Role of Protein Tyrosine Phosphatase Non-Receptor Type 7 in the Regulation of TNF-α Production in RAW 264.7 Macrophages

Protein tyrosine phosphatases play key roles in a diverse range of cellular processes such as differentiation, cell proliferation, apoptosis, immunological signaling, and cytoskeletal function. Protein tyrosine phosphatase non-receptor type 7 (PTPN7), a member of the phosphatase family, specifically inactivates mitogen-activated protein kinases (MAPKs). Here, we report that PTPN7 acts as a regulator of pro-inflammatory TNF-α production in RAW 264.7 cells that are stimulated with lipopolysaccharide (LPS) that acts as an endotoxin and elicits strong immune responses in animals. Stimulation of RAW 264.7 cells with LPS leads to a transient decrease in the levels of PTPN7 mRNA and protein. The overexpression of PTPN7 inhibits LPS-stimulated production of TNF-α. In addition, small interfering RNA (siRNA) analysis showed that knock-down of PTPN7 in RAW 264.7 cells increased TNF-α production. PTPN7 has a negative regulatory function to extracellular signal regulated kinase 1/2 (ERK1/2) and p38 that increase LPS-induced TNF-α production in macrophages. Thus, our data presents PTPN7 as a negative regulator of TNF-α expression and the inflammatory response in macrophages.


Introduction
Protein phosphorylation is a critical event in signal transduction, which regulates fundamental cellular processes such as differentiation, cell proliferation, apoptosis, immunological signaling, and cytoskeletal function [1]. Protein phosphorylation is regulated by the opposing actions of kinases and phosphatases, and, importantly, provides a means of regulating protein function. The regulated expression and activity of several protein tyrosine phosphatases (PTPs) in cells, in turn, control the duration and intensity of the activity of mitogen-activated protein kinase (MAPK), which determines the type of physiological response. The MAPK subfamily, including the c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERK), and p38, act as key inflammatory mediators in the mammalian innate immune system response [2,3]. In particular, the phosphorylation of MAPKs plays a critical role in the inflammatory response [4]. When stimulated with lipopolysaccharide (LPS), innate immune cells like macrophages release pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-a), interleukin-6 (IL-6), interleukin-12 (IL-12), monocyte chemotactic protein-1 (MCP-1), interferon-gamma (IFN-c), and interleukin-10 (IL-10), through complex signaling mechanisms [5,6]. The regulation of TNF-a expression is mediated by the adenosine/uridine-rich element (ARE) in the 39-untranslated region of the TNF-a mRNA that represses TNF-a expression post-transcriptionally [7,8]. MAPKs like p38, JNK, and ERK have been shown to target this ARE to increase TNF-a expression in response to LPS stimulation [9].
PTP-deficient mouse models have been used to identify the role of individual PTPs immune response regulation [10]. In LPSstimulated RAW 264.7 cells, the activity of dual-specificity phosphatase 1 (DUSP1) protein increases dramatically, reaching its maximal level between 1 and 2 h, and then decreasing thereafter [11]. Dusp1 2/2 macrophages have elevated p38 and JNK activity but unchanged ERK activity [12,13]. DUSP1 specifically inactivates JNK and p38 in vitro by dephosphorylating both phospho-Thr and phospho-Tyr residues of these kinases. Other DUSP members have also been identified as regulators of inflammation in innate immune cells [3]. We have previously shown that DUSP26, PTPRE, and PTPN3 are involved in the regulation of LPS-mediated inflammation [14,15,16]. While the mRNA levels of DUSP26 and PTPRE do not change after LPS treatment, PTPN3 mRNA levels increased rapidly after treatment [14,15,16]. Nonetheless, overexpression of these PTPs inhibits TNF-a production in RAW 264.7 cells and, therefore, may act as anti-inflammatory regulators.
In this study, we show that PTPN7 expression changes upon stimulation with LPS and that PTPN7 regulates TNF-a production in RAW 264.7 cells.

Cell Culture and Transfection
Mouse macrophage-like RAW 264.7 cells were maintained at 37uC in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad California) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and penicillin/streptomycin in the presence of 5% CO 2 . For transient transfection, 1.4610 6 cells were plated in each 60-mm cell culture plate, grown overnight, and transfected with DNA using FuGENE HD transfection reagent (Roche, Basel, Switzerland) according to the manufacturer's instructions.

Plasmid Construction
The N-terminal FLAG-tagged PTPN7 plasmid for expression in mammalian cells was constructed by inserting an NdeI-BamHI cDNA amplified by polymerase chain reaction into NdeI-BamHI
Enzyme Linked Immunosorbent Assay (ELISA) of TNF-a TNF-a protein concentrations were determined by standard sandwich ELISA using antibodies and standards obtained from BD Biosciences (San Diego, CA) and used according to manufacturer's instructions. Assays were performed on neat and diluted samples in duplicate on 96-well plates. Absorbance was measured by a microplate reader at 450 nm and concentrations were determined by comparison to a standard curve. All experiments were repeated at least three times.

In vitro Protein Phosphatase Assays
For in vitro phosphatase assays, RAW 264.7 cells were treated with or without LPS as described above and then harvested in PTP lysis buffer (0.5% NP-40, 0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% glycerol, 1 mM PMSF, and 1 mg/ml aprotinin) for 30 min at 4uC. Cleared cell lysates from centrifugation were immunoprecipitated with rabbit anti-PTPN7 or anti-IgG antibodies (Santa Cruz Biotechnology) followed by incubation with protein A/G agarose for 16 h at 4uC using rotation device. After incubation, immunoprecipi-

Immunoblotting Analysis
Samples were run in SDS-10% polyacrylamide gels and transferred to nitrocellulose membrane. The membrane was blocked in 5% nonfat skim milk and incubated with an appropriate antibody, followed by incubation with a secondary antibody conjugated to horseradish peroxidase. The immunoreactive bands were visualized using an ECL system (Pierce, Rockford, IL).

Reverse Transcription-polymerase Chain Reaction (RT-PCR)
Total RNAs were prepared from cells by Trizol (Roche) and reverse transcription was performed using M-MLV (Invitrogen). PCR for mouse PTPs was carried out using the primers listed in Table 1.

Quantitative Real-time PCR (qRT-PCR)
Total RNAs were isolated from RAW 264.7 cells by Accuzol (Bioneer Corporation, South Korea). cDNA amplification and polymerase chain reaction (PCR) were performed with Omnis-criptH RT Kit (Qiagen, Hilden, Germany) and iTaq TM Universal SYBRH Green Supermix (Bio-Rad, Hercules, CA). The following primer sets were used: PTPN7 mRNA (forward, 59-CAGAGA-CAGCTGCCAACTCCGG-39; reverse, 59-CCAT- . The data obtained from qRT-PCR were quantified using the comparative threshold (2 2[DDCt] ) method (Ct represents cycle threshold). Relative mRNA expression levels were normalized with GAPDH. The Ct value for each primer pairs was obtained from samples and averaged. Delta Ct value represented the calculated difference between the average Ct for the PTPN7 and the average Ct for GAPDH as the control for total starting RNA quantity. The delta-delta Ct method of calculation was the used to assess fold-change in gene expression relative to GAPDH gene.

PTPN7 Expression in Macrophages is Transiently Downregulated by LPS
Signaling pathways in innate immune cells are rapidly activated by phosphorylation, inducing a pro-inflammatory cytokine response to LPS of gram-negative bacteria, and are then downregulated by the dephosphorylation of signaling components, suggesting that phosphatases are involved in the regulation of inflammation. Therefore, transient regulation of phosphatases during LPS-mediated signaling might be critical for controlling kinase activity. We performed RT-PCR using gene-specific primers against the PTP genes using RNA purified from RAW 264.7 cells, stimulated with LPS for 1 or 3 h (Table 1). Among several PTPs found to be differentially expressed in response to LPS, PTPN7 was one whose expression was regulated upon exposure to LPS (Fig. 1). Interestingly, unlike other regulatory PTPs that were induced by LPS, PTPN7 expression was suppressed ( Table 2). PTPN7 expression decreased within 1 h, and then returned to a near basal level by 3 h of LPS stimulation. Using real-time-PCR, we further examined the kinetics of PTPN7 mRNA suppression upon exposure to LPS. Expression of PTPN7 mRNA was clearly suppressed when cells were treated with LPS for 2 h (Fig. 2), but returned to near basal levels when cells were treated with LPS for 24 h (Fig. 2A). When PTPN7 protein expression levels were analyzed by immunoblotting analysis using an anti-PTPN7 antibody, the protein expression pattern was similar to the data obtained from RT-PCR, even though protein expression seemed to be slightly delayed (Fig. 2B). Unlike PTPN7, DUSP1 was induced at early times of LPS treatment and then returned to basal levels after 2 h of LPS treatment. When the endogenous PTPN7 was immunoprecipitated from cells treated with LPS and its phosphatase activities were measured, PTPN7 phosphatase activity decreases in proportion to the decrease of PTPN7 expression (Fig. 2C). Taken together, these results suggest that LPS decreases expression of PTPN7 at an early stage of LPS treatment.

TNF-a Production is Dependent on PTPN7 Expression Levels
TNF-a production is one of the earliest pro-inflammatory responses from macrophages when stimulated by endotoxins such as LPS. Since LPS regulates PTPN7 expression in RAW 264.7 cells, we hypothesized that PTPN7 might be involved in the regulation of TNF-a expression. To investigate the effect of PTPN7 on TNF-a expression, we measured the time point changes of TNF-a and PTPN7 in response to LPS (Fig. 3A). TNFa levels increased sharply at early times of LPS treatment, then increased gradually and reached maximal point at 8 h after LPS treatment. The increase in TNF-a levels after 4 h of treatment coincides with the decrease in PTPN7 levels (Fig. 2B). To assess the effects of PTPN7 expression on TNF-a production, we analyzed the levels of TNF-a in RAW 264.7 cells transfected with a plasmid which coded for PTPN7 expression. RAW 264.7 cells transfected with a mammalian vector expressing FLAG-tagged PTPN7 were treated with 1 mg/ml LPS, and the effect of increased PTPN7 expression on TNF-a secretion in the medium was examined by ELISA. Compared to cells transfected with an empty plasmid, cells expressing FLAG-PTPN7 exhibited a 90% reduction in TNF-a production in response to LPS stimulation (Fig. 3B). To confirm that PTPN7 regulates TNF-a production, PTPN7-specific small interfering RNAs (siRNAs) were transfected into RAW 264.7 cells to knock-down PTPN7. Reduced levels of PTPN7 expression were confirmed by immunoblotting analysis (Fig. 3C). Following transfection with either non-targeting control siRNA or PTPN7-specific siRNAs (#1 and #2), RAW 264.7 cells were treated with LPS, and TNF-a levels in the medium were measured (Fig. 3D). As shown in Fig. 3D, knock-down of PTPN7 enhanced LPS-induced TNF-a production in RAW 264.7 cells. Taken together, these results suggest that PTPN7 inhibits LPSstimulated TNF-a induction in macrophages.

PTPN7 Regulates MAPK Signal Transduction
Since PTPN7 has been known to selectively dephosphorylate a phospho-tyrosine residue on the activation loop of the MAPKs ERK1/2 and p38 [25,26], we examined the effect of PTPN7 on MAPK signaling in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells transfected with either FLAG-tagged PTPN7 or empty plasmids were stimulated with LPS, and endogenous levels of phospho-JNK, phospho-p38, and phospho-ERK1/2 were assessed by immunoblotting with phospho-ERK1/2, phospho-JNK, and phospho-p38 antibodies. Compared to cells transfected with empty plasmid, the levels of phospho-ERK1/2 and phospho-p38 were reduced in PTPN7-transfected cells, whereas levels of phosphorylated JNK were not changed (Fig. 4A). In contrast, knock-down of PTPN7 enhanced levels of phopho-ERK1/2 and phopho-p38 in LPS-stimulated cells (Fig. 4B). Taken together, these results indicate that the activities of ERK1/2 and p38 were down-regulated by PTPN7 in LPS-stimulated macrophages. Therefore PTPN7 acts as a negative regulator of MAPKs, and particularly of ERK1/2 and p38 in macrophages.
The expression of PTPN7 was dramatically diminished soon after LPS stimulation, but gradually recovered to normal levels. Down-regulation of PTPN7 leads to the production of TNF-a, and an increase in MAPK phosphorylation in LPS-treated cells. However, unlike PTPN7, expression of other phosphatases, such as DUSP1, was enhanced in response to LPS stimulation. DUSP1, which was induced within 1 to 2 h of LPS treatment, downregulated JNK and p38 activities and TNF-a production in RAW 264.7 cells [11]. Therefore, both PTPN7 and DUSP1 inactivate MAPK signaling, even though there is a slight difference in substrate specificities. However, the expression patterns of these phosphatases in response to LPS are opposite: PTPN7 is suppressed after 2 h of LPS treatment while DUSP1 is induced at early times of LPS treatment. Why cells express conflicting responses to LPS is not yet clear. One possible explanation may be that PTPN7 has a role as an intrinsic inhibitor of MAPK signaling, and its down-regulation activates MAPK for TNF-a production. It is also possible that the kinetics of PTPN7 gene suppression might be different from that of DUSP1 gene induction in response to LPS as shown in Fig. 2B. Differential and time-dependent expression of these PTPs might be important to regulate inflammation. Another possibility is that other substrates for PTPN7 besides ERK and p38 might be involved in PTPN7-mediated inflammatory responses.
In summary, the present study shows that one important function of PTPN7 is the regulation of the inflammatory response in mouse macrophage cells. PTPN7 expression was potently reduced at early time points after LPS stimulation in RAW 264.7 cells, and is an important regulator of TNF-a production. PTPN7 also functions as a negative regulator of MAPK activity, especially of ERK1/2 and p38. Based on these data, we suggest that PTPN7 is one of key regulators of TNF-a expression and MAPK signal transduction in the inflammatory response of macrophages.