NF-κB/Rel, not STAT5, regulates nitric oxide synthase transcription in Apostichopus japonicus

Nitric oxide (NO) is an important signaling molecular in the immune system of all vertebrates and invertebrates for pathologic and physiologic process, and it is largely produced by inducible nitric oxide synthase (iNOS). To uncover key mechanisms regulating NOS expression in sea cucumber Apostichopus japonicus, we amplified a fragment of the NOS promoter by genome walking approach and characterized putative transcription factor binding motifs using luciferase assay. Transient transfection of EPC cells using 5′-deletion constructs linked to luciferase reporter revealed that the region −614/+39 contributed importantly to expression of the AjNOS gene, and the −614 bp of the 5′-flanking region of the AjNOS gene responded well to LPS. Analysis of the functional promoter region revealed the presence of two potential NF-κB (−375 bp to −366 bp, −76 bp to −67 bp) and three STAT binding sites (−284 bp to −276 bp, −95 bp to 87 bp, −81 bp to −73 bp). When luciferase reporter vector and expression vector co-transfected revealed that NF-κB/Rel, but not STAT5, activate the AjNOS promoter fragment. Furthermore, two truncated reporter vectors co-transfected with vector expressing NF-κB/Rel revealed that the first NF-κB binding site (−375 bp to −366 bp) was essential for the ability of this promoter to induce AjNOS transcription. In addition, blocking the AjRel by SN50 (NF-κB inhibitory peptide) depressed the AjNOS expression and NO production both in vivo and in vitro, respectively, revealing that AjRel might directly modulate AjNOS. All our findings confirmed that NF-κB dependent mechanisms regulating expression of AjNOS and suggested a means of linking NO production to the immune response.