Calcitriol and transforming growth factor-beta 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 (TGFbeta) 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 TGFbeta and calcitriol, which could be inhibited by cycloheximide. No significant effects on 5-LO transcription were observed with TGFbeta or calcitriol alone. However, treatment of the cells with either calcitriol or TGFbeta 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 TGFbeta but not the calcitriol effects, which indicated that there is a direct stimulation of 5-LO transcription by calcitriol in the presence of TGFbeta-induced proteins. In order to investigate the effects of TGFbeta/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/TGFbeta, 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 TGFbeta and calcitriol on transcript elongation and maturation were inhibited by cycloheximide. Our results show that induction of 5-LO mRNA by calcitriol and TGFbeta is due to a modest increase in 5-LO gene transcription and to the stimulation of transcript elongation and maturation.
Härle
D
D
Institute of Pharmaceutical Chemistry, University of Frankfurt, Germany.
Rådmark
O
O
Samuelsson
B
B
Steinhilber
D
D
D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics*; D001483:Base Sequence; D002117:Calcitriol / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D005091:Exons; D006207:Half-Life; D006801:Humans; D016133:Polymerase Chain Reaction; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
D017931:DNA Primers; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D003513:Cycloheximide; D001094:Arachidonate 5-Lipoxygenase; D002117:Calcitriol