Microarray Metanalysis and Gene Regulatory inference of LPS transactivation of macrophage 1--hydroxylase

MARTINELLI, R.; DAURELIO, L.D.; ESTEBAN, L.

Bariloche

Congreso; 5ta Conferencia Argentina de Bioinformática y Biología Computacional (VCAB2C); 2014

Asociación Argentina de Bioinformática y Biología Computacional (A2B2C)

Background. 25-Hydroxyvitamin-D can be activated to 1,25-dihydroxyvitamin-D3 [1,25(OH) 2 D3] by the rate-limiting enzyme 1- -hydroxylase. Particularly, in cells of the immune system this enzime is under control of immune stimuli. In pathological situations, such tuberculosis, this can lead to systemic excess of 1,25(OH) 2 D3 and hypercalcemia. Despite there are some studies of LPS transactivation of macrophage 1- -hydroxylase, all of them are focused on the most relevant transcriptional factors involved, but no systems approach was used to examine the complex interaction that involves the enzime regulation. Materials and methods. To make it, we employed microarray data from human macrophages, obtained from GEO (6), using ?macrophages? and ?LPS? as key words. The experiments made at least by triplicates (see Table 1). The meta-analysis was performed with INMEX (2). To perform di.erential expression analysis on individual data sets Benjamini-Hochberg?s False Discovery Rate (FDR) was settled. To combine p-values from multiple studies for information integration Fisher?s method was chosen. Enrichment in Pathways and Go analysis using hypergeometric test were done to get functionality information. List of genes significantly enriched in a particular pathway which contain to 1- -hydroxylase was selected. The list was feed with regulatory proteins and enzimes names detected in the lab involved in the 1- - hydroxylase response (4). The final list was loaded in Genemania (7). In order to improve the visualization and curate it after the network was deployed in Cytoscape (8). Results and discusion. As it was expected according to the literature, the Gene Differentially Expressed List was rich, among others in: Jak-STAT signaling pathway ,Transcriptional misregulation in cancer, Chemokine signaling pathway, and Toll-like receptor signaling pathway. Interesting the 1-hydroxylase gene mapped to tuberculosis pathway, one clinical association which gave the first insight of the extra-renal activity of this enzyme. The network show various hubs; Myd88, Stat1 and TIrap seem to be interesting from the 1- -hydroylase regulation. We found most of the transcription factors described before to interact with 1- -hydroxylase promoter, namely NFKB1, CREB, STAT1 , C/EBP and Jun. All of them possesses binding sites in the hydroxylase promoter. It was previously shown by transfection studies and gel shift assays that C/EBP (1,4,5) plays a role in 1-a-hydroxylase induction by direct binding to specific recognition sites in the promoter, whereas for STAT1 no such direct e.ects could be demonstrated. Cross-talk between the JAK-STAT, the NF-kappaB, and the p38 MAPK pathways should be explored. The new functional relationship of others proteins also were detected C/EBP - NFKB, C/EBP -Jun. This deserve further exploratory studies to confirm them.