Selenoprotein mRNA and selenoprotein expression and ROS levels
To determine the relative expression levels of selenoproteins mRNAs in mouse macrophages, total RNA was isolated from bone marrow-derived, normal macrophages and the expression of mRNA levels transcribed from all 24 mouse selenoprotein genes were analyzed by real-time PCR. As shown in , relatively high levels of selenoprotein mRNAs corresponding to GPx1, GPx4, TR1, Sep15, and selenoproteins P, R, K and T and SPS2 were expressed. Interestingly, the most highly expressed selenoprotein mRNA in macrophage, like that in T cells (see above), was GPx1 and a major role of this selenoenzyme is as an antioxidant. Similarly, Sep15, GPx4 and selenoproteins P, K and T were also highly expressed in macrophages.
Selenoprotein gene expression in macrophages
We deleted Trsp
in macrophage (ΔTrspM
) and examined the effects of Trsp
loss on selenoprotein expression and function. Initially, we demonstrated that Trsp
was no longer expressed in this cell type (). Selenoprotein expression was also examined in macrophages by incubating the bone marrow-derived macrophages from control and ΔTrspM
mice in the presence of 75
Se and electrophoresing a protein extract on a polyacrylamide gel (. left panel). Several selenoproteins whose mRNAs were also detected by real-time PCR were visualized on the gel, while the expression of selenoproteins in ΔTrspM
cells were almost completely abolished (, right panel). The low levels of Sec tRNA and selenoproteins observed in ΔTrspM
macrophages in are likely due to contamination of the bone marrow-derived macrophage preparations with non-macrophage cells as has been observed in studies involving other cell types(27)
The functions of only about half of selenoproteins have been characterized and most are oxidoreductases and/or antioxidants(31)
. Since selenoprotein-less T cells accumulate higher ROS levels than the corresponding control cells (see ), ROS levels in ΔTrspM
macrophages were also assessed by staining (). Resting selenoprotein-deficient macrophages manifested higher steady state ROS levels than control macrophages.
In vitro and in vivo inflammatory responses
Due to the highly significant role that macrophages have in initiating inflammation, it was important to assess the ability of the corresponding selenoprotein-deficient cells in the inflammation process. Macrophages generate cytokines in response to tissue injury and microbial infection. The fact that several inflammatory stimuli such as lipopolysaccharide (LPS) cause transient ROS accumulation as part of cellular signaling events(32,33)
, and several selenoproteins, e.g., GPx isoforms, are known to regulate inflammatory responses(34,35)
prompted us to examine the effect of selenoprotein deficiency and the resulting redox imbalance on macrophage inflammatory response. We initially treated control and ΔTrspM
macrophages with LPS and examined the degradation and replenishment of I Bα. It should be noted that the transcription factor NF-κB, and mitogen-activated protein kinase (ERK, JNK and p38) signaling pathways are essential for cellular responses to inflammatory stimuli and that both the degradation and replenishment of I Bα are indicative of NF-κB activation. However, in LPS-treated control and ΔTrspM
macrophages, the degradation and replenishment of IκBα occurred independently of selenoprotein status and induction of the active forms of the protein kinases (i.e., phosphorylated ERK, JNK and p38) was similar and therefore considered normal(22)
. Furthermore, comparison of LPS-induced inflammatory gene expression in the two bone marrow-derived macrophage groups and analyses of gene expression by real-time PCR revealed that the magnitude and kinetics of expression of the genes encoding the chemokines KC (Cxcl1
), macrophage inflammatory protein-2 (Cxcl2), and the cytokines tumor necrosis factor-α (Tnf-α
) and interleukin-1β (Il1b
) were similar(22)
We next turned our attention to examining the effects of selenoprotein deficiency in macrophages on the pathology of mice by exposing control and ΔTrspM
mice to various models of inflammatory response. The models used included zymosan-induced peritonitis, LPS endotoxemia and chemical irritant (12-O
-tetradecanoylphorbol-13-acetate [TPA]) dermatitis. In these models, control and ΔTrspM
mice had comparable rates of neutrophil infiltration, rates of mortality and levels of cytokine production and local edema formation. Since in vitro
and in vivo
inflammatory responses in both control and ΔTrspM
macrophages were similar, we have not shown the data herein, but refer the reader to the original work(22)
. Interestingly, the loss of selenoprotein expression and the deregulated ROS generation in ΔTrspM
macrophages did not appear to cause any detectable detrimental effects concerning inflammatory responses.
Macrophage microarray analysis
Since there were unexpectedly little or no distinguishable inflammatory phenotypes in ΔTrspM mice, it seemed reasonable that selenoproteins may have other functions in macrophages in addition to roles in inflammatory gene expression. To explore this possibility, an analysis of the global gene expression profiles was carried out in control and ΔTrspM macrophages. We used whole-genome DNA microarrays from mice to assess the expression levels of mRNAs in the two macrophage groups. Those genes that manifested a significantly higher or lower expression in ΔTrspM macrophages compared to control macrophages are shown in and are designated by those dots (designating mRNAs) that fell outside the lines above and below normal expression levels indicating significant changes.
Microarray analysis of ΔTrspM macrophages
The expression levels of those mRNAs that showed significant changes in selenoprotein-deficient macrophages were verified by real-time PCR analysis (). Interestingly, many genes that deviated significantly in their expression levels from normal in ΔTrspM macrophages were functionally related to the formation, remodeling cellular interaction with the extracellular matrix (ECM). Each of the ECM-related genes that we found to be significantly altered in ΔTrspM macrophages were upregulated compared to the corresponding control cells (). These included genes that encode ECM components (collagen chains, extracellular proteoglycans, and secreted glycoproteins; Col1a1, Col5a2, Bgn, and Ctgf), inhibitors of ECM proteolysis (metalloendopeptidase and serine-type endopeptidase inhibitors; Timp3, and Serpinh1), and ECM-induced cytoskeletal remodeling (actin binding proteins; Tagln, Enah, and Cald1).
Selenoprotein deficiency and macrophage invasiveness
The microarray analysis of ΔTrspM macrophages suggested that macrophage-ECM interactions were altered by the lack of selenoprotein expression in such a manner that matrix remodeling was decreased and the surrounding ECM was reinforced. Such effects might be expected to impair the migration of macrophages through the ECM and basement membrane. To test this possibility, we compared the invasiveness of control and ΔTrspM macrophages in a protein gel matrix. The migration of selenoprotein-deficient macrophages was dramatically reduced in the gel-laden transwell chambers (). We repeated this assay, but without the gel in the transwell chamber to assess whether the gel invasion phenotype arose from a cell-intrinsic motility defect. Surprisingly, the migration of ΔTrspM macrophages was slightly higher than control macrophages () suggesting that the changes observed in ECM-related gene expression in macrophages appeared to decrease the ability of the cell to migrate only in surroundings consisting of ECM components.
Analysis of ΔTrspM macrophage invasion in a protein gel matrix