Although known since 1922, vitamin D3 or cholecalciferol was identified only as recently as the late 1960s as the precursor of the active steroid 1,25-dihydroxyvitamin D3 or calcitriol, and this active steroid was known for its involvement in the metabolism of calcium—i.e., for its effects on bone mineralization. For this reason, children were administered fish oils, which are highly concentrated in vitamin D3. Food supplementation with vitamin D is common today and benefits the entire population.
Initially, the metabolism of vitamin D3 to calcitriol was thought to occur only in the liver and kidneys. However, the vitamin D receptor (VDR) for 1,25-dihydroxyvitamin D3 was more recently discovered in more than 30 tissues or organs including the skin, thus initiating new studies and reports on the activities of vitamin D metabolites and their regulation of important cellular skin functions.
Metabolism in Skin
The metabolism of vitamin D in the skin is a multi-step process that starts from 7-dehydrocholesterol.1-3 This is an important molecule in the synthesis of cholesterol and is present mainly in the stratum spinosum and stratum basale of the epidermis. 7-Dehydrocholesterol is a strong UV absorber with 3 λ max around 270 nm, 280 nm and 295 nm, and under UV irradiation, it is partly photolyzed to create previtamin D3 (see Figure 1). During prolonged exposure to the sun, the synthesis of previtamin D3 reaches a plateau at a concentration of about 10–15% of the original 7-dehydrocholesterol concentration.
In turn, previtamin D3 is rapidly isomerized into inactive vitamin D3, which then undergoes enzymatic hydroxylation in epidermal keratinocytes with the production of 1,25-dihydroxyvitamin D3. This calcitriol is an active steroid and mediates its effects by binding to the VDR. Under irradiation, other previtamin D3 derivatives also are formed, lumisterol and tachysterol, as well as other hydroxyl metabolites of vitamin D3, which are formed by the action of various hydrolases (see Figure 2).4
Therefore, it is obvious that UV exposure is a key factor in the production of vitamin D3 in the skin and that conditions affecting or limiting skin exposure to UV or skin absorption of UV may affect the formation of the vitamin D3 active metabolite—i.e., overuse of sunscreens or clothing; nursing home residents who remain indoors; seasonal and regional factors including latitude and altitude; time of the day; and skin pigmentation.5-7 The generation of calcitriol also is reported to be wavelength and UV dose dependent.2
Skin Cell Differentiation
Calcitriol is known to be involved in the differentiation and regulation of growth of keratinocytes.8 As such, analogues of 1,25-dihydroxyvitamin D3 are used topically to treat hyperproliferation in skin diseases such as psoriasis to provide anti-proliferative and pro-differentiating effects; the effectiveness of a specific wavelength to optimally generate calcitriol has been studied for this purpose.9
Similarly it is well-known that the production of 1,25-dihydroxyvitamin D3 influences the formation of the epidermal calcium gradient, which is involved in skin cell differentiation and in the formation of the envelope barrier.10-11 Calcium levels in the epidermis are the lowest in the stratum basale and the highest in the stratum granulosum, and calcium and calcitriol together regulate keratinocyte differentiation and induce involucrin, a protein important for the formation of the cornified envelope and for skin barrier function.12
Chronic low dose UV irradiation also stimulates the enzymes responsible for lipid synthesis in the skin—i.e., fatty acid synthase, serine palmitoyl transferase and HMG-CoA reductase—as well as the expression of filagrin and involucrin, in parallel with changes in vitamin D3 metabolism.13 Calcitriol and other hydroxylated derivatives of vitamin D exhibit this stimulating effect on keratinocyte differentiation and enhance the expression of proteins involved in barrier function.14 Obviously the stimulation of vitamin D production in the skin by suberythemal UV irradiation has an effect on the skin’s function.
Another recent report showed that 7-dehydrocholesterol by itself plays an important role in the formation of the cornified envelope of the skin, and on the maturation of the corneocytes.15
Protection from Microbial Attack
Interestingly, calcitriol also regulates immunocompetent cells. Skin is constantly exposed to microbes and constitutes an active barrier against pathogen attacks. The cutaneous production of antimicrobial peptides, such as defensins and cathelicidins, provides protection against infections16 as well as activates Toll-like receptors (TLRs), which trigger antimicrobial activity.
Low dose UV irradiation stimulates the expression of cathelicidin LL-37 and β-defensin 2 in the skin in parallel with an up-regulation of the cutaneous vitamin D3 system.13 Recent studies have demonstrated that the vitamin D3 metabolite is a major factor in the regulation of cathelicidin expression in keratinocytes through the induction of its precursor, hCAP18, by 1,25-dihydroxyvitamin D3.17 This molecule is also able to increase TLR-2 expression in keratinocytes.
Several skin diseases are associated with cathelicidin dysfunction. For instance, in atopic dermatitis and eczema, cathelicidin expression is reduced. In other diseases, cathelicidin expression is induced such as in rosacea or psoriasis, both with increased skin inflammation. Obviously, a sophisticated system operates between innate immunity and the vitamin D system. It is also interesting that the formation of 1,25-dihydroxyvitamin D3 in wound healing processes is regulated to amplify the antimicrobial response.16,18–20 Sebaceous glands also express cathelicidin, where it is inducible by 1,25-dihydroxyvitamin D3, suggesting a possible role of vitamin D metabolite in the defense of the follicle and in acne.21
UV Irradiation and Skin Damage
The contribution of vitamin D to protect keratinocytes against cell death induced by various stimuli and against senescence has been demonstrated on various models. Keratinocytes irradiated by ionizing radiation to simulate radiotherapy were treated with calcitriol, and an inhibition of caspase-dependent and independent programmed cell death was observed.22
Enhancement of the survival of cells under exposure to aggressive conditions has also been observed in vitro with the stimulation of protective Heat Shock Proteins by 7-dehydrocholesterol, the vitamin D3 precursor.23 In addition, protection brought by 1,25-dihydroxyvitamin D3 on skin survival under stressed conditions was also obtained in vitro with 7-dehydrocholesterol, which is active on the tumor suppressor protein p53 (the “guardian of the genome”) and on senescence-associated β-galactosidase, therefore contributing to the slowing of senescence in the keratinocytes.24
Calcitriol protects human skin cells from UV irradiation-induced apoptosis and from DNA damage by decreasing sunburn cells and cyclobutane pyrimidine dimers in surviving cells. In parallel, an increase in p53 expression and a reduction in the formation of nitric oxide and its derivatives were obtained with 1,25-dihydroxyvitamin D3 treatment in UV irradiated keratinocytes. This photoprotective effect of calcitriol has been observed in vitro and in vivo.25 It was also found that topically applied 7-dehydrocholesterol increases the minimal dose of UVB required to induce erythema.23Formula 1 and Formula 2 illustrate the use of 7-dehydrocholesterol in applications.
Besides psoriasis, vitamin D analogs are used to treat another severe skin condition, vitiligo—a pigmentary disorder characterized by the loss of functional melanocytes in the epidermis.26 A number of studies have reported that topical treatment with vitamin D compounds or their combination with UV light or corticosteroids enhances re-pigmentation in vitiligo.
For instance, 1,25-dihydroxyvitamin D3 was shown to increase the tyrosinase content and melanin formation in B16 mouse melanoma cells.27 Findings indicate that calcitriol promotes the advanced stage development of melanosomes and expression of endothelins in melanocyte precursors. Human keratinocytes produce endothelin-1, which is increased after UVB irradiation, and the endothelins secreted play an essential role in the maintenance of melanocyte proliferation and differentiation and pigmentation in the epidermis.28,29 Calcium regulation in melanocytes also regulates the supply of tyrosine for melanogenesis, and vitamin D analogs have been shown to restore calcium homeostasis and calcium flux in the melanocytes.30
Hair follicles are composed of epidermal keratinocytes and underlying dermal papilla cells, and these cells express the VDR. The VDR also is essential for hair follicle integrity, and the absence, loss or inactivation of the VDR leads to hair loss and the development of alopecia.31 In mice, the VDR is expressed throughout the basal layer of the interfollicular epidermis, the outer root sheath of the hair follicle, and at the periphery of sebaceous glands. During the murine hair cycle, the VDR expression in the hair follicle is stronger in anagen IV-VI and catagen phases than during telogen and early anagen phases, correlating with decreased proliferation and increased differentiation of the keratinocytes.32
Cytotoxic agents used in chemotherapy induce apoptosis of anagen hair bulbs in mice, and while pretreatment of the mice with topical 1,25-dihydroxyvitamin D3 did not prevent the chemotherapy-induced alopecia, the degree of apoptosis on follicle keratinocytes was reduced. In addition, hair shaft regrowth was improved as compared to non-pretreated mice.33-35
Mode of Action
The ability of calcitriol to generate biological responses is mediated through binding to the vitamin D3 receptor. Initially it was thought that the VDR was located in the cell nuclei like other steroid hormone receptors and was therefore involved in selective gene transcriptions for long-term genomic biological responses. However, rapid biological responses mediated by 1-25-dihydroxyvitamin D3 have also been obtained in minutes, which cannot be explained by nuclear VDR-regulating gene transcription. It appears that VDR is present not only in the cell nuclei, but also in the caveolae-enriched plasma membranes where it can generate rapid and direct responses through non-genomic mechanisms.36
An interesting approach to explain the various kinds of response is related to the chemical conformation of calcitriol, which provides exceptional flexibility and exhibits various structural shapes that can bind the receptor for either VDR-mediated nuclear responses or for rapid responses.37
7-Dehydrocholesterol, vitamin D3 and calcitriol—these molecules are involved in complex processes to protect and influence mechanisms in the skin such as: the skin envelope and barrier function, through cell differentiation; antimicrobial activation of antimicrobial peptides; protection against damage from UV irradiation; action on melanocytes and melanogenesis; and protection of the hair follicle. These benefits start with the 7-dehydrocholesterol naturally present in the epidermis—a natural precursor in vitamin D3 metabolism. Supplying 7-dehydrocholesterol to the skin in topical formulations is therefore an easy way to take advantage of these multiple activities.
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