PGE2 Promotes BM Hematopoietic Stem Cell Retention Via Stromal Lactate Production­­­­, cAMP and CXCL12/CXCR4 Regulation

The CXCR4/CXCL12 axis is essential for retention and protection from DNA damage of quiescent hematopoietic stem cells (HSC) in their bone marrow (BM) niches. Murine CXCR4⁺ HSC tightly adhere to BM stromal cells which functionally express cell surface CXCL12. Stress induces secretion of CXCL12 by BM stromal cells and its release to the circulation, mediating hematopoietic stem and progenitor cell (HSPC) egress, recruitment and clinical mobilization. Previously, we reported that Prostaglandin E2 (PGE2), highly produced by COX-2⁺ BM αSMA⁺ monocyte/macrophages, upregulates surface CXCR4 expression on enriched human CD34⁺ HSPC and their CXCL12 induced motility via cAMP activation in vitro. PGE2 inhibits intracellular reactive oxygen species (ROS) generation in HSPC and also increases membrane bound CXCL12 expression by BM stromal cells leading to HSC adhesion to their niche supporting cells in vivo, overall contributing to BM stem cell retention. We also found that elevation in cAMP activation promotes CXCL12 secretion from BM stromal cells, and another report has recently shown that lactate signaling via its major receptor HCA-1 inhibits cAMP.

Thus, we hypothesized that the major metabolite lactate, cAMP and PGE2 cross-regulate BM stem cell retention by modulating the CXCR4/CXCL12 axis. We found that both hematopoietic stem cells and BM stromal cells functionally express the lactate receptor HCA-1. Stimulation with PGE2 elevated lactate production by BM stromal cells and stimulation with a HCA-1 receptor agonist, or with lactate, both elevated membrane bound expression of CXCL12 on BM stromal cells. Moreover, since cAMP is elevated by PGE2 signaling whereas lactate signaling was shown to inhibit cAMP, we tested the role of cAMP in CXCL12 expression and secretion by BM stromal cells. We found that in vitro the cAMP enhancer forskolin increased CXCR4 expression by HSPC and in vivo forskolin administration reduced membrane bound CXCL12 levels and elevated CXCL12 secretion as expected. Conversely, in vivo forskolin co-administered with lactate, elevated membrane bound CXCL12 levels and reduced CXCL12 secretion, indicating that lactate limits cAMP elevation and promotes surface CXCL12 expression by BM stromal cells. In accordance,inhibition of cAMP under PGE2 stimulation both in vitro and in vivo, augmented membrane bound CXCL12 expression and inhibited CXCR4 upregulation, mimicking the effects of lactate. We found that PGE2 administration in vivo resulted in reduced CXCR4 expression on primitive BM HSPCs however in vitro PGE2 elevated CXCR4 expression on enriched HSPC. Our results suggest that PGE2 signaling in vivo induces secretion of the metabolite lactate by BM stromal cells, increasing membrane bound CXCL12 expression and reducing expression of CXCR4 on HSPC via cAMP inhibition.

Importantly, repeated in vivo administration of PGE2, lactate or its receptor HCA-1 agonist (once daily for 2 days), all reduced CXCR4 expression and steady state egress of HSPC to the bloodcirculation. Thus, PGE2 via downstream lactate secretion acts as a BM stem cell retaining factor. In accordance, we found that in vivo inhibition of PGE2 production by repeated (once daily for five days) injections of COX-2 inhibitors, such as Meloxicam led to HSPC mobilization. This mobilization was abrogated by co-administration of lactate, suggesting that in vivo inhibition of meloxicam induced CXCL12 secretion and release by lactate prevents HSPC mobilization. We found that in vivo COX-2 inhibition reduced membrane expression of CXCL12 by BM stromal cells and elevated surface CXCR4 expression by BM HSPC in a ROS dependent manner. Moreover, neutralization of CXCR4 or CXCL12 by specific antibodies, or ROS by its scavenger NAC, all blocked meloxicam induced stem and progenitor cell mobilization. These results reveal that COX-2 inhibition increased BM CXCL12 secretion and its release to the blood, upregulated CXCR4 leading to HSPC mobilization in a ROS and CXCL12 dependent manner.

In conclusion, our results reveal that PGE2 enhances both cAMP elevation and lactate secretion by BM stromal cells in the vicinity of hematopoietic stem cells. Lactate acts in an autocrine manner modulating surface CXCL12 expression by BM niche cells and reduced CXCR4 expression by hematopoietic stem cells via inhibition of cAMP, promoting retention and preservation of hematopoietic stem cells in their BM niches.