Anti-inflammatory activity of Wnt signaling in enteric nervous system: in vitro preliminary evidences in rat primary cultures
- Rosa Di Liddo†1Email author,
- Thomas Bertalot†1,
- Anne Schuster2,
- Sandra Schrenk2,
- Alessia Tasso1,
- Ilenia Zanusso1,
- Maria Teresa Conconi†1 and
- Karl Herbert Schäfer†2
© Di Liddo et al.; licensee BioMed Central. 2015
Received: 15 October 2014
Accepted: 14 January 2015
Published: 3 February 2015
In the last years, Wnt signaling was demonstrated to regulate inflammatory processes. In particular, an increased expression of Wnts and Frizzled receptors was reported in inflammatory bowel disease (IBD) and ulcerative colitis to exert both anti- and pro-inflammatory functions regulating the intestinal activated nuclear factor κB (NF-кB), TNFa release, and IL10 expression.
To investigate the role of Wnt pathway in the response of the enteric nervous system (ENS) to inflammation, neurons and glial cells from rat myenteric plexus were treated with exogenous Wnt3a and/or LPS with or without supporting neurotrophic factors such as basic fibroblast growth factor (bFGF), epithelial growth factor (EGF), and glial cell-derived neurotrophic factor (GDNF). The immunophenotypical characterization by flow cytometry and the protein and gene expression analysis by qPCR and Western blotting were carried out.
Flow cytometry and immunofluorescence staining evidenced that enteric neurons coexpressed Frizzled 9 and toll-like receptor 4 (TLR4) while glial cells were immunoreactive to TLR4 and Wnt3a suggesting that canonical Wnt signaling is active in ENS.
Under in vitro LPS treatment, Western blot analysis demonstrated an active cross talk between canonical Wnt signaling and NF-кB pathway that is essential to negatively control enteric neuronal response to inflammatory stimuli. Upon costimulation with LPS and Wnt3a, a significant anti-inflammatory activity was detected by RT-PCR based on an increased IL10 expression and a downregulation of pro-inflammatory cytokines TNFa, IL1B, and interleukin 6 (IL6). When the availability of neurotrophic factors in ENS cultures was abolished, a changed cell reactivity by Wnt signaling was observed at basal conditions and after LPS treatment.
The results of this study suggested the existence of neuronal surveillance through FZD9 and Wnt3a in enteric myenteric plexus. Moreover, experimental evidences were provided to clarify the correlation among soluble trophic factors, Wnt signaling, and anti-inflammatory protection of ENS.
In the last two decades, enormous progress has been made in the characterization of soluble factors that regulate gut functionality at physiological and inflamed conditions. Among signaling molecules, Wnt family proteins (Wnts) are reported to play a pivotal role in the development of gut [1,2] balancing the homeostasis of intestine epithelium . After the binding of Wnt ligands with the G protein-coupled receptor Frizzled (FZD) and single-span low-density lipoprotein receptor-related protein (LRP) [4,5], β-catenin is released from the large destruction “scaffolding” complex consisting of Axin, adenomatous polyposis coli (APC), casein kinase 1 (CK1), glycogen synthase kinase 3β (GSK3β) complex, and shuttles to the nucleus promoting the transcription of target genes [http://www.stanford.edu/~rnusse/wntwindow.html]. Moreover, under active inhibition induced by GSK3β or cross talked pathways [6-9], β-catenin is trapped at the plasma membrane or is tagged by phosphorylation for ubiquitin-mediated degradation .
Wnt ligands and Frizzled members have been largely studied in animal and human tissues [4,11]. Although the expression of Wnts, Frizzled, and Wnt-Frizzled binding specificities is well defined in epithelial and mesenchymal gut compartments , so far they have been poorly investigated in the enteric nervous system (ENS). The control of canonical Wnt pathway on intestinal tract has been first hypothesized observing β-catenin stabilization in familial and sporadic colon cancers . Furthermore, significant evidences have demonstrated that Wnt ligands control the proliferation, differentiation, and self-renewal of intestinal crypt progenitor cells [13-15], in both paracrine and autocrine fashion . As suggested by the increased expression of Wnts and Frizzled receptors in inflammatory bowel disease (IBD) and ulcerative colitis , Wnt signaling is involved in gut inflammation and exerts both anti- and pro-inflammatory functions. In particular, β-catenin has shown to i) negatively regulate intestinal NF-кB activity in bacterial-induced epithelial inflammation , ii) reduce TNFa release , and iii) induce the expression of IL10 and TGFB  while the activation of pro-inflammatory mediators seems to be correlated to noncanonical Wnt signaling .
The course of Wnt FZD signaling is dictated by the specificity of Wnt-FZD interactions, which is in turn governed by both cell type and stage of development [16,22-24]. Expressed in the dorsal neural tube at the time of neural crest emigration , Wnt3a activates β-catenin target genes that are involved in several processes such as the positioning/maturation of Paneth cells and the proliferation and differentiation of colonic stem cell compartment [3,26,27] but also the maintenance and expansion of enteric neural crest progenitor cells . As the release of Wnt3a increases in response to gut mucosa injury and inflammatory bowel diseases [29-31], the activation of canonical signaling pathway by Wnt3a could be hypothesized as an active mechanism in ENS.
Several studies have reported that Wnts preferentially activate the signaling pathway binding with defined members of FZD family according to a specific tissue distribution. Similarly to other Wnt ligands [32,33], a wide spectrum of FZD binding affinities for Wnt3a has been reported including FZD1 , FZD4, FZD5, and FZD8 . Among recently investigated Frizzled receptors, FZD9 has been detected early during the development of the mouse nervous system  and this expression pattern is highly conserved like in chicken and zebrafish . When FZD9 is activated by Wnt2, it leads to a Wnt/β-catenin signaling . Although FZD9 is widely expressed on both multipotent neuroepithelial precursor cells and neural-restricted precursors [36,38], it was so far not yet described in the ENS compartment.
Besides Wnt proteins, glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), fibroblast growth factors (FGF), epithelial growth factor (EGF), leukemia inhibitory factor (LIF), interleukin 6 (IL6), and lipopolysaccharide (LPS) from gram-negative enteric bacteria [39-44] modulate strongly the ENS or gut function. In case congenital or acquired defects reduce the bioavailability of enteric neurotrophic factors, a compromised ENS development [45-48] and a dysfunction of the immune system  are observed to promote the inhibition of GSK3β activity [50,51] and enterocolitis , suggesting that a possible cross talk between Wnt/β-catenin pathway and immune response could be involved.
In experimentally LPS-induced colitis, an increased enteric glia activation  and neuronal cell death due to an excessive stimulation of excitatory transmitters  are reported to occur. The endotoxin LPS is an essential membrane component of luminal microflora bacteria that interacts specifically with enteric responsive cells through toll-like receptor 4 (TLR4) [55-57] to regulate intestinal homeostasis. In neuronal compartments, low-dose LPS can contribute to maintain neuronal survival  and differentiation through the activation of NF-кB, while higher doses result in excessive stimulation of proinflammatory cytokines  that leads to neuronal toxicity . The involved intracellular signaling cascade includes the phosphorylation of mitogen-activated protein kinase (MAPK) and the migration of NF-κB into the nucleus . Studies performed on enterocytes  showed that the response to LPS triggers the inactivation of GSK3β through the activation of phosphatidylinositol 3 kinase (PI3K)/Akt pathway [63,64]. There is increasing evidence that the critical activators of NF-кB pathway, IKKα, and IKKβ interact differently with β-catenin by regulating its protein levels and cellular localization . In particular, unlike IKKβ that is predominantly cytoplasmic and activates NF-кB by phosphorylating IкBs under proinflammatory stimuli (TNFa, IL1, TLR agonists) , IKKα is detected in both nucleus and cytoplasm at resting state and inhibits β-catenin degradation mediated by Axin/APC/GSK3β, as well as it induces cyclin D1 expression that is a point of convergence between the Wnt/β-catenin and IкB pathways in mitogenic signaling . As soon as the IKK complex is activated in response to mitogens, inflammation, apoptosis, immune response, or cancer, IKKα is enhanced to shuttle from cytoplasmic to nuclear compartment where it phosphorylates nuclear p65 within the transactivation domains and promotes the transcriptional activity of NF-кB target genes. While IKKβ-dependent pathway is essential for activation of innate immunity, IKKα-dependent pathway is more important for the regulation of adaptive immunity .
Based on these evidences, the present study was focused on the expression and activity of Wnt signaling components such as FZD9 and Wnt3a during the ex vivo growth of postnatal ENS cells. In particular, standard settings and starvation of growth factors were used to simulate physiological and pathological conditions, respectively, as the availability of soluble factors is already demonstrated to control the structural integrity and functionality of ENS. Taking into consideration that canonical Wnt signaling controls stem cell proliferation/differentiation [68-70] and inflammation , the immunophenotypical characterization of ENS subpopulations , all components of LPS/TLR4 signaling  and β-catenin modulatory activity on NF-кB were explored to better define the ENS response to inflammation [73-75].
Isolation of cells from rat enteric nervous system
ENS cells (ENSc) were isolated from Sprague Dawley rats, 3 days old as previously described  and under Italian and German ethic committee authorization (CEASA 43/2012). The isolated cells were either cultured in standard (SM)  and basal conditions (BM) [Neuronal Base P (PAA, Cölbe, Germany), 1% L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin/streptomycin (Invitrogen Life Technology, Carlsbad, CA, USA) for 7 days before morphological analysis by optical microscopy, immunophenotypical characterization by flow cytometry (FCM) and Wnt signaling study by gene expression, Western blotting (WB), protein complex immunoprecipitation (Co-IP), and immunofluorescence assay (IF) were performed.
Immunophenotype characterization by FCM
Primary and secondary antibodies used for the immunophenotypical characterization of ENS cells
Mouse anti-rat NG2 FITC
Santa Cruz Biotechnology, Inc
Goat anti-rat Nanog PE
Mouse anti-rat CD34 PECy7
Rabbit anti-rat Sox2
Rabbit anti-rat Sox10
Santa Cruz Biotechnology, Inc
Rabbit anti-rat TLR4
Santa Cruz Biotechnology, Inc
Goat anti-rat Frizzled 9
Santa Cruz Biotechnology, Inc
Mouse anti-rat Nestin
Mouse anti-rat GFAP
Mouse anti-rat PAN neuronal
Rabbit anti-rat p75
Goat anti-mouse FITC
Santa Cruz Biotechnology, Inc
Goat anti-rabbit FITC
Santa Cruz Biotechnology, Inc
Bovine anti-goat FITC
Santa Cruz Biotechnology, Inc
Donkey anti-goat PE
Santa Cruz Biotechnology, Inc
ENS cells cultured for 7 days (T7) in SM or BM were fixed with BD Cytofix solution (BD Biosciences), for 20 min, at 4°C. All samples were double stained with primary goat anti-rat Frizzled 9 or rabbit anti-rat TLR4 (Santa Cruz Biotechnology, Inc, Dallas, TX, USA) and, then, indirectly conjugated with PE and FITC secondary antibodies (Santa Cruz Biotechnology, Inc), respectively. In parallel, single staining with primary rabbit anti-rat Wnt3a (Cell Signaling Technology, Inc, Danvers, MA, USA) and FITC-conjugated secondary antibody was performed. As negative controls, specimens stained only with FITC- and PE-conjugated secondary antibodies were prepared. After mounting with Fluoro-Gel II solution containing DAPI (EMS, Hatfield, PA, USA), the samples were analyzed using a Leica TCS SP5 confocal microscope (Leica, Wetzlar, Germany).
Formation of neurospheres under Wnt3a and LPS treatment
ENS cells were seeded at 16 × 103/cm2 and cultured for 14 days with or w/o 5 μg/mL LPS (Sigma-Aldrich) or 20 ng/mL Wnt3a (R&D System, Minneapolis, MN, USA). After 1 (T1), 7 (T7), and 14 (T14) days, the number of neurospheres was counted using an Olympus CKX 41 microscope (Olympus, Hamburg, Germany) equipped with a Moticam 2500 and Motic Images Plus 2.0 software (Motic, Wetzlar, Germany). In parallel, the diameter of neurospheres was measured using the image processing software ImageJ.
Wnt and LPS/TLR4 signaling pathway investigation
To investigate a possible interplay between Wnt and PI3K/Akt signaling pathways in ENSc, nuclear and cytoplasmic proteins were extracted from samples cultured in SM and BM and treated for 30 min, 1 h, and 2 h with 5 μg/mL LPS or 20 ng/mL Wnt3a. Proteins were obtained using NER PER Nuclear and Cytoplasmic Extraction Reagents kit and then quantified with BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocols. The separation of proteins was assessed by SDS/PAGE (Bio-Rad Laboratories, Inc, Hercules, CA, USA), and the immunoblot was carried out by overnight incubation, at 4°C, with primary rabbit anti-rat p(Ser9)-GSK3β (Cell Signaling Technology, Inc), mouse anti-rat p(Ser473)-Akt, β-catenin, p(Ser33)-β-catenin, NF-κB p50, and rabbit anti-rat NF-κB p65 (Santa Cruz Biotechnology, Inc) antibodies. The detection of target proteins was performed using peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (Bio-Rad Laboratories, Inc). The development of immunoreactivity was enhanced by chemiluminescence substrate (ECL) (Sigma-Aldrich) and then visualized by VersaDoc Imaging System. The protein expression level was normalized to housekeeping protein GAPDH (Millipore) or lamin B (Santa Cruz Biotechnology, Inc) and quantified using the image processing software ImageJ. Data were reported as ratio within target protein and relative housekeeping protein expression.
Co-IP: Wnt3a/Frizzled 9 (A) and NF-κB p65/β-catenin (B) binding assay
Total nuclear proteins were extracted from ENS cells cultured for 7 days (T7) using RIPA lysis buffer. (A) Recombinant Wnt3a protein (R&D System, Minneapolis, MN, USA) was added (200 ng/mL) to protein extracts and incubated overnight at 4°C. The immunoaffinity purification was performed as previously reported , using goat anti-rat Frizzled 9 (Santa Cruz Biotechnology, Inc) and rabbit anti-rat Wnt3a (Cell Signaling Technology, Inc) pre-immobilized onto Protein A Sepharose (Sigma-Aldrich). Western blot analysis was carried out using 4%–15% Mini PROTEAN® TGXTM Precast Gel (Bio-Rad Laboratories, Inc, Milan, Italy) and goat anti-rat Frizzled 9 (Santa Cruz Biotechnology, Inc) or rabbit anti-rat Wnt3a (Cell Signaling Technology, Inc). (B) For NF-κB p65/β-catenin binding assay, the immunoaffinity purification was performed using rabbit anti-rat NF-κB p65 or mouse anti-rat β-catenin (Santa Cruz Biotechnology, Inc), pre-immobilized onto Protein A Sepharose (Sigma-Aldrich). Both antibodies were used for Western blot analysis by 6.5% polyacrylamide gel (Bio-Rad Laboratories, Inc).
Gene expression study: RT-PCR and qPCR
Primer sequences for One-Step RT-PCR analysis
Glial cell-derived neurotrophic factor
Epidermal growth factor
Basic fibroblast growth factor
Nerve growth factor
Leukemia inhibitory factor
Primer sequences for qPCR analysis
Toll-like receptor 4
Wingless type MMTV integration site family, member 3A
Axis inhibition protein 2
Tumor necrosis factor α
Growth factors modulate the expression of FZD9 and TLR4 positive cells
Frizzled 9 is coexpressed with TLR4 in enteric neurons
Wnt3a and LPS enhance the proliferation of neurospheres
ENS-derived cells are usually cultured in vitro as aggregates known neurospheres that, including proliferating progenitors, neurons, and glial cells, mimic the in vivo niche of the gut. As growth factors and neurotrophic stimuli are demonstrated to be essential under in vivo settings for ENS functionality, they were used to study the regulation by Wnt signaling on the proliferation/differentiation of ENS cells under physiological conditions.
Canonical Wnt pathway is active in ENS cells and is modulated by growth factors
When the in vivo availability of soluble factors is restricted, the structural integrity of ENS is compromised with following inflammatory diseases. Using restricted culture conditions simulating a pathological disorder, we better defined the ENS response to inflammation.
BM conditions showed to specifically discriminate the effects of Wnt3a and LPS on ENS cells. Similar expression profiles of β-catenin, Akt, and GSK3β were observed in samples treated with Wnt3a and LPS (Figure 4B,C) suggesting an interplay between Wnt and NF-κB pathways. In fact, in both cases, the activation of Wnt signal involved the phosphorylation of GSK3β at Ser9, the nuclear translocation of β-catenin and NF-κB p65, and the phosphorylation tagging of β-catenin only at early phase of stimulation (T30′).
Wnt3a interferes with inflammatory ENS response
Exogenous Wnt3a exerts anti-inflammatory activity on ENSc
When intestinal homeostasis  is disrupted by severe inflammation, pathologic changes of ENS neurons [79-81] and glial cells  occur, compromising gut motility and secretion [73,82,83]. In concentration-dependent manner , LPS contributes to ENS plasticity  and targets enteric neuronal and glial populations through TLR4 [85,86] promoting the secretion of pro- and anti-inflammatory effectors [87,88]. Recently, higher gene expression of Wnt3a and FZD9 has been demonstrated in IBD disorders in comparison to healthy patients  suggesting the involvement of canonical Wnt signaling into gut inflammatory response.
The current study identified rat FZD9 as a novel marker associated with rat myenteric plexus and its ability to respond to Wnt3a ligand activating β-catenin signaling. It is known that in embryonic  and postnatal gut , the components of Wnt signaling are expressed in complex spatial and temporal patterns according to specific activities. Previous studies on Frizzled 9 in human and mouse have shown that it is highly expressed in the brain [21,91] and in neural precursor cells during developing nervous system [36,92]. Moreover, FZD9 protein has been localized at growth cones of regenerating adult spiral ganglion neurons  suggesting to be involved in neuronal response to damage.
As previously reported , the cultures of dissociated postnatal gut give rise to multipotent ENS progenitors generating in vitro neurons and glia. Based on FSC morphological discrimination and positive reactivity to anti-PAN neuronal marker by FCM analysis, the expression of FZD9 was demonstrated to characterize the neuronal compartment and to be preserved under BM and SM culture conditions.
During ENS development, there is a critical balance among migration, survival, proliferation, and differentiation of neural crest cells (NCCs). A fine control of these processes guarantees that a sufficient number of NCCs enter the foregut at the correct time while proliferation and differentiation are maintained at a balanced level at migration wavefront. It is known that the microenvironment plays a pivotal role to regulate the extent of ENS formation influencing on NCC number and gut colonization. Although neurotrophic factors are demonstrated to be essential for gut formation, the biological processes controlled by these factors are not yet completely elucidated [94,95]. In both BM and SM, a stem cell subset including enteric nervous stem cells (ENSCs) [96,97] is maintained during in vitro culturing. Numerous evidences suggest that germinal niches are adjacent to myenteric ganglia, and myenteric plexus-derived preparations are enriched of ENSCs. Using genetic fate mapping of Sox10, a neural crest cell marker , it has demonstrated that in postnatal gut, Sox10 positive cells can act as neuroprogenitor cells and regenerate neurons after damage. CD34 has been demonstrated to be expressed in hematopoietic stem cells [99,100], putative endothelial cells , and mesenchymal precursor cells . In the gut, CD34 expression has been reported either in interstitial Cajal cells together with cKit, CD44 , and ENSC  that, likely committed lineage cells [71,105-108], are commonly characterized by p75 [104,105] and Sox2 [106,109]. In our study, as previously shown by Hagl et al. , the expression of Nanog confirmed the existence of stem cells with high differentiative potentiality and the absence of cKit and CD44 led to consider CD34 as associated to an immature cell population rather than interstitial Cajal cells.
As suggested by the expression level of Sox10, p75, and GFAP, glial cells were stimulated under standard culture conditions while neuronal committed cells expressing Sox2, NG2 [110,111], and PAN neuronal marker were in vitro maintained and numerically increased when deprived of bFGF, EGF, GDNF, and NGF. These evidences suggested that a diminished availability of neurotrophic factors fostered neuronal commitment of progenitors but negatively controlled the cell differentiation process as shown by lacking neurosphere formation. In contrast, under standard culture conditions, glial differentiation was promoted more than neuronal and neurospheres were regularly formed.
Under Wnt3a (20 ng/mL) and LPS (5 μg/mL) stimulation, neurospheres were demonstrated to increase in number and size. After 1 day of culture, the neurosphere diameter was 10 μm bigger in LPS- and Wnt3a-treated samples than that observed in control. At T7, the number of neurospheres was doubled in LPS-treated samples compared to that detected under Wnt3a stimulation. Interestingly, a larger diameter of neurospheres was induced by Wnt3a with respect to LPS. The different proliferation effect could be indicative of a protective mechanism that, through the stimulation of immature cells but by the activation of different pathways, LPS  and Wnt3a  guaranteed a neural renewal.
Although FZD9 in human has been shown to bind Drosophila Wingless , the first evidence of its ability to activate Wnt/β-catenin signaling through the binding of Wnt2 ligand was obtained from Karasawa et al.  by an in vitro rat model.
The interaction between Wnts and Frizzled receptors is specific and differently involved in physiological and pathological conditions [3,113]. In our case, the demonstrated ability of FZD9 to bind Wnt3a and the presence of Wnt3a in glial cells provided the evidence of a possible protective role exerted by glia on enteric neurons through the activation of Wnt canonical pathway at physiological state or pathological conditions .
Wnt1 and Wnt3a are coexpressed at the dorsal midline of the developing neural tube and control the midbrain patterning and the formation of the paraxial mesoderm, respectively. When a deficiency of both Wnt1 and Wnt3a is observed, the development of neural crest derivatives is compromised suggesting that local Wnt signaling regulates the expansion of dorsal neural precursors. Despite the high number of mammalian Wnt and Frizzled members, a poor characterization of Wnt Frizzled binding specificities is reported.
In particular, it is known that canonical Wnt signaling controls crypt progenitor gene expression pattern . Recently, several studies have implied the involvement of canonical Wnt Frizzled signaling in inflammatory bowel disease (IBD) [21,31], as a significant increased expression of Wnt3a and Frizzled receptors are observed . In the current study, the nuclear translocation of β-catenin in rat ENS cells under stimulation with exogenous Wnt3a suggested that canonical Wnt pathway is active in postnatal myenteric plexus and Frizzled 9 could be involved as a specific ligand. The transcriptional regulation of FZD9 after the treatment with Wnt3a suggested that it might play a role in the context of inflammation. Only little is known about the expression and function of FZD9 in postnatal ENS. Among frizzled receptors, we demonstrated that FZD9 protein was significantly expressed on freshly extracted ENS cells and maintained in ex vivo cultured neuronal population. As previously reported , the regulation by Wnt signaling could be interpreted as protective and might be involved in the ENS defense. Activated Wnt3a/β-catenin signaling pathway is known to stimulate intestinal epithelial repair after wounding  and neural restricted precursor cell populations . In our study, the binding specificity of Frizzled 9 with Wnt3a was demonstrated in ENS compartment and suggested the involvement of FZD9 in neuronal response to inflammation in light of the evidence that a significant increase of Wnt3a and FZD9 is observed in inflammatory bowel diseases . The expression of TLR4 by the enteric neural cells highlighted the presence of a neural surveillance network that activates a cell-specific response against bacterial agents when the epithelial barrier permeability is altered . In the present study, the expression of both TLR4 and Frizzled 9 was detected in the neuronal population, while glial cells were characterized only by TLR4. Our evidences suggested that a neuronal subset was responsive to inflammatory agents through TLR-based mechanism and was regulated by Frizzled 9-mediated canonical Wnt signaling.
The cross regulation of Wnt and NF-кB signaling pathways has been shown to modify the biological effects of gene expression during development, immune function, inflammation, and carcinogenesis. A wide spectrum of phosphorylation/acetylation events and associating proteins is demonstrated to modulate the activation or inhibition of β-catenin, NF-кB, or their binding activity to DNA , independently on specific stimuli. In the last two decades, a lot of experimental data have reported that the interplay between Wnt signaling and NF-кB is expressed at different levels and is subject to variations in dependence of PI3K/Akt pathway, cell type specificity, and physiological/pathological conditions .
In the present work, we have demonstrated that canonical Wnt signaling is active in ENS and cross talks with NF-кB modulating the response to LPS. Phosphorylation events at serine 473 activate Akt and lock it at active conformation . In both standard and basal medium, the presence of nuclear β-catenin and NF-кB was observed in the absence of exogenous stimulation with Wnt3a and LPS and thus was correlated with active p(Ser473)-Akt.
As p(Ser9)-GSK3β was detected only in standard medium and p(Ser33)-β-catenin started to be expressed at T1h, we hypothesized that Akt controls phosphorylation events in resting cells promoting directly or indirectly both activation and degradation of β-catenin, besides nuclear translocation of NF-кB p65. It was demonstrated that Akt phosphorylates β-catenin at Ser552 leading first to its dissociation from cell-cell contacts and then to the increase of its binding to 14 3 3 proteins and its transcriptional activity . As GSK3β is active in resting cells and is inactivated through serine 9 phosphorylation induced by mitogens, growth factors, and several kinases, including Akt , we hypothesized that endogenous factors could contribute to regulated activation of Akt and control the stabilization of β-catenin by IKKα [65,67]. Wnt- and FGF-dependent PI3K Akt signaling pathways cross talk during a variety of cellular processes including neurogenesis  and both lead to GSK3β downregulation although depending on different phosphorylation events. In contrast to FGF signaling, the canonical Wnt is reported to be independent on Ser 9 phosphorylation. In the present work, we demonstrated that Wnt3a and LPS cross talk at the level of GSK3β and deactivate it by Ser9 phosphorylation, even if other phosphorylation events are not excluded because our in vitro model is not based on the inhibition of PI3K/Akt signaling pathway but only on the deprivation of main ENS trophic factors. This activity is hypothesized to be specifically dependent on the activation of Wnt and NF-кB signaling, probably modulated by GSK3β but not depending on exogenous growth factors such as bFGF and EGF as the evidences were taken from basal medium-treated samples. The concomitant presence of p(Ser33)-β-catenin and p(Ser9) GSK3β at early phase of Wnt3a and LPS stimulation suggested that alternative mechanisms of phosphorylation might occur promoting the negative control of β-catenin and thus its modulation. As the protein expression profile detected in standard and basal medium-treated samples showed a stronger accumulation of β-catenin at late phase of Wnt3a stimulation, GSK3β downregulation by simultaneous treatment with Wnt and growth factors is hypothesized to involve GSK3β pools differently modulated by Akt.
The critical role of Akt signaling for neuroprotection against deprivation of growth factors , oxidative stress , and ischemic injury  is evidenced from several in vitro studies on neuronal cell lines or primary cultures. Phosphorylation of Akt through phosphatidylinositol 3 kinase is essential to promote ENS precursor survival . Akt level expression was correlated with a constitutive activation of a neuroprotective mechanism as previously reported by Humbert et al. . Interestingly, in basal medium-treated samples, LPS response evidenced a superior expression level of nuclear β-catenin than that observed in ENS cells cultured in standard medium probably due to an interaction of Akt with GSK3β . As BM-based in vitro model could simulate pathological conditions, wherein the limited or absent supply of growth factors promotes gut inflammation, the higher nuclear translocation of β-catenin could be interpreted as a negative regulation of NF-кB p65 transcriptional activity.
In the present study, using Co-IP technique, we demonstrated that nuclear β-catenin and NF-кB p65 could complex together or are present as free proteins in resting ENS cells. The β-catenin/p65 complex and the free proteins resulted to be differently expressed in dependence on experimental conditions. It is already reported that β-catenin can physically interact with p65 and p50 subunits to inhibit the binding of NF-кB to DNA in IкBα-independent manner and in association with additional cellular factors . Interestingly, suppressed NF-кB activity and target gene expression were observed in cells expressing high level of β-catenin . Hwang et al.  have proposed a model of combinatorial binding of p65 and β-catenin showing that the specific transcriptional activity of β-catenin is regulated by protein-protein interaction with p65 at transcriptional level of their target genes. As the promoters of Wnt signaling target genes contain NF-кB binding elements, in the absence of specific stimulation, a constitutive binding of β-catenin/p65 complex to NF-кB sites is suggested to block β-catenin transcriptional activity. After Wnt signaling activation, the nuclear accumulation of β-catenin leads to the displacement of p65 from gene promoters and to the binding of β-catenin TCF transcriptional complex to DNA. Under LPS activation, IKKα-mediated phosphorylation of nuclear p65 subunit within the transactivation domains promotes the transient transcriptional activity of NF-кB target genes .
The major expression of β-catenin/NF-кB p65 complex in SM-treated samples was correlated to the greater accumulation of nuclear β-catenin and NF-кB p65 deriving from aspecific stimulation of NF-kB (or alternative NF-кB pathway) and Wnt signaling pathway exerted by trophic factors . Only the stimulation with LPS resulted into the activation of canonical NF-кB pathway, as demonstrated by the nuclear presence of NF-кB p50/p65. This evidence confirmed that a large diversity of signals might converge on degradation of IкBs, the known proteins that physically mask the nuclear localization signal (NLS) of NF-кB p65. However, the DNA binding activity of NF-кB to IкB sites, among a large excess of potential binding sites, is specifically controlled by NF-кB subunits, associated proteins, and binding to nuclear chromatin.
The specific responsiveness of ENS cells to Wnt3a or LPS stimulation was confirmed evaluating the transcriptional activity of nuclear β-catenin and p65 subunit in samples cultured in basal medium. Both canonical Wnt signaling and NF-кB pathway are expressed in ENS and control gut homeostasis . As the FZD9 expression was observed only in neuronal subpopulation while that of TLR4 resulted on both neurons and glial cells, we asked us which reciprocal regulation occurs between canonical Wnt pathway and NF-кB signaling in ENS neurons at physiological and inflamed conditions. The high level of GSK3β expression in brain tissue is likely due to its essential role in neuronal signaling. In neuronal cells, it is required for dendrite extension and synapse formation in newborns. Dysregulation of GSK3β expression leads to many pathological conditions, including neuronal dysfunction, Alzheimer’s disease , and Parkinson’s disease . Highly expressed in colorectal cancer [132,133], it has been shown to participate in NF-кB-mediated cell survival in pancreatic cancer . In cell culture studies, apoptosis is attenuated or fully abrogated by the inhibition of GSK3β in primary neurons  and neuronal cell lines . The Wnt pathway is involved in regulation of gut homeostasis. Under intestinal inflammation, the expression level of Wnt3a significantly increases  and neuronal apoptosis is observed .
Schaale et al.  described a regulatory role of Wnt signaling in inflammatory processes and reported that exogenous Wnt3a mediates anti-inflammatory effects in macrophages promoting the suppression of proinflammatory cytokines. Moreover, Li et al. demonstrated that Wnt signaling contributes to Alzheimer’s disease-related neurodegeneration by regulating neuroinflammation  and plays a crucial role in the expression of proinflammatory cytokines in the central nervous system . In our study, LPS treatment enhanced the gene expression of FZD9 and Wnt3a but reduced the level of TLR4 mRNA, suggesting a possible negative feedback scenario in which a positive regulation of Wnt pathway could be exerted by NF-кB signaling to restore homeostatic conditions. This hypothesis was further confirmed evaluating that the specific LPS-mediated inflammatory response was reversed in the presence of Wnt3a as demonstrated by a reduced expression of TNFa, IL6, and IL1B genes and, interestingly, an increased level of IL10 mRNA. As it is reported that LPS-treated enteric neurons respond stimulating the secretion of inflammatory cytokines such as IL6, TNFa  to activate glial cells that, in turn, release IL1B, the inhibitory activity exerted by Wnt3a could be interpreted as effective to control glial activation.
Taken together these considerations, we concluded that Wnt3a exerts a negative control of NF-кB transcriptional activity in rat myenteric plexus and contributes to ENS defense by the stimulation of IL10, TLR4, and FZD9 expression. In turn, LPS balances the inflammatory response stimulating Wnt signaling and enhancing the transcription level of FZD9 and Wnt3a.
The results of this study suggested the existence of neuronal surveillance through FZD9 and Wnt3a in enteric myenteric plexus. As detected in freshly isolated ENS cells, FZD9 was hypothesized to be expressed in constitutive manner for some homeostatic activities. Although the total number of FZD9 positive cells cultured under standard and basal conditions did not significantly change, the expression level of FZD9 was upregulated in response to GDNF, bFGF, and NGF, suggesting a possible involvement of Wnt signaling in neuronal and glial differentiation. Interestingly, under in vitro-simulated inflammation, Wnt signaling was demonstrated to exert an anti-inflammatory activity to negatively control NF-кB pathway.
Padua University (Grant ex60% Project) supported this work. We thank Prof. Pier Paolo Parnigotto and the Italian Association of Blood Donors (AVIS) of Veneto Region that contributed to acquire instrumental equipment (FACSCanto II cytometer). A special thanks to Silvia Barbon, Senthilkumar Rajendran, and Riccardo Sfriso for technical assistance.
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