In humans, the cutaneous distribution of type 1 isoenzyme was identified immuno- histochemically to be localized predominantly within sebaceous glands (8). The enzymatic activity is significantly higher, on the one hand in sebaceous glands of the face and scalp compared to nonacne-prone areas, and on the other hand in sebaceous glands in men than women (14). Type 1 isoenzyme was also found in epi- dermis, eccrine sweat glands, apocrine sweat glands (in normal ones as well as in people with osmidrosis), hair follicles (outer root sheath cells, dermal papilla cells, matrix cells), endothelial cells of small vessels, and in Schwann cells of cutaneous myelinated nerves (8).
Within hair follicles, type 2 isoenzyme was localized by prominent immuno- staining in the inner layer of outer root sheaths, inner root sheaths, and the infun- dibulum, as well as in sebaceous ducts. Regional studies showed the type 2 mRNA present in beard dermal papilla, but absent from occipital scalp and axillary dermal papilla. The type 2 isoenzyme in beard dermal papilla has a three-times higher activity than the type 1 5a-reductase present in the occipital scalp and axillary dermal papilla. The specific activity of 5a-reductase in the hair dermal papilla exceeded those in other hair follicle compartments (connective tissue sheaths and outer root sheaths) by a factor of at least 14 in the scalp and at least 80 in the
beard. The beard dermal papilla cells appeared to generate more DHT than those from nonbalding scalp hair follicles. However, the individual freshly isolated intact dermal papilla was shown to possess considerably different levels of ex vivo enzyme activities (14).
Taken together, while the type 1 5a-reductase has been definitely demon- strated in sebaceous glands, the isoenzyme distribution in hair follicle is less well defined, probably due to
1. local and temporal variation of enzyme activity,
2. utilization of different polyclonal/monoclonal antibodies, and
3. inadequate assessment of enough specimens.
More evidence is needed to better define the existence of the type 1 isoenzyme in hair follicles and the precise localization of the type 2 isoenzyme within the hair follicles (in outer root sheaths or in dermal papilla) (14).
In the following sections we refer extensively to three eminent publications demonstrating the localization of 5a-reductase in cells, in sebaceous follicles, and its enzymatic activity in epidermal and infrainfundibular keratinocytes.
Subcellular Localization and Expression of Type 1 5a-Reductase
Chen et al. (8) examined cutaneous expression and subcellular localization of type 1
5a-reductase in vitro by using human cell cultures (sebocytes, keratinocytes, fibro- blasts, dermal microvascular endothelial cells, hair dermal papilla cells, and mela- nocytes) with immunocytochemistry, western and northern blot studies.
With immunocytochemistry it was shown that type 1 5a-reductase is located in the cytoplasm of all examined cultured skin cell types and not in the nucleus as described by some authors. The expression could be broadly divided into three groups, in the order of decreasing intensity (Fig. 5):
1. adult facial sebocytes and neonatal foreskin keratinocytes,
2. adult nongenital keratinocytes (flank), occipital dermal papilla cells, occipital fibroblasts, and
3. beard dermal papilla cells, melanocytes, dermal microvascular endothelial cells, and chin fibroblasts.
Moreover, among sebocytes, the smaller, less differentiated sebocytes were more strongly stained than the larger, differentiated ones (Fig. 6).
Further findings of the study were:
1. The most abundant expression of type 1 5a-reductase is in neonatal foreskin keratinocytes, followed by adult facial sebocytes.
2. The strongest expression of type 1 5a-reductase among skin cells from adult subjects is in facial sebocytes.
3. The expression of type 1 5a-reductase in fibroblasts and keratinocytes is subject to regional variance.
4. Type 1 5a-reductase is present in dermal papilla cells of both beard and occipi- tal hair cells, whereas it is expressed more strongly in the latter.
5. Type 1 5a-reductase is expressed in cultured melanocytes. The significance of this interesting finding remains unknown.
The strongest expression of type 1 5a-reductase in sebocytes among adult skin cells examined confirmed the previous hypothesis of the authors that the sebaceous
FIGURE 5 (See color insert.) Immunocytochemistry using rabbit antiserum raised against human type 1 5a-reductase. (A) adult facial sebocytes, (B) neonatal foreskin keratinocytes, (C) adult nongenital keratinocytes (flank), (D) occipital dermal papilla cells, (E) beard dermal papilla cells, (F) occipital fibroblasts, (G) melanocytes, (H) dermal microvascular endothelial cells, and (I) chin fibroblasts. Scale bars = 50 mm. Source: From Ref. 8. Reproduced from Blackwell Publishing.
gland would be the main source of high concentration of tissue active androgens in the pilosebaceous unit.
The authors suggest in examining further, whether the expression of type 1
5a-reductase in keratinocytes decreases with aging or not, and the role of andro- gens in the physiological function of epidermis, such as maintenance of the integ- rity of skin barrier (8). Lately, testosterone has been shown to perturb epidermal permeability barrier homeostasis (22).
FIGURE 6 Immunocytochemistry of adult facial sebocytes from the fourth passage in culture. The smaller, less differentiated sebocytes are more strongly stained with type 1 5a-reductase antibody as compared with larger, well-differentiated ones. Scale bar ¼ 50 mm. Source: From Ref. 8. Reproduced from Blackwell Publishing.
Immunolocalization of 5a-Reductases in Sebaceous Follicles
Thiboutot et al. (21) determined immunohistochemically (hematoxylin-eosin staining) the distribution of 5a-reductase isoenzymes within sebaceous follicles and terminal hair follicles in acne lesions and normal skin. Higher levels of type 1 isoenzyme are detected in sebaceous glands derived from acne-prone areas of skin, such as the face, compared to the glands from other areas, such as the legs or arms. This seems to corre- late with the finding that the stimulatory effect of DHT on cell proliferation is more pro- minent on facial than on nonfacial sebocytes, as shown by Akamatsu et al. (23).
5a-Reductase Localization in Sebaceous Follicles of Normal Skin
In subjects with clinically normal skin, the majority of follicles contained a mild degree of hyperkeratosis. Monoclonal antibodies to type 1 5a-reductase specifically localized to the sebaceous glands of sebaceous follicles from normal skin (Fig. 7A). Reactivity was most pronounced at the periphery of the gland, that is, at the area of undifferentiated sebocytes in concomitance with the data by Chen et al. (8), but was noted throughout the sebaceous gland in most sections. No localization within hair follicles was noted (21).
Antibodies to type 2 5a-reductase localized to the companion layer (inner- most layer of the outer root sheath) of sebaceous follicles and sebaceous ducts (Fig. 7B). Immunoreactivity was also noted in the granular layer of the epidermis and in the sheath of myelinated cutaneous nerves (not shown). No staining within sebaceous glands was observed with type 2 antibody (21).
5a-Reductase Localization in Acne Lesions
The pattern of immunoreactivity in acne lesions was similar to that observed in normal sebaceous follicles. In sections of closed (Fig. 8A) and open comedones (Fig. 8B), type 1 immunoreactivity was noted only in remnants of sebaceous
FIGURE 7 Immunolocalization of 5a-reductase in sebaceous follicles from the back of a subject without acne. (A) Skin incubated with antibody to type 1 5a-reductase reveals specific localization within sebaceous glands (arrow) (original magnification 17). (B) Skin incubated with an antibody to type 2 5a-reductase reveals localization within the follicle and sebaceous duct (arrow), but not within the sebaceous gland (original magnification 42). Source: From Ref. 21. Reproduced from Archives of Dermatology.
FIGURE 8 (See color insert.) Immunolocalization of type 1 5a-reductase in comedones (hematoxylin and eosin). (A) Sections of closed comedones and (B) of open comedones reveal no localization of type 1 antibody within the comedone (original magnification 17). Of note is type 1 antibody localization in a sebaceous gland adjacent to an open comedone (arrow). Source: From Ref. 21. Reproduced from Archives of Dermatology.
glands and not within follicles. Type 2 antibody localized within the walls of both open and closed comedones, but not in the adjacent sebaceous gland, and extended into keratinized cells contained within the lumen (Fig. 9). In inflammatory lesions, type 1 immunoreactivity was noted specifically in sebaceous glands and not in fol- licles or any other epidermal or dermal structures (Fig. 10A and B). Immunoreactiv- ity was most pronounced within basal sebocytes and least evident in highly differentiated sebocytes in proximity to sebaceous ducts (Fig. 10C). Localization of type 2 antibody was observed within the granular layer of the epidermis, the companion layer of the follicle, and the sebaceous duct, but not in the sebaceous gland (Fig. 11A). Type 2 immunoreactivity was noted in endothelial cells adjacent to an inflammatory acne lesion (Figs. 11B and 12A) but not in endothelial cells in control sections of dermatitic skin (Fig. 12B).
An interesting observation is the localization of the type 2 isoenzyme of
5a-reductase in the walls of both open and closed comedones and in endothelial cells surrounding inflammatory acne lesions. This localization parallels are seen in similar regions of normal follicles. The significance of the localization of the type 2 isoenzyme within the walls of the open and closed comedones or endothelial cells in inflammatory acne lesions remains unknown. It has been suggested that andro- gens may play a role in the follicular hyperkeratinization seen in acne. Although a cause-and-effect relationship between androgens and follicular hyperkeratinization cannot be drawn from immunohistochemical findings, localization of 5a-reductase within the comedonal wall provides a rationale for research in this area (21).
Enzymatic Activity of 5a-Reductase in Epidermal and
Thiboutot et al. (24) determined the activity of 5a-reductase in keratinocytes excised and cultured from the infrainfundibulum and the epidermis of the forehead, using (1,2-3H) testosterone as substrate.
FIGURE 9 (See color insert.) Immunolocalization of type 2 5a-reductase in comedones. (A) Open comedone: type 2 antibody localizes within the comedone wall (large arrow) but not in the adjacent sebaceous gland (small arrow) (original magnification 17). (B) Open comedone: type 2 antibody localizes in the granular layer of the epidermis extending into the wall of the comedone (arrow) (original magnification 41). (C) Closed comedone: type 2 antibody localizes within the comedone wall (arrow) (original magnification 41). (D) Closed comedone: type 2 antibody localization extends into fully keratinized cells within the lumen of the comedone (arrow) (original magnification
83). Source: From Ref. 21. Reproduced from Archives of Dermatology.
Overall, the mean activity of 5a-reductase was not significantly different in the acne subjects compared to the nonacne subjects (Table 3). The mean enzyme activities in the acne groups were, however, slightly higher than in the groups without acne. When grouped by sex, males demonstrated significantly higher
5a-reductase activity in infrainfundibular keratinocytes compared to females. The activity of 5a-reductase was significantly higher in infrainfundibular kerati- nocytes compared to epidermal keratinocytes in all subject groups.
FIGURE 10 (See color insert.) Immunolocalization of type 1 5a-reductase in inflammatory acne lesions. (A) and (B) Type 1 antibody localizes specifically in sebaceous glands (arrows) (original magnification 17). (C) localization of type 1 antibody is most predominant in basal sebocytes and more undifferentiated sebocytes in the lateral portions of the gland (arrow) (original magnification
83). Source: From Ref. 21. Reproduced from Archives of Dermatology.
The activity of type 1 5a-reductase exhibits regional differences in isolated sebaceous glands. Increased activity of 5a-reductase could result in increased local concentrations of DHT, which may influence follicular keratinization. Although the data showed a tendency for a slight increase in enzyme activity in the acne group, these differences were not statistically significant.
FIGURE 11 (See color insert.) Immunolocalization of type 2 5a-reductase in inflammatory acne lesions. (A) Localization of type 2 antibody extends from the granular layer of the epidermis into the sebaceous duct (arrow), but not the sebaceous gland (original magnification 17). (B) Type 2 antibody localizes to endothelial cells within inflammatory lesions (arrow) (original magnification
17). Source: From Ref. 21. Reproduced from Archives of Dermatology.
FIGURE 12 (See color insert.) Type 2 reactivity in endothelial cells. (A) Type 2 antibody reactivity is noted in endothelial cells adjacent to an inflammatory acne lesion (arrow) (original magnification
17). (B) No localization of type 2 antibody is noted in endothelial cells in control sections of dermatitic skin (original magnification 41). Source: From Ref. 21. Reproduced from Archives of Dermatology.
These observations lead to the following interpretations:
1. Androgens may not be as important in follicular keratinization as inflammatory signals and peroxisome proliferator-activated receptors (PPAR) regulation of lipids.
2. The differences observed in enzyme activities between acne and nonacne groups may approach significance if the sample sizes were larger (24).
Although the study designed by Thiboutot et al. (24) could be optimized, generally, one would expect a more pronounced difference in enzymatic activity of
5a-reductase when comparing acne subjects with nonacne subjects. Open questions in this context could pertain to the mechanism of the regulation of enzymatic activity: do the 5a-reductases exist as zymogens to be proteolytically activated, or are there any endogenous inhibitors? Riboflavin, detected at high levels in the fol- licular duct, is suggested to play a certain role as an endogenous 5a-reductase inhibitor (25).
TABLE 3 Activity of 5a-Reductase in Keratinocytes