Alpha MSH In Cultured Melanoma Cells Biology Essay

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This study was designed to examine the expression of alpha-MSH in cultured melanoma cells which are highly pigmented(FM94), moderately pigmented (FM55) and unpigmented(FM3) in order to see whether there is a correlation between the expression levels of alpha-MSH and pigmentation in melanoma cells by employing cell culture and immunocytochemistry. In human skin pigmentation and hair follicle pigmentation this ligand receptor is believed to be a key regulator.

Melanoma cells that are highly pigmented were the most responsive to alpha MSH stimulation compared with those from unpigmented cell line. Kauser et al further did investigation on hair follicle melanocytes and found that hair follicle melanocytes that are from dark haired individuals (dark brown and black) have the most response to alpha-MSH stimulation whereas hair follicles of lighter colour were less responsive. There is evidence that supports this theory it has been shown that alpha MSH binding sites may be linked to hair colour. On human scalp hair bulbs derived from pigmented hair, high number of alpha MSH binding sites were identified whereas fewer binding sites were on hair bulbs derived from blond hair and were absent from bulbs derived from senile white hair.

Its now well documented that alpha-MSH acting via MC-1R stimulates melanogenesis by activating the adenylate cyclase system. Alpha-MSH has a proliferative effect on melanoma cells. Through its binding to the MC-1R the biological action of alpha MSH is mediated on melanoma cells. MC-1R is a G protein coupled receptor so when alpha-MSH binds to the MC-1R it activates a G protein which in turn activates adenylate cyclase and the resulting increased intracellular concentration of cAMP mediates the melanogenic and morphological responses to alpha MSH.

Alpha-MSH expression and its processing could signify vital control points for skin pigmentation. Evidence to prove this is given by the recent report of red hair and light skin in a patient with a nonsense mutation in the POMC gene that lead to a total absence of alpha-MSH (Krude et al. 1998). From previous observations it had been proposed that pigmentation phenotype is linked with melanocyte responsiveness to alpha-MSH. Red haired people’s melanocytes were completely unresponsive, failing to show melanogenic and dendritic responses to alpha-MSH (Hunt et al. 1996). MC-1R as a control point for pigmentation phenotype has been suggested from this evidence (Hunt et al. 1996) and this theory was consistent with the findings of several MC-1R variants in red haired individuals. (Valverde et al. 1995).

Control points that adjust ORS/hair bulb matrix melanocyte differentiation into pigment producing cells may function in the human hair follicle (Kauser et al. 2005). In addition people from northern Europe with red hair and fair skin that have been associated with MC-1R polymorphisms have also been reported in red haired individuals with African ancestry (McKenzie et al. 2003). Therefore implicating that there is a larger effect on hair colour than on skin colour from MC-1R polymorphisms (Kauser et al. 2005). It has been observed that in humans there can be distinct body site variation in hair colour for instance some males have pheomelanogenic beards and eumelanogenic scalp hair whereas there is extremely diminutive difference observed in the epidermis (Kauser et al. 2005).

In mice, ASP also regulates coat colour which is formed locally in the follicular papilla (FP) and to antagonize the effects of alpha-MSH it operates in a paracrine manner (Lu at al.1994; Blanchard et al. 1995; Siegrist et al. 1997; Suzuki et al. 1997.) In the human hair follicle a similar role may exist for ASP which may be responsible for the site specific variation in hair colour. In addition it has been suggested that attractin functions as an

additional receptor as ASP signalling in mice(Miller et al.1997; Dinulescu et al. 1998; Gunn et al .1999; Nagle et al. 1999). Consequently findings from Kauser et al that alpha MSH was not seen in melanogenically active hair bulb melanocytes and the recent discovery of ASP gene polymorphism strongly associated with dark hair and brown eyes (Kanetsky et al 2002) proposes that the regulation of human pigmentation does not dwell completely at MC-1R. Combined effects of other systems such as B-END, U-opiate R and ASP/attractin signalling may also regulate human hair pigmentation.

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More than 30 human MC1-R variants have been documented and were made known to be associated with pigmentation phenotypes and there is evidence of selection in several populations (reviewed by Rees 2000). There is an increased level of diversity in Europeans and a lot of variants affect MC-1R function. Among individuals with red hair and poor tanning ability certain of these variants (Arg142His, Arg151Cys, Arg160Trp and Asp294His) are common. It was later shown that these mutations result in decrease in intracellular cAMP levels in response to alpha-MSH (Franberg et al. 1998; Schioth et al. 1999) which in turn would inevitably lead to decrease in eumelanogenesis. Consequently this leads to an increase pheomelanin production which would explain not only the red hair phenotype but also the poor tanning ability in carrying these mutations.

Uncertainties have arisen as to whether the MC-1R is the solitary control point for the red hair phenotype. There have been studies in individuals that do not have red hair that show homozygous and compound heterozygous mutations of the MC-1R (Smith et al. 1998; Rees et al. 1999; Rees. 2000). Moreover there have been studies that showed melanocyte cultures from redheads that were unresponsive to alpha-MSH also did not respond to cAMP with increased levels of melanin content signifying that the unresponsiveness was due to factors downstream of the MC-1R (Hunt et al 1996)

In human melanoma cells in culture there appears to be very little doubt that alpha-MSH is able to stimulate melanogenesis. Nevertheless alpha MSH appears to act specifically to stimulate the synthesis of eumelanin rather than merely escalating the production of melanin. Basis for the increases in skin darkening in humans observed by Learner et al in 1960s could be explained by such an action and it is also in keeping with the observation that alpha MSH has a pigmentary role in human as it does in other mammals. To accept this observation we look at the origin of the alpha MSH. Unlike most mammals the human pituitary produces only small amounts of alpha-MSH and therefore it seems logical to suggest that alpha MSH is locally synthesized rather than of pituitary origin which is involved in the regulation of skin pigmentation.

This study has established that alpha-MSH has potent dendritogenic effects on melanoma cells. There is also evidence that alpha MSH stimulates melanocyte dendricity#9Hunt et al 1994a, c; Wakamatsu et al. 1997). The roles that alpha MSH plays in regulation of dendricity may be very important for pigmentary response as dendrites are significant for the formation of the epidermal-melanin unit and the transfer of melanin. It’s not clearly understood how alpha-MSH acts to promote melanocyte dendricity nevertheless this mechanism may be reliant upon activation of several intracellular pathways. cAMP appears to be likely play an important role and there is evidence to support this. cAMP increases actin disorganization hence stimulate melanocyte dendricity by acting on the GTP-binding proteins Rac and Rho( Busca et al. 1998; Scott and Cassidy. 1998;Busca and Balloti. 2000).

The acetylated form of alpha MSH is believed to be an important regulator of the pigmentary response in lower vertebrates and many mammals. Nonetheless desacetyl alpha-MSH is the principal form of alpha-MSH in human skin, as in the hypothalamus (Thody et al. 1983; Wakamatsu et al 1997). Desacetyl alpha-MSH binds to the human MC-1R with an affinity lower than that of acetylated alpha-MSH (Tsatmali et al. 1999a) and on these grounds is often dismiss as pigmentary importance. Current findings propose that deacetyl alpha-MSH acts as a partial agonist at the human MC1-R and consequently by opposing the actions of acetylated alpha-MSH could control melanogenic responses (Yukitake et al. 1999). On the melanophores of the Anolis lizard it has been revealed that deacetyl alpha-MSH acts in a comparable way to oppose the actions of alpha-MSH (McCormack et al. 1982).

This study was used as a model system to show that different cell lines have different pigmentation levels. Kauser et al have shown that in hair follicles the expression of alpha-MSH was prominent in the hair bulb matrix and outer root sheath of anagen vI hair follicles

Hair follicle melanocytes derived from dark haired individuals (dark brown and black) were the most responsive to alpha MSH stimulation compared with those from hair follicles of a lighter colour. In support of this it has been shown that alpha-MSH binding sites may be linked to hair colour; high number of alpha-MSH binding sites were detected on human scalp hair bulbs derived from pigmented hair and fewer binding sites were demonstrated on hair bulbs derived from blond hair and were absent from bulbs derived from senile white hair.

The responsiveness of EM to alpha-MSH stimulation is thought to reflect differences in the extent of peptide binding to MC-1R and its subsequent signalling activity. Unresponsiveness to these peptides is reported in EM derived from individuals with red hair and fair skin. Such control points that modulate ORS/hair bulb matrix melanocyte matrix melanocyte differentiation into pigment producing cells may operate in the human hair follicle. Furthermore MC-1R polymorphisms associated with red hair and fair skin in northern Europeans have also been reported in red haired individuals with African ancestry. However a pale complexion is not observed in these individuals suggesting that MC-1R polymorphisms may have greater effects on hair colour than on skin colour.

In human distinct body site variation in hair colour is observed;for example some males exhibit eumelnogenic scalp hair and have pheomelanogenic beards. BY comparison little difference is seen in the epidermis. In mice, coat colour is also regulated by ASP which is produced locally in the FP and acts as a paracrine manner to antagonize the effects of Alpha-MSH. A similar role may exist for ASP in the human hair follicle which may be responsible for the site specific variation in hair colour. Furthermore attractin has been proposed to function as an additional receptor as ASP signalling in mice. Thus our finding that alpha MSH was undetectable in melanogenically active hair bulb melanocytes and the recent detection of ASP gene polymorphism strongly associated with dark hair and borwn eyes suggest that the regulation of human pigmentation does not reside exclusively at MC-1R. Human hair pigmentation may be additionally regulated by combined effects of other systems such as B-END.U-opiate R and ASP/attractin signalling.

Its now well documented that alpha-MSH acting via MC-1R stimulates melanogenesis by activating the adenylate cyclase system. Alpha-MSH has a proliferative effect on melanoma cells. Through its binding to the MC-1R the biological action of alpha MSH is mediated on melanoma cells. MC-1R is a G protein coupled receptor so when alpha-MSH binds to the MC-1R it activates a G protein which in turn activates adenylate cyclase and the resulting increased intracellular concentration of cAMP mediates the melanogenic and morphological responses to alpha MSH.

In human melanoma cells in culture there appears to be very little doubt that alpha-MSH is able to stimulate melanogenesis. Nevertheless alpha MSH appears to act specifically to stimulate the synthesis of eumelanin rather than merely escalating the production of melanin. Basis for the increases in skin darkening in humans observed by Learner et al in 1960s could be explained by such an action and it is also in keeping with the observation that alpha MSH has a pigmentary role in human as it does in other mammals. To accept this observation we look at the origin of the alpha MSH. Unlike most mammals the human pituitary produces only small amounts of alpha-MSH and therefore it seems logical to suggest that alpha MSH is locally synthesized rather than of pituitary origin which is involved in the regulation of skin pigmentation.

This study has established that alpha MSH has potent dendritogenic effects on melanoma cells. Therefore alpha-MSH stimulated melanoma cells make more melanised pigment granules to assist more active transfer of this melanin to recipient keratinocytes. The alpha-MSH /MC1R system is confined to a subpopulation of less differentiated melanocytes located in the hair follicle and appeared to be down regulated in the highly pigmented and highly differentiated bulbar melanocyte sub population.

On the whole alpha MSH is able to promote follicular melanogenesis and up regulates melanogenesis, proliferation and dendritcity in less differentiated melanocyte subpopulations.

The role that Alpha MSH plays in regulation of dendricity may be very important for pigmentary response as dendrites are significant for the formation of the epidermal-melanin unit and the transfer of melanin. It’s not clearly understood how alpha-MSH acts to promote melanocyte dendricity nevertheless this mechanism may be reliant upon activation of several intracellular pathways. cAMP appears to be likely play an important role and there is evidence to support this. cAMP increases actin disorganization hence stimulate melanocyte dendricity by acting on the GTP-binding proteins Rac and Rho.

Evidence to prove this, is given by the recent report that showed a nonsense mutation in a patient’s POMC gene which resulted in a total absence of alpha-MSH of those with red hair and light skin

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