# GosB Inhibits Triacylglycerol Synthesis and Promotes Cell Survival in Mouse Mammary Epithelial Cells

Authors: Gaoxiao Xu; Saixing Duan; Jianye Hou; Zhongxin Wei; Guangwei Zhao
Journal: BioMed Research International
 (2017)

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## Abstract

It has been demonstrated that the activator protein related transcription factor Finkel-Biskis-Jinkins murine osteosarcoma B (GosB) is involved in preadipocyte differentiation and triacylglycerol synthesis. However, the role of GosB in regulating the synthesis of milk fatty acid in mouse mammary glands remains unclear. This research uncovered potentially new roles of GosB in suppressing milk fatty acid synthesis. Results revealed that GosB had the highest expression in lung tissue and showed a higher expression level during nonlactation than during lactation. GosB inhibited the expression of fatty acid synthase(FASN), stearoyl-CoA desaturase(SCD), fatty acid binding protein 4(FABP4), diacylglycerol acyltransferase 1(DGAT1), perilipin 2(PLIN2), perilipin 3(PLIN3), andC/EBPα in mouse mammary gland epithelial cells (MEC). In addition, GosB reduced cellular triglyceride content and the accumulation of lipid droplets; in particular, GosB enhanced saturated fatty acid concentration (C16:0 and C18:0). The PPARγ agonist, rosiglitazone (ROSI), promoted apoptosis and inhibited cell proliferation. GosB increased the expression ofBcl-2 and protected MEC from ROSI-induced apoptosis. Furthermore, MECs were protected from apoptosis through the GosB regulation of intracellular calcium concentrations. These findings suggest that GosB may regulate mammary epithelial cells milk fat synthesis and apoptosis via PPARγ in mouse mammary glands.


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## Body

## 1. Introduction

Milk fat is an important component of milk production and is necessary for neonatal growth and development [1]. Mammary gland epithelial cells which synthesize and secrete milk fat can be a cellular mode of research for mammary gland lipid synthesis mechanism [2]. The activator protein 1 (AP-1) family of transcription factors comprises various combinations of Fos (Gos, GosB, Fra-1, and Fra-2) and Jun (c-Jun, JunB, and JunD) which upon dimer formation regulate osteoblast differentiation and influence adipocyte commitment [3–5]. InGosB transgenic mice, the phenotype exhibits a decreased mass of adipose tissue, reduced adipocyte numbers, and abdominal fat concentrations [3, 6, 7]. The cultured bone marrow stromal cell fromGosB mice exhibited few adipocytes differentiated with only few and small lipid droplets. Additionally, the expression of PPARγ2, C/EBPα, and C/EBPβ, confirmed transcription factors associated with the adipocytic phenotype, is also decreased [3]. Overexpression of GosB in the multipotential stromal cell line ST2 inhibited adipogenic differentiation, binding to and altering the DNA-binding capacity of C/EBPβ [6, 8]. To better understand the molecular mechanisms behind the fat phenotype observed inGosB mice and to determine why adipocyte numbers are low inGosB mutants, we suspect that the proliferation and the apoptosis status of adipocyte could affect the mass of adipose tissue. It has been demonstrated that GosB not only is an important regulator of lipid synthesis but also has a role in cell survival in mammary cells [9, 10], as data revealed that it can mediate cell proliferation and apoptosis [11–13]. Overexpression of GosB in goat mammary gland epithelial cells increased cell viability [9, 10]. GosB protects rat embryo cells from apoptosis and exhibits a significantly higher survival occurring in the mitochondrial pathway dependent on caspase-3 and caspase-9 [13]. However, the role of GosB in milk fat synthesis and cell survival remains unknown in mouse mammary epithelial cells (MEC). It has been demonstrated that activation of PPARγ in human epithelial cells inhibits AP-1 DNA-binding activity due to competition for limited amounts of the transcriptional coactivator CREB-binding protein of PPARγ with AP-1 [14, 15]. Based upon the above facts we hypothesize that pretreatment of rosiglitazone may increase cellular triglyceride content and inhibit cell survival, and these effects may be regulated by GosB in mammary gland epithelial cells.


## 2. Materials and Methods

### 2.1. Tissue Collection

Brain, mammary gland, subcutaneous fat, skeletal muscle, heart, liver, spleen, lung, kidney, and small intestine tissues were collected from six nonlactating mice (C57BL/6J genetic background). In addition, mammary gland tissue samples were collected from six C57BL/6J mice on day 5 of lactation. All tissue samples were obtained under sterile conditions and washed with diethylpyrocarbonate (DEPC) treated water and then immediately frozen in liquid nitrogen.


### 2.2. Cell Culture and Treatment

The primary MEC were isolated from C57BL/6J mice on day 5 of lactation as previous described [16]. Briefly, mice were killed by cervical dislocation and tissues were removed, washed 3-4 times in PBS, and minced into 1 to 2 mm pieces. The tissue fragments were suspended in DMEM containing 2 mg collagenase/ml (type III), 20 mg BSA/ml, and 15 mM HEPES buffer, without serum, at a ratio of 12 mL medium/g wet weight of tissue. Enzymatic digestion was conducted at 37°C for 60–75 minutes. After removal of undigested tissue pieces and large clumps of cells by sedimentation, single cells and small clumps (2–4 cells) of cells were washed twice in CMF-PBS. Viability of the cells was determined prior to their use in the suspension agglutination assay by the try pan blue dye method. The cells were washed twice in DMEM supplemented with 13% FCS, 5 μg insulin/ml, 15 mM HEPES buffer, 100 μg streptomycin/ml, and 100 U penicillin/ml. The cells were plated in either plastic flasks or Petri dishes (Corning) at concentrations varying from 5 to 50 × 104 cells/cm2 to obtain cultures of different densities. Incubation was performed at 37°C in a 10% CO2–90% air atmosphere. The medium was replaced 36–40 hours after seeding with DMEM in which D-valine was substituted for L-valine.The GosB overexpression recombinant adenoviruses, Ad-GosB, and interference expression recombinant adenoviruses, Ad-siGosB, were prepared by our laboratory. The PPARγ specific ligand rosiglitazone (ROSI) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). When the MEC grew to approximately 80% confluence, the MEC were treated with (a) 10 μM ROSI; (b) 4 mM Ca2+; (c) Ad-GosB or Ad-siGosB, respectively; (d) Ad-GosB + ROSI; and (e) Ad-GosB + Ca2+. After incubation, the MEC were used for RNA extraction, Oil Red O staining assay, triglyceride content measurement, fatty acid analysis, cell proliferation, and apoptosis assay.


### 2.3. Total RNA Extraction and Quantitative Real-Time PCR

The MEC were lysed in 1 mL of Trizol Reagent (Invitrogen) and the total RNA was precipitated with an equal volume of isopropanol. The RNA pellet was washed twice with 75% ethanol and the cDNA was synthesized using a transcript first-strand cDNA synthesis kit (TaKaRa, Dalian, China). Quantitative real-time PCR (qPCR) primers are shown in Table S1 (in Supplementary Material available online athttps://doi.org/10.1155/2017/7394869). Three genes were used as internal control genes including glyceraldehyde-3-phosphate dehydrogenase(GAPDH), ubiquitously expressed transcript(UXT), and mitochondrial ribosomal protein L39(MRPL39). The relative expression level of qPCR data was analyzed by 2-ΔΔCt method.


### 2.4. Oil Red O Staining, Triglyceride Measurement, and Fatty Acid Analysis

The MEC were fixed for 20 min at room temperature with 4% paraformaldehyde. Cells were stained with Oil Red O solution (0.3% Oil Red O in 60% isopropanol) for 20 min. For quantification, Oil Red O staining was dissolved in isopropanol and absorbance was measured at 490 nm. Intracellular triglyceride content was assayed using a triglyceride assay kit (Applygen, China). Extraction and analyses of fatty acid (FA) were as previously described [17]. Total FA was extracted and methylated from approximately 100 mg of MEC using 2 mL of chloroform or methanol (3 : 1 vol/vol). Methylated samples were analyzed using gas chromatography-mass spectrometry (GC-MS) with an HP-5 column (Agilent Technologies). The relative ratios of FA were identified based on percentages of the total peak areas.


### 2.5. Cell Cycle Assay

Cell cycle analysis was performed using Cell Cycle Testing Kit (Multisciences, China). The MEC were grown in six-well plates (1 × 106 cells/well), harvested, and centrifuged at 800 g/min for 5 minutes. The supernatant was discarded, and the cells were washed once by cold phosphate buffered saline (PBS). Then the cells were resuspended using 1 mL of reagent A and 10 μL of reagent B, subsequently blended by vortexing for 10 seconds and incubated for 30 minutes, and then analysis was done using a flow cytometry (FACS Canto™ II, BD BioSciences, USA).


### 2.6. Cell Proliferation Assay

Cell proliferation was determined by using the Cell Counting Kit-8 (CCK-8) assay and EdU incorporation assay. For CCK-8 assay, the cells were plated into 96-well culture plates at a density of 1 × 104 cells/well in a 100 μL of culture medium per well. After 48 hours of culture, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 2 hours. The absorbance of each sample was detected using a microplate reader (Molecular Devices, USA) at 450 nm. Cell proliferation was also assessed by Cell-Light EdU DNA cell proliferation kit (RiboBio, Guangzhou, China), in accordance with the manufacturer’s instructions.


### 2.7. Cell Apoptosis Measurement by Annexin V-FITC/PI Staining Assays

After incubations, the MEC were washed with PBS three times and harvested by trypsinization, then washed with PBS again, and resuspended in 500μL 1x binding buffer. The cells were incubated for 10 minutes in the dark at room temperature in the presence of Annexin V-FITC (5 μL) and propidium iodide (PI) (10 μL, Vazyme, China). The cells were immediately analyzed using a flow cytometry.


### 2.8. Hoechst33342/PI Dual Staining Assays

Besides the Annexin V-FITC/PI staining assay measured by flow cytometry, cell apoptosis was also detected with Hoechst 33342 and PI double staining analysis (Solarbio, China). In brief, after transfection, the MEC were washed with PBS and subsequently stained with Hoechst 33342 (100μg/mL) for 15 min at room temperature. The cells were washed with PBS twice and incubated for 10 min at room temperature after 100 μg/mL PI was added. The presence of fluorescence was observed under a fluorescence microscope. Hoechst33342−/PI− cells (viable cells) appeared light blue; Hoechst33342+/PI− cells (early apoptotic cells) appeared with blue fragmentations; Hoechst33342+/PI− cells (late apoptotic cells) appeared with red fragmentations; PI+ cells (necrotic cells) appeared with debris signals.


### 2.9. Measurement of Intracellular Calcium

Upon closer examination of the cytosolic and mitochondrial calcium levels in myoblasts transfected with Ad-GosB or Ad-siGosB, the data indicate that activation of intracellular Ca2+ triggers cellular dysfunction and cell death [18, 19], and GosB promotes the formation of calcium deposits [6, 7]. Intracellular calcium trafficking of the cytoplasm and mitochondria were detected using Fluo-3 AM and Rhod-2 AM (2 μM, Molecular probes, Life Technologies), respectively. Cells were treated as described previously [9, 10] and screened by means of flow cytometry and the use of a confocal laser scanning microscope (Leica, Solms, Germany).


### 2.10. Statistical Analyses

The results were analyzed using SPSS 19.0 (SPSS Inc., Chicago, IL) and reported as means ± standard error of the means (SEM) of at least 3 independent experiments. The quantitative real-time PCR data were analyzed using the2-ΔΔCt method relative to the control, where Ct is the cycle threshold. Relative content of each FA is reported as a portion of the total FA, and the desaturation index was calculated as the ratio between the desaturation product and the sum of the product and the substrate [20]. Comparisons were performed by Student’st-test (unpaired and 2-tailed) for a one-way ANOVA. Significance was declared at P<0.05.


## 2.1. Tissue Collection

Brain, mammary gland, subcutaneous fat, skeletal muscle, heart, liver, spleen, lung, kidney, and small intestine tissues were collected from six nonlactating mice (C57BL/6J genetic background). In addition, mammary gland tissue samples were collected from six C57BL/6J mice on day 5 of lactation. All tissue samples were obtained under sterile conditions and washed with diethylpyrocarbonate (DEPC) treated water and then immediately frozen in liquid nitrogen.


## 2.2. Cell Culture and Treatment

The primary MEC were isolated from C57BL/6J mice on day 5 of lactation as previous described [16]. Briefly, mice were killed by cervical dislocation and tissues were removed, washed 3-4 times in PBS, and minced into 1 to 2 mm pieces. The tissue fragments were suspended in DMEM containing 2 mg collagenase/ml (type III), 20 mg BSA/ml, and 15 mM HEPES buffer, without serum, at a ratio of 12 mL medium/g wet weight of tissue. Enzymatic digestion was conducted at 37°C for 60–75 minutes. After removal of undigested tissue pieces and large clumps of cells by sedimentation, single cells and small clumps (2–4 cells) of cells were washed twice in CMF-PBS. Viability of the cells was determined prior to their use in the suspension agglutination assay by the try pan blue dye method. The cells were washed twice in DMEM supplemented with 13% FCS, 5 μg insulin/ml, 15 mM HEPES buffer, 100 μg streptomycin/ml, and 100 U penicillin/ml. The cells were plated in either plastic flasks or Petri dishes (Corning) at concentrations varying from 5 to 50 × 104 cells/cm2 to obtain cultures of different densities. Incubation was performed at 37°C in a 10% CO2–90% air atmosphere. The medium was replaced 36–40 hours after seeding with DMEM in which D-valine was substituted for L-valine.The GosB overexpression recombinant adenoviruses, Ad-GosB, and interference expression recombinant adenoviruses, Ad-siGosB, were prepared by our laboratory. The PPARγ specific ligand rosiglitazone (ROSI) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). When the MEC grew to approximately 80% confluence, the MEC were treated with (a) 10 μM ROSI; (b) 4 mM Ca2+; (c) Ad-GosB or Ad-siGosB, respectively; (d) Ad-GosB + ROSI; and (e) Ad-GosB + Ca2+. After incubation, the MEC were used for RNA extraction, Oil Red O staining assay, triglyceride content measurement, fatty acid analysis, cell proliferation, and apoptosis assay.


## 2.3. Total RNA Extraction and Quantitative Real-Time PCR

The MEC were lysed in 1 mL of Trizol Reagent (Invitrogen) and the total RNA was precipitated with an equal volume of isopropanol. The RNA pellet was washed twice with 75% ethanol and the cDNA was synthesized using a transcript first-strand cDNA synthesis kit (TaKaRa, Dalian, China). Quantitative real-time PCR (qPCR) primers are shown in Table S1 (in Supplementary Material available online athttps://doi.org/10.1155/2017/7394869). Three genes were used as internal control genes including glyceraldehyde-3-phosphate dehydrogenase(GAPDH), ubiquitously expressed transcript(UXT), and mitochondrial ribosomal protein L39(MRPL39). The relative expression level of qPCR data was analyzed by 2-ΔΔCt method.


## 2.4. Oil Red O Staining, Triglyceride Measurement, and Fatty Acid Analysis

The MEC were fixed for 20 min at room temperature with 4% paraformaldehyde. Cells were stained with Oil Red O solution (0.3% Oil Red O in 60% isopropanol) for 20 min. For quantification, Oil Red O staining was dissolved in isopropanol and absorbance was measured at 490 nm. Intracellular triglyceride content was assayed using a triglyceride assay kit (Applygen, China). Extraction and analyses of fatty acid (FA) were as previously described [17]. Total FA was extracted and methylated from approximately 100 mg of MEC using 2 mL of chloroform or methanol (3 : 1 vol/vol). Methylated samples were analyzed using gas chromatography-mass spectrometry (GC-MS) with an HP-5 column (Agilent Technologies). The relative ratios of FA were identified based on percentages of the total peak areas.


## 2.5. Cell Cycle Assay

Cell cycle analysis was performed using Cell Cycle Testing Kit (Multisciences, China). The MEC were grown in six-well plates (1 × 106 cells/well), harvested, and centrifuged at 800 g/min for 5 minutes. The supernatant was discarded, and the cells were washed once by cold phosphate buffered saline (PBS). Then the cells were resuspended using 1 mL of reagent A and 10 μL of reagent B, subsequently blended by vortexing for 10 seconds and incubated for 30 minutes, and then analysis was done using a flow cytometry (FACS Canto™ II, BD BioSciences, USA).


## 2.6. Cell Proliferation Assay

Cell proliferation was determined by using the Cell Counting Kit-8 (CCK-8) assay and EdU incorporation assay. For CCK-8 assay, the cells were plated into 96-well culture plates at a density of 1 × 104 cells/well in a 100 μL of culture medium per well. After 48 hours of culture, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 2 hours. The absorbance of each sample was detected using a microplate reader (Molecular Devices, USA) at 450 nm. Cell proliferation was also assessed by Cell-Light EdU DNA cell proliferation kit (RiboBio, Guangzhou, China), in accordance with the manufacturer’s instructions.


## 2.7. Cell Apoptosis Measurement by Annexin V-FITC/PI Staining Assays

After incubations, the MEC were washed with PBS three times and harvested by trypsinization, then washed with PBS again, and resuspended in 500μL 1x binding buffer. The cells were incubated for 10 minutes in the dark at room temperature in the presence of Annexin V-FITC (5 μL) and propidium iodide (PI) (10 μL, Vazyme, China). The cells were immediately analyzed using a flow cytometry.


## 2.8. Hoechst33342/PI Dual Staining Assays

Besides the Annexin V-FITC/PI staining assay measured by flow cytometry, cell apoptosis was also detected with Hoechst 33342 and PI double staining analysis (Solarbio, China). In brief, after transfection, the MEC were washed with PBS and subsequently stained with Hoechst 33342 (100μg/mL) for 15 min at room temperature. The cells were washed with PBS twice and incubated for 10 min at room temperature after 100 μg/mL PI was added. The presence of fluorescence was observed under a fluorescence microscope. Hoechst33342−/PI− cells (viable cells) appeared light blue; Hoechst33342+/PI− cells (early apoptotic cells) appeared with blue fragmentations; Hoechst33342+/PI− cells (late apoptotic cells) appeared with red fragmentations; PI+ cells (necrotic cells) appeared with debris signals.


## 2.9. Measurement of Intracellular Calcium

Upon closer examination of the cytosolic and mitochondrial calcium levels in myoblasts transfected with Ad-GosB or Ad-siGosB, the data indicate that activation of intracellular Ca2+ triggers cellular dysfunction and cell death [18, 19], and GosB promotes the formation of calcium deposits [6, 7]. Intracellular calcium trafficking of the cytoplasm and mitochondria were detected using Fluo-3 AM and Rhod-2 AM (2 μM, Molecular probes, Life Technologies), respectively. Cells were treated as described previously [9, 10] and screened by means of flow cytometry and the use of a confocal laser scanning microscope (Leica, Solms, Germany).


## 2.10. Statistical Analyses

The results were analyzed using SPSS 19.0 (SPSS Inc., Chicago, IL) and reported as means ± standard error of the means (SEM) of at least 3 independent experiments. The quantitative real-time PCR data were analyzed using the2-ΔΔCt method relative to the control, where Ct is the cycle threshold. Relative content of each FA is reported as a portion of the total FA, and the desaturation index was calculated as the ratio between the desaturation product and the sum of the product and the substrate [20]. Comparisons were performed by Student’st-test (unpaired and 2-tailed) for a one-way ANOVA. Significance was declared at P<0.05.


## 3. Results

### 3.1. Effects of GosB on the Expression of Genes Related to Milk Fat Synthesis

TheGosB was ubiquitously expressed in different tissues, and lung tissue had the highest expression ofGosB, followed by subcutaneous fat, mammary gland, and heart tissue (Figure 1(a)). Relative to the peak lactation, the expression level ofGosB during nonlactation increased by 2.3-fold (P<0.05, Figure 1(b)). Compared with the negative control cells, the expression ofGosB markedly increased after the cells were infected with overexpression recombinant adenoviruses Ad-GosB for 48 hours; theGosB expression was reduced significantly in MEC transfected with interference recombinant adenoviruses Ad-siGosB (P<0.01; Figure 1(c)).Figure 1
Effects of GosB on the expression of genes related to milk fat synthesis in cultured mouse mammary gland epithelial cells (MEC). (a) Relative mRNA levels of GosB in various mice tissues. (b) The mRNA expression of GosB in the mouse mammary gland on day 5 of lactation or nonlactation. (c) Relative mRNA levels ofGosB in MEC transfected with Ad-GosB or Ad-siGosB for 48 hours. The effect of GosB on the expression of genes related to (d) fatty acid (FA) uptake(LPL, CD36); (e) FA de novo synthesis and desaturation(SCD, FASN); (f) triacylglycerol (TAG) synthesis and hydrolysis(FABP4, AGPAT6, and DGAT1); (g) lipid-droplet formation(PLIN2, PLIN3); (h) transcription factors (C/EBPα andSREBF1) were analyzed by quantitative real-time PCR (qPCR). Values are means ± SEM for three individuals. ∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001. The abbreviations were shown in supplementary information.
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(h)The role of GosB in milk lipid synthesis was confirmed by measuring the mRNA expression of genes encoding proteins associated with triglyceride (TAG) synthesis, lipid-droplet formation, FA uptake and synthesis, and transcription factor in the overexpressing GosB cells compared with MEC infected with Ad-siGosB (Figures1(d)–1(h)). Relative to the control, qPCR results revealed that overexpression of GosB decreased the mRNA expression of stearoyl-CoA desaturase(SCD) to 57% (P < 0.05), fatty acid synthase(FASN) to 51% (P < 0.01), fatty acid binding protein 4(FABP4) to 58% (P < 0.05), diacylglycerol acyltransferase 1(DGAT1) to 49% (P < 0.01), perilipin 2(PLIN2) to 55% (P < 0.05), perilipin 3(PLIN3) to 50% (P < 0.05), and CCAAT/enhancer binding protein alpha(C/EBPα) to 61% (P < 0.05) but increased the expression of thrombospondin receptor(CD36) to 2.05-fold (P < 0.01). In contrast, the MEC transfected with Ad-siGosB exhibited marked upregulation ofFASN (1.87-fold;P < 0.05),FABP4 (1.54-fold;P< 0.05),DGAT1 (1.36-fold;P< 0.05),PLIN2 (1.84-fold;P < 0.01),PLIN3 (1.79-fold;P < 0.05), andC/EBPα (1.45-fold;P < 0.05) but downregulatedCD36(0.56-fold;P< 0.05). In addition, GosB had no effect on expression of lipoprotein lipase (LPL; P>0.05), sterol regulatory element-binding transcription factor 1 (SREBF1;P > 0.05), and 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6;P> 0.05).


### 3.2. Effects of GosB on Cellular Lipid-Droplets Accumulation and Triglyceride Synthesis

As shown in Figures1(a)–1(c), a decrease of lipid-droplets accumulation (to 72%;P < 0.05) and triglyceride (TAG) content (to 78%;P < 0.05) was seen in the MEC transfected with Ad-GosB (Figures 2(a)–2(c)). By contrast, the accumulation of lipid droplets and content of TAG in MEC transfected with Ad-siGosB increased to 123% and 129%, respectively (P< 0.05; Figures 2(a)–2(c)).Figure 2
Effects of GosB on cellular lipid-droplets accumulation, triglyceride synthesis, and fatty acid concentration in mouse mammary gland epithelial cells (MEC). The MEC were transfected with Ad-GosB or Ad-siGosB for 48 hours; then the lipid-droplets accumulation was determined by Oil Red O staining assays (a) and quantified by measuring absorbance at 490 nm (b), and the intracellular triglyceride content was also measured (c). (d and e) Concentrations of 16- to 18-carbon fatty acid (FA) in cells. Data are reported as ratios of total FA. Values are means ± SEM for three individuals.∗P<0.05. Unsaturated FA (UFA); saturated FA (SFA).
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(e)Compared with the control, the ratios of palmitoleate (C16:1), oleate (C18:1), and linoleate (C18:2) in MEC transfected with Ad-siGosB increased at the expense of palmitate (C16:0) and stearate (C18:0). Specifically, Ad-siGosB had a significant positive effect on unsaturated fatty acid (UFA) and resulted in increases of C16:1, C18:1, and C18:2 by 6.1, 3, and 1.2%, respectively, relative to the control (Figures2(d) and 2(e)). In contrast, GosB overexpression had a negative effect on C16:1, C18:1, and C18:2. The saturated FA (SFA) C16:0 and C18:0 increased in cells transfected with Ad-GosB (Figures 2(d) and 2(e)).


### 3.3. GosB Decreases PPARγ Expression in MEC

It has been demonstrated that the Gos-related antigen 2 (Fra-2) could directly bind to the PPARγ promoter and inhibit the expression of PPARγ [5]. Therefore, we also assess whether GosB could regulate the expression of PPARγ. We found GosB overexpression markedly repressed the expression ofPPARγ (P< 0.05), and thePPARγ increased significantly (P < 0.05) in the MEC transfected with Ad-siGosB (Figure 3(a)). The PPARγ gene was ubiquitously expressed in different tissues and was detected and highly expressed in mice mammary glands (Figure 3(b)). Relative to the nonlactation period, the expression level of PPARγ during lactation was greater (about 2.7-fold, Figure 3(c)). Furthermore, we analyzed whether GosB regulated the effect of PPARγ on milk fat synthesis. We found that the PPARγ specific ligand ROSI markedly promoted lipid-droplets accumulation and triglyceride synthesis, and this effect was abrogated byGosB overexpression (Figures 4(a)–4(c)).Figure 3
GosB suppresses PPARγ expression in cultured mouse mammary gland epithelial cells (MEC). (a) GosB decreases PPARγ expression in MEC. (b) Relative mRNA levels ofPPARγ in various mouse tissues. (c) The mRNA expression ofPPARγ in the mouse mammary gland on day 5 of lactation or nonlactation. The mRNA levels ofPPARγ were analyzed by qRT-PCR. Values are means ± SEM for three individuals. ∗P<0.05; ∗∗P<0.01.
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(c)Figure 4
GosB affects milk fat synthesis-induced by PPARγ. The mouse mammary gland epithelial cells (MEC) were transfected with Ad-GosB or Ad-siGosB for 48 hours, followed by exposure to 10 μM rosiglitazone (ROSI) for 24 hours. Then the lipid-droplets accumulation was determined by Oil Red O staining assays (a) and quantified by measuring absorbance at 490 nm (b), and the intracellular triglyceride content was also measured (c). Values are means ± SEM for three individuals. ∗P<0.05.
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### 3.4. PPARγ Mediates Antiapoptotic Effects of GosB

To assess whether the protective effects of GosB are dependent upon PPARγ, we pretreated MEC with ROSI to analyze the effects of GosB on cell survival in the presence or absence of Ad-GosB or Ad-siGosB. The Hoechst33342/PI and FITC-Annexin V/PI staining assays showed that the ROSI induced MEC apoptosis, and GosB overexpression protected MEC from ROSI-induced apoptosis (Figure 5). We found that ROSI inhibited cell proliferation in MEC, and this effect was inhibited by GosB overexpression (Figures 6(a) and 6(b)). The ROSI decreased the mRNA expression of cell proliferation related genescyclinD1 andPCNA, and this downregulation was inhibited by GosB in MEC (Figure 6(c)). Thus, these observations demonstrate that the prosurvival role of GosB in MEC is dependent on PPARγ.Figure 5
The GosB-dependent protection from rosiglitazone- (ROSI-) induced cell apoptosis in mouse mammary gland epithelial cells (MEC). The MEC were transfected with Ad-GosB or Ad-siGosB for 48 hours and subsequently exposed to 10μM rosiglitazone (ROSI) for 24 hours. Cell apoptosis was determined by Hoechst 33342/PI dual staining assays (a) and Annexin V-FITC/PI binding followed by flow cytometry (b). Values are means ± SEM for three individuals. Different lowercase letters (a, b) indicate significant (P< 0.05) differences.
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(c)Figure 6
Effects of GosB on cell proliferation in mouse mammary gland epithelial cells (MEC). The MEC were transfected with Ad-GosB or Ad-siGosB for 48 hours and subsequently exposed to 10μM rosiglitazone (ROSI) for 24 hours and then subjected to cell proliferation analysis by cell counting kit-8 (CKK-8) assay and EdU incorporation assay. Scale bar indicates 200 μm. Different lowercase (a–c) and uppercase letters (A–C) indicate significant (P< 0.05) differences.
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### 3.5. Overexpression of GosB Inhibits Mitochondrial Calcium Overload and Mitochondrial Apoptotic Pathway

Activation of intracellular Ca2+ triggers cellular dysfunction and death [18, 19, 21]; GosB regulates calcium deposition [6, 7]. In order to determine whether GosB protected MEC from apoptosis via affecting cellular Ca2+ we examined the mitochondrial calcium levels in MEC after being transfected with Ad-GosB for 48 hours. The mitochondrial Ca2+ was stained with Rhod-2 AM and detected using a laser scanning confocal microscope. GosB was found to regulate mitochondrial Ca2+ accumulation in MEC (Figure 7(a)). Overexpression of GosB decreased the mitochondrial calcium fluorescent intensity which remained at lower levels. Calcium overload subsequently activated the apoptotic cascade as evidenced by the inhibition of B-cell lymphoma(Bcl-2) and the activation of Bcl-2-associated X protein(Bax) (Figure 7(b)). Bcl-2 has been described as antiapoptotic protein and plays important roles in regulating apoptosis in mammary epithelial cells and breast cancer cells [22, 23]. We also found that calcium promoted the expression ofcytochrome c andcaspase-9, and this effect was specifically abrogated by GosB overexpression (Figure 7(c)). By contrast, cell apoptosis induced by acute increases in intracellular Ca2+ concentration was abolished by GosB overexpression (Figure 7(d)). These results demonstrate that GosB protects MEC from apoptosis and subsequent apoptotic events by a Ca2+-sensitive mechanism.Figure 7
The GosB prevents mitochondrial calcium overload and subsequent apoptotic events. (a) Confocal laser scanning microscopic examination of MEC loaded with Rhod-2 AM showing mitochondrial Ca2+. (b–d) The MEC were transfected with Ad-GosB for 48 hours and subsequently exposed to 4 mM CaCl2 for 24 hours. Cell apoptosis was determined by Annexin V-FITC/PI binding followed by flow cytometry. The mRNA expression was analyzed by qRT-PCR. Different lowercase (a–c) and uppercase letters (A–C) indicate significant (P< 0.05) differences.
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## 3.1. Effects of GosB on the Expression of Genes Related to Milk Fat Synthesis

TheGosB was ubiquitously expressed in different tissues, and lung tissue had the highest expression ofGosB, followed by subcutaneous fat, mammary gland, and heart tissue (Figure 1(a)). Relative to the peak lactation, the expression level ofGosB during nonlactation increased by 2.3-fold (P<0.05, Figure 1(b)). Compared with the negative control cells, the expression ofGosB markedly increased after the cells were infected with overexpression recombinant adenoviruses Ad-GosB for 48 hours; theGosB expression was reduced significantly in MEC transfected with interference recombinant adenoviruses Ad-siGosB (P<0.01; Figure 1(c)).Figure 1
Effects of GosB on the expression of genes related to milk fat synthesis in cultured mouse mammary gland epithelial cells (MEC). (a) Relative mRNA levels of GosB in various mice tissues. (b) The mRNA expression of GosB in the mouse mammary gland on day 5 of lactation or nonlactation. (c) Relative mRNA levels ofGosB in MEC transfected with Ad-GosB or Ad-siGosB for 48 hours. The effect of GosB on the expression of genes related to (d) fatty acid (FA) uptake(LPL, CD36); (e) FA de novo synthesis and desaturation(SCD, FASN); (f) triacylglycerol (TAG) synthesis and hydrolysis(FABP4, AGPAT6, and DGAT1); (g) lipid-droplet formation(PLIN2, PLIN3); (h) transcription factors (C/EBPα andSREBF1) were analyzed by quantitative real-time PCR (qPCR). Values are means ± SEM for three individuals. ∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001. The abbreviations were shown in supplementary information.
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(h)The role of GosB in milk lipid synthesis was confirmed by measuring the mRNA expression of genes encoding proteins associated with triglyceride (TAG) synthesis, lipid-droplet formation, FA uptake and synthesis, and transcription factor in the overexpressing GosB cells compared with MEC infected with Ad-siGosB (Figures1(d)–1(h)). Relative to the control, qPCR results revealed that overexpression of GosB decreased the mRNA expression of stearoyl-CoA desaturase(SCD) to 57% (P < 0.05), fatty acid synthase(FASN) to 51% (P < 0.01), fatty acid binding protein 4(FABP4) to 58% (P < 0.05), diacylglycerol acyltransferase 1(DGAT1) to 49% (P < 0.01), perilipin 2(PLIN2) to 55% (P < 0.05), perilipin 3(PLIN3) to 50% (P < 0.05), and CCAAT/enhancer binding protein alpha(C/EBPα) to 61% (P < 0.05) but increased the expression of thrombospondin receptor(CD36) to 2.05-fold (P < 0.01). In contrast, the MEC transfected with Ad-siGosB exhibited marked upregulation ofFASN (1.87-fold;P < 0.05),FABP4 (1.54-fold;P< 0.05),DGAT1 (1.36-fold;P< 0.05),PLIN2 (1.84-fold;P < 0.01),PLIN3 (1.79-fold;P < 0.05), andC/EBPα (1.45-fold;P < 0.05) but downregulatedCD36(0.56-fold;P< 0.05). In addition, GosB had no effect on expression of lipoprotein lipase (LPL; P>0.05), sterol regulatory element-binding transcription factor 1 (SREBF1;P > 0.05), and 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6;P> 0.05).


## 3.2. Effects of GosB on Cellular Lipid-Droplets Accumulation and Triglyceride Synthesis

As shown in Figures1(a)–1(c), a decrease of lipid-droplets accumulation (to 72%;P < 0.05) and triglyceride (TAG) content (to 78%;P < 0.05) was seen in the MEC transfected with Ad-GosB (Figures 2(a)–2(c)). By contrast, the accumulation of lipid droplets and content of TAG in MEC transfected with Ad-siGosB increased to 123% and 129%, respectively (P< 0.05; Figures 2(a)–2(c)).Figure 2
Effects of GosB on cellular lipid-droplets accumulation, triglyceride synthesis, and fatty acid concentration in mouse mammary gland epithelial cells (MEC). The MEC were transfected with Ad-GosB or Ad-siGosB for 48 hours; then the lipid-droplets accumulation was determined by Oil Red O staining assays (a) and quantified by measuring absorbance at 490 nm (b), and the intracellular triglyceride content was also measured (c). (d and e) Concentrations of 16- to 18-carbon fatty acid (FA) in cells. Data are reported as ratios of total FA. Values are means ± SEM for three individuals.∗P<0.05. Unsaturated FA (UFA); saturated FA (SFA).
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(e)Compared with the control, the ratios of palmitoleate (C16:1), oleate (C18:1), and linoleate (C18:2) in MEC transfected with Ad-siGosB increased at the expense of palmitate (C16:0) and stearate (C18:0). Specifically, Ad-siGosB had a significant positive effect on unsaturated fatty acid (UFA) and resulted in increases of C16:1, C18:1, and C18:2 by 6.1, 3, and 1.2%, respectively, relative to the control (Figures2(d) and 2(e)). In contrast, GosB overexpression had a negative effect on C16:1, C18:1, and C18:2. The saturated FA (SFA) C16:0 and C18:0 increased in cells transfected with Ad-GosB (Figures 2(d) and 2(e)).


## 3.3. GosB Decreases PPARγ Expression in MEC

It has been demonstrated that the Gos-related antigen 2 (Fra-2) could directly bind to the PPARγ promoter and inhibit the expression of PPARγ [5]. Therefore, we also assess whether GosB could regulate the expression of PPARγ. We found GosB overexpression markedly repressed the expression ofPPARγ (P< 0.05), and thePPARγ increased significantly (P < 0.05) in the MEC transfected with Ad-siGosB (Figure 3(a)). The PPARγ gene was ubiquitously expressed in different tissues and was detected and highly expressed in mice mammary glands (Figure 3(b)). Relative to the nonlactation period, the expression level of PPARγ during lactation was greater (about 2.7-fold, Figure 3(c)). Furthermore, we analyzed whether GosB regulated the effect of PPARγ on milk fat synthesis. We found that the PPARγ specific ligand ROSI markedly promoted lipid-droplets accumulation and triglyceride synthesis, and this effect was abrogated byGosB overexpression (Figures 4(a)–4(c)).Figure 3
GosB suppresses PPARγ expression in cultured mouse mammary gland epithelial cells (MEC). (a) GosB decreases PPARγ expression in MEC. (b) Relative mRNA levels ofPPARγ in various mouse tissues. (c) The mRNA expression ofPPARγ in the mouse mammary gland on day 5 of lactation or nonlactation. The mRNA levels ofPPARγ were analyzed by qRT-PCR. Values are means ± SEM for three individuals. ∗P<0.05; ∗∗P<0.01.
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GosB affects milk fat synthesis-induced by PPARγ. The mouse mammary gland epithelial cells (MEC) were transfected with Ad-GosB or Ad-siGosB for 48 hours, followed by exposure to 10 μM rosiglitazone (ROSI) for 24 hours. Then the lipid-droplets accumulation was determined by Oil Red O staining assays (a) and quantified by measuring absorbance at 490 nm (b), and the intracellular triglyceride content was also measured (c). Values are means ± SEM for three individuals. ∗P<0.05.
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## 3.4. PPARγ Mediates Antiapoptotic Effects of GosB

To assess whether the protective effects of GosB are dependent upon PPARγ, we pretreated MEC with ROSI to analyze the effects of GosB on cell survival in the presence or absence of Ad-GosB or Ad-siGosB. The Hoechst33342/PI and FITC-Annexin V/PI staining assays showed that the ROSI induced MEC apoptosis, and GosB overexpression protected MEC from ROSI-induced apoptosis (Figure 5). We found that ROSI inhibited cell proliferation in MEC, and this effect was inhibited by GosB overexpression (Figures 6(a) and 6(b)). The ROSI decreased the mRNA expression of cell proliferation related genescyclinD1 andPCNA, and this downregulation was inhibited by GosB in MEC (Figure 6(c)). Thus, these observations demonstrate that the prosurvival role of GosB in MEC is dependent on PPARγ.Figure 5
The GosB-dependent protection from rosiglitazone- (ROSI-) induced cell apoptosis in mouse mammary gland epithelial cells (MEC). The MEC were transfected with Ad-GosB or Ad-siGosB for 48 hours and subsequently exposed to 10μM rosiglitazone (ROSI) for 24 hours. Cell apoptosis was determined by Hoechst 33342/PI dual staining assays (a) and Annexin V-FITC/PI binding followed by flow cytometry (b). Values are means ± SEM for three individuals. Different lowercase letters (a, b) indicate significant (P< 0.05) differences.
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Effects of GosB on cell proliferation in mouse mammary gland epithelial cells (MEC). The MEC were transfected with Ad-GosB or Ad-siGosB for 48 hours and subsequently exposed to 10μM rosiglitazone (ROSI) for 24 hours and then subjected to cell proliferation analysis by cell counting kit-8 (CKK-8) assay and EdU incorporation assay. Scale bar indicates 200 μm. Different lowercase (a–c) and uppercase letters (A–C) indicate significant (P< 0.05) differences.
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## 3.5. Overexpression of GosB Inhibits Mitochondrial Calcium Overload and Mitochondrial Apoptotic Pathway

Activation of intracellular Ca2+ triggers cellular dysfunction and death [18, 19, 21]; GosB regulates calcium deposition [6, 7]. In order to determine whether GosB protected MEC from apoptosis via affecting cellular Ca2+ we examined the mitochondrial calcium levels in MEC after being transfected with Ad-GosB for 48 hours. The mitochondrial Ca2+ was stained with Rhod-2 AM and detected using a laser scanning confocal microscope. GosB was found to regulate mitochondrial Ca2+ accumulation in MEC (Figure 7(a)). Overexpression of GosB decreased the mitochondrial calcium fluorescent intensity which remained at lower levels. Calcium overload subsequently activated the apoptotic cascade as evidenced by the inhibition of B-cell lymphoma(Bcl-2) and the activation of Bcl-2-associated X protein(Bax) (Figure 7(b)). Bcl-2 has been described as antiapoptotic protein and plays important roles in regulating apoptosis in mammary epithelial cells and breast cancer cells [22, 23]. We also found that calcium promoted the expression ofcytochrome c andcaspase-9, and this effect was specifically abrogated by GosB overexpression (Figure 7(c)). By contrast, cell apoptosis induced by acute increases in intracellular Ca2+ concentration was abolished by GosB overexpression (Figure 7(d)). These results demonstrate that GosB protects MEC from apoptosis and subsequent apoptotic events by a Ca2+-sensitive mechanism.Figure 7
The GosB prevents mitochondrial calcium overload and subsequent apoptotic events. (a) Confocal laser scanning microscopic examination of MEC loaded with Rhod-2 AM showing mitochondrial Ca2+. (b–d) The MEC were transfected with Ad-GosB for 48 hours and subsequently exposed to 4 mM CaCl2 for 24 hours. Cell apoptosis was determined by Annexin V-FITC/PI binding followed by flow cytometry. The mRNA expression was analyzed by qRT-PCR. Different lowercase (a–c) and uppercase letters (A–C) indicate significant (P< 0.05) differences.
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## 4. Discussion

It has been well established that FASN plays an important role in de novo FA synthesis in human [24], mice [25], bovine [26], and goat [2] tissue. The expression of FASN also affects TAG synthesis and accumulation [27, 28]. The present study showed that GosB inhibited the expression ofFASN and suppressed lipid-droplet formation and accumulation of TAG. An unexpected upregulation ofCD36, an important gene responsible for FA uptake [29], was found in MEC overexpressed GosB. Studies have shown that the downregulation ofCD36 upon overexpression ofTHRSP, a major protein responsible for FA synthesis, was accompanied by an increase in FA synthesis [29, 30]. Therefore, our results suggested that the decrease in FA uptake may be caused by an increase in FA synthesis.The DGAT1 is involved in TAG synthesis during adipogenesis [31], and DGAT1-deficient mice show few and small lipid droplets in adipocytes [32]. The secretion of lipid droplets into milk occurs via a process that requires lipid-droplet formation-related genes PLIN2 and PLIN3 [33, 34]. In the present study, GosB inhibited the expression ofDGAT1,FABP4,PLIN2, andPLIN3 and decreased lipid-droplets accumulation and TAG content in MEC, suggesting that GosB may serve as a suppress or of TAG synthesis in the mouse mammary gland.The inhibition ofSCDexpression with GosB overexpression is consistent with data that GosB increased the unsaturated FA concentration. The SCD protein plays a key role in the synthesis of milk unsaturated fatty acid via catalysts C16:0 and C18:0 to C16:1 and C18:1 [35]. The C/EBPα is a key transcription factor involved in adipocyte differentiation [36], and GosB has been shown to decrease the expression of C/EBPα in the multipotential stromal cells [3]. Consistent with this, GosB in our study inhibited the mRNA expression ofPPARγ in MEC, suggesting that GosB may regulate milk fat synthesis through affecting transcription factor PPARγ functions. Our study showed direct evidence that GosB promotes de novo FA synthesis and TAG synthesis in mouse mammary epithelial cells. However, some limitations should be considered; for instance, the protein expression and enzymes activities should be measured, and the mechanisms for how GosB affects milk fat synthesis need to be further studied.The PPARγ is not only a master regulator of adipogenesis but also exhibits a role in cellular apoptosis signal transduction [37]. Previous studies have shown that Bcl-xL overexpression promotes prostate cancer cells survival which can be abolished by activating PPARγ [38]. PPARγ activation in breast cancer cells can inhibit cell growth and induce apoptosis through regulating the expression of caspase-9 and p53 [39]. The GosB triggers cell proliferation in mammary epithelial cells and induces a metastatic phenotype of breast cancer cells [40, 41]. Here, we indicated that the manipulation of PPARγ expression in MEC has significant effect on cell proliferation and apoptosis, showing a proapoptotic role of PPARγ in mammary gland. We also showed that GosB was found to decrease PPARγ expression and promote MEC survival by increasing the expression ofBcl-2. These findings demonstrate that GosB can promote mammary epithelial cells survival by a PPARγ-dependent mechanism.Calcium is a well-known intracellular messenger that modulates many aspects of cell life [21, 42, 43]. Intracellular Ca2+ accumulation activates the mitochondrial apoptotic pathway with high levels of Bax and activation of caspase-3 [44–46]. The GosB is a physiologically important regulator of osteoblast differentiation and increases bone formation and bone mass, leading to osteosclerosis [3, 6, 7]. Therefore, we speculated that GosB may have an antiapoptotic effect through regulating intracellular Ca2+ concentration. This notion was supported by the results that GosB overexpression decreased the mitochondrial calcium fluorescent intensity at lower levels and protected MEC from apoptosis triggered by calcium accumulation.In summary, our findings demonstrate that PPARγ agonist rosiglitazone increases cellular triglyceride content and inhibits cell survival, and these effects were abolished by GosB overexpression. Thus, GosB regulates mammary epithelial cells’ lipid synthesis and apoptosis via PPARγ in mouse mammary glands. Modulation of GosB expression in mammary glands may emerge as a potent tool to control the number of MEC and milk fat levels in mouse mammary glands.



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*Source: 7394869-2017-10-17.xml*