Faculty Bibliography

Expression of internal receptors for MSH is an important criterion for responsiveness to MSH by Cloudman melanoma cells (Orlow et al: J. Cell. Physiol., 142:129-136, 1990). Here, we show that internal and external receptors for MSH are of identical molecular weights (50-53 kDa) and share common antigenic determinants, indicating a structural relationship between the 2 populations of molecules. The internal receptors co-purified with a sub-cellular fraction highly enriched for small vesicles, many of which were coated. Ultraviolet B light (UVB) acted synergistically with MSH to increase tyrosinase activity and melanin content of cultured Cloudman melanoma cells, consistent with previous findings in the skin of mice and guinea pigs (Bolognia et al: J. Invest. Derm., 92:651-656, 1989). Preceding the rise in tyrosinase activity in cultured cells, UVB elicited a decrease in internal MSH binding sites and a concomitant increase in external sites. The time frame for the UVB effects on MSH receptors and melanogenesis, 48 hours, was similar to that for a response to solar radiation in humans. Together, the results indicate a key role for MSH receptors in the induction of melanogenesis by UVB and suggest a potential mechanism of action for UVB: redistribution of MSH receptors with a resultant increase in cellular responsiveness to MSH

Interactions between beta-melanotropin (MSH), interleukin 1-a (IL-1), and ultraviolet light (UV) were examined in Cloudman S91 mouse melanoma and RHEK human squamous carcinoma cell lines. The following points were established: 1) both cell lines produced IL-1 and their production was stimulated by exposure of the cells to UV; 2) both cell lines possessed high affinity binding sites for MSH, and their ability to bind MSH was modulated by IL-1; 3) IL-1 exhibited both stimulatory and inhibitory effects on MSH binding to Cloudman cells; and 4) the stimulatory effect of IL-1 on MSH binding to melanoma cells was reflected in enhanced cellular responsiveness to MSH regarding tyrosinase activity (E.C. 1.14.18.1) and melanin content. The findings raise the possibility that interactions between keratinocytes and melanocytes may be regulated by IL-1 and MSH, and suggest a possible mechanism for stimulation of cutaneous melanogenesis by solar radiation: enhancement of MSH receptor activity by induction of IL-1

Treatment of Cloudman S91 melanoma cells with retinoic acid (RA) inhibits MSH-induced tyrosinase activity and melanin formation [(1990) J. Invest. Dermatol. 94, 461-464]. We report here, however, that in spite of inhibiting MSH-induced pigmentation, RA treatment caused a marked increase in MSH binding capacity for both cell surface and internal MSH binding sites. The stimulation was dose- and time-dependent and reversible, with half-maximal effects seen at 2 microM RA. Stimulation of MSH binding was seen as early as 3 h after exposure of cells to RA. Cell surface and internal binding activity increased in concert. Scatchard analysis indicated that increased MSH binding resulted from a 3-4-fold increase in the number of sites with no significant difference in their affinity for MSH. It appears that in suppressing MSH-induced melanogenesis, RA elicited a compensatory up-regulation of the MSH receptor system

Retinoic acid, hexamethylene bisacetamide, sodium butyrate, and dimethylsulfoxide, four compounds which modulate phenotypic expression in a variety of neoplastic cell lines, all inhibited the induction of tyrosinase activity and melanogenesis by the combination of melanocyte-stimulating hormone and isobutylmethyxanthine in Cloudman S91 melanoma cells. Results were the same in assays of whole cells or in extracts made from them. Only retinoic acid, however, was effective at inhibiting the activation of dopachrome isomerase, another regulatory enzyme in melanogenesis. Despite inhibiting the effects of melanocyte-stimulating hormone (MSH) and isobutylmethylxanthine on tyrosinase activity, all of the agents tested increased the binding of MSH to intact cells. Ultrastructural analysis of treated cells following DOPA cytochemistry revealed that both retinoic acid and hexamethylene bisacetamide arrested melanosomal maturation at stage I-II. Retinoic acid resulted in a derangement of melanosomal structure. The specificity of these agents for preventing the induction of melanogenesis makes them powerful tools for the dissection of this complex cellular process

We describe a patient with two coexistent cutaneous eruptions: (1) trauma-induced bullae of the distal extremities and elbows and (2) multiple concentric gyrate lesions on the trunk and extremities, some of which became bullous. The gyrate lesions were stationary and nonpruritic. Biopsy of both types of lesions showed a subepidermal blister and a minimal inflammatory infiltrate. Direct immunofluorescence revealed linear deposition of IgG and C3 at the dermoepidermal junction and indirect immunofluorescence was negative. By immunoelectron microscopy, these immune deposits were localized to the lower lamina lucida. The eruption was not controlled despite high-dose (80 mg/d) oral administration of prednisone and required the addition of an oral administration of methotrexate (20 mg weekly). On further evaluation, an intraductal mammary carcinoma was detected. Following radiation therapy, the methotrexate and prednisone therapy were tapered without recurrence of the eruption during a follow-up period of 18 months

Melanocyte-stimulating hormone (MSH) induces melanogenesis in Cloudman mouse melanoma cells. The activities of two enzymes in the melanogenesis pathway, tyrosinase and dopachrome conversion factor, are increased as part of the induction process. Trans retinoic acid (RA), at concentrations as low as 0.1 nM, inhibited the induction of tyrosinase, dopachrome conversion factor, and melanogenesis, but had no effect on the basal levels of either enzyme or of cellular melanin content. Half-maximal effects of RA occurred at a concentration of 10 nM; maximal effects were observed at 1 microM. The effects of RA on melanogenesis were independent of its effects on cellular growth since one Cloudman line tested was growth-inhibited by RA and another was growth-stimulated by RA, but the induction of melanogenesis by MSH in both lines was inhibited by RA. Mixing experiments with cell lysates failed to demonstrate the induction of a tyrosinase inhibitor by RA. The effects of RA were not limited to MSH or to Cloudman melanoma cells since RA blocked cholera toxin-inducible melanogenesis in Cloudman cells, as well as the induction of tyrosinase activity by L-tyrosine in Bomirski hamster melanoma cells. The effects of RA were specific to melanogenesis, however, since RA did not interfere with MSH-induced changes in cellular morphology and growth. Thus, RA appears to be a new and potent tool for understanding mechanisms regulating induction of the pigmentary system

Cloudman S91 mouse melanoma cells express both external (plasma membrane) and internal binding sites for MSH. Using 125I-beta melanotropin (beta-MSH) as a probe, we report here an extensive series of studies on the biological relevance of these internal sites. Cells were swollen in a hypotonic buffer and lysed, and a particulate fraction was prepared by high-speed centrifugation. This fraction was incubated with 125I-beta-MSH with or without excess nonradioactive beta-MSH in the cold for 2 hours. The material was then layered onto a step-wise sucrose gradient (8-80%) and centrifuged (156,000g, 60 min); fractions were collected and counted in a gamma counter or assayed for various enzymatic activities. The following points were established: 1) Specific binding sites for MSH were observed sedimenting at an average density of 50% sucrose in amelanotic cells and at higher densities in melanotic cells. 2) These sites were similar in density to those observed when intact cells were labeled externally with 125I-beta-MSH and then warmed to promote internalization of the hormone. 3) Most of the internal binding sites were not as dense as fully melanized melanosomes. 4) In control experiments, the MSH binding sites were not found in cultured hepatoma cells. 5) Variant melanoma cells, which differed from the wild-type in their responses to MSH, had reduced expression of internal binding sites even though their ability to bind MSH to the outer cell surface appeared normal. (MSH-induced responses included changes in tyrosinase, dopa oxidase, and dopachrome conversion factor activities, melanization, proliferation, and morphology.) 6) Isobutylmethylxanthine, which enhanced cellular responsiveness to MSH, also enhanced expression of internal binding sites. The results indicate that expression of internal binding sites for MSH is an important criterion for cellular responsiveness to the hormone