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

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Abstract
Differentiation of the human monocytic cell line Mono Mac 6 with calcitriol plus transforming growth factor‐β (TGFβ) 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 TGFβ and calcitriol, which could be inhibited by cycloheximide. No significant effects on 5‐LO transcription were observed with TGFβ or calcitriol alone. However, treatment of the cells with either calcitriol or TGFβ 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 TGFβ but not the calcitriol effects, which indicated that there is a direct stimulation of 5‐LO transcription by calcitriol in the presence of TGFβ‐induced proteins. In order to investigate the effects of TGFβ/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/TGFβ, 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 TGFβ and calcitriol on transcript elongation and maturation were inhibited by cycloheximide. Our results show that induction of 5‐LO mRNA by calcitriol and TGFβ is due to a modest increase in 5‐LO gene transcription and to the stimulation of transcript elongation and maturation.

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