Transgenic overexpression of Tcfap2c/AP-2gamma results in liver failure and intestinal dysplasia

Abstract

Background: The transcription factor Tcfap2c has been demonstrated to be essential for various processes during mammalian development. It has been found to be upregulated in various undifferentiated tumors and is implicated with poor prognosis. Tcfap2c is reported to impinge on cellular proliferation, differentiation and apoptosis. However, the physiological consequences of Tcfap2c-expression remain largely unknown.

Methodology/principal findings: Therefore we established a gain of function model to analyze the role of Tcfap2c in development and disease. Induction of the transgene led to robust expression in all tissues (except brain and testis) and lead to rapid mortality within 3-7 days. In the liver cellular proliferation and apoptosis was detected. Accumulation of microvesicular lipid droplets and breakdown of major hepatic metabolism pathways resulted in steatosis. Serum analysis showed a dramatic increase of enzymes indicative for hepatic failure. After induction of Tcfap2c we identified a set of 447 common genes, which are deregulated in both liver and primary hepatocyte culture. Further analysis showed a prominent repression of the cytochrome p450 system, PPARA, Lipin1 and Lipin2. These data indicate that in the liver Tcfap2c represses pathways, which are responsible for fatty acid metabolism. In the intestine, Tcfap2c expression resulted in expansion of Sox9 positive and proliferative active epithelial progenitor cells resulting in dysplastic growth of mucosal crypt cells and loss of differentiated mucosa.

Conclusions: The transgenic mice show that ectopic expression of Tcfap2c is not tolerated. Due to the phenotype observed, iTcfap2c-mice represent a model system to study liver failure. In intestine, Tcfap2c induced cellular hyperplasia and suppressed terminal differentiation indicating that Tcfap2c serves as a repressor of differentiation and inducer of proliferation. This might be achieved by the Tcfap2c mediated activation of Sox9 known to be expressed in intestinal and hepatic stem/progenitor cell populations.

Figure 1. Generation of iTcfap2c-mice.

Schematic representation of transgenes used to produce Tcfap2c-inducible ES cells and mice. ES cells containing M2-rtTA expressed under control of the Rosa26 promoter were retargeted at the 3′ UTR of the Col1a1 locus by insertion of the murine Tcfap2c-cDNA under control of the tetracycline responsive element (TetOP) (pA, polyadenylation signal). (B) Western blot analysis of doxycyline (dox) dependent Tcfap2c induction in transgenic ES cells. (C–N) Immunohistochemical detection of Tcfap2c protein on sections from iTcfap2c-mice after dox-induction. Lack of Tcfap2c expression in untreated iTcfap2c mice in skin (P) and stomach (R) compared to dox induced iTcfap2c tissue (O,Q). Scale bars = 100 µm.

Figure 2. iTcfap2c-mice show strong morbidity in a dox-dependent manner.

(A) Kaplan-Meier plot showing survival after dox treatment. Green line: iTcfap2c-mice receiving dox via drinking water (dw) (0.1 mg/ml, n = 6). Blue and red line: iTcfap2c-mice receiving dox via intra peritoneal injection (i.p.) (blue: 1 mg/day, n = 33), (red: 0.5 mg/day n = 12). Black line: control mice (0.1 mg/ml dox by in dw for 20 days). (B) Weight of iTcfap2c (red) and ctrl animals (black), which received 0.5 mg dox i.p. per day (n = 12). (C–H) Biochemical blood serum analysis. Enzyme activity was measured as the concentration of substrate converted per minute (1 enzyme unit = 1 µmol substrate/min) in units/ liter. iTcfap2c (n = 9) and ctrl animals (n = 7) were induced with 1 mg/ml dox per day. Blood samples were collected the third day.

Figure 3. Tcfap2c increases proliferation and induces hepatic steatosis.

(A) Percentage of proliferating Ki67 positive hepatocytes in iTcfap2c (n = 5) and ctrl mice (n = 4) (B) Percentage of proliferating hepatocytes in cell culture, as the number of EdU positive cells. Error bars represent standard deviations. ***p≤0.0001, **p≤0.001 (C) Immunohistochemical staining of liver for Ki67 (proliferation, upper panel) and TUNEL staining positive (apoptosis, lower panel). (D) HE (middle panel) and Oil Red-O (lower panel) staining showing fat accumulation and microvisicular steatosis. (E) Tcfap2c immunohistochemistry (upper panel) and Oil-Red O staining (lower panel) of primary hepatocytes. (F) Transmission electron microscope images of iTcfap2c and ctrl liver. Hepatocytes from iTcfap2c mice are filled with lipid droplets (L); Dark and dense mitochondria in ctrl cells but bright mitochondria in iTcfap2c cells were evidence for mitochondria damage. Glycogen granules (G) were visible only in ctrl cells. Scale bars = 100 µm.

Figure 4. Genes regulated by Tcfap2c and deregulated pathways.

(A) Affymetrix microarray gene expression analysis performed with RNA extracted from livers of iTcfap2c (n = 4) and ctrl (n = 4) mice. (B) Heat map showing genes with at least twofold difference between any pair of samples from different classes. Red and blue indicate higher and lower relative expression, respectively. (C) Venn diagram; commonly regulated transcripts by Tcfap2c in vivo and in vitro are shown in the intersection part. (D) RT-PCR of RNA isolated from iTcfap2c and control liver (left column) and Hepatocytes (right colum using the genes indicated (E) Top 18 most significantly regulated pathways are presented based on common gene lists regulated by Tcfap2c.

Figure 5. Tcfap2c expression leads to dysplastic growth in the intestine.

Immunhistochemical detection of Tcfap2c expression on intestinal sections of iTcfap2c (A) and control mice (B). (C) Ratio of crypt length/villus length the area indicated. Length of villus was measured as the distance from the intestinal muscularis mucosa to the villus-apex; length of the crypts was measured as distance from muscularis mucosae to the bottom of the villus. ***p≤0.0001, **p≤0.001, Scale bar = 100 µm.

Figure 6. Tcfap2c induces proliferation of the intestine.

Sections of the intestine of iTcfap2c (A,B) and control (C) showing HE (upper panel) and Ki67 staining. Scale bars = 100 µm.

Figure 7. Induction of Tcfap2c results in expansion of the progenitor compartment.

(A) Sox9 staining of the intestine iTcfap2c (upper panel) and controls (lower panel). (B) Alcian blue-PAS staining of the intestine iTcfap2c (upper panel) and controls (lower panel). Arrowheads indicate Paneth cells, found at the bottom of the crypts by blue coloring of their granules. Scale bar = 100 µm.