Role of hemeoxygenase-1 in the inhibition of iNOS expression by nonsteroidal anti-inflammatory drugs in C6 glioma cells

Long term use of nonsteroidal anti-inflammatory drugs (NSAIDs) decreases the risk of the neurodegenerative disease of Alzheimer. Interestingly, neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease, have been associated with the expression of inducible nitric oxide synthase (iNOS) in glial cells. Furthermore, when iNOS expression is ablated in the Parkinson's disease model, neuronal cell death is reduced significantly. Since iNOS expression is harmful to neurons, it is important to understand what regulates the expression of iNOS. Inflammatory cytokines induce iNOS expression through activation of many regulatory kinases. These regulatory kinases activate transcription factors that are involved in transcription of the gene responsible for iNOS, called Nos2, in rat glial cells. Previous studies found that while NSAIDs inhibit the expression of iNOS, they induce hemeoxygenase-1 expression in rat glial cells. Hemeoxygenase-1 (HO-1) degrades heme into iron, bilirubin, and carbon monoxide. Carbon monoxide has been shown to decrease iNOS expression in glial cells. While previous studies have shown that NSAIDs decrease iNOS expression, the regulatory pathways of iNOS expression involved in this response in glial cells have not been studied previously. The purpose of this work was to investigate the mechanism by which NSAIDs suppress iNOS expression. The approach was to study the specific molecular mechanisms by which indomethacin and ibuprofen might modulate iNOS expression in vitro. The regulatory pathways for iNOS expression may provide targets for therapies to be used in prevention of neurodegenerative diseases.

This work confirmed that indomethacin and ibuprofen reduce the expression of iNOS at the protein level in glial cells. This study tested the hypothesis that HO-1 mediates the inhibition of iNOS expression by NSAIDs. Glial cells were treated with either indomethacin or ibuprofen in the presence and absence of cytokines to induce iNOS expression. Aim 1 studied the expression of iNOS protein and nitrite production by indomethacin or ibuprofen in cytokine stimulated C6 glial cells. Ibuprofen or indomethacin reduces the promoter activity of Nos2 in the rat C6 glial cells. Aim 2 evaluated the expression of HO-1 protein by indomethacin or ibuprofen in cytokine stimulated C6 glial cells. Indomethacin or ibuprofen induced HO-1 protein expression. Aim 3 examined whether the inhibition of iNOS expression by indomethacin or ibuprofen was mediated through the hemeoxygenase-1 end-products iron or carbon monoxide. An iron chelator reversed the inhibition of iNOS and Nos2 expression by indomethacin, but not by ibuprofen. Scavenging extracellular carbon monoxide did not affect the inhibition of iNOS expression in C6 cells. Aim 4 evaluated the effect of HO-1 protein expression on iNOS expression in indomethacin or ibuprofen treated C6 glial cells. Using small interefering ribonucleotide (siRNA) to suppress HO-1 protein expression, it was shown that indomethacin, but not ibuprofen, inhibition of iNOS was reversed in C6 cells as monitored by iNOS expression and Nos2 promoter activity. Thus, HO-1 protein is involved in the inhibition of of iNOS expression by indomethacin. Since ibuprofen did not inhibit Nos2 expression through HO-1, it led us to explore another pathway as a possible mechanism of action for ibuprofen inhibition of Nos2 expression. In contrast to indomethacin, we showed that ibuprofen inhibited NF-êB activation, a transcription factor critical for Nos2 expression. Since iNOS expression is associated with neurodegenerative diseases, understanding the targets of these two NSAIDs may be useful for future studies for therapies that prevent or treat neurodegenerative diseases.