Prevention of hepatic apoptosis and embryonic lethality in RelA/TNFR-1 double knockout mice

Abstract

Mice deficient in the nuclear factor kappaB (NF-kappaB)-transactivating gene RelA (p65) die at embryonic days 14-15 with massive liver apoptosis. In the adult liver, activation of the NF-kappaB heterodimer RelA/p50 can cause hepatocyte proliferation, apoptosis, or the induction of acute-phase response genes. We examined, during wild-type fetal liver development, the expression of the Rel family member proteins, as well as other proteins known to be important for NF-kappaB activation. We found these proteins and active NF-kappaB complexes in the developing liver from at least 2 days before the onset of lethality observed in RelA knockouts. This suggests that the timing of NF-kappaB activation is not related to the timing of lethality. We therefore hypothesized that, in the absence of RelA, embryos were sensitized to tumor necrosis factor (TNF) receptor 1 (TNFR-1)-mediated apoptosis. Thus, we generated mice that were deficient in both RelA and TNFR-1 to determine whether apoptotic signaling through TNFR-1 was responsible for the lethal phenotype. RelA/TNFR-1 double knockout mice survived embryonic development and were born with normal livers without evidence of increased hepatocyte apoptosis. These animals became runted shortly after birth and survived an average of 10 days, dying from acute hepatitis with an extensive hepatic infiltration of immature neutrophils. We conclude that neither RelA nor TNFR-1 is required for liver development and that RelA protects the embryonic liver from TNFR-1-mediated apoptotic signals. However, the absence of both TNFR-1 signaling and RelA activity in newborn mice makes these animals susceptible to endogenous hepatic infection.

Figure 1.

Protein expression in WT fetal liver development. Western blots using antibodies against RelA, IκBα, IκBβ, and TRAF-2 illustrate protein expression throughout liver development. Lanes 12–19: fetal liver protein from gestational days 12–19, respectively.

Figure 2.

Protein expression in the fetal livers of WT mice, RelA knockouts, and TNFR-1 knockouts. Western blots were performed with RelA, IκBα, IκBβ, and TRAF-2 antibodies. Lanes indicate fetal liver protein from E12, E13 and E14 for WT fetal livers, RelA^−/− fetal livers and TNFR-1^−/− fetal livers. RelA^−/− fetal livers illustrate absent RelA and IκBβ proteins, diminished IκBα protein, and unaltered TRAF-2 protein levels. WT and TNFR-1^−/− fetal livers express all proteins.

Figure 3.

NF-κB complex formation in the fetal liver. A: Nuclear extract from WT fetal liver at E12, E13, and E14, demonstrating both RelA/p50 heterodimers (top band) and p50 homodimers (bottom band). B: Nuclear extract from TNFR-1^−/− fetal livers at E12, E13, and E14, demonstrating both RelA/p50 heterodimers (top band) and p50 homodimers (bottom band). Supershift analysis was performed with E13 nuclear protein and anti-RelA antibody, anti-p50 antibody, anti-c-Rel antibody, anti-p52 antibody, or an irrelevant anti-STAT3 antibody, as indicated. Specificity of binding was determined by competition with 30-fold excess of unlabeled oligonucleotide binding sequence probe. C: Nuclear extract from RelA^−/− fetal livers at E12, E13, and E14, demonstrating only p50 homodimers. Supershift analysis was performed with E13 nuclear extracts and anti-RelA antibody, which did not cause a supershift, and with anti-p50 antibody, which shifted the entire band, as indicated.

Figure 4.

STAT3 binding in the fetal liver. Nuclear extracts were isolated from WT fetal livers (A), TNFR-1^−/− fetal livers (B), and RelA^−/− fetal livers (C) at E12, E13, and E14, demonstrating STAT3 binding at all time points in all animals examined.

Figure 5.

Histological analysis of WT and RelA^−/−/TNFR-1^−/− mice. All sections were stained with H&E. A: Liver section from a 2-day-old WT mouse showing normal hepatocytes and presence of focal areas of hematopoiesis. Original magnification, ×200. B: Liver section from a 2-day-old RelA^−/−/TNFR-1^−/− mouse showing normal hepatocytes and presence of focal areas of hematopoiesis. Original magnification, ×200. C: Liver section from an 8-day-old RelA^−/−/TNFR-1^−/− mouse illustrating the presence of massive neutrophilic infiltration. Original magnification, ×100. D: Higher magnification of the 8-day RelA^−/−/TNFR-1^−/− liver section, illustrating the immature neutrophils (band cells) responsible for the inflammation seen. Original magnification, ×640. E: Liver section from a 17-day-old RelA^−/−/TNFR-1^−/− mouse, demonstrating focal areas of necrosis. Original magnification, ×400. F: Lung lesion in a RelA^−/−/TNFR-1^−/− mouse killed at 16 days of age. The lesion was a solid mass that histologically resembled lobular pneumonia. The area was separated from the rest of the lung parenchyma (left side, showing alveoli) by connective tissue surrounded by inflammatory cells. The lesion itself (right side) contained closed and thickened alveoli, inflammatory cells, and cell necrosis. Original magnification, ×200. G: Spleen section from a 16-day-old WT mouse, showing normal germinal center formation. Original magnification, ×100. H: Spleen section from a 16-day-old RelA^−/−/TNFR-1^−/− mouse, demonstrating less defined white pulp and germinal follicle areas as well an increased numbers of megakaryocytes. Original magnification, ×200.

Figure 6.

Protein expression in livers of WT, TNFR-1^−/−, and RelA^−/−/TNFR-1^−/− newborn pups. Western blots for RelA, IκBα, IκBβ, and TRAF-2 were performed on liver extracts from 2-, 4-, and 7-day-old WT mice, TNFR-1^−/− mice, and RelA^−/−/TNFR-1^−/− mice as indicated. WT and TNFR-1^−/− mice express all proteins. RelA^−/−/TNFR-1^−/− mice have absent RelA and IκBβ proteins, diminished IκBα protein expression, and unaltered TRAF-2 protein levels.