Abstract :
[en] The focus of this thesis is the understanding of the molecular mechanisms related to the recently discovered anti-inflammatory function of vitamin D and its ability to suppress tumor growth and chronic inflammation. Our aim is the analysis of the modulatory effects of 1,25-dihydroxyvitamin D3 (1 ,25(OH)2D3 or 1,25-D3) on gene expression in THP-1 cells in a TNF- alpha-induced inflammatory state. This monocyte cell line was used as a model system for potential monocyte-endothelial interactions. For the gene expression profiling, the cells were treated with TNF-alpha and 1,25-D3 and analysed at three different time points, 1h, 4h and 24h, by using Illumina Human HT-12 v4.0 micro arrays to achieve specific changes in the mRNA expression levels. The validation of the micro array data was performed by RT-qPCR and resulted in gene clusters regulating cellular growth, cell development and cellular movement. Specifically, we were able to identify the genes BCL2, CCL2, MGAT3, NCF1, ORM1 and RAB37 and their expression levels were analysed in the presence and absence of vitamin D and TNF-alpha using quantitative PCR. Furthermore, the expression of the acute phase plasma alpha-globulin glycoprotein alpha-1-acid glycoprotein (AGP) encoded by ORM1 gene, and beta -1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase (Gnt-III) encoded by MGAT3 gene were investigated via quantitative western blot and ELISA experiments after treatment of cells with 1,25-D3 and TNF-alpha , where post-stimulation protein levels evolved correspondingly for the former protein.
Subsequent invasion assays showed an inhibitory effect on invasion efficacy of THP-1 cells after knockdown of ORM1 with specific siRNA. Moreover, microarray data and database knowledge were used to generate a network containing previously identified and validated target genes.
This candidate network is modeled computationally with a biochemical reaction-based Boolean model. It was further curated by an evolutionary algorithm-based pruning step removing unnecessary nodes and interactions, called MORGEN. This ultimately resulted in a model allowing the qualitative description of the observed gene expression patterns. In conclusion, 1,25-D3 affects a range of genes in THP-1 cells in TNF- alpha-induced-inflammatory state. We showed that it potentially alters transendothelial migration of monocytic cells, among other ways, via increase of AGP expression after exposure of the cells to both 1,25-D3 and TNF- alpha. Furthermore, the generated model could be reduced significantly via implementation of MORGEN. This promises a valuable new tool for network reduction, an essential step for generation of elaborated models with computationally feasible properties that help to understand the complex network and deregulation of signaling networks in different diseases.
