Introduction

UVR interacts with 7-dehydrocholesterol in the keratinocyte to form pre-vitamin D₃ which at body temperature, thermally isomerizes into vitamin D₃ (Holick, 1994). Vitamin D₃ metabolites, 25 hydroxyvitamin D₃ and 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) are also produced locally in skin cells (Bikle et al., 1986; Matsumoto et al., 1991; Lehmann et al., 2000, 2001), although their function in skin is not fully understood. The normal differentiation of keratinocytes is partly regulated by 1,25(OH)₂D₃ (Pillai et al., 1988a), as is hair follicle development (Reichrath, 2000) and, under some circumstances, 1,25(OH)₂D₃ can enhance pigmentary responses (Mason, 2000).

In earlier cell culture studies, we and others have shown that treatment with 1,25(OH)₂D₃ results in increased cornified envelope formation in keratinocytes (Holliday et al., 1997; Pillai et al., 1988a, 1988b) and in increased melanogenesis in melanocytes (Ranson et al., 1988). As two of the features of normal cellular responses to UVR in vivo as well as in vitro are increased cornification of keratinocytes and increased pigment production by melanocytes (Dissanayake et al., 1993), we were interested in the responses of skin cells to UVR in the presence of 1,25(OH)₂D₃. There has been some evidence to suggest that 1,25(OH)₂D₃ may enhance keratinocyte survival after UVR exposure (Koren et al., 2000b; Manggau et al., 2001). This is in contrast to the ability of 1,25(OH)₂D₃ to increase susceptibility of tumor cells to apoptosis-inducing agents (Kasukabe et al., 1987; Holliday et al., 1997; Koren et al., 2000a).

It is well accepted that UVR directly interacts with DNA causing DNA damage, for example, thymine dimer (TD) formation (Setlow, 1966). UVR induces high levels of the p53 tumor suppressor protein, mainly through post-translational stabilization of the protein (Hall et al., 1993). In turn, p53 activates the transcription of downstream genes responsible for cell cycle arrest. This growth arrest allows for repair of DNA damage, a process facilitated by increased p53 (Smith et al., 1995). However, p53 can also cause apoptosis of cells with excessive unrepaired DNA damage (Decraene et al., 2001). UVR also increases nitric oxide (NO) products which may contribute to UVR-induced DNA damage and inhibit DNA repair (Jaiswal et al., 2000; Bau et al., 2001). In human islet cells treated with cytokines and in rat hippocampus injected with lipopolysaccharide, 1,25(OH)₂D₃ had been reported to reduce apoptosis through a decrease in NO products (Garcion et al., 1998; Riachy et al., 2002).

A key question is whether 1,25(OH)₂D₃ allows cells with increased DNA damage to survive. Preliminary evidence indicates that this is not the case (Wong et al., 2004; De Haes et al., 2005). In this study, we show that UVR-induced cyclobutane pyrimidine dimers are reduced, not increased, after 1,25(OH)₂D₃ treatment and propose a novel mechanism for this involving enhanced elevation of nuclear p53 post-UVR and suppression of the NO pathway. Increased cell survival and reduced DNA damage were also seen in skin of UV-irradiated Skh:hr1 hairless mice after topical treatment with 1,25(OH)₂D₃. Thus, vitamin D production may be a photoprotective mechanism of the skin that increases skin cell survival after UVR without an increase in DNA damage.

Results

Cell numbers

The protective effect of 1,25(OH)₂D₃ and related compounds on UV-induced keratinocyte apoptosis has been described by a number of groups, including our own (Hanada et al., 1995; Holliday et al., 1997; Lee and Youn, 1998; Koren et al., 2000b; Manggau et al., 2001; De Haes et al., 2003, 2004; Wong et al., 2004), although the concentrations of 1,25(OH)₂D₃ used by some groups to achieve this were often high. When keratinocytes were pretreated with 1,25(OH)₂D₃, cell survival was significantly and dose-dependently increased from 681% (SEM) to 841% (P<0.01) (Figure 1). A protective effect by 1,25(OH)₂D₃ was seen in skin cells from more than 10 donors.

Figure 1.

1,25(OH)₂D₃ increases keratinocyte survival after UVR. Cells were preincubated with 1,25(OH)₂D₃ at doses ranging from 10^-11 to 10^-7 M or with vehicle for 24 hours before irradiation in buffer solution. 1,25(OH)₂D₃ was replaced after irradiation and cells counted 24 hours after UVR. Each point represents meanSEM of normalized data from six data points from two independent experiments. Significantly different from vehicle: ^**P<0.01, ^*P<0.05.

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Similar results were seen in separate experiments using keratinocytes derived from sun-exposed skin on the back of a female subject – keratinocyte survival 24 hours post-UVR was 4712% (SD) in vehicle-treated wells, this was increased to 6520% in keratinocytes treated with 10^-9 M 1,25(OH)₂D₃ (P<0.05) and 10036% in cells treated with 10^-8 M 1,25(OH)₂D₃ (P<0.01) (Figure S1). In separate experiments, a significant photoprotective effect on cell survival was also noted when 1,25(OH)₂D₃ was added immediately after UVR, and cell survival was not different to that in irradiated cells treated with 1,25(OH)₂D₃ 24 hours earlier. Percentage cell survival increased from 6112% (SD) to 8522% (P<0.01) in cells treated with 1,25(OH)₂D₃ for 24 hours before UV, and to 8117% (P<0.01) in cells treated immediately after irradiation only.

Treatment with a non-active analog, 1,25-dihydroxytachysterol₃, JB, (Norman et al., 2001) did not significantly alter keratinocyte numbers after UVR exposure in experiments where the active compound significantly enhanced cell survival after UVR (vehicle 6714% (SD), JB 6520%; P=not significant, Figure S2).

As noted earlier, the vitamin D hormone, 1,25(OH)₂D₃, enhances epidermal cell differentiation and inhibits proliferation. To investigate whether this reduced skin cell proliferation might explain the enhanced survival in cell numbers after UVR, we tested whether there was a correlation between the reduction in keratinocyte numbers induced by 1,25(OH)₂D₃ in the sham-irradiated group and the degree of protection from UVR-induced cell death. No significant correlations were found (r²=0.46, n=10, P>0.05) showing that the capacity of 1,25(OH)₂D₃ to prevent UVR-induced reductions in skin cell numbers is not explained by its ability to reduce cell proliferation (Figure S3).

Sunburn cells in mouse skin

Treatment with 1,25(OH)₂D₃ immediately post-UVR also reduced the number of sunburn cells (apoptotic keratinocytes) in Skh:hr 1 mice. The mean number of sunburn cells per linear millimeter in vehicle-treated skin was 2.00.7 (SD). This was reduced to 1.40.2 in mice treated with 1,25(OH)₂D₃ at a concentration of 22.8 pmol/cm² (P<0.05), and to 1.20.2 at a concentration of 45.6 pmol/cm² (P<0.01).

Thymine dimers

Exposure to UVR results in TD which can be detected in irradiated skin cells using a specific monoclonal antibody to TDs (Roza et al., 1988). Figure 2 shows very little staining in sham-irradiated control, but much increased staining after UVR in vehicle-treated wells. Figure 3a shows a dose-dependent decrease in TD 3 hours after UVR in 1,25(OH)₂D₃-treated keratinocytes from neonatal foreskins (Figure 3a). In separate experiments, similar reductions in cyclobutane pyrimidine dimers were seen in keratinocytes derived from the back of a female subject, where cyclobutane pyrimidine dimer-positive nuclei, as percent total nuclei, at 3 hours after irradiation were 5713% (SD) in vehicle-treated cultures but only 125% in keratinocytes treated with 10^-9 M 1,25(OH)₂D₃ (P<0.001) and 132% in cells treated with 10^-8 M 1,25(OH)₂D₃ (P<0.001, Figure S4).

Figure 2.

1,25(OH)₂D₃-induced reduction in TDs in keratinocytes after UVR. Keratinocytes were treated with vehicle or 1,25(OH)₂D₃ 24 hours before and immediately after irradiation. Cells were fixed 3 hours post-UVR and the level of TD measured by immunohistochemistry. A reduction in TD staining is shown with 1,25(OH)₂D₃ treatment. Dark arrows () point to TD-positive nuclei; open arrows () point to TD-negative nuclei. Bar=50 m. (a) Vehicle, (b) 1,25(OH)₂D₃ 10^-9 M, (c) 1,25(OH)₂D₃ 10^-9 M UVR, and (d) vehicle UVR.

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Figure 3.

Dose- and time- dependent reduction of UVR-induced TDs in Keratinocytes by 1,25(OH)₂D₃. (a) 1,25(OH)₂D₃-induced dose-dependent reduction in keratinocyte TDs after UVR, (b) and time course of reduction in UVR-induced keratinocyte TDs by 1,25(OH)₂D₃. (a) Keratinocytes were preincubated with 1,25(OH)₂D₃ at doses ranging from 10^-11 to 10^-8 M or with vehicle for 24 hours before irradiation in buffer solution. 1,25(OH)₂D₃ or vehicle was replaced after irradiation and immunohistochemistry performed at 3 hours. The number of positively stained nuclei for TD as a % of the total nuclei was calculated using light microscopy and image analysis. Results are presented as meanSD. The average value of the control in keratinocytes was 81.610.6%. Significantly different from vehicle: ^***P<0.001, ^**P<0.01. Significant difference between doses: ^###P<0.001, ^##P<0.01, ^#P<0.05. (b) Keratinocytes were preincubated with 1,25(OH)₂D₃ (10^-9 M) or with vehicle for 24 hours before irradiation in buffer solution. 1,25(OH)₂D₃ or vehicle was replaced after irradiation and cells fixed at the indicated time points for immunohistochemical detection of TD. Each point is normalized to the 0.5 hours time point and represents the pooled dataSEM from at least six data points from a minimum of two independent experiments. The average number of positive nuclei as a percent of total nuclei in the control was 30.39.6%. Significantly different from vehicle at the same time-point by Student's t-test: ^*P<0.05, ^**P<0.01.

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In a separate time course study, mean TDs were significantly reduced by 10^-9 M 1,25(OH)₂D₃ pretreatment at 0.5, 3 (P<0.05), and 6 hours (P<0.01) after UVR in keratinocytes. A similar reduction, although not statistically significant, was also observed at 24 hours after UVR (Figure 3b). No significant differences in TD were seen in the presence of 1,25(OH)₂D₃ or vehicle in cells examined immediately after UVR. Very few TD were present by 48 hours.

In separate experiments, treatment with 10^-9 M 1,25(OH)₂D₃ immediately post-UVR only, also significantly reduced TD at 3 hours from a mean of 186% (SD) in vehicle-treated cultures to 81%, (P<0.01). Treatment with the inactive analog, JB, did not reduce TD-positive nuclei as a percentage of total nuclei in keratinocytes. The respective values were vehicle, 196% (SD); 1,25(OH)₂D_3, 41% (P<0.01); JB, 1710% (P=not significant, Figure S5).

Recent studies by our group have shown a reduction in TD, measured 24 hours after UVR in the skin of Skh:hr 1 mice treated with topical 1,25(OH)₂D₃ 24 hours before and immediately after UVR (Dixon et al., 2005). In this study, the level of nuclear TD staining was also reduced in mouse skin treated only immediately post-UVR with 1,25(OH)₂D₃ compared with vehicle-treated mice, measured 24 hours after UVR (Figure 4). Mean TD-positive nuclei, as percent total nuclei, was 226% (SD) in vehicle-treated mouse skin, reduced to 93% in mouse skin treated with 1,25(OH)₂D₃ immediately post-UVR at a dose of 22.8 pmol/cm² (P<0.05).

Figure 4.

1,25(OH)2D3-induced reduction in TDs in Skh:hr1 mouse skin after UVR. Mice were exposed to three minimal erythemal doses SSUVR and treated topically with vehicle or 1,25(OH)₂D₃ immediately after irradiation. Skin sections were taken 24 hours post-UVR and the level of TD measured by immunohistochemistry. A reduction in TD staining is shown with 1,25(OH)₂D₃ treatment. Dark arrows () point to TD-positive nuclei; open arrows () point to TD-negative nuclei. Bar=50 m. (a) Isotype control, (b) vehicle, (c) vehicle UVR, (d) 1,25(OH)₂D₃ 22.8 pmol/cm², and (e) 1,25(OH)₂D₃ 22.8 pmol/cm² UVR.

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p53 expression

Only a few nuclei in sham-irradiated keratinocytes stained positively for p53 protein, with no differences between vehicle- or 1,25(OH)₂D₃-treated cultures. Nuclear staining increased markedly after UVR in all cultures, but to a much greater extent in cells treated with 1,25(OH)₂D₃ (Figure S6). There was no p53 staining in the cytoplasm and all p53 staining was expressed as positive nuclei as percent of total nuclei. As shown in Figure 5a, p53 staining at 3 hours post-UVR increased 3- to 5-fold in irradiated keratinocytes treated for 24 hours with 1,25(OH)₂D₃, compared with vehicle-treated cultures (P<0.05). By 6 hours after UVR, p53 staining was around 9-fold higher in the vehicle-treated keratinocytes than at 3 hours (P<0.001), but was still significantly higher in the cultures treated with 1,25(OH)₂D₃ compared with vehicle-treated keratinocytes at 6 hours (P<0.01). Where p53 expression and TD were measured in the same experiment, there was a reciprocal relationship between the increase in p53 and the decrease in TD (Figure 5b).

Figure 5.

Nuclear p53 protein expression after UVR and relationship with TDs in the presence of 1,25(OH)₂D₃. (a) 1,25(OH)₂D₃-induced increase in p53 protein expression in keratinocytes after UVR, (b) and reciprocal relationship between p53 and TD expression. Cells were preincubated with 1,25(OH)₂D₃ at 10^-9 or 10^-8 M or with vehicle for 24 hours before irradiation in buffer solution. 1,25(OH)₂D₃ or vehicle was replaced after irradiation and immunohistochemistry and image analysis performed at the indicated times. (a) An increase in p53 expression with 1,25(OH)₂D₃ treatment is shown at 3 and at 6 hours after UVR in keratinocytes. The meanSD for p53-positive nuclei as % total nuclei in vehicle-treated cells at 3 hours was 1.80.7%. Significantly different from 3 hours vehicle control: ^*P<0.05; significantly different from 6 hours vehicle control: ^##P<0.01. (b) An increase in p53 and concurrent decrease in TD is shown 6 hours after UVR in cells treated with 1,25(OH)₂D₃. The meanSD for positive nuclei as % total nuclei in vehicle-treated cells was 10.96.3% for p53 and 27.24.0% for TD. p53 expression significantly different from vehicle ^**P<0.01, ^*P<0.05; TD expression significantly different from vehicle ^###P<0.001, ^##P<0.01.

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NO pathways

UV irradiation increases NO and its products in skin cells (Bruch-Gerharz et al., 1998) and may play a role in enhancing the genotoxic effects of UVR (Suzuki and Inukai, 2006). The Griess reaction showed an increase in the stable metabolite of NO, nitrite, after UVR in all experiments. In sham-irradiated control keratinocytes, nitrite concentrations were 7020 mg/l (SD) nitrite as compared with 18040 mg/l nitrite after irradiation in control wells (P<0.05). There was a significant decrease in nitrite after UVR in cells which had been treated with 1,25(OH)₂D₃ (932% of the irradiated control, P<0.05). This decrease was similar to the reduction in nitrite seen with a NO synthase inhibitor, aminoguanidine, in the same experiment (882% of the irradiated control, significantly different from vehicle-treated cells, P<0.001).

Because both aminoguanidine and 1,25(OH)₂D₃ reduced nitrite in UV-irradiated keratinocytes, aminoguanidine and another NO synthase inhibitor L-N-monomethylarginine (L-NMMA) were used to treat keratinocytes immediately after irradiation. At 3 hours after irradiation, significant decreases in TD, similar to those with 1,25(OH)₂D₃ treatment alone, were observed (Figure 6). In separate experiments, there was no additional effect of L-NMMA in 1,25(OH)₂D₃-pretreated cultures. In pooled data from five experiments, the mean percentage TD-positive nuclei was 162% (SEM) in vehicle-treated keratinocytes and 91% in keratinocytes treated with 10^-9 M 1,25(OH)₂D₃ (P<0.01 vs vehicle); 101% in keratinocytes treated with 1.28 mM L-NMMA (P<0.01 vs vehicle) and 71% in keratinocytes treated with 10^-9 M 1,25(OH)₂D₃ and 1.28 mM L-NMMA (P<0.01 vs vehicle but not significant vs 1,25(OH)₂D₃ or L-NMMA alone).

Figure 6.

1,25(OH)₂D₃ and NO inhibitors L-NMMA and aminoguanidine, added after UVR induce a reduction in TDs. Keratinocytes were irradiated in buffer solution. 1,25(OH)₂D₃ or vehicle in medium was added to quadruplicate wells after irradiation whereas others received L-NMMA (1.28 mM) or aminoguanidine (2 mM). Cells were incubated in the above solutions for 3 hours post-UVR after which immunohistochemistry was performed. The number of positively stained nuclei for TD as a % of the total nuclei was calculated using light microscopy and image analysis. Significant differences compared to vehicle with Student's t-test are: ^**P<0.01, ^*P<0.05.

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Discussion

The results of this study show photoprotective effects of 1,25(OH)₂D₃ in vitro and in vivo. 1,25(OH)₂D₃ increased keratinocyte survival and decreased sunburn cells probably as a result of the reduction in detectable DNA damage in the form of TD from 30 minutes after UVR exposure. The reduction in TD was associated with an increase in p53 expression and a reduction in NO derivatives.

After the initial report of 1,25(OH)₂D₃ synthesis in keratinocytes (Bikle et al., 1986), several studies by Lehmann et al. (1999, 2000, 2001) have shown evidence of a complete pathway of vitamin D synthesis from 7-dehydrocholesterol to the active metabolite, 1,25(OH)₂D₃ in epidermal cells, skin equivalents, and living skin. Keratinocytes are prone to cell damage after UVR, resulting in cell cycle arrest or apoptotic cell death (Zhai et al., 1996; Lee and Youn, 1998). Treatment with 1,25(OH)₂D₃ improved cell survival after UVR compared with sham-irradiated cultures. The effect was concentration-dependent, and even occurred when the 1,25(OH)₂D₃ was added immediately after UVR. Increased cell survival was achieved at concentrations of 1,25(OH)₂D₃ that were much lower than those reported by other groups (Manggau et al., 2001; De Haes et al., 2003, 2004). The concentrations used in our study are likely to be achieved in skin. In general, basal 7-dehydrocholesterol content in cultured keratinocytes and skin equivalents is very low compared to that present in living skin (Nemanic et al., 1985). The low concentration of 7-dehydrocholesterol in cultured skin cells and the relatively long time required to produce 1,25(OH)₂D₃ after UVR in vitro or in vivo (Lehmann et al., 2001) help to explain the dramatic effects noted when 1,25(OH)₂D₃ was added to the system. UVB irradiation has been reported to induce expression of vitamin D-25-hydroxylase in keratinocytes in human skin equivalents (Lehmann et al., 1999). Based on the production rates demonstrated in human skin equivalents and in human skin using microdialysis (Lehmann et al., 2000, 2001), it can be calculated that local concentrations of 1,25(OH)₂D₃ in the order of 2–5 nM might be reasonably achieved. The photoprotective effects demonstrated in this study were achieved with concentrations of 1,25(OH)₂D₃ of this order of magnitude and even lower.

There have been preliminary reports of reduced sunburn cell formation in vivo after UVR in mouse skin treated with 1,25(OH)₂D₃ (Hanada et al., 1995; Lee and Youn, 1998). Confirming the cell culture studies, a dose-dependent decrease in sunburn cells in mice treated topically with 1,25(OH)₂D₃ was seen in this study. One potential concern with the 1,25(OH)₂D₃-induced increase in the survival of skin cells after UVR, is that the treatment might have allowed more cells with DNA damage to survive. In fact, a dose-dependent decrease in TD was seen, which is likely to have resulted in the survival of a greater number of cells. The reduction in TD was also confirmed in mice. It should be noted, however, that the majority of cells did not have DNA damage and the effects on survival were an improvement of a few percent. Furthermore, a reduction in TD at some time point after irradiation does not necessarily reduce mutation frequency after UVR, if for example, 1,25(OH)₂D₃ induces an error-prone DNA repair system. This cannot be readily tested in a short-term cell culture system. Nevertheless, 1,25(OH)₂D₃ has been shown to inhibit multistage chemical-induced skin carcinogenesis in mice (Wood et al., 1983), and more recently, novel analogs of vitamin D have been shown to have similar effects (Kensler et al., 2000). Furthermore, VDR^-/- mice have been reported to have enhanced sensitivity to chemically induced skin tumorigenesis compared to wild-type (VDR^+/+) mice (Zinser et al., 2002).

Two pieces of evidence indicate that the protective effect of 1,25(OH)₂D₃ cannot be merely owing to the compound acting as a UV absorber. These include the demonstration that even when 1,25(OH)₂D₃ was only added after irradiation, it still caused a significant reduction in keratinocyte losses and UVR-induced TD and the observation that the inactive compound JB failed to show any photoprotective activity, even though it absorbs in the UVB range, although to a lesser extent than 1,25(OH)₂D₃.

The p53 tumor suppressor gene is considered the "guardian of the genome". Following UVR, the amount of p53 protein in the nucleus of skin cells becomes elevated (Ouhtit et al., 2000). The elevated p53 induces cell cycle arrest, allowing DNA repair, and appears also to directly contribute to enhanced nucleotide excision repair (Smith et al., 1995; Li et al., 1996). If the DNA damage is excessive or unrepaired, the elevated p53 can also cause apoptosis by upregulation of Bax and/or downregulation of Bcl-2 expression (Ouhtit et al., 2000). UVR-induced apoptosis independent of p53 has also been described (Li et al., 1996). Previously, 1,25(OH)₂D₃, has been shown to have no effect on p53 expression in non-irradiated normal cultures of human keratinocytes (Sebag et al., 1994). A study that used a high dose of 1,25(OH)₂D₃ (10^-6 M) reported that surprisingly, 1,25(OH)₂D₃ treatment of irradiated keratinocytes led to an increase in Bcl-2 expression occurring in conjunction with a decrease in p53 expression following UVR (De Haes et al., 2004). The work presented here provides evidence that 1,25(OH)₂D₃ markedly enhances nuclear p53 protein expression in irradiated keratinocytes. Apart from dose effects, it is not clear why there is a divergence in results, as the biological systems in the two studies were similar. One possibility is that in this study, the keratinocytes were incubated without EGF and cholera toxin for 2 days to reduce overstimulation of cell signal pathways before irradiation.

The further increase in p53 with 1,25(OH)₂D₃ post-UVR may well be an important mechanism of enhanced DNA repair in the vitamin D-treated cells (Smith et al., 1995; Li et al., 1996). A decrease in UVR-induced DNA damage by the agent silibinin has also been reported to be associated with an increase in p53 accumulation in Skh:hr1 mice (Dhanalakshmi et al., 2004). The decrease in TD with 1,25(OH)₂D₃ was very marked. This is perhaps surprising as the rate of repair of pyrimidine dimers is relatively slow, with a half-life of around 7 hours (Mitchell et al., 1990; Yarosh and Yee, 1990; Katiyar, 2000), although the culture process may select for more rapidly replicating, repair-proficient cells (Mitchell et al., 1990). Interestingly, the study using topical silibinin before or immediately after UV exposure also reported comparable decreases in TD (76–86% reduction) at 1 hour after UVR. The authors speculated that the protective efficacy of silibinin against UV-induced tumor initiation might be owing to an inhibition in UV-induced DNA damage. The time course data reported here are consistent with this proposal. Metabolic induction of pyrimidine dimers has been reported (Lamola, 1971).

Studies by Ravid et al. (2002) and De Haes et al. (2003), as well as preliminary studies by our group (data not shown) have shown that photoprotection by 1,25(OH)₂D₃ involves inhibition of c-Jun-(NH)₂-terminal kinase activity, leading to increased cell survival after UVR. It has recently been reported that the prosurvival activity of p53 involves its ability to bind and inhibit the activity of c-Jun-(NH)₂-terminal kinase, and subsequent inhibition of its role in mitochondrial death signaling (Lo et al., 2004). This mechanism is consistent with our results which show upregulation of p53 by 1,25(OH)₂D₃.

Another mechanism may also contribute to increased DNA repair. NO and peroxynitrite are well known to be increased after UVR (Bruch-Gerharz et al., 1998). NO is converted to the toxic free radical, peroxynitrite (Beckman and Koppenol, 1996; Ischiropoulos, 1998). Recently, NO and NO donors have been shown to inhibit TD repair through the inhibition of TD excision (Jaiswal et al., 2000; Bau et al., 2001). Conversely, NO synthase inhibitors have been reported to enhance DNA repair in the presence of arsenite (Bau et al., 2001). Aminoguanidine and a second NO synthase inhibitor, L-NMMA, reduced TDs and nitrite in irradiated keratinocytes to an extent similar to that seen with 1,25(OH)₂D₃. L-NMMA, and 1,25(OH)₂D₃ did not augment each other's activity. These results, and those showing no reduction in TD immediately after UVR in the presence of 1,25(OH)₂D₃, are consistent with a hypothesis that the action of 1,25(OH)₂D₃ to reduce TD damage after UVR may be partly due to a decrease in NO products leading to an increase in TD repair. In this context, it is worth noting, although the evidence is circumstantial, that enhanced TD repair leads to reduced UVR-induced immunosuppression (Kripke et al., 1992) as does topical application of NO inhibitors (Kuchel et al., 2003). In a preliminary publication, we have reported that 1,25(OH)₂D₃ reduces UVR-induced immunosuppression in mice (Dixon et al., 2005).

The active metabolite of vitamin D is best known for its ability to enhance gut calcium and phosphate absorption to provide mineral for bone, although actions in many tissues are now established. The data presented here are consistent with the proposal that the production and accumulation of vitamin D in skin by UVR and its likely conversion locally over time to 1,25(OH)₂D₃ will enhance UVR-induced p53 protein expression and suppress NO and its derivatives, resulting in increased DNA repair. This photoprotective mechanism increases skin cell survival after UVR without an increase in DNA damage in vitro and in vivo. The possibility of using vitamin D compounds before or after UV irradiation to reduce skin damage is a potential avenue for investigation.

Materials and Methods

Materials

Tissue culture media and phosphate-buffered saline were purchased from TRACE Biosciences Pty. Ltd (Castle Hill, Australia). 1,25(OH)₂D₃ was provided by Roche Products (DeeWhy, Australia) and was also purchased from Sigma Chemical Company (St Louis, MO). Fetal bovine serum was from Commonwealth Serum Laboratories (Sydney, N.S.W., Australia). Multiwell plates, chamber-well slides, and glass coverslips were from Becton Dickinson Labware (Franklin Lakes, NJ), Nunc (Naperville, IL), and Menzel-Glaser (Braunschweig, Germany), respectively. Pronectin-coated culture plates were from ICN Biomedicals Australasia Pty. Ltd (Sydney, Australia). All other chemicals and hormones were obtained from Sigma (St Louis, MO), unless otherwise indicated.

Cell culture

The studies of human tissue were approved by the Human Research Ethics Committee of the University of Sydney and were conducted according to the Declaration of Helsinki Principles. All subjects or their legal guardians gave written informed consent. Keratinocytes were cultured from human neonatal foreskins from Caucasian donors as described previously by Gordon-Thomson et al. (2001) with some modifications. Keratinocytes were also grown from skin removed from the back of a female donor at elective surgery. Briefly, skin was washed in phosphate-buffered saline, cut into cubes, and immersed in 0.1% dispase (w/v) overnight at 4°C. The epidermal sheet was removed and incubated in 0.1% trypsin and 0.02% EDTA at 37°C until a cell suspension was formed. The resulting cell suspension was centrifuged and resuspended in low calcium medium (DMEM without calcium) with 30 mg/l penicillin, 50 mg/l streptomycin, 2.2 g/l NaHCO₃, 293 mg/l glutamine, 2.385 g/l (10 mM) N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer, 10 ng/ml EGF, 10^-10 M cholera toxin, 0.5 mg/l hydrocortisone, and 5% v/v heat-inactivated fetal bovine serum as described previously (Dissanayake et al., 1993).

Cells were used at passages 1–6. For experiments, cells were plated on poly-L-lysine-coated multiwell plates, chamber-well slides, or on glass coverslips and then treated with ethanol vehicle (0.1%) or 1,25(OH)₂D₃ at 10^-11 to 10^-8 M dissolved in ethanol 0.1% v/v, unless otherwise stated. For cell survival studies, keratinocytes were plated onto 24- or 96-well culture plates. For TD experiments, cells were plated onto poly-L-lysine-coated 5 mm glass coverslips in 96-well plates.

Experiments were carried out at approximately 60–80% confluence and the media changed to DMEM without EGF and cholera toxin for at least 2 days before experiments to allow cell signal pathways to normalize (McLeod et al., 1995). Pretreatment with vehicle or 1,25(OH)₂D₃ was for 24 hours, unless otherwise indicated.

Irradiation

The UV source consisted of a FS20T12 UVB lamp and a FL20SBL UVA lamp (Philips, Amsterdam, Holland) filtered through a 0.5 mm layer of cellulose tri-acetate (Eastman Chemical Products, Kingsport, TN) to remove wavelengths below 290 nm. This filter also markedly reduced irradiance of the more energetic UVB wavelengths (Figures S1–S7). Irradiance was 200 mJ/cm² UVB and 1,170 mJ/cm² UVA. Irradiance was checked regularly with an OL754 radiometer (Optronics Laboratories Inc., Orlando, FL) calibrated against the source just before irradiation. Media was changed to phosphate-buffered saline or to a Martinez buffer solution containing 145 mM NaCl, 5.5 mM KCl, 1.2 mM MgCl₂6H₂O, 1.2 mM NaH₂PO₄2H₂O, 7.5 mM Na-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 10 mM D-glucose, and 1 mM CaCl₂2H₂O as indicated. After irradiation, the buffer solution was replaced with media containing vehicle or 1,25(OH)₂D₃ for 24 hours unless otherwise stated, when remaining viable cells were counted or fixed for immunohistochemistry assays. Sham-irradiated cells were subjected to similar procedures but were shielded during the irradiation.

Cell counts

Cells were recovered by trypsinization at 24 hours after UVR unless otherwise stated and counted using a hemocytometer (Fuchs Rosenthall model) obtained from Weber Scientific International Ltd (Sussex, UK). Trypan blue exclusion was used to determine live cell numbers, which were used in results. Percentage cell survival was calculated by dividing the mean live cell number in irradiated wells by the mean live cell number in sham-irradiated control wells treated with the same dose of test substance.

Immunohistochemistry

Cells were fixed at 3 hours after UV irradiation, unless otherwise indicated. Fixation was achieved using 100% methanol at -20°C. H₂O₂ (1%) diluted in phosphate-buffered saline (v/v) was used to block endogenous peroxidase. Antigen retrieval involved nuclear DNA denaturation with 70 mM NaOH diluted in 70% ethanol, followed by proteolytic digestion with Proteinase K 1 g/ml. Non-specific staining was blocked with 50% normal human serum diluted in phosphate-buffered saline. Immunohistochemistry was performed using the Dakocytomation LSAB Plus kit (Glostrup, Denmark) with mouse monoclonal IgG1, lambda anti-TD antibody (Affitech, Oslo, Norway) for TD detection (Roza et al., 1988) or the D07 mouse monoclonal IgG2b, kappa antibody (Dakocytomation, Glostrup, Denmark) for detection of p53. To control for specificity of the primary antibody, isotype controls at the same protein concentration were used. This resulted in no staining. The chromogen diaminobenzidine was used for visualization of keratinocytes (Dakocytomation).

Image analysis

Slides were examined under a Zeiss-Axioplan light microscope at 10 original magnification. Areas from both sham-irradiated coverslips and UV-irradiated coverslips were randomly captured using a Sony Progressive color CCD camera (Sony, Tokyo, Japan) and transferred to a Zeiss KS400 (Carl Zeiss Vision, Munich, Germany) image analysis system.

Before capturing the images, light intensity was adjusted to a standard setting that remained the same throughout the image analysis process. One image was captured from each sham-irradiated coverslip and two from each UV-irradiated coverslip. The images taken from the sham-irradiated coverslips were used to set the thresholds for comparison of UV-irradiated coverslips. Two measurements were recorded from each image: total nuclei, and positively stained nuclei. The positively stained nuclei could be easily differentiated from all other nuclei according to their density and thus they were identified in the image analysis program by a combination of grayscale thresholding and manual editing. The parameters measured were area and grayscale density.

Griess assay

This assay quantifies nitrite (a stable metabolite of NO) in solution. The Griess assay is based on diazotization of sulfanilic acid with nitrite ions and the subsequent coupling of this product with diamine (Guevara et al., 1998). This results in a measurable pink metabolite with an absorption maximum of 540 nm. Griess reagent comprised 1% sulfanilamide (w/v), 3.5% phosphoric acid (v/v), and 0.1% beta-naphthylethylene diamine dihydrochloride (w/v), in double-distilled water.

Keratinocytes were pretreated for 24 hours with vehicle or 1,25(OH)₂D₃ diluted in cell culture media, then washed and irradiated or sham-irradiated in buffer as described above. After irradiation, known inhibitors of NO, L-N-monomethylarginine (L-NMMA) (1.28 mM in buffer) and aminoguanidine (2 mM in buffer) were added to the buffer solution and plates were incubated at 37°C for 30 minutes. An equal volume of Griess reagent was added to supernatant from keratinocytes. Plates were incubated for 15 minutes at room temperature, after which absorption at 550 nm was determined using a FLUOstar Galaxy microplate reader (BMG Labtechnologies (Mornington, Australia). The concentration of sodium nitrite was interpolated from the linear portion of a standard curve using sodium nitrite in double-distilled water as a reference.

Skh:hr1 hairless mice

All animal studies were approved by the Animal Ethics Committee of the University of Sydney. Mice were maintained in wire-topped plastic boxes at 23–25°C on compressed paper bedding from Fibrecycle Pty. Ltd (Mudgeeraba, Australia). Mice were fed Gordon Rat and Mouse Pellets (Yandeera, Australia) and tap water ad libitum. Mice were treated topically with vehicle only or 1,25(OH)₂D₃. Stock solutions of test compounds were dissolved in ethanol and diluted in a base lotion containing propylene glycol and water to a final solvent ratio of 2:1:1, respectively. Treatments were applied dorsally, immediately after irradiation. Skin samples were taken from UV-irradiated back skin 24 hours post-UVR, fixed, processed, and paraffin-embedded, and sections subjected to routine hematoxylin and eosin staining to visualize sunburn cells. The stained sections were examined under a Zeiss-Axioplan light microscope at 40 original magnification, and the number of sunburn cells recorded 1 per linear millimeter of skin. Immunohistochemical analysis was also carried out for detection of TD. Sections were de-paraffinized through graded alcohol and endogenous peroxidase blocked by 3% H₂O₂ in H₂O (v/v) for 5 minutes, followed by antigen retrieval with citrate buffer in a microwave oven for 15 minutes. Non-specific antibody blocking was achieved using 10% normal horse serum in Tris-buffered saline. Sections were incubated with the primary mouse monoclonal IgG₁ anti-TD antibody at a working concentration of 20 g/ml in combination with the Animal Research Kit (Dakocytomation) to minimize reactivity of the anti-mouse antibody with any endogenous immunoglobulin.

Statistical analysis

All experiments shown were repeated 2–6 times with similar results. Data are presented either as a representative single experiment or as normalized results from several pooled experiments. In each experiment, values were based on 3–5 separate wells or 3–5 mice per treatment group. Results are expressed as meansSD or SEM as indicated. Significant differences were determined with one-way analysis of variance followed by Dunnett's test using a GraphPad Instat statistical program (GraphPad Software Inc., San Diego, CA) or by Student's unpaired t-test where appropriate. Coefficients of variation of percentage cell survival were calculated using the coefficients of variation of each mean by the method of Colquhoun (1971).

Conflict of Interest

The authors state no conflict of interest.

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Acknowledgments

This work was supported by National Health and Medical Research Grant No. 960780, a N.S.W. Cancer Council Grant No. RG57/02, and by the Fred Bauer Grant from the Australasian College of Dermatologists. R. Gupta was the recipient of a University of Sydney Medical Foundation Scholarship. K.M. Dixon was the recipient of a N.S.W. Cancer Institute Research Scholar Award.

SUPPLEMENTARY MATERIAL

Figure S1. 1,25(OH)₂D₃ increases keratinocyte survival after UVR in human skin cells derived from the back of a female subject.

Figure S2. Increase in keratinocyte survival after UVR by 1,25(OH)₂D₃ but not by inactive compound JB.

Figure S3. No correlation between 1,25(OH)₂D₃-antiproliferative effect and improved cell survival after UV with 1,25(OH)₂D₃.

Figure S4. 1,25(OH)₂D₃-induced reduction in TDs after UVR in human keratinocytes derived from the back of a female subject.

Figure S5. Reduction in keratinocyte TDs after UVR by 1,25(OH)₂D₃ but not by inactive compound JB.

Figure S6. Further increase in UVR-induced p53 expression by 1,25(OH)₂D₃.

Figure S7. Output of UVR source used for in vitro studies.