The Effect of Cadmium on COX-1 and COX-2 Gene, Protein Expression, and Enzymatic Activity in THP-1 Macrophages

Biological Trace Element Research, Feb 2015

The aim of this study was to examine the effects of cadmium in concentrations relevant to those detected in human serum on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression at mRNA, protein, and enzyme activity levels in THP-1 macrophages. Macrophages were incubated with various cadmium chloride (CdCl2) solutions for 48 h at final concentrations of 5 nM, 20 nM, 200 nM, and 2 μM CdCl2. The mRNA expression and protein levels of COXs were analyzed with RT-PCR and Western blotting, respectively. Prostaglandin E2 (PGE2) and stable metabolite of thromboxane B2 (TXB2) concentrations in culture media were determined using ELISA method. Our study demonstrates that cadmium at the highest tested concentrations modulates COX-1 and COX-2 at mRNA level in THP-1 macrophages; however, the lower tested cadmium concentrations appear to inhibit COX-1 protein expression. PGE2 and TXB2 production is not altered by all tested Cd concentrations; however, the significant stimulation of PGE2 and TXB2 production is observed when macrophages are exposed to both cadmium and COX-2 selective inhibitor, NS-398. The stimulatory effect of cadmium on COXs at mRNA level is not reflected at protein and enzymatic activity levels, suggesting the existence of some posttranscriptional, translational, and posttranslational events that result in silencing of those genes’ expression.

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The Effect of Cadmium on COX-1 and COX-2 Gene, Protein Expression, and Enzymatic Activity in THP-1 Macrophages

Tomasz Olszowski 0 1 2 3 4 Izabela Gutowska 0 1 2 3 4 Irena Baranowska-Bosiacka 0 1 2 3 4 Katarzyna Piotrowska 0 1 2 3 4 Jan Korbecki 0 1 2 3 4 Mateusz Kurzawski 0 1 2 3 4 Dariusz Chlubek 0 1 2 3 4 0 I. Baranowska-Bosiacka ( 1 I. Gutowska Department of Biochemistry and Human Nutrition, Pomeranian Medical University , Broniewskiego 24 Str, 71-460 Szczecin , Poland 2 T. Olszowski Department of Hygiene and Epidemiology, Pomeranian Medical University , Powstancow Wlkp. 72 Av, 70-111 Szczecin , Poland 3 M. Kurzawski Department of Experimental and Clinical Pharmacology, Pomeranian Medical University , Powstancow Wlkp. 72 Av, 70-111 Szczecin , Poland 4 K. Piotrowska Department of Physiology, Pomeranian Medical University , Powstancow Wlkp. 72 Av, 70-111 Szczecin , Poland The aim of this study was to examine the effects of cadmium in concentrations relevant to those detected in human serum on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression at mRNA, protein, and enzyme activity levels in THP-1 macrophages. Macrophages were incubated with various cadmium chloride (CdCl2) solutions for 48 h at final concentrations of 5 nM, 20 nM, 200 nM, and 2 M CdCl2. The mRNA expression and protein levels of COXs were analyzed with RT-PCR and Western blotting, respectively. Prostaglandin E2 (PGE2) and stable metabolite of thromboxane B2 (TXB2) concentrations in culture media were determined using ELISA method. Our study demonstrates that cadmium at the highest tested concentrations modulates COX-1 and COX-2 at mRNA level in THP-1 macrophages; however, the lower tested cadmium concentrations appear to inhibit COX-1 protein expression. PGE2 and TXB2 production is not altered by all tested Cd concentrations; however, the significant stimulation of PGE2 and TXB2 production is observed when macrophages are exposed to both cadmium and COX-2 selective inhibitor, NS398. The stimulatory effect of cadmium on COXs at mRNA level is not reflected at protein and enzymatic activity levels, suggesting the existence of some posttranscriptional, translational, and posttranslational events that result in silencing of those genes' expression. - Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) are the bifunctional enzymes catalyzing the conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2) in two sequential reactions, the first being the generation of prostaglandin G2 (PGG2) (cyclooxygenase reaction) followed by the reduction of PGG2 to PGH2 (peroxidase reaction). The generated PGH2 is the precursor of biologically active prostanoids such as prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin I2 (PGI2), prostaglandin F2 (PGF2) and thromboxane A2 (TXA2) [1, 2]. COX-1 and COX-2 enzymes share 60 % identity in their amino acid sequences [3]. They exist as homodimers; each subunit consists of three domains, the epidermal growth factor domain, the membrane binding domain, and the catalytic domain containing the cyclooxygenase and peroxidase active sites [3]. COX-1, constitutively expressed in almost all cell types, but also inducible in some systems [4], was previously considered to be involved in physiological processes and playing no role in inflammation [4]. However, according to newer concept, COX-1 is also involved in inflammatory process [2, 5]; for example, COX-1 not only is responsible for the initial prostanoid response to inflammatory stimuli [6] but also contributes to the resolution of inflammation [4]. The human COX-1 gene (Ptgs1) expression is developmentally controlled and can be upregulated by tumor-promoting phorbol esters or growth factors [7]. The regulatory elements of this gene include SP1 binding site and activator protein-1 (AP-1) site [4, 7]; however, the transcriptional control of COX-1 gene expression was not well studied [7]. The products of COX-1 enzyme are thromboxane A2 (being metabolized to its stable metabolite, TXB2) and (PGE2 [6]. COX-2 is an enzyme highly inducible by pro-inflammatory cytokines, tumor promoters, mitogens, and growth factors in a variety of cell types, including monocytes [8], which results in increased prostaglandin release [5]. According to newer concept, COX-2 is the major contributor to prostanoid synthesis as inflammation progresses [6]. COX-2 gene (Ptgs2) contains several potential transcriptional regulatory elements in the 5flanking region: peroxisome proliferator response element (PPRE), two nuclear factor kappa B (NF-B) sites, one specificity protein 1 (Sp1) site, two cyclic AMP response elements (CRE), one nuclear factor for interleukin-6 expression (NFIL6) motif, two AP-1 sites, E-box, and TATA box [4, 7, 9]. Transcriptional regulation of COX-2 gene is very complex; it can involve numerous signaling pathways, and the mechanism varies depending on the specific stimulus and the cell type [7]. The main product of COX-2 enzyme is PGE2 [6]. Cadmium is a toxic and carcinogenic heavy metal that poses nowadays a serious threat to human health because it is u (...truncated)


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Tomasz Olszowski, Izabela Gutowska, Irena Baranowska-Bosiacka, Katarzyna Piotrowska, Jan Korbecki, Mateusz Kurzawski, Dariusz Chlubek. The Effect of Cadmium on COX-1 and COX-2 Gene, Protein Expression, and Enzymatic Activity in THP-1 Macrophages, Biological Trace Element Research, 2015, pp. 135-144, Volume 165, Issue 2, DOI: 10.1007/s12011-015-0234-6