CALCITRIOL AND TRANSFORMING-GROWTH-FACTOR-BETA UP-REGULATE 5-LIPOXYGENASE MESSENGER-RNA EXPRESSION BY INCREASING GENE-TRANSCRIPTION AND MESSENGER-RNA MATURATION

Differentiation of the human monocytic cell line Mono Mac 6 with calci triol 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 matur e 5-LO mRNA by reverse-transcriptase-PCR gave a 42-fold induction whic h was not due to alterations in 5-LO half life and which was only in p art due to an induction of gene transcription. There was an up to five fold 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. How ever, treatment of the cells with either calcitriol or TGF beta and ad dition of the corresponding second inducer lead to an about fourfold i nduction of primary transcript levels. Addition of cycloheximide toget her with the second inducer inhibited only the TGF beta but not the ca lcitriol effects, which indicated that there is a direct stimulation o f 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-transcr iption-PCR. Analysis of exons 1-5 indicated an about threefold inducti on of 5-LO transcripts by calcitriol/ TGF beta, respectively. However, when exons 6-14 were determined, more pronounced increments were foun d (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 a nd calcitriol on transcript elongation and maturation were inhibited b y cycloheximide. Our results show that induction of 5-LO mRNA by calci triol and TGF beta is due to a modest increase in 5-LO gene transcript ion and to the stimulation of transcript elongation and maturation.