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 January 3, 2017; First 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 2

Rapamycin, but not Torin-1, restores the loss of pigmentation in TSC protein complex–deficient melanocytes.

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Rapamycin, but not Torin-1, restores the loss of pigmentation in TSC pro...
(A–D) iHPMs stably expressing shTSC1 or control (Scr) were treated with rapamycin (Rapa) (20 nM) or vehicle control (DMSO) for 72 hours. (A) Immunoblot analysis shows recovery of MITF expression, suppression of mTORC1, and recovery of p-AKT levels in cells treated with rapamycin. (B) Pigmentation, (C) melanin content, and (D) TYR activity were also all rescued by rapamycin. (E–H) Entirely similar to the data shown in A–D, except using iHPMs with shTSC2 knockdown. (I–L) iHPMs stably expressing shTSC1, shTSC2, or control (Scr) were treated with Torin-1 (250 nM) or vehicle control (DMSO) for 72 hours. (I) Immunoblot analysis shows complete suppression of mTORC1 and mTORC2. (J) Pigmentation, (K) melanin content, and (L) TYR activity, none of which were rescued by Torin-1 treatment. (M–P) iHPMs were infected with control (Scr) or shRaptor or shRictor lentiviruses and selected with puromycin for 6 days. (M) Immunoblot analysis shows that Rictor knockdown reduced MITF, PGC-1α, and TYR expression. (N) Pigmentation, (O) melanin content, and (P) TYR activity, all of which show reduced pigmentation in Rictor-knockdown cells. Data in C, D, G, H, K, L, O, and P represent the mean ± SD from at least 3 independent experiments. *P < 0.05, by Student’s t test. The immunoblots presented in the figure panels include replicate samples run on parallel gels.