Proopiomelanocortin (POMC), the ACTH/melanocortin precursor, is secreted by human epidermal keratinocytes and melanocytes and stimulates melanogenesis.

Proopiomelanocortin (POMC) can be processed to ACTH and melanocortin peptides. However, processing is incomplete in some tissues, leading to POMC precursor release from cells. This study examined POMC processing in human skin and the effect of POMC on the melanocortin-1 receptor (MC-1R) and melanocyte regulation. POMC was secreted by both human epidermal keratinocytes (from 5 healthy donors) and matched epidermal melanocytes in culture. Much lower levels of alpha-MSH were secreted and only by the keratinocytes. Neither cell type released ACTH. Cell extracts contained significantly more ACTH than POMC, and alpha-MSH was detected only in keratinocytes. Nevertheless, the POMC processing components, prohormone convertases 1, 2 and regulatory protein 7B2, were detected in melanocytes and keratinocytes. In contrast, hair follicle melanocytes secreted both POMC and alpha-MSH, and this was enhanced in response to corticotrophin-releasing hormone (CRH) acting primarily through the CRH receptor 1. In cells stably transfected with the MC-1R, POMC stimulated cAMP, albeit with a lower potency than ACTH, alpha-MSH, and beta-MSH. POMC also increased melanogenesis and dendricity in human pigment cells. This release of POMC from skin cells and its functional activity at the MC-1R highlight the importance of POMC processing as a key regulatory event in the skin.

Modulation of the human hair follicle pigmentary unit by corticotropin-releasing hormone and urocortin peptides.

Human skin is a local source of corticotropin-releasing hormone (CRH) and expresses CRH and CRH receptors (CRH-R) at mRNA and protein levels. Epidermal melanocytes respond to CRH by induction of cAMP with up-regulation of pro-opiomelanocortin gene expression and subsequent production of adrenocorticotropin hormone. However, the role of CRH/CRH-R in melanocyte biology is complicated by the significant heterogeneity of cutaneous melanocyte subpopulations, from continuously active and UV-responsive melanocytes in epidermis to UV nonresponsive, hair growth cycle-coupled melanogenesis in hair follicles. In the present study we report that normal human scalp hair follicle melanocytes express CRH at the mRNA level. Furthermore, CRH, urocortin and CRH-R 1 and 2 were differentially expressed in follicular melanocytes, fibroblasts, and keratinocytes depending on anatomic location and differentiation status in situ and in vitro. Stimulation of follicular melanocytes with CRH and CRH peptides, modified for selectivity for CRH-R1 and/or CRH-R2, variably induced cell melanogenesis, dendricity, and proliferation. CRH-peptides also stimulated the expression and activity of Tyrosinase, and expression of Tyrosinase-related protein-1 and-2. However, a modified urocortin peptide highly selective for CRH-R2 down-regulated melanocyte differentiation phenotype. This study indicates that CRH peptides can differentially influence hair follicle melanocyte behavior not only via CRH-R1 signaling but also by complex cross-talk between CRH-R1 and CRH-R2.

Corticotropin releasing hormone and the skin.

Cotricotropin-releasing hormone (CRH) and related peptides are produced in skin that is dependent on species and anatomical location. Local peptide production is regulated by ultraviolet radiation (UVR), glucocorticoids and phase of the hair cycle. The skin also expresses the corresponding receptors (CRH-R1 and CRH-R2), with CRH-R1 being the major receptor in humans. CRH-R1 is expressed in epidermal and dermal compartments, and CRH-R2 predominantly in dermal structures. The gene coding for CRH-R1 generates multiple isoforms through a process modulated by UVR, cyclic adenosine monophosphate (cAMP) and phorbol 12-myristate 13-acetate. The phenotypic effects of CRH in human skin cells are largely mediated by CRH-R1alpha through increases in concentrations of cAMP, inositol triphosphate (IP3), or Ca2+ with subsequent activation of protein kinases A (PKA) and C (PKC) dependent pathways. CRH also modulates the activity of nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-kappaB), activator protein 1 (AP-1) and cAMP responsive element binding protein (CREB). The cellular functions affected by CRH depend on cell type and nutritional status and include modulation of differentiation program(s), proliferation, viability and immune activity. The accumulated evidence indicates that cutaneous CRH is also a component of a local structure organized similarly to the hypothalamo-pituitary-adrenal axis.

A fully functional proopiomelanocortin/melanocortin-1 receptor system regulates the differentiation of human scalp hair follicle melanocytes.

The proopiomelanocortin (POMC)-derived peptides, ACTH and alpha-MSH, are the principal mediators of human skin pigmentation via their action at the melanocortin-1 receptor (MC-1R). Recent data have demonstrated the existence of a functionally active beta-endorphin/mu-opiate receptor system in both epidermal and hair follicle melanocytes, whereby beta-endorphin can regulate melanogenesis, dendricity, and proliferation in these cells. However, a role for ACTH and alpha-MSH in the regulation of the human follicular pigmentary unit has not been determined. This study was designed to examine the involvement of ACTH and the alpha-MSH/MC-1R system in human follicular melanocyte biology. To address this question we employed RT-PCR and immunohisto/cytochemistry, and a functional role for these POMC peptides was assessed in follicular melanocyte cultures. Human scalp hair follicle melanocytes synthesized and processed POMC. ACTH and alpha-MSH in association with their processing enzymes and MC-1R are expressed in human follicular melanocytes at the message level in vitro and at the protein level both in situ and in vitro. The expression of the POMC/MC-1R receptor system was confined only to subpopulations of poorly and moderately differentiated melanocytes. In addition, functional studies revealed that ACTH and alpha-MSH are able to promote follicular melanocyte differentiation by up-regulating melanogenesis, dendricity, and proliferation in less differentiated melanocyte subpopulations. Thus, these findings suggest a role for these POMC peptides in regulating human hair follicle melanocyte differentiation.

A novel mechanism in control of human pigmentation by {beta}-melanocyte-stimulating hormone and 7-tetrahydrobiopterin.

The human skin holds the full machinery for pro-opiomelanocortin processing. The alpha-melanocyte-stimulating hormone (alpha-MSH)/melanocortin-1-receptor cascade has been implicated as a major player via the cAMP signal in the control of melanogenesis. Only very recently the beta-endorphin/mu-opiate receptor signal has been added to the list of regulators of melanocyte dendricity and melanin formation. In this context it was reported that (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)) can act as an allosteric inhibitor of tyrosinase, the key enzyme in melanogenesis, and this inhibition is reversible by both alpha- and beta-MSH. It was also shown earlier that 7BH(4), the isomer of 6BH(4), is twice as active in this inhibition reaction. However, as yet it is not known whether 7BH(4) is indeed present in loco in the melanosome. We here provide evidence that this isomer is present in this organelle in a concentration range up to 50 x 10(-6) M. Determination of beta-MSH in melanosomal extracts yielded 10 pg/mg protein. Moreover, we demonstrate reactivation of the 7BH(4)/tyrosinase inhibitor complex by beta-MSH, whereas alpha-MSH failed to do so. Furthermore, we show intra-melanosomal l-dopa formation from dopachrome by 7BH(4) in a concentration range up to 134 x 10(-6) M. Based on these results, we propose a new receptor-independent mechanism in the control of tyrosinase/melanogenesis by beta-MSH and the pterin 7BH(4).

Serotoninergic and melatoninergic systems are fully expressed in human skin.

We investigated the cutaneous expression of genes and enzymes responsible for the multistep conversion of tryptophan to serotonin and further to melatonin. Samples tested were human skin, normal and pathologic (basal cell carcinoma and melanoma), cultured normal epidermal and follicular melanocytes, melanoma cell lines, normal neonatal and adult epidermal and follicular keratinocytes, squamous cell carcinoma cells, and fibroblasts from dermis and follicular papilla. The majority of the samples showed simultaneous expression of the genes for tryptophan hydroxylase, arylalkylamine N-acetyltransferase (AANAT), and hydroxyindole-O-methyltransferase (HIOMT). The products of AANAT activity were identified by RP-HPLC with fluorimetric detection in human skin and in cultured normal and malignant melanocytes and immortalized keratinocytes; HIOMT activity was detected in human skin, keratinocytes, and melanoma cells. N-acetylserotonin (NAS) was detected by RP-HPLC in human skin extracts. NAS identity was confirmed further by LC/MS in keratinocytes. In conclusion, we provide evidence that the human skin expresses intrinsic serotonin and melatonin biosynthetic pathways.

beta-Endorphin as a regulator of human hair follicle melanocyte biology.

The pro-opiomelanocortin (POMC)-derived peptides, alpha-melanocyte-stimulating hormone, and adrenocorticotropic hormone, are important mediators of human skin pigmentation via action at the melanocortin-1 receptor. Recent data suggests that such a regulatory role also exists for the endogenous opiate, beta-endorphin (beta-END). A role for this beta-END in the regulation of follicular pigmentation, however, has not been determined. This study was designed to examine the involvement of the beta-END/mu-opiate receptor system in human follicular melanocyte biology. We employed RT-PCR, and immunohisto/cytochemistry and immunoelectron microscopy using beta-END and mu-opiate receptor specific antibodies and a functional role for beta-END was assessed by direct stimulation with the peptide. This study has demonstrated that human hair follicle melanocytes (HFM) express mRNA for the mu-opiate receptor and POMC. Furthermore, beta-END and its high affinity mu-opiate receptor are expressed at the protein level in glycoprotein100-positive follicular melanocytes and as a function of their anatomic location and differentiation status during the hair growth cycle. Functional studies revealed that beta-END is a modifier of HFM phenotype via its ability to upregulate melanogenesis, dendricity, and proliferation. These findings suggest a new regulatory role for beta-END in human HFM biology, providing a new research direction into the fundamental regulation of human hair pigmentation.

Regulation of human epidermal melanocyte biology by beta-endorphin.

beta-Endorphin is an opioid peptide cleaved from the precursor pro-hormone pro-opiomelanocortin, from which other peptides such as adrenocorticotropic hormone, beta-lipotropic hormone, and alpha-melanocyte-stimulating hormone are also derived. alpha-Melanocyte-stimulating hormone and adrenocorticotropic hormone are well documented to regulate human skin pigmentation via action at the melanocortin-1 receptor. Whereas plasma beta-endorphin is reported to increase after exposure to ultraviolet radiation, to date a functional role for beta-endorphin in the regulation of human epidermal melanocyte biology has not been demonstrated. This study was designed to examine the involvement of the beta-endorphin/mu-opiate receptor system in human epidermal melanocytes. To address this question we employed reverse transcription-polymerase chain reaction, and immunohistochemistry/cytochemistry and immunoelectron microscopy using beta-endorphin and mu-opiate receptor specific antibodies. A functional role for beta-endorphin was assessed in epidermal melanocyte cultures by direct stimulation with the peptide. This study demonstrated the expression of mu-opiate receptor mRNA in cultured epidermal melanocytes, as well as mRNA for pro-opiomelanocortin. In addition, we have shown that beta-endorphin and mu-opiate receptor are expressed at the protein level in situ in glycoprotein100-positive melanocytes. The expression of both beta-endorphin and mu-opiate receptor correlated positively with their differentiation status in vitro. Furthermore, immunoelectron microscopy studies revealed an association of beta-endorphin with melanosomes. Functional studies showed that beta-endorphin has potent melanogenic, mitogenic, and dendritogenic effects in cultured epidermal melanocytes deprived of any exogenous supply of pro-opiomelanocortin peptides. Thus, we report that human epidermal melanocytes express a fully functioning beta-endorphin/mu-opiate receptor system. In the absence of any data showing cross-talk between the mu-opiate receptor and the melanocortin-1 receptor, we conclude that the beta-endorphin/mu-opiate receptor system participates in the regulation of skin pigmentation.

Beta-endorphin: the forgotten hair follicle melanotropin.

The proopiomelanocortin (POMC) gene and protein are expressed principally in the pituitary and brain (e.g., hypothalamus). The POMC gene, protein, and derived peptides, however, can also be detected in the skin. It appears that POMC can also be processed in the skin, a tissue that also expresses the prohormone convertases PC1 and PC2 and 7B2 protein. All POMC peptides may be produced in the skin epidermis, dermis, and adnexa by epithelial cells, melanocytes, and mesenchymal cells (e.g., immune cells, fibroblasts, and endothelial cells), and can also be released from cutaneous sensory nerve endings (cf. Slominski et al, 2004).

Simple and rapid method to isolate and culture follicular papillae from human scalp hair follicles.

Study of the involvement of the hair follicle papilla in hair growth regulation was greatly facilitated by the isolation and cultivation of this tiny cluster of fibroblast-like cells in the rat vibrissae and in the human hair follicle. While isolation of the hair follicle papilla from the former is relatively straightforward, the current method to isolate the much smaller human hair follicle requires significant skill. Thus, the routine initiation of primary cultures of human scalp hair follicle papilla cells requires significant training, time, and commitment. In an attempt to simplify hair follicle papilla cell culture methodology for new laboratory personnel, we have made significant refinements to the current method. Our method requires only two simple manipulations to isolate hair follicle papilla from intact isolated hair follicles. This very rapid and easy method isolates clean and intact hair follicle papillae. Together with their attachment via scratching to the growth surface, the isolation and cultivation of this important hair follicle component can now be achieved easily by the laboratory newcomer. The method relies for its simplicity on the removal of the hair follicle papilla from the outside of the intact hair follicle rather than via internal manipulations from within the hair follicle.

Functional activity of serotoninergic and melatoninergic systems expressed in the skin.

We tested the expression of genes coding receptors of a cutaneous serotoninergic/melatoninergic system in whole human skin and in normal and pathologic cultured skin cells. Evaluation of serotonin (5HT), melatonin (MT), and melatonin-related receptors (MRR) showed expression of the isoforms 5HT2B, 5HT7, and MT1 genes in almost all the tested samples. Expression of other isoforms was less prevalent; 5HT2C, MRR, and MT2 were rarely detected. We also found novel isoforms for MT2, MRR, and 5HT2B and documented the process of RNA editing for 5HT2C. Testing for functional activity of these receptors with serotonin and melatonin (10(-14) to 10(-10) M) showed variable effects depending on cell type and culture conditions. Thus, serotonin stimulated proliferation of melanocytes in medium deprived of growth factors, while inhibiting cell growth in the presence of growth factors. Melatonin inhibited both apoptosis of HaCaT keratinocytes incubated in serum-free media, and proliferation of cells cultured in medium supplemented with serum. Melatonin also increased the numbers of viable fibroblasts incubated in serum free medium. N-acetylserotonin (NAS) and 5 methoxytryptamine (5MTT) were generally without effect on cell proliferation, with the exception of an inhibition of melanocyte proliferation at the higher 5MTT concentration of 10(-10) M. Thus, skin cells represent a true target for the products of the serotoninergic/melatoninergic cutaneous pathway with their actions modulating cell proliferation or viability.

In situ and in vitro evidence for DCoH/HNF-1 alpha transcription of tyrosinase in human skin melanocytes.

Human epidermal melanocytes hold the full capacity for autocrine de novo synthesis/regulation/recycling of the essential cofactor 6-tetrahydrobiopterin (6BH(4)) for conversion of L-phenylalanine via phenylalanine hydroxylase to L-tyrosine and for production of L-Dopa via tyrosine hydroxylase to initiate both pigmentation and catecholamine synthesis in these neural crest-derived cells. Earlier we have demonstrated pterin-4a-carbinolamine dehydratase (PCD) mRNA and enzyme activities in epidermal melanocytes and keratinocytes. This protein dimerises also the transcription factor hepatocyte nuclear factor 1 (HNF-1), leading to activation of multiple genes. This study demonstrates for the first time DCoH/HNF-1 alpha expression and transcriptional activity in human epidermal melanocytes in vitro and in situ and identified tyrosinase, the key enzyme for pigmentation, as a new transcriptional target. Specific binding of DCoH/HNF-1 complex to the human tyrosinase promoter was confirmed by gel shift analysis. These results provide a novel mechanism in the regulation of skin pigmentation.