Introduction and objectives The majority of inherited cases of pulmonary arterial hypertension (PAH) are caused by mutations that affect the Bone Morphogenetic Protein (BMP) receptor 2. Recently, patients with pulmonary veno-occlusive disease (PVOD), a rare subtype of pulmonary hypertension, were found to have mutations of EIF2AK4. This gene encodes the kinase GCN2, which regulates cellular responses to amino acid availability by phosphorylating the translation initiation factor eIF2a. Phosphorylation of eIF2a reduces protein synthesis and induces expression of a transcription factor called ATF4. It is unclear if BMP and GCN2 signalling are functionally related and so we set out to test this using the model organism Drosophila melanogaster.
Methods We examined the development of wing veins in Drosophila because this is known to depend on BMP signalling. We manipulated the expression of dGCN2 and its antagonistic phosphatase dPPP1R15 by RNA interference. Morphology of the wing veins was examined in adult flies and early developmental signalling was measured in pupating animals. Similar experiments performed in animals with defective BMP signalling tested for a genetic interaction between eIF2a phosphorylation and BMP signalling.
Results We observed that depletion of dGCN2 in the developing wing by RNA interference enhanced BMP signalling as evidenced by hyperphosphorylation of MAD, the Drosophila orthologue of SMAD1. This was associated with development of ectopic wing veins in the regions of RNAi expression. In contrast, knock down of the eIF2a phosphatase dPPP1R15 selectively impaired anterior crossvein formation. The effects of dPPP1R15 depletion were cell-autonomous suggesting an effect on downstream BMP signalling rather than on ligand secretion. The vein phenotypes were highly sensitive to manipulation of BMP signalling. Both in insect and mammalian cells, ATF4 expression inhibited BMP signalling suggesting a mechanism for the crosstalk between BMP and GCN2.
Conclusion We have discovered in Drosophila that GCN2 activation modulates BMP signalling. This effect is mediated, at least in part, by the downstream transcription factor ATF4, which inhibits the phosphorylation of MAD (insect SMAD1). Our findings indicate that this pathway is conserved between insects and mammals and this model may shed light on the pathogenesis of PAH and PVOD.
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