KRAS Affects the Lipid Composition by Regulating Mitochondrial Functions and MAPK Activation in Bovine Mammary Epithelial Cells

Abstract

Kirsten rat sarcoma viral oncogene homolog (KRAS), or guanosine triphosphatase KRAS, is a proto-oncogene that encodes the small guanosine triphosphatase transductor protein. Previous studies have found that KRAS can promote cytokine secretion, cell chemotaxis, and survival. However, its effects on milk fat synthesis in bovine mammary epithelial cells are unclear. In this study, the effects of KRAS inhibition on cell metabolism, autophagy, oxidative stress, endoplasmic reticulum stress, mitochondrial function, and lipid composition as well as the potential mechanisms were detected in an immortalized dairy cow mammary epithelial cell line (MAC-T). The results showed that inhibition of KRAS changed the lipid composition (especially the triglyceride level), mitochondrial functions, autophagy, and endoplasmic reticulum stress in cells. Moreover, KRAS inhibition regulated the levels of the mammalian target of rapamycin and mitogen-activated protein kinase (extracellular regulated protein kinases, c-Jun N-terminal kinases, p38) activation. These results indicated that regulation of KRAS would affect the synthesis and composition of milk fat. These results are also helpful for exploring the synthesis and secretion of milk fat at the molecular level and provide a theoretical basis for improving the percentage of fat in milk and the yield of milk from cows.

Keywords: KRAS; MAPK; bovine mammary epithelial cell; endoplasmic reticulum; lipid metabolism; milk fat; triglyceride.

Figure 1

Effects of Kirsten rat sarcoma viral oncogene homolog (KRAS) on cell metabolism in MAC-T cells. (A) Heatmap of some of the differential metabolites. In the figure, each column represents a sample, each row represents a metabolite, and the color indicates the relative level of metabolites expressed in the group. Red indicates that the metabolite is expressed at high levels, and blue indicates lower expression. (B) Volcano plot of the differentially expressed metabolites. Each dot in the figure represents a metabolite, and the size of the dot indicates the variable importance in the projection (VIP) value. The blue dots are significantly downregulated metabolites, and the red dots are significantly upregulated metabolites. (C) Pie chart based on HMDB chemical taxonomy (Super Class) counts for the differential metabolites identified. The color indicates different classes, and the area size indicates the number of metabolites. (D) Top 30 differential metabolites from the VIP plot based on the OPLS-DA models. (E) KEGG pathway enrichment of differential metabolites. Significant differences are represented with * (p < 0.05) and ** (p < 0.01).

Figure 2

Effects of Kirsten rat sarcoma viral oncogene homolog (KRAS) on lipid composition in MAC-T cells. (A) Heatmap of all the differential lipidomic metabolites after KRAS inhibition. In the figure, each column represents a sample, each row represents a metabolite, and the color indicates the relative level of metabolites expressed in the group. Red indicates that the metabolite is expressed at high levels, and blue indicates lower expression. (B) Volcano plot of the differentially lipidomic metabolites. Each dot in the figure represents a metabolite, and the size of the dot indicates the variable importance in the projection (VIP) value. The blue dots are significantly downregulated metabolites, and the red dots are significantly upregulated metabolites. (C) Identified differential lipidomic metabolites were classified. (D) Differential metabolites with VIP > 1.5 from the VIP plot based on the OPLS-DA models. (E) KEGG pathway enrichment of differential lipidomic metabolites. The size of bubbles in the figure represents the amount of differential metabolites, and the color of bubbles represents the p-values. Significant differences are represented with * (p < 0.05) and ** (p < 0.01).

Figure 3

Effects of Kirsten rat sarcoma viral oncogene homolog (KRAS) on the synthesis of milk fat in MAC-T cells. (A,B) Representative Bodipy staining images and relative changes in milk fat content with or without KRAS inhibition. Bar = 10 μm. The experiments were performed five times. The red line indicates the si-NC group, the blue line indicates the si-KRAS group. (C) Relative triglyceride (TG) level changes in MAC-T cells with or without KRAS inhibition. (D–F) Representative Western blot images of peroxisome proliferator-activated receptor gamma (PPARG), sterol regulatory element-binding protein (SREBP1), fatty acid-binding protein (FABP) 4, epidermal growth factor (EGF), EGF receptor (EGFR), IGF1 receptor (IGF1R), and transforming growth factor beta receptor type 1 (TGFβR1) and the relative protein level assays in MAC-T cells with or without KRAS inhibition. (G) Relative mRNA expression levels of xanthine dehydrogenase (XDH), CD36, FABP3, acyl-CoA synthetase long-chain family member 1 (ACSL1), acyl-CoA synthetase short-chain family member 2 (ACSS2), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), glycerol-3-phosphate acyltransferase (GPAM), lipin 1 (LPIN1), and 1-acylglycerol-3-phosphate O-acyltransferase 1 (AGPAT1) with or without KRAS inhibition in MAC-T cells. (H,I) Representative immunofluorescence images of PPARG and SREBP1 in MAC-T cells with or without KRAS inhibition. Bar = 10 μm. (J) Representative immunofluorescence images of Bodipy, FABP4, and CALNEXIN colocalization analysis in MAC-T cells with or without KRAS inhibition. Bar = 10 μm. The fluorescence signals of Hoechst, Bodipy, CALNEXIN, and FABP4 were represented by the blue, green, yellow, and red lines, respectively. Fluorescence colocalization was analyzed based on the white line in merged image. Significant differences are represented with * (p < 0.05) and ** (p < 0.01).

Figure 4

Effects of Kirsten rat sarcoma viral oncogene homolog (KRAS) on mitochondrial function in MAC-T cells. (A) Relative level of mitochondrial DNA (mtDNA) copy number in MAC-T cells with or without KRAS inhibition. (B) Relative ATP level in MAC-T cells with or without KRAS inhibition. (C) Relative mRNA expression levels of cytochrome c oxidase subunit 5B (COX5B), NADH:ubiquinone oxidoreductase core subunit S8 (NDUFS8), succinate dehydrogenase complex iron sulfur subunit B (SDHB), ATP synthase F1 subunit alpha (ATP5F1A), ubiquinol–cytochrome c reductase binding protein (UQCRB), nuclear respiratory factor 1 (NRF1), DNA polymerase gamma catalytic subunit (POLG), mitochondrial transcription factor A (TFAM), and mitochondrial transcription factor B1 (TFB1M) in MAC-T cells with or without KRAS inhibition. (D) Representative Hoechst, MitoTracker, and Bodipy fluorescence images in MAC-T cells with or without KRAS inhibition. Bar = 10 μm. (E,F) Representative Western blot images and relative protein expression levels of mitofusin (MFN)1, MFN2, optic atrophy 1 (OPA1), and dynamin-related protein 1 (DRP1) in MAC-T cells with or without KRAS inhibition. Significant differences are represented with * (p < 0.05) and ** (p < 0.01).

Figure 5

Effects of Kirsten rat sarcoma viral oncogene homolog (KRAS) on reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress in MAC-T cells. (A) Representative flow cytometry images of ROS content and relative dichlorofluorescein (DCFH) levels in MAC-T cells with or without KRAS inhibition. (B,C) Representative Western blot images and relative protein levels of glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), and C/EBP homologous protein (CHOP) in MAC-T cells with or without KRAS inhibition. The β-ACTIN loading controls were performed for CHOP on the same membrane. (D) Relative levels of catalase (CAT), glutathione (GSH), and total antioxidant capacity (T-AOC) in MAC-T cells with or without KRAS inhibition. (E,F) Representative flow cytometry images and apoptosis rate in MAC-T cells with or without KRAS inhibition. (G,H) Representative Western blot images and relative protein expression levels of CASPASE 3 and cleaved-CASPASE 3 in MAC-T cells with or without KRAS inhibition. Significant differences are represented with * (p < 0.05) and ** (p < 0.01).

Figure 6

Effects of Kirsten rat sarcoma viral oncogene homolog (KRAS) on autophagy in MAC-T cells. (A) Representative immunofluorescence images of microtubule-associated protein 1 light chain 3 beta (LC3B) in MAC-T cells with or without KRAS inhibition. Bar = 10 μm. (B–D) Representative Western blot images and relative protein levels of LC3B, mammalian target of rapamycin (mTOR), and p-mTOR in MAC-T. (E) Relative mRNA levels of beclin 1 (BECN1) and autophagy-related 7 (ATG7) in MAC-T cells with or without KRAS inhibition. (F) Representative immunofluorescence images and colocalization analysis of Bodipy, LC3B, and CALNEXIN in MAC-T cells with or without KRAS inhibition. Bar = 10 μm. The fluorescence signals of Hoechst, Bodipy, CALNEXIN, and LC3B were represented by the blue, green, yellow, and red lines, respectively. Fluorescence colocalization was analyzed based on the white line in merged image. Significant differences are represented with * (p < 0.05) and ** (p < 0.01).

Figure 7

Effects of Kirsten rat sarcoma viral oncogene homolog (KRAS) on the phosphorylation levels of mitogen-activated protein kinases (MAPKs) in MAC-T cells. Representative Western blotting images and phosphorylated/total levels of extracellular regulated protein kinase 1/2 (ERK1/2, (A)), c-Jun N-terminal kinase 1/2/3 (JNK1/2/3, (B)), and p38 (C) in MAC-T cells with or without KRAS inhibition. The α-TUBULIN loading controls were performed for p-p38 and p38 on the same membrane. Significant differences are represented with * (p < 0.05) and ** (p < 0.01).

Figure 8

Schematic diagram of the effects of Kirsten rat sarcoma viral oncogene homolog (KRAS) inhibition in MAC-T cells. After KRAS inhibition, the levels of triglycerides, mitochondrial DNA (mtDNA), ATP, reactive oxygen species (ROS), autophagy, and endoplasmic reticulum stress (ERS) were upregulated in MAC-T cells. Moreover, the levels of mitogen-activated protein kinase (extracellular regulated protein kinases, ERK; c-Jun N-terminal kinases, JNK; p38) activation were regulated after KRAS inhibition in MAC-T cells.