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Abstract
In the peripheral nerves, injury-induced cytokines and growth factors perform critical functions in the activation of both the MEK/ERK and JAK/STAT3 pathways. In this study, we determined that nerve injury-induced ERK activation was temporally correlated with STAT3 phosphorylation at the serine 727 residue. In cultured Schwann cells, we noted that ERK activation is required for the serine phosphorylation of STAT3 by neuropoietic cytokine interleukin-6 (IL-6). Serine phosphorylated STAT3 by IL-6 was transported into Schwann cell nuclei, thereby indicating that ERK may regulate the transcriptional activity of STAT3 via the induction of serine phosphorylation of STAT3. Neuregulin-1 (NRG) also induced the serine phosphorylation of STAT3 in an ERK-dependent fashion. In contrast with the IL-6 response, serine phosphorylated STAT3 induced by NRG was not detected in the nucleus, thus indicating the non-nuclear function of serine phosphorylated STAT3 in response to NRG. Finally, we determined that the inhibition of ERK prevented injury-induced serine phosphorylation of STAT3 in an ex-vivo explants culture of the sciatic nerves. Collectively, the results of this study show that ERK may be an upstream kinase for the serine phosphorylation of STAT3 induced by multiple stimuli in Schwann cells after peripheral nerve injury.
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Fig. 1.
Serine phosphorylation of STAT3 in the sciatic nerve following crush injury. (A, B) Frozen sections from control and injured sciatic nerves were immunostained with an antibody against serine phosphorylated STAT3 (pSTAT3(s)). Axons (arrowheads) and nuclei of SC-like cells (arrows) display a rapid induction in the serine phosphorylation of STAT3 at 30 min (A) and 60 min (B) after lesion. Scale bar; 30 μm. (C) Protein lysates were prepared from the injured sciatic nerves and analyzed with Western blot analysis for detection of serine phosphorylated STAT3 and pERK. (D) A quantitative analysis for result (C).
Fig. 2.
IL-6 induces serine phosphorylation of STAT3 in Schwann cells. RT4 schwannoma cells (A~D) and primary SCs (E, F) were serum starved overnight and then treated with IL-6 (50 ng/ml) for the indicated times. Total cell lysates were subjected to Western blot analysis using antibodies specific for phosphorylated proteins. (A, B) RT4 cells were treated with IL-6 for 15 min, and the cellular lysates were analyzed using the indicated antibodies. (B) A quantitative analysis of tyrosine phosphorylated STAT3 in result (A). (C, D) RT4 cells were treated with IL-6 for the indicated time in the presence or absence of U0126. (D) A quantitative analysis of serine phosphorylated STAT3 in result (C). (E, F) Primary SCs were treated with IL-6 for 15 min, and the total lysates were analyzed by Western blot analysis using the indicated antibodies. (F) A quantitative analysis of phosphorylated STAT3 in result (E).
Fig. 3.
NRG-1 induces serine phosphorylation of STAT3 in Schwann cells. (A, B) Primary SCs were serum starved overnight and then treated with NRG-1 (200 ng/ml) for the indicated times. Total cell lysates were subjected to Western blot analysis using antibodies specific for phosphorylated proteins. (B) A quantitative analysis of serine phosphorylated STAT3 in result (A). (C, D) Primary SCs were treated with NRG-1 for 15 min in the presence or absence of U0126 or DMSO, and cellular lysates were analyzed with Western blot analysis. (D) A quantitative analysis of serine phosphory-lated STAT3 in result (C).
Fig. 4.
Differential localization of phosphorylated STAT3 by IL-6 and NRG-1. (A) After 20 min of stimulation with IL-6, RT4 cells were fixed and immunostained for serine phosphorylated STAT3. Con; untreated control. (B) RT4 cells were pretreated with U0126 for 30 min and then stimulated with IL-6 for 20 min. Cells were immunostained for tyrosine or serine phosphorylated STAT3. Scale bar; 30 μm. (C) Primary SCs were treated with IL-6 or NRG for 20 min, and the cells were fixed and double immunostained for a SC marker (S100) and phosphorylated STAT3. Scale bar; 30 μm.
Fig. 5.
ERK inhibition reduces the serine phosphorylation of STAT3 in explant cultures of sciatic nerves. (A, B) Sciatic nerve explants were cultured for 6 or 24 h in the presence or absence of U0126, and then the protein lysates were analyzed with Western blot analysis using an antibody against serine phosphorylated STAT3 and anti-pERK antibody. (B) A quantitative analysis of serine phosphorylated STAT3 in result (A).
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