Generation of a New Congenic Mouse Strain with Enhanced Chymase Expression in Mast Cells

Mast cells are effector cells best known for their roles in IgE-associated allergy, but they also play a protective role in defense against pathogens. These cells express high levels of proteases including chymase, tryptase and carboxypeptidase. In the present study, we identified a congenic strain of C57BL/6 mice expressing an extraordinarily high level of chymases Mcp-2 and Mcp-4 in mast cells. The overexpression was associated with variant Mcp-2 and Mcp-4 genes originated from DBA/2 mice that also expressed high levels of the two enzymes. Real time PCR analysis revealed that Mcp-2 and Mcp-4 were selectively overexpressed as tryptases, Cpa3 and several other chymases were kept at normal levels. Reporter gene assays demonstrated that single-nucleotide polymorphisms (SNPs) in the promoter region of Mcp-2 gene may be partly responsible for the increased gene transcription. Our study provides a new model system to study the function of mast cell chymases. The data also suggest that expression of chymases differs considerably in different strains of mice and the increased chymase activity may be responsible for some unique phenotypes observed in DBA/2 mice.


Introduction
Mast cells are innate immune cells best known for their involvement in anaphylaxis, atopic asthma and other IgEassociated allergic disorders [1]. They also carry out a number of beneficial functions to the host including immune responses toward various pathogens. They are derived from hematopoietic stem cells and are widely distributed in tissues. Mast cells express a number of proteases including chymase, tryptase, and carbox-ypeptidaseA [2]. In mice, Mcp-1, -2, -4, -5, -9, and -10 are designated as chymases based on deduced amino acid sequences, whereas Mcp-6 and -7 are tryptases. These enzymes are stored in high amounts as active enzymes in mast cell secretory granules. Upon activation, massive fully active mast cell proteases are released through mast cell degranulation and elicit essential impacts on many physiological and pathological events which include extracellular matrix remodeling, extravascular coagulation, fibrinolysis, angiogenesis as well as antibacterial inflammatory responses [3].
Expressions of chymases are strictly regulated. At the level of transcriptional regulation, a well-documented transcription factor is Mitf. Direct or indirect binding of Mitf to the promoter element CANNTG can significantly enhance the expression of Mcp-2, -4, -5, -6, and -9 genes in C57BL/6 mice [4]. In addition to Mitf, bifunctional transcription factors C/EBPb and YY1 are thought to be responsible for the negative transcriptional regulation of Mcp-2 via intracellularly retained IL-15 [5,6]. In wild type bone marrowderived mast cells (BMMCs), C/EBPb is preferentially expressed over YY1 and binds to the Mcp-2 promoter. In contrast, in IL-15-deficient BMMCs, YY1 is dominantly expressed and binds to the Mcp-2 promoter, which allows hyper-transcription of the Mcp-2 gene [5]. Expression of chymases in mast cells is also known to be controlled at the post-transcriptional level. For example, an earlier study demonstrated that the half-life of the Mcp-2 transcript in mouse BMMCs was extended by 4-fold in the presence of IL-10 [7]. Together, expressions of chymases are regulated at multiple levels.
We previously generated a line of JAK2V617F transgenic mice that display polycythemia vera-like phenotypes [8]. Our most recent work demonstrated that the occurrence of PV-associated pruritus in these mice was associated with elevated levels of mast cells (Jin et al, unpublished). In this study, we identified a subpopulation of JAK2V617F transgenic mice that express very high levels of Mcp-2 and Mcp-4 in mast cells. However, this was found to be independent of JAK2V617F and due instead to the presence of Mcp-2 and Mcp-4 gene variants originated from DBA/2 mice. Our study thus provides a new line of congenic C57BL/6 mice with high expressions of specific chymases in mast cells.

Materials and Methods
Mice JAK2V617F transgenic mice were generated with a C57BL/ 66DBA/2 hybrid background and then crossed with wild type C57BL/6 mice for over 10 generations [8]. Wild-type C57BL/6 and DBA/2 mice were purchased from The Jackson Laboratory. Animals were housed in ventilated cages under standard conditions. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center.

Culture of Mast Cells
Bone marrow and peritoneal cavity cells from mice were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20% fetal bovine serum (FBS) and 1% each of conditioned media of cultured CHO cells overexpressing mIL-3 and mSCF. The resultant mast cells were analyzed after one month of culture initiation and maintained for up to four months with equal volumes of fresh medium added every 3 to 5 days. These cells were .95% pure based on positive staining for CD117 (c-Kit) and FceR1 upon flow cytometric analyses.

Proteomic Analyses
Protein identification was carried out by using the Mass Spectrometry and Proteomics core facility at the University of Oklahoma Health Sciences Center. In brief, proteins were separated on SDS gels, and protein bands were excised for digestion with trypsin. This was followed by HPLC separation with a Dionex UltiMate 3000 LC system and MS/MS analysis with an ABI MDS Sciex Qstar Elite mass spectrometer. MS/MS data was collected with the ABI Analyst QS 2.0 software and analyzed by using the Mascot search engine (Matrix Science) for protein identification against the 2011 SwissProt protein database.

Isolation of DNA and RNA
Genomic DNAs were purified from cultured mast cells and mouse tails by using the phenol/chloroform extraction method following digestion of samples with proteinase K. Total RNAs were isolated from cultured BMMCs by using the RNeasy Mini Kit (Qiagen), and single strand cDNAs were synthesized by using the QuantiTect reverse transcription kit from Qiagen.

Protein Expression and Antibody Production
DNA fragments encoding the mature Mcp-2 and Mcp-4 proteins without N-terminal signal sequences were amplified from mast cell single-strand cDNA by PCR with primer sets 59gaggagattattggtggtgttgagg plus 59-ggcttttcagctacttgctctttaa and 59gaggagattattggtggtgttgagt plus 59-ggcttttcactacttgccctttata, respectively. The PCR products were cloned into the pBluescript KS vector, and inserts were verified by DNA sequencing. This was followed by subcloning of the DNA inserts into a pT7 vector for protein expression as non-fusion proteins. Protein expression in recombinant E. coli cells was induced by 1 mM isopropyl b-D -1thiogalactopyranoside (IPTG). Both Mcp-2 and Mcp-4 proteins were found as prominent proteins in the inclusion body of the cells. We thus employed preparative SDS gels to purify them to near homogeneity. The purified proteins were used to immunize mice for generation of mouse anti-sera which were directly used for subsequent western blotting and immnuofluorescent cell staining.

SDS-PAGE and Western Blot Analyses
Cultured mast cells were collected and lysed in a buffer containing 25 mM b-glycerophosphate (pH 7.3), 5 mM EDTA, 2 mM EGTA, 5 mM b-mercaptoethanol, 1% Triton X-100, 0.1 M NaCl, and a protease inhibitor mixture or in 1X SDS sample buffer. Proteins were resolved on 10% or 12.5% SDS gels and then stained with Coomassie blue R-250 or transferred to polyvinylidenedifluoride (PVDF) for western blotting with anti-Mcp-2 and anti-Mcp-4 antibodies followed by horseradish peroxidase-conjugated secondary antibodies. Enhanced chemiluminescence signals were captured by using the FluorChem SP imaging system from Alpha Innotech.

Immunofluorescent Cell Staining
Cultured mast cells were spun onto glass slides by cytocentrifugation and fixed with 4% formaldehyde in PBS for 20 minutes. For antigen retrieval, fixed cells were treated with a buffer containing 10 mM sodium citrate (pH 6.0) and 0.05% Tween 20 for 40 minutes at 95-100uC. After rinsing with PBS, cells were probed with primary anti-Mcp-2 and Mcp-4 antibodies for 2 hours and then with a Cy3-conjugated anti-mouse secondary antibody for 1 hour. The nucleus was stained with 0.1 mg/ml Hoechst 33258. Fluorescence was visualized under 40X or 100X lens with an Olympus BX51 fluorescent microscope. Images were captured by using a DP71 digital camera.

Chymase Activity Assays
Cultured mast cells were collected and washed with ice-cold PBS. Following lysis in a buffer containing 25 mM Tris-HCl (pH 8.5), 1% Triton X-100, 5 mM EDTA and 0.1 M NaCl, cell extracts were cleared of insoluble materials by centrifugation at 18,0006g for 10 min. Chymase assays were performed with 0.375 mg/ml substrate N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Sigma-Aldrich) in 0.1 M Tris-HCl (pH 8.0). The reaction was allowed to proceed at room temperature, and absorbance was read at 405 nm using a nanodrop spectrophotometer at various time points. To calculate enzymatic activity, molar extinction coefficient 9.5610 3 /M/cm was used.

Degranulation of Mast Cells
Degranulation of mast cells was achieved by ligation of the highaffinity IgE receptor FceR1 via IgE. For this purpose, cultured mast cells were sensitized with 0.15 mg/ml of anti-DNP IgE (Sigma-Aldrich) in complete culture medium overnight at 37uC. Cells were then washed twice with and re-suspended in plain IMDM medium. This was followed by stimulation with 0.05 mg/ ml DNP-HSA for 30 min at 37uC. Cell and medium were then separated. Chymase activity in the medium and that remained in cells were determined as described above.

Real Time PCR Analyses
Total RNAs were isolated from cultured BMMCs by using RNeasy Mini Kit (Qiagen), and 1 mg RNA was then used to synthesize single-strand cDNA by using the QuantiTect reverse transcription kit from Qiagen. Real time PCR was performed in an IQ5 Multicolor Real-Time PCR Detection System using iQ SYBR Green Supermix (Bio-Rad). PCR amplifications were performed in triplicates, and the conditions were 95uC 200, 59uC 200, and 72uC 200 for 45 cycles. Melting curves were analyzed to confirm specific amplification of desired PCR, and the identities of final PCR products were verified by separation on agarose gels and by DNA sequencing. For quantification, standard curves were obtained by performing PCR with serial dilutions (covering 5 orders of magnitudes) of purified PCR products in salmon sperm DNA. Levels of transcripts were normalized against that of mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Cell Transfection and Report Gene Assays
For reporter gene assays, pGL3 luciferase reporter constructs together with the pRL-TK Renilla luciferase control vector were used to transfect mast cells and NIH3T3 cells. Transfection of mast cells was carried out by using the BTX Systems 600 Electro Cell Manipulator with a single 8.4 ms pulse with the setting of 800 mF, 350 V, and R1-13. Transfection of NIH3T3 cells was performed by using the Fugene 6 transfection reagent (Roche Applied Science). NIH3T3 cells were co-transfected with pcDNA or pcDNA3-Mitf-A. Luciferase activity was measured 24 hr after cell transfection by using the Dual-luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized against renilla luciferase activity.

Statistical Analysis
Statistical analyses were performed using the GraphPad Software. Differences between 2 groups of samples were assessed using t tests. p values less than 0.05 (2-tailed) are considered significant.

Identification of Markedly Increased Expressions of Mcp-2 and Mcp-4 in Mast Cells Derived from a Subpopulation of JAK2V617F Transgenic Mice
In a previous study, we generated a line of JAK2V617F transgenic mice that displayed phenotypes resembling polycythemia vera in humans [8]. The mice had an initial C57BL/66DBA/ 2 hybrid background but have been crossed with wild type C57BL/6 mice for over 10 generations. Theoretically, they have at least 99.95% C57BL/6 background. Our subsequent studies demonstrated that these mice developed pruritus associated with increased numbers of mast cells (Jin et al, unpublished). Interestingly, during our analyses of proteins extracted from cultured mast cells, we observed a very peculiar phenomenon. In Triton X-100 extracts of BMMCs from a subpopulation of transgenic mice, severe protein degradation occurred after a short incubation of cell extracts at room temperature even in the presence of protease inhibitors. Subsequently, only one major protein band with molecular size of 27 kDa was seen on SDS gels (Fig. 1A, left panel). When cells were extracted in the SDS gel sample buffer, protein degradations were eliminated but the 27 kDa band remained prominent, representing about 20% of total cellular proteins (Fig. 1A, right panel). The data suggest the presence of highly expressed proteins with possible protease activities. To identify the strongly expressed protein or proteins, in-gel trypsin digestion was conducted. This was followed by HPLC separation and MS/MS analyses. Searching of MS data against protein databases by using the Mascot search engine revealed mast cell protease Mcp-2 as by far the best hit with a score of 1612 in comparison with the second best hit actin with a score of 252. from DBA/2 mice perfectly matched those from our B6-cma mice, indicating that these variant genes indeed originated from DBA/2 mice. We further analyzed the expression of protein and chymase activity in mast cells from these mice. Protein staining, western blotting, and immunofluorescent cell staining revealed that BMMCs from DBA/2 mice showed essentially the same level of Mcp-2 and Mcp-4 overexpression as seen in B6-cma mice (Fig. 4). For comparison, we also analyzed mast cells derived from peritoneal cavity of mice and obtained similar results. Despite the strikingly different levels of Mcp-2 and Mcp-4 expressions, cultured mast cells derived from B6-cma and DBA/2 mice displayed morphologies highly similar to those obtained from control B6 mice (Fig. 4B, top panel). Chymase activity assays also showed expected results with DBA/2 and B6-cma mast cells showing much elevated activity over the control B6 mice cells (Fig. 5A). Mast cell proteases are known to be secreted upon stimulation. To verify the functionality of these overexpressed Mcp-2 and Mcp-4, we induced degranulation of mast cells derived   (Fig. 7A). When introduced into cultured BMMCs by electroporation, the Mcp-2Pv plasmid produced a significantly higher luciferase activity than the Mcp-2P plasmid, suggesting that the 3 SNPs in the promoter region contribute to the enhanced transcription activity (Fig. 7B). Although about 2fold increases in reporter gene activity are rather moderate, the data suggest that SNPs in the promoter region of Mcp-2 contribute to the increased expression of Mcp-2.
By analyzing DNA sequences in the putative promoter regions of the Mcp-2 and Mcp-4 genes, we found an extra CANNTG motif or E-box in both gene variants from B6-cma mice (see Fig. 2B). The E-box provides binding site for transcription factors including the microphthalmia-associated transcription factor (Mitf), a member of the basic helix-loop-helix leucine zipper protein family known to be involved in the regulation of mouse chymases [4]. To examine if this additional E-box facilitates Mitf transcription activity, we cloned the A isoform of Mitf from BMMCs of B6-cma mice into the pcDNA3 vector, and the cDNA insert was verified by sequencing. The Mitf-A construct was used to transfect NIH3T3 cells together with the Mcp-2P or Mcp-2Pv reporter gene constructs, and the plain pcDNA vector was used as control. Data in Fig. 7C demonstrates that co-expression of Mitf-A caused over 3-fold increases in reporter gene activity (p,0.001).    The overexpression is associated with gene variants originated from DBA/2 mice that were also found to express the chymases at the same level. We thus established useful mouse models to study the function of chymases and their implications in human diseases. B6-cma mice are superficially normal. Detailed phenotypic characterization of these mice in comparison with wild type C57BL/6 mice is under way.
Comparative studies of different strains of mice have provided important information about associations of specific genes with phenotypes. DBA/2 mice are more susceptible to the development of atherosclerosis [15][16][17]. However, the genes involved are not clear. We believe that overexpression of Mcp-2 and Mcp-4 chymases may play an important role in this process. Chymases are known as leucocyte chemoattractants and have been shown to induce apoptosis of vascular smooth muscle cells, endothelial cells, and macrophages [18][19][20][21], which all could contribute to plaque formation and stability. They can also convert angiotensin I into the proinflammatory, vasoactive angiotensin II [22]. Importantly, Mcp-4-positive mast cells are accumulated in atherosclerotic lesions, and they promote atherosclerosis by releasing proinflammatory cytokines [23]. Therefore, chymases are targets for cardiovascular diseases [24]. In fact, chymase inhibition reduces atherosclerotic plaque progression and improves plaque stability in ApoE2/2 mice [25]. Our congenic B6-cma mice thus provide an excellent system to study the involvement of chymase in atherosclerosis.
DBA/2 mice are also more susceptible to the development of autoimmune myocarditis [26,27]. This may also be related to increased activity of chymases. Murine mast cell chymases have been shown to be involved in many pathological events related to immune responses. An earlier study demonstrated a much reduced rate of autoimmune arthritis in Mcp-4 knockout mice induced by collagen and anti-collagen antibodies [28]. Mcp-2 and Mcp-4 have also been shown to play a protective role in the sepsis model induced by cecal ligation and puncture [6,29], and Mcp-4 has also been shown to protect the host from extensive allergic airway inflammation [30,31].  In consistence with the pathological role of chymase in murine models, studies have demonstrated strong associations of chymse with human diseases. Two major SNPs, one located in the promoter region and another in intron 2, have been identified. They have found to be associated with atopic skin disorders [32][33][34] and cardiovascular disease [35][36][37][38][39]. The mechanism underlying the association is not known. Our study indicates that gene variations or SNPs affect gene expression of chymases in mice. It is not known if these SNPs affect expression of the enzyme in human mast cells.
In the human genome, there is only one mast cell chymase which is encoded by the CMA1 gene, whereas mouse has 6 chymases including Mcp-1, 2, 4, 5, 9, and 10. Based on sequence similarity, Mcp-5 is the closest homolog to human chymase. However, Mcp-5 has elastase-rather than chymotrypsin-like substrate specificity [40]. Mcp-4 is the mouse chymase that has substrate specificity highly similar to that of human chymase [41,42]. Mcp-2, on the other hand, has been shown to lack enzymatic activity [43]. However, controversial results came from studies by Orinska et al demonstrating that deletion of IL-15 increases chymase activities through specific upregulation of Mcp-2 expression, but not the other chymases [6]. Therefore, further studies are needed to clarify this, and our study provided an excellent system for this.
Overexpression of Mcp-2 and Mcp-4 may be caused by altered transcription. Regulation of chymases at the transcriptional level has been extensively studied. A major transcription factor involved is Mitf that contains both basic helix-loop-helix and leucine zipper structural features. Binding of Mitf to promoter element CANNTG significantly enhanced the expression of mMCP-2, -4, -5, -6, and -9 genes in C57BL/6 mice [4]. Interestingly, our study revealed that SNPs cause generation of an extra CANNTG site in both Mcp-2 and Mcp-4 genes of the DBA/2 origin. However, reporter gene assays did not show any increased transcriptional activity due to this additional binding site (Fig. 7C). Bifunctional transcription factors C/EBPb and YY1 have also been implicated in the regulation of Mcp-2 gene expression and are thought to be responsible for the negative transcriptional regulation of Mcp-2 through intracellularly retained IL-15 [5,6]. C/EBPb is considered as a negative regulator of Mcp-2 expression. We searched consensus sequences for C/EBPb and YY1 binding sites within the putative promoter regions of Mcp-2 and Mcp-4 (see Fig. 2B). There are multiple putative C/EBPb and 4 YY1 binding sites in these regions. SNPs in the region did not change any of these sites in the Mcp-2 promoter but illuminated one consensus C/EBPb site from Mcp-4. Therefore, our analysis did not support a strong correlation of Mcp-2 expression with the C/EBPb binding. However, whether or not it contributes to the expression of Mcp-4 needs further investigation.
Expression of chymases in mast cells is controlled at the posttranscriptional and epigenetic levels. An earlier study demonstrated that the high steady-state level of the Mcp-5 transcript over those of Mcp-1, 2, and 4 is due to rapid turnover of transcribed mRNAs of the latter [7]. The transcripts of Mcp-2 and Mcp-4 but not Mcp-5 contain multiple UGXCCCC motifs in their 39-UTRs, and these potential cis-acting elements are thought to be responsible for the reduced stability of the Mcp-2 and Mcp-4 transcript [4,7]. However, analyses of coding DNA of mouse chymases in the GenBank database revealed that the 39-UTRs of Mcp-2 and Mcp-4 transcripts from DBA/2 mice has three and six such motifs, respectively, in comparison with three from each gene in C57BL/6 strain. Therefore, the difference in the numbers of Finally, the ongoing mouse genome project will provide invaluable data for us to dissect the differential regulation of chmyases in different strains of mice.

Author Contributions
Conceived and designed the experiments: ZJZ. Performed the experiments: XJ WZ KS WTH. Analyzed the data: XJ WTH ZJZ. Wrote the paper: XJ ZJZ.