Characterization of three growth hormone-responsive transcription factors preferentially expressed in adult female liver

Abstract

Plasma GH profiles regulate the sexually dimorphic expression of cytochromes P450 and many other genes in rat and mouse liver; however, the proximal transcriptional regulators of these genes are unknown. Presently, we characterize three liver transcription factors that are expressed in adult female rat and mouse liver at levels up to 16-fold [thymus high-mobility group box protein (Tox)], 73-fold [tripartite motif-containing 24 (Trim24)/transcription initiation factor-1alpha (TIF1alpha)], and 125-fold [cut-like 2 (Cutl2)/cut homeobox 2 (Cux2)] higher than in adult males, depending on the strain and species, with Tox expression only detected in mice. In rats, these sex differences first emerged at puberty, when the high prepubertal expression of Cutl2 and Trim24 was extinguished in males but was further increased in females. Rat hepatic expression of Cutl2 and Trim24 was abolished by hypophysectomy and, in the case of Cutl2, was restored to near-female levels by continuous GH replacement. Cutl2 and Trim24 were increased to female-like levels in livers of intact male rats and mice treated with GH continuously (female GH pattern), whereas Tox expression reached only about 40% of adult female levels. Expression of all three genes was also elevated to normal female levels or higher in male mice whose plasma GH profile was feminized secondary to somatostatin gene disruption. Cutl2 and Trim24 both responded to GH infusion in mice within 10-24 h and Tox within 4 d, as compared with at least 4-7 d required for the induced expression of several continuous GH-regulated cytochromes P450 and other female-specific hepatic genes. Cutl2, Trim24, and Tox were substantially up-regulated in livers of male mice deficient in either of two transcription factors implicated in GH regulation of liver sex specificity, namely, signal transducer and activator of transcription 5b (STAT5b) and hepatocyte nuclear factor 4alpha (HNF4alpha), with sex-specific expression being substantially reduced or lost in mice deficient in either nuclear factor. Cutl2 and Trim24 both display transcriptional repressor activity and could thus contribute to the loss of GH-regulated, male-specific liver gene expression seen in male mice deficient in STAT5b or HNF4alpha. Binding sites for Cutl1, whose DNA-binding specificity is close to that of Cutl2, were statistically overrepresented in STAT5b-dependent male-specific mouse genes, lending support to this hypothesis.

Figure 1. Expression of Cutl2 transcript variants in mouse liver

Panel A: schematic representation of three different mouse Cutl2 RNAs and qPCR primer pairs used to determine their expression. Cutl2 transcript variants, based on the February 2006 build of the mouse genome, are identified by their GenBank accession numbers. Exon/intron structure shown is not drawn to scale; exons 1A and 1B are separated by 165 nt and exons 1B and 1C by 1951 nts. Exons 1–3 are non-coding. Exon 2 is absent from the exon 1A-containing RNA. The forward primer for this transcript (CJ065459) traverses the exon 1A/exon 3 junction, as indicated. The primer pair amplifying portions of exons 22 and 23 does not discriminate between the three Cutl2 RNA transcripts and was used to assay mouse Cutl2 in a majority of the experiments presented in this study. Panel B: female/male expression ratios of individual Cutl2 transcripts in ICR mouse liver. The ratios were determined for transcripts containing exon 1A, exon 1C and for the exon 1B + exon 1C transcripts together (‘1B + 1C’), as indicated. Relative RNA levels were determined by qPCR analysis of liver RNA from wild-type male and female mice (n=5 per group). The expression levels of each RNA transcript were normalized to the 18S rRNA content of each liver and then calculated relative to the average expression of the corresponding RNA transcript in males. Data shown are mean ± SE. The calculated mean ratios are indicated above each bar. Levels of each Cutl2 transcript showed statistically significant differences between females and males (Student’s t-test, p<0.01).

Figure 2. Western blot analysis of liver Cutl2 protein

Nuclear extracts prepared from pooled mouse livers (panel A) or from individual rat livers (panel B) were resolved on a 6% SDS polyacrylamide gel and subjected to Western blot analysis with anti-Cutl2 antibody 356 in comparison to cDNA-expressed mouse Cutl2. Panel A: whole cell extracts (40 μg) from untransfected 293T cells (lane 1) or from 293T cells transfected with Cutl2 cDNA (lane 2); Cutl2 protein immunoprecipitated with anti-Cutl2 antibody 356 from liver nuclear extract (1 mg) prepared from pools of adult male (lane 3) and female (lane 4) mouse liver (n=8 livers/group); and adult male (lane 5) and female (lane 6) mouse liver nuclear extract (40 μg/lane). Panel B, portion of Western blot showing, in lanes 1–2: cell extracts (40 μg) from untransfected or Cutl2 cDNA-transfected 293T cells, respectively; lanes 3–14: liver nuclear extracts prepared from individual adult male (lanes 3–6) and female (lanes 7–10) rats and from adult male rats given a continuous GH infusion for 7 d (lanes 11–14). Authentic, cDNA-expressed Cutl2 protein (lane 2, panels A and B; arrow) runs close to the 204 kDa protein marker shown on the right.

Figure 3. Postnatal developmental profiles of rat liver Cutl2 and Trim24 RNAs in comparison with CYP2C11 and CYP2C12 RNAs

Expression of the 4 indicated RNAs was determined by qPCR analysis of liver RNA prepared from individual male and female rats ranging from 4 d to 12 wk of age. RNA levels were normalized to the 18S rRNA content of each liver and expressed as a percentage of the average level in 8-wk-old females (panels A, B, D) or 8-wk-old males (panel C), which were set to 100%. The data shown are mean ± SE for each group (n=3–6 livers).

Figure 4. Effect of hypophysectomy and continuous GH infusion on Cutl2 and Trim24 RNA in rat liver

Cutl2 and Trim24 RNA levels were determined by qPCR analysis of livers isolated from individual untreated (n=3 livers/group), sham-operated (n=2/group) and hypophysectomized (Hx, n=4/group) male and female rats and from hypophysectomized rats given a continuous GH infusion via an osmotic pump (+GH) for 7 d (n=3 males and n=1 female) (panel A and panel B). RNA levels were also determined in individual livers from untreated male (M) and female (F) rats (n=4–5/group) and control male rats treated with continuous GH via an osmotic mini-pump for 7 d (n=5) (panel C and panel D). Data were analyzed as in Fig. 3, except that the RNA levels (mean ± SE for individual livers) are shown relative to the average level in untreated females, which was set to 100%.

Figure 5. Response of Cutl2, Trim24 and Tox RNAs to continuous GH infusion in mice

Male ICR mice were implanted with osmotic mini-pumps delivering a continuous infusion of GH or vehicle control for time periods ranging from 10 h to 14 d. Individual livers were isolated and analyzed for Cutl2, Trim24 and Tox RNAs by qPCR. Data shown are based on the following number of individual mice per group: n=6–7 (each GH-treated time point) and n=2–3 (each vehicle-treated control group; sham). RNA levels (mean ± SE) were normalized to the 18S rRNA content of each liver and are presented relative to the average untreated female liver level, which was set to 1. All three RNAs showed stress-dependent responses to osmotic mini-pump implantation at the 10 h time point, as indicated by RNA increases in the vehicle-treated control group. The stress response of Tox RNA, evident at several of the early time points, was quite substantial and was indistinguishable for vehicle control and GH-treated livers at the 10h point (increase up to 80–95% of the untreated female control for both groups). Tox RNA data for the 10 h time point (arrow) was adjusted for the stress response by multiplying the relative expression levels in GH- and vehicle-receiving groups by the untreated male/vehicle control expression ratio.

Figure 6. Effect of Sst gene disruption on mouse liver expression of Cutl2, Trim24 and Tox

RNA levels were determined by qPCR analysis of individual livers from wild-type (WT) male (n=6) and female (n=3) mice and from Sst (somatostatin)-deficient (KO) male (n=5) and female (n=7) mice. RNA levels in individual livers were normalized to the 18S rRNA content and presented relative to the average RNA level in wild-type male mice, which was set at 1. Data shown are mean ± SE for each group. Data were analyzed using Student’s t-test: + and ++, p<0.05 and p<0.01, respectively, for somatostatin-deficient male (or female) vs. wild-type male (or female); * and **, p<0.05 and p<0.01, for wild-type (or somatostatin-deficient) female vs. wild-type (or somatostatin-deficient) male.

Figure 7. Effect of Stat5b gene disruption on mouse liver expression of Cutl2, Trim24 and Tox

RNA levels were determined by qPCR analysis of individual livers from wild-type (WT) male (n=6) and female (n=5) mice and STAT5b-deficient (KO) male (n=6) and female (n=7) mice. Data were analyzed as described in Fig. 6, with the wild-type male RNA level set to 1.0 for each gene, and graphed as mean ± SE for each group. Data were analyzed using Student’s t-test: + and ++, p<0.05 and p<0.01, respectively, for STAT5b-deficient male (or female) vs. wild-type male (or female); * and **, p<0.05 and p<0.01, respectively, for wild-type (or STAT5b-deficient) female vs. wild-type (or STAT5b-deficient) male.

Figure 8. Effect of Hnf4α gene disruption on mouse liver Cutl2, Trim24 and Tox RNA

RNA levels were assayed in livers of wild-type (WT) and HNF4α-deficient (KO) male and female mice by qPCR (n=8 livers/group). Data were analyzed as in Fig. 6, with the wild-type male level set to 1.0, and graphed as mean ± SE for each group. Data were analyzed using Student’s t-test: + and ++, p<0.05 and p<0.01, respectively, for HNF4α-deficient male (or female) vs. wild-type male (or female); * and **, p<0.05 and p<0.01, respectively, for wild-type (or HNF4α-deficient) female vs. wild-type (or HNF4α-deficient) male.