es. We show that Del-1, via its interaction with the αvβ3 integrin, promotes several critical functions in the niche, including HSC retention, hematopoietic progenitor cell cycle progression, and myeloid lineage commitment of HSCs. Del-1 thereby regulates myelopoiesis under steady-state conditions and in G-CSF-or inflammation-induced stress myelopoiesis, as well as myelopoiesis reconstitution under regenerative/transplantation conditions. Del-1 is hence a niche component that serves a juxtacrine homeostatic adaptation of the hematopoietic system in inflammation-related and regeneration myelopoiesis. ResultsDel-1 expression in the BM. First, we sought to investigate whether Del-1 is present in the BM. We initially studied the expression of the Del-1-encoding gene Edil3 in the BM niche and hematopoietic cell populations. We found that Edil3 mRNA expression was significantly higher in the endosteal region as compared with the central BM (cBM) ( Figure 1A), suggesting that Del-1 is enriched at the endosteal area of the BM. Analysis of sorted cells from CXCL12-GFP mice (33, 34) demonstrated that Edil3 was highly expressed integrin receptors (29-31). It consists of three N-terminal EGF-like repeats and two C-terminal discoidin I-like domains, and hence also is designated EGF-like repeats and discoidin-I-like domains-3 (EDIL3) (32). We have previously identified Del-1 as an endogenous modulator of leukocyte adhesion through interaction with integrin αLβ2 (LFA-1; CD11a/CD18) (29, 31). Moreover, Del-1 interacts with β3 integrin (CD61) via an Arg-Gly-Asp (RGD) motif on the second EGF-like repeat (30).In the present work, we observed that Del-1 is expressed by several major cellular components of the HSC niche, though not by hematopoietic progenitors. In particular, Del-1 is expressed by those niche cells that have a major role in the maintenance of HSCs, i.e., arteriolar endothelial cells and perivascular CAR cells (3,6,7,9,15). In addition, Del-1 is expressed by cells of the osteoblastic lineage that crucially mediate the engraftment of HSCs in the post-transplantation niche (3,17,18). This spatial distribution of Del-1 raised the possibility that it might be involved in the regulation of hematopoiesis. We addressed this hypothesis using in vivo models of steady-state, regenerative, and stress hematopoiesis and in vitro mechanistic approach-

The present study aims to predict the regiospecific glucuronidation of three dihydroxyflavones and seven mono-hydroxyflavones in human liver and intestinal microsomes using recombinant UGT isoforms. Seven mono-hydroxyflavones (or HFs), 2′-, 3′-, 4′-, 3-, 5-, 6-, and 7-hydroxyflavone, and three di-hydroxyflavones (or diHFs), 3,7-dihydroxyflavone (3,7-diHF), 3,5-dihydroxyflavone (3,5-diHF) and 3,4′-dihydroxyflavone (3,4′-diHF) were chosen and rates were measured at 2.5, 10 and 35 μM. The results indicated that the position of glucuronidation of three diHFs could be determined by using the UV spectra of relevant HFs. The results also indicated that UGT1A1, UGT1A7, UGT1A8, UGT1A9, UGT1A10 and UGT2B7 are the most important six UGT isoforms for metabolizing the chosen flavones. Regardless of isoforms used, 3-HF was always metabolized the fastest whereas 5-HF was usually metabolized the slowest, probably due to the formation of an intra-molecular hydrogen bond between 4-carbonyl and 5-OH group. Relevant UGT isoformspecific metabolism rates generally correlated well with the rates of glucuronidation in human intestinal and liver microsomes at each of the three tested concentrations. In conclusion, the glucuronidation "fingerprint" of seven selected mono-hydroxyflavones was affected by UGT isoforms used, positions of the −OH group, and the substrate concentrations, and the rates of glucuronidation by important recombinant UGTs correlated well with those obtained using human liver and intestinal microsomes.

Erythropoiesis must be tightly balanced to guarantee adequate oxygen delivery to all tissues in the body. This process relies predominantly on the hormone erythropoietin (EPO) and its transcription factor hypoxia inducible factor (HIF). Accumulating evidence suggests that oxygen-sensitive prolyl hydroxylases (PHDs) are important regulators of this entire system. Here, we describe a novel mouse line with conditional PHD2 inactivation (cKO P2) in renal EPO producing cells, neurons, and astrocytes that displayed excessive erythrocytosis because of severe overproduction of EPO, exclusively driven by HIF-2␣. In contrast, HIF-1␣ served as a protective factor, ensuring survival of cKO P2 mice with HCT values up to 86%. Using different genetic approaches, we show that simultaneous inactivation of PHD2 and HIF-1␣ resulted in a drastic PHD3 reduction with consequent overexpression of HIF-2␣-related genes, neurodegeneration, and lethality. Taken together, our results demonstrate for the first time that conditional loss of PHD2 in mice leads to HIF-2␣-dependent erythrocytosis, whereas HIF-1␣ protects these mice, providing a platform for developing new treatments of EPO-related disorders, such as anemia. (Blood.

We characterized the isoform specific glucuronidation of six isoflavones genistein, daidzein, glycitein, formononetin, biochanin A and prunetin using 12 expressed human UGTs and human intestinal and liver microsomes. The results indicated that these isoflavones are metabolized most rapidly at three different concentrations by one of these four UGT isoforms: UGT1A1, UGT1A8, UGT1A9 and UGT1A10. Furthermore, glycitein was usually metabolized the fastest whereas prunetin the slowest. Using the rates of metabolism by 12 UGT isoforms as a means to establish the metabolic "fingerprint", we found that each isoflavone had distinctive concentration-dependent patterns. Determination of kinetic parameters of glucuronidation using genistein and prunetin indicated that the distinct concentration-dependent metabolic patterns were the result of differences in K m and V max values. We then measured how well metabolic "fingerprinting" predicted metabolism of these isoflavones by human intestinal and liver microsomes. We found that the prediction was rather successful for five isoflavones in the liver microsomes, but not successful in the intestinal microsomes. We propose that a newly discovered UGT3A1 isoform capable of metabolizing phenols and estrogens might be responsible for the metabolism of isoflavones such as formononetin in humans. In conclusion, the first systematic study of metabolic "fingerprinting" of six common isoflavones showed that each isoflavone has UGT isoform-specific metabolic patterns that are concentration-dependent and predictive of metabolism of the isoflavones in liver microsomes.