Glial cell line-derived neurotrophic factor promotes barrier maturation and wound healing in intestinal epithelial cells in vitro

Abstract

Recent data suggest that neurotrophic factors from the enteric nervous system are involved in intestinal epithelial barrier regulation. In this context the glial cell line-derived neurotrophic factor (GDNF) was shown to affect gut barrier properties in vivo directly or indirectly by largely undefined processes in a model of inflammatory bowel disease (IBD). We further investigated the potential role and mechanisms of GDNF in the regulation of intestinal barrier functions. Immunostaining of human gut specimen showed positive GDNF staining in enteric neuronal plexus and in enterocytes. In Western blots of the intestinal epithelial cell lines Caco2 and HT29B6, significant amounts of GDNF were detected, suggesting that enterocytes represent an additional source of GDNF. Application of recombinant GDNF on Caco2 and HT29B6 cells for 24 h resulted in significant epithelial barrier stabilization in monolayers with immature barrier functions. Wound-healing assays showed a significantly faster closure of the wounded areas after GDNF application. GDNF augmented cAMP levels and led to significant inactivation of p38 MAPK in immature cells. Activation of p38 MAPK signaling by SB-202190 mimicked GDNF-induced barrier maturation, whereas the p38 MAPK activator anisomycin blocked GDNF-induced effects. Increasing cAMP levels had adverse effects on barrier maturation, as revealed by permeability measurements. However, increased cAMP augmented the proliferation rate in Caco2 cells, and GDNF-induced proliferation of epithelial cells was abrogated by the PKA inhibitor H89. Our data show that enterocytes represent an additional source of GDNF synthesis. GDNF contributes to wound healing in a cAMP/PKA-dependent manner and promotes barrier maturation in immature enterocytes cells by inactivation of p38 MAPK signaling.

The glial cell-line derived neurotrophic factor: a novel regulator of intestinal barrier function in health and disease

“…In an in vitro model the IEB tight junction proteins claudin 1, claudin 5, and occludin showed an augmented and more linear staining pattern following GDNF application, whereas overall concentrations of these proteins were not altered. This was shown to be mediated by the p38 MAPK pathway due to a significant dephosphorylation of p38 MAPK after the application of GDNF in vitro (36). The latter mechanism has been previously shown to stabilize the intestinal epithelial barrier (51).…”

“…Additionally, GDNF induced a cAMP/PKA-dependent increase of proliferation and augmented epithelial wound healing. An important observation was that under mature barrier conditions, as they are usually seen in the villi, GDNF did not alter IEB function or proliferation (36).…”

“…On the other hand, none of the proinflammatory cytokines stimulated an upregulation of GDNF (52). A recent study demonstrated that both EGCs and enterocytes express GDNF in significant amounts under physiological conditions (36). However, the stimuli leading to GDNF expression in enterocytes still remain unclear.…”

“…Additionally, GDNF prevented the hypoxia-induced downregulation of ZO1 (65). More evidence arguing for direct effects of GDNF on enterocytes derived from the observation that enterocytes express the RET receptor, as well as GDNFsusceptible receptors GFR␣1 and GFR␣2 (36,52). A recent study demonstrated that GDNF has direct effects on enterocytes, especially under conditions of immature barrier functions, which are usually seen in the crypts of the intestine.…”

Breastfeeding and the gut-brain axis: is there a role for melatonin?

“…DHA levels also positively correlate with many markers of immune function in infants, including when DHA has been added to formula feed (68). Both DHA (69) and GDNF (70) contribute to the maintenance of the gut barrier. However, stress-mediated increases in gut permeability in rodents are driven by increased NGF release from neighboring mast cells (71), suggesting that trophic factors may also contribute to the plasticity processes associated with increased gut permeability.…”