| Summary: | Oligodendrocytes (OL), the myelinating cells of the central nervous
system, extend processes to contact axons and wrap them in an insulative layer
of myelin. This series of studies was undertaken to examine the role of
extracellular signal-regulated protein kinases (ERKs) 1 and 2 in OL process
extension. First, it was determined that stimulation of mature primary bovine OL
with the phorbol ester PMA could induce both process extension and ERK1/2
activation. Furthermore, application of the MEK1 inhibitor PD 98059 was able to
both block PMA-induced process extension and reduce ERK1/2
phosphotransferase activity. Thus it appears that a threshold of ERK1/2
phosphotransferase activity is required for primary OL process extension.
To continue to elucidate the signalling cascades involved in OL process
extension, the Central-Glial 4 (CG-4) cell line was assessed for suitability as an
OL model. CG-4 are bipotential cells capable of differentiating into either
astrocytic or oligodendrocytic (CG-4 OL) cells. A multi-kinase Western blot
profile was conducted to compare the kinase expression patterns of primary rat
OL to CG-4 OL. Overall, the expression of a wide variety of kinases, including
conventional protein kinase C (PKC) isoforms, mitogen-activated protein kinases,
protein kinase A and protein kinase B were very similar between the two cell
types. However, some differences in kinase expression were detected.
Increased expression of focal adhesion kinase, PKC-ε and cyclin-dependent
kinase (CDK) 7 in CG-4 cells could be a function of the self-renewal capacity of
this cell line. Increased expression of Pak-α, PKC-δ and CDK5 in primary OL could explain why these primary cells can achieve a greater degree of
differentiation than CG-4 OL.
After verifying the suitability of the CG-4 cell line as an OL model, further
process extension studies were undertaken. It was found that transient ERK1/2
activation is required to prevent bipolar CG-4 cells from acquiring a multipolar
phenotype. This transient ERK1/2 activation was provided by addition of medium
containing B-104 mitogens. In B-104 mitogen-free medium, ERK1/2 was not
activated and the CG-4 cells acquired a multipolar phenotype. Furthermore,
PMA was able to activate ERK1/2 in lieu of B-104 mitogens, while at the same
time inhibiting the formation of a multipolar phenotype. To verify a role for
ERK1/2 activation in the inhibition of a multipolar phenotype, CG-4 cells were
exposed to B-104 mitogens in the presence of the MEK1/2 inhibitors PD 998059
or UO-126. Surprisingly, even pretreatment of the cells with either MEK inhibitor
could not induce the formation of multipolar processes. Western blots, however,
indicated that neither inhibitor was able to completely abolish ERK1/2 activity.
Therefore, it is possible that the MEK1/2 inhibitors were unable to reduce ERK1/2
activity below the level necessary to inhibit multipolar process formation.
In summary, ERK1/2 activation can both induce process extensions from
primary OL and inhibit the formation of a multipolar phenotype in CG-4 OL. This
could indicate that the CG-4 cell line does not make a suitable model for OL
signal transduction studies. Therefore, caution must be taken when applying the
results of signal transduction experiments conducted on CG-4 cells to primary
OL.
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