The ubiquitin ligase Nedd4-2 mediates the regulation of PepT2 by mTORC1 in bovine mammary epithelial cells

Peptide transporter 2 (PepT2) transports short peptides from the blood into bovine mammary epithelial cells (BMEC) to stimulate milk protein synthesis. Despite the fact that the effect of PepT2 is acknowledged in BMEC, little is known about its regulation. This study was completed to investigate the role of mammalian target of the rapamycin (mTOR) signaling in regulating the expression and function of PepT2 in BMEC. The regulation of PepT2 by mTOR in BMEC was studied in vitro using peptide transport assay, gene silencing, Western blot. The membrane expression of PepT2 and the uptake of β-Ala-Lys-N-7-amino-4-methylcoumarin-3-acetic acid (β-Ala-Lys-AMCA), a model dipeptide, in BMEC were reduced by rapamycin (a mTOR inhibitor) and silencing of either mTOR complex 1 (mTORC1) or mTOR complex 2 (mTORC2), stimulated by DEP domain-containing mTOR-interacting protein (DEPTOR, endogenous inhibitor of mTORC1 and mTORC2) silencing. The trafficking of PepT2 to the membrane and the uptake of β-Ala-Lys-AMCA was promoted by neuronal precursor cell-expressed developmentally down-regulated 4 isoform 2 (Nedd4-2) silencing. The effects of knockdown of mTORC1, but not mTORC2, on cell membrane expression and transport activity of PepT2 was abolished by Nedd4-2 silencing. With immunofluorescence staining, PepT2 was identified to be interacting with Nedd4-2. The Nedd4-2 expression and the interaction between PepT2 and Nedd4-2 was increased through mTORC1 knockdown, indicating an increased ubiquitination of PepT2. The results revealed that mTORC1 can regulate the expression and function of PepT2 through Nedd4-2 in BMEC.


Introduction
The supply of some free essential amino acids (EAA) in the blood is less than their output in milk proteins, and large amounts of peptide-bound amino acids (PBAA) can compensate for the shortage of EAA for milk protein synthesis (Bequette et al., 1999). PBAA represent an essential part of total amino acid flux across the portal-drained viscera and have been shown to contribute to milk protein synthesis (Mabjeesh et al., 2002). Our previous studies revealed that methionine (Met) containing peptides can be utilized to stimulate milk protein synthesis in bovine mammary epithelial cells (BMEC), and methionyl-methionine (MeteMet) has a higher efficiency in stimulating milk protein synthesis than free Met (Yang et al., 2015;Wang et al., 2018). Peptide transporter 2 (PepT2) acts an essential role in cellular uptake of di/tripeptides and peptidomimetic drugs in mammalian cells (Gilbert et al., 2008). Studies have shown that PepT2 is abundantly expressed in BMEC and the knockdown of PepT2 reduces the uptake of MeteMet and Met-Met-enhanced increase of as 1 -casein (CN) expression in BMEC (Yang et al., 2015;Wang et al., 2018;Groneberg et al., 2002). As PepT2 plays a great role in small peptide absorption, understanding the regulation of PepT2 is of great significance.
The mammalian target of the rapamycin (mTOR) signaling pathway controls many cellular processes that produce or consume energy and nutrients (Laplante and Sabatini, 2012), including regulation of protein synthesis responding to the nutrient level and hormones in dairy cows (Huang and Fingar, 2014). Moreover, mTOR is currently established as a modulator of amino acid transporters (Burgos et al., 2010). mTOR is a key component of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (Loewith et al., 2002).
The key difference between mTORC1 and mTORC2 is that mTORC1 comprises regulatory associated protein of mTOR (Raptor) whereas rapamycin-insensitive companion of mTOR (Rictor) is involved in mTORC2 (Peterson et al., 2009;Frias et al., 2006). Rosario et al. (2013) revealed mTORC1 and mTORC2 silencing significantly inhibits the trafficking and transport activity of system A and system L amino acid transporters in trophoblasts. However, the regulatory effect of mTOR signaling in the function of PepT2 in BMEC is still unclear.
Ubiquitination has been identified as a major regulatory mechanism of transporters (Acconcia et al., 2009). Ubiquitin-activating enzyme 3 (E3) is included in the last step of ubiquitination and transfers the ubiquitin to the target proteins (Acconcia et al., 2009;Wenzel et al., 2011). Neuronal precursor cell-expressed developmentally down-regulated 4 isoform 2 (Nedd4-2), participates in the ubiquitination of many nutrient transporters (Foot et al., 2017). Nedd4-2 regulates the plasma membrane expression of sodiumdependent neutral amino acid transporter 2 (SNAT2) and L-type amino acid transporter 1 (LAT1) and mediates the regulation of amino acid transporter by mTORC1 (Rosario et al., 2016).
The objective of this study was to define whether ubiquitination regulates the transport activity of PepT2 and find a link between the regulation of PepT2 by mTOR signaling and ubiquitination. In the study, we hypothesized that mTOR can modulate the expression and transport activity of PepT2 through Nedd4-2 in BMEC.

Culture of BMEC
The procedures of BMEC culture were described previously (Wang et al., 2018). The mammary gland tissues were acquired from 3 midlactation dairy cows at a local abattoir. The use of animal tissues was approved by the Institutional Animal Use Committee of Zhejiang University. Briefly, the minced tissues collected from bovine mammary gland were digested in trypsin (0.25%, Amesco, Solon, OH, USA) at 37 C for 30 min and subsequently in collagenase I (Amesco) and collagenase II (Amesco) for 4 h at 37 C. The digested tissue homogenate was filtrated and centrifuged at 300 g or 5 min, the cells were cultivated in plastic dishes containing DMEM/F12 with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), transferrin (Sigma, St. Louis, MO, USA), insulin (Sigma), prolactin (5 mg/mL, Sigma), hydrocortisone (1 mg/mL, Sigma) and epithelial growth factor (10 ng/mL, Sigma). After the cells were starved for 12 h and treated with rapamycin (100 ng/mL, Sangon, Shanghai, China) for 12 h, they were then harvested with RIPA cell lysate buffer (Beyotime, Jiangsu, China) or used to study peptide uptake. BMEC used in the study were between passages 5 and 8.

The siRNA transient transfection
The siRNA was transfected into BMEC with lipofectamine RNAimax (Invitrogen, Carlsbad, CA, USA). In brief, the siRNA (60 pmol/L) and lipofectamine RNAimax were diluted, mixed and incubated for 5 min. BMEC were harvested at 24 h after transfection. The sequences of siRNA are presented in Appendix Table 1.

Quantitative reverse transcription real-time PCR (qRT-PCR)
Total RNA was extracted from BMEC using RNA isolation kit (Sangon). After confirming the absorbance at A260 nm/A280 nm (within 1.8 to 2.1) and the RNA integrity by electrophoresis, it was transcribed into cDNA. The cDNA was synthesized with a reverse transcription kit (Takara, Otsu, Shiga, Japan). The relative mRNA abundance of target genes was quantified by SYBR PrimeScript reagent kit (Takara) and an ABI 7500 (Applied Biosystems, Singapore). The designed primers were present in Appendix Table 2, and the amplification efficiency was between 90% and 110%. The gene expression was normalized to b-actin and calculated by the 2 ÀDDCt method (Schmittgen and Livak, 2008).

Surface biotinylation
BMEC were treated with 100 mmol/L Nacetyl-Leu-Leunorleucinal for 2 h before harvest to avoid proteasomal degradation of ubiquitinated PepT2. BMEC were washed three times using PBS followed by incubating with sulfoeNHSeLC-biotin (1 mg/mL, Genescript, Nanjing, China) for 30 min on ice. Subsequently, the cells were quenched by PBS comprising 100 mmol/L glycine for 15 min at 4 C, lysed with ice-cold RIPA and centrifuged at 16,000Âg for 20 min. The supernatant was incubated with streptavidin-agarose beads for 2 h on ice to isolate the membrane proteins. The PepT2 at the cell membrane was detected by Western blot.
2.6. Immunofluorescence staining BMEC were cultured with 100 mmol/L Nacetyl-Leu-Leunorleucinal for 2 h and then fixed with 4% paraformaldehyde and blocked by 3% serum albumin for half an hour. The cells were incubated with a PepT2 antibody (Abcam, 1:50) overnight at 4 C. Subsequently, they were incubated with a FITC-conjugated secondary antibody (Sangon) for an hour in dark and counterstained with DAPI (2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, Sangon) for 10 min. Finally, they were evaluated by confocal laser scanning microscope (Nikon, Tokyo, Japan). b-Ala-Lys-AMCA, a coumarin-tagged peptide probe, was used to detect the uptake of dipeptide in BMEC (Wang et al., 2018). After the cells were cultivated at 12-well plates for 1 d, they were preincubated with KrebseRinger modified buffer (KRB) for 0.5 h, followed by incubation with KRB containing 25 mmol/L b-Ala-Lys-AMCA for 30 min at 37 C, the cells were washed 3 times with icecold KRB to stop the uptake of dipeptide and lysed with 1% triton-100. The uptake of b-Ala-Lys-AMCA in cells was detected by microplate reader (excitation at 350 nm, emission at 455 nm). Protein concentration was determined by BCA protein assay kit (Beyotime, Jiangsu, China). The uptake of b-Ala-Lys-AMCA in each well was standardized to the amount of protein.

Statistical analysis
Statistical analysis was examined with one-way ANOVA or unpaired Student's t-test by SAS software (SAS Institute, USA). Tukey's multiple range test was applied to detect the differences between means. Each experiment was repeated at least 3 times. Values are shown as the means and pooled SEM. P < 0.05 was considered as a significant difference.

mTOR regulates the membrane expression and peptide transport activity of PepT2
Treatment of BMEC with rapamycin significantly decreased the PepT2 membrane expression (Fig. 1A) and reduced the uptake of b-Ala-Lys-AMCA (Fig. 1B), but the total cellular expression of PepT2 was not affected (Appendix Fig. 1). The interference efficiency of DEPTOR siRNA was illustrated in Appendix Fig. 2. The siRNA 3 decreased DEPTOR mRNA abundance by 70%. In contrast, knockdown of DEPTOR using siRNA significantly increased the PepT2 membrane expression (Fig. 1C) and stimulated the uptake of b-Ala-Lys-AMCA in BMEC (Fig. 1D).

Nedd4-2 mediates the regulation of the membrane expression and peptide transport activity of PepT2 by mTORC1
The interference efficiency of Raptor siRNA was shown in Fig. S4. The siRNA 2 decreased Raptor mRNA abundance by almost 60% (Appendix Fig. 4). Compared with cells transfected with scramble siRNA, Raptor knockdown significantly decreased the PepT2 membrane expression (Fig. 3A) and the uptake of b-Ala-Lys-AMCA ( Fig. 3B) in BMEC. However, the simultaneous knockdown of Raptor and Nedd4-2 canceled the inhibitory effect of mTORC1 knockdown on the membrane expression of PepT2 (Fig. 3A) as well as the uptake of b-Ala-Lys-AMCA in BMEC (Fig. 3B).

Nedd4-2 is not required in the regulation of the membrane expression and peptide transport activity of PepT2 by mTORC2
The interference efficiency of Rictor siRNA was described in Appendix Fig. 5. The three siRNAs downregulated Rictor mRNA abundance in BMEC (Appendix Fig. 5). Similar to Raptor knockdown, knockdown of Rictor by siRNA 3 also significantly reduced the plasma membrane expression of PepT2 (Fig. 4A) and the uptake of b-Ala-Lys-AMCA (Fig. 4B) in BMEC. However, Nedd4-2 knockdown did not affect the reduction in the plasma membrane expression of PepT2 (Fig. 4A) and the uptake of b-Ala-Lys-AMCA induced by mTORC2 inhibition (Fig. 4B) in BMEC.

Discussion
PepT2 acts pivotal role in cellular uptake of di/tripeptides and peptidomimetic drugs in BMEC (Zhao and Lu, 2015;Wang et al., 2019). Hence, it is of great significance to understand the regulation of PepT2 in physiological and pathological processes. The present study revealed that PepT2 is regulated by mTOR signaling, and Nedd4-2 mediates the regulation of PepT2 by mTORC1 in BMEC.
The mTOR signaling pathway regulates protein translation and modulates cell growth and metabolism (Saxton and Sabatini, 2017;Xie et al., 2022). There is evidence suggesting that nutrient transporters are the downstream targets of mTOR signaling in mammalian cells (Roos et al., 2007). Rosario et al. (2013) found that mTORC1 and mTORC2 silencing inhibited the membrane expression and transport activity of system A and system L amino acid transporters in human trophoblast cells. The protein level of PepT2 in cell lysates was not affected after rapamycin treatment, but rapamycin decreased the membrane expression and transport activity of PepT2 in BMEC in the present study. The subcellular localization of PepT2 determines its function in peptide uptake. Our observations indicated that mTOR signaling can direct the trafficking of PepT2 to the plasma membrane, which leads to the increased activity of peptide uptake. Indeed, knockdown of DEP-TOR, an endogenous mTOR inhibitor, also enhanced the trafficking of PepT2 to the plasma membrane and promoted the uptake of b-Ala-Lys-AMCA, confirming the role of mTOR signaling in modulating the membrane expression of PepT2 and peptide transport activity in BMEC. As previous studies revealed that dipeptides can promote milk protein synthesis (Yang et al., 2015;Wang et al., 2018), we propose the activation of mTOR signaling pathway might increase milk protein synthesis by enhancing the dipeptide uptake in BMEC.
Whether mTORC1 and mTORC2 play roles in the regulation of PepT2 in BMEC was investigated by Raptor and Rictor silencing; the essential components of mTORC1 and mTORC2, respectively. Our results revealed that both mTORC1 inhibition by Raptor knockdown and mTORC2 inhibition by Rictor knockdown resulted in a marked decrease in plasma membrane expression of PepT2 as well as the uptake of b-Ala-Lys-AMCA. These observations indicated that both mTORC1 and mTORC2 are involved in the modulation of PepT2 function in BMEC.
C. Wang, F. Zhao, J. Liu et al. Animal Nutrition 10 (2022) 12e18 regulated by the Nedd4-2 (Boehmer et al., 2006). Nedd4-2mediated ubiquitination enhanced the trafficking of glutamate transporter between the membrane and cytoplasm and stimulated the transport activity of glutamate transporters (Zhang et al., 2017). In the present study, Nedd4-2 knockdown significantly increased membrane expression of PepT2 and inhibited the uptake of b-Ala-Lys-AMCA in BMEC, indicating that Nedd4-2 also participates the regulation of peptide uptake by modulating the membrane expression of PepT2 in BMEC.

Conclusions
In summary, mTOR signaling regulates peptide uptake by modulating the membrane expression of PepT2 and Nedd4-2 mediates the regulation of PepT2 by mTORC1, but not mTORC2, in BMEC. Our study provides new insight into molecular mechanisms of PepT2 regulation and milk protein synthesis by mTOR in BMEC.

Author contributions
Caihong Wang: Conceptualized the research, Conducted the experiments, Analyzed the data and wrote the paper. Fengqi Zhao: Revised the paper. Jianxin Liu: Revised the paper. Hongyun Liu: Revised the paper and had primary responsibility for the final content. All authors read and approved final manuscript.

Declaration of competing interest
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.