Expression of AFP and Rev-Erb A/Rev-Erb B and N-CoR in fetal rat liver, liver injury and liver regeneration

Abstract

Background: Alpha-fetoprotein (AFP) expression can resume in the adult liver under pathophysiological conditions. Orphan nuclear receptors were supposed to regulate AFP gene expression, in vitro. We were interested to study the expression of AFP and orphan nuclear receptors, in vivo.

Results: The expression of AFP gene and orphan nuclear receptors in the liver was examined in different rat models: (a) fetal liver (b) liver regeneration [partial hepatectomy (PH) with and without 2-acetyl-aminofluren treatment (2-AAF)], (c) acute liver damage [treatment with CCl4] and (d) acute phase reaction [treatment with turpentine oil]. After PH of 2-AAF treated rats, clusters of AFP positive cells occurred in the periportal region. In the Northern blot analysis, a positive hybridization signal for the full-length AFP-RNA was observed only in liver samples from 2-AAF treated rats after PH. In real-time PCR analysis, the full-length AFP-RNA was highly up regulated in the fetal liver (maximum at day 14: 21,500 fold); after PH of 2-AAF treated rats, the full-length AFP-RNA was also up regulated up to 400 fold (day 7 after PH). The orphan nuclear receptors were down regulated at nearly each time points in all models, also at time point of up regulation of the AFP gene.

Conclusion: Expression of "fetal" AFP could be demonstrated during liver development and during proliferation of the so-called oval cells. Changes of expression of orphan nuclear receptors, however, did not correlate with AFP expression. Other regulatory pathways were possibly involved in controlling AFP expression, in vivo.

Figure 1

Immunohistochemical detection of AFP in progenitor cells. Immunohistochemical detection of AFP in progenitor cells (oval cells) in the rat liver. The right panel represents a higher magnification of the portal field admitted by black box.

Figure 2

Detection of the oval cell typical AFP-RNA product by Northern blot analysis. Northern blot analysis was performed with liver RNA samples of 2-AAF treated rats after partial hepatectomy or after sham operation. The amplification product of the AFP1 primer pair was used as ₃₂P labelled probe. The first two lanes represent control samples of normal liver (Co) and 2-AAF fed animals (2-AAF-Co). In the middle RNA samples of sham operations at day 3 and day 7 are shown. The two lanes on the right site show RNA samples from day 3 and day 7 after partial hepatectomy of 2-AAF treated rats. In these samples, the oval cell typical product of 2.1 kb is detectable.

Figure 3

Expression of AFP gene and Rev-Erb A, Rev-Erb B and N-CoR in liver regeneration with 2-AAF treatment. After partial hepatectomy in 2-AAF treated rats, the expression of the AFP gene (A) and of Rev-Erb A, Rev-Erb B and N-CoR (B) were measured by real-time PCR. The mean values and the standard deviations of two different series are demonstrated. At day 7, the full-length AFP-RNA (AFP1) is maximally increased. The smaller variants of AFP-RNA (AFP2) showed no significant changes. Four days after the maximum increase of the full-length AFP-RNA Rev-Erb A is maximally decreased. Rev-Erb B is slightly decreased at each time point. N-CoR shows only slight changes in this model.

Figure 4

Expression of AFP gene and Rev-Erb A, Rev-Erb B and N-CoR in liver regeneration without 2-AAF treatment. After partial hepatectomy without 2-AAF treatment the expression of AFP gene (A) and of Rev-Erb A, Rev-Erb B and N-CoR (B) were measured by real-time PCR, in the rat liver. The mean values and the standard deviations of two different series are demonstrated. From 2 up to 16 hours, a down regulation of the smaller variants of AFP-RNA (AFP2) could be seen. Compared to 16 hours value an increase of the smaller splicing products up to -5.1 fold could be observed after 24 hours; afterwards, the level do not change up to 72 hours. The full-length AFP-RNA shows only slight changes. The genes of orphan nuclear receptors are down regulated at each time point. Rev-Erb A and Rev-Erb B are maximally down regulated after 16 hours similarly to the smaller variants of AFP-RNA (AFP2).

Figure 5

Expression of AFP gene and Rev-Erb A, Rev-Erb B and N-CoR in the rat model of acute liver damage. After CCl₄ treatment, the expression of the AFP gene (A) and of Rev-Erb A, Rev-Erb B and N-CoR (B) were measured by real-time PCR. The mean values and the standard deviations of two different series are shown. A decrease of full-length AFP-RNA (AFP1; – 4.5 fold) and the smaller splicing products (AFP2; -4.5 fold) could be seen after 12 hours; afterwards, a continuous increase could be observed of both products with a maximum after 72 hours (AFP1 up to 7.0 fold and AFP2 up to 4.8 fold). A continuous down regulation of Rev-Erb A with the maximum at 48 hours could be observed, 24 hour later both AFP products were maximally up regulated. Rev-Erb and N-CoR show only slight changes in this model.

Figure 6

Expression of AFP gene and Rev-Erb A, Rev-Erb B and N-CoR in fetal rat liver. The expression of the AFP gene (A) and of Rev-Erb A, Rev-Erb B and N-CoR (B) were measured by real-time PCR in the fetal rat liver. The full-length AFP-RNA reach the maximum at day 14 (21,500 fold); afterwards, a continuous decrease up to day 18 could be observed. Rev-Erb A and Rev-Erb B are down regulated at each time point, the maximum down regulation could be seen after 12 days. N-CoR was slightly down regulated at each time point (about 1.5 fold).

Figure 7

Expression of AFP gene and Rev-Erb A, Rev-Erb B and N-CoR during acute phase reaction. After turpentine oil treatment, the expression of AFP gene (A) and of Rev-Erb A, Rev-Erb B and N-CoR (B) were measured by real-time PCR in the rat liver. The mean values of the two different series are demonstrated in this Figure. In this model, only a slight up regulation of the AFP gene expression could be observed at 48 h. The strongest down regulation of Rev-Erb A occurs at 2, 24 and 36 hours. Compared to Rev-Erb A, Rev-Erb B gene expression was not significantly altered, e.g., decreased maximally by 4.5 fold at 24 hours.

Figure 8

Western blot analysis of AFP expression in the rat liver. The fetal AFP-RNA is translated into a 68 kDa and 70 kDa protein. The smaller AFP-RNA transcripts are translated into smaller protein products (58 kDa, 54 kDa and 44 kDa). The strongest expression of fetal AFP protein could be detected in the fetal liver. After PH and 2-AAF treatment, a clearly weaker expression of proteins with a size of 68 kDa and 70 kDa could be seen; but no smaller protein products could be observed. In the case of the normal PH and of the normal control (no co), beside a weak 68 kDa signal also a smaller protein product could be detected.