(Received for publication, June 6, 1994; and in revised form, November 28, 1994)
From the
Here we demonstrate that vascular cell adhesion molecule-1
(VCAM-1) is expressed in the developing central nervous system on
neuroepithelial cells, which are the precursors of neurons and glia. As
these cells differentiate, VCAM-1 is restricted to a subset of the
glial population. An understanding of mechanisms responsible for this
restricted pattern could provide insights into how lineage-specific
gene expression is maintained during neural differentiation. As a model
of neural differentiation, we turned to the P19 embryonic carcinoma
cell line, which in response to retinoic acid will differentiate along
a neural pathway. We show that VCAM-1 expression on the differentiating
P19 cells resembles that in the central nervous system. Transfection of
VCAM-1 gene promoter constructs into P19 cells revealed that the VCAM-1
gene is controlled sequentially by negative and positive elements
during differentiation. We present evidence that early during
differentiation, POU proteins block VCAM-1 gene activity; however,
later in differentiation coincident with the appearance of VCAM-1 the
pattern of POU proteins changes and the VCAM-1 gene promoter is
activated. This activation is mediated through the NFB/rel complex
p50/p65, which forms during P19 cell differentiation.
Vascular cell adhesion molecule-1 (VCAM-1) ()is a
member of the immunoglobulin superfamily (Osborn et al., 1989;
Elices et al., 1990; Rice et al., 1990). Classically,
in response to inflammatory cytokines VCAM-1 appears on the surface of
endothelial cells, and through interaction with integrin receptors
4
1 and
4
7 on leukocytes it mediates cell-cell
interactions that are important for targeting subsets of leukocytes to
sites of inflammation (Elices et al., 1990; Rice et
al., 1990; Erle et al., 1991; Freedman et al.,
1991, Miyake et al., 1991; Scheeren et al., 1991,
Shimizu et al., 1991; Ruegg et al., 1992). Recently,
we have found that VCAM-1 and
4
1 are also expressed during
development where they have a role mediating cell-cell interactions
that are important for skeletal myogenesis (Rosen et al.,
1992; Sheppard et al., 1994).
Here, we show that VCAM-1 is
expressed in the developing brain and spinal cord, again in the absence
of 4 integrins. VCAM-1 is confined to the ventricular zone of the
central nervous system (CNS), which contains a dividing population of
stem cells that will give rise to neurons and glia (Gilbert, 1988). As
these neuroepithelial cells differentiate, VCAM-1 is restricted to a
subset of the glial population; no expression was detected on the
neuronal lineage. Thus, VCAM-1 appears to be a lineage-specific marker
of neural differentiation. The glial population on which VCAM-1
persists is known as radial glia. The body of these cells is located in
the ventricular zone; however, they send projections across the
developing CNS, and differentiating neurons are thought to use these
projections as tracts for migration out of the ventricular zone (Rakic,
1972). VCAM-1(-) mice appear to die before it can be determined
whether there is normal CNS development. (
)Therefore, the
role of VCAM-1 in the developing CNS is uncertain. Despite the as yet
unestablished role of VCAM-1 in the developing CNS, its selective
expression on a subset of the glial lineage during CNS differentiation
indicates that it could be a useful lineagespecific marker of neural
differentiation.
A central question in neurobiology is how the different lineages arise from neuroepithelial cells and how their phenotypes are maintained. Studies of mechanisms controlling the pattern of VCAM-1 during neural differentiation could provide insight into how gene expression is controlled as neuroepithelial cells differentiate into radial glia.
Previously, we have cloned the
VCAM-1 gene promoter and characterized its activity in endothelial and
skeletal muscle cells (Iademarco et al., 1992, 1993). In
endothelial cells VCAM-1 gene expression is dependent upon inflammatory
cytokines for expression. We found that a series of octamers, which
bind the POU family of transcription factors (Rosenfeld et
al., 1991; Ruvkun and Finney, 1991; Wegner et al., 1993),
are negative elements that prevent VCAM-1 gene activity in unstimulated
endothelial cells. In response to tumor necrosis factor-,
B-like sites (Lenardo, 1989; Kieran et al., 1990;
Baeuerle and Baltimore, 1991; Schmid et al., 1991; Urban et al., 1991; Perkins et al., 1992; Ryseck et
al., 1992; Lernbecher et al. 1993; Wasserman, 1993) in
the promoter were activated. These sites overcame the negative effect
of the octamers resulting in transcription of the VCAM-1 gene.
Interestingly, the VCAM-1 gene
B sites were tissue-specific; they
were not active in lymphocytes, where such elements have classically
been studied (Iademarco et al., 1992). In skeletal muscle
cells we identified an enhancer located between the TATA box and
transcriptional initiation site that is responsible for a high level of
constitutive VCAM-1 expression in skeletal muscle; this enhancer did
not bind nuclear protein from endothelial cells, and it had no activity
in transfection assays in endothelial cells (Iademarco et al.,
1993).
To study VCAM-1 gene expression during neural
differentiation, we turned to the embryonic carcinoma cell line, P19,
which in response to retinoic acid will differentiate along a neural
pathway (Jones-Villeneuve at al., 1982). We show that the pattern of
VCAM-1 during P19 cell differentiation resembles that in the
differentiating CNS. Transfection of VCAM-1 gene promoter constructs
into P19 cells revealed that the gene is controlled sequentially by
negative elements and positive elements during differentiation. We
identify these elements as octamers and B sites; however, we show
that their pattern of activity in P19 cells is quite distinct from that
in endothelial cells. This is the first example that NF
B/rel
proteins could have a role in neural differentiation, and we suggest
that the combination of POU proteins and NF
B/rel proteins could be
important for regulating expression of genes that determine lineage
specificity.
For immunostaining of primary cultures and P19 populations, cells were plated on LabTek microslides and fixed and incubated with antibodies as described above. Primary cultures were immunostained 2 days after plating, whereas undifferentiated and differentiated P19 cells were immunostained at various times up to 14 days after plating.
Cerebral
hemispheres of mouse embryos were dissected at embryonic day 13 and
subjected to dissociation as described previously (Huettner and
Baughman, 1986). The isolated cells were plated on matrigel
(Collaborative Research)-coated LabTek microslides and maintained in
-minimal essential medium with 10% Nu-serum (Collaborative
Research), 2 mM glutamine, and 1 mg/ml glucose.
VCAM-1 gene promoter constructs fused to the chloramphenicol acetyltransferase gene (CAT) have been described previously (Iademarco et al., 1992). For stable transfections, 30 µg of reporter plasmid was cotransfected with 1 µg of pRSVNeo, which contains the Rous sarcoma virus long terminal repeat driving the neomycin resistance gene (Gorman et al., 1982), using the calcium phosphate technique as described previously (Iademarco et al., 1992). Forty-eight h after transfection, 400 µg/ml of G418 was added to the media. After 10 days, the resulting G418-resistant colonies were pooled. CAT activity was analyzed as described previously (Iademarco et al., 1992).
Total RNA was isolated as described previously (Rosen et al., 1992) from undifferentiated P19 cells and from P19 cells at different times after treatment with retinoic acid. RNA was subjected to Northern blot analysis for VCAM-1 and glyceraldehyde-3-phosphate dehydrogenase mRNAs as described (Rosen et al., 1992).
Figure 1: Co-expression of VCAM-1 and nestin on progenitor cells and radial glia in the ventricular zone of embryonic mouse brain. Cryostat sections through mouse brain at embryonic day 13 were incubated with a rat monoclonal antibody to VCAM-1 (A and B) or a mouse monoclonal antibody to nestin (C). Primary cultures of mouse telencephalon from embryonic day 13 were double immunostained with anti-VCAM-1 (D) and a mouse monoclonal antibody, RC2 (E). The box in panel A denotes the region seen at higher magnification in panels B and C. The filled arrows in panels D and E indicate the cell body of an RC2/VCAM-1-positive cell that exhibits characteristic radial glial morphology. Unfilled arrows indicate cells that immunostain for VCAM-1 but not for RC2. The bar in panel A is 100 µm in panel A, 15 µm in panels B and C, and 25 µm in panels D and E.
In an effort to confirm that radial glia express VCAM-1,
primary cultures of embryonic telencephalon were doubleimmunostained
for VCAM-1 and RC2, a marker of radial glia (Misson et al.,
1987). Fig. 1, D and E, show that VCAM-1 is
present on RC2-positive cells with characteristic radial glial
morphology. As in the brain, VCAM-1 appears to be concentrated on the
somata of these radial glia with lower levels apparent on the primary
projection. Only a subset of the VCAM-1-positive cells expressed RC2.
The VCAM-1-positive, RC2-negative cells probably represent progenitor
cells that have yet to differentiate into neurons or glia. Neither
VCAM-1 nor nestin was present on neurons that immunostained with TuJ1,
an antibody to class III -tubulin that is expressed early during
neuronal differentiation (Easter et al., 1993), nor were they
detected on cells expressing a second neuronal marker, neurofilament
(results not shown).
VCAM-1 was also found in the ventricular zone of the embryonic spinal cord (Fig. 2, D and E). As in the brain, VCAM-1 appeared to be concentrated on the surface of cell bodies, with lower concentrations on radial projections extending out of the ventricular zone. There was no overlap in immunostaining for VCAM-1 and TuJ1 (Fig. 2A), indicating that VCAM-1 is not expressed on neurons in the spinal cord. Cells in the ventricular zone of the spinal cord also immunostained for nestin, and nestin-positive projections were evident extending from the ventricular zone across the spinal cord (Fig. 2, B and C). As in the brain, we conclude that VCAM-1 is expressed on neural progenitor and radial glial cells in the spinal cord.
Figure 2: Co-expression of VCAM-1 and nestin on progenitor cells and radial glia in the ventricular zone of embryonic mouse spinal cord. Cryostat sections of mouse spinal cord at embryonic day 13 were immunostained with TuJ1 (A), Rat.401 (B and C), or anti-VCAM-1 (D and E). Boxes in panels B and D indicate regions shown at a higher magnification in panels C and E, respectively. Arrows indicate the midline. The bar in panel E is 100 µm in panels A, B, and D, and 25 µm in panels C and E.
Expression of VCAM-1 in the CNS is developmentally regulated, and no immunostaining was evident in the adult mouse CNS (results not shown). Radial glial cells are transient during development and eventually give rise to astrocytes (Hirano and Goldman, 1988). VCAM-1 was not found on cells that were positive for glial fibrillary protein (GFAP), which is a marker for astrocytes (results not shown), suggesting that VCAM-1 expression diminishes as radial glia take on properties of astrocytes. Factors responsible for the restricted pattern of VCAM-1 during neural differentiation could also control the activity of other genes that are important for lineage fate determination.
Figure 3: VCAM-1 and nestin are co-expressed on progenitor- and radial glial-like cells during differentiation of P19 cells. Undifferentiated P19 cells (A-D), cells 9 days after treatment with retinoic acid (A`-D`, E, and E`), and cells 14 days after treatment (F and G`) were immunostained with antisera to fibronectin (A and A`) or TuJ1 (D and D`). Cells were double immunostained with anti-VCAM-1 (B and B`) and Rat.401 (C and C`), anti-VCAM-1 (E), and RC2 (E`), or anti-VCAM-1 (F and G) and GFAP (F` and G`). The arrow in panels B` and C` indicates the same position. Long arrows in panels E and E` indicate VCAM-1/RC2-positive cells; the short arrow indicates a VCAM-1-positive, RC2-negative cell. The bar in panel A is 100 µm in panels F and F` and 25 µm in the other panels.
Immunostaining with TuJ1 demonstrated that a neuronal population of cells appears as a result of P19 cell differentiation; however, these cells do not express VCAM-1 or nestin (Fig. 3, D and D`). As in the primary cultures of embryonic brain (Fig. 1, D and E), there was overlap in the expression of VCAM-1 and RC2 (Fig. 3, E and E`, respectively), indicating that a portion of the VCAM-1-positive cells are radial glial-like. Since nestin is expressed by progenitor cells and radial glia, we conclude that the VCAM-1/nestin-positive population of cells, which are RC2-negative, are progenitor-like cells that have yet to differentiate into neurons and glia.
Immunostaining for VCAM-1 and nestin decreased by day 14 after treatment with retinoic acid (Fig. 3, F and G, and results not shown). The disappearance (Fig. 3, F and G) coincided with the appearance of the astrocyte marker GFAP (JonesVilleneuve et al., 1982); GFAP was not detected at day 9 (F` and G`, and results not shown). These results suggest that VCAM-1 expression diminishes as radial glia take on astrocyte-like properties, which is consistent with the pattern of expression observed in the CNS. The pattern of VCAM-1 during P19 cell differentiation then appears to resemble that observed in the developing CNS.
Figure 4: VCAM-1 mRNA expression is temporally regulated during differentiation of P19 cells. Twenty µg of total RNA from P19 cells at various times after treatment with retinoic acid was used for Northern blot analysis of VCAM-1 mRNA. Numbers indicate time after treatment with retinoic acid (RA). 28s and 18s indicate the position of migration of ribosomal RNA subunits. GAPDH, is glyceraldehyde 3-phosphate dehydrogenase mRNA. C2C12, indicates 20 µg of total RNA from a control mouse myoblast cell line C2C12, which we have shown previously expresses VCAM-1 mRNA (Rosen et al., 1992). Probes for VCAM-1 and GAPDH mRNAs were described previously (Rosen et al., 1992).
Retinoic acid-induced differentiation of P19 cells involves a 4-day exposure of the cells to retinoic acid followed by the subsequent removal of the morphogen. Thus, the fact that VCAM-1 mRNA was not detected until day 6, which is well after the removal of retinoic acid suggests that the effect of retinoic acid is indirect and involves a retinoic acid-triggered differentiation process.
Figure 5:
B sites between positions -130
and -68 bp in the VCAM-1 gene promoter are required for
transcriptional activation during P19 cell differentiation, and VCAM-1
gene octamers act as negative elements in undifferentiated P19 cells. A, a schematic diagram of the VCAM-1 gene promoter is shown at
the top (Iademarco et al., 1992, 1993). Oct indicates octamer elements that are binding sites for POU
proteins,
B indicates binding sites for NF
B/rel
proteins, and PSE is a position-specific enhancer that is
important for expression of VCAM-1 in muscle cells. 5` deletion mutants
of the VCAM-1 gene promoter fused to the CAT gene were stably
transfected into P19 cells, and the resulting colonies from each
transfection were pooled. Numbers in the construct name
indicate the amount of 5`-flanking sequence in each construct;
construction of these plasmids was described previously (Iademarco et al., 1992). CAT activity was compared in undifferentiated
cells(-) and cells 9 days after treatment with retinoic acid
(+). Note that deletion from position -130 to position
-68 bp, which removes the
B sites, eliminated promoter
activation. The results with each construct are representative of at
least three different assays with pooled colonies from two separate
stable transfection experiments. B, the VCAM-1 octamer is a
negative element in undifferentiated P19 cells. pTA-CAT-ATF contains a TATA box and an ATF site driving the CAT gene, and pTA-ATF-OCT-CAT and pTA-ATF-IgH-CAT contain the
VCAM-1 octamer at position -1554 bp and an octamer from the Ig
heavy chain gene enhancer (Iademarco et al., 1993). Ten µg
of the plasmids (2 µg of the RSVCAT control was
transfected) was transiently transfected into 10-cm dishes of
undifferentiated and differentiated (day 7 after treatment with
retinoic acid) p19 cells as described (Iademarco et al.,
1993); vector DNA was added to bring the total amount of DNA
transfected to 20 µg. CAT activity was determined 48 h after
transfection as described (Iademarco et al., 1993). pTA-CAT contains only a TATA box driving the CAT gene,
respectively (Iademarco et al.,
1993).
Figure 6: Activation of the VCAM-1 gene promoter during differentiation of P19 cells appears to involve two steps. P19 cells stably expressing VCAM-1 gene promoter constructs were treated with retinoic acid, and CAT activity was determined at various times after treatment. The pattern of CAT activity with 2.1VCAMCAT paralleled expression of the endogenous VCAM-1 gene: CAT activity did not increase until day 7 and it continued to increase at day 11. However, deletion to position -288 bp, which removes the octamers, changed this time course of promoter activation: CAT activity with this construct increased at day 2 and remained constant until day 11. Thus, the region of the VCAM-1 gene promoter between position -2.1 kb and -288 bp appears to act as a negative element until day 7 when this negative activity begins to diminish.
Unlike endothelial cells where the activity of
the VCAM-1 gene B sites is dependent upon cytokines such as tumor
necrosis factor-
, the
B sites were not activated in
undifferentiated P19 cells by tumor necrosis factor-
, nor did
tumor necrosis factor-
affect the constitutive activity of the
B sites in differentiated cells (results not shown). Likewise, the
pattern of endogenous VCAM-1 mRNA was unaffected by tumor necrosis
factor-
in undifferentiated or differentiated P19 cells.
Figure 7:
Binding of NFB/rel proteins to the
VCAM-1 gene
B sites during P19 cell differentiation. A,
VCAM-1 gene
B sites were used as probes in gel retardation assays
with nuclear extracts from undifferentiated P19 cells and cell either 2
or 9 days after treatment with retinoic acid. NS indicates a
nonspecific complex. Numbers are the days after treatment with
retinoic acid (RA). Competitor indicates that a
20-fold molar excess of unlabeled probe was included in the assay. ATF indicates that a control probe containing an ATF-binding
site was used in the assay. B, p50 and p65 interact with the
VCAM-1 kB sites in differentiated cells. One µl of the indicated
antisera were incubated were included in gel retardation. Note that
addition of anti-p50 resulted in a supershifted complex (arrow), whereas anti-p65 caused the loss of a complex. No
effect was seen with anti-Rel B or anti-c-rel. VCAM-1 and IgH indicate the octamer probes used in the
assays.
Figure 8:
Expression of NFB/rel proteins during
P19 cell differentiation. Antisera to p50, p65, relB, and c-rel were
used to immunostain undifferentiated(-) P19 cells and cells 2
days after retinoic acid treatment (+). Similar results were
obtained with cells 9 days after retinoic acid treatment (results not
shown). Note that p50 is evident constitutively in the nucleus, whereas
the level of p65 and relB in the nucleus increases after treatment with
retinoic acid; little or no nuclear staining for c-rel is evident. The bar in panel C is 25
µm.
It has been shown previously in mouse tissues that
expression of p50 alone is not sufficient for activation of B
sites (Lernbecher et al., 1993). Thus, it is likely that the
complexes of p50/65 and/or p50/relB are responsible for activation of
the VCAM-1 gene
B sites during P19 cell differentiation. Both of
these complexes have been shown previously to be involved in
transcriptional activation through
B sites (Lenardo, 1989;
Baeuerle and Baltimore, 1991; Ryseck et al., 1992; Lernbecher et al., 1993). In endothelial cells p65 is present
constitutively in the cytoplasm, and in response to inflammatory
cytokines it is translocated to the nucleus. In contrast, once p65 and
relB appear during P19 cell differentiation, they are present
constitutively in the nucleus. Thus, inflammatory cytokines are not
required for translocation of the proteins to the nucleus in p19 cells.
To determine which NFB/rel proteins bind the VCAM-1 gene
promoter in P19 cells, antibodies to NF
B/rel proteins were
included in gel retardation assays with VCAM-1 gene
B sites and
nuclear extract from retinoic acid-differentiated P19 cells. A
supershift was observed with anti-p50, and the loss of a complex was
evident with anti-p65-no effect was seen with anti-RelB or anti c-rel (Fig. 7B and results not shown). These results suggest
that it is the p50/p65 complex that binds to the VCAM-1 gene
B
sites, resulting in activation of the VCAM-1 gene during P19 cell
differentiation.
Figure 9: A change in the pattern of POU protein binding during P19 cell differentiation. A, the pattern of nuclear protein binding was compared with an octamer from the VCAM-1 gene promoter and an octamer from the Ig heavy chain gene enhancer (IgH), which has no negative activity in P19 cells (Fig. 5) using nuclear extracts from undifferentiated P19 cells(-) and cells either 2 or 9 days after treatment with retinoic acid (RA). Specific complexes are numbered 1-4. Note that the patterns with the two octamers are different with extract from undifferentiated cells, whereas they are similar with extract from cells at day 9. Also note that with extract from cells at day 2, complex 4 appears to be disappearing, whereas complex 3 is becoming evident, suggesting that the pattern at day 2 is in transition between that seen in undifferentiated cells and cells at day 9. B, competition assays with VCAM-1 and IgH octamers using nuclear extract from undifferentiated P19 cells. The indicated molar excess of unlabeled competitor probe (V = VCAM-1 and I = IgH) was included in the gel retardation assays. C, competition assays with VCAM-1 and IgH octamers using extract from differentiated P19 cells.
The pattern of VCAM-1 octamer complexes changed during P19 cell differentiation: 9 days after retinoic acid treatment a complex comigrating with complex 1 was apparent; however, complex 4 had disappeared and new complex migrating with complex 3 had formed (Fig. 9A). By this time, complexes with the IgH octamer were similar in mobility to those observed with the VCAM-1 octamer, and competition assays using unlabeled octamers suggest that the same complexes are formed with the two octamers, but, as with extract from undifferentiated cells, the IgH octamer is a higher affinity site than the VCAM-1 octamer with extract from differentiated cells (Fig. 9, B and C). The finding that, with extract from differentiated cells, the VCAM-1 octamer forms a complex that comigrates with complex 3 further supports the notion that formation of complex 3 correlates with a lack of octamer activity.
Next, the pattern of protein binding to the VCAM-1
gene octamers was examined in nuclear extracts at day 2 after treatment
with retinoic acid. By this time, the level of complex 4 had decreased
relative to that found with extract from undifferentiated cells,
whereas complex 3 had appeared, but its level was much lower than with
the day 9 extract (Fig. 9A). Therefore, the pattern of
binding to the VCAM-1 gene octamer at day 2 appears to be in transition
between that seen in undifferentiated cells and cells at day 9. This is
in contrast to results with the B sites where nuclear protein
binding increases either at or before day 2 (Fig. 7A).
Therefore, the increase in NF
B/rel protein binding and the
activation of
B sites appears to be a relatively early event
during P19 cells differentiation, whereas the change in the pattern of
POU protein binding and the loss of negative octamer activity seems to
occur relatively late.
Expression of VCAM-1 in the developing nervous system is quite restricted: it is confined to the ventricular zone of the embryonic brain and spinal cord where it is expressed on CNS progenitor cells and radial glia. This pattern of expression suggests that VCAM-1 could be a useful marker to follow neural differentiation. VCAM-1 has the added advantage over some of the other markers such as nestin that have been used to follow similar stages of neural differentiation in that it is a cell surface protein, and as such, it could be utilized to isolate uncommitted CNS cells and follow their differentiation in culture. As a lineage-specific marker of neural differentiation, VCAM-1 could provide insights into molecular events that dictate lineage fates of progenitor cells.
The embryonic carcinoma cell line P19 has been widely used as a model of neural differentiation. It has been shown previously that the neuroepithelial marker, nestin, is expressed in P19 cells that have been induced to differentiated along a neural pathway (Shimazaki et al., 1993), suggesting that differentiation of P19 cells into neurons and glia proceeds through neuroepithelial-like progenitor cells, as occurs in the CNS. Our results support this conclusion. We find a number of cells that are positive for VCAM-1 and nestin after differentiation of P19 cells with retinoic acid. With time, these proteins disappear from the differentiated P19 cell cultures, and this occurs with the onset of expression of GFAP, a marker for astrocytes in the adult CNS. Therefore, it appears that VCAM-1 and nestin are present on progenitor cells and radial glia and that their expression subsides as radial glia give rise to astrocytes.
We have found previously that B sites are responsible for
cytokine-dependent activation of the VCAM-1 gene promoter in
endothelial cells (Iademarco et al., 1992). Here we show that
these same sites are also important for expression of VCAM-1 during P19
cell differentiation; however, their pattern of activity is quite
different in P19 cells. This is the first evidence that NF
B/rel
proteins could have a role in neural differentiation. Activation of the
VCAM-1 gene
B sites is a relatively early event during P19 cell
differentiation occurring well before the VCAM-1 gene is actually
expressed. In contrast to endothelial cells, the
B sites are
constitutively active in the P19 cells. We found that three members of
the NF
B/rel family are present in the nucleus of P19 cells during
differentiation. p50 was present in both undifferentiated and
differentiating cells, whereas p65 and relB were only evident in the
differentiating cells. p50 alone is not sufficient to activate
B
sites in vivo; however, both p50/p65 and p50/RelB are potent
activators (Lernbecher et al., 1993). p50 and p65 interact
with the VCAM-1
B sites in gel retardation assays, whereas RelB
and c-rel do not, suggesting that the p50/p65 complex is responsible
for activation of the VCAM-1 gene during P19 cell differentiation.
Interestingly, when p65 appears during P19 cell differentiation, it is
concentrated in the nucleus. This is in contrast to endothelial cells
where it is present in the cytoplasm and only translocated to the
nucleus when cells are exposed to inflammatory cytokines. In
endothelial cells I
B binds to p65 retaining it in the cytoplasm,
inflammatory cytokines cause disruption of this interaction allowing
p65 to be translocated to the nucleus. Thus, I
B-like proteins
could be absent from or inactive in P19 cells leading to the
constitutive localization of p65 in the nucleus.
As noted above,
activation of B sites is an early event that precedes expression
of VCAM-1. Although activation of these sites is critical for the
subsequent expression of VCAM-1, it appears that the timing of VCAM-1
gene expression is ultimately controlled by the pattern of
octamer-binding POU proteins. We show that the pattern of POU protein
binding to the VCAM-1 gene octamers changes during P19 cell
differentiation and that this change is coincident with a loss of
octamer activity. We show that the lack of octamer activity correlates
with formation of a specific nuclear protein complex which we
designated 3: this complex is not apparent with extract from
undifferentiated P19 cells where the VCAM-1 octamer is active; however,
it is evident with a control IgH octamer that is inactive (other
complexes formed with the VCAM-1 and IgH octamers appear the same), and
a comigrating complex appears with the VCAM-1 octamer as it becomes
inactive during P19 cell differentiation. If these are all the same
complex, then why does the POU protein in this complex only bind to the
VCAM-1 octamer in differentiated P19 cells (it obviously binds to the
IgH octamer in undifferentiated cells)? One possible explanation
focuses on the difference in affinity between the VCAM-1 and IgH
octamers. The POU protein that forms complex 3 could have a relatively
higher affinity for the IgH octamer (the sequence of the two octamers
is slightly different) or the protein may not be in high enough
concentration in undifferentiated cells to bind efficiently to the
VCAM-1 octamer, which is clearly a lower affinity site than the IgH
octamer (nevertheless, it clearly binds the IgH octamer and not the
VCAM-1 octamer). During P19 cell differentiation, the level of this POU
protein could increase to the point where it binds efficiently to the
VCAM-1 octamer. It should be emphasized that although it is an
attractive hypothesis that complex 3 is the same in undifferentiated
and differentiated cells, this is not essential to explain how VCAM-1
octamers lose repressor activity during P19 cell differentiation (i.e. the POU protein responsible for repressor activity could
simply dissipate during differentiation; there is a clear change in the
pattern of protein complexes during P19 cell differentiation).
Several POU proteins have been shown to be expressed selectively in the CNS where they are thought to have roles in neural differentiation (Rosenfeld, 1991; Ruvkun and Finney, 1991; Wegner et al., 1993). As a model of neural differentiation, expression of POU proteins has been examined during P19 cell differentiation. One of these proteins, Oct-6 (also known as SCIP and Tst-1), is expressed in undifferentiated P19 cells, and its level of expression subsequently decreases during P19 cell differentiation (Meijer et al., 1990; He et al., 1991; Collarini et al., 1992). This decrease is gradual and is not complete until well after treatment with retinoic acid. Thus, the pattern of Oct-6 expression shows an inverse relationship to that of VCAM-1 during P19 cell differentiation, suggesting that it could negatively regulate VCAM-1 gene expression. In support of this possibility Oct-6 has been shown to be a transcriptional repressor in neural cells (He et al., 1991). Furthermore, it is thought to have a role in glial differentiation (Collarini et al., 1992), where VCAM-1 expression is developmentally regulated. Another POU protein that is expressed in a pattern similar to that of Oct-6 during P19 cell differentiation is Oct-3 (Okamoto et al., 1990; Rosner et al., 1990, 1991; Shimazaki et al., 1993). It has been demonstrated that the pattern of Oct-3 expression inversely correlates with that of nestin (which parallels that of VCAM-1) in differentiating P19 cells and that forced expression of Oct-3 results in a loss of nestin expression (Shimazaki et al., 1993). These properties suggest that Oct-3 and/or Oct-6 could be responsible for the negative activity of the VCAM-1 gene octamers during neural differentiation. However, other neural-specific POU proteins with repressor activity have also been described (Dent et al., 1991; Stoykova et al., 1992), indicating that several different POU proteins could mediate the inhibitory activity of the octamers in the VCAM-1 gene promoter observed in P19 cells.