Dermal fibroblasts pretreated with a sterol Delta7-reductase inhibitor produce 25-hydroxyvitamin D3 upon UVB irradiation.

As dermis is a physiological site of vitamin D3 photoproduction, the photo-endocrine vitamin D3 system was studied in dermal fibroblasts. Dermal fibroblasts contain the vitamin D receptor and induce 1alpha,25-dihydroxyvitamin D3-24-hydroxylase [CYP24] mRNA upon stimulation with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In addition, dermal fibroblasts contain mRNA of the vitamin D3-25-hydroxylases (CYP2R1 and CYP27A1). However, we could not detect any 25-hydroxyvitamin D3 [25OHD3]-1alpha-hydroxylase mRNA in dermal fibroblasts and no CYP24 mRNA was induced upon ultraviolet [UVB] irradiation, even when endogenous 7-dehydrocholesterol content was elevated by pretreatment with the sterol Delta7-reductase inhibitor BM15766. Nevertheless, dermal fibroblasts produce inactive vitamin D3 metabolites that can be activated by epidermal keratinocytes as CYP24 mRNA is induced in epidermal keratinocytes but not in dermal fibroblasts after transfer of medium or cellular suspensions from BM15766-pretreated, UVB-irradiated fibroblasts. This CYP24 induction was UVB-dose dependent and was inhibited by ketoconazole. As revealed in a competitive binding assay, BM15766-pretreated dermal fibroblasts are able to produce 25OHD3 upon UVB irradiation, but no 1,25(OH)2D3 was detected via combined high-performance liquid chromatography radioimmunoassay. The physiological relevance of dermal vitamin D3 photoproduction and its subsequent conversion into 25OHD3 remains elusive.

UVB-induced 1,25(OH)2D3 production and vitamin D activity in intestinal CaCo-2 cells and in THP-1 macrophages pretreated with a sterol Delta7-reductase inhibitor.

Epidermal keratinocytes are able to produce 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and induce vitamin D activity upon UVB irradiation. To find out whether this property is keratinocyte specific, we investigated this characteristic in two other cell types, namely intestinal CaCo-2 cells and the macrophage-like differentiated THP-1 cells. THP-1 macrophages and preconfluent CaCo-2 cells contain the vitamin D receptor (VDR), possess 25-hydroxylase (CYP2R1 and CYP27A1) and 1alpha-hydroxylase (CYP27B1) activity, and survive the low UVB doses essential for vitamin D3 photoproduction. Upon irradiation, 24-hydroxylase (CYP24) mRNA is induced in both cell types pretreated with the sterol Delta7-reductase inhibitor BM15766 whereby the 7-dehydrocholesterol (7-DHC) content was increased. Transfection studies in CaCo-2 cells with a vitamin D response element-containing construct revealed the involvement of the VDR in this UVB-dependent CYP24 induction. The CYP24 inducing activity in BM15766-pretreated UVB-irradiated CaCo-2 cells and THP-1 macrophages was identified as 1,25(OH)2D3 by combined high-performance liquid chromatography radioimmunoassay. Addition of vitamin D binding protein to the CaCo-2 cells attenuated UVB-induced CYP24 induction suggesting the possibility of a paracrine or autocrine role for the photoproduced 1,25(OH)2D3. In conclusion, preconfluent CaCo-2 cells and THP-1 macrophages are able to induce vitamin D activity upon UVB irradiation and hence combine all parts of the vitamin D photoendocrine system, a characteristic which is therefore not keratinocyte specific.

UVB-induced production of 1,25-dihydroxyvitamin D3 and vitamin D activity in human keratinocytes pretreated with a sterol delta7-reductase inhibitor.

The skin fulfills an important role in the vitamin D photo-endocrine system. Epidermis is not only the site of vitamin D3 photoproduction. In addition, epidermal keratinocytes contain the vitamin D receptor (VDR) and possess 25-hydroxylase and 1alpha-hydroxylase activity indicating that all components of the vitamin D system are present. We investigated whether these components cooperate in inducing vitamin D activity upon treatment with physiological UVB doses. Upon irradiation, 24-hydroxylase mRNA was induced in keratinocytes pretreated with a sterol Delta7-reductase inhibitor (BM15766) whereby the 7-dehydrocholesterol content increased by 300-fold. Transfection experiments with a vitamin D response element containing construct confirmed VDR-dependent gene activation. Furthermore, the UVB-dependent induction of 24-hydroxylase was blocked by the cytochrome-P450 inhibitor ketoconazole. The 24-hydroxylase inducing photoproduct was transferable to unirradiated keratinocytes by medium and cellular homogenates of UVB-irradiated, BM15766-pretreated cells and was identified as 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by high-performance liquid chromatography with tandem mass spectrometric detection. Addition of vitamin D binding protein blunted UVB-induced 24-hydroxylase suggesting the possibility of a paracrine or autocrine role for 1,25(OH)2D3. In conclusion, epidermal keratinocytes can produce vitamin D3, convert it to 1,25(OH)2D3 and respond to it upon UVB irradiation in the absence of exogenous 7-dehydrocholesterol and therefore contain a unique and complete photo-endocrine vitamin D system.

1,25-Dihydroxyvitamin D3 and analogues protect primary human keratinocytes against UVB-induced DNA damage.

Exposure to UVB irradiation is a major risk factor for the development of skin cancer. Therefore, it is important to identify agents that can offer protection against UVB-caused damage. Photocarcinogenesis is caused largely by mutations at sites of incorrectly repaired DNA photoproducts, of which the most common are the cyclobutane pyrimidine dimers (CPDs). In this study, we demonstrated that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] protects primary human keratinocytes against the induction of CPDs by UVB. This protection required pharmacologic doses 1,25(OH)2D3 and an incubation period of at least 8 h before irradiation. Furthermore, we provided arguments indicating that the anti-proliferative capacity of 1,25(OH)2D3 underlies its protective effect against UVB-induced DNA damage. Finally, we showed that 19-nor-14-epi-23-yne-1,25(OH)2D3 (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)2D3 (TX 527), two low-calcemic analogues of 1,25(OH)2D3, were even 100 times more potent than the parent molecule in inhibiting UVB-caused DNA damage. These molecules are therefore promising candidates for the chemoprevention of UVB-induced skin cancer.

Molecular pathways involved in the anti-apoptotic effect of 1,25-dihydroxyvitamin D3 in primary human keratinocytes.

We previously reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] protects primary human keratinocytes against ultraviolet (UV)B-induced apoptosis. Here, we confirmed the anti-apoptotic effect of 1,25(OH)2D3 in keratinocytes, using cisplatin and doxorubicin as apoptotic triggers. We further showed that 1,25(OH)2D3 activates two survival pathways in keratinocytes: the MEK/extracellular signal regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway. Activation of ERK and Akt by 1,25(OH)2D3 was transient, required a minimal dose of 10(-9) M and could be blocked by actinomycin D and cycloheximide. Moreover, inhibition of Akt or ERK activity with respectively a PI-3K inhibitor (LY294002) or MEK inhibitors (PD98059, UO126), partially or totally suppressed the anti-apoptotic capacity of 1,25(OH)2D3. Finally, 1,25(OH)2D3 changed the expression of different apoptosis regulators belonging to the Bcl-2 family. Indeed, 1,25(OH)2D3 treatment increased levels of the anti-apoptotic protein Bcl-2 and decreased levels of the pro-apoptotic proteins Bax and Bad in a time- and dose-dependent way. Induction of Bcl-2 by 1,25(OH)2D3 was further shown to be mediated by ERK and, to a lesser extent, by Akt. In conclusion, 1,25(OH)2D3 clearly protects keratinocytes against apoptosis (1) by activating the MEK/ERK and the PI-3K/Akt survival pathways and (2) by increasing the Bcl-2 to Bax and Bad ratio.

Two 14-epi analogues of 1,25-dihydroxyvitamin D3 protect human keratinocytes against the effects of UVB.

In search of photoprotective agents, we recently demonstrated a protective effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] against different events mediated by ultraviolet B (UVB) in human keratinocytes. Pharmacological doses of 1,25(OH)(2)D(3) were required to obtain significant UVB protection; however, these doses cannot be used in vivo due to the calcemic properties of 1,25(OH)(2)D(3). Therefore, we evaluated the photoprotective capacities of two low-calcemic 14-epi analogues of 1,25(OH)(2)D(3), 19-nor-14-epi-23-yne-1,25(OH)(2)D(3) (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3) (TX 527). Using cultured human keratinocytes, we investigated the influence of TX 522 and TX 527 on two hallmark events in UVB-irradiated keratinocytes: the induction of apoptosis and the production of interleukin-6 (IL-6). Treatment of the keratinocytes with TX 522 or TX 527, 24 h before irradiation, resulted in a significant and dose-dependent reduction of both UVB-induced apoptosis and IL-6 production. Both analogues were equally efficient in their anti-UVB effects and at least 100 times more potent than 1,25(OH)(2)D(3). We further demonstrated that metallothionein (MT) mRNA expression was clearly induced by 1,25(OH)(2)D(3) and both analogues. MT acts as a radical scavenger in oxygen-mediated UVB injury and its induction may therefore be relevant for the anti-UVB effects of 1,25(OH)(2)D(3) and both analogues. Taken together, these findings create new perspectives for the use of active vitamin D analogues as photoprotective agents.

1,25-Dihydroxyvitamin D3 inhibits ultraviolet B-induced apoptosis, Jun kinase activation, and interleukin-6 production in primary human keratinocytes.

We investigated the capacity of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] to protect human keratinocytes against the hazardous effects of ultraviolet B (UVB)-irradiation, recognized as the most important etiological factor in the development of skin cancer. Cytoprotective effects of 1,25(OH)(2)D(3) on UVB-irradiated keratinocytes were seen morphologically and quantified using a colorimetric survival assay. Moreover, 1,25(OH)(2)D(3) suppressed UVB-induced apoptotic cell death. An ELISA, detecting DNA-fragmentation, demonstrated that pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM for 24 h reduced UVB-stimulated apoptosis by 55-70%. This suppression required pharmacological concentrations 1,25(OH)(2)D(3) and a preincubation period of several hours. In addition, 1,25(OH)(2)D(3) also inhibited mitochondrial cytochrome c release (90%), a hallmark event of UVB-induced apoptosis. Furthermore, we demonstrated that 1,25(OH)(2)D(3) reduced two important mediators of the UV-response, namely, c-Jun-NH(2)-terminal kinase (JNK) activation and interleukin-6 (IL-6) production. As shown by Western blotting, pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM diminished UVB-stimulated JNK activation with more than 30%. 1,25(OH)(2)D(3) treatment (1 microM) reduced UVB-induced IL-6 mRNA expression and secretion with 75-90%. Taken together, these findings suggest the existence of a photoprotective effect of active vitamin D(3) and create new perspectives for the pharmacological use of active vitamin D compounds in the prevention of UVB-induced skin damage and carcinogenesis.