20,23‐dihydroxyvitamin D3, novel P450scc product, stimulates differentiation and inhibits proliferation and NF‐κB activity in human keratinocytes

Abstract

We have examined effects of the 20,23-dihydroxyvitamin D3 (20,23(OH)2D3), on differentiation and proliferation of human keratinocytes and the anti-inflammatory potential of 20,23(OH)2D3 from its action on nuclear factor-κB (NF-κB). 20,23(OH)2D3 inhibited growth of keratinocytes with a potency comparable to that for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Cell cycle analysis showed that this inhibition was associated with G1/G0 and G2/M arrests. 20,23(OH)2D3 stimulated production of involucrin mRNA and inhibited production of cytokeratin 14 mRNA in a manner similar to that seen for 1,25(OH)2D3. Flow cytometry showed that these effects were accompanied by increased involucrin protein expression, and an increase in the cell size and granularity. Silencing of the vitamin D receptor (VDR) by corresponding siRNA abolished the stimulatory effect on involucrin gene expression demonstrating an involvement of VDR in 20,23(OH)2D3 action. This mode of action was further substantiated by stimulation of CYP24 gene expression and stimulation of the CYP24 promoter-driven reporter gene activity. 20,23(OH)2D3 displayed several fold lower potency for induction of CYP24 gene expression than 1,25(OH)2D3. Finally, 20,23(OH)2D3 inhibited the transcriptional activity of NF-κB in keratinocytes as demonstrated by EMSA, NF-κB-driven reporter gene activity assays and measurements of translocation of p65 from the cytoplasm to the nucleus. These inhibitory effects were connected with stimulation of the expression of IκBα with subsequent sequestration of NF-κB in the cytoplasm and consequent attenuation of transcriptional activity. In summary, we have characterized 20,23(OH)2D3 as a novel secosteroidal regulator of keratinocytes proliferation and differentiation and a modifier of their immune activity.

Hydroxylation of CYP11A1-Derived Products of Vitamin D3 Metabolism by Human and Mouse CYP27B1

“…Along with the primary products of CYP11A1 action on vitamin D3, 17,20(OH) 2 D3, 20,22(OH) 2 D3, and 20,23(OH) 2 D3, we have a unique set of derivatives all containing a 20-hydroxyl group plus one additional hydroxyl group at every position on the side chain, except C21 (note that C26 and C27 are equivalent). All of these 20-hydroxy derivatives have been shown to be biologically active (Zbytek et al, 2008;Janjetovic et al, 2009Janjetovic et al, , 2010Janjetovic et al, , 2011Li et al, 2010;Slominski et al, 2010Slominski et al, , 2011Slominski et al, , 2012aTang et al, 2010a;Tuckey et al, 2011;Kim et al, 2012;Lu et al, 2012;Wang et al, 2012). The low catalytic efficiency of CYP27B1 toward 20(OH)D3 is greatly enhanced when the second hydroxyl group is added toward the end of the side chain, as seen for 24,20(OH) 2 D3, 20,25(OH) 2 D3, and 20,26(OH) 2 D3.…”

“…Via binding to the vitamin D receptor, these compounds can inhibit keratinocyte, melanoma, leukemia, breast, and liver cancer cell proliferation; promote differentiation; and display antiinflammatory activity by decreasing nuclear factor-ĸB activity (Zbytek et al, 2008;Janjetovic et al, 2009Janjetovic et al, , 2010Janjetovic et al, , 2011Li et al, 2010;Slominski et al, 2010Slominski et al, , 2011Slominski et al, , 2012aTang et al, 2010a;Tuckey et al, 2011;Kim et al, 2012;Lu et al, 2012;Wang et al, 2012). Importantly, all three of these derivatives lack calcemic activity in rodents, even at relatively high doses (Slominski et al, , 2013aWang et al, 2012).…”

Production of 22-Hydroxy Metabolites of Vitamin D3by Cytochrome P450scc (CYP11A1) and Analysis of Their Biological Activities on Skin Cells

“…They also display strong antiproliferative and prodifferentiation effects on a range of cells, including keratinocytes and melanoma and leukemia cells, and an inhibitory action on nuclear factor-B activity (Zbytek et al, 2008;Janjetovic et al, 2009Janjetovic et al, , 2010Slominski et al, 2010Slominski et al, , 2011, identifying 20(OH)D as a potential therapeutic or adjuvant agent for a number of hyperproliferative and inflammatory diseases. In this study, we have found that 22(OH)D 3 acting through the VDR also shows antiproliferative and, to a limited degree, prodifferentiation ( Fig.…”

“…The two major products of vitamin D 3 metabolism by P450scc, 20(OH)D 3 and 20,23(OH) 2 D 3 , have been tested on a range of skin and other cell types, and they have many but not all of the properties of the hormonally active form of vitamin D 3 , 1,25(OH) 2 D 3 , acting as partial receptor agonists (Zbytek et al, 2008;Janjetovic et al, 2009Janjetovic et al, , 2010Slominski et al, 2010;Tang et al, 2010a). Properties in common with 1,25(OH) 2 D 3 on skin cells include inhibition of proliferation and stimulation of differentiation of epidermal keratinocytes, and inhibition of nuclear factor-B activity via the stimulation of the expression of inhibitor of nuclear factor-B ␣.…”

Purified Mouse CYP27B1 Can Hydroxylate 20,23-Dihydroxyvitamin D3, Producing 1α,20,23-Trihydroxyvitamin D3, Which Has Altered Biological Activity

“…We have recently documented that the cytochrome P450scc-derived vitamin D analogs, 20(OH)D 3 and 20,23(OH) 2 D 3 , possess many of the properties of 1,25(OH) 2 D 3 such as the inhibition of proliferation, the promotion of differentiation, and suppression of inflammation, despite lacking the 1␣-hydroxyl group usually seen in vitamin D receptor agonists (Zbytek et al, 2008;Janjetovic et al, 2009Janjetovic et al, , 2010Slominski et al, 2010). Of particular note is our finding that 20(OH)D 3 lacks calcemic activity in rats but adding a 1␣-hydroxyl group to it partially returns the calcemic activity .…”