BMP-Akt pathway cooperativity in a Drosophila model of epithelial plasticity

Objective: Transforming Growth Factor-β (TGF-β) and Bone Morphogenetic Protein (BMP) cooperate with Akt signaling in many systems of epithelial plasticity (EP) and Epithelial-to-Mesenchymal Transition (EMT), such as palate formation and oral cancers. We address the mechanism of BMP-Akt pathway cooperativity using a simple Drosophila melanogaster model of EP in cell culture and thorax closure in vivo. The goal was to confirm BMP-Akt cooperativity in the system and rule out that Akt signaling affects BMP-induced Mad phosphorylation or nuclear translocation, hypothesizing that Akt signaling acts at the level of Mad complex transcriptional activation.

Method: Genes of the BMP (Drosophila Dpp) or Akt pathway were silenced by RNAi in KaBrü1D Drosophila epithelial cells along with +/- BMP stimulation (90min, 5d). Cell shape changes and Mad phosphorylation/nuclear translocation were monitored using immunocytochemistry (E-Cadherin, actin, p-Mad). The role of BMP (Dpp) and Akt pathway genes during thorax closure were examined, silencing genes by RNAi or expression of dominant-negative transgenes using the UAS-GAL4 system. Phenotypes were assessed using GFP and CellMask membrane labeling in dissected pupae and imaging the morphology of the adult thorax.

Result: RNAi silencing of Akt or the downstream TOR or Raptor suppressed BMP-induced cell elongation in culture and caused thorax closure defects in vivo, phenocopying suppression of BMP (Dpp) signaling. However, Mad was correctly phosphorylated and translocated into the nucleus. Double-silencing experiments to examine pathway synergy in vivo are in progress.

Conclusion: We confirmed BMP-Akt cooperativity during EP and ruled out that Akt signaling affects BMP-induced Mad phosphorylation or nuclear translocation. Therefore, we hypothesize that Akt signaling acts at the level of Mad complex transcriptional activation, which is currently being tested by ChIP-seq/RNA-seq in a collaboration with J. Zeitlinger (Stowers Institute) and J. Song (UCSF).