Circadian Corticosterone Levels Regulate and Integrate Hematopoietic Stem and Progenitor Cell Function and Bone Remodeling Via Notch1 Signaling

Abstract 840

Hematopoietic stem and progenitor cell (HSPC) regulation involves the integration of signals emanating from the nervous system and various bone marrow (BM) cells. Stress signals are produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis via secretion of corticosterone (Cort in mice is the equivalent to cortisol in humans). The major stress hormone, Cort, is secreted in a circadian manner and is known to cause bone loss in supraphysiologic levels. However, whether Cort plays a role in regulation of HSPC and their interactions with the BM microenvironment is currently unknown. Moreover, potentially undesired effects of clinical glucocorticoid administration on HSPC function have not been investigated. We hypothesized that Cort acts in a circadian and dose dependent manner to orchestrate and integrate HSPC function and bone turnover, which are essential for host defense and homeostasis. To determine the roles of circadian Cort oscillations and effects of aberrant Cort regulation on HSPC biology, we utilized CRFR1^-/- (R1KO) mice, which lack corticotropin releasing factor receptor 1, a known HPA axis receptor. These mice lack circadian Cort rhythms and have only residual plasma Cort levels. We found that R1KO mice have higher levels of hematopoietic progenitors and the more primitive SKL cells in their BM and blood compared to WT mice. Cort levels measured in the BM of R1KO mice were continuously low, contrasted to the normal and circadially oscillating levels detected in WT mice. As previously established, interactions of the chemokine SDF-1 (CXCL12), the pivotal regulator of stem cell migration and quiescence, with its major receptor CXCR4, are perturbed by various immune-mediated stress signals. Interestingly, the levels of SDF-1 were constantly high in the BM of R1KO mice with no typical circadian peaks. Furthermore, SKL cell release to the blood, expected to follow BM SDF-1-circadian oscillations, as previously shown by Frenette's group, was also constantly increased. Concomitantly, CXCR4 expression in R1KO SKL cells was reduced and the cells demonstrated higher proliferation rates with superior long-term repopulation capacity over WT transplanted cells. Rescue treatment of R1KO mice attained by adding Cort to their drinking water restored their BM SKL and SDF-1 levels to the basal values of WT mice. Increasing Cort levels continuously in the drinking water of WT mice diminished their BM phenotype, including long-term engraftment potential. In vitro cultures of normal BM cells yielded higher colony formation capacity when stimulated with low Cort levels (10^-8M). Conversely, high Cort levels (10^-5M) inhibited colony formation. Next, we examined microenvironmental components with HSPC support potential. R1KO mice had more primitive stromal cells (CD45^-/CD11b^-/CD29⁺/Sca-1⁺) in their BM. Expression of osteocalcin and osterix, genes characterizing mature osteoblasts, was reduced. Osteoclast maturation was also attenuated, as indicated by increased Gr-1^-/CD11b^-/c-Kit⁺ monocyte precursors and reduced transcription of the genes RANK and cathepsin K, both associated with mature osteoclasts and bone resorption. R1KO mice had fewer active osteoclasts in their bones and lower bone turnover rates as indicated by calcein/alizarin bone labeling. In addition, μCT analyses demonstrated a dramatic reduction in femoral trabecules and bone volume. Looking for mediating mechanisms, we found that R1KO BM mononuclear cells highly express Notch1 and its target gene Hes1, known to be involved in regulation of stem cell self renewal. Both genes reverted back to normal expression levels after adding Cort to the drinking water. The signaling Notch1 intracellular domain (NICD), a product of Notch1 cleavage by γ-secratase, was also upregulated in R1KO BM hematopoietic and stromal precursors. Finally, inhibition of Notch1 activation by adding the γ-secratase inhibitor DAPT to BM cultures prevented the stimulating effect of low Cort on colony formation, suggesting that this effect is mediated by Notch1 signaling. Taken together, our study suggests that abolishment of circadian Cort production coupled with continuous low Cort levels increases HSPC proliferation directly and indirectly via Notch1 signaling, while impairing bone maintenance and structure. We propose Cort oscillations and levels to be critical for integrating balanced bone turnover and HSPC function.