SOCS3 Inhibits STAT3 Activation in Primary Neurons and SH-SY5Y Cells.

<p><b>A</b>–<b>B</b>, Primary neurons (A) or SH-SY5Y cells (B) were infected with Lenti-control (−) or Lenti-SOCS3 (+). After infection, IL-6 plus sIL-6R or OSM was added to primary cortical neurons or SH-SY5Y cells for up to 1 h, and protein lysates were subjected to immunoblot analysis with antibodies against phosphorylated STAT3 Tyr705, total STAT3, SOCS3 and GAPDH. The densitometric ratios of P-STAT3 Tyr705 versus total STAT3 were calculated, and shown as Fold Increase. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001 compared to Lenti-control infected cells left untreated; ?p<0.001, and ??p<0.01 compared to Lenti-control infected cells treated with IL-6 plus IL-6R or OSM. <b>C</b>, SH-SY5Y cells were infected with control or shSOCS3 lentivirus, treated with OSM for 1 h, and total RNA was analyzed by qRT-PCR for SOCS3 expression. Graphic representation of the mean ± SEM of triplicate cultures in three separate experiments. *p<0.001 compared to control; **p<0.001 compared to OSM treatment of control lentivirus infected culture. <b>D</b>, After infection of SH-SY5Y cells with pGipz (control) or shSOCS3 lentivirus, IL-6 plus sIL-6R or OSM was added for the times indicated and cell lysates immunoblotted as described. The densitometric ratios of P-STAT3 Tyr705 versus total STAT3 were calculated, and shown as Fold Increase. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001 compared to control cultures infected with pGipz; ?p<0.001, and ??p<0.01 compared to IL-6 plus IL-6R or OSM treatment of pGipz lentivirus infected cultures.</p

Protective Role of STAT3 in NMDA and Glutamate-Induced Neuronal Death: Negative Regulatory Effect of SOCS3

<div><p>The present study investigates the involvement of the IL-6 family of cytokines, activation of the transcription factor Signal Transducer and Activator of Transcription-3 (STAT3), and the role of Suppressor Of Cytokine Signaling-3 (SOCS3) in regulating excitotoxic neuronal death <em>in vitro</em>. Biochemical evidence demonstrates that in primary cortical neurons and SH-SY5Y neuroblastoma cells, IL-6 cytokine family members, OSM and IL-6 plus the soluble IL-6R (IL-6/R), prevent NMDA and glutamate-induced neuronal toxicity. As well, OSM and IL-6/R induce tyrosine and serine phosphorylation of STAT3 in primary cortical neurons and SH-SY5Y cells. Studies using Pyridine 6 (P6), a pan-JAK inhibitor, demonstrate that the protective effect of OSM and IL-6/R on neuronal death is mediated by the JAK/STAT3 signaling pathway. In parallel to STAT3 phosphorylation, OSM and IL-6/R induce SOCS3 expression at the mRNA and protein level. P6 treatment inhibits SOCS3 expression, indicating that STAT3 is required for OSM and IL-6/R-induced SOCS3 expression. Lentiviral delivery of SOCS3, an inhibitor of STAT3 signaling, into primary neurons and SH-SY5Y cells inhibits OSM and IL-6/R-induced phosphorylation of STAT3, and also reverses the protective effect of OSM and IL-6/R on NMDA and glutamate-induced neurotoxicity in primary cortical neurons. In addition, treatment with IL-6 cytokines increases expression of the anti-apoptotic protein Bcl-xL and induces activation of the Akt signaling pathway, which are also negatively regulated by SOCS3 expression. Thus, IL-6/R and OSM-induced SOCS3 expression may be an important factor limiting the neuroprotective effects of activated STAT3 against NMDA and glutamate-induced neurotoxicity.</p> </div

Regulation of the Akt Signaling Pathway by IL-6 Cytokines and SOCS3.

<p><b>A</b>–<b>B</b>, Primary neurons (A) were treated with IL-6 (10 ng/ml) plus sIL-6R (25 ng/ml), or SH-SY5Y cells (B) were treated with IL-6 (10 ng/ml) plus sIL-6R (25 ng/ml) or OSM (10 ng/ml) in the absence or presence of P6 (0.5 µM) pretreatment for 1 h, and protein lysates subjected to immunoblot analyses with antibodies against P-Akt, total Akt and GAPDH. P6 was also present for indicated time points of cytokine exposure. The densitometric ratios of P-Akt versus total Akt were calculated, and shown as Fold Increase. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001, **p<0.01, and ***p<0.05 compared to control; ?p<0.001, &p<0.01, and ??p<0.05 compared to IL-6 plus IL-6R or OSM alone. <b>C</b>, SH-SY5Y cells were pretreated for 2 h with the PI3-Kinase inhibitors LY294002 (10 µM) or Wortmannin (1 µM), followed by treatment with IL-6 plus sIL-6R or OSM for 30 min with continued LY294002 or Wortmannin exposure, and protein levels of phosphorylated STAT3 Tyr705 and total STAT3 were analyzed. The densitometric ratios of P-STAT3 Tyr705 versus total STAT3 were calculated, and shown as Fold Increase. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001 compared to control. <b>D</b>, SH-SY5Y cells were infected with Lenti-control (−) or Lenti-SOCS3 (+). After infection, IL-6 plus sIL-6R or OSM was added to SH-SY5Y cells for the times indicated, and protein lysates subjected to immunoblot analyses with antibodies against P-Akt, total Akt, SOCS3 and GAPDH. The densitometric ratios of P-Akt versus total Akt were calculated, and shown as Fold Increase. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001 compared to Lenti-control lentivirus infected cultures left untreated; ?p<0.001 compared to IL-6 plus IL-6R or OSM treatment of control lentivirus infected cells.</p

Neuroprotective Effect of IL-6 Cytokines on NMDA and Glutamate-induced Neuronal Death.

<p>Primary cortical neurons or SH-SY5Y cells were pretreated with IL-11 (10 ng/ml), CNTF (10 ng/ml), IL-6 (10 ng/ml) plus sIL-6R (25 ng/ml) or OSM (10 ng/ml) for 1 h, and then grown in the presence of cytokine and NMDA (100 µM, <b>A</b>) and (1 mM, <b>C</b>) or glutamate (100 µM, <b>B</b>) and (1 mM, <b>D</b>) for an additional 24 h. Cell viability was determined by the MTT reduction assay. Graphic representation of the mean ± SEM of triplicate cultures in three separate experiments. ***p<0.001 compared to control; *p<0.05 and **p<0.01 compared to NMDA or glutamate only.</p

P6 Reverses the Protective Effect of IL-6 Cytokines on NMDA-induced Toxicity.

<p><b>A</b>–<b>B</b>, Primary cortical neurons (A) or SH-SY5Y cells (B) were pretreated with P6 (0.5 µM) for 1 h, and then grown in the presence of P6 and IL-6 plus sIL-6R or OSM for 1 h, and then in the presence of cytokine, P6, plus NMDA for an additional 24 h. Cell viability was determined by the MTT reduction assay. Graphic representation of the mean ± SEM of triplicate cultures in three separate experiments. ***p<0.001 compared to control; **p<0.001, *p<0.01, and ♦p<0.05 compared to NMDA; and ?p<0.001 and #p<0.01 compared to NMDA plus cytokines. <b>C</b>–<b>D</b>, P6 Inhibits IL-6 Cytokine-induced STAT3 Activation. Primary neurons (C) or SH-SY5Y cells (D) were grown with P6 for 1 h and where indicated, grown in the presence of OSM or IL-6 plus sIL-6R and P6. The levels of phosphorylated STAT3 Tyr705, phosphorylated STAT3 Ser727, and total STAT3 were analyzed at the indicated time points. The densitometric ratios of P-STAT3 Tyr705 or P-STAT3 Ser727 versus total STAT3 were calculated, and shown as Fold Increase. Graph represents the mean ± SEM of triplicate cultures in four separate experiments. *<i>p</i><0.001 and **p<0.01 compared to control; and ?p<0.001, #p<0.01, and &p<0.05 compared to IL-6 plus IL-6R or OSM alone.</p

Induction of SOCS3 Expression by IL-6 Cytokines in Primary Neurons and SH-SY5Y Cells.

<p><b>A,</b> Primary neurons were treated with IL-6 (10 ng/ml) plus sIL-6R (25 ng/ml) or OSM (10 ng/ml) for the times indicated, and total RNA was analyzed by qRT-PCR. Graphic representation of the mean ± SEM of triplicate cultures in three separate experiments. **p<0.001 compared to control. <b>B</b>, SH-SY5Y cells were treated with IL-6 (10 ng/ml) plus sIL-6R (25 ng/ml) or OSM (10 ng/ml) for the times indicated, and cell lysates immunoblotted with SOCS3 and GAPDH antibodies. <b>C</b>–<b>E,</b> Inhibition of the JAK/STAT Pathway Inhibits SOCS3 Expression. <b>C, D</b>, Primary cortical neurons (C) or SH-SY5Y cells (D) were pretreated with P6 (0.5 µM) for 1 h, and then treated with IL-6 plus sIL-6R or OSM in the presence of continued P6 exposure, and total mRNA was analyzed by qRT-PCR. Graphic representation of the mean ± SEM of triplicate cultures in three separate experiments. *p<0.001 compared to control; **p<0.001 compared to IL-6 plus IL-6R or OSM alone. <b>E</b>, SH-SY5Y cells were grown in the absence or presence of P6 pretreatment for 1 h and then grown in the presence of P6 and IL-6 plus sIL-6R or OSM and protein levels of SOCS3 were analyzed. The densitometric ratios of SOCS3 versus GAPDH were calculated. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001 compared to control; ?p<0.001 compared to IL-6 plus IL-6R or OSM alone.</p

Proposed Model for SOCS3 Contribution to Excitoxicity in Neurons. A

<p>, IL-6/R or OSM activates the JAK/STAT3 pathway, which increases Bcl-xL expression and causes activation of the Akt signaling pathway in a STAT3-dependent manner. Increased Bcl-xL expression and binding to Bax, as well as Akt activation inhibits NMDA or glutamate-induced neuronal death. <b>B</b>, IL-6/R and OSM also induce SOCS3 expression in a STAT3-dependent fashion. SOCS3, in turn, modulates IL-6/R or OSM induced Bcl-xL expression and Akt signaling pathway in a negative regulatory manner, which contributes, in part, to NMDA or glutamate-induced neuronal death.</p

Bcl-xL Expression is Regulated by IL-6 Cytokines and SOCS3.

<p><b>A</b>–<b>B,</b> Primary neurons (A) were treated with OSM (10 ng/ml) for the times indicated, or SH-SY5Y cells (B) were treated with IL-6 (10 ng/ml) plus sIL-6R (25 ng/ml) or OSM (10 ng/ml) for the times indicated. Protein lysates were prepared and subjected to immunoblot analyses with antibodies against Bcl-xL and GAPDH. The densitometric ratios of Bcl-xL versus GAPDH were calculated. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001, and **p<0.01 compared to control. <b>C</b>. Co-IP of Bax with Bcl-xL. Lysates from each sample were subjected to immunoprecipitation (IP) using antibody specific for Bax. IP samples were probed with antibody to Bcl-xL to demonstrate a specific interation between Bcl-xL and Bax. The densitometric ratios of co-immunoprecipitated Bcl-xL to immunoprecipitated Bax were calculated. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001, **p<0.01, and ***p<0.05 compared to control. <b>D</b>, SH-SY5Y cells were infected with pGipz or Lenti-shSOCS3. After infection, IL-6 plus sIL-6R was added for the times indicated, and cell lysates immunoblotted as described. The densitometric ratios of Bcl-xL versus GAPDH were calculated. Graph represents the mean ± SEM of triplicate cultures in three separate experiments. *<i>p</i><0.001, and **p<0.01 compared to pGipz-infected cultures; and &p<0.01 compared to IL-6 plus IL-6R treatment of pGipz-infected cultures.</p

IL-6 Cytokines Induce STAT3 Activation in Primary Neurons and SH-SY5Y Cells. A

<p>, Primary neurons were treated with IL-6 (10 ng/ml) plus sIL-6R (25 ng/ml) or OSM (10 ng/ml) for the times indicated. Protein lysates were prepared and subjected to immunoblot analyses with antibodies against phosphorylated STAT3 Tyr705, phosphorylated STAT3 Ser727, and total STAT3. <b>B</b>, SH-SY5Y cells were treated with IL-6 (10 ng/ml) plus sIL-6R (25 ng/ml) or OSM (10 ng/ml) for the times indicated, and cell lysates immunoblotted as described. The densitometric ratios of P-STAT3 Tyr705 or P-STAT3 Ser727 versus total STAT3 were calculated, and are shown as Fold Increase. Graphs represent the mean ± SEM for triplicate cultures in three separate experiments. *<i>p</i><0.001, #p<0.01, and **p<0.05 compared to control, untreated cultures.</p

Activation of STAT3 by TNF-α Treatment Induces <i>SOCS3</i> and <i>cIAP2</i> Expression.

<p><b>A</b>, U251-MG cells were stimulated with TNF-α (10 ng/ml) for the indicated times, lysed and immunoblotted with the indicated Ab. <b>B & C</b>, U87-MG cells were stimulated with TNF-α (10 ng/ml) for the indicated times. RNA was isolated, followed by generation of cDNA, and qRT-PCR was performed for the indicated genes. Data are shown as replicates of three and the experiment repeated with similar results observed. *, p<0.05.</p