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An evaluation of the biologic activity and vitamin D receptor binding affinity of the photoisomers of vitamin D3 and previtamin D3

https://doi.org/10.1016/S0955-2863(00)00077-2Get rights and content

Abstract

Skin is the site of previtamin D3 and vitamin D3 synthesis and their isomerization in response to ultraviolet irradiation. At present, little is known about the function of the photoisomers of previtamin D3 and vitamin D3 in skin cells. In this study we investigated the antiproliferative activity of the major photoisomers and their metabolites in cultured human keratinocytes by determining their influence on 3H-thymidine incorporation into DNA. Our results demonstrated that vitamin D3 and 25-hydroxytachysterol3 were effective in inhibiting 3H-thymidine incorporation at both 10−8 and 10−6 M in a dose-dependent manner. Lumisterol, tachysterol3, 5,6-trans-vitamin D3, and 25-hydroxy-5,6-transvitamin D3 only induced significant inhibition at 10−6 M. 25-Hydroxytachysterol3 was approximately 10- to 100-fold more active than tachysterol3. 7-Dehydrocholesterol was not active even at 10−6 M. The dissociation constants of vitamin D receptor (VDR) for 25-hydroxytachysterol3, 25-hydroxy-5,6-trans-vitamin D3, and 5,6-trans-vitamin D3 were 22, 58, and 560 nM, respectively. The dissociation constants for 7-dehydrocholesterol, tachysterol, and lumisterol were greater than 20 μM. In conclusion, vitamin D3, its photoisomers, and the photoisomers of previtamin D3 have antiproliferative activity in cultured human keratinocytes. However, the antiproliferative activity did not correlate with their binding affinity for VDR. The results suggest that some of the photoproducts may be metabolized to their 25-hydroxylated and 1α,25-dihydroxylated counterparts before acting on VDR. Alternatively, a different receptor may recognize these photoproducts or another mechanism may be involved in modulating the antiproliferative activity of the photoisomers examined.

Introduction

Vitamin D is absolutely essential for the maintenance of calcium homeostasis and for the development and maintenance of healthy bones in animals.1 There are two major sources of vitamin D; one is diet and the other is the cutaneous synthesis. However, because vitamin D is rare in foods, it is casual exposure to sunlight that is responsible for providing humans with most of their vitamin D requirement. During exposure to sunlight, the ultraviolet B (UVB) portion of the solar spectrum (295–315 nm) causes the photolysis of epidermal stores of 7-dehydrocholesterol (7-DHC) to previtamin D3. Previtamin D3 then thermoisomerizes to vitamin D3, a process that is facilitated by the lipid-membrane environment.2 After vitamin D3 is made in the skin or ingested in the diet, it must be hydroxylated at carbon positions 25 and 1 in the liver and kidney, respectively, to form 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], the active form of vitamin D3. 1α,25(OH)2D3 is responsible for promoting intestinal calcium absorption and the mobilization of calcium from bone for the purpose of maintaining a normal level of serum calcium. During the past 20 years, it has been recognized that 1α,25(OH)2D3 also can influence a variety of other biologic processes through its vitamin D receptor (VDR) in tissues other than those that regulate calcium metabolism, including skin. In cultured human keratinocytes, 1α,25(OH)2D3 inhibited basal cell proliferation and stimulated differentiation to form the cornified envelope.3, 4, 5

During chronic exposure to sunlight, both previtamin D3 and vitamin D3 in the skin can be photoisomerized to a variety of photoproducts, including 5,6-trans-vitamin D3, tachysterol, and lumisterol.6, 7 Because the concentrations of these photoproducts in skin cells are likely to be very high, it is possible that some of these photoproducts may have direct biologic properties on skin cells. Because tachysterol and 5,6-trans-vitamin D3, which are photoproducts of previtamin D3 and vitamin D3, respectively, have a pseudo-1-α-hydroxyl structure due to the 180-degree rotation of the 3-hydroxyl group during isomerization (Figure 1 ), it is possible that these analogs may act like 1α,25(OH)2D in the epidermis. It has been previously reported that 5,6-trans-vitamin D3 and tachysterol can mimic the intestinal calcium transport activity of 1α,25(OH)2D in anephric rats and that their 25-hydroxy derivatives were more active in the same assay system.8 Therefore, we evaluated the potential antiproliferative activity of the major photoproducts of previtamin D3 and vitamin D3 by determining their effects on 3H-thymidine incorporation into cultured normal human keratinocytes and compared the antiproliferative activity with their binding to VDR using a calf thymus cytosol receptor preparation.

Section snippets

Cell cultures

Normal human keratinocytes were obtained from neonatal foreskins as described previously.4, 9 Keratinocytes were plated and grown on lethally irradiated 3T3 fibroblast feeder layer in a serum-free basal medium supplemented with amino acids and growth factors as previously described.10

3H-thymidine incorporation

When the second-passage cells grown on 24-well plates reached approximately 40% confluency, they were fed with basal MCDB-153 medium supplemented with amino acids without growth factors.10 Two days later, cells

Results

Figure 2 demonstrates that 1α,25(OH)2D3 was highly effective in inhibiting 3H-thymidine incorporation in a dose-dependent manner as reported previously.10 At concentrations of 10−10, 10−8, and 10−6 M, 1α,25(OH)2D3 caused a 48 ± 2, 65 ± 3, and 85 ± 1% inhibition, respectively, in 3H-thymidine incorporation compared with the control. Lumisterol3, tachysterol3, 5,6-trans-vitamin D3, and 25(OH)5,6-trans-vitamin D3 at either 10−10 or 10−8 M did not cause any significant inhibition in 3H-thymidine

Discussion

Our results demonstrate that vitamin D3 itself and some of the photoisomers of vitamin D3 and previtamin D3 including tachysterol3, 5,6-trans-vitamin D3, and their 25-hydroxylated metabolites are active in inhibiting 3H-thymidine incorporation into cultured normal human keratinocytes (Figure 2). The activity found for vitamin D3 could be attributed to its metabolism to 1α,25(OH)2D3 in cultured keratinocytes, which have been shown to possess both vitamin D-25-hydroxylase and

Acknowledgements

The work was supported in part by grants RO1-AR36963, RO1-DK43690, and MO1RR00533 from the National Institutes of Health and from a generous gift from California Tan Inc. and the Heliotherapy, Light and Skin Research Center. The authors would like to thank David Jackson for preparation of the graphics.

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