CRISPR/Cas9-mediated Tryptophan Hydroxylase 1 Knockout Decreases Calcium Transportation in Goat Mammary Epithelial Cells

Background: Calcium is one of the major mineral nutrients in goat milk. Tryptophan hydroxylase1 (TPH1) is a rate-limiting enzyme catalyzing hydroxylation of L-tryptophan into 5-hydroxytryptamine (5-HT) essential for maintaining calcium homeostasis. The function of TPH1 and 5-HT in goat mammary calcium homeostasis is not well known. Methods: The CRISPR/Cas9-meidated TPH1 knockout goat mammary epithelial cells (GMEC) were constructed �rstly. Then the content of 5-HT, intracellular calcium level and abundance of key genes related to calcium transportation were evaluated and compared in wild-type GMEC, TPH1 knockout GMEC, to explore the impact of TPH1 on calcium transportation, respectively. Wild-type GMEC and TPH1 knockout GMEC were further treated with exogenous 5-HTP to con�rm the role of TPH1 in regulating calcium homeostasis in GMEC. 5-HT concentration was measured by enzyme-linked immunosorbent assay and uo-3 staining was used to determine intracellular calcium content. Results: The TPH1 knockout GMEC heterozygous clone with no off-target effects was obtained after transfection of the Cas9/sgRNA expression vector. The 5-HT synthesis and intracellular calcium level decreased in TPH1 gene knockout GMEC. The mRNA abundance of secretory-pathway Ca 2+ -ATPase1 (SPCA1) and plasma membrane Ca 2+ -ATPase1 (PMCA1) were up-regulated while the mRNA abundance of secretory-pathway Ca 2+ -ATPase2 (SPCA2) was down-regulated in TPH1 knockout GMEC. Up-regulation of parathyroid hormone-related peptide (PTHrP), a key regulator of mammary calcium metabolism, induced by 5-HTP were blocked by TPH1 gene knockout. The TPH1 knockout GMEC showed a lower sensitivity to 5-HTP induced elevation of calcium content. Conclusion: Results suggested that TPH1 plays an important role in regulating calcium homeostasis via PTHrP and calcium transportation related factors in GMEC.


Introduction
Calcium is one of the major mineral nutrients in dairy products.Calcium can regulate bone forming, maintain excitability of muscle system and nerver system, participating in the process of immune regulation and mediate normal life activities of cells [1][2][3].During lactation, a large amount of calcium was transported from plasma to mammary epithelial cells [4].The mammary gland generates a large transepithelial calcium gradient in favour of milk [5].The plasma membrane calcium e ux pumps PMCAs (plasma membrane Ca 2+ -ATPases), located on the apical membrane, was responsible for the transportation of Calcium from the epithelial cell to milk [6].SPCAs (secretory-pathway Ca 2+ -ATPases), another calcium pump given its role at Golgi, is also involved in the secretion of calcium into milk [7,8].
Despite this, pathways and mechanisms which underlie the movement of calcium across the mammary gland are still worth studying.
Tryptophan hydroxylase1 (TPH1) is a rate-limiting enzyme catalyzing hydroxylation of L-tryptophan into 5-hydroxytryptamine (5-HT) [9].As is reported widely, 5-HT, also named serotonin, plays an important role in regulating several aspects of lactation among species such as mammary tissue development, milk protein synthesis and lactation maintaining [10][11][12].More importantly, 5-HT in the mammary gland could affect the synthesis of Parathyroid hormone-related peptide (PTHrP) which has been recognized as a regulator of calcium metabolism, and thus maintain calcium homeostasis during lactation [13] [14].The previous studies showed that there was a positive correlation between the contents of 5-HT and PTHrP in goats and cows during pregnancy and lactation [15,16].
Global TPH1 knockout through Cre-loxP system has been carried out to discover novel genes, pathways and functions which serotonin modulates during lactation in mice [17] .Although TPH1 has a crucial effect on lactation, there is still no evidence of such endeavors coming out in goats or goat mammary epithelial cells.The novel clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing technique has been used successfully to build a permanent loss-of-function model [18][19][20][21].Because of its higher e ciency and exibility, CRISPR/Cas9 gene knockout system was chosen in this study to investigate the function of TPH1 on calcium metabolism in goat mammary gland.
The objective of this study is to evaluate the effect of TPH1 knockout through CRISPR/Cas9 technology on 5-HT synthesis, intracellular calcium content, expression levels of local calcium regulator and relative abundance of genes-related to calcium transportation.This study provides evidence about function of TPH1 and 5-HT in regulating calcium metabolism in dairy goats.

Animals and Cells
All experimental procedures with live goats used in this study were approved by the Animal Care and Use Committee of the Northwest A&F University, Yang Ling, China (permit number: 15-516, date: 2015-9-13).
GMEC were isolated from Xinong Saanen dairy goats at peak lactation (60 d after parturition) as described previously [22].In brief, a piece of tissue was dissected from mammary gland under sterile conditions and blood on the tissue was washed away with D-Hank's solution supplemented with 100 IU/ml penicillin and 100 IU/ml streptomycin.Then fatty and connective tissues were removed and the granular acinar tissue was cut into pieces and then cultured with complete medium.The composition of the medium is as follows: Dulbecco's modi ed eagle medium/nutrient mixture F-12 (DMEM/F12) medium (11320 − 033, Invitrogen Corporation, Waltham, MA) supplemented with insulin (5 µg/ml; Sigma-Aldrich, St. Louis, MO), penicillin and streptomycin (100 U/ml; 080092569, Harbin Pharmaceutical Group, Harbin, China), epidermal growth factor (10 ng/ml; PHG0311, Invitrogen), hydrocortisone (1 µg/ml; H0888, Sigma-Aldrich), and 10% fetal bovine serum (10099 − 141, Invitrogen).The GMEC were cultured to con uence in complete medium at 37 °C in a humidi ed atmosphere with 5% CO 2 .Culture medium was changed every 24 h.To promote lactogenesis, the cells were cultured in the basal medium with prolactin (L6520, 2 µg/mL, Sigma) for 48 h before performing the following experiments.

Cell Selection, Dna Extraction And T7en1 Cleavage Assay
Individual cell clones were isolated from the remaining cell poll by the limiting dilution method as described previously [25].Brie y, cells were diluted to one cell per 100 µL of medium, inoculated into 96well cell culture plates, and cultured for 10-14 days to obtain single clone colonies.When cells reached 90% con uence, half of the cells were collected and genomic DNA was extracted using the Universal Genomic DNA Kit (CW Biotech).The DNA fragments spanning the target site were PCR ampli ed using the test primers (Table 1; product size: 607 bp).The PCR products were puri ed by PCR Clean-Up Kit (AP-PCR-50, Axygen, CA, USA) according to the manufacturers' instructions.Puri ed DNA was annealed for T7EN1 cleavage assay [26] (M0302L, NEB, USA) and the enzyme digestion product was analyzed by agarose gel electrophoresis.Cleaved bands intensity were measured by ImageJ software (ImageLab, http://imagej.net).The genome editing e ciency was calculated by the formula: 100 ), where a is the intensity of the undigested PCR product and b and c are the intensities of each cleavage band.The PCR products of each single clone cell were cloned into pMD19-T vector.Each plasmid was randomly picked out and sent for sanger sequencing to assess sequence modi cations by Invitrogen (Shanghai, China).

Off-target Effects Analysis
Off-target (OT) sites were predicted using the online website tool Cas-OFFinder (http://www.rgenome.net/cas-onder/) [27].Mismatches ≤ 3 bp was used as criteria [28,29].Genomic DNA extracted from single clones were used as templates for off-target sites PCR.The primers for 10 offtarget site detections are shown in Suppl.Table 1.T7EN1 cleavage assay was used and the PCR products were inserted into pMD19-T vector for sequencing.
Cell Treatment With 5-htp 5-Hydroxytryptophan (5-HTP, Sigma-Aldrich) was dissolved in Dimethyl sulfoxide (DMSO) according to the manufacturers' instructions, and further dilutions were made in complete medium to reach a nal concentration.When wild type GMEC and TPH1 knockout GMEC reached approximately 70-80% con uence in lactogenic medium, they were treated with 0 µg/mL, 50 µg/mL or 100 µg/mL 5-HTP respectively.Cells were used for intracellular calcium analysis or collected for RNA isolation after 24 h incubation.After 48 h incubation cells were collected for protein extraction.

Measurement Of Intracellular Calcium
After 24 h incubation, the GMEC were, loaded with the uorescent Calcium indicator uo-3, Calcium imaging was done on a laser scanning confocal microscopy (Becton Dickinson, Inc.).Dissolve uo-3, AM (Solarbio, Beijing) to prepare 2 mM storage solution with anhydrous DMSO, then an equal volume of 20% pluronic F127 solution (Solarbio, Beijing) was added to uo-3, AM/DMSO solution.4 µm uo-3, AM working solution was prepared by diluting with Hanks balanced salt solution (HBSS, Solarbio, Beijing).Fluo-3, AM working solution was then added to the cells.After cultured at 37 ℃ for 20 minutes, 5 times the volume of HBSS containing 1% fetal bovine serum was added to GMEC for 40 minutes.The cells were washed 3 times and then resuscitated with HEPES buffer saline (Solarbio, Beijing) to make 1 × 10 5 cells/ml solution.Cells were cultured for 10 minutes and intracellular calcium tra cking was detected using Fluo-3, AM (4 µM, Solarbio) by a laser scanning confocal microscopy.The intensity of uorescence was measured by ImageJ software.
Mesurement Of 5-ht Synthesis 5-HT synthesis was assessed by the 5-HT concentration of medium which had or hand not been used to culture GEMC for 24 h.Medium concentration of 5-HT was analyzed by enzyme-linked immunosorbent assay (SHKXSM Co.Ltd., China) according to the manufacturer's instructions.Intra-assay CV (%) is less than 10% and Inter-assay CV(%) is less than 15%.Brie y, 50 µl of collected samples were added to the appropriate wells.Then, 100 µl of enzyme conjugate was added to standard wells and sample wells except the blank well, incubate for 60 min at 37 °C.After the microtiter plate was washed ve times, 50 µl of substrate A and B were added to each well and incubated for 15 min at 37 °C.Then, each well was added with a 50 µl stop solution.The absorbance of each sample at 450 nm wavelength was detected using a microplate reader within 15 min.

Quantitative Real-time Pcr
Total RNA was isolated using Trizol reagent (Takara Bio Inc., Japan) according to the manufacturer's instructions.The rst-strand complementary DNA was synthesized using the PrimeScript RT kit (Takara Bio Inc., Japan).Quantitative real time PCR (qPCR) primer sequences are shown in Table 1.Ubiquitously expressed transcript (UXT), ribosomal protein S9 (RPS9) and mitochondrial ribosomal protein L39 (MRPL39) were used as internal control genes [30].The qPCR was run in triplicate in a Bio-Rad master cycler using the SYBR Green PCR Master Mix (Takara, Japan) according to the manufacturer's protocol.
The qPCR data were analyzed using the 2 −ΔΔCt method.

Western Blot
Western Blot Western blot was performed as described previously [15].Cells were collected from different treatment groups, pelletedby centrifugation and lysedin RIPA buffer.Total protein was prepared and protein concentration was determined using the Bradford method.Proteins were then separated by SDSpolyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membranes and blocked with milk powder solution for 1.5 h at room temperature and overnight incubation with the primary antibody.The membranes were incubated with the primary antibody overnight.Anti-PTHrP, anti-TPH1, and anti-β-actin were purchased from (Abcam, Cambridge, MA).Then the membranes were washed with PBS-tween and incubated for 1.5 h with horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, MA).β-actin was used as a housekeeping protein.Protein bands were detected after treatment of SuperSignal West Femto agent of Thermo (Thermo Scienti c, Karlsruhe, Germany).

Statistical Analysis
All the data were presented as mean ± SEM of three independent experiments.T-test or one-way ANOVA was applied to analyze the difference between groups.P < 0.05 represented a signi cant difference.SPSS 19.0 statistics software (SPSS, Inc., Chicago, IL) and GraphPad Prism software 6.0 were used for data statistics and statistical mapping.

Cleavage E ciency analysis of sgRNAs and Single Cell Clone selection
Of the two sgRNAs, sgRNA1 targeted the anti-sense strand of the TPH1 exon1 while sgRNA2 targeted the sense strand of the TPH1 exon1(Fig.1A).Genomic DNA was extracted and performed T7EN1 assay after puromycin selection.As shown in Fig. 1B, sgRNA2 had 25.2% cleavage e ciency.Following selecting single clone cells from survival GMEC of sgRNA2 transfection, we obtained one single cell clone with 32.3% cleavage e ciency through T7EN1 assay (Fig. 1B).As the Sanger sequencing assay showed, there were two genotypes at the target site of single clone 1 (Fig. 1C).One allele showed 18 nucleotides deletion and 31 nucleotides insertion, the other allele showed 10 nucleotides deletion (Fig. 1C).Therefore, single clone 1 was chosen as the homozygous TPH1 knock clone in further experiments.
Off-target Effect Of Crispr/cas9 In Single Clone On the basis of the above single clone cell selection results, T7EN1 cleavage and sanger sequencing assay were performed to analysis off-target effect of single clone cell.Ten off-target sites (Fig. 2A) were chosen for examination and no off-targets were detected (Fig. 2B).
Knockout of TPH1 reduce serotonin synthesis and incracellular calcium content TPH1 is recognized as the main rate-limiting enzyme of 5-HT synthesis.Thus, western blot and enzymelinked immunosorbent assay were performed to evaluate content of TPH1 in cells and 5-HT in medium.

Knockout of TPH1 blocks 5-HTP induced PTHrP expression and calcium transportation
Previous study had reported that Parathyroid hormone-related protein (PTHrP) played an essential role in calcium mobilization and had a correation with 5-HTP.To determine whether knockout of TPH1 has in uences on the abundence of PTHrP and 5-HTP induced calcium accumulation, wild type GMEC and TPH1 knockout GMEC were treated with 5-HTP respectively.As is shown in Fig. 4A and Fig. 4B, knockout TPH1 decreased both the mRNA and protein abundance of PTHrP (P < 0.05).Treatment of 100 µM 5-HTP increased protein and mRNA level in wild type GMEC (P < 0.05) while showed no signi cant in uence on the expression of PTHrP in TPH1 knockout GMEC (P > 0.05) (Fig. 4C, 4D).In wild type GMEC, treatment of 100 µM 5-HTP decreased mRNA expression of PMCA1, SPCA1, SERCA2 (P < 0.05) and increased the mRNA expression of SPCA2 (P < 0.05) (Fig. 4E − 4H).In TPH1 knockout GMEC, treatment of 100 µM 5-HTP also decreased the mRNA expression of PMCA1 (P < 0.05) while had no signi cant in uence on the mRNA expression of SPCA1, SPCA2 and SERCA2 (P > 0.05) (Fig. 4E − 4H).Fluo-3 staining results showed that 5-HTP increased intracellular calcium contents in a dose dependent manner (Fig. 4I).Treament of 100 µM 5-HTP increased intracellular calcium levels by about 50% in TPH1 knockout GMEC (P < 0.05) and by about three times in wild type GMEC (P < 0.05).
Discussion 5-HT participates in numerous processes in the mammary gland, including tight junction permeability, PTHrP synthesis, and Calcium transport regulation [14,[31][32][33].TPH1 is responsible for hydroxylation to the 5' position to form the amino acid 5-hydroxytryptophan (5-HTP), which is the initial and rate-limiting step in the synthesis of the nonneuronal serotonin [34].Cognition of 5-HT system has been further deepened with the enrichment of TPH1 gene related researches [35,36].5-HT's neuronal and peripheral functions were separated through TPH1 knockout mice model [37].Besides, the functions of 5-HT in bone regulation and energy metabolism were also explained in detail with a TPH1 knockout models [38,39].In the present study, we analyzed the function of TPH1 on calcium metabolism in GMEC through CRISPR/Cas9 system.Knockout of TPH1 gene decreased both the transcriptional and translation level of TPH1.In the medium of TPH1 knockout cells, the concentration of 5-HT was lower compared with wild type cells, which indicating that TPH1 was necessary for the synthesis of 5-HT [40].Through 2D and 3D intracellular calcium imaging analysis, calcium content in GMEC decreased approximately 50% by TPH1 knockout,and this result was similar to the result of TPH1 knockout induced less bone resorption of mice [41].These results suggested that both 5-HT synthesis and calcium metabolism in GMEC were negatively affected in the TPH1 knockout GMEC model.
To further substantiate the speci c molecular components responsible for TPH1 affecting calcium transportation in GMEC, the expressions of calcium transportation related genes were detected.The plasma membrane calcium e ux pump PMCA1, responsible for the e ux of calcium from the epithelial cell [8], was increased in TPH1 knockout GMEC.Secretory pathway calcium pumps SPCA1 and SPCA2, involved in the secretion of calcium into milk, were up-regulated and down-regulated by TPH1 knockout, respectively.There was no in uence on the expression of endoplasmic reticulum calcium sequestration pump SERCA2 [42].However, in our previous study, only the upregulation of PMCA1 was detected when TPH1 was knocked down by small interfering RNA [15].The present study indicated a more realistic result that knockout TPH1 affected the expressions of those calcium transportation related genes, which led to the decrease in intracellular calcium content.
During lactation, PTHrP regulates calcium homeostasis via a feedback mechanism [43,44].5-HT, synthesized from 5-HTP, is also involved in processes including milk protein biogensis and calcium homeostasis [14,45].Supplementation of 5-HTP increased contents of circulating 5-HT, PTHrP and milk calcium around parturition in mice and cows [14,46,47].5-HT induced PTHrP expression in bovine and goat mammary epithelial cells, while knockout of TPH1 in mice reduces the expression of mammary PTHrP and the expression can be rescued by restoring 5-HT synthesis [14,15].Similarly, knockout of TPH1 in GEMC decreased mRNA expression and protein abundance of PTHrP in the present study.
Besides, the administration of 5-HTP increased intracellular calcium content in a dose dependent manner and rescued the expression of PTHrP.To interest us, then, TPH1 knockout appears to reduce the cell's sensitivity to 5-HTP.Intracellular calcium levels increased about threefold when wild-type GMEC were treated with 100 µM 5-HTP, but calcium levels in TPH1 knockout GMEC increased by only about 50% under the same treatment.Further detection of calcium transportation related genes' expression also showed that the response of TPH1 knockout GEMC to 5-HTP treatment was limited, except for PMCA1.
Combined with our simultaneous study, we have con rmed that 5-HTP can inhibit the expression of PMCA1 through miRNA-99a-3p axis in GMEC [48] and increase intracellular calcium content rather than through TPH1 axis.It is assumed that expression level of PTHrP, where there was no signi cant difference between wild type GEMC and 100 µM 5-HTP treated TPH1 gene knockout GEMC, mediates the phenomenon mentioned above.Despite the novel ndings, the mechanism still needs further study.
CRISPR/Cas9 technique has been used for generating gene modi ed animal model [49] and to perform in vivo study of genes, miRNAs and long non-conding RNAs [50][51][52].The CRISPR-associated protein 9, an endogenous endonuclease, directed by a single-guide RNA (sgRNA) causes a double strand break at the designed site rstly.Then, the cells tend to repair their DNA in two pathways [53]: non-homologous endjoining (NHEJ), which can cause random nucleotide insertion [54]; or homology-directed repair (HDR), which integrates a homologous DNA sequence [55].In the present study, we obtained a heterozygous TPH1 knockout clone using the NHEJ pathway.We noticed that although the double strand was broken at the same site, the difference occurred during the repair process: One allele showed 18 nucleotides deletion and 31 nucleotides insertion, the other allele showed 10 nucleotides deletion.Off-target effect is the major and common limitation in various gene editing techniques [56].In the present study, T7EN1 cleavage and sanger sequencing results showed that there was no off-target effect in the 10 predicted sites.Furthermore, TPH1 protein abundance was blocked in the THP1 knockout GMEC.These results indicate that the CRISPR/Cas9 system successfully knocked out the TPH1 gene.

Conclusions
In conclusion, we successfully obtained TPH1 gene heterozygous knockout GMEC by CRISPR/Cas9mediated gene editing system.The heterozygous knockout of TPH1 blocked the cellular 5-HT synthesis, decreased the content of intracellular calcium and affected the expression of genes responsible for the e ux of calcium from the epithelial cell.The expression of PTHrP, the key regulator of mammary calcium homeostasis, was decreased in TPH1 knockout GMEC and its expression can be rescued by 5-HTP treatment.The processes of 5-HTP induced expression of PTHrP and calcium content was limited by the expression of TPH1.These results suggest that TPH1 plays an essential role in regulating calcium homeostasis in GMEC.modes, the intensity of uorescence was measured by ImageJ software (D).E-H Total RNA was isolated using TRIzol reagent and the relative expression of genes related to calcium transportation was measured using real-time PCR.

Figures
Figure 4

Figure 1 Selection
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Figure 2 Analysis
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Figure 3 Effects
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