Genomics based analysis of interactions between developing B-lymphocytes and stromal cells reveal complex interactions and two-way communication.

BACKGROUND: The use of functional genomics has largely increased our understanding of cell biology and promises to help the development of systems biology needed to understand the complex order of events that regulates cellular differentiation in vivo. One model system clearly dependent on the integration of extra and intra cellular signals is the development of B-lymphocytes from hematopoietic stem cells in the bone marrow. This developmental pathway involves several defined differentiation stages associated with specific expression of genes including surface markers that can be used for the prospective isolation of the progenitor cells directly from the bone marrow to allow for ex vivo gene expression analysis. The developmental process can be simulated in vitro making it possible to dissect information about cell/cell communication as well as to address the relevance of communication pathways in a rather direct manner. Thus we believe that B-lymphocyte development represents a useful model system to take the first steps towards systems biology investigations in the bone marrow. RESULTS: In order to identify extra cellular signals that promote B lymphocyte development we created a database with approximately 400 receptor ligand pairs and software matching gene expression data from two cell populations to obtain information about possible communication pathways. Using this database and gene expression data from NIH3T3 cells (unable to support B cell development), OP-9 cells (strongly supportive of B cell development), pro-B and pre-B cells as well as mature peripheral B-lineage cells, we were able to identify a set of potential stage and stromal cell restricted communication pathways. Functional analysis of some of these potential ways of communication allowed us to identify BMP-4 as a potent stimulator of B-cell development in vitro. Further, the analysis suggested that there existed possibilities for progenitor B cells to send signals to the stroma. The functional consequences of this were investigated by co-culture experiments revealing that the co-incubation of stromal cells with B cell progenitors altered both the morphology and the gene expression pattern in the stromal cells. CONCLUSIONS: We believe that this gene expression data analysis method allows for the identification of functionally relevant interactions and therefore could be applied to other data sets to unravel novel communication pathways.

The CD53 and CEACAM-1 genes are genetic targets for early B cell factor.

Early B cell factor (EBF)-1 is a transcription factor known to be of critical importance for early B lymphocyte development. EBF-1 has been shown to directly interact with and regulate expression of a set of genes involved in the functional formation of the pre-B cell receptor, but the dramatic phenotype observed in the EBF-1-deficient mice suggests that several additional genes are activated by this protein. In order to identify additional target genes for EBF-1, we transduced a hematopoietic progenitor cell line, BaF/3, with an EBF-1-encoding retrovirus and investigated the induced gene expression pattern by micro-arrays. This analysis suggested that among others, the CD53 and the carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-1 genes both were induced by ectopic expression of EBF-1. Identification of the 5' end of the cDNA enabled the identification of promoter elements with functional binding sites for EBF-1 and ability to respond to EBF-1 expression in transient transfection assays. These data suggest that CD53 and CEACAM-1 are direct genetic targets for EBF-1, providing additional information concerning the activity of this crucial transcription factor in hematopoiesis.

The Cxcl12, periostin, and Ccl9 genes are direct targets for early B-cell factor in OP-9 stroma cells.

The development of blood cells from hematopoietic stem cells in the bone marrow is dependent on communication with bone marrow stroma cells, making these cells central for the appropriate regulation of hematopoiesis. To identify transcription factors that may play a role in gene regulation in stroma cells, we performed comparative gene expression analysis of fibroblastic NIH3T3 cells, unable to support hematopoiesis in vitro, and OP-9 stroma cells, highly efficient in this regard. These experiments revealed that transcription factors of the early B cell factor (EBF) family were highly expressed in OP-9 cells as compared with the NIH3T3 cells. To identify potential targets genes for EBF proteins in stroma cells, we overexpressed EBF in fibroblasts and analyzed the pattern of induced genes by microarray analysis. This revealed that EBF was able to up-regulate expression of among others the Cxcl12, Ccl9, and Periostin genes. The identification of relevant promoters revealed that they all contained functional EBF binding sites able to interact with EBF in OP-9 cells. Furthermore, ectopic expression of a dominant negative EBF protein or antisense EBF-1 RNA in OP-9 stroma cells resulted in reduced expression of these target genes. These data suggest that EBF proteins might have dual roles in hematopoiesis acting both as intrinsic regulators of B-lymphopoiesis and as regulators of genes in bone marrow stroma cells.

Immune status and inflammatory response differ locally and systemically in severe acute pancreatitis.

OBJECTIVE: Acute pancreatitis (AP) is an inflammatory disorder that develops a complex cascade of immunological events. The local and systemic immune status and inflammatory response might contribute to the understanding of underlying pathophysiological mechanisms and potential treatment. MATERIAL AND METHODS: Severe AP was induced by intraductal perfusion of 5% sodium taurodeoxycholate in rats. mRNA expression of cytokines and chemokines was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and NF-kappaB activation was assessed by electrophoretic mobility shift assay in fresh pancreatic acini and circulating monocytes 1, 3, 6 or 9 h after sham operation, induction of AP or N-acetylcysteine (NAC) pretreatment. Flow cytometry was performed on cells obtained from the peripheral blood. RESULTS: An inverse relationship in pancreatic and circulating monocytic NF-kappaB activation was detected 6 and 9 h after induction of AP. NAC further suppressed monocytic NF-kappaB activation induced by AP as seen 9 h after induction of AP. A marked constitutive increase in the expression of IL-6, CINC and MCP-1 was seen in pancreatic acini, whereas no change in mRNA expression of inflammatory mediators was observed in circulating monocytes 6 h after induction of AP. Flow cytometry further confirmed the altered function of circulating monocytes. CONCLUSIONS: The different immune status and inflammatory response in the pancreas and circulating monocytes improve the understanding of the mechanisms by which systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS) develop in severe AP. A potential therapeutic approach could be to restore the functional capacity of the immune system in AP. The use of an NF-kappaB inhibitor, preferentially reaching the local inflammatory foci, could be a potential future way of intervention.

Gene expression analysis suggests that EBF-1 and PPARgamma2 induce adipogenesis of NIH-3T3 cells with similar efficiency and kinetics.

Differentiation of multipotent mesenchymal stem cells into lipid-accumulating adipocytes is a physiological process induced by transcription factors in combination with hormonal stimulation. We have used Affymetrix microarrays to compare the adipogenic differentiation pathways of NIH-3T3 fibroblasts induced to undergo in vitro differentiation by ectopic expression of early B cell factor (EBF)-1 or peroxisome proliferator-activated receptor (PPAR)gamma2. These experiments revealed that commitment to the adipogenic pathway in the NIH-3T3 cells was not reflected in gene expression until 4 days after induction of differentiation. Furthermore, gene expression patterns at the earlier time points after stimulation indicated that EBF-1 and PPARgamma2 induced different sets of genes, while the similarities increased upon differentiation, and that several genes linked to adipocyte differentiation were also transiently induced in the vector-transduced cells. These data suggest that the initial activation of genes associated with adipocyte development is independent of commitment to the adipogenic pathway and that EBF-1 and PPARgamma2 induce adipocyte differentiation with comparable kinetics and efficiency.

Neuroblastoma and pre-B lymphoma cells share expression of key transcription factors but display tissue restricted target gene expression.

BACKGROUND: Transcription factors are frequently involved in the process of cellular transformation, and many malignancies are characterized by a distinct genetic event affecting a specific transcription factor. This probably reflects a tissue specific ability of transcription factors to contribute to the generation of cancer but very little is known about the precise mechanisms that governs these restricted effects. METHODS: To investigate this selectivity in target gene activation we compared the overall gene expression patterns by micro-array analysis and expression of target genes for the transcription factor EBF in lymphoma and neuroblastoma cells by RT-PCR. The presence of transcription factors in the different model cell lines was further investigated by EMSA analysis. RESULTS: In pre-B cells mb-1 and CD19 are regulate by EBF-1 in collaboration with Pax-5 and E-proteins. We here show that neuroblastoma cells express these three, for B cell development crucial transcription factors, but nevertheless fail to express detectable levels of their known target genes. Expression of mb-1 could, however, be induced in neuroblastoma cells after disruption of the chromatin structure by treatment with 5-azacytidine and Trichostatin A. CONCLUSION: These data suggest that transcription factors are able to selectively activate target genes in different tissues and that chromatin structure plays a key role in the regulation of this activity.

Olf/EBF proteins are expressed in neuroblastoma cells: potential regulators of the Chromogranin A and SCG10 promoters.

The childhood malignancy neuroblastoma is derived from developmentally arrested sympathetic nervous system precursor cells. To obtain further insight into the molecular processes involved in the formation of these tumors, we decided to investigate the functional role of Olf/EBF (O/E) transcription factors in human neuroblastoma cells. We here report that O/E-1 and O/E-2 are expressed at variable levels in neuroblastoma cell lines and that O/E proteins could be identified by electrophoretic mobility shift assays. To identify potential neuronal target genes for O/E proteins in neuroblastoma cells we investigated the ability of a set of neuronal promoters to interact with O/E-1 in electrophoretic mobility shift assays. This analysis suggested that the Chromogranin A (CgA) and SCG10 promoters both contained binding sites for O/E-1. O/E-1 was able to activate the CgA promoter in vivo and mutation of the O/E-1 binding site in the CgA promoter reduced the functional activity of the element to about 60% of the wild-type in neuroblastoma cells, supporting the idea that O/E proteins may be involved in the control of the CgA promoter. Furthermore, overexpression of O/E-1 in hippocampal progenitor cells led to neurite outgrowth, indicative of a role for O/E proteins in neuronal differentiation.

Pearson correlation analysis of microarray data allows for the identification of genetic targets for early B-cell factor.

B lymphocyte development is a complex biological process critically dependent on the transcription factor early B cell factor (EBF). To deepen understanding of the roles for EBF in this process, we have used Pearson correlation analysis to evaluate microarray data from a set of mouse B lymphoid cell lines representing different stages of development. Comparing the expression pattern of EBF to that of the other genes in the data set revealed that VpreB1, mb-1, and lambda5, all known target genes, presented high correlation values to EBF. High correlations were also seen for the VpreB3 and CD19 genes and biochemical as well as functional data supported that they are target genes for EBF even though the expression of CD19 was critically dependent of Pax-5. We also obtained evidence for extensive collaborative actions of EBF and E47 even though microarray analysis of hematopoetic progenitor cells ectopically expressing these proteins suggested that they activated only a subset of pre-B cell restricted genes.

RNA analysis of B cell lines arrested at defined stages of differentiation allows for an approximation of gene expression patterns during B cell development.

The development of a mature B lymphocyte from a bone marrow stem cell is a highly ordered process involving stages with defined features and gene expression patterns. To obtain a deeper understanding of the molecular genetics of this process, we have performed RNA expression analysis of a set of mouse B lineage cell lines representing defined stages of B cell development using Affymetrix microarrays. The cells were grouped based on their previously defined phenotypic features, and a gene expression pattern for each group of cell lines was established. The data indicated that the cell lines representing a defined stage generally presented a high similarity in overall expression profiles. Numerous genes could be identified as expressed with a restricted pattern using dCHIP-based, quantitative comparisons or presence/absence-based, probabilistic state analysis. These experiments provide a model for gene expression during B cell development, and the correctly identified expression patterns of a number of control genes suggest that a series of cell lines can be useful tools in the elucidation of the molecular genetics of a complex differentiation process.