Transforming growth factor (TGF-) signaling acts through Smad proteins to play fundamental roles in cell proliferation, differentiation, apoptosis, and metabolism. The Receptor associated Smads (R-Smads) interact with DNA and other nuclear proteins to regulate target gene transcription. Here, we demonstrate that the Caenorhabditis elegans R-Smad DAF-8 partners with the nuclear hormone receptor NHR-69, a C. elegans ortholog of mammalian hepatocyte nuclear factor 4 HNF4), to repress the
exp-2 potassium channel gene and increase insulin secretion. We find that NHR-69 associates with DAF-8 both in vivo and in vitro. Functionally,
daf-8 nhr-69 double mutants show defects in neuropeptide secretion and phenotypes consistent with reduced insulin signaling such as increased expression of the
sod-3 and
gst-10 genes and a longer life span. Expression of the
exp-2 gene, encoding a voltage-gated potassium channel, is synergistically increased in
daf-8 nhr-69 mutants compared to single mutants and wild-type worms. In turn,
exp-2 acts selectively in the ASI neurons to repress the secretion of the insulin-like peptide DAF-28. Importantly,
exp-2 mutation shortens the long life span of
daf-8 nhr-69 double mutants, demonstrating that
exp-2 is required downstream of DAF-8 and NHR-69. Finally, animals over-expressing NHR-69 specifically in DAF-28-secreting ASI neurons exhibit a lethargic, hypoglycemic phenotype that is rescued by exogenous glucose. We propose a model whereby DAF-8/R-Smad and NHR-69 negatively regulate the transcription of
exp-2 to promote neuronal DAF-28 secretion, thus demonstrating a physiological crosstalk between TGF- and HNF4-like signaling in C. elegans. NHR-69 and DAF-8 dependent regulation of
exp-2 and DAF-28 also provides a novel molecular mechanism that contributes to the previously recognized link between insulin and TGF- signaling in C. elegans.