The bone morphogenetic protein (BMP) family is a class of transforming growth factor (TGF-␤) superfamily molecules that have been implicated in neuronal differentiation. We studied the effects of BMP2 and glial cell line-derived neurotrophic factor (GDNF) on inducing differentiation of enteric neurons and the signal transduction pathways involved. Studies were performed using a novel murine fetal enteric neuronal cell line (IM-FEN) and primary enteric neurons. Enteric neurons were cultured in the presence of vehicle, GDNF (100 ng/ml), BMP2 (10 ng/ml), or both (GDNF ϩ BMP2), and differentiation was assessed by neurite length, markers of neuronal differentiation (neurofilament medium polypeptide and ␤-III-tubulin), and neurotransmitter expression [neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), choline acetyltransferase (ChAT) and Substance P]. BMP2 increased the differentiation of enteric neurons compared with vehicle and GDNF-treated neurons (P Ͻ 0.001). BMP2 increased the expression of the mature neuronal markers (P Ͻ 0.05). BMP2 promoted differentiation of NPY-, nNOS-, and TH-expressing neurons (P Ͻ 0.001) but had no effect on the expression of cholinergic neurons (ChAT, Substance P). Neurons cultured in the presence of BMP2 have higher numbers of TH-expressing neurons after exposure to 1-methyl 4-phenylpyridinium (MPP ϩ ) compared with those cultured with MPP ϩ alone (P Ͻ 0.01). The Smad signal transduction pathway has been implicated in TGF-␤ signaling. BMP2 induced phosphorylation of Smad1, and the effects of BMP2 on differentiation of enteric neurons were significantly reduced in the presence of Smad1 siRNA, implicating the role of Smad1 in BMP2-induced differentiation. The effects of BMP2 on catecholaminergic neurons may have therapeutic implications in gastrointestinal motility disturbances.immorto; glial cell line-derived neurotrophic factor; bone morphogenic protein 2; Smad1 ENTERIC NEURONS ARE DERIVED from vagal, sacral, and upper thoracic neural crest cells that migrate into the gut, where they proliferate and then differentiate into a variety of distinct cell types (2,17,22,23). Several growth factors influence the proliferation, differentiation, and survival of the enteric nervous system (ENS), including glial cell line-derived neurotrophic factor (GDNF), neurturin, neurotrophin-3, and Sonic hedgehog (14, 18). Bone morphogenetic proteins (BMPs), members of the transforming growth factor-␤ (TGF-␤) superfamily, are regulatory molecules that play critical roles in the formation and regionalization of the gut before its colonization by crest-derived cells (26,27). BMP2 and 4 continue to be expressed in the fetal gut and regulate the specification, migration, differentiation, and aggregation of enteric neurons (12,13,15,24).We have recently developed an enteric neuronal cell line that has characteristics similar to primary enteric neurons (4). We sought to determine the types of growth factors that would result in differentiation of this novel enteric neuronal cel...

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