Dual signaling role of the protein tyrosine phosphatase SHP-2 in regulating expression of acute-phase plasma proteins by interleukin-6 cytokine receptors in hepatic cells

Abstract

One of the major actions of interleukin-6 (IL-6) is the transcriptional activation of acute-phase plasma proteins (APP) genes in liver cells. Signaling by the IL-6 receptor is mediated through the signal transducing subunit gp130 and involves the activation of Janus-associated kinases (JAKs), signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein (MAP) kinase. Functional analysis of gp130 in rat hepatoma cells by using transduced chimeric G-CSFR-gp130 receptor constructs demonstrates that SHP-2, the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase, acts as a negative regulator of the JAK/STAT signaling in part by downregulating JAK activity, thereby indirectly moderating the induction of STAT3-dependent APP genes. This study shows that in hepatoma cells, the recruitment and tyrosine phosphorylation of SHP-2, but not SHC, is the primary signaling event associated with the activation of MAP kinases (ERK1/2) by gp130. Overexpression of truncated SHP-2 that lacks Grb2-interacting sites, but not the full-length catalytically inactive SHP-2, reduces ERK activation by IL-6, confirming the signal-mediating role of SHP-2. Activation of ERK1/2 is correlated with induction of the immediate-early response genes. Stimulation of the c-fos, c-jun, and egr-1 genes is essentially absent in cells expressing gp130 with a Y759F mutation, which is unable to recruit SHP-2. Interestingly, both JAK/STAT and SHP-2 pathways regulate the induction of the junB gene. Moreover, disengagement of SHP-2 from gp130 signaling not only enhances APP gene induction but also further reduces cell proliferation, in part correlated with the attenuated expression of immediate-early response genes. These results suggest that IL-6 regulation of APP genes is affected by SHP-2 in two ways: SHP-2 acts as a phosphatase on the JAK/STAT pathway and serves as linker to the MAP kinase pathway, which in turn moderates APP production.

FIG. 1

Expression of G-gp130(WT) and G-gp130(Y2F) in H-35 cells. (A) Aliquots of total-cell extracts (30 μg of protein) from parental H-35 cells, G-gp130(WT) cells, and G-gp130(Y2F) cells were separated on 7.5% polyacrylamide gels. After being transferred to a membrane, the proteins were reacted with anti-FLAG polyclonal antibodies. (B) Confluent monolayers of parental H-35 cells, G-gp130(WT) cells, and G-gp130(Y2F) cells in 10-cm-diameter dishes were treated for 10 min with G-CSF and then lysed. Proteins were immunoprecipitated (IP) with anti-FLAG monoclonal antibodies and analyzed on Western blots (WB) first with antiphosphotyrosine (PY) antibodies and then with anti-FLAG polyclonal antibodies. Ig, immunoglobulin. (C) G-gp130(WT) and G-gp130(Y2F) cells were treated for 10 min with G-CSF or IL-6. Half of the cell lysate was reacted with anti-SHP-2, and the other half was reacted with anti-FLAG. Immunoprecipitated proteins were analyzed by Western blotting first with antiphosphotyrosine and then with anti-SHP-2 antibodies.

FIG. 2

Time course of STAT3 and ERK activation. (A) H-35 cells in 10-cm-diameter dishes were treated with IL-6 for the times indicated and then lysed. Half of the lysate was reacted with anti-SHP-2 antibodies, and the other half was reacted with anti-gp130 antibodies. The two series of immunoprecipitates (IP) were analyzed under identical conditions by immunoblotting with antiphosphotyrosine (PY) antibodies and subsequently with anti-SHP-2 antibodies (top) or anti-gp130 (bottom). (B) Lysates from control and IL-6-treated culture were immunoprecipitated (First IP) with anti-gp130 antibodies. The supernatant lysate fractions were divided into two. Half was immunoprecipitated (Second IP) with anti-SHP-2 antibodies, and the other half was immunoprecipitated with anti-gp130 antibodies. In the latter immunoprecipitation, after binding to protein G-Sepharose, the resin was not washed but immediately boiled in sodium dodecyl sulfate buffer. Half of the first and all of the two second immunoprecipitates were separated on one sodium dodecyl sulfate–7.5% polyacrylamide gel. The blotted proteins were reacted first with antiphosphotyrosine and then with anti-gp130 antibodies. (C) Parental H-35, G-gp130(WT), and G-gp130(Y2F) cells in six-well culture plates were treated with IL-6, G-CSF, or insulin for the indicated times. Aliquots of whole-cell lysate (30 μg protein) were analyzed by immunoblotting for tyrosine-phosphorylated STAT3, STAT3, phosphorylated ERK1/2, and ERK in the same membrane.

FIG. 3

Interaction of Grb2 with SHP-2 but not with SHC by gp130 signaling. G-gp130(WT) cells and G-gp130(Y2F) cells in 10-cm-diameter dishes were treated for 10 min with medium alone, G-CSF, IL-6, or insulin. Half of the cell lysate was immunoprecipitated (IP) with anti-SHC antibodies, and the other half was immunoprecipitated with anti-Grb2 antibodies. Immunoprecipitated proteins were separated on a sodium dodecyl sulfate–10% polyacrylamide gel, and immunoblots were reacted with antiphosphotyrosine (PY) and then anti-Grb2 and anti-SHC (top) or antiphosphotyrosine, anti-SHP-2, and anti-Grb2 (bottom). Note that in H-35 cells the 52-kDa isoform of SHC predominates and the 66-kDa isoform is undetectable.

FIG. 4

Effect of overexpressed SHP-2 mutants on the activation of MAP kinases. HepG2 cells were transfected with expression vectors for GFP and SHP-2CS or SHP-2Δ. (A) GFP-negative and GFP-positive cells were selected by FACS and, after a 24-h reculture period, treated with IL-6 for 15 min. Equal amounts of cell lysate (30 μg protein) were separated on one sodium dodecyl sulfate–10% polyacrylamide gel. After protein transfer, the membrane was cut along the 60-kDa position (dotted line), the top part was reacted with antiphosphotyrosine (PY) STAT3, and the bottom part was reacted with antiphosphotyrosine ERK. Then both sections were reacted with anti-SHP-2 recognizing the N-terminal epitope, and the bottom section was reacted with anti-ERK. (B) HepG2 cells were transfected with expression vectors for GFP and SHP-2CS or SHP-2Δ. FACS-selected GFP-negative and GFP-positive cells (2 × 10⁵ per well in 24-well culture plates) were cultured overnight. After incubation for 8 h in serum-free medium, the cells were stimulated for 10 min with medium alone or with insulin or EGF. Equal amounts of whole-cell lysate were analyzed by Western blotting for phosphorylated ERK. (C) HepG2 cells in 15-cm-diameter dishes were transfected with expression vector for G-gp130(WT) (5 μg/ml) and Myc SHP-2CS or SHP-2Δ-Myc (5 μg/ml). After the cultures were divided into two and allowed to recover for 36 h, the cells were treated for 15 min with medium alone (control) or G-CSF. G-gp130 protein was immunoprecipitated (IP) with anti-FLAG antibodies and analyzed by Western blotting (WB). The membrane section with proteins of >100 kDa was reacted first with antiphosphotyrosine (PY) and then with anti-FLAG antibodies. The membrane section with proteins of <100 kDa was reacted with anti-Myc antibodies. The same ECL exposure for each section is reproduced; however, the portions showing SHP-2CS and SHP-2Δ have been rearranged due to the size difference of the proteins. (D) HepG2 cells in 10-cm-diameter dishes were transfected with the expression vectors for the Myc epitope-tagged versions of the indicated SHP-2 proteins (5 μg/ml). Subcultures were treated for 15 min with medium alone (control) or with IL-6. Proteins were immunoprecipitated with anti-Myc antibodies (IP:Myc) and analyzed by Western blotting first for reaction with antiphosphotyrosine antibodies (WB:PY) and then for reaction with anti-Myc antibodies (WB:Myc). The composite shows only the sections of the Myc-reacting proteins. Due to the high level of expressed proteins, the ECL reaction for anti-Myc is much shorter than for antiphosphotyrosine.

FIG. 5

Activation of immediate-response genes. (A) G-gp130(WT) and G-gp130(Y2F) cells were cultured in 15-cm-diameter dishes. After a 24-h serum deprivation, the cells were treated for 20 or 45 min with medium alone, G-CSF, or IL-6. Polyadenylated RNA (5 μg) was analyzed by Northern blot hybridization with the indicated probes. (B to D) G-gp130(WT) cells were pretreated with dimethyl sulfoxide (DMSO) or PD98059 (75 μM) and treated for 10 min (B), 20 min (C), or 2 h (D) with medium alone (Control) or medium containing G-CSF or IL-6 in the presence of dimethyl sulfoxide or PD98059. (B) Cell extracts were analyzed by immunoblotting for active STAT3 and ERK1/2 on a single membrane. The open arrow indicates the nonspecific band that demonstrates equal amounts of protein loading. (C) Total cellular RNA (5 μg) was subjected to Northern blot hybridization with the Egr-1 probe. (D) Total RNA (10 μg) was analyzed by Northern blot hybridization with the haptoglobin (HP) probe. EtBr, ethidium bromide.

FIG. 6

Suppression of ERK activation enhances Hp production. (A) Lysates of parental H-35 cells and H-35 cells stably transduced with the G-gp130 constructs were reacted with anti-FLAG antibodies. Immunoprecipitated proteins were immunoblotted with anti-FLAG antibodies. (B) Cell cultures transduced with the indicated receptors were treated for 48 h with IL-6 or G-CSF. The amount of Hp secreted during the second 24-h period was determined by immunoelectrophoresis, normalized to the cell number, and expressed relative to the values of the IL-6-treated cultures in each series (mean and standard deviation; n = 3 to 6). (C) G-gp130(133)WT and G-gp130(133)(Y2F) cultures were treated for 48 h with IL-6 or increasing doses of G-CSF in the presence of dimethyl sulfoxide (DMSO, DM) or 25 μM PD98059 (PD). Hp produced during the second 24-h treatment period was determined by Western blotting of 5 μl of culture medium. The band represents the immunoreactive Hp β subunit.

FIG. 7

Effect of gp130 signals on thymidine incorporation. (A) Cells in 96-well plates were maintained for 8 h in serum-free medium and then treated for 16 h with serum-free medium containing no additives, G-CSF, or IL-6 followed by 8 h with [³H]thymidine. Values for ³H incorporations (cpm/culture) are shown (mean and standard deviation; n = 5). (B) HepG2 cells were transfected with expression vector for GFP and G-gp130(WT) or G-gp130(Y2F) (1 μg/ml each) and plasmid DNA carrier (18 μg/ml). GFP-positive and GFP-negative cells were selected by FACS. From each preparation, an aliquot of 10⁵ cells was plated in one well of 24-well plates and the remaining cells were distributed in aliquots of 2.5 × 10⁴ cells in 96-well cluster plates. After overnight recovery, the cells in the 24-well plates were lysed and equal amounts of whole-cell lysate were analyzed by Western blotting for anti-FLAG-reactive proteins (top). The cells in the 96-well plates were treated with medium alone or with G-CSF or IL-6 and processed for [³H]thymidine incorporation as in panel A. The values determined in each culture were expressed relative to the mean value calculated for the medium control in each series (set to 100%) (mean and standard deviation; n = 3 to 5). ∗, P < 0.05; ∗∗, P < 0.005.

FIG. 8

Proliferation is reduced by the action of gp130. (A) Cells (∼2 × 10⁴/cm²) were cultured in six-well dishes for 48 h in serum-free medium. Then they were treated with medium containing 10% serum alone or G-CSF or IL-6 in addition. After 2 days, the treatment media were replaced by fresh media. Two days later, cell numbers were determined and calculated per 10 cm² of culture area (mean and standard deviation; n = 3). The cell number at the onset of the experiment is indicated by a hatched bar. (B) (Top) Extracts of parental H-35 cells and cultures of three independent clonal lines of G-gp130(WT) and G-gp130(Y2F) cells were immunoprecipitated with anti-FLAG antibodies and analyzed by Western blotting for anti-FLAG reactive proteins. (Bottom) The same cells were analyzed for proliferation in response to G-CSF treatment as the cells in panel A. In each series, the values for the treated cell cultures were calculated relative to the mean cell number determined for the medium control cultures (set to 100%) (mean and standard deviation; n = 3). ∗, P < 0.05; ∗∗, P < 0.005. (C) G-gp130(WT) and G-gp130(Y2F) cells in six-well dishes were cultured for 0, 24, and 48 h in medium containing 1% serum in the presence of G-CSF. Total cellular RNA (10 μg) were analyzed by Northern blot hybridization for haptoglobin mRNA. An autoradiogram after a 6-h exposure is shown. EtBr, ethidium bromide.