Much as Krox-20 promotes the transition from the immature Schwann cell phenotype toward myelination, we find that c-Jun acts in a reverse way to promote the immature Schwann cell state at the expense of myelination. c-Jun, at physiological expression levels and apparently without N-terminal phosphorylation, inhibits myelin gene activation by Krox-20 or cAMP elevation. Enforced c-Jun expression also inhibits myelination in cocultures. When early myelinating cells are released from axon-associated promyelin signals and placed in culture, the resulting c-Jun activation drives the cells back to the immature state. Also in vivo, reactivation of c-Jun after injury of neonatal nerves pushes early myelinating cells toward demyelination. In previous work, we also showed that c-Jun is important for events that characterize the immature but not myelinating state, namely cell death and proliferation (Parkinson et al., 2004
The most striking abnormality in early postnatal nerves in conditional c-Jun–null mice is the failure of myelinating cells to dedifferentiate normally after injury. It is therefore likely that the negative influence of c-Jun on myelin differentiation that we have documented is, in vivo, more important for pushing the dedifferentiation program in injured or pathological nerves than for the regulation of myelination during normal development.
Consistent with the involvement of Krox-20 and c-Jun in antagonistic programs, expression of these factors is mutually exclusive: immature cells express relatively high levels of c-Jun and low levels of Krox-20, switching to high levels of Krox-20 and low levels of c-Jun in myelinating cells. In cut or crushed nerves, Krox-20 falls and c-Jun is reexpressed at high levels as cells transit back to the immature state. Furthermore, c-Jun and Krox-20 show a cross-antagonistic functional relationship because expression of Krox-20 suppresses c-Jun and enforced expression of c-Jun suppresses Krox-20.
c-Jun is therefore a transcription factor that negatively regulates the myelinating Schwann cell phenotype, representing a signal which functionally stands in opposition to the network of promyelin transcription factors, which includes Oct-6, Brn2, NFκB, Sox-10, and Nab1 and 2, in addition to the key role of Krox-20 (Topilko et al., 1994
; Nagarajan et al., 2001
; Jaegle et al., 2003
; Le et al., 2005b
; Ghislain and Charnay, 2006
; LeBlanc et al., 2006
). Negative regulation of myelin differentiation is likely to emerge as a major aspect of Schwann cell biology, with particular importance for plasticity, demyelinating pathology, and responses to injury and regeneration, in addition to developmental regulation. It is also likely that the molecular machinery of myelin suppression will turn out to be equally complex to that of its promyelin counterpart. Thus, inducible nitric oxide synthase, Sox-2, Erk1/2 activation, and Notch signaling have all been implicated in negative control of myelin differentiation (Harrisingh et al., 2004
; Ogata et al., 2004
; Le et al., 2005a
; Agthong et al., 2006
; Woodhoo, A., M. Duran, K.R. Jessen, and R. Mirsky. 2004. Differentiation
. Abstr. 119). Similarly, the p38 MAPK signaling pathway, although important for events immediately before myelination, also accelerates demyelination (Fragoso et al., 2003
; unpublished data), and delayed demyelination is seen in mice with genetic inactivation of Toll receptors, nitric oxide synthase, phospholipase A2, and matrix metalloprotease 9 (Levy et al., 2001
; De et al., 2003
; Shubayev et al., 2006
; Boivin et al., 2007
). In future work, it will be important to learn about the functional interactions between the different signaling systems that are emerging as potential myelin suppressors.
We find that Krox-20 represses both c-Jun and Sox-2 and that, in the absence of Krox-20, c-Jun is highly expressed in Schwann cells without the need for specific extrinsic signaling. Therefore, the loss of Krox-20 that follows nerve cut or crush is likely to allow the cells to resume their constitutive c-Jun expression. This will help push the cells toward dedifferentiation and promote proliferation as seen in Wallerian degeneration. A similar mechanism is likely to be operative during the dedifferentiation and proliferation that takes place when Krox-20 is genetically inactivated in adult nerves without axon transection (Decker et al., 2006
). The way in which c-Jun and Sox-2 interact during these events remains to be examined.
Although we have shown previously that in Schwann cells, as in other cell types, c-Jun has a role in controlling proliferation (Parkinson et al., 2004
), this effect can be clearly dissociated from the negative regulation of myelin differentiation. Several of the present experiments demonstrate this, including those in which genetic removal of c-Jun amplifies myelin gene expression in response to Krox-20 or cAMP and where myelin gene activation by these signals is inhibited by enforced expression of Jun or JNK activation by MKK7. It is also seen when clearance of Krox-20 and myelin proteins from early myelinating cells that have been removed from axons and placed in DM is accelerated by c-Jun or JunAA. In all these cases, Jun inhibits myelination or drives demyelination under conditions where Schwann cells are not dividing.
Suppression of myelination by c-Jun can also be dissociated from the classical N-terminal phosphorylation of c-Jun typically performed by JNK. Rather, suppression of the myelin phenotype appears to depend on the levels of the c-Jun protein itself. This can be seen from the fact that Jun(Ala), in which all potential N-terminal phosphorylation sites are absent, suppressed Krox-20–induced myelin gene expression as effectively as Jun(Asp), in which the N-terminal phosphorylation sites have been mutated to aspartic acid to mimic the active phosphorylated c-Jun. It is also evident from the observation that normal c-Jun and nonphosphorylatable c-Jun (in cells from JunAA
mice) appear to be equally effective at driving down myelin gene expression in experiments where neonatal myelinating cells are removed from axonal contact and placed in culture. This is in line with results from other systems, where phosphorylation of c-Jun by JNK is not required for all its actions (Behrens et al., 1999
; Raivich and Behrens, 2006
). The JNK pathway may instead function to control c-Jun protein levels, in part by modulating the activity of several transcription factors that together regulate the c
promoter (Besirli et al., 2005
), which is a situation that would explain the effects of the JNK activator MKK7 in our experiments.
In injured nerves, loss of myelin differentiation is a beneficial response that helps axonal regeneration and repair. But the loss of myelin differentiation in demyelinating neuropathies, such as Charcot-Marie-Tooth 1A and Guillain-Barré syndrome, is unhelpful and leads to disability. From a clinical standpoint, it will be important to determine whether c-Jun is a component of the mechanism that pushes myelinating cells toward demyelination in these pathological conditions because this would open new avenues for clinical intervention.