GLI2 induces genomic instability in human keratinocytes by inhibiting apoptosis.

Abnormal Sonic Hedgehog signalling leads to increased transcriptional activation of its downstream effector, glioma 2 (GLI2), which is implicated in the pathogenesis of a variety of human cancers. However, the mechanisms underlying the tumorigenic role of GLI2 remain elusive. We demonstrate that overexpression of GLI2-ß isoform, which lacks the N-terminal repressor domain (GLI2ΔN) in human keratinocytes is sufficient to induce numerical and structural chromosomal aberrations, including tetraploidy/aneuploidy and chromosomal translocations. This is coupled with suppression of cell cycle regulators p21(WAF1/CIP1) and 14-3-3σ, and strong induction of anti-apoptotic signalling, resulting in a reduction in the ability to eliminate genomically abnormal cells. Overexpression of GLI2ΔN also rendered human keratinocytes resistant to UVB-mediated apoptosis, whereas inhibition of B-cell lymphoma 2 (BCL-2) restored endogenous (genomic instability (GIN)) and exogenous (UVB) DNA damage-induced apoptosis. Thus, we propose that ectopic expression of GLI2 profoundly affects the genomic integrity of human epithelial cells and contributes to the survival of progenies with genomic alterations by deregulating cell cycle proteins and disabling the apoptotic mechanisms responsible for their elimination. This study reveals a novel role for GLI2 in promoting GIN, a hallmark of human tumors, and identifies potential mechanisms that may provide new opportunities for the design of novel forms of cancer therapeutic strategies.

The mTOR inhibitor rapamycin opposes carcinogenic changes to epidermal Akt1/PKBα isoform signaling.

Epidermal squamous cell carcinoma (SCC) is the most aggressive non-melanoma skin cancer and is dramatically increased in patients undergoing immunosuppression following solid organ transplantation, contributing substantially to morbidity and mortality. Recent clinical studies show that use of the mammalian target of rapamycin (mTOR) inhibitor rapamycin as a post-transplantation immunosuppressive significantly reduces SCC occurrence compared with other immunosuppressives, though the mechanism is not fully understood. We show that rapamycin selectively upregulates epidermal Akt1, while failing to upregulate epidermal Akt2. Rapamycin increases epidermal Akt1 phosphorylation via inhibition of the mTOR complex 1-dependent regulation of insulin receptor substrate-1. Epidermal Akt1 is commonly downregulated in SCC while Akt2 is upregulated. We now demonstrate similar Akt1 downregulation and Akt2 upregulation by ultraviolet (UV) radiation, the most important skin carcinogen. Hence, rapamycin's upregulation of Akt1 signaling could potentially oppose the effects of UV radiation and/or tumor-associated changes on Akt1 signaling. We show in skin culture that rapamycin does enhance restoration of Akt1 phosphorylation in skin recovering from UV radiation, suggesting a mechanism for rapamycin's antitumor activity in epidermis in spite of its efficient immunosuppressive properties.

Functional role of beta 1 integrin-mediated signalling in the human hair follicle.

Integrins are transmembrane adhesion proteins that convey critical topobiological information and exert crucial signalling functions. In skin and hair follicle biology, beta1 integrins and their ligands are of particular interest. It is not yet known whether beta1 integrins play any role in the regulation of human hair growth and the expression pattern of beta1 integrin in the human pilosebaceous unit remains ill-defined. Here, we show that pilosebaceous immunoreactivity for beta1 integrin is most prominent in the outermost layer of the outer root sheath and the surrounding connective tissue sheath of human scalp hair follicles in situ and in vitro. Sites of beta1 integrin immunoreactivity co-express fibronectin and tenascin-C. Contrary to previous reports, beta1 integrin immunoreactivity in situ was not significantly upregulated in the human bulge region. Functionally, two beta1 integrin-activating antibodies (12G10, TS2/16) and ligand-mimicking RGD peptides promoted the growth of microdissected, organ-cultured human scalp hair follicles in vitro and inhibited spontaneous hair follicle regression. This supports the concept that beta1 integrin-mediated signalling is also important in human hair growth control. The physiologically relevant organ culture assay employed here is a potential research tool for exploring whether targeted stimulation of beta1 integrin-mediated signalling is a suitable candidate for human hair loss management.

Ectopic expression of c-Myc in the skin affects the hair growth cycle and causes an enlargement of the sebaceous gland.

BACKGROUND: The hair follicle continually undergoes dynamic remodelling in a cyclical manner involving tightly coordinated patterns of cell proliferation, differentiation and apoptosis. The oncoprotein c-Myc is a key regulator of these events in epidermal keratinocytes, but its importance in the hair growth cycle has not previously been determined. OBJECTIVES: To determine the role of c-Myc in the hair growth cycle. METHODS: We characterized the hair follicle phenotype of transgenic mice that permit expression of a switchable form of c-Myc (c-Myc-ER) in the suprabasal epithelial layers of the epidermis and hair follicle. RESULTS: c-Myc activation increased epithelial cell proliferation in the outer root sheath and distal hair follicle, without any substantial alteration in levels of apoptosis. Moreover, chronic c-Myc activation resulted in marked desynchronization of the murine hair growth cycle, uncoupling of hair cycle-related skin thickness and enlargement of the sebaceous gland. CONCLUSIONS: These data implicate c-Myc in the control of hair growth cycling and hair cycle-related epidermal and sebaceous gland homeostasis. We suggest that c-Myc may be activating follicular stem cells either directly or indirectly and that this has important implications for control of the 'hair cycle clock', hair growth and epidermal maintenance.

Adrenomedullin: expression and possible role in human skin and hair growth.

BACKGROUND: Adrenomedullin (AM) is a regulatory peptide that is synthesized and secreted by a wide number of cells and tissues. AM is a potent vasodilator, but also exerts other functions, such as regulating cell growth and antimicrobial defence. Two receptors, L1 and calcitonin receptor-like receptor (CRLR), which are able to bind AM, have been cloned and characterized. OBJECTIVES: To investigate expression of AM protein and its receptors in human skin and during different stages of the human hair cycle and, moreover, because of the suggested antimicrobial function of AM in skin, to investigate AM immunoreactivity (IR) in inflammatory acne lesions compared with healthy pilosebaceous follicles. METHODS: We used immunohistochemistry to determine the distribution of AM and its receptors in human skin and during different stages of the human hair cycle. AM IR in inflammatory acne lesions was investigated to evaluate the antimicrobial function of the protein, and hair follicle cultures were performed to examine the role of AM in differentiation and proliferation of hair follicle keratinocytes. RESULTS: Strong IR for AM and its receptors was present in the suprabasal epidermis, in the melanocytes of the epidermis, and in sweat and sebaceous glands. In the hair follicle, AM protein was strongly expressed in the basal and suprabasal layers of the hair bulb and the proximal outer root sheath (ORS). In the distal ORS, AM expression was increasingly suprabasal, especially in proximity to the bulge region where the basal cell layer was free of IR. IR for the CRLR revealed a similar expression pattern to that seen for AM. In contrast, L1 IR showed a suprabasal pattern of IR throughout the ORS. Similar expression of AM and its receptors was observed in catagen and early anagen follicles. AM expression was not markedly upregulated in acne lesions, suggesting a minor role for this antimicrobial peptide in acne. Despite its well-documented mitogenic effects, particularly in oral and skin keratinocytes, AM had no significant effect on hair follicle growth in vitro. CONCLUSIONS: AM and its receptors are expressed in human hair follicles, and both AM and its receptors are colocalized in the same compartments and cell types of the skin. This finding is consistent with the proposed autocrine/paracrine mechanism in the physiology of AM.

Human beta defensin-1 and -2 expression in human pilosebaceous units: upregulation in acne vulgaris lesions.

A rich residential microflora is harboured by the distal outer root sheath of the hair follicle and the hair canal - normally without causing skin diseases. Although the basic mechanisms involved in the development of inflammation during acne vulgaris remain unclear, microbial agents might play an important role in this process. In this study we have analyzed by in situ hybridization and immunohistochemistry the expression patterns of two antimicrobial peptides, human beta defensin-1 and human beta defensin-2, in healthy human hair follicles as well as in perilesional and intralesional skin of acne vulgaris lesions such as comedones, papules, and pustules. Strong defensin-1 and defensin-2 immunoreactivity was found in all suprabasal layers of the epidermis, the distal outer root sheath of the hair follicle, and the pilosebaceous duct. Marked defensin-1 and defensin-2 immunoreactivity was also found in the sebaceous gland and in the basal layer of the central outer root sheath including the bulge region. The majority of acne biopsies displayed a marked upregulation of defensin-2 immunoreactivity in the lesional and perilesional epithelium - in particular in pustules - and a less marked upregulation of defensin-1 immunoreactivity. The upregulation of beta-defensin expression in acne vulgaris lesions compared to controls suggests that beta-defensins may be involved in the pathogenesis of acne vulgaris.

Contrasting localization of c-Myc with other Myc superfamily transcription factors in the human hair follicle and during the hair growth cycle.

The mammalian hair follicle is a highly dynamic skin appendage that undergoes repeated cycles of growth and regression, involving closely co-ordinated regulation of cell proliferation, differentiation, and apoptosis. The Myc superfamily of transcription factors have been strongly implicated in the regulation of these processes in many tissues. Using immunohistochemistry, we have investigated the patterns of c-Myc, N-Myc, Max, and Mad1-4 expression at different stages of the human hair growth cycle. N-Myc, Max, Mad1, and Mad3 immunoreactivity was detected in the epidermis and the epithelium of both anagen and telogen hair follicles. Three distinct patterns of hair follicle c-Myc immunoreactivity were observed. In the infundibulum, c-Myc staining was predominantly in the basal layers, with little detectable immunoreactivity in the terminally differentiating suprabasal layers; this pattern was similar to that seen in the epidermis. In contrast, c-Myc expression in the follicle bulb was found both in the proliferating germinative epithelial cells and in the terminally differentiating matrix cells that give rise to the hair fiber. Finally, intense c-Myc immunoreactivity was detected in the bulge region of the outer root sheath. Using the C8/144B antibody as a bulge marker, we confirmed that c-Myc immunoreactivity in the outer root sheath correlates with the putative hair follicle stem cell compartment. c-Myc expression in the bulge was independent of the hair growth cycle stage. Our data suggest that Myc superfamily members serve different functions in separate epithelial compartments of the hair follicle and may play an important role in determining cell fate within the putative stem cell compartment.

Cyclical changes in rat vibrissa follicles maintained In vitro.

In mammals hair growth is cyclical; however, the factors that regulate the hair growth cycle are still poorly understood. The recent development of methods for culturing hair follicles in vitro has proved an important tool to investigate many aspects of the regulation of hair follicle growth. At present, however, these models are based on the culture of anagen hair follicles and have only partially been used to address the cyclical nature of hair growth. In this study we have made use of the fact that in rodents the hair growth cycle is synchronized, well characterized, and relatively short. We have isolated vibrissa follicles from 12 d old rats and confirmed by histology that these follicles are in the anagen stage of their first hair growth cycle. We have then maintained these follicles in vitro, on Gelfoam supports, for up to 23 d (35 d of age) and compared their histology with in vivo follicles from equivalent age littermates. We observed that 12 d old follicles maintained in vitro for up to 23 d show changes in morphology that suggest that cultured rat vibrissa follicles retain cyclical activity in vitro. Cyclical changes in hair follicle morphology were only seen in follicles maintained on gelfoam supports and moreover, hair follicle size appears to be a key feature in determining the ability of the follicle to cycle in vitro. All follicles that showed cyclical changes in vitro, however, appeared to remain blocked in pro-anagen. These data suggest that the vibrissa follicle is a in vitro good model system with which to investigate hair cycle control. J Invest Dermatol 115:1152-1155 2000

The role of IGF-I in human skin and its appendages: morphogen as well as mitogen?

Previous studies have investigated the expression of insulin-like growth factor-I (IGF-I) and its receptor in cultured skin cells or in whole skin. In order to fully understand the role of IGF-I in the skin and its appendages, however, a comprehensive study that details the expression of IGF-I and the IGF-I receptor in sections of human skin is needed. Therefore, we now report an immunocytochemical and in situ hybridization localization study of the cell types expressing IGF-I and its receptor in human adult skin and its appendages. We have observed that (i) dermal fibroblasts produce IGF-I, (ii) the epidermal basal keratinocytes are IGF-I negative but IGF-I receptor positive, and (iii) the keratinocytes of the stratum granulosum produce IGF-I. These observations indicate either that the mitogenesis of the basal keratinocytes is regulated by IGF-I expressed both in the dermis and in the stratum granulosum, or that dermal fibroblasts are responsible for sequestering IGF-I to the basal keratinocytes and that the stratum granulosum-derived IGF-I may be an autocrine regulator of epidermal differentiation. The distribution of IGF-I and its receptor in the hair follicle indicates that IGF-I may be a morphogen, not a mitogen, at those sites, because their proliferating cells, but not their differentiating cells, are IGF-I receptor negative. Further, IGF-I receptor expression by the dermal papilla appears to be switched off during the transition from anagen to catagen, which implies a regulatory role for IGF-I during the hair growth cycle.

Characterization of LHTric-1, a new monospecific monoclonal antibody to the trichocyte keratin Ha1.

The hair follicle is a heterogeneous tissue involving differentiation of both hair forming (trichocyte) and non-hair forming (root sheath) cells; while there are many antibody markers available which can determine the distribution of 'soft' epithelial keratins, fewer have been described which are truly monospecific for hair specific 'hard' keratins. We employed the proven strategy of raising monoclonal antibodies to a short synthetic peptide from the carboxy-terminal sequence of mouse Ha1 and report here the successful production of a monospecific monoclonal antibody which we have called LHTric-1. We have characterized the antibody using immunostaining on rat and human tissues and by immunoblotting against an extract of human follicles. The antibody cross-reacted between rat and human tissue but did not stain formalin-fixed tissue. LHTric-1 localized very specifically to the pre-cortical region of the hair follicle in early anagen and to pre-cortical cells in the upper bulb in anagen. Telogen follicles did not react. LHTric-1 immunoreacted within tongue and nail, staining being restricted to the mid-line above the connective tissue core in tongue and to the suprabasal layers of the nail matrix. The antibody did not react with the fully keratinized hair or nail plate. Finally, in immunoblotting, LHTric-1 reacted with a single band of 44 kDa, suggesting that a single protein was recognized. We conclude that this antibody, by virtue of its known antigen sequence specificity, will be useful in research into the formation of hair and nail in normal and diseased states.

Effects of interleukins, colony-stimulating factor and tumour necrosis factor on human hair follicle growth in vitro: a possible role for interleukin-1 and tumour necrosis factor-alpha in alopecia areata.

The immune system may be involved in the regulation of normal hair follicle growth as well as in the pathogenesis of some hair diseases. Immunomodulatory cytokines not only act as mediators of immunity and inflammation but also regulate cell proliferation and differentiation and, as such, may play an important part in regulating hair growth. We have investigated the effects of a number of interleukins (IL), colony stimulating factors and tumour necrosis factors (TNF) on hair follicle growth in vitro. Dose-response studies showed that IL-1 alpha, IL-1 beta and TNF-alpha were potent inhibitors of hair follicle growth. The histology of hair follicles maintained with inhibitory doses of IL-1 alpha, IL-1 beta and TNF-alpha showed similar changes in hair follicle morphology, resulting in the formation of dystrophic anagen hair follicles. These changes in histology were characterized by the condensation and distortion of the dermal papilla, marked vacuolation of the hair follicle matrix, abnormal keratinization of the follicle bulb and inner root sheath, disruption of follicular melanocytes and the presence of melanin granules within the dermal papilla. Moreover, these changes in hair follicle morphology are similar to those reported in alopecia areata and suggest that IL-1 alpha, IL-1 beta and TNF-alpha may play an important part in the pathophysiology of inflammatory hair disease.

Whole hair follicle culture.

In this article the authors have reviewed the historical background behind the organ culture of whole hair follicles. The methods developed by the authors and others for the isolation and whole organ maintenance of hair follicles from both human and other species are described. How whole organ models have been used to further understanding of the biology of the hair follicle and how they may be used in the future are discussed.

In the absence of streptomycin, minoxidil potentiates the mitogenic effects of fetal calf serum, insulin-like growth factor 1, and platelet-derived growth factor on NIH 3T3 fibroblasts in a K+ channel-dependent fashion.

There is considerable evidence to suggest that the opening of K+ channels plays an important role in stimulating mitogenesis. K+ channel blockers have been shown to inhibit mitogenesis in vitro, mitogens increase cytosolic membrane K+ channel permeability, K+ channel openers stimulate hair growth in vivo, and the Ras/Raf signal transduction pathway induces K+ channel activity. Paradoxically, however, K+ channel openers such as minoxidil have been reported in vitro not to modulate, or even to inhibit, mitogenesis in a range of cell types. Only untherapeutic concentrations have stimulated mitogenesis. These experiments, however, appear to have been carried out in the presence of aminoglycoside antibiotics, which inhibit potassium channel activity. We now report that in the absence of aminoglycoside antibiotics, minoxidil at 10 microg/ml (0.05 mM) causes a significant stimulation of proliferation of NIH 3T3 fibroblasts maintained over a 10-d period in 5% fetal calf serum-supplemented medium. Further, we show that in the presence of 100 microg streptomycin per ml, minoxidil at 10 microg/ml produces an initial inhibition of proliferation, which apparently confirms, in NIH 3T3 fibroblasts, that the inhibition of mitogenesis by minoxidil in the presence of streptomycin is an artifact. The potentiation of NIH 3T3 cell growth by minoxidil can be attributed to the opening of potassium channels, because the potassium channel blocker tolbutamide (5 mM) or combinations of the blockers tolbutamide (1 mM)/tetraethylammonium (2 mM) or glibenclamide (1 microM)/apamin (10 nM) block the minoxidil-induced stimulation of growth. We also demonstrate that minoxidil is able to significantly potentiate the mitogenic effects of both platelet-derived growth factor and insulin-like growth factor 1 on NIH 3T3 fibroblasts in the presence of CPSR-2 (a cytokine free serum substitute). Thus we have shown that minoxidil potentiates the mitogenic effects of fetal calf serum in vitro on NIH 3T3 fibroblasts by opening potassium channels and is also able to potentiate the mitogenic effects of the growth factors platelet-derived growth factor and insulin-like growth factor 1.

The isolation and maintenance of the human pilosebaceous unit.

We have previously developed methods for the isolation and maintenance of human sebaceous glands and hair follicles. However, in long-term culture the maintenance of both is suboptimal. This may be due to a lack of stem cells, which are thought to be located in the bulge area of the hair follicle, and this region is not present in either model. Isolation of the entire pilosebaceous unit would retain this region, and may lead to improved maintenance of both structures. We describe a method for the isolation of viable, individual, pilosebaceous units by microdissection from human scalp face-lift skin. The viability of isolated pilosebaceous units has been determined by light microscopy, patterns of DNA synthesis by [methyl-3H] thymidine autoradiography, and lipogenesis by [U-14C] acetate uptake into lipids. When maintained for 7 days in supplemented Williams E medium, isolated pilosebaceous units showed a significant increase in length. This was due to the production of a keratinized hair fibre which grew at the in vivo rate of 0.3 mm/day. Light microscopy and [methyl-3H] thymidine autoradiography confirmed that after 7 days maintenance the hair follicle retained apparently normal morphology and patterns of DNA synthesis, However, the morphology of the sebaceous gland on maintenance was more variable, generally showing luminal keratinization. Moreover [methyl-3H] thymidine autoradiography of sebaceous glands showed a marked reduction on maintenance. The rates and patterns of lipogenesis by the whole pilosebaceous unit were, respectively, lower and different from those seen with isolated human sebaceous glands; this indicates that the bulk of pilosebaceous lipogenesis is derived from the hair follicle. Rates of recovery of [14C] from 2 mM-[U-14C] sodium acetate into thin-layer chromatography plates after 7 days maintenance decreased, although this was not statistically significant, indicating that rates of lipogenesis may fall on maintenance. Pilosebaceous units were maintained for 7 days on Gelfoam (an absorbable gelatin sponge) at the media-air interface. Initial results show a marked improvement in sebaceous gland morphology. It is possible, therefore, to obtain viable human pilosebaceous units by microdissection, and to maintain them in vitro for up to 7 days, with apparently full retention of hair follicle function, but only partial retention of sebaceous gland function.

Human hair growth in vitro: a model for the study of hair follicle biology.

The factors that regulate hair follicle growth are still poorly understood. In vitro models may be useful in elucidating some aspects of hair follicle biology. We have developed an in vitro human hair growth model that enables us to maintain isolated human hair follicles for up to 10 days, during which time they continue to grow at an in vivo rate producing a keratinised hair fibre. We have shown that epidermal growth factor (EGF) in our system mimics the in vivo depilatory action of EGF in sheep, and suggest that this occurs as a result of EGF stimulating outer root sheath (ORS) cell proliferation which results in the disruption of normal mechanisms of cell-cell interaction in the hair follicle. We identify transforming growth factor-beta (TGF-beta) as a possible negative regulator of hair follicle growth and show that physiological levels of insulin-like growth factor-I (IGF-I) can support the same rates of hair follicle growth as supraphysiological levels of insulin. Furthermore, in the absence of insulin hair follicles show premature entry into a catagen-like state. This is prevented by physiological levels of IGF-I. Finally we demonstrate that the hair follicle is an aerobic glycolytic, glutaminolytic tissue and discuss the possible implications of this metabolism.

Effects of insulin and insulin-like growth factors on cultured human hair follicles: IGF-I at physiologic concentrations is an important regulator of hair follicle growth in vitro.

Insulin stimulated hair follicle growth in a dose-dependent manner over the range of 0.01 to 100 micrograms/ml. Maximum rates of hair follicle growth were observed when follicles were maintained in medium containing 10 micrograms/ml insulin, which is supraphysiologic. Hair follicles maintained in the absence of insulin or at physiologic levels showed premature entry into a catagen-like state. Insulin-like growth factor (IGF)-I and -II had no significant effect on hair follicle growth when maintained in the presence of 10 micrograms/ml insulin. However, in the absence of insulin, both IGF-I (0.01-100 ng/ml) and IGF-II (0.01-100 ng/ml) stimulated hair follicle growth in a dose-dependent manner. IGF-I was more potent than either insulin or IGF-II, stimulating maximum rates of hair follicle growth at 10 ng/ml, whereas IGF-II gave maximum stimulation at 100 ng/ml. The rates of hair follicle growth stimulated by 10 ng/ml IGF-I were identical to those stimulated by 10 micrograms/ml insulin. IGF-II (100 ng/ml), however, was unable to stimulate hair follicle growth to the same extent as insulin. Both IGF-I (10 ng/ml) and IGF-II (100 ng/ml) were more potent than insulin at preventing hair follicles from entering into a catagen-like state. Growth hormone had no effect on hair follicle growth or morphology in the absence of insulin. These data suggest that in vitro IGF-I may be an important physiologic regulator of hair growth and possibly the hair growth cycle. Moreover, the removal of insulin from tissue culture medium may be a useful method of generating large numbers of catagen hair follicles for further in vitro studies.

Effects of EGF on the morphology and patterns of DNA synthesis in isolated human hair follicles.

We have previously reported that human hair grows at a normal rate in vitro for up to 10 d. We have also reported that, on gross observation, epidermal growth factor appears to induce a catagen-like effect on cultured hair follicles, but we have not characterized the details of this. We now report that when isolated human hair follicles are maintained in the presence of epidermal growth factor, the rate of hair follicle elongation is significantly stimulated but hair fiber production is inhibited. Light microscopy showed that epidermal growth factor stimulated a thickening and vacuolation of the cells of the lower outer root sheath of the hair follicle and that the matrix cells of the hair follicle underwent an upward migration resulting in the formation of a 'club hair'-like structure that remained connected to the dermal papilla by a thin strand of epithelial cells. [Methyl-3H] thymidine autoradiography was carried out to investigate the patterns of DNA synthesis and showed that epidermal growth factor inhibited DNA synthesis in the hair follicle matrix cells but dramatically stimulated DNA synthesis in the outer root sheath. We conclude from these studies that epidermal growth factor may be inducing an artificial 'catagen-like' effect by stimulating outer root sheath proliferation, which uncouples the normal patterns of proliferation and migration that occur in the anagen hair follicle and that result in an anagen-to-catagen-like transition. Moreover, these results also suggest that, under certain conditions, outer root sheath cells in the hair follicle may be capable of downward migration.

The human hair follicle engages in glutaminolysis and aerobic glycolysis: implications for skin, splanchnic and neoplastic metabolism.

On maintenance in supplemented Williams E medium, human hair follicles grow at the normal rate, and retain their normal anagen morphology, for up to 10 days. This permits us to study their metabolism under near-physiological conditions. The ATP content of freshly isolated follicles was 124.4 +/- 10.6 pmol/follicle (mean +/- SEM; n = 50). The energy charge was 0.81 +/- 0.08 and the glycogen content 2.3 +/- 0.3 nmol/follicle. These did not alter significantly during any metabolic studies, which were performed for up to 6 h in supplemented Williams E medium. We found that the major fuel was glucose, which at physiological concentrations yields 5.47 +/- 0.77 nmol ATP/follicle/h, but 90% of the glucose was metabolised to lactate, and only 10% oxidised. Glutamine was also an important fuel, generating 2.16 +/- 0.33 nmol ATP/follicle/h, but this too was largely metabolised to lactate rather than oxidised. Lipid fuels such as palmitate or beta-hydroxybutyrate only yielded 0.72 +/- 0.15 and 0.72 +/- 0.14 nmol ATP/follicle/h, respectively, and their oxidation did not inhibit glucose utilisation. No glucose-fatty acid cycle operates in the hair follicle, therefore, but a glucose-glutamine cycle does, since the presence of glutamine will inhibit glucose utilisation.

Prolonged maintenance of human hair follicles in vitro in a serum-free medium.

We have previously reported the in vitro growth of human hair follicles for up to 4 days in a partially defined medium containing serum. We now report the prolonged in vitro growth of isolated human hair follicles for at least 9 days. This was achieved after analysis of the contribution of certain components of the original medium and, by a process of elimination, deriving a completely defined medium supplemented only with antibiotics, L-glutamine, insulin and hydrocortisone. We have shown, by [methyl-3H] thymidine autoradiography, that the hair follicles grown in this medium maintain an in vivo pattern of DNA synthesis, and that the gross morphology and histology of these maintained hair follicles remains similar to that of freshly isolated hair follicles. We have also shown that the patterns of keratin synthesis, as determined by [35S] methionine labelling, do not alter with maintenance.