Conditional telomerase induction causes proliferation of hair follicle stem cells

Kavita Y. Sarin, Peggie Cheung, Daniel Gilison, Eunice Lee, Ruth I. Tennen, Estee Wang, Maja K. Artandi, Anthony E. Oro & Steven E. Artandi

Introduction

Telomerase, or more specifically, its protein component TERT, is traditionally known to maintain the length of telomeres. Through this mechanism, it plays a role in the increased proliferative capability of stem cells, progenitor cells, as well as cancer cells. Sarin et. al., however, found that TERT can promote the proliferation of resting stem cells through a non-canonical pathway. They used the conditional transgenic induction of TERT to promote robust hair growth in mouse epithelium. They found that TERT causes a rapid transition between telogen (the resting phase of the hair follicle cycle) and anagen (the active phase). More specifically, TERT overexpression stimulates the proliferation of quiescent stem cells in the hair follicle bulge region. This function does not require the RNA component of telomerase, which codes for telomere extension, and thus operates through a mechanism independent of its traditional function of maintaining telomere length.

Methods and Results

Sarin et. al. obtained protein extracts from wild-type mouse skin, and analyzed the first and second postnatal hair cycles using the telomere repeat amplification protocol (TRAP assay). They detected strong telomerase activity during the anagen phases, and none during the telogen phases (Fig. 1b).

a, A schematic diagram of hair follicle cycling. b, TRAP on non-transgenic (Non-Tg) skin samples at day 4, 10, 16, 19, 21, 28, 34 and 52 (anagen, A; catagen, C; telogen, T). c, d, Northern blot analysis and TRAP on skin extracts from i-TERT and wild type (WT) mice at day 50. e, Doxycycline-treated mice at day 70, Non-Tg (+ doxy) and i-TERT (+ doxy) mice. f, H&E skin sections at 20 magnification. -doxy indicates no doxycycline treatment; asterisk indicates telogen hair follicle; arrow indicates anagen hair follicle. g, RNA in situ hybridization for TERT mRNA and immunofluorescence for K14 in i-TERT (+ doxy) skin (asterisk indicates autofluorescence). h, RNA in situ hybridization for TERT mRNA (blue) in an i-TERT(+ doxy) skin section (right panel) and WT anagen skin section (left panel) (asterisk indicates dermal papilla).

To determine whether TERT expression can induce a transition from telogen to anagen phase, they injected induced TERT (i-TERT) mice with doxycycline after their hair follicles had entered the telogen phase (Fig. 2), and took tissue samples at regular intervals for 12 days. TERT mRNA and telomerase activity were found to increase from day 3 to day 9 (Fig. 2a).

a, Northern blot (left) and TRAP assay (right) show increased TERT expression and telomerase activity after nine days of doxycycline treatment. b, H&E stain showing that follicles in i-TERT mice entered anagen (black arrows) by day 9, whereas Non-Tg controls remained in telogen (asterisks). c, Hair growth was observed only in i-TERT mice with doxycycline treatment (+ doxy), but not in i-TERT mice (- doxy) or Non-Tg littermates.

Since the activation of stem cells in the bulge section of hair follicles is critical to the initiation of anagen cycles, they hypothesized that TERT’s effects on the hair follicle cycle are mediated through stem cells. To test this hypothesis, they marked hair follicle bulge stem cells with repeated injections of BrdU followed by a long chase period. During the second telogen, the mice were biopsied, switched to doxycycline (a TERT inducer) drinking water and biopsied again between days 80 and 100. Label retaining cells (LRCs) were visualized by double immunostaining with antibodies against BrdU and CD34, a cell surface marker for hair follicle stem cells. LRCs were present in similar numbers in both i-TERT and non-transgenic mice at day 55, before the switch to doxycycline containing water.
After five weeks of doxycycline treatment, the BrdU label in CD34 stem cells was retained at similar levels in non-transgenic mice. In contrast, the BrdU label was significantly weakened in the CD34 cell population in the bulge in i-TERT mice (Fig. 3a, b). Despite the loss of the BrdU label, the CD34 cells remained in similar numbers in the bulge, suggesting that stem cells divide but likely self-renew to maintain the CD34 population when exposed to TERT. The conclusion is drawn that TERT causes hair follicle bulge cells to proliferate, diluting the BrdU label from the quiescent stem cell population.

a, Immunofluorescence for BrdU (red) and CD34 (green) shows maintenance of LRCs in non-transgenic (Non-Tg) group, but dramatic loss of label in i-TERT mice after doxycycline (doxy) treatment (pre-doxy, day 55; post-doxy, day 90). Asterisk indicates autofluorescence of hair. b, Quantification of LRC data from a, showing the fraction of CD34⁺ cells that are also BrdU⁺. Data for i-TERT mice (black bars, n = 4 mice) and non-transgenic mice (grey bars, n = 3 mice), pre-doxy (- ) and post-doxy (+ ) treatment are shown. c, LRC analysis from wholemounts of epidermis from the tail of mice labelled with BrdU at day 10, switched to doxy at day 40 and analysed at day 65 (BrdU, red; K14, green); B indicates bulge and SG indicates sebaceous gland. d, Immunofluorescence using Ki-67 (red) to mark proliferating cells and K14 (green) to identify basal layer of skin. e, Quantification of proliferation index in d as Ki-67⁺ cells per 100 m length of basal layer (each comparison n = 2 mice). i-TERT mice (black bar) and non-transgenic mice (grey bars). f, GFP epifluorescence co-stained with CD34 (inset, confocal microscopy) in skin section from an actin–GFP mouse. g, RNA in situ analysis for TERT mRNA in i-TERT(+ doxy) mouse skin (inset, TERT mRNA expression (cytoplasmic) overlaps in bulge with LRCs, marked by BrdU (nuclear)). h, H&E sections from K5tTA⁺;tetop-TERT⁺(- doxy) (bottom) and Non-Tg (top) mice, at 20 magnification. Error bars indicate standard deviation. P values derived from Student's t-test.

To establish the theory that TERT in this case operates through a non-canonical pathway (that is, without TERC, the RNA component of telomerase), they intercrossed TERC +/- mice with i-TERT alleles. They found that conditional activation of TERT induced anagen in five out of five i-TERT+, TERC- mice (Fig. 4a).

a, H&E sections showing TERT-induced anagen in mice with TERC^+/+, TERC^+/- and TERC^-/- backgrounds, at 20 magnification. b, Skin samples from i-TERT TERC^-/- mice (+ doxy) lacked telomerase activity by TRAP (left panel) and lacked TERC expression by RT–PCR (right panel). Minus sign indicates negative control lacking reverse transcriptase.

Critique

Sarin et. al. demonstrate an effective examination of their hypothesis. Using TRAP, Northern blotting, immunofluorescence, and tissue analysis, they provide multiple methods to create a thorough support of their conclusion. They also considered possible side effects, and created additional experiments to account for them. For instance, they considered whether abnormalities in the hair follicle cycle could be causing the altered phenotype. Overall, they convincingly showed that TERT causes the proliferation of hair follicle stem cells, and that a rapid transition from telogen to anogen phase results.

There are just a few minor weaknesses in their paper. First, there ideally should be more than five mice used to demonstrate the non-canonical pathway of TERT. Also, in a few cases, they make assumptions. The words “probably” and “likely” show up in isolated situations, such as when they say that the stem cells “probably self-renew to maintain the CD34 population”. Obviously, not all assumptions can be supported by today’s experiments, but this is just something to watch out for.