Bodo Lehmann

Vitamin D and skin: new aspects for dermatology.

Abstract:  It has been shown that epidermal keratinocytes have the capacity for the UVB‐induced photochemical conversion of 7‐dehydrocholesterol to vitamin D3, and also for the enzymatically controlled hydroxylation of the photolysis product. This metabolic loop results in the formation of the biologically active final product 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3, calcitriol). The epidermal synthesis of calcitriol is of fundamental relevance because calcitriol regulates important cellular functions in keratinocytes and immunocompetent cells. Because of their anti‐proliferative and prodifferentiating effects, calcitriol and other vitamin D analogs are highly efficient in the treatment of psoriasis vulgaris. In addition, the known therapeutic effect of UVB light therapy in the treatment of psoriasis may, at least in part, be mediated via UVB‐induced synthesis of calcitriol. Increasing evidence now indicates that cutaneous vitamin D synthesis is of great importance for the prevention of a broad variety of diseases, including various malignancies. It has been postulated that cancer mortality could be reduced via careful UV exposure or, more safely, via oral substitution with vitamin D. These new findings must be taken into account when establishing new sun protection guidelines for the prevention of skin cancer. In addition, better understanding of the metabolism of vitamin D in the skin has opened up new perspectives for the therapeutic application of vitamin D analogs, e.g. in inflammatory skin diseases.

The Vitamin D3 Pathway in Human Skin and its Role for Regulation of Biological Processes

Abstract The skin is the only tissue yet known in which the complete ultraviolet-B (UV-B)–induced pathway from 7-dehydrocholesterol to hormonally active calcitriol (1α,25-dihydroxyvitamin D3) occurs under physiological conditions. Epidermal synthesis of calcitriol could be of fundamental relevance because calcitriol regulates important cellular functions in keratinocytes and immunocompetent cells. Because of their antiproliferative and prodifferentiating effects, calcitriol and other vitamin D analogs are highly efficient in the treatment of psoriasis vulgaris. The known antipsoriatic effect of UV-B light could, at least in part, be mediated via UV-B–induced synthesis of calcitriol. In addition, mounting evidence indicates that cutaneous vitamin D3 synthesis is of high importance for the prevention of a broad variety of diseases, including various malignancies. New but controversially discussed sun-protection guidelines were established for the prevention of internal cancers. A better understanding of the metabolism of vitamin D in the skin opens new perspectives for therapeutic applications of vitamin D analogs.

Vitamin D metabolism

Irradiation of human skin with ultraviolet B (280–320 nm) initiates the photochemical conversion of 7‐dehydrocholesterol via previtamin D3 to vitamin D3. Vitamin D3 needs for its activation two hydroxylation steps in the liver and kidney. The final product, hormonally active 1α,25‐dihydroxyvitamin D3 (calcitriol), arrives via the circulation to its target tissues and acts in a genomic or nongenomic manner. It has been found that human skin irradiated with ultraviolet B also is able to produce calcitriol in substantial amounts. This cutaneous vitamin D3 pathway is unique and, most likely, of considerable relevance for healthy and diseased skin. It is well known that topical application of calcitriol and its analogs can improve hyperproliferative skin diseases. Some studies have convincingly demonstrated that calcitriol and other vitamin D analogs may also be used for the treatment of immunological, inflammatory, and infectious skin diseases. More recently, it has been found that calcitriol or vitamin D analogs have photoprotective effects and can reduce UV‐induced deoxyribonucleic acid damage.

Role of the vitamin D3 pathway in healthy and diseased skin--facts, contradictions and hypotheses.

Abstract:  Irradiation of human keratinocytes with UVB (280–320 nm) in vitro and in vivo activates the metabolism of 7‐dehydrocholesterol to hormonally active calcitriol. The production of calcitriol in the skin strongly depends on the photosynthesis of vitamin D3 which is biologically inactive in the first instance. Vitamin D3 serves as the starting substrate for two subsequent enzymatic hydroxylation steps in epidermal keratinocytes. Both the amount of vitamin D3 and the activity of anabolic and catabolic vitamin D hydroxylases determine the cutaneous level of calcitriol. The hormonally active metabolite of vitamin D3 regulates a huge number of genes in keratinocytes, and thus acts in an autocrine and/or paracrine manner. This local pathway of vitamin D3 is unique, but its relevance for healthy and diseased skin is widely unknown, yet. Experimental findings implicate several questions: ( 1 ) Is UVB‐induced formation of calcitriol involved in regulation of growth and differentaition of epidermal cells as well as immunological and skin protective processes? ( 2 ) What endogenous and exogenous factors including drugs affect the cutaneous vitamin D3 pathway? From a therapeutical point of view, it has been known for a long time that topical application of calcitriol and its analogs can improve hyperproliferative skin diseases like psoriasis. In spite of many encouraging studies in recent years, the fields of the routinely therapeutical application of calcitriol or vitamin D analogs in dermatology (e.g. treatment of immunological, inflammatory, malignancies and infectious skin diseases) have not been intensified. Why is that?

Selection and Validation of Candidate Housekeeping Genes for Studies of Human Keratinocytes—Review and Recommendations

Gene expression analysis using real-time PCR has become an integral part of biomedical research. Appropriate data normalization based on stably expressed housekeeping genes is crucial for reliable results. Thus, candidate housekeeping genes require careful evaluation with regard to the individual experimental system before being selected for studies of human keratinocytes. Future research may be based on published data, as provided by Minner and Poumay in this issue.

UVB-induced Conversion of 7-Dehydrocholesterol to 1α,25-Dihydroxyvitamin D3 (Calcitriol) in the Human Keratinocyte Line HaCaT¶

Abstract We have previously shown that keratinocytes in vitro can convert biologically inactive vitamin D3 to the hormone calcitriol. The present study was initiated to test whether ultraviolet B (UVB)-induced photolysis of provitamin D3 (7-dehydrocholesterol, [7-DHC]) which results in the formation of vitamin D3 also leads to the generation of calcitriol in keratinocytes. Submerged monolayers of HaCaT keratinocytes were preincubated with 7-DHC (25 μM) at 37°C and irradiated with monochromatic UVB at different wavelengths (effective UV-doses: 7.5–60 mJ/cm2), or a narrow-band fluorescent lamp Philips TL-01 (UVB-doses: 125–1500 mJ/cm2). Irradiation with both sources of UVB resulted in the generation of different amounts of previtamin D3 in our in vitro model followed by time-dependent isomerization to vitamin D3 and consecutive formation of calcitriol in the picomolar range. Unirradiated cultures or cultures exposed to wavelengths >315 nm generated no or only trace amounts of calcitriol. The conversion of vitamin D3 generated after UVB irradiation to calcitriol is inhibited by ketoconazole indicating the involvement of P450 mixed function oxidases in this chemical reaction. The generation of calcitriol was wavelength- and UVB dose dependent and reached approximately 18-fold higher levels after irradiation at 297 nm than at 310 nm (effective UVB dose: 30 mJ/cm2). Hence, keratinocytes may be a potential source of biologically active calcitriol within epidermis, when irradiated with therapeutical doses of UVB.

Extrarenal sites of calcitriol synthesis: the particular role of the skin.

Calcitriol (1alpha,25(OH)2D3), the hormonally active form of vitamin D3 (D3) is produced by a cascade of reactions, including photochemical D3 synthesis in the skin and subsequent hydroxylation at the C-25 atom in the liver and finally at C-1alpha position in the kidney. However, there is substantial evidence for additional extrarenal sites of calcitriol synthesis. In vitro, many nonrenal cells, including bone, placenta, prostata, keratinocytes, macrophages, T-lymphocytes and several cancer cells (e.g., from lung, prostata and skin) can enzymatically convert calcidiol (25OHD3) to 1alpha,25(OH)2D3. We have demonstrated that keratinocytes of the skin have unique properties in the D3 pathway; they are not only capable of producing D3 from 7-dehydrocholesterol (7-DHC), but also generate 1alpha,25(OH)2D3 from the substrates 25OHD3, lalpha-hydroxyvitamin D3 (1alpha-OHD3) and even D3. It is evident that keratinocytes possess at least 1alpha-OH-, 25OH- and 24OHase activity, which is necessary for enzymatic conversion of D3 to 1alpha,25(OH)2D3, and the 24OHase activity for initiation of catabolism of 1alpha,25(OH)2D3 to more polar metabolites. Thus, the skin is apparently the only extrarenal tissue where the complete UVB-induced pathway from 7-DHC to 1alpha,25(OH)2D3 takes place under physiological circumstances.

Conversion of vitamin D3 to 1α, 25-dihydroxyvitamin D3 in human skin equivalents

Abstract: These results demonstrate for the first time that human keratinocytes under in vivo‐like conditions have the capacity of the enzymatic hydroxylation of vitamin D3 to hormonally active calcitriol (1α,25(OH)2D3). Supplementation of the culture medium with bovine serum albumin (BSA) up to 1.5% (w/v) amplifies the conversion of vitamin D3 to 1α,25(OH)2D3. The maximum turnover rate of this reaction at 780 nM vitamin D3 in presence of 1.0% (w/v) BSA amounts to approximately 3 pmol 1α,25(OH)2D3 per 106 cells after 6 h of incubation. The hydroxylation of vitamin D3 to 1α,25(OH)2D3 is inhibited by the P‐450 oxidase inhibitor ketoconazole. The generation of 1α,25(OH)2D3 from vitamin D3 has an apparent Michaelis constant (Km) of 2.3×10−6 M. The intrinsic conversion of vitamin D3 to biologically active 1α,25(OH)2D3 may be of importance for the regulation of proliferation and differentiation of keratinocytes.

Human keratinocyte line HaCaT metabolizes 1α-hydroxyvitamin D3 and vitamin D3 to 1α,25-dihydroxyvitamin D3 (calcitriol)

Abstract Cultured human keratinocytes have the property to hydroxylate exogenous 25-hydroxyvitamin D 3 (25OHD 3 ) at the C-1 α position thus producing 1 α ,25-dihydroxyvitamin D 3 (1 α ,25(OH) 2 D 3 ). In this study we investigated whether keratinocytes can also hydroxylate vitamin D 3 and one of its metabolites at the C-25 position. We could demonstrate that HaCaT keratinocytes can metabolize 1 α -hydroxyvitamin D 3 (1 α -OHD 3 ) and vitamin D 3 to 1 α ,25(OH) 2 D 3 . Identification of the generated product as 1 α ,25(OH) 2 D 3 was based on its elution pattern in two different high performance liquid chromatography systems, on its specific binding in a calf thymus receptor assay and on its gas chromatography–mass spectrometry characteristics. The hydroxylation of vitamin D 3 to 1 α ,25(OH) 2 D 3 was dose- and time-dependent. Bovine serum albumin added up to 1.5% (w/v) to the culture medium greatly increased the hydroxylation rates. These results show that HaCaT cells have the capacity to hydroxylate vitamin D 3 at the C-1/25 positions. The generation of endogenous 1 α ,25(OH) 2 D 3 from vitamin D 3 within the skin may indicate a novel pathway which is of importance for the regulation of epidermal cell growth and differentiation.

The UVB-induced synthesis of vitamin D3 and 1α,25-dihydroxyvitamin D3 (calcitriol) in organotypic cultures of keratinocytes: Effectiveness of the narrowband Philips TL-01 lamp (311 nm)

Both calcitriol and UVB radiation exert potent antipsoriatic effects. We hypothesize that the therapeutical effect of UVB radiation may be attributed at least in part to UVB-triggered cutaneous synthesis of calcitriol. The optimum wavelength for initiation of the vitamin D(3) pathway was found to be in the range of 300+/-5 nm in vitro and in vivo. The narrowband Philips TL-01 lamp which is commonly used as UVB source for phototherapy of psoriasis has maximum spectral irradiance at around 311 nm which is presumed to be, however, of lesser importance in photochemical activation of the vitamin D(3) pathway. The aim of this study was to compare the vitamin D(3) and calcitriol-inducing potential of UVB from the TL-01 lamp with that of monochromatic UVB at 300+/-2.5 nm and 310+/-2.5 nm in organotypic cultures of keratinocytes supplemented with 25 microM 7-DHC. We found that maximum calcitriol-generating capacity of the TL-01 lamp at 500 mJ/cm(2) and 16 h after irradiation still amounts up to 44% of that found after monochromatic irradiation at 300+/-2.5 nm and 30 mJ/cm(2). Thus, the antipsoriatic effect of UVB emitted from the TL-01 lamp may, at least in part, based on the antiproliferative and prodifferentiative action of newly synthesized calcitriol on epidermal keratinocytes.