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* Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA;
Center for Devices and Radiological Health, Food and Drug Administration, Rockville, Maryland, USA; and
Center for Food Safety and Applied Nutrition, U. S. Food and Drug Administration, College Park, Maryland, USA
1Correspondence: National Institutes of Health, Laboratory of Cell Biology, Bldg. 37, Rm. 2132, Bethesda, MD 20892-4254, USA. E-mail: hearingv{at}nih.gov
SPECIFIC AIMS
To better understand the role of melanin in protecting the skin against UV radiation and subsequent photocarcinogenesis. We previously reported that levels of melanin correlated inversely with amounts of DNA damage induced by UV in normal human skin of different racial/ethnic groups. In this study, we further investigated DNA damage in the upper and lower epidermal layers in various types of skin before and after exposure to UV, and measured subsequent apoptosis and phosphorylation of p53.
PRINCIPAL FINDINGS
1. DNA damage in different types of skin
UV radiation of human skin results in two major types of DNA lesions: 6,4-photoproducts (6,4PP) and cyclobutane pyrimidine dimers (CPD). In this study, we measured levels of 6,4PP and CPD and determined their distribution in skin of varying racial/ethnic origin following a single exposure to a 1 minimal erythemal dose (MED) of UV. The 1 MED dose of UV was on average 
3 times higher in dark skin than in fair skin, and was 30% higher in intermediate skin than in fair skin. Despite that, overall levels of 6,4PP were significantly lower in dark skin immediately after UV exposure compared with fair skin (not shown). Immediately after UV, DNA damage was significantly lower in the lower epidermis than in the upper epidermis in intermediate and in dark skin, but not in fair skin, which suggests that the upper epidermis has a photoprotective effect against UV-induced 6,4PP and that this effect is more remarkable in darker skin.
Figure 1
A shows representative images of DNA damage as CPD for subjects with fair, intermediate and dark skin. CPD damage density in the lower epidermis of fair skin did not differ significantly from that in the upper epidermis immediately after UV radiation, or 1 d or 7 d later. However, in intermediate skin and more dramatically in dark skin, CPD damage in the lower epidermis was significantly less than in the upper epidermis at 7 min, 1 d and 7 d after UV exposure. Consistent with the 6,4PP damage, levels of CPD damage in the lower epidermis were higher in fair skin after UV than in dark skin at all time points examined, despite the fact that the darker skin had received up to 3-fold more UV to reach 1 MED. These results show that UV penetrates deeper in less pigmented skin.
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6,4PP and CPD damage in total, upper and lower epidermis were inversely correlated with melanin content (data not shown), which indicates that darker skin incurs less DNA damage in the lower epidermis while DNA damage in the upper epidermis is similar among racial/ethnic groups after exposure to UV.
Since DNA damage immediately after 1 MED UV was significant, even deep in the epidermis, we investigated CPD damage in melanocytes (Fig. 1B
, identified by costaining for tyrosinase). The % melanocytes with detectable levels of CPD was significantly higher in fair skin (86%) than in intermediate (30%) and in dark skin (11%) and the constitutive melanin content of the skin correlated inversely with the % melanocytes with DNA damage immediately after UV (r=.588, P<0.001).
2. Melanin facilitates the induction of apoptosis by UV
We used the TUNEL assay to measure apoptotic cells in the skin of different racial/ethnic groups after exposure to UV. Surprisingly, 7-fold more apoptotic cells were observed in dark skin than in fair skin after 1 MED UV (not shown), although the DNA damage in dark skin was significantly less than in fair and intermediate skin, as noted above. To rule out that the higher physical UV dose at 1 MED used for dark skin increased TUNEL-positive cells, we performed a similar study but using an approximately identical dose (180–200 J/m2 UV). TUNEL-positive apoptotic cells were observed at more than 5-fold higher levels in dark skin 1 d after that same dose of UV than in fair skin (Fig. 2
A). Increased levels of melanin correlated directly with the number of TUNEL-positive cells at 1 d and at 7 d after UV exposure, demonstrating that skin containing more melanin undergoes significantly more apoptosis in response to a single low UV dose.
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We hypothesized that it might be the melanin within keratinocytes that is involved in the increased apoptosis after UV exposure. To examine this, we used reconstructed 3-dimensional human skin equivalents (termed MelanoDerm) containing melanocytes derived from Black, Asian or White donors and keratinocytes from one Hispanic donor. The differences in melanin distribution are significant among these composites and they represent typical skin morphology (including pigmentation) of those types of skin. The MelanoDerm cultures were unirradiated or were UVB-irradiated at 25 or 50 J/m2 and were then fixed and embedded 2 d later. TUNEL assays showed that significantly more apoptotic cells were found in Black skin equivalents than in Asian or White skin equivalents at both UV doses (Fig. 2B
).
3. Phosphorylation of p53 and caspase-3 activation after UV exposure
The oncogene p53 plays important roles in responses to UV-induced DNA damage and induction of DNA repair. There is an overall nuclear accumulation of p53 in response to UV. More than 13 sites of p53 are known to be phosphorylated, one of them being a critical site at Ser-46, which is associated with the induction of apoptosis. Thus, we investigated the effects of UV on the accumulation of p53 in nuclei and its phosphorylation at Ser-46 in skin. More p53 accumulated in the nuclei of cells in fair skin than in dark skin at 1 d and at 7 d after UV exposure. However, phosphorylation of p53 at Ser-46 was not seen in fair skin, whereas it was readily seen in dark skin 1 d after UV (Fig. 2C
). The nuclear expression of p53 phosphorylated at Ser-46 colocalized with TUNEL-positive cells.
CONCLUSIONS AND SIGNIFICANCE
DNA damage (6,4PP and CPD) in skin from different racial/ethnic groups after exposure to 1 MED UV was significantly greater in fair skin than in dark skin and constitutive melanin content correlated inversely with that DNA damage (summarized in Fig. 3
). DNA damage in the upper epidermis immediately after UV exposure was similar among racial/ethnic groups but levels of DNA damage in the lower concentration of the epidermis was inversely proportional to the melanin content. Taken together, we conclude that the upper epidermis of dark skin is significantly more photoprotective for the deeper tissue against UV damage than that of fair skin.
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The localization of TUNEL-positive cells in the middle to upper layers of the epidermis following UV exposure suggests that they are keratinocytes rather than melanocytes, and the mechanism(s) underlying their formation is of great interest. Pulse lasers cause highly selective injury to cells containing melanosomes suggesting that the UV energy absorbed by melanin in the upper epidermis causes photothermolysis (heat damage) to pigmented cells. We investigated the appearance of TUNEL-positive cells in skin from various racial/ethnic groups after UV and in skin equivalents after UV exposure to test the hypothesis that the melanin content is responsible for the apoptosis. Cells containing melanin in the upper epidermis of dark skin tended to undergo more apoptosis after UV than do those of fair skin. Thus, the presence of melanin facilitates the apoptotic effect of UV on cells but whether that results from photothermolysis or whether other properties of melanins are involved will require further study.
Our study also demonstrates that the nuclear accumulation of p53 is less in dark skin than in fair skin, suggesting that the overall activation of p53 following UV-induced DNA damage is greater in fair skin. The sustained activation of p53 may also in part cause the higher incidence of photocarcinogenesis in fair skin. Phosphorylation of p53 at Ser-46, which is associated with the induction of apoptosis, occurred at low levels in fair skin after low doses of UV exposure but was significant in dark skin, suggesting that p53 phosphorylation site is involved in UV-induced apoptosis in epidermis with abundant levels of melanin.
In summary, we propose two major mechanisms which underlie the dramatic differences in photocarcinogenesis of light vs. dark skin. First, UV-induced DNA damage in the lower epidermis (which contains keratinocyte stem cells and melanocytes) is not effectively prevented in fair skin because of the low melanin content in the upper (and lower) epidermis. DNA damage in the upper epidermis is quite similar among all types of skin, which indicates that epidermal pigmentation is an efficient UV filter for underlying cells. Second, UV-induced apoptosis was virtually absent in fair skin after low UV doses, but was significant in dark skin, facilitating the effective removal of UV-damaged cells in dark skin. Virtually all epidermal cells had significant DNA damage in fair skin but only 1% of them became apoptotic whereas less than 50% of epidermal cells in dark skin had significant DNA damage, yet 5% of those cells were apoptotic. The combination of relatively low DNA damage and efficient removal of UV-damaged cells contributes to the decreased incidence of skin cancer in darker skin.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5725fje
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