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

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.


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].

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.
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).

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% CO 2 . 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 cotransfected 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% CO 2 . 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 TransIntro TM 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.

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).

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 Taq TM 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 -∆∆Ct model [25].

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.

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.

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.
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.

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.

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.

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.

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.

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

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). 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).

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

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).

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).

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

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).

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.

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

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.

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.

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.