Christof Dame

Regulation of Clock-Controlled Genes in Mammals.

The complexity of tissue- and day time-specific regulation of thousands of clock-controlled genes (CCGs) suggests that many regulatory mechanisms contribute to the transcriptional output of the circadian clock. We aim to predict these mechanisms using a large scale promoter analysis of CCGs. Our study is based on a meta-analysis of DNA-array data from rodent tissues. We searched in the promoter regions of 2065 CCGs for highly overrepresented transcription factor binding sites. In order to compensate the relatively high GC-content of CCG promoters, a novel background model to avoid a bias towards GC-rich motifs was employed. We found that many of the transcription factors with overrepresented binding sites in CCG promoters exhibit themselves circadian rhythms. Among the predicted factors are known regulators such as CLOCK∶BMAL1, DBP, HLF, E4BP4, CREB, RORα and the recently described regulators HSF1, STAT3, SP1 and HNF-4α. As additional promising candidates of circadian transcriptional regulators PAX-4, C/EBP, EVI-1, IRF, E2F, AP-1, HIF-1 and NF-Y were identified. Moreover, GC-rich motifs (SP1, EGR, ZF5, AP-2, WT1, NRF-1) and AT-rich motifs (MEF-2, HMGIY, HNF-1, OCT-1) are significantly overrepresented in promoter regions of CCGs. Putative tissue-specific binding sites such as HNF-3 for liver, NKX2.5 for heart or Myogenin for skeletal muscle were found. The regulation of the erythropoietin (Epo) gene was analysed, which exhibits many binding sites for circadian regulators. We provide experimental evidence for its circadian regulated expression in the adult murine kidney. Basing on a comprehensive literature search we integrate our predictions into a regulatory network of core clock and clock-controlled genes. Our large scale analysis of the CCG promoters reveals the complexity and extensiveness of the circadian regulation in mammals. Results of this study point to connections of the circadian clock to other functional systems including metabolism, endocrine regulation and pharmacokinetics.

Megakaryocytes constitute a functional component of a plasma cell niche in the bone marrow

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.

Primary and secondary thrombocytosis in childhood

This review summarizes current data on the pathomechanisms and clinical aspects of primary and secondary thrombocytosis in childhood. Primary thrombocytosis is extremely rare in childhood, mostly diagnosed at the beginning of the second decade of life. As in adults, the criteria of the Polycythemia Vera Group are appropriate to diagnose primary thrombocytosis. The pathomechansims of non‐familial forms are complex and include spontaneous formation of megakaryopoietic progenitors and increased sensitivity to thrombopoietin (Tpo). Familial forms can be caused by mutations in Tpo or Tpo receptor (c‐mpl) genes. These mutations result in overexpression of Tpo, sustained intracellular signalling or disturbed regulation of circulating Tpo. Treatment of primary thrombocytosis is not recommended if platelet counts are <1500/nl and bleeding or thrombosis did not occur in patients history. In severe cases, decision on treatment should weigh potential risks of treatment options (hydroxyurea, anagrelide) against expected benefits for preventing thrombosis or haemorrhage. Secondary thrombocytosis is frequent in children, in particular in the first decade of life. Hepatic Tpo production is stimulated in acute response reaction to a variety of disorders. Thrombosis prophylaxis is not required, even at platelet counts >1000/nl, except for cases with additional prothrombotic risk factors.

Erythropoietin gene expression in different areas of the developing human central nervous system

UNLABELLED Evidence from cell culture and animal experiments suggests a neuroprotective and neurotrophic function of erythropoietin (EPO). We have quantitated the distribution of EPO mRNA expression in the developing human central nervous system (CNS). PATIENTS AND METHODS Up to seven biopsies from different areas of the CNS of four preterm fetuses (gestational age 23-37 weeks) were obtained at routine postmortem examinations. EPO mRNA was quantitated by competitive PCR in samples from the CNS, the kidneys, and the liver where the EPO gene is predominantly expressed at this gestational age. RESULTS EPO mRNA was most abundant in one sample from the cerebellum (0.29 amol/microg total RNA [amol=10(-18)mol]) and two from the pituitary gland (0.23 amol/microg total RNA), but levels varied considerably. EPO mRNA in the cortex cerebri (median 0.12 amol/microg total RNA; n=4) dominated over the expression in the corpora amygdala (median 0.05 amol/microg total RNA; n=4), the hippocampus (median 0.03 amol/microg total RNA; n=4), or the basal ganglia (median 0.01 amol/microg total RNA; n=3). Only little EPO mRNA (<0.01 and 0.06 amol/microg total RNA) was found in the spinal cord. EPO mRNA levels in the cerebellum, pituitary gland, or the cerebral cortex were within the same range as in the liver (0.03-1.67 amol/microg total RNA; n=4), or the kidneys (0.06-0.79 amol/microg total RNA; n=4). CONCLUSION We found the EPO gene expressed throughout the fetal human CNS. Our data provide the basis to discuss a function for EPO in the brain of humans as well.

Screening for mutations in transcription factors in a Czech cohort of 170 patients with congenital and early-onset hypothyroidism: identification of a novel PAX8 mutation in dominantly inherited early-onset non-autoimmune hypothyroidism.

OBJECTIVE Mutations in NKX2.1, NKX2.5, FOXE1 and PAX8 genes, encoding for transcription factors involved in the development of the thyroid gland, have been identified in a minority of patients with syndromic and non-syndromic congenital hypothyroidism (CH). DESIGN In a phenotype-selected cohort of 170 Czech paediatric and adolescent patients with non-goitre CH, including thyroid dysgenesis, or non-goitre early-onset hypothyroidism, PAX8, NKX2.1, NKX2.5, FOXE1 and HHEX genes were analysed for mutations. METHODS NKX2.1, NKX2.5, FOXE1 and HHEX genes were directly sequenced in patients with syndromic CH. PAX8 mutational screening was performed in all 170 patients by single-stranded conformation polymorphism, followed by direct sequencing of samples with abnormal findings. The R52P PAX8 mutation was functionally characterized by DNA binding studies. RESULTS We identified a novel PAX8 mutation R52P, dominantly inherited in a three-generation pedigree and leading to non-congenital, early-onset, non-goitre, non-autoimmune hypothyroidism with gradual postnatal regression of the thyroid size and function. The R52P PAX8 mutation results in the substitution of a highly conserved residue of the DNA-binding domain with a loss-of-function effect. CONCLUSIONS The very low frequency of genetic defects in a population-based cohort of children affected by non-goitre congenital and early-onset hypothyroidism, even in a phenotype-focussed screening study, suggests the pathogenetic role of either non-classic genetic mechanisms or the involvement of genes unknown so far. Identification of a novel PAX8 mutation in a particular variant of non-congenital early-onset hypothyroidism indicates a key function of PAX8 in the postnatal growth and functional maintenance of the thyroid gland.

Hypoxia-Inducible Factor 1α Determines Gastric Cancer Chemosensitivity via Modulation of p53 and NF-κB

Background Reduced chemosensitivity of solid cancer cells represents a pivotal obstacle in clinical oncology. Hence, the molecular characterization of pathways regulating chemosensitivity is a central prerequisite to improve cancer therapy. The hypoxia-inducible factor HIF-1α has been linked to chemosensitivity while the underlying molecular mechanisms remain largely elusive. Therefore, we comprehensively analysed HIF-1αs role in determining chemosensitivity focussing on responsible molecular pathways. Methodology and Principal Findings RNA interference was applied to inactivate HIF-1α or p53 in the human gastric cancer cell lines AGS and MKN28. The chemotherapeutic agents 5-fluorouracil and cisplatin were used and chemosensitivity was assessed by cell proliferation assays as well as determination of cell cycle distribution and apoptosis. Expression of p53 and p53 target proteins was analyzed by western blot. NF-κB activity was characterized by means of electrophoretic mobility shift assay. Inactivation of HIF-1α in gastric cancer cells resulted in robust elevation of chemosensitivity. Accordingly, HIF-1α-competent cells displayed a significant reduction of chemotherapy-induced senescence and apoptosis. Remarkably, this phenotype was completely absent in p53 mutant cells while inactivation of p53 per se did not affect chemosensitivity. HIF-1α markedly suppressed chemotherapy-induced activation of p53 and p21 as well as the retinoblastoma protein, eventually resulting in cell cycle arrest. Reduced formation of reactive oxygen species in HIF-1α-competent cells was identified as the molecular mechanism of HIF-1α-mediated inhibition of p53. Furthermore, loss of HIF-1α abrogated, in a p53-dependent manner, chemotherapy-induced DNA-binding of NF-κB and expression of anti-apoptotic NF-κB target genes. Accordingly, reconstitution of the NF-κB subunit p65 reversed the increased chemosensitivity of HIF-1α-deficient cells. Conclusion and Significance In summary, we identified HIF-1α as a potent regulator of p53 and NF-κB activity under conditions of genotoxic stress. We conclude that p53 mutations in human tumors hold the potential to confound the efficacy of HIF-1-inhibitors in cancer therapy.

Pathogenic Long-Lived Plasma Cells and Their Survival Niches in Autoimmunity, Malignancy, and Allergy

Long-lived plasma cells survive in a protected microenvironment for years or even a lifetime and provide humoral memory by establishing persistent Ab titers. Long-lived autoreactive, malignant, and allergen-specific plasma cells are likewise protected in their survival niche and are refractory to immunosuppression, B cell depletion, and irradiation. Their elimination remains an essential therapeutic challenge. Recent data indicate that long-lived plasma cells reside in a multicomponent plasma cell niche with a stable mesenchymal and a dynamic hematopoietic component, both providing essential soluble and membrane-bound survival factors. Alternative niches with different hematopoietic cell components compensate fluctuations of single cell types but may also harbor distinct plasma cell subsets. In this Brief Review, we discuss conventional therapies in autoimmunity and multiple myeloma in comparison with novel drugs that target plasma cells and their niches. In the future, such strategies may enable the specific depletion of pathogenic plasma cells while leaving the protective humoral memory intact.

Thrombopoietin gene expression in the developing human central nervous system.

Thrombopoietin gene expression in the human adult central nervous system (CNS) appears to be locally restricted. The aim of this study was to identify areas of thrombopoietin expression in the developing human CNS, and to compare the thrombopoietin mRNA content in the CNS to that in liver and kidneys as major sites of thrombopoietin production. Thrombopoietin protein concentrations in the cerebrospinal fluid (CSF) were measured by ELISA. In 14 fetuses and neonates with perinatal death, thrombopoietin mRNA expression was measured by competitive RT-PCR. Thrombopoietin mRNA was expressed in 29 of 32 specimens taken from the CNS. The following ranking of the intensity of expression in the CNS was possible: Spinal cord=cerebellum=cortex>>pituitary gland>>>brain stem=corpora amygdala=hippocampus. Whereas in the latter three tissues only trace amounts of thrombopoietin transcripts were detectable, thrombopoietin mRNA levels in the spinal cord were comparable to levels in liver and kidney. Thrombopoietin protein concentrations in CSF ranged between 41 and 75 pg/ml. In the developing human CNS, the thrombopoietin gene is abundantly expressed. Considering that thrombopoietin contains a neurotrophic sequence, it may well play a role in neuronal cell biology.

Erythropoietin-receptor gene regulation in neuronal cells.

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.

Safety of Early High-Dose Recombinant Erythropoietin for Neuroprotection in Very Preterm Infants.

OBJECTIVE To investigate the safety and short term outcome of high dose recombinant human erythropoietin (rhEpo) given shortly after birth and subsequently over the first 2 days for neuroprotection to very preterm infants. STUDY DESIGN Randomized, double masked phase II trial. Preterm infants (gestational age 26 0/7-31 6/7 weeks) were given rhEpo (nt = 229; 3000 U/kg body weight) or NaCl 0.9% (nc = 214) intravenously at 3, 12-18, and 36-42 hours after birth. RESULTS There were no relevant differences between the groups for short-term outcomes such as mortality, retinopathy of prematurity, intraventricular hemorrhage, sepsis, necrotizing enterocolitis, and bronchopulmonary dysplasia. At day 7-10, we found significantly higher hematocrit values, reticulocyte, and white blood cell counts, and a lower platelet count in the rhEpo group. CONCLUSIONS Early high-dose rhEpo administration to very premature infants is safe and causes no excess in mortality or major adverse events. TRIAL REGISTRATION ClinicalTrials.gov: NCT00413946.