Owing to its ability to induce growth arrest and differentiation of keratinocytes, 1alpha,25-dihydroxyvitamin D3 and its analogs are useful for the treatment of hyperproliferative skin diseases, such as psoriasis vulgaris. It has been implicated that the 1alpha,25-dihydroxyvitamin D3-induced differentiation of keratinocytes is mediated, at least in part, by the formation of ceramides; however, ceramides have also been identified to induce apoptosis in many cells, including keratinocytes. Therefore, it was of interest to investigate the influence of 1alpha,25-dihydroxyvitamin D3 on apoptosis in keratinocytes. Most interestingly, physiological concentrations of 1alpha,25-dihydroxyvitamin D3 did not induce apoptosis in keratinocytes, despite the formation of ceramides. Moreover, 1alpha,25-dihydroxyvitamin D3 appeared cytoprotective and made keratinocytes resistant to apoptosis induced by ceramides, ultraviolet irradiation, or tumor necrosis factor-alpha. The cytoprotective effect was accompanied by the formation of the sphingolipid breakdown product sphingosine-1-phosphate, which prevented apoptosis in analogy to 1alpha,25-dihydroxyvitamin D3. The effect of 1alpha,25-dihydroxyvitamin D3 was specific as the almost inactive precursor cholecalciferol neither induced sphingosine-1-phosphate formation nor prevented cells from apoptosis. Besides this, the cytoprotective aptitude of 1alpha,25-dihydroxyvitamin D3 was completely abolished by the sphingosine kinase inhibitor N,N-dimethylsphingosine, which blocked sphingosine-1-phosphate formation. Moreover, sphingosine-1-phosphate was able to restore the cytoprotective effect of 1alpha,25-dihydroxyvitamin D3 in the presence of N,N-dimethylsphingosine. Taken together, here we report for the first time that 1alpha,25-dihydroxyvitamin D3 protects keratinocytes from apoptosis and additionally this cytoprotection is mediated via the formation of sphingosine-1-phosphate.
Sphingosine-1-phosphate (SPP) has been proposed to act both as an intracellular second messenger and as an extracellular mediator via specific cell surface receptors. Based on the increasing diverse cellular roles methods to quantify endogenous and exogenous SPP are highly required. Here, we report a rapid HPLC method that allows quantification of SPP in the picomolar range even in complex biological systems. A two-step lipid extraction serves to separate SPP from most interfering phospholipids and sphingolipids. Importantly, dihydrosphingosine-1-phosphate (dihydro-SPP), not detectable in all cultured cells and biological samples in considerable amounts, possesses equal extraction properties and therefore is an ideal internal standard. Following extraction SPP and dihydro-SPP are converted to fluorescent isoindol derivatives by ortho-phthaldialdehyde (OPA) and separated by HPLC using a gradient program with methanol and 0.07 M K2HPO4 as eluents. With this procedure we were able to obtain reproducible measurements of SPP over a broad range from 0.5 pM to 0.2 nM. The identity of SPP and dihydro-SPP was confirmed by the use of the ion pair reagent tetraammoniumsulfate, which induced a shift of both peaks but did not alter peak areas. Moreover, enzymatic conversions to sphingosine and sphinganine by bovine intestinal mucosa alkaline phosphatase (AP) excluded the existence of overlapping compounds. Levels of SPP were determined in a variety of biological samples like serum, thrombocytes, primary keratinocytes and several cell lines. Furthermore, we were able to detect increases of intracellular SPP levels in human keratinocytes after exposure to 1alpha,25-dihydroxyvitamin D3(1,25-(OH)2D3) for which a stimulation of sphingosine kinase activity has been recognized.
1alpha,25-Dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] has been shown to induce cell growth arrest and to possess differentiation-inducing behaviour in both primary melanocytes and melanoma cell lines. Moreover, in several melanoma cell lines it has been demonstrated that the antiproliferative action is accompanied by an increase in apoptosis. In contrast, here we show that physiological concentrations of 1,25-(OH)(2)D(3) did not induce apoptosis in primary melanocytes despite a cell growth inhibitory effect. Furthermore, treatment with 1,25-(OH)(2)D(3) made melanocytes resistant to several inductors of programmed cell death, including tumour necrosis factor-alpha and ultraviolet radiation. The antiapoptotic effect of 1,25-(OH)(2)D(3) was completely abolished by the addition of N,N-dimethylsphingosine, which blocks the formation of the sphingolipid degradation product sphingosine 1-phosphate (S1P), suggesting a crucial role for this sphingolipid in 1,25-(OH)(2)D(3)-mediated cytoprotection. Indeed, stimulation of melanocytes with S1P also resulted in an antiapoptotic action. In addition, S1P induced cell growth arrest of human melanocytes. This was an unexpected finding, as S1P is generally known as a potent mitogenic molecule in a variety of cells, including fibroblasts. As both 1,25-(OH)(2)D(3) and S1P have been identified to modify the Bcl-2/Bax ratio in epithelial cells, we also measured the expressions of these proteins; however, treatment of melanocytes with either 1,25-(OH)(2)D(3) or S1P did not alter the Bcl-2/Bax ratio. In conclusion, 1,25-(OH)(2)D(3) was shown to protect human melanocytes from apoptosis by formation of S1P, which is opposite to its apoptotic action in diverse melanoma cell lines.
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