Molecular evaluation of vitamin D3 receptor agonists designed for topical treatment of skin diseases

MC903 (calcipotriol) is a synthetic, low calcemic analog of the nuclear hormone 1alpha,25-dihydroxyvitamin D3 and used in the treatment of psoriasis. The beneficial effects of MC903 on psoriasis are based on gene regulatory events. The genomic actions of 1alpha,25-dihydroxyvitamin D3 and its analogs are primarily mediated by a complex of the vitamin D3 receptor and the retinoid X receptor bound to a 1alpha,25-dihydroxyvitamin D3 response element that can be considered as the molecular switch of 1alpha,25-dihydroxyvitamin D3 signaling. In this study, the interaction of MC903 and two new analogs, GS1500 and EB1213, with this molecular switch was compared with that of 1alpha,25-dihydroxyvitamin D3. In DNA-dependent limited protease digestion assays, ligand-dependent gel shift assays and mammalian-one-hybrid assays, all four ligands appeared to be equally sensitive VDR agonists that activated vitamin D3 receptor-retinoid X receptor-1alpha,25-dihydroxyvitamin D3 response element complexes at a concentration of approximately 0.2 nM. The analyzed VDR agonists, however, also showed individual molecular properties, such as a reduced sensitivity in HaCaT cells (MC903), a selectivity for DNA-bound vitamin D3 receptor-retinoid X receptor heterodimers (GS1500) and a long-lasting stabilization of vitamin D3 receptor-retinoid X receptor-1alpha,25-dihydroxyvitamin D3 response element complexes (EB1213). This molecular evaluation demonstrated that the sensitivity in activating the vitamin D3 receptor is already optimal for MC903, but the analog may not be ideal in keeping the receptor active and in selectively triggering 1alpha,25-dihydroxyvitamin D3 signaling pathways.     

Stimulation of human melanocytes by vitamin D3 possibly mediates skin pigmentation after sun exposure

We found an increased amount of immunoreactive tyrosinase in human melanocytes after 6-d culturing with vitamin D3 (cholecalciferol). Most of these melanocytes became more dendritic and swollen in a fashion similar to that noted in the skin after ultraviolet irradiation. However, 7-dehydrocholesterol (pro-vitamin D3) or 1 alpha,25-dihydroxy-vitamin D3 (activated vitamin D3) were found to have little effect on the same system. Because vitamin D3 is known to be photochemically converted from pro-vitamin D3 in the skin by ultraviolet irradiation, the mechanism of human skin pigmentation after ultraviolet irradiation, thus far unknown, may be at least partly explained by this stimulating effect of vitamin D3 on melanocytes.     

Neue Aspekte zur Bedeutung des Vitamin-D3-Stoffwechsels in der Haut

Epidermal keratinocytes are the site of both UVB-induced photochemical conversion of 7-dehydrocholesterol to vitamin D(3) (25 OHD(3)) and the enzymatically controlled hydroxylation via 25-hydroxyvitamin D(3) to the biologically active final product 1alpha,25-dihydroxy vitamin D(3) (1alpha,25(OH)(2)D(3), calcitriol). The epidermal synthesis of calcitriol is of fundamental relevance because calcitriol regulates important cellular functions in keratinocytes and dermal immunocompetent cells. Calcitriol and other vitamin D-analogues are effective in the treatment of psoriasis because of their anti-proliferative and pro-differentiation effects. One mechanism for UVB-light therapy in psoriasis could be the induction of calcitriol synthesis. A better understanding of the metabolism of vitamin D(3) in the skin opens new perspectives for potential therapeutic applications of vitamin D analogues in inflammatory skin diseases. Further studies investigating the role of vitamin D(3) metabolism in the prevention of malignant skin disorders are needed.     

1α,25(OH)2 vitamin D3 increases intracellular calcium in human keratinocytes

Vitamin D3 metabolites have been found to improve psoriasis but their mechanism of action is not clear. Keratinocyte proliferation and differentiation are known to be dependent on calcium concentrations in vitro. The aim of this study was to examine whether 1 alpha,25(OH)2 vitamin D3 had any direct effect on intracellular free calcium concentrations in cultured keratinocytes. A response to 1 alpha,25(OH)2 vitamin D3 was seen in 88% of monolayers of normal human keratinocytes attached to glass coverslips. An increase in intracellular free calcium was seen in 80% of the reactive cultures, with over half the responses occurring within 30 s of exposure to 1 alpha,25(OH)2 vitamin D3 and the remainder occurring within minutes. Responses could be seen at physiological concentrations of 1 alpha,25(OH)2 vitamin D3 and were not blocked by the protein synthesis inhibitor cycloheximide. The response to 1 alpha,25(OH)2 vitamin D3 took the form of rapid transient increases in intracellular free calcium in 29 out of 59 coverslips. The basal intracellular free calcium was calculated to be 245 +/- 47 nM rising to a maximum of 834 +/- 267 nM (mean +/- SEM; n = 20) following exposure to 1 alpha,25(OH)2 vitamin D3. We conclude that 1 alpha,25(OH)2 vitamin D3 acts directly on keratinocytes to increase intracellular free calcium and that this may be relevant to its mechanism of action in psoriasis.     

An open study of vitamin D3 treatment in psoriasis vulgaris

Active forms of vitamin D3, 1 alpha-hydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3, were administered in an open-design study to 40 patients with psoriasis vulgaris in three ways: to 17 patients 1 alpha-hydroxyvitamin D3 was given orally at a dose of 1.0 micrograms/day for 6 months, to four patients 1 alpha,25-dihydroxyvitamin D3 was given orally at a dose of 0.5 microgram/day for 6 months, and 19 patients were given 1 alpha,25-dihydroxyvitamin D3 applied topically at concentration of 0.5 microgram/g of base for 8 weeks. Improvement was observed at the end of the individual study periods in 13 (76%) patients in Group 1 with a mean period of treatment (+/- SD) of 2.7 +/- 0.6 months, in one patient in Group 2 at 3 months after the start of treatment, and in 16 (84%) patients in Group 3 when the chemical was applied for 3.3 +/- 1.2 weeks. No side-effects were observed in any of these trials. These data suggest that psoriasis may respond to active metabolites of vitamin D3 and that abnormalities in vitamin D metabolism or in responsiveness of the skin cells to active metabolites of vitamin D may be involved in the pathogenesis of this skin disease.     

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

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 (1alpha,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 1alpha,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 1alpha,25(OH)2D3 per 10(6) cells after 6 h of incubation. The hydroxylation of vitamin D3 to 1alpha,25(OH)2D3 is inhibited by the P-450 oxidase inhibitor ketoconazole. The generation of 1alpha,25(OH)2D3 from vitamin D3 has an apparent Michaelis constant (Km) of 2.3x10(-6) M. The intrinsic conversion of vitamin D3 to biologically active 1alpha,25(OH)2D3 may be of importance for the regulation of proliferation and differentiation of keratinocytes.     

Transcriptional profiling of keratinocytes reveals a vitamin D-regulated epidermal differentiation network

1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] regulates mineral homeostasis and exhibits potent anti-proliferative, prodifferentiative, and immunomodulatory activities. It mediates these effects by binding to the vitamin D receptor (VDR), which belongs to the superfamily of steroid/thyroid hormone nuclear receptors. As a result of keratinocyte differentiation and anti-proliferation activities, 1,25(OH)(2)D(3) and its synthetic analogs are therapeutically effective in psoriasis and show promise for the treatment of actinic keratosis and squamous cell carcinoma. To elucidate the VDR signaling pathway in keratinocytes, we examined the gene expression profile with 1,25(OH)(2)D(3) treatment using oligonucleotide microarrays. Out of the 12,600 genes investigated, 82 were upregulated and 16 were downregulated and many of these were involved in differentiation, proliferation, and immune response. We have identified three vitamin D-responsive chromosomal loci (1p36, 19q13, and 6p25) and show the induction of various class II tumor suppressor/growth-regulatory genes in response to 1,25(OH)(2)D(3). Finally, quantitative differences in gene expression revealed a vitamin D-regulated differentiation network and identified peptidylarginine deiminases, kallikreins, serine proteinase inhibitor family members, Kruppel-like factor 4, and c-fos as vitamin D-responsive genes, whose protein products may play an important role in epidermal differentiation in normal and diseased state.     

1,25-dihydroxy vitamin D3 regulates cutaneous innate immune function

Hormonally active vitamin D3 – 1,25‐dihydroxyvitamin D3 (1,25D3) – acts as a signalling molecule in cutaneous immunity. In this study we investigated if Toll‐like‐receptor (TLR) function and antimicrobial peptide (AMP) expression are controlled by 1,25D3 in keratinocytes. The AMP cathelicidin and TLR cofactor CD14 were known to be induced by 1,25D3, and analysis of TLR2 expression revealed this also was increased by 1,25D3. Topical 1,25D3 application to human skin confirmed these results, showing increased cathelicidin, CD14 and TLR2 by immunostaining. Furthermore, the presence of 1,25D3 enabled human keratinocytes to respond to Malp2 (a TLR2/6 ligand) with increased cathelicidin production which was inhibited by neutralizing antibody to TLR2. 1,25D3 also increased the ability of keratinocytes to kill Staphylococcus aureus. Interestingly, keratinocytes surrounding human skin wounds increased expression of CD14 and showed a previously known increase in cathelicidin AMP. Thus, we hypothesized that 1,25D3 was also a signalling molecule during skin injury. Supporting this, we found that CYP27B1, the enzyme that converts 25‐hydroxy vitamin D3 (25D3) to active 1,25D3, was significantly increased in wounds and induced in response to factors in the wound micromilieu such as TGFβ₁ or TLR stimulation. Blocking the vitamin D receptor, inhibiting CYP27B1 enzymatic activity, or limiting 25D3 in culture each prevented TGFβ₁ from inducing cathelicidin, CD14 or TLR2. Furthermore, mice deficient in CYP27B1 failed to increase CD14 in vivo following injury. Thus, this investigation demonstrates how injury initiates the innate immune response; 25D3 is activated to 1,25D3 by enzymatic conversion, a process triggered by microbial products or host factors such as TGFβ₁. The increase in 1,25D3 then directly increases cathelicidin release and enables responsiveness to microbial products through induction of TLR cofactor CD14.     

Suppression of vitamin D receptor and induction of retinoid X receptor alpha expression during squamous differentiation of cultured keratinocytes

To gain more insight in the role of the vitamin D system in epidermal differentiation, we studied the expression of the vitamin D receptor and its heterodimeric partner retinoid X receptor alpha in cultured normal human keratinocytes during squamous differentiation, as triggered by different approaches. Northern and western blot analysis allowed us to investigate mRNA and protein levels of these nuclear receptors and of markers for growth control (c-myc, cyclin D1, p21WAF1) and differentiation (keratinocyte transglutaminase, small proline rich proteins). Growing cells to postconfluence was a potent stimulus for growth arrest and differentiation with concomitant suppression of vitamin D receptor and induction of retinoid X receptor alpha, at both the mRNA and the protein level. These changes could be prevented by concomitant treatment with epidermal growth factor or keratinocyte growth factor. Subjecting the cells to a calcium switch leading to stratification and differentiation lowered vitamin D receptor protein levels without affecting vitamin D receptor mRNA and induced both retinoid X receptor alpha mRNA and protein. Interferon-gamma and the phorbolester 12-O-tetradecanoyl phorbol 13-acetate, two well-known inducers of keratinocyte differentiation, both inhibited vitamin D receptor expression but only interferon-gamma induced retinoid X receptor alpha. The decreased vitamin D receptor expression was accompanied by reduced vitamin D responsiveness (as assessed by 24-hydroxylase mRNA induction) in postconfluent, high calcium, and 12-O-tetradecanoyl phorbol 13-acetate treated keratinocytes but not with interferon-gamma treatment. Taken together, our results associate vitamin D receptor expression with undifferentiated, proliferating keratinocytes, whereas retinoid X receptor alpha expression appears to be related to the differentiated phenotype. Therefore, proliferating and differentiating keratinocytes may be differentially targeted by active vitamin D metabolites.     

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.     

Effects of 1 alpha,25-dihydroxyvitamin D3 on the transglutaminase activity of transformed mouse epidermal cells in culture

Induction of the transglutaminase activity of a transformed mouse epidermal cell line (PAM 212 cells) by 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2-D3), the active form of vitamin D3, was investigated. Addition of 1 alpha,25-(OH)2-D3 to a culture medium stimulated twice the transglutaminase activity at a concentration of 10(-7) M, but vitamin D3, prostaglandin E1, E2, and F2 alpha failed to show this induction. Phorbol 12-o-tetradecanoylphorbol-13-acetate (TPA) and dexamethasone also induced an increase in transglutaminase activity. Exposure to both 1 alpha,25-(OH)2-D3 and retinoic acid caused remarkably synergistic effects on the induction of transglutaminase in the PAM 212 cells. In contrast, simultaneous addition of 1 alpha,25-(OH)2-D3 and TPA was antagonistic and resulted in less than additive induction. Vitamin D3 also showed a similar but lesser effect. These results suggest that 1 alpha,25-(OH)2-D3 induces the transglutaminase activity via mechanisms disparate from those of retinoic acid and modifies epidermal differentiation.     

Vitamin D and the skin

Vitamin D is a fat-soluble nutrient that humans obtain through the diet and by synthesis in the skin upon exposure to ultraviolet B. Vitamin D is then converted by the liver to 25-hydroxyvitamin D, its major circulating form. This form is the best indicator of vitamin D nutritional status and is easily measured. Under the influence of parathyroid hormone, the kidney then converts 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D, the biologically active, hormonal form of the nutrient that is important in the metabolism of calcium and phosphorus and is critical in building and maintaining healthy bones. Many cell types outside of the skeletal system, including various cells in the skin, also express the vitamin D receptor. In addition, many cell types convert circulating 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D for local use. This metabolite has been shown to exert potent effects on cellular differentiation, cellular proliferation, and immune regulation. It is theorized that by these mechanisms vitamin D and its analogues are effective treatment options for psoriasis and other skin diseases. Insufficient vitamin D nutritional status has been associated with a host of other diseases, most notably cancer. There is evidence that supplementation with vitamin D reduces the overall incidence of cancer, although current evidence is insufficient to prove a causative effect. Sunscreen use blocks the ability of the skin to photosynthesize vitamin D, although the effect this has on the vitamin D status of the general population is unclear.     

Sweat gland epithelial and myoepithelial cells are vitamin D targets

Nuclear receptor binding of 1,25(OH)(2)-vitamin D(3) (vitamin D) in skin keratinocytes of epidermis, hair sheaths and sebaceous glands was discovered through receptor microscopic autoradiography. Extended experiments with (3)H-1,25(OH)(2)-vitamin D(3) and its analog (3)H-oxacalcitriol (OCT) now demonstrate nuclear receptor binding in sweat gland epithelium of secretory coils and ducts as well as in myoepithelial cells, as studied in paws of nude mice after i.v. injection. The results suggest genomic regulation of cell proliferation and differentiation, as well as of secretory and excretory functions, indicating potential therapies for impaired secretion as in hypohidrosis of aged and diseased skin.     

Demonstration of UVB-induced synthesis of 1α,25-dihydroxyvitamin D<Subscript>3</Subscript> (calcitriol) in human skin by microdialysis

Cutaneous vitamin D(3) (VD(3)) is generated by UVB-induced photolysis of 7-dehydrocholesterol (7-DHC). VD(3) then undergoes sequential hydroxylation to calcidiol (25-OHD(3)) in the liver and to hormonally active calcitriol (1 alpha,25-(OH)(2)D(3)) in the kidney. Recently, we have described the epidermal VD(3) metabolic pathway by demonstrating the autochthonous formation of calcitriol in cultured keratinocytes. In this study we sought to determine whether photolysis of 7-DHC induced by irradiation of human skin with monochromatic UVB at 300 nm results in epidermal synthesis of calcitriol in vivo. Using a microdialysis technique we demonstrated that UVB irradiation results in a dose- and time-dependent increase in the calcitriol concentration in the extracellular fluid of UVB-irradiated skin. Topical treatment of skin with an ointment containing 2% ketoconazole immediately after irradiation suppressed UVB-induced intraepidermal calcitriol synthesis. This study demonstrates for the first time UVB-triggered synthesis of calcitriol in human skin in vivo. The link between UVB irradiation and synthesis of calcitriol in the skin may be of great importance for regulation of biological processes such as cell growth, differentiation, apoptosis and immunological reactions.     

Ergocalciferol promotes in vivo differentiation of keratinocytes and reduces photodamage caused by ultraviolet irradiation in hairless mice

BACKGROUND: Ergocalciferol (VD(2)) is usually administered orally and it is metabolized to produce its biologically active metabolites in the liver and kidney. Active vitamin D is a well-known potent regulator of cell growth and differentiation. PURPOSE: Active vitamin D such as 1,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) prevents photodamage, including wrinkles and morphologic alterations. However, its clinical and cosmetic use is limited because of its potent, associated effect on calcium metabolism. We examined the efficacy of vitamin D analogues with few adverse effects for preventing skin photodamage. METHOD: Topical application of VD(2) to hairless mouse dorsal skin, and exposure to solar-simulating ultraviolet (UV) radiation at a dose of 10.8 J/cm(2) (UVA) were performed for 15 weeks, five times a week on weekdays. At the end of the final irradiation, histological and analytical studies were performed. RESULTS: Topical application of VD(2) significantly prevented wrinkle formation and abnormal accumulation of extracellular matrix components. In addition, VD(2) suppressed excessive secretion of IL-6 induced by UV irradiation in cultured human normal keratinocytes, in a dose-dependent manner. CONCLUSION: VD(2) promoted keratinocytes differentiation in the epidermis and showed diverse physiological effects, the same as the active form of VD(3). The results suggested that the suppression of skin photodamage involved the promotion of keratinocytes differentiation and suppression of IL-6 secretion induced by exposure to UV. Topical application of VD(2) may become an effective means to suppress solar UV-induced human skin damage.     

Analysis of the vitamin D system in cutaneous squamous cell carcinomas

BACKGROUND: Increasing evidence points at an important function of vitamin D metabolites for growth regulation in various tissues, and new vitamin D analogs are interesting candidates for the treatment of malignancies, including squamous cell carcinomas (SCC). METHODS: We have analyzed expression of vitamin D receptor (VDR), vitamin D-25-hydroxylase (25-OHase), 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-OHase), and 1,25-dihydroxyvitamin D-24-hydroxylase (24-OHase) in SCC. RESULTS: Intensity of VDR immunoreactivity was increased in SCCs as compared to normal human skin. VDR staining did not correlate with histological type or grading, nor with markers for proliferation, differentiation, or apoptotic cells. Incubation of SCC cell lines (SCL-1, SCL-2) with calcitriol resulted in a dose-dependent suppression of cell proliferation (approximately up to 30%) in vitro, as measured by a tetrazolium salt (WST-1)-based colorimetric assay. RNA levels for VDR, 25-OHase, 1 alpha-OHase, and 24-OHase were significantly elevated in SCCs as compared to HS, as measured by real-time polymerase chain reaction. CONCLUSIONS: Our findings demonstrate that modulation of VDR expression and local synthesis or metabolism of vitamin D metabolites may be of importance for growth regulation of SCCs. Additionally, SCCs represent potential targets for therapy with new vitamin D analogs that exert little calcemic side effects or for pharmacological modulation of calcitriol synthesis/metabolism in these tumors.     

Image Gallery: Immunohistochemical detection of parvovirus B19 VP2 in periflexural primary infection in an adult female patient

Deficiency of vitamin D is associated with an increased risk of cancer. Although its importance in malignant melanoma is debated, vitamin D does appear to suppress the growth of the non‐melanoma skin tumours basal cell carcinoma and squamous cell carcinoma. This study from Santiago, Chile, and Boston, USA, looked at whether the vitamin D receptor in melanoma cells was lost in comparison with mole cells. Both are formed from cells called melanocytes; the difference is that the melanocytes are malignant in melanoma, benign in moles. Melanoma samples were taken from 54 patients and compared with 55 samples of moles from patients with no history of melanoma or abnormal (“dysplastic”) moles. The melanoma samples were looked at down the microscope and classified according to standard measures such as thickness, depth of invasion into the skin, ulceration, etc. They were specially stained to show up the vitamin D receptor. The intensity of staining, and the numbers of cells that showed vitamin D receptor in both the nucleus and cytoplasm, were assessed. There was significant loss of the vitamin D receptor in the cytoplasm, though not the nucleus, of melanoma cells in comparison to mole cells. This was associated with the thickness and staging of the melanoma in the skin: the thicker the tumour, the more vitamin D receptor was lost. Similar findings have been reported in carcinomas of the prostate, stomach and pancreas. The degree of loss of vitamin D receptor may help decide which patients might benefit from taking vitamin D.