Gial cell line-derived neutrotrophic factor expression in proliferating intestinal smooth muscle cells is important for enteric neuron survival

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Han, Tian Yu
Enteric nervous system , ISMC proliferation , intestinal inflammation , TNBS-induced colitis , GDNF , myenteric plexus , Enteric neuron
Normal intestinal functions are coordinated by enteric neurons within the enteric nervous system (ENS). In the embryonic and neonatal gut, enteric neuron survival is dependent on the expression of glial cell line-derived neurotrophic factor (GDNF) from its targets of innervation - the intestinal smooth muscle cells (ISMC). In the inflamed adult intestine, enteric neuron loss is immediately followed by ISMC proliferation, resulting in severe disruption of normal intestinal functions. Although GDNF can support the survival of postnatal enteric neurons, whether adult ISMC can secrete GDNF and support neuron survival is unclear. Results from qPCR analysis showed that freshly isolated adult ISMC have acquired the ability to express GDNF at the onset of proliferation, in vitro. Western blot analysis indicates that GDNF continues to be upregulated in ISMC at Passage 2 (P2), but its expression is decreased after long periods of proliferation at Passage 10 (P10). A neuron survival bioassay suggests that GDNF expression is correlated with enteric neuron survival. Results showed that P2 ISMC or conditioned media (CM) - but not P10 ISMC and CM, significantly increased enteric neuron survival. In subsequent experiments, the RET tyrosine kinase inhibitor vandetanib was used to block GDNF receptor-ligand interactions, and anti-GDNF neutralizing antibody was used to sequester soluble GDNF within the culture media. Both methods were successful at decreasing myenteric neuron survival. Furthermore, abolishing GDNF expression in P2 ISMC with GDNF siRNA also resulted in a decreased myenteric neuron survival. The above observations suggest that ISMC-derived GDNF is important in supporting myenteric neuron survival in vitro.
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