Lone Frier Bovin

Analysis of the epidermal growth factor receptor specific transcriptome: Effect of receptor expression level and an activating mutation

Overexpression or expression of activating mutations of the epidermal growth factor receptor (EGFR) is common in cancer and correlates with neoplastic progression. The present study employed Affymetrix® oligonucleotide arrays to profile genes induced by ligand‐activated EGFR with the receptor either moderately expressed or overexpressed at an in‐itself transforming level. These changes were compared to those induced by the naturally occurring constitutively active variant EGFRvIII. This study provides novel insight on the activities and mechanisms of EGFRvIII and EGFR mediated transformation, as genes encoding proteins with functions in promoting cell proliferation, invasion, antiapoptosis, and angiogenesis featured prominently in the EGFRvIII‐ and EGFR‐expressing cells. Surprisingly, it was found that ligand‐activated EGFR induced the expression of a large group of genes known to be inducible by interferons. Expression of this module was absent in the EGFRvIII‐expressing cell line and the parental cell line. Treatment with the specific EGFR inhibitor AG1478 indicated that the regulations were primary, receptor‐mediated events. Furthermore, activation of this module correlated with activation of STAT1 and STAT3. The results thus demonstrate that ligand‐activated EGFR at different expression levels results in different kinetics of signaling and induction of gene expression. In addition, the constitutively active variant EGFRvIII seems to activate only a subset of signal pathways and induce a subset of genes as compared to the ligand‐activated EGFR.

Massive parallel gene expression profiling of RINm5F pancreatic islet β‐cells stimulated with interleukin‐1β

Interleukin 1 (IL‐1) is a pleiotropic cytokine with the potential to kill pancreatic β‐cells, and this unique property is thought to be involved in the pathogenesis of type I diabetes mellitus. We therefore determined the quantitative expression of 24000 mRNAs of RINm5F, an insulinoma cell line derived from rat pancreatic β‐cells, before and after challenge with 30 and 1000 pg/ml of recombinant human IL‐1β. The highest concentration resulted in decreased insulin production and cell death over a period of 4 days. Using three different time points, 2, 4 and 24 hours after challenge, we found that 146 full‐length genes and a large number of expressed sequence tags were differentially regulated 3‐fold or more. Most of the differentially regulated transcripts have not previously been described to be regulated by IL‐1β in β‐cells. We have analysed the expression data and sorted the genes into groups according to functional relations on the basis of knowledge of the structure or function ascribed to the individual genes. Many of the differentially regulated genes are known to play a role in immune‐ and stress‐related pathways as well as in insulin secretion and vesicle trafficking, e.g. α‐endosulfine and K+ channel Kir6.2 are differentially regulated. A number of transcripts in the biosynthesis pathway for cholesterol are also differentially regulated.

Sulphatide and its precursor galactosylceramide influence the production of cytokines in human mononuclear cells

Sulphatide is expressed in the central and peripheral neural system, in islets of Langerhans, and in tissues affected by late diabetic complications. Autoantibodies to sulphatide are present in patients with insulin‐dependent diabetes and the Guillain‐Barré syndrome. Cytokines influence these disease processes, and we therefore studied whether sulphatide and its precursor galactosylceramide (gal‐cer) influence the in vitro production of cytokines by blood mononuclear cells (MNC) originating from 15 healthy persons. Using lipopolysaccharide (LPS)‐stimulated cells, sulphatide increased the IL‐2 production (163±17% of controls without sulphatide, p=0.02), and gal‐cer increased the IL‐1α production (145±13%, p=0.006), whereas neither gal‐cer nor sulphatide had an effect on the production of IL‐6, IL‐10 or TNFα. When stimulating cells with phytohaemagglutinin (PHA), sulphatide decreased the production of IL‐6 (88±5%, p=0.009), IL‐10 (66±3%, p=0.000003), and TNFα (75±9%, p=0.02). Galcer, however, increased the production of IL‐6 (188±13%, p=0.000006), and decreased the production of TNFp (80±6%, p=0.007). Neither galcer nor sulphatide had an effect on the production of IL‐2 or IFNy from PHA‐stimulated cells. Northern blot analysis using an IL‐6 probe similarly showed an increased amount of IL‐6 mRNA after galcer incubation (range 469%‐150%, n=3) of PHA‐stimulated control. Thus, sulphatide and galcer influence the production of several cytokines thought to be involved in immunoinflammatory disease processes.

In vitro production of cytokines is influenced by sulfatide and its precursor galactosylceramide

Effects of sulfatide and its precursor galactosylceramide (gal‐cer) on the kinetics of production of cytokines were studied. In human mononuclear leucocytes, gal‐cer but not sulfatide induced significantly increased amounts of interleukin (IL)‐1β, IL‐6 and tumor necrosis factor (TNF) mRNA. In phytohemagglutinin‐stimulated cultures, gal‐cer increased the levels of IL‐1β and IL‐6 mRNA and secreted IL‐1β and IL‐6, while sulfatide decreased the amounts of IL‐6 mRNA and secreted IL‐6. Gal‐cer also increased TNF secretion. In lipopolysaccharide‐stimulated cells, sulfatide but not gal‐cer decreased the secretion of IL‐1β and IL‐10, a potent suppressor of production of many cytokines. Thus, sulfatide and gal‐cer affect cytokine production differently, most likely at the level of gene expression. This may have implications in diseases where inflammatory cytokines play a pathogenic role.

Gene expression profiling in autoimmune diseases: Chronic inflammation or disease specific patterns?

A central issue in autoimmune disease is whether the underlying inflammation is a repeated stereotypical process or whether disease specific gene expression is involved. To shed light on this, we analysed whether genes previously found to be differentially regulated in rheumatoid arthritis (RA) patients and healthy individuals were specific for the arthritic process or likewise altered in other chronic inflammatory diseases such as chronic autoimmune thyroiditis (Hashimotos thyroiditis, HT) and inflammatory bowel disease (IBD). Using qPCR for 18 RA-discriminative genes, there were no significant differences in peripheral blood mononuclear cell (MNC) gene expression patterns between 15 newly diagnosed HT patients and 15 matched healthy controls. However, the MNC expression levels of five genes were significantly upregulated in 25 IBD patients, compared to 18 matched healthy controls (CD14, FACL2, FCN1, RNASE2, VNN2). There was concordance in the directional change for all genes between IBD and RA patients, i.e. increased expression compared to controls. These data show that one third of the genes significantly upregulated in MNC from RA patients were upregulated in patients with other chronic immunoinflammatory diseases, but only if accompanied by pronounced systemic manifestations. This suggests that at least some of the genes activated in RA are predominantly or solely related to general and disease-nonspecific autoimmune processes.