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. 2023 Jan 3:101:skac412.
doi: 10.1093/jas/skac412.

C/EBPα promotes triacylglycerol synthesis via regulating PPARG promoter activity in goat mammary epithelial cells

Huibin Tian  1 , Jun Luo  1 , Peng Guo  1 , Chun Li  1 , Xueying Zhang  2
Affiliations

C/EBPα promotes triacylglycerol synthesis via regulating PPARG promoter activity in goat mammary epithelial cells

Huibin Tian et al. J Anim Sci. .

Abstract

CCAAT/enhancer binding protein α (C/EBPα) is the key transcription factor involved in lipid metabolism, however, the role of C/EBPα in milk fat synthesis of dairy goats remains unknown. The objective of the present research was to clarify the function of C/EBPα in goat mammary epithelial cells (GMECs) and its impact on peroxisome proliferator-activated receptor gamma (PPARG) promoter activity. In this study, C/EBPα overexpression increased its mRNA and protein levels by 42-fold and 6-fold, respectively. In contrast, transfecting siRNA _targeting C/EBPα decreased its mRNA level to 20% and protein abundance to 80% of the basal level. The contents of lipid droplets, triacylglycerol (TAG), and cholesterol were increased (P < 0.05) in C/EBPα-overexpressing GMECs, and knockdown of C/EBPα led to the opposite results. Overexpression of C/EBPα significantly increased the expression levels of genes involved in TAG synthesis (AGPAT6, DGAT2, P < 0.01), lipid droplet formation (PLIN2, P < 0.01), and fatty acid synthesis (FADS2, P < 0.05; ELOVL6, P < 0.01). Knockdown of C/EBPα decreased (P < 0.05) the expression levels of AGPAT6, DGAT1, DGAT2, PLIN2, FADS2, and ELOVL6. C/EBPα upregulated the expression level of PPARG (P < 0.05), and four C/EBPα binding regions were identified in the PPARG promoter at -1,112 to -1,102 bp, -734 to -724 bp, -248 to -238 bp, and -119 to -109 bp. Knockdown of C/EBPα reduced (P < 0.05) the PPARG promoter activity when the C/EBPα binding regions were mutated at -1,112 to -1,102 bp, -734 to -724 bp, and -248 to -238 bp locations of the promoter. However, the promoter activity did not change when the mutation was located at -119 bp. In conclusion, our results suggest that C/EBPα can promote TAG synthesis in GMECs through its effects on mRNA abundance of genes related to lipid metabolism and regulation of the PPARG promoter activity via C/EBPα binding regions.

Keywords: CCAAT/enhancer binding protein α; goat mammary epithelial cells; lactation; transcriptional regulation; triacylglycerol synthesis.

Plain language summary

Goat milk is beneficial for human health because of its nutritional value, especially milk fat, which is plentiful in goat milk. The molecular mechanism of milk fat synthesis is of great importance for developing processing technology and using genetic approaches to improve goat milk quality. The purpose of this study was to identify the role of CCAAT/enhancer binding protein α (C/EBPα) in goat mammary gland epithelial cells (GMECs) to provide support for understanding the mechanism of milk fat synthesis. Overexpression of C/EBPα increased the contents of lipid droplets, triacylglycerol, and cholesterol in GMECs. The expression levels of genes related to lipid metabolism were influenced after C/EBPα overexpression or inhibition. The promoter transcriptional activity of peroxisome proliferator-activated receptor gamma, which is the key transcription factor in milk fat synthesis, was regulated by C/EBPα through its binding regions. Our results indicate that C/EBPα affects lipid metabolism in GMECs by regulating PPARG transcription. This study provides support for milk fat synthesis regulation and improvement of milk quality in goats.

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Conflict of interest statement

The authors declare no real or perceived conflicts of interest.

Figures

Figure 1.
Figure 1.
Detection of C/EBPα overexpression and knockdown efficiency in GMECs. (A) The expression level of C/EBPα after transfection of the overexpression vector in GMECs. (B, C) Protein level and abundance analysis of C/EBPα in cells transfected with overexpression vector. (D) mRNA expression level of C/EBPα in siRNA-transfected GMECs. (E, F) Protein level and abundance analysis of C/EBPα in knockdown and control group. pcDNA3.1-CTR, empty overexpression vector; pcDNA3.1-C/EBPα, C/EBPα overexpression vector; siSCR, negative control of small interference RNA; siC/EBPα, small interference RNA of C/EBPα. Values are presented as means ± SEM, **P < 0.01 vs. control.
Figure 2.
Figure 2.
C/EBPα affected the lipid droplet formation in GMECs. (A) Lipid droplets staining of GMECs after C/EBPα overexpression or inhibition. (B, C) The relative immunofluorescence intensity of lipid droplets. Values are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. control.
Figure 3.
Figure 3.
Cellular TAG and cholesterol contents in GMECs after C/EBPα overexpression or knockdown. (A, B) The contents of TAG and cholesterol in GMECs after C/EBPα overexpression. (C, D) The contents of TAG and cholesterol in C/EBPα knockdown GMECs. Values are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. control.
Figure 4.
Figure 4.
The expression levels of genes related to lipid metabolism in GMECs. Expression levels of genes related to (A, D) TAG synthesis (AGPAT6, DGAT1, DGAT2), (B, E) lipid droplet formation (PLIN2, ATGL), and (C, F) fatty acid synthesis (FADS2, ELOVL6). Values are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. control.
Figure 5.
Figure 5.
C/EBPα overexpression or inhibition affected PPARG mRNA expression, and PPARG promoter activity was regulated by C/EBPα binding regions in GMECs. The mRNA level of PPARG was influenced by (A) overexpression or (B) knockdown of C/EBPα. (C) The basic activity of PPARG promoter in GMECs. (D) The C/EBPα binding regions analysis in PPARG promoter sequence. (E) Effect of different C/EBPα binding regions mutation on PPARG promoter activity after C/EBPα inhibition. Firefly luciferase activity was examined and normalized to renilla luciferase activity. C/EBPα represents different locations of C/EBPα binding regions. TSS, transcription start site. The number −1,112, −734, −248, and −119 represent four different C/EBPα binding regions at −1,112 to −1,102 bp, −734 to −724 bp, −248 to −238 bp, and −119 to −109 bp locations of the promoter, respectively. Values are presented as means ± SEM. *P < 0.05, ** P < 0.01 vs. control.
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