Katja Fiedler

MYC stimulates EZH2 expression by repression of its negative regulator miR-26a

The MYC oncogene, which is commonly mutated/amplified in tumors, represents an important regulator of cell growth because of its ability to induce both proliferation and apoptosis. Recent evidence links MYC to altered miRNA expression, thereby suggesting that MYC-regulated miRNAs might contribute to tumorigenesis. To further analyze the impact of MYC-regulated miRNAs, we investigated a murine lymphoma model harboring the MYC transgene in a Tet-off system to control its expression. Microarray-based miRNA expression profiling revealed both known and novel MYC targets. Among the miRNAs repressed by MYC, we identified the potential tumor suppressor miR-26a, which possessed the ability to attenuate proliferation in MYC-dependent cells. Interestingly, miR-26a was also found to be deregulated in primary human Burkitt lymphoma samples, thereby probably being of clinical relevance. Although today only few miRNA targets have been identified in human disease, we could show that ectopic expression of miR-26a influenced cell cycle progression by targeting the bona fide oncogene EZH2, a Polycomb protein and global regulator of gene expression yet unknown to be regulated by miRNAs. Thus, in addition to directly targeting protein-coding genes, MYC modulates genes important to oncogenesis via deregulation of miRNAs, thereby vitally contributing to MYC-induced lymphomagenesis.

Hepatic activation of IKK/NFκB signaling induces liver fibrosis via macrophage-mediated chronic inflammation.

Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor‐κB (NF‐κB) system is activated in response to several of these stresses, we hypothesized that NF‐κB activation in hepatocytes may contribute to fibrosis development. To activate the NF‐κB signaling pathway in a time‐ and cell‐type‐specific manner in the liver, we crossed transgenic mice carrying the tetracycline‐responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double‐transgenic mice displayed doxycycline‐regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF‐κB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up‐regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate‐liposomes attenuated NF‐κB‐induced liver fibrosis in a liver‐injury‐independent manner. Conclusion: Our results revealed that hepatic activation of IKK/NF‐κB is sufficient to induce liver fibrosis by way of macrophage‐mediated chronic inflammation. Therefore, agents controlling the hepatic NF‐κB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases. (HEPATOLOGY 2012;56:1117–1128)

Neutrophil development and function critically depend on Bruton tyrosine kinase in a mouse model of X-linked agammaglobulinemia

Bruton tyrosine kinase (Btk) is essential for B cell development and function and also appears to be important for myeloid cells. The bone marrow of Btk-deficient mice shows enhanced granulopoiesis compared with that of wild-type mice. In purified granulocyte-monocyte-progenitors (GMP) from Btk-deficient mice, the development of granulocytes is favored at the expense of monocytes. However, Btk-deficient neutrophils are impaired in maturation and function. Using bone marrow chimeras, we show that this defect is cell-intrinsic to neutrophils. In GMP and neutrophils, Btk plays a role in GM-CSF- and Toll-like receptor-induced differentiation. Molecular analyses revealed that expression of the lineage-determining transcription factors C/EBPα, C/EBPβ, and PU.1, depends on Btk. In addition, expression of several granule proteins, including myeloperoxidase, neutrophilic granule protein, gelatinase and neutrophil elastase, is Btk-dependent. In the Arthus reaction, an acute inflammatory response, neutrophil migration into tissues, edema formation, and hemorrhage are significantly reduced in Btk-deficient animals. Together, our findings implicate Btk as an important regulator of neutrophilic granulocyte maturation and function in vivo.

Long-Term IKK2/NF-κB Signaling in Pancreatic β-Cells Induces Immune-Mediated Diabetes

Type 1 diabetes is a multifactorial inflammatory disease in genetically susceptible individuals characterized by progressive autoimmune destruction of pancreatic β-cells initiated by yet unknown factors. Although animal models of type 1 diabetes have substantially increased our understanding of disease pathogenesis, heterogeneity seen in human patients cannot be reflected by a single model and calls for additional models covering different aspects of human pathophysiology. Inhibitor of κB kinase (IKK)/nuclear factor-κB (NF-κB) signaling is a master regulator of inflammation; however, its role in diabetes pathogenesis is controversially discussed by studies using different inhibition approaches. To investigate the potential diabetogenic effects of NF-κB in β-cells, we generated a gain-of-function model allowing conditional IKK2/NF-κB activation in β-cells. A transgenic mouse model that expresses a constitutively active mutant of human IKK2 dependent on Pdx-1 promoter activity (IKK2-CAPdx-1) spontaneously develops full-blown immune-mediated diabetes with insulitis, hyperglycemia, and hypoinsulinemia. Disease development involves a gene expression program mimicking virus-induced diabetes and allergic inflammatory responses as well as increased major histocompatibility complex class I/II expression by β-cells that could collectively promote diabetes development. Potential novel diabetes candidate genes were also identified. Interestingly, animals successfully recovered from diabetes upon transgene inactivation. Our data give the first direct evidence that β-cell–specific IKK2/NF-κB activation is a potential trigger of immune-mediated diabetes. Moreover, IKK2-CAPdx-1 mice provide a novel tool for studying critical checkpoints in diabetes pathogenesis and mechanisms governing β-cell degeneration/regeneration.

Octamer-dependent transcription in T cells is mediated by NFAT and NF-κB

The transcriptional co-activator BOB.1/OBF.1 was originally identified in B cells and is constitutively expressed throughout B cell development. BOB.1/OBF.1 associates with the transcription factors Oct1 and Oct2, thereby enhancing octamer-dependent transcription. In contrast, in T cells, BOB.1/OBF.1 expression is inducible by treatment of cells with PMA/Ionomycin or by antigen receptor engagement, indicating a marked difference in the regulation of BOB.1/OBF.1 expression in B versus T cells. The molecular mechanisms underlying the differential expression of BOB.1/OBF.1 in T and B cells remain largely unknown. Therefore, the present study focuses on mechanisms controlling the transcriptional regulation of BOB.1/OBF.1 and Oct2 in T cells. We show that both calcineurin- and NF-κB-inhibitors efficiently attenuate the expression of BOB.1/OBF.1 and Oct2 in T cells. In silico analyses of the BOB.1/OBF.1 promoter revealed the presence of previously unappreciated combined NFAT/NF-κB sites. An array of genetic and biochemical analyses illustrates the involvement of the Ca2+/calmodulin-dependent phosphatase calcineurin as well as NFAT and NF-κB transcription factors in the transcriptional regulation of octamer-dependent transcription in T cells. Conclusively, impaired expression of BOB.1/OBF.1 and Oct2 and therefore a hampered octamer-dependent transcription may participate in T cell-mediated immunodeficiency caused by the deletion of NFAT or NF-κB transcription factors.

Cardiac-Specific Activation of IKK2 Leads to Defects in Heart Development and Embryonic Lethality.

The transcription factor NF-κB has been associated with a range of pathological conditions of the heart, mainly based on its function as a master regulator of inflammation and pro-survival factor. Here, we addressed the question what effects activation of NF-κB can have during murine heart development. We expressed a constitutively active (CA) mutant of IKK2, the kinase activating canonical NF-κB signaling, specifically in cardiomyocytes under the control of the α-myosin heavy chain promoter. Expression of IKK2-CA resulted in embryonic lethality around E13. Embryos showed defects in compact zone formation and the contractile apparatus, and overall were characterized by widespread inflammation with infiltration of myeloid cells. Gene expression analysis suggested an interferon type I signature, with increased expression of interferon regulatory factors. While apoptosis of cardiomyocytes was only increased at later stages, their proliferation was decreased early on, providing an explanation for the disturbed compact zone formation. Mechanistically, this could be explained by activation of the JAK/STAT axis and increased expression of the cell cycle inhibitor p21. A rescue experiment with an IκBα superrepressor demonstrated that the phenotype was dependent on NF-κB. We conclude that activation of NF-κB is detrimental during normal heart development due to excessive activation of pro-inflammatory pathways.

Mechanisms Controlling Hematopoiesis

Hematopoiesis – the generation of blood cells that proceeds mainly in the bone marrow is a well-controlled process constantly occurring throughout the live of the mammalian organism. Generally, blood cells are relatively short-lived cells with a life span ranging from few hours to several weeks causing the need for a sustained replenishment of functional erythroid, lymphoid and myeloid cells. The development of mature hematopoietic cells in a hierarchical manner from a pluripotent hematopoietic stem cell over multipotent progenitors that further develop to oligopotent and then to lineage-restricted progenitors requires several control mechanisms at different levels. Transcription factors important for the expression of lineage-specific genes play a major role in the regulation of hematopoietic stem cell maintenance as well as hematopoietic lineage decision. Moreover, the discovery of so-called master transcription factors determining the fate of a terminally differentiated cell population indicates on one side the coordinated processes of hematopoietic cell differentiation but on the other side the complex mechanisms of transcriptional activation and/or repression of specific genes. However, what in turn regulates the expression of transcription factors that finally determine the lineage and differentiation choice of a certain progenitor or immature cell? Is the development into one or another cell type a definitive event or is there some plasticity observed? Which factors are necessary and which sufficient for hematopoietic cell differentiation? These and several other important questions concerning the regulation of development and differentiation of blood cells will be discussed. This chapter summarizes the current knowledge about cell intrinsic, environmental as well as epigenetic mechanisms involved in the control of hematopoiesis under homeostatic as well as infectious conditions.