Selenoproteins regulate macrophage invasiveness and extracellular matrix-related gene expression
© Carlson et al; licensee BioMed Central Ltd. 2009
Received: 28 April 2009
Accepted: 28 October 2009
Published: 28 October 2009
Selenium, a micronutrient whose deficiency in diet causes immune dysfunction and inflammatory disorders, is thought to exert its physiological effects mostly in the form of selenium-containing proteins (selenoproteins). Incorporation of selenium into the amino acid selenocysteine (Sec), and subsequently into selenoproteins is mediated by Sec tRNA[Ser]Sec.
To define macrophage-specific selenoprotein functions, we generated mice with the Sec tRNA[Ser]Sec gene specifically deleted in myeloid cells. These mutant mice were devoid of the "selenoproteome" in macrophages, yet exhibited largely normal inflammatory responses. However, selenoprotein deficiency led to aberrant expression of extracellular matrix-related genes, and diminished migration of macrophages in a protein gel matrix.
Selenium status may affect immune defense and tissue homeostasis through its effect on selenoprotein expression and the trafficking of tissue macrophages.
Macrophages, a class of myeloid leukocytes with phagocytic activity and inflammatory signaling properties, play a pivotal role in antimicrobial defense and tissue homeostasis [1–3]. These tissue-resident immune cells express receptors that detect the presence of signature molecules associated with microbial infection and tissue damage [4, 5]. Such receptors signal to induce macrophage production of cytokines and other inflammatory mediators that effect pathogen clearance and tissue repair. Many cell-intrinsic and -extrinsic regulatory mechanisms act to limit inflammatory signaling in macrophages, thereby preventing excessive, self-destructive responses of the cell. Furthermore, the spatial and temporal turnover of macrophages in tissue is subject to dynamic control by the local microenvironment and metabolic state. Uncontrolled macrophage recruitment and activation is associated with development of rheumatic, cardiovascular, metabolic, and neoplastic disorders.
Selenium is a dietary trace element that exerts both beneficial and adverse effects on health depending on the specific chemical form and dose. Deficiency or excess of dietary selenium has been linked to immune dysfunction and inflammatory disorders [6, 7] although the precise molecular mechanisms remain to be determined. In living organisms, selenium either exists as low-molecular weight compounds such as selenite, selenomethionine, methylselenol or selenomethylselenocysteine, or is assimilated into selenium-containing proteins (selenoproteins) by way of the amino acid selenocysteine (Sec). Apart from nonspecific selenium incorporation into protein, the pathway of Sec and selenoprotein biosynthesis requires a unique tRNA, Sec tRNA[Ser]Sec [8, 9].
Using a conditional gene knockout strategy, we have shown that ablation of Sec tRNA[Ser]Sec results in the loss of expression of the whole selenoprotein set, or selenoproteome, in hepatocytes , and T cells . Therefore, cell type-specific deletion of the Sec tRNA[Ser]Sec gene (Trsp) offers a useful way to examine the physiological effects of selenium in the form of selenoproteins in relation to the function of a desired cell type. In this paper, we present data from a study of mice lacking Sec tRNA[Ser]Sec in macrophages and describe the molecular and cellular abnormalities caused by the ablation of macrophage selenoprotein expression.
Selenoprotein expression in macrophages
Selenoprotein expression in macrophages was further examined by labeling BMDMs with 75Se, and analyzing labeled selenoproteins by gel electrophoresis . BMDMs expressed a distinct set of selenoproteins, whose repertoire was largely unchanged by LPS activation (Figure 1C). However, the amount of TR1 was substantially increased, whereas that of GPx1 was modestly decreased in LPS-treated macrophages, which was verified by immunoblotting with antibodies specific to individual selenoproteins (Figure 1D). In addition to TR1 and GPx1, several other selenoproteins with high mRNA abundance in macrophages were detected as 75Se-labeled protein bands on the gel.
Ablation of Trsp and selenoprotein expression in macrophages
To study the macrophage-specific functions of selenoproteins, we generated mutant mice in which deletion of floxed (fl) alleles of Trsp is driven by Cre recombinase expressed under the control of the lysozyme M (LysM) promoter. LysMCre expression is restricted to macrophages and other leukocyte subpopulations of myeloid origin .Trspfl/fl-LysMCre (ΔTrsp M ) mice were born at Mendelian ratios, survived to adulthood, and displayed no spontaneous pathology under the specific pathogen-free condition (data not shown).
Inflammatory responses in ΔTrsp M macrophages and mice
Changes in gene expression in ΔTrsp M macrophages
The most salient feature of the genes showing aberrant expression in selenoprotein-deficient macrophages was that many were functionally related to the formation and remodeling of and cellular interaction with the extracellular matrix (ECM). In both resting and LPS-stimulated ΔTrsp M macrophages, these ECM-related genes were expressed at higher levels relative to control cells (Figure 5C), and included those that encode ECM components (collagen chains, extracellular proteoglycans, and secreted glycoproteins; Col1a1, Col5a2, Bgn, Ctgf and Sparc), 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). A few other genes were detected as being expressed at lower levels in LPS-treated ΔTrsp M macrophages, but their repertoire did not point to any particular shared attributes (Figure 5C).
Effects of selenoprotein deficiency on macrophage invasiveness
The immune regulatory effects exerted by dietary selenium are complex, and appear to be determined by multiple variables associated not only with the micronutrient but also with the immune system. Despite the epidemiological link between selenium deficiency and inflammatory disorders, little insight has been gained as to the role of selenium and selenoproteins in the pathology, and the cell types wherein they serve protective and self-destructive functions. Inflammation is driven by multiple types of leukocytes and parenchymal cells of the immune system. We previously reported that T cells lacking Sec tRNA[Ser]Sec and selenoprotein expression were defective in T cell receptor-induced proliferation, a key step for activating T cell-mediated immune responses . In the current study, we determined the selenoproteins abundantly expressed in resting and activated macrophages, and identified a role for selenoproteins in macrophage gene regulation and invasive behavior.
Redox regulation is an inherent component of inflammatory signaling in macrophages . In accord with the antioxidant activities of many selenoproteins, we observed elevated ROS generation in selenoprotein-deficient macrophages. However, this ROS dysregulation did not lead to overt phenotypes in the mouse models of inflammation that were used in this study. These results may imply that the levels of ROS and oxidative damage increased in ΔTrsp M macrophages are subthreshold for effects on inflammatory responses. Alternatively, there may exist fail-safe mechanisms in macrophages that act to maintain inflammatory events in the absence of selenoprotein function. It was indeed shown by others that cytoprotective enzymes that are induced in a manner dependent on the transcription factor NF-E2-related factor 2 can counter oxidative stress in Sec tRNA[Ser]Sec-deficient cells and thereby compensate for the loss of selenoproteins . Whichever the case may be, selenoprotein function is likely dispensable for the onset as well as the termination of inflammatory responses, at least under the specific experimental settings employed in our tests. Of note, the selenoprotein deficiency of ΔTrsp M macrophages resulted in increased expression of ECM-related genes. Therefore, although the precise mechanism remains to be determined, selenoproteins serve nonredundant gene regulatory functions in macrophages.
Cell migration in vivo entails proteolytic remodeling of ECM. Matrix metalloproteinases (MMPs) and other groups of protein-degrading enzymes are well known for their role in cellular ECM invasion. Amongst the ECM-related genes overexpressed in ΔTrsp M macrophages are Tagln and Timp3, whose protein products were shown to inhibit the expression or activity of MMPs and thus suppress cell invasive activity [22, 23]. In keeping with the high expression of these genes, ΔTrsp M macrophages displayed greatly diminished invasion in a protein gel matrix. Other ECM-related genes identified in our DNA microarray experiments are also likely to contribute to this phenotype. Invasive macrophages play a central pathogenic role in certain chronic inflammatory lesions: most notably foam cells in atherosclerotic plaques, and tumor-associated macrophages. Therefore, ΔTrsp M mice may show an altered severity or kinetics of disease in as-yet-unexplored experimental conditions, and serve as animal models of chronic human diseases that are associated with selenium deficiency.
Selenium and selenoproteins may regulate immunity and tissue homeostasis through ECM-related gene expression and macrophage invasiveness.
Mice and primary macrophages
A C57BL/6 mouse line carrying floxed Trsp (Trspfl/fl; designated as control) was described previously . A transgenic C57BL/6 line carrying the Lysozyme M-Cre transgene  was from the Jackson Laboratory. These lines were mated to obtain ΔTrsp M mice. The maintenance and care of all mice were conducted under IACUC-approved protocols and in accordance with the National Institutes of Health institutional guidelines under the expert direction of Dr. John Dennis (NCI, NIH, Bethesda, MD, U.S.A.). BMDMs and PEMs were prepared and cultured as described . The extent of Trsp deletion in each cell preparation were determined by qPCR analysis of the floxed region of the gene.
75Se-labeling and analysis of selenoproteins
Macrophages were incubated with 25 μCi/ml of 75Se for 24 h, lysed, the resulting protein extractions electrophoresed on gels, gels stained with Coomassie Blue R-250, vacuum dried and exposed to a PhosphorImager as described [14, 26].
Protein, mRNA, and ROS analysis
The level of IκBα, ERK, JNK, and p38 activation was determined by immunoblotting with antibodies specific to total and phosphorylated proteins (Cell Signaling Technology). Secreted proteins in culture media were assayed by SearchLight protein array analysis (Pierce). Total RNA was isolated using Trizol (Invitrogen). Microarray analysis was performed with GeneChip Mouse 430 2.0 Array (Affymetrix) at the Molecular Technology Microarray Laboratory (Frederick, MD). The data were normalized and statistically analyzed using software tools (Expression Console; Affymetrix) provided by the National Cancer Institute, Center for Cancer Research in collaboration with the National Institutes of Health, Center for Information Technology, Bioinformatics and Molecular Analysis Section. All microarray data discussed in this paper are available at the GEO repository at NCBI under the accession number GSE15610. Genes showing significantly different expression levels (p < 0.01) in WT versus ΔTrsp M macrophages in three independent hybridizations were chosen for validation by real-time qPCR, which was performed as described . ROS were measured by flow cytometry and confocal microscopy using oxidation sensitive dye DCFDA as described .
Inflammatory response in vivo
LPS endotoxemia was induced by intraperitoneal injection of Escherichia coli 055:B5 LPS (50 mg/kg; Sigma) in phosphate-buffered saline (PBS). For acute edema formation, the right auricle was irritated by topical treatment with 2 μg of TPA in 20 μl of acetone. For control irritation, 20 μl of acetone was applied to the left auricle of the same animal. Ear thickness was measured using a dial thickness gauge (Mitutoyo). Changes in ear thickness were determined base on the following formula: Ear swelling on day X after TPA treatment = (thickness of the right ear on day X - thickness of the right ear on day 0) - (thickness of the left ear on day X - thickness of the left ear on day 0). To induce acute neutrophil infiltration, 0.5 mg of zymosan (Sigma) in 0.5 ml of PBS was injected intraperitoneally. Peritoneal exudate was recovered 4 h following injection and the number of neutrophils in the exudate was counted under bright-field microscopy.
Cell invasion and migration assay
3 × 105 BMDMs in 200 μl of culture medium containing 0.5% fetal bovine serum were loaded onto the upper chamber of either a protein gel-coated BD BioCoat Matrigel Invasion Chamber (pore size, 8.0 μm; BD Biosciences) or a non-coated transwell plate (pore size, 8.0 μm; Corning). For invasion assay, 600 μl of culture medium containing 10% fetal bovine serum was placed in the lower well, and cells were incubated for 24 hr. The lower surface of the membrane was stained with 0.09% crystal violet and macrophages counted under bright-field microscopy. The numbers of macrophages in three independent image fields of the same size were counted to obtain the means and standard deviations.
P-values between a pair of datasets were obtained from two-tailed Student's t-test using GraphPad Prism 4.0. Gene expression data are shown as the average ± standard deviation.
This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, and Center for Cancer Research, and in addition, a specific grant from the Office of Dietary Supplements, NCI, NIH and the Cutaneous Biology Research Center through the Massachusetts General Hospital/Shiseido Co. Ltd. Agreement to JMP and NIH grants awarded to VNG.
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