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12-O-tetradecanoylphorbol-13-acetate activates hair follicle melanocytes for hair pigmentation via Wnt/β-catenin signaling

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Abstract

Melanocyte stem cells (McSCs) undergo cyclical activation and quiescence together with hair follicle stem cells (HFSCs). This process is strictly controlled by the autonomous and extrinsic signaling environment. However, the modulation of factors important for the activation of McSCs for hair pigmentation remains unclear. 12-O-tetradecanoylphorbol-13-acetate (TPA) mimics vital signaling pathways involved in melanocyte growth and melanogenesis in vitro. To investigate whether TPA regulates quiescent McSCs for hair pigmentation, we topically smeared TPA on 7-week-old mouse dorsal skin and found that TPA stimulated hair growth and hair matrix pigmentation. These changes were associated with a significant increase in the number of hair bulb melanocytes. Moreover, in the TPA-treated group, hair bulge McSCs and hair bulb melanoblasts actively proliferated. At the molecular level, nuclear β-catenin, a key factor of Wnt/β-catenin signaling, was highly synthesized in melanocytes and keratinocytes in TPA-induced hair bulbs. Inhibition of Wnt/β-catenin signaling by injecting Dickkopf1 plasmids into TPA-treated skin decreased hair matrix pigmentation and inhibited the proliferation and differentiation of McSCs. Our findings suggest that the topical application of TPA stimulates the proliferation and differentiation of McSCs and their progeny for hair matrix pigmentation by activating Wnt/β-catenin signaling. This might provide a useful experimental model for the study of signals controlling the activation of McSCs.

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Abbreviations

McSCs:

Melanocyte stem cells

TPA:

12-O-tetradecanoylphorbol-13-acetate

sHG:

Secondary hair germ

HFSCs:

Hair follicle stem cells

TGF-β:

Transforming growth factor-β

PKC:

Protein kinase C

DKK1:

Dickkopf1

W:

Week

PBS:

Phosphate-buffered saline

BrdUrd:

Bromodeoxyuridine

H&E:

Hematoxylin and eosin

MITF:

Microphthalmia-associated transcription factor

DCT:

Dopachrome tautomerase

TRP1:

Tyrosinase-related protein 1

TYR:

Tyrosinase

EGFP:

Enhanced green fluorescent protein

CMV:

Cytomegalovirus

SD:

Standard deviation

IRS:

Inner root sheath

PHB:

Periphery of hair bulb

HFs:

Hair follicles

HS:

Hair shaft

HB:

Hair bulb

Epi:

Epidermis

SG:

Sebaceous gland

HM:

Hair matrix

MC:

Melanocyte

KCs:

Keratinocytes

Nβ-cat:

Nuclear β-catenin

DP:

Dermal papilla

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Acknowledgements

We thank Dr. Tung-Tien Sun for AE15 antibodies. Mingxing Lei is supported by Project funded by China Postdoctoral Science Foundation (2016M590866), Fundamental Research Funds for the central Universities (106112015CDJRC231206) and Special Funding for Postdoctoral Research Projects in Chongqing (Xm2015093).

Author information

Authors and Affiliations

  1. Department of Dermatology, Daping Hospital, The Third Military Medical University, Chongqing, 400038, China

    Weiming Qiu, Hui Tang, Hongtao Yan & Jinjin Wu

  2. Department of Dermatology, Wuhan General Hospital of Guangzhou Military Area Command, Wuhan, 430070, China

    Weiming Qiu

  3. Department of Cell Biology, The Third Military Medical University, Chongqing, 400038, China

    Haiying Guo & Xiaohua Lian

  4. “111” Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing, 400044, China

    Mingxing Lei

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  1. Weiming Qiu
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Correspondence to Xiaohua Lian or Jinjin Wu.

Additional information

This work was supported by grants from the National Nature Science Foundation of China (grant number: 30972645).

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Fig. S1

Detection of hair size and differentiation after TPA treatment. a Bar chart depicting the maximum diameter of HB and HS from H&E-stained skin tissues from various groups of mice (HB hair bulb, HS hair shaft). *P < 0.05. b–d’’ Immunostaining detection of AE15 synthesis in control (b–d), T4W (b’–d’) and 14 W (b’’–d’’) hair follicles (HFs). Similar AE15 location in the T4W and 14 W HFs. Nuclei were stained with DAPI. Bars 100 μm (DOCX 623 kb)

Fig. S2

DCT + Brdu + cells in hair bulge. *P < 0.05. (DOCX 1999 kb)

Fig. S3

Detection of nuclear β-catenin in TPA- (b–b’’) or acetone- (control, a–a’’) treated melanoblasts by immunostaining. White arrows indicate β-catenin in cytomembrane (red), whereas yellow arrows indicate β-catenin in cell nuclei (red). Nuclei were stained with DAPI (blue). Bars 100 μm (DOCX 59 kb)

Fig. S4

Detection of green fluorescent protein (GFP) in various plasmid-treated groups. a T3W + N1: TPA-treated for 3 weeks plus N1-plasmid-treated. b T3W + DKK1: TPA-treated for 3 weeks plus DKK1-plasmid-treated. a’ T4W + N1: TPA-treated for 4 weeks plus N1-plasmid-treated. b’ T4W + DKK1: TPA-treated for 4 weeks plus DKK1-plasmid-treated. White arrows indicates cells synthesizing GFP. Nuclei were stained blue with DAPI. Bars 100 μm (DOCX 3760 kb)

Fig. S5

Decreased synthesis of nuclear β-catenin after T4W + DKK1 treatment. a–b’’ Immunostaining detection of both DCT and β-catenin synthesis in T4W + N1 (a, b), T4W + DKK1 (a’, b’) and 14 W HFs (a’’, b’’). c Representation showing the synthesis of both DCT and β-catenin in various groups. d Number of nuclear β-catenin + DCT+ cells and nuclear β-catenin + DCT- cells in the various groups (HB hair bulb, HM hair matrix, Nβ-cat nuclear β-catenin). Yellow arrows indicate cells synthesizing both DCT and nuclear β-catenin. White arrows indicate cells synthesizing nuclear β-catenin only. Nuclei were stained blue with DAPI. # P > 0.05, *P < 0.05. Bars 100 μm (DOCX 1255 kb)

Fig. S6

Detection of hair size and differentiation after T4W + DKK1 treatment. a Bar chart depicting the width of HB and HS in T4W + N1, T4W + DKK1 and 14 W HFs (HB hair bulb, HS hair shaft). *P < 0.05, # P > 0.05, no significant differences. b–d’’ Immunostaining analysis of AE15 in T4W + N1 (b–d), T4W + DKK1 (b’–d’) and 14 W HFs (b’’–d’’). Similar pattern of AE15 in the T4W + N1, T4W + DKK1 and 14 W HFs. White arrows indicate AE15-positive cells. Nuclei were stained blue with DAPI. Bars 100 μm (DOCX 715 kb)

Fig. S7

DCT + Brdu+ cells in hair bulge. *P < 0.05 (DOCX 59 kb)

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Qiu, W., Tang, H., Guo, H. et al. 12-O-tetradecanoylphorbol-13-acetate activates hair follicle melanocytes for hair pigmentation via Wnt/β-catenin signaling. Cell Tissue Res 366, 329–340 (2016). https://doi.org/10.1007/s00441-016-2450-6

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