Partial proteasome inhibitors induce hair follicle growth by stabilizing β-catenin

Abstract

The activation of tissue stem cells from their quiescent state represents the initial step in the complex process of organ regeneration and tissue repair. While the identity and location of tissue stem cells are becoming known, how key regulators control the balance of activation and quiescence remains mysterious. The vertebrate hair is an ideal model system where hair cycling between growth and resting phases is precisely regulated by morphogen signaling pathways, but how these events are coordinated to promote orderly signaling in a spatial and temporal manner remains unclear. Here, we show that hair cycle timing depends on regulated stability of signaling substrates by the ubiquitin-proteasome system. Topical application of partial proteasomal inhibitors (PaPIs) inhibits epidermal and dermal proteasome activity throughout the hair cycle. PaPIs prevent the destruction of the key anagen signal β-catenin, resulting in more rapid hair growth and dramatically shortened telogen. We show that PaPIs induce excess β-catenin, act similarly to the GSK3β antagonist LiCl, and antagonize Dickopf-related protein-mediated inhibition of anagen. PaPIs thus represent a novel class of hair growth agents that act through transiently modifying the balance of stem cell activation and quiescence pathways.

Keywords: Hair growth; Proteasome; Stem cells; Tissue regeneration; Wnt signaling.

Figure 1

Topically applied PaPIs partially inhibit the proteasome. (A, B): Fluorescent micrographs of Ub^G76VGFP-expressing mice where GFP fluorescence indicates proteasome inhibition. (A): Control vehicle-treated skin sections (10 μM sections) and (B) PaPI-treated sections showing relatively uniform distribution of proteasome inhibition. (C, D): Quantitation of inhibition by Western blot for GFP in two independent samples of vehicle (V) or PaPI (P)-treated tissue or in explanted keratinocytes (in vitro). WT mice lack the Ub^G76VGFP allele. (D): Quantitation of PaPI effect by densitometry. Abbreviations: GFP, green fluorescent protein; PaPI, partial proteasomal inhibitor.

Figure 2

PaPIs induce murine hair growth. PaPI-treated murine skin induces a shortened telogen. (A, B): H+E sections and gross images of vehicle and PaPI-treated skin showing marked hair growth. Hair cycle staging was determined in part by changes in skin pigmentation. Treatment started during the first anagen in postnatal day 28 female B6 mice. (C): Quantitation of the number of mice in anagen and telogen of PaPI treatment using two different topical PaPI treatments. Note marked inhibition of telogen and mild extension of anagen in PaPI-treated skin. (D, E): Normal differentiation in PaPI-treated skin. Differentiation markers keratin 10 (D, green) and keratin 14 (E, green) reveal no abnormalities at Day 47. (F): Cleaved caspase 3 staining shows no difference in PaPI-treated and vehicle-treated skin samples at Day 52. (G): PaPI-treated skin reveals a lot more proliferation compared to vehicle-treated skin at Day 52. Abbreviation: PaPI, partial proteasomal inhibitor.

Figure 3

PaPIs inappropriately accumulate β-catenin in the hair germ/bulge. (A): Immunoperoxidase staining of β-catenin protein (brown). Little difference in levels of β-catenin between vehicle and PaPI-treated skin during catagen. (B): In contrast, during telogen, there are increased levels of β-catenin in PaPI-treated skin as compared to vehicle treatment. This difference is magnified in late telogen with marked accumulation of β-catenin in the hair germ (arrow, and in magnified view) as the hair enters anagen. β-catenin accumulation occurs in the bulge and hair germ of treated skin. Abbreviation: PaPI, partial proteasomal inhibitor.

Figure 4

Wild-type β-catenin with PaPIs accelerates anagen. (A): Injection of adenovirus-expressing wild-type β-catenin subcutaneously rapidly accelerates the onset of anagen with the application of PaPIs (left and middle) but not vehicle control (right most). Note the onset of anagen only occurs at the injection site. (B): β-catenin accumulation occurs in the hair follicle bulge. Despite β-catenin adenovirus, little accumulation occurs in mice that were injected with the adenovirus only. However, with PaPI treatment, marked β-catenin accumulation occurs and the hair enters anagen. Abbreviation: PaPI, partial proteasomal inhibitor.

Figure 5

PaPIs act downstream of DKK. To identify where in the Wnt pathway PaPIs act, Wnt modulators were tested. (A): The number of animals in anagen were measured after topical treatment with LiCl, an inhibitor of GSK3β and control NaCl. LiCl dramatically shortens telogen. (B, C): Histology of animals showing anagen stage hair follicles in LiCl-treated mice. (D): Animals injected with adenovirus-expressing DKK cease anagen and enter catagentelogen (histology below). (E): Animals treated with PaPIs and DKK remained in late anagen, indicating PaPIs act downstream of DKK (n = 3). Abbreviations: DKK, Dickkopf-related protein 1; PaPI, partial proteasomal inhibitor.