Enzymology of the hair follicle

ARTICLE

Androgenetic alopecia (AGA) is the most common type of hair loss in men and women. This continuous process results in a type of alopecia that follows a definite pattern in those individuals who are genetically predisposed. Although clinically different, the pathogenetic pathways leading to this type of hair loss are thought to be similar in both sexes [1]. A genetic predisposition is a feature of AGA, but the predisposing genes are still unknown. Our understanding, however, of the hormonal effects on hair growth is far more advanced, and with this article the present knowledge of steroidogenic isoenzymes within the human hair follicle and their role in the pathogenesis of AGA will be reviewed.

Androgen metabolism and hair growth

Androgens are required in males for the development of normal genitalia in utero, and with the beginning of puberty they become necessary for the libido and secondary sexual characteristics as well as for maturation and growth of the male muscle mass. Moreover, they are involved in the pathogenesis of hair disorders such as hirsutism and AGA. More than 50 years ago Hamilton observed that men who were castrated did not develop AGA [2]. Therefore it was concluded that the growth of hair follicles is in some parts of the body androgen-dependent (Table I). At present it is unknown how androgens exert their paradoxical effect on the growth of HF in different body sites, and which genes are involved. However, Hamilton already showed that AGA can be triggered in castrated men by injecting testosterone.

The synthesis of androgens is complex because it occurs in several organs, each of which has its own pecularities. The androgen metabolism (AM) of adrenals and gonads and the influence of the pituitary gland are beyond the scope of this review and are described in detail elsewere [3].

The pathway of androgens begins with cholesterol which is converted to pregnenolone. Following alpha-hydroxylation at the C17-position, the action of the enzyme C17-20 lyase cleaves distal carbon moities, leaving a C 19 carbon steroid with a C-17 ketone in the distal ring. These "17-ketosteroids" make up a group of relatively weak androgens, such as dehydroepiandrosterone (DHEA), defined by their relatively low affinity to the androgen receptor (AR). Approximately 75% of DHEA and 95% of DHEA-S is derived from the adrenal gland. These weak androgens, however, can be enzymatically converted to more potent androgens such as T which is the major circulating androgen. In the HF the principal pathways involved in conversion of weak to more potent androgens are through activity of the enzymes 3beta-hydroxysteroid dehydrogenase/delta^5=>4-isomerase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD). In most target organs T can be further metabolized to DHT via the action of 5alpha-reductases 5alpha-R (Fig. 1). The affinity of DHT to the AR is approx. five-fold higher than that of T. Potent androgens such as T or DHT can be removed by conversion to weaker androgens, or they can be metabolized via the aromatase to estrogens, or they can be glucuronidated to form androgen conjugates that are more rapidly cleared from the circulation. Remarkably, some target tissues show enhanced AM and androgen sensitivity [3]. Circulating DHEA-S may be more rapidly metabolized to DHEA via steroid sulfatase (STS), DHEA would be, in turn, more rapidly converted to androstenedione if increased 3beta-HSD activity is present. Androstenedione would be converted to T if 17beta-HSD activity is present. If target cells convert weak androgens at an accelerated pace, then there will be enhanced conversion of T to DHT. Another reason for the increased sensitivity of a target to androgens is believed to be an increase in the number of AR.

Only a small fraction of androgens exist as free steroids in the circulation, with an equilibrium between free androgen hormone and protein-bound androgens. The most important protein for androgen binding is the sex-hormone binding globulin (SHBG). Normally approx. 70% of T is bound to SHBG, 19% to albumin and only the remainder is unbound. Whether the bound fractions are still metabolically active is a matter of controversy, but the binding of androgens to SHBG is an important factor of AM because it acts somehow as a "sink" for circulating T.

To sum up, the AM is highly complex and can be tuned at various points, e.g. the amount of weak androgens present for conversion to more potent androgens, the repertoire of metabolizing enzymes present in target cells, the ratio of conversion and reconversion, the concentration of SHBG in the serum, the affinity of androgens to the AR.

The principal elements of the androgen metabolism can be summarized as follows:

Androgen-dependent processes are not the result of the summation of the activity of individual metabolites, but are solely due to the binding of DHT and translocation of the receptor to the nucleus. This concept has been discussed for the development of benign prostate hyperplasia [4], and is most likely valid for AGA as well. Therefore DHT-dependent cell functions will only be initiated or will be amplified if:

i: enough weak androgens are present for conversion;

ii: more potent androgens are formed via the action of 5alpha-R;

iii: the enzymatic activity of androgen inactivating enzymes is rather low: e.g. aromatase;

iv: reconversion of weaker steroids to DHT takes place;

v: functionally active AR are present in high numbers.

Steroidogenic enzymes within the hair follicle

Steroid sulfatase (STS)

The skin is capable of synthesizing active androgens, such as DHT, from the systemic precursor DHEA-S. The first step in this route is the desulfatation of DHEA-S by the enzyme steroid sulfatase (STS). Because DHEA is further metabolized to androstendione, T and in the hair follicle (HF) to DHT [5], STS is an important enzyme for the conversion of the weak adrenal androgens to more potent androgens in the periphery. DHEA-S is believed to maintain axillary hair and is thought to be involved in the pathogenesis of hirsutism [6] in women. In women, however, excess of DHEA-S or the STS metabolite is believed to be involved in several androgen-dependent processes such as acne and AGA [40]. Remarkably, DHEA-S and DHEA plasma levels seem to correlate with balding in young men [42] indicating that STS may play a role in the pathogenesis of AGA, and recently we were able to show that the dermal papilla (DP) is the predominant site of STS expression, and that STS can be inhibited by estrone sulfamate [5]. Therefore STS within the human hair follicle appears to be an interesting pharmaceutical target to treat AGA or hirsutism.

3beta-hydroxysteroid dehydrogenase/delta^{5 => 4}-isomerase (3beta-HSD)

The 3beta-HSD isoenzymes catalyze an obligatory step in the biosynthesis of androgens, estrogens, mineralocorticoids and glucocorticoids. The two 3beta-HSD isoforms are expressed in a tissue-specific manner involving separate mechanisms of regulation. The structures of several cDNAs encoding 3beta-HSD isoenzymes have been characterized in human and several other vertebrate species.

The importance of the 3beta-HSD in male steroid hormone physiology is underscored by a genetically determined deficiency that is transmitted as an autosomal recessive trait and is characterized by varying degrees of salt wasting. All mutations were detected in the type II 3beta-HSD gene, which is expressed almost exclusively in the adrenals and gonads. No mutation was detected in the type I 3beta-HSD gene, which is believed to be expressed in peripheral tissues. Whether hair growth is affected in these individuals has not been investigated so far, but because of the importance of 3beta-HSD in the AM and increased activity in AGA [11], this question warrants further investigation. Today we know that 3beta-HSD activity is present in hair follicles and sebaceous glands because homogenates of the isolated sebaceous glands (ex vivo) exhibited 17beta-HSD and 5alphaR activities as well as high 3beta-HSD/D^{5 => 4}-i activity, which was highest in the sebaceous glands isolated from HF affected by AGA [11]. Immunohistochemical studies confirmed these results showing that 3beta-HSD/D^{5 =>4}-i is located in the sebaceous gland and only the type 1 3beta-HSD/D^{5 =>4}-i is believed to be present in the skin [12]. However, plucked HF (without sebaceous gland) ex vivo also exhibit marked 3beta-HSD/D^{5 =>4}-i activity [22] and detectable mRNA in outer root sheath keratinocytes [15]. We were able to show that in contrast to the ORS and CTS, the DP exclusively metabolizes androstendiol to T, thus indicating 3beta-HSD activity. Which kind of HF cells express a specific type of 3beta-HSD/D^{5 =>4}-i isoenzyme is unknown.

17beta-hydroxysteroid dehydrogenases (17beta-HSD)

Isoenzymes of 17beta-HSD regulate levels of bioactive androgens and estrogens in a variety of tissues. At present five isoenzymes of 17beta-HSD that differ in tissue expression and requirements of cofactors such as NADPH for type III 17beta-HSD, and NAD(+) for type 2 17beta-HSD are known (Table III). The importance of the type 3 enzyme in male steroid hormone physiology is underscored by the genetic disease 17beta-HSD deficiency. Mutations in the type 3 17beta-HSD gene impair the formation of testosterone in the fetal testis and give rise to genetic males with normal male Wolffian duct structures but female external genitalia very similar to the abnormalities seen in 5alpha-R deficiency. These individuals are usually brought up as females, but at puberty there is a striking rise in testosterone levels and they change their social sex. To date, more than 18 recessive mutations have been identified, giving rise to different clinical phenotypes. The potential significance of 17beta-HSD isoenzymes in AGA is underscored by the observations of Hodgins et al. [16] who plucked hair follicles from young adults not yet expressing AGA but with a strong family history of baldness, and found two populations, one with high 17beta-HSD activity and one with low enzyme activity. This study suggests that low enzyme activity may be related to lesser degrees of balding. Therefore, linkage of the genes coding for the 17beta-HSD isoenzymes and AGA warrants further investigations.

Very early on it was shown that plucked human HF or HF from the stump-tailed macaques express considerable 17beta-HSD activity due to the principle metabolite of T being androstenedione (Fig. 1). The isoenzyme-specific expression pattern in different parts of the HF has so far not been investigated in detail mainly because of technical problems. Only one study describes the type 1 and 2 17beta-HSD in the epithelial parts of the HF [15]. These results are in line with our studies where we were able to show that in contrast to the CTS and RS the DP exhibits only little 17betaHSD activity [17].

5alpha-reductases (5alpha-R)

The microsomal enzyme steroid 5alpha-R is responsible for the conversion of testosterone into the more potent androgen DHT and the conversion of androstenedione to 5alpha-androstanedione (Fig. 1). 5a-R deficiency is a rare autosomal recessive trait that was first described by Nowakowski and Lenz [18], although, without etiological characterization which was not possible at that time. In 1974 it became clear that these individuals lack functional 5alpha-R [19] and today we know that the type 2 5alpha-R is lacking [55]. In typical cases, a 46, XY male who has testes, normal plasma T and low DHT levels is observed. Interestingly, no or minimal beard growth or AGA is seen in these men. These observations together with the finding that both humans and stump-tailed macaques have beard and frontal scalp HF with higher 5alpha-R activity than HF from the occiput [23, 24] indicates that the type 2 5alpha-R is crucially involved in the pathogenesis of androgen-dependent hair growth and that the inhibition of this isoenzyme is therefore a rational approach for treatment.

Today two distinct isoforms designated type 1 and type 2 have been cloned. Subsequently it has been shown that both isoenzymes have distinct molecular, biochemical and tissue expression characteristics (Table IV). In humans, mutations in the gene coding for the type 1 5alpha-R have not been reported. In mice, however, a mutation in this gene will cause early fetal death because of estrogen excess in utero.

Early studies have revealed that plucked human hair follicles (HF) are able to convert testosterone (T) to the more potent androgen dihydrotestosterone (DHT) via the action of the enzyme 5alpha-R [27]. Recently, the presence of both type 1 and type 2 5alpha-R within human HF has been demonstrated [25], but the intrafollicular localization of both isoenzymes remains controversial. Special attention has been paid to the DP and several authors have tried to localize both isoenzymes within the DP. So far, for the examination of the androgen metabolism in the hair follicle, cultivated primary cell lines of papilla cells [26-29], keratinocytes, and dermal reticular fibroblasts [30] have been used. Some authors were unable to find considerable 5alpha-R activity in occipital scalp DP [47, 48, 50], whereas others found this enzyme in beard and occipital scalp DP [32, 33]. Recently we were able to show that the main metabolic activity of type 2 5alpha-R can be detected in intact occipital scalp and beard DP [34]. These results are in contrast to the above mentioned results measuring the testosterone consumption in DP cell cultures, but are in line with the observation of type 2 5alpha-R activity below the HF isthmus [35] and with the detection of type 2 5alpha-R mRNA in DP cells [15].

Aromatase

The cytochrome P450 aromatase (P450arom) enzyme is required for bioconversion of androgens to estrogens. Only a single human gene encoding P450arom (CYP19) has been isolated. Mutations in the CYP19 gene do occur occasionally and result in aromatase deficiency. Girls show pseudohermaphrodism at birth which is sometimes corrected by surgical repair of the external genitalia, including a clitoridectomy. Males are rather tall with eunuchoid skeletal proportions. Their bone age is retarded and osteopenia can be observed, indicating that estrogens are crucially important for bone development. At puberty females will develop hirsutism due to an androgen excess and in theory females and males might develop early onset AGA. However, this question has not been investigated so far. Localizing the aromatase in the external root sheath of anagen HF suggests that aromatase may have a function in the intrafollicular AM by converting potent androgens to less potent estrogens in order to avoid potentially harmful androgen-mediated effects on androgen-dependent HF. This concept is supported by the fact that women taking aromatase inhibitors for the treatment of breast cancer will often experience AGA-like hair loss. Moreover, in both men and women aromatase activity has been shown to be diminished in HF affected by AGA [24].

CONCLUSION

Summary and future perspectives

AGA can be defined as a DHT-dependent process with continuous miniaturization of sensitive HF. So far the predisposing genes for AGA are unknown and we do not understand the molecular steps involved in androgen-dependent beard growth versus androgen-dependent hair loss. However, the local AM plays a central role in the intrafollicular conversion of weak androgens such as DHEAS to more potent androgens such as T or DHT. Within the HF the DP plays a central role by exhibiting an array of important steroidogenic isoenzymes. Provided that the DP cell triggers and regulates the growth of HF, this physiological role may be reflected by metabolic differences which could account for differences in androgen sensitivity as observed in HF from different body sites, and in conditions such as male pattern baldness. The observation of STS, 3betaHSD and type 2 5alpha-R-activity within the DP could be a clue in the understanding of the regulation of androgen action in the human hair follicle by local androgen modification at the target cell level. Therefore, in the future some of the intrafollicular steroidogenic enzymes are potential pharmaceutical targets for the treatment of AGA or hirsutism.

Abbreviations

AGA: androgenetic alopecia
AM: androgen metabolism
AR: androgen receptor
DHEA-S: dihydroepiandrosterone sulfate
DHEA: dihydroepiandrosterone
DHT: dihydrotestosterone
T: testosterone
DP: dermal papilla
17betaE: 17beta-estradiol
E2: estradiol
HF: hair follicle
3beta-HSD/delta^{5 => 4}-i: 3beta-hydroxysteroid dehydrogenase/delta^{5 =>4}-isomerase
17beta-HSD: 17beta-hydroxysteroid dehydrogenase
3alpha-HSD: 3alpha-hydroxysteroid dehydrogenase
5alpha-R: 5alpha-reductase
STS: steroid sulfatase
P450arom: aromatase

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