Do androgens influence hair growth by altering the paracrine factors secreted by dermal papilla cells?

ARTICLE

Androgens are the main regulator of changes in human hair growth [1]. One of the first signs of puberty in both sexes is the gradual replacement of tiny vellus hairs in the pubic and axillary areas with longer, more pigmented intermediate forms and eventually the large, dark terminal hair characteristic of adulthood [2, 3]. These changes parallel the rise in plasma androgens in both sexes [4, 5]. Later, similar changes occur on the face, chest, upper pubic triangle and the limbs of men readily distinguishing the adult male [6, 7]. In complete contrast, androgens progressively inhibit large terminal scalp follicles in individuals with a genetic predisposition so that they are gradually replaced by tiny vellus ones causing male pattern baldness or androgenetic alopecia [8-11]. The absence of the characteristic increased hair growth in adults or any male pattern baldness in individuals with deficient androgen receptors i.e. androgen insensitivity syndrome confirms the essential role of androgens and androgen receptors in the regulation of human hair growth [12]. The paradoxically different responses of hair follicles to similar circulating levels of androgens (reviewed in [1] and [13]) clearly demonstrated by bald men with bushy beards indicates that the particular effect produced by androgens depends on factors within the individual follicles themselves. How this occurs is not fully understood, but one strong possibility is that androgens alter the paracrine factors produced by dermal papilla cells and hence alter the cellular activity of the follicles various cell types. This is currently the subject of experimental investigation.

Mode of action of androgens in hair follicles

The current hypothesis for the mode of action of androgens in hair follicles focuses on the dermal papilla. The mesenchyme-derived dermal papilla plays an important regulatory role in the follicle, altering many parameters and determining the type of hair produced [14, 15]. This is believed to occur by the production of paracrine growth factors and extracellular matrix proteins. During the beginning of anagen, the growth phase of the hair cycle, hair follicles appear to recapitulate follicular embryogenesis when a new hair is being formed [13]. Since steroids act via the mesenchyme in many developing steroid-dependent tissues such as the prostate [16], Randall has proposed that androgens act on the hair follicle via the dermal papilla [13, 17]. In this hypothesis, summarised in Figure 1, androgens enter the follicle via the dermal papilla's capillaries, bind to androgen receptors within the dermal papilla cells and trigger the expression of hormone responsive genes. This then alters the paracrine factors produced by the dermal papilla cells which regulate the growth and activity of the other cell types in the follicle (Table I). These paracrine factors could be soluble mitogenic factors or extracellular matrix components.

Androgens alter the size of the hair produced and, therefore, the size of the sheaths surrounding the hair and the dermal papilla at its base. In addition hair pigmentation is also altered during responses to androgens and larger follicles require a greater vascular supply. This means that several cell types are potential targets for these factors. They include: the follicular keratinocytes which form the hair itself and the various layers of the outer and inner root sheaths; the melanocytes, which produce the pigment which gives the follicle its colour; and the endothelial cells of the blood vessel capillaries. Factors could also act in an autocrine or paracrine manner on the dermal papilla cells themselves.

This model of androgens acting directly on the regulatory dermal papilla and then indirectly on the other cell types does seem very plausible. Androgens have such widely differing effects on hair follicles even within the same person that it is difficult to conceive of the responses being so well controlled if each cell type had to react directly to androgens.

The hypothesis has received a great deal of experimental support. Androgen receptors have been located in dermal papilla cells of hair follicles by immunohistochemistry, although the distribution reported elsewhere in the follicle varies with a monoclonal antibody detecting no epithelial cell staining [18] while a polyclonal antibody study reported staining also in the outer root sheath [19]. Specific high affinity, low capacity androgen receptors have been identified in cultured dermal papilla cells derived from androgen target follicles such as beard [20] and balding scalp [21].

Studies of androgen metabolism by dermal papilla cells also provide strong corroboration. Beard cells metabolise testosterone to 5alpha-dihydrotestosterone [22, 23] unlike either pubic or axillary cells [24]. This corresponds to the absence of beard growth but presence of axillary and the female pubic pattern hair growth in patients with 5alpha-reductase deficiency [25]. As well as supporting the current hypothesis for androgen action in the hair follicle (Fig. 1), these studies confirm that cultured dermal papilla cells retain characteristics in vitro that reflect the androgen responses of their parent follicle in vivo.

Secretion of mitogenic factors

Currently, research is focussing on the soluble mitogenic factors secreted by dermal papilla cells. A particular aim of our research is to determine whether androgens can alter the production of mitogenic factors produced by dermal papilla cells. A number of bioassays have been carried out involving co-culture of dermal papilla cells and other cell types or collecting media in which dermal papilla cells have been grown, "conditioned media", and assessing the capacity of this conditioned media to promote cell growth in other cells. These have shown that human dermal papilla cells secrete soluble, proteinaceous factors which are mitogenic for other dermal papilla cells [17, 26], outer root sheath cells [27, 28], transformed epidermal keratinocytes [29] and endothelial cells [30]. These mitogenic factors can cross at least some species as human dermal papilla cell media have the capacity to stimulate growth of rat whisker cells [31].

When the effect of androgens on mitogenic capacity was assessed, testosterone was found to stimulate the mitogenic capacity of beard cells for outer root sheath cells [28] and beard dermal papilla cells [26], as would be predicted by the hypothesis. Interestingly, testosterone had no effect on the mitogenic capacity of non-balding scalp cells and only beard dermal papilla cells were able to respond to the factors (Fig. 2). The effect of testosterone was not seen when the same range of testosterone concentrations were added to media previously conditioned by beard cells in the absence of testosterone (Fig. 2). This demonstrates that the androgenic effect was due to the androgen-potentiated synthesis of a mitogenic factor or factors rather than an interaction of testosterone with an existing paracrine factor in the conditioned media. The restriction of the response to beard dermal papilla cells could reflect a different receptor on beard cells or that scalp cells were already responding at their full capacity to the mitogenic factors. This suggests that an autocrine mechanism of producing paracrine factors for beard cells is involved in beard cell growth in response to testosterone. Certainly, the size of the dermal papilla has been shown to be proportional to the size of the hair [32] and this appears to involve an alteration in the number of dermal papilla cell numbers as well as the amount of extracellular matrix present [33]. A need to alter the autocrine production of growth factors after androgen stimulation could account for the slow response of follicles to androgens which often takes many years to have a full effect [6-10].

When dermal papilla conditioned media was assayed on a standard line of keratinocytes, physiological levels of testosterone again stimulated the production of greater mitogenic ability by beard, but not non-balding scalp cells [34]. Interestingly, when dermal papilla cells from androgen-inhibited balding scalp follicles from both men [29] and the stump-tailed macaque [35, 36] were investigated the mitogenic capacity assayed on keratinocytes was inhibited by physiological levels of testosterone. All these markedly contrasting responses to testosterone in vitro reflect the paradoxical in vivo responses and provide further strong support for the model of androgen action (Fig. 1).

Identification of paracrine factors

Research is now focussing on identifying specific paracrine factors secreted by cultured dermal papilla cells and determining whether their production is altered by the androgen responsiveness of the parent follicle in vivo or by androgen in vitro. A range of growth factors and cytokines have been implicated in hair growth (reviewed in [37-39]). Several of these have been shown to be produced by cultured dermal papilla cells from androgen-independent follicles either by measurement of protein production by ELISA of conditioned media or by examining their expression of mRNA by RT-PCR. One of the most studied of these is insulin like growth factor-I, IGF-I [40], a potent mitogen which plays an important role in maintaining anagen in cultured human scalp follicles in vitro [41] and which causes abnormal patterns of growth and differentiation of hair follicles [42] when its effects are blocked in the IGF-I receptor deficient knockout mouse. Itami and colleagues [28] have identified the expression of mRNA for IGF-I in beard dermal papilla cells and confirmed its importance by blocking the mitogenic effect of dermal papilla cells on co-cultured outer root sheath cells with an antibody to IGF-I.

Hepatocyte growth factor, HGF (also known as scatter factor), is a potent mitogen, morphogen and motogen for epithelial cells which is produced by mesenchyme cells during embryogenesis [43]. It is also expressed by dermal papilla cells [44, 45]. HGF also stimulates the growth of mouse hair follicles in culture [46], but the effects reported on isolated human hair growth vary depending on the group [39, 47]. Vascular endothelial growth factor, VEGF, is a major regulator of angiogenesis and vascular permeability which is also secreted [48] and expressed by cultured human dermal papilla cells [49, 50]. So many other growth factors and cytokines have been implicated in hair follicle growth that there is insufficient space to consider them here; they have recently been reviewed elsewhere [37-39, 51]. Studies on factors altered by androgens are more limited, although they are a prime focus in our laboratory. Itami, Takayasu and colleagues have found that physiological levels of testosterone in vitro stimulated increased IGF-I mRNA expression by beard dermal papilla cells [28].

In our studies of human dermal papilla cells we have employed a particular experimental design to investigate the effects of androgens in vivo and in vitro. We have investigated in vivo effects of androgens on androgen potentiated hair growth by comparing beard dermal papilla cells with control, non-balding scalp cells and also the inhibitory effects of androgens by comparing balding scalp cells with non-balding ones. The effects of androgens in vitro have been assessed by incubating dermal papilla cells from the three types of follicles in the presence, or absence, of 10 nM testosterone for 24 hrs.

When HGF was investigated testosterone in vitro had no effect on its expression by any cell type. However, beard cells expressed much more HGF than non-balding scalp cells and expression was virtually undetectable in balding scalp cells. This suggests that HGF could be important in maintaining large follicles and that its levels in androgen-dependent follicles may be altered by exposure to androgens in vivo [45]. Results with the vascular regulator VEGF were markedly different; both beard and non-balding scalp cells secreted the same amounts of VEGF into the media and expressed the same amounts of mRNA [48, 50]. However, this is not too surprising as both types of follicles were producing terminal hairs necessitating a good blood supply.

Since androgens alter the pigmentation of follicles we have also investigated the role of stem cell factor (SCF,
c-kit ligand, steel factor, mast cell growth factor) known to play important roles in the development of epidermal [52, 53] and hair pigmentation [54, 55]. Dermal papilla cells from both non-balding scalp and beard follicles secreted SCF [56] implicating the dermal papilla as the local source of SCF for hair follicle melanocytes. Adult human scalp hair follicle melanocytes do express the receptor for SCF, c-kit [57]. Interestingly, although androgen in vitro had no effect, beard cells secreted more SCF than non-balding scalp which may indicate that androgens had increased the production of SCF by facial dermal papilla cells to cause the darkening of facial hair in the transformation of a boy's vellus facial hair to a full adult beard.

Recently, an interesting study of cultured human dermal papilla cells by Professor Takayasu's group has shown that they express the protease nexin-1 and that its expression by cells from balding follicles is inhibited by androgen in vitro [58]. Protease nexin-1, also known as glia-derived nexin-1, is a potent inhibitor of serine proteases, such as thrombin, urokinase and plasmin, thereby regulating cellular growth and differentiation in many tissues [59]. Since its effect in other tissues is modulated by extracellular matrix components such as type IV collagen [60] which dermal papilla cells also produce [61], alterations in protease nexin-1 production by dermal papilla cells could result in changed production of such extracellular matrix components by the dermal papilla. These could act as paracrine signals between the dermal papilla cells and the other follicular components as proposed in Figure 1 and may play an important role as alterations in the size of the dermal papilla are known to correlate with the size of the hair produced by the follicle [32, 33].

CONCLUSION

The original question being addressed was whether androgens influence hair growth by altering the paracrine factors produced by dermal papilla cells. There is certainly strong experimental support for the hypothesis that androgens alter follicle size by acting via the dermal papilla. Dermal papilla cells from both human and primate follicles secrete paracrine factors in culture which stimulate the growth of many cell types from the follicle. Importantly, androgens in vitro increase or inhibit the production of mitogenic factors by human and macaque cells in line with the parent follicle's response to androgens in vivo. Thus, the evidence so far is that androgens may influence hair growth in this way. The identification of specific growth factors and enzymes, some of which appear to be altered by androgens either in vivo or in vitro supports this view. Further analysis of factors whose production is altered by androgens may lead to the development of novel regimens for the treatment of androgen-potentiated hair disorders.

Acknowledgements

The assistance of Mr Chris Bowers with the figures and Mrs Christine Dove with the preparation of the manuscript is gratefully acknowledged. The research in this paper was supported by grants to Professor Randall and Dr Messenger from the Medical Research Council (G8610976 SB; G9108798 SB) and to Professor Randall from Kanebo Ltd., Japan and the Trimill Trust. Dr Kato and Ms de Oliveria were visiting research scientists in Professor Randall's laboratory supported by the Japanese Ministry of Health and CAPES, Brazil, respectively.

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