The Aryl Hydrocarbon Receptor Influences Multiple Stages of Megakaryocyte Differentiation

Abstract 1298

Previous microarray analyses (n=3) uncovered evidence that aryl hydrocarbon receptor (AHR) mRNA increased 4–6 fold during megakaryocytic (Mk) differentiation as compared to isogenic granulocytic cultures (Lindsey et al. Blood, 2010) and identified AHR as a novel regulator of Mk polyploidization and differentiation (Lindsey et al. Brit J of Haem, 2011). Best known as a mediator of toxicological signals, we now provide unpublished data suggesting AHR impacts several additional aspects of Mk differentiation, including the initial “decision” of hematopoietic stem cells (HSCs) to differentiate toward the Mk lineage, as well as platelet function. In our current work, we first investigated if the absence of AHR signaling within the bone marrow results in Mk polyploidization defects. We found that after 7 days of ex vivo expansion, AHR-null mice had 59.5% fewer Mks ≥128n compared to WT littermates (n=3; p=0.011). In separate experiments, treatment of murine progenitor cells (n=4) with 10 nM TCDD (Dioxin, a prototypic AHR ligand) generated polyploid CD41-expressing cells within 1 day of ex vivo expansion, indicating that AHR ligands can stimulate ex vivo expansion toward the Mk lineage in the absence of cytokines such as thrombopoietin (TPO). As one might expect, TCDD is not as effective as TPO; 18.5% of the Mks treated with TCDD were polyploid by day 7, as compared to 31.5% of the Mks treated with TPO (n=3, p=0.004). Adding TCDD (n=3) did not significantly enhance Mk differentiation in response to TPO (35.4% vs 31.5%; p=0.377). Ex vivo expanding murine progenitor cells with 10 mM of the AHR inhibitor 6',2',4'-trimethoxyflavone (TMF) resulted in 37% fewer highly polyploid (≥32n) Mks by day 7 (n=3, p=0.017), effectively blocking the effects of TPO on Mk differentiation and suggesting that AHR activation is downstream of TPO signaling. Further underscoring a role for AHR during the initial differentiation “decisions” of HSCs, AHR antagonists promote HSC expansion (Boitano et al. Science, 2010) and treatments with AHR agonists deplete the HSC pool within the bone marrow (Singh et al. Carcinogenesis, 2009). HSCs reside in hypoxic niches within the bone marrow and move toward areas of increasing oxygen tension as the differentiate (Laluppa et al. Exp Hematol, 1998), suggesting to many that HSCs prefer areas of hypoxia and that HIF-1α may play a role in this process. Preliminary data shows a 3.1-fold decrease in AHR protein level under hypoxic conditions at a time when HIF-1α expression increases by 2.1-fold. AHR and HIF-1α expression is mediated by the same nuclear chaperone, HIF-1β, suggesting AHR and HIF-1α competition for HIF-1β may serve as a molecular switch by which hematopoietic cells respond to differences in oxygen levels. AHR-null mice bleed 5.3 times longer and lose three times as much blood volume than WT mice in bleeding time assays. While AHR-null mice had 9% fewer platelets and 10.4% fewer reticulated, young RNA-containing platelets than WT mice, we felt that this was not enough to explain the drastic bleeding phenotype in AHR-null mice. In agreement with our hypothesis that AHR impacts platelet function, others have suggested AHR is critical for blood clotting during Oryzias latipes embryogenesis (Kawamura et al. Zoolog Sci, 2002). Platelet function is mediated by both outside-in and inside-out signaling; defects in one or both of these signaling cascades result in bleeding disorders. In the current study, platelets from AHR-null mice bind fibrinogen as well or better than WT platelets (n=3), suggesting that AHR is not involved in inside-out platelet signaling. Based on these findings and work that shows AHR regulates vav2, a critical mediator of platelet outside-in signaling (Pearce et al. JBC, 2004), we pursued the possibility that AHR mediates platelet outside-in signaling. In agreement with a role in outside-in signaling, platelets from AHR-null mice demonstrate defective aggregation in response to collagen as compared to WT platelets. Current work seeks to further investigate the role of AHR in outside-in signaling using spreading assays. Ideally, new therapeutic approaches for Mk/platelet diseases should target specific biological events such as the initial “decisions” leading to Mk expansion from HSCs, Mk polyploidization, or platelet function. By impacting all of these processes, AHR is quickly becoming a very interesting therapeutic target and definitely warrants further investigation.