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Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on May 9, 2008; DOI: 10.1124/jpet.107.133702

0022-3565/08/3262-514-522$20.00
JPET 326:514-522, 2008
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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

Compounds That Increase or Mimic Cyclic Adenosine Monophosphate Enhance Tristetraprolin Degradation in Lipopolysaccharide-Treated Murine J774 Macrophages

Ulla Jalonen, Erja-Leena Paukkeri, and Eeva Moilanen

The Immunopharmacology Research Group, Medical School, University of Tampere and Research Unit, Tampere University Hospital, Tampere, Finland

Received for publication November 6, 2007
Accepted May 8, 2008.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Tristetraprolin (TTP) is a trans-acting factor that can regulate mRNA stability by binding to the cis-acting AU-rich element (ARE) in the 3'-untranslated region in mRNAs of certain transiently expressed genes. The best-studied target of TTP is tumor necrosis factor (TNF)-{alpha}. By binding to ARE, TTP increases the degradation of TNF-{alpha} mRNA, thereby reducing the expression of TNF-{alpha}. We examined the effects of cAMP analogs and the cAMP-elevating agents forskolin and β2-agonists on lipopolysaccharide (LPS)-induced TTP mRNA and protein expression by quantitative real-time reverse transcriptase-polymerase chain reaction and Western blotting in activated macrophages. All of these agents caused a slight increase in LPS-induced expression of TTP mRNA. However, TTP protein levels were significantly reduced when the cells were treated with the combination of LPS and cAMP-elevating agent compared with LPS alone. Proteasome inhibitors MG132 (N-[(phenylmethoxy)-carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide) and lactacystin increased TTP protein levels and abolished the effects of cAMP-enhancing compounds on TTP protein levels. The results suggest that mediators and drugs that enhance intracellular cAMP reduce TTP expression in macrophages exposed to inflammatory stimuli by increasing TTP degradation through the proteasome pathway.

Post-transcriptional regulation of gene expression by cis-acting elements and trans-acting factors has been an object of growing interest in recent years. One of the trans-acting factors regulating the stability of transiently expressed mRNAs is tristetraprolin (TTP), also known as Nup475, TIS11, G0S24, or Zfp36 (Blackshear, 2002Go). The cis-acting element to which TTP binds resides in the AU-rich element (ARE) in the 3'-untranslated region of mRNAs of many inflammatory and other transiently expressed genes. The stability of an increasing number of transcripts has been shown to be regulated by TTP in studies with overexpression of TTP or by knockout (KO) or knockdown of TTP expression. Most studies have focused on the post-transcriptional regulation of tumor necrosis factor (TNF)-{alpha} mRNA. TTP can promote the deadenylation and decay of TNF-{alpha} mRNA by binding to the conserved ARE in the 3'-untranslated region of TNF-{alpha} mRNA (Lai et al., 1999Go; Lai and Blackshear, 2001Go), thereby decreasing the amount of proinflammatory cytokine TNF-{alpha}. TTP KO mice developed a complex inflammatory syndrome characterized by arthritis, autoimmunity, cachexia, and myeloid hyperplasia due to increased levels of TNF-{alpha} (Taylor et al., 1996Go; Carballo et al., 1998Go).

Other suggested and/or partially confirmed targets of TTP include inflammatory modulators such as granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-2, IL-3, IL-6, IL-12, macrophage inflammatory protein-2, macrophage inflammatory protein-3{alpha}, inducible nitric oxide synthase, and cyclooxygenase-2 (Carballo et al., 2000Go; Stoecklin et al., 2000Go, 2001Go; Sawaoka et al., 2003Go; Linker et al., 2005Go; Ogilvie et al., 2005Go; Jalonen et al., 2006Go). The most recent members in the group of mRNAs possibly regulated post-transcriptionally by TTP include suppressor of cytokine signaling 3 (SOCS3), transforming growth factor β1, and transcription factor E47 (Chang et al., 2007Go; Ehlting et al., 2007Go; Frasca et al., 2007Go). However, studies using cells derived from TTP KO mice have not confirmed TTP as a sole regulator of mRNA stability of these genes. For instance, in the case of SOCS3, the results showed that in NIH 3T3 cells, down-regulation of TTP by small interfering RNA and TTP overexpression affected SOCS3 mRNA stability, but SOCS3 mRNA half-life was similar in cells from wild-type and TTP KO mice (Ehlting et al., 2007Go).

In addition, there is increasing evidence that the p38 mitogen-activated protein kinase (MAPK) pathway is a crucial regulator of the expression, stability, and function of TTP (Mahtani et al., 2001Go; Brook et al., 2006Go; Hitti et al., 2006Go). Phosphorylation of particular serine residues of TTP increases binding of 14-3-3 proteins, thereby excluding TTP from stress granules and inactivating TTP and increasing the stability of target mRNAs (Johnson et al., 2002Go; Chrestensen et al., 2004Go; Stoecklin et al., 2004Go). In addition to stress granules, other sites where degradation of ARE-containing mRNAs has been shown to occur are the processing body (Franks and Lykke-Andersen, 2007Go; Stoecklin and Anderson, 2007Go), the exosome (Chen et al., 2001Go), and the proteasome (Laroia et al., 1999Go). Micro-RNAs have also been reported to have a role in TTP-mediated mRNA degradation (Jing et al., 2005Go). Whatever the site of mRNA degradation is, TTP protein has been proposed to be degraded by the proteasome (Brook et al., 2006Go; Deleault et al., 2008Go).

We have recently shown that β2-agonists, cAMP analogs, and forskolin increase TTP mRNA and protein expression in resting cells, possibly by increasing the activation of transcription factor activator protein 2 (Jalonen et al., 2007Go). In the present study, we found that TTP protein expression was differently regulated by cAMP-increasing compounds in cells exposed to LPS (which mimics the inflammatory situation) than in our earlier study in resting cells. Even though TTP mRNA amounts were increased, the TTP protein levels decreased when J774 macrophages were treated with a combination of LPS and cAMP-elevating agents. A possible mechanism of these effects is the increased degradation of TTP protein through the proteasome pathway because the inhibition of proteasome activity by proteasome inhibitors MG132 and lactacystin inhibited the decrease in TTP protein levels found with the combination of LPS and cAMP-elevating agents compared with treatment with LPS alone.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Materials. Reagents were purchased as follows: forskolin, MG132, and SB203580 from Tocris Cookson Inc. (Ellisville, MO), and ubiquitin aldehyde was from Boston Biochem (Cambridge, MA). All other reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.

Cell Culture. The murine J774 macrophages (European Collection of Cell Cultures, Porton Down, Wiltshire, UK) were maintained in an atmosphere of 5% carbon dioxide at 37°C in Dulbecco's modified Eagle's medium with UltraGlutamine 1 (Lonza Verviers SPRL, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (Lonza Verviers SPRL), penicillin (100 units/ml), streptomycin (100 µg/ml), and amphotericin B (250 ng/ml) (Invitrogen, Paisley, UK). Cells were seeded on six- or 24-well plates and grown to confluence before the experiments. Tested compounds were added to the cell culture at the same time as LPS, unless otherwise stated.

Cell Viability Test. Cell viability was tested using the Cell Proliferation Kit II (Roche Diagnostics, Mannheim, Germany). Cells were incubated with LPS (1 ng/ml) or LPS (1 ng/ml) and forskolin (100 µM) for 12 h before the addition of the sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate labeling reagent (final concentration, 0.3 mg/ml) and N-methyl dibenzopyrazine methyl sulfate (2.5 µg/ml). Then, cells were incubated for 3 h, and the amount of formazan accumulated in the growth medium was assessed spectrophotometrically. No difference in cell viability between treated and untreated cells was detected. Triton X-treated cells were used as a positive control resulting in 97% reduction in cell viability.

RNA Extraction and Quantitative Real-Time Reverse Transcriptase-PCR. The protocol for RNA extraction and quantitative real-time reverse transcriptase (RT)-PCR has been described by Jalonen et al. (2005Go). Primers and TaqMan 6-carboxyfluorescein (Fam)-TAMRA probes (Table 1) for TTP, TNF-{alpha}, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed using Primer Express Software (Applied Biosystems, Foster City, CA) and supplied by Metabion (Martinsried, Germany).


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  		TABLE 1 Primer and probe sequences

 

Western Blotting. The protocol for Western blotting has been described by Jalonen et al. (2005Go). When preparing cell lysates for ubiquitin Western blotting, lysis buffer also contained ubiquitin aldehyde (20 µg/ml) and MG132 (25 µM) to prevent deubiquitinylation of the sample. The gels were loaded with 35 µg of protein for TTP and actin Western blots and 240 µg for ubiquitin Western blotting. Actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), the mouse TTP antibody (Cao et al., 2004Go) was kindly provided by Dr. Perry Blackshear (National Institute of Environmental Health Sciences, Research Triangle Park, NC), ubiquitin antibody was from Zymed Laboratories (South San Francisco, CA), p38 mitogen-activated protein kinase and phospho-p38 mitogen-activated protein kinase (Thr180/Tyr182) antibodies were from Cell Signaling Technology Inc. (Danvers, MA). The bound antibodies were detected using SuperSignal West Dura or Femto chemiluminescent substrate for horseradish peroxidase detection (Pierce Chemical, Cramlington, Northumberland, UK) and the FluorChem 8800 imaging system (Alpha Innotech, San Leandro, CA). Actin was used as a loading control, and it was detected from the same membrane as TTP after stripping the membrane. The chemiluminescent signals were measured with FluorChem software, version 3.1.

Statistics. Results are expressed as the mean ± S.E.M. The significance of differences was calculated by analysis of variance supported by Dunnett's adjusted significance levels. A difference between treatment groups was considered significant when p < 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
cAMP Analogs, Forskolin, and β2-Agonists Decreased LPS-Induced TTP Protein Expression in J774 Macrophages. In resting cells, low levels of TTP protein were detected. LPS (1 ng/ml) induced transient expression of TTP protein, which enhanced rapidly, remained relatively constant for 4 to 9 h, and declined thereafter (data not shown). After incubation with LPS (1 ng/ml) for 9 h, TTP protein amount was increased 5-fold in J774 macrophages compared with resting cells (Fig. 1A). cAMP analogs N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate sodium salt (db-cAMP; 1 mM) and 8-bromoadenosine 3',5'-cyclic monophosphate sodium salt (8-Br-cAMP; 1 mM) decreased LPS-induced TTP protein expression by 40% (Fig. 1A). At lower concentrations (0.1 mM), db-cAMP was less efficient than 8-Br-cAMP in decreasing TTP protein levels. Likewise, forskolin (an activator of adenylate cyclase; 100 µM) decreased LPS-induced TTP protein levels by 45% when measured after 9 h of incubation (Fig. 1B). In addition to the cAMP analogs and forskolin, β2-agonists salbutamol and terbutaline (0.3 and 1 µM) decreased LPS-induced TTP protein levels by 41 to 50% when measured after 9-h incubation (Fig. 1C).


Figure 1
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  		Fig. 1. Effects of cAMP analogs, forskolin, and β2-agonists on LPS-induced TTP protein expression in J774 macrophages. The cells were treated with LPS (1 ng/ml) and indicated concentrations of 8-Br-cAMP or db-cAMP (A), forskolin (B), or salbutamol or terbutaline (C) for 9 h. Proteins were extracted, and TTP and actin proteins were measured by Western blot. The TTP protein level in the LPS-treated samples was set at 100, and the other values were related to that. The blot is a representative of six (in A and C) or three (in B) with similar results. Values are mean ± S.E.M. (n = 6 in A and C, n = 3 in B). **, p < 0.01 when compared with the LPS-treated samples.

 

cAMP Analogs, Forskolin, and Salbutamol Increased LPS-Induced TTP mRNA Levels in J774 Macrophages. Low levels of TTP mRNA were found in unstimulated cells, and that was significantly increased when LPS (1 ng/ml) was added into the culture. J774 macrophages were incubated with LPS (1 ng/ml) and 8-Br-cAMP or db-cAMP (0.1 or 1 mM) for 1 h (Fig. 2A). 8-Br-cAMP (0.1 and 1 mM) increased TTP mRNA levels by 49 and 79%, respectively, compared with cells treated with LPS only. db-cAMP (0.1 and 1 mM) had a similar effect and enhanced TTP mRNA levels by 37 and 93% compared with LPS-treated cells. When the cells were incubated with LPS (1 ng/ml) in combination with forskolin (100 µM), TTP mRNA levels were higher than when treated with LPS (1 ng/ml) alone (Fig. 2B). The difference between the treatments was statistically significant after 1- and 2-h incubation. A slight increase in TTP mRNA levels was also observed when the cells were treated with LPS (1 ng/ml) and increasing concentrations (0.1–3 µM) of salbutamol for 1 h (Fig. 2C). Salbutamol (3 µM) increased LPS-induced TTP mRNA levels by 35%.


Figure 2
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  		Fig. 2. Effects of cAMP analogs, forskolin, and salbutamol on LPS-induced TTP mRNA expression in J774 macrophages. A, LPS (1 ng/ml) and indicated concentrations of 8-Br-cAMP or db-cAMP were used to stimulate macrophages for 1 h, after which total RNA was extracted. Quantitative RT-PCR was used to measure TTP mRNA, and the values were normalized to GAPDH mRNA. The mean of TTP mRNA levels in the LPS-treated samples was set at 100, and the other values were related to that. Values are mean ± S.E.M. (n = 3). **, p < 0.01 and *, p < 0.05 when compared with the LPS-treated samples. B, cells were stimulated with LPS (1 ng/ml) in combination with forskolin (100 µM). Total RNA was extracted at the time points indicated. Quantitative RT-PCR was used to measure TTP mRNA, and the values were normalized to GAPDH mRNA. The mean of TTP mRNA levels in the LPS-treated samples at the 1-h time point was set at 100, and the other values were related to that. Values are mean ± S.E.M. (n = 3). **, p < 0.01 when compared with the LPS-treated samples in each time point. C, LPS (1 ng/ml) and indicated concentrations of salbutamol were used to stimulate macrophages for 1 h, after which total RNA was extracted. Quantitative RT-PCR was used to measure TTP mRNA, and the values were normalized to GAPDH mRNA. The TTP mRNA level in the LPS-treated samples was set at 100, and the other values were related to that. Values are mean ± S.E.M. (n = 3). **, p < 0.01 and *, p < 0.05 when compared with the LPS-treated samples.

 

Forskolin Did Not Affect TTP mRNA Half-Life in LPS-Treated J774 Macrophages. To find out the reasons for the differences between mRNA and protein expression, we first examined the degradation rate of TTP mRNA by actinomycin D assay (Fig. 3). Cells were treated with LPS (1 ng/ml) in the presence or absence of forskolin (100 µM) for 1 h; thereafter, actinomycin D (0.5 µg/ml) was added to the culture to inhibit transcription. Total RNA was extracted 2, 4, and 5 h after addition of actinomycin D, and the TTP mRNA levels were measured. Forskolin did not alter the TTP mRNA degradation rate, and the half-life of TTP mRNA with both treatments was approximately 4.5 h.


Figure 3
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  		Fig. 3. Effects of forskolin on TTP mRNA decay in LPS-treated J774 macrophages. The cells were treated with LPS (1 ng/ml) with or without forskolin (100 µM) for 1 h before actinomycin D (0.5 µg/ml) was added to the culture. Total RNA was extracted 2, 4, and 5 h after actinomycin D addition, and quantitative PCR was used to detect TTP mRNA. GAPDH mRNA was measured for normalization. The mean of TTP mRNA levels in the LPS-treated samples at the time of actinomycin D addition was set at 100, and the other values were related to that. Values are mean ± S.E.M. (n = 3).

 

Forskolin, cAMP Analogs, and β2-Agonists Decreased LPS-Induced TTP Protein Expression in a Time-Dependent Manner. Because forskolin had no effect on TTP mRNA half-life, we examined its effects on TTP protein expression after different incubation times (Fig. 4A). Macrophages were incubated with LPS (1 ng/ml) with or without forskolin (100 µM), and proteins were extracted after 4, 6, 9, and 12 h of culture. With both treatments, TTP protein levels were almost equal when measured after 4-h incubation. After 6-h incubation, TTP protein levels were 10% lower in forskolin-treated cells. After 9- and 12-h incubations, LPS-induced TTP protein levels were 51 and 57% lower in forskolin-treated cells, respectively. In addition, TTP mRNA levels were measured at the same time points (Fig. 4B). As expected, after 4-h incubation, TTP mRNA levels were higher in forskolin-treated cells. In contrast, after 6-, 9-, and 12-h incubation, the TTP mRNA levels in LPS + forskolin-treated cells were similar to those in cells treated with LPS only.


Figure 4
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  		Fig. 4. Effects of forskolin on TTP protein and mRNA expression in LPS-treated J774 macrophages. The cells were incubated with LPS (1 ng/ml) with or without forskolin (100 µM) for 4, 6, 9, or 12 h, after which proteins (A) or total RNA (B) were extracted. A, TTP and actin proteins were measured by Western blot. The TTP protein levels in the LPS- and LPS + forskolin-treated samples at 4 h were set at 100, and the other values gained with the same treatment were related to those. The blot is a representative of three with similar results. Values are mean ± S.E.M. (n = 3). C, control. **, p < 0.01 when compared between the LPS- and LPS + forskolin-treated samples. B, quantitative PCR was used to detect TTP mRNA, and GAPDH mRNA was measured for normalization. The mean of TTP mRNA levels in the LPS-treated samples at the 4-h time point was set at 100, and the other values were related to that. Values are mean ± S.E.M. (n = 4). **, p < 0.01 and N.S. (ns) when compared between the LPS- and LPS + forskolin-treated samples.

 
Thereafter, we investigated the effects of cAMP analogs and the β2-agonist on TTP protein levels (Fig. 5). The cells were treated with LPS (1 ng/ml) with or without the cAMP analogs or β2-agonist. Proteins were extracted after 4- and 9-h incubation, and TTP protein was detected by Western blot. After 4-h incubation, LPS-induced TTP protein levels were approximately similar in control cells and in cells treated with 8-Br-cAMP, db-cAMP, or salbutamol. After 9-h incubation, all of the three compounds significantly decreased the level of TTP protein expression. The cAMP analogs 8-Br-cAMP and db-cAMP decreased TTP protein levels after 9-h incubation by 52 and 40%, respectively. Likewise, salbutamol decreased the amount of TTP protein by 41%. In conclusion, the results suggest that cAMP analogs and cAMP-elevating agents increase the rate of TTP protein degradation.


Figure 5
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  		Fig. 5. The effects of cAMP analogs and β2-agonist on TTP protein expression in LPS-treated J774 macrophages. The cells were incubated with LPS (1 ng/ml) with or without 8-Br-cAMP (1 mM) (A), db-cAMP (1 mM) (B), or salbutamol (1 µM) (C) for 4 or 9 h, after which proteins were extracted. TTP and actin proteins were measured by Western blot. The TTP protein levels in the LPS-treated samples at the 4-h time point were set at 100, and the other values were related to those. C, control. The blot is a representative of six with similar results. Values are mean ± S.E.M. (n = 6). **, p < 0.01 and *, p < 0.05 when compared between the LPS and LPS + cAMP analog or LPS + β2-agonist-treated samples at each time point.

 

Proteasome Inhibitors Abolished Differences between TTP Protein Levels Extracted from LPS- and LPS + Forskolin-Treated J774 Macrophages. We used two inhibitors of proteasome, lactacystin and MG132, to investigate whether the proteasome-mediated degradation of TTP protein is increased with forskolin in LPS-treated cells. First, we wanted to confirm that lactacystin, an inhibitor of 20S and 26S proteasomes, has a functional effect as a proteasome inhibitor in the cell culture conditions used. When protein degradation through the proteasome is inhibited, ubiquitinated proteins accumulate in the cells. Therefore, we examined the effects of lactacystin on the levels of ubiquitinated cellular proteins (Fig. 6A). The cells were first treated with or without LPS (10 ng/ml) for 8 h, and then lactacystin (10 µM) was added to inhibit the activity of proteasome. Total proteins were extracted as described under Materials and Methods after 16-h incubation with lactacystin. Ubiquitinated proteins were detected with ubiquitin antibody. Addition of lactacystin to untreated cells increased the amount of ubiquitinated proteins as can be seen in Fig. 6A, lanes 1 and 2. An even greater increase of ubiquitinated proteins in cells by lactacystin could be seen in samples extracted from LPS-treated cells (see Fig. 6A, lanes 3 and 4).


Figure 6
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  		Fig. 6. Effects of proteasome inhibitors on TTP protein expression in J774 macrophages. A, cells were incubated with or without LPS (10 ng/ml) for 8 h before the addition of lactacystin (10 µM). Proteins were extracted 16 h after the addition of lactacystin, and protein ubiquitination was analyzed by Western blot. The blot is a representative of three with similar results. B, J774 macrophages were treated with LPS (1 ng/ml) with or without forskolin (100 µM) for 5 h, and then lactacystin (20 µM) was added. Incubation was continued for 4 h, after which proteins were extracted. TTP and actin proteins were measured by Western blot. The TTP protein level in the LPS-treated samples was set at 100, and the other values were related to that. The blot is a representative of three with similar results. Values are mean ± S.E.M. (n = 3). **, p < 0.01 and N.S. (ns) when compared with the LPS- and LPS + lactacystin-treated samples. C, J774 macrophages were incubated with LPS (1 ng/ml) with or without forskolin (100 µM) for 6 h before MG132 (10 µM) was added, and incubation was continued for another 6 h. Proteins were extracted, and TTP and actin protein were detected by Western blot. The TTP protein level in the LPS-treated samples was set at 100, and the other values were related to that. The blot is a representative of three with similar results. Values are mean ± S.E.M. (n = 3). **, p < 0.01 and N.S. (ns) when compared with the LPS- and LPS + MG132-treated samples.

 
J774 macrophages were incubated for 5 h with LPS (1 ng/ml) in the presence or absence of forskolin (100 µM) before lactacystin (20 µM) was added into the culture (Fig. 6B). Incubations were continued for 4 h, after which proteins were extracted and detected by Western blot. Addition of lactacystin increased LPS-induced TTP protein levels by 68%, suggesting that the degradation of TTP protein was decreased during inhibition of the proteasome. In the absence of lactacystin, forskolin reduced the amount of LPS-induced TTP protein by 45%. When lactacystin was added to the culture, forskolin had no effect on TTP protein levels.

Another inhibitor of proteasome, MG132, was used to assess the effect of forskolin on TTP protein degradation in a similar experiment (Fig. 6C). The cells were first incubated with LPS (1 ng/ml) with or without forskolin (100 µM) for 6 h before MG132 (10 µM) was added. Incubation was continued for another 6 h. After protein extraction, TTP and actin proteins were detected by Western blot. MG132 increased TTP protein levels of LPS-treated samples more than 10-fold, indicating that without the inhibitor of proteasome large amounts of TTP protein are degraded by the proteasome. In the absence of MG132, forskolin decreased LPS-induced protein levels by 55%. In the presence of MG132, forskolin did not alter TTP protein levels.

LPS and LPS + Forskolin Activated MAPK p38 at 30 min but Not at 9 h. Activation of p38 by phosphorylation was studied by Western blot (Fig. 7A). J774 macrophages were treated with LPS (1 ng/ml) with or without forskolin (100 µM) for 30 min or 9 h, after which proteins were extracted, and phosphorylated and total p38 were detected by Western blot. Thirty-minute incubation with LPS or LPS + forskolin increased the amount of phosphorylated p38 3 or 4-fold, respectively, compared with control. After 9-h incubation, phospho-p38 levels were very low, and there were no differences between the treatments.


Figure 7
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  		Fig. 7. Effects of LPS and forskolin on p38 activation and the suppressive effect of p38 inhibitor SB203580 on TTP mRNA expression in J774 macrophages. A, cells were treated with LPS (1 ng/ml) and forskolin (100 µM) for 30 min or 9 h. Proteins were extracted, and phospho-p38 (pp38), total p38, and actin proteins were measured by Western blot. The phospho-p38 protein level in the LPS-treated samples was set at 100, and the other values were related to that. The blot is a representative of four with similar results. Values are mean ± S.E.M. (n = 4). **, p < 0.01 when compared with the control samples at the 30-min time point. B, cells were incubated with LPS (1 ng/ml), forskolin (100 µM), and p38 inhibitor SB203580 (1 µM) as indicated for 1 h, after which total RNA was extracted. Quantitative RT-PCR was used to measure TTP mRNA, and the values were normalized to GAPDH mRNA. The mean of TTP mRNA levels in the LPS-treated samples was set at 100, and the other values were related to that. Values are mean ± S.E.M. (n = 4). **, p < 0.01 when compared with the LPS-treated or LPS + forskolin-treated sample.

 
The p38 pathway is one of the regulators of TTP transcription. To find out whether the inhibition of p38 activation affects the mRNA levels of TTP, we treated the J774 macrophages with different combinations of LPS (1 ng/ml), forskolin (100 µM), and p38 inhibitor SB203580 (1 µM) (Fig. 7B). Total RNA was extracted after 1-h incubation, and TTP mRNA was measured by quantitative PCR. Both LPS-induced and LPS + forskolin-induced TTP mRNA levels were inhibited by the p38 inhibitor SB203580 by 51 to 53%, indicating that p38 regulates TTP mRNA expression both in LPS and LPS + forskolin-treated cells.

Forskolin Decreased TNF-{alpha} mRNA Decay in LPS-Treated J774 Macrophages. Because TNF-{alpha} mRNA is a well known target of TTP, and its decay is increased by TTP, we measured TNF-{alpha} mRNA decay by actinomycin D assay at a time point when the forskolin-induced reduction in TTP protein amount was most significant (Fig. 8). Macrophages were treated with LPS (1 ng/ml) in the presence or absence of forskolin (100 µM) for 9 h; thereafter, actinomycin D was added. Total RNA was extracted at 60 and 90 min after addition of actinomycin D, and TTP mRNA levels were measured. As seen in Fig. 8, TNF-{alpha} mRNA decay was slower in LPS + forskolin-treated cells that in cells exposed to LPS only.


Figure 8
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  		Fig. 8. Effects of forskolin on TNF-{alpha} mRNA decay in LPS-treated J774 macrophages. The cells were treated with LPS (1 ng/ml) with or without forskolin (100 µM) for 9 h before actinomycin D (0.5 µg/ml) was added to the culture. Total RNA was extracted 60 and 90 min after actinomycin D addition, and quantitative PCR was used to detect TNF-{alpha} mRNA. GAPDH mRNA was measured for normalization. The mean of TNF-{alpha} mRNA levels at the time of actinomycin D addition was set at 100, and the other values were related to that. Values are mean ± S.E.M. (n = 4). **, p < 0.01 and *, p < 0.05 when compared with the LPS-treated samples.

 


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The principal finding in the present study was that cAMP analogs, forskolin, and β2-agonists decreased LPS-induced TTP protein expression by increasing the rate of TTP degradation via proteasome. The role of p38 in the regulation of TTP expression was also studied, indicating a role for p38 in the regulation of TTP mRNA expression at early time points. The results suggest a novel mechanism by which mediators or drugs that increase intracellular cAMP concentrations may participate in the up-regulation of the expression of inflammatory genes.

Experiments on TTP KO mice and cells derived from them have shown that TTP is a physiological regulator to reduce mRNA stability and, hence, the expression of two important proinflammatory cytokines, TNF-{alpha} and GM-CSF (Taylor et al., 1996Go; Carballo et al., 1998Go, 2000Go). Due to increased levels of TNF-{alpha} and GM-CSF, the TTP KO mice suffer from arthritis-like symptoms, autoimmunity, and myeloid hyperplasia (Taylor et al., 1996Go; Phillips et al., 2004Go). In this study, we showed that forskolin reduced LPS-induced TTP protein levels significantly after 9-h incubation (i.e., by approximately 50% based on the Western blot results). Forskolin also reduced TNF-{alpha} mRNA decay, suggesting that the forskolin-induced changes in TTP levels may be functionally significant. In our earlier study, down-regulation of TTP expression by small interfering RNA by approximately 50% resulted in increased release of TNF-{alpha} and some other cytokines from activated macrophages (Jalonen et al., 2006Go). That result further supports that down-regulation of TTP expression by the degree found in the present study may have significant effects in macrophages.

In addition to arthritis, recent studies suggest that TTP has a potential role in cancer and in obesity-related metabolic complications (Bouchard et al., 2007Go; Carrick and Blackshear, 2007Go). The possibility to regulate TTP expression to treat these conditions with future drugs requires intensive study of the regulation TTP expression and the effects of TTP over- and underexpression. Only little is known about the pharmacological regulation of TTP expression. Dexamethasone has been shown to increase TTP mRNA and protein levels in resting human A549 lung epithelial cells and in rat lung tissue (Smoak and Cidlowski, 2006Go). On the other hand, dexamethasone reduced LPS-induced TTP expression in J774 macrophages (Jalonen et al., 2005Go). Interferons have been reported to up-regulate TTP expression, which in turn results in down-regulation of several proinflammatory genes (Sauer et al., 2006Go). Cinnamon extract and cinnamon polyphenols have been shown to increase both the protein and mRNA levels of TTP in mouse adipocytes (Cao et al., 2007bGo). In addition, green tea, which is also associated with anti-inflammatory effects, increased the levels of TTP mRNA in rat liver and skeletal muscle (Cao et al., 2007aGo).

We recently reported that the expression of TTP mRNA and protein could be induced by β2-agonists, cAMP analogs, and forskolin in resting macrophages, and that was probably mediated by increased activator protein 2 activation (Jalonen et al., 2007Go). Here, we have shown that when these compounds were given in combination with LPS, which mimics the inflammatory situation, TTP protein levels were significantly decreased. The decrease in TTP protein amounts could be prevented by treating the cells with proteasome inhibitors MG132 and lactacystin. The observed decrease in TTP protein levels in cells treated with LPS in combination with cAMP-elevating agents compared with treatment with LPS alone may be caused by enhanced activity of the proteasome activated by cAMP. This assumption is supported by earlier findings that phosphorylation of an ATPase in the 19S cap of the mammalian proteasome, Rpt6, by cAMP-dependent protein kinase A enhances proteasome activity (Zhang et al., 2007Go). cAMP seems to regulate the expression of TTP differently in resting cells and in cells exposed to inflammatory stimulus. It is interesting to note that very similar results have been reported with glucocorticoid dexamethasone. Dexamethasone, in combination with LPS, decreased TTP expression in mouse macrophages (Jalonen et al., 2005Go), but in resting human A549 lung epithelial cells and in rat lung tissues (in the absence of inflammatory stimuli), dexamethasone increased TTP mRNA and protein levels (Smoak and Cidlowski, 2006Go).

In contrast to TTP mRNA, which is transiently expressed, TTP protein has been regarded to be fairly stable, and the stability is regulated by p38 MAPK and its downstream target, MAPK-activated protein kinase 2, and extracellular signal-regulated kinase (Cao et al., 2004Go; Brook et al., 2006Go; Hitti et al., 2006Go; Deleault et al., 2008Go). It has been suggested that dephosphorylation of TTP directs TTP to the proteasome by an unknown mechanism to be rapidly degraded (Brook et al., 2006Go; Deleault et al., 2008Go). TTP protein sequence contains three PEST domains (proline, glutamic acid, serine, and threonine) that target proteins for degradation by proteasome, but studies with mutated domains have not yet shown the functional activity of these domains (Rigby et al., 2005Go). Here, we provide further evidence that TTP protein expression is down-regulated by degradation through proteasome by showing that two proteasome inhibitors (lactacystin and MG132) enhanced TTP protein levels. Phosphorylation of p38 was also examined at 30 min and 9 h to gain mechanistic information on the regulation of TTP expression. The results suggest that at early time points, when p38 is activated by phosphorylation, it activates TTP transcription. At later time points, when p38 has been inactivated by phosphatases, the phosphorylation status of TTP also may decrease, enabling the degradation of TTP protein. We suggest that the degradation through the proteasome is an inducible mechanism to withdraw TTP from the cells and serves as a regulatory mechanism that enhances the inflammatory reaction by limiting the duration of TTP expression. Although the expression of TTP is regulated in a similar manner in resting mouse and human macrophages by cAMP-elevating agents (Jalonen et al., 2007Go), the results described in the present study require confirmation in human macrophages. Many factors increase intracellular cAMP levels through G-protein-coupled receptors in cell membrane, indicating that several other factors than those tested in the present study may also increase TTP protein degradation in a similar manner.

The data in the present study suggest that activation of cAMP-mediated mechanisms by cAMP analogs, forskolin, and β2-agonists decreases LPS-induced TTP protein expression in macrophages possibly through the activation of proteasome. The results described here provide a mechanism by which the expression of TTP can be turned off at sites of inflammation. β2-Agonists have been reported to have anti-inflammatory effects in various in vitro conditions but these actions do not clearly translate into the in vivo situation, e.g., in inflamed lung tissue in patients with asthma (Sitkauskiene and Sakalauskas, 2005Go). The mechanism described in the present study and in our earlier study (Jalonen et al., 2007Go) could, at least partly, explain the discrepancy between these in vitro and in vivo findings. In vivo, in situations with an ongoing inflammation, the expression of TTP can be down-regulated by β2-agonists. In the absence of an LPS-like inflammatory stimulus, these agents could increase the amount of TTP. Actually, the increased TTP degradation may be involved in the enhancement of asthmatic inflammation sometimes seen in asthma patients treated with β2-agonists only without concomitant inhaled anti-inflammatory steroids. Our results suggest that compounds that increase or mimic cAMP decrease LPS-induced TTP protein expression, possibly by directing TTP for degradation through the proteasome, which is likely to enhance the expression of factors regulated by TTP.


   Acknowledgements
 
We thank Salla Hietakangas, Meiju Kukkonen, and Eeva Lahtinen for excellent technical assistance and Heli Määttä for skillful secretarial help.


   Footnotes
 
This work was supported by Tampere Graduate School in Biomedicine and Biotechnology, Medical Research Fund of Tampere University Hospital, Academy of Finland, Pirkanmaa Regional Fund of the Finnish Cultural Foundation, and Emil Aaltonen Foundation.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.133702.

ABBREVIATIONS: TTP, tristetraprolin; ARE, AU-rich element; KO, knockout; TNF, tumor necrosis factor; GM-CSF, granulocyte macrophage colony-stimulating factor; IL, interleukin; SOCS3, suppressor of cytokine signaling 3; MAPK, mitogen-activated protein kinase; LPS, lipopolysaccharide; MG132, N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)-1H-imidazole; PCR, polymerase chain reaction; RT, reverse transcriptase; TAMRA, 6-carboxytetramethylrhodamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; db-cAMP, N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate sodium salt; 8-Br-cAMP, 8-bromoadenosine 3',5'-cyclic monophosphate sodium salt.

Address correspondence to: Dr. Eeva Moilanen, Medical School, Immunopharmacology, FIN-33014 University of Tampere, Finland. E-mail: eeva.moilanen{at}uta.fi


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

Blackshear PJ (2002) Tristetraprolin and other CCCH tandem zinc-finger proteins in the regulation of mRNA turnover. Biochem Soc Trans 30: 945–952.[CrossRef][Medline]
Bouchard L, Tchernof A, Deshaies Y, Marceau S, Lescelleur O, Biron S, and Vohl MC (2007) ZFP36: a promising candidate gene for obesity-related metabolic complications identified by converging genomics. Obes Surg 17: 372–382.[CrossRef][Medline]
Brook M, Tchen CR, Santalucia T, McIlrath J, Arthur JSC, Saklatvala J, and Clark AR (2006) Posttranslational regulation of tristetraprolin subcellular localization and protein stability by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase pathways. Mol Cell Biol 26: 2408–2418.[Abstract/Free Full Text]
Cao H, Kelly MA, Kari F, Dawson HD, Urban JF Jr, Coves S, Roussel AM, and Anderson RA (2007a) Green tea increases anti-inflammatory tristetraprolin and decreases pro-inflammatory tumor necrosis factor mRNA levels in rats. J Inflamm 4: 1.[CrossRef]
Cao H, Polansky MM, and Anderson RA (2007b) Cinnamon extract and polyphenols affect the expression of tristetraprolin, insulin receptor, and glucose transporter 4 in mouse 3T3–L1 adipocytes. Arch Biochem Biophys 459: 214–222.[CrossRef][Medline]
Cao H, Tuttle JS, and Blackshear PJ (2004) Immunological characterization of tristetraprolin as a low abundance, inducible, stable cytosolic protein. J Biol Chem 279: 21489–21499.[Abstract/Free Full Text]
Carballo E, Lai WS, and Blackshear PJ (2000) Evidence that tristetraprolin is a physiological regulator of granulocyte-macrophage colony-stimulating factor messenger RNA deadenylation and stability. Blood 95: 1891–1899.[Abstract/Free Full Text]
Carballo E, Lai WS, and Blackshear PJ (1998) Feedback inhibition of macrophage tumor necrosis factor-{alpha} production by tristetraprolin. Science 281: 1001–1005.[Abstract/Free Full Text]
Carrick DM and Blackshear PJ (2007) Comparative expression of tristetraprolin (TTP) family member transcripts in normal human tissues and cancer cell lines. Arch Biochem Biophys 462: 278–285.[CrossRef][Medline]
Chang X, Fan Y, Karyala S, Schwemberger S, Tomlinson CR, Sartor MA, and Puga A (2007) Ligand-independent regulation of transforming growth factor β1 expression and cell cycle progression by the aryl hydrocarbon receptor. Mol Cell Biol 27: 6127–6139.[Abstract/Free Full Text]
Chen CY, Gherzi R, Ong SE, Chan EL, Raijmakers R, Pruijn GJM, Stoecklin G, Moroni C, Mann M, and Karin M (2001) AU binding proteins recruit the exosome to degrade ARE-containing mRNAs. Cell 107: 451–464.[CrossRef][Medline]
Chrestensen CA, Schroeder MJ, Shabanowitz J, Hunt DF, Pelo JW, Worthington MT, and Sturgill TW (2004) MAPKAP kinase 2 phosphorylates tristetraprolin on in vivo sites including Ser178, a site required for 14-3-3 binding. J Biol Chem 279: 10176–10184.[Abstract/Free Full Text]
Deleault KM, Skinner SJ, and Brooks SA (2008) Tristetraprolin regulates TNF-{alpha} mRNA stability via a proteasome dependent mechanism involving the combined action of the ERK and p38 pathways. Mol Immunol 45: 13–24.[CrossRef][Medline]
Ehlting C, Lai WS, Schaper F, Brenndörfer ED, Matthes RJ, Heinrich PC, Ludwig S, Blackshear PJ, Gaestel M, Häussinger D, et al. (2007) Regulation of suppressor of cytokine signaling 3 (SOCS3) mRNA stability by TNF-{alpha} involves activation of the MKK6/p38MAPK/MK2 cascade. J Immunol 178: 2813–2826.[Abstract/Free Full Text]
Franks TM and Lykke-Andersen J (2007) TTP and BRF proteins nucleate processing body formation to silence mRNAs with AU-rich elements. Genes Dev 21: 719–735.[Abstract/Free Full Text]
Frasca D, Landin AM, Alvarez JP, Blackshear PJ, Riley RL, and Blomberg BB (2007) Tristetraprolin, a negative regulator of mRNA stability, is increased in old B cells and is involved in the degradation of E47 mRNA. J Immunol 179: 918–927.[Abstract/Free Full Text]
Hitti E, Iakovleva T, Brook M, Deppenmeier S, Gruber AD, Radzioch D, Clark AR, Blackshear PJ, Kotlyarov A, and Gaestel M (2006) Mitogen-activated protein kinase-activated protein kinase 2 regulates tumor necrosis factor mRNA stability and translation mainly by altering tristetraprolin expression, stability, and binding to adenine/uridine-rich element. Mol Cell Biol 26: 2399–2407.[Abstract/Free Full Text]
Jalonen U, Lahti A, Korhonen R, Kankaanranta H, and Moilanen E (2005) Inhibition of tristetraprolin expression by dexamethasone in activated macrophages. Biochem Pharmacol 69: 733–740.[CrossRef][Medline]
Jalonen U, Leppänen T, Kankaanranta H, and Moilanen E (2007) Salbutamol increases tristetraprolin expression in macrophages. Life Sci 81: 1651–1658.[CrossRef][Medline]
Jalonen U, Nieminen R, Vuolteenaho K, Kankaanranta H, and Moilanen E (2006) Down-regulation of tristetraprolin expression results in enhanced IL-12 and MIP-2 production and reduced MIP-3{alpha} synthesis in activated macrophages. Mediators Inflamm 2006: 40691.[Medline]
Jing Q, Huang S, Guth S, Zarubin T, Motoyama A, Chen J, Di Padova F, Lin SC, Gram H, and Han J (2005) Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell 120: 623–634.[CrossRef][Medline]
Johnson BA, Stehn JR, Yaffe MB, and Blackwell TK (2002) Cytoplasmic localization of tristetraprolin involves 14-3-3-dependent and -independent mechanisms. J Biol Chem 277: 18029–18036.[Abstract/Free Full Text]
Lai WS and Blackshear PJ (2001) Interactions of CCCH zinc finger proteins with mRNA: tristetraprolin-mediated AU-rich element-dependent mRNA degradation can occur in the absence of a poly(A) tail. J Biol Chem 276: 23144–23154.[Abstract/Free Full Text]
Lai WS, Carballo E, Strum JR, Kennington EA, Phillips RS, and Blackshear PJ (1999) Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA. Mol Cell Biol 19: 4311–4323.[Abstract/Free Full Text]
Laroia G, Cuesta R, Brewer G, and Schneider RJ (1999) Control of mRNA decay by heat shock-ubiquitin-proteasome pathway. Science 284: 499–502.[Abstract/Free Full Text]
Linker K, Pautz A, Fechir M, Hubrich T, Greeve J, and Kleinert H (2005) Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HUR. Nucleic Acids Res 33: 4813–4827.[Abstract/Free Full Text]
Mahtani KR, Brook M, Dean JL, Sully G, Saklatvala J, and Clark AR (2001) Mitogen-activated protein kinase p38 controls the expression and posttranslational modification of tristetraprolin, a regulator of tumor necrosis factor alpha mRNA stability. Mol Cell Biol 21: 6461–6469.[Abstract/Free Full Text]
Ogilvie RL, Abelson M, Hau HH, Vlasova I, Blackshear PJ, and Bohjanen PR (2005) Tristetraprolin down-regulates IL-2 gene expression through AU-rich element-mediated mRNA decay. J Immunol 174: 953–961.[Abstract/Free Full Text]
Phillips K, Kedersha N, Shen L, Blackshear PJ, and Anderson P (2004) Arthritis suppressor genes TIA-1 and TTP dampen the expression of tumor necrosis factor {alpha}, cyclooxygenase 2, and inflammatory arthritis. Proc Natl Acad Sci U S A 101: 2011–2016.[Abstract/Free Full Text]
Rigby WF, Roy K, Collins J, Rigby S, Connolly JE, Bloch DB, and Brooks SA (2005) Structure/function analysis of tristetraprolin (TTP): p38 stress-activated protein kinase and lipopolysaccharide stimulation do not alter TTP function. J Immunol 174: 7883–7893.[Abstract/Free Full Text]
Sauer I, Schaljo B, Vogl C, Gattermeier I, Kolbe T, Müller M, Blackshear PJ, and Kovarik P (2006) Interferons limit inflammatory responses by induction of tristetraprolin. Blood 107: 4790–4797.[Abstract/Free Full Text]
Sawaoka H, Dixon DA, Oates JA, and Boutaud O (2003) Tristetraprolin binds to the 3'-untranslated region of cyclooxygenase-2 mRNA. A polyadenylation variant in a cancer cell line lacks the binding site. J Biol Chem 278: 13928–13935.[Abstract/Free Full Text]
Sitkauskiene B and Sakalauskas R (2005) The role of β2-adrenergic receptors in inflammation and allergy. Curr Drug Targets Inflamm Allergy 4: 157–162.[CrossRef][Medline]
Smoak K and Cidlowski JA (2006) Glucocorticoids regulate tristetraprolin synthesis and posttranscriptionally regulate tumor necrosis factor alpha inflammatory signaling. Mol Cell Biol 26: 9126–9135.[Abstract/Free Full Text]
Stoecklin G and Anderson P (2007) In a tight spot: ARE-mRNAs at processing bodies. Genes Dev 21: 627–631.[Free Full Text]
Stoecklin G, Ming XF, Looser R, and Moroni C (2000) Somatic mRNA turnover mutants implicate tristetraprolin in the interleukin-3 mRNA degradation pathway. Mol Cell Biol 20: 3753–3763.[Abstract/Free Full Text]
Stoecklin G, Stoeckle P, Lu M, Muehlemann O, and Moroni C (2001) Cellular mutants define a common mRNA degradation pathway targeting cytokine AU-rich elements. RNA 7: 1578–1588.[Abstract]
Stoecklin G, Stubbs T, Kedersha N, Wax S, Rigby WFC, Blackwell TK, and Anderson P (2004) MK2-induced tristetraprolin: 14-3-3 complexes prevent stress granule association and ARE-mRNA decay. EMBO J 23: 1313–1324.[CrossRef][Medline]
Taylor GA, Carballo E, Lee DM, Lai WS, Thompson MJ, Patel DD, Schenkman DI, Gilkeson GS, Broxmeyer HE, Haynes BF, et al. (1996) A pathogenetic role for TNF{alpha} in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. Immunity 4: 445–454.[CrossRef][Medline]
Zhang F, Hu Y, Huang P, Toleman CA, Paterson AJ, and Kudlow JE (2007) Proteasome function is regulated by cyclic AMP-dependent protein kinase through phosphorylation of Rpt6. J Biol Chem 282: 22460–22471.[Abstract/Free Full Text]





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