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Bodó1,
Arno Kromminga,
Erzsébet Gáspár,
Katja Meyer,
Michal A. Zmijewski,
Andrzej Slominski,
Björn E. Wenzel and
Ralf Paus
Department of Dermatology (N.v.B., E.B., E.G., K.M., R.P.) and Cell and Immunobiological Laboratory (B.E.W.), Department of Medicine I, University of Lübeck, D-23538 Lübeck, Germany; Institute for Immunology, Clinical Pathology, and Molecular Medicine (A.K.), D-22339 Hamburg, Germany; Department of Pathology and Laboratory Medicine and Center for Cancer Research (M.A.Z., A.S.), University of Tennessee, Memphis, Tennessee 38163; Agricultural and Molecular Research Institute (E.B.), College of Nyíregyháza, H-4400 Nyíregyháza, Hungary; and School of Translational Medicine (R.P.), University of Manchester, Manchester M13 9PL, United Kingdom
Address all correspondence and requests for reprints to: Ralf Paus, M.D., Department of Dermatology, University Hospital Schleswig-Holstein, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany. E-mail: ralf.paus{at}uk-sh.de.
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Abstract |
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Objective: Our objective was to asses the impact of T3/T4 on human HF in vitro.
Methods: Human anagen HFs were isolated from skin obtained from females undergoing facelift surgery. HFs from euthyroid females between 40 and 69 yr (average, 56 yr) were cultured and treated with T3/T4.
Results: Studying microdissected, organ-cultured normal human scalp HFs, we show here that T4 up-regulates the proliferation of hair matrix keratinocytes, whereas their apoptosis is down-regulated by T3 and T4. T4 also prolongs the duration of the hair growth phase (anagen) in vitro, possibly due to the down-regulation of TGF-β2, the key anagen-inhibitory growth factor. Because we show here that human HFs transcribe deiodinase genes (D2 and D3), they may be capable of converting T4 to T3. Intrafollicular immunoreactivity for the recognized thyroid hormone-responsive keratins cytokeratin (CK) 6 and CK14 is significantly modulated by T3 and T4 (CK6 is enhanced, CK14 down-regulated). Both T3 and T4 also significantly stimulate intrafollicular melanin synthesis.
Conclusions: Thus, we present the first evidence that human HFs are direct targets of thyroid hormones and demonstrate that T3 and/or T4 modulate multiple hair biology parameters, ranging from HF cycling to pigmentation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although these hair effects may well reflect a direct modulatory influence of TH on human hair follicle (HF) cycling (2, 4) and/or on hair keratin expression (14), this remains to be documented. Also, it remains to be conclusively shown that TH affects human HF pigmentation. Because thyroid dysfunction is associated with multiple secondary endocrine abnormalities, e.g. up-regulation of serum TSH as a defining feature of hypothyroidism (15) and changes of insulin serum level (16, 17), these may have caused the clinically observed abnormalities of hair growth and pigmentation. Thus, evidence is still missing that TH directly alter human HF growth, pigmentation, and/or cycling.
Human HFs underlie a lifelong cyclic regression and regeneration, the so-called hair cycle (18). After completion of their morphogenesis, HFs enter the so-called catagen (organ involution driven by controlled apoptosis) phase, followed by a stage of relative rest (telogen) (18). After telogen, HFs enter the active growth phase (anagen), which is associated with pigmented hair shaft formation (18).
That human scalp HFs do express TH receptors (TR) on the gene and protein level (TRβ1) and that T3 reportedly prolongs the survival of microdissected, organ-cultured human scalp HFs (19) encourages one to exploit this model (20) to further dissect the direct effects of TH on human HFs. Therefore, we have employed this as an excellent, physiologically relevant preclinical test system for probing the effects of TH on human HF growth, cycling, and/or pigmentation.
This model also allows us to dissect differences between T3 and T4, which is actively transported into cells and then transformed into T3 by intracellular deiodinases (21), with respect to human HF biology. Because the expression of TRs in human HFs had already been convincingly documented by others (19, 22), we have studied here only deiodinase expression (23).
This was complemented with an analysis of keratin immunoreactivity (IR) in situ, because the keratins cytokeratin (CK) 14 and CK6 genes display a TH-responsive element (TRE) that upon ligand binding to TR reportedly down-regulated transcription of these keratin genes (24, 25, 26). Finally, TGF-β2 is recognized as a central hair cycle-modulatory growth factor for human HFs, operating as a key terminator of anagen (27, 28, 29). Previously, we had shown that other steroid hormones (i.e. retinoic acid) exploit TGF-β2 to mediate, at least in part, their hair growth-inhibitory, anagen-shortening effects (30). Given that retinoic acid is a heterodimerization partner of THs (31), we concluded our analyses by also assessing whether or not T3/T4 treatment alters the intrafollicular IR for TGF-β2.
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Materials and Methods |
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The study was approved by the Institutional Research Ethics Committee and adhered to Declaration of Helsinki guidelines. Human anagen HFs were isolated from skin obtained from females undergoing facelift surgery. We cultured HFs from euthyroid females between 40 and 69 yr (average, 56 yr). Isolated HFs were maintained in 24-multiwell plates in serum-free Williams’ E medium (Biochrom, Cambridge, UK) supplemented with 2 mmol/liter I-glutamine (Invitrogen, Paisley, UK), 10 ng/ml hydrocortisone (Sigma-Aldrich, Taufkirchen, Germany), 10 µg/ml insulin (Sigma), and antibiotics (Sigma). After 24 h preincubation, HFs were treated with vehicle (Williams’ E medium)/THs (Sigma) T3 (concentrations of 1 pM, 100 pM, or 10 nM)/T4 (concentrations of 10 nM, 100 nM, or 1 µM) for either 5 or 9 d. Normal T3 and T4 serum levels are 0.92–2.7 nM and 56–154 nM, respectively (32).
Hair shaft elongation, quantitative hair cycle histomorphometry, and histology
Hair shaft length measurements of vehicle/T3/T4-treated HFs were performed every second day on individual HFs using a Zeiss inverted binocular microscope with an eyepiece measuring graticule.
Seven-micrometer-thick cryostat sections of cultured HFs were fixed in acetone, air dried, and processed for histology. Masson-Fontana histochemistry was used for studying HF morphology as well as visualizing melanin pigment. HF cycle staging was carried out according to previously defined morphological criteria, and the percentage of HFs in anagen and early, mid, or late catagen was determined. Densitometric measurement of melanin staining intensity was performed with ImageJ software (National Institutes of Health, Bethesda, MD).
Quantitative immunohistochemistry (Ki67/TUNEL, CK14, CK6, and TGF-β2)
To evaluate apoptotic cells in colocalization with a proliferation marker Ki-67, a Ki-67/terminal dUTP nick-end labeling (TUNEL) double-staining method was used. Cryostat sections were fixed in paraformaldehide and ethanol-acetic acid (2:1) and labeled with a digoxigenin-deoxy-UTP (ApopTag fluorescein in situ apoptosis detection kit; Intergen, Purchase, NY) in the presence of terminal deoxynucleotidyl transferase, followed by incubation with a mouse anti-Ki-67 antiserum (1:20 in PBS overnight at 4 C; Dako, Glostrup, Denmark). TUNEL-positive cells were visualized by an antidigoxigenin fluorescein isothiocyanate-conjugated antibody (ApopTag kit), whereas Ki-67 was detected by a rhodamine-labeled goat antimouse antibody (Jackson ImmunoResearch, West Grove, PA). Negative controls were performed by omitting terminal deoxynucleotidyl transferase and the Ki-67 antibody. Counterstaining was performed with 4',6-diamidino-2-phenylindole (DAPI) (Roche Molecular Biochemicals GmbH, Mannheim, Germany). Proliferating matrix/epidermal keratinocytes of normal human skin and frozen sections of murine spleen were used as positive control tissues for the Ki-67/TUNEL reaction, respectively.
Quantitative immunohistomorphometry was performed as described previously; Ki-67-, TUNEL-, or DAPI-positive cells were counted in a previously defined reference region of the HF matrix, and the percentage of Ki-67/TUNEL-positive cells was determined.
For the detection of CK14, the peroxidase-based avidin-biotin complex method (Vector Laboratories, Burlingame, CA) was used. After fixation in acetone, blocking of endogenous peroxidases (3% H2O2) and preincubation with goat serum [10% in Tris-buffered saline (TBS); Dako] cryosections were incubated with monoclonal mouse antihuman CK14 antibody (1:200 in TBS overnight at 4 C; Sigma). Cryosections were stained with biotinylated goat antimouse IgG (1:200 for 45 min at room temperature; Beckmann Coulter, Marseille, France) as secondary antibody and then with an avidin-biotin kit (Vector) followed by 3-amino-9-ethylcarbazole substrate-chromogen system (Dako). As negative controls, the primary antibodies were omitted, and human skin (epidermis) was used as a positive control. Counterstaining was performed with Meyer’s hematoxylin.
To investigate CK6 and TGF-β2 expression, acetone-fixed cryosections were pretreated with goat serum (10% in TBS; Dako; only for CK6). Cryoslides were incubated first with the primary antibodies against CK6 (mouse antihuman, 1:10, overnight at 4C; PROGEN, Heidelberg, Germany) and TGF-β2 (rabbit antihuman, 1:50, overnight at 4 C; Santa Cruz Biotechnology Inc., Santa Cruz, CA) and then with rhodamine-conjugated goat antimouse (for CK6, 1:200 in TBS for 45 min at room temperature; Jackson ImmunoResearch) and fluorescein isothiocyanate-labeled goat antirabbit (for TGF-β2, 1:200 in TBS for 45 min at room temperature; Jackson ImmunoResearch) secondary antibodies. Counterstaining was performed with DAPI (Roche Molecular Biochemicals).
Densitometric measurement of staining intensities were performed using the Image J software (National Institutes of Health).
Semiquantitative RT-PCR for deiodinases type 2 (D2) and 3 (D3)
For the semiquantitative PCR analysis of expression of D2 (accession no. AF093774) and D3 (accession no. NM 001362), total RNA was isolated from microdissected HFs using the RNA easy kit (QIAGEN, Hilden, Germany), and 0.5 µg of total RNA was reverse-transcribed with SuperScript First-Strand Synthesis System (Applied Biosystems, Foster City, CA). The quality and quantity of cDNA in all samples were standardized by the amplification of housekeeping gene GAPDH as described previously (33). The PCR conditions for D2 and D3 amplification were as follows: initial denaturation at 95 C for 2 min followed by 30 cycles of denaturation at 94 C for 30 sec, annealing at 60 C for 30 sec, and elongation 72 C for 30 sec. The final elongation step was at 72 C for 5 min. The primers used for amplification were described previously (34) and purchased from Integrated DNA Technology Inc., (Coralville, IA). For detection of D2, PCR was repeated with a second pair of primers and 0.5 µl of reaction mixture from the first round. PCR products were visualized on 2% agarose gel with ethidium bromide. Data of D2 and D3 expression were normalized to the expression of GAPDH of the same sample. Nontemplate control (by omitting RNA) was used as negative control.
Free T3 (fT3) and fT4 immunoassay
For investigating whether HFs are able to produce T3 and whether possible endogenous T3 production can be stimulated by TSH, furthermore whether HFs are able to convert T4 to T3, HFs were washed several times and treated for 48 hours with T4 (100 nM) and TSH (100 mU/ml, Sigma-Aldrich). The THs fT3 and fT4 was measured by an electrochemiluminescent immunoassay on an automated Modular Analytics E170 according to the recommendation of the manufacturer (Roche Diagnostics, Mannheim, Germany). HFs were washed several times and cultured for 48 h. Serum-free supernatant was collected for the analysis. The sensitivity of the assays was 0.260 pg/ml (0.400 pmol/liter) for fT3 and 0.23 pg/ml (0.30 pmol/liter) for fT4.
Statistical analysis
Statistical analysis was performed using the Mann-Whitney U Test and/ or two-tailed paired t test. P values <0.05 were regarded as significant differences.
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Results |
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First we studied by quantitative immunohistomorphometry of key proliferation and apoptosis parameters (Ki67/TUNEL) whether T3 (1 pM, 100 pM, and 10 nM) and/or T4 (10, 100, and 1000 nM) modulate human hair matrix keratinocyte proliferation and apoptosis when added directly to the serum-free medium of microdissected, organ-cultured normal human scalp HFs that were in the stage of maximal growth during HF cycling (anagen VI). As shown in Fig. 1
, A–D, the proliferation of hair matrix keratinocytes was significantly stimulated by T4, whereas the proliferation-modulatory effects of the T3 concentrations tested did not reach the level of significance. However, both T3 and T4 significantly reduced the number of TUNEL-positive matrix keratinocytes in defined reference areas (Fig. 1, A–D).
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However, during the relatively short HF organ culture period (9 d), these effects on hair matrix keratinocyte proliferation/apoptosis did not result in marked alterations of actual hair shaft morphology (Fig. 2) or hair shaft production: When measuring the rate of hair shaft elongation in vitro, this remained essentially unaltered by TH. Compared with vehicle controls, significant, reproducible, hair shaft growth-promoting effects of either T3 or T4 (Fig. 2) were not detectable.
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Clinically, the most important effect that hair loss-inhibitory/hair growth-promoting agents can have is to prolong the duration of anagen, which is indistinguishable from an inhibition of catagen development (18). Therefore, we next tested by quantitative hair cycle histomorphometry whether TH-treated human HFs showed any evidence of such an effect. Indeed, this was the case. Both T3 and T4 increased the number of anagen and decreased the number of catagen HFs after either 5 (Fig. 3A) or 9 d in organ culture (Fig. 3B). This provides the first direct evidence that THs are potent modulators of human scalp HF cycling, at least in vitro.
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Next we tested whether the anagen prolongation by TH is associated with an altered intrafollicular expression of TGF-β, the best-studied endogenous human hair growth inhibitor (27, 28, 29). Although substantial interindividual variations in intrafollicular TGF-β2 IR intensity and pattern were noted (data not shown), quantitative immunohistomorphometry revealed that TH treatment results in a discrete but significant reduction of TGF-β2 IR in the proximal hair bulb epithelium (P < 0.05) (Fig. 3
, C–E). This suggests that TH down-regulates in situ protein expression for a key hair growth-inhibitory endogenous growth factor in the proliferatively most active epithelial hair shaft factory, the anagen hair bulb.
T3 and T4 differentially modulate intrafollicular keratin expression
Next, we assessed TH actions on the intrafollicular protein expression for two cytokeratins with recognized TH-responding elements (24, 25, 26), namely human CK14 and CK6. Interestingly, CK6 IR is significantly increased after treatment with 1 pM T3 and 10 nM T4 and 1 µM T4 (P < 0.05) (Fig. 4, A–C and H), whereas quantitative immunohistomorphometry for CK14 IR showed its expression to be decreased in all TH-treated groups (P < 0.001) (Fig. 4, D–G).
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As revealed by quantitative Masson-Fontana histochemistry, both T3 and T4 significantly stimulate human HF melanin synthesis, with supraphysiological concentrations of T4 showing the strongest stimulation of HF melanogenesis (Fig. 5, A and B1–D1). Although high-power magnification suggested that TH synthesis not only stimulates the total hair bulb melanin content but also appeared to stimulate HF melanocyte dendricity (Fig. 5, B2, C2, and D2), the strength of the melanin-associated histochemical signals hindered definitive confirmation of this intriguing observation.
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In the current study, both T4 and T3 exerted (partially differential) effects on treated HFs. This already suggests that T4 can indeed be transported into human HF cells in organ culture and is here intracellularly deiodinated to T3. To confirm the intrafollicular presence of deiodinases, we finally studied by RT-PCR whether D2 (which transforms T4 into T3 by outer ring deiodinization) (21) and/or whether D3 (which transforms T4 into inactive, rT3) (21) are transcribed in human scalp HFs. As shown in Fig. 6A, specific mRNA for both D2 and D3 are indeed expressed in microdissected human HFs derived from three different individuals. To test whether T4 deiodination occurs in HF, we measured fT3 in culture supernatant after 48 h T4 treatment (Fig. 6B). Compared with the vehicle (which has a value at the detection or baseline limit, suggesting that HFs are not able to produce detectable levels of T3), we observed a significantly higher fT3 level after T4 treatment. This indirect result indicates that D2 may be functionally active. We also asked whether the main regulator of TH synthesis (TSH) may influence T3 levels in HFs, but we could not detect any significant alteration (Fig. 6B). Together with the functional data obtained with T4 stimulation listed above, this further supports the concept that human scalp HFs have indeed the enzymatic capacity to transform T4 into T3.
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Discussion |
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Because both T3 and T4 alter key parameters of human HF biology in organ culture and because HFs transcribe deiodinase genes, it is likely that human HFs can convert T4 to T3 (just like all TH-sensitive target tissues) (21), in line with the previous demonstration of D2 and D3 transcripts in cultured human skin fibroblasts, melanocytes. and keratinocytes (34). However, the subtle differences observed here between both THs in the human HF biology parameters that are significantly modulated by T3 vs. T4 (see e.g. Figs. 1 and 5) raise the possibility that T4 also unfolds separate activities that are independent of its conversion to T3 (35). However, TSH stimulation of organ-cultured HFs did not result in a significant alteration of the new fT3 level detectable in the medium.
Because the promoter regions of CK6 and CK14 host TRE (24, 25, 26), our finding that T3 and T4 modulate the intraepithelial protein expression of these TH-responsive keratins CK6 and CK14 in a differential manner (Fig. 4) suggests that the effects of TH on human HFs are mediated via the classical pathway characterized by intranuclear interaction of TH-TR complexes with TRE-containing promoters (24, 25, 26). However, the very low TH doses at which some effects were seen (see Figs. 1, 3, and 4) raise the question whether they were actually mediated by recently demonstrated (35) TH membrane-bound receptors. In view of the increasing awareness of nuclear TR-independent, nongenomic signaling mechanisms of TH (whose investigation clearly was outside the scope of the current study), (36) it now also needs to be systematically investigated whether T4 and T3 differ not only in their stimulation of classical intrafollicular nuclear TR but also in the nongenomic, TR-independent signaling events each of these TH elicits/modulates in human scalp HFs.
Although the paucity of available human scalp HFs precluded systematic dose-response studies, our findings suggest that the predominant direct effect of both physiological and supraphysiological T3 and/or T4 concentrations in human hair growth control is that of a hair growth-promoting agent: 1) THs prolong anagen duration (i.e. retard spontaneous catagen development) (Fig. 3), T4 stimulates hair matrix keratinocyte proliferation (Fig. 1, A and D), and 3) matrix keratinocyte apoptosis is inhibited by T3 and T4 (Fig, 1, A, C, and D).
The anagen prolongation documented here may correspond to the prolonged HF survival previously seen by Billoni et al. (19). Interestingly, the hair growth-promoting effects of TH were not associated with significant stimulatory effects of either T3 or T4 on hair shaft growth. This confirms corresponding findings of Billoni et al. (19) and might simply be explained by the relatively short culture period, which may be too short to allow detection of subtle TH effects on hair shaft formation.
In this context, it needs to be kept in mind that the assay employed here (re)creates a severely hypothyroid state in vitro, because HF organ culture is performed with serum-free William’s E medium supplemented only with insulin and hydrocortisone (20). Thus, HFs could only have been stimulated by residual quantities of endogenous TH already bound within the HFs before microdissection and organ culture. Such residual endogenous TH likely are further reduced with every change of medium (every 2 d) and are expected to quickly lose activity with increasing culture time. Nevertheless, even in the absence of extrafollicular TH, as long as human scalp HFs remain in anagen, their hair shaft production in vitro progresses at almost the normal speed seen in vivo (37, 38), as can be seen here in the vehicle control groups (Figs. 2 and 3). This suggests that, despite the modulatory effects of TH on CK6 and CK14, extrafollicular THs are dispensable for normal hair shaft production under assay conditions. Naturally, this does not exclude a long-term role for TH in human hair shaft production in vivo, especially because hair shaft abnormalities have been clinically reported in both hypo- and hyperthyroid patients (1, 9, 10).
Our preclinical data help to explain the previously unclear pathogenesis of the telogen effluvium observed in hypothyroid patients, where a relative TH deficiency has been proposed to cause premature catagen induction and reduced hair matrix proliferation (2, 3, 4). Our demonstration that addition of exogenous TH to human HFs cultured in the absence of systemic TH prolongs anagen and stimulates hair matrix keratinocyte proliferation and inhibits their apoptosis provides the first direct evidence for the validity of this old postulate. Our findings also fit well with the reportedly increased hair matrix proliferation in hyperthyroid patients in vivo (3). Unfortunately, amputated, organ-cultured human scalp anagen VI hair bulbs do not run through full cycles in vitro and decay shortly after catagen development (30, 37, 38, 39). Therefore, this assay cannot clarify another old, as yet unconfirmed hypothesis that, conversely, hyperthyroidism may also lead to effluvium due to the induction of faster HF cycling (1, 9, 10, 11).
The current study provides at least one important pointer to the molecular mechanisms by which TH may exert their anagen-prolonging effects: THs down-regulate the intrafollicular protein expression of TGF-β2 (Fig. 3C), one of the recognized key catagen inducers during human HF cycling, which also inhibits proliferation and stimulates apoptosis of human hair matrix keratinocytes in situ (27). Suppression of intrafollicular TGF-β2-mediated signaling by THs, therefore, constitutes at least one conceivable pathway by which THs may prolong anagen, stimulate proliferation, and inhibit apoptosis in the human hair matrix. Such an inhibitory effect of THs on intrafollicular TGF-β2 expression would be in striking contrast to that of another important, hair growth-modulatory steroid hormone, all-trans retinoic acid, which we had previously shown exerts its hair growth-inhibitory, catagen-promoting effects on organ-cultured human HFs at least in part via up-regulating TGF-β2 (30). However, given the many direct TH target genes that display a TRE in their promoter region and the numerous additional, TRE-negative genes that nevertheless are regulated by TH (40) as well as nonclassical, receptor-independent TH activities (35, 36), multiple additional pathways and molecular targets by which TH may impact on human HF biology must now be considered and systematically explored.
Finally, we show that both T3 and T4 significantly stimulate intrafollicular melanin synthesis (Fig. 5, A–D). Because quantitative Masson-Fontana histochemistry was compared only between anagen VI HFs from test and control groups (intrafollicular melanogenesis is strictly coupled to anagen, and HF pigmentation declines sharply early during the anagen-catagen transformation of HFs (41, 42), this pigmentation-stimulatory effect cannot have been a simple reflection of the anagen-prolonging effects of TH. Thus, this is the first evidence that THs can directly alter human HF pigmentation and, to the best of our knowledge, also the first indication that THs can stimulate melanogenesis in the mammalian system in situ. These findings are in line with the recently reported darkening of gray/white hair in patients with increased exogenous T3 (13). Although the underlying mechanism remains to be dissected, this further illustrates how well-suited organ-cultured human scalp HFs are as a clinically relevant, highly instructive, yet still under-exploited discovery tool for the identification of new functions for ancient steroid hormones in cutaneous endocrinology.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online August 26, 2008
1 N.v.B. and E.B. contributed equally. 
Abbreviations: CK, Cytokeratin; DAPI, 4',6-diamidino-2-phenylindole; fT3, free T3; HF, hair follicle; IR, immunoreactivity; TBS, Tris-buffered saline; TH, thyroid hormone; TR, TH receptor; TRE, TH-responsive element; TUNEL, terminal dUTP nick-end labeling.
Received February 5, 2008.
Accepted August 20, 2008.
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References |
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is a potent inducer of catagen-like changes in cultured human anagen hair follicles. Br J Dermatol 152:623–631[CrossRef][Medline]
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0.01 (mean ± SEM). B, Cryosections of cultured, vehicle-, and 10 nM T3/100 nM T4-treated HFs were double labeled with Ki-67 (red)/TUNEL (green) staining. Ki-67/TUNEL-positive cells were counted below a line marking the end of the dermal papilla (indicated in red). DP, Dermal papilla; MK, matrix keratinocytes.
