genomewide characterization of host-pathogen interactions by ...

Maren Depke
Results
Liver Gene Expression Pattern in a Mouse Psychological Stress Model
molecule Nnmt was up-regulated selectively after repeated stress. To verify the biological
relevance of an altered redox homeostasis, protein carbonylation was analyzed as a marker of
oxidative stress. Remarkably, increased protein carbonyl content of plasmatic and hepatic
proteins was detectable already immediately after acute stress, indicating a rapid and significant
protein damage by increased formation of reactive oxygen species (data not shown). After
chronic psychological stress the protein-carbonyl content had returned to the level seen in nonstressed
control mice (data not shown).
Finally, the expression of Glrx (glutaredoxin) and Gsta (glutathione S-transferase) was
significantly increased in the liver of chronically stressed mice indicating that compensatory antioxidative
mechanisms were induced. This may explain why the carbonyl content of proteins in
plasma and liver in repeatedly stressed mice had declined to the normal level observed in nonstressed
control mice (data not shown).
Repeated stress-induced apoptosis in the liver
Several genes that were regulated in the liver of repeatedly stressed mice provided hints for
increased cell death. By comparing the mRNA profile of liver homogenates of acutely and
repeatedly stressed mice a highly similar pattern of altered gene expression of cell cycle and
apoptosis-related genes was observed. In both acutely and chronically stressed animals,
increased mRNA levels compared to non-stressed controls were detected for Tnfrsf1b, Cdkn1a,
Cebpb, Igfbp1, and Gadd45b (Fig. R.1.8 A). Other genes, such as Fos and Jun, which were induced
immediately after acute stress exposure, did not reveal prolonged high mRNA expression
(Fig. R.1.8 A, B), whereas yet another group, including Ccnd1 and Xbp1, were selectively
repressed after the ninth stress session (Fig. R.1.8 B). Using TUNEL techniques, induction of
hepatocyte apoptosis was not detectable immediately after one single acute stress exposure
(Fig. R.1.8 D), but high numbers of dead cells in livers after repeated stress exposure (Fig. R.1.8 E)
compared with control mice (Fig. R.1.8 C) were documented. However, the liver mass after the
ninth stress session was only slightly decreased compared to non-stressed control animals
(Fig. R.1.8 F). This may be caused by induction of repair processes for which heightened hepatic
expression of IL-6 receptor and Ptp4a1 after chronic stress are indications.
Fig. R.1.8. Differential regulation of cell death associated genes in liver of stressed mice.
Genes resulting from a search for “apoptosis AND liver”, “apoptosis AND hepatocyte”, “cell death AND liver” and “cell death AND
hepatocyte” using Ingenuity Pathway Analysis (IPA, Ingenuity Systems, www.ingenuity.com) were collected in a list. All genes of this
list which displayed differential regulation in liver after acute and/or chronic stress exposure were added to a new user-defined
pathway as analysis nodes and interactions between these selected genes were drawn using the “Connect” tool of IPA. Edge lines that
are continuous represent direct interactions, whereas broken lines indicate indirect influences. The pathway was overlaid with
expression data from (A) acute stress and from (B) chronic stress. Red indicates increased and green decreased expression after stress
exposure compared with the control. Intensity of the node color represents the degree of up (red) and down (green) regulation in the
stressed livers. Different shapes of the nodes indicate the functional classes of the gene products (e. g. vertical
ellipse − transmembrane receptor; horizontal ellipse − transcription regulator; vertical rectangle − G-protein coupled receptor;
horizontal rectangle − ligand-dependent nuclear receptor; trapezium − transporter; triangle − phosphatase; inverted triangle − kinase;
vertical rhomb − enzyme; horizontal rhomb − peptidase; quadrat − cytokine; double lined circle − group or complex; single lined
circle − other).
The level of apoptosis TUNEL assay (x400): compared to the control (C), livers of mice exposed to acute stress did no display any
significant increase in the level of apoptosis (D). However, high numbers of apoptotic hepatocytes were detected in repeatedly
stressed mice (E). Each picture is representative for n = 9 mice/group. Increased apoptosis did not alter liver mass (F), even after the
9th stress cycle (black box plots) compared with non-stressed mice (white box plots) n = 9 mice/group; data representative for least 2
independent experiments.
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