Fig 1.
CDCA markedly repressed AKR1D1 expression in human hepatoma HepG2 cells.
HepG2 cells were treated with CDCA (25μM) or vehicle DMSO (0.1%) for 30h, followed by detection of the mRNA levels of (A) AKR1D1, (B) CYP7A1 and (C) CYP8B1 by real-time PCR, and (D) protein levels of AKR1D1, CYP7A1 and CYP8B1 by Western blotting. (E) quantification of AKR1D1, (F) CYP7A1, and (G) CYP8B1 protein levels in (D). The data are presented as mean ± SD of at least three separate experiments or treatments. The Student’s t-test was applied to pair-wise comparison. *p<0.05, **p<0.01.
Fig 2.
CDCA repressed AKR1D1 expression in time and dose-dependent manners in HepG2 cells.
(A) HepG2 cells were treated with either CDCA (25 μM) or vehicle DMSO for 8, 24 or 30 hrs, followed by detection of AKR1D1 mRNA with real-time PCR. (B) HepG2 cells were treated with various concentrations of CDCA (0, 5, 25, 50, 100 μM) for 30 hrs, followed by detection of AKR1D1 mRNA expression with real-time PCR. **p<0.01 with the Student’s t-test for pair-wise comparison.
Fig 3.
Akr1d1 expression was severely repressed by CDCA in vivo in mice.
Two groups of mice (n = 6/group) were treated with CDCA (5mg/kg) or vehicle propanediol through intraperitoneal injection twice a day for 3 days. Twelve hours after the last injection, mice were euthanized and liver tissues were harvested and processed for mRNA and protein analyses. (A) the effects of CDCA treatment on Akr1d1, (B) Cyp7a1 and (C) Cyp8b1 mRNA levels detected by real-time PCR. (D) the effects of CDCA treatment on Akr1d1, Cyp7a1 and Cyp8b1 protein expression detected by Western blotting, and (E) quantification of Akr1d1, (F) Cyp7a1 and (G) Cyp8b1 protein levels in (). The data are presented as mean ± SD of the groups. The Student’s t-test was applied to pair-wise comparison. *p<0.05, **p<0.01.
Fig 4.
CA significantly upregulated AKR1D1 expression in human HepG2 cells.
HepG2 cells were treated with CA (25μM) or vehicle DMSO (0.1%) for 30h, followed by detection of the mRNA levels of (A) AKR1D1, (B) CYP7A1 and (C) CYP8B1 by real-time PCR, and the protein levels of (D) AKR1D1, CYP7A1 and CYP8B1 detected by Western blotting. (E) quantification of AKR1D1, (F) CPY7A1 and (G) CYP8B1 protein levels in (D). The data are presented as mean ± SD of at least three separate experiments or treatments. The Student’s t-test was applied to pair-wise comparison. *p<0.05.
Fig 5.
CA modulated AKR1D1 expression in time and dose-dependent manners in HepG2 cells.
(A) HepG2 cells were treated with either CA (25 μM) or vehicle DMSO for 8, 24 or 30 hrs, followed by detection of AKR1D1 mRNA with real-time PCR. (B) HepG2 cells were treated with various concentrations of CA (0, 5, 25, 50, 100, 200 μM) for 30 hrs, followed by detection of AKR1D1 mRNA expression with real-time PCR. *p<0.05 with the Student’s t-test for pair-wise comparison.
Fig 6.
Akr1d1 expression was induced by CA in vivo in mice.
Two groups of mice (n = 6/group) were treated with CA (5mg/kg) or vehicle propanediol through intraperitoneal injection twice a day for 3 days. Twelve hours after the last injection, mice were euthanized and liver tissues were harvested and processed for mRNA and protein analyses. (A) the effects of CA treatment on Akr1d1, (B) Cyp7a1 and (C) Cyp8b1 mRNA levels detected by real-time PCR. (D) the effects of CA treatment on Akr1d1, Cyp7a1 and Cyp8b1 protein expression detected by Western blotting, and (E) quantification of Akr1d1, (F) Cyp7a1 and (G) Cyp8b1 protein levels in (D). The data are presented as mean ± SD of the groups. The Student’s t-test was applied to pair-wise comparison. *p<0.05.
Fig 7.
FXR signaling was not involved in regulating AKR1D1 by bile acids in HepG2 cells.
(A) HepG2 cells were treated with FXR agonist GW4064 (1μM) or vehicle DMSO (0.1%) for 30h, followed by detection of AKR1D1 and BSEP mRNA levels by real-time PCR. (B) HepG2 cells were transfected with FXRα1, FXRα2 or vector, followed by treatment with GW4064 (1μM) for 30h. The expression levels of AKR1D1 and BSEP were detected by real-time PCR. The Student’s t-test was applied to pair-wise comparison. One-way ANOVA was applied to analyze data with multiple groups, followed by Tukey post-hoc test for multiple comparisons. ** p<0.01.
Fig 8.
FXR signaling was not involved in regulating AKR1D1 by bile acids in vivo in mice.
(A) two groups of mice (n = 6/group) were treated with GW4064 (5mg/kg) or vehicle propanediol through intraperitoneal injection twice a day for 3 days. The expression levels of Akr1d1 and Bsep were quantified by real-time PCR and (B) Western blot. (C) quantification of Akr1d1 and (D) Bsep protein levels in (B). (E) the expression levels of Akr1d1 and Bsep in wt and FXR-knockout (FXR-/-) mice were determined by real-time PCR and (F) Western blot. (G) quantification of Akr1d1 and (H) Bsep protein levels in (F). The data are presented as mean ± SD of the groups of mice. The Student’s t-test was applied to pair-wise comparison. ** p<0.01.
Fig 9.
CDCA and CA differentially modulated the MAPK/JNK signaling pathway.
HepG2 cells were reversely transfected with 45 signaling pathway element reporter plasmids, followed by treatment of transfected cells with CDCA (25μM), CA (25μM) or vehicle DMSO (0.1%) for 30 h. The luciferase activities were detected with the Dual Luciferase Assays. The data are presented as mean ± SD of at least three separate experiments. One-way ANOVA was applied to analyze data with multiple groups, followed by Tukey post-hoc test for multiple comparisons. * p<0.05 and ** p<0.01.
Fig 10.
Inhibition of MAPK/JNK signaling pathway abolished CDCA-mediated regulation of AKR1D1.
(A) HepG2 cells were treated with CDCA (25μM) in the absence or presence of MAPK/JNK inhibitor SP600125 (1μM), MAPK/ERK1/2 inhibitor PD98059 (5μM) or vehicle for 30 hrs, followed by detection of AKR1D1 mRNA by real-time PCR and (B) AKR1D1 protein by Western blot. (C) quantification of AKR1D1 protein levels in (B). One-way ANOVA was applied to analyze the data, followed by Tukey post-hoc test for multiple comparisons. * p<0.05 and ** p<0.01.
Fig 11.
Inhibition of MAPK/JNK signaling pathway abolished CA-mediated regulation of AKR1D1.
(A) HepG2 cells were treated with CA (50μM) in the absence or presence of MAPK/JNK inhibitor SP600125 (1μM), MAPK/ERK1/2 inhibitor PD98059 (5μM) or vehicle for 30 hrs, followed by detection of AKR1D1 mRNA by real-time PCR and (B) AKR1D1 protein by Western blot. (C) quantification of AKR1D1 protein levels in (B). One-way ANOVA was applied to analyze the data, followed by Tukey post-hoc test for multiple comparisons. * p<0.05.
Fig 12.
Functions and regulation of AKR1D1 in bile acid synthesis and steroid hormone metabolism.
In the bile acid synthesis pathway, bile acid intermediate 7α-hydroxy-4-cholesten-3-one can take one of the two routes in subsequent steps. If the intermediate is acted upon by AKR1D1, the ultimate product is CDCA. If the intermediate is acted upon by CYP8B1, followed by AKR1D1, the ultimate product is CA. In this study, we demonstrated that CDCA and CA regulated AKR1D1 through a negative and positive feedback mechanism, respectively. In addition to bile acid synthesis, AKR1D1 is also involved in steroid hormone metabolism. 5β-reduction by AKR1D1 is a common transformation and major deactivation pathway for many steroid hormones. Therefore, AKR1D1 plays a critical role in regulating and maintaining the homeostasis of steroid hormones. Thus bile acids crosstalk with steroid hormone signaling pathways through modulating AKR1D1 expression. Plus (+) and minus (-) indicated positive and negative feedback regulation, respectively.