p38α Activates Purine Metabolism to Initiate Hematopoietic Stem Cell Cycling

Hematopoietic stem cells (HSCs) maintain quiescence by activating specific metabolic pathways, including glycolysis. However, how stress hematopoiesis, including bone marrow transplantation (BMT), induces metabolic changes in HSCs remains unclear. Here, we report a critical role for the p38MAPK family isoform p38α in initiating HSC proliferation during stress hematopoiesis in mouse. First, we identified p38α as the major p38MAPK isozyme highly expressed in HSCs and we also performed conditional knockout of p38α in mice. This mouse showed no overt difference relative to wild type mouse. However, treatment of p38α-deficient mice with 5-FU exhibited defective recovery of hematopoiesis, and the survival rate were lower in p38α-deficient mice than wild-type mice (42.9%, N=7, p38α-deficient mice, vs 100%, wild-type mice, N=6, p=0.03) and loss of p38α in HSCs showed a defective transplantation capacity in primary and secondary transplantation. To gain further insight into p38MAPK function during hematological stress, we evaluated the time course of p38MAPK activation in stressful contexts by intracellular flow cytometry. We found that p38MAPK was immediately phosphorylated in HSCs after hematological stress and returned to normal in a short period, suggesting that p38α functions rapidly after hematological stresses to activate downstream events. To identify events downstream of p38α after hematological stress, we initially evaluated mechanisms such as homing, apoptosis, and ROS generation immediately after BMT. However, defects seen in p38α-deficient HSCs after hematological stress could not be explained by these mechanisms. Therefore we next focused on cell cycle. In CFSE assay, p38α loss resulted in defective recovery from hematological stress and a delay in initiating cycling of HSCs. In addition, p38α-deficient HSCs showed lower BrdU incorporation in vivo (p=0.045) and EdU incorporation in vitro (p=0.003). Transcriptome analysis of transplanted wild-type or p38α-deficient HSCs suggested that p38α-deficient HSCs showed lower enrichment of genes related to HSC-related markers and proliferation. Taken together, loss of p38α resulted in defective HSC cell cycle progression in stressed settings such as transplantation. Given that altered metabolic activities can change cell cycle status, we asked whether p38α regulation of a particular metabolic pathway could initiate HSC cycling under stress conditions. To do so, we collected p38α-deficient or wild-type LSK cells either at steady state or after BMT and extracted metabolites for metabolome analysis using mass spectrometry. Among metabolites surveyed, we focused on changes in glycine and aspartic acid, which are required for purine biosynthesis. Levels of both increased in p38α-deficient as compared with wild-type LSK cells after BMT. Also, mice transplanted with p38α-deficient compared with wild-type LSK cells showed lower levels of allantoin, a product of purine catabolism. These findings suggest that p38α loss suppresses purine metabolism during stress hematopoiesis. Next, we evaluated mRNAs encoding key enzymes functioning in purine metabolism by qPCR. Expression of both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), and guanosine monophosphate synthetase (GMPS) was significantly decreased in p38α-deficient HSCs relative to wild-type HSCs on day 1 after BMT. To assess how changes in purine metabolism could affect the HSC response to stress, we treated HSCs with cytokines in the presence or absence of mycophenolic acid (MPA), an IMPDH2 inhibitor. MPA treatment significantly suppressed colony formation capacity of HSCs in a dose-dependent manner. Also, EdU incorporation into HSCs was reduced by MPA dose-dependently. Finally, isolated HSCs were cultured with or without MPA for 3 days and then transplanted into recipients along with competitor cells. PB chimerism was dose-dependently decreased in recipients of MPA-treated cells. These findings suggest that purine metabolism directly maintains proliferation capacity of HSCs in stress conditions. In summary, expression of purine-synthesizing enzymes decreased in p38α-deficient HSCs after transplantation, an activity correlated with defective cell cycle progression in vitro and in vivo. Overall, this is the first report of p38α-regulated changes in purine metabolism associated with HSC stress and cell cycle initiation.