The Effect of Cadmium on COX-1 and COX-2 Gene, Protein Expression, and Enzymatic Activity in THP-1 Macrophages
Tomasz Olszowski
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Izabela Gutowska
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Irena Baranowska-Bosiacka
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Katarzyna Piotrowska
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Jan Korbecki
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Mateusz Kurzawski
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Dariusz Chlubek
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I. Baranowska-Bosiacka (
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I. Gutowska Department of Biochemistry and Human Nutrition, Pomeranian Medical University
,
Broniewskiego 24 Str, 71-460 Szczecin
,
Poland
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T. Olszowski Department of Hygiene and Epidemiology, Pomeranian Medical University
,
Powstancow Wlkp. 72 Av, 70-111 Szczecin
,
Poland
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M. Kurzawski Department of Experimental and Clinical Pharmacology, Pomeranian Medical University
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Powstancow Wlkp. 72 Av, 70-111 Szczecin
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Poland
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K. Piotrowska Department of Physiology, Pomeranian Medical University
,
Powstancow Wlkp. 72 Av, 70-111 Szczecin
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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.
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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)