In Megakaryocytic Differentiation, the Aryl Hydrocarbon Receptor (AhR) Transcription Factor Regulates Hes1 Expression and Megakaryocytic Polyploidization.

Abstract 2519

Poster Board II-496

Understanding the mechanisms underlying megakaryocytic (Mk) differentiation and maturation is vital to the discovery of novel approaches to treating Mk and platelet disorders such as thrombocytopenia, megakaryoblastic leukemia, and thrombocythemia. The number of platelets released is proportional to the amount of DNA present in a given Mk, so insights into the molecular basis of Mk polyploidization could inspire improved ex vivo culturing methods to promote Mk commitment, expansion, and differentiation, leading to improved autologous transfusion protocols to offset thrombocytopenia associated with HSC transplants following high-dose chemotherapy or MDS progression. Microarray analyses on ex vivo Mk-differentiated primary human CD34⁺ cells showed that mRNA levels of the Aryl Hydrocarbon Receptor (AhR) increased during Mk differentiation and was elevated 4–6 fold in Mks compared to isogenic granulocytic cultures. These data were further confirmed by quantitative(Q)-RT-PCR analysis of differentiating Mks derived from primary human CD34⁺ cells as well as from CHRF cells (human megakaryoblastic leukemia). We have shown that CHRF cells are a valid model of human Mk differentiation (Fuhrken PG et al. Exp Hematol, 2007; 35:476–489). Thus, we hypothesized that AhR may act as a novel Mk transcription factor, possibly by influencing or regulating Mk polyploidization. Known as a “toxin sensor”, AhR is involved in the mechanism of action of environmental toxins, likely by altering cell cycle regulation. Epidemiological studies of toxic waste spills and Vietnam veterans suggest that exposure to known AhR ligands may result in increased platelet counts proportional to dioxin exposure level (Webb K et al. Am J Ind Med, 1987;11:685–691, Michalek JE Arch Environ Health, 2001; 56:396–405). These studies offer the intriguing possibility that AhR activation modulates megakaryocyte differentiation and/or platelet production. Interestingly, AhR influences the differentiation of other myeloid lineages including monocytes (Hayashi S et al. Carcinogenesis, 1995; 16:1403–1409) and is upregulated after leukocyte activation (Crawford RB et al. Mol Pharmacol, 1997; 52:921–927). Western blot analyses determined that although initially expressed in both the cytoplasm and nucleus, AhR became solely nuclear in differentiating CHRF cells. EMSA analysis using CHRF nuclear extracts demonstrated that AhR binding to a consensus binding sequence increased as megakaryopoiesis progressed (n=3). Increased AhR-DNA binding during CHRF Mk differentiation correlated with 4.6-fold increased mRNA expression of the AhR transcriptional target Hes1 (n=3, p<0.005), a known cell-cycle regulator and mediator of notch signaling. In order to examine the functional role of AhR in megakaryopoiesis, we generated 3 independent AhR knockdown (KD) CHRF cell lines. Depending on the day of culture, AhR-KD CHRF cell lines differentiated into Mk cells expressed 2-3 fold less AhR mRNA (n=3; p<0.02), 40–60% less AhR protein (n=3), 2.7 times less Hes1 mRNA (n=3; p=0.018), displayed Mk-ploidy distributions shifted towards lower ploidy classes, and were incapable of reaching higher ploidy classes (i.e., ≥32n) seen in control cells. Ploidy levels on day 7 (maximal ploidy in control cells) were 3-fold lower in AhR-KD CHRF cells (n=3; p=0.012 or p=0.005 depending on KD cell line). AhR KD resulted in increased DNA synthesis of low ploidy (<8n; n=3; p<0.05) without influencing apoptosis (n=3, p=0.37). These data suggest that AhR may regulate the cell cycle differently in Mks compared to other cell types, where loss of AhR results in cell cycle blockage and increased apoptosis. As such, AhR deregulation provides a mechanistic explanation for chemical-induced thrombocytopenia, including chemotherapy, and suggests that AhR agonists may provide novel therapies for megakaryoblastic leukemia. AhR-mediated expression of Hes1, an established regulator of the Notch signaling pathway, provides a novel molecular model of endomitotic entry and Mk polyploidization; in drosophila, Notch cell-cycle regulation controls the initial switch toward endomitosis.