In stem cell and progenitor cell compartments3–5
, TERT serves an important role in keeping telomeres sufficiently long and stable to prevent the adverse consequences of dysfunctional telomeres on cell viability and chromosomal stability6–8
. However, the need for expression of TERT in tissue stem cells and progenitor cells with long telomeres is less clear, especially in laboratory mice, whose telomeres are significantly longer than those of humans (40–60kb vs. 5–15kb). Moreover, recent findings indicate that TERT promotes tumor development even in settings of ample telomere reserve, although the mechanisms underlying these telomere length-independent activities of TERT remain unclear9–13
. We therefore hypothesized that TERT may exert effects in stem cell and progenitor cell compartments that could explain both its regulation during lineage development and its poorly understood telomere length-independent activities.
To test this hypothesis, we turned to the mammalian hair follicle, an organ that harbors tightly regulated multipotent stem cells and that cycles between telogen and anagen14
. Initiation of a new anagen cycle depends upon activation of a small number of quiescent stem cells that reside in the bulge, a niche at the follicle base15–19
. These activated stem cells proliferate and differentiate into progenitor cells (matrix cells) that give rise to the differentiated lineages that comprise the hair shaft and root sheaths. This period of hair synthesis ceases when the new section of the anagen follicle is remodeled through apoptotic regression (catagen), resulting in another telogen phase (). To understand how telomerase is regulated during mouse hair follicle cycling, we analyzed telomerase activity in mouse skin, exploiting the fact that hair follicle cycles are synchronized for the first two postnatal periods of hair follicle growth, approximately 8 weeks20
. Protein extracts from wild type mouse skin were obtained between postnatal days 4 and 52, to allow analysis of both the first and second postnatal hair cycles using the Telomere Repeat Amplification Protocol (TRAP assay). Telomerase activity strongly correlated with the first and second anagen phases and was not detected during telogen phases (). These data indicate that, in mouse hair follicles, as in human21
, telomerase is associated with the anagen phase, a period of intense progenitor cell activity.
Figure 1 Conditional activation of TERT promotes the anagen phase of the hair follicle cycle. a, Diagram of hair follicle cycling b, TRAP on Non-Tg skin samples at days 4, 10 (anagen-A), 16 (catagen-C), 19, 21 (telogen-T), 28, 34 (A), and 52 (T). c–d, (more ...)
To determine if TERT can modulate adult tissue stem and progenitor cell function, we engineered a conditional TERT transgenic system in mice using a tetracycline-regulated approach22
. The mouse TERT cDNA was cloned under control of a tetracycline responsive promoter (tetop-TERT+
). To drive expression of TERT, we chose a CMV enhancer/β-actin promoter (actin-rtTA+
) because this element was previously shown to be active in stem cells23
and in many epithelial tissues, including skin24,25
mice were intercrossed with actin-rtTA+
mice to generate actin-rtTA+
(termed inducible TERT or i-TERT) mice. To induce expression of TERT, i-TERT mice were administered drinking water containing the tetracycline analogue, doxycycline. Northern blot and TRAP assay confirmed doxycycline-dependent induction of the TERT transgene and increased telomerase activity in the skin of i-TERT mice () as well as in other tissues (data not shown). Remarkably, within several weeks of doxycycline treatment, the coats of i-TERT mice were significantly altered, reminiscent of mice with known mutations that affect hair follicle cycling26,27
To determine if abnormalities in hair follicle cycling might underlie the altered hair phenotype, we analyzed skin biopsies from i-TERT mice after doxycycline administration beginning at day 21. Hair follicles were appropriately in anagen at day 28 in i-TERT mice on and off doxycycline, and in littermate controls. By day 50, follicles from i-TERT mice off doxycycline and from non-transgenic mice had exited anagen and were in the second post-natal telogen phase. In marked contrast, hair follicles from i-TERT mice on doxycycline were consistently in anagen at day 50 (). This effect was doxycycline-dependent and occurred with 100% penetrance in i-TERT mice (18/18 in anagen) (chi square analysis, p<0.0001 for i-TERT on vs. off doxycycline, Table S1). RNA in situ hybridization revealed a pan-epithelial expression pattern of transgenic TERT in skin that included the Keratin-14+ compartment, but spared the dermal papilla, indicating that transgenic TERT mRNA is expressed principally in hair follicle and skin epithelium ( & ). Together, these data show that conditional induction of TERT in adult hair follicle epithelium promotes the anagen phase.
Figure 3 TERT activates stem cells, depleting BrdU label from LRCs. a, Immunofluorescence for BrdU (red) and CD34 (green) shows maintenance of LRCs in Non-Tg group, but dramatic loss of label in i-TERT mice after doxy treatment (pre-doxy = day 55, post-doxy= day (more ...)
To determine if expression of TERT is sufficient to induce a transition from telogen to anagen, i-TERT mice were treated with doxycycline after hair follicles had entered the prolonged second telogen (day 40 in FVB mice (; data not shown)) and were biopsied at regular intervals for 12 days. TERT mRNA and telomerase activity progressively increased from day 3 through day 9 (). Whereas hair follicles from non-transgenic mice remained in telogen during the duration of the time course, follicles from i-TERT mice treated with doxycycline entered anagen by day 9 and were in mid-anagen20
by day 12 () (chi-square analysis, p=0.005, Table S2). The kinetics of anagen entry closely followed the timing of TERT induction. To determine if induction of anagen by TERT enabled hair growth, i-TERT mice were treated with doxycycline-drinking water beginning at day 45 and shaved at day 55. Fourteen days after shaving, i-TERT mice administered doxycycline showed significant hair growth as compared with both i-TERT mice off doxycycline and non-transgenic controls, in which hair did not grow during this interval (). Strikingly, these results show that induction of TERT in telogen follicles is sufficient to initiate a transition to the anagen phase and promote new hair synthesis.
Figure 2 TERT induction triggers a rapid transition from telogen to anagen. a, Northern blot (left) and TRAP assay (right) show increased TERT expression and telomerase activity by nine days of doxycycline treatment. b, Follicles in i-TERT mice entered anagen (more ...)
Because activation of bulge stem cells is integral to the initiation of a new anagen cycle15,16,18
, we hypothesized that TERT’s effects on the hair follicle cycle might be mediated through the stem cell compartment. To address this hypothesis, we employed a label retaining technique that has been used successfully to mark hair follicle bulge stem cells by repeated injections of BrdU followed by a long chase period15
. Cohorts of i-TERT mice and non-transgenic controls were injected with BrdU at 10 days of age. During the second telogen, mice in each group were biopsied, switched to doxycycline 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 membrane marker for hair follicle stem cells28,29
. LRCs were present in similar numbers in both i-TERT and non-transgenic mice at age 55 days, before the switch to doxycycline water (approximately 0.6 BrdU+ cells/CD34+ cell). After five weeks of doxycycline treatment, BrdU label in CD34+ stem cells was retained in non-transgenic mice at comparable levels, consistent with previous observations that BrdU label persists in quiescent bulge cells for more than six months17
. In marked contrast, BrdU label was profoundly depleted in the CD34+ cell population in the bulge by induction of TERT in i-TERT mice (76% reduction in BrdU+ cells/CD34+ cell, p<0.0001) (). Despite the loss of BrdU label, CD34+ cells in the bulge remained in similar numbers, indicating that, under the influence of TERT, stem cells divide but likely self-renew to maintain the CD34+ population. A similar reduction in LRCs in i-TERT mice was seen in epidermal wholemounts, corroborating the effects seen in dorsal skin sections (). These data show that TERT causes hair follicle bulge cells to proliferate, diluting BrdU label from this quiescent stem cell population.
To determine if TERT more broadly enhances keratinocyte proliferation, we measured the proliferation index in the basal layer of the interfollicular epidermis (). Despite expression of transgenic TERT mRNA in this compartment, proliferation was not substantially altered in the basal layer in i-TERT mice compared to non-transgenic littermates in anagen (4.2 Ki-67+ cells/100μm for i-TERT day 50 compared to 4.3 Ki-67+ cells/100μm for non-transgenic day 28) (). Furthermore, we found no changes in structure, differentiation, or signaling in either hair follicle or interfollicular epidermis in i-TERT mice (Fig. S2, S3). We therefore conclude that the principle effects of TERT in this system occur through activation of quiescent hair follicle stem cells.
To understand if these results are consistent with a direct effect for TERT on stem cells, we asked if transgenic TERT is expressed in the stem cell compartment. We found that the promoter element used to direct rtTA expression is strongly active in CD34+ bulge cells in actin-GFP mice24
(). Furthermore, TERT mRNA was co-expressed with BrdU in LRCs in the bulge region in i-TERT mice (). While induction of anagen can occur through signals from the dermal papilla30
, the lack of detectable levels of TERT mRNA in the dermal papilla () makes it unlikely in this case. To confirm that TERT exerts its effect through the epithelium, we intercrossed tetop-TERT mice with a transgenic mouse in which the Keratin-5 promoter drives expression of the tetracycline transactivator (tTA) in the basal layer and outer root sheath (K5-tTA, tet off configuration)18
. Compound K5-tTA+
mice were bred on doxycycline and weaned off doxycycline-drinking water at day 21 to induce the TERT transgene. Expression of TERT mRNA in skin epithelium (data not shown) induced anagen in 5/5 K5-tTA+
mice, whereas all littermate control biopsies were in telogen (6/6, p=0.0009 by Chi square analysis)(). These data show that TERT’s effects in promoting anagen are intrinsic to the K5 compartment of the skin epithelium, the layer where the hair follicle stem cells reside.
These effects for TERT in facilitating a switch from telogen to anagen could occur through TERT’s established role in telomere synthesis or through a novel mechanism, independent of its known enzymatic function. Telomere synthesis requires both TERT and TERC; therefore, if the effects of TERT are retained in a TERC−/− background, telomere extension cannot be required for stem cell activation events mediated by TERT. To answer this question, TERC+/− mice were intercrossed with inducible TERT alleles to derive cohorts of i-TERT mice that were TERC+/+, TERC+/− and TERC−/−. Mice in each group were treated with doxycycline beginning in telogen at day 40. Histological analysis at day 55 revealed that conditional activation of TERT induced anagen in 5/5 i-TERT; TERC−/− mice () (p=0.0003 for i-TERT; TERC−/− mice on doxycycline vs. i-TERT mice off doxycycline, Table S3). These results were identical to those obtained from i-TERT mice in either TERC+/+ or TERC+/− backgrounds (6/6 in anagen), indicating that TERC is not required for TERT to induce anagen. TRAP assays and RT-PCR analysis confirmed the absence of telomerase activity and TERC RNA in the skin of i-TERT; TERC−/− mice (). These data prove that the mechanism by which TERT triggers hair follicles to transition from telogen to anagen does not require telomere synthesis.
Figure 4 TERT's activity in facilitating a transition from telogen to anagen is independent of its function in telomere synthesis. a, TERT induced anagen in mice with TERC+/+, TERC+/−, and TERC−/− backgrounds, H&E 20x. b, Skin samples (more ...)
Here we show that conditional activation of TERT protein in mouse skin is sufficient to induce proliferation of hair follicle stem cells and that this function is independent of its role in telomere synthesis. In this transgenic context, either altered timing of TERT expression or higher protein levels may be important in mediating these new activities for TERT. Our data encourage further studies to understand the role of the endogenous TERT protein in stem cells, including loss-of-function studies and experiments designed to determine if TERT's function in stem cell proliferation is cell autonomous. We propose that as TERT is dynamically regulated during the course of stem and progenitor cell maturation, or during advancing stages of cancer development, TERT may directly support these processes. These data provide a novel framework for understanding the recently identified telomere length-independent functions of TERT9–12
and suggest new strategies for manipulating TERT for therapeutic purposes in treating disorders associated with tissue injury and aging.