J A S O N M . B U T L E R & S H A H I N R A F I I

Stem and progenitor cells of the blood system reside in the safe haven of their immediate surroundings — a micro­

environment that contains a complex net­work of ‘niche’ cells. Interaction with niche cells prevents unauthorized egress of these haematopoietic stem and progenitor cells into the circulation, whereas their mature counter­parts are readily launched there. How the various niche cells decide to relocate some cells but not others, to meet the demand for blood cells under normal and stressful conditions, is not fully understood. In a paper published on Nature’s website today, Hoggatt et al.1 provide one notable clue.

The authors show that interfering with the interaction between the messenger lipid molecule prostaglandin E2 (PGE2) and its G­protein­coupled receptor EP4 stimulates the mobilization of haemato poietic stem cells and haematopoietic progenitor cells (HSCs/HPCs) from the bone marrow to the circula­tion. This effect was evident following deletion of the EP4 gene in mice; using EP4 antagonists; and after lowering PGE2 production by treat­ing mice with non­steroidal anti­inflammatory drugs (NSAIDs), including aspirin, ibuprofen and meloxicam.

Individually, NSAIDs and EP4 antagonists were marginally effective in forcing HSC/HPC egress, but their effect was consider­ably improved when they were used together with other mobilizing agents — G­CSF and a CXCR4 antagonist. Most importantly, the authors report such enhanced effects of NSAIDs, alone or in combination with G­CSF in non­human primates and human subjects. This sets the stage for clinical studies to inter­rogate the potential of PGE2 for optimizing HSC/HPC mobilization protocols as part of therapies involving myeloablation, which involves the destruction of diseased blood cells followed by induced production of new blood cells from the stem­cell pool.

The mechanism by which PGE2–EP4 signal­ling regulates blood­cell trafficking is complex

and involves balancing HSC/HPC mobilization both directly, through cell­specific signalling, and indirectly, by activating stromal cells of the niche. Together with earlier work2,3 show­ing that, by contrast, short­term exposure of haematopoietic cells to PGE2 increases their migration and engraftment, these findings qualify the PGE2–EP4 signalling hub as a gate­keeper of the haematopoietic microenviron­ment (Fig. 1).

Uncovering the molecular pathways involved in the efficient mobilization and engraftment of HSCs/HPCs has major impli­cations for the treatment of various cancers with myeloablative therapies4,5. High blood levels of a range of factors — including G­CSF, CXCR4 antagonists, numerous CC and CXC chemokines4, VEGF­A and PlGF (ref. 5) — were known to stimulate the mobilization of HSCs/HPCs localized to specific haemato­poietic niches. However, the identity of factors that could modulate both mobilization

and engraftment of these cells were not known. PGE2 is one such factor, and, by interact­

ing with EP4, it regulates bidirectional traf­ficking of HSCs/HPCs. Indeed, the effects of PGE2 could be biphasic. Brief stimulation of HSCs/HPCs with this molecule augments engraftment and reconstitution of blood cells by accelerating haematopoiesis6. This function of PGE2 is mediated by activation of the Wnt signalling pathway to control proliferation and programmed death of HSCs/HPCs7. Non­stop stimulation, however, may inhibit HPC expansion and recovery of haematopoiesis8, as well as prevent the trafficking of HSCs/HPCs, partly by CXCR4 downregulation. Therefore, capitalizing on the potential of the PGE2–EP4 signalling pathway to augment haematopoiesis may require a two­pronged approach in which EP4 is initially activated to trigger migration and engraftment of HSCs/HPCs, and then inhibited to encourage HPC expansion and haematopoietic reconstitution.

An intriguing question is exactly how NSAIDs modulate trafficking of the HSCs/HPCs within their microenvironment. EP4 is expressed by bone cells called osteoclasts and osteoblasts; by endothelial cells, which line the blood vessels; by subsets of haemato poietic cells; and most probably by peri vascular cells, which occur in peripheral blood ves­sels. Hoggatt et al. show that treatment with NSAIDs results in thinning and loss of the cells of the bone lineage, diminishing their capa city to retain HSCs/HPCs within the bone marrow. Moreover, they find that NSAIDs decrease bone­derived osteopontin, a molecule that

S T E M C E L L S

Painkillers caught in blood-cell trafficking Haematopoietic stem and progenitor cells move from the bone marrow into the circulation to replenish normal blood-cell levels. Inhibiting a prostaglandin-mediated signalling pathway may promote this process.

Steady state

Bone cells

Stem-cell niche

HSCHPC

Endothelial cell

Blood vessel

a Mobilizationb

Perivascularstromal cell

EP4

PGE2

PGE2

G-CSF,CXCR4

antagonists

NSAIDs

Figure 1 | NSAIDs are gatekeepers of the haematopoietic microenvironment. a, Prostaglandin E2 (PGE2) can bind to its receptor EP4 on the surface of haematopoietic stem and progenitor cells (HSCs/HPCs), as well as on the surface of niche cells within the HSC/HPC microenvironment, which include perivascular stromal cells, endothelial cells that line blood vessels and cells of the bone lineage. The signalling cascade initiated by PGE2–EP4 interaction inhibits mobilization of HSCs/HPCs into the circulation. b, Hoggatt et al.1 show that NSAIDs, by inhibiting PGE2–EP4 interactions and in part by thinning of the bone cells, can synergize with the mobilizing agents G­CSF and CXCR4 antagonists. This promotes egress of HSCs/HPCs into the circulation by weakening their tethering to the niche cells.

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regulates mobilization of haematopoietic cells. These painkillers might therefore primarily target bone cells to decrease the tethering of HSCs to stromal cells.

Nonetheless, PGE2 might also induce mobilization by directly affecting perivascu­lar and endothelial cells. Notably, sinusoidal endothelial cells line small blood vessels called sinusoids. The sinusoids, which form branch­ing structures in much of the bone­marrow vasculature, are not mere passive conduits for delivering oxygen and nutrients, but rather represent specialized blood vessels that form an instructive ‘vascular niche’ in the haemato­poietic organs. Normally, activated endothelial cells produce defined blood­related growth factors (angio crine factors) to balance the self­renewal and differentiation of HSCs9–16. One angiocrine factor is PGE2, through which endothelial cells could modulate HSC/HPC trafficking. Indeed, during recovery from myelo ablative therapy, the induction of angio crine factors — including PGE2 — in endo thelial cells regulates trafficking and regeneration of HSC/HPCs11–14. Unravelling the mechanism by which PGE2 polices the trafficking of HSCs/HPCs within the complex network of intertwined niche cells will require the deletion of EP4 in specific niche cells, including endothelial cells, perivascular

cells and cells of the bone lineage. Notwithstanding the potential benefits of

NSAIDs, these drugs may act as a double­edged sword during haematopoietic recovery. Because activation of PGE2–EP4 signalling promotes blood­vessel formation, NSAIDs might interfere with the regeneration of endothelial cells, thus impairing haematopoi­etic reconstitution. These complex issues could also be addressed by differentially deleting EP4 in various bone­marrow stromal niche cells, specifically endothelial cells during recovery from myeloablation. In addition, selective deletion of prostaglandin­E synthase enzymes in various niche cells will define the specific niche cells that functionally deploy PGE2 to activate HSCs/HPCs.

The present findings are relevant to whether NSAIDs can restore the mobilization of HSCs/HPCs in patients who are resistant to treat­ment with G­CSF and CXCR4 antagonists. It is plausible that resistance to these factors is not due to intrinsic HSC/HPC defects, but rather reflects impaired instructive functions of haematopoietic niche cells. Thus, NSAIDs could pave the way for defining alternative routes to safely mobilize HSCs/HPCs through manipulation of haematopoietic niche cells. These observations also shed light on the mechanism by which NSAIDs may contribute

to protection against cardiovascular disorders, by mobilizing haemangiocytes — HPCs that promote organ­specific formation of func­tional blood vessels17. ■

Jason M. Butler and Shahin Rafii are in Weill Medical College, Cornell University, and the Howard Hughes Medical Institute, New York, New York 10065, USA.e-mail: srafii@med.cornell.edu

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3. North, T. E. et al. Nature 447, 1007–1011 (2007).4. Bonig, H. & Papayannopoulou, T. Leukemia 27,

24–31 (2013).5. Hattori, K. et al. Nature Med. 8, 841–849 (2002).6. Goessling, W. et al. Cell Stem Cell 8, 445–458

(2011).7. Goessling, W. et al. Cell 136, 1136–1147 (2009).8. Hoggatt, J. & Pelus, L. M. Leukemia 24, 1993–2002

(2010).9. Butler, J. M. et al. Cell Stem Cell 6, 251–264 (2010).10. Hooper, A. T. et al. Cell Stem Cell 4, 263–274

(2009).11. Kobayashi, H. et al. Nature Cell Biol. 12, 1046–1056

(2010).12. Butler, J. M. et al. Blood 120, 1344–1347 (2012).13. Heissig, B. et al. Cell 109, 625–637 (2002).14. Kimura, Y. et al. PLoS ONE 6, e26918 (2011).15. Ding, L. et al. Nature 481, 457–462 (2012). 16. Ding, L. et al. Nature http://dx.doi.org/10.1038/

nature11885 (2013). 17. Jin, D. K. et al. Nature Med. 12, 557–567 (2006).

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