Regulation of pigmentation in human epidermal melanocytes by functional high-affinity beta-melanocyte-stimulating hormone/melanocortin-4 receptor signaling.
2009
Abstract
To date, the principal receptor considered to regulate human pigmentation is the melanocortin-1 receptor (MC1-R) via induction of the cAMP/protein kinase A pathway by the melanocortins alpha-MSH and ACTH. In this context, it is noteworthy that beta-MSH can also induce melanogenesis, although it has a low affinity for the MC1-R, whereas the preferred receptor for this melanocortin is the MC4-R. Because beta-MSH is present in the epidermal compartment, it was of interest to ascertain whether functioning MC4-Rs are present in human epidermal keratinocytes and melanocytes. Our results provide evidence that the MC4-R is expressed in situ and in vitro throughout the human epidermis at the mRNA and protein level using RT-PCR, Western blotting, and double immunofluorescence staining. Moreover, radioligand binding studies yielded high-affinity receptors for beta-MSH on epidermal melanocytes (3600 receptors per cell), undifferentiated keratinocytes (7200 receptors per cell), and differentiated keratinocytes (72,700 receptors per cell), indicating that MC4-R expression correlates with epidermal differentiation. Importantly, increased melanogenesis after stimulation of the beta-MSH/cAMP/microphthalmia-associated transcription factor/tyrosinase cascade proved the functionality of this signal in melanocytes, which was attenuated in the presence of the specific MC4-R antagonist HS014. In summary, our results imply an important role for the beta-MSH/MC4-R cascade in human melanocyte biology, although the function and purpose of this signal in keratinocytes needs further elucidation.
Discussion
It has recently been shown that the POMC-derived peptide MSH is expressed throughout the human epidermis in keratinocytes and melanocytes as well as within its specific organelle (melanosome). This melanocortin is capable of inducing melanogenesis in vitro as well as in vivo, whereas binding proteins for -MSH on both keratinocytes and Cloudman melanoma cells have been shown earlier. Here it is noteworthy that these proteins have not been further characterized. Recently, the presence of MC4-R has been reported in dermal papilla cells, and a reduced gene expression in the skin of patients with vitiligo indicated the presence of MC4-R in this tissue. Because Schioth et al. concluded that the receptor with the strongest affinity for -MSH is the MC4-R, we asked the question whether functioning -MSH receptors are present in the epidermal compartment. Our data demonstrate that the MC4-R is indeed expressed throughout the entire epidermis at the mRNA and protein level. In situ immunofluorescence staining indicates a definite increase in expression with keratinocyte differentiation. This observation is supported by invitro immunostaining as well as MSH binding studies, demonstrating high-affinity -MSH binding sites on melanocytes, undifferentiated keratinocytes, and differentiated keratinocytes, with 3,600, 7,200, and 72,700 receptors per cell, respectively. Despite receptor numbers being higher on keratinocytes, the specific affinity for -MSH is much lower compared with melanocytes. One possible explanation for the decrease in binding affinity could be that an increase in Calcium concentration can dramatically increase the affinity of the -MSH binding to its receptors. Because Ca levels increase in the upper layers of the epidermis, the requirement for a high-affinity receptor could be reduced. However, the function for the -MSH /MC4-R cascade in keratinocytes remains to be established.
Although the function in keratinocytes is still unclear, the data presented herein provide unambiguous proof that the -MSH/MC4-R signal provokes melanogenesis in melanocytes. Taking into consideration that these receptors stimulate the cAMP/MITF/tyrosinase cascade in association with increased melanogenesis, which is attenuated by the specific antagonist HS014, we conclude that the presence of a functioning -MSH/MC4-R axis is a major contributor in the regulation of melanogenesis. Previously, MC1-R densities on melanocytes after NDP--MSH binding studies yielded only 700 low-affinity receptors per melanocyte, as reported by Donatien et al., compared with 3600 high-affinity MC4-Rper cell, as shown above. At this point, it is tempting to invite a critical reevaluation on the role of the MC1-R as the major melanin inducing system in melanocytes.
Design, Synthesis, and Biological Studies of Efficient Multivalent Melanotropin Ligands: Tools toward Melanoma Diagnosis and Treatment.
2011
Abstract
To achieve early detection and specific cancer treatment, we propose the use of multivalent interactions in which a series of binding events leads to increased affinity and consequently to selectivity. Using melanotropin (Melanotan II) ligands, our aim is to target melanoma cells which overexpress melanocortin receptors. In this study, we report the design and efficient synthesis of new trivalent ligands bearing MSH ligands. Evaluation of these multimers on a cell model engineered to overexpress melanocortin 4 receptors (MC4R) showed up to a 350-fold increase in binding compared to the monomer, resulting in a trivalent construct with nanomolar affinity starting from a micromolar affinity ligand. Cyclic adenosine monophosphate (cAMP) production was also investigated, leading to more insights into the effects of multivalent compounds on transduction mechanisms.
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