mTORC1 activity negatively regulates human hair follicle growth and pigmentation

Abstract

Dysregulation of the activity of the mechanistic target of rapamycin complex 1 (mTORC1) is commonly linked to aging, cancer, and genetic disorders such as tuberous sclerosis (TS), a rare neurodevelopmental multisystemic disease characterized by benign tumors, seizures, and intellectual disability. Although patches of white hair on the scalp (poliosis) are considered as early signs of TS, the underlying molecular mechanisms and potential involvement of mTORC1 in hair depigmentation remain unclear. Here, we have used healthy, organ-cultured human scalp hair follicles (HFs) to interrogate the role of mTORC1 in a prototypic human (mini-)organ. Gray/white HFs exhibit high mTORC1 activity, while mTORC1 inhibition by rapamycin stimulated HF growth and pigmentation, even in gray/white HFs that still contained some surviving melanocytes. Mechanistically, this occurred via increased intrafollicular production of the melanotropic hormone, α-MSH. In contrast, knockdown of intrafollicular TSC2, a negative regulator of mTORC1, significantly reduced HF pigmentation. Our findings introduce mTORC1 activity as an important negative regulator of human HF growth and pigmentation and suggest that pharmacological mTORC1 inhibition could become a novel strategy in the management of hair loss and depigmentation disorders.

Keywords: alpha-MSH/MC1R; mTORC1; melanocyte; rapamycin; tuberous sclerosis.

Figure 1. mTORC1 activity is significantly upregulated while TSC2 protein expression is not altered in gray/white human anagen hair follicles in vivo

1. Quantitative analysis of phosphorylated S6 (p‐S6; mTORC1 activity read‐out) immunoreactivity. N = 11–15 pigmented or gray HFs from four different donors.

2. Representative images of p‐S6 immunofluorescence.

3. Quantitative analysis of TSC2 protein levels. N = 11–14 pigmented or gray HFs from four different donors.

4. Representative images of TSC2 immunofluorescence.

Data information: Only anagen VI HFs were investigated and analyzed immediately after surgery. Analyses were performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Student's t‐test, **P < 0.01. Scale bar: 50 μm. Samples from each donor represented by a different color. Nuclei stained with DAPI. Source data are available online for this figure.

Figure 2. mTORC1 inhibition stimulates human scalp hair follicle growth and pigmentation through MC1R activation

1. Quantitative analysis of phosphorylated S6 (p‐S6; mTORC1 activity read‐out) immunoreactivity. N = 23–30 anagen VI HFs from eight different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

2. Representative images of p‐S6 immunofluorescence.

3. Hair cycle staging was performed using Ki‐67 and Masson–Fontana histochemistry (Kloepper et al, 2010). Mean ± SEM; N = 36–39 HFs per group from six different donors treated with Rapamycin 20 ng/ml or vehicle (control) for 7 days.

4. Representative fluorescence images of Ki‐67 and bright‐field microscopic images of Masson–Fontana.

5. Quantitative histomorphometry of melanin production by Masson–Fontana histochemistry. N = 28–30 anagen VI HFs from eight different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

6. Representative bright‐field microscopic images of Masson–Fontana histochemistry.

7. Quantitative analysis of tyrosinase activity. N = 18–21 anagen VI HFs from five different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

8. Representative images of tyrosinase activity immunofluorescence.

9. Quantitative analysis of gp100 expression. N = 24–26 anagen VI HFs from eight different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

10. Representative images of gp100 immunofluorescence.

11. Quantitative analysis of melanocyte dendricity. N = 22–24 anagen VI HFs from six different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

12. Representative images of gp100 immunofluorescence.

13. Quantitative analysis of α‐MSH expression. N = 17–20 anagen VI HFs from five different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

14. Representative images of α‐MSH immunofluorescence.

15. Quantitative analysis of phosphorylated S6 (p‐S6; mTORC1 activity read‐out) immunoreactivity. N = 8–10 anagen VI HFs from two different donors treated with Rapamycin 20 ng/ml, 2 μg/ml Agouti, Rapamycin + Agouti or untreated (vehicle) for 7 days.

16. Representative images of p‐S6 immunofluorescence.

17. Quantitative histomorphometry of melanin production by Masson–Fontana histochemistry. N = 8–11 anagen VI HFs from two different donors treated with Rapamycin 20 ng/ml, 2 μg/ml Agouti, Rapamycin + Agouti or untreated (vehicle) for 7 days.

18. Representative bright‐field microscopic images of Masson–Fontana histochemistry.

Data information: Only anagen VI HFs (except for C and D where all HFs were analyzed) were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, unpaired Student's t‐test (A, C, E, G, I, K, M), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and one‐way ANOVA (O, Q). Scale bar: 50 μm. Samples from each donor represented by a different color. Nuclei stained with DAPI. Source data are available online for this figure.

Figure EV1. mTORC1 inhibition did not affect MITF expression or the number and proliferation state of melanocytes in human scalp hair follicles

1. Quantitative analysis of Ki‐67⁺ cell number. N = 40–48 anagen VI HFs from eight different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

2. Representative images of Ki‐67 immunofluorescence.

3. Quantitative immunohistomorphometry of the number of gp100⁺ cells. N = 22–29 anagen VI HFs from eight different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

4. Representative images of gp100 immunofluorescence.

5. Quantitative analysis of gp100⁺/Ki‐67⁺ cell number. N = 13–17 anagen VI HFs from four different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

6. Representative images of gp100/Ki‐67 immunofluorescence. Yellow arrows indicate gp100⁺/Ki‐67⁺ cells.

7. Quantitative analysis of MITF expression. N = 17–20 anagen VI HFs from five different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

8. Representative images of MITF immunofluorescence. Yellow arrows indicate MITF⁺ cells.

9. Quantitative analysis of MITF phosphorylation (pMITF). N = 12–16 anagen VI HFs from four different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

10. Representative images of pMITF immunofluorescence.

Data information: Only anagen VI HFs (except for A and B where all HFs were analyzed) were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Student's t‐test (A, C, G, I) or Mann–Whitney U‐test (E). Scale bar: 50 μm. Samples from each donor represented by a different color. Nuclei stained with DAPI. Source data are available online for this figure.

Figure 3. mTORC1 overactivation by TSC2 silencing ex vivo inhibits human scalp hair follicle pigmentation

1. A, B

   Quantitative analysis of TSC2 mRNA (A) and protein expression (B). N = 8–10 anagen VI HFs from four different donors treated with siTSC2 or nontargeting oligos (NTO) for 6 days.

2. C

   Representative images of TSC2 immunofluorescence.

3. D

   Quantitative analysis of phosphorylated S6 (p‐S6; mTORC1 activity read‐out) immunoreactivity. N = 2–3 anagen VI HFs from one donor treated with siTSC2 or nontargeting oligos for 6 days.

4. E

   Representative images of p‐S6 immunofluorescence.

5. F

   Quantitative histomorphometry of melanin production. N = 9–10 anagen VI HFs from four different donors treated with siTSC2 or nontargeting oligos for 6 days.

6. G

   Representative bright‐field microscopic images of Masson–Fontana histochemistry.

7. H

   Quantitative analysis of tyrosinase activity. N = 8–9 anagen VI HFs from four different donors treated with siTSC2 or nontargeting oligos for 6 days.

8. I

   Representative images of tyrosinase activity immunofluorescence.

9. J

   Quantitative analysis of α‐MSH expression. N = 8–9 anagen VI HFs from four different donors treated with siTSC2 or nontargeting oligos for 6 days.

10. K

    Representative images of α‐MSH immunofluorescence.

Data information: Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t‐test, *P < 0.05, **P < 0.01, ****P < 0.0001. Scale bar: 50 μm. Samples from each donor are represented by a different color. Nuclei stained with DAPI. Source data are available online for this figure.

Figure EV2. mTORC1 overactivation does not affect melanocyte dendricity and number in human scalp hair follicles

1. Hair cycle staging was performed using Ki‐67 and Masson–Fontana histochemistry. Mean ± SEM; N = 21–22 HFs per group from four different donors treated with siTSC2 or nontargeting oligos for 6 days; Unpaired Student's t‐test.

2. Representative fluorescence images of Ki‐67 and bright‐field microscopic images of Masson–Fontana.

3. Quantitative analysis of MITF expression. N = 9 anagen VI HFs from four different donors treated with siTSC2 or nontargeting oligos (NTO) for 6 days.

4. Representative images of MITF immunofluorescence. White arrows show MITF⁺ cells.

5. Quantitative analysis of MITF phosphorylation (pMITF). N = 8 anagen VI HFs from four different donors treated with siTSC2 or nontargeting oligos for 6 days.

6. Representative images of pMITF immunofluorescence.

7. Quantitative analysis of gp100 expression. N = 10 anagen VI HFs from four different donors treated with siTSC2 or nontargeting oligos for 6 days.

8. Representative images of gp100 immunofluorescence.

9. Quantitative analysis of gp100⁺ cell number. N = 9 anagen VI HFs from four different donors treated with siTSC2 or nontargeting oligos for 6 days.

10. Representative images of gp100 immunofluorescence.

11. Quantitative analysis of melanocyte dendricity. N = 10 anagen VI HFs from four different donors treated with siTSC2 or nontargeting oligos for 6 days.

Data information: Only anagen VI HFs (except for A and B where all HFs were analyzed) were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Student's t‐test. Scale bar: 50 μm. Samples from each donor represented by a different color. Nuclei stained with DAPI.

Figure 4. Rapamycin stimulates repigmentation‐associated parameters in gray/white anagen scalp hair follicles of responding donors

1. Quantitative analysis of phosphorylated S6 (p‐S6; mTORC1 activity read‐out) immunoreactivity. N = 9–10 gray anagen VI HFs from three different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

2. Representative fluorescence images of p‐S6 immunofluorescence.

3. Hair cycle staging was performed using Ki‐67 and Masson–Fontana histochemistry. Mean ± SEM; N = 16–17 gray HFs per group from three different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

4. Representative fluorescence images of Ki‐67 and bright‐field microscopic images of Masson–Fontana.

5. Quantitative histomorphometry of melanin production by Masson–Fontana histochemistry. N = 7–9 gray anagen VI HFs from three different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

6. Representative pictures of Masson–Fontana histochemistry.

7. Quantitative analysis of tyrosinase activity. N = 7 gray anagen VI HFs from three different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

8. Representative images of tyrosinase activity immunofluorescence.

9. Quantitative analysis of α‐MSH expression in defined reference area within the bulb. N = 5–7 gray anagen VI HFs from three different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

10. Representative images of α‐MSH immunofluorescence.

Data information: Only anagen VI HFs (except for C and D where all HFs were analyzed) were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Student's t‐test (A, C) or Mann–Whitney U‐test (E, G, I), *P < 0.05, ***P < 0.001. Scale bar: 50 μm. Samples from each donor represented by a different color. Nuclei stained with DAPI. Source data are available online for this figure.

Figure EV3. mTORC1 inhibition stimulates repigmentation of gray/white human scalp hair follicles only in certain hair follicles

1. Quantitative histomorphometry of melanin production by Masson–Fontana histochemistry in defined reference area in the bulb. N = 16 gray anagen VI HFs from five different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

2. Representative bright‐field microscopy images of Masson–Fontana histochemistry.

3. Quantitative analysis of MITF expression in defined reference area in the bulb. N = 15–17 gray anagen VI HFs from five different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

4. Representative images of MITF immunofluorescence. White arrows showed MITF⁺ cells.

5. Quantitative analysis of MITF phosphorylation (pMITF) in defined reference area in the bulb. N = 16 gray anagen VI HFs from five different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

6. Representative fluorescence images of pMITF immunofluorescence. White arrows showed pMITF⁺ cells.

7. Quantitative analysis of tyrosinase activity in defined reference area in the bulb. N = 12–13 gray anagen VI HFs from four different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

8. Representative images of tyrosinase activity immunofluorescence.

9. Quantitative analysis of gp100 expression in defined reference area within the bulb. N = 15–16 gray anagen VI HFs from five different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

10. Representative images of gp100 immunofluorescence.

11. Quantitative analysis of α‐MSH expression in defined reference area within the bulb. N = 10–12 gray anagen VI HFs from five different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

12. Representative images of α‐MSH immunofluorescence.

Data information: Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Mann–Whitney U‐test (A) or Student's t‐test (C, E, G, I, K), *P < 0.05. Scale bar: 50 μm. Samples from each donor represented by a different color. Nuclei stained with DAPI.

Figure EV4. mTORC1 inhibition does not significantly alter melanocyte number, proliferation state, and dendricity in gray/white human scalp gray hair follicles

1. Quantitative immunohistomorphometry of the number of gp100⁺ cells. N = 14 gray anagen VI HFs from four different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

2. Representative images of gp100 immunofluorescence. Arrows indicate gp100⁺ cells.

3. Quantitative analysis of gp100⁺/ki‐67⁺ cell number. N = 9 gray anagen VI HFs from three different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

4. Representative images of gp100/ki‐67 immunofluorescence. Arrows indicate gp100⁺Ki‐67⁺ cells.

5. Quantitative analysis of melanocyte dendricity. N = 10–12 gray anagen VI HFs from four different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.

Data information: Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Mann–Whitney U‐test. Scale bar: 50 μm. Samples from each donor represented by a different color. Nuclei stained with DAPI.