Protein tyrosine phosphatase PTP-RR regulates corticosteroid sensitivity

Background We have recently reported that protein phosphate 2A (PP2A) inactivation resulted in increased phosphorylation of the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase 1 (JNK1) and glucocorticoid receptors (GR) at Ser226, thereby reducing GR nuclear translocation and causing corticosteroid insensitivity in severe asthmatics. Protein tyrosine phosphatases (PTPs) are also known to be critically involved in the regulation of MAPKs, such as JNK and therefore potentially associated with GR function. The aim of study was to elucidate the involvement of MAPK-PTPs (PTP-RR, PTP-N5 and PTP-N7), which can dephosphorylate MAPKs, in the regulation of corticosteroid sensitivity. Methods Corticosteroid sensitivity, GR nuclear translocation, phosphorylation levels of GR-Ser226, JNK1 and PP2A catalytic subunit (PP2AC)-Tyr307 and protein expression levels and activities of PTP-RR and PP2AC were evaluated in U937 cells and/or peripheral blood mononuclear cells (PBMCs). Knock-down effects of MAPK-PTPs using siRNA were also evaluated. Results Knock-down of PTP-RR, but not of PTP-N5 or PTP-N7 impaired corticosteroid sensitivity, induced GR-Ser226 phosphorylation and reduced GR nuclear translocation. Under IL-2/IL-4-induced corticosteroid insensitivity, PTP-RR expression, activity and associations with JNK1 and GR were reduced but PTP-RR activity was restored by formoterol. Also in PBMCs from severe asthmatic patients, PTP-RR and JNK1 expression were reduced and GR-Ser226 phosphorylation increased. Furthermore, PTP-RR was associated with PP2A. PTP-RR reduction enhanced PP2AC-Tyr307 phosphorylation leading to impairment of PP2A expression and activity. Conclusions We demonstrated that with corticosteroid insensitivity PTP-RR fails to reduce phosphorylation of JNK1 and GR-Ser226, resulting in down-regulation of GR nuclear translocation. Reduced PTP-RR may represent a novel cause of corticosteroid insensitivity in severe asthmatics. Electronic supplementary material The online version of this article (doi:10.1186/s12931-016-0349-0) contains supplementary material, which is available to authorized users.


Background
Currently most patients with asthma are well controlled with the regular use of inhaled corticosteroids (ICS) with or without long-acting β 2 -adrenoceptor agonists (LABAs). Even though corticosteroids exert multifaceted anti-inflammatory effects, they are not disease-modifying and poorly effective in severe asthmatics who present a more corticosteroid insensitive phenotype [1]. Hence, elucidating the molecular mechanisms of corticosteroid insensitivity could lead a more effective therapeutic approach for severe asthma.
Defective function of glucocorticoid receptors (GR), such as decreased nuclear translocation, protein expression, ligand binding and binding to glucocorticoidresponse elements (GRE) may be involved in corticosteroid insensitivity in severe asthma [2]. One possible cause of impaired GR nuclear translocation is the phosphorylation of GR by mitogen-activated protein kinases (MAPKs) [2][3][4][5]. In patients with severe asthma increased phosphorylation of GR and subsequent reduced nuclear translocation have been reported [2,5,6]. However, the molecular mechanisms of GR phosphorylation that lead to GR nuclear translocation have not yet been elucidated. Phosphorylation of Ser 203 and Ser 211 have been shown to induce GR activation [7][8][9] whereas Ser 226 phosphorylation leads to inhibition of GR function [10][11][12]. Recently, we found that GR is hyperphosphorylated at Ser 226 possibly via the action of c-Jun N-terminal kinase 1 (JNK1) in peripheral blood mononuclear cells (PBMCs) from severe asthmatics and in a corticosteroid insensitive PBMC model [13]. This is supported by previous findings that phosphorylation of Ser 226 by JNK contributes to impairment of GR transcriptional activation [11] and GR shuttling [10]. Thus, GR-Ser 226 phosphorylation might be involved in reduction of GR nuclear translocation.
Protein tyrosine phosphatases (PTPs) which regulate the level of tyrosine phosphorylation play a critical role in regulating signaling molecules such as mitogenactivated protein kinases (MAPKs) [14]. At least 107 PTPs are now recognized and they are classified into four groups based on the amino acid sequences of their catalytic domains [15,16]. Tyrosine-specific classical PTPs which are classified into class I cysteine-based PTPs are further divided into two subgroups: receptorlike transmembrane PTPs; R1-R8 and non-transmembrane PTPs; NT1-NT9 [14]. The R7, one of receptor-like PTPs subgroup, termed 'MAPK-PTPs' , consists of PTP-N5 [also known as striatal-enriched phosphatase (STEP)], PTP-N7 [also known as hematopoietic PTP (HePTP)] and PTP-RR [also known as PCPTP1 or STEP-like PTP (PTP-SL)]. PTP-N5, PTP-N7 and PTP-RR have all been reported to down-regulate MAPKs [17]. Among dual-specific phosphatases (DUSPs), another class I cysteine-based PTPs, MAPK phosphatases such as DUSP1 and DUSP4 are also involved in the regulation of MAPKs [16].
We have recently found that protein phosphatase 2A (PP2A), a serine/threonine phosphatase, regulates corticosteroid sensitivity by dephosphorylation of JNK1 and GR-Ser 226 , and PP2A located in the cell membrane/cytoplasm may be inactivated by phosphorylation of Tyr 307 [13,18], suggesting that other PTPs may be also involved in the regulation of corticosteroid sensitivity. We therefore hypothesized that reduction of MAPK-PTPs might also enhance JNK1 and GR-Ser 226 phosphorylation resulting in impairment of GR nuclear translocation and corticosteroid sensitivity. In this study, we focus on 'R7 PTPs (PTP-N5, −N7 and -RR)' , but not other MAPK phoshatases (DUSPs) which are mainly localized in the nucleus [19], and show that PTP-RR is down-regulated in PBMCs from severe asthmatics and in an in vitro corticosteroid insensitive cellular model. In addition, knockdown of PTP-RR (using siRNA) reduced GR nuclear translocation and corticosteroid sensitivity concomitantly with enhanced phosphorylation of JNK1 and GR-Ser 226 .

Subjects
PBMCs obtained from 10 patients with severe asthma (SA) [20,21] and 8 age-matched healthy volunteers (HV) from the Royal Brompton Hospital (London, UK) were isolated from whole blood using AccuSPIN (Sigma-Aldrich). As our previous data showed corticosteroid sensitivity in PBMCs from non-severe asthmatics was normal range [4,5] without reduction of PP2A activity (unpublished data), we focused on the comparison between HV and SA in this study. Six of 10 subjects in SA and 5 of 8 subjects in HV were analyzed from samples used previously [13]. The characteristics of subjects are shown in Table 1. The severity of asthma was defined by GINA guideline [22]. All patients and healthy volunteers recruited for this study were never smokers. This study was approved by the local ethics committee of Royal Brompton and Harefield NHS Trust (07_Q0404_31) and written informed consent was obtained from each patient or volunteer.

Cells
The human monocytic cell line U937 was obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells were cultured in complete growth medium (RPMI 1640; Sigma) supplemented with 10 % fetal bovine serum (FBS) and 1 % L-glutamine at 37°C in a humidified atmosphere with 5 % CO 2 . Cell viability was assessed microscopically by trypan blue staining.

Cell lysis, immunoprecipiation, and western blotting
Cell protein extracts were prepared using modified RIPA buffer (50 mM Tris HCL pH 7.4, 0.5-1.0 % NP-40, 0.25 % Na-deoxycholate, 150 mM NaCl with freshly added complete protease (Roche, Mannheim, Germany)), as described previously [13]. Phosphatase inhibitor (Active Motif, Rixensart, Belgium) and MG-132, a proteasome inhibitor, were used as needed. Nuclear extraction was performed using the Nuclear Extraction Active Motif kit (Active Motif ). Protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad). Immunoprecipitation was conducted with anti-PTP-RR Ab (Abcam) or anti-PP2A Ab (Bethyl, Montgomery, TX). Protein extracts or immunoprecipitates were separated by SDS-PAGE (Invitrogen, Paisley, UK) and detected by Western Blot analysis using chemiluminescence (ECL Plus; GE Healthcare, Chalfont St. Giles, UK). β-actin expression was used as a control for protein loading as needed.

Corticosteroid sensitivity
Cells were treated with dexamethasone (10 −6 M or 10 −11 M) for 45 min, followed by TNFα (10 ng/ml) stimulation overnight. The ability of dexamethasone to inhibit TNFα-induced CXCL8 release was determined in cell medium by sandwich ELISA according to the manufacturer's instructions (R&D Systems Europe). IC 50 values for dexamethasone on CXCL8 release were calculated using the computer program Prism 4.0 (GraphPad Software Inc., San Diego, CA) and were used as a marker for steroid sensitivity (Dex-IC 50 ).

Glucocorticoid receptor nuclear translocation
Cells were treated with dexamethasone (10 −7 M) for 1 h. Nuclear and cytoplasmic GR were measured by Western Blot. TATA-binding protein (TBP) (for nuclear protein) or α-tubulin (for cytoplasmic protein) expression was used as a control for protein loading. The ratio of nuclear GR to TBP was used as an index of GR nuclear translocation.

Protein phosphatase activity
Phosphatase activity was assayed by using the Senso-Lyte™ MFP Protein Phosphatase Assay Kit (AnaSpec, San Jose, CA) [13]. Immunopurified PTP-RR or PP2A were added into assay buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM DTT for PTP-RR; 40 mM Tris HCl pH 8.4, 34 mM MgCl 2 , 4 mM EDTA, 4 mM DTT for PP2A) and incubated with 3-Omethylfluorescein phosphate (MFP). Phosphatase activity was measured as a phosphatase-induced dephosphorylation which was monitored by measuring the fluorescence of MFP product. PTP-RR or PP2A immunoprecipitates were equally divided into two parts, with half used for phosphatase assay and the other half for target protein detection by Western Blot analysis. Phosphatase activity was normalized to the protein expression level calculated as band-density.

Statistical analysis
Comparisons of two groups of data were performed using Mann-Whitney U test or paired t-test. Pearson's or Spearman's correlation coefficients were also calculated. Other data were analyzed by ANOVA with post hoc test adjusted for multiple comparisons (Bonferroni test or Newman-Keuls test), as appropriate. The difference was considered statistically significant if P < 0.05. Descriptive statistics were expressed as the mean ± SEM.

PTP-RR regulates PP2A function
Co-immunoprecipitation studies showed that PTP-RR is also associated with PP2A, suggesting a possible role of PTP-RR in the regulation of corticosteroid sensitivity by dephosphorylation of JNK1 and GR-Ser 226 as shown previously for PP2A [13]. In fact, the association between PTP-RR and PP2A was reduced by 48 % after costimulation with IL-2/IL-4 (Fig. 3a). Additionally, PTP-RR KD enhanced PP2A catalytic subunit (PP2A C )-Tyr 307 phosphorylation (Fig. 3b), which is required for downregulation of PP2A activity [24]. In addition, reduced PP2A activity and protein expression were observed after PTP-RR KD ( Fig. 3c and d). On the other hand, PP2A KD did not change PTP-RR activity or expression ( Fig. 3e and f ).

PTP-RR is reduced in PBMCs from severe asthmatics
Consistent with the IL-2/IL-4-induced corticosteroid insensitive U937 model, PTP-RR expression in PBMCs from severe asthmatics was significantly lower than those from healthy volunteers (Fig. 4a). In line with the findings observed in PTP-RR KD study, PTP-RR expression levels in PBMCs from severe asthmatics and healthy volunteers negatively correlated with phosphorylation levels of PP2A C -Tyr 307 and positively correlated with PP2A activity and expression (panel (i) to (iii) in Fig. 4b). Previously, we have shown that GR from severe asthmatic patients is hyperphosphorylated at Ser 226 and associated with increased JNK1 phosphorylation [13]. Interestingly, we found in the present study that PTP-RR expression levels in PBMCs were negatively correlated with phosphorylation levels of GR-Ser 226 and JNK1, suggesting an association between PTP-RR and the GR/JNK1 complex (panel (iv) and (v) in Fig. 4b). Regarding PTP-RR activity in PBMCs, we could not collect enough samples from patients because a lot of PBMCs were needed to detect the phosphatase activity in IP-PTP-RR. We confirmed that co-stimulation with IL-2/ IL-4 reduced IP-PTP-RR activity in PBMCs from healthy volunteers (by up to 30 %) as observed in U937 cells (data not shown).

Discussion
Our study has revealed a novel role for PTP-RR in the regulation of corticosteroid sensitivity by modulating GR nuclear translocation after corticosteroid treatment. Importantly, we confirmed that PTP-RR is down-regulated in PBMCs from severe asthmatics and in an in vitro corticosteroid insensitive cellular model induced by costimulation with IL-2/IL-4. In this model, PTP-RR was dissociated from GR/JNK1 complex leading to hyperphosphorylation of JNK1 and GR-Ser 226 . In addition, we found that impaired PTP-RR causes down-regulation of PP2A, which is also involved in JNK1 and GR-Ser 226 phosphorylation by favoring of PP2A C -Tyr 307 phosphorylation. Thus, it seems that PTP-RR might regulate corticosteroid sensitivity both directly and indirectly via modulation of PP2A activity (Fig. 6).
Reduction of GR nuclear translocation is one of causes of corticosteroid insensitivity in severe asthma [2,4]. In fact, dexamethasone-induced translocation of GR into the nucleus is reduced in PBMCs from patients with severe asthma and this correlates with other indices of defective corticosteroid sensitivity in these patients [6]. Also in IL-2/IL-4-induced corticosteroid insensitive model, reduced GR nuclear translocation has been reported [4,23]. We demonstrated that knocking-down PTP-RR reduces GR nuclear translocation with concomitant induction of corticosteroid insensitivity, and that PTP-RR expression was reduced in severe asthma and in the IL-2/IL-4-induced corticosteroid insensitive model. Our results suggest that PTP-RR might be a key molecule involved in the regulation of GR nuclear translocation in severe asthma.
Inhibition of PTP-RR by knockdown and coimmunoprecipitation studies indicated that PTP-RR is present in a GR/JNK1 complex and associated with dephosphorylation of GR-Ser 226 and JNK1. We also examined the involvement of other MAPK-PTPs, PTP-N5 and PTP-N7 in phosphorylation of JNK1 and GR-Ser 226 . In U937 cells that were knocked down for PTP-N5 or PTP-N7, the phosphorylation levels of JNK1 were also enhanced, however neither PTP-N5 nor PTP-N7 knockdowns increased GR-Ser 226 phosphorylation. SP600125, a JNK inhibitor partially reduced phosphorylation of GR-Ser 226 in IL-2/IL-4-induced corticosteroid insensitive model [18], suggesting that other signals (kinases and/or phosphatases) may be involved in PTP-RR-mediated regulation of GR-Ser 226 phosphorylation.
It has been reported that PTP-RR inactivates extracellular signal-regulated kinase (ERK)1/2 by dephosphorylation of tyrosine residues [25] and that PTP-RR associates with ERK1/2 and p38 through a kinase interaction motif (KIM) required for phosphorylation of PTP-RR by these kinases [26]. However, it was not previously established whether PTP-RR could dephosphorylate JNK1. We have recently found that a serine/ threonine phosphatase, protein phosphatase 2A (PP2A) also dephosphorylates JNK1 and GR-Ser 226 [13]. As we have shown an association between PTP-RR and PP2A (Fig. 3a), PTP-RR might dephosphorylate JNK1 and GR-Ser 226 in collaboration with PP2A. Another possibility is that PTP-RR indirectly regulates phosphorylation levels of JNK1 and GR-Ser 226 via PP2A. PP2A catalytic subunit, a regulator of PP2A complexes and activity [27], is phosphorylated at Tyr 307 residue [28], resulting in reduction of PP2A activity [24]. We confirmed that increased phosphorylation of PP2A C -Tyr 307 after knockdown of PTP-RR, but not of PTP-N5 and PTP-N7 leads to a decrease in PP2A activity (Fig. 3b, c, and Additional file 1: Figure S1). On the other hand, PP2A knockdown did not affect PTP-RR activity, expression ( Fig. 3e and f ) and association with JNK1 (data not shown), suggesting that PTP-RR could be associated with JNK1, independently of PP2A. Thus, PTP-RR, but not PTP-N5 and PTP-N7 regulates dephosphorylation of JNK1 and GR-Ser 226 directly and/or indirectly via PP2A. Regarding the mechanism of PTP-RR down-regulation, MG-132, a proteasome inhibitor, abrogated IL-2/IL-4-induced reduction of PTP-RR protein expression, suggesting that a reduction in PTP-RR protein stability in severe asthma could be taking place. Recently, several studies demonstrated Tyr 307 phosphorylation of PP2A is regulated via protein tyrosine phosphatase 1B (PTP1B) [29,30]. Together with our findings, the conclusion can be drawn that several phosphatases with the ability to dephosphorylate tyrosine residues might control cellular functions in collaboration with PP2A.
PTP-RR is expressed predominantly in brain, and regulates cerebellar neurons and motor coordination [31]. In addition, PTP-RR has been also detected in other nonneuronal tissues, such as cartilage and intestinal mucosa, and regulates bone morphogenesis and tumor genesis, respectively [32,33]. Importantly, it seems that PTP-RR negatively regulates tumor progression in neoplastic disorders such as cervical cancer or colorectal cancer [32,34]. We have revealed for the first time that PTP-RR is also expressed in PBMCs and the U937 human monocytic cell line, and shows reduced expression in severe asthma and IL-2/IL-4-induced corticosteroid insensitivity, respectively. This suggests that PTP-RR might be a potential novel therapeutic target for various diseases with reduced corticosteroid sensitivity including severe asthma. PTP-RR was activated by formoterol although this effect was independent of the β 2 -adrenoceptor function. We have previously observed that formoterol was able to restore PP2A activity independently of β 2 -adrenoceptor, however the mechanism of action remains unknown [18]. Furthermore, as LABAs are reported to provide beneficial effects in exacerbation of allergic airway disease by activating PP2A [35], PTP-RR activation by LABAs may also contribute to their beneficial effects on asthma control.

Conclusions
We have reported for the first time that PTP-RR is reduced in severe asthmatics and that in corticosteroid insensitive condition, impaired PTP-RR causes phosphorylation of JNK1 and GR-Ser 226 directly and/or indirectly via PP2A, resulting in less corticosteroidinduced nuclear translocation of GR. Thus, PTP-RR is a novel therapeutic target for the restoration of corticosteroid sensitivity in severe asthma.

Additional file
Additional file 1: Figure S1.