The interaction of Hedgehog and mTor signaling in healthy hepatocytes

Introduction:

The Hedgehog (Hh) pathway is one of the central morphogenetic pathways. It plays a key role in embryonic development and is a regulator of regeneration and stem cell function in adult tissues. Abnormal Hh Signaling contributes to different diseases like hepatic steatosis and cancer. Studies showed various interactions of the Hh pathway and the signaling of the mechanistic target of rapamycin (mTor) in different cancer types. The mTor pathway is an important mediator between nutritional signals and their appropriate cellular responses. However, the mechanisms behind the interaction of Hh and mTor Signaling are poorly understood, particularly in healthy tissue. In our studies, we demonstrate synergistic effects of both pathways in controlling healthy liver metabolism.

Methods:

Primary hepatocytes from C57Bl6/N mice were treated with the Hh inhibitor Cyclopamine, the mTorC1 inhibitor Rapamycin, the mTorC1/C2 inhibitor Torin and the combinations thereof. The cells were analyzed using Western Blots, qPCR and Seahorse technology.

Results:

Our experiments show synergistic inhibition by Cyclopamine and Rapamycin of p70S6 phosphorylation, a downstream kinase of mTorC1. Cyclopamine alone and Rapamycin alone have no or only a weak influence on the phosphorylation, respectively. When we repress mTorC1 and C2 with Torin, we see the same effect as with Cyclopamine and Rapamycin. However, the addition of Cyclopamine to Torin has no further influence on the phosphorylation state. In addition, we see this effect also in the phosphorylation state of Rictor, an mTorC2 component. Moreover, although the inhibition of the Hh pathway alone has no impact on the functionality of mitochondria, our Seahorse analyses show an increased inhibition of the electron transport chain in cells incubated with Rapamycin and Cyclopamine compared to Rapamycin alone.

Conclusion:

We suppose that the minor impact of Rapamycin on mTor pathway activity is an effect of the feedback mechanism via mTorC2. Therefore, our results suggest a possible interaction of the Hh pathway with mTorC2 signaling. We hypothesize that the Hh pathway may influence the energy metabolism and other cellular responses through the modulation of mTor signaling. This should be considered as potential side effects when using Hh pathway inhibitors as part of therapeutic intervention in cancerous diseases.