Fig 1.
Effects of nutrients and hormones on SOAT1 mRNA accumulation in EECs and hepatocytes.
(A) The mRNA accumulations of SOAT1 during embryogenesis in YSM of Japanese quail. (B-I) EECs and hepatocytes were treated for 24 hours with different concentrations of a fatty acid, OA (oleic acid) or PA (palmitic acid); L(low) = 10 μM; H (high) = 100 μM, or glucose (3.3, 5.5 or 17.5 mM), or hormones (estrogen L = 1.8 nM, H = 18 nM; Dex L = 1 μm, H = 10 μm, glucagon L = 10 nM, H = 100 nM; IBMX L = 0.05 mM, H = 0.5 mM). SOAT1 mRNA accumulation was measured by Real-Time PCR and normalized to β-actin mRNA accumulation. Data were expressed as mean ± S.E.M. (n = 6) Statistical significance was determined by one-way analysis of variance. Tukey‘s test was used to evaluate differences between means. Control value was set as 1. Different letters indicate a significant difference (P≤0.05).
Fig 2.
Positive regulation of transcription and enzymatic function of SOAT1.
(A-D) Effects of forskolin and dibutyryl-cyclic AMP (db-cAMP) on SOAT1 mRNA accumulations in EECs and hepatocytes. EECs and hepatocytes were treated with different concentrations of forskolin or db-cAMP for 24 hours. SOAT1 mRNA accumulation was measured and normalized to β-actin mRNA. (n = 4) (E-F) H89 inhibited IBMX-induced SOAT1 mRNA accumulations in EECs and hepatocytes. SOAT1 mRNA accumulation was increased by 0.5 mM IBMX treatment for 24 hours, whereas the PKA inhibitor, H89 (10 μM) abolished the IBMX-mediated increase of SOAT1. (n = 6) (G-H) The IBMX treatment increased PKA activity in EECs and hepatocytes. The cells were treated with 0.5 mM IBMX with or without 10 μM H89 for 24 hours. The cell lysates were assayed for PKA activity using the PepTag assay. Typical gel patterns are represented (three independent experiments) for EECs and hepatocytes with statistical analysis of the densitometric analysis represented by the graphs (quantified by ImageQuant software). (I) Fluorescence examination of SOAT1 activity by applying NBD-cholesterol as the substrate. EECs were incubated with 10 μg/mL of NBD-cholesterol at 37°C for 30 mins or 1 hour. Green indicated the cholesterol ester inside of EECs and blue indicated cell nucleus stained with DAPI. (J-K) The fluorescent intensity was quantified by ImageJ software. EECs were treated with or without db-cAMP for 24 hours. EECs were then incubated with 10 μg/mL of NBD-cholesterol for 30 min or 1 hour and examined by confocal microscopy. (J) Fluorescent intensity of total area. K: Fluorescent intensity per area of lipid droplets in EECs. (n = 4) Statistical significance was determined by one-way analysis of variance. Tukey‘s test was used to evaluate differences between means. Control value was set as 1. Different letters indicate a significant difference (P≤0.05). (L) The SOAT1 protein levels after db-cAMP or IBMX treatments. EECs were treated with 0.5 or 1 mM db-cAMP or 0.5 mM IBMX for 48 hours and protein levels were examined by Western blotting. The β-actin was used as internal control. Statistical significance was determined by one-way analysis of variance. Dunnett's multiple comparisons test was used to evaluate differences between means. Data were expressed as means ± S.E.M. (n = 8) and different letters indicate the significant difference (P≤0.05). (M-N) The effects of H89 with or without db-cAMP and IBMX treatments on NBD-cholesterol uptake and storage. (M) EECs were pre-treated with or without H89 (10 μM) for 2 hours, and added db-cAMP (1 mM) or IBMX (0.5 mM) for 24 hours. Cells were then incubated with 10 μg/mL NBD-cholesterol for one hour, fixed with 4% paraformaldehyde and finally examined by confocal microscopy. Green indicated the cholesterol ester inside of EECs and blue indicated cell nucleus stained with DAPI. Scale bar = 50 μm. (N) The quantitation of fluorescent intensity on NBD-cholesterol uptake and storage by ImageJ software in cells treated with H89 with or without IBMX or db-cAMP. Data were expressed as means ± S.E.M. (n = 4) Statistical significance among more than three different experimental groups was determined by one-way analysis of variance. Tukey‘s test was used to evaluate differences between means. Different letters indicate a significant difference (P≤0.05).
Table 1.
The chicken primers for real-time PCR quantification.
Table 2.
Conditions and primers for serial promoter region deletion.
Table 3.
Promoter mutation primer sets.
Fig 3.
The importance of functional responsive elements CRE (cAMP-responsive element) on the SOAT1 promoter.
(A) Effect of IBMX and the PKA inhibitor, H89 on the induction of SOAT1 promoter activity. Promoter activities were normalized for transfection efficiency using the renilla luciferase activity. (n = 5) (B) Effect of IBMX on the induction of serially deleted promoter regions of SOAT1. The 293T cells were transfected with variable length constructs of the SOAT1 promoter and promoter activities were assayed after 24 hours of IBMX treatment. The pGL3 was a luciferase vector without SOAT1 promoter insertion, and was used as a negative control. (n = 6) (C) Diagram of wild type and mutated cAMP-responsive element on the SOAT1 promoter construct in a luciferase vector. (D) Effect of IBMX and H89 on the induction of promoter activity by the mutated CREB in the SOAT1 promoter. The 293T cells were transfected with normal or mutated SOAT1 promoter, and promoter activities were assayed after 0.5 mM IBMX and 10 μM H89 (PKA inhibitor) treatments for 24 hours. (n = 6) Data were expressed as means ± S.E.M. (n = three independent experiments) Statistical significance was determined by one-way analysis of variance. Tukey's test was used to evaluate differences between means. Control value was set as 1. Different letters indicate a significant difference (P≤0.05).
Fig 4.
H89 inhibited IBMX-induced transcription factors mRNA accumulations in EECs and hepatocytes.
Cells were treated with 0.5 mM IBMX alone for 24 h, CREB1 and CREBBP mRNA accumulations were increased, whereas the H89 (PKA inhibitor, 10 μM) abolished the increased mRNA accumulations mediated by IBMX in both culture systems. Data were expressed as means ± S.E.M. (n = 6) Statistical significance was determined by one-way analysis of variance. Tukey's test was used to evaluate differences between means. Control value was set as 1. Different letters indicate a significant difference (P≤0.05).
Fig 5.
The illustration of YSM structure and the positive regulation of SOAT1 enhancement by a cAMP-dependent pathway in avian EECs.
AC, adenylyl cyclase; CRE, cAMP-response element; CREB1, cAMP responsive element binding protein; CREBBP, phosphorylated CREB binding protein; CEBPRE, C/EBP (CCAAT-enhancer-binding protein) response element; db-cAMP, dibutyryl-cAMP (a cAMP analog); MAM, mitochondria-associated membrane; PKA, protein kinase A; R-regulatory units, C-catalytic units. (Fig 5 was drawn by Han-Jen Lin).