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. 2008 Nov 15;112(10):4098-108.
doi: 10.1182/blood-2008-03-148726. Epub 2008 Aug 20.

"VSports手机版" PTP1B is a negative regulator of interleukin 4-induced STAT6 signaling

Xiaoqing Lu  1 , Raquel Malumbres, Benjamin Shields, Xiaoyu Jiang, Kristopher A Sarosiek, Yasodha Natkunam, Tony Tiganis, Izidore S Lossos
Affiliations

PTP1B is a negative regulator of interleukin 4-induced STAT6 signaling

Xiaoqing Lu et al. Blood. .

Abstract

Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed enzyme shown to negatively regulate multiple tyrosine phosphorylation-dependent signaling pathways. PTP1B can modulate cytokine signaling pathways by dephosphorylating JAK2, TYK2, and STAT5a/b. Herein, we report that phosphorylated STAT6 may serve as a cytoplasmic substrate for PTP1B. Overexpression of PTP1B led to STAT6 dephosphorylation and the suppression of STAT6 transcriptional activity, whereas PTP1B knockdown or deficiency augmented IL-4-induced STAT6 signaling. Pretreatment of these cells with the PTK inhibitor staurosporine led to sustained STAT6 phosphorylation consistent with STAT6 serving as a direct substrate of PTP1B. Furthermore, PTP1B-D181A "substrate-trapping" mutants formed stable complexes with phosphorylated STAT6 in a cellular context and endogenous PTP1B and STAT6 interacted in an interleukin 4 (IL-4)-inducible manner. We delineate a new negative regulatory loop of IL-4-JAK-STAT6 signaling. We demonstrate that IL-4 induces PTP1B mRNA expression in a phosphatidylinositol 3-kinase-dependent manner and enhances PTP1B protein stability to suppress IL-4-induced STAT6 signaling. Finally, we show that PTP1B expression may be preferentially elevated in activated B cell-like diffuse large B-cell lymphomas. These observations identify a novel regulatory loop for the regulation of IL-4-induced STAT6 signaling that may have important implications in both neoplastic and inflammatory processes VSports手机版. .

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Figures

Figure 1
Figure 1
PTP1B dephosphorylates IL-4–induced STAT6. (A,B) HEK293 cells were transfected with PTP1B (A) or PTP-PEST (B) plasmids and either STAT6 plasmid or empty vector. Forty-eight hours after transfection, cells were stimulated with IL-4 (100 U/mL) for 30 minutes. As a control, nontransfected HEK293 cells were similarly stimulated (A). Cellular proteins from unstimulated and IL-4–stimulated cells were resolved by SDS-PAGE and immunoblotted for tyrosine-phosphorylated STAT6 (pSTAT6), STAT6, PTP1B, or PTP-PEST and actin. The result is representative of 3 independent experiments.
Figure 2
Figure 2
PTP1B-D181A colocalizes and traps pSTAT6 in the cytoplasm. Control vector-transfected HeLa cells (A) or cells transiently expressing PTP1B D181A mutant (B) were stimulated with IL-4 (100 U/mL) for 15 minutes, fixed in 4% formaldehyde, and stained with DAPI and antibodies to pSTAT6 and PTP1B as described in “Methods” in “Microscopy.” Cells were visualized by Zeiss LSM510 confocal microscope with a plan-apochromat 40×/1.3 NA oil-immersion lens, 364, 488, and 555 nm laser lines, for DAPI, Alexa Fluor 488 detection of anti-STAT6, and Alexa Fluor 555 detection of anti-PTP1B, respectively. Images were acquired using 1024 × 1024 pixel scan size, 12-bit digitization, and 4-frame averaging. The measurements were performed as described in “Methods” in “Microscopy.” (C) Four-fold magnification of individual cells from experimental conditions shown in panels A and B. (A-C) Slides were viewed with a Zeiss LSM 510/UV confocal Axiovert 200M microscope (Zeiss, Thornwood, NY) using a Zeiss plan-apochrome oil-immersion lens at 40×/1.3 NA and ProLong Gold antifade reagent (Invitrogen, Eugene, OR) as mounting medium. Images were acquired using a Zeiss LSM510/UV confocal scanner, and were processed with Zeiss LSM510 AIM 3.2 SP2 confocal microscope software and Adobe Photoshop version 7.0 (Adobe, San Jose, CA). (D) Pixel intensity corresponding to pSTAT6 was quantified as described in “Methods” in “Microscopy” in 175 control HeLa cells and 175 HeLa cells expressing the PTP1B D181A mutant. The results shown are mean plus or minus SE; P < .001. The results in panels A, B, and D are representative of 3 independent experiments.
Figure 3
Figure 3
STAT6 is the physiologic substrate of PTP1B. (A) HEK293 cells were transfected with Myc-tagged STAT6 and PTP1B or PTP1B D181A plasmids. At 48 hours after transfection, cells were stimulated with IL-4 (100 U/mL) for 30 minutes. Cellular lysates were extracted and subjected to immunoprecipitation with anti-MYC antibody. (B) HeLa cells were transfected with either SMART pool siRNA for PTP1B or scrambled control (200 pmol). At 72 hours after transfection, the cells were stimulated with IL-4 (100 U/mL) for 15 to 120 minutes. Cellular lysates were extracted at indicated time points and blotted for pSTAT6, STAT6, PTP1B, and actin. Mean relative densitometry of pSTAT6/STAT6 ratio from 3 independent experiments is depicted. The value in specimen siRNA-PTP1B at time point 0 was arbitrarily defined as 1. (C) HeLa cells were transfected with either SMART pool siRNA for PTP1B or scrambled control (200 pmol). At 48 hours after transfection, the cells were stimulated with IL-4 (100 U/mL) for 15 minutes and fixed in 4% formaldehyde and stained with 7AAD (nuclear staining) and antibodies to pSTAT6 and PTP1B. Slides were viewed with a Zeiss LSM510/UV confocal Axiovert 200M microscope (Zeiss, Thornwood, NJ) using a Zeiss plan-apochrome oil-immersion lens at 40×/1.3 NA and ProLong Gold antifade reagent (Invitrogen, Eugene, OR) as mounting medium. Images were acquired using a Zeiss LSM510/UV confocal scanner, and were processed with Zeiss LSM510 AIM 3.2 SP2 confocal microscope software and Adobe Photoshop version 7.0 (Adobe, San Jose, CA). (D) IL-4 induces enhanced STAT6 phosphorylation in PTP1B-deficient cells. Mouse embryo fibroblasts from PTP1B-deficient (−/−) and wild-type (+/+) mice were serum starved for 4 hours and then stimulated with 50 ng/mL IL-4 for the indicated times. Activation of STAT6 and AKT was assessed with phosphotyrosine-specific STAT6 and phosphoserine-specific AKT antibodies. Equal loading was confirmed by immunoblotting with an actin antibody. Representative results of 3 independent experiments are shown.
Figure 4
Figure 4
Dephosphorylation of pSTAT6 by PTP1B is independent of PTP1B effects on JAK1. (A) HEK293 cells were transfected with STAT6 plasmid and either PTP1B plasmid or empty vector. Forty-eight hours after transfection, cells were stimulated with IL-4 (100 U/mL) for 30 minutes. Cellular lysates were extracted and subjected to immunoprecipitation with anti-JAK1 or JAK2 antibodies followed by anti- pTyr or anti-JAK1 or JAK2 Western immunoblotting. Corresponding cellular lysates were also immunoblotted with STAT6 and pSTAT6-specific antibodies. (B) HeLa cells were either left unstimulated or were stimulated with IL-4 (100 U/mL) for 30 minutes. Cellular lysates were extracted and subjected to immunoprecipitation with anti- JAK1 and PTP1B followed by anti-PTP1B and anti-JAK1 immunoblotting. (C) HeLa cells were transfected with either SMART pool of siRNA for PTP1B or scrambled control (200 pmol). At 72 hours after transfection, the cells were stimulated with IL-4 (100 U/mL) for 15 to 45 minutes with or without the addition of staurosporine starting 15 minutes after IL-4 stimulation. Cellular proteins were immunoblotted for pSTAT6, STAT6, and actin at the indicated times. Representative results of 3 independent experiments are shown.
Figure 5
Figure 5
PTP1B interacts with the transactivation domain of STAT6. (A) Schematic representation of STAT6 and its mutant missing the C-terminal transactivation domain (STAT6ΔC). (B,C) HEK293 cells were transfected with MYC-fused STAT6 or STAT6 ΔC plasmids and vector control, PTP1B, or PTP1B D181A plasmids. At 48 hours after transfection, cells were stimulated with IL-4 (100 U/mL) for 30 minutes. Cellular lysates were extracted and subjected to immunoprecipitation with (B) anti-MYC or (C) anti-PTP1B antibodies followed by anti-PTP1B and anti-STAT6 Western blotting. (D) HEK293 cells were transfected with PTP-PEST, PTP-PEST-D199A, STAT6 plasmids, and vector control. At 48 hours after transfection, cells were stimulated with IL-4 (100 U/mL) for 30 minutes. Cellular lysates were extracted and subjected to immunoprecipitation with anti–PTP-PEST or anti-STAT6 followed by blotting with anti-STAT6 and anti–PTP-PEST antibodies, respectively. (E) HeLa and OCILY10 cells were stimulated with IL-4 (100 U/mL) for 30 minutes. Cellular lysates were extracted and subjected to immunoprecipitation with anti-STAT6 followed by anti-PTP1B or anti-STAT6 blotting. (F). STAT6 and PTP1B were transiently expressed in the HEK293 cells, as described in “Methods” in “Cell transfections.” At 48 hours after transfection, the cells were left untreated or incubated for 30 minutes with sodium orthovanadate (2 mM). The cells were then stimulated with IL-4 (100 U/mL) for 30 minutes, and cellular lysates were extracted and subjected to immunoprecipitation with anti-MYC followed by blotting with either anti-PTP1B or anti-STAT6 antibodies. Representative results of 3 independent experiments are shown in panels B-F.
Figure 6
Figure 6
PTP1B inhibits IL-4–induced gene transcription. (A) Mock or PTP1B encoding plasmids were cotransfected into HEK293 cells with the STAT6-driven luciferase reporter-construct C/EBP-N4 and STAT6 and pRL plasmids. Luciferase activity was determined 48 hours after transfection in either unstimulated cells or cells that had been stimulated with IL-4 (100 U/mL) for 6 hours. Numbers refer to relative luciferase activities, the average value obtained for the reporter without activator being taken as 1. Numbers are means and SD of 3 independent experiments, each performed in triplicate. (B) VAL cells were transiently transfected with either control pIRES-hrGFPII vector or pIRES-hrGFPII PTP1B vectors as described in “Methods” in “Cell transfections.” At 48 hours after transfection, cells were sorted for GFP expression and incubated with or without IL-4 (100 U/mL) for 6 hours. RNA was extracted, and CD23 and PTP1B RNA expression was measured by real-time RT-PCR in triplicate. The result is representative of 3 independent experiments.
Figure 7
Figure 7
PTP1B mRNA and protein levels are increased by IL-4 stimulation. (A) DLBCL cell lines RCK8 or VAL (not shown) were stimulated with IL-4 (100 U/mL) for 3, 6, and 12 hours. RNA was extracted and PTP1B mRNA was measured by real-time RT-PCR in triplicate. In addition, cells lysates were prepared and PTP1B and actin were immunoblotted. (B) To monitor for the effect of IL-4 on protein stability, pulse-chase experiments were performed as described in “Methods” in “Pulse-chase immunoprecipitation assays.” Briefly, RCK8 cells were starved of methionine and then labeled with [;35S]methionine for 2 hours, after which they were either left unstimulated or stimulated with IL-4 (100 U/mL) for up to 24 hours and incorporated [35S]methionine monitored in PTP1B immunoprecipitates by autoradiography. MD indicates mean densitometry of 3 independent experiments. The value at time point 0 was arbitrarily defined as 1. (C) VAL DLBCL cells were transfected with control or STAT6 siRNA. At 48 hours after siRNA transfection, the cells were stimulated with IL-4 for 6 hours. RNA was extracted, and PTP1B and CD23 RNA expression was measured by real-time RT-PCR in triplicate. Cells lysates were immunoblotted for STAT6 and actin. (D) VAL DLBCL cells were grown in complete media supplemented with Ly294002 (10 μM) or PD98059 (30 μM) for 30 minutes. The cells were stimulated with IL-4 for 6 hours, RNA was extracted, and PTP1B RNA expression was measured by real-time RT-PCR in triplicate. (E) RCK8 DLBCL cells were grown in complete media either without or with IL-4 (100 U/mL) for 15 hours. The cells were then stimulated with IL-4 (100 U/mL) for 30 minutes, and cellular lysates were extracted and blotted for pSTAT6, STAT6, PTP1B, and actin. (F) RCK8 DLBCL cells were grown in complete media either without (green) or with IL-4 (100 U/mL; red) for 15 hours. The cells were stained with phycoerythrin-conjugated antihuman IL-4R antibody or appropriate anti isotype control antibody (black) and analyzed by flow cytometry. Representative results from 3 independent experiments are shown.
Figure 8
Figure 8
Coordinated regulation of STAT6 by PTP1B and TCPTP and PTP1B expression in DLBCL. (A) HeLa cells were transfected with either SMART pool siRNA for PTP1B, TCPTP, or scrambled control (200 pmol). At 72 hours after transfection, the cells were stimulated with IL-4 (100 U/mL) for 15 minutes. Cytoplasmic and nuclear lysates were extracted and blotted for tyrosine-phosphorylated STAT6 (pSTAT6), STAT6, PTP1B, TCPTP, actin, and nucleolin. (B) Immunohistochemical staining for PTP1B in tonsils and DLBCL. Low magnification image of normal tonsil sections shows PTP1B-specific staining mainly outside germinal centers (original magnification ×40); PTP1B staining is localized to the cytoplasm. Also demonstrated are representative examples of PTP1B immunostaining in DLBCL (original magnification ×400). Images of immunohistologic staining were acquired using a Nikon Eclipse E400 microscope (Nikon, Tokyo, Japan) and a Nikon DS-L1 digital camera. (C) Hierarchical cluster analysis of immunohistologic data. The expression patterns of 7 proteins (LMO2, HGAL, CD10, BCL6, MUM1/IRF4 (MUM1), BCL2, and PTP1B) in 80 cases of DLBCL are shown. Positive staining is indicated in red, lack of staining in green, and uninformative data in white. PTP1B protein expression is clustered on the same branch of the dendrogram as nongerminal-center proteins MUM1 and BCL2 and away from germinal-center proteins HGAL, BCL6, LMO2, and CD10.
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