Elsevier

Nitric Oxide

Volume 8, Issue 2, March 2003, Pages 95-102
Nitric Oxide

Comparison of the signal transduction pathways for the induction of gene expression of nitric oxide synthase-2 in response to two different stimuli

https://doi.org/10.1016/S1089-8603(02)00164-7Get rights and content

Abstract

Human optic nerve astrocytes induce nitric oxide synthase-2 (NOS-2) in vitro in response to cytokines (interferon-γ/interleukin-1β) and elevated hydrostatic pressure. Using relatively specific inhibitors, we have compared induction of NOS-2 in response to these two stimuli to determine whether the same or different signal transduction pathways participate in the responses. Using SN50 and CAGE, which inhibit the NFκB pathway, the induction of NOS-2 in response to both cytokines and elevated hydrostatic pressure was blocked. Using SB202190 and SB203580, which inhibit p38 mitogen-activated protein kinase, only the response to cytokines was blocked. In contrast, when inhibitors of epidermal growth factor receptor tyrosine kinase AG 82 and AG 18 were used, the induction of NOS-2 in response to pressure, but not in response to cytokines, was blocked. Signal transduction pathways presumably regulate the synthesis of NOS-2 through downstream events that induce transcription of the NOS-2 gene. Our data suggest that activation of different sites in the promoter region of the NOS-2 gene is needed for these different stimuli to induce NOS-2.

Section snippets

Human optic nerve head astrocyte cultures

Twelve human eyes from donors (aged 22–49 years) with no history of eye disease were obtained within 24 h after death from eye banks throughout the United States. The eyes were stored at 4 °C and processed within 8 h of enucleation. Primary lamina cribrosa astrocyte cultures were derived as previously described [18]. The posterior pole of the eye was dissected and the optic nerve head was freed from sclera and other neighboring tissues. The optic nerve head was sliced sagittally and under a

Results

The immunoblots in Fig. 1, Fig. 2, Fig. 3, all demonstrate that the combination of cytokines, IFNγ/IL-1β, causes a marked increase in the cellular content of NOS-2 protein in human optic nerve astrocytes in vitro. Similarly, when these astrocytes are exposed to elevated hydrostatic pressure, there are also marked increases in NOS-2 protein.

The immunoblot in Fig. 1A also shows the effects of the inhibitor of NFκB, SN-50, on the appearance of NOS-2 protein in human optic nerve astrocytes treated

Discussion

Our results confirm that several independent signal transduction pathways participate in the induction of the gene expression of NOS-2. We interpret our results to indicate that, in response to specific stimuli, downstream products of the NFκB pathway, the p38 MAP kinase pathway, and the EGFR tyrosine kinase pathway contribute to activation of the promotor region of the NOS-2 gene. We can distinguish at least one signal transduction pathway that is common for two different stimuli and two

Acknowledgements

This work was supported in part by a grant from NIH (EY 12017).

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