Hepatic Hedgehog signaling contributes to the regulation of IGF1 and IGFBP1 serum levels

Abstract

Background: Hedgehog signaling plays an important role in embryonic development, organogenesis and cancer. In the adult liver, Hedgehog signaling in non-parenchymal cells has been found to play a role in certain disease states such as fibrosis and cirrhosis. However, whether the Hedgehog pathway is active in mature healthy hepatocytes and is of significance to liver function are controversial.

Findings: Two types of mice with distinct conditional hepatic deletion of the Smoothened gene, an essential co-receptor protein of the Hedgehog pathway, were generated for investigating the role of Hedgehog signaling in mature hepatocytes. The knockout animals (KO) were inconspicuous and healthy with no changes in serum transaminases, but showed a slower weight gain. The liver was smaller, but presented a normal architecture and cellular composition. By quantitative RT-PCR the downregulation of the expression of Indian hedgehog (Ihh) and the Gli3 transcription factor could be demonstrated in healthy mature hepatocytes from these mice, whereas Patched1 was upregulated. Strong alterations in gene expression were also observed for the IGF axis. While expression of Igf1 was downregulated, that of Igfbp1 was upregulated in the livers of both genders. Corresponding changes in the serum levels of both proteins could be detected by ELISA. By activating and inhibiting the transcriptional output of Hedgehog signaling in cultured hepatocytes through siRNAs against Ptch1 and Gli3, respectively, in combination with a ChIP assay evidence was collected indicating that Igf1 expression is directly dependent on the activator function of Gli3. In contrast, the mRNA level of Igfbp1 appears to be controlled through the repressor function of Gli3, while that of Igfbp2 and Igfbp3 did not change. Interestingly, body weight of the transgenic mice correlated well with IGF-I levels in both genders and also with IGFBP-1 levels in females, whereas it did not correlate with serum growth hormone levels.

Conclusions: Our results demonstrate for the first time that Hedgehog signaling is active in healthy mature mouse hepatocytes and that it has considerable importance for IGF-I homeostasis in the circulation. These findings may have various implications for mouse physiology including the regulation of body weight and size, glucose homeostasis and reproductive capacity.

Figure 1

Phenotypic features induced by hepatocyte-specific ablation of Smoothened in transgenic SAC mice. Photographs of (A): male SAC mice and (B): female SAC mice show two WT mice (left) and two KO mice (right). Comparison of body weight of (C): male SAC-WT (black squares) (n = 4) and SAC-KO (open circles) (n = 5) mice and (D): female SAC-WT (black squares) (n = 3) and SAC-KO (open circles) (n = 4) mice. Values are presented as the means ± SEM; *, p < 0.05.

Figure 2

Expression of markers for non-parenchymal cells in transgenic SAC mice. qRT-PCR analyses of (A): Acta2, Gfap and Emr1; (B): Pkm2, Krt19 and Cd34 in liver tissue of 12 weeks old male SAC-WT (white bars) (n = 13) and SAC-KO (black bars) (n = 11) mice. Values are presented as the means ± SEM. Immunohistochemistry in liver sections from male SAC-WT and SAC-KO mice of (C): GFAP (present only in HSC’s), (D): PKM2 (Is present in all non-parenchymal cell types. Hepatocytes in SAC-KO mice show a very light cytoplasmic staining). Bar: 100 μm and 20 μM.

Figure 3

Expression changes of Hedgehog signaling components. qRT-PCR analyses of hedgehog members in hepatocytes freshly isolated from 12-week-old male SAC-WT (white bars) (n = 8-17) and SAC-KO (black bars) (n = 8-13) mice: (A): Smo, Ihh and Shh; (B): Boc, Cdo and Ptch1; (C): Hhip1, Fu and Sufu, and the transcription factors (D): Gli1, Gli2 and Gli3. Values are presented as relative means ± SEM; *, p < 0.05, **, p < 0.01. Immunohistochemical staining of (E): Indian Hedgehog and (F): GLI3 in liver sections from male transgenic SAC-WT and SAC-KO mice at the age of 12 weeks. Scale bars: 100 μM; 50 μM and 25 μM.

Figure 4

Changes of IGF axis members in the liver and serum. Quantitative RT-PCR analyses of (A, B): Igf1 and (C, D): Igfbp1, Igfbp2 and Igfbp3 mRNA levels in hepatocytes isolated from 12-week-old male SAC-WT (white bars) (n = 7) and SAC-KO (black bars) (n = 7) (left diagrams) and female SAC-WT (white bars) (n = 7) and SAC-KO (black bars) (n = 5) (right diagrams) mice relative to that of β-actin. Values are presented as the means ± SEM; *, p < 0.05. (E, F): Serum analyses of circulating IGF-I and IGFBP-1 in male SAC-WT (white bars) (n = 9) and SAC-KO (black bars) (n = 8) mice and female SAC-WT (white bars) (n = 11) and SAC-KO (black bars) (n = 13) mice. Values are presented as the means ± SEM; *, p < 0.05.

Figure 5

RNA interference, ELISA and ChIP experiments. (A): Relative expression of Smo, Gli1, Gli2 and Gli3 determined by qRT-PCR analyses in cultured hepatocytes of male C57BL/6-N mice in response to transfection of Smo siRNA (black bars) (n = 8) compared to nonsense transfection (white bars) (n = 8) after 48 h of incubation. Values are presented as relative means ± SEM; *, p < 0.05. (B): Relative expression of Ptch1, Gli3, Igf1 and Igfbp1 determined by qRT-PCR analyses in cultured hepatocytes of male C57BL/6-N mice in response to transfection with Ptch1 siRNA (black bars) compared to nonsense transfection (white bars) after 48 h (n = 10-12) and 72 h (n = 5-6) of incubation. Values are presented as relative means ± SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (C): Relative expression of Gli3 determined by qRT-PCR analyses in cultured hepatocytes of male C57BL/6-N mice in response to transfection of Gli3 siRNA (black bars) (n = 11) compared to nonsense transfection (white bars) (n = 11) after 72 h of incubation and relative expression of Igf1 and Igfbp1 in C57BL/6 N-hepatocytes after 72 h of incubation with Gli3 siRNA (black bars) (n = 8) compared to nonsense transfection (white bars) (n = 8). Values are presented as relative means ± SEM; *, p < 0.05; **, p < 0.01. (D): Supernatant analyses of secreted IGF-I and IGFBP-1 in cultured hepatocytes of male C57BL/6-N mice in response to transfection of Gli3 siRNA (black bars) (n = 6) compared to nonsense transfection (white bars) (n = 6) after 72 h of incubation. Values are presented as the means ± SEM; *, p < 0.05. (E): GLI3 binding site at -405 bp of the Igf1 promoter according to MotifMap. (F): qRT-PCR analyses of precipitated DNA. DNA immunoprecipitated with GLI3 Antibody (black), Histone H3 Antibody as positive control (grey), and IgG as a negative control (white) calculated relative to input.