Cutaneous Distribution

15 May

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

Infrainfundibular  Keratinocytes

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).

Discussion

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

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