Dissection of a Hypoxia-induced, Nitric Oxide-mediated Signaling Cascade

Pascale F. Dijkers and Patrick H. O’Farrell

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143-2200

Monitoring Editor: Thomas D. Fox

Befitting oxygen’s keyrole in life’s processes, hypoxia engages multiple signalingsystems that evoke pervasive adaptations. Using surrogate geneticsin a powerful biological model, we dissect a poorly understoodhypoxia-sensing and signal transduction system. Hypoxia triggersNO-dependent accumulation of cyclic GMP and translocation ofcytoplasmic GFP-Relish (an NF ${kappa}$ B/Rel transcription factor) tothe nucleus in Drosophila S2 cells. An enzyme capable of eliminatingNO interrupted signaling specifically when it was targeted tothe mitochondria, arguing for a mitochondrial NO signal. Longpretreatment with an inhibitor of nitric oxide synthase (NOS),L-NAME, blocked signaling. However, addition shortly beforehypoxia was without affect, suggesting that signaling is supportedby the prior action of NOS and is independent of NOS actionduring hypoxia. We implicated the glutathione adduct, GSNO,as a signaling mediator by showing that overexpression of thecytoplasmic enzyme catalyzing its destruction, GSNOR, blockssignaling while knockdown of this activity caused reporter translocationin the absence of hypoxia. In downstream steps, cGMP accumulated,and calcium-dependent signaling was subsequently activated viacGMP-dependent channels. These findings reveal the use of unconventionalsteps in an NO pathway involved in sensing hypoxia and initiatingsignaling.

Address correspondence to: Patrick H. O’Farrell (ofarrell{at}cgl.ucsf.edu)