Human Hair Follicle Melanocytes Research Articles

Stress-sensing in the human greying hair follicle: Ataxia Telangiectasia Mutated (ATM) depletion in hair bulb melanocytes in canities-prone scalp

Canities (or hair greying) is an age-linked loss of the natural pigment called melanin from hair. While the specific cause(s) underlying the loss of melanogenically-active melanocytes from the anagen hair bulbs of affected human scalp remains unclear, oxidative stress sensing appears to be a key factor involved. In this study, we examined the follicular melanin unit in variably pigmented follicles from the aging human scalp of healthy individuals (22–70 years). Over 20 markers were selected within the following categories: melanocyte-specific, apoptosis, cell cycle, DNA repair/damage, senescence and oxidative stress. As expected, a reduction in melanocyte-specific markers in proportion to the extent of canities was observed. A major finding of our study was the intense and highly specific nuclear expression of Ataxia Telangiectasia Mutated (ATM) protein within melanocytes in anagen hair follicle bulbs. ATM is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks and functions as an important sensor of reactive oxygen species (ROS) in human cells. The incidence and expression level of ATM correlated with pigmentary status in canities-affected hair follicles. Moreover, increased staining of the redox-associated markers 8-OHdG, GADD45 and GP-1 were also detected within isolated bulbar melanocytes, although this change was not clearly associated with donor age or canities extent. Surprisingly, we were unable to detect any specific change in the expression of other markers of oxidative stress, senescence or DNA damage/repair in the canities-affected melanocytes compared to surrounding bulbar keratinocytes. By contrast, several markers showed distinct expression of markers for oxidative stress and apoptosis/differentiation in the inner root sheath (IRS) as well as other parts of the hair follicle. Using our in vitro model of primary human scalp hair follicle melanocytes, we showed that ATM expression increased after incubation with the pro-oxidant hydrogen peroxide (H2O2). In addition, this ATM increase was prevented by pre-incubation of cells with antioxidants. The relationship between ATM and redox stress sensing was further evidenced as we observed that the inhibition of ATM expression by chemical inhibition promoted the loss of melanocyte viability induced by oxidative stress. Taken together these new findings illustrate the key role of ATM in the protection of human hair follicle melanocytes from oxidative stress/damage within the human scalp hair bulb. In conclusion, these results highlight the remarkable complexity and role of redox sensing in the status of human hair follicle growth, differentiation and pigmentation.

The biology of human hair greying.

Hair greying (canities) is one of the earliest, most visible ageing-associated phenomena, whose modulation by genetic, psychoemotional, oxidative, senescence-associated, metabolic and nutritional factors has long attracted skin biologists, dermatologists, and industry. Greying is of profound psychological and commercial relevance in increasingly ageing populations. In addition, the onset and perpetuation of defective melanin production in the human anagen hair follicle pigmentary unit (HFPU) provides a superb model for interrogating the molecular mechanisms of ageing in a complex human mini-organ, and greying-associated defects in bulge melanocyte stem cells (MSCs) represent an intriguing system of neural crest-derived stem cell senescence. Here, we emphasize that human greying invariably begins with the gradual decline in melanogenesis, including reduced tyrosinase activity, defective melanosome transfer and apoptosis of HFPU melanocytes, and is thus a primary event of the anagen hair bulb, not the bulge. Eventually, the bulge MSC pool becomes depleted as well, at which stage greying becomes largely irreversible. There is still no universally accepted model of human hair greying, and the extent of genetic contributions to greying remains unclear. However, oxidative damage likely is a crucial driver of greying via its disruption of HFPU melanocyte survival, MSC maintenance, and of the enzymatic apparatus of melanogenesis itself. While neuroendocrine factors [e.g. alpha melanocyte-stimulating hormone (α-MSH), adrenocorticotropic hormone (ACTH), ß-endorphin, corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH)], and micropthalmia-associated transcription factor (MITF) are well-known regulators of human hair follicle melanocytes and melanogenesis, how exactly these and other factors [e.g. thyroid hormones, hepatocyte growth factor (HGF), P-cadherin, peripheral clock activity] modulate greying requires more detailed study. Other important open questions include how HFPU melanocytes age intrinsically, how psychoemotional stress impacts this process, and how current insights into the gerontobiology of the human HFPU can best be translated into retardation or reversal of greying.

Exploring the potential effect and mechanisms of protocatechuic acid on human hair follicle melanocytes.

This study aims to evaluate the effect of protocatechuic acid (PCA) on human hair follicle melanocytes (HFM). Normal primary HFM were isolated and cultured till logarithmic period of second passage, then treated with different concentrations of PCA (0.1-200 μmol L-1) to study the cell proliferation, melanin contents, tyrosinase activity and protein and mRNA expression of melanogenic genes (tyrosinase-related protein 1 (TRP-1), tyrosinase-related protein 2 (TRP-2), and microphthalmia-associated transcription factor (MITF)) in the cultured HFM. In addition, we have also measured the contents of superoxide dismutase (SOD) and glutathione (GSH) in PCA treated HFM. Vitamin C was used as a positive control. The result showed that PCA can decrease the synthesis of melanin and the tyrosinase activity with IC50 = 8.9 μmol L-1 and IC50 = 6.4 μmol L-1, respectively, at the treatment time of 24 hours, without inducing any cytotoxicity in HFM cells. In addition, the mRNA transcription and protein expression levels of TRP-1, TRP-2 and MITF significantly decreased with a dose-dependent manner after 24-hour PCA treated in HFM cells. Furthermore, PCA has significantly increased the SOD and GSH activity in a dose-dependent manner for 24-hour PCA treatment. This study suggested that PCA has an inhibitory effect on the production of melanin through down-regulation of the expression of melanogenesis-related protein and the effect of anti-oxidation, which could be useful for the therapy of melanin overproduction or skin whitening.

285 A new scalp treatment for anti-ageing hair

Hair aging is commonly defined as hair graying, hair loss and fragility. Today, hair care treatments fighting against hair ageing focus directly on the hair fibers. However, as well as the skin, the scalp needs care. Indeed, the scalp contains hair follicles that produces hair fibers. The scalp directly provides nutrition to the hair follicles and consequently to the hair fibers. By taking care of the scalp, the hair quality can be improved. We screened multiple botanical extracts and discovered one having anti-inflammatory and anti-oxidative properties along with reducing UVB-induced DNA fragmentation and increasing repair in keratinocytes (comet assay, thymine dimers, Gadd45). One active ingredient was particularly selected for its ability to reduce activation of a specific stress sensor in human hair follicle melanocytes. To assess the anti-aging hair potential of this selected plant extracts in more realistic conditions of use, we designed in vivo protocols to monitor graying (chromametry), hair growth (Phototrichogram) and fiber mechanical properties (tensile stress-strain) on hair neo-fibers, over a six-month period, to have numerous and long enough growing hair fibers for assessment (at least 6cm from the root). This newly designed protocol allows to observe that after six months, hair color became increasingly white (+5%), hair loss was increased (+13%) and break extension and post-yield gradient of hair fibers was modified (+3% and -5% respectively). Contrariwise, the ingredient stabilized hair loss and preserved natural hair pigmentation and strength during the study. In conclusion, this study on emerging fibers shows that it is possible to follow in vivo perceivable benefits of a protecting scalp treatment on the hair structure and function. However, the clinical study should be long enough to have neo-fibers to evaluate hair mechanical properties and color.

T-oligos as differential modulators of human scalp hair growth and pigmentation: a new “time lapse system” for studying human skin and hair follicle biology in vitro?

Small DNA oligonucleotides homologous to the 3' overhang of human telomeres, called T-oligos, stimulate pigmentation in human epidermal melanocytes in vitro and in vivo. They induce UV-mimetic effects in the absence of DNA-damage, however, it is unknown how T-oligos affect human hair follicle keratinocyte and melanocyte functions in situ. Here, we present the first evidence that these oligonucleotides are powerful modulators of pigmentation and growth of microdissected, organ-cultured human scalp hair follicles. Hair follicles were incubated with T-oligo or vehicle control and were then assessed for changes in hair shaft length, follicle morphology, pigmentation, proliferation and apoptosis. After only 48 h, T-oligos induced a fourfold increase in pigmentation of human anagen VI hair bulbs, while hair matrix keratinocyte proliferation was reduced by 65%, without apparent changes in hair bulb cell apoptosis. This corresponded well with a significant inhibition of hair shaft elongation, which was not accompanied by premature catagen induction in anagen VI hair follicles. These diametrically opposed effects of T-oligos on human hair follicle melanocytes (stimulation of melanogenesis) versus human hair bulb keratinocytes (inhibition of proliferation) in situ illustrate that human hair follicle organ culture offers an excellent tool for T-oligo research. They suggest that T-oligos deserve to be further explored for the management of clinical hair growth and pigmentation disorders, and raise the possibility that this model may offer a unique "time lapse system" for studying skin and hair follicle biology and DNA repair strategies under physiologically relevant conditions.

A Fully Functional Proopiomelanocortin/Melanocortin-1 Receptor System Regulates the Differentiation of Human Scalp Hair Follicle Melanocytes

The proopiomelanocortin (POMC)-derived peptides, ACTH and alpha-MSH, are the principal mediators of human skin pigmentation via their action at the melanocortin-1 receptor (MC-1R). Recent data have demonstrated the existence of a functionally active beta-endorphin/mu-opiate receptor system in both epidermal and hair follicle melanocytes, whereby beta-endorphin can regulate melanogenesis, dendricity, and proliferation in these cells. However, a role for ACTH and alpha-MSH in the regulation of the human follicular pigmentary unit has not been determined. This study was designed to examine the involvement of ACTH and the alpha-MSH/MC-1R system in human follicular melanocyte biology. To address this question we employed RT-PCR and immunohisto/cytochemistry, and a functional role for these POMC peptides was assessed in follicular melanocyte cultures. Human scalp hair follicle melanocytes synthesized and processed POMC. ACTH and alpha-MSH in association with their processing enzymes and MC-1R are expressed in human follicular melanocytes at the message level in vitro and at the protein level both in situ and in vitro. The expression of the POMC/MC-1R receptor system was confined only to subpopulations of poorly and moderately differentiated melanocytes. In addition, functional studies revealed that ACTH and alpha-MSH are able to promote follicular melanocyte differentiation by up-regulating melanogenesis, dendricity, and proliferation in less differentiated melanocyte subpopulations. Thus, these findings suggest a role for these POMC peptides in regulating human hair follicle melanocyte differentiation.

Expression and regulation of pro‐opiomelanocortin‐derived peptides in human hair growth and pigmentation

Human skin provides for the local cleavage of pro‐opiomelanocortin (POMC) to yield multiple peptide products, e.g. α‐MSH, ACTH and β‐endorphin (β‐end). α‐MSH and ACTH are well documented to regulate human skin pigmentation. We have recently shown that human epidermal melanocytes express a fully functioning β‐end/µ‐opiate receptor system, and that β‐end has potent melanogenic, mitogenic and dendritogenic effects in cultured epidermal melanocytes. However, little is known about their role in human hair follicle (HF) biology. We have characterized the expression and regulation of β‐end, ACTH, α‐MSH and their receptors in human haired scalp and in cultured HF melanocytes, keratinocytes and fibroblasts by immunochemistry, detection of mRNA transcripts and by assessment of potential melanogenic, dendritogenic and mitogenic effects of these peptides in vitro. These studies show that the POMC peptide system is expressed in HF cells as a function of their anatomic location and differentiation status during the hair growth cycle. All three peptides exhibit similar melanogenic, mitogenic and dendritogenic effects in cultured HF melanocytes. Moreover, while the in situ expression profiles of ACTH and α‐MSH are similar, they differ strikingly from β‐end for all follicular cell populations. One such example is the persistent expression of the former in the hair inductive follicular papilla (FP) throughout the entire hair cycle (but are expressed variably in HF keratinocytes and melanocytes). However, β‐end expression is low to undetectable in the FP during hair growth. In toto, these results indicate that POMC peptides are involved in regulating human HF growth and pigmentation and that alterations in POMC homeostasis may be contribute to pathology.

A ‘hot’ new twist to hair biology – involvement of vanilloid receptor‐1 signaling in human hair growth control

The functional role of VR1, which we and others have recently identified on several epithelial and mesenchymal human skin cell populations, was investigated in the human hair follicle (HF), as a prototypic epithelial–mesenchymal interaction system. VR1 immunoreactivity was confined to distinct epithelial compartments of HFs in anagen and catagen, while dermal papilla fibroblasts and HF melanocytes were VR1 negative. In organ culture, VR1 activation by capsaicin resulted in a dose‐dependent and VR1‐specific inhibition of hair shaft elongation, suppression of proliferation, promotion of apoptosis, and induction of catagen transformation, possibly due to upregulation of a potent hair growth inhibitor TGFβ2. Cultured outer root sheath (ORS), as well as HaCaT, keratinocytes also expressed functional VR1, whose stimulation inhibited proliferation, induced apoptosis, and elevated intracellular calcium concentration. Finally, VR1 stimulation of cultured ORS keratinocytes upregulated the expression of recognized endogenous hair growth inhibitors (IL‐1β and TGFβ2) and downregulated the expression of stimulators (HGF, IGF‐1, and SCF), while key differentiation markers (CK17, CK14, filaggrin, and involucrin) remained unaffected. In conclusion, VR1 is a significant novel player in human hair growth control underscoring that its physiological functions in human skin far extend beyond sensory neuron‐coupled nociception.

Beta-Endorphin as a regulator of human hair follicle melanocyte biology.

The pro-opiomelanocortin (POMC)-derived peptides, alpha-melanocyte-stimulating hormone, and adrenocorticotropic hormone, are important mediators of human skin pigmentation via action at the melanocortin-1 receptor. Recent data suggests that such a regulatory role also exists for the endogenous opiate, beta-endorphin (beta-END). A role for this beta-END in the regulation of follicular pigmentation, however, has not been determined. This study was designed to examine the involvement of the beta-END/mu-opiate receptor system in human follicular melanocyte biology. We employed RT-PCR, and immunohisto/cytochemistry and immunoelectron microscopy using beta-END and mu-opiate receptor specific antibodies and a functional role for beta-END was assessed by direct stimulation with the peptide. This study has demonstrated that human hair follicle melanocytes (HFM) express mRNA for the mu-opiate receptor and POMC. Furthermore, beta-END and its high affinity mu-opiate receptor are expressed at the protein level in glycoprotein100-positive follicular melanocytes and as a function of their anatomic location and differentiation status during the hair growth cycle. Functional studies revealed that beta-END is a modifier of HFM phenotype via its ability to upregulate melanogenesis, dendricity, and proliferation. These findings suggest a new regulatory role for beta-END in human HFM biology, providing a new research direction into the fundamental regulation of human hair pigmentation.

Melanocyte subpopulation turnover during the human hair cycle: an immunohistochemical study.

The human hair cycle is characterized by successive phases of growth and involution that imply tissue regression and regeneration. As a consequence, the hair melanin unit has to be renewed in a cyclic manner. Actually, the behavior of human hair follicle melanocytes throughout the hair cycle has been poorly studied. Thus, the origin of melanocytes present in the bulb after human hair regeneration is still not clarified, and neither are the events that control the melanin biosynthesis activity in the human hair bulb. In this study, we showed at the cellular level that in human pigmented hair follicles, the expression of tyrosinase and tyrosinase-related protein-1 (TRP-1) was detectable during the anagen phases III/IV through VI, only in those melanocytes which were located in the bulb. During the catagen phase, the two evaluated melanogenic enzymes were detectable no more, although melanocytes were still present in the preceding bulbar area. The epithelial column of catagen follicles and the capsule of telogen follicles also contained inactive melanocytes as evidenced by pMel-17 labeling. At the induction of a new anagen hair follicle, some melanocytes were committed to cell division, but only when located in the nascent bulb close to the dermal papilla. Our results emphasize the close relationship between melanogenesis and the hair cycle and suggest that in humans, melanogenesis is restricted to anagen hair follicles not because of the regulation of tyrosinase activity, but because of melanogenic enzyme expression, e.g., tyrosinase and TRP-1. Furthermore, the fact that in the newly developing anagen hair follicles, cell-division commitment and tyrosinase and TRP-1 expression were observed in melanocytes only when located in the nascent bulb suggests a highly regio-specific melanocyte stimulation in early the anagen phase.

Isolation and long-term culture of human hair-follicle melanocytes.

We report a method to establish long-term cultures of melanocytes derived from human hair follicles. Normal human scalp was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by collagenase treatment. Hair-follicle cell suspensions were prepared by trypsin/ethylenediamine tetraacetic acid treatment and cultured in a mixture of Eagle's minimum essential medium (supplemented with 12-O-tetradecanoyl-phorbol-13-acetate and cholera toxin) and keratinocyte serum-free medium. After contaminating fibroblasts and keratinocytes were removed, cells with two distinct morphologies remained. These included large, dendritic and deeply pigmented cells, which did not proliferate and which disappeared by the third passage, and small bipolar cells, which initially were unpigmented, proliferated very rapidly, and became pigmented after the addition of 3-isobutyl-1-methylxanthine to the culture medium. Both cell types were melanocytes as confirmed by electron microscopy and by staining with antibodies to S-100, GD3, and melanosomal antigens. The availability of cultured hair-follicle melanocytes wil facilitate investigations of the role of these cells in normal and abnormal hair biology.