Selective eicosanoid formation during HL‐60 macrophage differentiation

Abstract

Earlier studies on HL-60 cells induced to differentiate into macrophages by phorbol esters have shown a selective stimulation of thromboxane (Tx) formation from endoperoxide prostaglandin (PG) H,, indicating that Tx synthesis is regulated at the level of Tx synthase (TxS), one of the peripheral enzymes of the PGH-synthase pathway. We now report on the regulation of TxS during HL-60 macrophage differentiation using monoclonal anti-TxS serum and comparing turnover rates of TxS and its biological activity with those of other enzymes of arachidonic acid metabolism. Western-blot analysis, enzyme-linked immunosorbent assay, immunohistochemical staining and [35S]methioninelabeling experiments suggested a phorbol-ester-dependent early induction of synthesis of TxS.[35S]Methionine incorporation into TxS was stimulated within 4 h after initiation of differentiation and was associated with a major rise in the TxS catalytical activity. Pulse-chase experiments showed a half life for the TxS protein of 16.4 h in both control and phorbol-ester-treated cells. The biological half life of TxS was 10.5 h, as determined by PGH, incorporation into TxB, after cycloheximide treatment. In contrast, the biological half lives of PGH synthase, prostacyclin synthase and 5-lipoxygenase were significantly shorter and were 3, 2.5 and 2.5 h, respectively. These results reveal that Tx synthesis in macrophages is mediated by at least two distinct mechanisms; a protein-kinase-cdependent induction of de novo synthesis of TxS and the selective resistance of the enzyme against the activity of protein kinase C.The activity of enzymes that catalyze the conversion of arachidonic acid and the other C,, fatty acids into biologically active eicosanoids, has to be rigorously controlled to ensure cell-type-specific eicosanoid synthesis [ 11. Recent studies on proliferating fibroblasts [2-41, differentiating hematopoietic cells [5][6][7][8] and other cells [9-131 largely focused on two key enzymes that initiate the major pathways of arachidonic acid metabolism, the PGH synthase and the 5-lipoxygenase. Regulation of the activity of these enzymes has been shown to be rather complex and involves transcriptional, translational and several post-translational events including activation and suicide-type inactivation reactions Mechanisms that might regulate the activities of enzymes catalyzing the conversion of the short-lived intermediates of the PGH (PG, prostaglandin) synthase reaction, P G R , or the 5-lipoxygenase reaction, LT&, into biologically active eicosanoids have received less attention. Several of the peripheral branch-point enzymes of the PGH-synthase pathway, including thromboxane synthase (TxS) and the prostacyclin synthase, deserve attention because Tx and prostacyclin are not [14-171. constitutively formed in mammalian cells and show tissue specificity [ 181. For Tx, tissue specificity is particularly striking; only two cell types of the hematopoietic system appear to be major sources of Tx, i.e. monocytes/differentiating macrophages and circulati...

Candida albicansHyr1p Confers Resistance to Neutrophil Killing and Is a Potential Vaccine Target

“…We also performed candidal killing assays using the HL-60 cell line, which can be differentiated into either neutrophil-like cells or macrophage-like cells [13, 14]. Similar to that of freshly harvested human neutrophils, conditional expression of HYR1 reduced killing of C. albicans blastospores by both HL-60-derived neutrophils (Figure 1 C ) and macrophages (Figure 1 D ) in vitro.…”

Reorientation of prostanoid production accompanies ?activation? of adult microglial cells in culture

“…The finding that the LPS-induced expression of COX-2 (the rate-limiting enzyme for the synthesis of both prostanoids) was much more pronounced in the more ''activated'' cultures suggests that the availability of the COX product and common precursor PGH 2 was also increased. The concomitant findings that the activity of TX-synthase was never enhanced after LPS treatment and was, if anything, lower in 4-day than in 2-day-old cultures, as well as the knowledge that the Km of PGH 2 for TX-synthase is lower than for PGE-synthase (Shen and Tai, 1986;Nüsing et al, 1993;Smith et al, 1993) suggest that in ''activated'' microglia stimulated with LPS, TX-synthase may be rapidly saturated, and more Fig. 3.…”

Thromboxane synthase and TP receptor mRNA in rat kidney and brain: effects of salt intake and ANG II

“…The protein for TxA 2 -S is expressed in renal microvessels, interstitial fibroblasts, and macrophages (36,40), and in the distal nephron of the kidney (37). Isolated glomeruli and glomerular mesangial cells and podocytes release TxB 2 and therefore presumably express TxA 2 -S (29).…”