65 phosphorylation at basal conditions and in response to NT (Fig. 6C). Moreover, NT-stimulated NF-κB-driven luciferase activity and IL-8 secretion were reduced after SM19712 treatment or βARR2 silencing (Fig. 6, D and E). We next examined the outcome of βARR1 and βARR2 silencing in the expression of other inflammatory genes by NT. Quantitative PCR analysis performed on cDNA collected from NCM460-NTR1 after 2 h of NT exposure demonstrated reduced TNF-α and IL-1β mRNA levels in βARR2-silenced cells, whereas βARR1 silencing only reduced TNF-α mRNA (Fig. 6F).

FIGURE 6.

FIGURE 6.

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Inhibition of NTR1 endocytosis and ECE-1-dependent resensitization attenuates NF-κB signaling. A and B, NCM460-NTR1 cells were treated with SM19712 (10 μm) 30 min prior to NT treatment or transfected with siRNA specific for scrambled siRNA (si-Control) and siRNA specific for βARR1 (si-βARR1) or siRNA specific forβARR2 (si-βARR2) 48 h prior to NT treatment. The cells were treated with NT (100 nm) for 1.5 h. Equal amounts of proteins were subjected to Western blot analysis using anti-phospho-p65 antibody (p-p65) (A) or anti-phospho-IκBα antibody (p-IκBα) (B) or anti-β-tubulin to ensure equal loading. C, NCM460-NTR1 cells were subjected to the same treatment as above. The nuclei from different treatment groups were isolated and quantified. Equal amounts of nuclear lysates were used to quantify the concentration of p65 by ELISA. D, NCM460-NTR1 cells were transfected with plasmids expressing NF-κB-driven luciferase and control luciferase 48 h prior to NT treatment. The cells were treated as stated above, and the luciferase activity was measured by luminometer. E, NCM460-NTR1 cells were pretreated with SM19712 (10 μm) for 30 min or transfected with scrambled siRNA (si-Control) and siRNA specific for βARR1 (si-βARR1) or βARR2 (si-βARR2) 48 h before NT (100 nm) treatment for 6 h. Culture media were collected, and IL-8 secretion was analyzed with ELISA. F, NCM460-NTR1 cells were treated with SM19712 and siRNA transfection as mentioned above and underwent NT (100 nm) stimulation for 2 h. Transcription levels of TNF-α and IL-1β of different treatment groups were analyzed by quantitative PCR. *, p < 0.05 when compared with treatment control.

DISCUSSION

Previous studies have shown that activated NTR1 internalizes by a βARR-dependent process (21–24). We have reported that NTR1 couples to proinflammatory pathways in human colonocytes, including NF-κB and MAP kinase activation and secretion of neutrophil chemoattractants, such as IL-8 (14, 15). However, the fate of NT/NTR1 complex following NT stimulation in colonocytes is not known, and whether NT/NTR1 internalization and recycling are associated with NT-induced proinflammatory and proliferative responses in these cells remains to be elucidated. Our present results in human colonocytes show that NT induces trafficking of NTR1, βARR1, and βARR2 to early endosomes containing ECE-1 and that NTR1 then slowly recycles back to the plasma membrane. βARRs are required for the proinflammatory and proliferative signaling of NT, including activation of ERK1/2, JNK, and p65 and generation of IL-8. By degrading NT in acidified endosomes, ECE-1 mediates recycling of NTR1, where resensitization of plasma membrane signaling is necessary for the sustained proinflammatory actions of NT. Thus, we propose a mechanism (Fig. 7) in which NT-induced proinflammatory signaling responses involve a βARRs-dependent endosomal mechanism and sustained NT-induced MAP kinase and NF-κB activation require ECE-1-dependent NTR1 receptor recycling.

FIGURE 7.

FIGURE 7.

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Proposed mechanism by which βARRs and endosomal ECE-1 regulate NT-induced MAP kinase and NF-κB activity in human colonocytes. Step 1, NT binding on NTR1 induces G protein receptor kinases (GRKs) phosphorylation and β-arrestins (βARRs) membrane translocation. Step 2, NTR1 is translocated with both β-arrestin-1 (βARR1) and β-arrestin-2 (βARR2) to early endosomes. Step 3, endosomal ECE-1 degrades NT from the NT-NTR1 complex in acidified endosomes. Step 4, free NTR1 is transported back to the plasma membrane in recycling endosomes for resensitization. Step 5, βARR1 promotes MAP kinase phosphorylation. Step 6, βARR2 is involved in IκB-α phosphorylation and NF-κB p65 nuclear translocation.

In human NCM460-NTR1 colonocytes, βARRs and NTR1 were translocated to early endosomes (Fig. 1), confirming previous evidence in nonintestinal cells that NTR1 is a class B GPCR (21, 22). In addition to interfering with G protein-dependent signaling and facilitating receptor endocytosis, βARRs recruit receptors and MAP kinases to assemble “signalsomes” that can transmit sustained ERK1/2 signals to particular subcellular locations (35–38). This enhances subsequent ERK phosphorylation, thereby activating βARR-dependent activation of the ERK cascade (30, 61), leading to cell proliferation and proinflammatory signaling (35–37, 62). Indeed, our results indicated that NT-induced MAP kinase and NF-κB activation are attenuated after βARR1 and βARR2 gene silencing (Figs. 5 and 6).

Structural and in vivo evidence indicate that βARR1 and βARR2 are, to some extent, functionally redundant (63, 64). However, we observed a stronger attenuation in NTR1-dependent ERK and JNK phosphorylation after βARR1 silencing compared with βARR2 knockdown (Fig. 5, B–D). In contrast, NF-κB p65 phosphorylation and IL-8 production were more strongly reduced in βARR2-silenced colonocytes compared with βARR1-silenced cells (Fig. 6, A, E, and F). Moreover, NT-induced IκB-α phosphorylation, NF-κB-driven luciferase activity, and p65 translocation were only reduced after βARR2 silencing (Fig. 6, B–D). These results suggest that NTR1 signaling is βARRs-dependent and that βARR1 and βARR2 may play different roles in promoting inflammatory signal transduction pathways in the context of NTR1 activation.

Our results suggest that only βARR1 but not βARR2 is required for NT-induced activation of ERK1/2, yet βARR2 appears to be involved in NT-induced NF-κB activation. Because evidence indicates that in many signaling pathways NF-κB activation involves ERK1/2, our finding of an independent association of βARR1 and βARR2 with ERK1/2 and NF-κB signaling pathways may appear contradictory. However, recent studies have identified ERK-independent NF-κB activation in rheumatoid synovial fibroblasts and hepatocellular HepG2 cells (65, 66). Although our results suggest that βARR2-associated NF-κB activation is ERK-independent, the mechanism(s) involved in the participation of βARRs in NTR1-associated MAP kinase and NF-κB activation in colonocytes are not entirely clear. Moreover, βARR1 and βARR2 have distinct roles in other systems. For example, previous studies have shown that βARR1 can form signalsomes with MAP kinases, such as c-Src, ERK, and JNK3 (30, 32–34), whereas βARR2 recruits CARMA3, a scaffold protein involved in lysophosphatidic acid-induced NF-κB activation and subsequent IL-6 production (67). Moreover, the NF-κB inhibitor IκB-α co-immunoprecipitates with βARR2 in mammalian cells and a yeast two-hybrid system (62, 68). We have also shown that NT-induced ERK activation is Ras-GTPase-related and NF-κB activation is Rho-GTPase-dependent (9, 14), although both signaling cascades are involved in IL-8 secretion (9). Therefore, additional studies are required to further elucidate the involvement of the two β-arrestin isoforms in proinflammatory signaling in colonocytes.

We demonstrated that in human NCM460-NTR1 colonocytes, NTR1 was translocated to ECE-1-containing acidified early endosomes along with βARR1 and βARR2 (Figs. 1 and 2). Endosomal ECE-1 regulates intracellular signaling at two levels. ECE-1 promotes receptor recycling and sensitization of plasma membrane signaling of receptors for substance P and calcitonin gene-related peptide (42, 44). On the other hand, endosomal ECE-1 also terminates signaling by destabilizing the endosomal MAP kinase signalosome after ligand degradation in acidified early endosomes (48). Our results indicate that recycling of NTR1 promoted by ECE-1 may be more important for sustained NT-induced proinflammatory signaling (Figs. 4, 5, C and D, and 6). Similarly to NTR1, neurokinin-1 receptors, the receptor of substance P, is a class B GPCR (21). When stimulated by high ligand concentrations, both NTR1 and neurokinin-1 receptors are translocated to perinuclear sorting endosomes by Rab5a, which is present in early endosomes and remains in perinuclear sorting endosomes for more than 60 min (25, 69), whereas when low concentrations of substance P are used, neurokinin-1 receptor is rapidly recycled from early endosomes (69). It is likely that in human colonocytes, inhibition of ECE-1 activity promotes the translocation of NTR1 to perinuclear sorting endosomes and depletes NTR1 on plasma membrane (25), thus reducing NT-associated signaling.

In the present study, we found that NT induced JNK phosphorylation via a pathway that involved NTR1 internalization and recycling, whereas inhibition of NT-induced JNK activation leads to reduced cell proliferation in colonocytes (Fig. 5, D and E). Increased JNK phosphorylation is evident in the inflamed colonic mucosa of patients with inflammatory bowel disease (70, 71), whereas NTR1 has been associated with mucosal healing following colitis (7), suggesting that JNK activation in response to NT in colonocytes may be involved in the pathophysiology of mucosal healing.

In summary, NT-induced MAP kinase signaling and NF-κB activation in human nontransformed colonocytes is regulated by NTR1 internalization and recycling. Both MAP kinase and NF-κB activities are βARRs-dependent, and rapid recycling of internalized NTR1 from early endosomes by ECE-1 is essential for signaling transduction. Further investigation is needed to address whether NTR1 internalization and recycling participate in the proinflammatory mechanisms by which NTR1 modulates colitis.

*

This work was supported, in whole or in part, by National Institutes of Health Grants DK60729, P50 DK 64539 (to C. P.), DK43207, and DK39957 (to N. W. B.). This work was also supported by National Health and Medical Research Council Australia Fellowship 633033 (to N. W. B.), and the Blinder Research Foundation for Crohn's Disease and the Eli and Edythe Broad Chair (to C. P.). This work was also supported in part by National Institutes of Health Grants JCCC/P30 CA016042 and CURE/P30 DK041301.

2
The abbreviations used are:
NT
neurotensin
βARR
β-arrestin
EEA1
early endosome antigen 1
GPCR
G protein-coupled receptor
NTR1
neurotensin-receptor 1
ECE-1
endothelin-converting enzyme-1
rhECE-1
recombinant human ECE-1.

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