c-Jun is a negative regulator of myelination - PubMed (original) (raw)

c-Jun is a negative regulator of myelination

David B Parkinson et al. J Cell Biol. 2008.

Abstract

Schwann cell myelination depends on Krox-20/Egr2 and other promyelin transcription factors that are activated by axonal signals and control the generation of myelin-forming cells. Myelin-forming cells remain remarkably plastic and can revert to the immature phenotype, a process which is seen in injured nerves and demyelinating neuropathies. We report that c-Jun is an important regulator of this plasticity. At physiological levels, c-Jun inhibits myelin gene activation by Krox-20 or cyclic adenosine monophosphate. c-Jun also drives myelinating cells back to the immature state in transected nerves in vivo. Enforced c-Jun expression inhibits myelination in cocultures. Furthermore, c-Jun and Krox-20 show a cross-antagonistic functional relationship. c-Jun therefore negatively regulates the myelinating Schwann cell phenotype, representing a signal that functionally stands in opposition to the promyelin transcription factors. Negative regulation of myelination is likely to have significant implications for three areas of Schwann cell biology: the molecular analysis of plasticity, demyelinating pathologies, and the response of peripheral nerves to injury.

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Figures

Figure 1.

Figure 1.

Components of the c-Jun pathway are regulated during development, Wallerian degeneration, and in vitro differentiation. A–D show Western blots. (A) Down-regulation of phospho–c-Jun and c-Jun proteins in sciatic nerve from embryonic day (E) 18 to adult. (B) Rapid up-regulation of JNK–c-Jun pathway components at 20 min (20′)–12 h after nerve cut when compared with contralateral control (Con). (C) Strong increase in phospho–c-Jun at 1, 2, and 3 d after transection (Cut) compared with the contralateral control (Con). (D) Activation of cAMP-related pathways (1 mM db-cAMP for 24 h) decreases c-Jun and phospho–c-Jun and increases the myelin proteins Krox-20 and periaxin, whereas phospho-ERK1/2 levels are unchanged. (E–H) Double immunolabeling of controls (E and F) and cells treated with 1 mM db-cAMP for 3 d (G and H), with phospho–c-Jun and P0 antibodies. Note that cAMP induces P0 and suppresses phospho–c-Jun levels in the P0-positive cells. Bar, 15 μm.

Figure 2.

Figure 2.

Genetic removal of c-Jun amplifies Krox-20 or cAMP-induced myelin protein expression. (A) Western blot showing that c-Jun is absent from _Jun_fl/fl cells infected with CRE-expressing adenovirus. The blot also compares periaxin in control (Con) and _c-Jun_–null cells (CRE) infected with GFP control adenovirus (GFP) or a Krox-20/GFP virus (K20). Note high periaxin levels in Krox-20–infected _c-Jun_–null cells (CRE). (B–E) c-Jun control (cJun con) and _c-Jun_–null mouse Schwann cells 2 d after infection with Krox-20/GFP adenovirus. Note that Krox-20 induces much higher levels of P0 protein in _c-Jun_–null cells (D and E) than in control cells (B and C). The reason why P0 levels in the Krox-20–expressing control cells appear low in this picture (C) compared with other comparable experiments (e.g., Fig. 4 I) is that exposure had to be reduced (equally for C and E) to avoid overexposure in E. (F and G) P0 protein expression in control cells P0 (cJun con) and c_-Jun_–null mouse Schwann cells after 3 d of exposure to db-cAMP/NRG-1. Note that cAMP/NRG-1 induces substantially higher P0 levels in cells without c-Jun. Bars, 15 μm.

Figure 3.

Figure 3.

Persistent expression of c-Jun inhibits Krox-20–induced myelin protein expression. (A and B) Cotransfection of Krox-20/GFP with Jun(Asp) or with Jun(Ala) inhibits Krox-20–mediated induction of periaxin and P0. K20/EV represents cells cotransfected with Krox-20 and control vector. (C–F) P0 in situ experiment showing that cotransfection of Krox-20/GFP with Jun(Ala) inhibits Krox-20–mediated induction of P0 mRNA. C and D are controls, and the arrows show a cell coexpressing Krox-20 and a control vector where Krox-20 has induced P0 mRNA. Arrows in E and F show a cell coexpressing Krox-20 and Jun(Ala) where Jun(Ala) has inhibited Krox-induced P0 expression. Bar, 15 μm. (G) Percentages of GFP-positive cells that also express P0 mRNA in cells cotransfected with the constructs indicated. Error bars show one standard deviation of the mean.

Figure 4.

Figure 4.

MKK7 inhibits myelin gene expression in a c-Jun–dependent way. (A) Western blot showing that MKK7, presumably by activating JNK, inhibits Krox-20–induced myelin protein expression. The cells were coinfected with adenoviruses expressing the constructs indicated. (B) Western blot of cells infected with adenoviruses expressing control LacZ or activated MKK7 to activate JNK. Note that periaxin induced by 2 d of exposure to 1 mM of db-cAMP in LacZ control cells is inhibited by MKK7 expression. (C–F) MKK7 activates c-Jun even in the presence of Krox-20. C and E show that Krox-20 coinfected with a control LacZ-expressing adenovirus suppresses c-Jun levels. D and F show that when Krox-20 is coexpressed with MKK7, high c-Jun levels are maintained. (G) MKK7-mediated suppression of myelin gene expression depends on c-Jun. In normal cells (cJun con), Krox-20–induced periaxin expression (K20/LacZ) is suppressed by MKK7 (K20/MKK7). This suppression does not occur when this experiment is repeated in cells without c-Jun (cJun null). Error bars show one standard deviation of the mean. (H–K) Reactivation of JNK/c-Jun in Krox-20–expressing cells that already synthesize P0 abolishes P0 protein expression. Retrovirally infected cells already expressing Krox-20 and P0 were infected with either LacZ control (H and I) or MKK7-expressing (J and K) adenoviruses. Cells were labeled with either LacZ and P0 (H and I) or MKK7 and P0 (J and K) antibodies. Note down-regulation of P0 protein in Krox-20–expressing cells infected with MKK7 adenovirus. Bars, 15 μm.

Figure 5.

Figure 5.

c-Jun expression inhibits myelination in Schwann cell/DRG neuron cocultures. (A and B) c-Jun is down-regulated in myelinating cells. Arrows show c-Jun–negative nuclei in periaxin-positive (red) myelinating cells. Numerous other cells that are not forming myelin remain c-Jun positive (green). (C) Western blot showing expression of endogenous c-Jun and cJunER fusion protein in purified Schwann cells retrovirally infected with either control empty vector (BP2) or vector expressing the cJunER fusion protein (BP2/cJunER). (D) The number of myelinating segments in cocultures is reduced by c-Jun expression (cJun ER). Error bars show one standard deviation of the mean. (E and F) In cocultures, c-Jun is reactivated in myelinating cells induced to demyelinate by high concentrations of NRG-1. (E) Control cultures with arrows showing c-Jun–negative nuclei associated with periaxin-positive (red) myelinating cells. Arrowhead shows c-Jun (green) in a nucleus not associated with myelinating cells. (F) Cultures exposed to 200 ng/ml NRG-1 for 3 d. Arrow shows activation of c-Jun in a degenerating myelin internode and arrowhead indicates c-Jun in a nucleus of a cell not engaged in myelin formation. Bars, 15 μm.

Figure 6.

Figure 6.

Neonatal myelinating cells dedifferentiate slowly in the absence of c-Jun. (A and B) Comparison of P0 expression in control cells from c-Jun_fl/fl/CRE_−(cJun con) mice and cells without c-Jun from _c-Jun_fl/fl/CRE+ (cJun null) mice prepared at P5 and cultured in DM containing 20 ng/ml NRG-1 for 4 d. (A) Cells with high myelination-related levels of P0 have largely disappeared from control cultures. (B) _c-Jun_–null cultures retain numerous cells with high P0, two of which are illustrated. (C–F) Similar delay in the loss of periaxin in cells prepared at P2 from nerves of _cJun_–null mice cultured for 2 d (see Materials and methods). (C and D) Control cultures are c-Jun positive and periaxin has largely disappeared. (E and F) _c-Jun_–null cultures are c-Jun negative and retain a large number of strongly periaxin-positive cells. (G and H) Similar experiments with cells from JunAA mice. These cells lose periaxin expression at the same rate as control cells, indicating that the major N-terminal phosphorylation sites of c-Jun are not needed for suppression of periaxin. Bar, 15 μm. (I) Quantification of the results illustrated in C–H. The graph shows the number of periaxin-positive cells (relative to the number 3 h after plating) in c-Jun control, _c-Jun_–null and JunAA cells after 2, 4, and 6 d in DM ± 20 ng/ml NRG-1. (J) mRNA for myelin genes disappears only slowly from c-Jun–null cells. Schwann cells purified from control, c-Jun–null, and JunAA P3 mice were plated for 48 h in DM before RNA was extracted and levels of myelin markers were analyzed by quantitative PCR. Relative expression levels of c-Jun, MBP, periaxin, and P0 (MPZ) were obtained by normalizing samples to GAPDH. The data were then expressed as a percentage of the maximum (1) for each of the four genes. The difference in MBP, P0, and periaxin mRNA levels between c-Jun control and null mice was significant (P < 0.01). The same applies to the differences between JunAA and null mice. The dataset for each gene was analyzed by one-way ANOVA and Tukey's Multiple Comparison test where appropriate (using GraphPad Prism Software). Error bars show one standard deviation of the mean. AU, arbitrary units.

Figure 7.

Figure 7.

c-Jun drives dedifferentiation in vivo. (A) MBP immunolabeling of sciatic nerve sections showing delayed loss of myelin in _c-Jun_–null nerves compared with controls, 3 d after transection of nerves of 5-d-old mice. Bar, 10 μm. (B) Quantification of the delay in myelin disappearance by quantitative image analysis of MBP-immunolabeled sections (comparable to those shown in A) 2, 3, and 5 d after injury (expressed as percentage of MPB+ area in uncut P5 nerve). In every case, the difference between c-Jun–null and control nerves is significant (P < 0.01). (C) Electron micrographs showing _c-Jun_–null and control nerves from 5-d-old mice, intact and 3 d after injury as indicated. Note preservation of rounded or partially collapsed myelin sheaths in _c-Jun_–null nerves. Bar, 4 μm. (D) Counts of myelin sheaths (rounded or collapsed) in c_-Jun_–null and control nerves 3 and 5 d after injury (3 d, P < 0.05; 5 d, P < 0.01). Error bars show standard deviation of the mean.

Figure 8.

Figure 8.

Cross-inhibitory relationship between c-Jun and Krox-20. (A and B) Cells cotransfected with empty GFP vector (to visualize transfected cells) and an empty control vector (EV; A) or a Jun(Asp) vector (B). Both cultures were then treated with 1 mM db-cAMP for 2 d to induce Krox-20 and were immunolabeled for Krox-20. In A, arrows point to induced Krox-20 in nuclei of GFP-positive control cells (yellow nuclei of Krox-20–positive GFP-positive cells). In B, no Krox-20 is induced (arrows) in cells containing Jun(Asp). Arrowheads in both panels indicate untransfected cells that have been induced to express Krox-20 by db-cAMP as controls for induction. (C) Activation of JNK inhibits induction of Krox-20. Western blot of cells infected with adenovirus expressing control LacZ or virus expressing activated MKK7 to activate JNK is shown. Note that the Krox-20 and periaxin induced by 2 d of exposure to 1 mM db-cAMP in LacZ control cells is inhibited by MKK7 expression. Note also that MKK7 elevates c-Jun in the presence of db-cAMP. (D–G) In _c-Jun_–null cells, loss of Krox-20 expression is significantly delayed. Double immunolabeling of c-Jun control cells (D and E) and _c-Jun_–null cells (F and G) for Krox-20 (red) and periaxin (green) after 2 d in culture in DM containing 20 ng/ml NRG-1 is shown. Note that Krox-20 has disappeared from the control cells, whereas many _c-Jun_–null cells still have Krox-20–positive nuclei (G, arrows). Note that _c-Jun_–null Krox-20–positive cells are also periaxin positive (F, arrows), whereas control cells have lost periaxin expression (D). Bars, 15 μm.

Figure 9.

Figure 9.

The JNK–c-Jun pathway does not suppress Oct-6. (A) Western blot of Schwann cells infected with adenoviruses expressing control LacZ or activated MKK7 to activate JNK. Note comparable induction of Oct-6 (by 1 mM db-cAMP for 24 h) in control cells and MKK7 cells. (B–E) Teased E17 sciatic nerve stained with HOECHST dye showing that Oct-6 and phospho–c-Jun are coexpressed in immature Schwann cells. (B and C) Double immunolabeling for phospho–c-Jun (P-cJun; red) and Oct-6 (green) in C shows coexpression in Schwann cell nuclei. (D and E) Double immunolabeling for phospho–c-Jun (red) and Krox-20 (green) in E shows phospho–c-Jun alone. Bar, 15 μm.

Figure 10.

Figure 10.

Regulation of Sox-2 by Krox-20 and c-Jun. (A–D) Immunolabeling of teased newborn sciatic nerve. Nuclear c-Jun is in premyelinating cells (Parkinson et al., 2004), most of which also express Sox-2 (overlay). (E) db-cAMP down-regulates Sox-2 protein. Western blot of control untreated cells (Con) and cells treated with 1 mM db-cAMP for 3 d (cAMP) is shown. Note down-regulation of c-Jun and Sox-2 and increase in Oct-6 and Krox-20. (F) Krox-20 down-regulates Sox-2 and c-Jun. Western blot of cells 3 d after infection with either GFP control (GFP) or Krox-20 (K20) adenoviruses. (G–N) c-Jun and Sox-2 are coexpressed after nerve injury. Double immunolabeling of a teased uncut nerve (G–J) and of the distal stump of a nerve 3 d after transection (K–N). Note that neither c-Jun nor Sox-2 are expressed in intact nerve, whereas both factors are activated by injury and found in the same nuclei (arrows). Bars, 15 μm. (O) Sox-2 protein is reduced in _c-Jun_–null cells. Western blot of mouse _Jun_fl/fl Schwann cells infected with either GFP control (GFP) or CRE-recombinase (CRE) adenoviruses. Note reduced levels of Sox-2 in CRE recombined cells. (P) Inhibition of JNK reduces Sox-2 protein levels. Western blot for Sox-2 in control cells (Con) and cells treated for 2 d with 30 μM SP600125 JNK inhibitor (+SP6). (Q) Western blot showing that enforced Krox-20 expression suppresses residual Sox-2 in c-Jun–null cells. Control cells (Con) and _c-Jun_–negative cells (CRE) were infected with control adenovirus (GFP) or adenovirus expressing Krox-20 (K20). Note the two-step reduction in Sox-2, first by removing c-Jun alone (GFP and CRE) and second by expressing Krox-20 from _c-Jun_–null cells (K20 and CRE).

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