1α,25-dihydroxyvitamin D₃-induced myeloid cell differentiation is regulated by a vitamin D receptor-phosphatidylinositol 3-kinase signaling complex

1α,25-dihydroxyvitamin D₃ (D₃) promotes the maturation of myeloid cells and surface expressions of CD14 and CD11b, markers of cell differentiation in response to D₃. To examine how these responses are regulated, THP-1 cells were grown in serum-free medium and incubated with D₃. This was associated with rapid and transient increases in phosphatidylinositol 3-kinase (PI 3-kinase) activity. Furthermore, induction of CD14 expression in response to D₃ was abrogated by (a) the PI 3-kinase inhibitors LY294002 and wortmannin; (b) antisense oligonucleotides to mRNA for the p110 catalytic subunit of PI 3-kinase; and (c) a dominant negative mutant of PI 3-kinase. In THP-1 cells, induction of CD11b expression by D₃ was also abrogated by LY294002 and wortmannin. Similarly, LY294002 and wortmannin inhibited D₃-induced expression of both CD14 and CD11b in peripheral blood monocytes. In contrast to CD14 and CD11b, hormone-induced expression of the Cdk inhibitor p21 in THP-1 cells was unaffected by either wortmannin or LY294002. These findings suggest that PI 3-kinase selectively regulates D₃-induced monocyte differentiation, independent of any effects on p21. Pretreatment of THP-1 cells with antisense oligonucleotides to the vitamin D receptor (VDR) mRNA abrogated both activation of PI 3-kinase in response to D₃ and hormone-induced CD14 expression. Moreover, both Western blots and in vitro kinase assays carried out on immunoprecipitates of the VDR showed that D₃ treatment brought about formation of a complex containing both PI 3-kinase and the VDR. These findings reveal a novel, nongenomic mechanism of hormone action regulating monocyte differentiation, in which vitamin D₃ activates a VDR- and PI 3-kinase-dependent signaling pathway.