Krox-20 inhibits Jun-NH2-terminal kinase/c-Jun to control Schwann cell proliferation and death

Abstract

The transcription factor Krox-20 controls Schwann cell myelination. Schwann cells in Krox-20 null mice fail to myelinate, and unlike myelinating Schwann cells, continue to proliferate and are susceptible to death. We find that enforced Krox-20 expression in Schwann cells cell-autonomously inactivates the proliferative response of Schwann cells to the major axonal mitogen beta-neuregulin-1 and the death response to TGFbeta or serum deprivation. Even in 3T3 fibroblasts, Krox-20 not only blocks proliferation and death but also activates the myelin genes periaxin and protein zero, showing properties in common with master regulatory genes in other cell types. Significantly, a major function of Krox-20 is to suppress the c-Jun NH2-terminal protein kinase (JNK)-c-Jun pathway, activation of which is required for both proliferation and death. Thus, Krox-20 can coordinately control suppression of mitogenic and death responses. Krox-20 also up-regulates the scaffold protein JNK-interacting protein 1 (JIP-1). We propose this as a possible component of the mechanism by which Krox-20 regulates JNK activity during Schwann cell development.

Figure 1.

Krox-20 inhibits DNA synthesis in Schwann cells. (A) The proliferation of Schwann cells, as measured by BrdU incorporation, in response to NRG-1 is inhibited by Krox-20. Schwann cells retrovirally infected with empty vector (BP) or vector expressing Krox-20 (K20) were treated with medium alone (Control) or with medium supplemented with 20 ng/ml NRG-1. The error bars represent one SD of the mean. (B) Inhibition of BrdU incorporation in cells expressing Krox-20. Cells infected with adenovirus coexpressing GFP and Krox-20 in control (−NRG-1) and 20 ng/ml NRG-1–treated cells. Note that GFP/Krox-20–positive cells are BrdU negative. Conversely, BrdU-positive cells are GFP negative (arrows). Bar, 20 μm. (C and D) Krox-20 expression inhibits cyclin D1 induction in response to NRG-1. (C) The graph shows percentage of adenovirally infected GFP/cyclin D1–positive cells 6 h after addition of 20 ng/ml NRG-1 in GFP control (GFP) and GFP/Krox-20 (Krox-20)–expressing cells. The error bars represent one SD of the mean. (D) Western blot of cyclin D1 in GFP control (GFP) and GFP/Krox-20 (Krox-20) infected Schwann cells exposed to DM alone (Con) or DM containing 20 ng/ml NRG-1 for 6 h. (E–I) Krox-20 expression increases levels of p27 protein in Schwann cells. (E–H) Immunolabeling of GFP (E and G) and GFP/Krox-20 (F and H) infected Schwann cells with p27 antibody. Arrows indicate GFP/Krox-20 cells with elevated p27 protein. Bar, 20 μm. (I) Western blot using p27 antibody of Schwann cells infected with either empty vector (BP) or Krox-20 (K20)–expressing retrovirus.

Figure 2.

Krox-20 inhibits apoptosis of Schwann cells. (A and B) Krox-20 inhibits TGFβ-dependent apoptosis of Schwann cells. Survival assays for immunopanned Schwann cells infected with GFP or GFP/Krox-20 (Krox-20)–expressing adenovirus. Cells were treated for 24 h with TGFβ + TNFα (A) or TGFβ alone (B) for 48 h and the number of surviving GFP-positive cells counted. (C) Krox-20 inhibits Schwann cell death after serum withdrawal. Survival assay of cells infected with GFP or GFP/Krox-20 (Krox-20)–expressing adenovirus. Surviving GFP-positive cells were counted 72 h after serum withdrawal. The error bars represent one SD of the mean.

Figure 3.

Krox-20 regulates proliferation, survival and myelin gene expression in 3T3 fibroblasts. (A) Krox-20 expression inhibits PDGF-induced proliferation of Swiss 3T3 cells. Shown are the percentage of BrdU-positive cells in empty vector (BP) and Krox-20–expressing (K20) cells in the absence (Control) and presence (+PDGF) of 5 ng/ml PDGF. The error bars represent one SD of the mean. (B) Krox-20 expression in Swiss 3T3 cells inhibits cyclin D1 induction in response to PDGF. Shown are control (BP) and Krox-20 (K20) infected cells, treated for 6 h with 5 ng/ml PDGF. The cells were double immunolabeled with Krox-20 (K20) and cyclin D1 (CycD1) antibodies, and counterstained with Hoechst dye (Ho). Bar, 20 μm. (C) Krox-20 expression inhibits apoptosis of NIH 3T3 cells in serum-free medium. The graph shows the percentage of TUNEL-positive cells in control (BP) and Krox-20 (K20)–expressing cells 48 h after serum withdrawal. The error bars represent one SD of the mean. (D–G) Krox-20 activates periaxin and P₀ myelin protein expression in Swiss 3T3 cells. Swiss 3T3 cells were retrovirally infected with either empty vector (BP; D and F) or Krox-20 (K20; E and G), and immunolabeled with antibody against periaxin (Prx; D and E) or P₀ (F and G). Cells were counterstained with Hoechst dye (Ho) to reveal nuclei. Bar, 20 μm. (H) Western blot of control (BP) or Krox-20 (K20) infected cells showing elevated periaxin and P₀ protein levels.

Figure 3.

Krox-20 regulates proliferation, survival and myelin gene expression in 3T3 fibroblasts. (A) Krox-20 expression inhibits PDGF-induced proliferation of Swiss 3T3 cells. Shown are the percentage of BrdU-positive cells in empty vector (BP) and Krox-20–expressing (K20) cells in the absence (Control) and presence (+PDGF) of 5 ng/ml PDGF. The error bars represent one SD of the mean. (B) Krox-20 expression in Swiss 3T3 cells inhibits cyclin D1 induction in response to PDGF. Shown are control (BP) and Krox-20 (K20) infected cells, treated for 6 h with 5 ng/ml PDGF. The cells were double immunolabeled with Krox-20 (K20) and cyclin D1 (CycD1) antibodies, and counterstained with Hoechst dye (Ho). Bar, 20 μm. (C) Krox-20 expression inhibits apoptosis of NIH 3T3 cells in serum-free medium. The graph shows the percentage of TUNEL-positive cells in control (BP) and Krox-20 (K20)–expressing cells 48 h after serum withdrawal. The error bars represent one SD of the mean. (D–G) Krox-20 activates periaxin and P₀ myelin protein expression in Swiss 3T3 cells. Swiss 3T3 cells were retrovirally infected with either empty vector (BP; D and F) or Krox-20 (K20; E and G), and immunolabeled with antibody against periaxin (Prx; D and E) or P₀ (F and G). Cells were counterstained with Hoechst dye (Ho) to reveal nuclei. Bar, 20 μm. (H) Western blot of control (BP) or Krox-20 (K20) infected cells showing elevated periaxin and P₀ protein levels.

Figure 4.

Krox-20 does not inactivate NRG-1 or TGFβ signaling. (A) Western blot analysis of control (GFP) and Krox-20 adenovirally infected Schwann cells, showing levels of ErbB2 and ErbB3 protein, together with activation (i.e., phosphorylation) of ERK1/2 and Akt in DM only and 10 min after addition of 20 ng/ml NRG-1. Shown also are the corresponding levels of periaxin in control cells and in cells expressing Krox-20. (B) NRG-1 receptor levels are not altered in Krox-20−/− nerves. Western blot of ErbB2 and ErbB3 in sciatic nerve from P10 wild-type (+/+), Krox-20 heterozygous (+/−) and Krox-20 null (−/−) animals. (C–F) SMAD2 and SMAD4 proteins translocate normally to the nucleus in response to TGFβ in Krox-20–expressing Schwann cells. Note intense nuclear labeling in cells infected with Krox-20–expressing adenovirus, treated for 1 h with 10 ng/ml TGFβ1 and immunolabeled with antibodies to either SMAD2 (C and D) or SMAD4 (E and F). Bar, 20 μm.

Figure 5.

Krox-20 inhibits the JNK–c-Jun pathway in vitro and in vivo. (A) Western blot (long exposure) showing suppression of phospho–c-Jun (P-cJun), c-Jun protein (cJun), and phospho-JNK1/2 (P-JNK1/2) in cells infected with Krox-20/GFP adenovirus for 48 h and maintained in DM alone. GFP indicates control cells infected with empty vector. (B–E) Krox-20 expression in Schwann cells inhibits NRG-1– (B and C) and TGFβ-dependent (D and E) activation of the JNK–c-Jun pathway. (B and D) Western blot of Schwann cells, 20 h after adenoviral infection, treated with 20 ng/ml NRG-1 (B) or 10 ng/ml TGFβ (D). Note inhibition of c-Jun phosphorylation by Krox-20 after growth factor addition compared with GFP control, whereas c-Jun protein levels are unaffected. (C and E) Western of Schwann cells 48 h after infection, treated with NRG-1 (C) or TGFβ (E). At this time point, both phospho–c-Jun and c-Jun proteins are suppressed by Krox-20 as compared with control cells. Either β-tubulin or GAPDH were used as loading controls. (F–M) c-Jun and Ser63 phospho–c-Jun immunolabeling of Schwann cells infected with either GFP (F, H, J, and L) or GFP/Krox-20 (G, I, K, and M) and treated 48 h after infection with NRG-1 for 30 min. Both c-Jun and phospho–c-Jun levels are reduced in response to NRG-1 in Krox-20–expressing cells compared with GFP control infected cells (arrows). (N and O) Teased preparation of sciatic nerve at E17, before myelination double labeled with c-Jun and Krox-20 antibodies. Hoechst nuclear dye (Ho) is used to visualize nuclei. Note that most/all nuclei contain c-Jun, whereas Krox-20 is not yet present. (P and Q) Teased preparation of sciatic nerve at P1 when many cells are myelinating. The nerves are double immunolabeled with Krox-20 (K20) and c-Jun (P) or phospho–c-Jun (PSer63c-Jun; Q) antibodies. Note that nuclei expressing Krox-20 do not contain c-Jun or phospho–c-Jun. Bar, 20 μm.

Figure 6.

Inhibition of the JNK pathway inhibits NRG-1–dependent proliferation of Schwann cells. (A) Western blot showing inhibition of NRG-1–induced JNK and c-Jun phosphorylation by 30 μM SP600125 (SP6). (B) Percentage of BrdU-positive Schwann cells in DM only (Cont.) and in medium containing 20 ng/ml NRG-1 in the absence or presence of increasing concentrations of the JNK inhibitor SP600125 as indicated. The error bars represent one SD of the mean. (C) Expression of the JBD of JIP-1 inhibits NRG-1–induced c-Jun phosphorylation, but not ERK1/2 activation. (D) Percentage of BrdU-positive Schwann cells infected with control virus expressing GFP only (GFP) or JBD-expressing adenovirus in the absence (Cont.) or presence of 20 ng/ml NRG-1. The error bars represent one SD of the mean.

Figure 7.

Expression of MEKK1 or MKK7D and the resulting activation of c-Jun phosphorylation restores vulnerability to death in cells expressing Krox-20. (A) Western blot showing induction of c-Jun phosphorylation in Schwann cells coexpressing Krox-20 and either MEKK1 (K20/MEKK1) or MKK7D (K20/MKK7D) as compared with control (K20/LacZ) cells. (B) Serum withdrawal survival assay of Krox-20/LacZ, Krox-20/MEKK1 or Krox-20/MKK7D–expressing Schwann cells. The death protection offered by Krox-20 alone (Fig. 2 C) is not seen in Krox-20/MEKK1 or Krox-20/MKK7D– expressing cells. The error bars represent one SD of the mean.

Figure 8.

Regulation of JIP-1 during sciatic development. (A) Western blot of GFP and Krox-20/GFP (K20) adenovirally infected Schwann cells showing increased levels of JIP-1 in Krox-20–expressing cells. (B) JIP-1 protein levels are reduced, and c-Jun levels increased, in sciatic nerve from P9 and P12 Krox-20 null animals (−/−) compared with wild type (+/+). (C) Semi-quantitative PCR analysis of JIP-1 mRNA expression in sciatic nerve during development from embryonic day 14 (E14) to P12 and at P12, 5 d after nerve cut (P12/CUT). (D) Expression of JIP-1 inhibits c-Jun phosphorylation in Schwann cells. Shown are the percentage of Ser63 phospho–c-Jun–positive cells for cells cotransfected with either GFP-expressing plasmid, for identification of transfected cells, plus empty vector (GFP/EV) or GFP plus JIP-1 (GFP/JIP-1)–expressing plasmids. The error bars represent one SD of the mean. (E) Western blot showing that, unlike Krox-20, JIP-1 expression does not reduce c-Jun protein levels in Schwann cells. (F and G) Immunolabeling of P12 sciatic nerve showing phase (F) and JIP-1 immunofluorescence (G). Arrows indicate localization of JIP-1 in the paranodal loops of myelinating Schwann cells.

Figure 8.

Regulation of JIP-1 during sciatic development. (A) Western blot of GFP and Krox-20/GFP (K20) adenovirally infected Schwann cells showing increased levels of JIP-1 in Krox-20–expressing cells. (B) JIP-1 protein levels are reduced, and c-Jun levels increased, in sciatic nerve from P9 and P12 Krox-20 null animals (−/−) compared with wild type (+/+). (C) Semi-quantitative PCR analysis of JIP-1 mRNA expression in sciatic nerve during development from embryonic day 14 (E14) to P12 and at P12, 5 d after nerve cut (P12/CUT). (D) Expression of JIP-1 inhibits c-Jun phosphorylation in Schwann cells. Shown are the percentage of Ser63 phospho–c-Jun–positive cells for cells cotransfected with either GFP-expressing plasmid, for identification of transfected cells, plus empty vector (GFP/EV) or GFP plus JIP-1 (GFP/JIP-1)–expressing plasmids. The error bars represent one SD of the mean. (E) Western blot showing that, unlike Krox-20, JIP-1 expression does not reduce c-Jun protein levels in Schwann cells. (F and G) Immunolabeling of P12 sciatic nerve showing phase (F) and JIP-1 immunofluorescence (G). Arrows indicate localization of JIP-1 in the paranodal loops of myelinating Schwann cells.