From the
Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06510 and the
Molecular and Cellular Pharmacology, ISIS Pharmaceuticals, Carlsbad, California 92008
Received for publication, November 21, 2002 , and in revised form, April 10, 2003.
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ABSTRACT |
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INTRODUCTION |
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In the case of IL-6 or IL-11, cytokine binds to cell surface-associated receptor subunits ( chains) that lack signaling domains. Ligand binding induces association of these
chains with gp130 and causes gp130 homodimer formation. OnM initiates signals by binding directly to gp130 and the OnM/gp130 complex induces formation of either a LIFR
-gp130 receptor complex or an OnMR
-gp130 receptor complex (5, 6). Binding of LIF to LIFR
chain results in the formation of the LIFR
-gp130 receptor complex. Each of these clustered receptors initiates signaling via transphosphorylation of receptor-associated cytosolic tyrosine kinases of the Janus kinase family, namely Janus kinase (JAK)1, JAK2, and tyrosine kinase 2 (TYK2) (7). Once activated, these kinases phosphorylate gp130 on several intracellular tyrosine residues, providing docking sites for various cytoplasmic proteins containing Src homology 2 (SH-2) domains. For example, signal transducer and activator of transcription (STAT3) binds to four different phosphorylated tyrosine residues in the intracellular region of gp130 (Tyr767, Tyr814, Tyr905, and Tyr915); two of these tyrosine residues (Tyr905 and Tyr915) also contribute to the binding of STAT1 (8, 9). Phosphorylation of Tyr residue 759 leads to recruitment of SH-2 domain-containing tyrosine phosphatase 2 (SHP-2) (1014). STAT and SHP-2 molecules bound to gp130 subsequently become tyrosine phosphorylated by receptor-associated JAKs. Phospho-STAT proteins form homo- and/or heterodimers which translocate to the nucleus where they act as transcriptional activators of cytokine-inducible genes. SHP-2 is thought to provide regulatory feedback through its phosphatase activity but also acts as a gp130-associated linker/adapter protein, binding Grb2/Sos and initiating the activation of a mitogen-activated protein kinase (MAPK) pathway independent of its tyrosine phosphatase activity (11, 15). The same phosphotyrosine residue that binds SHP-2 also recruits suppressor of cytokine signaling protein (SOCS-3). OnM, but not IL-6 or LIF, induces recruitment and activation of phosphatidylinositol 3-kinase (PI-3-kinase) which, in turn results, in membrane association phosphorylation, and activation of PI-3 dependent kinases and of protein kinase B/AKT (16). This response is initiated by phosphorylation of Tyr residue 861 in the OnMR
chain (16).
Activation of signal transduction pathways through gp130 are transient, implying that JAK/STAT signal transduction pathway activated through gp130 is under negative control. As noted above, SHP-2 is a potential regulator of this pathway (17, 18). SHP-2 is an ubiquitiously expressed and highly conserved enzyme. JAK-mediated tyrosine phosphorylation of SHP-2 is believed to increase its catalytic activity (19). SHP-2 can down-regulate gp130-mediated signaling upon association with the phosphorylated Tyr residue 759 of gp130 by exerting tyrosine phosphatase activity both on other tyrosine residues of the receptor and possibly on associated JAK proteins (11, 12, 14). Mutation of Tyr residue 759 to Phe in gp130 inhibits SHP-2 binding to gp130 and leads to elevated and prolonged activation of JAKs and STATs. SOCS proteins have also been implicated in the down-regulation of gp130 signaling (10, 20, 21). Overexpression of SOCS-1 or SOCS-3 inhibits IL-6- and LIF-induced differentiation of murine monocytic leukemia M1 cells and LIF induction of a STAT3-responsive reporter construct in 293T fibroblast (22). SOCS-1 protein interacts directly with JAKs by binding to their activation loop in a phosphorylation-dependent manner (20). In contrast, SOCS-3 binds directly to Tyr residue 759 on gp130 (10, 12), and this interaction is necessary for the inhibitory effect of overexpressed SOCS-3, but not of SOCS-1, on both STAT1 and STAT3 activation in HepG2 cells (10). SOCS-3 mRNA is rapidly and transiently induced by IL-6 and LIF, and LIF-stimulated SOCS-3 gene expression is at least in part is mediated by STAT3 and STAT1 (2326). In this way, SOCS-3 acts as a negative regulator of its own gene expression.
We have previously shown that in cultured human umbilical vein endothelial cells (HUVEC) OnM and IL-11 rapidly but transiently activate STAT1, STAT3, p42, and p44 MAPK (27). Tyr residue 759 of gp130 signaling domain was demonstrated to be necessary for this transience of activation of STAT1 in HUVEC (27), implying that the short duration of active signaling through gp130 is mediated by SHP-2 or SOCS-3. In the present study, we show that stimulation with OnM desensitizes HUVEC as well as human aortic smooth muscle cells (HASMC) to further activation by OnM. Desensitization can occur in trans, affecting other gp130-signaling cytokines, and also involves Tyr residue 759 in gp130. However, our data indicate that this desensitization is not mediated by either SOCS-3 or SHP-2, the only two proteins to date shown to bind to this site.
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EXPERIMENTAL PROCEDURES |
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Mouse monoclonal antibody (Ab) to -actin was obtained from Sigma. Rabbit polyclonal Abs to STAT1, phosphotyrosine STAT1, STAT3, phosphotyrosine STAT3, phosphoserine AKT, phosphotyrosine TYK2, and to phosphothreonine/phosphotyrosine-p42/p44 MAPK were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Rabbit polyclonal Ab to SOCS-3 was purchased from Immuno-Biological Laboratory Co., LTD. (Japan). Mouse monoclonal Ab to SHP-2 and mouse monoclonal Ab to JAK1 were purchased from BD Pharmingen (Los Angeles, CA). Rabbit polyclonal Ab to phosphotyrosine JAK1 was purchased from Biosource International, Inc. (Camarillo, CA). Mouse monoclonal Ab to hemagglutinin (HA) was purchased from Roche Applied Science. Rabbit polyclonal Ab to gp130 and mouse monoclonal Ab to phosphotyrosine were purchased from Upstate Biotechnology (Lake Placid, NY).
Human umbilical vein EC were isolated from discarded umbilical cords as previously described (28, 29) under a protocol approved by the Yale Human Investigation Committee. ECs from 35 cords were pooled and serially cultured on gelatin (J. T. Baker, Phillipsburg, NJ)-coated tissue culture plastic at 37 °C in 5% CO2-humidified air in Medium 199 containing 20% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin (all from Invitrogen) 50 µg/ml of fibroblast growth factor-1 (Collaborative Research/Becton Dickinson, Bedford, MA) and 100 µg/ml of porcine intestinal heparin (Sigma). Confluent cultures were serially passaged, and cells were typically used at the second or third subculture. Such cultures are uniformly positive for von Willebrand factor and CD31 and lack detectable contamination by CD45-expressing leukocytes.
HASMC were isolated from aorta removed from donor human heart prior to transplantation under a protocol approved by Yale Human Investigation Committee. The intimal cell layer and adventitial layer were peeled off, and the medial layer, containing smooth muscle cells, was dissected into small fragments, which were explanted on gelatin-coated tissue culture plastic at 37 °C in 5% CO2-humidified air in Medium 199 containing 20% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin to permit outgrowth of HASMC. Confluent HASMC cultures were serially passaged in the same media, and cells were typically used at the second or third subculture. Such cells are uniformly positive for smooth muscle specific -actin.
The Phoenix-Ampho packaging cell line for production of high titer amphotropic retroviruses was obtained from Dr. G. Nolan (Stanford University, Stanford, CA) and cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin.
To perform pretreatment experiments, cells were pretreated with OnM for various times as indicated. The medium was removed, and cells were then treated with either OnM, LIF, or IL-11 for the times indicated.
RNA Isolation and Quantitative Real-time RT-PCRTotal RNA was isolated from HUVEC with Qiagen Rneasy mini kit (Qiagen Inc., Valencia, CA) as recommended by the supplier. Total RNA was quantitated by OD at 260 using a Du-64 spectrophotometer (Beckman, Columbia, MD). Using equal amount of total RNA (200 ng) from HUVEC, stimulated under various conditions, mRNA was primed with random hexamers, and complementary DNA (cDNA) was synthesized from mRNA by TaqMan reverse transcription with MultiScribe reverse transcriptase (PE Applied Biosystems, Foster, CT) according to the manufacturer's description. The final cDNA product was used for subsequent cDNA amplification by polymerase chain reaction.
cDNA was amplified and quantitated by using SYBR Green PCR reagents from PE Applied Biosystems according to the manufacturer's instructions. Briefly, the cDNA for SOCS-3 and GAPDH were amplified by AmpliTag Gold DNA polymerase using specific primers, which were synthesized by Yale HHMI/Keck oligonucleotide synthetic facility (Yale University School of Medicine, New Haven, CT). The cDNA for GAPDH was amplified by using a specific forward primer (5'-GAAGGTGAAGGTCGGAGTC-3') and a specific reverse primer (5'-GAAGATGGTGATGGGATTTC-3'). The following specific primers were used to amplify SOCS-3 cDNA: a forward primer, 5'-GGCCACTCTTCAGCATCTC-3' and a reverse primer, 5'-ATCGTACTGGTCCAGGAACTC-3'. The PCR reaction mixture (final volume 25 µl) contained 5 µl of cDNA, 1 µl of 10 µM forward primer, 1 µl of 10 µM reverse primer, 2.5 µl of PCR 10x SYBR Green PCR buffer, 3 µl of 25mM MgCl2, 2 µl of dNTP mix (2.5 mM dATP, 2.5 mM dCTP, 2.5 mM dGTP, and 5 mM dUTP), 0.25 µl of AmpErase UNG (1 unit/µl uracil-N-glycosylase), 0.125 µl of AmpliTag Gold DNA polymerase (5 units/µl AmpliTag Gold DNA polymerase), and 10.125 µl of H2O. The PCR reaction was performed in triplicate (3 wells of C96 well plate). The reaction was amplified with iCycler iQ Multicolor Real Time PCR Detector (Bio-Rad) for 37 cycles with melting at 94 °C for 30 s, an annealing at 58 °C for 30 s, and extension at 72 °C for 1 min in iCycler iQ PCR 96-well plates (Bio-Rad).
The relative quantification values for the SOCS-3 gene expression were calculated from the accurate CT, which is the PCR cycle at which an increase in reporter fluorescence from SYBR Green dye can be first detected obtained above a baseline signal. CT values for GAPDH cDNA were subtracted from CT values for SOCS-3 cDNA for each well to calculate -CT. The triplicate
-CT values for each sample were averaged. To calculate the fold induction of SOCS-3 mRNA in cells treated with cytokines over control cells, the averaged
-CT values calculated for control cells was subtracted from
-CT values calculated for cytokine-treated cells to calculate
-CT. Then, the fold induction for each well was calculated by using 2(
-CT) formula. The fold induction value for triplicate wells was averaged, and data are presented as the mean ± S.E. of triplicate wells.
ImmunoblottingFollowing the indicated treatment, confluent EC cultures were washed twice with ice-cold phosphate-buffered saline (PBS) containing 1 mM sodium orthovanadate and 1 mM sodium fluoride and lysed with ice-cold radioimmune precipitation assay lysis buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM pefabloc, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mg/ml benzamidine, 1 mM sodium orthovanadate, 1 mM sodium fluoride). Cell lysates were clarified by centrifugation at 10,000 x g for 15 min, and protein concentrations of the supernatant were determined by using a Bio-Rad assay kit (Bio-Rad). Clarified lysates were prepared for SDS-polyacrylamide electrophoresis (PAGE) by adding an equal volume of 2x SDS-PAGE sample buffer (100 mM Tris-Cl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromphenol blue, 20% glycerol) and heating the mixture in a boiling water bath for 3 min. 20 µg of protein were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Immobilon P, Millipore, Bedford, MA) by electrophoresis. After blocking with TBS-T (10 mM Tris-HCl, pH 8.0, 0.150 mM NaCl, 0.05% Tween 20) containing 5% nonfat milk for 1 h at room temperature, the membranes were incubated with blocking solution containing the indicated Ab overnight at 4 °C. Membranes were washed and incubated with a horseradish peroxidase (HRP)-conjugated detecting reagent specific for the primary Ab (Jackson Immuno Research, West Grove, PA), and HRP activity was detected using an enhanced chemiluminescence kit according to the manufacturer's instructions (Pierce). Exposed films were scanned using a laser densitometer (Fast Scan, Series 300, Molecular Dynamics, Sunnyvale, CA).
gp130 was immunoprecipitated and P-gp130 was detected as described previously (27). For immunoprecipitation prior to immunoblotting of P-JAK1, 500 µg of total cell lysate was precleared by addition of 50 µl of GammaBind G Sepharose beads (Amersham Biosciences) with continual incubation on a rotator at 4 °C for 90 min. The beads were removed from the precleared lysates by centrifugation. To form specific immune complexes, 2.5 µg of anti-JAK1 Ab was added to the precleared lysate, which was then incubated for 3 h on a rotator at 4 °C. To collect specific immune complexes, 50 µl of GammaBind G Sepharose beads were added and the sample was incubated on a rotator at 4 °C overnight at which time beads were collected by centrifugation at 13,000 x g for 1 min. The beads containing immune complexes were washed five times with PBS, the immune complexes were solubilized from the beads by addition of 1x SDS-PAGE sample buffer and heated in a boiling water bath for 5 min. Aliquots were resolved by SDS-PAGE and immunoblotted for total JAK1 and for P-JAK1, as described above.
Direct Immunofluorescence and FACS AnalysisFor immunostaining, HUVEC were washed with Hanks-buffered saline solution and incubated for 1 min with trypsin/EDTA. Detached cells were collected and washed twice with ice-cold PBS containing 1% bovine serum albumin (BSA), and incubated with specific phycoerythrin(PE)-conjugated mouse monoclonal Ab to gp130 (R&D Systems) or control PE-conjugated mouse IgG (Jackson Immuno Research) for 30 min at 4 °C. Cells were washed twice with PBS/1% BSA and fixed with 2% paraformaldehyde. After fixation, cells were analyzed by FACS using a FACSort and Lysis II software (BD Biosciences, San Jose, CA). Corrected mean fluorescence values were calculated as follows: for each treatment the mean fluorescence value for control PE-conjugated mouse IgG was subtracted from the mean fluorescence value for the specific PE-conjugated Ab to gp130.
Construction of Retroviral Vectors Expressing Enhanced Green Fluorescent Protein (EGFP), PDGFR-gp130, and (
446474)SHP-2Retroviral expression vectors coding for chimeric receptors containing the extracellular domains of human PDGFR
receptor and transmembrane and cytosolic portions of wild-type gp130 (gp130(Tyr759)) or mutant gp130 (gp130(Y759F)), in which Tyr residue 759 was replaced with Phe, were constructed as described previously (27). Retroviral expression vectors coding for [
446474]SHP-2 was constructed as follows. Human SHP-2 cDNA coding for SHP-2, which lacks amino acids 446474 ([
446474]SHP-2) in expression vector pBluescript KS was kindly provided by Dr. A. Bennett (Yale University School of Medicine, New Haven, CT). The cDNA was isolated by PCR amplification, and a sequence coding for HA was tagged into 5'-end of the cDNA. The BamH1-EcoR1 fragment containing cDNA coding [
446474]SHP-2-HA was subcloned into the LZRSpBMN-Z retroviral vector using 5' BamH1 and 3' EcoR1 cloning sites. The LZRSpBMN-Z retroviral vector and LZRSpBMN-Z retroviral vector containing EGFP was kindly provided from Dr. A. Bothwell (Yale University School of Medicine, New Haven, CT) (30). These retroviral vectors containing EGFP, PDGFR
-gp130(Tyr759), and PDGFR
-gp130(Y759F), and [
446474]SHP-2-HA were directly transfected into the Phoenix-Amphoteric packaging cell line using LipofectAMINE Plus reagent (Invitrogen). Two days after transfection Phoenix cells were selected in media containing puromycin (1 µg/ml) (Sigma), and puromycin-resistant cells were used to condition medium, providing a source of retroviral stock.
Transduction of HUVEC was accomplished by serial infections over 2 weeks without drug selection as previously described (30). A round of viral infections in the presence of polybrene (8 µg/ml) (Sigma) was performed for 5 h with HUVEC in primary culture. The normal growth medium was replaced, and cells were maintained overnight. The infection was repeated the next day. After the second round of infection, cells were passaged, and then the process of double infection was repeated. Using this protocol the percentage of HUVEC expressing transduced genes is routinely >95%.
Antisense Oligonucleotide Transfection of HASMCSpecific antisense oligonucleotides for human STAT3, human SHP-2, and mismatch controls were designed and synthesized by ISIS Pharmaceuticals (Carlsbad, CA). The following oligonucleotides were used: SHP-2 antisense (ISIS 103918, 5'-CGCGATGTCATGTTCCTCCC-3'); scramble control antisense for SHP-2 (ISIS 12307, 5'-TCGTCCAGGCCTCGTTCACT-3'); STAT3 antisense (ISIS 113176, 5'-GCTCCAGCATCTGCTGCTTC-3'); mismatch control antisense for STAT3 (ISIS 129987, 5'-GCTCCAATACCCGTTGCTTC-3'); SOCS3 antisense (ISIS 267676, 5'-GCAGCTGGGTGACTTTCTCA-3'). To transfect HASMC with oligonucleotides, cells were grown to 70% confluency in 6-well plates and then incubated with either 200 nM of STAT3 or mismatch control or 3 µM of SHP-2 antisense or mismatch control antisense or 500 nM SOCS-3 antisense or the mismatch control along with 20 µl of FuGENE 6 transfection reagent according to the manufacturer's instructions (Roche Applied Science). After 48 h, the transfection was repeated, and cells were used 48 h after the second round of transfection.
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RESULTS |
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The induction of SOCS-3 mRNA by gp130-signaling cytokines is mediated by STAT proteins (24, 25). We have previously shown that in cultured HUVEC, OnM stimulates phosphorylation of gp130, STAT1, STAT3, and p42 and p44 MAPK in a time-dependent manner, and this phosphorylation is seen as early as 2.5 min, is maximal at 1015 min, and has largely disappeared by 60 min (31). We next examined whether OnM treatment of HUVEC can desensitize these signaling events measured as ability to reactivate these signaling pathways following pretreatment. HUVEC were either untreated or treated with OnM for 1 h after which the medium was removed, and cells were treated with OnM for 15 min. In cells preincubated with medium, Western blotting demonstrated that initial treatment with OnM stimulates phosphorylation of STAT1 Tyr residue 701, phosphorylation of STAT3 Tyr residue 705, and phosphorylation of p42 and p44 MAPK Thr residue 202 and Tyr residue 204 (Fig. 2A). These experiments also demonstrated phosphorylation of AKT Ser residue 473 (Fig. 2A). Phosphorylated forms of STAT1, p42 and p44 MAPK, and AKT were no longer detectable after 1 h stimulation with OnM (Fig. 2A, preincubation with OnM, control). HUVEC that were preincubated with OnM did not respond to retreatment with OnM, i.e. there was no phosphorylation of STAT1, p42 and p44 MAPK, or AKT in response to fresh OnM (Fig. 2A, preincubation with OnM). Desensitization persisted in HUVEC in the presence of OnM for at least 24 h (Fig. 2B). Similar results were observed when cells were washed three times with Hanks-buffered saline solution after pretreatment with OnM (data not shown).
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We explored the basis of STAT protein desensitization caused by OnM treatment. Transphosphorylation of receptor-associated JAK1 and TYK2 is an early event initiated upon binding of OnM to its receptor (7). Once phosphorylated and activated, these kinases phosphorylate gp130 on several intracellular tyrosine residues, providing docking sites for STATs. Therefore, we next determined whether pretreatment with OnM would inhibit tyrosine phosphorylation of TYK2, JAK1, and gp130 induced by fresh OnM. Tyrosine phosphorylation of TYK2 can be assessed by direct immunoblotting of lysates. However, we were unable to detect OnM-induced tyrosine phosphorylation of JAK1 and gp130 by this approach and to increase the sensitivity of the assay, we immunoprecipitated JAK1 and gp130 before immunoblotting with phosphotyrosine JAK1 Ab to detect P-JAK1 or phosphotyrosine Ab to detect P-gp130 (Fig. 2C), respectively. In the absence of stimulation, HUVEC express no detectable tyrosine phosphorylation of TYK2, JAK1, or gp130. However, tyrosine phosphorylation of TYK2, tyrosine phosphorylation of JAK1 on Tyr residues 1022 and 1023, and tyrosine phosphorylation of gp130 were detected after 15 min of stimulation with OnM (Fig. 2C). Pretreatment of HUVEC with OnM completely blocked tyrosine phosphorylation of TYK2, JAK1, and gp130 induced by fresh OnM (Fig. 2C). Thus desensitization appears to be mediated at the level of the receptor signaling complex.
Next, we examined whether OnM caused desensitization to other gp130-signaling cytokines. Like OnM, IL-11 (Fig. 3A) and LIF (Fig. 3B) stimulate phosphorylation of STAT1 at Tyr residue 701 and phosphorylation of STAT3 Tyr residue 705 and phosphorylated forms of STAT1 was no longer detectable after 1 h stimulation with IL-11 (Fig. 3A) and LIF (Fig. 3B). Pretreatment with OnM inhibited OnM- and IL-11-mediated phosphorylation of STAT1 and STAT3 (Fig. 3A). Similarly, pretreatment with OnM inhibited LIF-mediated phosphorylation of STAT1 and STAT3 (Fig. 3B). These experiments show that treatment of HUVEC with OnM desensitized these cells not only to OnM but also to other cytokines, which use gp130 to transduce signals. These data are also consistent with our finding that pretreatment with OnM also inhibited the induction of SOCS-3 mRNA by LIF and IL-11 (Fig. 1C).
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OnM Desensitization Is Mediated in Trans and Involves gp130 Tyr Residue 759 Because biological effects of OnM-mediated desensitization largely involves gp130, we examined whether cross-desensitization of gp130 responses by OnM could result from down-regulation and disappearance of gp130 from the surface of HUVEC. To test this, HUVEC were untreated or treated with OnM for various times, and surface expression of gp130 was detected by direct immunofluorescence followed by FACS analysis. OnM had no effect on gp130 expression at any time examined between 1 and 24 h (Fig. 4). To further rule out a direct effect on gp130 as the basis of desensitization, we examined whether OnM could inhibit signaling via a transduced chimeric receptor involving the extracellular domains of PDGFR and cytoplasmic domains of gp130. This chimeric receptor allowed us to turn on the gp130 signaling pathway independently of endogenous gp130 through addition of PDGFBB. As we have shown previously (27), stably transduced HUVEC with retroviruses encoding PDGFR
-gp130(Tyr759) respond to OnM or PDGFBB by increasing tyrosine phosphorylation of STAT1 (Fig. 5A). PDGFR
-gp130(Tyr759)-transduced cells, which were preincubated with OnM did not respond to retreatment with either OnM or PDG-FBB (Fig. 5A). In other words, binding of OnM to endogenous gp130 cross-desensitized PDGFR
-gp130(Tyr759)-transduced cells to further stimulation with PDGFBB. These experiments show that desensitization does not result from down-regulation and disappearance of gp130 from the surface of HUVEC, and is mediated in trans, affecting receptors that do not bind OnM.
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Tyr residue 759 on the intracellular region of gp130 leads to recruitment and activation of tyrosine phosphatase SHP-2 and SOCS-3 (10, 12). Both SHP-2 and SOCS-3 are negative regulators of gp130 signaling (10, 1214). Mutation of Tyr residue in gp130 results in an enhanced and prolonged STAT1 and STAT3 activation (13, 14, 27). To further explore the mechanism of desensitization, we determined whether Tyr residue 759 in gp130 plays a role in desensitization induced by OnM. To do so, we created a PDGFR-gp130 chimeric receptor, in which the extracellular domains of PDGFR
were combined with intracellular domains of gp130, which contains a mutated non-functional SHP-2 docking site. We have previously shown that PDGFBB-induced tyrosine phosphorylation of STAT1 remained elevated for up to 120 min in PDGFR
-gp130(Y759F) cells, a response which is prolonged compared with that of PDGFR
-gp130(Tyr759) transductants (27). Thus, preventing the recruitment of SHP-2 by the Y759F mutation in gp130 maintains the activation of STAT proteins. Like PDGFR
-gp130(Tyr759) transductants, treatment of PDGFR
-gp130(Y759F)-transduced cells with either OnM or PDGFBB induced tyrosine phosphorylation of STAT1 (Fig. 5B). Also similar to PDGFR
-gp130(Tyr759) transductants, pretreatment of PDGFR
-gp130(Y759F)-transduced cells with OnM completely inhibited OnM-mediated tyrosine phosphorylation of STAT1 (Fig. 5B). However, unlike PDGFR
-gp130(Tyr759) transductants, OnM did not block PDGFBB-mediated tyrosine phosphorylation of STAT1 in PDGFR
-gp130(Y759F)-transduced cells (Fig. 5B). These findings suggest that Tyr residue 759 in gp130, which binds and interacts with SHP-2 and SOCS-3, is necessary for trans desensitization induced by OnM.
Desensitization Induced by OnM Is Not Mediated by SHP-2 Next we examined the functional role of SHP-2 in desensitization induced by OnM by three independent approaches. First, we stably transduced HUVEC with retroviruses encoding either EGFP or catalytically inactive SHP-2, [446474]SHP-2-HA, which can act as a dominant negative mutant. Monoclonal Ab to HA or SHP-2 was used to confirm the expression of [
446474]SHP-2-HA in transduced cells (Fig. 6A). [
446474]SHP-2-HA was only detected in cells transduced with [
446474]SHP-2-HA (Fig. 6A, upper panel). Single protein bands were detected in cells transduced with EGFP gene corresponding to endogenous levels of SHP-2 by using Ab to SHP-2 (Fig. 6A, lower panel). This contrasts with the appearance of an additional protein band of slightly higher molecular weight detected in cells transduced with the [
446474]SHP-2-HA gene (Fig. 6A, lower panel). Pretreatment with OnM blocked tyrosine phosphorylation induced by OnM in both EGFP-transduced cells and [
446474]SHP-2-HA-transduced cells (Fig. 6B). Therefore, overexpression of truncated SHP-2 that lacks the phosphatase catalytic domain did not prevent desensitization in response to OnM. However, we are not certain that SHP-2 activation was effectively blocked because the time course of STAT1 phosphorylation was not altered (data not shown).
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Second, SHP-2 has been shown to be a calpeptin-sensitive phosphatase as calpeptin interferes with the catalytic activity of SHP-2 in vitro and with SHP-2 signaling in vivo (32). Therefore, to further investigate the role of SHP-2 in mediating desensitization, we tested the effects of calpeptin on desensitization-induced by OnM. Preincubation of HUVEC with calpeptin (100 µM) did not inhibit OnM-induced desensitization (Fig. 6C). Also, calpeptin did not change the time course of STAT1 tyrosine phosphorylation in response to OnM (data not shown).
Finally to determine whether SHP-2 is necessary for OnM-mediated desensitization, we used an antisense oligonucleotide approach to inhibit endogenous levels of SHP-2 protein. We have found that inhibition of SHP-2 expression by antisense oligonucleotide in HUVEC is less effective than in HASMC, so we extended these experiments to examine this cell type. HASMC were transfected with either scramble control antisense oligonucleotide or specific SHP-2 antisense oligonucleotide (Fig. 7). The SHP-2 antisense significantly blocked the expression of SHP-2 protein in HASMC (Fig. 7). We used these transfectants to compare the time course of STAT1 tyrosine phosphorylation in response to OnM in cells transfected with control antisense with that of cells transfected with SHP-2 antisense. In scrambled control transfectants, increased tyrosine phosphorylation of STAT1 after addition of OnM was transient and completely gone by 60 min, similar to untransfected HASMC (Fig. 7A). However, tyrsoine phosphorylation of STAT1 remained elevated for up to 1 h after stimulation with OnM in cells transfected with SHP-2 antisense (Fig. 7A). Next, we tested whether OnM pretreatment inhibits tyrosine phosphorylation of STAT1 in response to subsequent OnM treatment in cells that lack SHP-2 (Fig. 7B). Similar to HUVEC (Fig. 2) after 1 h of OnM treatment scrambled control-transfected HASMC are refractory to restimulation by OnM, measured as failure to further re-induce tyrosine phosphorylation of STAT1 (Fig. 7B). Similarly, HASMC transfected with SHP-2 antisense and pretreated with OnM also could not be re-induced to phosphorylate STAT1 by subsequent stimulation with OnM (Fig. 7B). On the bases of these three experimental approaches we conclude that SHP-2 does not play a role in desensitization induced by OnM in vascular cells.
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SOCS-3 Is Not Sufficient or Necessary for DesensitizationTo evaluate the role of SOCS-3 in desensitization induced by OnM, we first assessed the induction of SOCS-3 mRNA and protein by OnM in PDGFR-gp130-transduced cells (Fig. 8). By immunoblotting, OnM could induce SOCS-3 protein in PDGFR
-gp130(Tyr759) transductants but that PDGFR
-gp130(Y759F) transductants basally express SOCS-3 protein at levels similar to those induced by OnM and that OnM caused no further increase (Fig. 8A). By using a quantitative real time RT-PCR method, we found that, similar to untransduced HUVEC, pretreatment with OnM significantly induced SOCS-3 mRNA levels in PDGFR
-gp130(Tyr759) transductants (Fig. 8B). However, consistent with their protein expression, untreated PDGFR
-gp130(Y759F)-transduced cells already express elevated levels of SOCS-3 mRNA compared with the levels in untreated PDGFR
-gp130(Tyr759) transduced cells (Fig. 8B). Significantly, OnM treatment further increased SOCS-3 mRNA levels above that in untreated in PDGFR
-gp130(Y759F)-transduced cells (Fig. 8B), showing that elevated levels of SOCS-3 protein is not sufficient to cause desensitization of HUVEC to OnM.
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SOCS-3 is a STAT-dependent gene (24, 25). To determine whether induction of SOCS-3 protein by OnM is necessary for OnM-mediated desensitization, we used STAT3 antisense to inhibit SOCS-3 induction by OnM. HASMC were transfected with either control mismatched antisense or specific STAT3 antisense (Fig. 9A). Transfection with STAT3 antisense completely blocked the expression of STAT3 protein in HASMC (Fig. 9A). Although STAT3 antisense had no effect on the expression of STAT1 protein, phosphorylation of STAT1 by OnM was partially blocked in the presence of STAT3 antisense (Fig. 9A). More importantly, STAT3 antisense treatment completely inhibited OnM-mediated SOCS-3 induction (Fig. 9B). We used STAT3 antisense-transfected HASMC to assess whether STAT3 and SOCS-3 are necessary for desensitization. Pretreatment of STAT3 antisense transfected HASMC with OnM still inhibited tyrosine phosphorylation of STAT1 induced by OnM despite the absence of SOCS-3 protein (Fig. 9A). These results indicated that SOCS-3 is not necessary for desensitization induced by OnM, and further suggest that this role is not dependent upon any other STAT3-inducible gene products.
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It is also possible that inhibition of either SOCS-3 or SHP-2 is not sufficient to block desensitization and that simultaneous inhibition of both SOCS-3 and SHP-2 is required to prevent desensitization. To test this hypothesis, HASMC were transfected with either control antisense or combination of specific STAT3 antisense and SHP-2 antisense (Fig. 10). Transfection with STAT3 antisense and SHP-2 antisense completely blocked the expression of STAT3 protein (Fig. 10A), SHP-2 protein (Fig. 10A), and OnM-mediated SOCS-3 induction (Fig. 10B). Pretreatment of HASMC, which lack both SHP-2 and SOCS-3, with OnM inhibited tyrosine phosphorylation of STAT1 induced by OnM (Fig. 10A).
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While STAT3 antisense is effective at reducing SOCS-3 expression, it remains possible that this approach could have additional effects that complicate the interpretation of the data. In a final experiment we used SOCS-3 antisense to directly target SOCS-3 induction by OnM (Fig. 11). HASMC were transfected with either control antisense or combination of specific SOCS-3 antisense and SHP-2 antisense. Each treatment was effective on its own and the combination resulted in effective down-regulation of both proteins simultaneously (Fig. 11). Once again, blocking expression of both SOCS-3 and SHP-2 did not prevent desensitization induced by OnM (Fig. 11A).
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DISCUSSION |
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The kinetics and magnitude of cytokine signal transduction are tightly regulated (33). For the IL-6 family of cytokines, two basic processes have emerged. One process is the modulation of the expression levels of gp130 (23, 34). Baumann and co-workers (23) demonstrated that in human fibroblast or epithelial cells, gp130 levels decreased to 50% by 2 h of OnM treatment followed by recovery of the original level as determined by immunoblotting. In our study, treatment with OnM for various times up to 24 h does not alter the surface expression of gp130 in HUVEC. Moreover, ligand-induced gp130 internalization could not explain the desensitization of chimeric receptor that do not contain gp130. The second process involves the recruitment of inhibitory molecules suppressor such as SHP protein, SOCS protein, and the protein inhibitors of activated STATs (PIAS). PIAS proteins directly interact with STATs (35) and would not be expected to inhibit tyrosine phosphorylation of gp130 or JAKs and are thus unlikely to contribute to the desensitization process described here. SHP and PIAS proteins are constitutively expressed, whereas SOCS proteins are essentially absent in unstimulated cells and rapidly induced by various cytokines including IL-6 family of cytokines (23, 26). SOCS proteins are largely regulated at the transcriptional level by activated STATs (24, 25) and thus serve as negative regulator of their own expression (20). Minvielle and co-workers (26) demonstrated that among the SOCS family members, only SOCS-3 mRNA was strongly and rapidly induced by OnM and new SOCS-3 protein was present within 1 h and rapidly declined thereafter in human melanoma cell line A375. In fibroblasts and epithelial cells, SOCS-3 protein detected by immunoblotting peaked at1hofOnM treatment (23). By using SOCS-3 expression vectors for and pulse-chase experiments in COS-7 cells, Schaper and co-workers (36) showed that the SOCS-3 protein half-life is about 90 min. Consistent with these findings, we demonstrated that SOCS-3 mRNA is also rapidly and transiently induced by OnM in human vascular cells. However, we found that SOCS-3 protein levels remained elevated for up to 36 h after treatment with OnM in HUVEC. By overexpressing SOCS proteins, it has been shown that these molecules act as a negative regulator of gp130 signaling (22, 26). For example, constitutive expression of SOCS-3 protein completely abolished the activation of JAK-STAT signaling pathway in response to OnM in human melanoma cell line A375 (26). Recently, N. Kaur et al. (37) demonstrated that exposure of neuroblastoma cells to CNTF causes desensitization to CNTF and other gp130 receptor cytokines. They demonstrated that these cells constitutively express significant amounts of SOCS-1 and SOCS-3 protein, suggesting that CNTF-mediated desensitization is not caused by SOCS-1 or SOCS-3. There are no published reports that demonstrate the effects of SOCS-3 overexpression on the gp130 signaling in vascular cells. We have demonstrated that PDGFR-gp130(Y759F)-transduced HUVEC constitutively express levels of SOCS-3 protein comparable to that induced by OnM. However, OnM treatment of these cells still results in an increase in tyrosine phosphorylation of STAT1. Therefore, unlike other cells types, HUVEC can respond to OnM in the presence of SOCS-3, and induction of SOCS-3 cannot account for induction of desensitization. It is possible that other members of SOCS family proteins that are induced by OnM could bind to the Tyr residue 759 of gp130 and mediate desensitization. By using STAT3 antisense, although we were able to eliminate STAT3 protein expression in HASMC, we did not inhibit the desensitization induced by OnM. This result makes it unlikely that other inducible proteins play a role in the desensitization process.
The functional role of protein-tyrosine phosphatase SHP-2 in signal transduction in hepatoma cells has been assessed indirectly by preventing recruitment of SHP-2 to gp130 or by overexpressing enzymatically inactive SHP-2 mutants (1014, 27). The data indicated that activation of SHP-2 via gp130 is required for MAPK activation. Baumann and co-workers (11) demonstrated that overexpression of a catalytically inactive SHP-2 in hepatoma cells, containing the two SH2 domains but lacking the phosphorylation domain and the four potential Grb2 binding site, did not enhance the STAT3 phosphorylation in response to IL-6, but did inhibit MAPK activation in response to this cytokine. Overexpression of a different catalytically inactive SHP2, in which Cys residue 463 was replaced with Ser, did not affect the magnitude of STAT3 phosphorylation in response to IL-6 in these cells and had no affect on MAPK activation (11). In the present study, we demonstrated that overexpression of a catalytically inactive SHP-2 containing the two SH2 domains but lacking the phosphatase domain by retroviral transduction did not inhibit the magnitude or the kinetics of STAT phosphorylation in response to OnM. Also, overexpression of this catalytically inactive SHP-2 did not inhibit desensitization induced by OnM. A similar lack of effect was noted with calpeptin treatment. Experimentally, it is not possible to know for sure whether these observations rule out a role for SHP-2 phosphatase activity or reflect inadequate inhibition. We also used specific SHP-2 antisense, which effectively reduced constitutive expression of SHP-2 in HASMC and further excluded a role for SHP-2 in mediating desensitization induced by OnM.
Cumulatively, these experiments suggest that there must be a third regulatory protein that bind to gp130 phosphotyrosine residue 759. This mediator could be an as yet uncharacterized member of the SHP family or would act through a novel mechanism. Further investigation will be necessary to identify and characterize this regulatory protein.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Boyer Center for Molecular Medicine, Room 454, 295 Congress Ave., New Haven, CT 06510, Tel.: 203-737-2292; Fax: 203-737-2293; E-mail: jordan.pober{at}yale.edu.
1 The abbreviations used are: OnM, oncostatin M; Ab, antibody; CNTF, ciliary neurotrophic factor; EC, endothelial cells; EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting; FGF-1, fibroblast growth factor-1; HA, hemagglutinin; HASMC, human aortic smooth muscle cells; HUVEC, human umbilical vein endothelial cells; JAK, Janus kinase; LIF, leukemia inhibitory factor; LIFR, leukemia inhibitory factor receptor
; MAPK, mitogen-activated protein kinase; OnMR
, oncostatin M receptor
; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; PDGFR
, platelet-derived growth factor receptor
; PI-3, phosphatidylinositol 3; PE, phycoerythrin; PIAS, protein inhibitor of activated STATs; SHP, Src homology 2 domain-containing tyrosine phosphatase; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TYK2, tyrosine kinase 2.
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ACKNOWLEDGMENTS |
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REFERENCES |
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