Modulation of Bovine Wnt Signaling Pathway Genes by Cowpea Phenolic Extract

The Wingless (Wnt) signaling pathway is a conserved pathway with essential roles in cellular and biological processes in mammals. Wnt signal transduction has been implicated in inflammation, innate immunity and homeostasis via Toll-like receptor and NF-κB pathways. Plant bioactive compounds are capable of modulating the Wnt signalling pathway, which can be either a canonical (β-Catenin dependent) or non-canonical (β-Catenin independent) mechanism. This study evaluated the effect of cowpea phenolic extract (CPE) on the expression and modulation of genes of the Wnt signaling pathway in cow blood. Whole blood collected from six Holstein-Friesian cows was treated with 10 μg/ml of the extract, and evaluated for packed cell volume (PCV), total count and viability of cells, and white blood cell differential count before and after treatment. Cowpea phenolic extract agonist activity in blood was measured using a Bovine toll-like receptor (TLR) 2, and TLR 4 ELISA kit. Total RNA was isolated from the blood cell pellet, reverse transcribed and used for real-time PCR to detect expression of 84 genes on the Cow Wnt signaling pathway array. The total cell-associated β-Catenin level was measured using a commercial ELISA kit. There was no treatment effect on PCV, total cell and viability (P > 0.05). The percentage of mononuclear cells were influenced by treatment, % monocytes (P = 0.0136) decreased and % lymphocytes (P = 0.0114) increased. Treatment with CPE activated cow blood cells, increased TLR2 release and total β-Catenin levels (6 ng/ml, P < 0.05), but TLR4 was not detected. Polyphenols from cowpea modulated the expression of Wnt signalling genes, especially canonical β-Catenin mediated pathway genes. Modulation of Wingless gene expression may be an important mechanism by which polyphenols in cowpea feed impact cellular immune response and homeostasis. Thus, further studies are needed to determine the association of CPE-mediated Wnt gene modulation on blood leucocytes subpopulations and animal health.


Introduction
Cowpea (Vigna unguiculata L.Walp) is an important annual legume plant used for food.It is cultivated and consumed largely in Africa, Asia, and South America.Cowpea seed grains are rich in protein: 27% (Gupta et al., 2010).Cowpea with a comparable yield and digestibility to alfafa (Cook, 2005), has high nutritive value, a crude protein of 22%, and are fed to animals as forage, hay or silage (Etana et al., 2013).Using cowpea as protein supplement feed has been recommended for ruminants fed low-quality roughages for improved productivity (Etana et al., 2013;Gwanzura et al., 2012).Cowpea was used as one of the summer legumes for cow and calves on Bermudagrass creep grazing pastures (Pitman et al., 2015).Supplementing small ruminant feed with cowpea forage enhanced feed intake, increased average daily gain, nitrogen intake and digestibility and fecal nitrogen output (Baloyi et al., 2008;Koralagama et al., 2008).Use of cowpea as summer finishing forage in cattle resulted in greater marbling score and increased dressing percentage and consumer steak preference (Schmidt et al., 2013).
Cowpea contains phenolic compounds including phenol acids, flavonoids, and tannins (Cai et al., 2003).Due to their antioxidant, anti-inflammatory, and anticarcinogenic effects, these bioactive compounds pose an added health benefit to humans and animals (Lee & Yen, 2006;Balsano & Alisi, 2009;Folmer et al., 2014;Ojwang et al., 2015).In animal studies, flavonoids (Middleton et al., 2000) and condensed tannins (Min et al., 2004) have been reported to have benefits for ruminant health and productivity.For example, feeding tannin-rich forages to animals attenuates blotting, increases their resistance to gastrointestinal nematodes (Min & Hart, 2003;Min et al., 2004) and improves nutrition through increased availability of high-quality proteins to the small intestines (Barry et al., 2001).The health promoting benefits of cowpeas for human consumption have been studied using human cell lines and laboratory animal models against chronic diseases such as cancer (Gutiérrez-Uribe et al., 2011).The evaluation of cowpeas and their bioactive properties for use in livestock health has received less attention.Dietary bioactive substances influence animal health and well-being via activation of conserved gene pathways including the Wingless (Wnt) pathway (Logan & Nusse, 2004;Tarapore et al., 2012).
The Wnt gene family is comprised of numerous genes encoding highly conserved secreted glycoproteins related to the Wnt-1 proto-oncogene and to Drosophila wingless gene products (Wodarz & Nusse, 1998).Secreted Wnt proteins serve as ligands for Frizzled (Fzd) family membrane receptors together with co-receptors lipoprotein receptor-related protein (LRP) 5 and 6 to initiate signaling.Currently, 19 Wnt and 10 Fzd genes have been identified (Miller, 2002;He et al., 2004).The absence of Wnt protein secretion results in Axin/glycogen synthase kinase 3 β (GSK3β)/adenomatous polyposis coli (APC) complex degradation of phosphorylated cytoplasm β-Catenin (Liu et al., 2005).In vertebrates, the Wnt signaling pathway regulates various cellular processes such as cell fate, polarity, proliferation, movement, and stem cells hematopoiesis (Clevers & Nusse, 2012).Wnt proteins control cellular functions through two distinct pathways, canonical and non-canonical pathways; the former is β-Catenin dependent but the latter is not.The non-canonical pathway is subcategorized into two, planar cell polarity (PCP) and Wnt/Ca 2+ pathways (Logan & Nusse, 2004).
Wnt signaling lies at the heart of numerous biological processes including embryonic development, adipogenesis, myogenesis, disease progression as well as impacting meat quality (marbling) and production.Furthermore, Wnt signal transduction has been implicated in inflammation and innate immunity via Toll-like receptor and NF-κB pathways (Umar et al., 2009;Sun et al., 2005;Duan et al., 2007).Previous reports mostly but not limited to studies in cancerous cells have shown the effect of plant bioactive compounds in modulating the Wnt/B-catenin signaling pathway (Tarapore et al., 2012).The expression and regulation of Wnt signaling pathway genes in the peripheral blood cells of healthy cattle in response to phenolic extract fractions of cowpea have not been reported to the authors' knowledge.In this current study, the role of cowpea phenolic extracts in modulating the expression of 84 genes in the cow Wnt signaling pathway was investigated in bovine peripheral blood.

Animal Sampling and Blood Collection
Six clinically healthy age-matched Holstein-Friesian cows (Body weight = 1662±79 kg) selected from the herd at the North Carolina Agricultural and Technical State University dairy farm were used.All protocol used were approved by the Institutional Animal Care and Use Committee.The animals used were under no medical and feed treatment during the study period.Twenty milliliters (20 mL) of blood were collected from the jugular vein of the cows in the morning prior to feeding and placed into vaccutainer tubes containing Acid Citrate Dextrose anticoagulant (BD Biosciences, San Jose, CA).Blood samples were kept on ice and processed with two hours.

Cowpea Phenolic Compounds Extraction
The Mississippi silver variety of cowpea, a widely adapted variety with early and uniform maturity, resistant to Fusarium wilt, root knot nematodes, and other viruses was used for the study.Cowpea seeds were planted in pots (dimension are 6.3-inch depth × 6.5-inch diameter) in the greenhouse.Fresh leaves (100 g) were sampled from 30-day old plants and oven dried at 55 o C for 24 hours.The procedure followed for preparation of cowpea polyphenolic extracts was described by Adjei-Fremah, Jackai and Worku (2015).Total phenolic compound (TPC) was measured with the Folin-Ciocalteu method using gallic acid as standard (Singleton et al., 1999).

Evaluation of Endotoxin Levels
All Solvents and diluents used in this study were tested for endotoxin with the ToxinSensor TM Chromogenic LAL Endotoxin Assay Kit following the manufacturer's protocol (GenScript, Piscataway, NJ).Ultrapure distilled water (Invitrogen, Camarillo, CA) had low endotoxin levels of 0.01 EU/ml and was used for the preparation of all solutions.

CPE Treatment of Bovine Peripheral Blood
Bovine whole blood (10 7 cells/mL, viable cells) was treated with 10 ug/mL CPE, and Untreated control samples were maintained in PBS.CPE-treated and control samples were incubated for 30 minutes at 37 o C, 95% humidity and 5% CO 2 .The treatment conditions followed was described by Worku and Morris, (2009).After incubation, the samples were centrifuged at 700 g for 5 mins at 4 o C. The supernatant (plasma) was transferred into new tubes, and stored at -80 o C until further analysis.Plasma was also used to evaluate secretion of bovine Toll-like receptor 2 (TLR2) using a commercial ELISA kit.Tri-reagent (Sigma-Aldrich, St Louis, MO) was added to the cell pellet for total RNA isolation.

Blood Cell Analysis
Total cell count and viability in blood samples before and after CPE treatment was determined in the diluted blood (1:1000 PBS).Diluted samples were mixed with Trypan Blue and counts were performed using the TC20 automated cell counter (Bio-Rad, Hercules, CA).Blood packed cell volume (PCV) was determined using a microhematocrit centrifuge (Damon/IEC division).Thin blood smears were made on sterile glass slides prepared in triplicate for each animal.Wright staining was performed for histologic cell staining and white blood differentiation count.Cells were identified microscopically and enumerated.Counts of one hundred cells were evaluated for different white blood cell types.

Cell Lysate Preparation
To detect cellular secreted total β-Catenin levels, cell lysate was prepared after treatment of blood with CPE.The cell lysate preparation procedure followed was described by Adjei-Fremah et al. (2015).Blood cells were washed twice with PBS and then lysed using cell lysis buffer (Cell Signaling technology, Danvers, MA).The mixture was sonicated briefly on ice for 5 minutes.The samples were centrifuged at 700 g for 5 mins at 4 o C and supernatant (cell lysate) removed for detection and estimation of total β-Catenin levels.

Total β-Catenin Quantification
The β-Catenin (total) ELISA kit (Invitrogen, Camarillo, CA) was used to measure β-Catenin protein levels in the cell lysate for CPE-treated and control groups to determine canonical Wnt pathway activation, following manufacturer's protocol.The Enzyme Linked-Immuno-Sorbent Assay kit contains a monoclonal antibody specific for β-Catenin and detects and quantifies β-Catenin levels regardless of its phosphorylation state.

Detection and Quantification of TLR2 and TLR4 Secretion
A commercial bovine toll-like receptor 2 (TLR2) and TLR4 ELISA kit (CUSABIO) were used for detection and quantification of TLR2 and TLR4 concentration respectively in plasma of CPE-treated and control samples.Plasma sample (200 ul) was concentrated using the ProteoExtract TM protein precipitation kit (CALBIOCHEM).Concentrated samples were dissolved in 200 ul PBS for the detection of TLR2 and TLR4 as recommended by the manufacturer's (CUSABIO).

RNA Isolation and Quantitative RT-PCR
Total RNA was isolated using the Tri-reagent method (Sigma-Aldrich St. Louis, MO).The concentration and purity of RNA were measured with a Nanodrop spectrophotometer (Thermo-Scientific, Waltham, MA).The integrity of the isolated RNA was determined with Bioanalyzer (Agilent).Total RNA of the individual samples (0.5 µg each, RIN > 7) were pooled for cDNA synthesis using the RT 2 first strand kit (Qiagen).The cow Wnt signaling RT² Profiler™ PCR Array (Qiagen) with 84 test genes related to Wnt-mediated signals transduction was used.The genes profiled included genes associated with the canonical Wnt signaling, planar cell polarity, Wnt /Ca 2+ signaling and Wnt signaling negative regulation.Real-time PCR was performed using RT 2 SYBR Green Mastermix (Qiagen) on the CFX Connect real time system (Bio-rad).Real-time PCR workflow was per manufacturer's manual (Qiagen).All reactions were performed in triplicate.Real-time data analysis normalization was performed with Ct value of GAPDH and fold change in gene expression was calculated using the 2 -ΔΔCT method (Livak & Schmittgen, 2001).The student's t-test was used to determine the statistical significance of fold change in genes expressed.

Statistical Analysis
All data were analyzed using SAS 9.3 version (SAS Institute, Cary, NC).One way Analysis of variance (ANOVA) was performed on PCV, total cell and viability counts, white blood cell differential counts, total β-Catenin data and P-value < 0.05 was considered significant.All results are presented as mean ± SD.

CPE Treatment of Bovine Cells
Treatment of bovine blood ex vivo with CPE had no effect on total cell count and viability (P > 0.05).The average PCV observed was 32% and was not affected by treatment.However, the CPE treatment significantly changed the proportion of mononuclear cells in whole blood.Treatment significantly increased the percent lymphocytes (P = 0.0114) and decreased the % of monocytes (P = 0.0136) compared to the control group (Table 1).The percentage of neutrophils in blood was unaffected by treatment although animal variation in % neutrophils was observed after treatment with CPE (P = 0.0032).Secretion/release of TLR in plasma occurred following exposure to CPE.Treatment resulted in detection of TLR2 (51.56 ng/ml) in cow blood.Bovine TLR2 was not detected in the untreated control samples (Figure 1).Furthermore, bovine TLR4 was not detected in plasma of both CPE-treated and control groups.

Effect of CPE Exposure on Activation of the Wnt Signaling Pathway
The Wnt signaling pathway activation was observed at the level of mRNA transcription in ex vivo CPE-treated bovine blood cells.Out of the 84 genes on the cow Wnt signaling pathway array, 60 genes were detected in untreated cow blood.Sixteen genes on the array were not detected; DKK3, DVL1, FGF4, FOSL1, FZD7, FZD8, SFRP1, WISP1, Wnt1, Wnt11, Wnt2b, Wnt5a, Wnt5b, Wnt7b, Dkk2 and KREMENS.Gene description, average Ct values, and fold changes for all 84 genes on the array are found on Appendix 1. Forty-two genes were expressed in both CPE and control group (Figure 2).Note.* P-value < 0.05.

Total β-Catenin Levels
Cellular associated β-Catenin protein was detected in bovine blood (Figure 3).However, increased β-Catenin levels (6 ng/ml, P < 0.05) were detected after CPE treatment relative to control group.There was variation in the β-Catenin levels among the different animals used (P = 0.0084).The assay used is unable to distinguish between active β-Catenin from phosphorylated B-catenin-It detects both in response to Wnt signaling.Thus, CPE increased the total cellular β-Catenin released in the bovine blood.TWS119 or Wnt3a.Their study demonstrated that for naïve-to-effector T cell differentiation in human T lymphocytes, the Wnt/β-Catenin system may act as a negative regulator (Muralidharan et al., 2011).Wnt signaling regulation of various aspects of hematopoiesis including stem cell establishment, regeneration, maintenance of homeostasis, and differentiation of cells in blood has been reported (Lento et al., 2013).Wnt signaling controlled early embryonic hematopoiesis in mice and dysregulated β-catenin has been implicated in leukemia (Kabiri et al., 2014).The hematopoietic effect of Wnt activation may be related to changes in leukocyte concentration and this was evident in our study with activation of cells and subsequent expansion of lymphocytes numbers, and fold increase in cell growth and proliferation genes expression after CPE treatment.
Treatment of bovine blood with CPE activated the cells, had no effect on PCV, total cell count and viability of cells.Our results indicated cell activation and increased mRNA transcriptional activity following CPE treatment.Plant-derived bioactive compounds such as tannins have been shown to activate blood cells such as gamma delta T cells, one of the primary lymphocytes that are key to innate immune response in bovine (Holderness et al., 2007).Our results provide insight into CPE activation of bovine blood cells and subsequently Wnt signaling.Further research is needed to elucidate the role CPE on activation of Wnt signaling in subpopulations of blood lymphocytes.
In cancerous cells, plant-derived phenolic compounds have been reported to inhibit Wnt signaling pathway (reviewed by Teiten et al., 2012).However, in this current study, we elucidated the potential role CPE on Wnt signaling in healthy bovine peripheral blood cells.Our results demonstrated that CPE induces Wnt signaling, increase β-Catenin expression levels, and functions to promote lymphocytes proliferation.Zhang et al. (2010), studied the effect of flavonoids from Herba epimedii on Wnt signaling using healthy human bone marrow-derived mesenchymal stem cells.Flavonoids from Herba epimedii were shown to induce Wnt signaling and an increase in β-Catenin expression was reported.Thus, CPE, activates and modulate Wnt signaling genes and may involve a crosstalk with the TLR pathway.

Conclusion
Cowpea serves as a source of nutrition for man and animals.The CPE extract evaluated in this study modulates a highly conserved signaling pathway essential to homeostasis and health.In the study we showed the ex vivo effect of cowpea-derived phenolic extract stimulates TLR2 release and modulate Wnt signaling, a key cellular pathway functioning in cell fate, specification, polarity, proliferation, movement and maintenance of homeostasis.Treatment of bovine peripheral blood cells with CPE specifically activated the canonical pathway which is β-Catenin dependent whiles inhibiting the expression of gene associated with the planar cell polarity and Wnt/Ca 2+ signaling pathways.The significant role of Wnt signaling in cellular and biological processes, elevates the potentials of cowpea as a feed supplement in the animal system for enhanced nutrition, development and health.

Table 1 .
Analysis of bovine peripheral blood in Cowpea Phenolic Extract-treated and control Note.CPE = Cowpea Phenolic extract; PBS = Phosphate Buffer Saline; * for P < 0.05; NS: P > 0.05; a,b Values within a row with different superscripts differ significantly at P < 0.05.

Table 2 .
Differentially expressed Wnt signalling pathway genes