Hes1 Expression Impacts Megakaryocytic Polyploidization and May Contribute to the Platelet Defects Found In Aryl Hydrocarbon Receptor (AhR)-Null Mice.

Abstract 2612

Microarray analyses (n = 3) from our lab uncovered that Aryl hydrocarbon receptor (AhR) mRNA increased 4–6 fold in Megakaryocytes (Mks) compared to isogenic granulocytic cultures and identified the Aryl hydrocarbon receptor as a novel transcriptional activator of megakaryocytic differentiation and maturation (Lindsey et al. Blood, 2009). (Q)-RT-PCR and Western Blot analyses confirmed these findings in differentiating Mks derived from primary human CD34⁺ cells and differentiating CHRF cells (human megakaryoblastic leukemia cell line). Unpublished data from our lab found that AhR-null mice (Schmidt et al. Proc Natl Acad Sci, 1996) exhibited a 9% decrease in platelet counts compared to WT mice. There were also 10.4% fewer reticulated young, RNA-containing platelets in AhR-null mice compared to WT mice, indicating a defect in either platelet generation or Mk maturation. We examined platelet functionality and found that while the average bleeding time for WT mice was approximately 1.5 minutes, AhR-null mice bled on average 8 minutes, or roughly 5.3 times longer. We also calculated the volume of blood loss and found that AhR-null mice lost 3 times as much blood during these assays as WT mice. To investigate if these platelet defects were due to abnormal Mk maturation, we examined the level of steady-state polyploidization of Mks residing within the murine bone marrow niche and found that compared to WT mice, AhR-null mice had ca. 25% fewer high ploidy (≥ 32n) Mks, but more Mks in the lower ploidy classes of 8n and 16n. Together, these data suggest that AhR-null mice exhibit platelet defects of both number and function, and steady-state Mks from AhR-null mice are less polyploid than WT mice. Previous EMSA experiments (n = 4) had found that AhR transcriptional activity, measured by the binding of AhR to a consensus DNA binding site, increased during Mk polyploidization and that decreased Mk ploidy correlated to a 2.7 fold decrease in expression of Hes1, an AhR target gene (n = 3; p= 0.018). Therefore, we hypothesized that AhR impacts Mk ploidy through transcriptional regulation of Hes1. In our current work, EMSA experiments (n = 3) showed a 3.5 fold increase of AhR binding to oligonucleotides corresponding to a putative AhR binding site within the Hes1 promoter, indicating that AhR could transcriptionaly regulate Hes1 during megakaryopoiesis. As an important component and regulator of the Notch signaling cascade, Hes1 regulates the cell cycle and cellular quiescence (Sang et al. Science, 2008), but its role during normal Mk differentiation remains unexplored. Interestingly, mRNA expression of Dlk1, a Hes1 transcriptional target, is increased in CD34⁺ cells isolated from MDS patients compared to CD34⁺ cells from normal individuals (Sakajiri et al. Leukemia, 2005), suggesting that Hes1 target genes may be deregulated in megakaryoblastic leukemia. To determine if Hes1 could be involved in Mk differentiation, we performed (Q)-RT-PCR experiments on differentiating Mks, granulocytes, and erythrocytes ex vivo expanded from primary human CD34⁺ cells. We found that by day 12 Hes1 expression increases 2.8 fold in Mks, but decreases more than 2.9 fold in isogenic granulocytes (n = 2 for each; p = 0.044). Hes1 expression appears to remain unchanged during erythroid development (n = 2). To investigate the functional impact of Hes1 during megakaryopoiesis, we used RNAi-mediated knockdown of Hes1 to generate two independent KD-Hes1 cell lines. These cell lines express 68 and 59 percent less Hes1 mRNA (n = 3; p = 0.008 for Hes1-A and p = 0.015 for Hes1-B), 31 and 47 percent less Hes1 protein (n = 3), without impacting AhR expression (n = 3; p = 0.46). Similar to knocking down AhR, Mk-ploidy distributions in KD-Hes1 cells were shifted towards lower ploidy classes, and were incapable of reaching higher ploidy classes (i.e., ≥ 32n) seen in control cells. Although there was little impact in undifferentiated CHRF cells, ploidy levels on day 7 (maximal ploidy in control cells) were between 25.3 and 26.7 percent less than control cells depending on the cell line (n = 3; p < 0.05 for both cell lines). These experiments genetically link Hes1 and AhR and suggest that AhR regulation of Hes1 during megakaryopoiesis mediates megakaryocytic polyploidization. As such, we provide a novel molecular model of endomitotic entry and Mk polyploidization, while also providing a mechanistic explanation for the platelet defects found in AhR null mice.