ABSTRACT
In spite of intensive research, our understanding of the regulation of expression of 5-LO (the key enzyme in the leukotriene metabolism) remains fragmentary. We investigated the effects of dexamethasone on the expression of this gene in a binary model consisting of two clones of the human mast cell line HMC-1, one with a 5-LO-negative and the other with a 5-LO-positive phenotype, respectively. When dexamethasone was included in the culture medium at a physiologically relevant concentration, biosynthesis of 5-LO derivatives increased considerably not only in the 5-LO-negative HMC-1 cells (approx 10-fold) but also in the 5-LO-positive cells, characterized by an already substantial enzyme activity. Consistently, Northern blot analysis revealed that a dramatic increase in the abundance of 5-LO mRNA occurred when the cells were exposed to dexamethasone. Likewise, a significant increase in the immunoreactive 5-LO protein was detected by Western blotting. In contrast, dexamethasone seemed to have no effect on the expression of two other genes of pivotal importance in leukotriene biosynthesis, viz. FLAP and LTC(4) synthase. We conclude that in human mast cells glucocorticoids effectively and selectively upregulate the expression of 5-LO.
Subject(s)
Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/metabolism , Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Mast Cells/drug effects , Mast Cells/enzymology , 5-Lipoxygenase-Activating Proteins , Carrier Proteins/genetics , Clone Cells , Gene Expression Regulation, Enzymologic/drug effects , Glutathione Transferase/genetics , Humans , Leukotrienes/biosynthesis , Membrane Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Up-Regulation/drug effectsABSTRACT
Prostaglandins play an important role in reproduction, particularly in pregnancy and parturition. We investigated the expression of prostaglandin endoperoxide H synthase (PGHS) 1 and 2, key enzymes in prostaglandin biosynthesis, in the human placenta at term. Northern blot analysis of placenta total and poly(A)+ RNA failed to detect significant levels of PGHS-1 mRNA, using a human PGHS-1 specific DNA probe. In contrast, definite PGHS-2 mRNA bands, with a calculated size of about 4.5 kb, were observed in Northern blots of placenta poly(A)+ RNA, using a PGHS-2 specific DNA probe. Moreover, PGHS-1 and PGHS-2 expression was assessed at the translational level by Western blot analysis. Thus, using three highly specific antibodies, we found a selective expression of PGHS-2 immunoreactive protein. These results indicate that PGHS-2 is the PGHS isoform prevalently expressed in the human placenta at term and support the hypothesis that PGHS-2 plays a prominent role in the maintenance of pregnancy, in line with the proposed anti-apoptotic and growth promoting properties of this enzyme.
