Induction of hypoxia-inducible factor-1α 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 hemorrhagic shock. Substance P (SP) is the major peptide encoded by the immune/hemopoietic modulator gene, preprotachykinin-1 (PPT-I). Differential gene expression using a microarray showed that SP reduced hypoxia-inducible factor-1 alpha (HIF-1 alpha) mRNA levels in BM stroma. Because long-term hypoxia induced the expression of PPT-I in BM mononuclear cells, we used timeline studies to determine whether PPT-I is central to the biologic responses of BM stroma subjected to 30-min hypoxia (pO(2) = 35 mm Hg) followed by reoxygenation. 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 release correlated with down-regulation of HIF-1 alpha to baseline. A direct role for SP in HIF-1 alpha expression was demonstrated as follows: 1) transient 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 of SP. Reoxygenation activated the PPT-1 promoter with concomitant nuclear translocation of HIF-1 alpha that can bind to the respective consensus sequences within the PPT-1 promoter. SP reversed active caspase-3, an indicator of apoptosis and erythropoiesis, to homeostasis level after reoxygenation of 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 caspase-3 in BM stroma subjected to reoxygenation.