Activation of MAP Kinase (ERK1/2) in human neonatal colonic enteric nervous system Authors

The aim of this study was to examine MAP kinase (ERK1/2) activation in the human neonatal colonic enteric nervous system. For this, we investigated by immunocytochemistry the cellular localization of phosphorylated ERK1/2 (P-ERK) in a series of normal human colon samples removed from newborns and in patients with intestinal obstruction such as Hirschsprung’s disease (HSCR), stenosis and atresia. We checked the presence of P-ERK in the 3 distinct histological layers of normal colon. P-ERK was detected in the colonic mucosa, in the enteric nervous system and in endothelial cells. In the mucosa from normal colon, PERK was detected at the upper part of the crypt, while P-ERK activation in epithelial cells is altered in HSCR, stenosis and atresia. In the normal colon, strong P-ERK staining was detected in myenteric and submucosal enteric plexuses. Using confocal microscopy analyses, we observed that P-ERK staining was localized in enteric glial cells and not in enteric neurons. Strong P-ERK staining was also observed in plexuses from stenosis and atresia whereas in HSCR, hypertrophic nerve fibres were not stained. in se rm -0 03 23 86 4, v er si on 1 23 S ep 2 00 8 The motility of the gastrointestinal tract is ensured by the correct coordination of the visceral smooth muscle cells and the autonomous enteric nervous system. The enteric nervous system (ENS) originates from neural crest cells that migrate from the dorsal region of the neural tube and colonize the whole gut to establish its innervation. At week 4, the neural crest-derived cells migrate into the gut mesoderm layer and colonize the gut along a craniocaudal wave. These cells reach the hindgut by week 7, form two cluster rings and finally will differentiate into enteric neurons and glial cells. Intestinal obstructions in infants comprise a wide group of heterogeneous diseases with clinical symptoms ranging from simple constipation to intestinal occlusion. Intestinal obstruction may be due to mechanical diseases such as stenosis, stricture, atresia or to nonmechanical diseases of which Hirschsprung disease (HSCR) is a particular case. HSCR is due to an absence of the enteric nervous system along certain lengths of the bowel. In addition to the absence of ganglion cells, which constitutes the diagnostic criteria, HSCR is characterized by the presence of numerous hypertrophied nerve fibres. Despite the low prevalence, 1/5,000 live births, HSCR is the best described of the congenital neural abnormalities of the intestinal tract and has been widely studied. The discovery that HSCR was due to a failure of migration of cells derived from the neural crest cells, has lead to the identification of molecular and cellular events essential for the normal development of the enteric nervous system (ENS). MAPK extracellular signal-regulated-kinases, ERK1 (p44) and ERK2 (p42) are pivotal compounds of intracellular phosphorylation cascades from the cytoplasm to the nucleus recruited for growth factor signal transduction. Several signalling pathway ligands such as Fibroblast growth factor (FGF), Epidermal growth factor (EGF), Platelet-derived growth factor (PDGF) or Transforming growth factor β (TGFβ) are know to initiate receptor tyrosine kinase (RTK) and mitogen-activated protein kinase (MAPK) activation. After extracellular stimulation, ERK1/2 are both activated by dual phosphorylation (termed P-ERK) in se rm -0 03 23 86 4, v er si on 1 23 S ep 2 00 8 by the mitogen-activated kinase MEK1 and 2. Such phosphorylation strongly increases ERK1/2 activity, induces its nuclear translocation and subsequently triggers specific gene expression programs. In addition, activated ERK1/2 in neurons can stay in the cytoplasm to act locally. Since published studies concerning MAPK activation report the detection of ERK1/2 in cultured intestinal epithelial cell lines, central neurons and prospective and nascent hepatic endoderm of mouse embryo, we investigated the cellular localization of P-ERK by immunocytochemistry and immunofluorescence in the 3 distinct histological layers of normal human colon samples, paying particular attention to enteric plexuses. Also, we checked the presence of P-ERK in the enteric nervous system in cases of intestinal obstruction such as HSCR, stenosis and atresia. Materials and Methods Human tissues Tissue samples were obtained from 19 newborns, aged 12 days to 2-months old and are summarised in Table 1. Normal samples were right colon specimens from 5 neonates undergoing ileocolic resection for congenital cystic duplication of the terminal ileum. In eight patients, after HSCR was diagnosed, rectosigmoïdectomy was performed and full-thickness large bowel specimens were collected in the aganglionic as well as in ganglionated zones. Full-thickness large bowel samples were also isolated from six patients undergoing segmental left colectomy for mechanical stenosis, in 4 cases of post-necrotizing enterocolitis (NEC) stenosis and 2 cases of atresia. in se rm -0 03 23 86 4, v er si on 1 23 S ep 2 00 8 Immunohistochemistry Paraffin embedded sections (3 μm thick) were immunostained by standardized automated procedures using a Dako autostainer (Universal staining system, Dakocytomation, Trappes, France), as previously described. Antigen retrieval was achieved by heating at 98°C for 1h in 1mM EDTA (pH 9.0). P-ERK, (#4376, Clone 20G11, Cell Signaling, USA), a rabbit polyclonal antibody which detects phosphorylated forms of ERK1 and ERK2 MAP Kinases was used diluted 1:100 as primary antibody. Immunohistochemistry control experiments were performed by excluding the primary antibody (data not shown). In the competition experiments, phospho-p44/42 MAPK blocking peptide (Cell Signalling, Boston, MA, USA) was added to the primary antibody incubation, diluted 1:50. For immunofluorescence, de-paraffined slides after antigen retrieval (1h at 98°C in 1mM EDTA, pH 9,0) were incubated 30 min at room temperature with P-ERK (1:100 dilution) and one of the primary antibodies: HuC/D (mouse, clone 16A11, Abcam, UK) diluted 1:20, synaptophysin (mouse, clone SY38, DAKO, Denmark) diluted 1:10, c-Kit (mouse, clone CD117, Zymed, USA) diluted 1:100, glial marker S100 (mouse, clone 4C4.9, Clinisciences, France) diluted 1:100 and α-Smooth Muscle Actin (mouse, clone 1A4, Sigma, USA) diluted 1:400. After washing, slides were incubated 30 min with Alexa 488 anti-mouse (Invitrogen, France, 1:2000 dilution) and Alexa 555 anti-rabbit (Invitrogen, France, 1:2000 dilution). Cells were rinsed again, mounted in FluorSave reagent (Calbiochem, Germany). Tissues were visualized under a laser confocal microsope (Leica SP2). Fluorescence from each channel was captured sequentially to avoid cross-talk between channels. Excitation of Alexa 488 was from an argon laser (488 nm) and emitted light was collected through a band pass filter (512 nm). Alexa 555 was from a helium-neon laser (excitation 555 nm) and light collected via a band pass filter (beyond 560 nm). No cross-talk between channels was detected with these settings. in se rm -0 03 23 86 4, v er si on 1 23 S ep 2 00 8