sn-1,2-Diacylglycerols and phorbol diesters stimulate thromboxane synthesis by de novo synthesis of prostaglandin H synthase in human promyelocytic leukemia cells.

Abstract

We studied the regulation of thromboxane (TX) synthesis in promyelocytic leukemia cells during macrophage differentiation.Cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) showed rates of TXB2 synthesis from exogenous arachidonic acid that exceeded that of control cells by a factor of up to 81. Cells treated with sn-1,2-dioctanoylglycerol (diC8) showed similarly high TXB2 synthesis rates when diC8 was added concomitantly with a subthreshold concentration of TPA or when given in multiple doses. These activities depended on de novo synthesis of prostaglandin H (PGH) synthase because: (a) microsomal PGH synthase activity showed large increases in V.ax values, and (b) mass measurements of PGH synthase revealed the presence of PGH synthase in differentiating cells whereas the enzyme was undetectable in control cells. These results indicate that macrophage differentiation is associated with stimulation of TXB2 synthesis that requires both activation of protein kinase C and de novo synthesis of PGH synthase.

Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence.

“…During catalysis, the synthase undergoes a "suicide" reaction that may be a crude mechanism for limiting product formation (20,21). Moreover, hormones, growth factors, and phorbol 12-myristate 13-acetate can alter the prostaglandin-synthetic capacities of cultured cells (22)(23)(24)(25)(26) and tissues (27,28) (32) (residues 278-302) are underlined with dashes. The putative signal peptide (residues 1-24) is indicated by bold underline; N-glycosylation sites (residues 104, 144, and 410) are marked with filled triangles; and the aspirin-acetylated serine (residue 530) is marked with an open diamond.…”

Selective eicosanoid formation during HL‐60 macrophage differentiation

“…In previous studies of HL-60 cells [3,6,7], we and others have provided evidence that activation of protein kinase C Table 1. Quantification of TxS in control and TPA-treated HL-60 cells, HL-60 cells were cultured, then TPA or 1.25% Me,SO added as described previously [3].…”

“…Eicosanoids, 12-U-tetradecanoylphorbol-13-acetate (TPA) and all other reagents were obtained from sources previously described [3,6,19,201.…”

“…While platelet TxS has been the subject of many recent pharmacological and clinical studies aimed at reducing Tx formation during platelet aggregation [22], mechanisms that might regulate transcription and translation of the enzyme remain largely to be defined. Earlier studies on differentiating HL-60 cells or peritoneal macrophages provided circumstantial evidence that TxS is subject to regulation in response to activation of protein kinase C [6], calcitriol [23] or inflammatory stimuli [24]. In this report, we focused on the regulation of TxS in phorbol-ester-treated HL-60 cells to study mechanisms of the selective up-regulation of Tx synthesis during macrophage differentiation.…”

“…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.…”