TSC1 and TSC2 regulate cilia length and canonical Hedgehog signaling via different mechanisms

Søren Tvorup Christensen,Lisbeth Birk Møller,Thomas Rosengren,Lotte Bang Pedersen,Lasse Jonsgaard Larsen

doi:10.1007/s00018-018-2761-8

Abstract

Primary cilia are sensory organelles that coordinate multiple cellular signaling pathways, including Hedgehog (HH), Wingless/Int (WNT) and Transforming Growth Factor-β (TGF-β) signaling. Similarly, primary cilia have been implicated in regulation of mTOR signaling, in which Tuberous Sclerosis Complex proteins 1 and 2 (TSC1/2) negatively regulate protein synthesis by inactivating the mTOR complex 1 (mTORC1) at energy limiting states. Here we report that TSC1 and TSC2 regulate Smoothened (SMO)-dependent HH signaling in mouse embryonic fibroblasts (MEFs). Reduced SMO-dependent expression of Gli1 was demonstrated in both Tsc1−/− and Tsc2−/− cells, and we found that Tsc1 is required for TGF-β induced phosphorylation of SMAD2/3 and subsequent expression of the HH signaling effector and transcription factor GLI2. Hedgehog signaling was restored in Tsc1−/− cells after exogenous expression of Gli2, whereas rapamycin restored HH signaling in Tsc2−/− cells. Furthermore, we observed that Tsc1−/− MEFs display significantly elongated cilia, whereas cilia in Tsc2−/− MEFs were shorter than normal. The elongated cilium phenotype of Tsc1−/− MEFs is likely due to increased mTORC1-dependent autophagic flux observed in these cells, as both the autophagic flux and the cilia length phenotype was restored by rapamycin. In addition, ciliary length control in Tsc1−/− MEFs was also influenced by reduced expression of Gli2, which compromised expression of Wnt5a that normally promotes cilia disassembly. In summary, our results support distinct functions of Tsc1 and Tsc2 in cellular signaling as the two genes affect ciliary length control and HH signaling via different mechanisms.

Highlights

Mutations in the tumor suppressor genes encoding TSC1 (Hamartin) and TSC2 (Tuberin) cause a multisystemic tumor syndrome termed tuberous sclerosis complex (TSC)
Tsc1−/− and Tsc2−/− mouse embryonic fibroblasts (MEFs) were starved (0.5% fetal bovine serum, FBS) for 48 h and analyzed by SDS-PAGE and western blotting (WB) for the expression of Tsc1 and Tsc2, and the phosphorylation status of S6, which is a marker for mTOR complex 1 (mTORC1) activity (Fig. 1a)
The heterodimeric TSC complex that consists of TSC1 (Hamartin) and TSC2 (Tuberin) plays a critical role in coordinating extracellular signals with intracellular energy status to control cell proliferation and growth [48]

Summary

Introduction

Mutations in the tumor suppressor genes encoding TSC1 (Hamartin) and TSC2 (Tuberin) cause a multisystemic tumor syndrome termed tuberous sclerosis complex (TSC). TSC1 and TSC2 were originally found to form a heterodimeric complex that acts as a switch for turning off mTOR signaling by inactivating the mTOR complex 1 (mTORC1). The heterodimeric TSC complex negatively regulates mTORC1 activity via the GTPase activity of TSC2, towards the small G-protein RHEB (Ras homologue enriched in brain) [5]. In the presence of nutrients, mTORC1 is activated and promotes cell growth, including protein synthesis and energy storage. TSC1 was demonstrated to be required for the proper activation of the transforming growth factor-β (TGF-β)-SMAD2/3 pathway in HeLa cells [11]. Despite these advances, the roles of TSC1 and TSC2 in regulating signaling pathways independently of mTORC1/2 remain poorly understood

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