Mobilizing transit-amplifying cell-derived ectopic progenitors prevents hair loss from chemotherapy or radiation therapy

Abstract

Genotoxicity-induced hair loss from chemotherapy and radiotherapy is often encountered in cancer treatment, and there is a lack of effective treatment. In growing hair follicles (HF), quiescent stem cells (SC) are maintained in the bulge region and hair bulbs at the base contain rapidly dividing, yet genotoxicity-sensitive transit-amplifying cells (TAC) that maintain hair growth. How genotoxicity-induced HF injury is repaired remains unclear. We report here that HF mobilize ectopic progenitors from distinct TAC compartments for regeneration in adaptation to the severity of dystrophy induced by ionizing radiation (IR). Specifically, after low-dose IR, keratin5⁺ basal hair bulb progenitors, rather than bulge SC, were quickly activated to replenish matrix cells and regenerated all concentric layers of HF, demonstrating their plasticity. After high-dose IR, when both matrix and hair bulb cells were depleted, the surviving outer root sheath cells rapidly acquired a SC-like state and fueled HF regeneration. Their progeny then homed back to SC niche and supported new cycles of HF growth. We also revealed that IR induced HF dystrophy and hair loss and suppressed WNT signaling in a p53- and dose-dependent manner. Augmenting WNT signaling attenuated the suppressive effect of p53 and enhanced ectopic progenitor proliferation after genotoxic injury, thereby preventing both IR- and cyclophosphamide-induced alopecia. Hence, targeted activation of TAC-derived progenitor cells, rather than quiescent bulge SC, for anagen HF repair can be a potential approach to prevent hair loss from chemotherapy and radiotherapy.

Introduction

Chemotherapy and radiotherapy are widely employed in the treatment of various cancers (1,2). Both ionizing radiation (IR) and a substantial proportion of chemotherapeutic agents exert their treatment effects against cancer cells by inducing DNA damage (1–3). Despite extensive efforts made to minimize off-target injuries, damage to normal tissues is still inevitable (2,4–6). Hair loss is a common side effect (7–9). It brings psychosocial stress, compromises patients’ sense of personal identity and even jeopardizes the willingness for treatment (8,10). Prevention of such hair loss is still an unmet clinical need (7,8).

Hair follicles (HFs) are a dynamic organ that undergo life-long growth cycles, consisting of anagen (active growth), catagen (regression) and telogen (relative rest) phases (Fig. 1A) (9). Both telogen and anagen HFs share an upper permanent segment, spanning from the follicular infundibulum to the bulge (Fig. 1A) (9,11–13). The structures below the bulge are not permanent (Fig. 1A) (9,11–13). In telogen, the lower segment shrinks to a minimum structure of secondary hair germ (SHG) (9,11,12). In anagen, the lower segment expands dramatically into a long cylinder where distinct populations of transit amplifying cells (TACs) reside. Among them, outer root sheath (ORS) cells, located immediately below the bulge, are connected with an enlarged hair bulb where hair matrix germinative cells surrounding the dermal papilla (DP) actively multiply to generate concentric cellular layers of distinct differentiations to support hair elongation (9,13–15). Since at any given time the majority of human scalp HFs are in anagen (9), this highly proliferative nature makes anagen HFs one of the most sensitive organs to genotoxic injury (7,16,17).

Figure 1.

Dystrophic changes and regenerative activities in HFs after IR exposure. A, Mouse hair cycle and introduction of IR injury. Bg: bulge; Mx: matrix; PD: postnatal day; PW: postnatal week; SG: sebaceous gland. B, IR-induced hair loss. (n=10 in each dose). C-E, Histology and quantification of HF lengths and matrix cell numbers. F and G, Apoptosis detected by TUNEL staining and quantification of apoptotic matrix cells. H and I, Cell proliferation mapped by BrdU and quantification of BrdU⁺ matrix cells. J, Apoptosis detected by cleaved caspase-3. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparison test. Blue star * p<0.05, 2Gy vs 0Gy; green star * p<0.05, 5.5Gy vs 0Gy; # p<0.05, 2Gy vs 5.5Gy. Error bars represent mean ± S.E.M. Dashed line: DP. Scale bar=75µm in (C), 25µm in (F) (H)(J).

The slow-cycling long-lived HF stem cells (HFSCs) are constantly preserved in the bulge around hair cycles (11,12,18). In addition to these quiescent bulge SCs (BgSCs), telogen HFs house another population of more active SCs in SHG (SHG stem cells (ShgSCs)) (11,12,19,20). During transition from telogen to anagen, ShgSCs proliferate first to support the formation of the initial hair bulbs (11,12,19). This is followed by the activation of quiescent BgSCs a few days later, which contribute to the upper ORS (11,12,19). In contrast, the highly expanded lower segment of anagen HFs lacks long-lived SCs (9,18).

With the presence of TACs only in the lower segment, how are anagen HFs repaired to resume the ongoing anagen hair growth following genotoxic injuries? Since quiescent BgSCs are relatively resistant to genotoxic insults (21), such repair might be driven by BgSCs. However, it would require complete HF involution and lengthy resetting of the hair cycle. This possibility is contrasted by the fact that DNA-damaged anagen HFs often bypass telogen involution and resume hair production without cycle resetting (8,17). Such observations suggest that, in contrast to the telogen-to-anagen regeneration that relies on the activation of BgSCs and ShgSCs, anagen HFs can mobilize mother progenitor cells for repair.

In this work, we attempt to explore the mechanisms and map the progenitor sources underlying the regenerative responses of anagen HF repair following ionizing radiation (IR) injury. We provide evidence that HFs are able to employ ectopic progenitor cells from distinct TAC compartments for repair in adaptation to the severity of genotoxic damage. We also demonstrate that efficient mobilization of TAC-derived ectopic progenitor cells for anagen HF repair can be a strategy to prevent hair loss from chemotherapy and radiotherapy.

Materials and Methods

Mice

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University. K5^CreER mice were provided by Chen CM (22), Lgr5^{EGFP-Ires-CreERT2} mice were from Clevers H (23), and K19^CreER mice were from Gu G (24). p53 null mice, Ctnnb1^flox/flox mice (25) and R26LSL^tdTomato mice were from Jackson lab. C57BL/6 mice were from Taiwan National Laboratory Animal Center. For IR and invasive experiments, animals were anesthetized by Tiletamine-Zolazepam (Telazol®).

Radiation exposure

The dorsal hair of female mice at postnatal day 30 was carefully shaved by an electric shaver. Around two days later when dorsal HFs were in early full anagen (~postnatal day 32), single doses (2Gy or 5.5Gy) of gamma irradiation were given from the dorsal side by a ¹³⁷Cs source (dose rate 3.37Gy/min, gamma irradiator IBL 637 from CIS Bio International, France). For comparison, littermate control with the same genetic background was used. Mice were consistently irradiated in the afternoon.

Lineage tracing experiment

To label basal cells and BgSCs, K5^CreER/+; R26LSL^tdTomato/+ and K19^CreER/+; R26LSL^tdTomato/+ mice received a single intraperitoneal injection of tamoxifen (TAM; Sigma) (0.1mg/g of body weight) 24hrs prior to irradiation. To label cells in the lower segment of epithelial strand at 5.5 Gy of IR, Lgr5^{EGFP-Ires-CreERT2/+}; R26LSL^tdTomato/+ mice received a single dose of tamoxifen (0.05mg/g of body weight) at 48hrs after radiation.

Inhibition of Wnt/β-catenin signaling

Inhibition of Wnt/β-catenin signaling in the epithelium was achieved by the conditional deletion of Ctnnb1 in K5^CreER/CreER; Ctnnb1^flox/flox mice by tamoxifen (0.1mg/g body weight) at 6 and 12hrs after irradiation or by using specific inhibitors IWR1 and IWP2 (Sigma). Both IWR1 and IWP2 were reconstituted in DMSO, and DMSO was used as a control. Mice were subcutaneously injected with IWR1 and IWP2 at 48, 72 and 96hrs post irradiation, totaling 12.5µg/g body weight for each dose. Skin was harvested at day 5 for examination.

Histology, immunostaining and TUNEL staining

Skin specimens were fixed at 4°C overnight either in formalin-acetic-alcohol solution for paraffin embedment or 4% paraformaldehyde for OCT (Sakura Finetek) embedment. Specimens were sectioned and stained with hematoxylin and eosin (H&E). Apoptotic cells were detected by DeadEnd™ Fluorometric TUNEL System (Promega). Cryosections were used to visualize tdTomato fluorescence of lineage tracing. Immunohistochemistry and immunofluorescence staining were performed with routine antigen retrieval as suggested by the antibody manufacturers. Super Sensitive™ IHC Detection Systems (BioGenex) were used for the detection of horseradish peroxidase activity. The antibodies used were described in Supplementary Table S1.

Image acquisition and quantification

All fluorescent images were acquired on the confocal microscope (SP5, Leica). To quantify BrdU⁺ and TUNEL⁺ cells, we acquired 1024×1024 pixels sequential scans with a 63x oil immersion objective lens (1.4 NA). Hair matrix was counted as epithelial cells below the top of DP. Germinative cells are epithelial cells adjacent to DP, and basal hair bulb cells are basal cells located on the outer surface of the hair bulb abutting dermal sheaths.

Quantification of γ-H2AX foci

DNA double-strand breaks were determined by γ-H2AX foci as previously described (26). Briefly, the 3-dimensional fluorescent images were reconstituted by approximately 10-15 images of serial 2-dimensional Z-stacks of confocal images. Data were analyzed using the software MetaMorph 7.7. Foci within Hoechst-stained nucleus were scored by the value of pixels after the focus threshold was set manually. 5 pixels were considered as a standard for single DNA double-strand breaks based on the few discrete γ-H2AX foci generated in the nucleus of unirradiated control specimen. Because extensive γ-H2AX foci appeared after irradiation, individual foci could not be distinguished accurately. We compared the total value of positive pixels within the nucleus in selected cells with the standard 5 pixels/focus to estimate the number of foci in an entire nuclear region.

RNA-sequencing analysis

Keratinocytes of irradiated skin were collected at different time points after irradiation. Total RNA from keratinocytes was extracted using TRI reagent® solution (Thermo) for the following RNA-seq library preparation and sequencing. All procedures were carried out according to the protocol from Illumina. The libraries were sequenced on the Illumina NextSeq 500 platform using 75 single-end base pair strategy, and 10 million reads per sample were generated. Gene Ontology (GO) and KEGG analysis were performed using DAVID (https://david.ncifcrf.gov/) (27).

Quantitative RT-PCR

Total RNA from keratinocytes of irradiated skin collected at different time points was extracted using TRI reagent® solution (Thermo). The RNA was reverse transcribed into cDNA using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo). In bead implantation experiment, HF epithelial cells were isolated from the HFs surrounding Wnt3a and BSA-soaked beads, and the RNA was extracted and cDNA was amplified by using REPLI-g WTA single cell kit (QIAGEN). Quantitative PCR analysis was performed using SYBR Green qPCR Master Mix (Thermo) on an ABI 7900HT Real-Time PCR System (Applied Biosystems). Sequences of gene-specific primers used were described in Supplementary Table S2.

FACS

The dorsal skin of seven-week-old female mice were used for the sorting of inactive ShgSCs. For the sorting of activated ShgSCs, dorsal hairs of seven-week-old female mice were plucked to activate ShgSCs 2 days before skin specimen collection (28). The dorsal skin of 32-day-old mice were irradiated with 5.5Gy of IR and skin specimen was collected at 72hr. Cell preparation for FACS was performed as described (29). The following antibodies were used: CD34-FITC (eBioscience, 11-0341, 1:50), Sca1-PE-Cy7 (eBioscience, 25-5981, 1:50) and p-cadherin-PE (R&D systems, FAB761P, 1:100). FACSAria III sorter equipped with Diva software (BD, Bioscience, San Jose, CA) was used for sorting. Total RNA from sorted cells was extracted using MessageBOOSTER™ cDNA Synthesis from Cell Lysates Kit (Epicentre).

Protein administration experiment

Intracutaneous implantation of protein-coated beads was performed as described (30). Human Wnt3a recombinant protein (R&D systems) was re-suspended in 1mg/ml BSA solution at 1mg/ml. Affinity affi-gel blue gel beads (Bio-Rad) were then suspended in 5µl protein solution, either control (1mg/ml BSA) or experimental (1mg/ml Wnt3a), at 4°C for 2hrs. Approximately 100 beads were implanted into skin immediately after irradiation. Mice were sacrificed at different time points post-irradiation. To visualize HFs, skin specimens were dehydrated in ethanol with graded concentrations and then immersed in xylene until it became transparent. The skin specimens were then further processed for histological examination and immunofluorescent staining.

Statistical analysis

Statistical comparison was performed using the software Prism (GraphPad). An unpaired Student’s t-test was used to compare data sets with two groups. To compare three or more groups, we performed One-Way ANOVA followed by Bonferroni’s multiple comparison. Data were presented as mean ± standard error. P-values were considered significant when less than 0.05.

Results

Dose-dependent HF dystrophy after IR and two tempospatially distinct regenerative attempts

To characterize the effect of IR, mice were irradiated with 2Gy and 5.5Gy on postnatal day 32 when dorsal HFs were in early full anagen (Fig. 1A). In five to ten days, there was a dose-dependent hair loss and HF dystrophy (Fig. 1B and C; Supplementary Fig. S1A). At 2Gy, the initial reduction of matrix cells and HF shortening were recovered by days 3 and 4 (Fig. 1C-E). At 5.5Gy, HFs progressively shrank to slender epithelial strands by day 3, followed by restoration of anagen morphology and length between days 5 and 7 (Fig. 1C and D). Around day 10, HFs of the unirradiated, 2Gy and 5.5Gy groups entered catagen, indicated by decreased cell proliferation and increased apoptosis (Fig. 1C, F-I). Afterwards, HFs could resume a new anagen in 2Gy and 5.5Gy groups (Supplementary Fig. S1A), indicating the hair loss was transient.

We then analyzed cell death and proliferation. TUNEL staining and cleaved caspase-3 staining were performed to detect apoptosis and both assays exhibited a similar trend (Fig. 1F, G, J). Apoptosis was more extensive and persistent after 5.5Gy than 2Gy (Fig. 1F, G, J). After 2Gy exposure, cell proliferation almost entirely halted after 6hrs and then increased at 12 and 48hrs (Fig. 1H and I). This early proliferative response successfully restored hair bulb structures and HF length between 72 and 96hrs (Fig. 1C and D). After 5.5Gy exposure, a similar regenerative response, albeit with lower proliferation, was observed between 12 and 48hrs (Fig. 1H and I). However, proliferation almost entirely ceased again at 72hrs. Progressive HF shrinkage between 0 to 72hrs indicated failure of the early regenerative attempt to compensate for the more severe cell death (Fig. 1C, F, G, J). Importantly, a second late proliferative attempt was observed in the lower tip of HFs at 96hrs (Fig. 1H and I) and led to HF elongation and restoration of hair bulbs (Fig. 1C- E).

Next, we examined whether these regenerative attempts led to production of mature hair shafts. At 2Gy, differentiation toward inner root sheath (IRS) and hair cortex was only transiently disrupted between 36 and 48hrs (Supplementary Fig. S1B). At 5.5Gy, a lengthier and more severe disruption was induced, yet differentiation was eventually restored (Supplementary Fig. S1C).

These results show that, depending on the severity of IR damage, HFs activate two distinct regenerative responses: early regenerative attempt between 12 and 48hrs and late regenerative attempt after 72hrs.

K5⁺ basal hair bulb keratinocytes preferentially proliferate during the early regenerative attempt

Next, we tried to identify cells contributing to early regenerative attempts. In normal hair bulbs, keratin 5 (K5) is exclusively expressed in basal cells (Fig. 2A, 0hr). However, 12 to 36hrs after 2Gy exposure, K5⁺ cells extended into the suprabasal positions (Fig. 2A and B). TUNEL showed that, after 2Gy, apoptosis was more prominent in suprabasal K5^negative matrix and germinative cells than K5⁺ cells (Fig. 2A, C, and D; Supplementary Fig. S2A), and that apoptosis of K5⁺ cells only slightly increased (Fig. 2C). BrdU pulse labeling showed these K5⁺ cells were proliferative (Fig. 2E, yellow arrowheads, and F). During the same period, proliferation of K5^negative suprabasal matrix cells nearly ceased at 6hrs and was progressively restored only by 48hrs (Fig. 2E and G). Analysis of double-stranded DNA breaks by γ-H2AX expression also showed faster repair of DNA damage in K5⁺ basal hair bulb cells (Supplementary Fig. S2B and B’). Together, basal K5⁺ cells are more resistant to IR than suprabasal K5^negative matrix and germinative cells, and become proliferative to support early regenerative attempts.

Figure 2.

Apoptosis, proliferation, and DNA damage of hair bulb cells during the early regenerative attempt. Mice were pulse labeled with BrdU 1hr before sampling. A, Double staining with K5 and TUNEL. B-D, Quantification of K5 and TUNEL expression in (A). B, Proportion of K5⁺ cells in the matrix. C, Proportion of K5⁺ TUNEL⁺ matrix cells. More severe apoptosis of K5⁺ cells was observed in 5.5Gy than 2Gy. D, Proportion of K5^negative TUNEL⁺ matrix cells. E, Double staining with K5 and BrdU. F and G, Quantification of K5 and BrdU expression in (E). F, Proportion of K5⁺ BrdU⁺ matrix cells. G, Proportion of K5^negative BrdU⁺ matrix cells. Compared to 2Gy, 5.5Gy of IR induced more prolonged reduction of proliferation in K5^negative matrix cells. H, Quantification of γ-H2AX foci in basal hair bulb cells. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparison test. Blue star * p<0.05, 2Gy vs 0Gy; green star * p<0.05, 5.5Gy vs 0Gy; # p<0.05, 2Gy vs. 5.5Gy. Error bars represent mean ± S.E.M. Dashed line: DP. Scale bar=25µm.

5.5Gy exposure induced a similar increase in suprabasal K5⁺ cells (Fig. 2A and B), yet their proliferation was delayed compared to the 2Gy group (Fig. 2E and F). Notably, higher apoptosis in both K5⁺ and K5^negative cells was detected after 5.5Gy during the early regenerative attempt (Fig. 2A, white arrowheads, C and D). K5⁺ cells also showed more persistent expression of DNA damage markers (Fig. 2H; Supplementary Fig. S2C and C’). These data suggest that K5⁺ cells and their progeny sustain catastrophic IR injury. Without sufficient resupply from the K5⁺ compartment, K5^negative matrix cells, which also proliferate poorly (Fig. 2E and G), undergo eventual collapse, leading to failure of the early regenerative attempt.

K5⁺ basal hair bulb cells replenish germinative population and contribute to all layers of recovered HFs

To further confirm the cellular source for early regenerative attempts (Fig.3A), we lineage traced K5⁺ cells in K5^CreER; R26LSL^tdTomato mice (Fig. 3B). Injection with tamoxifen 24hrs prior to IR allowed successful and exclusive labeling of basal, but not germinative cells in the hair bulb (Fig. 3B, 0hr). After 2Gy exposure, labeled cells expanded into suprabasal positions at 12hr (Fig. 3B, yellow arrowhead), replenished the germinative compartment (Fig. 3B, white arrowheads) and contributed to all layers of repaired hair bulbs, including hair shafts (Fig. 3B, white arrow), between 24 and 36hrs. In control HFs, most of the labeled cells remained in the basal position even after 36hrs (Fig. 3B). Quantitatively, progeny of K5⁺ lineage progressively increased, accounting for about 60% of all matrix cells at 36hrs (Fig. 3C). The data underscore that, following IR, K5⁺ basal hair bulb cells display high lineage plasticity.

Figure 3.

Cell origin for the early regenerative attempt after 2Gy of IR. A, Possible cell origin. B and C, Lineage tracing in K5^CreER; R26LSL^tdTomato mice and quantification of labeled cells in the hair matrix. B, After 2Gy, labeled cells expanded suprabasally (yellow arrowhead), replenished germinative cells (white arrowheads) and formed hew hair shafts (white arrow). Tam: tamoxifen. C, Proportion of tdTomato⁺ matrix cells. Statistical significance was determined by Student’s t test. * p<0.05. Error bars represent mean ± S.E.M. D, Analysis of BgSC apoptosis by TUNEL. E, Continuous BrdU labeling for analysis of BgSC proliferation. F, Lineage tracing in K19^CreER; R26LSL^tdTomato mice. Dashed line: DP; Bg: bulge. Scale bar=25µm.

Next, we determined the contribution of BgSCs. Neither apoptosis (Fig. 3D) nor proliferation (Fig. 3E) was detected in the bulge. Lineage tracing in K19^CreER; R26LSL^tdTomato mice did not show BgSC expansion (Fig. 3F). The results indicate that BgSCs survive 2Gy IR injury but do not actively contribute to early regenerative attempts.

K5⁺ ORS cells are remodeled into the epithelial strand during the late regenerative attempt

The results above show that the remaining short epithelial strand at 72hrs serves as a platform for the late regenerative attempt. To determine the origin of the epithelial strand, we performed lineage tracing in K5^CreER; R26LSL^tdTomato mice (Fig. 4A) following tamoxifen administration 24hrs prior to IR. This protocol labeled basal cells in the ORS in addition to basal cells of the bulb (Fig. 4A). As HFs regressed between 24 and 72hrs, a larger portion of the epithelial strand became composed of the K5⁺ progeny (Fig. 4A). Since K5⁺ basal hair bulb cells fail to survive 5.5Gy injury, we conclude that the epithelial strand originates from the K5⁺ ORS cells that are more radioresistant, and that basal ORS progeny fuel successful HF regeneration during late regenerative attempts (Fig. 4A).

Figure 4.

Remodeling of ORS cells for the late regenerative attempt after 5.5Gy of IR. A, Lineage tracing in K5^CreER; R26LSL^tdTomato mice. B, Cell proliferation mapped by pulse BrdU labeling. The p-cadherin⁺ lower tip cells started to proliferate at 84hrs (white arrowheads) and regenerated new hair bulb at 96hrs. C, Analysis of BgSC apoptosis by TUNEL. D, Continuous BrdU labeling showed that BgSCs did not proliferate until day 5 (white arrowheads). E, Lineage tracing of BgSCs in K19^CreER; R26LSL^tdTomato mice. Labeled BgSC progeny did not move out of the bulge until day 5. F, Immunofluorescence of p-cadherin and Sox9. Similar to SHG (yellow arrowheads), p-cadherin⁺ lower tip cells were also positive for Sox9 (white arrowheads). G, Lgr5 promoter-driven EGFP expression in Lgr5^{EGFP-Ires-CreERT2} mice. The lower tip cells were positive for EGFP. H, Expression of ShgSC specific genes. Similar to activated ShgSCs, several specific genes were upregulated in lower tip cells at 72hrs. I, Possible cell origin for late regenerative attempts. J, Lineage tracing in Lgr5^{EGFP-Ires-CreERT2}; R26LSL^tdTomato mice. Error bars represent mean ± S.E.M. Scale bar=25µm.

Lower tip cells acquire a stem cell-like property and undergo stepwise activation with BgSCs for the late regenerative attempt

After 5.5Gy, cell proliferation was largely halted at 72hrs (Figs. 1H and I, 4B). By 84hrs, proliferation was first resumed in p-cadherin⁺ lower tip cells (Fig. 4B, white arrowheads). At 96hrs, more lower tip cells proliferated as they continued to regenerate new hair bulbs (Fig. 4B). We did not detect apoptosis of BgSCs (Fig. 4C). Continuous BrdU labeling showed that BgSCs did not start proliferating until day 5 post-IR (Fig. 4D, white arrowheads), when the new hair bulb already regenerated (Fig. 1C). Lineage tracing of BgSCs in K19^CreER; R26LSL^tdTomato mice showed that BgSCs contributed to the suprabulbar ORS cells right below the bulge, but not to the regenerated hair bulbs (Fig. 4E). These results indicate that the lower tip cells fuel the initial late regenerative attempt. This parallels the stepwise activation of epithelial progenitor populations during physiological telogen-to-anagen transition, when ShgSCs, rather than quiescent BgSCs, fuel early HF growth (12,19).

We considered that cells in the lower segment of the epithelial strand acquire SC-like characteristics. We compared the epithelial strand structure of 5.5Gy-irradiated anagen HFs with normal telogen HFs. Typical BgSC markers, CD34 and K15 (31,32) , were still maintained in the bulge (Supplementary Fig. 3A). However, cells at the lower end of the epithelial strand were CD34/K15 double-negative, yet positive for p-cadherin, Sox9 and Lgr5 promoter activity, markers of the normal telogen ShgSCs (Fig. 4F and G; Supplementary Fig. 3A) (12,20,33). Lack of AE13, AE15 and K75 keratin expression shows that lower epithelial strand cells do not prematurely differentiate toward inner anagen HF structures (Supplementary Fig. 3A). We then FACS-isolated these lower tip cells using a Sca1^low CD34^low p-cadherin^high marker profile (Supplementary Fig. 3B) (12), and examined the expression of known ShgSC genes (12). We found Ccnb1, Clca1, Sox4 and Sox6 were also upregulated in the sorted lower tip cells at 72hrs (Fig. 4H). Taken together, these results show that following 5.5Gy IR injury, ORS cells were remodeled into the epithelial strand whose lower tip cells acquired a ShgSC-like progenitor property.

To strengthen the evidence for the cellular dynamics noted above (Fig. 4I), we performed additional lineage tracing using Lgr5^{EGFP-Ires-CreERT2} mice, which allowed for specific tracing of the lower tip cells (Fig. 4J; Supplementary Fig. S4). Consistent with the prior report (34), in unirradiated Lgr5^{EGFP-Ires-CreERT2} mice, ORS cells and suprabasal hair bulb cells were labeled after tamoxifen induction (left panel, Fig. 4J). In the 5.5Gy group, although the epithelial strand was negative for Lgr5 by immunostaining (Supplementary Fig. S3A), its lower portion was distinctly positive for the Lgr5 promoter activity (Fig. 4G). Twenty four hours after tamoxifen injection to Lgr5^{EGFP-Ires-CreERT2}; R26LSL^tdTomato mice at 48hrs after 5.5Gy, labeled cells were exclusively found in the lower portion of the epithelial strands of all HFs examined (n=50; right panel, Fig. 4J), with higher incidence in the lower tip. These labeled Lgr5 progeny contributed to the ORS, the entire hair bulb and hair shafts of the repaired HFs (right panel, Fig. 4J; Supplementary Fig. S4).

Further tracing showed that the progeny of labeled Lgr5 cells homed back to the SHG and bulge when repaired HFs eventually transitioned to telogen (d17, right panel, Fig. 4J), and that in the next cycle they formed the new lower segment of the anagen HF (d35, right panel, Fig. 4J). These results reveal expanded plasticity of ORS cells following IR and underscore IR-induced acquisition of SC properties.

Genotoxic injury disrupts WNT signaling that is required for late regenerative attempt

Because HF regeneration from ORS-derived progenitors represents a novel repair mechanism, we aimed to clarify its molecular basis. First, we compared epithelial cell transcriptomes at different time points before and after 5.5Gy of IR by RNA-sequencing. Gene ontology (GO) enrichment analysis revealed several biological processes altered following IR (Fig. 5A). We screened for signaling pathways downregulated between 0 and 24hrs and upregulated between 24 and 72hrs. We found that WNT and hedgehog pathways fit this trend (Supplementary Table S3). Hedgehog signaling has been shown to be downregulated by cyclophosphamide in growing HFs (35).

Figure 5.

WNT signaling is suppressed by IR and its reactivation is required for late regenerative attempts. A, Cellular processes affected by 5.5Gy based on the comparative GO-enrichment analysis of the transcriptomes of the epithelial cells. B and C, Quantitative RT-PCR and immunofluorescence for Lef1 expression after 5.5Gy of IR. D, Immunofluorescence of β-catenin. Nuclear localization of β-catenin was detected in the lower tips between 72 and 96hrs. E and F, Quantitative RT-PCR and immunofluorescence for Wnt3a after 5.5Gy of IR. G, Inhibitor for WNT secretion and WNT signaling by IWP2 and IWR1, respectively. H, Inhibition of epithelial WNT/β-catenin signaling by ablating Ctnnb1 in keratinocytes of K5^CreER; Ctnnb1^f/f mice. * p<0.05, compared to 0hr, by Student’s t test. Error bars represent mean ± S.E.M. Scale bar=25µm in (C) (D) (F), 75µm in (G) (H).

WNT signaling is activated in ShgSCs at the onset of physiological anagen and is crucial for normal hair cycle progression (36–38). The transcript and protein levels of Lef1, a downstream mediator of WNT signaling in HFs, were prominently suppressed after IR and restored only by 96hrs post-IR (Fig. 5B and C). This was accompanied by an increase in nuclear β-catenin in the lower tip cells 72-96hrs post-IR (Fig. 5D). WNT ligands are secreted by HF epithelium and help to maintain proper epithelial-mesenchymal interaction during anagen (36,39–41). We found that Wnt3a levels were diminished between 24 and 48hrs post-IR, but later rebounded from 72hrs onward at the base of the epithelial strand (Fig. 5E and F). These results show that 5.5Gy disrupts WNT signaling and that its reactivation correlates with late regenerative attempts. In comparison, after 2Gy exposure, levels of Wnt3a and Lef1 were suppressed only transiently and partially (Supplementary Fig. S5A-D). Therefore, higher IR doses induce more severe suppression of WNT signaling.

To determine whether WNT signaling reactivation is required for late regenerative attempts, we pharmacologically disrupted WNT ligand secretion with IWR1 and IWP2 inhibitors and found that the late regenerative attempt was attenuated (Fig. 5G). We also disrupted β-catenin in K5^CreER; Ctnnb1^flox/flox mice after 5.5Gy of IR and found that the late regenerative attempt was abolished (Fig. 5H). Therefore, reactivation of WNT signaling is essential for the late regenerative attempt. This parallels the requirement for WNT signaling during normal HFSC activation upon telogen-to-anagen transition (36,38)

p53 is required for IR-induced HF dystrophy and suppression of WNT signaling

p53 has been shown to mediate pathological changes of chemotherapy-induced hair loss and IR-induced injury to other organs (42,43). Its role in IR-induced HF dystrophy remains unclear. We found that p53 was induced by IR in hair bulbs, but not DP, and that p53 expression was more extensive and persistent at 5.5Gy than 2Gy (Fig. 6A). Furthermore, matrix apoptosis, suppression of proliferation, HF shrinkage and hair loss were not induced in p53 null mice by IR (Fig. 6B-E; Supplementary Fig. S6A-C). In wild-type mice, IR increased expression of downstream target genes of p53, Noxa, Bax, and p21, whereas Puma expression was slightly decreased from 6 to 48hr and transiently elevated at 72hr (Fig. 6F). In p53 null mice, the baseline expression of Bax and p21 was higher than wild-type mice while Noxa and Puma were lower than wild-type mice (Fig. 6F). After IR, compared to wild-type mice, decreased expression of Bax, Noxa and Puma was observed in p53 null mice and p21 was only slightly increased at 24hr. This might help to explain the decreased apoptosis and unsuppressed cell proliferation after IR in p53 null mice.

Figure 6.

WNT ligand production and WNT signaling are not inhibited in p53 null mice after 5.5Gy of IR. A, Immunohistochemistry for p53. B, Apoptosis detected by TUNEL staining. C, Cell proliferation mapped by BrdU pulse labeling. D, IR-induced hair loss was not observed in p53 null mice 5 days after IR. E, Histology showed no dystrophic changes in HFs of p53 null mice after IR. F, Quantitative RT-PCR for Bax, Noxa, Puma, and p21 expression in epithelial cells of p53 WT and p53 null mice after 5.5Gy of IR, respectively. G, Quantitative RT-PCR for Wnt3a and Lef1 expression in epithelial cells of p53 WT and p53 null mice after 5.5Gy of IR. H, Immunofluorescence for Wnt3a. I, Immunofluorescence for Lef1. Dashed line: DP. Scale bar=75µm. Blue star * p<0.05, compared to 0hr of p53 WT; green star * p<0.05, compared to 0hr of p53 null; # p<0.05, p53 WT vs p53 null. Error bars represent mean ± S.E.M. WT: wild type.

Comparison of 2Gy and 5.5Gy-induced injuries revealed a correlation between the duration of p53 activation and more severe and persistent WNT signaling suppression, suggesting that p53 might be involved in mediating IR-induced WNT suppression. We found that the basal mRNA expression of Wnt3a and Lef1 at 0hr was significantly higher in p53 null mice than in p53 wild-type mice (Fig. 6G). The higher WNT signaling activity in p53 null mice is consistent with the prior report showing the inhibition of WNT signaling by p53 (44). In p53 wild-type mice, both Wnt3a and Lef1 expression were prominently suppressed by 5.5Gy of IR and were restored again at 72hrs and 96hrs, respectively. In p53 null mice, although the Wnt3a expression was decreased after IR, it was still higher than that in p53 wild-type mice at 6, 24 and 96hrs. Lef1 expression was not downregulated until 24hrs but its levels in p53 null mice were much higher than that of p53 wild-type at all time points. Consistent with this, we found that protein expression of Wnt3a and Lef1 was not suppressed by IR in p53 null mice (Fig. 6H and I). Judging from Lef1 expression, WNT signaling was maintained at a higher level in p53 null mice and this might contribute in part to the attenuated HF dystrophy after IR.

Local delivery of WNT ligands prevents IR and cyclophosphamide-induced alopecia by enhancing ectopic progenitor cell proliferation

Since WNT ligands produced by anagen HF epithelium are known to maintain anagen progression (36,38,39) and since WNT signaling undergoes early suppression after IR, we posited that maintaining WNT signaling might promote HF regeneration, in part by bypassing the suppressive effect of p53 on cell proliferation. To test this, we delivered Wnt3a-soaked beads into the 5.5Gy irradiated skin (Fig. 7A). We found that hairs continued to emerge in the Wnt3a-treated skin (Fig. 7A; Supplementary Fig. S6D) with reduced HF dystrophy and faster restoration of anagen structures (Fig. 7B; Supplementary Fig. S6E and F). Wnt3a treatment preserved Lef1 expression (Supplementary Fig. S7A), but did not prevent p53 activation and apoptosis (Fig. 7C and D; Supplementary Fig. S7B). The latter was only slightly decreased at 24hrs post-IR (Fig. 7C and D). Analysis of gene expression of HF epithelial cells from Wnt3a-treated and control (BSA)-treated skin showed that Wnt3a treatment significantly inhibited the upregulation of Bax, Noxa, and p21, but increased the expression of Puma after IR (Fig.7 E). The upregulation of proapoptotic Puma with downregulation of proapoptotic Bax and Noxa might help to explain that Wnt3a did not largely suppress IR-induced apoptosis (Fig. 7D). The suppression of p21 expression, an important p53 target that induces cell cycle arrest, by Wnt3a treatment (Fig.7E) might promote regenerative proliferation. Indeed, compared to control, an early increase of basal K5⁺ cell proliferation was observed at 24hrs and cell proliferation within the hair matrix was maintained at a higher level for at least 72hrs (Fig. 7F, white arrows, and G; Supplementary Fig. S7C). Importantly, the number of matrix cells was increased in the Wnt3a-treated HF (Supplementary Fig. S7D).

Figure 7.

Enhancement of WNT signaling prevents IR and cyclophosphamide-induced alopecia by boosting the early regenerative attempt. A, Subcutaneous injection of Wnt3a-soaked beads (blue beads) maintained hair growth after 5.5Gy of IR. Dashed circles indicate bead-implanted area. B, Histology of Wnt3a and BSA-treated HFs. C and D, Apoptosis detected by TUNEL and quantification of TUNEL⁺ matrix cells. * p<0.05. E, Quantitative RT-PCR for p53 transcriptional targets in HF epithelial cells in Wnt3a-treated skin. Blue star* p<0.05, compared to 0hr of BSA treatment; green star* p<0.05, compared to 0hr of Wnt3a treatment. F and G, Pulse BrdU labeling and quantification of K5⁺ BrdU⁺ matrix cells. Wnt3a increased K5⁺ cell proliferation (white arrows). * p<0.05. H, Cyclophosphamide (CYP) treatment. Hair loss was observed 5 days after treatment. I, Wnt3a-soaked beads (blue beads) maintained hair growth in their vicinity after CYP treatment. J, Anagen HF repair. Depiction of cell origin and cell dynamics for early and late regenerative attempts for anagen HF repair following IR injury. BSA: bovine serum albumin. Dashed lines in (C) (F) indicate DP. Scale bar=75µm in (B), 25µm in (C) (F).

We also tested whether this approach can prevent chemotherapy-induced alopecia. Cyclophosphamide (CYP) administered on postnatal day 32 also induced extensive hair loss on day 5 (Fig. 7H). Similarly, local delivery of Wnt3a-soaked beads reduced hair loss after CYP treatment (Fig. 7I). These results demonstrate that maintaining WNT signaling can prevent hair loss from genotoxic injury by enhancing the regenerative cell proliferation of K5⁺ basal hair bulb progenitors.

Discussion

Our results show that, rather than deploying long-lived bulge SCs, IR-damaged anagen HFs mobilize extra-bulge TAC-derived progenitor cells for repair without having to reset their hair cycle (Fig. 7J). This ability of HFs to mobilize distinct extra-bulge progenitors for anagen HF repair also suggests a new therapeutic strategy for hair loss that relies on TACs.

In hair bulbs, germinative and basal bulb cells are thought to represent two distinct lineages within the TAC population (14). Proliferative germinative cells fuel hair shaft elongation (14,45) while basal hair bulb cells (also referred to as lower proximal cup cells) are speculated to maintain the shape of hair bulbs and not to contribute to the inner HF structures (14). We show that K5⁺ basal cells serve as SCs for rapid repair of the HF bulb, and that their concealed plasticity is quickly unveiled upon injury. Previously, colony-forming epithelial SCs were shown to be present in the hair bulbs (46). It is likely that K5⁺ basal hair bulb cells can be a source of colony-forming cells. The employment of local multipotent progenitors within the TACs shortens the time needed for regeneration and this strategy is advantageous over regeneration via BgSCs. The employment of BgSCs would require intricate signaling relays and create a significant time delay during their downward migration toward injured hair bulbs.

We also revealed a novel role for ORS cells of the TAC pool for regeneration. HFs suffering from a more dystrophic change regenerate from the surviving ORS cells. Although our lineage studies are not able to directly differentiate between K5⁺ basal bulb cells and K5⁺ basal ORS cells, the two cell populations are known to be distinct in origin and function during anagen (14). K5⁺ hair bulb cells broadly apoptose after 5.5Gy, indicating that radioresistant K5⁺ ORS cells are the most likely origin for the epithelial strand. Although targeted depletion of SCs can allow differentiated cells to gain SC-like properties upon migration into the physiological SC niche (47,48), it remains unknown if acquisition of SC-like properties can occur ectopically outside the physiological SC niche. Here we showed that ORS cells can directly acquire a SC-like state ectopically after IR. Since DP cells are able to convert interfollicular keratinocytes into follicular cells for HF neogenesis upon close contact (49), the close proximity of lower tip cells to DP suggested that DP cells might play an important role here by either directly reprogramming ORS cells into a SC-like state or providing signals for the dedifferentiation of ORS cells through short-range interaction.

The lower tip cells in the epithelial strand not only express markers characteristic of ShgSCs, but also behave like them. Similar to the telogen-to-anagen transition (12), the late regenerative attempt displays the step-wise activation of progenitor cells: lower tip cells are activated first to regenerate hair bulbs, followed by the activation of BgSCs to resupply upper ORS. Their contribution to the entire hair bulb and all bulb-derived differentiated structures confirms their multipotency. Furthermore, their progeny home back to the SC niche at the end of the repaired hair cycle and contribute to the new hair bulb and ORS during the following physiological hair growth cycle.

The activation of p53, a central mediator of pathological changes following IR injuries in other organs (43,50), is required for chemotherapy-induced HF dystrophy (42). We revealed its key role in the response of HFs to IR and also uncovered a complicated crosstalk between IR, p53 and WNT signaling. We found that IR induces p53 expression in the hair bulb in a dose-dependent manner. In p53 null mice, neither apoptosis nor dystrophy was induced, indicating the essential role of p53 in IR-induced hair loss. Since WNT signaling is not inhibited by IR in p53 null mice, our results suggest that p53 may have dual roles in both inducing apoptosis and suppressing regeneration-promoting WNT signaling following IR injury. We demonstrated that augmenting WNT signaling could attenuate the suppressive effect of p53 on cell proliferation, likely through inhibition of p53-responsive upregulation of p21, to enhance the regenerative program of ectopic progenitors from TACs following the injury of IR and cyclophosphamide. Because the effect of applied Wnt3a protein is transient and localized, it potentially represents a new, low-risk clinical strategy to reduce hair loss after genotoxic injury.

Abbreviations

BgSC

bulge stem cell

DP

dermal papilla

HF

hair follicle

HFSC

hair follicle stem cell

IR

ionizing radiation

IRS

inner root sheath

ORS

outer root sheath

SC

stem cell

SHG

secondary hair germ

ShgSC

secondary hair germ stem cell

TAC

transit amplifying cell.