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Background: During mammalian gestation, fetal circadian rhythms are thought to be mainly controlled by maternal signals. In humans, the initiation and activity of central and peripheral circadian clocks is largely unknown. This study aimed to elucidate the developmental clock properties in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were obtained from (a) preterm infants, subgrouped according to birth weight or gestational age classification, and (b) term infants (in total: n= 60). In vitro clock activity was determined by using live bioluminescence recording of a luciferase reporter gene under circadian control over 120 h. In addition, core clock and clock-associated gene expression were quantified using NanoString technology. Results: Peripheral clock activity was detected, regardless of prematurity and birth weight classification. The mean period, amplitude, and phase of circadian oscillations were not significantly associated with gestational age or birth weight classification. Conclusions: Peripheral clock activity can be demonstrated in HUVECs from both preterm and term infants without significant developmental differences in the period, amplitude, and phase of oscillations. This model may be useful to identify perturbation factors of proper development and entrainment of neonatal circadian clock activity. Impact:

We established a model system for analyzing the peripheral clock in preterm and term HUVECs.

In HUVECs, the peripheral clock exhibits functional in vitro activity independent of gestational age or birth weight categories.

In this model system, neither significant developmental differences exist in the period, amplitude, and phase, nor in the expression of circadian core clock and clock-associated genes.

Entrainment and proper function of the circadian clock deserve attention in neonatal intensive care.

Generation of circadian rhythms is cell-autonomous and relies on a transcription/translation feedback loop controlled by a family of circadian clock transcription factor activators including CLOCK, BMAL1 and repressors such as CRY1 and CRY2. The aim of the present study was to examine both the molecular mechanism and the hemopoietic implication of circadian erythropoietin expression. Mutant mice with homozygous deletion of the core circadian clock genes cryptochromes 1and 2(Cry-null) were used to elucidate circadian erythropoietin regulation. Wild-type control mice exhibited a significant difference in kidney erythropoietinmRNA expression between circadian times 06 and 18. In parallel, a significantly higher number of erythropoietin-producing cells in the kidney (by RNAscope®) and significantly higher levels of circulating erythropoietin protein (by ELISA) were detected at circadian time 18. Such changes were abolished in Cry-null mice and were independent from oxygen tension, oxygen saturation, or expression of hypoxia-inducible factor 2 alpha, indicating that circadian erythropoietin expression is transcriptionally regulated by CRY1 and CRY2. Reporter gene assays showed that the CLOCK/BMAL1 heterodimer activated an E-box element in the 5’ erythropoietinpromoter. RNAscope® in situhybridization confirmed the presence of Bmal1in erythropoietin-producing cells of the kidney. In Cry-null mice, a significantly reduced number of reticulocytes was found while erythrocyte numbers and hematocrit were unchanged. Thus, circadian erythropoietin regulation in the normoxic adult murine kidney is transcriptionally controlled by master circadian activators CLOCK/BMAL1, and repressors CRY1/CRY2. These findings may have implications for kidney physiology and disease, laboratory diagnostics, and anemia therapy.

Supplemental Digital Content is available in the text.This study aimed to examine whether the transfusion of donor blood products, abnormal coagulation or inflammation increase the risk of venous thromboembolism (VTE) associated with central venous catheters (CVC) in neonates. A retrospective case-control study including 25 neonates with CVC-associated VTE and tightly matched controls with CVC, but without VTE was performed. The frequency of (i) abnormal coagulation screens, (ii) increased inflammatory marker proteins before catheter insertion, or (iii) catheter-associated blood stream infection did not differ between cases and controls. No difference was found in the number or type of transfusions within the last day before VTE. However, the total number of transfusions in the time period between catheter placement and VTE diagnosis (median 6.5 d) was significantly higher (P<0.001) in cases (44 red blood cell, 61 plasma, and 18 platelet transfusions) compared with an equal median time period of 7 days postcatheter insertion in controls (26/24/11). In conclusion, intensive transfusion treatment (through a peripheral line) after CVC insertion was associated with a higher risk of VTE (odds ratio 7.58; 95% confidence interval, 0.84-68.46), suggesting that transfusion of adult donor blood products into the cellular and plasmatic hemostatic system of the neonate increases the risk for CVC–associated VTE.

Thrombopoietin (Tpo), which primarily regulates megakaryopoiesis, and its receptor (c-Mpl) are expressed in the brain, where Tpo exhibits proapototic effects on neurons. In the present study, we investigated the implication of Tpo in experimental pneumococcal meningitis. Following intrathecal infection with the encapsulated Streptococcus pneumoniae strain D39, we observed upregulation of Tpo mRNA expression at 12 h and 24 h in brain homogenates of wild-type C57BL/6 mice. c-Mpl mRNA expression was upregulated at 12 h and returned to baseline at 24 h. Compared to wild-type mice, mutants with homozygous Tpo receptor ablation (c-Mpl–/–) displayed reduced microglial activation and neuronal apoptosis in the dentate gyrus. Concentrations of bacteria in blood or cerebrospinal fluid (CSF), as well as CSF pleocytosis, were not significantly different between wild-type and c-Mpl–/–mice. In human postmortem brain, Tpo protein was colocalized to macrophages during encephalitis. In murine primary microglia and RAW264.7 macrophages, upregulation of Tpo mRNA was induced by D39-conditioned medium but not by bacterial lipopeptide or by medium conditioned by pneumococcal mutants defective in hydrogen peroxide formation (spxB) or pneumolysin (pln). We conclude that Tpo acts as a mediator of neuronal damage in bacterial meningitis.

ABSTRACTThrombopoietin (Tpo), which primarily regulates megakaryopoiesis, and its receptor (c-Mpl) are expressed in the brain, where Tpo exhibits proapototic effects on neurons. In the present study, we investigated the implication of Tpoin experimental pneumococcal meningitis. Following intrathecal infection with the encapsulated Streptococcus pneumoniaestrain D39, we observed upregulation of TpomRNA expression at 12 h and 24 h in brain homogenates of wild-type C57BL/6 mice. c-MplmRNA expression was upregulated at 12 h and returned to baseline at 24 h. Compared to wild-type mice, mutants with homozygous Tpo receptor ablation (c-Mpl−/−) displayed reduced microglial activation and neuronal apoptosis in the dentate gyrus. Concentrations of bacteria in blood or cerebrospinal fluid (CSF), as well as CSF pleocytosis, were not significantly different between wild-type and c-Mpl−/−mice. In human postmortem brain, Tpo protein was colocalized to macrophages during encephalitis. In murine primary microglia and RAW264.7 macrophages, upregulation of TpomRNA was induced by D39-conditioned medium but not by bacterial lipopeptide or by medium conditioned by pneumococcal mutants defective in hydrogen peroxide formation (ΔspxB) or pneumolysin (Δpln). We conclude that Tpo acts as a mediator of neuronal damage in bacterial meningitis.

Long-lived plasma cells in the bone marrow produce memory antibodies that provide immune protection persisting for decades after infection or vaccination but can also contribute to autoimmune and allergic diseases. However, the composition of the microenvironmental niches that are important for the generation and maintenance of these cells is only poorly understood. Here, we demonstrate that, within the bone marrow, plasma cells interact with the platelet precursors (megakaryocytes), which produce the prominent plasma cell survival factors APRIL (a proliferation-inducing ligand) and IL-6 (interleukin-6). Accordingly, reduced numbers of immature and mature plasma cells are found in the bone marrow of mice deficient for the thrombopoietin receptor (c-mpl) that show impaired megakaryopoiesis. After immunization, accumulation of antigen-specific plasma cells in the bone marrow is disturbed in these mice. Vice versa, injection of thrombopoietin allows the accumulation and persistence of a larger number of plasma cells generated in the course of a specific immune response in wild-type mice. These results demonstrate that megakaryocytes constitute an important component of the niche for long-lived plasma cells in the bone marrow.

Because erythropoietin (Epo) is intensively studied as neuroprotective agent, Epo receptor (EpoR) regulation in neurons is of particular interest. Herein, we investigated molecular mechanisms of EpoR regulation in neuronal cells including the role of GATA transcription factors. First, developmental downregulation of EpoR expression in murine brain was observed. A differential expression pattern of the Gata factors was found in these specimens as well as in murine adult neural stem cells (NSC) and primary rat neurons, astrocytes, and microglia. Human SH-SY5Y cells served as a model to analyze EpoR regulation. In vitro binding of GATA-2, -3, and -4 to the 5'-flanking region was demonstrated. In reporter gene assays, the activity of a region containing two GATA binding sites was significantly induced when these GATA factors were overexpressed. However, GATA factors alone did not affect endogenous EpoR expression. Importantly, EpoR transcripts have doubled under hypoxia. Furthermore, we analyzed the methylation pattern close to the GATA motifs. Indeed, demethylation with 5-Aza-2'-deoxycytidine (Aza) resulted in upregulation of EpoR mRNA. Additionally, several CpGs were mostly nonmethylated in SH-SY5Y cells, but methylated in specific regions of the human adult brain. Thus, methylation may be involved in developmental EpoR downregulation.

Because erythropoietin (Epo) is intensively studied as neuroprotective agent, Epo receptor (EpoR) regulation in neurons is of particular interest. Herein, we investigated molecular mechanisms of EpoRregulation in neuronal cells including the role of GATA transcription factors. First, developmental downregulation of EpoRexpression in murine brain was observed. A differential expression pattern of the Gatafactors was found in these specimens as well as in murine adult neural stem cells (NSC) and primary rat neurons, astrocytes, and microglia. Human SH-SY5Y cells served as a model to analyze EpoRregulation. In vitrobinding of GATA-2, -3, and -4 to the 5′-flanking region was demonstrated. In reporter gene assays, the activity of a region containing two GATA binding sites was significantly induced when these GATA factors were overexpressed. However, GATA factors alone did not affect endogenous EpoRexpression. Importantly, EpoRtranscripts have doubled under hypoxia. Furthermore, we analyzed the methylation pattern close to the GATA motifs. Indeed, demethylation with 5-Aza-2′-deoxycytidine (Aza) resulted in upregulation of EpoRmRNA. Additionally, several CpGs were mostly nonmethylated in SH-SY5Y cells, but methylated in specific regions of the human adult brain. Thus, methylation may be involved in developmental EpoRdownregulation.

Molecular mechanisms for the developmental stage and tissue-specific regulation of the erythropoietin (EPO)gene are poorly understood. Recent findings indicate a role of the Wilms tumor suppressor, Wt1, in the formation of the hematopoietic system. Herein, we tested the hypothesis that Wt1 is a transcriptional regulator of the EPOgene. Binding of the transcriptionally competent Wt1(–KTS) isoform to the minimal EPOpromoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. Under normoxia, EPO expression was significantly increased in HEK 293 and HepG2 cells with forced expression of Wt1(–KTS). A reporter construct harboring the 117-bp minimal human EPOpromoter was activated up to 20-fold by transient cotransfection of Wt1(–KTS) in different cell lines. Mutation of the Wt1 binding site in the EPOpromoter abrogated this stimulatory effect of the Wt1(–KTS) protein. Hepatic EpomRNA expression was significantly reduced in embryonic mice with homozygous Wt1deletion. Furthermore, Wt1 and EPO were colocalized in hepatocytes of the liver and in neuronal cells of the dorsal root ganglia in developing mice. Both proteins were also detected in Sertoli cells of the adult murine testis. In conclusion, we identified Wt1(–KTS) as a novel transcriptional activator for the tissue-specific expression of the EPOgene.

Molecular mechanisms for the developmental stage and tissue-specific regulation of the erythropoietin (EPO) gene are poorly understood. Recent findings indicate a role of the Wilms tumor suppressor, Wt1, in the formation of the hematopoietic system. Herein, we tested the hypothesis that Wt1 is a transcriptional regulator of the EPO gene. Binding of the transcriptionally competent Wt1(–KTS) isoform to the minimal EPO promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. Under normoxia, EPO expression was significantly increased in HEK 293 and HepG2 cells with forced expression of Wt1(–KTS). A reporter construct harboring the 117-bp minimal human EPO promoter was activated up to 20-fold by transient cotransfection of Wt1(–KTS) in different cell lines. Mutation of the Wt1 binding site in the EPO promoter abrogated this stimulatory effect of the Wt1(–KTS) protein. Hepatic Epo mRNA expression was significantly reduced in embryonic mice with homozygous Wt1 deletion. Furthermore, Wt1 and EPO were colocalized in hepatocytes of the liver and in neuronal cells of the dorsal root ganglia in developing mice. Both proteins were also detected in Sertoli cells of the adult murine testis. In conclusion, we identified Wt1(–KTS) as a novel transcriptional activator for the tissue-specific expression of the EPO gene.

We observed a 12-year-old boy with acute hepatitis and associated aplastic anemia (AA), where parvovirus B19 genome was repeatedly detected in liver and bone marrow biopsies, but not in blood samples. We conclude that: (1) B19 infection may be underdiagnosed as the causative agent responsible for acute hepatitis and associated AA if no organ-specific diagnostic tests are applied; (2) B19 deoxyribonucleic acid (DNA) can persist in the liver; (3) during the acute phase of hepatitis, extramedullary hematopoiesis may be involved in the susceptibility for hepatic B19 infection.

The objective of this study is to determine whether the fetus compensates for fetal alloimmune thrombocytopenia (FAITP) by increasing serum concentrations of thrombopoietin (TPO) and interleukin-11 (IL-11). TPO and IL-11 concentrations were measured in cord blood sera of 12 neonates with FAITP, and 35 preterm and 25 term controls. TPO concentrations in the 12 patients (median 129 pg/mL, range 73 to 325 pg/mL) were similar to those of 35 healthy preterm neonates (median 183 pg/mL, range 71 to 290 pg/mL) and the 25 term controls (median 180 pg/mL, range 93 to 302 pg/mL), although the platelet counts were significantly lower in FAITP. TPO concentrations did not correlate with the platelet counts, platelet nadir after birth, or time to recovery of normal platelet count. IL-11 reached detectable concentrations only in four patients with FAITP (median 54 pg/mL). After birth, these patients had a more rapid recovery of the platelet count. Cord blood serum concentrations of TPO are not significantly elevated in FAITP and do not predict the severity of the thrombocytopenia. Elevated IL-11 may signal a more rapid platelet recovery in FAITP.

We present clinical and neuropathological findings in a female infant with Yunis-Varon syndrome (YVS) comprising absence of thumbs and halluces, aphalangia of fingers and toes, hypoplasia of clavicles, severely undermineralized skeleton (especially skull), microcephaly, and multiple nonskeletal anomalies. The patient also had a Dandy-Walker malformation, hydrocephalus, and hypertension, which were not reported previously in YVS. The infant excreted an abnormal unidentified oligosaccharide. The patient died at day 108 with severe neurological impairment. Autopsy showed prominent intraneuronal inclusions with vacuolar degeneration, mainly in the thalamic, dentate nuclei, cerebellar cortex, and inferior olivary nuclei. No storage phenomena were observed in other tissues. These findings strongly suggest that a lysosomal storage disorder is involved in the pathogenesis of Yunis-Varon syndrome. Am. J. Med. Genet. 95:157–160, 2000. © 2000 Wiley-Liss, Inc.

Thrombopoietin (TPO) regulates megakaryopoiesis and platelet production. In the adult, TPO is mainly produced by the liver and the kidneys. This study focuses on fetal and neonatal TPO mRNA expression. In 26 human fetuses and preterm neonates, samples from liver, kidney, spleen, lung, and bone marrow were extracted for total RNA. We measured platelet counts, TPO serum concentrations by enzyme-linked immunosorbent assay, and TPO mRNA contents by reverse transcription/competitive polymerase chain reaction. TPO mRNA concentrations per microgram total RNA were similar in liver, spleen, and bone marrow, slightly lower in kidney, and significantly lower in lung. When related to gram tissue, TPO mRNA levels were highest in the liver. Considering the total amount of TPO mRNA produced in liver, kidney, and spleen, the liver accounted for 95.3%. No correlations between TPO mRNA expression and serum TPO concentration, blood platelet count, or gestational age were observed. In conclusion, the liver is the primary site of TPO gene expression in human fetuses and neonates. The spleen may contribute to TPO production during fetal life. Like in the adult, TPO mRNA is expressed in fetal bone marrow.

Thrombopoietin (TPO) regulates megakaryopoiesis and platelet production. In the adult, TPO is mainly produced by the liver and the kidneys. This study focuses on fetal and neonatal TPO mRNA expression. In 26 human fetuses and preterm neonates, samples from liver, kidney, spleen, lung, and bone marrow were extracted for total RNA. We measured platelet counts, TPO serum concentrations by enzyme-linked immunosorbent assay, and TPO mRNA contents by reverse transcription/competitive polymerase chain reaction. TPO mRNA concentrations per microgram total RNA were similar in liver, spleen, and bone marrow, slightly lower in kidney, and significantly lower in lung. When related to gram tissue, TPO mRNA levels were highest in the liver. Considering the total amount of TPO mRNA produced in liver, kidney, and spleen, the liver accounted for 95.3%. No correlations between TPO mRNA expression and serum TPO concentration, blood platelet count, or gestational age were observed. In conclusion, the liver is the primary site of TPO gene expression in human fetuses and neonates. The spleen may contribute to TPO production during fetal life. Like in the adult, TPO mRNA is expressed in fetal bone marrow.

Based on animal experiments, a switch of the erythropoietin (EPO) production site from the liver in the fetus to the kidneys in the adult has been postulated. To study the switch in humans, we have quantitated EPO mRNA expression in liver, kidney, spleen, and bone marrow of human fetuses and neonates by means of a competitive polymerase chain reaction (PCR). Tissue samples from 66 routine postmortem examinations were obtained. EPO mRNA was expressed in 97% of the tissue specimen derived from the liver (n = 66) and in 93% of those from the kidneys (17 weeks of gestation until 18 months after birth; n = 59). For the first time the EPO gene was found expressed in vivo in human spleen (96% of 64 samples) and in fetal and neonatal bone marrow (81% of 21 samples). EPO mRNA expression in the kidneys increased significantly beyond 30 weeks of gestation (P < .05). Although there was a slight decrease in EPO mRNA content per g liver tissue towards birth, the liver accounted for about 80% of the total body EPO mRNA. The contribution of the spleen and bone marrow were minor compared with liver and kidneys. Our results indicate that in humans the liver is the primary site of EPO gene expression not only in fetal, but also in neonatal life. A significant increase of renal EPO mRNA expression after 30 weeks of gestation might indicate the beginning switch. © 1998 by The American Society of Hematology.

Based on animal experiments, a switch of the erythropoietin (EPO) production site from the liver in the fetus to the kidneys in the adult has been postulated. To study the switch in humans, we have quantitated EPO mRNA expression in liver, kidney, spleen, and bone marrow of human fetuses and neonates by means of a competitive polymerase chain reaction (PCR). Tissue samples from 66 routine postmortem examinations were obtained. EPO mRNA was expressed in 97% of the tissue specimen derived from the liver (n = 66) and in 93% of those from the kidneys (17 weeks of gestation until 18 months after birth; n = 59). For the first time the EPO gene was found expressed in vivo in human spleen (96% of 64 samples) and in fetal and neonatal bone marrow (81% of 21 samples). EPO mRNA expression in the kidneys increased significantly beyond 30 weeks of gestation (P< .05). Although there was a slight decrease in EPO mRNA content per g liver tissue towards birth, the liver accounted for about 80% of the total body EPO mRNA. The contribution of the spleen and bone marrow were minor compared with liver and kidneys. Our results indicate that in humans the liver is the primary site of EPO gene expression not only in fetal, but also in neonatal life. A significant increase of renal EPO mRNA expression after 30 weeks of gestation might indicate the beginning switch.

No abstract.

Thrombocytopenia affects 20–35% of patients admitted to Neonatal Intensive Care Units (NICUs). Platelet transfusions are usually administered to thrombocytopenic neonates at higher thresholds than those used for older children or adults, although there is a paucity of evidence to guide these decisions.In this study, we used a web-based survey to investigate the platelet transfusion thresholds used in level 1 NICUs (equivalent to level 3 in the US) in German-speaking European countries (Austria, Germany and Switzerland, AUT/GER/SUI). This survey was identical to the one previously used to investigate the transfusion practices of US neonatologists, thus allowing for a direct comparison of both populations. Eleven common clinical case scenarios of thrombocytopenia in preterm or term neonates were described and neonatologists were asked at which platelet count they would order a transfusion. For each case scenario, the median and the most frequently selected thresholds were determined. The Mann-Whitney-U-test was applied to compare the distribution of platelet transfusion thresholds between AUT/GER/SUI and US neonatologists. Univariate cumulative logit models (proportional odds model) were used to evaluate the differences between AUT/GER/SUI and US practices. In this analysis, an odds ratio >1 indicates an increased odd to select a higher threshold.At least one neonatologist from 100 of the 171 (58%) eligible level 1 NICUs (AUT n=2; GER n=92; SUI n=6) participated in the survey, for a total of 144 neonatologists. Their answers were compared with those of 1006 U.S. neonatologists previously surveyed. In 9 of the 11 scenarios, AUT/GER/SUI neonatologists selected substantially lower platelet transfusion thresholds than US neonatologists (P<.0001). For example, in a preterm infant (27 weeks of gestation and 950g) who was clinically stable and not bleeding at two days of life, the median platelet transfusion threshold was 30×109/L in AUT/GER/SUI vs. 50×109/L in the US (P<.0001). If the same infant had an intracranial hemorrhage, the most frequent threshold among AUT/GER/SUI neonatologists increased to 50×109/L, while most US neonatologists rose their trigger to 100×109/L (P<.0001). To quantify the differences in transfusion practices between the two groups of physicians, we then calculated (for each case scenario) the odds of US neonatologists selecting a higher transfusion category than their European colleagues. In nine of the eleven scenarios the odds ratios were between 3.25 and 5.05, reflecting the high likelihood that a US neonatologist would choose a higher transfusion threshold than an AUT/GER/SUI neonatologist facing the same patient. Only in two case scenarios (term infant with alloimmune thrombocytopenia and premature infant prior to lumbar puncture) the odds ratios were <2, reflecting more similar (although still significantly different) responses. Finally, in order to estimate the clinical impact of the observed differences, we recorded the platelet counts of every neonate admitted to our NICU over a six month period, and determined whether they would receive a platelet transfusion by extrapolating the answers of AUT/GER/SUI vs. US neonatologists to the first clinical vignette. Using this approach, we estimated that 18 out of 1000 neonates would be transfused if AUT/GER/SUI patterns were applied, compared to 33 transfused infants if US patterns were applied (1.8-fold increase).This first international comparative survey on platelet transfusion practices in neonates revealed substantially higher transfusion thresholds in the US than in AUT/GER/SUI. These differences might have substantial clinical implications, since platelet transfusions are associated with higher neonatal mortality and morbidity. In neonates with necrotizing enterocolitis and thrombocytopenia, receiving a higher number of platelet transfusions has been associated with a higher incidence of short bowel syndrome and cholestasis, and there is evidence to support the hypothesis that platelet transfusions can worsen bowel injury. Thus, well-designed clinical studies are needed to address the risks/benefits of these different approaches.Josephson: Immucor: Speakers Bureau.

Abstract 3356

No relevant conflicts of interest to declare.

Abstract 3610

The Wilms’ tumor suppressor, Wt1, has been implicated in the stage-specific quiescence and differentiation of hematopoietic progenitor cells. Since Wt1 deficiency compromises the proliferation and differentiation of erythroid blast-forming units (BFU-E) and colony-forming units (CFU-E), we analyzed the potential role of the transcriptionally active Wt1 isoform, Wt1(-KTS), in regulating the expression of the erythropoietin receptor (EpoR). CD117-positive hematopoietic progenitor cells isolated from the liver of Wt1-deficient murine embryos (Wt1−/−) at 11.5 dpc exhibited a 10-fold lower proliferative responsiveness to recombinant erythropoietin than CD117-positive cells from wild-type embryos (Wt1+/+), or embryos with heterozygous Wt1-deletion (Wt1+/−). Benzidine staining revealed a reduced fraction of hemoglobin-containing cells in Wt1-deficient cells in comparison to Wt1+/+ cells after 48 hrs of culture of CD117-positive cells in the presence of 6 U rhEpo/ml medium. Consistently, expression of the EpoR was significantly reduced in hematopoietic progenitor cells that lack Wt1 compared to Wt1+/+ cells. The decrease of EpoR mRNA was not due to a general down-regulation of gene expression in Wt1−/− CD117-positive cells, since levels of c-kit and c-mpl expression were normal. In the heart of wild-type and Wt1−/− embryos, which was analyzed as a non-hematopoietic tissue expressing both EpoR und Wt1, no difference in Epo-R expression was found. Transient transfection of Wt1(-KTS) into human hepatoma-derived HepG2 cells increased EpoR transcripts approximately 2-fold. A luciferase reporter plasmid carrying 309 bp of the proximal human EpoR promoter was stimulated 8-fold by co-transfection with Wt1(-KTS). The responsible cis-element in the EpoR promoter was identified by mutation analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. In summary, we identified the EpoR gene to be the first downstream target of Wt1 within the hematopoietic cytokine receptor superfamily. Our data indicate that activation of the EpoR gene by Wt1 is a critical mechanism in normal murine hematopoiesis. However, our data do not support a role for Wt1 in EpoR expression in non-erythoid tissues, such as the heart.

The Wilms' tumor suppressor, Wt1, has been implicated in the stage-specific quiescence and differentiation of hematopoietic progenitor cells. Since Wt1deficiency compromises the proliferation and differentiation of erythroid blast-forming units (BFU-E) and colony-forming units (CFU-E), we analyzed the potential role of the transcriptionally active Wt1 isoform, Wt1(-KTS), in regulating the expression of the erythropoietin receptor (EpoR). CD117-positive hematopoietic progenitor cells isolated from the liver of Wt1-deficient murine embryos (Wt1−/−) at 11.5 dpc exhibited a 10-fold lower proliferative responsiveness to recombinant erythropoietin than CD117-positive cells from wild-type embryos (Wt1+/+), or embryos with heterozygous Wt1-deletion (Wt1+/−). Benzidine staining revealed a reduced fraction of hemoglobin-containing cells in Wt1-deficient cells in comparison to Wt1+/+cells after 48 hrs of culture of CD117-positive cells in the presence of 6 U rhEpo/ml medium. Consistently, expression of the EpoRwas significantly reduced in hematopoietic progenitor cells that lack Wt1compared to Wt1+/+cells. The decrease of EpoRmRNA was not due to a general down-regulation of gene expression in Wt1−/−CD117-positive cells, since levels of c-kitand c-mplexpression were normal. In the heart of wild-type and Wt1−/−embryos, which was analyzed as a non-hematopoietic tissue expressing both EpoRund Wt1, no difference in Epo-Rexpression was found. Transient transfection of Wt1(-KTS) into human hepatoma-derived HepG2 cells increased EpoRtranscripts approximately 2-fold. A luciferase reporter plasmid carrying 309 bp of the proximal human EpoRpromoter was stimulated 8-fold by co-transfection with Wt1(-KTS). The responsible cis-element in the EpoRpromoter was identified by mutation analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. In summary, we identified the EpoRgene to be the first downstream target of Wt1 within the hematopoietic cytokine receptor superfamily. Our data indicate that activation of the EpoRgene by Wt1 is a critical mechanism in normal murine hematopoiesis. However, our data do not support a role for Wt1 in EpoRexpression in non-erythoid tissues, such as the heart.