Inhibition of cGMP-dependent protein kinase reduces the response to sonic hedgehog in neuralized embryoid bodies.

Previously, we have shown that increasing the intracellular cGMP concentration enhances the sonic hedgehog (Shh) response in neural plate cells. The use of two mouse embryonic stem (ES) cell lines allowed a highly sensitive and reproducible quantification of the Shh response in neuralized embryoid bodies. Here we demonstrate that the specific, membrane-permeable cGMP-dependent protein kinase G-Ialpha (PKG-Ialpha) inhibitor DT-2 prevents an efficient Shh response, indicating that the effects of cGMP on the Shh response are mediated via PKG. We also demonstrate that the PKG acts upon the Shh response upstream of the Ptc1 promoter, which is up-regulated invariably and early in response to Shh, significantly limiting the targets for PKG phosphorylation to molecules involved in the early steps of the Shh response. These effects of cGMP and PKG are antagonistic to those of cAMP and PKA, and thus provide a mechanism by which the sensitivity of cells to the effects of Shh can be regulated, by modulating the intracellular cyclic nucleotide concentration.