Reagents and chemical products

PAF (C-16), calpeptin and rabbit anti-N-Terminal PAFR were from Cayman Chemicals (Ann Arbor, MI, USA). Thapsigargin, PTP1B inhibitor, AG490, Jak2 inhibitor IV and Jak2 inihitor III (SD-1029) were from Calbiochem (San Diego, CA, USA). BSA for cell-culture, cOmplete, Mini Protease Inhibitor Tablets and ATP for luciferase assays were from Roche Diagnostics (Laval, QC, Canada). Dulbecco’s Modified Eagle Medium (DMEM, high glucose), GST-FER, 4′,6-diamidino-2-phenylindol dilactate (DAPI), Fluoro3 and D-Luciferin Na+ Salt were from Invitrogen (Burlington, ON, Canada). Fetal bovine serum (FBS) was from PAA Laboratories (Etobicoke, ON, Canada). TransIT-LT1 transfection reagent was from Mirius Bio (Medicorp Montréal, QC, Canada). Antibodies: Rabbit anti-Jak2 for microscopy (clone C20), rabbit anti-PTP1B (clone H135) (eptiope 301–435), mouse anti-PTP1B (H135) (eptiope 301–435) were from Santa-Cruz (LaJolla, CA, USA). Goat anti-PTP1B (epitope 2–321) was from R&D Systems, Minneapolis, MN. Anti-Src and pTyr 527 Src were from Cell Signalling (New England Biolabs Ltd., Pickering, ON, Canada). Rabbit anti-Jak2 and rabbit anti-phospho-Jak2 were from EMD Millipore (Mississauga, ON, Canada). Mouse anti-CD83 and mouse anti-CD86 were from BD Pharmingen, Mississauga, ON, Canada. Calpain Fluorometric Activity Assay Kit was from Biovision (Milpitas, CA, USA). Poly(Glu-:Tyr) [4:1], Gly-Gly, anti-FLAG and BSA essentially IgG-free were from Sigma-Aldrich, (Oakville, ON, Canada). Amersham ECL Select Western Blotting Detection Reagent, G-25 Sephadex PD-10 and GSH-sepharose beads, nitrocellulose membranes (Hybond ECL) were from GE (GE Heathcare, Piscataway, NJ, USA). Vectashield Mounting Media was from Vector Laboratories (Burlington, ON, Canada). Guanidine was from Fisher Scientific, (Nepean, ON, Canada) and p-Nitrophenyl phosphate (pNPP) was from ProteoChem (Loves Park, IL, USA). RPMI 1640 was from Wisent (Saint-Bruno, QC, Canada). RhGM-CSF and rhIL-4 were from Peprotech (Rocky Hill, NJ, USA)

Plasmids

Clones from E-coli populations with human PTP1B received from Open Biosystems (Ge Dharmacon, Lafayette, CO, USA) were isolated and DNA was extracted. PCR products were inserted in the pcDNA3 poly-cloning site after KpnI/NheI vector digestion. PTP1B sequence was also cloned in pCDNA3-FLAG, pGFP2-N3(h)_linker (a gift of Dr. C. Le Gouill, U. Montréal, Montreal, Canada) or pCDNA3-Venus. Integrity of the construct was confirmed by sequencing (Centre d'innovation Génome Québec and McGill University, Montreal, Canada). Jak2 and kinase-dead mutant K882E Jak2 were a gift from Dr Roy J. Duhé (University of Mississippi, USA). pGL3-IL-6 plasmid was described elsewhere [23].

siRNAs sequences

We used a mix of 3 siRNA duplexes that we designed using siRNA Wizard Guideline (http://www.sirnawizard.com) and Ambion website. First one is against the mRNA region 338–357 (5'-AUAGGUACAGAGACGUCAGUU-3'), the second, against region 702–720 (5'-CCAAGAAACUCG AGAGAUC-3') and the last one, against the region 2038–2056 (5'-AUCCUCAGGUAGUACUGGGU U-3'). SiRNAs duplexes, with a UU overhang and siRNAs controls (mission siRNA Universal Negative control #1) were from Sigma-Aldrich (Oakville, ON, Canada).

Cell culture

HEK-293 (CRL. 1573, American Type Culture Collection (ATCC), Rockville, USA) stably transfected with pIRES_puro_PAFR_HA (HEK-PAFR) were grown in DMEM, 5% FBS at 37°C 5%C0₂ in a humidified atmosphere with puromycin, 5μg/ml, penicillin, 60 μg/ml and streptomycin, 100 μg/ml.

Monocyte-derived dendritic cells (Mo-DCs) were generated from monocytes obtained from healthy volunteers after informed, written consent in accordance with protocol #93–04 approved by the Comité d'Éthique de la recherche chez l'Humain (Human ethics research committee), Université de Sherbrooke. Monocytes were isolated from venous peripheral blood after enrichment by dextran sedimentation followed by purification by density gradient centrifugation on Ficoll-Hypaque and after adhesion on autologous serum-coated Petri dishes. Following intensive washings, adherent cells were recovered and cultured for 7 days in RPMI 1640 supplemented with 10%FBS, rhGM-CSF (20 ng/mL) and rhIL-4 (20 ng/mL) at 1.6x10⁶ cells/ml in a 6-well plate, 3 ml/well. Medium was changed after 24–48h by removing half of the volume and adding the same volume of new medium. At day 5, the medium was changed by removing 1.5mL, but refilling with medium containing only 10ng/mL of each cytokine. Finally, at the end of day 6, 3ml of RPMI 5% FBS without any cytokine was added to begin the starvation of the cells. On day 7, cells were collected and stimulated as described below. Where mentioned, cells were transfected with siRNAs: at day 4. 75μl of a 2μM siRNA solution (in RPMI 1640) containing 2.3μL TransiT LT1 was added to 4.8x10⁶ cells (1 well of a 6 wells-plate, 1.5mL) and incubated at 37C, 5% CO₂. Thereby, the siRNA final concentration is 100nM. After 6h, 1.5mL of fresh medium with 20ng/ml of each cytokine and 10%FBS was added. A second transfection of siRNAs was done 30h after the first one: 75μl of a solution of 1.53μM siRNA in RPMI 1640 containing 2.3μL TransiT LT was added to 4.8x10⁶ cells (1 well of a 6-wells plate, 1.5mL) and incubated at 37C, 5% CO₂. Thereby, the siRNA final concentration is 76nM. After 6h, 1.5mL of fresh medium with 10ng/ml of each cytokine and 10%FBS was added.

Transient transfection and luciferase assays

HEK-PAFR were plated 12 hours before transfection in 24-wells plates. Cells were transiently transfected with 200ng of pcDNA3-hPTP1B and 40ng of pGL3-hIL-6 per well using 0,75μl of TransIT LT1 transfection reagent according to the manufacturer’s instructions. After 8h in DMEM 5% FBS without puromycin, medium was changed and cells were incubated in DMEM 0.2% BSA for 16h. The cells were lysed 6h after stimulation by PAF at indicated concentrations. Where mentioned, cells were pre-treated for 20min with the inhibitors at indicated concentrations. Luciferase activity in lysates was measured as described before [43] using a Sirius luminometer (Berthold detector Systems, Montreal, QC, Canada).

Western blot analysis

HEK-PAFR were plated 12 hours before transfection in 6-well plates. Where mentioned, cells were transiently transfected with 625ng of pcDNA3-human WT or mutant D181A PTP1B per well using 3μl of TransIT LT1 transfection reagent according to the manufacturer’s instructions. After 8h in DMEM 5% FBS without puromycin, medium was changed and cells were left in DMEM 0.2% BSA for 16h. Cells were then stimulated with 100nM PAF for indicated times and the reaction was stopped on ice with ice-cold PBS containing 4μM Na₃VO₄ and 20mM NaF. Cells were scraped, centrifuged and lysed by adding ice-cold lysis buffer containing inhibitors (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% TRITON X-100, 2mM NaF, 4mM Na₃VO₄, 10nM calyculin A and cOmplete, Mini Protease Inhibitor Tablet) for 20 min on ice before being frozen at -80°C.

When the samples were thawed, they were centrifuged, 4x loading buffer (40% glycerol, 258mM Tris-HCl pH 6.8, 8% SDS, 0,008% bromophenol blue, 20% 2-mercaptoetanol) was added to the supernatants, proteins were separated on 10% SDS-PAGE and subsequently transferred to a nitrocellulose membrane (Hybond, GE). Membranes were blocked with 5% milk in Tris-buffered saline (TBS) with 0.1%Tween 20 (TBS-T) before overnight blotting at 4°C in TBS-T 5% milk with appropriate antibodies. For phospho-proteins, membranes were washed 3x5min in TBS-T after blocking in milk, and blocked again for 20min in TBS-T 5% BSA before overnight blotting at 4°C in BSA. Finally, HRP-coupled secondary antibody (Cell Signalling) was added for 1h, protein expression was revealed by ECL chemiluminescence detected with Versadoc (BioRad). Signal intensity was analyzed with ImageJ 1.43X software (National Institutes of Health, Bethesda, MD, USA).

Microscopy

HEK-PAFR were grown overnight on poly-L-lysine coated coverslips in DMEM, 5%FBS, transfected with 400ng Venus-tagged pCDNA3-D181A-hPTPN1 and 1.5μL Transit LT1. After 24h, cells were starved 7h in DMEM 0.2% BSA and stimulated with 100nM PAF for indicated times. Cells were fixed with 2.5% paraformaldehyde (PFA) for 15min at room temperature (RT), then placed in 0.5% (v/v) Triton X-100 in PBS for 10min RT, blocked 20min in PBS 3%BSA, 0.1%Triton X-100 and incubated overnight, 4°C, in PBS, 1%BSA, 0.1% Triton X-100 containing anti-Jak2 antibodies (Abs) (C20, Santa-Cruz, 1:200). Controls with rabbit isotype Abs were performed in parallel. The coverslips were washed 3 times and incubated 1h, RT, in PBS, 2% BSA, 0.1% Triton X-100 containing anti-rabbit-Cy5 Abs (1:500, Jackson Immunoresearch). After washing, the coverslips were mounted on slides using Vectashield containing DAPI. Acquisitions were performed with an Plan Apo 60x oil immersion objective NA 1.42 on inverted spectral scanning confocal microscope FV1000 (Olympus, Tokyo, Japan). Images were acquired the same day, typically from 4 different optical fields for each experimental condition using identical settings of the instrument. Images were analyzed with FluoView software (Olympus). The percentage of colocalized pixels (P) was calculated

In-gel PTP assays

In-gel PTP assays were performed as described elsewhere [44–45]. Briefly, 2x10⁶ cells were starved for 3h in serum-free medium with 0.2%BSA and stimulated for indicated times. Stimulation was stopped with PBS containing NaF (final concentration 20mM) and Na₄VO₃ (final concentration 5mM). Cells were collected and lysed 20min on ice with 80μL in-gel PTP lysis buffer (0.5% NP-40, 50mM HEPES (pH 7.4), 150mM NaCl, 1mM EDTA, 2mM EGTA, 1mM Na₄VO₃ 1mM NaF, cOmplete, Mini Protease Inhibitor Tablet) and then centrifuged. 27μL of 4x loading buffer was added to cleared lysate. Lysates were frozen until the assay was performed. For immunoprecipitation, cleared lysates were incubated 30min at 4°C with Protein-A sepharose beads and centrifuged. Supernatants were incubated 3h with rabbit anti-PTP1B Abs (1.4μg) or rabbit isotype control Abs before addition of 25μL of a solution 50% protein-A sepharose beads. Lysates were then incubated for 2h at 4°C. Beads were recovered by centrifugation (3000rpm, one minute) and subsequently rinsed three times with ice-cold TBS-T. Beads were resuspended in 2x loading loading buffer.

Poly(Glu-:Tyr) [4:1] (1.2mg) was labelled overnight at RT using GSH-Sepharose beads- coupled GST-FER and 0.5mCi [y-³²P] ATP (3000 Ci/mmol) in incubation buffer (50mM immidazole, pH 7.2, 30mM MgCl₂, 10mM MnCl₂, 10mM DTT, 1mM Na₃VO₄, 1% Triton and 0.1% 2-mercaptoethanol). Poly(Glu-:Tyr) [4:1] was then purified on G-25 Sephadex PD-10 by collecting 1mL fractions eluted with a 50mM immidazol buffer (pH 7.2) and quantification was performed using a LKB-Wallac β-counter. Fraction with the highest activity was aliquoted and stored at -20°C.

In-gel PTP assays were performed by copolymerizing the labelled poly(Glu-:Tyr) at a ratio of 1.5x10⁶cpm for 20mL in a 10% acrylamide SDS-PAGE gel, overnight. 25μg of proteins from HEK-PAFR lysates and 15μg from Mo-DCs lysate were used and an equivalent amount of immunoprecipited PTP1B was used. After migration, gels were fixed (50mM Tris-HCl, pH 8.0, 20% isopropanol) overnight, washed 2x30min in washing buffer (50m Tris-HCl pH 8.0, 0,3% 2-mercaptoéthanol) and left for 90min in renaturing buffer (50mM Tris-HCl, pH8.0, 0,3% 2-mercaptoethanol 6M guanidine). After that, gels were washed 2x1h in assay buffer (50mM Tris-HCl, pH 8.0, 0,04% (v/v) Tween 40, 1mM EDTA, 0.3% 2-mercaptoethanol), incubated 1h in assay buffer with 3mM DTT and changed once again for the assay buffer with 3mM DTT. Gels were incubated overnight at RT, stained with Coomassie Blue and dried under vacuum. Dried gels were exposed to Hyperfilm MP (Amersham Pharmacia Biotech). Signal intensity and protein loading obtained with Coomassie blue staining was quantified by densitometry with Epson Expression 1680 Scanner and analyzed with ImageJ 1.43X software, NIH. Data are presented as normalized density. This value is calculated in two steps. First a value is calculated with the ratio of the density for the band X with a molecular weight Y over the density obtained with the Coomassie blue staining for the entire lane. Similarly, a value is calculated for the band with the same molecular weight found in unstimulated cells. Secondly, the normalized density is calculated with the ratio of the calculated value for the band X, found in step one, over the calculated value obtained for the corresponding band in unstimulated cells.

Colorimetric PTP assays

Colorimetric PTP assays were performed with a protocol adapted from T. McAvoy and C. Nairn [46]. Pre-treated and stimulated HEK-PAFR were lysed 20min on ice with lysis buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10mM NaF, 1 mM Na₃VO₄, 10μM Calyculin A and cOmplete, Mini Protease Inhibitor Tablet) and frozen at -80°C. PTP1B was immunoprecipitated from cleared lysates (at least 1mg of lysate) as described for in-gel PTP assays. Instead of being resuspended in loading buffer, beads were resuspended in 130μL PTP buffer (25mM HEPES, pH7.4, 0.1mM EDTA, 100μg/mL BSA, 0.01% Tween 20, 1mM DTT), distributed in 96-wells plate (30μL/wells) and incubated 10min at RT. After that, 20μL of substrate is added (pNPP, 95mg/mL in PTP buffer). Plates are incubated at 30°C under mild agitation. Optical density (OD) is taken every 10min at 405nm (reference 800nm) until the signal is saturated. OD is plotted as function of time and dephosphorylation rates are taken in linear portion of the curve. Hydrolysis rate is calculated with an extinction coefficient of 18000M^-1cm^-1.

Calpain assays

Calpain activity was determined with Fluorometric Activity Assay Kit according to the manufacturer's instructions. Briefly, 1 plate with 6-wells of HEK-PAFR or 2x10⁶ DCs was pre-treated and stimulated as mentioned above, then lysed in 100μL of lysis buffer 1min before centrifugation. Protein concentration was determined by the Bradford assay (Pierce). 80–100μg of DCs lysate or 200μg of HEK-PAFR lysate was used for determination of calpain activity. Calpain activity levels were determined by fluorescence (Excitation wavelength: 355nm, Emission wavelength: 405nm) read with a PerkinElmer Fusion spectrometer.

Calcium assays

HEK-PAFR were plated in 96-well plates and grown until confluence. Cells were incubated 30min with 5μM Fluo3 calcium probe and washed 3x with HBSS 0,5% BSA without calcium. Cells were then incubated for 20min in HBSS 0.2%BSA with Jak2 inhibitor IV or SD-1029. CaCl₂ (2mM final) or buffer without CaCl₂ was added to cells, after reading of the fluorescence basal levels, the cells were stimulated as indicated. Minimal and maximal fluorescence was determined by addition of EGTA (100mM) or Triton X-1000 (0.5% final), respectively. Data were obtained by acquiring fluorescence with a 140ms integration time with 20s between each reading taken on an Ascent Fluoroskan (Thermo Scientific).

Flow cytometry analysis

Mo-DCs were collected, fixed with 2% PFA for 15min RT and non-specific binding sites were blocked by incubating cells for 30min in PBS 2%BSA containing human IgG (5μg/mL). Cells were stained with mouse anti-CD83 Abs (1:200) or with mouse anti-CD86 Abs (1:200), 1h at RT or overnight with rabbit anti-N-terminal PAFR Abs (1:250) in PBS 2% BSA, IgG 1μg/mL. After washes with PBS, cells were stained with secondary antibody coupled to Cy5 for 45min. After washing, cells were analyzed on a FACSCalibur flow cytometer using the CellQuestPro software.

RNA isolation and real-time quantitative PCR

RNA was obtained using Trizol reagent (Invitrogen, Burlington, ON, Canada) according to the manufacturer’s instructions. After quantification, 1.0 μg of RNA was converted to cDNA, QuantiTect Reverse Transcription Kit, according to the manufacturer's instruction. GAPDH, IL-6 and PTP1B expression was measured using real-time PCR performed on a Rotor-Gene 3000 (Corbett Research, Kirkland, QC, Canada) as described previously [13]. The following oligonucleotide primer sets were obtained from IDT (Coralville, Ind, USA): human GAPDH: forward, 5'-GATGACATCAAGAAGGTG GTGAA-3' /reverse, 5'-GTCTTACTCCTTGGAGGCCATGT-3'; human IL-6: forward, 5'-GTGTGA AAGCAGCAAAGAGGC-3'/reverse, 5'-CTGGAGTACTCTAGGTATAC; human PTP1B: forward, 5'-acagagtgatggagaaaggttc-3'/reverse, 5'-ctcgAgtttcttgggttgtaag-3'. Gene expression was normalized with GAPDH mRNA content and differences were calculated with the delta-delta (ΔΔ)Ct method according to the following formula: (ΔΔCt = [(Ct G.O.I.Ctl −Ct HK.G.Ctl) − (Ct G.O.I.STIM. −Ct HK.G.STIM.)]. Comparison of the expression of each gene between its control and stimulated/siRNA transfected states was determined by ΔΔCt. Results were then transformed into fold variation measurements: fold increase -2ΔΔCt.

Statistical analysis

Statistical significance was calculated using Prism 6 software (GraphPad Software, San Diego Calif, USA). For analysis of differences between experimental groups, we used Student's t-test, paired one-way and paired two-way ANOVA with appropriate post-test, as described in figure legends. Values of P ≤ 0.05 were considered statistically significant.

PAF regulates PTP1B activity

Previous studies have shown that PAF stimulation can induce IL-6 production in a PLC- PLA₂- [17] 5-lipoxygensase- [18] and calcium-dependent [17, 19] manner. Our laboratory has shown that PAF activates Jak2 and Tyk2 leading to STAT1, STAT2, STAT3 and STAT5 tyrosine phosphorylation [16, 31]. Others have shown that STAT1 and STAT5 could modulate IL-6 production either by directly binding to the promoter [29] or via induction [25] or interaction [27] with other transcription factors that bind the hIL-6 promoter [28,30]. Taking into consideration these data, we decided to investigate the role of the regulation of the Jak/STAT pathway in PAF-induced IL-6 production in immature Mo-DCs (iMo-DCs). Given the importance of PTPs, in particular PTP1B, in modulation of Jak2, Tyk2 and STAT activity [35], we first assessed whether PAF modulated PTP activity. Global representation of PAF-induced PTP activation was studied by in-gel PTP assays. iMo-DCs (Fig 1A) or HEK-PAFR (S1 Fig) were stimulated with PAF, for indicated times, and PTP activity was measured as described in Material and Methods. In both cell types, PAF induced an increase in PTP activity in a time-dependent manner. Compilation of normalized densities obtained from different donors shows that PAF stimulation increased the activity of several PTPs with molecular weights of approximately 55, 43 and 37kDa and more modestly, compared to unstimulated controls, ones with molecular weights of 72kDa. This PAF-stimulated phosphatase activity could be due to full length (50kDa) PTP1B and its cleaved segments (43 and 37kDa), in addition to activation of other PTPs. As we were interested in the modulation of the Jak/STAT pathway and PTP1B is a phosphatase associated with Jak2 modulation, we pursued this avenue.

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Fig 1. PAF induces PTP1B activity.

A) Representative autoradiography with lysates of iMo-DCs stimulated with PAF. iMo-DCs (2x10⁶ cells) were starved for 3h in serum-free medium with 0.2% BSA and stimulated with 10nM PAF for indicated times. Cells were lysed with in-gel PTP lysis buffer and 15μg of whole cell lysate was loaded onto a 10% acrylamide SDS-PAGE gel, copolymerized with labeled poly(Glu-:Tyr). B) Graph summarizing the normalized intensity of bands according to the molecular weight and stimulation time. Data presented are mean ±S.E.M of normalized intensity (as described in Materials & Methods) for 3 independent experiments. C) Representative autoradiography obtained with PTP1B immunoprecipitated from Mo-DCs, stimulated with 10nM PAF for indicated times. Data are representative of at least 3 independent experiments.

https://doi.org/10.1371/journal.pone.0180336.g001

We immunoprecipitated PTP1B from lysates of iMo-DCs stimulated with 10nM PAF. In-gel PTP assays showed that PTP1B activity was transiently increased in iMo-DCs 5 min post-stimulation with PAF and then decreased at 10 and 15 min (Fig 1C). Our data indicate that PAF could modulate PTP activation in different cell types in a time-dependent manner and that PTP1B is among the PTPs stimulated by PAF.

PTP1B regulates PAF-induced IL-6 mRNA levels in DCs

Previous studies from our laboratory had demonstrated that PAF can be involved in polarization of lymphocytes toward a Th17 phenotype in a IL-6-dependent manner, via IL-6 production by Langerhans cells, a subtype of DCs [13]. We therefore studied the potential involvement of PTP1B in PAF-induced IL-6 mRNA levels in iMo-DCs by using iMo-DCs transfected with control (siCTRL) or PTP1B-specific (siPTP1B) siRNA.

First, we ensured that the siRNAs did not activate the iMo-DCs or induce maturation, both associated with a decrease in PAFR expression [47] and with induction of different pathways that could affect the interpretation of our results. Cell surface expression of CD83, a maturation marker, CD86, a differentiation marker, and PAFR was not significantly affected by transfection with siCTRL or siPTP1B (Fig 2A), confirming that any difference in results between the two populations was not due to a difference in the cell activation state or PAFR expression. Secondly, we tested the efficiency and specificity of these siRNA. siRNAs against PTP1B caused a 30% decrease in PTP1B mRNA (Fig 2B) and protein (Fig 2C) levels compared to siCTRL. In addition, the closely related phosphatase TC-PTP was not modulated by the siRNAs we used (Fig 2D).

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Fig 2. PTP1B regulates PAF-induced IL-6 mRNA levels in human immature Mo-DCs.

iMo-DCs were transfected with siRNAs, either control (siCTRL) or against PTP1B (siPTP1B) at day 4 and 5 before being collected at day 7 for experiments. A) Mean fluorescence Intensity (MFI) of surface expression of CD83, CD86 and PAFR. At day 7, iMo-DCs, transfected with siCTRL or siPTP1B, were collected, fixed and stained with antibodies against indicated markers. Cell surface expression was analyzed on a FACSCalibur flow cytometer. Data presented are mean±S.E.M of 5 independent experiments B) iMo-DCs were lysed in Trizol and RNA was extracted and converted to cDNA, as described in Materials and Methods. GAPDH and PTP1B expression was measured using real-time PCR. Results are presented as 2^ΔΔCT relative to siCTRL. Data presented are in Box & Whisker graph (Min to Max) of 5 independent experiments. Significance was established with Student's t-test. C) Representative blot of 6 experiments performed: PTP1B levels in siCTRL and siPTP1B treated iMo-DCs. At day 7, iMo-DCs were lysed and whole cell lysates were loaded onto SDS-PAGE, transferred to a nitrocellulose membrane and blotted with goat anti-PTP1B, epitope 2–321, or mouse anti-PTP1B, epitope 301–435, antibodies. Blots were scanned and variations in PTP1B levels normalized with vinculin are represented in a graph as fold change over siCTLR cells. Data presented are in Floating bar (Min to Max) with line at the mean of 3–5 independent experiments. Significance was established with Kruskal-Wallis test with Dunn's multiple comparaison test. C) Representative blot of TC-PTP levels in untreated, siCTRL- or siPTP1B-treated iMo-DCs. TC-PTP levels were determined by blotting with an anti-TC-PTP antibody. Variations in TC-PTP levels normalized with vinculin are represented in a graph as fold change over siCTLR cells. Data presented are in Floating bar (Min to Max) with line at the mean of 3–5 independent experiments. D) iMo-DCs, transfected with siCTRL or siPTP1B were stimulated for 5hrs with vehicle or PAF (10nM), then lysed in Trizol and RNA was extracted and converted to cDNA. GAPDH and IL-6 expression was measured using real-time PCR. Results are presented as 2^ΔΔCTrelative to unstimulated cells. Data presented are mean±S.E.M of 6 independent experiments. Significance was established with paired two-way anova with Sidak post-test.: *p<0.05, ***p<0.01 and &:p< 0.05 over unstimulated cells with the same pre-treatment.

https://doi.org/10.1371/journal.pone.0180336.g002

When PAF-induced IL-6 mRNA levels were measured in siRNA-transfected iMo-DCs, a significant increase of IL-6 mRNA levels was observed in iMo-DCs transfected with siPTP1B compared to siCTRL-transfected cells (Fig 2E). These results indicate that PAF-induced IL-6 mRNA expression in iMo-DCs was regulated by PTP1B.

PTP1B regulates PAF-induced hIL-6 promoter activation in a Jak2-dependent manner

Previous studies from our group have demonstrated that Tyk2 and Jak2 are activated by PAFR stimulation, whereas others have shown that PTP1B dephosphorylates both these kinases [16, 31, 35]. Our previous data had also indicated that Jak2 activation by PAFR may result in negative regulation of PAFR signaling [31]. We therefore concentrated on this kinase in conjunction with PTP1B activation. We first examined whether PTP1B could modulate PAF-induced IL-6 promoter activation in HEK-PAFR and whether Jak2 had a role in this modulation.

Fig 3A and 3B show, first, that Jak2 inhibition alone, by use of the pharmacological inhibitor AG490, or transfection of a dominant negative Jak2 (KD Jak2) caused a decrease in PAF-induced IL-6 promoter activity. Secondly, as found in iMo-DCs, PTP1B negatively modulated PAF-induced IL-6 promoter activity. In addition, both Jak2 dominant negative mutant and AG490 partially, but significantly, reversed the inhibition by PTP1B of PAF-stimulated IL-6 transcription, indicating that Jak2 had a role in PTP phosphatase activity, since the decrease caused by the transfection of a phosphatase inactive mutant (D181A PTP1B) was not reversed by the KD Jak2. This indicates that the increase caused by co-transfection of the KD Jak2 in PTP1B-transfected cells needs a catalytically active form of PTP1B. This suggests that Jak2 could be upstream of PTP1B and that Jak2 may have a dual function in PAF-stimulated IL-6 mRNA expression.

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Fig 3. PTP1B regulates PAF-induced hIL-6 modulation in a Jak2-dependent manner.

A) HEK-PAFR were transiently co-transfected with hIL-6 promoter luciferase constructs and PTP1B or vector control (pcDNA-3). After starvation, cells were pre-treated for 20min with 5μM AG490 before a 6h stimulation with PAF (100nM) or vehicle. B) HEK-PAFR were transiently co-transfected with hIL-6 promoter/luciferase construct and WT or D181A mutant PTP1B, and/or kinase-dead mutant Jak2 or control vectors. After starvation, cells were stimulated for 6h with PAF (100nM) or vehicle (EtOH) and IL-6 promoter activity was established. C) HEK-PAFR were transfected with hIL-6 promoter/luciferase construct and vector control. After starvation, cells were pre-treated for 20min with 4μM PTP1B inhibitor before a 6h stimulation with PAF (100nM) or vehicle and IL-6 promoter activity was determined. For all luciferase assays, results are presented as fold induction of IL-6 promoter activity induced by PAF over vehicle control. The data presented are mean±S.E.M of at least 4 experiments performed in triplicate. Significance was established with paired two-way anova with Sidak post-test. *: p<0.05; ***:p<0.001 and &: p<0.05 &&&: p<0.001 compared to conditions without overexpression or inhibition of PTP1B.

https://doi.org/10.1371/journal.pone.0180336.g003

To confirm that the effect of PTP1B seen on PAF-induced hIL-6 activation depended on phosphatase activity and was not only due to overexpression, luciferase assays were performed using PTP1B inhibitor as pre-treatment, combined with AG490 or its vehicle (Fig 3C). As expected, inhibition of PTP1B increased PAF-induced hIL-6 promoter activation. AG490 significantly decreased PAF-induced promoter activity in vehicle and PTP1B inhibitor pre-treated cells, suggesting that Jak2 was needed for a maximal transduction in both cases. Moreover, when cells treated with AG490 were co-incubated with the PTP1B inhibitor, the PAF-induced hIL-6 promoter activity was partially rescued, suggesting that the decrease caused by AG490 depended to some extent on PTP1B activity. These results suggest that Jak2 kinase activity is important for PAF-induced transactivation of hIL-6 promoter, that PTP1B regulates this transactivation in a phosphatase-dependent manner and that this modulation partly depends on Jak2 activity that could be upstream of PTP1B activity.

To test this hypothesis, we therefore investigated whether PTP1B could modulate Jak2 activation. We examined whether PTP1B had an effect on Jak2 phosporylation in PAF-stimulated cells using HEK-PAFR cells transfected with control vector or WT PTP1B. As shown in Fig 4A, PAF induced a rapid phosphorylation of Jak2, within 2 min post-stimulation, which then decreased after 5 min. A compilation of 5 experiments, Fig 4B, shows that mutant PTP1B increases phosphorylation levels of Jak2 in unstimulated cells, indicating that PTP1B could be involved regulation of basal activation state of this kinase. On the other hand, transfection of WT or D181A PTP1B did not significantly alter the PAF-induced phosphorylation levels of Jak2 after PAF stimulation. These results suggest that PTP1B does not participate in PAF-induced Jak2 activation, indicating that, possibly, it may be Jak2 which had a role in modulating PTP1B activity.

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Fig 4. PTP1B does not modulate PAF-induced Jak2 phosphorylation.

A) Representative blot of experiments conducted with HEK-PAFR transfected with Flag-tagged wt or D181A PTP1B or control vector for 8h before overnight starvation in DMEM, 0.2% BSA. Cells were stimulated for indicated times with PAF (100nM) or vehicle. Cells were lysed and western blots were performed. B) Blots were scanned and the variation in Jak2 phosphorylation (pJak2 Tyr 1007/1008) is represented as ratio of phosphorylation in stimulated cells over control unstimulated cells. Data presented are mean±S.E.M of 5 independent experiments. Significance was established with paired two-way anova with Sidak post-test. *: p<0.05.

https://doi.org/10.1371/journal.pone.0180336.g004

We then investigated whether the two molecules could co-localize, either in the basal state or after PAF stimulation. Using HEK-PAFR cells transfected with the Venus-tagged substrate-trapping mutant D181 PTP1B and Abs agains Jak2, we found a low level of PTP1B and Jak2 co-localization in the basal state, but after PAF stimulation, there was a rapid and transient increase in co-localized pixels at 2 min, which was no longer visible after 5 min of stimulation (Fig 5A & 5B). Given that we used a trapping mutant which should not release its substrate, if bound by the PTP domain, this suggests, in agreeement with results obtained in Fig 4 that Jak2 and PTP1B may interact, either through other domains than the PTP domain or indirectly, via other proteins. Western blot experiments combined with microscopy suggest that PTP1B does not directly regulate Jak2 activation but is found in the same transitory signaling complexes. Maximal down-regulation of PAF-induced IL-6 promoter activity by PTP1B seems to depends on Jak2, suggesting that PTP1B acts downstream of Jak2.

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Fig 5. PTP1B is found in the same complex as Jak2.

A) HEK-PAFR, grown on poly-L-lysine coated coverslips, were transfected with Venus-tagged D181A-PTP1B then stimulated, 30h later, with vehicle or with 100nM PAF for 2 or 5min before permeabilization with 0,5% Triton X-100. The cells were stained overnight with rabbit anti-Jak2 antibodies or control isotype (not shown), followed by anti-rabbit-Cy5 staining. Nuclei were stained with DAPI and cells were analyzed by confocal microscopy. Results are representative of 4 independent experiments, with at least 4 pictures from different fields in each experiment. Scale bar = 5μm. B) Variation of colocalization is presented as % of colocalizated pixels at different time points where each dot represents a cell. Data presented are mean ±S.E.M of 4 independent experiments. Significance was established with one-way anova, Tukey's Multiple Comparison post-test *: p<0.05.

https://doi.org/10.1371/journal.pone.0180336.g005

Next, the involvement of Jak2 in PAF-induced PTP1B activation was tested by colorimetric PTP assays using pNPP as substrate. PTP1B was immunoprecipitated from HEK-PAFR which had been pre-treated for 30min with 2μM of Jak2 inhibitor IV and then stimulated with 100nM PAF for indicated times. Fig 6 shows that Jak2 inhibitor significantly reduced PAF-induced PTP activation. Effectively, PAF induced a significant increase in PTP1B activity and this, as soon as 2 min post-stimulation in control cells and activity remained significantly higher than basal state for 10min. In Jak2 inhibitor IV-pre-treated cells, PAF did not induce a significant increase in PTP1B activity, compared to basal activity in unstimulated cells. Even if basal levels of pre-treated cells were higher than those found in control cells, the pNPP hydrolysis rate of PTP1B in Jak2 inhibitor IV-pre-treated cells was slightly lower (p = 0.16) at 2min and significantly lower at 5min post-stimulation, than the rate in vehicle pre-treated cells stimulated with PAF for the same time periods. Results obtained with Jak2 inhibitor IV were confirmed using 200nM SD-1029 (another Jak2 inhibitor) as pretreatment for HEK-PAFR stimulated with PAF for 5 or 10min (S2 Fig). These data suggest that Jak2 is involved in the activation of PTP1B during PAF stimulation in a kinase-dependent manner, and also suggest the involvement of Jak2 in regulating the basal activity of this phosphatase.

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Fig 6. Jak2 is involved in PAF-induced PTP1B activity.

Graphs represent hydrolysis rate of pNPP by PTP1B immunoprecipitated from HEK-PAFR stimulated with 100nM PAF for indicated times, after pre-treatment with 2μM Jak2 inhibitor IV. PTP1B was immuoprecipitated from HEK-PAFR, which had been starved overnight in DMEM 0.2% BSA, pre-treated and stimulated, as indicated, before being incubated with pNPP. Data presented are mean ±S.E.M of pNPP hydrolysis rate for at least 3 independent experiments. Significance was established with paired two-way anova with Sidak post-test. *p<0.05, ** p<0.01 and &:p< 0.05 over unstimulated cells with the same pre-treatment.

https://doi.org/10.1371/journal.pone.0180336.g006

Jak2 regulates PTP1B cleavage

It has been shown that Jak2 and calpain could be involved in the same signaling pathways [48]. Given that calpain is also known to modulate PTP1B activity [49–51], we investigated whether Jak2 modulated PAF-induced PTP1B activity in a calpain-dependent manner. First, we tested whether PAF could induce cleavage of PTP1B and whether Jak2 could be involved in this cleavage. This question was addressed by pre-treating HEK-PAFR for 20min with 2μM Jak2 inhibitor IV, or vehicle, and stimulating the cells with 100nM PAF, for indicated times. Western blots were performed with 2 different antibodies directed against 2 different regions of PTP1B, the sequences encompassing the cleavage site of oxidized PTP1B between Glu76-Ala 77 and the putative cleavage site of unoxidized PTP1B found between residues 360–381 [50–51]. In Fig 7A, we show that, for both antibodies, PAF induced a decrease in the major band, found around 50kDa and this, in a time-dependent manner, indicating that cleavage had occurred. A decrease can be observed as soon as 2 min post-stimulation with a significant decrease at 5 min. This decrease is maintained until 30 min post stimulation. The PTP1B levels, for the 50kDa band, were significantly higher in Jak2 inhibitor IV-treated cells at 5 and 10 min post-stimulation compared to control cells and this for both antibodies (Fig 7A). Same results were obtained in SD-1029 pre-treated cells stimulated 5 or 10 min with 100nM PAF (S3 Fig). When more lysate was loaded on the gel, a band under the 50kDa major band was detected with the antibody against the C-terminal part of PTP1B (Fig 7B). This shorter isoform was faintly detected under basal conditions in control cells, increased in expression within 2 min after stimulation, and was significantly increased after 5 min of stimulation. The elevated levels were maintained for all tested times. In Jak2 inhibitor pre-treated cells, this isoform of PTP1B was slightly higher at basal levels than in control cells but the PAF-induced increase was not seen. The increase in detected levels for this isoform correlated with the decreased levels for the 50kDa band in control cells (Spearman r = -0,4212, P = 0,0454) but not in Jak2 inhibitor pre-treated cells (Spearman r = -0.2731 P = 0.2579), suggesting that Jak2 could be important in the generation of this isoform, which seems to be the 42kDa PTP1B fragment generated by calpain described by other groups [49–51]. Since the decrease in the 50kDa band correlated with the appearance of the smaller fragment, for the rest of this article, the cleavage of PTP1B will be followed by the decrease of the 50kDa band considering that this decrease is more easily detected by both the antibodies used. Results obtained, here, suggest that PAF induces PTP1B cleavage in a Jak2-dependent manner.

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Fig 7. Jak2 regulates PTP1B cleavage.

HEK-PAFR were starved overnight and pre-treated with vehicle or 2μM Jak2 inhibitor IV for 20min prior to stimulation with 100nM PAF, for indicated times. Whole cell lysates were loaded onto SDS-PAGE, transferred to nitrocellulose membranes and blotted overnight with indicated antibodies: mouse anti-PTP1B (epitope 301–435) or goat anti-PTP1B (epitope 2–321) and PTP1B expression was followed over time. A) Representative western blots of PTP1B levels are shown and in the lower panels data are presented as relative intensity of PTP1B expression over vinculin and reported as fold change, at indicated times. B) Representative western blot of PTP1B levels, that had been over-exposed in order to show the cleaved fragment. In the right panel data are presented as relative intensity of the cleaved PTP1B fragment, over vinculin expression and reported as fold change at indicated times. Data presented are mean±S.E.M. of at least 5 experiments. Significance was established with paired two-way anova with Sidak post-test: *p<0.05; &p<0.05 compared to unstimulated cells.

https://doi.org/10.1371/journal.pone.0180336.g007

Next we determined if these cleaved fragments could indeed have a functional role by performing in-gel phosphatase assays with PTP1B immunoprecipitated from HEK-PAFR (Fig 8A & 8B) or Mo-DC lysates (Fig 8C & 8D). In order to clearly detect the fragments, we loaded more proteins (30ug of total protein against 15ug for Fig 1C) and thus the full length PTP1B activity is satturated and no modulation is visible. For bands with a molecular weight lower than 50 kDa, in both cell types, PAF induced a significant increase in PTP activity, measured after stimulation of 5 and 10 min. This increase was singnificantly lower when cells were pre-treated with the Jak2 inhibitor, SD-1029. These results that the cleaved fragments, observed after PAF stimulation, increase in phosphatase activity and could have a functional importance in PAF-induced signaling pathways. Since calpain has been found to be both a positive and negative regulator of PTP1B activity [49–51], we decided to further investigate the involvement of this protease in PAF-induced IL-6 production and in PAF-induced PTP1B activation.

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Fig 8. Jak2 regulates the activity of the cleaved fragments of PTP1B.

A) Representative autoradiography obtained with PTP1B, immunoprecipitated from HEK-PAFR, stimulated with 100nM PAF for indicated times, after a 20 min pre-treatment with 200nMSD-1029 or its vehicle. Data are representative of 3 independent experiments. B) Graph summarizing the normalized intensity of bands with a molecular weight lower than 50 kDa and stimulation times. Data presented are mean±S.E.M. of normalized intensity (as described in Materials & Methods) for 3 independent experiments. C) Representative autoradiography obtained with PTP1B, immunoprecipitated from Mo-CD, stimulated with 10nM PAF for indicated times, after a 20 min pre-treatment with 200nMSD-1029 or its vehicle. Data are representative of 4 independent experiments. D) Graph summarizing the normalized intensity of bands with a molecular weight lower than 50 kDa and stimulation times. Data presented are mean±S.E.M. of normalized intensity (as described in Materials & Methods) for 4 independent experiments.

https://doi.org/10.1371/journal.pone.0180336.g008

PAF regulates PTP1B activity in a calpain-dependent manner

First, the involvement of calpain in PAF-induced hIL6-promoter activation and in PTP1B-mediated down-regulation of the promoter was tested by luciferase assays. HEK-PAFR were transfected with WT PTP1B or control vector and pre-treated 20 min with vehicle or calpeptine, a calpain inhibitor, and stimulated or not with 100nM PAF. Calpeptine, at both concentrations used, decreased PAF-induced hIL-6 promoter activity to 63,3±11.1% and 58.0±5.2% of activity in control cells (Fig 9A). In PTP1B-transfected cells, the PAF-induced promoter activity reached 51.0±8.9% of hIL-6 promoter activity obtained in control vector-transfected cells. When PTP1B-transfected cells were pre-treated with calpeptine, the PAF-induced hIL-6 promoter activation was partially rescued, since the promoter activity resembled that of calpeptine-treated cells in the absence of PTP1B overexpression (62.8±7.4%, at 350nM calpeptine, and 63.5±10.8%, at 70nM, of the PAF-induced hIL-6 promoter activity observed in control cells). This was an increase of around 25% when compared to untreated PTP1B-transfected cells. This suggests that, in HEK-PAFR stimulated with PAF, calpain is involved in maximal transactivation of the IL-6 promoter but is also important for maximal down-regulation hIL-6 promoter by PTP1B.

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Fig 9. PAF regulates PTP1B activity in a calpain-dependent manner.

A) HEK-PAFR were transiently co-transfected with hIL-6 promoter luciferase constructs, WT PTP1B or control vectors for 8h before overnight starvation in DMEM 0.2% BSA. After a 20min pre-treatment with 70 or 350nM calpeptin or vehicle, cells were stimulated for 6h with PAF (100nM) or vehicle and luciferase activity was measured. Luciferase activity of cells transfected with the control vector or WT PTP1B, pre-treated with the vehicle for calpeptin and stimulated with PAF was set at 100% and compared with the activity of cells treated with the two concentrations of calpeptin. The data presented are mean±S.E.M of at least 3 independent experiments performed in triplicate. Significance was established with paired two-way anova with Sidak post-test. &: p<0.05 &&: p<0.01. B) Hydrolysis rate of pNPP by PTP1B immunoprecipitated from HEK-PAFR stimulated with 100nM PAF for indicated times after pre-treatment with 70nM calpeptine or its vehicle. PTP1B was immunoprecipitated from HEK-PAFR, which had been starved overnight in DMEM 0.2% BSA, pre-treated and stimulated as indicated. Data presented are mean ±S.E.M of hydrolysis rate of pNPP for at least 3 experiments. Significance was established with paired two-way anova with Sidak post-test: ** p<0.01 and & p< 0.05 && <0.01 over unstimulated cells with the same pre-treatment. C) Representative western blot of PTP1B levels. HEK-PAFR were starved overnight, then pre-treated with 70nM calpeptin or vehicle for 20min prior to stimulation with 100nM PAF for indicated times. Whole cell lysates were loaded onto SDS-PAGE, transferred onto nitrocellulose membrane and blotted overnight with goat anti-PTP1B, epitope 2–321. In the lower panel data are presented as relative intensity of PTP1B expression over vinculin and reported as fold change at indicated times. Data presented are mean±S.E.M of 6 independent experiments. Significance was established with paired two-way anova with Sidak post-test: *p<0.05, &:p< 0.05 over unstimulated cells with the same pre-treatment.

https://doi.org/10.1371/journal.pone.0180336.g009

Next, we tested whether calpain is involved in PAF-induced PTP1B activation. Colorimetric assays of PTP1B activity were performed with PTP1B immunoprecipitated from HEK-PAFR pre-treated with calpeptin or with vehicle and stimulated with PAF for indicated times. Calpeptin inhibited, in a time-dependent manner, PAF-induced PTP1B activation (Fig 9B). In the cells pre-treated with the inhibitor, PTP1B activity was at 67.8±25.4% (at 5 min) and 54.18±14.6% (at 10 min) of control cells. To ensure that activation of PTP1B could be correlated to cleavage of PTP1B by calpain, PTP1B levels were measured by western blot. Lysates from HEK-PAFR pre-treated with calpeptin or vehicle and stimulated with PAF, for different times, were studied. In control cells, levels of PTP1B detected after stimulation were significantly lower than those in unstimulated cells (Fig 9C). However, in cells stimulated with PAF, PTP1B levels detected in calpeptin pre-treated cells were approximately 40% higher at 5 min and 45% higher at 10 min, compared to control cells. Similar results were obtained with anti-PTP1B directed against C-terminal tail (S4 Fig). Thus, results indicate that PAF induced a decrease in PTP1B 50kDa levels and this decrease was abrogated by the calpain inhibitor. Together, these results suggest that, by activating calpain, PAF induces a cleavage of PTP1B and an increase in its phosphatase activity, engendering a negative feedback for hIL-6 promoter activation. Next, we investigated whether the PAF-induced calpain activation could be under control of Jak2, given that inhibition of this kinase also inhibits the cleavage of PTP1B, as does inhibition of calpain.

Jak2 regulates PTP1B activity in a calpain-dependent manner

To explore the possibility that Jak2 modulates calpain activity, calpain assays using a fluorescent substrate were performed. HEK-PAFR were pre-treated with Jak2 inhibitor IV (Fig 10A) or SD-1029 (S5 Fig) for 20min before stimulation with 100nM PAF for indicated times. As seen in Figs 10A and S5, PAF transiently activated calpain activity with a peak at 5 min post-stimulation and a return to basal activity at 10 min. Pre-treatment with either of the inhibitors abrogated PAF-mediated calpain activation indicating a role of Jak2 in PAF-induced activation of calpain at early stimulation times. Given that PAF activates PTP1B in iMo-DCs, involvement of Jak2 in calpain activation was also investigated in these cells. iMo-DCs were incubated for 20 min with Jak2 inhibitor IV or vehicle, then stimulated with PAF for indicated times (Fig 10B). A rapid and transient increase in calpain activity, with a peak at 5 min post-stimulation, was observed in PAF-stimulated control cells, while no significant variation in calpain activity was found when cells were pre-incubated with the Jak2 inhibitor.

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Fig 10. Jak2 is involved PAF-induced calpain activity.

HEK-PAFR were starved overnight A) or B) Mo-DCs were starved for 5h, then pre-treated with vehicle or 2μM Jak2 inhibitor IV for 20min prior to stimulation with PAF for indicated times and lysis with Fluorometric Activity Assay Kit lysis buffer. Assays were performed according to manufacturer's instructions. Variations in calpain activity levels are presented as ratio of fluorescence of stimulated cells over untreated, unstimulated cells. Data presented are in Floating bar (Min to Max) with line at the mean of 3–4 independent experiments, for HEK-PAFR and 3 different donors, for iMo-DCs. Significance was established with paired two-way anova with Sidak post-test.*p<0.05, &:p< 0.05, &&: p<0.01 over unstimulated cells with the same pre-treatment.

https://doi.org/10.1371/journal.pone.0180336.g010

Jak2 regulates PTP1B in a calcium-dependent manner

It is well known that calpain activation is regulated via the modulation of intracellular calcium concentrations ([Ca²⁺]_i). Additionally, some studies show the involvement of Jak2 in [Ca²⁺]_i regulation [52]. Hence, we studied whether Jak2 could regulate [Ca²⁺]_i in PAF-stimulated cells and thus regulate PTP1B activity. HEK-PAFR were loaded with Fluo 3, pre-treated with Jak2 inhibitor IV or SD-1029 and stimulated with PAF. Changes in calcium levels were registered until 110sec post-stimulation. PAF induced a rapid increase in [Ca2+]i in control cells: 5s after stimulation, the [Ca²⁺]_i was 2432.9±315.1nM over the basal [Ca²⁺]_i (Fig 11A). On the other hand, in cells pre-treated with the Jak2 inhibitor IV or SD-1029, the [Ca²⁺]_i reached only 1157.3±165.3nM and 596.6±171.8nM, respectively, over basal levels, a significant decrease compared to control cells. For all conditions, the [Ca²⁺]_i rapidly decreased to similar levels around 35 sec. Pre-treatment with the Jak2 inhibitors did not significantly affect [Ca²⁺]_i in unstimulated cells and did not completely abrogate PAF-induced increase in [Ca²⁺]_i. This latter point suggests that Jak2 is not essential for calcium response but rather that it is involved in the modulation of its intensity.

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Fig 11. Jak2 regulates PTP1B in a calcium-dependent manner.

A) HEK-PAFR were starved for 5h, incubated 30min with 5μM Fluo3 and pre-treated with vehicle, Jak2 inhibitor IV or SD-1029 for 20min in HBSS 0.2%BSA prior to stimulation with 100nM PAF in presence of 2mM CaCl₂, for indicated times. Minimal and maximal fluorescence was determined by the addition of EGTA (100mM) or Triton X-100 (0,5% final), respectively. Data presented are mean±S.E.M of fold increase of [Ca²⁺]_i over unstimulated cells obtained from 3–4 independent experiments. Significance was established with paired two-way anova with Sidak post-test: *p<0.05, &p< 0.05 over unstimulated cells with the same pre-treatment. B) Phosphatase activity was measured via the hydrolysis rate of pNPP by PTP1B. HEK-PAFR were treated with Jak2 inhibitor IV or vehicle. These cells were then stimulated with vehicle, 200nM thapsigargin or 100nM PAF or co-stimulated with PAF and thapsigargin or PAF and vehicle. PTP1B was immunoprecipitated and incubated with pNPP. Data presented are mean ±S.E.M of hydrolysis rate from 4 independent experiments. Significance was established with paired two-way anova with Sidak post-test: ** p<0.01 and & p< 0.05 && p<0.01, over unstimulated cells with the same pre-treatment.

https://doi.org/10.1371/journal.pone.0180336.g011

Next, we confirmed that modulation of PTP1B activity by Jak2 depended on calcium. PTP1B activity assays were performed using Jak2 inhibitor IV- or vehicle-pre-treated cells, which were then stimulated with 200nM thapsigargin or 100nM PAF or co-stimulated with PAF and thapsigargin. Co-stimulations were done to test whether the addition of thapsigargin could rescue PTP1B activity. Fig 10B shows that thaspsigargin alone increased pNPP hydrolysis rate by PTP1B, indicating that calcium mobilization alone is enough to influence PTP1B activation. This thapsigargin-mediated increase in pNPP hydrolysis rate was not affected by the Jak2 inhibitor IV. When cells were stimulated for 5 min with PAF, no difference in PTP1B activity was found between cells stimulated with PAF alone and those co-stimulated with thapsigargin. The hydrolysis rate, as described in Fig 2, was reduced in cells pre-treated with Jak2 inhibitor IV and stimulated 5min with PAF, but PTP1B activity was rescued when cells were co-stimulated with thapsigargin. These results suggest that Jak2 inhibitor IV-mediated decrease in calcium influx could cause the decrease seen in PAF-induced PTP1B activity in Jak2 inhibitor pre-treated cells. The PTP1B activity measured in Jak2 inhibitor IV-treated cells co-stimulated with PAF and thapsigargin was increased compared to co-stimulated cells without the Jak2 inhibitor IV, or compared to cells stimulated with PAF alone, suggesting that PAF could probably fine-tune PTP1B activity by other pathways than Jak2.