TGF-β Signaling Is Transiently Activated in Stem Cells during the Telogen→Anagen Transition
To determine whether and how TGF-βs might affect HF regeneration, we first verified the specificity of our anti-phospho-Smad2 antibodies. Upon TGF-β exposure in vitro, primary wild-type (WT) mouse epidermal keratinocytes (1°MKs) exhibit phosphorylation and nuclear translocation of Smad2, which can be detected by immunoblotting of nuclear extracts and immunofluorescence. This band and nuclear staining were absent in the equivalent 1°MKs from Tgfbr2fl/fl
cKO) mice whose skin epithelium lacks TβRII, an essential component of the TGF-β receptor (Figures S1A and S1B available online; Guasch et al., 2007
). Using WT and TβRII
-cKO mice, we then turned to in vivo analyses.
During normal homeostasis, epidermis displayed little or no signs of active TGF-β signaling (not shown). Postnatally, the first signs of TGF-β signaling appeared in telogen, when pSmad2+ nuclei were detected in HG cells adjacent to the DP (). As follicles began cycling, nuclear pSmad2+ cells also appeared in CD34+ HFSCs at the bulge base. By the time full anagen was reached, pSmad2 immunofluorescence was no longer detected in HFSCs (Figure S1C).
TGF-β Signaling Is Transiently Activated in HFSCs at the Telogen→Anagen Transition of the New Hair Cycle
The second telogen (marked by two rather than one club hairs) lasted about 4 weeks in control mice. During early and mid-telogen phases, TGF-β activity was weak. As telogen ended, pSmad2 reappeared in the P-cadherin+ HG and lower bulge (). Notably, pSmad2 was detected ~5 days before this proliferative activity was seen within the HG (). Similar to the first hair cycle, most HG cells in early anagen exhibited TGF-β signaling, but as the expanding HG engulfed the DP and formed the matrix, pSmad2 waned.
To further examine TGF-β signaling, we engineered a lentivirus harboring a canonical TGF-β-reporter driven by Smad2/3-Smad4 binding sites (). In vitro, reporter expression (NLS-mRFP+
) was detected only in TGF-β-stimulated, transduced (H2B-CFP+
) 1°MKs from WT and not in TβRII
-cKO mice (; shown are data for TGF-β2). When transduced by in utero infection into embryonic skin epithelium (Beronja et al., 2010
) and then monitored in adult mice, the TGF-β-reporter was active selectively in the WT HG, beginning at late telogen and continuing into early anagen (). Underscoring reporter specificity, similarly transduced TβRII-
cKO animals showed no signs of reporter activity. These data revealed that nuclear pSmad2 at the telogen→anagen transition is accompanied by pSmad2-mediated active transcription in the HG.
Stem Cell Activation in Hair Follicles Is Delayed when TGF-β Signaling Is Lost
The TGF-β/pSmad2 signaling pattern upon anagen onset paralleled the two-step activation of HFSCs described previously (Greco et al., 2009
). To determine whether TGF-β signaling functions in HFSC activation, we analyzed this process in TβRII
-cKO mice. By 22 days, control HFs had already entered the first anagen and showed proliferating (Ki67+
) HG cells, while TβRII
-cKO HFs were still in telogen (). Because the first WT telogen lasts only 1–2 days, the phenotypic consequences of losing TGF-β signaling to SC activation and tissue regeneration were best visualized by waiting until HFs had entered the extended second telogen. At this time, we clipped hair coats of sex-matched littermates and then followed the emergence of new hairs as the next cycle began ().
Mice whose Skin Epithelium Is Deficient in TGF-β Signaling Show a Delay in HFSC Activation and Hair Cycle Entry
In the second telogen, hair growth was so delayed in TβRII-cKO mice that 75% coat recovery occurred ~1 month later than normal (). Despite this delay, the final lengths of the four hair types were normal (), as were HF morphologies and established markers (). Thus, once TβRII-deficient SCs were eventually activated, they executed what appeared to be an otherwise normal hair cycle.
The Dermal Papillae Produce a Paracrine TGF-β2 Signal to Activate Canonical TGF-β-Mediated Transcription in Hair Follicle Stem Cells
cells appeared at the interface between HG and DP, and after the extended crosstalk period that is required for HFSC activation, we sought to identify the TGF-β ligands involved and the cell population expressing them. A survey of existing microarray data indicated that in adult skin, TGFβ2
mRNAs might be enriched in DP relative to HG or HFSCs (Greco et al., 2009
). Support for this came from real-time quantitative polymerase chain reaction (RT-qPCR) of mRNAs isolated from purified cell populations of late-telogen phase, Lef1-RFP
transgenic skins. TGFβ2
mRNAs selectively were enriched in DP relative to other IFE and HF populations, whereas TGFβ1
mRNA expression was low and showed no specific enrichment ( and S2A).
TGF-β2 Is Produced by DP, Activates pSmad2 in HFSCs, and Participates in the Telogen→Anagen Transition
Immunofluorescence analyses were consistent with these findings. Of the three TGF-βs, only TGF-β2 protein appeared in HFs toward the end of telogen ( and S2B). Intercellular TGF-β2 first accumulated at the ECM interface between DP and HG, but by anagen onset, it intensified in the DP, with weaker staining in HGs. Although latent TGF-β complexes can reside within ECM (ten Dijke and Arthur, 2007
), TGF-β2 signal intensity in the DP coincided with nuclear pSmad2 in the HG.
To test whether TGF-β2 signaling can precociously activate resting HFSCs, we coinjected recombinant, active TGF-β2 with fluorescent beads into mid-telogen skin dermis, i.e., well before the normal appearance of endogenous TGF-β2 in DP (see experimental design in ). In TβRII-heterozygous (WT) mice, TGF-β2 injections resulted in Smad2 phosphorylation not only in the HG, but also in IFE and some dermal cells (Figure S2C). By contrast, TβRII-cKO skin epithelium was refractory to TGF-β2- mediated Smad2 activation, while dermal cells responded. This result indicated that the effects of DP-derived TGF-β2 on precocious pSmad2 induction rely upon the HG’s ability to respond through the canonical receptors. With our TGF-β-reporter mice, we further demonstrated that the signaling involves canonical pSmad2-mediated transcriptional activation (Figure S2D).
TGF-β2 Induces Proliferation in Quiescent Hair Follicle Stem Cells and Propels Them into a Tissue-Regenerating Mode
Bromodeoxyuridine (BrdU) administration and S-phase cell quantification revealed that HGs of WT HFs reacted to TGF-β2 by displaying both nuclear pSmad2 and proliferation ( and S2C). Although this further substantiated the sequential order of events we had observed in the normal hair cycle (see ), it contrasted with TGF-β’s established negative growth effects on epidermal cells, which was also reflected in decreased BrdU incorporation within IFE (). These results indicate that IFE and HF progenitors interpret TGF-β2 differently and further underscore the specificity of the proliferative response of HFSCs to TGF-β2.
TGF-β2’s stimulatory effects depended upon close proximity of HFs to the bead injection site and were followed by a precocious hair cycle (, S2E, and S2F). This differed from similar injections with FGF7, another DP-derived factor that stimulated HG proliferation, but did not kindle a new hair cycle (Greco et al., 2009
). Taken together, our findings indicate that in normal homeostasis, DP utilizes TGF-β2 in short-range signaling to stimulate HFSCs at the bulge base. The outcome is canonical TGF-β receptor/pSmad2 transcription that in some way enables them to achieve the activating threshold necessary to flip the telogen→anagen transition switch.
BMP Signaling Is Prolonged when Hair Follicle Stem Cells Cannot Respond to TGF-β2
Throughout the resting phase, high BMP signaling maintains HFSCs in a quiescent state and must be overcome to promote new tissue growth (Andl et al., 2004
; Blanpain et al., 2004
; Kobielak et al., 2007
; Plikus et al., 2008
; Rendl et al., 2008
; Greco et al., 2009
; Hsu et al., 2011
). The appearance of TGF-β2 protein in the DP toward the end of telogen coincided with the timing of enhanced mRNAs encoding DP-derived BMP inhibitory factors, including Sostdc1
(Greco et al., 2009
). Given the seemingly opposing effects of TGF-β2 and BMPs on HFSC quiescence, we wondered whether TGF-β2 might be impacting the BMP pathway, and if so how.
To address this possibility, we first evaluated the consequences of loss of TGF-β signaling on BMP receptor signaling in HFSCs (). In WT HFSCs, nuclear anti-phospho-Smad1/5/8 immunostaining, a sign of active BMP signaling, was observed throughout early and mid-telogen, but waned toward the end of telogen, coincident with the appearance of pSmad2 (; see also ). However, in TβRII-cKO HFSCs, pSmad1/5/8 remained high throughout this time (), consistent with the notion that TGF-β signaling negatively affects BMP signaling.
TGF-β Stimulates Colony Formation of Primary HFSCs In Vitro and Dampens BMP Signaling in HFSCs In Vivo
To further explore TGF-β2’s effects, we cultured epidermal 1°MKs, where the antiproliferative effects of TGF-βs are well established (Siegel and Massagué, 2003
). Indeed, all three TGF-βs promoted comparable Smad2 phosphorylation and induced growth arrest in a monophasic, dose-dependent fashion (Figures S3A–S3E). Consistent with its lower binding affinity to TβRII (Cheifetz et al., 1987
; Massagué et al., 1992
), ~10× more TGF-β2 (10 versus 1 pM) was necessary to achieve growth inhibition (Figure S3E). Recombinant TGF-βs had no effects on TβRII-
cKO 1°MKs, thereby confirming the specificity of the response.
Next, we tested cultured HFSCs, purified by fluorescence-activated cell sorting (FACS) of mid-telogen HFs (). At higher concentrations (50–100 pM), TGF-βs reduced both number and size of HFSC colonies. However, at lower concentrations (~10 pM) where epidermal 1°MKs were still growth restricted, HFSC colonies grew larger. This effect was highly reproducible and not seen in TβRII-null HFSCs (). In good agreement with our in vivo observations, these results further unveiled the dose dependency of TGF-β’s stimulatory effects on HFSCs.
Consistent with HFSC quiescence in and out of the niche in vivo, nuclear pSmad1/5/8 staining was also greater in nascent colonies of HFSCs than in 1°MKs (Figure S3F). This was true for both WT and TβRII-null cells, but not Bmpr1a-cKO cells, which showed no signs of BMP signaling. Moreover, pSmad1/5/8 staining in WT HFSCs was reduced by exposure to TGF-β2 (Figure S3G).
Further substantiating the antagonistic effects of TGF-β2 on the BMP pathway, we found that BMP4’s effects were monophasic and could be partially relieved by TGF-β2 in a TβRII-dependent manner (). Importantly, neither BMP4 nor TGF-β2 affected the colony size in Bmpr1a-cKO HFSCs (). Moreover, even though TGF-β2 on its own no longer exerted effects on colony size as HFSCs were passaged, TGF-β2 still counteracted the negative effects of BMPs that were added exogenously to HFSC cultures (Figure S3H). Thus, the stimulatory growth effects of TGF-βs appeared to be restricted to cells exhibiting active BMP signaling.
To further pursue the notion that TGF-β2 acts positively to overcome BMP inhibitory thresholds and activate HFSCs, we compared TGF-β2’s effects to Noggin, an established and potent BMP antagonist (Botchkarev et al., 2001
; Plikus et al., 2008
). In early telogen, neither Noggin nor TGF-β2 were sufficient to overpower the exceedingly high BMPs within the HFSC niche and dermis. However, in mid-telogen, both Noggin and TGF-β2 precociously diminished nuclear pSmad1/5/8 and induced HG proliferation (). As endogenous TGF-β2 signaling and BMP signaling blockers rose and dermal BMP waves waned at telogen’s end, these effects were less pronounced. Notably, although probably not physiologically relevant, TGF-β1 and TGF-β3 behaved similarly, indicating that it is TGF-β availability and not TGF-β2 per se that is important in the process ().
In contrast to Noggin, TGF-β2’s effects were seen only in WT and not TβRII-cKO mice. This result was important as it demonstrated that the opposing effects of TGF-β2 on BMP signaling are mediated directly through the TGF-β receptor pathway rather than cross-counter effects on their respective receptors. Moreover, these data provided compelling complementary evidence that the atypically long telogen of TβRII-cKO mice arises from protracted BMP signaling in HFSCs.
Evidence Favoring a TGF-β Target Gene in Hair Follicle Stem Cell Activation
To further probe the antagonistic interaction between TGF-β2 and BMP signaling, we engineered and tested a BMP-reporter lentivirus (). In vitro, transduced WT and TβRII-null, but not Bmpr1a-null, 1°MKs displayed BMP-reporter activity in response to BMP4. In vivo, WT HFSCs showed concomitant downregulation of both BMP-reporter activity and pSmad1/5/8 at telogen’s end. By contrast, TβRII-cKO HFSCs sustained BMP-reporter activity and pSmad1/5/8 during this time.
Evidence Favoring a TGF-β Target Gene Mechanism for Dampening BMP Signaling rather than pSmad2/3-Mediated Competition for Limiting Smad4
Importantly, our BMP-reporter was specific, and in the absence of BMPs, TGF-β2 had little or no effect on its expression (). In contrast, BMP4 strongly stimulated BMP-reporter (ZsGreen) expression. TGF-β2 dampened this activity specifically in WT and not in TβRII-cKO 1°MKs. Moreover, similar results were obtained with cultured, FACS-purified HFSCs. However, HFSCs displayed active BMP signaling in vitro, and hence TGF-β2 alone was sufficient to reduce BMP-reporter activity in HFSCs (). Based upon these results, TGF-β2 appeared to counteract not only extrinsic but also intrinsic BMP signaling.
The dampening effects on BMP signaling were paralleled at the mRNA level and appeared relatively late (12–24 hr) after TGF-β2 treatment (). With 1°MK, this effect was even more obvious when they were pretreated with TGF-β2 prior to adding BMP4 (). Because the reporter differs from control vector only by multimerized Smad1/5/8 binding sites, these results further bolstered the notion that TGF-β2 was specifically affecting canonical BMP signaling at the transcriptional level.
One way of achieving these effects might be through competition between pSmad2/3 and pSmad1/5/8 for a limiting amount of the Smad4 cofactor. Such mechanisms should be refractory to drugs such as cycloheximide, which effectively blocks protein synthesis within minutes in epidermal keratinocytes (Rice and Green, 1979
). However, 10–100 μg/ml cycloheximide nearly abrogated the inhibitory effects of TGF-β2 on BMP-reporter expression (; shown are data for 10 μg/ml). Thus, TGF-β2’s antagonistic effects appeared to require new protein synthesis.
Further evidence against a Smad4 competition mechanism came from testing whether simultaneous activation of Smad2/3 interfered with the availability of transcriptional cofactor Smad4 for Smad1/5/8. Immunoblot analysis of nuclear and cytoplasmic lysates from TGF-β2, BMP, and TGF-β2/BMP-costimulated (short-term) cells showed that BMP4 stimulated phosphorylation and nuclear accumulation of Smad1/5/8 and that this did not change upon TGF-β2 costimulation (). Conversely, TGF-β2-enhanced phosphorylation and nuclear accumulation of Smad2/3 did not change with BMP4 costimulation. Notably, although nuclear:cytoplasmic ratios of Smad4 increased upon BMP4/TGF-β2 costimulation, there appeared to be ample Smad4 to accommodate both pathways. This was further demonstrated by coimmunoprecipitating Smad4 with Smad1 antibodies. As shown in , complex levels were not diminished by BMP4/TGF-β2 costimulation.
A Search for Relevant TGF-β Target Genes Yields Tmeff1, Encoding an Antagonist of the BMP Pathway
In searching for an alternative transcriptional mechanism that could explain TGF-β2’s antagonistic effects on BMP signaling, we wondered whether one of pSmad2/3-Smad4’s target genes might encode a negative regulator of BMP signaling. To test this hypothesis, we purified and transcriptionally profiled HFSC mRNAs at times prior to and during peak TGF-β2 expression in the DP.
Microarray profiling and comparative analyses were performed on duplicate sets of HG (YFP+
), bulge HFSCs (YFP+
), and total (YFP+
) cells FACS-purified from two pairs of late-telogen/early-anagen female TβRII-
Het (WT) and cKO littermates (Figures S4A and S4B). As expected, the HG displayed more changes over these times than the other cell populations. Concomitant with TGF-β2 expression, 341 probes (292 genes) were specifically elevated by ≥2× in the WT HG relative to the bulge or total YFP+
cells. Shown in Table S1, this “activated HG signature” differed from prior HG array data (Greco et al., 2009
) in that it was rich in, e.g., cell cycle genes associated with stem cell activation.
In comparing “preactivated” TβRII-
null and WT HGs, only 40 of these genes were downregulated in TβRII-
null HGs. By contrast, in comparing their “activated” states, 252 genes were downregulated by ≥2× in the TβRII-
null HG (Table S2), and 239 of these were among the “activated HG signature.” Although some changes could be more closely linked to HG activation and not specifically to the absence of TGF-β2 signaling, 63 of these genes have evolutionally conserved Smad2/3-Smad4 sequence motifs within the body of the gene or 5,000 bp 5′ upstream (ECR browser [Ovcharenko et al., 2004
]) (Table S3).
Several interesting findings emerged from these comparisons (, S4C, and S5D). TβRII
ablation did not affect the master transcription factors required for HFSC/HG maintenance. This was true even for Nfatc1, which is known to play a role in HFSC quiescence downstream of BMP signaling (Horsley et al., 2008
). Rather, genes relating to (1) BMP inhibition, (2) cell cycle stimulation, and (3) HF lineage-associated transcription were featured among the genes downregulated in late-telogen/early-anagen TβRII
-cKO versus WT HG. Notably and in contrast to cell cycle genes, inhibitors of BMP signaling remained on the shortlist of putative TGF-β target genes: Bambi
(3.7×), and Tmeff1
Tmeff1 Is a Direct Target Gene of TGF-β Signaling and Expresses in the Hair Germ at the Telogen→Anagen Transition
After validating the microarray data by RT-qPCR, we examined temporal expression of BMP inhibitor genes after TGF-β2 stimulation in vitro (). Bambi
has been reported as a direct target of TGF-β-Smad signaling (Sekiya et al., 2004
) and has previously been identified as a possible participant in DP-HG crosstalk (Greco et al., 2009
). However, of the three, Tmeff1
mRNA was not only the sixth most changed putative TGF-β target gene in the TβRII-
cKO HG signature, but it also displayed more pronounced and sustained induction upon TGF-β2 treatment (). Therefore, we focused on this hitherto unexplored gene in skin for our remaining studies.
To test whether Tmeff1’s putative Smad2/3 binding site is indeed utilized, we conducted chromatin immunoprecipitation (ChIP) with Smad2/3 antibodies. Notably, Smad2/3 binding was enriched on the 5′ UTR of the Tmeff1 gene of WT but not TβRII-null 1°MK (). Moreover, this enrichment was observed only when WT cells were stimulated with TGF-β2.
Further evidence linking Tmeff1 to TGF-β2 signaling came from protein analyses, where Tmeff1 protein displayed kinetics that paralleled Tmeff1 gene induction in vitro (). In vivo, Tmeff1 protein appeared concomitantly with pSmad2+ cells in (1) HGs activated during normal homeostasis and (2) HGs precociously activated upon intradermal TGF-β injections (, and S4E). Finally, when TβRII-cKO HFSCs eventually initiated their next hair cycle, the activated HGs showed no signs of either Tmeff1 or pSmad2 (, S4F, and S4G). These findings link Tmeff1 directly to TGF-β2 signaling and not merely SC activation.
Functional Evidence that Tmeff1 Lowers the BMP Threshold for Stem Cell Activation
To investigate whether Tmeff1 is functionally relevant to the TGF-β2-mediated downregulation of BMP signaling, we performed lentiviral Tmeff1-shRNA-mediated gene knockdown experiments in BMP-reporter-transduced 1°MK. 1°MK were exposed to BMP4/TGF-β2 as before, so that control cells would maintain TGF-β2 signaling-dependent suppression of BMP-reporter activity. In striking contrast to scrambled-shRNA where BMP-reporter (ZsGreen) remained silent, cells transduced with Tmeff1-shRNA were green (). By contrast, albeit somewhat less effective at knocking down their targets, Bambi and Bmper-shRNA were considerably less potent at rescuing BMP-reporter activity (Figures S5A and S5B).
Tmeff1 Mediates the Dampening Effect of TGF-β2 on BMP Signaling
In BMP4/TGF-β2-treated cells transduced with Tmeff1-shRNA, endogenous Tmeff1 expression was kept low, and BMP-reporter activity remained elevated. In contrast, Tmeff1 levels did not affect TGF-β-reporter activity or Smad2 phosphorylation (Figures S5C and S5E). Moreover, Tmeff1 appeared to be a key mediator of the specific antagonistic effects of TGF-β2 on BMP-reporter activity, because introducing mCherry- or FLAG-tagged Tmeff1 into BMP4-stimulated cells resulted in strong suppression of both Smad1/5/8 phosphorylation and BMP-reporter activity (, S5D, and S5F).
To assess the physiological relevance of Tmeff1, we employed our powerful in utero lentiviral delivery system, which enables stable, efficient, and selective shRNA-knockdown in skin epithelium (Beronja et al., 2010
). We transduced mice with two different Tmeff1-
shRNAs and a scrambled control shRNA. Both Tmeff1-
shRNAs behaved similarly, ruling out off-target effects. For the purposes here, we show representative skin sections from mice transduced with the most effective Tmeff1-
At P21, when HFs were in their short first-telogen, Tmeff1 protein was detected only in HGs of scrambled-shRNA- and not in HGs of Tmeff1-shRNA-transduced mice (). When adult mice were shaved at the start of their first postnatal anagen (P23–P24), scrambled-shRNA-transduced hair coats regrew with kinetics similar to uninfected littermates, whereas Tmeff1-shRNA-transduced animals exhibited a significant delay in hair regrowth (). By P43, when scrambled-shRNA-transduced HFs had already entered into their second-telogen (two hairs/HF), Tmeff1-shRNA-transduced HFs were still in the midst of their first anagen (). Although the extent of these differences varied somewhat with individual infections, the trend was consistent, underscoring Tmeff1 as a downstream target of TGF-β signaling and important mediator of SC activation at the telogen→anagen transition.