Transcriptional activation and translocation of ancient NOS during immune response

NOS is the key component of the NO system, which plays an indispensable role in many physiologic and immunologic processes; however, the process that underlies the activation of ancient NOSs and their functions remains unclear. Expression of Crassostrea gigas NOS (CgNOS) mRNA in hemocytes was examined after stimulating oysters with LPS and TNF-α. Expression level of CgNOS mRNA was increased significantly, by 2.61-fold (P < 0.05), at 24 h poststimulation. A positive CgNOS signal was detected via immunoprecipitation, and only one protein was detected in oyster hemocytes. Shifting and supershifting bands were observed in EMSAs between the CgNOS promoter and the transcription factors CgNF-κB1 and Cg-signal transducer and activator of transcription (STAT). CgNF-κB1 was detected in the nucleus only at 12 h, whereas CgSTAT was observed in the cytoplasm and nucleus at 12 and 24 h. Expression levels of tyrosine-protein kinase receptor Tie-1, phosphatidylinositide phosphatase SAC2, phosphatidylinositol-4-phosphate 5-kinase type-1α, diacylglycerol kinase θ, LPS-induced TNF-α factor-like protein, cAMP-dependent transcription factor-2, NF-κB1, and STAT6 were significantly elevated in a transcriptome analysis after 12 h of LPS and TNF-α stimulation. An immunoreactive CgNOS signal was observed in both the cell membrane and cytoplasm at 12 h, whereas it was mainly localized to the cytoplasm at 24 h post-LPS and -TNF-α stimulation. These findings revealed that CgNOS could be transcriptionally activated by CgNF-κB1 and CgSTAT via the PI3K-Akt pathway, similar to what occurs for iNOS, but CgNOS translocated to the cytoplasm, similar to neuronal NOS, to modulate downstream signals during an immune defense. These results collectively provide crucial knowledge about the evolution of NOS structure and function.-Jiang, Q., Liu, Z., Zhou, Z., Wang, L., Wang, L., Yue, F., Wang, J., Wang, H., Song, L. Transcriptional activation and translocation of ancient NOS during immune response.