Thromboxane Synthase Expression Research Articles

Characterization of the Complete Genomic Structure of Human Thromboxane Synthase Gene and Functional Analysis of Its Promoter

Thromboxane A2(TXA) is a potent vasoconstrictor and mediator of platelet aggregation. Thromboxane synthase (TXAS), which catalyzes the biosynthesis of TXA, is a member of the cytochrome P450 superfamily. We report here the complete genomic structure of the human TXAS gene. The gene contains 13 exons and is 193 kb long, the largest P450 gene ever isolated. The physical localization of the TXAS gene on the genetic map of human chromosome 7 was established. A major transcription start site is located at a motif similar to the initiator element where the transcription factor TFII-I binds. We have sequenced up to 1.6 kb of the 5′-flanking region of the TXAS gene and characterized the promoter activity in a human megakaryocyte cell line and endothelial cells. Our results demonstrated that the nucleotides −248/+137 relative to the major transcription start site conferred a full promoter activity of the TXAS gene. This region was regulated negatively by cis-elements located between −1562 and −248. Moreover, the regions outside −1562/+137 might control tissue-specific TXAS expression.

Thromboxane synthase expression co-localizes with infiltrating macrophages in renal allograft biopsies

Thromboxane synthase (TS) catalyzes the formation of the eicosanoid thromboxane (TxA 2 ), which is a potent vasoconstrictor and activator of platelet aggregation. It has a widespread distribution, including hemopoietic cells (platelets, macrophages, monocytes) and many tissues, such as lung, liver, kidney, and spleen [1]. Cultured renal cells including mesangial cells, tubular epithelia, and medullary interstitial cells have also been found to produce TxA 2 [2]. TxA 2 has been proposed as a potential mediator of cyclosporine nephrotoxicity in renal allografts. In this light, the TS inhibitor, CGS 13080, has been used to attenuate the decrease in renal function caused by cyclosporine, but with mixed results [4, 5]. In human renal allografts undergoing acute or chronic rejection, we found thromboxane synthase expression in cells which appear to be infiltrating macrophages or monocytes by morphology [3]. This study describes an immunohistochemical technique utilized to confirm whether parenchymal renal cells or macrophages account for this TS enzyme expression. This method is based in histochemical double-labeling technique utilizing the immunoperoxidase avidin/biotin system and two monoclonal antibodies: Kon 7 and KP1. Kon 7 is a mouse monoclonal antibody directed against TS [2]. KP1 is a mouse anti-human monoclonal antibody directed against the CD68 antigen present in a lysosomal fraction of human lung macrophages and identifies macrophages and other phagocytic mononuclear cells [6].

THROMBOXANE SYNTHASE EXPRESSION IN RENAL TRANSPLANT PATIENTS WITH REJECTION1

Thromboxane synthase (TS) catalyzes the formation of thromboxane (TxA2) in monocytes/macrophages, platelets, and various tissues. TxA2 is likely to play a role in graft dysfunction due to its vasoconstrictive and platelet aggregatory properties. We studied the expression of TS in 7 normal native kidneys, 29 consecutive renal allograft biopsies (performed for rising serum creatinine, n = 23, and delayed graft function, n = 6), and one transplant nephrectomy specimen with severe acute rejection. TS expression was determined by immunocytochemistry using a monoclonal antibody against human TS, Kon-7. Histologic grading of the transplant biopsy specimens was based on the Banff classification. The degree of TS staining was graded in the glomeruli, interstitium, tubules and vessels from 0 to 3+. Of 29 biopsies, 13 had chronic nephropathy (CN), 6 had acute rejection (AR) with chronic nephropathy (AR/CN), 4 had acute rejection (AR), and 6 had acute tubular necrosis (ATN). TS staining of native kidneys showed sporadic interstitial cells. The biopsy and transplant nephrectomy specimens showed significant staining, predominantly in the glomeruli and interstitium. Positively staining cells appeared to be of macrophage/monocyte lineage by morphology. The mean glomerular staining grade was significantly increased in specimens with AR (2.3 +/- 0.9) and the mean interstitial staining was increased in specimens with AR/CN (2.2 +/- 0.9). Follow-up renal function 6 months post-biopsy showed that patients with higher TS staining grades had a faster decline in graft function. In conclusion, TS expression is increased in patients with acute rejection with or without chronic nephropathy and is associated with more rapid deterioration in function.

THROMBOXANE SYNTHASE EXPRESSION IN RENAL TRANSPLANT PATIENTS WITH REJECTION1

Thromboxane synthase (TS) catalyzes the formation of thromboxane (TxA2) in monocytes/macrophages, platelets, and various tissues. TxA2 is likely to play a role in graft dysfunction due to its vasoconstrictive and platelet aggregatory properties. We studied the expression of TS in 7 normal native kidneys, 29 consecutive renal allograft biopsies (performed for rising serum creatinine, n=23, and delayed graft function, n=6), and one transplant nephrectomy specimen with severe acute rejection. TS expression was determined by immunocytochemistry using a monoclonal antibody against human TS, Kon-7. Histologic grading of the transplant biopsy specimens was based on the Banff classification. The degree of TS staining was graded in the glomeruli, interstitium, tubules and vessels from 0 to 3+. Of 29 biopsies, 13 had chronic nephropathy (CN), 6 had acute rejection (AR) with chronic nephropathy (AR/CN), 4 had acute rejection (AR), and 6 had acute tubular necrosis (ATN). TS staining of native kidneys showed sporadic interstitial cells. The biopsy and transplant nephrectomy specimens showed significant staining, predominantly in the glomeruli and interstitium. Positively staining cells appeared to be of macrophage/monocyte lineage by morphology. The mean glomerular staining grade was significantly increased in specimens with AR (2.3±0.9) and the mean interstitial staining was increased in specimens with AR/CN (2.2±0.9). Follow-up renal function 6 months post-biopsy showed that patients with higher TS staining grades had a faster decline in graft function. In conclusion, TS expression is increased in patients with acute rejection with or without chronic nephropathy and is associated with more rapid deterioration in function.

Abundant expression of thromboxane synthase in rat macrophages

The cloned cDNA for rat thromboxane (TX) synthase with a size of 1851 bp contained a 1599-bp open reading frame which encoded a 533-amino acid protein sharing 79.7% identity with human TX synthase. RNA blot analysis was carried out with rat cells and tissues. Rat peritoneal macrophages most abundantly expressed mRNA for TX synthase, and its level was not changed by in vivo stimulation of casein. Bone marrow, spleen, lung and thymus also expressed the TX synthase gene. These findings suggest the possibility that TXA 2 plays a role in the immune system.

Induction of thromboxane synthase and prostaglandin endoperoxide synthase mRNAs in human erythroleukemia cells by phorbol ester

The effects of 12- O-tetradecanoyl-phorbol-13-acetate (TPA) on the mRNA levels of two enzymes, thromboxane synthase (TXS) and prostaglandin endoperoxide synthase (PES), responsible for the synthesis of thromboxane A 2 from arachidonic acid, were studied in human crythroleukemia (HEL) cells by RNA blot analysis. TPA induced both TXS and PES mRNAs in HEL cells in a dose-dependent manner at 36 h. The half-maximal and maximal effects for the induction of both mRNAs ware at approximately 3×10 −9 M and at 10 −8 M, respectively. TXS and PES mRNA levels increased in a time-dependent fashion by TPA, and reached to 7- and 3.5-fold of the control, respectively after 48 h of TPA treatment. These results suggest that expression of TXS and PES genes in HEL cells were simultaneously stimulated by TPA.