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Aging, The Molecular Concepts

7.2. ACTIVATION OF THE cAMP SIGNALING PATHWAY BY α-MSH AND CHOLERA TOXIN: A TOPSY-TURVY RESPONSE

α-MSH was recently detected in non-pituitary tissues including human skin. α-MSH synthesis and release increases in keratinocytes and melanocytes after exposure to UVB light (reviewed in 8, 9). Human epidermal melanocytes respond to α-MSH and other agents that target the cAMP pathway, such as cholera toxin and forskolin (5, 7, 10-13) by increasing melanin production and melanosome aggregation. These agents have different molecular targets in the cAMP pathway, including the melanocortin 1 receptor (MC1R) for α-MSH, a Gα protein for cholera toxin, and adenylate cyclase itself in the case of forskolin. Subsequently, the enzyme adenylate cyclase is activated with accumulation of the second messenger cAMP.It has been found that prolonged exposure to cholera toxin or α-MSH causes melanocytes isolated from skin types IV-VI to increase their size (Fig. 7.2) and to accumulate high levels of brown/black melanin (Figs. 7.2B and 27-4A). Melanin accumulation eventually results in loss of the proliferative capacity of the heavily pigmented melanocytes (2, 5, 6). However, α-MSH or cholera toxin causes little or no eumelanin accumulation in melanocytes isolated from skin types I and II. Importantly, these melanocytes are able to undergo many more rounds of DNA synthesis than their heavily pigmented counterparts (2). The above results are in agreement with published data (14), demonstrating that melanocytes from different skin types have dissimilar rates of proliferation, and levels and types of melanin accumulated in response to α-MSH. Transcription of the melanogenic enzymes tyrosinase and tyrosinase-related protein 1 (TRP-1) results from binding of the transcription factor microphthalmia (MITF) to a 10-bp sequence termed the M-box (15). cAMP elevating agents increase MITF expression and stimulate its binding to the M-box containing a CATGTG motif and an E-box in the tyrosinase promoter (16, 17). MITF protein levels are induced after 3 h of forskolin treatment and return to basal levels after 24 h (17).

Fig. 7.1.
Fig. 7.1. WRN melanocytes and age-mached controls.
A: melanocytes from a normal adult donor.
B: melanocytes from WRN patient. The melanocyte cultures were obtained from skin biopsies as described in ref. 22.

Fig. 7.2.
Fig. 7.2. Morphological changes and hyperpigmentation in terminally differentiated melanocytes.
A: proliferating melanocytes isolated from Black skin.
B: the same culture, after 4 weeks in medium supplemented with 10nM cholera toxin (2).

It has been observed that treatment with cAMP inducers causes MITF message levels to increase up to 7-10 days and then decline below the original basal levels. Cos7 cells (lane 6) have been used as a negative control. Therefore, in response to cAMP inducers, MITF appears to undergo an acute and long-term response. The early response is characterized by a rapid increase, followed by an equally rapid decrease in transcript levels, whereas prolonged exposure to cAMP causes a slow but sustained increase, which begins to decrease when the cells have accumulated large amounts of melanin. These results suggest a high degree of complexity in the regulation of MITF promoter activity.

Interestingly, although the levels of tyrosinase protein increase in melanocytes from dark skin (2), it was found no significant differences in MITF message level in melanocytes from either light or dark skin (Haddad & Medrano, unpublished results). It is possible that post-translational modifications of MITF protein, such as phosphorylation by PKA or by other protein kinases, may be differentially regulated in melanocytes from different skin colors. Alternatively, or in addition, MITF protein could be more readily degraded by its association with the ubiquitin-conjugating enzyme hUBC9, which targets MITF for degradation by the ubiquitin-proteasome pathway (18), in melanocytes from skin types I/II. Increased degradation of MITF protein would result in decreased levels of tyrosinase and TRP-1.

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