From the Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
Received for publication, October 16, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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Induction of myelin genes occurs around
birth in the last stage of Schwann cells differentiation and is
reactivated in case of nerve injury. Previous studies showed that
activation of the gp130 receptor system, using as ligand interleukin-6
fused to its soluble receptor (IL6RIL6), causes induction of myelin
genes such as myelin basic protein (MBP) and myelin protein zero (Po) in embryonic dorsal root ganglia Schwann cells. We also reported that
in murine melanoma B16/F10.9 cells, IL6RIL6 causes a shut-off of
melanogenesis mediated by a down-regulation of the paired-homeodomain factor Pax3. The present work demonstrates that these IL6RIL6-treated F10.9 cells undergo transdifferentiation to a myelinating glial phenotype characterized by induction of the transcriptional activities of both Po and MBP promoters and accumulation of myelin gene products. For both Po and MBP promoters, a repression by Pax3 and stimulation by
Sox10 can be demonstrated. Because after IL6RIL6-treatment, Pax3
disappears from the F10.9 cells (as it does in mature myelinating Schwann cells) whereas the level of Sox10 rather increases, we modulated the relative level of these factors and show their
involvement in the induction of myelin gene expression by IL6RIL6.
In addition, however, we show that a C/G-rich CACC box in the Po
promoter is required for activation by IL6RIL6, as well as by ectopic
Sox10, and identify a Kruppel-type zinc finger factor acting through this CACC box, which stimulates Po promoter activity.
Axonal myelination is a function of specialized glial cells,
oligodendrocytes in the brain and myelinating Schwann cells
(SC)1 in peripheral nerves,
whose impairment in demyelinating diseases affects nerve function and
integrity. Maturation of cells producing myelin occurs around birth and
needs to be reactivated for repair of nerve injury (1). The
developmental stages from neural crest-derived precursors to
mature myelinating SC were defined by specific gene markers (see Refs.
2-5 for reviews). At early stages, embryonic SC express low affinity
nerve growth factor receptor, neural cell adhesion molecule
(N-CAM), glial fibrillary acidic protein (GFAP), the
transcription factors paired homeodomain Pax3, and later POU domain Octamer-6 (Oct-6)/suppressed cAMP-inducible POU (SCIP). This
phenotype also characterizes the adult non-myelinating SC and reappears
after nerve injury. Maturation of myelinating SC is marked by
disappearance of these early markers, including Pax3, and the induction
of myelin genes such as myelin basic protein (MBP) and protein zero (Po
or myelin protein zero). Promoters driving glial-specific expression of
Po (6-8) and of MBP (9-14) were identified along with regulatory
transcription factors (see Ref. 15 for review). Thus, the high
mobility group (HMG) domain Sox10, already present in early neural
crest cells, activates the Po promoter (16). Krox20/Egr2, a late marker
of myelinating SC differentiation required for myelination (17),
stimulates expression of several myelin genes (18, 19). Conversely,
Pax3 represses expression of MBP (20), and so does SCIP (21), in line
with the need for of Pax3 and SCIP to decrease during the terminal
differentiation of myelinating SC.
Among extracellular factors acting on SC, only a few could be shown to
activate myelin gene expression. The main SC growth factors,
neuregulins nerve-derived factor (NDF)/glial growth factor (GGF)
(22), stimulate proliferation at early SC differentiation stages but
have negative effects on the induction of MBP and Po genes (23).
Similarly, fibroblast growth factor or transforming growth factor- We show here another system in which the gp130 activator IL6RIL6
switches on the expression of myelin genes and causes
transdifferentiation of melanoma cells toward a glial phenotype. The
murine melanoma B16/F10.9 cells undergo terminal growth arrest when
exposed to the combination of IL-6 with its agonistic soluble IL-6
receptor (39) or to the IL6RIL6 chimera (34). There is a silencing of
the melanogenic pathway because of a profound reduction in Pax3, which
then down-regulates the microphtalmia-associated transcription factor
(MITF) gene and, in turn, the tyrosinase activity (40). The present
work demonstrates that IL6RIL6 subsequently induces expression of the
myelin Po and MBP mRNAs through promoter-mediated transcriptional
activation. This melanoma provides a new model to study the
transcription factors involved in the regulation of myelin genes
expression and their induction by IL-6 family cytokines.
Cell Cultures and Cytokines--
Murine B16 melanoma metastatic
clone F10.9 cells (41, 42) were cultured as a monolayer at 37 °C,
5% CO2, in Dulbecco's modified Eagle's medium with 8%
fetal calf serum (Biolabs, Bet Ha-Emek, Ness Ziona, Israel),
supplemented with glutamine, penicillin, and streptomycin. Cells were
subcultured every 3 days at 10-30% confluency. Fused IL6RIL6
chimera was produced as described (34, 35) using mammalian
Chinese hamster ovary cells and immunoaffinity purification of the
secreted 85-kDa protein (Interpharm, Israel).
RT-PCR, Northern Blots, and Western Blots--
Total RNA was
extracted with Tri-Reagent (Molecular Research Center), as
recommended by the manufacturer. For RT-PCR, RNA samples (about 2 µg/assay) were reverse-transcribed with SuperscriptII (Invitrogen
Molecular Biology) in the presence of oligo(dT) in 20 µl, and
2 µl of the RT reaction was used for amplification with
Taq polymerase. The primers used to amplify specific mouse cDNAs were as follows: Pax3 (accession number NM_008781),
forward (F): 414-438 and reverse (R): 837-861; Sox10 (AF047043), F: 924-948 and R: 1435-1459; glial fibrillary acidic protein (L27219; rat), F: 1301-1605 and R: 2175-2194; Po (mRNA sequence
reconstituted from M62857-60), F: 231-255 and R: 690-714; and MBP
(M15060), F: 136-156 and R: 466-485. Glyceraldehyde
3'-phosphodehydrogenase (G3'PDH) primers
(Clontech) were used to verify RNA loading.
Amplification conditions were 94 °C (1 min), 52-58 °C (45 s),
72 °C (1 min) for 29 cycles or for 22 cycles (G3'PDH). The sequence
of PCR fragments was verified on DNA analyzer 3700 (PE Applied
Biosystems, Hitachi). Real time PCR for Pax3 and Sox10 was carried out
in a LightCycler (Roche Diagnostics) as recommended by the
manufacturer. Northern blots were probed with a MBP cDNA fragment
cloned, sequenced, and radiolabeled with [ cDNA Expression Vectors--
RT-PCR with F10.9 total RNA was
used as described (40) to amplify the entire coding sequences of Pax3c
cDNA (NM_008781; nt 255-1759) and Sox10 cDNA (AF047043; nt
39-1339). The cDNAs were cloned in the pcDNA3 expression
vector (Invitrogen). The plasmid pAdRSV Krox20G (a gift of Drs. P. Charnay and P. Topilko, Paris, France) contains 5.2 kbp of Krox20 mouse
genomic DNA sequence (including the complete protein coding sequence)
in 3' of Rous sarcoma virus long terminal repeat and in 5' of the pIX
gene polyadenylation domain.
We prepared permanently transfected clones of F10.9 cells where Sox10
or Pax3 gene expression can be increased following addition of
tetracycline (or doxycyclin) to the growth medium (Tet-on system). We
first selected F10.9 cell clones expressing the recombinant reverse
tetracycline repressor rtTA isolated from the pTet-On regulator
plasmid (Clontech) cloned in pEFIRES-puro,
using puromycin as before (40). The clones were screened for rtTA
activity, wild-type growth rate, and response to IL6RIL6 by
morphological change. In the selected clone cells, we introduced the
vector pSV2Hygro, together with pBI vectors containing a bidirectional Tet-regulated element (Clontech). In the pBI
vectors, downstream to the Tet-regulated element, we cloned the
cDNA for the green fluorescent protein, enhanced green fluorescent
protein (Clontech), either alone (control) or with
Sox10 or Pax3 cDNA (in the opposite direction). F10.9-rtTA cell
clones growing in the presence of hygromycin and becoming fluorescent
after treatment with doxycycline (200 ng/ml) were selected. Expression
of the ectopic mRNAs was verified by RT-PCR, using the vector
reverse primer 5' ACTCACCCTGAAGTTCTCAG and forward primers Sox10
(AF047043; nt 924-948) or Pax3 (NM_008781; nt 850-880).
Promoter Reporter Gene Constructs--
The MBP reporter plasmid
pBG1b (9) (a gift of Dr. C. Kioussi, San Diego, CA) was excised with
BglII and BspeI and blunt-ended to obtain
the 5' flanking MBP gene sequence
Site-directed mutagenesis was done by using an Expand high fidelity PCR
system (Roche Molecular Biochemicals). Two primers in opposite
orientation, each one carrying half of the mutated site in its 5', were
used to amplify the mutated plasmid in a single PCR reaction. Typical
conditions for the PCR reaction were according to the manufacturer's
protocol. Amplification conditions were 95 °C (3 min) for one cycle;
94 °C (15 s (sec), 58 °C (30 s), and 68 °C (4 min) for 10 cycles; 94 °C (15 s), 58 °C (30 s), and 68 °C (4 min) for 15 cycles; and 72 °C (7 min) for 1 cycle. The PCR product was
precipitated with ethanol and then phosphorylated by T4 kinase. The
phosphorylated fragment was self-ligated by T4 DNA ligase and digested
with restriction enzyme DpnI to eliminate the non-mutated
template. The mutated plasmid was cloned and amplified in
Escherichia coli (DH5- Cell Transfections--
For transfections, growing F10.9 cells
were seeded in 6-cm Nunc plates. After 24 h, each well received 2 ml of a mixture containing a constant amount of 3.3 µg of DNA
composed of 0.5 µg of the Po or MBP promoter-luciferase pGL3
plasmids, 100 ng of pSV40-Renilla luciferase plasmid (Promega)
and completed with empty pCDNA3, alone or with Sox10 or Pax3
expression vectors, plus 20 µl of LipofectAMINE (Invitrogen), all in
medium without antibiotics. After culture for 12 h at 37 °C,
each transfected plate was split into 10 wells of 6-well plates, and
half the wells were treated with IL6RIL6, and half were left untreated.
After further culture for 72 h, the dual luciferase assay system
kit (Promega) was used to measure luciferase activities according to
the manufacturer's protocol. Results were calculated from
quadruplicate wells.
Electrophoretic Mobility Shift Assays--
Nuclear extracts were
prepared from F10.9 cells grown in 9-cm dishes for different times in
the presence or absence of IL6RIL6 (300 ng/ml). The cell monolayer,
washed with PBS (minus calcium, magnesium), was scraped with a rubber
policeman in 2 ml of phosphate-buffered saline into Eppendorf tubes.
Pellets recovered by centrifugation at 3000 rpm were frozen and stored
in liquid nitrogen until use. Pellets were thawed on ice and
homogenized in hypotonic Buffer A (4 volumes per volume pellet) by
mixing five times with pipette, left 10 min on ice, and centrifuged for
10 min at 4 °C at 5000 rpm. The pellet compacted by a 10-s spin at
14000 rpm was resuspended in 2.5 volumes (relative to original cell
pellet) of Buffer B by mixing five times with pipette, left on ice for
10 min, and centrifuged 14000 rpm for 10 min at 4 °C. The
supernatants (nuclear extracts) were kept at One-hybrid System--
The MATCHMAKER one-hybrid system from
Clontech was used according to the manufacturer's
protocols. Complementary synthetic oligonucleotides containing the CACC
box sequence Induction of Myelin Gene Products following Loss of Melanogenesis
in Melanoma Cells--
As previously reported (40), treatment of the
melanoma F10.9 cells by the gp130 activator IL6RIL6 induces a
transition in morphology from epithelial-like cells to elongated cells
with extended processes that align to form cell tracks. Concomitantly to the cell shape changes, the IL6RIL6-treated cells stopped releasing melanin pigment in the medium, and the rate-limiting melanogenic enzyme
tyrosinase decreased. We have demonstrated (40) that the loss of
tyrosinase upon IL6RIL6 treatment results from the decrease in the
transcription factor MITF, decrease that is itself mainly because of
the loss of Pax3 from the cells. Fig.
1A shows that following the
Pax3 RNA down-regulation, there was a strong induction of MBP
transcripts in the IL6RIL6-treated melanoma cells. A progressive
accumulation of MBP mRNA was observed from 12 to 48 h (Fig.
1B). Transcripts for myelin Po, which is the most abundant component specifically found in the peripheral nerve myelin made by
Schwann cells, were strongly induced by IL6RIL6 in the F10.9 cells
starting from 24 h (Fig.
2C). The CNPase protein,
another constant component of myelin (43), was similarly induced by IL6RIL6 in the F10.9 melanoma cells (Fig. 2D). There was
also an induction of PMP-22 and of galactocerebroside (GalC; not
shown). The F10.9 melanoma cell response to IL6RIL6 can be defined as a
transdifferentiation, because the cells transit from a melanocytic phenotype, which is enhanced if the cells are treated by forskolin (40), to a phenotype characteristic of myelinating Schwann cells.
Transcriptional Activation of Myelin Po and MBP Gene Promoters in
IL6RIL6-treated F10.9 Melanoma Cells: Roles of Pax3 and Sox10--
To
determine whether the induction of the MBP and Po mRNAs following
IL6RIL6 treatment results from transcriptional gene activation, we
cloned the 5' flanking
The B16/F10.9 cells express endogenous Sox10, an HMG factor found to be
important in both melanocytic (45-48) and SC differentiation (15, 16,
49). An increase in Sox10 RNA was observed by RT-PCR with RNA extracted
72 h after IL6RIL6 addition to the cells (Fig. 2E).
This induction was ascertained by real-time PCR (Light Cycler), which
indicated an increase in Sox10 transcripts of 2- and 5.5-fold, respectively, at 48 and 72 h post-IL6RIL6 addition. The amount of
Sox10 protein measured by immunoblots in nuclear extracts of F10.9
cells showed also an increase, which contrasts with the decrease in
Pax3 protein (Fig. 2D). We reported before (40) that this
increase in Sox10 is not seen during the first day after IL6RIL6
treatment, where Sox10 was even lower than in untreated cultures, but
becomes apparent at days 2 and 3 amounting to a 50% increase in the
level of Sox10 protein in the treated cell nuclei.
Co-transfections with Sox10 expression vectors showed that Sox10 is
positively involved in the activation of the MBP promoter in the F10.9
melanoma cells non-treated with IL6RIL6 (Fig.
3A). In the same experiment,
the Pax3 vector again strongly repressed the MBP promoter. Induction by
Sox10 was observed with the three MBP constructs tested, including the
shortest construct containing only the proximal Modulation of the Concentrations of Pax3 and Sox10 in Stably
Transfected F10.9 Cells Regulates the Level of Cellular Myelin Po
mRNA--
To further evaluate the contributions of Pax3 and Sox10
on the induction of Po mRNA, the F10.9 cells were permanently
transformed with vectors containing Pax3 and Sox10 cDNAs placed
under the control of a tetracycline-regulated element. The levels of
Pax3 and Sox10 transcripts were measured by semiquantitative RT-PCR in
the cells treated by doxycycline or IL6RIL6 or both (Fig.
4A), and in parallel we
measured the amount of myelin Po mRNA. In cells that overexpressed
Pax3 when treated with doxycycline, the level of Po RNA induced by
IL6RIL6 was decreased by about 50% as compared with the level induced
in the absence of doxycyclin (Fig. 4B). The basal level of
Po RNA was also very low in the Pax3 expressers. Cells harboring a
Tet-regulated enhanced green fluorescent protein control vector had the
same endogenous Po mRNA induction with or without doxycycline
(noted as 100% on the graph in Fig. 4B). These
results confirm that the induction of the Po mRNA by IL6RIL6 is
impaired if Pax3 is artificially re-increased in the IL6RIL6-treated cells, in line with the hypothesis that the down-regulation of Pax3 by
the gp130 activator accounts for a significant part of the accumulation
of the myelin gene product.
Conversely, with a clone that overexpressed ectopic Sox10 in response
to doxycycline (Fig. 4A), we observed an increase of Po
mRNA (Fig. 4B). However, under conditions where the
increase in total Sox10 RNA caused by doxycycline was comparable with
that in IL6RIL6-treated cells (Fig. 4A), the increase in Po
was less than the one induced by IL6RIL6 treatment (Fig.
4B). Thus, the increase in Sox10 caused by IL6RIL6 could
account for part, but probably not all, of the induction of the Po
mRNA by IL6RIL6 treatment. This conclusion was confirmed when we
next examined the Po promoter sequences required for the
transcriptional activation by IL6RIL6.
Sequences Required for the Activation of the Po Promoter by
IL6RIL6: Involvement of Sox10 Sites and of a CACC Box--
The rat Po
promoter sequence mediating tissue-specific expression of Po contains a
number of binding sites for known transcription factors (8, 16), some
of which are indicated in Fig.
5A. Po promoter constructs
with sequences from
The induction of Po promoter activity by ectopic Sox10 was, as
expected, abrogated by mutations of the Sox10 sites B and C or mutation
of site C' (Fig. 6A).
Interestingly, the A Kruppel-type Factor Is Involved in the Function of the
In search of a factor that could act through this element of the Po
promoter, we used a one-hybrid selection approach in yeast. A cDNA
library prepared from IL6RIL6-treated F10.9 cells was screened for
clones encoding a protein that binds a target reporter construct containing the
ZBP-99 (797 amino acids) has been described (51) as a member of a class
of transcription factors that contain a characteristic array of four
Kruppel-type zinc fingers and bind to CACC boxes, such as that in the
gastrin gene promoter whose sequence and length (CCCCCCACCCCGCCCC)
resembles that of the
Mutation of the Krox20 Also Synergizes with Sox10 to Activate the Myelin Po
Promoter but Does Not Act through the
Ectopic expression of Krox20 in the F10.9 cells induced the Po-300
promoter by itself (Table II). Moreover,
a marked synergistic effect was observed when both Krox20 and Sox10
were expressed together in the melanoma cells (Table II). The
importance of Sox10 for the response to Krox20 was further seen when
Sox10 sites B and C were mutated (Table
III). However, when the The gp130 activator IL6RIL6 activates myelin gene expression in
embryonic Schwann cells (36, 37). We show here that IL6RIL6 induces
expression of the myelin genes in the murine B16/F10.9 melanoma cells.
These cells undergo a transdifferentiation, with loss of their
melanocytic phenotype characterized by MITF and tyrosinase expression
(40) and acquisition of a myelinating Schwann cell phenotype. Melanoma
and nevus cells can exhibit such phenotypic changes in vivo
(53, 54). The inducible transdifferentiation of the F10.9 cells may
recapitulate molecular switches that operate when neural crest-derived
progenitors develop into either melanocytic or glial lineages
(55, 56) and may help identify them. We investigated here
mechanisms involved in the transcriptional activation of promoters from
two myelin genes, Po and MBP. One of the switches that seems to affect
both MITF and myelin gene promoters is a profound down-regulation of
Pax3 observed following IL6RIL6 treatment. The Pax3 RNA was decreased
at 12 h after IL6RIL6, followed by a decrease in the protein (40).
Part of this effect may be on stability of Pax3 RNA, because its
half-life decreased from 6 to 3 h, 1 day after IL6RIL6 (not
shown). Pax3 is an activator of the MITF gene (57), and we have
demonstrated that the loss of Pax3 caused by IL6RIL6 is responsible for
the silencing of the MITF promoter, because its activity can be
restored by ectopic expression of Pax3 (40). As shown here, expression
of Pax3 in the same F10.9 cells conversely represses the ability of
IL6RIL6 to activate the promoters of the myelin Po and MBP genes. An
inhibition of MBP gene induction by Pax3 had been described in SC and
in neuroblastoma (20). This is the first report of a similar repression of the Po gene promoter. It is of interest that the down-regulation of
Pax3 by the gp130 activator IL6RIL6 is also observed in cultures of
embryonic DRG and derived SC, in which Po and MBP gene products are
induced (37). Because the decrease in Pax3 is a constant feature of
terminal differentiation of myelinating SC (5), this may be an
important mechanism by which gp130 signaling enhances myelin gene expression.
Pax3 acts in synergy with Sox10 to activate the MITF promoter, and
mutations in any of these three genes cause some form of Waardenburg
syndrome with melanocytic deficiencies (45-48). Contrasting with the
strong decrease in Pax3 caused by IL6RIL6 in F10.9 cells, the Sox10
protein is rather increased after IL6RIL6 treatment for 2-3 days. It
may seem paradoxical that IL6RIL6, which inhibits melanocytic
differentiation of the cells, causes an increase in Sox10, a factor
needed for melanogenesis (45-48). However, we have shown that, in
IL6RIL6-treated F10.9 cells, Sox10 overexpression does not restore MITF
and does not synergize with Pax3 as it does in the untreated F10.9
cells (40). This suggests that the lowering of Pax3 relative to Sox10,
and the deregulation of their synergistic interaction, prevent Sox10
from activating MITF and melanogenesis. It may also free Sox10, making
it available for acting on other genes. The repression effect exerted
by Pax3 on the Po Increasing the levels of Sox10 in F10.9 cells transfected by
Tet-regulated expression plasmids led to an induction of Po mRNA. However, this induction was always lower than that produced by IL6RIL6.
This is consistent with many lines of evidence that Sox10 does not work
alone in the regulation of myelin gene expression. Thus, in embryonic
SC precursors, Sox10 is present long before myelin gene products start
to be made (49) indicating that other changes are needed. Although
Sox10 activates the transcriptional activity of the Po and MBP
promoters in the F10.9 melanoma cells, as it does in N2A neuroblastoma
cells (16, 50), Sox10 fails to activate MBP promoters in HeLa cells
(58). This suggests that HeLa cells lack a co-factor for Sox10 or
contain an inhibitor. In general, proteins of the Sox family are known
to affect DNA topology (DNA bending) and to require partner factors to
activate transcription (59). Such partners of Sox10, besides Pax3, are, for example, SCIP/Oct-6 (49) and Sp1 (60). SCIP, like Pax3, is an
inhibitor that is down-regulated before SC activate myelin gene
expression (5). For Sp1, a binding site at One such element is a long CACC box, which is functionally defined by
the In addition to Pax3, Sox10 and ZBP-99 we examined the role of
Egr-2/Krox20 in the activation of the Po gene in F10.9 melanoma cells.
Krox20 is required for the onset of myelination during SC development
(17), and it controls a large array of SC genes including Po/myelin
protein zero (19). Activation of the Po promoter by Krox20 was seen in
SC (18) but not in neuroblastoma (16), and synergism of Krox20 and
Sox10 was reported on the Connexin-32 promoter (57). In the F10.9 cell
system, Krox20 expression activates the Po promoter and exhibits a
strong synergism with Sox10. The effect of Krox20 was not related to
the There is growing evidence for a major role of the IL-6 cytokine family
in the synthesis of myelin. In vivo, the inactivation of the
IL-6 family gp130 receptor in newborn mice was shown to impair nerve
myelination (38). Using the potent gp130 activator IL6RIL6, we reported
in vivo stimulation of myelination in regenerating sciatic
nerves (37) and in vitro activation of myelin gene
expression in premyelinating Schwann cells from embryonic DRG (36, 37). The present melanoma cell system provides a new model to investigate the multiple mechanisms that activate myelin gene transcription in
response to the IL-6-type stimulus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
inhibit Po gene expression (24). Axonal contacts are thought to provide
positive stimuli for myelin gene expression in SC cultures (25) and in
transected nerves (26). The molecules mediating these axonal cues are
not well known. Intracellular cyclic AMP elevation (e.g. by
forskolin) induces Po and MBP (25, 27) whereas it represses Pax3 (20).
Activating effects of forskolin were seen on the promoters of the genes
encoding myelin proteins Po (6), MBP (28, 29), and peripheral
myelin protein-22 (PMP-22) (30). Hormones, such as progesterone and
glucocorticosteroids, also stimulate Po and PMP-22 promoter activities
(31, 32). A third group of factors are IL-6 family cytokines that
activate the gp130 receptor system. As a prototype of this large family (see Ref. 33 for review), we have used a recombinant protein IL6RIL6
resulting from the fusion of IL-6 to its soluble IL-6 receptor (sIL-6R)
that activates the gp130 signaling pathway in many cell types and has a
high affinity for gp130 (34, 35). We showed that this gp130 activator
potently stimulates the expression of MBP and Po mRNAs and proteins
in cultures of mouse E14 embryonic dorsal root ganglia (DRG), as well
as in derived SC (36, 37). IL6RIL6 strongly down-regulated the
expression of Pax3 in these embryonic cells. Moreover, IL6RIL6 enhanced
in vivo the number of myelinated fibers in regenerating
sciatic nerve (37). Others observed that IL6RIL6 induces MBP RNA in
brain cells, as well.2
The physiological relevance of these data is supported by the fact that
in mice the conditional inactivation of gp130 after birth causes loss
of myelin sheaths and SC defects in peripheral nerves (38).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP by
random priming with the Rediprime-II kit (Amersham Biosciences). Western blots with rabbit anti-Sox10 antibody (CeMines) and anti-Pax3 (Geneka Biotechnology Inc.) antibodies (both at 1/2000 dilution) and
with anti-CNPase mouse monoclonal antibody (1/300 dilution; Sigma) were
as detailed elsewhere (40). Anti-extracellular signal-regulated kinase
(ERK) 1/2 antibodies were used as control (gift of R. Seger, Weizmann Institute).
1320/+33. The fragment was cloned
in front of the luciferase coding sequence in the pGL3 basic plasmid
(Promega) cut by XhoI and HindIII, blunt-ended, to create the plasmid pGL3MBP/
1.3. The latter was cut with
BglII (in 5') and PstI (in the MBP sequence) and
closed by ligation to create pGL3MBP/
0.65 or was cut with Ecl136-1
and PvuII to create pGL3MBP/
105. The rat Po promoter (6)
was generated by genomic PCR, using the following primers: F,
5'-GACATTATCCCTCCCATCCCCTTATTTCCC-3'; and R,
5'-GCCCAGAGCGTCTGT-GGGGTGGAGAGAGCG-3'. After end polishing, the
amplified fragment (genomic sequence
912/+45 relative to the
start site) was cloned upstream of luciferase in pGL3 basic cut with
SmaI to create pGL3Po-912. For pGL3Po-500, Po gene sequence
500/+45 was made by PCR with pGL3Po-912 as template and primers F,
5'-GGGGACGCGTCCAGGATGCAGGGAGATG-3', with a
MluI site (underlined); and R, 5'-
GGGGAAGCTTGCCCAGAGCGTCTGTGGG-3', with a
HindIII site. The MluI/HindIII PCR
fragment was inserted in pGL3 basic cut by MluI/HindIII. The other 5' deletion plasmids were
similarly prepared. For 3' deletions of the Po promoter, the pGL3Po-300
plasmid was used as template for PCR amplification between primer F,
5'-GGGGGCTAGCTCTATCCCTCAGAGAAGT-3', with a
NheI site; and R,
5'-GGGGAGGCCTCCCCTGGATCCCCAGCAT-3' or 5'-GGGGAGGCCTGGGGCATTGTATACTCTG-3', with a
StuI site. The amplified fragments contain sequences
299/
30 or
299/
137 of the Po promoter. All 3' deletions were
made with the same method and were introduced in front of the minimal
herpes simian virus (HSV)-TK promoter (
73/+57) in the PGL3-TK
plasmid cut by NheI/StuI. PGL3-TK was made by
using as template a construct containing the
155/+57 sequence of
HSV-TK in front of the luciferase gene in PGL3 basic. We amplified by
PCR a large fragment of this plasmid by using primer F,
5'-GGGGGCTAGCAGGCCTAACACGCAGATGCAGTCGG
containing in 5' NheI (bold) and StuI
(underlined) sites in front of the TK sequence from
73. The reverse
primer 5'-TCTGGCATGCGAGAATCTCACGC-3' overlapped the
unique SphI site of the vector. The PCR fragment cut
by NheI/SphI was cloned in PGL3 basic vector cut
by NheI/SphI.
strain) competent cells.
70 °C. Buffer A
contained 10 mM Hepes, pH 7.9, 10 mM NaCl, 5%
glycerol, 2 mM EDTA, 2 mM EGTA, 50 mM NaF and was completed at the last moment with 1 mM dithiothreitol, 10 mM sodium molybdate, 0.1 mM sodium orthovanadate, and a mix of protease inhibitors
(Calbiochem) diluted 1/50 in Buffer A. Buffer B is the same as
Buffer A with 400 mM NaCl. Complementary oligonucleotides
of Po promoter 176/
151 segment (see Fig. 6D) were
5'-labeled with [
-32P]ATP (104 cpm/fmol)
and polynucleotide kinase. After annealing, the double-stranded oligonucleotide was purified on a non-denaturing 8% polyacrylamide gel. About 20,000 cpm of the oligonucleotide probe (20 fmol) was incubated with 2 µl of nuclear extracts for 20 min on ice in a final
volume of 20 µl. The incubation buffer final composition was 20 mM Hepes, pH 7.9, 60 mM NaCl, 1 mM
dithiothreitol, 5% glycerol, 5 mM MgCl2, 0.1 mM ZnCl2, and 100 µg/ml bovine serum albumin, with, respectively, 2 and 0.1 µg of poly(dG)-poly(dC) and
poly(dI)-poly(dC) alternate copolymers per assay (Roche Molecular
Biochemicals). For competition, 2 pmol of cold wild-type
176/
151
probe or oligonucleotides with mutated CACC site were used as follows:
5'-TGTGTCCCTAGATCTACCTACCCAGA-3' (
168/
161 mutant) or
5'-TGTGTCCCCCGGGCCCCCTACCCAGA-3' (
165/
163 mutant).
174/
156 of the myelin Po promoter
cTGTCCCCCCACCCCCCTACa were placed in four tandem repeats upstream of
the pHis-1 and pLAcZ1 plasmids. Target reporter strains of
Saccharomyces cerevisiae YM4271 were obtained after
transformation with these plasmids and tested for minimal background
growth in minimal medium lacking histidine and containing calibrated
concentrations of 3-amino-1,2,4-triazole (3-AT). A cDNA library
(minimal length 400 bp) was prepared from RNA extracted from F10.9
melanoma cells that had been treated for 48 h with IL6RIL6. The
amplified and tailed cDNAs were used for recombination-mediated cloning in yeast with the SmaI-linearized pGADT7-Rec plasmid
to form fusion products with the Gal4 activation domain upon
transformation into the target reporter yeast strain. Large positive
yeast colonies growing in the minimal synthetic dropout (SD)
medium lacking histidine and leucine and supplemented with 30 mM 3-AT were selected. Plasmids isolated from the yeast
colonies by cloning into E. coli DH5
were tested
individually in the yeast target reporter strain. Screening was done in
the histidine- and leucine-free selective medium for colonies that grew
as efficiently in the presence of at least 30 mM 3-AT as in
the absence of 3-AT, thereby eliminating false-positive clones.
Candidate plasmids were isolated and sequenced from 5' and 3' ends of
the insert. Specific primers were prepared for RT-PCR with RNA from
F10.9 cells treated or not with IL6RIL6 (for ZBP-99, forward,
5'-GAGGACACATAGTGGAGAAAAGCC-3'; reverse, 5'-TTTCTACTGAATAACTATGCATGT-3'). Constructs with full-length open reading frames were made in pcDNA3 expression vector and used for
transfections as above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Induction of glial cell markers in F10.9
melanoma cells treated with IL6RIL6. A, RT-PCR on F10.9
total cell RNA with primers for Pax3 versus MBP mRNAs.
G3'PDH amplification to verify equal RNA loading is shown. Where
indicated (+), cells were treated with IL6RIL6 (140 ng/ml) for the
specified time. B, Northern blot with total RNA (15 µg/lane) from cells treated with IL6RIL6 (280 ng/ml) for
48 h, or non-treated, was first hybridized with
[32P]dCTP-labeled MBP cDNA and then with cDNA
from 18 S ribosome RNA. The graph shows the intensity of the
MBP band normalized for that of 18 S RNA, from scanning of complete
blot. C, RT-PCR as in A with primers for Po
mRNA and G3'PDH. D, Western blot containing F10.9 cell
RIPA extracts (50 µg protein/lane) reacted with a
monoclonal antibody against the CNPase myelin component and, after
erasing, with anti-extracellular signal-regulated kinase (ERK) 1/2
antibodies to verify equal protein loading.
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Fig. 2.
Ectopic expression of Pax3 inhibits Po and
MBP promoter activities; down-regulation of Pax3 with IL6RIL6 treatment
may account for promoter induction. A, F10.9
cells co-transfected with MBP promoter ( 1.3kb/+33), luciferase
reporter (0.5 µg), and different amounts of pcDNA3-Pax3 plasmid
(with empty pcDNA3 to keep DNA constant at 3.3 µg). Promoter
activity, expressed as firefly luciferase values, normalized on Renilla
luciferase values, is shown at 48 h with 350 ng/ml IL6RIL6
(black bars) or untreated (open bars).
B, same with Po promoter
912/+45, in the same transfection
experiment. C, plot of the -fold increase by IL6RIL6 and its
repression by different ratios of pcDNA3-Pax3 over the reporter
plasmid in three experiments of transfection with Po
912/+45 or MBP
1.3 kb/+33 promoters. D, Western blots of nuclear extracts
from F10.9 (72 h without or with IL6RIL6 treatment, 140 ng/ml) reacted
successively with Sox10 antibodies and after stripping, with anti-Pax3
antibodies. E, RT-PCR for Sox10 RNA in F10.9 cells without
and with IL6RIL6 for 72 h.
1320/+33 region of the murine MBP proximal promoter (9) in front of the luciferase reporter gene. This region of
the promoter confers tissue-specific expression in cell lines and in
myelinating oligodendrocytes in vivo (44). Transfection of
the F10.9 cells with this reporter gene demonstrated that the MBP gene
undergoes a transcriptional activation of about 5-fold in response to
the IL6RIL6 stimulus (Fig. 2A). For the myelin Po gene, the
5' flanking sequences
912 to +45 of the rat gene, which confer
specific expression in Schwann cells (6, 7), were cloned in the
luciferase reporter gene and transfected into F10.9 cells. IL6RIL6
induced 10-15-fold the activity of this myelin Po promoter construct
(Fig. 2B). Because one of the major effect of IL6RIL6 in the
transdifferentiating F10.9 cells is the decrease of Pax3 protein (Fig.
2D) (40), we investigated if Pax3 may be involved in the
regulation of the myelin Po gene promoter activity, as reported for the
MBP gene in primary Schwann cells (20). The F10.9 cells were
transfected with a Pax3 expression vector in addition to the MBP and Po
promoter reporter genes. The IL6RIL6-dependent induction of
the promoter activities for both MBP (Fig. 2A) and Po (Fig.
2B) was inhibited upon expression of Pax3. Increasing doses
of Pax3 cDNA produced parallel reductions in the effect of IL6RIL6
on the myelin Po and MBP gene promoters (Fig. 2C). Thus the
repressor effect of Pax3 on the MBP gene is operating in the melanoma
F10.9 cells and can now be extended to the peripheral myelin Po gene,
as well. Up to 80% inhibition by Pax3 was seen with the
912/+45 Po
promoter construct (Fig. 2B), and a
dose-dependent decrease could also be observed with a
300/+45 Po promoter construct (data not shown). Inspection of the rat
Po promoter sequence does not indicate the presence of Pax3 binding
sites, leaving the possibility that Pax3 acts indirectly. In any event,
this repression effect on the myelin Po and the MBP promoters strongly
suggests that the down-regulation of Pax3 by IL6RIL6 contributes
significantly to the induction of the myelin gene expression.
105/+33 fragment of
the MBP promoter (Fig. 3A). A further shortened construct,
70/+33, failed, however, to respond to either Sox10 or IL6RIL6 (not
shown). These experiments show that MBP transcription in F10.9 cells is
dependent on Sox10, as found very recently (50) in oligodendrocytes by
in vivo and in vitro experiments. Likewise,
transfection of Sox10 into the F10.9 melanoma stimulated the Po
promoter transcriptional activation in a dose-dependent
manner in the absence of IL6RIL6 (Fig. 3B). At high ratios
of Sox10 over the Po reporter the activation was close to that produced
by IL6RIL6. A truncated form of Sox10 (E188X) was not comparably active
(not shown). In the cells treated by IL6RIL6, Sox10 did not increase
the Po promoter activity (Fig. 3B) and did not increase the
MBP promoter activity (not shown). Hence, in these cells, Sox10 appears
to be a limiting factor in the activity of the MBP and Po gene, unless
the cells have been treated by the gp130 activator.
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Fig. 3.
Ectopic expression of Sox10 induces MBP and
Po promoter activity in F10.9 cells non-treated with IL6RIL6.
A, luciferase activity in cells transfected with 0.5 µg of
luciferase constructs with different length of MBP promoter, together
with control vector (2.5 µg), into non-treated F10.9 cells
(empty bars) or IL6RIL6-treated cells (gray
bars). Right panel, pcDNA3-Sox10 was added to
reporter constructs (point-filled bars, Sox10/MBP-luciferase
plasmid ratio 5:1) in cells without IL6RIL6. Left panel, the
effect of co-transfection with pcDNA3-Pax3 is shown. B,
plot of the -fold induction of Po promoter activity in F10.9 cells
co-transfected with the Po promoter ( 300/+45) luciferase construct
and either only control expression vector pcDNA3 in 5:1 ratio or
with various amounts of pcDNA3-Sox10. Filled circles,
F10.9 cells without IL6RIL6; open circles, IL6RIL6-treated
cells. Luciferase was normalized as in Fig. 2A.
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Fig. 4.
Modulation of Pax3 and Sox10 concentrations
in F10.9 melanoma cells regulates the level of cellular myelin Po
mRNA. A, clones of F10.9 cells that were stably
transfected with Pax3 (left panel) or Sox10 (right
panel) cDNAs placed under the control of the
Tetracycline-regulated element were treated as indicated (+) with
doxycycline (1 µg/ml) or with IL6RIL6 (350 ng/ml) or with both. After
48 h RNA was extracted from the cultures, and total cellular Pax3
or Sox10 RNA was measured by RT-PCR, as well as Pax3 or Sox10 RNAs
specifically originating from the Tet-regulated ectopic genes.
B, the level of cellular Po mRNA was determined by
scanning RT-PCR bands intensity using Tet-regulated clones containing
cDNAs for enhanced green fluorescent protein (control, open
diamonds), Sox10 (open squares), or Pax3 (open
triangles), cultured 48 h without and with indicated amounts
of doxycycline and for Pax3 also with IL6RIL6 and doxycycline
(closed triangles). The Po mRNA level in control cells
treated with IL6RIL6 was taken as 100%.
912,
500 or
300 bp to +45 all gave
10-15-fold activation by IL6RIL6 (Fig. 5B). Further 5'
deletions from
300 to
136 resulted in a progressive loss of
induction by IL6RIL6, most of the decrease occurring between
222 and
137 (Fig. 5C). By further deletion from
136 to
63, the
basal activity of the promoter decreased steeply probably because of
the loss of the two CAAT boxes (Fig. 5, A and C).
Several 3' deletions were made and inserted upstream of a
minimal TK promoter. As shown in Table
I, a TK construct containing the Po
300
to
137 sequence was highly inducible by IL6RIL6, although its overall activity was lower than the
300/
30 construct. The
222/
137 segment of the Po promoter (Fig. 5A, dotted
lines) contains the Sox10 sites C (
204/
198), C' (
193/
187),
and B (
147/
140). These sites, as characterized by Peirano et
al. (16), bind bacterially produced Sox10 and mediate the rat Po
promoter activation in cells stimulated to express Sox10 by
doxycycline. We therefore examined the effect of mutating these sites
as part of a series of linker scanning mutations in the
300 to
137
region (relevant mutants indicated in Fig. 5D). Mutation of
the Sox10 site C' by itself caused a 77% reduction in the
IL6RIL6-induced activity of the promoter and markedly reduced the -fold
induction (Table I). Mutation of the Sox10 C and B sites also reduced
the induction by IL6RIL6, the reduction being greater when both sites
were mutated (Table I). However, we found that additional mutations
located between the Sox10 C' and B sites also affected the activity of the Po promoter and its inducibility by IL6RIL6. In particular, mutating the C/G-rich Sp1-like site or CACC box at
168/
161
(mutation of CCCACCCC; see Fig. 5D) consistently reduced the
activity and response to IL6RIL6 in the Po
300/+45, Po
222/+45, and
Po TK
300/
137 constructs (Table I). The sequences at and around
168/
161 do not resemble a Sox10 site, indicating that the induction
by IL6RIL6 is not only because of an effect on Sox10.
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Fig. 5.
Analysis of Po promoter response to IL6RIL6
in F10.9 cells. A, schematic view of rat genomic
fragment representing the 5' flanking nucleotides 600/+45 of the Po
gene (6, 16) with predicted transcription factor binding sites.
B, three Po promoter segments (from indicated 5' and ending
at +45) cloned in firefly luciferase pGL3 plasmid were transfected into
F10.9 cells followed by 72 h of culture without treatment
(black bars) or with 350 ng/ml IL6RIL6 (crossed
bars). The luciferase values, normalized as in Fig. 2A,
are shown (-fold induction by IL6RIL6 given above the
bars). C, a series of 5' deletions from
500 to
63 (all ending at +45) were assayed as in B. The basal
activity in non-treated cells (dotted line) and the -fold
induction by IL6RIL6 (squares, full line) is
plotted against the position of the 5' deletion. The black
bar shows the DNA domain most important for induction with its
corresponding region in scheme A. D, mutations
performed in Po promoter fragment
264/
136 are shown
under the sequence.
IL6RIL6 response of myelin gene Po promoter mutants
168/
161 CACC box mutation decreased the
induction by ectopic Sox10 as it did for the IL6RIL6 inducer (Fig.
6A, right panel). Changing just the central CAC
(
165/
163) of this element into GGG also reduced the activity of the
Po promoter in response to IL6RIL6 and Sox10 (Table I), as did a
mutation of
176/
169, which shortens the C/G-rich arm (Fig.
6A). These results strongly suggest a model in which Sox10
acts with one or more cooperative factor binding to the CACC box and/or
its C/G-rich arms.
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Fig. 6.
The CACC box ( 168/
161) affects IL6RIL6-
and Sox10-induced Po promoter activity. Sequence-specific factor
attachment to this CACC box is modulated by IL6RIL6. A,
F10.9 cells co-transfected with the Po promoter
300/+45 luciferase
construct or different Po promoter mutants (0.5 µg), with the
expression vector pcDNA3-Sox10 (left panel,
Sox10/Po-luciferase plasmids ratio 5:1; right panel, 2:1).
The luciferase activity in cells treated by IL6RIL6 (normalized as in
Fig. 2A) was taken as 100 (black bars).
Empty bars, no IL6RIL6; point-filled bars, with
Sox10; oblique stripes, with Sox10 and IL6RIL6 treatment.
B, binding of sequence-specific factors to the CACC box in
F10.9 extracts, demonstrated by electrophoretic mobility shift assays.
Oligonucleotide representing sequence
176/
151 of the Po promoter,
32P-end labeled, was incubated with nuclear extracts of
F10.9 cells treated with IL6RIL6 (300 ng/ml) or non-treated for 48 h, as indicated. The first two lanes show induction of
complex C and reduction of complex A with IL6RIL6 treatment. The
next two lanes show specific binding to the CACC box,
complexes A, B, and C disappearing upon competition with a 100-fold
excess of cold probe but persisting when CACC box sequence
168/
161
in the cold competitor is mutated (mutation indicated in Fig.
5D).
168/
161 CACC Box in the Po
Promoter--
Electrophoretic mobility shift assays with a
175/
151
probe demonstrated binding of two slow migrating complexes (A and B) and one fast migrating complex (C), which were specifically competed by
the unlabeled probe but not when that probe had the
168/
161 mutation destroying the CACC box (Fig. 6B, lanes 3 versus 4). Using probes with the
165/
163 CAC
mutated to GGG or TTT also affected the formation of these
complexes (not shown). Although the complexes were seen with nuclear
extracts of untreated cells, IL6RIL6 produced a significant change in
the pattern of complex formation, with a consistent increase in complex
C but a decrease in complex A (Fig. 6B, lanes 1 versus 2).
174/
156 sequence of the Po promoter. Screening of
about 0.5 × 106 clones, followed by re-screening
under stringent conditions of yeast growth (see "Materials and
Methods"), identified a clone with a 3.6-kb insert. This clone was
found by sequencing to contain the entire open reading frame of a
cDNA designated ZBP-99 (accession number XM_129403), and RT-PCR
indicated that its corresponding mRNA was significantly increased
following treatment of the F10.9 cells by IL6RIL6 (Fig.
7A).
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Fig. 7.
CACC box-specific factor ZBP-99 stimulates Po
promoter activity and is increased by IL6RIL6. A,
RT-PCR with primers for ZBP-99 on non-saturating amounts (0.25 µg/ml)
of total cell RNA from F10.9 cells, either untreated ( ) or treated
(+) for 24 h with IL6RIL6 (140 ng/ml). G3'PDH amplification is
shown as control of equal RNA loading. B, F10.9 cells
co-transfected with Po promoter
300/+45 luciferase construct (0.3 µg) and pcDNA3-ZBP99, 3 µg, full-length (black bars)
or truncated after amino acid 599 (gray bars), and 0.6 µg
of pcDNA3 carrier. In control group (white bars), cells
were transfected with 3.6 µg of pcDNA3 only. Where indicated,
cells were co-transfected with pcDNA3-Sox10, 0.6 µg, and 3 µg
of pcDNA3 (white bars) or 3 µg of pcDNA3-ZBP99 or
truncated ZBP99 (black and gray bars,
respectively). Cells were either non-treated (NT) or
IL6RIL6-treated. C, transfections with pcDNA3-ZBP99
comparing
300/+45 Po promoter constructs with either wild-type
sequence, with the
168/
161 CACC box mutation (P1 mut),
or with Sox10 site B and site C mutations (Sox10mut).
Luciferase activity was expressed as in Fig. 2A.
171/
161 element in the Po promoter (see Fig.
5D). Although ZBP-99 was described as an inhibitor of
transcription (51), we found that it stimulates the myelin Po promoter
activity (Fig. 7B). When expressed in the F10.9 melanoma
cells, the full-length ZBP-99 stimulated by itself the transcriptional
activity of the Po-300 promoter, whereas a truncated form of ZBP-99 had
no effect (Fig. 7B). The combination of Sox10 with ZBP-99
produced a 10.2-fold stimulation of the Po promoter, above the
additional effect of each factor alone, and similar to that seen
following IL6RIL6 treatment of the cells (Fig. 7B). The
truncated ZBP-99, lacking its C-terminal domain, was inactive or
slightly inhibitory even with Sox10 or IL6RIL6.
168/
161 CACC box in the Po
300/+45 promoter
(designated as P1 mutation) strongly reduced the activity of ZBP-99,
indicating the functional role of this CACC box for ZBP-99 action, as
seen for the IL6RIL6-dependent response (Fig.
7C). In addition, the effect of ZBP-99 was also abolished
when the Sox10 B and C sites in the Po promoter were mutated,
supporting a cooperative action between the CACC box factor and Sox10
in the activation of the Po promoter (Fig. 7C).
168/
161
Element--
In addition to ZBP-99, which is positively involved in
the activity of the Po promoter and increases after IL6RIL6 treatment, other proteins appear to bind to the
175/
171 probe and contribute to the three DNA-protein complexes observed. DNA-protein UV
cross-linking experiments (not shown) indicated that other proteins are
present in complexes A and B than in complex C, which increases after IL6RIL6. Because Krox-10/Egr-2 has been shown to be required for myelination in SC (17) and binds to C/G-rich Sp1-like sequences (52),
we examined whether it may be one of the proteins acting on the
168/
161 element, possibly through the C/G-rich arms of this CACC box.
168/
161
sequence was mutated, the stimulation by Krox20 was still observed, as well as the Krox20-Sox10 synergism, although the effect of Sox10 was
reduced (Table II). Thus, the site of Krox20 action must be elsewhere.
Indeed, mutation of the upstream CCCCACCCC element (
247/
240; see
Fig. 5D) reduced the effect of Krox20 more than those of
Sox10 or IL6RIL6 (Table III). Another C/G-rich element, GGGGGAGG
(
220/
213), also affected the response to Krox20 with less effects
on the other inducers (Table III). Krox20 may, therefore, work
through several distinct upstream sites. The mutation analysis further
showed that the effect of IL6RIL6 on Po promoter activity correlates
less with the effect of Krox20 than with the response to Sox10. We also
did not find that IL6RIL6 treatment significantly changed the level of
Krox20 RNA already present in these F10.9 cells (not shown). Instead,
the
168/
161 CACC box appears to be the main element required, in
addition to its surrounding Sox10 binding sites, for the activation of
the Po promoter by IL6RIL6, as well as by Sox10 in the F10.9 melanoma
cells.
Effect of Krox20 alone or with Sox10 on the Po promoter
Mutations affecting the Po promoter response to IL6RIL6, Sox10, and
Krox20
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
912/+45 promoter occurs without apparent Pax3 DNA
binding site and may be indirect, possibly because of protein-protein
interactions between Pax3 and factors (such as Sox10) acting on the
myelin gene promoters.
55/
50 in the rat Po
promoter was reported as required for the basal Po promoter activity
(8), but this site is not in the region that we find essential for the
response to IL6RIL6. We, therefore, searched in the
300/
137 region
of the Po promoter for sequence elements that are positively involved
in the response to IL6RIL6 and to Sox10. Our analysis indicates that
activation by IL6RIL6 and by Sox10 requires the Sox10 binding sites C
(
204/
198), C' (
193/
187), and B (
147/
141) defined by Peirano
et al. (16) but requires in addition other critical
transcriptional elements around these sites.
168/
161 mutation inhibiting the Po promoter response to
IL6RIL6, as well as to Sox10. Several DNA-protein complexes are formed
on a probe containing this CACC box sequence, and some of the complexes
are increased following IL6RIL6 treatment whereas other may decrease.
We identified a protein that functionally interacts with this CACC box
sequence used as cis-acting transcriptional element in a
one-hybrid yeast selection system, as transcription factor ZBP-99. The
mRNA for ZBP-99 is increased in the F10.9 cells following treatment
with IL6RIL6. ZBP-99 had been isolated previously as
binding a long CACC box from the gastrin gene promoter and shown to
belong to a novel family of proteins defined by an array of four
Kruppel-type zinc fingers (51). ZBP-99 was reported to inhibit
transcription of the Ornithine decarboxylase gene similarly to its
homologous factor ZBP-89/BFCOL1 (51, 61). However, ZBP-89 has also been
shown to activate transcription of the p21/waf1 gene through
cooperation with histone acetyltransferase p300 in HT29 cells (51).
Transfection of the F10.9 cells with ZBP-99 stimulated the activity of
the Po promoter, and its action was potentiated when co-transfected
with Sox10. Moreover, ZBP-99 was not active on a Po promoter in which
Sox10 sites B and C were mutated. These results demonstrate that a
protein, acting through the CACC box, stimulates Po promoter activity
and mediates a cooperative action of Sox10 with the CACC box element.
The increased expression of the Kruppel-type factor ZBP-99 that is
observed in the IL6RIL6-treated melanoma cells may be one of the
mechanisms activating the Po promoter in these cells. However, complex
interactions with other transcription factors and regulatory proteins
are likely to be involved in the function of the CACC box and its
interplay with the Sox10 sites.
168/
161 CACC box, but we identified other G/C-rich sequences
that may mediate its action on the Po promoter. The untreated F10.9
cells already contain Krox20 mRNA, and IL6RIL6 did not
significantly increase its level, although other types of modulations
affecting Krox20 functions are not excluded. In embryonic neuroglial
cell cultures from DRG of rat E14 embryos, we did
find3 that IL6RIL6 causes a
strong increase in Krox20 mRNA and protein, in correlation with the
induction of MBP and Po mRNAs.
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ACKNOWLEDGEMENTS |
---|
The assistance of Rosalie Kaufmann, Raya Zwang, Lia Chazin, Perla Federman, Osnat Raccach, and Zippi Marks is gratefully acknowledged. We thank Dr. Dalia Gurari for developing and analyzing the Tet-on system. We thank Drs. P. Charnay and P. Topilko for the gift of Krox20 expression vector and helpful discussions. We thank Drs. C. Kioussi and P. Gruss for the MBP gene clone.
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FOOTNOTES |
---|
* This work was supported by Interpharm (Weizmann Industrial Park, Israel) and Ares Serono Group (Geneva, Switzerland).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
972-8-9342101; Fax: 972-8-9344108; E-mail:
michel.revel@weizmann.ac.il.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M210569200
2 M. Pizzi, personal communication.
3 P. L. Zhang, J. Chebath, and M. Revel, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: SC, Schwann cell(s); MBP, myelin basic protein; Po, protein zero; IL, interleukin; IL6R, IL-6 receptor; IL6RIL6, IL-6 fused to its soluble receptor; DRG, dorsal root ganglia; MITF, microphtalmia-associated transcription factor; RT, reverse transcription; F, forward; R, reverse; G3'PDH, glyceraldehyde 3'-phosphodehydrogenase; nt, nucleotides; 3-AT, 3-amino-1,2,4-triazole; TK, thymidine kinase.
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REFERENCES |
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