Thymic Peptides Differentially Modulate Human Hair Follicle Growth

hair follicle inner root sheath outer root sheath prothymosin alpha thymosin β4 thymic peptide thymulin Topical preparations containing thymus-derived protein extracts have long been claimed to stimulate human hair growth (Renner et al., 1986Renner D. Schuster D. Heim M.E. Experiences using the “thymu-skin” hair cure for the prevention of alopecia in cytostatic treatment.Onkologie. 1986; 5: 285-286Crossref Google Scholar; Sawaya and Shapiro, 2000Sawaya M.E. Shapiro J. Alopecia: unapproved treatments or indications.Clin Dermatol. 2000; 18: 177-186Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), but there is still no convincing evidence available that they really modulate human hair growth. Despite their historical name, most thymic peptides (TPs), such as thymosin β4 (TB4), prothymosin alpha (PTMA), and thymulin (TYL), are much more widely distributed than in the thymic epithelium where they were first identified (Kato et al., 1981Kato K. Ikeyama S. Takaoki M. et al.Epithelial cell components immunoreact with anti-serum thymic factor (FTS) antibodies: possible association with intermediate-sized filaments.Cell. 1981; 24: 885-895Abstract Full Text PDF PubMed Scopus (33) Google Scholar; Moll et al., 1996Moll J. Schmid P. Sansig G. et al.The pattern of prothymosin alpha gene expression coincides with that of myc oncogenes during mouse embryogenesis.Histochem J. 1996; 28: 45-52Crossref PubMed Scopus (8) Google Scholar; Folch et al., 2010Folch H. Villegas J.V. Leyan V. et al.Immunohistochemical evidences showing the presence of thymulin containing cells located in involuted thymus and in peripheral lymphoid organs.Biol Res. 2010; 43: 291-298Crossref PubMed Scopus (6) Google Scholar; Supplementary Information S1 online). The PTMA gene is prominently expressed in many rodent tissues (Moll et al., 1996Moll J. Schmid P. Sansig G. et al.The pattern of prothymosin alpha gene expression coincides with that of myc oncogenes during mouse embryogenesis.Histochem J. 1996; 28: 45-52Crossref PubMed Scopus (8) Google Scholar), including fetal murine hair follicles (HFs). PTMA is involved in many cellular processes such as apoptosis, chromatin remodeling, and transcriptional regulation (reviewed in Hannappel and Huff, 2003Hannappel E. Huff T. The thymosins. Prothymosin alpha, parathymosin and beta-thymosins: structure and function.Vitam Horm. 2003; 66: 257-296Crossref PubMed Scopus (88) Google Scholar); Supplementary Information S1 online). TYL has been claimed to be thymus specific, although TYL immunoreactivity has also been reported in murine epidermis and skin appendages (Kato et al., 1981Kato K. Ikeyama S. Takaoki M. et al.Epithelial cell components immunoreact with anti-serum thymic factor (FTS) antibodies: possible association with intermediate-sized filaments.Cell. 1981; 24: 885-895Abstract Full Text PDF PubMed Scopus (33) Google Scholar) and in murine spleen (Folch et al., 2010Folch H. Villegas J.V. Leyan V. et al.Immunohistochemical evidences showing the presence of thymulin containing cells located in involuted thymus and in peripheral lymphoid organs.Biol Res. 2010; 43: 291-298Crossref PubMed Scopus (6) Google Scholar). Interactions between TYL and the neuroendocrine system have been reported to lead to a release of prolactin, thyrotropin, and ACTH (Hadley et al., 1997Hadley A.J. Rantle C.M. Buckingham J.C. Thymulin stimulates corticotrophin release and cyclic nucleotide formation in the rat anterior pituitary gland.Neuroimmunomodulation. 1997; 4: 62-69PubMed Google Scholar; Brown et al., 1998Brown O.A. Sosa Y.E. Bolognani F. et al.Thymulin stimulates prolactin and thyrotropin release in an age-related manner.Mech Ageing Dev. 1998; 104: 249-262Crossref PubMed Scopus (18) Google Scholar). As these neurohormones are well appreciated as regulators of human HF growth, cycling, and pigmentation (Paus, 2011Paus R. A neuroendocrinological perspective on human hair follicle pigmentation.Pigment Cell Melanoma Res. 2011; 24: 89-106Crossref PubMed Scopus (45) Google Scholar), it is particularly interesting to investigate TYL in a hair research context. Download .pdf (.53 MB) Help with pdf files Supplementary Information Topically applied TB4 reportedly enhances hair growth in rats and mice and stimulates early differentiation of rat vibrissae epithelial progenitor cells (Philp et al., 2004Philp D. Nguyen M. Scheremeta B. et al.Thymosin beta4 increases hair growth by activation of hair follicle stem cells.FASEB J. 2004; 18: 385-387Crossref PubMed Google Scholar), whereas it is unknown whether TB4 impacts on human hair growth. Therefore, we wished to clarify in the current study whether human scalp HFs express any TPs, and whether selected TPs exert measurable effects on human scalp HFs in organ culture (Kloepper et al., 2010Kloepper J.E. Sugawara K. Al-Nuaimi Y. et al.Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follcile organ culture.Exp Dermatol. 2010; 19: 305-312Crossref PubMed Scopus (100) Google Scholar). First, we asked whether human HF epithelium expresses any defined TPs on the gene and/or protein level. As no gene coding for TYL has been identified (see Supplementary Information S1 online), only TB4 and PTMA gene transcription could be examined. As shown in Figure 1a, strong and specific mRNA signals for both genes were identified, thus confirming that human anagen HF epithelium expresses both TB4 and PTMA. By immunohistochemistry or immunofluorescence (Supplementary Information S2 online), strong PTMA immunoreactivity was detected in the hair matrix, the cortex, and inner and outer root sheaths (IRS and ORS, respectively) of the lower HF epithelium and, more weakly, in the dermal papilla (Figure 1b). Interestingly, TB4-like immunoreactivity was only found in the nuclei of the IRS, ORS, and cuticle. Strong TYL immunoreactivity was detected in Henle's layer of the IRS, in the ORS, and in the hair shaft cortex (Figure 1b). Expression of these TPs on the gene and/or protein level suggests the hypothesis that endogenous, intrafollicularly produced TPs are used by human HFs to regulate some of their functions. This hypothesis was tested in serum-free human HF organ culture (Supplementary Information S2 online). We first studied whether thymosin alpha 1, a peptide comprising amino acids 2–29 of PTMA, (Supplementary Information S1 online), TYL, and TB4 modulate hair shaft production (i.e., the hair shaft elongation rate) in vitro. Cultivation of HFs with 10pgml−1 TYL for 7 days in three independent experiments from three different human donors resulted in an increased hair shaft growth rate compared with vehicle-treated HFs (Figure 2a). In contrast, hair shaft elongation rates of HFs treated with 100 or 1,000ngml−1 thymosin alpha 1 for 7 days were slightly lower than those of control HFs (Figure 2a). In TB4-treated HFs, hair shaft production was 10–20% lower than in the vehicle control (Figure 2a and Supplementary Figure S1 online). Next, we studied by quantitative hair cycle histomorphometry (Kloepper et al., 2010Kloepper J.E. Sugawara K. Al-Nuaimi Y. et al.Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follcile organ culture.Exp Dermatol. 2010; 19: 305-312Crossref PubMed Scopus (100) Google Scholar) whether the tested TPs had an effect on the transformation of anagen VI HFs into the regression stage of the hair cycle (catagen). This transformation is the clinically most relevant parameter one can study in HF organ culture, as any prolongation effect on anagen would be expected to correlate with a reduction of telogen effluvium in vivo (Cotsarelis and Millar, 2001Cotsarelis G. Millar S.E. Towards a molecular understanding of hair loss and its treatment.Trends Mol Med. 2001; 7: 293-301Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar; Paus and Foitzik, 2004Paus R. Foitzik K. In search of the “hair cycle clock”: a guided tour.Differentiation. 2004; 72: 489-511Crossref PubMed Scopus (232) Google Scholar; Kloepper et al., 2010Kloepper J.E. Sugawara K. Al-Nuaimi Y. et al.Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follcile organ culture.Exp Dermatol. 2010; 19: 305-312Crossref PubMed Scopus (100) Google Scholar). Moreover, the effect of each peptide on the hair cycle was further assessed by determining the hair cycle score in each treatment condition (Figure 2b and c; Supplementary Information S2 and Supplementary Figure S1b online). These analyses showed that HFs treated with 10pgml−1 TYL for 7 and 9 days stayed longer in anagen VI than vehicle-treated controls (Figure 2b, Supplementary Figure S1b online). By two-tailed Student's t-test, these differences did not come up as significant in the 7-day treatment group where the effect of TYL 10 on the distribution of anagen vs. catagen had a P-value of 0.06. Hair cycle score analysis further indicated that treatment with TYL for 7 and 9 days inhibited the progression of HFs from anagen to catagen (Figure 2c, Supplementary Figure S1b online). This anagen-prolonging effect of TYL (which reached significance in the 9 day cultures) was independently corroborated by the demonstration that, compared with vehicle controls, treatment of HFs with 10pgml−1 TYL for 7 days increased the number of Ki67+ cells in the hair matrix of anagen VI HFs, whereas the number of TUNEL+ (i.e., apoptotic) cells was reduced (Figure 2d). Despite its slight growth-inhibiting effect, treatment with 1,000ngml−1 thymosin alpha 1 for 7 days did not markedly change HF cycling in vitro in three independent experiments with HFs from three different patients (Figure 2d). Interestingly, there even was a stimulatory effect on hair matrix keratinocyte proliferation of anagen HFs (Supplementary Figure S2 online). Instead, treatment with 1,000ngml−1 TB4 for 7 days shortened the duration of anagen and prematurely induced catagen (Figure 2b and c), yet did not affect the number of Ki67+ hair matrix cells, if only anagen HFs were compared between test and control groups (Supplementary Figure S2 online). In conclusion, our pilot study presents evidence that selected TPs are produced by human HF epithelium and operate as direct modulators of human HF growth; this is—to our knowledge—previously unreported. In addition, these initial findings in human scalp HFs suggest that selected TPs may be exploited therapeutically for stimulating, and also for inhibiting, human hair growth. Namely, we identify TYL as a clinically interesting candidate hair growth stimulator. The varying response of the limited number of donors from whom HFs were available for study suggests substantial interindividual variations in the response of human HFs to TP stimulation. Although the mechanisms of action through which selected TPs exert their hair growth–modulatory properties remain to be dissected (Bock-Marquette et al., 2004Bock-Marquette I. Saxena A. White M.D. et al.Thymosin beta4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair.Nature. 2004; 432: 466-472Crossref PubMed Scopus (571) Google Scholar; Mosoian et al., 2010Mosoian A. Teixeira A. Burns C.S. et al.Prothymosin-a inhibits HIV-1 via Toll-like receptor mediated zype I interferon induction.Proc Natl Acad Sci USA. 2010; 107: 10178-10183Crossref PubMed Scopus (75) Google Scholar), our data support the idea that the tested TPs are indeed important regulators of human HF biology (see Supplementary Information S3 online). We gratefully acknowledge Nadine Dörwald, Antje Winter-Keil, and Astrid Becker for excellent technical assistance. This study was supported in part by a research grant from the German Federal Ministry of Economics and Technology (AiF Programme, ProInno-II-Project KF0670201UL8) to RP. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid