15 May

The pilosebaceous duct  is lined  by a stratified, squamous epithelium, and  consists of keratinocytes that undergo a similar, but distinct, terminal differentiation process compared to  the  keratinocytes  that  comprise the  interfollicular epidermis.  The upper fifth  of  the  duct   is  identical to  the  interfollicular portion; however, the remaining portion, the infrainfundibulum, is histologically different. In the infrain- fundibulum, the granular layer is frequently absent and the keratinocytes contain a tremendous amount of glycogen. In addition, the stratum corneum is only two  to three  layers  thick  and  will  desquamate into  the  central  part  of the  pilosebaceous duct  where mixing  with  sebum and  bacteria occurs  (1).

Ductal   hypercornification results initially   in  the  formation of  microcome- dones, eventually leading to the production of a comedonal lesion  resulting from the  accumulation of corneocytes in the pilosebaceous duct.  This is believed to be the result  of enhanced keratinocyte proliferation, enhanced desquamation, inadequate squame separation, or  a combination of factors  thereof.  Individuals with   acne  have   more   cornified material  in  their   ducts   when compared with control  subjects  (60).

Organ Culture of the  Infrainfundibulum

Keratinocyte cell culture of the  interfollicular epidermis is well-established, and procedures are  available to  expand cell  populations  in  serum containing and serum-free medium in the presence and  absence  of fibroblast-feeder support (61 –

63). In addition, three-dimensional models are also well-characterized to simulate a living  skin equivalent. Using  these  models, it is possible to monitor the cultures

to  examine the  effects  on  keratinocyte proliferation and  differentiation using   a variety of end-point markers.

In contrast, the culture of the infrainfundibulum is less established and  only one  laboratory has  reported on  the  use  of the  infundibula as  a model  for  acne (64 – 66). Using  a procedure similar to  that  used  for  isolating sebaceous glands, pilosebaceous ducts  can  be  isolated by  micro-dissection of keratomed layers  of skin.  Interestingly,  it  was  found that   only  subjects   with   high   rates   of  sebum secretion contained pilosebaceous ducts  large  enough for  positive identification and  isolation.

Isolated infundibula have  been  successfully cultured in serum-free medium containing essential growth factors  for up  to seven  days  (66). During this  culture period, morphology of the  duct  is retained, although desquamated corneocytes are not eliminated via the follicular canal (Fig. 8). Rather,  the keratinocytes/corneo- cytes accumulate in the lumen, whereas the keratinocytes express markers consist- ent with  those  found in the basal  and  suprabasal layers.

In the presence of 1 ng/mL of IL-1a, the infundibula were found to hypercor- nify resulting in a darkly stained appearance (Fig. 8). The resulting scaling was his- tologically similar to the changes seen in a comedonal lesion in vivo and addition of an  excessive  amount of IL-1a receptor blocker  prevented this  hypercornification process  from  occurring. This  is of note,  as IL-1a  is found in high  concentrations in  open   comedones  (67 – 69).  The  addition  of  5 ng/mL  EGF  or  transforming

FIGURE 8    Cross-sections of infundibula isolated and maintained in vitro. Infundibula from human pilosebaceous follicles can be isolated and cultured in vitro with retention of viability and architecture as a model for acne. (A) Freshly  isolated infundibulum depicting stratified squamous epithelium  with dispersed fibroblasts  in dermal portion. (B) Infundibulum maintained in vitro for seven days depicting visible stratum granulosum and stratum corneum in the central  regions. (C) Infundibulum maintained in vitro for seven days in the presence of 1 ng/mL Il-1a depicting scaling phenotype in central lumen. (D) Infundibulum  maintained in vitro for seven days  in the  presence of 5 ng/mL epidermal growth factor depicting  loss of architecture. Source: From Ref. 66.

growth factor-a resulted in a disorganized appearance of infundibular morphology, although individual cellular  integrity was retained. Looking  at the retinoids, 13-cis- retinoic  acid (1 mM) supplemented into the medium resulted in a significant fall in the rate of DNA synthesis after seven days maintenance which  can be interpreted as an inhibition of new  ductal cell growth.

In a subsequent report, the effects of inflammatory cytokines on the infundi- bulum were  evaluated. The addition of 100 U of interferon-g, 10 ng/mL of tumor necrosis   factor-a  or  10 ng/mL  IL-6  resulted in  the  expression  of  intercellular adhesion molecule-1 which  was  not seen with  IL-1a treatment.

Although the organ  culture of the infundibulum suffers  from  the same  con- straints as sebaceous gland organ  culture, the advantages over keratinocyte mono- layer  culture lend  support as  an  additional model   for  the  understanding and treatment of acne.

As  previously  mentioned,  keratinocyte cell  culture  procedures  are  well- established and it is beyond the scope of this chapter to disclose  the numerous refer- ences  related to epidermal biology.  However, the  use  of keratinocyte monolayer models in  further understanding acne  is  reported in  subsequent areas  of  this review.


Enzyme Assays for In Vitro Androgen Metabolism

Human skin is a collection  of androgen-responsive tissues,  which  have been recog- nized to  contain a  wide   variety of  enzymes capable of  metabolizing  topically applied drugs and  endogenous substrates supplied by the body.

One  of the  most  important classes  of hormones affecting  the  pilosebaceous appendage, and  more specifically  the sebaceous glands, is the sex steroids secreted by the testes  and  ovaries in response to pituitary gonadotrophic hormones (70). In general, androgens stimulate sebaceous gland activity,  whereas estrogens have  a suppressive function (70,71). Androgens are  the  best-known stimulators of  the sebaceous glands, and  are  responsible for  the  development and  enlargement  of the  glands  that   occur   at  puberty  (72).  Castration  results  in  sebaceous  gland atrophy accompanied by an overall  decrease in sebum production (73). The most effective   androgens  possess a  17b-hydroxyl group  that  includes   testosterone,

5a-DHT, 5a-androstene-3b, and  17b-diol.

The  enzymes involved in  androgen metabolism in  follicles  that  have  been identified using biochemical and histological techniques include 3b-hydroxysteroid dehydrogenase (3b-HSD), 17b-HSD, and 5a-reductase. A schematic diagram of the pathways of cutaneous androgen metabolism and the converting enzymes involved has been effectively  conveyed by Chen et al. (74) in Figure  9.

The reduction of testosterone to DHT by the enzyme 5a-reductase is one of the  most  important reactions involved in androgen metabolism. DHT is a more potent androgen than testosterone due to its greater affinity for the androgen recep- tor and  enhanced stability. DHT is generally considered responsible for increased sebum production, as increased local formation of DHT has  been  documented in acne (75) and  by increasing the size of the sebaceous gland  (73). Two subtypes of

5a-reductase have  been identified.

The Type I isozyme has an optimal pH of 6 to 9 and  has been found localized in  skin  from  the  scalp,  chest,  dermal papilla, and  sebaceous gland  regions (76).

FIGURE 9    Pathways of cutaneous androgen metabolism and the converting enzymes. Abbreviations: DHEAS,  dehydroepiandrosterone  sulfate;  17b-HSD, 17b-hydroxysteroid  dehydrogenase. Source: From Ref. 74.

Activity  was found to be greater in acne-bearing facial skin and in sebaceous glands than in other cells of similar location.  Activity  was also higher in glands in the facial region than glands in other bodily regions. In a subsequent study (77), keratinocytes cultured from  the infrainfundibulum demonstrated greater  5a-reductase activity than   those   obtained  from   the   interfollicular  epidermis.  Overall   activity   was highest in  the  sebaceous  gland   followed by  the  duct,   infrainfundibulum, and lastly  the epidermis. Acne-bearing skin was  found to produce 2 to 20 times  more DHT than normal skin, and facial skin produced more DHT than normal back skin.

The  Type  II  isozyme  has  an  acidic   pH   optimum and   is  present in  the epididymis, seminal vesicles,  prostate, and  the  inner  root  sheath of the  terminal hair  follicle (78).

Specific  target   assays   for  5a-reductase are  problematic  in  nature as  the enzyme is  difficult  to  extract  and  the  substrate testosterone is  water insoluble. Nonetheless,  several   assays   are  available  to  monitor androgen  metabolism in cell/tissue homogenates or  in  living  cell/tissue culture systems. The  enzymatic assay  for conversion of testosterone to DHT involves adding testosterone (usually radiolabeled)  together  with   a  cell  or  tissue   homogenate  in  the   presence  of NADPH. Inhibitors  can  be  added and  after  a  set  period of  time,  the  steroids formed are isolated and  separated either  by TLC, HPLC, or GC (79).

Complementary to using  skin homogenates, activity  of enzymes involved in androgen modulation can be assessed using  cell monolayer cultures and living skin equivalents.  Although  keratinocyte methods  for  cultivation  of  each  are  well- established, cell culture may be of limited use for evaluating water-insoluble com- pounds or formulations. For the latter,  the use of a reconstituted three-dimensional living  skin model  has been reported by Bernard et al. (80) and  Slivka (81).

Harris et al. (82) reported on  the  expression of 5a-reductase in  COS cells, whereas  Sugimoto et  al.  (83)  constructed  5a-reductase  expression vectors   that were  transfected into  a human adrenal carcinoma cell line.  Type  I 5a-reductase was  strongly inhibited by the  chloride salts  of cadmium, copper, and  zinc  (inhi- bition  constant or Ki  less than  5 mM), moderately by nickel  and  iron  (Ki  less than

250 mM), and  no inhibition by manganese, magnesium, calcium,  or lithium. Only the  Type  II isozyme was  inhibited by  copper at  11 mM. Additionally, minimal effects  were   seen   when   the  chloride  counter  ion  of  zinc  was   replaced with sulfate,  chloride, or acetate,  with  acetate  being  the least effective.

In general, 5a-reductase inhibitors are classified  into two categories: steroidal and nonsteroidal, and there is very little overlap in efficacy between the two different isozymes. In fact, many  of the Type II inhibitors have little to no effect on sebum pro- duction in vivo. This is possibly the result  because the two enzymes have  only 50% homology in amino acid sequences. The gene for Type I isozyme is located on chromo- some 5, whereas the gene for Type II isozyme is located  on chromosome 2 (84).

Using  foreskin  keratinocytes (85), testosterone was  utilized as a substrate in the production of 5a-reduced metabolites including DHT (5a-reductase), androste- nedione (17b-HSD Type II), androsterone, and androstanediol (3a-HSD activity).  A similar pattern was reported with fibroblasts, although scalp skin fibroblasts metab- olized  testosterone only to a limited extent.  Using  cultured keratinocytes obtained from  the infrainfundibulum and  epidermis (86), Thiboutot et al. found that  mean enzyme activities were slightly  higher in the acne group, but not statistically differ- ent from the control  group. Overall  activity  was greatest in the infrainfundibulum.

Altenburger and  Kissel (87) cultured the human keratinocyte cell line HaCaT as a model  to express the  enzyme systems involved in testosterone metabolism, which  were  subsequently identified by LC/MS. HaCaT cells were  found to predo- minantly express the Subtype I isoform  of 5a-reductase. Similar studies have  been reported utilizing keratinocyte cultures derived from  breast  skin  (88), in cultured fibroblasts (89), and  living  skin equivalents (90).

Assays for the  Rate-Limiting Enzymes Involved in Cholesterol and Fatty Acid  Synthesis

In   the   formation   of   squalene  and    cholesterol,   3-hydroxy-3-methylglutaryl- coenzyme A (HMG-CoA) is the  rate-limiting enzyme involved in the  cholesterol synthesis pathway. As reported by Shapiro et al. (91) and  Smythe  et al. (92), radio- labeled HMG-CoA is added to skin homogenates containing NADP (as a necessary cofactor) along with glucose-6-phosphate and glucose-6-phosphate dehydrogenase to form radiolabeled mevalonate. The reaction then involves a spontaneous conver- sion of mevalonate to mevalonolactone, which  can then  be separated by TLC and quantified.

Using  this  assay  with  isolated sebaceous gland homogenates, Smythe  et al. (92) reported that  HMG-CoA activity  decreased with  subject  age,  and  enzyme activity  was inactivated via phosphorylation.

In the  study of fatty  acid  synthesis, acetyl-coenzyme A carboxylase (acetyl- CoA) has been identified as a rate-limiting step. This assay  involves adding radio- labeled bicarbonate to skin  homogenates in the presence of ATP and  acetyl-CoA. Total radioactivity incorporated is then  quantified via direct  scintillation counting.

All of the enzyme assays  described are amenable for high throughput screen- ing to assess  compounds that  may  modulate lipid  synthesis.

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