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Identification and functional analysis of candidate gene VPS28 for milk fat in bovine mammary epithelial cells
Biochemical and Biophysical Research Communications 510 (2019) 606e613
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Identification and functional analysis of candidate gene VPS28 for milk fat in bovine mammary epithelial cells Lily Liu a, b, Qin Zhang b, * a
College of Life Sciences, Southwest Forestry University, Kunming, Yunnan, 650224, China Key Laboratory of Animal Genetics and Breeding of Ministry of Agriculture, National Engineering Laboratory of Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 December 2018 Accepted 4 January 2019 Available online 8 February 2019
In a previous genome-wide association study on milk production traits in Chinese Holstein population, we revealed VPS28 gene was highly expressed in mammary gland tissue and a 58C > T mutation in 5’UTR of it was significantly associated with milk fat content traits. In this study, we explored the effect of this 58C > T mutation on VPS28, and found it could significantly decrease promoter activity of VPS28 by reducing transcription factor binding sites. To identify the potential functional SNP involved, we performed RNAi experiment in BMECs, the results showed that VPS28 knockdown could increase the expression of ADFP and CD36, lead accumulation of ubiquitinated proteins, long chain fatty acids and triglyceride, and decrease the proteasome activity. Therefore, our study demonstrates that the 58C > T mutation could facilitate milk fat synthesis in two ways. The one is involved in ESCRTs signaling, it could directly lead an accumulation of ubqiuitinated membrane proteins to promote the long chain fatty acids uptake to incorporation into TG. The other is involved in ubiquitination-proteasome system, it could indirectly lead a dysfunction of proteasome to accumulate the ubqiuitinated proteins to promote TG synthesis. In conclusion, our study demonstrates that VPS28 could be a strong candidate gene for milk fat content traits, and in particular, the 58C > T mutation in 5’-UTR of VPS28 could be a functional mutation for its effects on milk fat content. © 2019 Published by Elsevier Inc.
Keywords: Dairy cattle Milk production traits VPS28 gene Ubiquitination Molecular mechanism of the regulation
1. Introduction Milk fat is one of the most economically important traits in dairy cattle, and identification and functional analysis of milk fat candidate genes are important for comprehending the real genetic mechanisms. In previous genome-wide association study (GWAS) and the subsequent analysis of the novel variants revealed by targeted sequencing of GWAS loci ([1,2]), we found VPS28 gene was highly expressed in mammary gland tissue and a 58C > T mutation in 5’-UTR of it was significantly associated with milk fat content traits. In the mammals, VPS28 as one of the class E VPS (Vacuolar protein sorting 28 homolog) proteins is a major component for ESCRTs (Endosomal sorting complexes required for transport) which contain four multi-protein complexes (ESCRT-0, ESCRT-Ⅰ,
* Corresponding author. Professor College of Animal Science and Technology, China Agricultural University, 2 Yuanmingyuan West Road, Beijing, 100193, China. E-mail address: [email protected] (Q. Zhang). https://doi.org/10.1016/j.bbrc.2019.01.016 0006-291X/© 2019 Published by Elsevier Inc.
ESCRT-Ⅱ, ESCRT-Ⅲ) ([3e5]). The ESCRTs have a clear role in lysosomal degradation of ubiquitinated membrane proteins and proetaesome degradation of ubiquitinated cytoplasmic proteins ([6,7]). Later works established that genetic mutants of ESCRTs could lead an accumulation of uiquitinated proteins and result in some aberrant phenotype ([8,9]). As noted above, we posed a hypothesis that the regulation of VPS28 gene on milk fat synthesis was mediated by ubiquitiation. In this aim, we firstly tested the effect of the 58C > T mutation on promoter activity of VPS28 via a dual-luciferase reporter system and the effect on transcription factor binding via gel retardation assays. We subsequently investigated the relationship among VPS28, ubiquitination and milk fat synthesis by knocking down VPS28 in BMECs (bovine primary mammary epithelial cells). Our results suggest that ubiquitination may be an important new area of study for milk fat synthesis regulation in bovine, and provide a valuable information for elucidating the genetic basis of milk fat traits.
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2. Materials & methods 2.1. Animals All of the cows in our study were fed under consistent environmental conditions. The entire procedure for the collection of the mammary tissues and blood samples from cows was performed in strict accordance with the protocol approved by the Animal Welfare Committee of China Agricultural University (Permit Number is DK996). 2.2. Cells culture Cell experiments were performed using HEK293T cells and primary BMECs. BMECs were separated from the mammary gland tissues of a healthy Holstein dairy cow in middle lactation, following the procedure previously described by Zhao et al. ([10]). Chemicals were purchased from Life Technologies (Carlsbad, CA, USA) unless noted otherwise. All cells were seeded on plastic cell culture plates at 37 C in 5% CO2. HEK 293T cells were cultured in DMEM with 10 kU/mL penicillin, 10 mg/mL streptomycin, and 10% fetal bovine serum (FBS). BMECs were grown in DMEM/F12 with 10 kU/mL penicillin, 10 mg/mL streptomycin, and 10% FBSsupplemented with 1% ITS -G (1 mg/mL Insulin, 0.55 mg/mL Transferrin, 0.67 mg/L Selenium Solution) to promote lactogenesis. When cells reached 80% confluence, cells were cultured in basal media and then treatments were applied. 2.3. Construction of plasmid, transfection and dual luciferase reporter assays According to promoter sequence data for the bovine VPS28 gene, a pair of specific primers containing Bgl Ⅱ and Kpn Ⅰrestriction enzyme cutting sites was designed for amplification of the bovine VPS28 gene promoter sequence using Oligo 6.0 software (forward 50 -CGGAggtaccTGCACCAGGATAAGCCCAGA-30 and reverse 50 -TTTTagatctGAGTGGCTGGGATCCCGTGA-30 ), designated VPS28-Wild and VPS28-Mutation. PCR was performed under the following conditions: after denaturation at 95 C for 5 min, DNA amplification was performed for 32 cycles at 95 C for 30 s, 65 C for 30 s, and 72 C for 1 min, with a final extension at 72 C for 7 min. The obtained PCR product was purified using the Omega E.Z.N.A. cycle Pure Kit (Omega, USA).Next, the PGL4.14 vector was transformed into E. coli DH5a competent cells for amplification and then isolated using an AxyPrep Plasmid Miniprep Kit (Axygen, USA). Wild-type VPS28 or its mutant-type fragment were synthesized and cloned into pGL4.14 vector (Promega, Mannheim, Germany) with Bgl Ⅱ and Kpn Ⅰ, and were termed VPS28-Wt and VPS28-Mut, respectively. HEK 293T cells were seeded in 24-well plates, and were transfected with VPS28-Wt or VPS28-Mut reporter plasmin, and pRL-TK reporter plasmin (Promega, Mannheim, Germany) using Lipofectamine™ LTX & PLUS Transfection Reagent. According to the Promega protocol, cells were harvested after 24 h, and then firefly and renilla luciferase activities were assayed using the Dual Luciferase Assay system (Promega, Mannheim, Germany) with the Infinite M200 Reader (Tecan, Switzerland). Renilla luciferase activity was normalized to firefly luciferase activity.
Nuclear
extracts
were
prepared
following
manufacturer's instructions (Pierce, Rockford, United States). The BCA Protein Assay Kit was used to measure the protein concentration in the extract (Pierce, Rockford, United States), which was then stored at 80 C. Commercially synthesized oligo-nucleotide probes corresponding to VPS28 promoter were used to detect the interaction of nuclear proteins and VPS28 promotor. A 24 bp probe covering the VPS28 promoter sequences between nt-69 to 46 was used for EMSA. The sequences of VPS28 wild-type probe (VPS28-Wt, 50 -gcctgtcactcCtccagtggctgc-30 ) and the sequences of mutant-type probe (VPS28-Mut, 50 -gcctgtcactcTtccagtggctgc-30 ) are giving in the parentheses. EMSA was performed using EMSA kit following the manufacturer's instructions (Thermo Biotech, United States). 2.5. BMECs transfection and VPS28 knockdown analysis We used the bovine VPS28 (NM-001035504) sequence for designing effective siRNAs to against bovine VPS28. The three artificial double-stranded siRNAs (GenePharma Corporation, Shanghai, China) (shown in Fig. S1) were transfected BMECs with the X-treme GENE siRNA Transfection Reagent (Roche, Penzberg, Germany) at a molar ratio of 1:10. After 72 h, transfection efficiency was normalized against FAM expression labeled in the ends of siRNA, and mRNA expression of VPS28 was determined by qRT-PCR and western blot. Total RNA was isolated from BMECs and converted into cDNA. Transcript copies were calculated relative to those of the housekeeping gene GAPDH following amplification of gene-specific sequences in triplicate by qRT-PCR. Results are presented as mean ± SD. Total cell lysates were prepared in the presence of protein inhibitors 72 h after tranfection. Bovine VPS28 were detected on western blots with goat anti-bovine VPS28 primary antibodies, in combination with anti-b-actin as secondary antibody (Santa Cruz, Texas, United States). 2.6. Western blots analysis BMEC cells were lysed for 30 min at 4 C in a cold lysis buffer, then broken by ultrasonic wave and centrifuged at 12,000 g for 20 min. The supernatants were collected and the protein was quantified using a BCA protein assay kit (Pierce, Rockford, USA). CD36 (FAT/CD36, Abcam, Cambridge, United States), ADFP (Adipose differentiation related protein, Abcam, Cambridge, United States) and UB (Ubiquitin, Santa Cruz, Texas, United States) levels were detected by western blot with cow anti-bovine, and anti-b-actin as secondary antibody (Santa Cruz, Texas, United States). 2.7. Cellular TG content and fatty acids composition quantification For quantifying the cellular TG content, BMEC cells were broken by the ultrasonic wave with 0.2e0.3 ml cold saline, and then the homogenate was analyzed by the TG assay Kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions. The fluorescence intensity was measured at 546 nm with Infinite M200 Reader (Tecan, Switzerland). And the BMECs fatty acids composition was quantified by high performance liquid chromatography (HPLC). 2.8. Proteasome inhibitor treatment and proteasome activities analysis
2.4. Preparation of cell lysates and electrophoretic mobility shift assays (EMSA) BMECs
607
the
To appraise the regulation mechanism of the VPS28 on the milk fat synthesis is mediated by the UPS, we cultured the cells in
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present of 10 mM proteasome inhibitor Epoxomicin (Epo) (Enzo Life Sciences, Alexis Biochemicals, Lausen, Switzerland) for 24 h, Epo was dissolved in dimethyl sulfoxide (DMSO). In a separate experiment, cells were treated with 0.1% DMSO that served as control. From the normalized protein level, the three proteasome activities were measured using the Proteasome-Glo™ ChymotrypsinLike, Caspase-Like and Trypsin-Like Cell-Based Assays (Promega, Mannheim, Germany) according to the manufacturer's instructions. Fluorogenic substrates of the chymotrypsin-like, caspase-like, and trypsin-like active sites were replaced with the luminogenic substrates Suc-LLVY-aminoluciferin (aLuc), Z-nLPnLD-aLuc, and Z-LRRaLuc, respectively. The fluorescence intensity was measured with Infinite M200 Reader (Tecan, Switzerland). 2.9. Microscopy BMECs was collected and fixed simultaneously with 2.5% glutaraldehyde that was postfixed in 1% osmium tetroxide, than washed by sodium cacodylate buffer, dehydrated with gradient alcohol and embedded in Epon 812. Semithin sections (1 mm) were cut, stained by methylene blue, and localized by a microscope. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined under JEM-1400 electron microscope (JEOL, Japan). 2.10. Statistical analysis Each treatment was performed in triplicate. All data were compared via analysis of variance and multiple testing using the R-package (R v3.02). Differences were declared significant at P < 0.05 and P < 0.01. For the purposes of proteins discussed in the manuscript proteins had to be found in six groups, and have 1.2 or 0.83 fold changes in addition to P < 0.05 and P < 0.01.
binding sites, and suggested that it may be a functional mutation to decrease VPS28 mRNA expression.
3.2. VPS28 knockdown in BMECs To look for whether the VPS28 contributed directly to milk fat synthesis, as the first step, we attempted to knock down VPS28 with siRNA in BMECs. For the same transcript, cumulative action of multiple siRNAs targeting different regions could produce a higher knockdown than the single siRNA on their own ([11]). Therefore, we transfected four tandem siRNA constructs (siRNA12, siRNA13, siRNA23, siRNA123) into BMECs and compared their interference efficient with individual siRNA. As shown in Fig. 1D, all of tandem constructs showed a higher VPS28 knockdown activity in the range of 43%e75%, we selected the most efficient tandem constructs, siRNA 23 (75%), for the subsequent evaluation. As expected, VPS28 protein level was down-regulated by 70% relative to the control in BMECs (Fig. 1E).
3.3. VPS28 knockdown increases the level of ubiquitinated proteins and decreased the proteasome activity in BMECs VPS28 as a subunit of ESCRTs play a crucial role in ubiquitinmediated degradation of proteins ([6,7]). We examined the ubiquitinated proteins level to determine the regulation of VPS28. As shown in Fig. 2A, a significantly increase in ubiquitinated proteins level was observed in BMECs, supporting that VPS28 knockdown was involved in accumulation of ubiquitinated proteins in BMECs. Alteration of ubiquitinated proteins indicates a mechanism that VPS28 could regulate proteasome activity in BMECs. We examined it and found (as shown in Fig. 2B) the three activities of proteasome (Chymotreypsin-like activity, Caspaselike activity, Trypsin-like activity) were decreased markedly in BMECs.
3. Results 3.4. VPS28 knockdown increases milk fat synthesis in BMECs 3.1. 58C > T mutation reduces the mRNA expression of VPS28 through decreasing transcription factor binding sites To detect the effect of 58C > T mutation in 5’-UTR of VPS28 on the promoter activity, we constructed two recombinant vectors carrying the VPS28 promoter corresponding to this SNP, and then measured the promoter activity of wide type (CC-wide) and mutant type (TT-mutant) VPS28 segments using a dual-luciferase reporter system. As shown in Fig. 1A, the recombinant promoter plasmids exhibited a stronger luciferase response than the negative control (pGL4.14 vector), and the wild type displays a stronger luciferase response than the mutant type (P < 0.05). These results indicate the 58C > T mutation could significantly reduce the promoter activity of VPS28. To confirm the causality of the 58C > T mutation for decreasing promoter activity of VPS28, we predicted the transcription factor binding sites of this SNP using TFSEARCH (http://molsun1.cbrc.aist. go.jp/research/db/TFSEARCH.html), and found this -58C > T mutation could influence the binding sites for transcription factors (as shown in Table 1). And then we performed EMSA to identify this prediction. As shown in Fig. 1B, the nuclear extracts from BMECs were incubated with biotin labeled wild-type or mutant-type probe, and showed a higher affinity for the probe with the wildtype allele (C) than the probe with the mutant-type allele (T). These results indicated that the 58C > T mutation could decrease the promoter activity of VPS28 by reducing the transcript factors
Ubiquitin-mediated degradation of membrane proteins is crucial for attenuation of receptor-mediated signaling pathways. CD36 (Cluster of differentiation 36) as a membrane scavenger receptor was identified as a fatty acid receptor, and ubiquitinated CD36 could facilitate the long chain fatty acids uptake ([12e14]). We examined ubiquitinated CD36 level in BMECs. The western blot analysis (shown in Fig. 3A) showed that VPS28 knockdown leaded to a 1.8-fold increased of ubiquitinated CD36 in BMECs. These results suggested triglyceride (TG) level could be altered by VPS28 knockdown through increasing ubiquitinated CD36 level. And then, the BMECs lysates were quantified for all long chain fatty acids by HPLC. As shown in Fig. 3B, most of long chain fatty acids levels were increased significantly following an increase in ubiquitinated CD36 in BMECs. ADFP (Adipose differentiation-related protein) is a specific marker for lipid droplet, the expression level of it is accordance with abundance of lipid droplets in cell ([15,16]). We next examined the ADFP expression in BMECs, as shown in Fig. 3C, the ADFP level had a 2.6-fold increase, and in parallel, the TG content was increased by 3.6-fold above untreated BMECs (Fig. 3D). And then we examined the general architecture of VPS28 knockdown BMECs at the resolution of the light microscope. Compared with the control (Fig. 3E), VPS28 knockdown BMECs contained strikingly large lipid droplets and many luminal spaces were completely filled with aggregated lipid (Fig. 3F).
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Fig. 1. Effects of -58C > T mutation on the promoter activity of VPS28, and siRNA-mediated VPS28 knockdown in BMECs. A: Relative promoter activities of VPS28, CC-wild is the wild-type promoter vector, TT-mutant is the mutant-type promoter vector. Promoter activities were detected using a dual-luciferase reporter system. B: Characterization of the protein binding site for the SNP site, C/T (chr14:1693701), revealed in EMSA experiments. Line 1, 2 ¼ blank; Line 3 ¼ BMECs nuclear extract þ biotin-labeled Probe-wild type; Line 4 ¼ BMECs nuclear extract þ biotin-labeled Probe-mutant type; Line 5 ¼ BMECs nuclear extract þ100 times of cold Probe-wide type; Line 6: BMECs nuclear extract þ100 times of cold Probe-mutant type. C: BMECs were transfected with tandem siRNA23 constructs, the efficiency were normalized to FAM expression. D: The VPS28 mRNA expression relative to the negative control sample, which was defined as 100% VPS28 expression. E: The VPS28 protein expression level in BMECs that were transfected with siRNA23. Data are expressed as means ± SEM. Error bars denote SEM.* indicates the difference is significant (P < 0.05).
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L. Liu, Q. Zhang / Biochemical and Biophysical Research Communications 510 (2019) 606e613 Table 1 Prediction of the transcription factor binding sites (TFBSs) of 58C > T mutation by TFSEARCH. SNPs
Mutation
TFBSs
Value
-58 C > T
C/T
ADR1/NRF2
89.5
Fig. 2. Effects of VPS28 knockdown on ubiquitinated proteins level and proteasome activity in BMECs. A: Effect of VPS28 knockdown on ubiquitinated proteins level. B: Effect of VPS28 knockdown on three proteasome activities, chymotreypsin-like, caspase-like, and trypsin-like in BMECs. 23 and NTC: BMECs were transfected with SiRNA23 or not. UB: ubiquitinated proteins. Data are averages of three replicates. The error or bars denote SEM. * indicates the difference is significant (P < 0.05).
3.5. The proteasome inhibitor epoxomicin reduces proteasome activity and enhances accumulation of ubiquitinated proteins and TG in BMECs Epoxomicin is a special proteasome inhibitor, and we found that inhibition of proteosomal activity with it could result in significant decrease in PSMB5 expression (Proteasome Subunit Beta 5) (Fig. 4A) and chymotreypsin-like activity (Fig. 4B). And in parallel, ubiquitinated proteins (Fig. 4C) and TG (Fig. 4D) were enhanced by epoxomicin inhibitor.
4. Discussion In our previous study, VPS28 gene was found to be highly expressed in dairy mammary gland tissues, and there was a 58C > T mutation in its 5’-UTR region which had a significant association with milk fat content traits ([1,2]). However, additional studies are needed to further discern the molecular machinery of this potential functional SNP in regulating milk fat synthesis. Our current study is an effort in this direction. To uncover the regulation of the 58C > T mutation of VPS28 on milk fat synthesis, we firstly used promoter activity analysis and EMSA to confirm this mutation could decrease VPS28 expression through reducing binding sites of transcription factors. Thus, we confirmed that the effect of the 58C > T mutation on the milk fat synthesis could be caused by the decreased mRNA expression of VPS28. Then, we performed RNAi in BMECs to identify the effect of this 58C > T mutation on milk fat synthesis. Our findings found that VPS28 knockdown could increase the expression of ubiquitinated CD36 and ADFP, lead accumulation of the long chain fatty acids, TG and ubiquitinated proteins in BMECs. CD36 could facilitate the long chain fatty acid uptake and utilization ([17,18]), and a high CD36 level is proposed to contribute to lipid accumulation ([9,19]). Jill et al. has shown that CD36 level could be
regulated by ubiquitination ([20]). It is consistent with our observation that VPS28 knockdown could increase the long chain fatty acids uptake through an accumulation of ubiquitinated CD36. ADFP is a specific marker for lipid droplet and its expression level is accordance with abundance of lipid droplets in cell ([15,16]). Our study had similarly found connections between ADFP and TG content which were both significantly increased by VPS28 knockdown in BMECs. Thus, our finding indicated that VPS28 knockdown could promote milk fat synthesis through increasing the long chain fatty acids uptake in BMEC, and it may be mediated by ubiquitination. This study may be the first demonstration of ubiquitiation regulating milk fat synthesis in bovine. To further verify our hypothesis, we detected ubiquitination level and proteasome activity in VPS28 knockdown BMECs. We demonstrated that VPS28 deficiency could increase ubiquitination level, and lead proteasome dysfunction in BMECs, and these were similarly to the results following proteasome inhibitor treatment. Our data were consistent with other studies that VPS28 could regulate degradation of ubiquitinated membrane proteins ([5,7,21e24]), and plays an important role in degradation of ubiquitinated cytoplasmic proteins with proeteasome ([6,7]). As a result, our data suggest that VPS28 could regulate milk fat synthesis via two pathways. The one is involved in ESCRTs signaling, it could directly lead an accumulation of ubqiuitinated membrane proteins to promote the long chain fatty acids uptake to incorporation into TG. The other is involved in ubiquitination-proteasome system, it could indirectly lead a proteasome dysfunction to accumulate the ubqiuitinated proteins to promote TG synthesis. In conclusion, we demonstrated that the 58C > T mutation in 5’-UTR of VPS28 could reduce the promoter activity to decrease VPS28 expression, and then regulate milk fat synthesis through ubiquitination. The mutant T allele of this SNP associated with the higher milk fat content could be explained by ESCRTs signaling and ubiquitination proteasom system. Therefore, VPS28 could be a
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Fig. 3. Effects of VPS28 knockdown in BMECs. A: Effect of VPS28 knockdown on ubiquitinated CD36 expression. B: Effect of VPS28 knockdown on long chain fatty acids levels. C: Effect of VPS28 knockdown on ADFP expression. D: Effect of VPS28 knockdown on TG level. E: Electron micrographs of BMECs, a: Electron micrographs of BMECs, b: Electron micrographs of VPS28 knockdown BMECs. siRNA23 þ/: BMECs were transfected with siRNA23 or not. Data are averages of three replicates. The error or bars denote SEM. * indicates the difference is significant (P < 0.05).
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Fig. 4. Effects of epoxomicin inhibitor on PSMB5 expression, chymotreypsin-like activity, and accumulation of ubiquitinated proteins and TG in BMECs. A: Effect of epoxomicin inhibitor on PSMB5 expression. B: Effect of epoxomicin inhibitor on chymotreypsin-like relative activity. C: Effect of epoxomicin inhibitor on ubiquitinated proteins level. D: Effect of epoxomicin inhibitor on TG level. Epo: epoxomicin. UB: ubiquitinated proteins. Data are averages of three replicates. The error or bars denote SEM. * indicates the difference is significant (P < 0.05).
functional gene for milk fat content, and in particular, the 58C > T mutation in 5’-UTR of VPS28 could be a functional mutation for its effects on milk fat content. Our study also provides evidence of a novel relationship between milk fat synthesis and ubiquitination, suggesting that this axis may be an important new area of study for milk fat synthesis regulation in bovine.
no roles in the study design, data collection and analysis, decision to publish or preparation of the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.01.016.
Acknowledgments Transparency document This work was financially supported by the National Major Development Program of Transgenic Breeding (2014ZX0800953B), the National Natural Science Foundations of China (31201772), the National Science and Technology Programs of China (2013AA102504), the Beijing Dairy Industry Innovation Team and the Program for Changjiang Scholar and Innovation Research Team in University (IRT1191), the thesis of "Scientific and Technological Innovation Team Construction Project for Protection and Utilization of Under-forest Biological Resources (51400666)". The funders had
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Identification and functional analysis of candidate gene VPS28 for milk fat in bovine mammary epithelial cells Lily Liu a, b, Qin Zhang b, * a
College of Life Sciences, Southwest Forestry University, Kunming, Yunnan, 650224, China Key Laboratory of Animal Genetics and Breeding of Ministry of Agriculture, National Engineering Laboratory of Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 December 2018 Accepted 4 January 2019 Available online 8 February 2019
In a previous genome-wide association study on milk production traits in Chinese Holstein population, we revealed VPS28 gene was highly expressed in mammary gland tissue and a 58C > T mutation in 5’UTR of it was significantly associated with milk fat content traits. In this study, we explored the effect of this 58C > T mutation on VPS28, and found it could significantly decrease promoter activity of VPS28 by reducing transcription factor binding sites. To identify the potential functional SNP involved, we performed RNAi experiment in BMECs, the results showed that VPS28 knockdown could increase the expression of ADFP and CD36, lead accumulation of ubiquitinated proteins, long chain fatty acids and triglyceride, and decrease the proteasome activity. Therefore, our study demonstrates that the 58C > T mutation could facilitate milk fat synthesis in two ways. The one is involved in ESCRTs signaling, it could directly lead an accumulation of ubqiuitinated membrane proteins to promote the long chain fatty acids uptake to incorporation into TG. The other is involved in ubiquitination-proteasome system, it could indirectly lead a dysfunction of proteasome to accumulate the ubqiuitinated proteins to promote TG synthesis. In conclusion, our study demonstrates that VPS28 could be a strong candidate gene for milk fat content traits, and in particular, the 58C > T mutation in 5’-UTR of VPS28 could be a functional mutation for its effects on milk fat content. © 2019 Published by Elsevier Inc.
Keywords: Dairy cattle Milk production traits VPS28 gene Ubiquitination Molecular mechanism of the regulation
1. Introduction Milk fat is one of the most economically important traits in dairy cattle, and identification and functional analysis of milk fat candidate genes are important for comprehending the real genetic mechanisms. In previous genome-wide association study (GWAS) and the subsequent analysis of the novel variants revealed by targeted sequencing of GWAS loci ([1,2]), we found VPS28 gene was highly expressed in mammary gland tissue and a 58C > T mutation in 5’-UTR of it was significantly associated with milk fat content traits. In the mammals, VPS28 as one of the class E VPS (Vacuolar protein sorting 28 homolog) proteins is a major component for ESCRTs (Endosomal sorting complexes required for transport) which contain four multi-protein complexes (ESCRT-0, ESCRT-Ⅰ,
* Corresponding author. Professor College of Animal Science and Technology, China Agricultural University, 2 Yuanmingyuan West Road, Beijing, 100193, China. E-mail address: [email protected] (Q. Zhang). https://doi.org/10.1016/j.bbrc.2019.01.016 0006-291X/© 2019 Published by Elsevier Inc.
ESCRT-Ⅱ, ESCRT-Ⅲ) ([3e5]). The ESCRTs have a clear role in lysosomal degradation of ubiquitinated membrane proteins and proetaesome degradation of ubiquitinated cytoplasmic proteins ([6,7]). Later works established that genetic mutants of ESCRTs could lead an accumulation of uiquitinated proteins and result in some aberrant phenotype ([8,9]). As noted above, we posed a hypothesis that the regulation of VPS28 gene on milk fat synthesis was mediated by ubiquitiation. In this aim, we firstly tested the effect of the 58C > T mutation on promoter activity of VPS28 via a dual-luciferase reporter system and the effect on transcription factor binding via gel retardation assays. We subsequently investigated the relationship among VPS28, ubiquitination and milk fat synthesis by knocking down VPS28 in BMECs (bovine primary mammary epithelial cells). Our results suggest that ubiquitination may be an important new area of study for milk fat synthesis regulation in bovine, and provide a valuable information for elucidating the genetic basis of milk fat traits.
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2. Materials & methods 2.1. Animals All of the cows in our study were fed under consistent environmental conditions. The entire procedure for the collection of the mammary tissues and blood samples from cows was performed in strict accordance with the protocol approved by the Animal Welfare Committee of China Agricultural University (Permit Number is DK996). 2.2. Cells culture Cell experiments were performed using HEK293T cells and primary BMECs. BMECs were separated from the mammary gland tissues of a healthy Holstein dairy cow in middle lactation, following the procedure previously described by Zhao et al. ([10]). Chemicals were purchased from Life Technologies (Carlsbad, CA, USA) unless noted otherwise. All cells were seeded on plastic cell culture plates at 37 C in 5% CO2. HEK 293T cells were cultured in DMEM with 10 kU/mL penicillin, 10 mg/mL streptomycin, and 10% fetal bovine serum (FBS). BMECs were grown in DMEM/F12 with 10 kU/mL penicillin, 10 mg/mL streptomycin, and 10% FBSsupplemented with 1% ITS -G (1 mg/mL Insulin, 0.55 mg/mL Transferrin, 0.67 mg/L Selenium Solution) to promote lactogenesis. When cells reached 80% confluence, cells were cultured in basal media and then treatments were applied. 2.3. Construction of plasmid, transfection and dual luciferase reporter assays According to promoter sequence data for the bovine VPS28 gene, a pair of specific primers containing Bgl Ⅱ and Kpn Ⅰrestriction enzyme cutting sites was designed for amplification of the bovine VPS28 gene promoter sequence using Oligo 6.0 software (forward 50 -CGGAggtaccTGCACCAGGATAAGCCCAGA-30 and reverse 50 -TTTTagatctGAGTGGCTGGGATCCCGTGA-30 ), designated VPS28-Wild and VPS28-Mutation. PCR was performed under the following conditions: after denaturation at 95 C for 5 min, DNA amplification was performed for 32 cycles at 95 C for 30 s, 65 C for 30 s, and 72 C for 1 min, with a final extension at 72 C for 7 min. The obtained PCR product was purified using the Omega E.Z.N.A. cycle Pure Kit (Omega, USA).Next, the PGL4.14 vector was transformed into E. coli DH5a competent cells for amplification and then isolated using an AxyPrep Plasmid Miniprep Kit (Axygen, USA). Wild-type VPS28 or its mutant-type fragment were synthesized and cloned into pGL4.14 vector (Promega, Mannheim, Germany) with Bgl Ⅱ and Kpn Ⅰ, and were termed VPS28-Wt and VPS28-Mut, respectively. HEK 293T cells were seeded in 24-well plates, and were transfected with VPS28-Wt or VPS28-Mut reporter plasmin, and pRL-TK reporter plasmin (Promega, Mannheim, Germany) using Lipofectamine™ LTX & PLUS Transfection Reagent. According to the Promega protocol, cells were harvested after 24 h, and then firefly and renilla luciferase activities were assayed using the Dual Luciferase Assay system (Promega, Mannheim, Germany) with the Infinite M200 Reader (Tecan, Switzerland). Renilla luciferase activity was normalized to firefly luciferase activity.
Nuclear
extracts
were
prepared
following
manufacturer's instructions (Pierce, Rockford, United States). The BCA Protein Assay Kit was used to measure the protein concentration in the extract (Pierce, Rockford, United States), which was then stored at 80 C. Commercially synthesized oligo-nucleotide probes corresponding to VPS28 promoter were used to detect the interaction of nuclear proteins and VPS28 promotor. A 24 bp probe covering the VPS28 promoter sequences between nt-69 to 46 was used for EMSA. The sequences of VPS28 wild-type probe (VPS28-Wt, 50 -gcctgtcactcCtccagtggctgc-30 ) and the sequences of mutant-type probe (VPS28-Mut, 50 -gcctgtcactcTtccagtggctgc-30 ) are giving in the parentheses. EMSA was performed using EMSA kit following the manufacturer's instructions (Thermo Biotech, United States). 2.5. BMECs transfection and VPS28 knockdown analysis We used the bovine VPS28 (NM-001035504) sequence for designing effective siRNAs to against bovine VPS28. The three artificial double-stranded siRNAs (GenePharma Corporation, Shanghai, China) (shown in Fig. S1) were transfected BMECs with the X-treme GENE siRNA Transfection Reagent (Roche, Penzberg, Germany) at a molar ratio of 1:10. After 72 h, transfection efficiency was normalized against FAM expression labeled in the ends of siRNA, and mRNA expression of VPS28 was determined by qRT-PCR and western blot. Total RNA was isolated from BMECs and converted into cDNA. Transcript copies were calculated relative to those of the housekeeping gene GAPDH following amplification of gene-specific sequences in triplicate by qRT-PCR. Results are presented as mean ± SD. Total cell lysates were prepared in the presence of protein inhibitors 72 h after tranfection. Bovine VPS28 were detected on western blots with goat anti-bovine VPS28 primary antibodies, in combination with anti-b-actin as secondary antibody (Santa Cruz, Texas, United States). 2.6. Western blots analysis BMEC cells were lysed for 30 min at 4 C in a cold lysis buffer, then broken by ultrasonic wave and centrifuged at 12,000 g for 20 min. The supernatants were collected and the protein was quantified using a BCA protein assay kit (Pierce, Rockford, USA). CD36 (FAT/CD36, Abcam, Cambridge, United States), ADFP (Adipose differentiation related protein, Abcam, Cambridge, United States) and UB (Ubiquitin, Santa Cruz, Texas, United States) levels were detected by western blot with cow anti-bovine, and anti-b-actin as secondary antibody (Santa Cruz, Texas, United States). 2.7. Cellular TG content and fatty acids composition quantification For quantifying the cellular TG content, BMEC cells were broken by the ultrasonic wave with 0.2e0.3 ml cold saline, and then the homogenate was analyzed by the TG assay Kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions. The fluorescence intensity was measured at 546 nm with Infinite M200 Reader (Tecan, Switzerland). And the BMECs fatty acids composition was quantified by high performance liquid chromatography (HPLC). 2.8. Proteasome inhibitor treatment and proteasome activities analysis
2.4. Preparation of cell lysates and electrophoretic mobility shift assays (EMSA) BMECs
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the
To appraise the regulation mechanism of the VPS28 on the milk fat synthesis is mediated by the UPS, we cultured the cells in
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present of 10 mM proteasome inhibitor Epoxomicin (Epo) (Enzo Life Sciences, Alexis Biochemicals, Lausen, Switzerland) for 24 h, Epo was dissolved in dimethyl sulfoxide (DMSO). In a separate experiment, cells were treated with 0.1% DMSO that served as control. From the normalized protein level, the three proteasome activities were measured using the Proteasome-Glo™ ChymotrypsinLike, Caspase-Like and Trypsin-Like Cell-Based Assays (Promega, Mannheim, Germany) according to the manufacturer's instructions. Fluorogenic substrates of the chymotrypsin-like, caspase-like, and trypsin-like active sites were replaced with the luminogenic substrates Suc-LLVY-aminoluciferin (aLuc), Z-nLPnLD-aLuc, and Z-LRRaLuc, respectively. The fluorescence intensity was measured with Infinite M200 Reader (Tecan, Switzerland). 2.9. Microscopy BMECs was collected and fixed simultaneously with 2.5% glutaraldehyde that was postfixed in 1% osmium tetroxide, than washed by sodium cacodylate buffer, dehydrated with gradient alcohol and embedded in Epon 812. Semithin sections (1 mm) were cut, stained by methylene blue, and localized by a microscope. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined under JEM-1400 electron microscope (JEOL, Japan). 2.10. Statistical analysis Each treatment was performed in triplicate. All data were compared via analysis of variance and multiple testing using the R-package (R v3.02). Differences were declared significant at P < 0.05 and P < 0.01. For the purposes of proteins discussed in the manuscript proteins had to be found in six groups, and have 1.2 or 0.83 fold changes in addition to P < 0.05 and P < 0.01.
binding sites, and suggested that it may be a functional mutation to decrease VPS28 mRNA expression.
3.2. VPS28 knockdown in BMECs To look for whether the VPS28 contributed directly to milk fat synthesis, as the first step, we attempted to knock down VPS28 with siRNA in BMECs. For the same transcript, cumulative action of multiple siRNAs targeting different regions could produce a higher knockdown than the single siRNA on their own ([11]). Therefore, we transfected four tandem siRNA constructs (siRNA12, siRNA13, siRNA23, siRNA123) into BMECs and compared their interference efficient with individual siRNA. As shown in Fig. 1D, all of tandem constructs showed a higher VPS28 knockdown activity in the range of 43%e75%, we selected the most efficient tandem constructs, siRNA 23 (75%), for the subsequent evaluation. As expected, VPS28 protein level was down-regulated by 70% relative to the control in BMECs (Fig. 1E).
3.3. VPS28 knockdown increases the level of ubiquitinated proteins and decreased the proteasome activity in BMECs VPS28 as a subunit of ESCRTs play a crucial role in ubiquitinmediated degradation of proteins ([6,7]). We examined the ubiquitinated proteins level to determine the regulation of VPS28. As shown in Fig. 2A, a significantly increase in ubiquitinated proteins level was observed in BMECs, supporting that VPS28 knockdown was involved in accumulation of ubiquitinated proteins in BMECs. Alteration of ubiquitinated proteins indicates a mechanism that VPS28 could regulate proteasome activity in BMECs. We examined it and found (as shown in Fig. 2B) the three activities of proteasome (Chymotreypsin-like activity, Caspaselike activity, Trypsin-like activity) were decreased markedly in BMECs.
3. Results 3.4. VPS28 knockdown increases milk fat synthesis in BMECs 3.1. 58C > T mutation reduces the mRNA expression of VPS28 through decreasing transcription factor binding sites To detect the effect of 58C > T mutation in 5’-UTR of VPS28 on the promoter activity, we constructed two recombinant vectors carrying the VPS28 promoter corresponding to this SNP, and then measured the promoter activity of wide type (CC-wide) and mutant type (TT-mutant) VPS28 segments using a dual-luciferase reporter system. As shown in Fig. 1A, the recombinant promoter plasmids exhibited a stronger luciferase response than the negative control (pGL4.14 vector), and the wild type displays a stronger luciferase response than the mutant type (P < 0.05). These results indicate the 58C > T mutation could significantly reduce the promoter activity of VPS28. To confirm the causality of the 58C > T mutation for decreasing promoter activity of VPS28, we predicted the transcription factor binding sites of this SNP using TFSEARCH (http://molsun1.cbrc.aist. go.jp/research/db/TFSEARCH.html), and found this -58C > T mutation could influence the binding sites for transcription factors (as shown in Table 1). And then we performed EMSA to identify this prediction. As shown in Fig. 1B, the nuclear extracts from BMECs were incubated with biotin labeled wild-type or mutant-type probe, and showed a higher affinity for the probe with the wildtype allele (C) than the probe with the mutant-type allele (T). These results indicated that the 58C > T mutation could decrease the promoter activity of VPS28 by reducing the transcript factors
Ubiquitin-mediated degradation of membrane proteins is crucial for attenuation of receptor-mediated signaling pathways. CD36 (Cluster of differentiation 36) as a membrane scavenger receptor was identified as a fatty acid receptor, and ubiquitinated CD36 could facilitate the long chain fatty acids uptake ([12e14]). We examined ubiquitinated CD36 level in BMECs. The western blot analysis (shown in Fig. 3A) showed that VPS28 knockdown leaded to a 1.8-fold increased of ubiquitinated CD36 in BMECs. These results suggested triglyceride (TG) level could be altered by VPS28 knockdown through increasing ubiquitinated CD36 level. And then, the BMECs lysates were quantified for all long chain fatty acids by HPLC. As shown in Fig. 3B, most of long chain fatty acids levels were increased significantly following an increase in ubiquitinated CD36 in BMECs. ADFP (Adipose differentiation-related protein) is a specific marker for lipid droplet, the expression level of it is accordance with abundance of lipid droplets in cell ([15,16]). We next examined the ADFP expression in BMECs, as shown in Fig. 3C, the ADFP level had a 2.6-fold increase, and in parallel, the TG content was increased by 3.6-fold above untreated BMECs (Fig. 3D). And then we examined the general architecture of VPS28 knockdown BMECs at the resolution of the light microscope. Compared with the control (Fig. 3E), VPS28 knockdown BMECs contained strikingly large lipid droplets and many luminal spaces were completely filled with aggregated lipid (Fig. 3F).
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Fig. 1. Effects of -58C > T mutation on the promoter activity of VPS28, and siRNA-mediated VPS28 knockdown in BMECs. A: Relative promoter activities of VPS28, CC-wild is the wild-type promoter vector, TT-mutant is the mutant-type promoter vector. Promoter activities were detected using a dual-luciferase reporter system. B: Characterization of the protein binding site for the SNP site, C/T (chr14:1693701), revealed in EMSA experiments. Line 1, 2 ¼ blank; Line 3 ¼ BMECs nuclear extract þ biotin-labeled Probe-wild type; Line 4 ¼ BMECs nuclear extract þ biotin-labeled Probe-mutant type; Line 5 ¼ BMECs nuclear extract þ100 times of cold Probe-wide type; Line 6: BMECs nuclear extract þ100 times of cold Probe-mutant type. C: BMECs were transfected with tandem siRNA23 constructs, the efficiency were normalized to FAM expression. D: The VPS28 mRNA expression relative to the negative control sample, which was defined as 100% VPS28 expression. E: The VPS28 protein expression level in BMECs that were transfected with siRNA23. Data are expressed as means ± SEM. Error bars denote SEM.* indicates the difference is significant (P < 0.05).
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Mutation
TFBSs
Value
-58 C > T
C/T
ADR1/NRF2
89.5
Fig. 2. Effects of VPS28 knockdown on ubiquitinated proteins level and proteasome activity in BMECs. A: Effect of VPS28 knockdown on ubiquitinated proteins level. B: Effect of VPS28 knockdown on three proteasome activities, chymotreypsin-like, caspase-like, and trypsin-like in BMECs. 23 and NTC: BMECs were transfected with SiRNA23 or not. UB: ubiquitinated proteins. Data are averages of three replicates. The error or bars denote SEM. * indicates the difference is significant (P < 0.05).
3.5. The proteasome inhibitor epoxomicin reduces proteasome activity and enhances accumulation of ubiquitinated proteins and TG in BMECs Epoxomicin is a special proteasome inhibitor, and we found that inhibition of proteosomal activity with it could result in significant decrease in PSMB5 expression (Proteasome Subunit Beta 5) (Fig. 4A) and chymotreypsin-like activity (Fig. 4B). And in parallel, ubiquitinated proteins (Fig. 4C) and TG (Fig. 4D) were enhanced by epoxomicin inhibitor.
4. Discussion In our previous study, VPS28 gene was found to be highly expressed in dairy mammary gland tissues, and there was a 58C > T mutation in its 5’-UTR region which had a significant association with milk fat content traits ([1,2]). However, additional studies are needed to further discern the molecular machinery of this potential functional SNP in regulating milk fat synthesis. Our current study is an effort in this direction. To uncover the regulation of the 58C > T mutation of VPS28 on milk fat synthesis, we firstly used promoter activity analysis and EMSA to confirm this mutation could decrease VPS28 expression through reducing binding sites of transcription factors. Thus, we confirmed that the effect of the 58C > T mutation on the milk fat synthesis could be caused by the decreased mRNA expression of VPS28. Then, we performed RNAi in BMECs to identify the effect of this 58C > T mutation on milk fat synthesis. Our findings found that VPS28 knockdown could increase the expression of ubiquitinated CD36 and ADFP, lead accumulation of the long chain fatty acids, TG and ubiquitinated proteins in BMECs. CD36 could facilitate the long chain fatty acid uptake and utilization ([17,18]), and a high CD36 level is proposed to contribute to lipid accumulation ([9,19]). Jill et al. has shown that CD36 level could be
regulated by ubiquitination ([20]). It is consistent with our observation that VPS28 knockdown could increase the long chain fatty acids uptake through an accumulation of ubiquitinated CD36. ADFP is a specific marker for lipid droplet and its expression level is accordance with abundance of lipid droplets in cell ([15,16]). Our study had similarly found connections between ADFP and TG content which were both significantly increased by VPS28 knockdown in BMECs. Thus, our finding indicated that VPS28 knockdown could promote milk fat synthesis through increasing the long chain fatty acids uptake in BMEC, and it may be mediated by ubiquitination. This study may be the first demonstration of ubiquitiation regulating milk fat synthesis in bovine. To further verify our hypothesis, we detected ubiquitination level and proteasome activity in VPS28 knockdown BMECs. We demonstrated that VPS28 deficiency could increase ubiquitination level, and lead proteasome dysfunction in BMECs, and these were similarly to the results following proteasome inhibitor treatment. Our data were consistent with other studies that VPS28 could regulate degradation of ubiquitinated membrane proteins ([5,7,21e24]), and plays an important role in degradation of ubiquitinated cytoplasmic proteins with proeteasome ([6,7]). As a result, our data suggest that VPS28 could regulate milk fat synthesis via two pathways. The one is involved in ESCRTs signaling, it could directly lead an accumulation of ubqiuitinated membrane proteins to promote the long chain fatty acids uptake to incorporation into TG. The other is involved in ubiquitination-proteasome system, it could indirectly lead a proteasome dysfunction to accumulate the ubqiuitinated proteins to promote TG synthesis. In conclusion, we demonstrated that the 58C > T mutation in 5’-UTR of VPS28 could reduce the promoter activity to decrease VPS28 expression, and then regulate milk fat synthesis through ubiquitination. The mutant T allele of this SNP associated with the higher milk fat content could be explained by ESCRTs signaling and ubiquitination proteasom system. Therefore, VPS28 could be a
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Fig. 3. Effects of VPS28 knockdown in BMECs. A: Effect of VPS28 knockdown on ubiquitinated CD36 expression. B: Effect of VPS28 knockdown on long chain fatty acids levels. C: Effect of VPS28 knockdown on ADFP expression. D: Effect of VPS28 knockdown on TG level. E: Electron micrographs of BMECs, a: Electron micrographs of BMECs, b: Electron micrographs of VPS28 knockdown BMECs. siRNA23 þ/: BMECs were transfected with siRNA23 or not. Data are averages of three replicates. The error or bars denote SEM. * indicates the difference is significant (P < 0.05).
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Fig. 4. Effects of epoxomicin inhibitor on PSMB5 expression, chymotreypsin-like activity, and accumulation of ubiquitinated proteins and TG in BMECs. A: Effect of epoxomicin inhibitor on PSMB5 expression. B: Effect of epoxomicin inhibitor on chymotreypsin-like relative activity. C: Effect of epoxomicin inhibitor on ubiquitinated proteins level. D: Effect of epoxomicin inhibitor on TG level. Epo: epoxomicin. UB: ubiquitinated proteins. Data are averages of three replicates. The error or bars denote SEM. * indicates the difference is significant (P < 0.05).
functional gene for milk fat content, and in particular, the 58C > T mutation in 5’-UTR of VPS28 could be a functional mutation for its effects on milk fat content. Our study also provides evidence of a novel relationship between milk fat synthesis and ubiquitination, suggesting that this axis may be an important new area of study for milk fat synthesis regulation in bovine.
no roles in the study design, data collection and analysis, decision to publish or preparation of the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.01.016.
Acknowledgments Transparency document This work was financially supported by the National Major Development Program of Transgenic Breeding (2014ZX0800953B), the National Natural Science Foundations of China (31201772), the National Science and Technology Programs of China (2013AA102504), the Beijing Dairy Industry Innovation Team and the Program for Changjiang Scholar and Innovation Research Team in University (IRT1191), the thesis of "Scientific and Technological Innovation Team Construction Project for Protection and Utilization of Under-forest Biological Resources (51400666)". The funders had
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