27-Hydroxycholesterol Induces Hematopoietic Stem Cell Mobilization and Extramedullary Hematopoiesis Mediated By Estrogen Receptor Alpha

In adults, hematopoietic stem cells (HSCs) reside primarily in the bone marrow and their number is tightly regulated under steady state conditions. However, acute demands on the hematopoietic system promote HSC division and mobilization to extramedullary tissues such as the spleen, to increase production of blood cells. While the mechanisms that regulate HSC numbers and residence in the bone marrow under steady-state conditions have been extensively characterized, the mechanisms that activate HSCs in response to acute hematopoietic demands are less well understood. We have previously reported that extramedullary hematopoiesis (EMH) is induced during pregnancy when maternal blood volume expands rapidly. This requires HSC division and mobilization, processes that depend upon estrogen receptor α (ERα) in HSCs. Signaling through this nuclear hormone receptor can be triggered by sex hormones, such as 17β-estradiol (E2), as well as 27-hydroxycholesterol (27HC), which is the first identified endogenous ER ligand other than sex hormones. However, it has been unclear whether 27HC has a physiological role that is effected through ERα signaling in normal mice.

Here we show that treatment of mice with E2, which increases during pregnancy, induced HSC division in the bone marrow but did not increase HSC number in the spleen, indicating that E2 treatment does not induce HSC mobilization. In contrast, treatment with the alternative endogenous ERα ligand, 27HC, increased HSC number in the spleen and induced EMH, but not HSC division in the bone marrow, indicating a role in inducing HSC mobilization. The effect of 27HC on HSC mobilization was nullified by deletion of Esr1 (the gene that encodes ERα) in hematopoietic cells using Vav1-icre ; Esr1^fl/fl mice, indicating that 27HC-induced HSC mobilization is dependent on ERα.

To test whether 27HC acts directly on HSCs, we competitively transplanted Vav1-icre ; Esr1^fl/fl donor bone marrow cells along with wild-type competitor bone marrow cells and treated the recipient mice with 27HC four months after the transplantation. 27HC treated mice had significantly lower frequencies of donor-derived (Esr1- deficient) HSCs in the spleen as compared to vehicle-treated mice. This indicates that Esr1- deficient HSCs were at a disadvantage compared to wild-type HSCs in the same mice for mobilization in response to 27HC. ERα thus acts cell-autonomously within HSCs to promote mobilization in response to 27HC.

27HC is generated directly from cholesterol by the sterol hydroxylase, Cyp27a1, and plasma 27HC levels correlate with total cholesterol levels. It has been reported that mice with defects in cholesterol efflux exhibit increased mobilization of hematopoietic stem and progenitor cells (HSPCs) associated with increased serum granulocyte colony-stimulating factor (G-CSF) levels. However, we observed that G-CSF deficiency using Csf3^-/- mice did not affect the magnitude of the increase in mobilized HSCs in response to 27HC treatment. Together, 27HC and G-CSF co-treatment additively increased the numbers of colony-forming HSPCs in the blood. Therefore, 27HC and G-CSF likely act through distinct mechanisms.

During pregnancy, 27HC levels increased in HSPCs as a result of Cyp27a1. Cyp27a1 -deficient mice had significantly reduced 27HC levels but, under steady-state conditions, Cyp27a1 deficiency did not affect the numbers of HSCs and hematopoietic cells in both bone marrow and spleen. However, during pregnancy, Cyp27a1 -deficient mice had significantly reduced HSC mobilization and EMH, while the increased rate of HSC division and hematopoiesis in the bone marrow was not affected. In contrast, Cyp27a1 deficiency did not affect HSC mobilization and EMH in response to blood loss or G-CSF treatment. Distinct hematopoietic stresses thus induce EMH through different mechanisms. Taken together, these results indicate that two different endogenous ERα ligands, E2 and 27HC, work together to promote EMH during pregnancy, revealing a collaboration of hormone and lipid signaling as well as a physiological function for 27HC in normal mice.