SEBUM

12 May

Sebum Production
Sebum  is an oily liquid containing triglycerides, free fatty acids,  wax esters,  squa- lene, and  a little cholesterol produced by the gland.  It is modified by bacteria that hydrolyze triglycerides to produce free fatty  acids,  thus  a sample of skin  surface lipids   has  a  different composition compared to  sebum produced by  the  gland (Table 2).

The  delay  between sebum synthesis, as  measured by  the  incorporation  of injected  14-C acetate  into forehead skin of four healthy male  subjects,  and  the sub- sequent excretion of radiolabeled sebum was determined to be eight days (56). This was similar to the five days  reported for the delay  between the onset of fasting  and initial  change  in composition of skin surface  lipids  reported by Pochi et al. in 1970 (57). However, the  overall  process  from  sebocyte cell division to  cell rupture  is longer,  about  14 days  (58). In the  beard and  scalp  follicles,  it is believed that  the hairs  act as a wick  to facilitate  the passage of sebum to the surface  of the skin. In follicles,  which  possess a vellus  hair,  the  sebum may  pool  and  form  a follicular reservoir (59). Thus,  the rate of sebum excretion onto the skin surface  is a function of the rate  of sebocyte proliferation, lipid  synthesis, cell lysis, and  the rate  of flow through the follicular  reservoir.

Sebum  secretion is increased around puberty under the influence of andro- gens,  concomitant with  sebaceous gland enlargement. In  human males,  sebum secretion continues until  the age of 80, but  in females  it drops significantly in the decade  after   menopause  (60).  In  elderly  individuals,  sebaceous glands  may undergo hyperplasia, but  this  does  not  seem  to  result  in  an  increase in  sebum output (61).

Function of Sebum

In animals, sebum may  provide odor:  it contains pheromones and  may  also con- dition the  hair.  However, in humans, the  function of sebum is unclear (5) and  it has  been  speculated that  the sebaceous glands are vestigial (20). There  is now  an increasing  body   of   evidence  indicating  that   the   sebaceous  gland  and   the components  of  sebum  play   a   crucial   role   in   the   homeostasis  of  skin   (6). Sebum  transports vitamin E, a lipophilic antioxidant, to the skin  surface  where it may  play  a key role in protecting skin  surface  lipids  from  peroxidation (62). Gly- cerol, a major component of sebaceous triglycerides, may play a role in maintaining epidermal barrier function, since asebic mice lacking sebum and, therefore, glycerol have a poorly functioning epidermal barrier (63). Other proposed functions include antibacterial, lubrication, and/or to  provide precursor substrates for  epidermal metabolism and  synthesis of lipids  and/or vitamin D (5). In equine follicles,  the sebaceous gland and  sebum are  required to  regulate growth of the  hair  sheath (64) and  an  intact  sebaceous gland in  sheep   and  human terminal follicles  was shown to be required for the  inner  root  sheath breakdown (65,66). Whether this holds  true for human sebaceous and vellus follicles, remains to be determined. Alo- pecia  is present in asebic  mice  with  hypoplastic sebaceous glands, that  may  also indicate a role of the sebaceous gland in hair  follicle growth (67).

It seems  unlikely that lack of sebum is a causative factor in the production of dry  skin.  The  distribution of sebaceous glands and  amount of sebum produced does   not  correlate  with   dry   skin.  Dry  skin  in  the  elderly  is,  apparently,  not related to sebum output (68), nor does a low sebum output in prepubertal children lead  to dry  skin  (69). In addition, subjective self-assessment of skin  type  as dry, normal, or oily does  not always correlate with  the amount of sebum as measured using  a sebumeter (70).

Lipogenesis

Human sebum is quite distinct in composition and biological  complexity compared to that  of other  animals, and  also  compared to epidermal lipids  synthesized by keratinocytes. It is unique in containing high  levels  of squalene and  characteristic free fatty  acids,  for which  the rate-limiting enzymes of the biosynthetic pathways are  3-hydroxy-3-methylglutaryl (HMG)  CoA  reductase and  acetyl  CoA  carboxy- lase,  respectively. Both  enzymes are  inactivated by  phosphorylation through a cAMP-activated protein kinase.  Comparison of the  kinetic  parameters for  these enzymes with  those  previously described in the  literature found in other  organs, indicates that  the  sebaceous gland  enzymes have  similar affinities  for substrates and  similar responses to  allosteric effectors  such  as  citrate.  However, there  are key points of differences and  these  will be discussed in turn.

Squalene Biosynthesis

Human sebum contains a high percentage of squalene, in contrast to the epidermal lipids,  which  contain a higher proportion of cholesterol. In the sebaceous glands, therefore, the  cholesterol biosynthetic pathway appears to  be  partly arrested in the steps  after the production of squalene. This may be due  to low activity  of squa- lene  epoxidase, or other  enzyme of cholesterol biosynthesis, or the  availability of substrates. For example, a ready supply of acetate  appears to direct  lipogenesis toward squalene biosynthesis, while  glucose,  glutamine, and  isoleucine preferen- tially  result  in  more  triacylglyceride production  in  vitro  (71). Limiting levels  of

NADPH had  previously been suggested, but this is not believed to be responsible for the low levels of cholesterol produced. Since squalene is unique to sebum, it is often used  as a marker to differentiate sebaceous lipids  from  epidermal lipids.

The  activity  of HMG  CoA  reductase in  sebocytes regulates the  amount of squalene produced. This can be reduced by 200 mg/mL of low-density lipoproteins (LDL) in isolated glands (72), similar to LDL downregulation of cholesterol pro- duction in  fibroblasts, which   may  work  via  HMG  CoA  reductase suppression. Cholesterol itself is a weak  repressor of HMG CoA reductase, with  the oxygenated cholesterol derivatives mediating suppression of the  enzyme. Thus,  low  concen- trations of 25-hydroxycholesterol (2 mg/mL) did not have an effect on squalene syn- thesis  in sebocytes. However, synthesis was  reduced by 65% by a 10-fold  higher concentration of 25-hydroxycholesterol (73). Mevalonate, the  end  product of the reaction catalyzed by HMG  CoA reductase, can downregulate the  reaction and  a structurally  related compound,  mevinolin (lovastatin), inhibits the  synthesis  of squalene and  cholesterol in isolated sebaceous glands (74).

Other  compounds that  inhibit  cholesterol synthesis such  as oral  aluminum nicotinate and  clofibrate  had  no effect on sebum production (75). Eicosa-5:8:11:14- tetraynoic  acid,   given   orally,   suppressed  squalene  synthesis  by  13%  to  64% between subjects  (76), but its site of action  has never  been determined.

Free Fatty Acids

Sebum  contains numerous fatty  acids  and  many  are  quite  unique in  structure, including a wide  variety of straight, branched, saturated, and  unsaturated  fatty acids  (77). In human sebum, about  27% of fatty  acids  chains  are  saturated, with the  greatest proportion being  unsaturated (68%). The  vast  majority of these  are monounsaturated (64%)  and   about   4%  are  diunsaturated,  with   the  remainder being composed of fatty acid chains  longer  than  22 carbons (78). Monounsaturated fatty acids of sebum have a double bond  usually inserted at position D9. However, in human sebum there  is an unusual placing of the double bond  at D6 to produce sapienic acid.  Sapienic  acid  is a very  abundant and  important monounsaturated fatty  acid  with  16-carbons and  a cis double bond  located at the sixth  carbon  from the carboxyl  terminal. This fatty acid has been implicated in acne and  is produced by an enzyme unique to sebaceous glands, the D6 desaturase, which  has  recently been isolated from human skin, and its expression is demonstrated in human sebac- eous  glands (79). This  is the  first  time  that  a sebaceous gland-specific functional marker has been demonstrated.

In mice,  a similar enzyme has  been  located  in sebaceous glands producing unsaturation between carbons 9 and  10, in other  words, a D9 desaturase. Loss of expression of this  enzyme in  mice  results in  profound effects  with  hypoplastic sebaceous glands and  an asebic  phenotype (80). Although the reason for the pre- sence of these  unique fatty acids  is not understood, it is speculated that  they  may impart fluidity on  the  sebum allowing it  to  reach  the  skin  surface.  An  area  of active  interest is, whether subtle  changes in these  lipids  occur  causing blockage in the  duct  leading to skin  diseases such  as acne  (81) or not.  The ratio  of D6:D9 fatty  acids  depends on the rate  of sebum secretion—in prepubertal children, and in sebum from the vernix caeosa, as well as elderly individuals the D9 fatty acid pre- dominates. The  D6 form  is  found most  commonly in  sebum from  adults and increases concomittantly with  sebum excretion rate.  This is thought to be due  to a higher level  of the  D6 desaturase enzyme located  in  differentiating sebocytes,

compared to the enzyme responsible for producing the D9 unsaturated fatty acids, which  predominates in undifferentiated  sebocytes (82). Thus,  as sebum secretion increases, the D6 fatty  acid increases and  dilutes the D9 fraction.  Since high  levels of sebum production are  implicated in  acne,  the  increased content of sapienate may  be relevant to the pathogenesis of this disease.

Diunsaturated fatty acids in sebum have double bonds at positions D5 and D8 or  at  positions D7 and  D10. The  major  form  of  dienoic   acid  was  identified as

18:2 D5:8 and  named sebaleic  acid.  This  is presumably synthesized by  action  of the D6 desaturase on palmitic acid (16:0) to produce 16:1 D6, which  then undergoes further desaturation at the  5,6 position following chain  elongation to 18 carbons (83). Sebaleic  acid  is thought to  be  a major  component of sebaceous membrane phospholipids and  this may explain the increased levels associated with  increased rates  of sebum excretion and  acne.  Another important dienoic  acid  of sebum is linoleic  acid  (18:2 D9,12), and  the  levels  are  inversely related to sebum excretion, with  lower  levels  being  found in  subjects  with  high  sebum excretion rates  (84). As the cells enlarge during differentiation, they may need  additional diunsaturated fatty  acids  for membrane synthesis and  presumably synthesize sebaleic  acid  as a subsititute for  linoleic  acid,  thus   explaining the  change in  ratio  of  these  fatty acids    accompanying   high    sebum   excretion   (81).   There    is   evidence  that linoleic  acid  from  sebum is incorporated into epidermal acylceramides, but  when levels  of sebum are  low  it  is  replaced by  sapienic acid,  and  this  could  impair barrier function.

There is evidence to suggest that the types  of fatty acids  synthesized by indi- viduals is controlled by genotype (85) and  is unchanged by fluctuations in diet  or metabolism. Site to site  variations in  free  fatty  acids  in  skin  surface  lipids  have also  been  reported (86). It is important to note  that  the  types  of fatty  acids  syn- thesized seem  to  vary  significantly during  the  life  of  an  individual: there   are several   reported  examples of  this.   In  addition  to  those   mentioned  earlier,   a similar effect is seen  with  some  branched chain  fatty  acids  (82). Although human fatty  acids  are  predominantly straight chain,  branched chain  components were detected with  gas  chromatography, and  saturated fatty  acids  of human sebum were  found to  contain methyl branches on  one  or  more  of the  even  numbered carbon  atoms  throughout the  chain  (87). The  terminally isobranched 15 and  17 carbon  species,  often  comprising wax  esters,  occur  in  higher proportions in  the sebum of young children, when sebum secretion is low,  and  in lower  quantities

in adult sebum (88).

Another enzyme involved in fatty  acid  synthesis is acetyl  CoA carboxylase and this controls the rate-limiting step of this biosynthetic pathway. It exists in differ- ent isoforms, but it is not known whether any of these forms predominates in sebo- cytes. These cells incorporate fatty acids into triglycerides and wax esters. Palmitate, palmitoleate, stearate, and  oleate  seem  to be the main  fatty  acids  used  for further esterification in the gland  (89). Of all the fatty acids  investigated, linoleic  acid was unique, since  it was  preferentially broken down to 2-carbon  units  to fuel  further fatty acid synthesis, such as palmitic and  oleic acids, and  squalene synthesis.

The  processes of lipid  biosynthesis and  terminal differentiation in  human sebocytes  are  quite   well  understood, but  the  pathways that   control   holocrine secretion are  still  unknown. Immortalized humans (SZ95) sebocytes were  found to  exhibit   DNA  fragmentation after  a  six-hours culture  followed by  increased lactate  dehydrogenase release  after  24 hours, indicating cell damage, indicating at least in culture this cell line that  lipid  release  is accompanied by apoptosis (90).

Regulation of Pilosebaceous Unit Activity

There are many  controlling influences on the pilosebaceous unit (Fig. 3). The devel- opment of model  systems for the sebaceous gland and  duct  has  made it easier  to study these  pathways in vitro.

Androgens

Perhaps the most  profound and  well-known effect of hormones on the pilosebac- eous  unit  is the one caused by androgens, more  specifically  in causing sebaceous gland enlargement, sebocyte  proliferation, and  lipid  metabolism (91,92). It is well established that  the increase in lipid  production and  enlargement of the sebaceous glands at puberty is attributable to an increase in circulating androgens from  the testes  in  males,  the  ovaries   in  females,   and   the  adrenal glands of  both  sexes. Androgen-insensitive subjects  do not  produce sebum (93). Furthermore, the local application of testosterone to skin resulted in a 15-fold increase in sebum excretion rate,  although admittedly this  was  a small  study in which  2% testosterone cream was  applied to  the  foreheads of prepubertal boys.  In  fact,  three  subjects  failed to  respond,  but   this   study does   demonstrate  that   skin   can  respond to  local androgens (94).

Androgen sensitivity of pilosebaceous units  has been confirmed by the posi- tive detection of androgen receptors in pilosebaceous units  by immunohistochem- istry   (Fig.  4)  (95 – 97)  and   by  their   isolation  from   sebaceous  gland   cells  (98). However, it has  not  been  possible to  demonstrate  differences in  receptor levels that   explain  androgen  dependent  dermatoses,  such   as  acne   or  the   different rates  of sebum production in different individuals. There  is a strong  correlation between sebum excretion rate and  acne grade (99,100), but it has not been possible

FIGURE 3    A  schematic  representation  of  potential   pathways  in  sebocytes  that  can   regulate lipogenesis. Abbreviations: CAMP, cyclic adenosine monophosphate; CRH, corticotrophic  releasing hormone; EGF,  epidermal growth factor;  FGF,  fibroblast growth factor;  GH, growth hormone; IGF, insulin-like growth factors;  KGF, keratinocyte growth factor; MSH, melanocyte stimulating  hormone; PPARs, peroxisome proliferator-activated receptors; TGF, transforming growth factor.

FIGURE 4    Immunohistochemical  labeling   of  a   cross-section  through   the   infundibulum   of  a sebaceous follicle stained with a  monoclonal antibody  for androgen receptors. Positively  stained nuclei are  dark gray.

to relate  the rate of sebum production to differences in androgen receptor levels in different acne patients.

Androgen receptor antagonists, such as oral cyproterone acetate  and spirono- lactone  compete with  androgens for the androgen receptor-binding site (101), and while   both  compounds effectively   decrease sebum production, they  also  have feminizing  side-effects. Since  their   effect  is  not  specific   for  skin,  their   use  is restricted to women. Antiandrogens also decrease follicular impactions (see later) (102), but this may be secondary to a reduction in sebum flow or a change in sebac- eous  lipid  composition.

An important feature of the effect of androgens on pilosebaceous unit metab- olism is the metabolic conversion of the androgens themselves, and much  work has been done  in the last decade to define  the pathways of androgen metabolism. It is now  clear  that  pilosebaceous units  possess all the  steroid metabolizing enzymes needed to convert dehydroepiandrosterone to the most potent androgen, dihydro- testosterone (DHT),  including 3-b-hydroxsteroid dehydrogenase (103 – 105), and

5-a-reductase (5-a-R)  (106). These  latter  two  enzymes exist  in  several  isoforms: type  2 isozyme of 17 b-hydroxy steroid dehydrogenase ( b-HSD)  is predominant in sebaceous glands and  type  1 5a-R is highest in sebaceous glands (107,108), iso- lated   and   cultured  infundibular  keratinocytes, and   in  epidermis  (109).  This would explain why  5a-R II inhibitors such as finasteride, do not produce a signifi- cant reduction in sebum output (93).

Furthermore, sebocytes have  the  biosynthetic capacity to produce their  own androgens  from   cholesterol through  the   cytochrome  P450  side-chain  cleavage system (P450scc) (110). Along with its cofactors, adrenodoxin, adrenodoxin reductase, and  the  transcription factor,  steroidogenic factor  1, P450scc converts cholesterol to pregnenolone, which  is also the  precursor for estrogen synthesis. Positive  staining using   antibodies to  these  proteins was  demonstrated in  hair  follicles  in  human facial skin, and biochemical activity was demonstrated in vitro confirming that sebac- eous glands are locally steroidogenic with  the capacity to produce the highly  potent DHT from cholesterol. The conclusion from this body of work is that the skin has its own capacity to metabolize androgens suggesting that the skin exercises local control over the ultimate effects of circulating androgens on the target  tissue.

Genes  for lipid  biosynthesis are regulated in sebaceous glands by androgen- dependent transcription (111). Topical  application of 0.005% DHT  to hamster ear activated  the  steroid  regulating  element  (SRE)  binding  proteins in  sebaceous glands, with   an  upregulation  of  the  mRNA   expression for  enzymes involved in   lipid   production,  including  HMG   CoA   reductase  and   synthase,  glycerol

3-phosphate  acyltransferase, and  stearoyl CoA  desaturase. SREs are  present in the promoter regions of the genes  for these  enzymes (112).

Estrogens

In contrast to the stimulatory effect of androgens on the sebaceous glands, estrogens and compounds that have estrogenic activity,  such as phenol red (113), reduce lipo- genesis  in vitro. In vivo, the effect of estrogens is contradictory, although at pharma- cological  doses  estrogens are sebosuppressive in rat  preputial gland (114) and  in humans also  producing feminizing side  effects  (115). This  action  of estrogens is indirect  and   occurs   by  inhibiting  the  adrenals  and   gonads  via  the  pituitary, thereby  reducing  the  production  of  androgens.  During  hormonal  treatments, such  as  hormone replacement therapy (HRT),  the  effect  on  skin  surface   lipids depends on  the  predominant hormone given.  Skin  surface  lipids  are  increased during combined HRT, possibly reflecting stimulatory effects  of the  progestogen component  on  sebaceous  gland  activity,   while   estrogen  alone   has   a  sebum- suppressive action  (116).

Local effects of estrogens have  been demonstrated (115). Thus,  ethinyl estra- diol  is  capable of  reducing sebum secretion when applied to  the  forehead. In addition, sebum secretion was  reduced at sites  on the  forehead distal  to the  site of application. In agreement with  a local  effect,  both  a and  b  receptor subtypes for estrogens are detected in sebaceous glands of human terminal follicles in both basal   and   partially  differentiated  sebocytes (117).  There   was   also  widespread expression of estrogen receptor b in the terminal hair follicle, where it was localized to the  nuclei  of the  outer  root  sheath, epithelial matrix,  dermal papilla cells, and in the cells of the bulge  (118).

Retinoids

Retinoids exert  dramatic, dose-dependent  effects  on  sebocytes  in  vitro  and   in vivo.  The effect of retinoids is mediated through nuclear receptors [retinoic  acid receptor (RAR), retinoid X receptor (RXR)] expressed in  sebocytes (119), and  in vitro,  sebocytes respond differently to retinoids depending upon whether RAR or RXR agonists are given.  Activation of RAR by all-trans-retinoic acid (all-trans-RA) mediated the  antiproliferative and  antidifferentiation effects,  while  RXR agonists caused differentiation and  only weak  proliferation in rat preputial cells (120).

Vitamin  A was essential for the proliferation, lipogenesis, and  differentiation of immortalized human sebocytes in vitro  (121), but  these  cells showed different responses to other  retinoids and  the  effects  were  dose  dependent. In delipidized serum (i.e., in the  absence  of vitamin A), isotretinoin [13-cis-retinoic  acid  (13-cis-

27

RA)] and  acitretin at concentrations    10

M enhanced sebocyte proliferation and

lipid synthesis, whereas at concentrations .1027M – sebocyte proliferation was inhi- bited  (121). In contrast, with  cells cultured in media containing vitamin A, further addition of either  isotretinoin or to a lesser  extent,  all-trans-RA  at concentrations ranging from  1028M to 1025M inhibited cell proliferation and  lipid  synthesis, and altered  keratin expression (122).  For  13-cis-RA  to  affect  sebocyte   proliferation,

differentiation, and  lipogenesis, it must  apparently be first isomerized to all-trans-

RA (123), so it is perhaps not surprising that these  retinoids show  similar effects.

Other  culture systems support the inhibitory role for retinoids in sebaceous glands.   Retinoids   significantly   inhibited   proliferation   in    the    rank    order

13-cis-RA . all-trans-RA       acitretin in immortalized sebocytes (124) and  in sebac- eous  gland organ  culture, 13-cis-RA caused a significant decrease in lipogenesis over  seven  days  (113,125), and  reduced thymidine uptake in pilosebaceous duct organ  culture (126).

Clinically,  13-cis-RA is given  orally  for severe  acne at doses  of 1 mg/kg/day for four  months and  produces a rapid reduction in sebum excretion, for example, down to 20% of its former  level within four  weeks  (127). This is due  to sebaceous gland atrophy as demonstrated by histological methods (128) but  the mechanism involved is unknown. 13-cis-RA and  the  derivatives, 3,4-didehydroretinoic acid, and   3,4-didehydroretinol  are   potent  competitive  inhibitors  of  the   oxidative

3 a-HSD, potentially downregulating the  production of potent androgens, DHT, and  androstandione, in  vitro  (129). 13-cis-RA does  not  however have  any  effect on 5-a-R responsible for the production of DHT (107). There is also a strong  increase in cellular  retinoic  acid binding protein II expression in the sebaceous gland, and to some extent, in the infundibular structures compared with the level of expression in the epidermis in biopsies obtained from patients treated with  13-cis-RA. Strongest staining was always found in layers of suprabasal sebocytes lacking  lipid  droplets, and  staining was  absent  in the basal  layers  (130).

Vitamin D

The effect of topical  vitamin D on human sebocytes and  sebum secretion has  not been  documented, although receptors for vitamin D have  been  demonstrated in the  nuclei  of basal  sebocytes (131,132). In a culture system of hamster auricular sebocytes, 1-a,25-dihydroxyvitamin D3, decreased the triglyceride level by 34.3%, but  augmented the accumulation of wax  esters  by 30%, with  no difference in the level of cholesterol produced (133).

Peroxisome Proliferator-Activated Receptors

Peroxisome proliferator-activated receptors (PPARs)  are  a subfamily of so-called orphan receptors belonging to the nonsteroid receptor family  of nuclear hormone receptors.  Several   major   types   of  PPARs   have   been   identified.  Activation  of PPARs regulates many  of the lipid metabolic genes contained in peroxisomes, mito- chondria, and  microsomes, with  the  net  effect of stimulating lipogenesis. Adipo- cytes,  for  example, are  induced to  differentiate and   accumulate lipid   through PPARg2. Therefore, there  is interest in PPARs functioning as regulators of lipogen- esis in sebocytes (134,135). Certainly in rat preputial sebocytes, PPARg is selectively expressed and  has  been  implicated in  the  regulation of sebocyte  differentiation (136). Fatty  acids  are PPAR ligands and  act as regulators of lipogenesis (137). For example, linoleic  acid  incubation for 48 hours in immortalized sebocytes induced abundant lipid  synthesis. Furthermore, the effect of androgens appears to be addi- tive with  the PPAR pathways: androgens appear to upregulate downstream PPAR expression, which  in turn  influences terminal differentiation of sebocytes (137,138). PPARs can also form heterodimers with RXRs and there is some evidence that there is cooperation between RXR agonists and  PPAR agonists in sebocyte growth and development (139). This is a complex area  and  the role of these  receptors in lipid

production in sebocytes is just beginning to be understood with  the help  of model systems.

Thyroid Hormones

Thyroid  hormones  exert   an   effect   on   sebum  secretion  since   thyroidectomy decreases the  rate  of  sebum  secretion in  rats  and   administration of  thyroxine reverses  this   effect   (140).  Increases  in   sebum  secretion  rate   were   observed when thyroxine was  given  to hypothyroid patients (141), and  immunohistochem- ical localization of thyroid hormone nuclear receptors has  been  demonstrated in human scalp  follicles  in the  nuclei  of the  outer  root  sheath cells,  dermal papilla cells, sheath cells, and  sebaceous gland  cells (142). Other  than  this, little attention has been paid  to the effect of thyroid hormones on sebum secretion.

Melanocortins

Melanocortins are  small  peptides, including a-melanocyte stimulating hormone (aMSH) and  adrenocorticotrophic hormone (ACTH),  known to regulate immune functions, lipid  metabolism, and  pigmentation pathways. These  small  peptides are  derived from  a larger  precursor, proopiomelanocortin (POMC),  a 241 amino acid  peptide secreted by the pituitary, which  is cleaved to liberate  the melanocor- tins. Evidence has existed  since the 1970s demonstrating that  the sebaceous gland is influenced by pituitary secretions. For example, removal of the posterior pituitary in rats  significantly reduced lipid  secretion from  rat preputial glands, addition of aMSH stimulated a  dose-dependent  increase in  sebum  secretion in  rats  (143), and    patients   with    hypopituitarism   show    reduced   sebum   secretion  (141). The expression of POMC-derived peptides in hair  follicles  in mice  varies  during the  hair  cycle,  with   high  levels  of  b-endorphin expressed during anagen and catagen (144).

The biological  effects of melanocortins are  mediated through specific  trans- membrane  receptors, known  as  melanocortin  receptors, which   are  coupled  to G-proteins to modulate intracellular levels of cAMP. To date, five receptor subtypes (MCR1 – 5) have  been  cloned  (145) and,  of these,  MCR-1 was  detected in human sebaceous glands using  immunostaining (146), and  in  an  immortalized human sebocyte  cell line  (147). MCR-5  expression was  demonstrated in  microdissected human sebaceous glands (148), but  could  not  be detected using  immunostaining of sections.  Specific transcripts for MCR-1, but  not for MCR-5, were  present in an immortalized sebocyte  cell line and  these sebocytes responded to aMSH by modu- lating interleukin-8 (IL-8) secretion (147). In rat preputial cells, MCR-5 was detected perhaps explaining the earlier  observations of the effect of melanocortin on these cells  (143); and  in  transgenic mice,  targeted disruption of MCR-5  gave  marked reduction of  sebum secretion (149). Other   investigators showed  that  sebocytes derived from   normal human  facial  skin  were   stimulated  to  differentiate and produce prominent lipid   droplets  when  POMC-derived peptides,  aMSH,  and ACTH  were  added, and  this  correlated with  an increase in expression of MCR-5 (150). Together, these  data  support the regulation of human sebum production by melanocortins and  may  offer an explanation for the link between stress  and  acne, with  high  circulating levels  of ACTH  acting  directly on  the  sebaceous gland  to mediate sebum production (148).

Growth Factors and Neuropeptides

EGF, TGF-a, basic FGF, and keratinocyte growth factor (KGF) all inhibit  lipogenesis and   with   the  exception  of  KGF  are  mitogenic  in  cultured  hamster sebocytes (133,151). In  organ  culture of human sebaceous glands, EGF produced a  dose- dependent inhibition of lipid  synthesis and  inhibited DNA  synthesis (152), and removal of EGF from  the  medium caused an  increase in  the  rate  of lipogenesis without  affecting   cell  turnover  (113).  In  vivo,  the  presence  of  EGF  inhibited the   sebaceous  gland  differentiation  in   sheep   (153),  and   in   hamster  ears,   it stimulated the sebaceous glands to proliferate (154). Receptors for EGF are found in sebaceous glands (155). Based on this evidence, Kealey and colleagues proposed that EGF may be, in part, responsible for sebaceous gland  atrophy observed during acne lesion formation (7,152). EGF can also induce changes in sebum composition. Ridden (156), reported that  there  was  a  fall  in  the  amount of  squalene and  a corresponding rise  in  cholesterol, although  this  was  not  corroborated by  other researchers (157).

Sebocyte proliferation is stimulated by insulin, thyroid-stimulating hormone, and  hydrocortisone (158). In rat  preputial cells, growth hormone (GH)  increased sebocyte   differentiation  and   was   more   potent than   either   insulin-like growth factors  (IGFs) or insulin. Furthermore, GH enhanced the effect of DHT on sebocyte differentiation perhaps complementing the effect of androgens in increasing sebum during puberty. IGFs, in contrast, had  a greater effect on increasing DNA synthesis compared to  GH,  and  had  no  effect  on  the  response of the  cell  to  DHT  (159), although a role for IGF in causing the  observed increase in sebum production at puberty has not been  ruled out.  This suggests that  antagonists of IGF may  offer a way  of decreasing sebum synthesis.

Corticotrophic-releasing  hormone  (CRH),  a  key  regulator  in  the   central nervous system, has been implicated in sebum production (160). CRH and its recep- tor are expressed in cultured sebocytes, where the hormone appears to upregulate expression of 3 b-HSD—a  key  enzyme in  the  androgen biosynthetic pathways. Therefore, CRH  could  indirectly influence lipid  synthesis by modifying the  local production of androgens.

Incubation of sebaceous glands in  organ  culture with  neuropeptides  (e.g., calcitonin gene-related peptide, substance P, vasoactive intestinal polypeptide, or neuropeptide Y), or NGF  up  to a final  concentration of 1027M showed that  only substance P had  any  effect on sebaceous gland  ultrastructure as observed in the electron   microscope,  and   this  effect  was   dose   dependent (161).  Skin  samples cultured with   substance P  showed  sebaceous glands  with   an  increased gland area  (in sections)  compared with  control  samples. The  sebocytes were  engorged with  numerous lipid  droplets.

Cytokines

Expression of IL-1a and  b was  demonstrated in sebaceous glands by immunohis- tochemistry (162), and  mRNA  for IL-1a was  detected in cultured sebocytes (163). No change in staining pattern was  observed when sebum was  extracted from  the samples, indicating that  IL-1 is not  associated with  sebum. The  function of IL-1 in normal sebaceous glands is unclear, although in vitro IL-1a and  tumor necrosis factor  a, inhibited sebaceous lipogenesis in  sebaceous gland organ  culture and induced de-differentiation of human sebocytes into a keratinocyte-like phenotype (152). In organ  culture, IL-1a at 1 ng/mL promoted ductal cornification (164) and

upregulated mRNA  and  protein synthesis of vascular endothelial growth factor (VEGF) (165). Thus  in follicular  diseases, liberation of IL-1 into  the  dermis may contribute toward inflammation and  increased levels of VEGF by follicular  kerati- nocytes  can stimulate angiogenesis. In acne, high levels of IL-1a bioactivity existed in comedonal contents (166).

SECTION  THREE:  DISEASES

Disorders Affecting Pilosebaceous Unit Biology

Various  disorders affect pilosebaceous units,  although these diseases are rarely  life threatening. Three types  of skin cysts exist: epidermoid cysts result  from squamous metaplasia of a damaged sebaceous gland, while  trichilemmal cysts and  steatocys- toma  are  both  genetically determined structural aberrations of the  pilosebaceous duct.  Accumulation of material in the follicular  lumen results in distension of the follicle,  leading to the  formation of noninflamed lesions  that  are  typical  of acne, which  is the most  common follicular  disease. In terminal follicles, plugging of the pilosebaceous  duct   may   occur   and   result   in   keratosis  pilaris.   Inflammation around follicles  is  often  seen  at  the  skin  surface,   for  example in  folliculitis   or acne.  In  folliculitis,   there   is  extensive  colonization  of  the  follicular   lumen  by microflora.

Acne

Acne is the most  common follicular disorder and  accounts for the majority of der- matological visits  by patients between the  ages  of 15 to 45 years,  with  treatment costs  likely  to  exceed  $1 billion  in  the  United States  (167). Problems associated with  follicles often  occur  on the face and  thus  can cause  great  psychological pro- blems  for the sufferer. Four  main  factors  contribute to acne: increased sebum pro- duction,  ductal  hypercornification,  increased  bacterial  activity    in   the   duct, and  inflammation. The  changes that  occur  in  the  pilosebaceous biology  will  be reviewed briefly.

Changes in the  Pilosebaceous Unit in Acne

There  is no doubt that  sebum plays  an important etiological role in acne (99,168 –

170). Patients with  acne secrete  more  sebum than  unaffected individuals and  sebo- suppressive treatments alleviate acne: the greater the inhibition the more profound the clinical response. In addition to elevated sebum excretion, it is well-established that  acne  is associated histologically and  clinically  with  hypercornification of the duct  epithelium (7,38,171). Ductal  hypercornification results initially  in  the  for- mation of microcomedones and  eventually comedones, a process  termed comedo- genesis.  On the  basis  of a detailed histological study Kligman (7) concluded that comedogenesis began in the infrainfundibulum and was closely followed by hyper- keratosis of the  sebaceous duct.  This  is now  generally accepted to  a reasonable description of what  is likely to happen.

Essentially, microcomedones are those  follicles containing “impactions,” “follicular casts,”  or  “sebaceous filaments” of corneocytes, bacteria,  sebum, and vellus   hairs   within  the  follicular   lumen.  There   are  no  clinical  signs   of  these changes occurring, although they  are more  common in patients with  acne (7,172). Microcomedones may  develop either  into  noninflamed, or  inflamed lesions,  or even   resolve   (173).  The  contents  of  the   normal  infundibulum  from   nonacne

FIGURE 5    Environmental scanning  electron micrograph of a  microcomedone removed from  a pilosebaceous duct. Keratinocytes are  seen covering  multiple hairs.

control  patients were easy to extrude and  contained numerous empty spaces  in the cells which  appeared to have  lost their  contents and  had  no clear cytoskeleton. In contrast, the contents of follicles from  acne patients were  hard to extrude and  the cells were  firm and  compact (7).

An  extensive ultrastructural  study  on  the  cornified material  in  follicular impactions was  carried out  by  Al  Baghdadi (174). He  showed that  corneocytes are arranged into three  layers  in the follicular  impaction: a loosely  attached outer layer;  a densely packed middle layer;  and  a further loosely  attached layer  in the middle of the  cast. Lipid  droplets occur  inside  the  cornified cells (38,175) and  as a result  the  corneocytes become  balloon-like in  appearance. Abnormal lamellar inclusions of lipid  have  been  described as a marker for abnormal keratinization (176). Scanning electron  microscopy of comedonal contents revealed a complex system of  numerous  internal channels or  “canaliculi” composed  of  concentric lamellae of corneocytes enclosing bacteria and  sebum (177) (Fig. 5).

The overall  composition of follicular casts was reported to be water (20 – 60%), lipids  (20%), proteins (15%), and microorganisms (178). Bladon et al. (179) analyzed the protein content of comedones and found both epidermal keratins and degraded keratins to be present in the comedones.

Biochemical Changes in the  Infundibulum During Comedogenesis

The process  of comedogenesis is thought to be due  to hyperproliferation of ductal keratinocytes, inadequate separation of the ductal corneocytes, or a combination of both  factors,  resulting in microcomedones. Hyperproliferation of basal  keratino- cytes  in  acne  has  been  demonstrated  (180,181)  and   correlates with   keratin 16 expression, a marker for hyperproliferation suprabasally (182). Aldana et al. (183) proposed a cycling  of normal follicles  through different levels  of expression  of Ki-67 (a proliferation marker) and  keratin 16 and  that  these  represent different stages  of development of the microcomedone. The point  at which  both  Ki-67 and keratin 16 are  coexpressed by  the  follicle  is  when the  follicle  is  susceptible to comedogenic changes (184).

A more  extensive investigation of adhesion in follicles is required. In epider- mis, intercellular adhesion is mediated by lipids,  cellular  adhesion molecules, and desmosomes. At the present time, the relative importance of follicular  intercellular lipids  in normal and  acne  follicles  is unclear due  to the  experimental difficulties encountered  obtaining sufficient   material for  analysis. Hypercornification from the  retention of  desmosomes was  described to  be  a  contributory factor  to  the formation of acne lesions.  However, no differences were detected between staining for these  epitopes in the wall of normal and  comedonal follicles (29).

CONCLUSION

Although it is clear that we now  know  a great  deal about  the pilosebaceous unit  at the  structural, biochemical, and  physiological levels,  there  are  still  considerable gaps  in  our  understanding. Much  of the  early  work  was  descriptive biology  of the structure obtained using  immunohistochemical studies. Over  the last decade, several  in vitro  models systems (isolated organ  culture, cultured sebocytes) have made it easier  to study the cellular  processes in the follicle, including sebum pro- duction and  the  effect  of inhibitors and  stimulators. There  is also  now  a  good deal  of information about  the  chemistry and  biosynthesis of sebum components, and  we also have a much  better  understanding of the cell biology  of the pilosebac- eous  duct.  Even  so, we are still unable to precisely define  the cause  of one of the most  common skin  diseases, namely acne,  which  affects  a very  large  proportion of the population globally.

ACKNOWLEDGMENTS

The  author wishes to  acknowledge and  thank Professor W  Cunliffe  for  kindly allowing the  use  of  Figure   1B, and   the  journal  “Retinoids and   Lipid   Soluble Vitamins in Clinical  Practice” for kindly allowing the use of Figures  1A, 2, and  3.

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