Neuronal nitric oxide synthase is required for erythropoietin stimulated erythropoiesis in mice.

Introduction: Erythropoietin (EPO), produced in the kidney in a hypoxia responsive manner, is required for red blood cell production. In non-erythroid tissue, EPO increases endothelial cell production of nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) that regulates vascular tone to improve oxygen delivery. This contributes to EPO cardioprotective activity in mouse models. Nitric oxide treatment in mice shifts hematopoiesis toward the erythroid lineage, increases red blood cell production and total hemoglobin. In erythroid cells, nitric oxide can also be generated by hydroxyurea metabolism that may contribute to hydroxyurea induction of fetal hemoglobin. We find that during erythroid differentiation, EPO induces neuronal nitric oxide synthase (nNOS) and that neuronal nitric oxide synthase is required for normal erythropoietic response. Methods: Wild type (WT) mice and mice with targeted deletion of nNOS (nNOS-/-) and eNOS (eNOS-/-) were assessed for EPO stimulated erythropoietic response. Bone marrow erythropoietic activity was assessed in culture by EPO dependent erythroid colony assay and in vivo by bone marrow transplantation into recipient WT mice. Contribution of nNOS to EPO stimulated cell proliferation was assessed in EPO dependent erythroid cells and in primary human erythroid progenitor cell cultures. Results: EPO treatment increased hematocrit similarly in WT and eNOS-/- mice and showed a lower increase in hematocrit nNOS-/- mice. Erythroid colony assays from bone marrow cells were comparable in number from wild type, eNOS-/- and nNOS-/- mice at low EPO concentration. Colony number increased at high EPO concentration is seen only in cultures from bone marrow cells of wild type and eNOS-/- mice but not from nNOS-/- mice. Colony size with high EPO treatment also exhibited a marked increase in erythroid cultures from wild type and eNOS-/- mice but not from nNOS-/- mice. Bone marrow transplant from nNOS-/- mice into immunodeficient mice showed engraftment at comparable levels to WT bone marrow transplant. With EPO treatment, the increase in hematocrit was blunted in recipient mice that received with nNOS-/- donor marrow compared with recipient mice that received WT donor marrow. In erythroid cell cultures, addition of nNOS inhibitor resulted in decreased EPO dependent proliferation mediated in part by decreased EPO receptor expression, and decreased proliferation of hemin induced differentiating erythroid cells. Discussion: EPO treatment in mice and in corresponding cultures of bone marrow erythropoiesis suggest an intrinsic defect in erythropoietic response of nNOS-/- mice to high EPO stimulation. Transplantation of bone marrow from donor WT or nNOS-/- mice into recipient WT mice showed that EPO treatment post-transplant recapitulated the response of donor mice. Culture studies suggest nNOS regulation of EPO dependent erythroid cell proliferation, expression of EPO receptor and cell cycle associated genes, and AKT activation. These data provide evidence that nitric oxide modulates EPO dose dependent erythropoietic response.

PAMMELA: Policy Administration Methodology using Machine Learning.

In recent years, Attribute-Based Access Control (ABAC) has become quite popular and effective for enforcing access control in dynamic and collaborative environments. Implementation of ABAC requires the creation of a set of attribute-based rules which cumulatively form a policy. Designing an ABAC policy ab initio demands a substantial amount of effort from the system administrator. Moreover, organizational changes may necessitate the inclusion of new rules in an already deployed policy. In such a case, re-mining the entire ABAC policy requires a considerable amount of time and administrative effort. Instead, it is better to incrementally augment the policy. In this paper, we propose PAMMELA, a Policy Administration Methodology using Machine Learning to assist system administrators in creating new ABAC policies as well as augmenting existing policies. PAMMELA can generate a new policy for an organization by learning the rules of a policy currently enforced in a similar organization. For policy augmentation, new rules are inferred based on the knowledge gathered from the existing rules. A detailed experimental evaluation shows that the proposed approach is both efficient and effective.

Erythropoietin Non-hematopoietic Tissue Response and Regulation of Metabolism During Diet Induced Obesity.

Erythropoietin (EPO) receptor (EPOR) determines EPO response. High level EPOR on erythroid progenitor cells gives rise to EPO regulated production of red blood cells. Animal models provide evidence for EPO activity in non-hematopoietic tissue mediated by EPOR expression. Beyond erythropoiesis, EPO activity includes neuroprotection in brain ischemia and trauma, endothelial nitric oxide production and cardioprotection, skeletal muscle wound healing, and context dependent bone remodeling affecting bone repair or bone loss. This review highlights examples of EPO protective activity in select non-hematopoietic tissue with emphasis on metabolic response mediated by EPOR expression in fat and brain and sex-specific regulation of fat mass and inflammation associated with diet induced obesity. Endogenous EPO maintains glucose and insulin tolerance and protects against fat mass accumulation and inflammation. Accompanying the increase in erythropoiesis with EPO treatment is improved glucose tolerance and insulin response. During high fat diet feeding, EPO also decreases fat mass accumulation in male mice. The increased white adipose tissue inflammation and macrophage infiltration associated with diet induced obesity are also reduced with EPO treatment with a shift toward an anti-inflammatory state and decreased inflammatory cytokine production. In female mice the protective effect of estrogen against obesity supersedes EPO regulation of fat mass and inflammation, and requires estrogen receptor alpha activity. In brain, EPOR expression in the hypothalamus localizes to proopiomelanocortin neurons in the arcuate nucleus that promotes a lean phenotype. EPO stimulation of proopiomelanocortin neurons increases STAT3 signaling and production of proopiomelanocortin. Cerebral EPO contributes to metabolic response, and elevated brain EPO reduces fat mass and hypothalamus inflammation during diet induced obesity in male mice without affecting EPO stimulated erythropoiesis. Ovariectomy abrogates the sex-specific metabolic response of brain EPO. The sex-dimorphic EPO metabolic response associated with fat mass accumulation and inflammation during diet induced obesity provide evidence for crosstalk between estrogen and EPO in their anti-obesity potential in female mice mediated in part via tissue specific response in brain and white adipose tissue. Endogenous and exogenous EPO response in non-hematopoietic tissue demonstrated in animal models suggests additional activity by which EPO treatment may affect human health beyond increased erythropoiesis.

Erythropoietin-Induced Changes in Bone and Bone Marrow in Mouse Models of Diet-Induced Obesity.

Obesity remodels bone and increases bone marrow adipocytes (BMAT), which negatively regulate hematopoiesis and bone. Reduced BMAT could restore altered hematopoiesis and bone features. We analyzed the potential of erythropoietin (EPO), the cytokine required for erythropoiesis, to inhibit BMAT in C57BL6/J mice fed four weeks of a high-fat diet (HFD). Acute EPO administration markedly decreased BMAT in regular chow diet (RCD) and HFD-fed mice, without affecting whole body fat mass. Micro-CT analysis showed EPO reduced trabecular bone in RCD- and HFD-fed mice, but EPO-treated HFD-fed mice maintained cortical bone mineral density and cortical bone volume, which was reduced on RCD. Despite achieving similar increased hematocrits with BMAT loss in RCD- and HFD-fed mice treated with EPO, decreased bone marrow cellularity was only observed in RCD-fed mice concomitant with an increasing percentage of bone marrow erythroid cells. In contrast, in HFD-fed mice, EPO increased endothelial cells and stromal progenitors with a trend toward the normalization of marrow homeostasis. EPO administration increased c-terminal FGF23 and intact serum FGF23 only in HFD-fed mice. These data demonstrate the distinct EPO responses of bone and marrow in normal and obese states, accompanying EPO-induced loss of BMAT.

Sex-specific brain erythropoietin regulation of mouse metabolism and hypothalamic inflammation.

The blood hormone erythropoietin (EPO), upon binding to its receptor (EpoR), modulates high-fat diet-induced (HFD-induced) obesity in mice, improves glucose tolerance, and prevents white adipose tissue inflammation. Transgenic mice with constitutive overexpression of human EPO solely in the brain (Tg21) were used to assess the neuroendocrine EPO effect without increasing the hematocrit. Male Tg21 mice resisted HFD-induced weight gain; showed lower serum adrenocorticotropic hormone, corticosterone, and C-reactive protein levels; and prevented myeloid cell recruitment to the hypothalamus compared with WT male mice. HFD-induced hypothalamic inflammation (HI) and microglial activation were higher in male mice, and Tg21 male mice exhibited a lower increase in HI than WT male mice. Physiological EPO function in the brain also showed sexual dimorphism in regulating HFD response. Female estrogen production blocked reduced weight gain and HI. Targeted deletion of EpoR gene expression in neuronal cells worsened HFD-induced glucose intolerance in both male and female mice but increased weight gain and HI in the hypothalamus in male mice only. Both male and female Tg21 mice kept on normal chow and HFD showed significantly improved glycemic control. Our data indicate that cerebral EPO regulates weight gain and HI in a sex-dependent response, distinct from EPO regulation of glycemic control, and independent of erythropoietic EPO response.

Erythropoietin modulates bone marrow stromal cell differentiation.

Erythropoietin is essential for bone marrow erythropoiesis and erythropoietin receptor on non-erythroid cells including bone marrow stromal cells suggests systemic effects of erythropoietin. Tg6 mice with chronic erythropoietin overexpression have a high hematocrit, reduced trabecular and cortical bone and bone marrow adipocytes, and decreased bone morphogenic protein 2 driven ectopic bone and adipocyte formation. Erythropoietin treatment (1 200 IU·kg-1) for 10 days similarly exhibit increased hematocrit, reduced bone and bone marrow adipocytes without increased osteoclasts, and reduced bone morphogenic protein signaling in the bone marrow. Interestingly, endogenous erythropoietin is required for normal differentiation of bone marrow stromal cells to osteoblasts and bone marrow adipocytes. ΔEpoRE mice with erythroid restricted erythropoietin receptor exhibit reduced trabecular bone, increased bone marrow adipocytes, and decreased bone morphogenic protein 2 ectopic bone formation. Erythropoietin treated ΔEpoRE mice achieved hematocrit similar to wild-type mice without reduced bone, suggesting that bone reduction with erythropoietin treatment is associated with non-erythropoietic erythropoietin response. Bone marrow stromal cells from wild-type, Tg6, and ΔEpoRE-mice were transplanted into immunodeficient mice to assess development into a bone/marrow organ. Like endogenous bone formation, Tg6 bone marrow cells exhibited reduced differentiation to bone and adipocytes indicating that high erythropoietin inhibits osteogenesis and adipogenesis, while ΔEpoRE bone marrow cells formed ectopic bones with reduced trabecular regions and increased adipocytes, indicating that loss of erythropoietin signaling favors adipogenesis at the expense of osteogenesis. In summary, endogenous erythropoietin signaling regulates bone marrow stromal cell fate and aberrant erythropoietin levels result in their impaired differentiation.

Compensatory mechanisms in myoglobin deficient mice preserve NO homeostasis.

The mechanism for nitric oxide (NO) generation from reduction of nitrate (NO3-) and nitrite (NO2-) has gained increasing attention due to the potential beneficial effects of NO in cardiovascular diseases and exercise performance. We have previously shown in rodents that skeletal muscle is the major nitrate reservoir in the body and that exercise enhances the nitrate reduction pathway in the muscle tissue and have proposed that nitrate in muscle originates from diet, the futile cycle of nitric oxide synthase 1 (NOS1) and/or oxidation of NO by oxymyoglobin. In the present study, we tested the hypothesis that lack of myoglobin expression would decrease nitrate levels in skeletal muscle. We observed a modest but significant decrease of nitrate level in skeletal muscle of myoglobin deficient mice compared to littermate control mice (17.3 vs 12.8 nmol/g). In contrast, a NOS inhibitor, L-NAME or a low nitrite/nitrate diet treatment led to more pronounced decreases of nitrate levels in the skeletal muscle of both control and myoglobin deficient mice. Nitrite levels in the skeletal muscle of both types of mice were similar (0.48 vs 0.42 nmol/g). We also analyzed the expression of several proteins that are closely related to NO metabolism to examine the mechanism by which nitrate and nitrite levels are preserved in the absence of myoglobin. Western blot analyses suggest that the protein levels of xanthine oxidoreductase and sialin, a nitrate transporter, both increased in the skeletal muscle of myoglobin deficient mice. These results are compatible with our previously reported model of nitrate production in muscle and suggest that myoglobin deficiency activates compensatory mechanisms to sustain NO homeostasis.

Vasopressin stimulates the proliferation and differentiation of red blood cell precursors and improves recovery from anemia.

Arginine vasopressin (AVP) made by hypothalamic neurons is released into the circulation to stimulate water resorption by the kidneys and restore water balance after blood loss. Patients who lack this antidiuretic hormone suffer from central diabetes insipidus. We observed that many of these patients were anemic and asked whether AVP might play a role in red blood cell (RBC) production. We found that all three AVP receptors are expressed in human and mouse hematopoietic stem and progenitor cells. The AVPR1B appears to play the most important role in regulating erythropoiesis in both human and mouse cells. AVP increases phosphorylation of signal transducer and activator of transcription 5, as erythropoietin (EPO) does. After sublethal irradiation, AVP-deficient Brattleboro rats showed delayed recovery of RBC numbers compared to control rats. In mouse models of anemia (induced by bleeding, irradiation, or increased destruction of circulating RBCs), AVP increased the number of circulating RBCs independently of EPO. In these models, AVP appears to jump-start peripheral blood cell replenishment until EPO can take over. We suggest that specific AVPR1B agonists might be used to induce fast RBC production after bleeding, drug toxicity, or chemotherapy.

Erythropoietin and Hypothalamic-Pituitary Axis.

Erythropoietin (EPO), known primarily for its erythropoietic activity, is commonly used clinically to treat anemia of chronic kidney disease. However, the expression of EPO receptor (EpoR) beyond erythroid tissue provides for potential extrahematopoietic effects of EPO, including EPO regulation of metabolic homeostasis (Zhang et al., 2014). Small clinical studies have shown that EPO treatment in patients with end-stage renal disease improved glycemic control and insulin sensitivity. Studies in animal models have shown that EPO regulation of metabolism is mainly attributed to its response in fat, and the hypothalamus-pituitary axis (Dey et al., 2016; Dey, Scullen, & Noguchi, 2015; Teng, Gavrilova, et al., 2011; Wang et al., 2013) and is not dependent on its hematopoietic activity. EpoR expression in the hypothalamus is localized to the neurons expressing proopiomelanocortin (POMC) in the arcuate nucleus region, the most important site in the brain for the regulation of physiological energy expenditure. EPO treatment increases POMC production in anorexigenic POMC neurons in the hypothalamus. In the pituitary, EPO modulates the secretion of the POMC-derived peptide, adrenocorticotropic hormone (ACTH) that regulates physiological and metabolic stress response. With EPO produced by cells in the brain, such as astrocytes, and with EPO-stimulated POMC expression in the hypothalamus and EPO-inhibited ACTH secretion in the pituitary, EPO signaling contributes to the hypothalamic-pituitary axis as a major regulator of glucose metabolism and energy homeostasis.

Non-hematopoietic effects of endogenous erythropoietin on lean mass and body weight regulation.

OBJECTIVE: To investigate the concurrent relationships between human plasma erythropoietin concentrations and energy expenditure (EE), body composition, plasma leptin concentrations, and associations with weight change. METHODS: Plasma to measure erythropoietin and leptin; data for body composition; 24-h EE measured in a whole-room calorimeter; and 75 g oral glucose tolerance testing were available from 109 full-heritage Pima Indians (55% male) from a larger study designed to understand the causes of obesity. Seventy-nine subjects had data for weight at a later visit (mean follow-up = 4.3 ± 1.9 years) to calculate percent weight change per year. RESULTS: Erythropoietin, adjusted for covariates, correlated with 24-h EE (r = 0.26, P = 0.007), sleeping EE (r = 0.29, P = 0.003), fat-free mass (r = 0.19, P = 0.05), and fat mass (r = 0.27, P = 0.005), but not insulin or glucose measures. The association of erythropoietin with 24-h EE was fully mediated by fat-free mass. Erythropoietin associated with leptin in women (ρ = 0.36, P = 0.01), but not in men (P = 0.9), independently from fat mass. The association of erythropoietin with percent weight change per year was in opposing directions (interaction: P = 0.002) in males (r = -0.35, P = 0.02) versus females (r = 0.37, P = 0.02). CONCLUSIONS: Non-hematopoietic endogenous erythropoietin action may be involved in body weight regulation in opposing directions in men and women, i.e., weight loss in men and weight gain in women.

Erythropoietin regulates POMC expression via STAT3 and potentiates leptin response.

The arcuate nucleus of the hypothalamus is essential for metabolic homeostasis and responds to leptin by producing several neuropeptides including proopiomelanocortin (POMC). We previously reported that high-dose erythropoietin (Epo) treatment in mice while increasing hematocrit reduced body weight, fat mass, and food intake and increased energy expenditure. Moreover, we showed that mice with Epo receptor (EpoR) restricted to erythroid cells (ΔEpoRE) became obese and exhibited decreased energy expenditure. Epo/EpoR signaling was found to promote hypothalamus POMC expression independently from leptin. Herein we used WT and ΔEpoRE mice and hypothalamus-derived neural culture system to study the signaling pathways activated by Epo in POMC neurons. We show that Epo stimulation activated STAT3 signaling and upregulated POMC expression in WT neural cultures. ΔEpoRE mice hypothalamus showed reduced POMC levels and lower STAT3 phosphorylation, with and without leptin treatment, compared to in vivo and ex vivo WT controls. Collectively, these data show that Epo regulates hypothalamus POMC expression via STAT3 activation, and provide a previously unrecognized link between Epo and leptin response.

Interaction with WDR5 promotes target gene recognition and tumorigenesis by MYC.

MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies. The oncogenic potential of MYC stems from its ability to bind regulatory sequences in thousands of target genes, which depends on interaction of MYC with its obligate partner, MAX. Here, we show that broad association of MYC with chromatin also depends on interaction with the WD40-repeat protein WDR5. MYC binds WDR5 via an evolutionarily conserved "MYC box IIIb" motif that engages a shallow, hydrophobic cleft on the surface of WDR5. Structure-guided mutations in MYC that disrupt interaction with WDR5 attenuate binding of MYC at ∼80% of its chromosomal locations and disable its ability to promote induced pluripotent stem cell formation and drive tumorigenesis. Our data reveal WDR5 as a key determinant for MYC recruitment to chromatin and uncover a tractable target for the discovery of anticancer therapies against MYC-driven tumors.

Skeletal muscle as an endogenous nitrate reservoir.

The nitric oxide synthase (NOS) family of enzymes form nitric oxide (NO) from arginine in the presence of oxygen. At reduced oxygen availability NO is also generated from nitrate in a two step process by bacterial and mammalian molybdopterin proteins, and also directly from nitrite by a variety of five-coordinated ferrous hemoproteins. The mammalian NO cycle also involves direct oxidation of NO to nitrite, and both NO and nitrite to nitrate by oxy-ferrous hemoproteins. The liver and blood are considered the sites of active mammalian NO metabolism and nitrite and nitrate concentrations in the liver and blood of several mammalian species, including human, have been determined. However, the large tissue mass of skeletal muscle had not been generally considered in the analysis of the NO cycle, in spite of its long-known presence of significant levels of active neuronal NOS (nNOS or NOS1). We hypothesized that skeletal muscle participates in the NO cycle and, due to its NO oxidizing heme protein, oxymyoglobin has high concentrations of nitrate ions. We measured nitrite and nitrate concentrations in rat and mouse leg skeletal muscle and found unusually high concentrations of nitrate but similar levels of nitrite, when compared to the liver. The nitrate reservoir in muscle is easily accessible via the bloodstream and therefore nitrate is available for transport to internal organs where it can be reduced to nitrite and NO. Nitrate levels in skeletal muscle and blood in nNOS(-/-) mice were dramatically lower when compared with controls, which support further our hypothesis. Although the nitrate reductase activity of xanthine oxidoreductase in muscle is less than that of liver, the residual activity in muscle could be very important in view of its total mass and the high basal level of nitrate. We suggest that skeletal muscle participates in overall NO metabolism, serving as a nitrate reservoir, for direct formation of nitrite and NO, and for determining levels of nitrate in other organs.

Erythropoietin negatively regulates pituitary ACTH secretion.

Erythropoietin (Epo) and Epo-receptor (EpoR) signaling, in addition to its classical role in erythropoiesis, exhibit a protective response in non-hematopoietic tissues. Mice with EpoR expression restricted to only hematopoietic tissues (ΔEpoRE), become obese, have low energy expenditure, and are glucose intolerant and insulin resistant. In the arcuate nucleus of the mouse hypothalamus, EpoR expression co-localizes in proopiomelanocortin (POMC) neurons. In vivo high-dose Epo treatment increases hypothalamus POMC, reduces food intake and fat mass accumulation. Here we report that Epo treatment also decreases plasma concentration of the pituitary derived POMC peptide, adrenocorticotropic hormone (ACTH). Conversely, ΔEpoRE mice show reduced hypothalamus POMC and high plasma concentrations of ACTH. In the pituitary, POMC is synthesized in the corticotroph cells, and here we examine Epo effect on pituitary POMC expression using the AtT-20 mouse corticotroph pituitary cell line. In AtT-20 cells, enzyme immunoassay analysis showed that Epo inhibits ACTH secretion. This effect is post-translational, as Epo treatment did not affect POMC mRNA expression but increased intracellular levels of ACTH peptide. Moreover, Epo reduced the basal intracellular calcium (Ca(2+)) levels, suggesting an effect in the Ca(2+)-signaling pathway. In summary, our studies suggest a novel regulatory pathway of ACTH secretion in the pituitary via EpoR-signaling. The higher plasma ACTH level in ΔEpoRE mice also suggests a possible mechanism of deregulated pituitary function with loss of Epo-signaling.

Erythropoietin's inhibiting impact on hepcidin expression occurs indirectly.

Under conditions of accelerated erythropoiesis, elevated erythropoietin (Epo) levels are associated with inhibition of hepcidin synthesis, a response that ultimately increases iron availability to meet the enhanced iron needs of erythropoietic cells. In the search for erythroid regulators of hepcidin, many candidates have been proposed, including Epo itself. We aimed to test whether direct interaction between Epo and the liver is required to regulate hepcidin. We found that prolonged administration of high doses of Epo in mice leads to great inhibition of liver hepcidin mRNA levels, and concomitant induction of the hepcidin inhibitor erythroferrone (ERFE). Epo treatment also resulted in liver iron mobilization, mediated by increased ferroportin activity and accompanied by reduced ferritin levels and increased TfR1 expression. The same inhibitory effect was observed in mice that do not express the homodimeric Epo receptor (EpoR) in liver cells because EpoR expression is restricted to erythroid cells. Similarly, liver signaling pathways involved in hepcidin regulation were not influenced by the presence or absence of hepatic EpoR. Moreover, Epo analogs, possibly interacting with the postulated heterodimeric ß common EpoR, did not affect hepcidin expression. These findings were supported by the lack of inhibition on hepcidin found in hepatoma cells exposed to various concentrations of Epo for different periods of times. Our results demonstrate that hepcidin suppression does not require the direct binding of Epo to its liver receptors and rather suggest that the role of Epo is to stimulate the synthesis of the erythroid regulator ERFE in erythroblasts, which ultimately downregulates hepcidin.

Erythropoietin action in stress response, tissue maintenance and metabolism.

Erythropoietin (EPO) regulation of red blood cell production and its induction at reduced oxygen tension provides for the important erythropoietic response to ischemic stress. The cloning and production of recombinant human EPO has led to its clinical use in patients with anemia for two and half decades and has facilitated studies of EPO action. Reports of animal and cell models of ischemic stress in vitro and injury suggest potential EPO benefit beyond red blood cell production including vascular endothelial response to increase nitric oxide production, which facilitates oxygen delivery to brain, heart and other non-hematopoietic tissues. This review discusses these and other reports of EPO action beyond red blood cell production, including EPO response affecting metabolism and obesity in animal models. Observations of EPO activity in cell and animal model systems, including mice with tissue specific deletion of EPO receptor (EpoR), suggest the potential for EPO response in metabolism and disease.

The TAL1/SCL transcription factor regulates cell cycle progression and proliferation in differentiating murine bone marrow monocyte precursors.

Monocytopoiesis involves the stepwise differentiation in the bone marrow (BM) of common myeloid precursors (CMPs) to monocytes. The basic helix-loop-helix transcription factor TAL1/SCL plays a critical role in other hematopoietic lineages, and while it had been reported to be expressed by BM-derived macrophages, its role in monocytopoiesis had not been elucidated. Using cell explant models of monocyte/macrophage (MM) differentiation, one originating with CMPs and the other from more committed precursors, we characterized the phenotypic and molecular consequences of inactivation of Tal1 expression ex vivo. While Tal1 knockout had minimal effects on cell survival and slightly accelerated terminal differentiation, it profoundly inhibited cell proliferation and decreased entry into and traversal of the G(1) and S phases. In conjunction, steady-state levels of p16(Ink4a) mRNA were increased and those of Gata2 mRNA decreased. Chromatin immunoprecipitation analysis demonstrated the association of Tal1 and E47, one of its E protein DNA-binding partners, with an E box-GATA sequence element in intron 4 of the Gata2 gene and with three E boxes upstream of p16(Ink4a). Finally, wild-type Tal1, but not a DNA binding-defective mutant, rescued the proliferative defect in Tal1-null MM precursors. These results document the importance of this transcription factor in cell cycle progression and proliferation during monocytopoiesis and the requirement for direct DNA binding in these processes.

Dichloroacetate- and trichloroacetate-induced phagocytic activation and production of oxidative stress in the hepatic tissues of mice after acute exposure.

Dichoroacetate (DCA) and trichloroacetate (TCA) are by-products formed during chlorination of the drinking water and were found to be hepatotoxic and hepatocarcinogenic in rodents. In this study, the abilities of the compounds to induce oxidative stress and phagocytic activation have been studied in B6C3F1 mice. Groups of mice were administered 300 mg/kg of either DCA or TCA, p.o, and were sacrificed after 6 or 12 h. Peritoneal lavage cells (PLCs) were isolated and assayed for superoxide anion (SA) production, and hepatic tissues were assayed for the production of SA, lipid peroxidation (LP), and DNA-single strand breaks (SSBs). TCA resulted in significant production of SA in the PLCs, and in the production of SA, LP, and DNA-SSBs in the hepatic tissues, 12 h after dosing, as compared with the control. DCA administration, on the other hand, resulted in significant increases in the productions of LP and DNA-SSBs in the hepatic tissues at both time points, and in SA production in PLCs and hepatic tissues, 6 h after dosing. However, DCA-induced increases in SA production in PLC and hepatic tissues declined at the 12-h time point, reaching control level in the hepatic tissues. These results may implicate the contribution of phagocytic activation to the induction of oxidative stress in the hepatic tissues and also the role of SA production in the induction of LP and/or DNA damage in those tissues, in response to the compounds. The results also suggest studying the involvement of these mechanisms in the long-term hepatotoxicity/hepatocarcinogencity of the compounds.