Synergism of IL10R and TLR9 signaling affects gene expression, proliferation and metabolism in B cells: A comparative study of STAT3/NF-kB and c-Myc mediated effects
by Maren Feist
Date of Examination:2016-09-19
Date of issue:2016-10-11
Advisor:Prof. Dr. Dieter Kube
Referee:Prof. Dr. Detlef Doenecke
Referee:Prof. Dr. Jörg Großhans
Persistent Address:
http://dx.doi.org/10.53846/goediss-5867
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Abstract
English
The interplay of different signaling pathways activated by factors of the cellular environment and expression the proto-oncogene c-Myc (MYC) defines B cell fate in the germinal center (GC). Importantly, the same signaling networks are chronically active in different types of B-cell lymphoma. While the effects of aberrantly expressed MYC on cell proliferation and metabolism were intensively studied, less is known about the cross talk of pathways, like NF-kB and JAK/STAT signaling, in the context of B cell proliferation and metabolic reprogramming. Therefore, the aim of the study was to analyze (i) how chronic active factors of the GC microenvironment and their interaction networks can reprogram global gene expression (gGE) and metabolism of resting B cells to support B cell proliferation, (ii) what is common with MYC overexpression and (iii) whether this is important for lymphoma. Thus the P493-6 B cell line, carrying a conditional MYC expression construct, was stimulated with random combinations of GC derived factors as -IgM, CD40L, IGF1, CpG and IL10 to model different signaling pathway and Myc activation on the same genetic background. Linear regression analysis of gGE and metabolome changes in stimulated Myc depleted cells revealed qualitative similar but quantitative different changes on these parameters by the different stimulations. Thereby, greatest changes where observed after IL10+CpG stimulation due to a strong synergy on gGE and metabolome associated with sustained cell cycle entry and cell proliferation. Using small molecule inhibitors and RNAi mediated knockdown a dependency of IL10+CpG induced proliferation on NF-kB and JAK/STAT3 signaling was revealed. Furthermore, simultaneous binding of STAT3 and p65 to proximal promoter of the cell cycle regulator CDK4 but also the aspartate amino transaminase GOT2 was detected by chromatin immunoprecipitation. The increase in gGE mediated by IL10+CpG stimulation resembled Myc induced gene expression changes accompanied by a comparable cell proliferation but different glutamine metabolism. Herein, GOT2 was essential for cell proliferation of both conditions. However, glutamine tracing, respiration analysis and rescue experiments revealed distinct dependencies of proliferation on glutamine derived metabolites: In IL10+CpG treated cells a strong dependency of cell proliferation on glutamine derived aspartate and nucleotides was observed, whereas in MYC overexpressing cells a-ketoglutarate was most important for respiration and proliferation. Using CA-46, OCI-LY3 and L-428 lymphoma cell lines the important role of GOT2 for Myc and STAT3/NF-kB dependent proliferation but also the differences in glutamine usage in context of these pathways could be confirmed. Last of all, a high expression of GOT2 in proliferating centroblasts but also primary B cell lymphoma could be shown. Thereby, activated B cell like diffuse large B cell lymphoma (DLBCL) were characterized by higher GOT2 expression as germinal center like DLBCL. More importantly high GOT2 expression was associated with a worse clinical outcome of RCHOP treated DLBCL patients. Overall, a new role for IL10R and TLR9 signaling and the interaction of STAT3 and NF-kB signaling in gGE, proliferation and metabolism of B cells was identified that might also be important for lymphomagenesis.
Keywords: B cell; proliferation; metabolism; STAT3; NF-kB; c-Myc