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

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.Electronic supplementary materialThe online version of this article (10.1007/s00018-018-2761-8) contains supplementary material, which is available to authorized users.

Primary Cilia as Signaling Hubs in Health and Disease

“…[208] Growth factor and morphogen signaling are highly context dependent. [212] USP8 is also activated by EGF, PDGF, and FGF signaling. [15,19,22] A primary cilium can express various receptors for growth factors and morphogens.…”

Crosstalk of Hedgehog and mTORC1 Pathways

“…Thus, Tsc1 KD in zebrafish leads to phenotype characteristics of ciliary mutants [ 4 ] and mouse embryonic fibroblasts (MEFs) lacking either Tsc1 or Tsc2 display a phenotype of enhanced ciliary formation [ 5 ]. Our own results also indicate a connection between mTORC1 and the primary cilium, as we observed an elongated ciliary phenotype in cells lacking Tsc1 and a shortened ciliary phenotype in cells lacking Tsc2 [ 6 ].…”

“…We have recently demonstrated that mutations in either Tsc1 or Tsc2 inhibit Hh signaling [ 6 ]. We demonstrated that reduced Hh signaling in Tsc1 -defective MEFs is due to a reduced amount of the Hh initiation transcription factor Gli2.…”

“…The factors GLI2-FL and GLI3-FL are subsequently, by phosphorylation, activated (GLI2-A and GLI3-A) and translocated to the nucleus, and transcriptional activation of the Hh target genes including GLI1 and PTCH are initiated [ 33 , 34 , 35 ]. Expression of GLI1 is routinely used as a marker for Hh activity [ 6 ].…”

Effect of Electroacupuncture Treatment at Dazhui (GV14) and Mingmen (GV4) Modulates the PI3K/AKT/mTOR Signaling Pathway in Rats after Spinal Cord Injury

“…mTORC2 is related to the construction of the cell skeleton and cell movement, while mTORC1 is the main regulator of cell proliferation, apoptosis, and autophagy [21]. mTORC1 could be activated by Ras homology enriched in brain (Rheb) enrichment, and AKT could phosphorylate tuberous sclerosis complex (TSC1/2), which reduces negative regulation to Rheb enrichment from TSC1/2 [11,22]. AKT also could activate mTORC1 by phosphorylating PRAS40 to reduce competitive binding of PRAS40 and mTORC1 [23].…”