Characterization of the pro-inflammatory signaling induced by protein acetylation in microglia

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Abstract

Protein acetylation regulates the extent of inflammatory responses and disturbances in protein acetylation have been proposed to play an important role in inflammatory and neurodegenerative diseases. We have recently observed that histone deacetylase inhibitors, such as trichostatin A (TSA) and SAHA, strongly potentiate the LPS induced inflammatory response in several rat and mouse inflammatory models. Our aim here was to characterise pro-inflammatory signaling mediated via increased protein acetylation and protein phosphorylation in microglial N9 cells. First we observed that TSA induced pro-inflammatory response was independent of the different Toll-like receptors activated, since LPS, flagellin and unmethylated CpG oligonucleotides, equally potentiated IL-6 secretion from N9 microglia. Next we compared the protein acetylation induced potentiation to that induced by okadaic acid, a well-known inducer of pro-inflammatory responses. The time scale of the IL-6 responses showed that the effects of okadaic acid were clearly early-response effects appearing as soon as 6 h after exposure, whereas TSA evoked a significant inhibition in IL-6 secretion up to 12 h but after that it induced an exponential increase in cytokine and nitric oxide production up to 24 h. It seems that okadaic acid induces an early moderate response and TSA a late but exponential potentiation of microglial inflammatory responses. The pro-inflammatory responses of TSA and okadaic acid were both dependent on NF-κB signaling but independent on the DNA-binding capacity of nuclear NF-κB complexes. Interestingly, we observed that the transactivation of the NF-κB-Luc reporter gene was clearly activated during TSA induced pro-inflammatory potentiation. Our studies imply that the potentiation of the inflammatory response by increased acetylation is due to the enhancement of transactivation of NF-κB driven inflammatory genes. Our studies on signaling pathways revealed that PI3K inhibitors LY294002 and Wortmannin blocked the TSA induced pro-inflammatory response but surprisingly did not affect the okadaic acid induced response. Furthermore, LY294002 did not inhibit DNA-binding activity of NF-κB but still inhibited NF-κB-Luc reporter gene transactivation. These results indicate that PI 3-kinase regulates the transactivation efficiency of NF-κB-dependent transcription rather than transduction of NF-κB signaling.

Introduction

Disturbances in the regulation of protein acetylation have been proposed to play an important role in the pathogenesis of several diseases, such as cancer (Marks et al., 2004, Hess-Stumpp, 2005), diabetes (Gray and de Meyts, 2005), neurodegenerative disorders (Mattson, 2003, Langley et al., 2005), as well as being involved in several inflammatory diseases (Chung et al., 2003, Rahman et al., 2004, Adcock et al., 2005). Treatment with histone deacetylase inhibitors have shown promising therapeutic results in some nervous system disorders, such as gliomas (Eyupoglu et al., 2005), polyglutamine diseases (Taylor and Fischbeck, 2002, Hockly et al., 2003), and several other neurodegenerative diseases (see Langley et al., 2005).

Inflammation is involved in several neurodegenerative diseases (Gonzalez-Scarano and Baltuch, 1999, Minghetti, 2005). Recent publications have highlighted how protein acetylation prominently modulates the inflammatory responses (Rahman et al., 2004, Barnes et al., 2005, Ito et al., 2005). We have recently observed that histone deacetylase inhibitors, such as trichostatin A and SAHA, strongly potentiate the LPS-induced inflammatory response in several rat and mouse inflammatory models in cell and slice cultures (Suuronen et al., 2003). The inflammatory responses are also modified by MAPK signaling (Shanley et al., 2001, Avdi et al., 2002). Okadaic acid, an inhibitor of protein phosphatases PP2A and PP1, increases protein phosphorylation and simultaneously potentiates the inflammatory responses (Tebo and Hamilton, 1994, Shanley et al., 2001).

In the present study, we compared the potentiation of inflammatory responses induced via the “hyperacetylation” or “hyperphosphorylation”, i.e. either TSA induced or okadaic acid induced pro-inflammatory responses. Our results show that there are diverse potentiation mechanisms behind the pro-inflammatory responses mediated via acetylation and phosphorylation signaling pathways. The okadaic acid induced pro-inflammatory reaction was shown to be an early-response event whereas the TSA induced response was clearly a late event. However, both responses could be inhibited by dexamethasone and the NF-κB inhibitor. Surprisingly, the PI3K inhibitor, LY294002, blocked only the TSA induced pro-inflammatory response but did not affect the okadaic acid induced response. Our results also indicate that TSA potentiates the LPS-induced inflammatory response in N9 microglial cells by increasing the transactivation efficiency of NF-κB complexes.

Section snippets

Chemicals

Lipopolysaccharide (LPS) used in all experiments was from E. coli 055:B5 lyophilized powder (L 6529 from Sigma). To compare the LPS purity effect, the Ultra Pure LPS purified from E. coli 0111:B4 strain (InvivoGen, San Diego, USA) was used. Pam3CSK4 synthetic lipoprotein, SLTA (lipoteichoid acid from Staphylococcus aureus), zymosan, flagellin and ODN1826 were from InvivoGen (San Diego, USA). Okadaic acid, LY294002, Wortmannin, H-7 dihydrochloride, H-89 dihydrochloride, herbimycin A, JAK3

TSA induced pro-inflammatory response is independent on different Toll-like receptors activated

LPS activates the inflammatory signaling through the Toll-like receptors (Kirschning and Bauer, 2001). First we analyzed whether TSA could enhance the inflammatory response mediated by different Toll-like receptors. It is known that commercially available LPS preparations contain other stimulating compounds, such as flagellin as impurities. We treated N9 microglia with our ordinary LPS (Sigma) and Ultra-pure LPS (InvivoGen). Fig. 1 shows that ordinary LPS induced a higher IL-6 cytokine response

Discussion

The significance of protein acetylation in signal processing has been compared to that of protein phosphorylation (Kouzarides, 2000) and both of these processes are also involved in the modification of inflammatory responses. Okadaic acid, an inhibitor of protein phosphatases PP2A and PP1, enhances protein phosphorylation in cells and simultaneously potentiates the inflammatory responses (Tebo and Hamilton, 1994, Shanley et al., 2001). The potentiation of inflammatory reactions is mediated via

Acknowledgements

This study was supported by grants from the Academy of Finland (A.S.), University Hospital of Kuopio (EVO 5510) and the University of Kuopio (A.S., T.S. and J.H.), Finland. The authors thank Dr. Ewen MacDonald for checking the language of the manuscript and Mrs. Airi Boman for technical assistance.

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    Both authors contributed equally to this article.

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