Dipeptidyl peptidase 4 (DPP4) truncated factors manifest distinct regulatory functions compared to their full length forms and DPP4 is altered by, and modulates stem cell response to, extra physiologic oxygen shock/stress (EPHOSS)

Hematopoietic stem cells (HSC) reside in hypoxic niches (∼1-4% O2), yet, HSC studies are typically performed using cells isolated in ambient air (∼20% O2). Inhibiting ambient air exposure/harm of cells, which we termed Extra Physiologic Oxygen Shock/Stress (EPHOSS), by collecting/processing human cord blood (hCB) or mouse bone marrow (mBM) stem cells in hypoxia (3% O2), enhances recovery of phenotypic/functional long-term repopulating HSC (LT-HSC) and is mechanistically linked, in part, to the mitochondrial permeability transition pore (MPTP), Reactive Oxygen Species (ROS) and cyclophilin D. We hypothesized that Dipeptidyl Peptidase 4 (DPP4), an enzyme that N terminally cleaves and modifies the function of select proteins leading to alterations in homing/engraftment of HSC, may be altered by EPHOSS and involved in EPHOSS effects on HSC. Proteomic and bioinformatic analysis identified 1) many unexpected intracellular/secreted proteins with DPP4 truncation (T) sites and 2) specific, as well as overlapping, modifications in phosphorylated and differential protein signaling of T-cytokines (GM-CSF and IL-3) compared to their full length (FL) forms leading to diversified regulation of signaling/function in normal and leukemic cells. T-GM-CSF and T-IL-3 had enhanced receptor binding compared to their FL forms with significant, and reciprocal, blunting of functional activity of both factors in vitro and in vivo. To investigate effects of DPP4 on EPHOSS, and vice versa, mBM was harvested (air/ hypoxia) with a DPP4 inhibitor (DPA), or from DPP4 K/O mice. This resulted in significant increases in the number of phenotypic LT-HSC (p=.017) in air, suggesting that DPP4 inhibition blunts EPHOSS mediated loss of phenotypic LT-HSC. Also, the percentage of DPP4+ cells was increased in primitive fractions of mBM or hCB, (LSK ∼15%, LSKCD150 40%, CD34+CD38- ∼10%, CD34+CD38-CD45RA-CD90+CD49F+ ∼40% p=.007) and further enhanced 15- 20% when cells are isolated in hypoxia (p=.005). Unexpectedly, LT-HSC ROS levels (mitochondrial/total) were not diminished in DPA or DPP4 K/O groups harvested in air despite the increase in phenotypic LT-HSC over air harvest alone, suggesting a non ROS/MPTP mechanism. In conclusion, DPP4 expression/activity serves heretofore unknown roles in the regulation/signaling of multiple protein types as well as cellular responses to EPHOSS and hematopoiesis.