Next Article in Journal
Bioactive Nutrient Retention during Thermal-Assisted Hydration of Lupins
Previous Article in Journal
A Novel Electrochemiluminescence Immunosensor Based on Resonance Energy Transfer between g-CN and NU-1000(Zr) for Ultrasensitive Detection of Ochratoxin A in Coffee
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Foods
Volume 12
Issue 4
10.3390/foods12040708
foods-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

CEBPA-Regulated Expression of SOCS1 Suppresses Milk Protein Synthesis through mTOR and JAK2-STAT5 Signaling Pathways in Buffalo Mammary Epithelial Cells

by
Xinyang Fan
†,
Lihua Qiu
†,
Wei Zhu
†,
Lige Huang
,
Xingtiao Tu
and
Yongwang Miao
*
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Foods 2023, 12(4), 708; https://doi.org/10.3390/foods12040708
Submission received: 21 November 2022 / Revised: 15 December 2022 / Accepted: 1 February 2023 / Published: 6 February 2023
(This article belongs to the Section Dairy)
Browse Figures
Review Reports Versions Notes

Abstract

:
Milk protein content is a key quality indicator of milk, and therefore elucidating its synthesis mechanism has been the focus of research in recent years. Suppressor of cytokine signaling 1 (SOCS1) is an important inhibitor of cytokine signaling pathways that can inhibit milk protein synthesis in mice. However, it remains elusive whether SOCS1 plays roles in the milk protein synthesis in the buffalo mammary gland. In this study, we found that the mRNA and protein expression levels of SOCS1 in buffalo mammary tissue during the dry-off period was significantly lower than those during lactation. Overexpression and knockdown experiments of SOCS1 showed that it influenced the expression and phosphorylation of multiple key factors in the mTOR and JAK2–STAT5 signaling pathways in buffalo mammary epithelial cells (BuMECs). Consistently, intracellular milk protein content was significantly decreased in cells with SOCS1 overexpression, while it increased significantly in the cells with SOCS1 knockdown. The CCAAT/enhancer binding protein α (CEBPA) could enhance the mRNA and protein expression of SOCS1 and its promoter activity in BuMECs, but this effect was eliminated when CEBPA and NF-κB binding sites were deleted. Therefore, CEBPA was determined to promote SOCS1 transcription via the CEBPA and NF-κB binding sites located in the SOCS1 promoter. Our data indicate that buffalo SOCS1 plays a significant role in affecting milk protein synthesis through the mTOR and JAK2-STAT5 signaling pathways, and its expression is directly regulated by CEBPA. These results improve our understanding of the regulation mechanism of buffalo milk protein synthesis.

1. Introduction

The synthesis and secretion of milk is the most important function of the mammalian mammary gland, and it is one of the most metabolically demanding stages in mammalian life [1,2]. Milk not only provides the newborn with necessary nutrients, but also with a complex repertoire of agents required for healthy development [3]. As the primary solid components of milk, milk proteins mainly include the caseins and whey proteins, which are synthesized in mammary epithelial cells and mainly by ribosomes in the rough endoplasmic reticulum [4]. Milk proteins contain a large number of essential amino acids, which is essential to meet the nutritional needs of newborns, so milk protein content can serve as a quality indicator for milk [5]. The synthesis of milk protein is regulated by a variety of signaling pathways and hormones at multiple levels. The mammalian target of rapamycin (mTOR) pathway and janus kinase 2/signal transducer and activator of transcription 5 (JAK2–STAT5) pathway are well known to be the crucial signaling pathways involved in regulating milk protein synthesis in the mammary gland [5]. Studies have shown that multiple genes are associated with the regulation of milk protein synthesis in cows, such as ULF1, TWF1 and NUCKS1 [1,6,7].
The suppressor of cytokine signaling (SOCS) family proteins, the critical inhibitors of cytokine signaling pathways, play an important role in mammary gland development, along with the JAK-STAT pathway [8]. In the mammary gland, the SOCS inhibit the ability of prolactin (PRL) to stimulate milk synthesis [9]. As an important member of this family, SOCS1 is involved in mammary gland development and a variety of cytokine signal transduction [10]. In mouse mammary epithelial cells, the expression of SOCS1 can be induced by PRL, suggesting that SOCS1 is implicated in PRL-stimulated regulation of cell signaling [11]. Meanwhile, the evidence in vivo confirms that SOCS1 attenuates the prolactin receptor (PRLR) signaling through negative feedback during pregnancy and lactation [9]. A previous study reported that several SNPs close to SOCS1 are significantly associated with milk protein yield in dairy cattle [8]. In addition, the deletion of SOCS1 restores milk protein expression in PRLR-deficient mice [10]. These findings reveal that SOCS1 plays a key role in the regulation of milk protein synthesis.
The CCAAT/enhancer binding proteins (CEBPs) are a family of transcription factors implicated in the growth and differentiation of mammary epithelial cells [12]. They contain highly conserved basic leucine zipper motifs that mediate dimerization and DNA binding at their carboxyl terminus [13]. CEBPs are differentially expressed throughout mammary gland development, and they can bind to the promoter of CSN2 to regulate its expression [14]. CEBPA, the first member of this family to be identified, is regulated by lactogenic hormones in mammary epithelial cells. Although lactogenic hormones have no effect on CEBPA protein level, they down-regulate the DNA binding activity of CEBPA [12]. In addition, CEBPA is an important lipogenic transcription factor that targets FASN and CD36 related to lipid synthesis [15,16]. In bovine mammary epithelial cells, CEBPA is involved in the regulation of milk fat synthesis [17]. CEBPA was found to directly activate miR-29b expression, while miR-29b induced SOCS1 expression via promoter demethylation [18]. However, direct regulation of SOCS1 by CEBPA has not been reported.
At present, buffalo milk production accounts for 13% of the world’s total milk production, and water buffalo has become the second largest source of milk in the world [19,20]. Compared with cow milk, buffalo milk has a higher content of milk protein. Although SOCS1 has been proven to play essential roles in milk protein synthesis in mice, its functions in milk protein synthesis, and the interaction mechanism with CEBPA in buffalo mammary gland are unclear. We hypothesized that SOCS1 could regulate the network associated with buffalo milk protein synthesis and that its expression could be regulated by CEBPA. The purpose of this study was to determine the role of SOCS1 in buffalo milk protein synthesis and to clarify the interaction mechanism between this protein and CEBPA through functional experiments at the cellular level. This study can lay a foundation for elucidating the regulation of buffalo milk protein synthesis.

2. Materials and Methods

2.1. Animals and Sampling

Eight healthy female Binglangjiang buffalo (river type) aged 4 years old in the same management conditions were selected for tissue sample collection. The samples from the mammary gland, liver, ovary, lungs, rumen, cerebellum, kidney, brain, heart, pituitary, spleen and muscle were collected from 4 buffalo in lactation (60 d postpartum) after slaughter. In addition, the mammary gland biopsies were conducted during the dry-off period (60 d before parturition) from other 4 buffalo by a previously described surgical procedure [21]. All tissue samples were obtained rapidly under sterile conditions and frozen instantly in liquid nitrogen.

2.2. Vector Construction and Small RNA Synthesis

The overexpression plasmid EGFP-SOCS1 of buffalo SOCS1 (accession No. XM_006079909) was constructed using pEGFP-N1 vector (Clontech Laboratories, Inc., Palo Alto, CA, USA) by PCR with a pair of specific primers containing Xho I and Hind III restriction site (forward: 5′-CTCGAGATGGTAGCACACAACCAGGT-3′; reverse: 5′-AAGCTTGATCTGGAAGGGGAAGGAGC-3′). To knock down the buffalo SOCS1, one pair of short hairpin RNA (shRNA) targeting buffalo SOCS1 were designed using the software BLOCK-iT RNAi Designer (http://rnaidesigner.invitrogen.com/rnaiexpress/, accessed on 19 June 2022) (Table S1). The shRNA was annealed and further ligated into the vector pLKO.1-TRC to construct the recombinant plasmids (pLKO.1-SOCS1). The obtained vectors were verified by sequencing, and then purified with EndoFree Maxi Plasmid Kit (QIAGEN, Valencia, CA, USA).
In order to investigate the regulatory effect of CEBPA on SOCS1, we also constructed the overexpression vector EGFP-CEBPA by the same method. The primer sequences were as follows: forward: 5′-CTCGAGATGGAGTCGGCCGACTTCTA-3′; reverse: 5′-AAGCTTCGCGCAGTTGCCCATGGCCT-3′. In addition, the small interfering RNA (siRNA) targeting CEBPA (siCEBPA) and negative control siRNA (siNC) were designed and further synthesized by Shanghai Sangon Biotech Company (Table S1).

2.3. Cell Preparation and Treatment

The 293T cells were cultivated in the medium composed of Dulbecco’s modified Eagle medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 2% penicillin/streptomycin (Gibco), maintained at 37 °C in 5% CO2. The process for lentivirus generation of shRNA was performed as previously reported [22]. Briefly, when the confluence of cultured 293T cells reached 80%, pLKO.1-shSOCS1, psPAX2 and pMD2.G were co-transfected into 293T cells through TransIntro™ EL Transfection Reagent (TransGen Biotech, Beijing, China) for generating lentiviral particles (shSOCS1) in accordance with the manufacturer’s instructions. The 293T cells were co-transfected with pLKO.1-TRC, psPAX2 and pMD2G as the non-interfered control group (shNC). They were all measured by the serial dilution method for the infection titer of concentrated lentivirus particles.
Buffalo mammary epithelial cells (BuMECs) were isolated from the mammary gland tissue of lactating Binglangjiang buffalo (60 d postpartum) and purified based on the differential sensitivity of the cells to trypsin digestion as previously described by our group [22,23]. The BuMECs purified and identified by cytokeratin 18 (Sigma, Louis, MO, USA) were cultivated and expanded to passage five in a basal medium composed of DMEM (Gibco), 10% fetal bovine serum (Gibco), 100 μg/mL penicillin/streptomycin (Gibco) and various cytokines, including 5 μg/mL insulin (Sigma), 2 μg/mL hydrocortisone (Sigma) and 100 ng/mL epidermal growth factor (Sigma), and maintained at 37 °C under 5% CO2. When the cell confluence reached 80% in a 6-well cell culture plate, the cells were cultured for 24 h in a basal medium supplemented with 2 μg/mL prolactin (Sigma). Subsequently, these cells were transfected with EGFP-SOCS1 (3 μg), EGFP-CEBPA (3 μg), siCEBPA (60 nM) and the corresponding negative controls (EGFP and siNC) according to the manufacturer’s protocol of TransIntroTM EL transfection reagent (TransGen Biotech, Beijing, China). At the same time, they were transduced into shSOCS1 and shNC to knock down SOCS1. Through pre-experiments, it was determined that the best results were obtained when the cells were collected 48 h after treatment. Therefore, the cells of each treatment group were harvested 48 h later for gene and protein expression analysis.

2.4. Protein Subcellular Localization Analysis

The pEGFP-SOCS1 was transfected into BuMECs for subcellular localization analysis of SOCS1. After 48 h of transfection, the mitochondria and nucleus of the cells were stained with Mito-Tracker Red CMXRos (Beyotime, Shanghai, China) and Hoechst 33342 (Beyotime), respectively, according to the instructions. After the staining solution was removed, observations were made with a confocal laser scanning microscope (Olympus, Tokyo, Japan).

2.5. Quantitative PCR (qPCR) Detection of Expression

Total RNA was extracted from transfected cells using the TRIzol (Invitrogen) and reverse transcription was performed by following the instructions of a RT reagent kit with gDNA Eraser (Takara, Dalian, China) for subsequent analysis of mRNA expression. The primers for qPCR were designed using Primer Premier 5.0 [24], and the primer sequences are listed in Table S2. The qPCR assay was performed using the TB Green® Premix Ex TaqTM II (TaKaRa) on a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). The purity of PCR products was confirmed by melting curve analysis. The efficiency of amplification was determined utilizing LinRegPCR (www.linregpcr.nl, accessed on 20 May 2022; Table S2). The geometric mean of the Ct values of ACTB, GAPDH and RPS23 was used as the endogenous control for mRNA expression analysis. The relative expression of mRNA was analyzed using the 2–ΔΔCt model [25].

2.6. Protein Extraction and Western Blotting

The proteins of mammary gland tissue and transfected BuMECs were collected by trypsin digestion and lysed in RIPA buffer (Beyotime) containing 1% PMSF (Beyotime) and 1% phosphatase inhibitor cocktail (Roche, Shanghai, China). After quantifying their concentration using the BCA assay kit (Beyotime), equal amounts of protein samples (approximately 25 μg of total protein) were electrophoresed in SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, Burlington, MA, USA). The membranes were probed with primary polyclonal rabbit anti-SOCS1 (1:1000; abs131478, Absin, Shanghai, China), rabbit anti-β-casein (1:1000; bs-0466R, Bioss, Beijing, China), rabbit anti-PI3K (1:1000; bs-10657R, Bioss), rabbit anti-Phospho-PI3K (1:1000; bs-6417R, Bioss), rabbit anti-AKT1 (1:1000; bs-0115R, Bioss), rabbit anti-Phospho-AKT1 (1:1000; bs-10133R, Bioss), rabbit anti-mTOR (1:1000; bs-1992R, Bioss), rabbit anti-Phospho-mTOR (1:1000; bs-3495R, Bioss), rabbit anti-STAT5 (1:1000; bs-1142R, Bioss), rabbit anti-Phospho-STAT5 (1:1000; bs-5619R, Bioss), rabbit anti-JAK2 (1:1000; bs-23003R, Bioss), rabbit anti-Phospho-JAK2 (1:1000; #3771, Cell Signaling Technology, Danvers, MA, USA) and monoclonal mouse anti-β-actin (1:6000; HC201, TransGen Biotech, Beijing, China) at 4 °C overnight. The species reactivity of these antibodies is all for cow. Next, the membranes were further incubated with polyclonal goat anti-rabbit IgG (1:5000; #2491145, Millipore, Burlington, MA, USA) and polyclonal goat anti-mouse IgG (1:5000; #2517746, Millipore), and the immunoreactive bands were visualized using the chemiluminescent ECL Western blot detection system (Pierce, Rockford, IL, USA). Protein abundance was calculated by Alpha View SA (ProteinSimple, San Jose, CA, USA).

2.7. SOCS1 Promoter Cloning and Deletion Analysis

The different fragments of the SOCS1 promoter (GenBank no. NC_059180) derived from primers that hybridize at positions −1999, −1734, −1364, −961, −614 and −77, coupled with a common downstream primer at +105 with Xho Ι and Hind III sites (Table S3), were prepared by PCR from mixed genomic DNA, which was isolated from blood samples of 4 buffalo. They were subcloned into pGL4 vector (Promega, Madison, WI, USA) with Xho Ι/Hind III restriction enzyme to generate multiple luciferase reporter vectors (pGL-(−1999/+105), pGL-(−1734/+105), pGL-(−1364/+105), pGL-(−961/+105), pGL-(−614/+105) and pGL-(−77/+105)). The putative transcription factor binding sites were analyzed using the JASPAR database (http://jaspar.genereg.net/, accessed on 25 April 2022), AliBaba2.1 (http://gene-regulation.com/pub/programs/alibaba2/, accessed on 25 April 2022) and hTFtarget (http://bioinfo.life.hust.edu.cn/hTFtarget#!/prediction, accessed on 25 April 2022).

2.8. Dual-Luciferase Activity Assay

The luciferase reporter vectors containing different-length fragments of the SOCS1 promoter were transfected into the BuMECs for luciferase expression to determine the core promoter region. Moreover, BuMECs were co-transfected with EGFP-CEBPA or siCEBPA and 5′ progressive deletion of pGL-SOCS1 plasmids. After 48 h, luciferase assays were carried out using the Dual-Glo luciferase assay system kit (Promega). The firefly luciferase activity was normalized to the activity of Renilla luciferase (pRL-TK; Promega), and the ratio of pGL-SOCS1 to pRL-TK was 30:1.

2.9. Statistical Analyses

The results were presented as mean ± SEM for each group from three independent experiments. GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA) was used for statistical analysis and visualization. Statistical significance of observed differences between the two groups was assessed using a two-tailed Student’s t-test, and p values < 0.05 were considered as significant.

3. Results

3.1. SOCS1 Differential Expression in Various Tissues

We measured the mRNA abundance of SOCS1 in 12 tissues of lactating buffalo (Figure 1A) and observed that SOCS1 was expressed in all examined tissues, with the highest levels in the muscle, followed by the spleen, pituitary, heart, brain and kidney. The lowest mRNA abundance among the examined tissues was found in the mammary gland. Furthermore, we also examined the differential expression of buffalo SOCS1 mRNA and its encoded protein in the mammary gland during different physiological stages. The results showed that the mRNA (p < 0.01) and protein expression (p < 0.01) of buffalo SOCS1 were significantly lower in lactation than that in the dry-off period (Figure 1B–D), indicating that SOCS1 is involved in the lactation process in buffalo.

3.2. Nuclear–Cytosolic Localization of Buffalo SOCS1

The results of purified BuMECs identified by cytokeratin 18 are shown in Supplementary Figure S1. The recombinant vector of EGFP-SOCS1 was transfected into BuMECs to determine the distribution of buffalo SOCS1. The results of transfection showed that most of the green fluorescent protein (GFP) of EGFP-SOCS1 coincided with the red fluorescence of the mitochondria; some of the GFP overlapped with the blue fluorescence of the nucleus (Figure 2). The above results suggest that the SOCS1 protein is mainly distributed in the cytoplasm of BuMECs, but some are also distributed in the nucleus.

3.3. SOCS1 Suppresses the Milk Protein Synthesis in BuMECs

To understand the impact of SOCS1 on milk protein synthesis, EGFP-SOCS1 or shSOCS1 were transfected into BuMECs. Compared with the negative controls, the expression level of SOCS1 in BuMECs increased 106-fold after treatment with EGFP-SOCS1 (p < 0.05), while the expression level of SOCS1 in cells treated with shSOCS1 decreased by 67.6% (p < 0.05) (Figure 3A,B). For the effect of SOCS1 on milk protein synthesis, we examined the expression changes of CSN2 and CSN3 in BuMECs. Compared with the negative controls, EGFP-SOCS1 markedly reduced the mRNA abundance of CSN2 (p < 0.01) and CSN3 (p < 0.01) (Figure 3C), whereas shSOCS1 significantly increased the expression of CSN2 (p < 0.01) and CSN3 (p < 0.01) (Figure 3D). CSN2 (β-casein) is the second most abundant protein in cow’s milk and can be employed as an indicator of milk protein synthesis in mammary epithelial cells [26]. Thus, after analyzing the expression of CSN2 at the mRNA level, we also examined the protein level of CSN2 to confirm the regulatory effect of SOCS1 on the milk protein synthesis. The results displayed that the expression of CSN2 protein in the group treated with EGFP-SOCS1 decreased significantly compared to that in the EGFP group (p < 0.01). On the contrary, the expression of CSN2 was significantly up-regulated in the group treated with shSOCS1 (p < 0.05) (Figure 3E,F).

3.4. SOCS1 Inhibits the Activation of the mTOR and JAK2-STAT5 Pathways

To further determine the function of SOCS1 in milk protein synthesis, the expression changes of genes related to the mTOR and JAK2-STAT5 pathways were investigated when SOCS1 was overexpressed and knocked down in BuMECs. We examined the changes in the expression of central genes, including genes in the upstream pathway (PI3K, AKT1, mTOR) and genes in the downstream pathway (4EBP1, EIF4E, S6K1) of mTOR signaling, as well as genes (PRLR, JAK2, STAT5a, STAT5b, ELF5) in JAK2-STAT5 signaling. The results showed that the SOCS1 overexpression significantly decreased the mRNA abundance of PI3K (p < 0.01), mTOR (p < 0.01) and EIF4E (p < 0.01) in BuMECs (Figure 4A,B). In contrast, the treatment with shSOCS1 significantly increased the mRNA expression of PI3K (p < 0.05), AKT1 (p < 0.01), mTOR (p < 0.05) and EIF4E (p < 0.05) in mTOR signaling pathways (Figure 4D,E). However, the mRNA expression of 4EBP1 and S6K1 (p > 0.05) did not change after transfection with EGFP-SOCS1 or shSOCS1 in BuMECs. Moreover, the mRNA expression of genes involved in the JAK2-STAT5 pathway, PRLR (p < 0.001), STAT5a (p < 0.05), STAT5b (p < 0.01) and ELF5 (p < 0.05) were significantly decreased upon the up-regulation of SOCS1 (Figure 4C). The down-regulation of SOCS1 caused significantly increased expression of PRLR (p < 0.01), JAK2 (p < 0.01), STAT5b (p < 0.01) and ELF5 (p < 0.05) (Figure 4F). Importantly, Western blot detection showed that SOCS1 overexpression decreased the protein levels of PI3K, p-PI3K, p-AKT1, mTOR, p-mTOR, p-JAK2, STAT5 and p-STAT5 in BuMECs, whereas SOCS1 knockdown had the opposite effects (Figure 4G–J). However, the protein levels of AKT1 and JAK2 remained almost unchanged. These data revealed that SOCS1 affects milk protein synthesis through the inhibition of the mTOR and JAK2-STAT5 signaling pathways.

3.5. CEBPA Is Required for Induction of SOCS1 Expression

To investigate the role of CEBPA on the expression of SOCS1, the changes in promoter activity, mRNA and protein expression of SOCS1 when CEBPA was overexpressed or knocked down were analyzed. It was found that CEBPA overexpression dramatically increased the luciferase activity of the pGL-(−1999/+105) (p < 0.01), whereas the luciferase level of the pGL-(−1999/+105) transfected with siCEBPA was significantly inhibited relative to siNC (p < 0.01) (Figure 5A). The SOCS1 mRNA expression was up-regulated by CEBPA overexpression (p < 0.01), but its expression was markedly down-regulated by CEBPA inhibition (p < 0.05) (Figure 5B). In addition, the protein expression level of SOCS1 was consistent with the trend of mRNA expression after CEBPA overexpression or knockdown in BuMECs (Figure 5C,D).

3.6. Identification of the Core Promoter Region of Buffalo SOCS1

To determine the core promoter region of buffalo SOCS1, pGL-SOCS1 plasmids with 5′ progressive deletions were constructed (Figure S2) and transfected directly into BuMECs for the expression of luciferase. Luciferase detection showed that compared with other deletion constructs, the pGL-(−1364/+105) had the highest luciferase activity (Figure 6A). The deletion from −1999 bp to −1364 bp showed a significant increase in luciferase activity, indicating that this region may contain some negative regulatory elements. The deletion from −1364 bp to −961 bp showed a dramatic decrease in luciferase activity (p < 0.05), suggesting that some important positive regulatory elements were deleted. When the deletion reached −614 bp, we found that the luciferase activity was significantly reduced (p < 0.01). Subsequently, with the deletion of the 5′ flanking region, the activity continued to decrease until it was similar to that of the empty vector pGL4 (p < 0.05). The above results reveal that the promoter region ranging from −1364 to +105 is the proximal core promoter region of buffalo SOCS1. Analysis of transcription factor response elements in the core promoter region demonstrated that there are two CEBPA (from −1346 to −1336 and −1085 to −1076) and two NF-κB binding sites (from −931 to −921 and −672 to −662) in this region (Figure 6B).

3.7. CEBPA and NF-κB Binding Sites Are Responsible for Induction of SOCS1 Promoter Activity by CEBPA

To identify which cis-regulatory elements are responsible for CEBPA-mediated SOCS1 regulation, we assessed the effect of CEBPA on the promoter activity of SOCS1 with different lengths in BuMECs. Compared with the control (EGFP), the overexpression of CEBPA effectively enhanced the activity of promoters containing CEBPA and NF-κB binding sites (Figure 7A). Meanwhile, its knockdown significantly reduced the luciferase activity of recombinant vectors containing these cis-regulatory elements (Figure 7B). However, CEBPA had no effect on the activity of promoters that did not contain CEBPA and NF-κB binding sites. Collectively, these results indicated that CEBPA promotes SOCS1 transcription via the CEBPA and NF-κB binding sites located in the SOCS1 promoter in BuMECs.

4. Discussion

Lactation is a complex and dynamic biological process that is an important part of the mammalian reproductive process [27]. Lactation traits are extremely important economic traits in dairy production, which include milk protein content, milk protein rate, milk fat content, milk fat rate and milk yield, and so on. Milk protein is an important source of dietary protein for humans, and its content is a key economic indicator used to evaluate milk quality and process characteristics [28,29,30]. SOCS1 has been revealed to be involved in the regulation of milk protein synthesis in mice, but data on it in buffalo are extremely scarce. In the present study, it was found that SOCS1 was highly expressed in the muscle and spleen of buffalo. In addition, this gene was moderately expressed in the lung, which is also consistent with the findings in pigs [31]. Cytokines play a crucial role in mammary gland development, which is reflected by their different expression in the mammary gland at various physiological periods [32]. As a cytokine signaling suppressor, SOCS1 expression has been reported to decrease from pregnancy to lactation in the mammary gland of cows [8]. Consistent with this, both mRNA and protein expression levels of SOCS1 in the mammary gland of buffalo during lactation in this study were markedly lower than that in the dry-off period, suggesting that SOCS1 is closely associated with buffalo mammary gland development and lactation.
The inhibition of JAK2 by SOCS1 is achieved by blocking substrate access to the JAK2 kinase via a fragment of 24 amino acids called the kinase inhibition region. SOCS1 recruits phosphorylated Tyr-1007 in the activation loop of JAK2 through its SH2 domain, thereby inhibiting the catalytic activity of JAK2 [33]. Meanwhile, SOCS1 can directly bind and target phosphorylated JAK2 for degradation through its E3 ubiquitin-ligase-like activity [34]. These processes take place in the cytoplasm of the cell. In addition, SOCS1 has been reported to have a nuclear localization signal, and recent studies have shown that it is detected in the nucleus, where it interacts with nuclear factor-κB (NF-κB), and is also part of the DNA damage response [35,36]. Our data show that buffalo SOCS1 is mainly distributed in the cytoplasm and partially in the nucleus of BuMECs, which is consistent with previous findings, revealing that SOCS1 interacts with JAK2 in the cytoplasm and also functions in the nucleus of BuMECs.
Previous studies have shown that SOCS1 is a key physiological attenuator of PRLR signaling, suggesting that SOCS1 can inhibit the expression or activity of PRLR and its downstream proteins [9]. In human 293 cells, the overexpression of SOCS1 resulted in a significant decrease in PRLR-mediated phosphorylation of JAK2 and STAT5, and eliminated the ability of PRLR to induce activation of the CSN2 promoter [37]. Consistently, the overexpression of SOCS1 here reduced the mRNA and protein abundance of CSN2, accompanied by the decreased phosphorylation of JAK2 and STAT5, while the knockdown of this gene had the opposite effect in BuMECs. It is worth noting that ELF5 also plays a role in activating STAT5, and its encoding gene is the target gene of STAT5 [38]. In this experiment, the overexpression of SOCS1 decreased the expression of ELF5, whereas the knockdown of this gene increased the expression of ELF5. We speculate that since SOCS1 altered the expression and phosphorylation of STAT5 protein, thereby indirectly regulating ELF5 expression in BuMECs. Intriguingly, we have further confirmed that SOCS1 can affect the protein expression levels of PI3K, p-PI3K, p-AKT1, mTOR and p-mTOR. Evidence suggests that phosphorylation of insulin receptor substrate 1 (IRS1) activates the PI3K-AKT-mTOR signaling pathway, and SOCS proteins are important mediators of IRS1 degradation, and target IRS1 through interaction with SOCS box motif [39,40]. Therefore, we speculate that buffalo SOCS1 may affect the PI3K-AKT-mTOR signaling pathway through IRS1. Overall, SOCS1 inhibits the synthesis of buffalo milk proteins by affecting the mTOR and JAK2-STAT5 pathways.
A previous study confirmed that STAT5 induces CEBPA expression during basophil and mast cell development [41]. As an essential transcription factor, CEBPA takes part in many biological processes, such as negative regulation of cell cycle progression, cell differentiation and apoptosis [42]. In patients with acute myeloid leukemia, SOCS1 expression is negatively correlated with mutant CEBPA [43]. It has also been found that CEBPA indirectly induces the expression of SOCS1 through miR-29b [18]. These findings suggest that CEBPA is closely associated with the expression of SOCS1. To date, whether CEBPA directly regulates the expression of buffalo SOCS1 and its regulatory mechanism remain unclear. The results in this study showed that the overexpression of CEBPA led to increased activity of SOCS1 promoter, while the knockdown of CEBPA had the opposite effect. Furthermore, the mRNA and protein abundance of SOCS1 varied with CEBPA expression, suggesting that CEBPA can regulate the expression of buffalo SOCS1. This promoter deletion experiment demonstrated that the overexpression of CEBPA enhanced the activity of SOCS1 promoter fragments representing the promoter regions between −1734/+105, −1364/+105 and −961/+105 bp. Sequence analysis of the SOCS1 promoter identified two potential CEBPA binding sites in the promoter regions between −1734/+105 and −1364/+105. Therefore, CEBPA can bind to these sites and thus promote the SOCS1 expression. Notably, although the fragment of SOCS1 promoter from −961 to +105 bp did not contain the CEBPA binding site, up-regulation of CEBPA still enhanced its activity, suggesting that CEBPA indirectly regulates the activity of SOCS1 promoter. We identified two NF-κB binding sites in this fragment of the promoter. As proposed by Paz-Priel et al., CEBPA can induce NF-κB expression by binding to its promoter [44]. In addition, SOCS1 may be induced by NF-κB in mice [45]. Therefore, it is suggested that CEBPA can also indirectly regulate the expression of SOCS1 in an NF-κB-dependent manner. This was further confirmed here by the knockdown experiment of CEBPA in BuMECs. This work reveals that buffalo SOCS1 is a target gene of CEBPA, and that the binding sites of CEBPA and NF-κB are essential elements for CEBPA-mediated transcriptional regulation of SOCS1.

5. Conclusions

Our results suggest that buffalo SOCS1 can inhibit milk protein synthesis through the mTOR and JAK2-STAT5 pathways in BuMECs. The promoter region of SOCS1 ranged from −1364 to +105 contains CEBPA and NF-κB binding sites, and was identified as the proximal core promoter. CEBPA regulates the transcription of buffalo SOCS1 through CEBPA and NF-κB binding sites in the SOCS1 promoter. This study can provide a basis for elucidating the genetic basis and regulatory mechanism of buffalo milk protein traits.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods12040708/s1, Figure S1: Images of buffalo mammary epithelial cells incubated with anti-cytokeratin 18 monoclonal antibody (A × 200, B × 400); Figure S2: Double-enzyme digestion of 5′ progressive deletion of pGL-SOCS1 recombinant plasmid. M, DL2000 Marker; 1–6, the fragments of SOCS1 promoter: −77~+105 bp, −614~+105 bp, −961~+105 bp, −1364~+105 bp, −1734~+105 bp and −1999~+105 bp; Table S1: Information on chemically synthesized miRNAs; Table S2: The primer of qPCR; Table S3: Primers used for isolation and deletion of buffalo SOCS1 promoter constructs.

Author Contributions

Conceptualization, X.F., L.Q. and Y.M.; methodology, software, validation, X.F., L.Q. and W.Z.; data curation, L.Q., W.Z., L.H. and X.T.; writing—original draft preparation, X.F.; writing—review and editing, X.F. and Y.M.; visualization, X.F. and W.Z.; project administration, Y.M.; funding acquisition, Y.M. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the National Natural Science Foundation of China (Grant nos. 32260822 and 31760659) and the Natural Science Foundation Key Project of Yunnan Province, China (Grant nos. 2014FA032 and 2007C0003Z).

Institutional Review Board Statement

Sample collection in the described experiments of this study was approved by the Animal Care and Use Committee of Yunnan Agricultural University (No. YNAU2019llwyh019).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Li, C.; Li, L.; Ali, I.; Kuang, M.; Wang, X.; Wang, G. UFL1 regulates milk protein and fat synthesis-related gene expression of bovine mammary epithelial cells probably via the mTOR signaling pathway. Vitr. Cell. Dev. Anim. 2021, 57, 550–559. [Google Scholar] [CrossRef]
  2. Li, H.; Liu, X.; Wang, Z.; Lin, X.; Yan, Z.; Cao, Q.; Zhao, M.; Shi, K. MEN1/Menin regulates milk protein synthesis through mTOR signaling in mammary epithelial cells. Sci. Rep. 2017, 7, 5479. [Google Scholar] [CrossRef] [PubMed]
  3. Hue-Beauvais, C.; Faulconnier, Y.; Charlier, M.; Leroux, C. Nutritional regulation of mammary gland development and milk synthesis in animal models and dairy species. Genes 2021, 12, 523. [Google Scholar] [CrossRef] [PubMed]
  4. van Lieshout, G.A.A.; Lambers, T.T.; Bragt, M.C.E.; Hettinga, K.A. How processing may affect milk protein digestion and overall physiological outcomes: A systematic review. Crit. Rev. Food Sci. 2020, 60, 2422–2445. [Google Scholar] [CrossRef]
  5. Bionaz, M.; Loor, J.J. Gene networks driving bovine mammary protein synthesis during the lactation cycle. Bioinform. Biol. Insights 2011, 5, 83–98. [Google Scholar] [CrossRef]
  6. Li, L.; Liu, L.; Qu, B.; Li, X.; Gao, X.; Zhang, M. Twinfilin 1 enhances milk bio-synthesis and proliferation of bovine mammary epithelial cells via the mTOR signaling pathway. Biochem. Biophys. Res. Commun. 2017, 492, 289–294. [Google Scholar] [CrossRef] [PubMed]
  7. Yuan, X.; Zhang, M.; Ao, J.; Zhen, Z.; Gao, X.; Li, M. NUCKS1 is a novel regulator of milk synthesis in and proliferation of mammary epithelial cells via the mTOR signaling pathway. J. Cell. Physiol. 2019, 234, 15825–15835. [Google Scholar] [CrossRef]
  8. Arun, S.J.; Thomson, P.C.; Sheehy, P.A.; Khatkar, M.S.; Raadsma, H.W.; Williamson, P. Targeted analysis reveals an important role of JAK-STAT-SOCS genes for milk production traits in Australian dairy cattle. Front. Genet. 2015, 6, 342. [Google Scholar] [CrossRef]
  9. Sutherland, K.D.; Lindeman, G.J.; Visvader, J.E. Knocking off SOCS genes in the mammary gland. Cell Cycle 2007, 6, 799–803. [Google Scholar] [CrossRef]
  10. Lindeman, G.J.; Wittlin, S.; Lada, H.; Naylor, M.J.; Santamaria, M.; Zhang, J.; Starr, R.; Hilton, D.J.; Alexander, W.S.; Ormandy, C.J.; et al. SOCS1 deficiency results in accelerated mammary gland development and rescues lactation in prolactin receptor-deficient mice. Genes Dev. 2001, 15, 1631–1636. [Google Scholar] [CrossRef] [Green Version]
  11. Motta, M.; Accornero, P.; Baratta, M. Leptin and prolactin modulate the expression of SOCS-1 in association with interleukin-6 and tumor necrosis factor-α in mammary cells: A role in differentiated secretory epithelium. Regul. Pept. 2004, 121, 163–170. [Google Scholar] [CrossRef]
  12. Cella, N.; Chiquet-Ehrismann, R.; Hynes, N.E. Lactogenic hormones and Tenascin-C regulate C/EBPα and β in mammary epithelial cells. J. Cell. Biochem. 2000, 76, 394–403. [Google Scholar] [CrossRef]
  13. Rosen, J.M.; Wyszomierski, S.L.; Hadsell, D. Regulation of milk protein gene expression. Annu. Rev. Nutr. 1999, 19, 407–436. [Google Scholar] [CrossRef]
  14. Seagroves, T.N.; Krnacik, S.; Raught, B.; Gay, J.; Burgess-Beusse, B.; Darlington, G.J.; Rosen, J.M. C/EBPβ, but not C/EBPα, is essential for ductal morphogenesis, lobuloalveolar proliferation, and functional differentiation in the mouse mammary gland. Genes Dev. 1998, 12, 1917–1928. [Google Scholar] [CrossRef]
  15. Lee, M.; Song, S.J.; Choi, M.; Yu, R.; Park, T. IL-7 receptor deletion ameliorates diet-induced obesity and insulin resistance in mice. Diabetologia 2015, 58, 2361–2370. [Google Scholar] [CrossRef] [PubMed]
  16. Ahsani, M.; Mohammadabadi, M.R.; Fozi, M.A.; Koshkooieh, A.E.; Khezri, A.; Babenko, O.I.; Bushtruk, M.V.; Tkachenko, S.V.; Stavetska, R.V.; Klopenko, N.I. Effect of roasted soybean and canola seeds on peroxisome proliferator-activated receptors gamma (PPARG) gene expression and cattle milk characteristics. Iran. J. Appl. Anim. Sci. 2019, 9, 635–642. [Google Scholar]
  17. Wang, Y.; Guo, W.; Tang, K.; Wang, Y.; Zan, L.; Yang, W. Bta-miR-34b regulates milk fat biosynthesis by targeting mRNA decapping enzyme 1A (DCP1A) in cultured bovine mammary epithelial cells. J. Anim. Sci. 2019, 97, 3823–3831. [Google Scholar] [CrossRef]
  18. Yan, B.; Guo, Q.; Fu, F.; Wang, Z.; Yin, Z.; Wei, Y.; Yang, J. The role of miR-29b in cancer: Regulation, function, and signaling. Onco Targets Ther. 2015, 8, 539–548. [Google Scholar] [CrossRef] [PubMed]
  19. Basilicata, M.; Pepe, G.; Adesso, S.; Ostacolo, C.; Sala, M.; Sommella, E.; Scala, M.; Messore, A.; Autore, G.; Marzocco, S.; et al. Antioxidant properties of buffalo-milk dairy products: A β-Lg peptide released after gastrointestinal digestion of buffalo Ricotta cheese reduces oxidative stress in intestinal epithelial cells. Int. J. Mol. Sci. 2018, 19, 1955. [Google Scholar] [CrossRef]
  20. Khan, I.T.; Nadeem, M.; Imran, M.; Asif, M.; Khan, M.K.; Din, A.; Ullah, R. Triglyceride, fatty acid profile and antioxidant characteristics of low melting point fractions of Buffalo Milk fat. Lipids Health Dis. 2019, 18, 59. [Google Scholar] [CrossRef]
  21. Farr, V.C.; Stelwagen, K.; Cate, L.R.; Molenaar, A.J.; Mcfadden, T.B.; Davis, S.R. An improved method for the routine biopsy of bovine mammary tissue. J. Dairy Sci. 1996, 79, 543–549. [Google Scholar] [CrossRef] [PubMed]
  22. Fan, X.; Zhu, W.; Qiu, L.; Zhang, G.; Zhang, Y.; Miao, Y. Elongase of very long chain fatty acids 6 (ELOVL6) promotes lipid synthesis in buffalo mammary epithelial cells. J. Anim. Physiol. Anim. Nutr. 2022, 106, 1–11. [Google Scholar] [CrossRef] [PubMed]
  23. Fan, X.; Qiu, L.; Teng, X.; Zhang, Y.; Miao, Y. Effect of INSIG1 on the milk fat synthesis of buffalo mammary epithelial cells. J. Dairy Res. 2020, 87, 349–355. [Google Scholar] [CrossRef] [PubMed]
  24. Lalitha, S. Primer Premier 5. Biotech. Softw. Internet Rep. 2000, 1, 270–272. [Google Scholar] [CrossRef]
  25. Bustin, S.A.; Benes, V.; Garson, J.A.; Hellemans, J.; Huggett, J.; Kubista, M.; Mueller, R.; Nolan, T.; Pfaffl, M.W.; Shipley, G.L.; et al. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 2009, 55, 611–622. [Google Scholar] [CrossRef]
  26. Chatchatee, P.; Järvinen, K.M.; Bardina, L.; Vila, L.; Beyer, K.; Sampson, H.A. Identification of IgE and IgG binding epitopes on β- and κ-casein in cow’s milk allergic patients. Clin. Exp. Allergy 2001, 31, 1256–1262. [Google Scholar] [CrossRef] [PubMed]
  27. Capuco, A.V.; Akers, R.M. The origin and evolution of lactation. J. Biol. 2009, 8, 37. [Google Scholar] [CrossRef]
  28. Pegolo, S.; Yu, H.; Morota, G.; Bisutti, V.; Rosa, G.J.M.; Bittante, G.; Cecchinato, A. Structural equation modeling for unraveling the multivariate genomic architecture of milk proteins in dairy cattle. J. Dairy Sci. 2021, 104, 5705–5718. [Google Scholar] [CrossRef]
  29. Anderson, J.L.; Schingoethe, D.J.; Kalscheur, K.F.; Hippen, A.R. Evaluation of dried and wet distillers grains included at two concentrations in the diets of lactating dairy cows. J. Dairy Sci. 2006, 89, 3133–3142. [Google Scholar] [CrossRef]
  30. Jenkins, T.C.; McGuire, M.A. Major advances in nutrition: Impact on milk composition. J. Dairy Sci. 2006, 89, 1302–1310. [Google Scholar] [CrossRef] [PubMed]
  31. Delgado-Ortega, M.; Melo, S.; Meurens, F. Expression of SOCS1-7 and CIS mRNA in porcine tissues. Vet. Immunol. Immunopathol. 2011, 144, 493–498. [Google Scholar] [CrossRef]
  32. Alluwaimi, A.M.; Cullor, J.S. Cytokines gene expression patterns of bovine milk during middle and late stages of lactation. J. Vet. Med. B 2022, 49, 105–110. [Google Scholar] [CrossRef] [PubMed]
  33. Ali, S.; Nouhi, Z.; Chughtai, N.; Ali, S. SHP-2 regulates SOCS-1-mediated Janus kinase-2 ubiquitination/degradation downstream of the prolactin receptor. J. Biol. Chem. 2003, 278, 52021–52031. [Google Scholar] [CrossRef] [PubMed]
  34. Kamizono, S.; Hanada, T.; Yasukawa, H.; Minoguchi, S.; Kato, R.; Minoguchi, M.; Hattori, K.; Hatakeyama, S.; Yada, M.; Morita, S.; et al. The SOCS box of SOCS-1 accelerates ubiquitin-dependent proteolysis of TEL-JAK2. J. Biol. Chem. 2001, 276, 12530–12538. [Google Scholar] [CrossRef] [PubMed]
  35. Sharma, J.; Larkin, J. Therapeutic implication of SOCS1 modulation in the treatment of autoimmunity and cancer. Front. Pharmacol. 2019, 10, 324. [Google Scholar] [CrossRef]
  36. Zimmer, J.; Weitnauer, M.; Boutin, S.; Küblbeck, G.; Thiele, S.; Walker, P.; Lasitschka, F.; Lunding, L.; Orinska, Z.; Vock, C.; et al. Nuclear localization of suppressor of cytokine signaling-1 regulates local immunity in the lung. Front. Immunol. 2016, 7, 514. [Google Scholar] [CrossRef]
  37. Tomic, S.; Chughtai, N.; Ali, S. SOCS-1, -2, -3: Selective targets and functions downstream of the prolactin receptor. Mol. Cell. Endocrinol. 1999, 158, 45–54. [Google Scholar] [CrossRef]
  38. Igelmann, S.; Neubauer, H.; Ferbeyre, G. STAT3 and STAT5 activation in solid cancers. Cancers 2019, 11, 1428. [Google Scholar] [CrossRef]
  39. Law, N.C.; White, M.F.; Hunzicker-Dunn, M.E. G protein-coupled receptors (GPCRs) that signal via protein kinase A (PKA) cross-talk at insulin receptor substrate 1 (IRS1) to activate the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. J. Biol. Chem. 2016, 291, 27160–27169. [Google Scholar] [CrossRef]
  40. Ueki, K.; Kondo, T.; Kahn, C.R. Suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 cause insulin resistance through inhibition of tyrosine phosphorylation of insulin receptor substrate proteins by discrete mechanisms. Mol. Cell. Biol. 2004, 24, 5434–5446. [Google Scholar] [CrossRef]
  41. Sasaki, H.; Kurotaki, D.; Tamura, T. Regulation of basophil and mast cell development by transcription factors. Allergol. Int. 2016, 65, 127–134. [Google Scholar] [CrossRef] [Green Version]
  42. Barbagallo, D.; Condorelli, A.G.; Piro, S.; Parrinello, N.; Fløyel, T.; Ragusa, M.; Rabuazzo, A.M.; Størling, J.; Purrello, F.; Di Pietro, C.; et al. CEBPA exerts a specific and biologically important proapoptotic role in pancreatic β cells through its downstream network targets. Mol. Biol. Cell 2014, 25, 2333–2341. [Google Scholar] [CrossRef] [PubMed]
  43. Hou, H.; Lu, J.; Lin, T.; Tsai, C.; Chou, W.; Lin, C.; Kuo, Y.; Liu, C.; Tseng, M.; Chiang, Y.; et al. Clinico-biological significance of suppressor of cytokine signaling 1 expression in acute myeloid leukemia. Blood Cancer J. 2017, 7, e588. [Google Scholar] [CrossRef] [PubMed]
  44. Paz-Priel, I.; Houng, S.; Dooher, J.; Friedman, A.D. C/EBPα and C/EBPα oncoproteins regulate nfkb1 and displace histone deacetylases from NF-κB p50 homodimers to induce NF-κB target genes. Blood 2011, 117, 4085–4094. [Google Scholar] [CrossRef] [PubMed]
  45. Chan, H.C.; Ke, L.Y.; Chang, L.L.; Liu, C.C.; Hung, Y.H.; Lin, C.H.; Li, R.N.; Tsai, W.C.; Liu, H.W.; Yen, J.H. Suppressor of cytokine signaling 1 gene expression and polymorphisms in systemic lupus erythematosus. Lupus 2010, 19, 696–702. [Google Scholar] [CrossRef]
Foods 12 00708 g001 550
Figure 1. Differential expression of SOCS1 in various tissues of buffalo. (A) Differential mRNA expression of SOCS1 in 12 buffalo tissues. (B) Differential mRNA expression of buffalo SOCS1 in the mammary gland between lactation and dry-off period. (C,D) Differential expression of buffalo SOCS1 protein in the mammary gland between lactation and dry-off period. Data are presented as means ± SEM for three individual cultures; asterisks indicate differences between dry-off and lactation: ** p < 0.01.
Figure 1. Differential expression of SOCS1 in various tissues of buffalo. (A) Differential mRNA expression of SOCS1 in 12 buffalo tissues. (B) Differential mRNA expression of buffalo SOCS1 in the mammary gland between lactation and dry-off period. (C,D) Differential expression of buffalo SOCS1 protein in the mammary gland between lactation and dry-off period. Data are presented as means ± SEM for three individual cultures; asterisks indicate differences between dry-off and lactation: ** p < 0.01.
Foods 12 00708 g001
Foods 12 00708 g002 550
Figure 2. Immunofluorescent staining of nucleus (blue), mitochondria (red) or EGFP-SOCS1 (green) and confocal microscopy analysis in BuMECs. (A,B) Nucleus and mitochondria stained with Hoechst 33342 and Mito-Tracker, respectively; (C) green fluorescent protein was expressed by EGFP-SOCS1; (D) merged overlaid nucleus and EGFP-SOCS1; (E) merged overlaid mitochondria and EGFP-SOCS1; (F) merged overlaid nucleus, mitochondria and EGFP-SOCS1.
Figure 2. Immunofluorescent staining of nucleus (blue), mitochondria (red) or EGFP-SOCS1 (green) and confocal microscopy analysis in BuMECs. (A,B) Nucleus and mitochondria stained with Hoechst 33342 and Mito-Tracker, respectively; (C) green fluorescent protein was expressed by EGFP-SOCS1; (D) merged overlaid nucleus and EGFP-SOCS1; (E) merged overlaid mitochondria and EGFP-SOCS1; (F) merged overlaid nucleus, mitochondria and EGFP-SOCS1.
Foods 12 00708 g002
Foods 12 00708 g003 550
Figure 3. Effect of SOCS1 on milk protein synthesis in buffalo mammary epithelial cells (BuMECs). (A,B) The expressions of SOCS1 in BuMECs treated with the EGFP-SOCS1, shSOCS1 and corresponding negative controls (EGFP and shNC). (C,D) Effects of EGFP-SOCS1 or shSOCS1 on the expression of CSN2 and CSN3. (E,F) Effects of EGFP-SOCS1 or shSOCS1 on the expression level of CSN2 protein. Data are presented as means ± SEM for three individual cultures; * p < 0.05, ** p < 0.01.
Figure 3. Effect of SOCS1 on milk protein synthesis in buffalo mammary epithelial cells (BuMECs). (A,B) The expressions of SOCS1 in BuMECs treated with the EGFP-SOCS1, shSOCS1 and corresponding negative controls (EGFP and shNC). (C,D) Effects of EGFP-SOCS1 or shSOCS1 on the expression of CSN2 and CSN3. (E,F) Effects of EGFP-SOCS1 or shSOCS1 on the expression level of CSN2 protein. Data are presented as means ± SEM for three individual cultures; * p < 0.05, ** p < 0.01.
Foods 12 00708 g003
Foods 12 00708 g004 550
Figure 4. Effect of SOCS1 on the expression of genes and proteins related to the mTOR and JAK2-STAT5 pathways in BuMECs. (A,D) Effects of EGFP-SOCS1 or shSOCS1 on the expression of genes related to the upstream pathway of mTOR signaling. (B,E) Effects of EGFP-SOCS1 or shSOCS1 on the expression of genes related to the downstream pathway of mTOR signaling. (C,F) Effects of EGFP-SOCS1 or shSOCS1 on the expression of genes participating in the JAK2-STAT5 signaling. (G–J) Effects of EGFP-SOCS1 or shSOCS1 on the expression of proteins related to the mTOR and JAK2-STAT5 pathways. Data are presented as means ± SEM for three individual cultures; * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 4. Effect of SOCS1 on the expression of genes and proteins related to the mTOR and JAK2-STAT5 pathways in BuMECs. (A,D) Effects of EGFP-SOCS1 or shSOCS1 on the expression of genes related to the upstream pathway of mTOR signaling. (B,E) Effects of EGFP-SOCS1 or shSOCS1 on the expression of genes related to the downstream pathway of mTOR signaling. (C,F) Effects of EGFP-SOCS1 or shSOCS1 on the expression of genes participating in the JAK2-STAT5 signaling. (G–J) Effects of EGFP-SOCS1 or shSOCS1 on the expression of proteins related to the mTOR and JAK2-STAT5 pathways. Data are presented as means ± SEM for three individual cultures; * p < 0.05, ** p < 0.01, *** p < 0.001.
Foods 12 00708 g004
Foods 12 00708 g005 550
Figure 5. CEBPA promotes the transcription and expression of SOCS1 in buffalo mammary epithelial cells (BuMECs). (A) The BuMECs were co-transfected with the vector pGL-(−1999/+105) of SOCS1 promoter and either the EGFP-CEBPA or EGFP vector, or either the siCEBPA or siNC. (B–D) The mRNA and protein expression of SOCS1 in BuMECs following transfection with EGFP-CEBPA or siCEBPA, as well as their corresponding negative controls. Data are presented as means ± SEM for three individual cultures; * p < 0.05, ** p < 0.01.
Figure 5. CEBPA promotes the transcription and expression of SOCS1 in buffalo mammary epithelial cells (BuMECs). (A) The BuMECs were co-transfected with the vector pGL-(−1999/+105) of SOCS1 promoter and either the EGFP-CEBPA or EGFP vector, or either the siCEBPA or siNC. (B–D) The mRNA and protein expression of SOCS1 in BuMECs following transfection with EGFP-CEBPA or siCEBPA, as well as their corresponding negative controls. Data are presented as means ± SEM for three individual cultures; * p < 0.05, ** p < 0.01.
Foods 12 00708 g005
Foods 12 00708 g006 550
Figure 6. Identification of the core promoter region of SOCS1 and analysis of transcription factor binding sites. (A) Relative luciferase activity (Firefly: Renilla) after 48 h transfection with 5′ progressive deletions of pGL-SOCS1 plasmids (pGL-(−1999/+105), pGL-(−1734/+105), pGL-(−1364/+105), pGL-(−961/+105), pGL-(−614/+105) and pGL-(−77/+105)). (B) Predicted transcription factor binding sites in the core promoter region of buffalo SOCS1. Data are presented as means ± SEM for three individual cultures; * p < 0.05, ** p < 0.01.
Figure 6. Identification of the core promoter region of SOCS1 and analysis of transcription factor binding sites. (A) Relative luciferase activity (Firefly: Renilla) after 48 h transfection with 5′ progressive deletions of pGL-SOCS1 plasmids (pGL-(−1999/+105), pGL-(−1734/+105), pGL-(−1364/+105), pGL-(−961/+105), pGL-(−614/+105) and pGL-(−77/+105)). (B) Predicted transcription factor binding sites in the core promoter region of buffalo SOCS1. Data are presented as means ± SEM for three individual cultures; * p < 0.05, ** p < 0.01.
Foods 12 00708 g006
Foods 12 00708 g007 550
Figure 7. Identification of the cis-regulatory elements of CEBPA regulating the transcriptional activity of buffalo SOCS1 in BuMECs. (A) The effect of CEBPA overexpression on the activity of SOCS1 promoters of different lengths. (B) The effect of CEBPA knockdown on the activity of SOCS1 promoters of different lengths. Yellow rectangles represent CEBPA binding sites and orange pentagons represent NF-κB binding sites. Data are presented as means ± SEM for three individual cultures; * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 7. Identification of the cis-regulatory elements of CEBPA regulating the transcriptional activity of buffalo SOCS1 in BuMECs. (A) The effect of CEBPA overexpression on the activity of SOCS1 promoters of different lengths. (B) The effect of CEBPA knockdown on the activity of SOCS1 promoters of different lengths. Yellow rectangles represent CEBPA binding sites and orange pentagons represent NF-κB binding sites. Data are presented as means ± SEM for three individual cultures; * p < 0.05, ** p < 0.01, *** p < 0.001.
Foods 12 00708 g007
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Fan, X.; Qiu, L.; Zhu, W.; Huang, L.; Tu, X.; Miao, Y. CEBPA-Regulated Expression of SOCS1 Suppresses Milk Protein Synthesis through mTOR and JAK2-STAT5 Signaling Pathways in Buffalo Mammary Epithelial Cells. Foods 2023, 12, 708. https://doi.org/10.3390/foods12040708

AMA Style

Fan X, Qiu L, Zhu W, Huang L, Tu X, Miao Y. CEBPA-Regulated Expression of SOCS1 Suppresses Milk Protein Synthesis through mTOR and JAK2-STAT5 Signaling Pathways in Buffalo Mammary Epithelial Cells. Foods. 2023; 12(4):708. https://doi.org/10.3390/foods12040708

Chicago/Turabian Style

Fan, Xinyang, Lihua Qiu, Wei Zhu, Lige Huang, Xingtiao Tu, and Yongwang Miao. 2023. "CEBPA-Regulated Expression of SOCS1 Suppresses Milk Protein Synthesis through mTOR and JAK2-STAT5 Signaling Pathways in Buffalo Mammary Epithelial Cells" Foods 12, no. 4: 708. https://doi.org/10.3390/foods12040708

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop