Tuberous sclerosis complex inactivation disrupts melanogenesis via mTORC1 activation
Juxiang Cao, … , Hans R. Widlund, David J. Kwiatkowski
Juxiang Cao, … , Hans R. Widlund, David J. Kwiatkowski
Published December 5, 2016
Citation Information: J Clin Invest. 2017;127(1):349-364. https://doi.org/10.1172/JCI84262.
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Research Article Dermatology Genetics

Tuberous sclerosis complex inactivation disrupts melanogenesis via mTORC1 activation

Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor-suppressor gene syndrome caused by inactivating mutations in either TSC1 or TSC2, and the TSC protein complex is an essential regulator of mTOR complex 1 (mTORC1). Patients with TSC develop hypomelanotic macules (white spots), but the molecular mechanisms underlying their formation are not fully characterized. Using human primary melanocytes and a highly pigmented melanoma cell line, we demonstrate that reduced expression of either TSC1 or TSC2 causes reduced pigmentation through mTORC1 activation, which results in hyperactivation of glycogen synthase kinase 3β (GSK3β), followed by phosphorylation of and loss of β-catenin from the nucleus, thereby reducing expression of microphthalmia-associated transcription factor (MITF), and subsequent reductions in tyrosinase and other genes required for melanogenesis. Genetic suppression or pharmacological inhibition of this signaling cascade at multiple levels restored pigmentation. Importantly, primary melanocytes isolated from hypomelanotic macules from 6 patients with TSC all exhibited reduced TSC2 protein expression, and 1 culture showed biallelic mutation in TSC2, one of which was germline and the second acquired in the melanocytes of the hypomelanotic macule. These findings indicate that the TSC/mTORC1/AKT/GSK3β/β-catenin/MITF axis plays a central role in regulating melanogenesis. Interventions that enhance or diminish mTORC1 activity or other nodes in this pathway in melanocytes could potentially modulate pigment production.

Authors

Juxiang Cao, Magdalena E. Tyburczy, Joel Moss, Thomas N. Darling, Hans R. Widlund, David J. Kwiatkowski

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Figure 3

Disruption of the TSC protein complex leads to GSK3β activation and reduced β-catenin and MITF transcription in human primary melanocytes.

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Disruption of the TSC protein complex leads to GSK3β activation and redu...
(A) Immunoblot analysis of iHPMs expressing shTSC1 or shTSC2 or control (Scr) shows reduced p-AKT (Ser473/Thr308), reduced p-GSK3β (Ser9), increased GSK3β, reduced β-catenin and increased p–β-catenin (Ser33/Ser37/Thr41). (B) Immunoblot analysis of iHPMs expressing shTSC1 or shTSC2 or control (Scr), treated with rapamycin (20 nM) or vehicle (DMSO) for 72 hours shows recovery of β-catenin levels. (C) Immunoblot analysis of cytosolic (Cyt) and nuclear (Nuc) fractions shows loss of nuclear β-catenin in the TSC1- and TSC2-knockdown cells. Topo IIβ and β-tubulin served as controls for the nucleus and cytosol, respectively. (D) Entirely similar to the data shown in C, except that cells on the right were treated with rapamycin for 4 days, and β-catenin was seen again in the nuclear fraction. (E) Relative luciferase expression in iHPMs stably expressing shTSC1 or shTSC2 or control (Scr), transfected with TK-TOP– or TK-FOP–driven luciferase. (F) Entirely similar to the data shown in E, except that iHPMs were treated with rapamycin (20 nM) or vehicle (DMSO) for 72 hours prior to analysis. (G) MITF promoter activity of iHPMs with TSC1 or TSC2 knockdown, treated with or without 20 nM rapamycin and measured using pGL3-luciferase empty vector or MITF-PGL3-luciferase. (H) Cells treated as in G were analyzed by qPCR for MITF and TYR mRNA levels. Data in E–H represent the mean ± SD from at least 3 independent experiments. *P < 0.05, by Student’s t test. The immunoblots presented in the figure include replicate samples run on parallel gels.