Liu Rong, Li Qing-zhang, Huang Jian-guo, Lu Li-min, and Gao Xue-jun

Key Laboratory of Dairy Science of Education Ministry, Northeast Agricultural University, Harbin 150030, China

Introduction

Vaccaria segetalisis a well known Chinese herb.Vaccaria segetalisor its crude extracts as feed additives possess diverse pharmacological effects including anti-oxidant and increase mammary gland development and lactation of dairy cow, especially at mid- and late-lactation periods (Wanet al., 2011).It is well documented that many genes are involved in these activities in our previous studies. Effects ofVaccaria segetalison cell proliferation, lactation and anti-oxidant have also been extensively studied,showing that the promotion of cell proliferation and lactation involve several gene overexpressions (Tonget al., 2011). Whereas,Vaccaria segetalisis a multitarget agent, it is conceivable that more mechanisms for its anti-oxidant and promotion of cell proliferation activity might remain to be discovered.

Proteomics has emerged as a powerful technique for the quantitative and qualitative mapping of the whole nuclear extracts proteome under the treatment of abnormal conditions (Shenet al., 2009). In this study,we used a two-dimensional gel electrophoresis (2-DE)-based on proteomic approach to identify differentially expressed nuclear proteins in the crude extracts ofVaccaria segetalis-treated DCMECs in an attempt to further elucidate molecular mechanisms ofVaccaria segetalisin increasing mammary gland development and lactation of the dairy cow.

Materials and Methods

Chemicals and reagents

TheVaccaria segetaliswas purchased from Baofeng Drugstore, Harbin. All the reagents used in 2-DE were bought from GE Healthcare Corporation (Uppsala,Sweden). HaltTMProtease inhibitor cocktail EDTAFree was from Thermo Scientific (San Jose, CA,USA). DMEM/Ham's F-12 base and fetal dairy cow serum was from GIBCO BRL (Life Technologies,Burlington, ON, Canada).

Experimental animals

Dairy cow mammary gland tissues were obtained from lactating Holstein cows after parturition, which conditions were fine for lactation efficiency and physiological functions, procedures towards animals were approved by the Animal Care Committee of the University of Northeast Agricultural University.

Primary culture of DCMECs

The cells were cultured in DF-12 medium growth fluid added with 10% FBS and 5 μg · mL-1insulin, 100 U · mL-1penicillin, and 100 μg · mL-1streptomycin.Cytokeratin-18 was identified by immuno fluorescence(Chenet al., 2009).

Cell growth activity assay

Mammary epithelial cells (3×105cells/well) were incubated with 450 μL culture medium in 24-well plate and incubated 1 day at 37℃ in a CO2incubator.The cell was then changed to DF-12 containing 0.5%FBS and the cultures were treated with 5 mg · mL-1Vaccaria segetaliswater decoction. After incubation for different time (0, 6, 12, 24, 36, 48, 60, 72 h,respectively), cell growth curve were assessed using CASY cytoanalyzer System (Scharfe System GmbH, Reutlingen, Germany) at every time point.Numbers of the viable of cells were used for cell proliferation assay and construction of the cell growth curve and percentage of viable cells was used to analyze the cell activities. All the experiments were performed in triplicate.

Nuclear protein extracts

For nuclear proteins extraction, cells were seeded onto 100 mm diameter petridishes and maintained in DF-12 containing 10% FBS for 24 h. The medium was then changed to DF-12 containing 0.5% FBS for 12 h and the cultures were treated with 2 mg · mL-1Vaccaria segetaliswater decoction for 24 h. Nuclear extracts were prepared as the followings. Briefly, the medium was removed and the monolayer were rinsed twice with cold phosphate-buffered saline (PBS) and then scrapped in cold PBS. The cell suspensions were transferred to micro-centrifuge tubes and pelleted at 4℃ for 10min at 500×g. Cells were resuspended in hypotonic buffer (10 mmol · L-1HEPES pH7.9, 1.5 mmol · L-1MgCl2, 10 mmol · L-1KCl, 0.5 mmol · L-1DTT and 0.2 mmol · L-1PMSF) and centrifuged for 5 min at 500×g at 4℃. The supernatant was discarded and the cells were resuspended in hypotonic buffer and allowed to swell in ice for 30 min. Lysis progression was monitored by addition of Trypan Blue to an aliquot of the cell suspensions. The nuclei were then pelleted (3 000×g) for 15min at 4℃. The supernatant was discarded and the nuclei were resuspended in lowsalt buffer (20 mmol · L-1HEPES pH7.9, 25% glycerol,1.5 mmol · L-1MgCl2, 20 mmol · L-1KCl, 0.2 mmol · L-1EDTA, 0.5 mmol · L-1DTT and 0.2 mmol · L-1PMSF).Gentle dropwise addition of high-salt buffer (20 mmol · L-1HEPES pH7.9, 25% glycerol, 1.5 mmol · L-1MgCl2, 1.2 mol · L-1KCl, 0.2 mmol · L-1EDTA, 0.5 mmol · L-1DTT and 0.2 mmol · L-1PMSF) and incubation for 30 min in ice were used to release proteins from the nuclei. Cellular debris was removed by centrifugation (20 000×g) for 30 min at 4℃ and the supernatant (nuclear extract) was stored at -80℃.Protein concentrations were determined using the protein assay kit (GE Healthcare, USA). Protein concentration in these extracts was determined using the Bradford assay.

Two-dimensional gel electrophoresis (2-DE)and image analysis

2-DE was performed with IPGphor isoelectric focusing(IEF) and electrophoresis units (GE Healthcare, CA,USA). Protein sample was mixed with rehydration solution (8 mol · L-1urea, 4% CHAPS, 0.5% IPG buffer) to a final volume of 350 μL. Precast 18 cm immobilized pH gradient (IPG) strips were rehydrated for overnight. IEF was performed in a step-and-hold pattern of 200 V for 2 h, 500 V for 1.5 h and 1 000 V for 1 h. Then a gradient pattern was used to reach 1 000 V for 1 h and 8 000 V for 2 h, followed by a step-and-hold pattern of 8 000 V for 6.5 h and a holding pattern of 500 V. After the first dimensional run, the proteins were reduced (6 mol · L-1urea, 30%glycerol, 2% SDS and 50 mmol · L-1Tris-HCl, pH 6.8)and alkylated for 15 min in the same buffer containing 135 mmol · L-1iodoacetamide instead of DTT. Then,proteins were separated in the second dimension on homemade 10% SDS polyacrylamide gels using the Ettan DALTsix Electrophoresis Unit (GE Healthcare,CA, USA). Analytical gels were stained with silver nitrate and preparative gels were stained. Gels were scanned using a Powerlook 2 100 XL (Umax, USA)and analyzed with Image Master 2D analysis software(GE Healthcare, CA, USA). Data was obtained in triplicates, normalized and expressed as percentages of all the valid spots to account for differences in protein loading and staining. Only those spots that changed consistently and significantly (more than 1.5-fold)were selected for mass spectrometry analysis (Gorget al., 2004).

The In-Gel digestion and the MALDI-TOF MS/MS analysis

Protein spots were excised from gels and the in-gel digestion was performed as described previously(Bjorhallet al., 2005). The mixture was then immediately spotted onto a 384 well MALDI target plate and dried at room temperature. Tandem MALDI mass spectrometry was conducted using a 4800 MALDITOF/TOF (Applied Biosystems, Framingham, MA,USA) mass spectrometer. The resulting peak lists were submitted for database sequence searches using MASCOT v2.1.03 software (Matrix Science,UK). Database search were carried out using the following parameters: type of search, MS/MS ion search; enzyme, trypsin and allowance of one missed cleavage. Carbamido-methylation was selected as a fixed modification, and oxidation of methionine was allowed to be variable. The peptide and frag-ment mass tolerance were set at 100 mg · L-1and ± 0.5 ku,respectively. Proteins with probability based on MOWSE(molecular weight search) scores (P＜0.05) were considered to be positively identified. The acquired MS/MS spectra were compared against the BOVIN International Protein Index protein sequence database using the TurboSEQUEST program in the BioWorks 3.1 software suite (Thermo). Subcellular classifications were performed using gene ontology annotation(GOA; http://www.ebi.ac.uk/GOA/) according to the accession numbers of the proteins in Uniprot.

The RNA extraction and the RT-PCR analysis

Total RNA was extracted using TRIZOL reagent according to manufacturer's protocol. For cDNA synthesis, 2 μg of total RNA was used to generate cDNA in each sample using the RTase and random hexamers from ExScript TM reagent kit (TaKaRa,Dalian, China) according to manufacturer's instructions. After reverse transcription, the reaction was carried out on ABI PRISM 7300 RT-PCR System(Framingham, MA, USA). All the RT-PCR reactions were performed at 95℃ for 10 s, followed by 40 cycles at 95℃ for 5 s, 60℃ for 31 s using two-step RT-PCR. Product purity was confirmed by melting curve analysis. All the RT-PCR assays were performed in duplicate in three independent experiments.β-actin was amplified as an intrinsic control (Table 1). Data was expressed as fold induction over control as defined for each experiment.

Verification in dairy cow mammary glands of different lactation levels

Total RNA was isolated from dairy cow mammary glands of different lactation levels, cDNA synthesis,and the RT-PCR steps as previously.

Statistical analysis

Results were expressed as mean±SEM. Individual differences among groups were analyzed usingt-test by Sigma Plot 9.0 and differences of *P＜0.05 were considered significant, while **P＜0.01 were considered extremely significant.

Table 1Primers and sequences for the RT-PCR analysis

Results

Cell proliferation

The CASY-TT Analyzer System was used to count the alive cells and to draw a cellular growth curve.With the increasing of cell culture time, number of cells in treatment group were keeping increased,relatively compared to control, cell proliferation reached stabilization after 48 h. The cell activity of all time points got agreement. This revealed that the water decoction fromVaccaria segetaliscould increase cell proliferation (Fig.1).

Fig.1 Growth curve of DCMECs treated with water decoction from Vaccaria segetalis

The results showed that adding the water decoction fromVaccaria segetaliscaused an increase in cell numbers (statistical significance is shown as *P＜0.05 were considered significant, while **P＜0.01 were considered extremely significant), cell proliferation reached stabilization after 48 h. The differences of cell activities at all time points got the agreement.

Differential expressions of proteins

The 2DE-based proteomic analysis was performed to identify differentially expressed proteins in the DCMECs treated with 5 mg · mL-1Vaccariae segetaliswater decoction for 24 h. Three gels per sample were processed simultaneously and analyzed using Image Master 2-D Platinum Software 6.0. A total of 35 protein spots were found to be differentially expressed between treated groups and control groups.Five proteins were successfully identified by MALDITOF-MS (Fig.2A and B and Table 2). For the five up-regulated proteins, mRNA expression levels were assessed by quantitative RT-PCR. Results showed that in treated cells, the mRNA expression level of each tested gene was up-regulated (Fig.2C), which was comparable to the tendency of their protein expression changes.

Fig.2 Differentially expressed proteins

Verification in dairy cow mammary glands of different lactation levels

Five genes were differently expressed in these dairy cows of different lactation levels (Fig.3). The highest expression level was in tissues of high-yielding dairy cows, followed by low-yielding cows, and the lowest were prenatal cows. The results could be drawn from the above, and these five genes were associated with lactation.

Table 2Identification of differentially expressed protein spots in treated DCMECs

Fig.3 Expression of different genes in cows of different lactation levels

Discussion

In previous study we have verified antioxidant,proliferative and galactopoietic activity properties of the water decoction ofVaccaria segetalis, and in this experiment we further identified the differentially expressed proteins in DCMECs in response to this water decoction ofVaccaria segetalistreatment using a 2-DE based proteomic approach. Five up-regulated expressed proteins were successfully identified.These proteins involve in DNA transcription, mRNA splicing and translation, protein folding, degradation and modification, antioxidant and cell proliferation.Their roles in the metabolic regulation activity of the water decoction fromVaccaria segetalisare discussed below. Protein ETHE1, also called the HSCO, may function as a nuclear-cytoplasmic shuttling protein that binds transcription factor RELA/NFKB3 in the nucleus and exports it to the cytoplasm. It suppresses p53-induced apoptosis by preventing nuclear localization of the RELA and the HSCO and inhibits function of the NF-κB by binding to the RELA and accelerating its export from the nucleus and by increasing ubiquitylation and degradation of p53, the HSCO reduces p53 protein levels (Higashitsiuet al., 2007). The HSCO is a cofactor that increases the deacetylase activity of the HDAC1 toward p53, leading to suppression of apoptosis (Tirantiet al., 2006).

Parkinson disease 7, also known as the PARK7 or protein DJ-1, belongs to the peptidase C56 family of proteins. This is essential for cell-growth promoting activity and transforming activity. It acts as a positive regulator of androgen receptor-dependent transcription and may also function as a redoxsensitive chaperone, as a sensor for oxidative stress,and it apparently protects neurons against oxidative stress and cell death. The DJ-1 has been shown to interact with EFCAB6 and protein inhibitor of activated STAT2 (Dieguez-Acunaet al., 2010). The DJ-1 is preferentially expressed in the testis and moderately in other tissues, and it is translocated from the cytoplasm to nuclei during the cell cycle after mitogen stimulation, suggesting that DJ-1 has a growth-related function (Takahashiet al., 2001).

Proteasome subunit alpha type-2 is a multicatalytic proteinase complex with a highly ordered ringshaped 20S core structure. Proteasomes are distributed throughout eukaryotic cells at a high concentration and cleave peptides in an ATP/ubiquitin-dependent process in a nonlysosomal pathway. An essential function of a modified proteasome, the immunoproteasome is the processing of class I MHC peptides. This gene encodes a member of the peptidase T1A family, which is a 20S core alpha subunit (Wohlres-Vianaet al.,2011). Destructive functions of the proteasome,ubiquitin-dependent proteolytic activity, are significant for activator localization, activator destruction, coactivator/repressor destruction and PIC disassembly.Non-proteolytic functions of the proteasome are important for recruitment of activators and coactivators to promoters, ubiquitin-dependent histone modification,transcription elongation and possibly maturation of mRNA via the facilitation of mRNA export from the nucleus to the cytoplasm (Kwaket al., 2011).

The SUMO-activating enzyme subunit 1, this heterodimer acted as an E1 ligase for the SUMO1, the SUMO2, the SUMO3, and probably the SUMO4. It mediates the ATP-dependent activation of the SUMO proteins followed by the formation of a thioester bond between a SUMO protein and a conserved active site cysteine residue on UBA2/SAE2 (Boggioet al., 2004).The SUMO modification pathway is a key regulator of numerous cellular activities, including transcriptional regulation, nuclear transport, maintenance of genome integrity, and signal transduction (Stehmeieret al.,2009). The SUMO is first activated by the E1-activating enzyme, and the Aos1/Uba2 heterodimer; subsequently transferred to the unique E2 enzyme, Ubc9;and conjugated to substrates in a reaction that is catalyzed by a variety of E3 enzymes such as the PIAS family members (Johnsonet al., 2001).

Synaptic vesicle membrane protein VAT-1 homolog belongs to the quinone oxidoreductase subfamily of zinc-containing alcohol dehydrogenase proteins. The functions of the VAT-1 in vertebrates remain to be defined. The VAT-1, as well as the murine VAT-1 homolog has the ATPase activity (Hayesset al.,1998). A dehydrogenase/reductase activity has been suggested since the VAT-1 has sequence homology with this enzyme family and the ability to bind nicotinic nucleotide compounds, but this remains unproven.Recent studies suggest a role for the VAT-1 in human keratinocyte physiology as well as in glioma invasion.In cancer cells, the VAT-1 is localized in the cytosol,mainly in the nuclear periphery region, and to discrete ruffle-like membrane structures (Mertschet al., 2009).In conclusion, the proteomic approach was to identify proteins involved in the action mechanisms of the water decoction fromVaccaria segetalisin DCMECs. All the five identified proteins had been implicated in the metabolic regulation activities ofVaccaria segetalis. Up-regulations of protein ETHE1,VAT-1, PARK7, PSMA2, and SAE1 in DCMECs positively contribute to the activities ofVaccaria segetalis. Findings of this study provided novel molecular mechanisms genes for the metabolic regulation activity ofVaccaria segetalisin lactation management.

Bjorhall K, Miliotis T, Davidsson P. 2005. Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples.Proteomics, 5(1): 307-317.

Boggio R, Colombo R, HayR T,et al. 2004. A mechanism for inhibiting the SUMO pathway.Molecular Cell, 16: 549-561.

Chen J H, Tong H L, Li Q Z,et al. 2009. Establishment of dairy cow mammary gland epithelial cell line.Chinese Journal of Animal and Veterinary Sciences, 40: 743-747.

Dieguez-Acuna F, Kodama S, Okubo Y,et al. 2010. Proteomics identifies multipotent and low oncogenic risk stem cells of the spleen.The International Journal of Biochemistry and Cell Biology, 42:1651-1660.

Higashitsuji H, Higashitsuji H, Masuda T,et al. 2007. Enhanced deacetylation of p53 by the anti-apoptotic protein HSCO in association with histone deacetylase.The Journal of Biological Chemistry,282, 18: 13716-13725.

Hayess K, Kraft R, Sachsinger J,et al. 1998. Mammalian protein homologous to VAT-1 ofTorpedo californica: isolation fromEhrlich ascitestumor cells, biochemical characterization, and organization of its gene.Cellular Biochemistry, 69(3): 304-315.

Johnson E S, Gupta A A. 2001. An E3-like factor that promotes SUMO conjugation to the yeast septins.Cell, 106: 735-744.

Kwak J, Workman J L, Lee D. 2011. The proteasome and its regulatory roles in gene expression.Gene Regulatory Mechanisms, 1809:8-96.

Mertsch S, Becker M, Lichota A,et al. 2009. Vesicle amine transport protein-1 (VAT-1) is unregulated in glioblastomas and promotes migration.Neuropathol Appl Neurobiol, 35(4): 342-352.

Shen L M, Liu Q, Ni J Z,et al. 2009. A proteomic investigation into the human cervical cancer cell line hela treated with dicitratoytterbium(iii) complex.Chemico-Biological Interactions, 181: 455-462.

Stehmeier P, Muller S. 2009. Regulation of p53 family members by the ubiquitin-like SUMO system.DNA Repair, 8: 491-498.

Wohlres-Viana S, Pereira M M, Viana J H M,et al. 2011. Comparison of gene expression inBos indicusandBos taurusembryos producedin vivoorin vitro.Livestock Science, 140: 62-67.

Takahashi K, Taira T, Niki T,et al. 2001. DJ-1 positively regulates the androgen receptor by impairing the binding of piasxα to the receptor.The Journal of Biological Chemistry, 276, 40: 37556-37563.

Tong H L, Gao X J, Li Q Z,et al. 2011. Metabolic regulation of mammary gland epithelial cells of dairy cow by galactopoietic compound isolated from vaccariae segetalis.Agricultural Sciences in China, 10(7): 1106-1116.

Tiranti V, Briem E, Lamantea E,et al. 2006. ETHE1 mutations are specific to ethylmalonic encephalopathy.J Med Genet, 43: 340-346.

Wan Z Y, Gao X J, Li Q Z,et al. 2011. Influence on cellular signal transduction pathway in dairy cow mammary gland epithelial cells by galactopoietic compound isolated fromVaccariae segetalis.Agricultural Sciences in China, 10(4): 619-630.