The role of nuclear factor kappaB in late-gestation liver development in the rat

Abstract

During the last 3 days of fetal development in the rodent, a burst of hepatocyte proliferation results in a tripling of liver size. Despite stimulation of mitogenesis via multiple signaling pathways, including some that are considered stress response pathways, little apoptosis accompanies this cell growth. Given the accepted role of nuclear factor kappaB (NF-kappaB) in preventing hepatocellular apoptosis during proliferation in mid-development, we predicted that NF-kappaB would be functional during the period of rapid growth during late gestation in the rat. NF-kappaB binding in electrophoretic mobility shift assays was low in embryonic day (E) 19 liver nuclear extracts relative to adult liver nuclear extracts. An additional band that was present in E19 liver was purified and identified as nucleolin. Tumor necrosis factor alpha (TNF-alpha) administration to E19 embryos in utero produced minimal induction of NF-kappaB p50 homodimers and p50/p65 heterodimers, yet baseline apoptosis was not affected. Although p65 was present in E19 hepatocyte cytoplasm in amounts comparable to adult liver, we observed little translocation of p65 to the liver nuclei following TNF-alpha administration. Additionally, expression of several NF-kappaB-responsive genes remained minimally induced in E19 liver following TNF-alpha treatment. In conclusion, although the NF-kappaB components are present in late-gestation fetal liver, NF-kappaB as a transcription factor is relatively inactive and unresponsive to TNF-alpha. Given this finding and the high level of proliferation in late-gestation fetal liver, we predict that alternative antiapoptotic mechanisms are active during this period of rapid hepatic growth.

Fig. 1.

Apoptosis in adult and fetal liver. Adult and E19 rat livers were analyzed for frequency of apoptosis (arrows) by TUNEL staining (fluorescein). Cells were counterstained with propidium iodide. Images were acquired at 600× magnification. E, embryonic day.

Fig. 2.

NF-κB activity in liver as assessed via EMSA. (A) NF-κB activity in nuclear extracts from rat liver at the indicated developmental ages was determined by the ability of the extract to retard migration of a radio-labeled consensus NF-κB oligonucleotide in an EMSA. (B) The specificity of binding to the consensus sequence was tested by adding an excess (×40, ×80, ×160) of cold NF-κB oligonucleotide or unrelated oligonucleotide containing an AP-1 or OCT1 binding site (×160) to nuclear extracts from rat liver obtained 30 minutes after PH. (C) PH and E19 rat liver nuclear extracts were incubated with antibodies for specific NF-κB subunits to identify proteins contributing to the binding complexes. Although not shown here, the antibody to RelB did not produce a supershift. E, embryonic day; PND, postnatal day; NF-κB, nuclear factor κB; PH, partial hepatectomy.

Fig. 3.

EMSA on nucleolin-depleted fetal liver nuclear extract. E19 rat liver nuclear extract was incubated with water (control), Protein A-Sepharose, a series (1 hour each) of immobilized rabbit immunoglobulin G antibody (IgG 1-3×), or a series (1 hour each) of immobilized nucleolin antibody (1-3×). The unbound extract was used for EMSA for NF-κ>B to determine whether immunodepletion of nucleolin reduced the intensity of the lower band.

Fig. 4.

Extranuclear protein expression of p50 and p65 in E19 and adult rat liver. Adult and E19 postnuclear liver extracts (in duplicate) were separated via SDS-PAGE and immunoblotted for p50 and p65. E, embryonic day.

Fig. 5.

Ontogeny of cellular localization of p65 in rat liver. Cryosections of liver from E13-15, E19, PND1, and adult rats were immunofluorescently stained for p65. Omission of primary antibody produced a faint ubiquitous haze similar to that seen in Fig. 6. Images were acquired at 600× magnification. Insets were acquired at 1000× magnification and were further enlarged for presentation. E, embryonic day; PND, postnatal day.

Fig. 6.

NF-κB activation in E19 and adult liver after TNF-α treatment. Livers from E19 or adult rats were collected 30 minutes following TNF-α treatment and nuclear extracts were used (A) for an EMSA for NF-κB or (B) to detect p65 via immunoblotting. Parallel samples of frozen liver sections were immunofluorescently stained for p65. Adult liver incubated with the secondary antibody and no primary antibody was used to determine the specificity of binding of the primary antibody for p65. Images were acquired at 600× magnification. TNF-α, tumor necrosis factor α; E, embryonic day; PND, postnatal day; PBS, phosphate-buffered saline.

Fig. 7.

Apoptosis in E19 fetal liver. Livers from E19 rats injected intraperitoneally with phosphate-buffered saline or TNF-α (25 μg/kg) were collected 4 hours after treatment and homogenized. Samples were electrophoresed, and an immunoblot for PARP was performed to detect apoptosis. Cleaved PARP, which migrated at 91.5 kd, was present in the positive control (etopiside-treated HL60 cells). Other controls for the immunoblot included extracts of untreated HL60 cells and purified PARP from bovine thymus. PARP, poly(ADP-ribose)polymerase; E, embryonic day; PBS, phosphate-buffered saline; TNF-α, tumor necrosis factor α.

Fig. 8.

Expression of NF-κB-responsive genes in livers following TNF-α treatment. Ribonuclease protection assays were used to analyze the gene expression pattern in adult and E19 livers 1 hour after phosphate-buffered saline or TNF-α treatment (25 μg/kg). (A) Representative autoradiographs. (B) Results of the densitometric analysis of autoradiographs. Genes that showed an age effect, treatment effect, or an interaction between age and treatment as determined via two-way ANOVA were analyzed using a Bonferroni post hoc test: adult versus E19 control (*P < .05), or control (white boxes) versus the corresponding TNF-α-treated group (black boxes) (**P < .05). Densitometry results were first expressed as a ratio of the gene to GAPDH expression. These data were then normalized to the highest value so that the results could be expressed on a scale of 0 to 1. The results are reported as the mean ± SEM. Two-sided significance testing was used. A P value of less than .05 was considered significant. E, embryonic day; TNF-α, tumor necrosis factor α.