Walter Siegrist

INTERACTIONS OF ALPHA -MELANOTROPIN AND AGOUTI ON B16 MELANOMA CELLS : EVIDENCE FOR INVERSE AGONISM OF AGOUTI

alpha-Melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin) and agouti control the switch between eumelanin and pheomelanin synthesis in mammalian melanocytes. Here we investigated interactions between alpha-MSH, agouti protein, cAMP elevating agents and phorbol ester on mouse B16 melanoma cells. Agouti (Kd 3.7 nmol/l) and alpha-MSH (Kd 2.3 nmol/l) had similar affinities to the MC1 melanocortin receptor. Both alpha-MSH and agouti induced MC1 receptor down-regulation. Agouti antagonized melanogenesis induced by alpha-MSH, forskolin, cholera toxin (CT), and pertussis toxin (PT). It also reduced the constitutive melanin formation of long-term cultures. Cell proliferation was inhibited by agouti (43% at 100 nM). This effect was reversed by alpha-MSH, forskolin, or CT. B16-G4F cells, a cell variant that lacks the MC1 receptor, did not respond to agouti. From these results we conclude that agouti shows the characteristics of an inverse agonist acting through the MC1 receptor.

Radioreceptor Assay for α;-Msh Using Mouse B16 Melanoma Cells

A radioreceptor assay for a-MSH is described which is based on cultured llpouse B16 melanoma cells and bioactive monoiodinated [Nle 1-a-MSH tracer. The assay was used (1) to study the binding characteristics of a-MSH to 816 cells, (2) to determine the relative binding activity of MSH peptides, and (3) to measure MSH in tissue extracts. The association of a-MSH to 816 cells reached a stable plateau after 3 h at 15OC. At 25O or 37OC, the binding was transient and at O-lOC, the association was very slow. The hormone-receptor complex was relatively stable between Oo and 15OC whereas a 50% dissociation was reached after 90 min at 25OC and after 35 min at 37OC. The mean KD for a-MSH of four saturation experiments was 1.3 nM and the number of receptors 9570 per cell. 1,lOPhenanthroline had a stabilizing effect in the binding assay when used at a 0.3 mM concentration. From the MSH peptides tested in the binding assay, some showed similar potencies in three bioassays (tyrosinase, melanin and Anolis skin), whereas others displayed considerably

MELANIN-CONCENTRATING HORMONE BINDING TO MOUSE MELANOMA CELLS IN VITRO

An analogue of human melanin‐concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr13 was replaced by Phe and Val19 by Tyr. The resulting monoiodinated [125I][Phe13,Tyr19]‐MCH radioligand was biologically active and led to the discovery of high‐affinity binding sites on mouse B16‐F1, G4F and G4F‐7 melanoma cells. Saturation binding analysis with G4F‐7 cells revealed 1090 MCH receptors per cell and a K D of 1.18 × 10−10 mol/l. Receptors for MCH were also found on rat PC12 phaeochromocytoma cells, human RE melanoma cells and COS‐7 cells. Competition binding analyses with other peptides such as α‐MSH, NPY and PACAP demonstrated that MCH receptor binding is specific. rANF(1–28) was found to be a weak competitor of MCH, indicating topological similarities between MCH and rANF(1–28) when interacting with MCH receptors.

Effect of melanin concentrating hormone on pigment and adrenal cells in vitro

Highly purified synthetic salmonid melanin concentrating hormone (MCH) and some analogs were investigated for their ability to concentrate the pigment in scale melanophores of the Chinese grass carp, Ctenopharyngodon idellus, to produce melanin dispersion in frog or lizard melanophores and to inhibit alpha-MSH in its action on mouse melanoma and rat adrenal glomerulosa cells in vitro. In the grass carp, MCH produced half-maximal pigment aggregation at 6 X 10(-11) M and its oxidized form at 7 X 10(-11) M. Replacement of the two methionines at position 3 and 6 with norvaline lowered the potency by a factor of 2.7 and with propargylglycine by a factor of about 7. Linear, Cys5,14-Acm-protected MCH was a full agonist of MCH but with a 345-fold lower potency. Iodinated MCH showed similar, low activity. In tetrapods, salmonid MCH and its analogs displayed only marginal pigment dispersion at concentrations greater than 10(-5) M. Alkali-treatment of MCH increased the pigment-dispersing potency by a factor of about 30 whereas the activity for pigment aggregation in the grass carp was destroyed. At high concentrations (10(-6), 10(-5) M) MCH also stimulated tyrosinase activity in B-16 mouse melanoma cells but did not modify the effects of alpha-MSH in this system. By contrast, when tested on rat adrenal glomerulosa cells, salmonid MCH had no effect alone but at a concentration of greater than 10(-10) M it slightly reduced corticosterone production by an alpha-MSH concentration of 10(-7) M. Aldosterone production was not affected and MCH did not influence the response to ACTH.

Biologically active monoiodinated α-MSH derivatives for receptor binding studies using human melanoma cells

Three different monoiodinated radioligands of alpha-MSH (alpha-melanocyte-stimulating hormone) were compared in a binding assay with human D10 melanoma cells: [Tyr(125I)2]-alpha-MSH, [Tyr(125I)2,NIe4]-alpha-MSH, and [Tyr(125I)2,NIe4,D-Phe7]-alpha-MSH. They were prepared either by the classical chloramine T method or by the Enzymobead method. A simple and rapid purification scheme was developed consisting of a primary separation on reversed-phase C18 silica cartridges immediately after the iodination, followed by HPLC purification before each binding experiment. Biological testing of the three radioligands showed that they all retained high melanotropic activity in the B16 melanin assay and the Anolis melanophore assay. However, in human D10 melanoma cells, [Tyr(125I)2,NIe4]-alpha-MSH led to a high degree of non-specific binding to the cells which could not be displaced by excess alpha-MSH and only partially by [NIe4]-alpha-MSH. The [Tyr(125I)2,NIe4,D-Phe7]-alpha-MSH tracer gave similar results but with a much lower proportion of non-specific binding. On the other hand, [Tyr(125I)2]-alpha-MSH proved to be an excellent radioligand whose non-specific binding to the D10 cells was not higher than 20% of the total binding.

Melanocortins and their implication in melanoma

The melanocortins (MCs), that is, the melanocyte-stimulating hormones (MSHs) and ACTH, are a group of related peptides containing the typical melanotropin core sequence, His-Phe-Arg-Trp, and are derived from a common precursor, pro-opiomelanocortin. They are pleiotropic molecules that occur in the pituitary, some brain regions, and also in several peripheral tissues, and they exert a variety of physiologic functions. Their effect on melanogenesis in the skin is well established, but their role in melanocyte and melanoma cell proliferation and metastasis is less clear. The recent cloning of five types of MC receptors (MC1-5), new studies on the regulation of these receptors, the discovery of a naturally occurring MSH antagonist, the agouti protein, and the finding that melanocytes and melanoma cells exclusively express MC1 receptors have laid the basis for the future development of specific MC ligands, which may become useful for melanoma diagnosis and eventually therapy.

Region- and stage-specific patterns of melanocortin receptor ontogeny in rat central nervous system, cranial nerve ganglia and sympathetic ganglia

Observations on developmental actions of melanotropic peptides in nervous system have been difficult to interpret in the absence of data on receptor ontogeny. We investigated binding of [125I]Nle4,D-Phe7-alpha-MSH ([125I]NDP) in developing Long Evans rats from gestational day (E) 13 by quantitative autoradiography. Regional [125I]NDP binding characteristics were assessed by competition experiments in early postnatal brain. The study revealed region- and stage-specific, often transient ontogenetic patterns. Sympathetic ganglia exhibit high [125I]NDP binding from E13, with a peak in superior cervical ganglion at E16-E18. The first central [125I]NDP binding sites transiently appear in parts of thalamus between E13 and E15. The early fetal period is characterized by prominent peaks of receptor density in somatosensory and viscerosensory nuclei (trigeminal sensory nuclei, solitary tract nucleus), paralleled by receptor expression in 5th, 7th, 9th and 10th cranial nerve ganglia. During late fetal life, receptor density peaks in dorsal motor nucleus of vagus and inferior olive; binding sites transiently appear in cerebellum. Caudate-putamen, nucleus accumbens, olfactory tubercle and septohippocampal nucleus show a high perinatal maximum. Starting with late fetal piriform cortex, [125I]NDP binding peaks sequentially in cerebral cortical areas, with highest levels in entorhinal cortex. Preoptic, septal, hypothalamic and amygdaloid areas known for elevated receptor densities in adulthood, exhibit a slow, peri- and postnatal receptor ontogeny. Temporal relations to regional developmental processes support the idea of a role of melanocortins during ontogeny.

Receptors for melanocyte-stimulating hormone on melanoma cells.

Most studies of melanocyte-stimulating hormone (MSH) receptors on melanoma cells are based on in uitro model systems, such as B16 or Cloudman S91 mouse melanoma and, more recently, certain human melanoma cell lines. The advantage of using established cell lines is the ease of culturing them in large quantity and the reproducibility of hormonal responses. Also, amelanotic variants of these cells can be isolated which may be better models for metastatic forms. Our present knowledge of MSH effects on melanoma cells stems from studies on (1) proliferation and growth inhibition of these cells in uitro and in uiuo, (2) MSHreceptor binding in uitro and in uiuo, (3) activation of adenylate cyclase and protein kinases, (4) changes in protein phosphorylation, RNA, and protein synthesis in uitro, (5 ) stimulation of tyrosinase and melanin formation in vifro and in uiuo, and (6) cloning and expression of the MSH receptor. These studies form the basis for the potential application of MSH peptides to melanoma tumor diagnosis and therapy. The following review covers some of the relevant findings in these different areas.

Homologous Regulation of the MSH Receptor in Melanoma Cells

We have examined the mechanism of homologous regulation of MSH receptor binding and receptor-mediated adenylate cyclase activation in three human and two mouse melanoma cell lines. Pretreatment with alpha-MSH resulted in a time- and dose-dependent up-regulation of MSH receptors in human D10 and 205 melanoma cells whereas in human HBL and in mouse B16-F1 and Cloudman S91 cells alpha-MSH induced receptor down-regulation. Up-regulation of receptors was maximal after a 24-h incubation period and an alpha-MSH concentration of 100 nM (EC50 = 2.4 nM). The increase in alpha-MSH binding was independent of adenylate cyclase activation and protein synthesis and appeared to be caused by recruitment of spare receptors. The structural requirements of the peptide for triggering this process differed from those found in receptor-binding analyses. Receptor down-regulation was maximal after 12 h and hence more rapid than up-regulation. In B16-F1 cells, 10 nM alpha-MSH caused the disappearance of 85-90% of the MSH receptors, the EC50 of 0.23 nM lying exactly between that for alpha-MSH-induced melanogenesis (0.027 nM) and the dissociation constant of receptor binding (1.31 nM). Down-regulation in B16-F1 cells appears to be the consequence of receptor internalization following MSH binding and seems to be initiated during an early step in MSH signalling, preceding the activation of adenylate cyclase and the cAMP signal. Receptor up- and down-regulation were not accompanied by an alteration in affinity to alpha-MSH, as demonstrated by Scatchard analysis of the binding curves.

QUANTIFICATION OF MSH RECEPTORS ON MOUSE MELANOMA TISSUE BY RECEPTOR AUTORADIOGRAPHY

MSH receptors on mouse melanoma tissue sections were quantified by receptor autoradiography, yielding results which were very similar to those obtained by a conventional receptor binding assay with isolated cells. In order to minimize non-specific binding, it proved to be crucial to use a radioactive monoiodinated MSH radioligand retaining full biological activity and to apply the binding conditions developed for isolated cells to the incubation of whole tissue sections. The displacement curves obtained after quantitative analysis of autoradiograms from tissue sections yielded a KD-value of the same order of magnitude (0.58 nM; average of n = 7 experiments) as those obtained in the normal binding assay with isolated cells (1.2 nM; average of n = 10 experiments). Similarly, receptor numbers per cell on tissue sections (14,700; n = 7) did not differ markedly from those determined with isolated cells (10,500; n = 10). These results demonstrate that receptor autoradiography can be applied to the quantification of peptide hormone receptors on peripheral tissues.