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. 2006;2006(6):40691.
doi: 10.1155/MI/2006/40691.

Down-regulation of tristetraprolin expression results in enhanced IL-12 and MIP-2 production and reduced MIP-3alpha synthesis in activated macrophages

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Down-regulation of tristetraprolin expression results in enhanced IL-12 and MIP-2 production and reduced MIP-3alpha synthesis in activated macrophages

Ulla Jalonen et al. Mediators Inflamm. 2006.

Abstract

In inflammation, the post-transcriptional regulation of transiently expressed genes provides a potential therapeutic target. Tristetraprolin (TTP) is of the factors regulating decay of cytokine mRNAs. The aim of the present study was to identify cytokines whose expression is regulated by TTP. We established a TTP knock-down cell line by expressing shRNA against TTP (shTTP cell line). A cytokine antibody array was used to measure cytokine production in macrophages exposed to lipopolysaccharide (LPS). Cytokines IL-6, IL-12, TNF-alpha, and MIP-2 (a homologue to human IL-8) were expressed at higher levels whereas MIP-3alpha was produced at lower levels in LPS-treated shTTP cells than in control cells suggesting that the expression of these cytokines is regulated by TTP. The present data provide IL-12, MIP-2, and MIP-3alpha as novel inflammatory cytokine targets for TTP-mediated mRNA decay and stress the role of TTP in the regulation of the inflammatory process.

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Figures

Figure 1
Figure 1
Down-regulation of TTP expression and enhancement of TNF-α production in shTTP cells. (a) shNEG and shTTP cells were stimulated with LPS (100 ng/mL) and proteins were extracted after 6 h of incubation. TTP and actin were detected by Western blot. The blot is a representative of three blots with similar results. (b) shNEG and shTTP cells were stimulated with LPS (100 ng/mL) for 1 h. Thereafter the medium was changed and the cells were incubated for another 48 h. TNF-α concentrations in the culture media were measured by ELISA. Values are mean ± SEM (n = 3). ** = P < .01.
Figure 1
Figure 1
Down-regulation of TTP expression and enhancement of TNF-α production in shTTP cells. (a) shNEG and shTTP cells were stimulated with LPS (100 ng/mL) and proteins were extracted after 6 h of incubation. TTP and actin were detected by Western blot. The blot is a representative of three blots with similar results. (b) shNEG and shTTP cells were stimulated with LPS (100 ng/mL) for 1 h. Thereafter the medium was changed and the cells were incubated for another 48 h. TNF-α concentrations in the culture media were measured by ELISA. Values are mean ± SEM (n = 3). ** = P < .01.
Figure 2
Figure 2
Cytokine antibody array. (a) A schematic diagram of the Mouse Cytokine Antibody Array III shows the locations of controls and the duplicate spots of cytokines. (b)–(f) Images of the membranes treated with cell culture media from the following experiments: (b) culture medium without cells, (c) shNEG cells, 49 h incubation, (d) shNEG cells, stimulated for 1 h with LPS (100 ng/mL) and incubated thereafter for 48 h, (e) shTTP cells, 49 h incubation, (f) shTTP cells, stimulated for 1 h with LPS (100 ng/mL) and incubated thereafter for 48 h. Representative membranes of three with similar results are shown. POS = positive control, NEG = negative control.
Figure 2
Figure 2
Cytokine antibody array. (a) A schematic diagram of the Mouse Cytokine Antibody Array III shows the locations of controls and the duplicate spots of cytokines. (b)–(f) Images of the membranes treated with cell culture media from the following experiments: (b) culture medium without cells, (c) shNEG cells, 49 h incubation, (d) shNEG cells, stimulated for 1 h with LPS (100 ng/mL) and incubated thereafter for 48 h, (e) shTTP cells, 49 h incubation, (f) shTTP cells, stimulated for 1 h with LPS (100 ng/mL) and incubated thereafter for 48 h. Representative membranes of three with similar results are shown. POS = positive control, NEG = negative control.
Figure 2
Figure 2
Cytokine antibody array. (a) A schematic diagram of the Mouse Cytokine Antibody Array III shows the locations of controls and the duplicate spots of cytokines. (b)–(f) Images of the membranes treated with cell culture media from the following experiments: (b) culture medium without cells, (c) shNEG cells, 49 h incubation, (d) shNEG cells, stimulated for 1 h with LPS (100 ng/mL) and incubated thereafter for 48 h, (e) shTTP cells, 49 h incubation, (f) shTTP cells, stimulated for 1 h with LPS (100 ng/mL) and incubated thereafter for 48 h. Representative membranes of three with similar results are shown. POS = positive control, NEG = negative control.
Figure 2
Figure 2
Cytokine antibody array. (a) A schematic diagram of the Mouse Cytokine Antibody Array III shows the locations of controls and the duplicate spots of cytokines. (b)–(f) Images of the membranes treated with cell culture media from the following experiments: (b) culture medium without cells, (c) shNEG cells, 49 h incubation, (d) shNEG cells, stimulated for 1 h with LPS (100 ng/mL) and incubated thereafter for 48 h, (e) shTTP cells, 49 h incubation, (f) shTTP cells, stimulated for 1 h with LPS (100 ng/mL) and incubated thereafter for 48 h. Representative membranes of three with similar results are shown. POS = positive control, NEG = negative control.
Figure 2
Figure 2
Cytokine antibody array. (a) A schematic diagram of the Mouse Cytokine Antibody Array III shows the locations of controls and the duplicate spots of cytokines. (b)–(f) Images of the membranes treated with cell culture media from the following experiments: (b) culture medium without cells, (c) shNEG cells, 49 h incubation, (d) shNEG cells, stimulated for 1 h with LPS (100 ng/mL) and incubated thereafter for 48 h, (e) shTTP cells, 49 h incubation, (f) shTTP cells, stimulated for 1 h with LPS (100 ng/mL) and incubated thereafter for 48 h. Representative membranes of three with similar results are shown. POS = positive control, NEG = negative control.
Figure 2
Figure 2
Cytokine antibody array. (a) A schematic diagram of the Mouse Cytokine Antibody Array III shows the locations of controls and the duplicate spots of cytokines. (b)–(f) Images of the membranes treated with cell culture media from the following experiments: (b) culture medium without cells, (c) shNEG cells, 49 h incubation, (d) shNEG cells, stimulated for 1 h with LPS (100 ng/mL) and incubated thereafter for 48 h, (e) shTTP cells, 49 h incubation, (f) shTTP cells, stimulated for 1 h with LPS (100 ng/mL) and incubated thereafter for 48 h. Representative membranes of three with similar results are shown. POS = positive control, NEG = negative control.
Figure 3
Figure 3
TTP down-regulation (shTTP cell line) enhanced the production of IL-6, IL-12, MIP-2, and TNF-α in response to LPS. Cytokine antibody array membranes were incubated in medium control (M) or in media from cultures of TTP knock-down cells (shTTP cell line) or control cells (shNEG cell line) after treatment with or without LPS (100 ng/mL). Values are arbitrary units compared to positive controls (100) and are presented as mean ± SEM (n = 3). ** = P < .01, * = P < .05.
Figure 4
Figure 4
TTP down-regulation (shTTP cell line) reduced the production of MIP-3α and IL-12 p40 subunit in response to LPS. Cytokine antibody array membranes were incubated in medium control (M) or in media from cell cultures of TTP knock-down cells (shTTP cell line) or control cells (shNEG cell line) after treatment with or without LPS (100 ng/mL). Values are arbitrary units compared to positive controls (100) and are presented as mean ± SEM (n = 3). ** = P < .01.

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