Mycoplasma hyopneumoniae Induces Grap, Gadd45β, and secreted phosphoprotein 1 Gene Expression as Part of the Inflammatory Response in RAW264.7 Cells

Genes related to Mycoplasma hyopneumoniae-induced inflammation were identified using the gene-fishing technology, an improved method for identifying differentially expressed genes (DEGs) using an annealing control primer (ACP) system in RAW264.7 cells. After treatment with M. hyopneumo-niae, 16 DEGs were expressed in RAW264.7 cells using a pre-screening system. Among these 16 DEGs, 11 DEGs (DEGs 1, 4, 5–10, 12-15) were selected and sequenced directly, revealing that DEG12 (Grap), DEG14 (Gadd45), and DEG15 (secreted phosphoprotein 1) were related to inflammatory cytokines. This is the first report that intact M. hyopneumoniae induces the expression of Grap, Gadd 45β, and secreted phosphoprotein 1 in RAW264.7 cells. Subsequently, these genes may be targets for screening novel inhibitors of the mycoplasmal inflammatory response.


NTRODUCTION
Mycoplasma hyopneumoniae is the etiological agent of mycoplasmal pneumonia in swine, and has high morbidity and low mortality. The bacteria colonize ciliated respiratory epithelial cells and inhibit cell function by inducing an inflammatory response. Previously, we showed that the activation of phospholipase C (PLC) causes the loss of cilia (DeBey and Ross, 1994;Park et al., 2002), and our findings suggested that the receptors for pathogenic M. hyopneumoniae are coupled to G,o. Upon binding to these receptors, the G protein stimulates the PLC pathway to increase [Ca 2 +1 through increased Ca 2 + release from the endoplasmic reticulum (ER). We also confirmed that polyclonal antibodies produced by mycoplasma vaccination do not prevent M.
hyopneumonia -induced increases in [Ca 2 +1 i (Hwang et al., 2006). In addition, pathogenic M. hyopneumoniae induces some pro-inflammatory cytokines, including interleukin (IL)-1~, tumor necrosis factor (TN F)-a, and IL-6 in the bronchoalveolar lavage of pigs (Asai et al., Correspondence to: Seung-Chun Park, Laboratory of Veterinary Pharmacokinetics & Pharmacodynamics, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Korea E-mail: parksch@knu.ac.kr 119 1993. Because pathogenic M. hyopneumoniae induces many inflammatory cytokines, leading to chronic respiratory disease with high morbidity, it is of interest to find a new screening method for the development of novel therapeutic agents in vitro.
Many genes must be expressed in a stage-specific manner to ensure the inflammatory response. To date, however, the pathogenic functions of only a few have been characterized. Therefore, to further understand the molecular basis of inflammation, it is necessary to identify and characterize differentially expressed genes (DEGs) in detail. To identify novel genes related to mycoplasmal inflammation, we used an annealing control primer (ACP) system to investigate mRNA expression profiles in RAW264.7 cells with or without exposure to intact M. hyopneumoniae (Hwang et al., 2003.

MATERIALS AND METHODS
Mycoplasmas. Pathogenic M. hyopneumoniae was obtained from the American Type Culture Collection (ATCC 25934). The mycoplasma was cultured to the logarithmic phase in Friis medium (Friis, 1975) and harvested by centrifugation at 15,000 g for 30 min at 4°C. The mycoplasma pellets were collected and washed three times in 50 ml of cold phosphate-buffered saline (PBS), with an additional centrifugation step at 15,000 g for 15 min. The final pellets were dispersed in PBS by passing them through a 27-gauge needle. The number of whole mycoplasma cells collected from a 200-ml culture aliquot (3.4 ± 1.7 x 10 11 CCU [color changing units]/ml, n = 7) was determined using serial dilutions in tubes containing Friis medium. Cell density (1 x 10 11 CCU/ml corresponded to 0.3 mg protein/ml) was measured using the bicinchoninic acid method (Pierce, Rockford, IL).
First strand cDNA synthesis. To identify first strand cDNA synthesis, total mRNA extracted from RAW264.7 cells exposed to intact M. hyopneumoniae (0.3 mg/ml) was used to synthesize first-strand cDNAs using reverse transcriptase. Fig. 1 shows the electrophoresis of total RNA extracted from treated and non-treated RAW264.7 cells. Reverse transcription was performed for 1. addition of 80 fll of ultra-purified water for GeneFishing ™ PCR, and stored at -20°C until further use.
ACP-based GeneFishing™ PCR. DEGs were screened using an ACP-based PCR method  using GeneFishing™ DEG kits (Seegene, Seoul, Korea). Briefly, second-strand cDNA was synthesized at 50°C during one cycle of first-stage PCR in a final reaction volume of 20 fll containing 3-5 fll (about 50 ng) of diluted first-strand cDNA, 1 fll of dT-ACP2 (10 flM), 1 fll of 10 flM arbitrary ACP, and 10 fll of 2 x Master Mix (Seegene). The PCR protocol for second-strand synthesis consisted of 94°C for 1 min, followed by 50°C for 3 min, and 72°C for 1 min. After second-strand DNA synthesis was completed, the second-stage PCR amplification protocol consisted of 40 cycles of 94°C for 40 s, followed by 65°C for 40 s, and 72°C for 40 s, with a final 5-min extension at 72°C. The amplified PCR products were separated on 2% agarose gels stained with ethidium bromide.
Cloning and direct sequencing. The differentially expressed bands were re-amplified and extracted from the gel using a GENCLEAN® II Kit (Q-Bio gene, Carlsbad, CA, USA), and sequenced (one end sequencing) directly using an ABI PRISM® 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using universal primer (5'-GTC TAC CAG GCA TTC GCT TCA T-3'). The sequences were analyzed using BLASTX search program at the National Center for Biotechnology Information (NCBI) GenBank.

RESULTS
To identify DEGs related to the inflammatory response by intact pathogenic M. hyopneumoniae in RAW264.7 cells, we compared mRNA expression profiles in RAW264.7 cells with or without exposure to intact M. hyopneumoniae for 18 h. Total RNAs were isolated from treated and non-treated cells and the RNA concentration was 0.69 and 0.72 flg/ml in the non-treated and treated RAW264.7 cells, respectively, and it was used for the first-strand cDNA synthesis (Fig. 1). Using 20 arbitrary ACP primers for the first-strand cDNA from intact form-treated cells, DEGs were screened using an ACP-based PCR method (Kim et a/., 2004b) using GeneFishing™ DEG kits (Seegene, Seoul, Korea).
were down-regulated and 6 genes (DEG 1, 7, 12, 13, 14, and 15) were up-regulated in M. hyopneumoniaetreated RAW264.7 cells (Fig. 2). 11 obvious bands were selected to identify DEGs related to the inflammatory response in RAW264.7 cells (Fig. 3). The DEGs were re-amplified and extracted from the gel and directly sequenced by using kit described above. Sequence analysis showed that nine DEGs were known genes (Table 1). Although DEG 6 and DEG 9 had not been characterized, their fUll-length cDNA or expressed sequence tag (EST) sequence had been deposited in the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/; Table 1).  Fig. 2. Bands from the secondary extraction were sequenced directly and the results were summarized in Table 1. using the ACP-based differential display RT-PCR (Gen-eFishing DEG screening technology). Olignucletide and cDNA microarray methods can be used for large scale gene expression analysis of thousands of genes at a time, however, real-time RT-PCR assay is more accurate quantitative analysis of small numbers of candidate genes. The ACP-based RT-PCR technology which has recently been developed has provided new insights into the efficacious analysis of gene expression between tissues and cell lines. This method substantially improved the specificity and sensitivity of PCR through elimination of false positives and poor reproducibility of previous DEG discovery methods such as cDNA microarray, thereby allowing real products to be amplified (Hwang et al., 2003;Kim et al., 2004b).

DISCUSSION
highly expressed. Of these DEGs, EST and sequence analyses suggested that DEG12, DEG14, and DEG15 are isomers of Grap, growth arrest and DNA-damageinducible 45 gamma (Gadd45y), and secreted phosphoprotein 1 or osteopontin, respectively. The remaining DEGs observed during the inflammatory response have not been characterized in detail. Shiraiwa et al. (2004) reported that the Grap gene might have a role in the pathogenesis of Sjogren's syndrome, an autoimmune disease in which the body's immune system mistakenly attacks its own moistureproducing glands (Shiraiwa et al., 2004). Shen et al. (2002) showed that Grap inhibited cell proliferation, IL-2 production, and c-fos induction in response to mitogenic T-cell receptor (TCR) stimulation mediated by the Ras/Erk pathway, but not by JNKlSAPK or p38 mitogen-activated protein (MAP) kinase pathways (Shen et After treatment with intact M. hyopneumoniae, DEG1, DEG7, DEG12, DEG13, DEG14, and DEG15 were al., 2002).
To our knowledge, no data are available regarding the role of Grap in bacterial pathogenesis. Fig. 1 show that Grap was strongly expressed in M. hyopneumoniaetreated RAW264.7 cells, suggesting that Grap expression is a good indicator for the initial screening of bioactive materials to prevent M. hyopneumoniae infection.
Unlike Grap, Gadd45~ activates TCR signaling or inflammatory cytokines in T cells, such as IL-12 and IL-18, by activating the p38 and JNKlSAPK MAP kinase pathways (Lu et al., 2001). Gadd45~-deficient mice showed an impaired T helper type 1 response to Listeria monocytogenes infection (Lu et al., 2004a). Based on these reports, Gadd45~ may also be activated in the acute inflammatory response to bacterial infection, which is consistent with our results showing that pathogenic M. hyopneumoniae activates the Gadd45~ gene in RAW264.7 cells.
Mycoplamsa could induce the up-regulation of secreted phosphoprotein 1 (Osteopontin). Osteopontin plays an important role in lung pathologies, such as abnormal pulmonary granuloma formation (O'Regan et al., 2001). In addition, Serlin et al. (2006) reported that IL-1~ induces osteopontin expression in pulmonary fibroblasts (Serlin et al., 2006). Recently, Kohan et al. (2007) reported that osteopontin expression in the lungs increased in a murine model of allergen-induced chronic airway remodeling, suggesting a role for this cytokine in airway remodeling in asthma (Kohan et al., 2007). In a previous report (Hwang et al., 2008b), it was found that intact pathogenic M. hyopneumoniae induced the transcription of pro-inflammatory cytokines, such as cyclooxygenase (COX)-2, TNF-a, IL-1~ , IL-6, and inducible NO synthase (iNOS) in RAW264.7 cells (Hwang et al., 2008b). In the same cells, mycoplasma induced osteopontin gene expression, suggesting that IL-1 ~ induces the osteopontin gene, as do pulmonary fibroblasts. Therefore, this gene may also be a candidate for screening for inhibitors of M. hyopneumoniae infection.
In conclusion, we have searched for differentially expressed anti-inflammatory genes that are induced in RAW264.7 cells after exposure to intact M. hyopneumoniae. Our findings suggest that DEG 12 (Grap), DEG 14 (Gadd45fJ), and DEG 15 (secreted phosphoprotein 1) are attractive novel target genes for screening anti-inflammatory agents in M. hyopneumoniae-treated RAW264.7 cells.