Suppressor of cytokine signaling-3 suppresses the ability of activated signal transducer and activator of transcription-3 to stimulate neurite growth in rat primary sensory neurons

Abstract

The actions of the neuropoietic cytokines are mediated by the gp130 receptor, which activates several signaling molecules including the transcription factor STAT3 (signal transducer and activator of transcription), which, in turn, is subject to feedback inhibition by SOCS3 (suppressor of cytokine signaling). Activation of the gp130 receptor has been implicated in axonal growth particularly during regeneration, but the specific contribution of STAT3 is the subject of conflicting reports. Measurements of SOCS3 mRNA in rat dorsal root ganglia showed a significant induction in this inhibitory molecule after peripheral nerve injury. The functions of STAT3 and SOCS3 in adult rat primary sensory neurons were investigated in vitro through transduction of lentiviruses yielding a conditionally activated STAT3, native SOCS3, or a mutant SOCS3 with dominant-negative actions. The SOCS3 construct was effective in inhibiting tyrosine phosphorylation of STAT3 in a neuroblastoma cell line and in blocking nuclear accumulation of endogenous STAT3 or of the conditionally activated STAT3 chimera in primary sensory neurons. In such neurons, transduction and activation of STAT3 enhanced neurite growth, transduction with SOCS3 reduced neurite outgrowth, and transduction with mutant SOCS3 enhanced neurite growth, at least under basal conditions. In conclusion, STAT3 signaling is beneficial to axonal growth through activating transcription of unidentified genes, and SOCS3 is detrimental to axonal growth through inhibition of STAT3 and/or other transcription factors.

Figure 1.

Induction of SOCS3 mRNA in injured neurons. SOCS mRNAs in DRGs removed 6–96 h after sciatic nerve transection and cultures of DRG cells enriched for neurons are shown. a, Real-time PCR measurements. For each time point and each SOCS, the ratio of SOCS mRNA to 18S RNA in DRGs ipsilateral to the sciatic nerve transection was normalized to the ratio in contralateral DRGs (mean ± SD; n = 3 animals; ∗p < 0.05 vs contralateral DRGs by Student's t test). b, c, Photomicrographs of in situ hybridization preparations to illustrate the presence of SOCS3 mRNA in DRG neurons ipsilateral (c) but not contralateral (b) to nerve injury. Scale bar, 50 μm. d, e, Real-time PCR measurements of SOCS and cytokine mRNAs in DRG neurons after cell dissociation and cell culture. For each condition and each SOCS or cytokine mRNA, the ratio of SOCS mRNA to 18S RNA in DRGs was normalized to the ratio in normal DRGs frozen immediately after removal. 1, Freshly harvested whole DRGs; 2, cell preparation enriched for neurons several hours later after dissection, dissociation, and centrifugation; 3, neurons after an additional 20 h in culture in serum-free medium (means; n = 3 animals).

Figure 2.

Influence of SOCS3 and mSOCS3 on signal transduction in SH-SY5Y neuroblastoma cells. a, Western blot analysis of STAT3 and tyrosine-phosphorylated STAT3 (pSTAT3). Cells were transduced with LV-GFP, LV-SOCS3, or LV-mSOCS3, maintained in serum-free medium, and subsequently stimulated by 20 pm CNTF for 30 min. Total cell lysates were subjected to SDS-PAGE, and membranes were processed with antibodies to pySTAT3 or STAT3. b, Ratio of phosphorylated STAT3 signal to total STAT3 signal. Open bars, Without CNTF; filled bars, with CNTF. c, Western blot analysis of phosphorylated ERK (pERK) and ERK in cells treated as in a. d, Ratio of phosphorylated ERK signal to ERK signal. Open bars, Without CNTF; filled bars, with CNTF.

Figure 3.

Influence of excess SOCS3 on STAT 3 phosphorylation in DRG neurons. Adult rat DRG neurons were infected by lentivirus constructs for 24 h, cultured for another 48 h, and processed for immunocytochemistry. a, Tetramethylrhodamine isothiocyanate immunocytochemistry with antibody to tyrosine-phosphorylated STAT3. b, GFP fluorescence to detect neurons successfully transduced with the SOCS3-GFP construct. c, Merged image of a and b. Note that the two neurons with GFP have no pySTAT3 immunoreactivity in their nuclei, whereas the two neurons without GFP have strong pySTAT3 immunoreactivity in their nuclei. d, Quantification of pySTAT3 immunoreactivity in cells transduced with SOCS3-GFP. For each neuron, the ratio of nuclear to cytoplasmic pySTAT3 immunoreactivity is plotted against the intensity of GFP fluorescence. The curve fits pySTAT measurement versus ln (GFP measurement) by least squares (r = 0.4; p < 0.01 that the two measurements are correlated). e, As for d, except that cells were stimulated by 2 nm CNTF for 40 min before fixation. The curve fits pySTAT measurement vs ln (GFP measurement) by least squares (r = 0.5; p < 0.01 that the two measurements are correlated). Scale bar, 50 μm.

Figure 4.

Nuclear translocation of the STAT3ER complex in response to 4HT. a, Characterization of the plasmid construct, which was cloned into the LV. Components include long terminal repeats (LTR), the ψ encapsidation signal, cytomegalic virus (CMV) promoter, STAT3, modified ligand binding site of ER, IRES, GFP, and polyadenylation tail. b–d, Cell transduced with STAT3ER. Green fluorescence (b) reflects ER immunoreactivity (used to visualize STAT3ER), and red fluorescence (c) reflects β3-tubulin immunoreactivity (used to visualize neurons). The images are merged in d. Without stimulation of 4HT, ER immunoreactivity is absent from the nucleus, although abundant in the cytoplasm. e–g, As for b–d, except that 4HT has been added for 40 h. Note that this neuron has ER immunoreactivity in the nucleus. h–k, A cell cotransduced with STAT3ER (blue fluorescence; h) and SOCS3/GFP (GFP fluorescence; i) and immunostained for β3-tubulin (red fluorescence; j). The merged image is in k. Note the absence of STAT3ER in the nucleus despite the addition of 4HT. l, Quantification of nuclear translocation of STAT3ER in DRG neurons in response to 4HT stimulation (means ± SEM; n = 4 cultures; ∗p = 5 × 10⁻⁶ by Student's t test). Scale bar, 25 μm.

Figure 5.

Influence of activation of STAT3 on neurite outgrowth in rat DRG neurons. a–d, Neurons were infected with LV-GFP (a, c) or LV-STAT3-ER (b, d) and cultured for 40 h in the absence (a, b) or presence (c, d) of 4HT. β3-Tubulin immunoreactivity (red) is used to detect neurons, GFP fluorescence is used to detect LV-GFP-transduced cells, and ER immunoreactivity (green, FITC) is used to detect LV-STAT3ER-transduced cells. Note that neurite outgrowth is enhanced by the activated STAT3ER complex. e, Frequency histogram to show the percentage of neurons with neurites in each of the three length ranges (n = 1840–3471 neurons per group; p = 5 × 10⁻¹⁸, STAT3ER without 4HT vs STAT3ER with 4HT by χ² analysis). f, Neurite lengths in four groups of neurons transduced with GFP or with STAT3ER and with or without 4HT. Median and 95% confidence levels are plotted. Scale bar, 50 μm.

Figure 6.

Influence of SOCS3 and mSOCS3 on neurite outgrowth in rat DRG neurons. In a–d and f, neurons from DRGs ipsilateral to the side of sciatic nerve transection 3 d previously were infected by lentivirus for 24 h and cultured for an additional 20 h. In e and g, neurons from DRGs associated with an intact sciatic nerve were infected by lentivirus for 24 h and cultured for an additional 44 h. a, Immunocytochemistry for β3-tubulin (red). b, GFP fluorescence to detect neurons successfully transduced with LV-SOCS3-GFP. c, Merged images of a and b. Note the paucity of neurites in neurons transduced with SOCS3-GFP. d, Frequency histogram for conditioned neurons showing percentages with neurite length in each of three given ranges (n = 219–425 neurons per group; p = 1 × 10⁻¹⁶, SOCS3 vs GFP by χ² analysis). e, Frequency histogram for unconditioned neurons showing percentages with neurite length in each of three given ranges (n = 325–410 neurons per group; p = 6 × 10⁻²⁷, SOCS3 vs GFP by χ² analysis; p = 0.01, mSOCS3 vs GFP by χ² analysis). f, g, Neurite lengths for conditioned (f) and unconditioned (g) neurons transduced with GFP, SOCS3, or mSOCS3. Median lengths and 95% confidence levels are plotted. Scale bar, 100 μm.