Induction of hypoxia-inducible factor-1 alpha and activation of caspase-3 in hypoxia-reoxygenated bone marrow stroma is negatively regulated by the delayed production of substance P

The bone marrow (BM), which is the major site of immune cell development in the adult, responds to different stimuli such as inflammation and hemorrha gic shock. Substance P (SP) is the major peptide encoded by the immune/hemo poietic modulator gene, preprotachykinin-1 (PPT-I). Differential gene expre ssion using a microarray showed that SP reduced hypoxia-inducible factor-1 alpha (HIF-1 alpha) mRNA levels in BM stroma. Because long-term hypoxia ind uced the expression of PPT-I in BM mononuclear cells, we used timeline stud ies to determine whether PPT-I is central to the biologic responses of BM s troma subjected to 30-min hypoxia (pO(2) = 35 mm Hg) followed by reoxygenat ion. HIF-1 alpha mRNA and protein levels were increased up to 12 h. At this time, beta -PPT-1 mRNA was detected with the release of SP at 16 h. SP rel ease correlated with down-regulation of HIF-1 alpha to baseline. A direct r ole for SP in HIF-1 alpha expression was demonstrated as follows: 1) transi ent knockout of beta -PPT-I showed an increase in HIF-1 alpha expression up to 48 h of reoxygenation; and 2) HIF-1 alpha expression remained baseline during reoxygenation when stroma was subjected to hypoxia in the presence o f SP. Reoxygenation activated the PPT-1 promoter with concomitant nuclear t ranslocation of HIF-1 alpha that can bind to the respective consensus seque nces within the PPT-1 promoter. SP reversed active caspase-3, an indicator of apoptosis and erythropoiesis, to homeostasis level after reoxygenation o f hypoxic stroma. The results show that during reoxgenation the PPT-1 gene acts as a negative regulator on the expression of HIF-1 alpha and active ca spase-3 in BM stroma subjected to reoxygenation.