Calcitriol and transforming growth factor-β upregulate 5-lipoxygenase mRNA expression by increasing gene transcription and mRNA maturation

Differentiation of the human monocytic cell line Mono Mac 6 with calcitriol plus transforming growth factor-beta (TGF beta) strongly induces 5-lipoxygenase (5-LO) mRNA and protein expression. The mechanism of 5-LO mRNA induction by these agents was investigated. Analysis of mature 5-LO mRNA by reverse-transcriptase-PCR gave a 42-fold induction which was not due to alterations in 5-LO half life and which was only in part due to an induction of gene transcription. There was an up to fivefold increase in 5-LO primary transcripts by TGF beta and calcitriol, which could be inhibited by cycloheximide. No significant effects on 5-LO transcription were observed with TGF beta or calcitriol alone. However, treatment of the cells with either calcitriol or TGF beta and addition of the corresponding second inducer lead to an about fourfold induction of primary transcript levels. Addition of cycloheximide together with the second inducer inhibited only the TGF beta but not the calcitriol effects, which indicated that there is a direct stimulation of 5-LO transcription by calcitriol in the presence of TGF beta-induced proteins. In order to investigate the effects of TGF beta/calcitriol on 5-LO transcript, elongation and maturation, the relative changes in immature and mature 5-LO RNA species were analyzed by reverse-transcription-PCR. Analysis of exons 1-5 indicated an about threefold induction of 5-LO transcripts by calcitriol/ TGF beta, respectively. However, when exons 6-14 were determined, more pronounced increments were found (3.6-12-fold). Selective analysis of polyadenylated and spliced 5-LO mRNA species gave a 42-fold induction. The effects of both TGF beta and calcitriol on transcript elongation and maturation were inhibited by cycloheximide. Our results show that induction of 5-LO mRNA by calcitriol and TGF beta is due to a modest increase in 5-LO gene transcription and to the stimulation of transcript elongation and maturation.