NF-κB Participates in Mouse Hair Cycle Control and Plays Distinct Roles in the Various Pelage Hair Follicle Types.

The transcription factor NF-κB controls key features of hair follicle (HF) development, but the role of NF-κB in adult HF cycle regulation remains obscure. Using NF-κB reporter mouse models, strong NF-κB activity was detected in the secondary hair germ of late telogen and early anagen HFs, suggesting a potential role for NF-κB in HF stem/progenitor cell activation during anagen induction. At mid-anagen, NF-κB activity was observed in the inner root sheath and unilaterally clustered in the HF matrix, which indicates that NF-κB activity is also involved in hair fiber morphogenesis during HF cycling. A mouse model with inducible NF-κB suppression in the epithelium revealed pelage hair-type-dependent functions of NF-κB in cycling HFs. NF-κB participates in telogen-anagen transition in awl and zigzag HFs, and is required for zigzag hair bending and guard HF cycling. Interestingly, zigzag hair shaft bending depends on noncanonical NF-κB signaling, which previously has only been associated with lymphoid cell biology. Furthermore, loss of guard HF cycling suggests that in this particular hair type, NF-κB is indispensable for stem cell activation, maintenance, and/or growth.

Human hair follicle organ culture: theory, application and perspectives.

For almost a quarter of a century, ex vivo studies of human scalp hair follicles (HFs) have permitted major advances in hair research, spanning diverse fields such as chronobiology, endocrinology, immunology, metabolism, mitochondrial biology, neurobiology, pharmacology, pigmentation and stem cell biology. Despite this, a comprehensive methodological guide to serum-free human HF organ culture (HFOC) that facilitates the selection and analysis of standard HF biological parameters and points out both research opportunities and pitfalls to newcomers to the field is still lacking. The current methods review aims to close an important gap in the literature and attempts to promote standardisation of human HFOC. We provide basic information outlining the establishment of HFOC through to detailed descriptions of the analysis of standard read-out parameters alongside practical examples. The guide closes by pointing out how serum-free HFOC can be utilised optimally to obtain previously inaccessible insights into human HF biology and pathology that are of interest to experimental dermatologists, geneticists, developmental biologists and (neuro-) endocrinologists alike and by highlighting novel applications of the model, including gene silencing and gene expression profiling of defined, laser capture-microdissected HF compartments.

N(1)-methylspermidine, a stable spermidine analog, prolongs anagen and regulates epithelial stem cell functions in human hair follicles.

Spermidine (Spd), the prototypic polyamine, has been shown to be essential for hair follicle (HF) growth. However, Spd can be readily converted into other polyamines, and is physiologically unstable. Therefore, to assess its individual functions on HFs, we used the metabolically stable Spd analog N(1)-methylspermidine (N(1)-MeSpd). N(1)-MeSpd was confirmed to be a metabolically stable compound, with a half life of 90 h. 0.5 µM N(1)-MeSpd strongly prolonged anagen and decreased cell apoptosis in HFs in culture after 6 days, accompanied by specific stimulation of the expression of the epithelial stem cell-associated keratin, K15. N(1)-MeSpd also reduced lactate dehydrogenase activity in the culture supernatant, a parameter of cell death and cell lysis. N(1)-MeSpd diminished intracellular reactive oxygen species production in cultured keratinocytes, and reduced tumor necrosis factor-α, interleukin (IL)-1ß and IL-6 gene and protein expression after lipopolysaccharide stimulation. This suggests that some effects of N(1)-MeSpd may be mediated by anti-oxidative and anti-inflammatory effects. These additional properties of N(1)-MeSpd could be clinically important for the treatment of inflammatory alopecias and inflammatory scalp diseases.

Mitochondrial function in murine skin epithelium is crucial for hair follicle morphogenesis and epithelial-mesenchymal interactions.

Here, we studied how epithelial energy metabolism impacts overall skin development by selectively deleting intraepithelial mtDNA in mice by ablating a key maintenance factor (Tfam(EKO)), which induces loss of function of the electron transport chain (ETC). Quantitative (immuno)histomorphometry demonstrated that Tfam(EKO) mice showed significantly reduced hair follicle (HF) density and morphogenesis, fewer intrafollicular keratin15+ epithelial progenitor cells, increased apoptosis, and reduced proliferation. Tfam(EKO) mice also displayed premature entry into (aborted) HF cycling by apoptosis-driven HF regression (catagen). Ultrastructurally, Tfam(EKO) mice exhibited severe HF dystrophy, pigmentary abnormalities, and telogen-like condensed dermal papillae. Epithelial HF progenitor cell differentiation (Plet1, Lrig1 Lef1, and ß-catenin), sebaceous gland development (adipophilin, Scd1, and oil red), and key mediators/markers of epithelial-mesenchymal interactions during skin morphogenesis (NCAM, versican, and alkaline phosphatase) were all severely altered in Tfam(EKO) mice. Moreover, the number of mast cells, major histocompatibility complex class II+, or CD11b+ immunocytes in the skin mesenchyme was increased, and essentially no subcutis developed. Therefore, in contrast to their epidermal counterparts, pilosebaceous unit stem cells depend on a functional ETC. Most importantly, our findings point toward a frontier in skin biology: the coupling of HF keratinocyte mitochondrial function with the epithelial-mesenchymal interactions that drive overall development of the skin and its appendages.

Overexpression of epigen during embryonic development induces reversible, epidermal growth factor receptor-dependent sebaceous gland hyperplasia.

The epidermal growth factor receptor (EGFR) system is a key regulator of epithelial development and homeostasis. Its functions in the sebaceous gland (SG), however, remain poorly characterized. In this study, using a transgenic mouse line with tissue-specific and inducible expression of the EGFR ligand epigen, we showed that increased activation of the EGFR in skin keratinocytes results in enlarged SGs and increased sebum production. The phenotype can be reverted by interrupting transgene expression and is EGFR dependent, as gland size and sebum levels return to normal values after crossing to the EGFR-impaired mouse line Wa5. Intriguingly, however, the SG enlargement appears only if EGFR activation occurs before birth. Importantly, the enlarged sebaceous glands are associated with an increased expression of the transcription factor MYC and of the transmembrane proteins LRIG1, an established negative-feedback regulator of the EGFR/ERBB tyrosine kinase receptors and a stem cell marker. Our findings identify EGFR signaling as a major pathway determining SG activity and suggest a functional relationship between the EGFR/ERBB system and MYC/LRIG1 in the commitment of stem cells toward specific progenitor cell types, with implications for our understanding of their role in tissue development, homeostasis, and disease.

From frog integument to human skin: dermatological perspectives from frog skin biology.

For over a century, frogs have been studied across various scientific fields, including physiology, embryology, neuroscience, (neuro)endocrinology, ecology, genetics, behavioural science, evolution, drug development, and conservation biology. In some cases, frog skin has proven very successful as a research model, for example aiding in the study of ion transport through tight epithelia, where it has served as a model for the vertebrate distal renal tubule and mammalian epithelia. However, it has rarely been considered in comparative studies involving human skin. Yet, despite certain notable adaptations that have enabled frogs to survive in both aquatic and terrestrial environments, frog skin has many features in common with human skin. Here we present a comprehensive overview of frog (and toad) skin ontogeny, anatomy, cytology, neuroendocrinology and immunology, with special attention to its unique adaptations as well as to its similarities with the mammalian integument, including human skin. We hope to provide a valuable reference point and a source of inspiration for both amphibian investigators and mammalian researchers studying the structural and functional properties of the largest organ of the vertebrate body.

A meeting of two chronobiological systems: circadian proteins Period1 and BMAL1 modulate the human hair cycle clock.

The hair follicle (HF) is a continuously remodeled mini organ that cycles between growth (anagen), regression (catagen), and relative quiescence (telogen). As the anagen-to-catagen transformation of microdissected human scalp HFs can be observed in organ culture, it permits the study of the unknown controls of autonomous, rhythmic tissue remodeling of the HF, which intersects developmental, chronobiological, and growth-regulatory mechanisms. The hypothesis that the peripheral clock system is involved in hair cycle control, i.e., the anagen-to-catagen transformation, was tested. Here we show that in the absence of central clock influences, isolated, organ-cultured human HFs show circadian changes in the gene and protein expression of core clock genes (CLOCK, BMAL1, and Period1) and clock-controlled genes (c-Myc, NR1D1, and CDKN1A), with Period1 expression being hair cycle dependent. Knockdown of either BMAL1 or Period1 in human anagen HFs significantly prolonged anagen. This provides evidence that peripheral core clock genes modulate human HF cycling and are an integral component of the human hair cycle clock. Specifically, our study identifies BMAL1 and Period1 as potential therapeutic targets for modulating human hair growth.

A practical guide for the study of human and murine sebaceous glands in situ.

The skin of most mammals is characterised by the presence of sebaceous glands (SGs), whose predominant constituent cell population is sebocytes, that is, lipid-producing epithelial cells, which develop from the hair follicle. Besides holocrine sebum production (which contributes 90% of skin surface lipids), multiple additional SG functions have emerged. These range from antimicrobial peptide production and immunomodulation, via lipid and hormone synthesis/metabolism, to the provision of an epithelial progenitor cell reservoir. Therefore, in addition to its involvement in common skin diseases (e.g. acne vulgaris), the unfolding diversity of SG functions, both in skin health and disease, has raised interest in this integral component of the pilosebaceous unit. This practical guide provides an introduction to SG biology and to relevant SG histochemical and immunohistochemical techniques, with emphasis placed on in situ evaluation methods that can be easily employed. We propose a range of simple, established markers, which are particularly instructive when addressing specific SG research questions in the two most commonly investigated species in SG research, humans and mice. To facilitate the development of reproducible analysis techniques for the in situ evaluation of SGs, this methods review concludes by suggesting quantitative (immuno-)histomorphometric methods for standardised SG evaluation.

Thyrotropin-releasing hormone (TRH) promotes wound re-epithelialisation in frog and human skin.

There remains a critical need for new therapeutics that promote wound healing in patients suffering from chronic skin wounds. This is, in part, due to a shortage of simple, physiologically and clinically relevant test systems for investigating candidate agents. The skin of amphibians possesses a remarkable regenerative capacity, which remains insufficiently explored for clinical purposes. Combining comparative biology with a translational medicine approach, we report the development and application of a simple ex vivo frog (Xenopus tropicalis) skin organ culture system that permits exploration of the effects of amphibian skin-derived agents on re-epithelialisation in both frog and human skin. Using this amphibian model, we identify thyrotropin-releasing hormone (TRH) as a novel stimulant of epidermal regeneration. Moving to a complementary human ex vivo wounded skin assay, we demonstrate that the effects of TRH are conserved across the amphibian-mammalian divide: TRH stimulates wound closure and formation of neo-epidermis in organ-cultured human skin, accompanied by increased keratinocyte proliferation and wound healing-associated differentiation (cytokeratin 6 expression). Thus, TRH represents a novel, clinically relevant neuroendocrine wound repair promoter that deserves further exploration. These complementary frog and human skin ex vivo assays encourage a comparative biology approach in future wound healing research so as to facilitate the rapid identification and preclinical testing of novel, evolutionarily conserved, and clinically relevant wound healing promoters.

Cannabinoid receptor 1 controls human mucosal-type mast cell degranulation and maturation in situ.

BACKGROUND: Because many chronic inflammatory and allergic disorders are intimately linked to excessive mast cell (MC) numbers and activation, it is clinically important to understand the physiologic mechanisms preventing excess MC accumulation/degranulation in normal human tissues. OBJECTIVE: Because endocannabinoids are increasingly recognized as neuroendocrine regulators of MC biology, we investigated how cannabinoid receptor (CB) 1 signaling affects human mucosal-type mast cells (hMMCs). METHODS: Using organ-cultured nasal polyps as a surrogate tissue for human bronchial mucosa, we investigated how CB1 stimulation, inhibition, or knockdown affects hMMC biology using quantitative (immuno)histomorphometry and electron microscopy. RESULTS: Kit(+) hMMCs express functional CB1 in situ. Blockade of CB1 signaling (with the specific CB1 antagonist N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide [AM251] or CB1 gene knockdown) enhanced hMMC degranulation and increased total numbers without affecting their proliferation in situ. This suggests that inhibiting CB1 signaling induces hMMC maturation from resident progenitor cells within human mucosal stroma. hMMC maturation was induced at least in part through upregulating stem cell factor production. Both the prototypic endocannabinoid anandamide and the CB1-selective agonist arachidonyl-2-chloroethylamide effectively counteracted secretagogue-triggered excessive hMMC degranulation. CONCLUSIONS: The current serum-free nasal polyp organ culture model allows physiologically and clinically relevant insights into the biology and pharmacologic responses of primary hMMCs in situ. In human airway mucosa hMMC activation and maturation are subject to a potent inhibitory endocannabinoid tone through CB1 stimulation. This invites one to target the endocannabinoid system in human airway mucosa as a novel strategy in the future management of allergic diseases.

Topobiology of human pigmentation: P-cadherin selectively stimulates hair follicle melanogenesis.

P-cadherin serves as a major topobiological cue in mammalian epithelium. In human hair follicles (HFs), it is prominently expressed in the inner hair matrix that harbors the HF pigmentary unit. However, the role of P-cadherin in normal human pigmentation remains unknown. As patients with mutations in the gene that encodes P-cadherin show hypotrichosis and fair hair, we explored the hypothesis that P-cadherin may control HF pigmentation. When P-cadherin was silenced in melanogenically active organ-cultured human scalp HFs, this significantly reduced HF melanogenesis and tyrosinase activity as well as gene and/or protein expression of gp100, stem cell factor, c-Kit, and microphthalmia-associated transcription factor (MITF), both in situ and in isolated human HF melanocytes. Instead, epidermal pigmentation was unaffected by P-cadherin knockdown in organ-cultured human skin. In hair matrix keratinocytes, P-cadherin silencing reduced plasma membrane ß-catenin, whereas glycogen synthase kinase 3 beta (GSK3ß) and phospho-ß-catenin expression were significantly upregulated. This suggests that P-cadherin-GSK3ß/Wnt signaling is required for maintaining the expression of MITF to sustain intrafollicular melanogenesis. Thus, P-cadherin-mediated signaling is a melanocyte subtype-specific topobiological regulator of normal human pigmentation, possibly via GSK3ß-mediated canonical Wnt signaling.

ß1 integrin signaling maintains human epithelial progenitor cell survival in situ and controls proliferation, apoptosis and migration of their progeny.

ß1 integrin regulates multiple epithelial cell functions by connecting cells with the extracellular matrix (ECM). While ß1 integrin-mediated signaling in murine epithelial stem cells is well-studied, its role in human adult epithelial progenitor cells (ePCs) in situ remains to be defined. Using microdissected, organ-cultured human scalp hair follicles (HFs) as a clinically relevant model for studying human ePCs within their natural topobiological habitat, ß1 integrin-mediated signaling in ePC biology was explored by ß1 integrin siRNA silencing, specific ß1 integrin-binding antibodies and pharmacological inhibition of integrin-linked kinase (ILK), a key component of the integrin-induced signaling cascade. ß1 integrin knock down reduced keratin 15 (K15) expression as well as the proliferation of outer root sheath keratinocytes (ORSKs). Embedding of HF epithelium into an ECM rich in ß1 integrin ligands that mimic the HF mesenchyme significantly enhanced proliferation and migration of ORSKs, while K15 and CD200 gene and protein expression were inhibited. Employing ECM-embedded ß1 integrin-activating or -inhibiting antibodies allowed to identify functionally distinct human ePC subpopulations in different compartments of the HF epithelium. The ß1 integrin-inhibitory antibody reduced ß1 integrin expression in situ and selectively enhanced proliferation of bulge ePCs, while the ß1 integrin-stimulating antibody decreased hair matrix keratinocyte apoptosis and enhanced transferrin receptor (CD71) immunoreactivity, a marker of transit amplifying cells, but did not affect bulge ePC proliferation. That the putative ILK inhibitor QLT0267 significantly reduced ORSK migration and proliferation and induced massive ORSK apoptosis suggests a key role for ILK in mediating the ß1 integrin effects. Taken together, these findings demonstrate that ePCs in human HFs require ß1 integrin-mediated signaling for survival, adhesion, and migration, and that different human HF ePC subpopulations differ in their response to ß1 integrin signaling. These insights may be exploited for cell-based regenerative medicine strategies that employ human HF-derived ePCs.

P-cadherin regulates human hair growth and cycling via canonical Wnt signaling and transforming growth factor-ß2.

P-cadherin is a key component of epithelial adherens junctions, and it is prominently expressed in the hair follicle (HF) matrix. Loss-of-function mutations in CDH3, which encodes P-cadherin, result in hypotrichosis with juvenile macular dystrophy (HJMD), an autosomal recessive disorder featuring sparse and short hair. Here, we attempted to recapitulate some aspects of HJMD in vitro by transfecting normal, organ-cultured human scalp HFs with lipofectamine and CDH3-specific or scrambled control siRNAs. As in HJMD patients, P-cadherin silencing inhibited hair shaft growth, prematurely induced HF regression (catagen), and inhibited hair matrix keratinocyte proliferation. In situ, membrane ß-catenin expression and transcription of the ß-catenin target gene, axin2, were significantly reduced, whereas glycogen synthase kinase 3 ß (GSK3ß) and phospho-ß-catenin immunoreactivity were increased. These effects were partially reversed by inhibiting GSK3ß. P-cadherin silencing reduced the expression of the anagen-promoting growth factor, IGF-1, whereas that of transforming growth factor ß 2 (TGFß2; catagen promoter) was enhanced. Neutralizing TGFß antagonized the catagen-promoting effects of P-cadherin silencing. In summary, we introduce human HFs as an attractive preclinical model for studying the functions of P-cadherin in human epithelial biology and pathology. This model demonstrates that cadherins can be successfully knocked down in an intact human organ in vitro, and shows that P-cadherin is needed for anagen maintenance by regulating canonical Wnt signaling and suppressing TGFß2.

The mitochondrial electron transport chain is dispensable for proliferation and differentiation of epidermal progenitor cells.

Tissue stem cells and germ line or embryonic stem cells were shown to have reduced oxidative metabolism, which was proposed to be an adaptive mechanism to reduce damage accumulation caused by reactive oxygen species. However, an alternate explanation is that stem cells are less dependent on specialized cytoplasmic functions compared with differentiated cells, therefore, having a high nuclear-to-cytoplasmic volume ratio and consequently a low mitochondrial content. To determine whether stem cells rely or not on mitochondrial respiration, we selectively ablated the electron transport chain in the basal layer of the epidermis, which includes the epidermal progenitor/stem cells (EPSCs). This was achieved using a loxP-flanked mitochondrial transcription factor A (Tfam) allele in conjunction with a keratin 14 Cre transgene. The epidermis of these animals (Tfam(EKO)) showed a profound depletion of mitochondrial DNA and complete absence of respiratory chain complexes. However, despite a short lifespan due to malnutrition, epidermal development and skin barrier function were not impaired. Differentiation of epidermal layers was normal and no proliferation defect or major increase of apoptosis could be observed. In contrast, mice with an epidermal ablation of prohibitin-2, a scaffold protein in the inner mitochondrial membrane, displayed a dramatic phenotype observable already in utero, with severely impaired skin architecture and barrier function, ultimately causing death from dehydration shortly after birth. In conclusion, we here provide unequivocal evidence that EPSCs, and probably tissue stem cells in general, are independent of the mitochondrial respiratory chain, but still require a functional dynamic mitochondrial compartment.

Visualization and characterisation of defined hair follicle compartments by Fourier transform infrared (FTIR) imaging without labelling.

BACKGROUND: To visualise and characterise skin architecture, the tissue usually has to be destroyed and labelled. OBJECTIVES: The use of Fourier transform infrared (FTIR) spectroscopy as a label-free, minimally sample destructive method to define hair follicular structure has been explored and demonstrated in this paper. METHODS: Human scalp skin cryosections were imaged using FTIR microscopy and the data was subsequently analysed with N-FINDR spectral unmixing algorithm. RESULTS: This resulted in an excellent distinction of known hair follicle tissue layers, which could be discerned based on their molecular structure. CONCLUSION: The development of a minimally sample-destructive, label-free spectroscopy based technique that can differentiate layers of cells in the dermal papilla and connective tissue sheath in the mesenchyme of the hair follicle paves the way forward to identifying spectral markers important in wound healing and stem cell therapies.

Can the hair follicle become a model for studying selected aspects of human ocular immune privilege?

Immune privilege (IP) is important in maintaining ocular health. Understanding the mechanism underlying this dynamic state would assist in treating inflammatory eye diseases. Despite substantial progress in defining eye IP mechanisms, because of the scarcity of human ocular tissue for research purposes, most of what we know about ocular IP is based on rodent models (of unclear relevance to human eye immunology) and on cultured human eye-derived cells that cannot faithfully mirror the complex cell-tissue interactions that underlie normal human ocular IP in situ. Therefore, accessible, instructive, and clinically relevant human in vitro models are needed for exploring the general principles of why and how IP collapses under clinically relevant experimental conditions and how it can be protected or even restored therapeutically. Among the few human IP sites, the easily accessible and abundantly available hair follicle (HF) may offer one such surrogate model. There are excellent human HF organ culture systems for the study of HF IP in situ that instructively complement in vivo autoimmunity research in the human system. In this article, we delineate that the human eye and HF, despite their obvious differences, share key molecular and cellular mechanisms for maintaining IP. We argue that, therefore, human scalp HFs can provide an unconventional, but highly instructive, accessible, easily manipulated, and clinically relevant preclinical model for selected aspects of ocular IP. This essay is an attempt to encourage professional eye researchers to turn their attention, with appropriate caveats, to this candidate surrogate model for ocular IP in the human system.

Exploring the "hair growth-wound healing connection": anagen phase promotes wound re-epithelialization.

When the skin is damaged, a variety of cell types must migrate, proliferate, and differentiate to reform a functional barrier to the external environment. Recent studies have shown that progenitor cells residing in hair follicles (HFs) are able to contribute to this re-epithelialization of wounds in vivo. However, the influence of the hair cycle on wound healing has not previously been addressed. Here, we have exploited spontaneous postnatal hair-cycle synchronicity in mice to systematically examine the influence of the different hair-cycle stages on murine skin wound healing. We report significant acceleration of healing during the anagen phase of HF cycling in vivo, associated with alterations in epithelial, endothelial, and inflammatory cell types. Intriguingly, gene profiling data reveal a clear correlation between the transcription of genes beneficial for wound healing and those upregulated during the anagen phase of the hair cycle in unwounded skin. These findings, which demonstrate a previously unappreciated association between HF cycling and wound healing, reveal numerous molecular correlates for further investigation.