In the human body, the skin is the largest organ, with it being a complicated epithelial and mesenchymal tissue. It consists of an epidermis which is multilayered as well as structures such as sebaceous and sweat glands, hair follicles, a dermis consisting of elastic and collagen fibres. There is also a layer of subcutaneous fat. There has been a discovery of over 1000 disease entities involved with the skin such as eczema, psoriasis, melanoma and urticaria . Around 15% of a human adult's total body weight is accounted for by the skin with a surface area of approximately 2m2. The skin consists of three layers; the epidermis, the dermis and the hypodermis.
The outer layer of the skin is known as the epidermis, which is a stratified squamous epithelium , where 95% of its cells are keratinocytes. The remaining cells in the epidermis are the melanocytes, merkel and langerhans cells. The role of the epidermis is to provide a defence barrier against environments of an inhospitable nature . The epidermis can be divided into four layers, in order from outer layer to deepest layer; stratum corneum (the cornified layer), stratum lucidum (the clear/translucent layer), stratum granulosum (the granular layer), stratum spinosum (the spinous layer) and the stratum basale (the basal layer) .
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There is a single layer of keratinocytes in the basal layer, where daughter cells can be produced by them to terminal differentiation via proliferation, resulting in the forming of the cornified layer , which can take around 40 days, however this can be shorter in various diseases, such psoriasis. About ten layers of corneocytes that are flattened make up the cornified layer .
From the neural crest, dendritic cells can be derived which are known as melanocytes, which are also found in the basal layer. Melanosomes, which are subcellular organelles, transport melanin, which are synthesised by melanocytes, to the neighbouring basal keratinocytes. In order to prevent harm to the nuclei of the basal keratinocytes from ultraviolet radiation, a 'melanin cap' is formed by the melanosomes . Melanosome size and number, as well as melanin's nature determine skin colour or pigmentation . Langerhans cells are derived from the bone marrow and are antigen presenting dendritic cells founds in the epidermis. Sensory information is transmitted from the skin to the sensory nerves by merkel cells found in the epidermis .
The dermis is the layer beneath the epidermis, and it's thickness depends on the area of the body. For example, on the eyelid, the dermis is thin, whilst the dermis is thick on a person's back. The dermis consists of two layers; the papillary dermis and the reticular dermis. The papillary dermis is in contact with the basement membrane zone, which provides adhesion between the epidermis and dermis, where skin blistering can occur due to defects . Blood vessels as well as sensory nerve endings are richly supplied to the papillary dermis. The reticular dermis is in contact with the hypodermis and is the main component of the dermis. The structure of the skin, as described above, can be seen in Figure 1.
Figure 1. Diagrammatic representation of the skin's architecture in light and dark pigmented skin types. Key: SC = Stratum Corneum, SG = Stratum Granulosum, SS = Stratum Spinosum, SB = Stratum Basale, BM = Basement Membrane, D= Dermis. Modified from Busca and Ballotti, 2000.
Interstitial components, such as elastic and collagen fibres, and cellular components, such as fibroblasts and plasma and mast cells, are what make up the composition of the dermis. Collagen accounts for around 70% of the dermis' dry weight , where types I and III are predominant . The predominant cell type, however, is fibroblasts in the dermis, which are derived from the mesenchyme.
The hypodermis is the deepest layer of the skin consisting of lipocytes. The function of the hypodermis is to connect the skin to the bone and muscle, thus supplying the bone with nerves and blood vessels. The arrangement of these is in fat lobules, where the fibrous septae separates one from another. The connection between the dermis and the hypodermis is strengthened by fibre bundles originally from the dermis. Around 80% of the entire body fat is found within the hypodermis in those individuals who are not obese .
As very briefly mentioned previously, melanocytes function is dependent upon for pigmentation. These cells from melanoblasts during embryological development. Each basal melanocyte is connected functionally to the dermal fibroblasts as well as to the basal keratinocytes. These three cell types interact and communicate with each other in order to regulate the skin's phenotype and function through the secreted factors and receptors in addition to cell to cell contact .
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Stem cell keratinocytes and basal melanocytes has a slow proliferation rate in normal circumstances, however the upper basal keratinocytes have a much rapid proliferation rate, which carries them towards the skin's surface alongside the ingested melanin thus forming a barrier. Therefore the skin's colour is not personified by only the melanin found in melanocytes, but also in a conjunction with pigment found in the superficial layers of the skin .
Currently, pigmentation is known to be regulated in a direct or indirect fashion by over 125 different genes, with this number potentially rising 150-200 in less than another 100 years . Out of them genes, the one's whose function is understood, a lot of them affect processes that are involved in development which are critical for melanoblasts. Some genes regulate melanocytes' differentiation and survival whilst others control processes affecting pigmentation. Melanosomes functions or biogenesis is affected by more than 25 of the genes. Some critical enzymes involved in the control of pigmentation include tyrosinase, tyrosinase related protein 1 (TRP-1) and DCT (DOPAchrometautomerase). If these enzymes are mutated, melanins which are synthesis could be affected in terms of their quantity and quality. Some critical structural proteins required for the melanosomes to mature structurally include Pmel17 and MART1 . Mutations in proteins that are involved in the sorting of melanosome proteins can result inherited disorders of hypopigmentary nature .
This critical appraisal will look at in further detail the different types of pigmentation, constitutive and facultative, and how they are regulated, including the roles of MC-1R, cyclic AMP (cAMP) pathway Agouti Signalling Protein, MITF and ultraviolet radiation. Also covered in this piece of work is CRH's and Î²-endorphin's roles in regulation of human skin pigmentation.
Within the melanosomes, melanins are synthesises via the previously mentioned enzymes. The reaction which limits the melanogenesis' rate becomes catalysed by tyrosinase, as is tyrosinase's hydroxylation resulting in 3,4-dihydroxyphenylalanine (DOPA), along with DOPA oxidising into DOPAquinone. The oxidation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) take place in mice due to TRP-1, however this same activity doesn't take place in humans . DOPAchrome is isomerised into DHICA by DCT . In human, there is productions of two types of melanin, eumelanin which is black or brown and pheomelanin which is yellow or red. Tyrosinase is essential for the synthesis of both types of melanin, whilst TRP1 and DCT more for the synthesis of eumelanin .
With regards to skin pigmentation, there are two types; Constitutive pigmentation and Facultative pigmentation.
Regulation of Constitutive Pigmentation
Depending upon the racial and ethnic background of the person, the colour of human skin varies from extremely light to extremely dark. Several major chaperones, melanin, oxyhaemoglobin and deoxyhaemoglobin and carotenoids determine the colour of human skin .
In 1954, the first observation was made with regards to the pigmentary system of the skin by Szabo when an immunohistochemical technique was used to test tyrosinase's enzymatic activity via staining of tissues , where Caucasian skin was examined at first followed by other colours of skin . Along with various other studies as well as Szabo's it was shown that in different human skin types had similar melanocytes densities as well as distribution in similar body areas . They also found that there is less melanin content in lighter skin, with melanosomes which are pigmented poorly being clustered above the nuclei within keratinocytes. There is more melanin present in darker skin, with the distribution of melanosomes that are pigmented heavily being individual in keratinocytes rather than clustered, which increases light absorption.
The density of melanocytes is different in distinct parts of the body. For example, the skin on an individual's palms or soles is lighter in comparison to others parts of the body. Environmental factors can affect the density of constitutive melanocytes in the skin, including ultraviolet radiation (UVR), where the density can be increase by 3 or 4 times of the norm. Another environmental factor that can increase the density are toxic compounds, for example hydroquinone, resulting in the destruction of melanocytes. In increase of decreased melanocyte densities, pigmentary disorders which are inherited can result, for example freckles or vitiligo respectively .
Due to Bcl2's high expression, epidermal keratinocytes are resistant to apoptosis as they have a slow proliferation rate in normal circumstances . It has been shown that the palms and soles dermis have a high level of Dickkopf-1 (DKK1) secretion which causes the Wnt/Î²-catenin signalling pathway to become inhibited via the suppression of the growth function of melanocytes, thus inhibiting the melanogenic pathway. This can have effects on some transcriptional regulators, for example microphthalmia transcription factor (MITF), to some downstream melanogenic proteins. Epidermal Keratinocytes also become affected by DKK1 as melanin uptake is diminished, resulting in a skin phenotype which is a lot thicker with less pigmentation .
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Melanocortin 1 Receptor (MC-1R), which is domain receptor of seven transmembranes which binds to pro-opiomelanocortin peptides due to it being coupled with Î±s G-protein , is a major skin pigment phenotype determinant. It regulated the quality and quantity of melanin production . Two agonists regulate MC-1R function, which are Î± melanocyte stimulating hormone (Î±-MSH) and adrenocorticotropic hormone (ACTH). An antagonist called Agouti signalling protein (ASP) also regulates MC-1R function. When Î±-MSH or ACTH activate MC-1R, melanogenic cascade expression is stimulated, resulting in stimulation of eumelanin synthesis. This can be reversed by ASP, resulting in stimulation of pheomelanin production. MC-1R gene expression can be upregulated by Î±-MSH and ACTH, which act in a positive feedback loop .
Melanogenesis can be defined as the biosynthetic pathway of melanin in living cells, which is a complex process with multiple steps which involves substrates, specific enzymes already mentioned and various cofactors commencing with phenylalanine and/or tyrosinase resulting in melanin deposition on the melanosome's protein matrix . The understanding of melanogenesis was greatly increased in the 1950's onwards by Fitzpatrick et al .
During the cycle of hair growth in Agouti mice, melanogensis regulation occurs quantitatively as well as qualitatively. Pheomelanins are produced instead of eumelanins in the anagen phase, a switch incurred by the melanocytes in the hair follicles, causing a yellow band on top of a brown background. The regulation of this switch involves extension and agouti loci products that encode MC-1R and ASP respectively . When a ligand binds to MC-1R it activates, resulting in activation of adenylyl cyclase by the Î±s G-protein, causing an increase in the intracellular cAMP significantly . If the extension locus incurs any mutations, the MC-1R reception will become non functional, therefore adenylate cyclase will remain inactive in Î±-MSH presence, meaning mice will have a yellow coat colour. The MC-1R receptor is bound by ASP, which results in the Î±-MSH effects being antagonised, which includes the adenylate cyclase activation caused by the Î±-MSH .
There is lots of evidence which shows that Î±-MSH, ACTH and cAMP have key roles in skin pigmentation regulation in humans. For example, Î±-MSH hypersecretrion has been reported to cause skin hyperpigmentation . Patients with severe obesity and hair pigmentation which is red have been shown to have pro opiomelanocortin gene mutations .
In human melanocytes that have been cultured, dendricity and melanogenesis are upregulated by the pro opiomelanocortin peptides . Pharmalogical cAMP can also mimic these effects . All of this clearly suggests that Î±-MSH, ACTH and cAMP have a vital role in melanogenesis regulation.
Role of cAMP
It is suggested that cAMP has a pivotal role in melanogenic enzymes activity / expression regulation. This is because the enzyme activity of melanogenesis is diminished much more than TRP-1 and DCT. The melanogenic effects of the pro opiomelanocortin peptides seem to be mediated via the cAMP pathway upregulation through the activation of MC-1R as well as adenylate cyclase.
Within the cell, cAMP binds to protein kinase A (PKA), enabling activation of the catalytic subunit . PKA phosphorylates its substrates, then translocating to the nucleus, phosphorylating cAMP responsive element binding protein (CREB) transcription factors family. Specific genes have their expression activated by this family of proteins, which contains consensus cAMP responsive element (CRE) sequences within their promoters . CREB-binding protein is also phosphorylated by PKA, where PKA dependent gene expression is required in order for the interaction with the family of CREB proteins .
MITF, which is a helix loop helix transcription factor has been shown to be encoded by the mi locus. This is due to mice which have the mi mutation have a coat colour which is diluted, have white spots, or entire pigmentation loss. They can also have a microphthalmic phenotype caused by ocular development defects . Within melanocytes, mast cells, pigment cells in the retina and osteoclasts, MITF has been found to be expressed . Also, the lack of melanocytes seems to be the cause of the defective pigmentation in mi mice. It has been established that in the development and survival of melanocytes, MITF plays a key role, which is confirmed by the cloning of the MITF homologue within humans . Patients who have type II Waardenburg syndrome have been known to have mutations in MITF present, where there is defective pigmentation in the skin, hair and eyes, as well as hearing alterations .
It was shown by Bertolotto et al that there is some sort of connection between the cAMP pathway with MITF. In normal melanocytes and B16 melanoma cells, MITF expression was shown to be increased by cAMP . Tyrosinase expression stimulation that is induced by cAMP requires MITF as shown by a MITF missing the transactivation domain by a dominant negative mutation .
Figure 2 below shows the mechanism of action of cAMP melanogenesis regulation involving MITF as a signal transducer. In the diagram, we see that PKA becomes activated by cAMP. PKA then goes on to phosphorylate and activate CREB. CREB binds to CRE after it is activated. The CRE is in the microphthalmia promoter, which upregulates its transcription. Microphthalmia expression is therefore increased, which leads to amplified binding of microphthalmia to the M box motif found in the tyrosinase promoter. Tyrosinase expression is increased, as is the upregulation of the synthesis of melanin.
Figure 2. cAMP induced melanogenesis regulation mechanism involving MITF
Agouti Signalling Protein
In cultured human melanocytes, eumelanin synthesis and the activity of tyrosinase is inhibited by ASP. TRP-1 and the expression of tyrosinase is also reduced by ASP . Because of tyrosinase's slightest inhibition of activity as well as to the near loss of the expression of TRP-1 and DCT, ASP decreases eumelanin synthesis. It has been found that genes were downregulated by ASP founds in tyrosinase and DCT, as well as upregulated genes which have some association with a basic helix loop helix transcription factor (ITF2) . This shows that ITF2 may have a role in melanogenesis regulation, particularly in the previously mentioned switch of eumelanin to pheomelanin.
Regulation of Facultative Pigmentation
Physiological regulation that causes an increase in skin colour can be defined as facultative skin pigmentation. There are lots of factors that regulated facultative skin colour, including ultraviolet (UV) which is also known as the tanning reaction which occurs in fish as well as humans . The skin's response to UV radiation is kinetically complex causing tanning of the skin over a period of several weeks .
The effects of UVR can be divided into acute and chronic effects. The acute effects include erythema (sunburn), tanning and immunosuppression. The skin's visible pigmentation is added to by UV causing erythema of the skin itself. There are three different stages of tanning, two of which occur rapidly, immediate and persistent tanning, and one that takes time in order for it to develop, delayed tanning. Immunosuppression can result in a decreased number and function of antigen presenting langerhans cells, as well morphological changes.
The chronic effects of UVR include photoaging and photocarcinogenesis. In photoaging, wrinkles and freckles start to appear on the skin, where there is a leather type appearance. Photocarcinogenesis can be caused due to the indirect damage of DNA by reactive oxygen species generation.
There are three types of UVR:
UVA - which is 320 - 400nm long. It's the longest wave from all the types of UVR and can penetrate deeply into the dermis.
UBV - is 280 - 320nm long. It can penetrate the epidermis and is 100 fold more energetic and mutagenic.
UVC - is 200 - 280nm long but does not reach the surface of Earth.
Immediate & Persistent Tanning
The reaction of immediate tanning can occur almost instantaneously, within a few minutes after being exposed to UV, where it still persists several hours later . Persistent tanning is a separate second stage of the tanning reaction whereupon it occurs within a few hours after being exposed to UV, with it still being persistent several days later . Melanin and/or melanogenic precursors oxidation and polymerisation is thought to be behind both immediate and persistent tanning. The responses of both these types of tanning are greater to UVA than to UVB. Immediate tanning has a colour of gray to black whilst persistent tanning appears brown.It has been shown that one week after being exposed to UV, there is very little production of more melanin .
Reported in 1986 was that immediate tanning can be educed by UVA in epidermal sheets . Honigsmann's results suggested that existing melanin or melanin precursors chemical oxidation is reflected upon by immediate tanning rather than pigment granules physiological movement. Reactive oxygen species are able to cause the oxidation of tyrosine as well as DOPA to melanin which occurs in immediate tanning . Also, pigmentation's UVA induction is dependent upon melanin which is soluble and there are two different types of melanin absorption which are involved in UVA photoxidation.
The reaction of delayed tanning has a developmental time of more than several days . Skin tanning appears to peak one week after being exposed to UV, after which tanning appears to diminish for the next ten weeks, but doesn't return to the constitutive level after that time. Within the same time frame, Asian skin pigmentation increase is relatively small. Therefore there is a higher level of hyperplasia in skin that contains smaller levels of constitutive pigment, playing somewhat of a protective role in the response to UV than did the increased pigmentation the skin types that are lighter . Skin pigmentation increase over a long term caused by UV are due to lots of physiological facts being regulated by UV, affecting the growth and / or differentiation of melanocytes. Pigmentation is also stimulated by DNA damage caused by UV exposure .
The levels of eumelanin and pheomelanin slowly increase together after being exposed to UV on human skin . This shows that by UV, they are not regulated separately. In various ethnic origins skin pigmentation, there are around two fold differences in melanin's chemical content and the melanosomes distribution and size of the particle are important to the visible colour of skin . In comparison with skin which is protected to skin which is constantly radiated with UV, there is only less than a two-fold increase again. All of this shows that aside from the quantity of melanin, other factors are necessary for skin pigmentation.
An increase in their expression of Î±-MSH and ACTH is a response by epidermal keratinocytes and melanocytes to UV exposure. This MC-1R's function and expression to become upregulated, increasing the response of melanocytes to melanocortins. The weakly functioning MC-1R variants can be located in people with fair skin with red hair who have more pheomelanin with an inability to get a tan. The expression of Endothelin-1 by keratinocytes is enhanced by UV, thus enhancing MC-1R's expression also, but endothelin-1 works via its own receptor on the melanocytes. The secretion of interleukin-1 by keratinocytes is also caused by UV, stimulating endothelin-1, Î±-MSH and ACTH secretion by keratinocytes . In keratinocytes, p53's stimulation by the exposure to UV causes increased POMC gene expression resulting in an increase in Î±-MSH secretion as well as MC-1R function stimulation in the neighbouring melanocytes .
It was reported for the first time by Kauser et al that Î²-endorphin and the Âµ-opiate receptor system is expressed in epidermal melanocytes, they're associated closely with melanosomes, and that in melanocyte biology regulation, this system is active due to its pigmentation, dendricity and proliferation upregulating ability . In the epidermal melanocytes and keratinocytes, the presence of both aforementioned ligand and receptor gives a platform for both autocrine and paracrine mechanisms for the regulation of melanocyte behaviour. Î²-endorphin and Î²-lipotropic levels are raised being exposed to UVR , further suggesting that Î²-endorphin has a role in epidermal melanocytes. Kauser et al also showed that Î²-endorphin that has been supplied exogenously can cause an increase in melanogenesis and proliferation in epidermal melanocyte cultures . Î²-endorphin also has an association with melanosomes suggesting that melanogenesis might be regulated locally in the secretory granule.
Corticotropin Releasing Hormone
It has been established that Corticotropin Releasing Hormone (CRH) does have some sort of role in pigmentation . MC-1R action as well as the Âµ-opiate receptor moderates melanocytes behaviour in hair follicles where there is complete expression of the pro opiomelanocortin system within the pigmentary unit . The expression of CRH is low is different melanogenic zones, whilst there is differentiated distribution of melanocytes in the same area . CRH Receptor 1 seems to be more important in follicular melanocytes for the stimulation of melanogenesis, proliferation and dendricity. However, the role of CRH is in skin pigmentation is not 100% full established, and is an area that definitely required further research in order to gain some clarification.
As people get older, pigmentation continues to increase until adolescence or adulthood. Once they've reached this point, pigmented lesions often begin to appear, and the hair starts to turn gray. It was found in 1979 that the quantity of melanocytes was decreased by about 10% with each decade the age went up by . This was confirmed by two other researches , where one was carried out on darker skin also . Another study compared very fair skin with Caucasian skin, finding that melanocyte density was greatly enhanced following continuous exposure to sunlight in the darker skin, however langerhans cell densities were decreased in the same time frame in both skin types after being exposed to UV.. It was proposed by Stierner et al that being exposed to UV might have some role in the development of melanoma in both exposed and protected skin, as well as being exposed to aberrant UV profoundly can be a lot more harmful than normal exposure , which has since been confirmed by various other studies .
Disruption of Regulation
From time to time, different types of pigmentary disorders can occur due to disturbance of the normal regulation of skin pigmentation. Tyrosine function regulation lays importance on intracellular pH as catalytic functions are affected by the intramelanosomal pH as well as melanosomal protein delivery requiring the sorting pathway to have the right pH gradient . It is also considered that pigment production is regulating in some part by intracellular pH in different types of skin depending upon the racial or ethnic origin .
Every single form of albinism is caused by tyrosine dysfunction or other types of melanogenic proteins, which can cause skin pigmentation to be blemished. Another pigmentary disorder is Hermansky - Pudlak syndrome . This disorder have pleiotropic clinical effects .
Pigmentary disorders which are caused by the acquiring of melanin involve the skin becoming lighter or darker. Skin colour which reduced is normally caused from epidermal melanin content declining. The skin may become darker due to an extremely large number of melanin being produced due to there being a an enhanced quantity of melanocytes, however it can also be due to melanin distribution becoming abnormal.
To summarise, in different skin colours and racial backgrounds, the density of melanocytes is near identical. The quantity and distribution of melanin is largely dependent upon for constitutive skin pigmentation. Less DNA damage occurs to melanocytes present in darker skin than those present in lighter skin. The activity of melanogenesis increases in darker skin in a more efficient manner than in lighter skin.
The determination of constitutive skin pigmentation is achieved by:
Melanoblasts migration during development
Melanoblasts differentiation and survival to melanocytes
Melanosomal enzymes and their structural components expression and function
Eumelanin and pheomelanin synthesis
Melanosome transport to the dendrites
Melanosomes being transferred into keratinocytes
Melanin distribution in the skin's suprabasal layers.
MITF seems to respond to UV pretty quickly, with a response after 1 to 2 days. Some melanosomal proteins respond slower, such as tyrosinase, TRP-1 and DCT, with a response being elicited after about 1 week, where 3 weeks later an increase in the synthesis in melanin can be observed, whereas melanocyte density is increased around 4 to 5 weeks.
Cyclic AMP causes the complex induction of intracellular processes which seem to be interconnected. The sub-pathway where PKA is activated, MITF is upregulated as is some of the enzymes involved in melanogenesis, causes melanogenesis stimulation. A cAMP activated pathway, through ERK activation inducing the degradation and phosphorylation of MITF, regulated melanogenesis negatively, where PKA is independent upon.
The skin's melanin distribution plays a key role in pigmentation that is visible. After around 1 week, the existing pigments migration towards the epidermal surface is increased, after which newly synthesis melanin restores the balance in the distribution of pigment around 4 to 5 weeks later. It is also apparent that when the distribution in the content of melanin undergoes minor changes, it can result in major changes in visible pigmentation, affecting constitutive pigmentation as well as facultative pigmentation or the responses to being exposed to UV.
After reading through the literature to carry out this critical appraisal, it is evident that lots of studies have opposing and conflicting results as well as conclusions which may be incoherent, where the same group of authors may diverge from something which they have previously stated. This is most probably due to lots of variants when conducting these types of in vivo and in situ physiological studies. These variants most definitely include:
UV source types
How much dose amount and frequency that has been applied
The sites which have been exposed and their locations
The point in time which is assessed after being exposed to UV
The history of the subjects, and whether they have been exposed to the same / higher levels of UV conducted in the experiments previously
The capacity of an individual DNA repairing
Very importantly, the racial and / or ethnic origin of the subjects.
There are many areas which require clarification in field, which are definite area of potential future research. For example; Is melanocyte function affected eumelanin against pheomelanin production? As facultative pigmentation is increased, is there further protection against damage caused by UV? Does DNA repair have a role in reducing the skin's long term damage?
Also, photocarcinogenesis understanding needs to be enhanced, some of the parameters that are critical to it, and some strategies on how to reduce its risks. Research into which pathways participate in melanogenesis induced by UV and MSH still seems to be under way. Any advances could help in discovery of new potential ways of treating certain pigmentary disorders.
The regulating mechanisms in the synthesis on melanin aren't understood as clearly as required, where it's been speculate that research into this may lead to topical melanogens discovery that can cause the production of melanin when UV irradiation is absent, which causes photo aging as well as some skin cancers.
Further studies are necessary regarding the pigmentary role of Î²-endorphin, which seems to be the forgotten melanocortin with regards to pigmentation . Similarly, the role of CRH in pigmentation also needs to be enhanced further. It is also shown that some hormones play a role in regulation of pigmentation including some oestrogens and androgens, which are areas that could used for further research to increase our understanding.