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Volume 270, Number 8, Issue of February 24, 1995 pp. 3710-3719
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Control of Vascular Cell Adhesion Molecule-1 Gene Promoter Activity during Neural Differentiation (*)

(Received for publication, June 6, 1994; and in revised form, November 28, 1994)

Allan M. Sheppard ,  Jay J. McQuillan ,  Michael F. Iademarco (§),  ,  Douglas C. Dean (¶)

From the Departments of Medicine and Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
Discussion
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 NFkappaB/rel complex p50/p65, which forms during P19 cell differentiation.

INTRODUCTION

Vascular cell adhesion molecule-1 (VCAM-1) (^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 alpha4beta1 and alpha4beta7 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 alpha4beta1 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 alpha4 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. (^2)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-alpha, kappaB-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 kappaB 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 kappaB 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 NFkappaB/rel proteins could have a role in neural differentiation, and we suggest that the combination of POU proteins and NFkappaB/rel proteins could be important for regulating expression of genes that determine lineage specificity.

MATERIALS AND METHODS

Antibodies

M/K-1 (Miyake, et al., 1991a; diluted 1:1) and anti-NCAM-110 (Pharmigen; diluted 1:5) are rat anti-mouse monoclonal antibodies to VCAM-1. Rat.401 (anti-nestin) (Hockfield and McKay, 1985; diluted 1:100), TuJ1 (Easter et al., 1993; diluted 1:1000), and RC2 (Misson et al., 1987; undiluted hybridoma supernatant) are mouse monoclonal antibodies. Anti-fibronectin (Collaborative Research; diluted 1:75), anti-GFAP (Eng et al., 1971; undiluted sera), and antibodies to NFkappaB/rel family members p50, p65, c-rel, and relB (Santa Cruz Biotechnology; diluted 1:500) are rabbit antisera. Fluorescein or rhodamine goat anti-mouse, goat anti-rat (Boehinger Mannheim; diluted 1:200) and rhodamine donkey anti-rabbit (Jackson Immunochemicals; diluted 1:200) were used as secondary antibodies.

Immunohistology

Timed pregnant mice and adult mice were obtained from a colony maintained by the laboratory. The presence of a vaginal plug was used to define embryonic day 0; birth occurred early on embryonic day 19. Whole embryos were frozen in isopentane that had been chilled in liquid nitrogen. Ten-µm sections were cut on a cryostat, mounted on Superfrost Plus slides (Fisher), and stored at -70 °C until needed. Sections were fixed in methanol at -20 °C and incubated with primary antibodies (either separately or in combination) diluted in PBS containing 2% fish gel (Sigma) at 4 °C overnight. Then, sections were washed with PBS and incubated with the appropriate secondary antibodies, diluted in PBS containing 2% fish gel, for 1 h at room temperature. After further washing, sections were mounted in Vectashield (Vector Laboratories) and observed with epifluorescent illumination. Similar patterns of immunostaining were seen with each of the anti-VCAM-1 monoclonal antibodies.

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.

Cell Culture, Plasmid Constructs, Transfections, and RNA Analysis

P19 cells were maintained in alpha-minimal essential medium containing 2.5% fetal calf serum and 7.5% calf serum. For differentiation, cells were trypsinized and placed in non-tissue culture-treated Petri dishes containing Dulbecco's modified Eagle's medium with 2% fetal bovine serum and 3 times 10M retinoic acid. After 4 days, the resulting aggregates of cells were trypsinized and plated on tissue culture dishes or on LabTek microslides for immunostaining.

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 alpha-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).

Gel Retardation Assays

Nuclear protein extracts were prepared from undifferentiated and retinoic acid-treated P19 cells using a modified Dignam protocol (Dignam et al., 1983). Briefly, 1 times 10^9 cells were harvested and rinsed with PBS. The pelleted cells were resuspended in 15 ml of lysis buffer (20 mM HEPES pH 6.8, 5 mM KCl, 5 mM MgCl(2), 0.25 M sucrose, 0.05% Nonidet P-40, 4 mM phenylmethylsulfonyl fluoride, and 10 mM dithiothreitol). Cells were lysed using a Dounce homogenizer, and nuclear proteins were extracted in a solution of 20 mM HEPES pH 7.9, 20% glycerol, 0.35 M KCl, 0.1 mM EGTA, 0.5 mM EDTA, 0.01% aprotinin, 0.01% leupeptin, and 4 mM phenylmethylsulfonyl fluoride. Single-stranded oligonucleotides were annealed in 10 mM KCl then purified by polyacrylamide gel electrophoresis. Gel-purified, double-stranded oligonucleotides were labeled on their 5` ends with P using polynucleotide kinase and used as probes in gel retardation assays. For gel retardation assays, nuclear extract was incubated with P-labeled probe for 30 min on ice in 4% glycerol, 1 mM EDTA, 5 mM dithiothreitol, 10 mM Tris-HCl, pH 7.5, 100 µg/ml bovine serum albumin, and 2 µg of poly(dI-dC). Oligonucleotide probes containing VCAM-1 gene kappaB sites and octamers, IgH gene octamers, and the activating transcription factor (ATF)-binding site from the human fibronectin gene promoter have been described previously (Iademarco et al., 1993).

RESULTS

Expression of VCAM-1 on CNS Progenitor Cells and Radial Glia

Sections of embryonic mouse brain were immunostained with rat anti-mouse antibodies to VCAM-1. Adjacent sections were immunostained with Rat.401, an antibody to the intermediate filament protein nestin, which is a recognized marker of progenitor cells and radial glia cells in the developing CNS (Hockfield and McKay, 1985). Fig. 1, A and B, shows that VCAM-1 is expressed in the ventricular zone of embryonic brain. VCAM-1 is concentrated on the surface of cell bodies in this region. However, there is additional immunostaining for VCAM-1 on cellular projections extending from the ventricular region; these projections are indicative of radial glia whose somata also reside in the ventricular zone. Cells in the ventricular zone also expressed nestin, and nestin is evident on radial glial projections extending out of the ventricular zone (Fig. 1C). These results suggest that VCAM-1 and nestin are coexpressed on progenitor cells and radial glia in the ventricular zone.

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 beta-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.

P19 Embryonic Carcinoma Cells as a Model System for Examining Molecular Mechanisms Controlling VCAM-1 Expression during Neural Differentiation

In an effort to determine how expression of VCAM-1 may be controlled in the developing CNS, we turned to the embryonic carcinoma cell line P19 which can be induced by treatment with retinoic acid to differentiate along a neural pathway (Jones-Villeneuve et al., 1982). Little or no fibronectin matrix was detected in undifferentiated P19 cells (Fig. 3A); however, an extensive matrix was evident 9 days after treatment with retinoic acid (Fig. 3A`). There was little or no immunostaining for VCAM-1 or nestin in undifferentiated cells (Fig. 3, B and C); however, a significant number of cells expressed these proteins by 9 days after treatment with retinoic acid (Fig. 3, B` and C`).

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.

Temporal Regulation of VCAM-1 mRNA Levels during P19 Cell Differentiation

To determine whether VCAM-1 expression is controlled at the level of mRNA during P19 cell differentiation, RNA was isolated from P19 cells at various times after treatment with retinoic acid, and VCAM-1 mRNA levels were analyzed by Northern blot (Fig. 4B). As with the protein, no VCAM-1 mRNA was detected in undifferentiated cells. VCAM-1 mRNA first appeared at day 6 after treatment with retinoic acid, and the level of the message increased at day 9. However, there was a subsequent decrease in message level at day 12 and again at day 14. This pattern of VCAM-1 mRNA then mirrors that of the protein, which is not evident until after day 4 and which dissipates by day 14 as GFAP-positive astrocytes appear (Fig. 3, B, B`, F, and G, and results not shown). The level of VCAM-1 mRNA at day 9 was as high or higher than that in the C2C12 myoblast cell line (Fig. 4), which we have shown previously expresses VCAM-1 mRNA (Rosen et al., 1992).

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.

Activation of the VCAM-1 Gene Promoter during P19 Cell Differentiation

A VCAM-1 gene promoter-CAT construct, 2.1VCAMCAT, which contains 2.1 kb of VCAM-1 gene 5`-flanking sequence driving the CAT gene (Iademarco et al., 1992), was transfected into P19 cells to determine if the increase in VCAM-1 mRNA that occurs upon P19 cell differentiation is a result of an increase in VCAM-1 gene promoter activity. Little CAT activity was detected in undifferentiated cells; however, as with endogenous VCAM-1 and VCAM-1 mRNA, CAT activity increased at day 7 after treatment with retinoic acid (Fig. 5A). Thus, the increase in VCAM-1 expression that occurs during P19 cell differentiation is due at least in part to an increase in VCAM-1 gene promoter activity.

Figure 5: kappaB 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, kappaB indicates binding sites for NFkappaB/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 kappaB 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).


Positive and Negative Elements Control VCAM-1 Gene Promoter Activity during P19 Cell Differentiation

The activity of VCAM-1 gene 5` deletion mutants was compared to that of the full-length promoter (2.1VCAMCAT) in a series of transfection assays. None of the constructs showed activity in undifferentiated P19 cells (Fig. 5A). However, deletion from position -2.1 kb to position -288 bp (288VCAMCAT) altered the time course of promoter activation: instead of promoter activity rising at day 7 after retinoic acid treatment, as occurs with 2.1VCAMCAT and the endogenous VCAM-1 mRNA and protein, 288VCAMCAT activity increased after only 2 days of treatment with retinoic acid and remained constant until day 11 (Fig. 6). One explanation for such results is that an element or elements in the first 288 bp of the VCAM-1 gene promoter is activated 2 days after treatment with retinoic acid, but sequences between positions -2.1 kb and -288 bp act as a negative element, delaying transcription until day 6-7. Therefore, the pattern of VCAM-1 expression during P19 cell differentiation could be a composite of opposing activities that are mediated by two separate regions of the VCAM-1 gene promoter. To test this possibility, the experiments described below were preformed.

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.


kappaB Sites Mediate Transcriptional Activation of the VCAM-1 Gene

First, we addressed the mechanism through which sequences in the first 288 bp of the VCAM-1 gene promoter activate transcription during P19 cell differentiation. To identify promoter elements that are responsible for the activation of 288VCAMCAT, VCAM-1 gene promoter constructs with additional 5` deletions were tested in transfection assays in P19 cells. Deletion from position -288 to -130 bp had no effect on the activation that occurred in response to retinoic acid treatment (Fig. 5A). There are two kappaB sites in the VCAM-1 gene promoter located at positions -77 and -63 bp (Iademarco et al., 1992). Both of these sites are required for the activation of VCAM-1 gene expression by tumor necrosis factor-alpha in endothelial cells. A subsequent deletion to position -68 bp, which removes the upstream site, blocked activation during P19 cell differentiation. The kappaB sites are the only detectable elements located between position -130 and -68 bp in P19 cells, thus they are sufficient for activation of the VCAM-1 gene promoter during P19 cell differentiation. The lack of activity of 32VCAMCAT, which contains only the first 32 bp of VCAM-1 gene 5`-flanking sequence, suggests that the position-specific enhancer located between the TATA box and transcriptional start site, which is critical for VCAM-1 gene expression in skeletal muscle cells (Iademarco et al., 1993), is not active in P19 cells. These results demonstrate that kappaB sites are sufficient for activation of the VCAM-1 gene promoter during P19 cell differentiation, and they suggest that the kappaB sites are also responsible for the activation of -288VCAMCAT at day 2 of treatment with retinoic acid. However, it is conceivable that another element located between position -288 and the kappaB sites could mediate the early activation of -288VCAMCAT; nevertheless, by the time the intact promoter and the endogenous gene are activated, the kappaB sites are sufficient for promoter activation.

Unlike endothelial cells where the activity of the VCAM-1 gene kappaB sites is dependent upon cytokines such as tumor necrosis factor-alpha, the kappaB sites were not activated in undifferentiated P19 cells by tumor necrosis factor-alpha, nor did tumor necrosis factor-alpha affect the constitutive activity of the kappaB sites in differentiated cells (results not shown). Likewise, the pattern of endogenous VCAM-1 mRNA was unaffected by tumor necrosis factor-alpha in undifferentiated or differentiated P19 cells.

Activation of kappaB Sites Correlates with Increased Binding of NFkappaB/rel Proteins

VCAM-1 gene kappaB sites were used in gel retardation assays with nuclear extracts from undifferentiated P19 cells and from cells either 2 or 9 days after treatment with retinoic acid. No binding to the kappaB sites was observed with extracts from undifferentiated cells; however, binding was apparent 2 days after treatment with retinoic acid, and it persisted and appeared to increase at day 9 (Fig. 7A). The more slowly migrating complex with extracts from cells treated for either 2 or 9 days with retinoic acid was observed reproducibility; however, the more rapidly migrating complexes were variable with both extracts (they could represent partially degraded products or partial occupancy of the kappaB sites within the probe). There was a reproducible slight increase in the more slowly migrating complex from day 2 to 9. Most importantly, it should be emphasized that no binding to the kappaB sites was ever detected with extracts from undifferentiated cells where the sites are inactive, and protein binding correlates with the activation of the sites that occurs during differentiation. As a control, binding of nuclear protein to an ATF site, which interacts with a ubiquitous family of proteins (Brindle and Montminy, 1992), was compared in the different extracts. Similar ATF site binding activity was seen with extracts from undifferentiated cells and day 2 cells; slightly less binding activity was seen with the day 9 extract (Fig. 7A).

Figure 7: Binding of NFkappaB/rel proteins to the VCAM-1 gene kappaB sites during P19 cell differentiation. A, VCAM-1 gene kappaB 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.


Appearance of p50/p65 and p50/relB during P19 Cell Differentiation

To determine which NFkappaB/rel family members are expressed during P19 cell differentiation, undifferentiated and retinoic acid-treated P19 cells were immunostained with antibodies to p50, p65, relB, and c-rel. p50 was found in the nucleus of both undifferentiated and retinoic acid-treated cells (Fig. 8, A and B, respectively). Neither p65 nor relB was evident in undifferentiated cells (Fig. 8, C and E, respectively); however, both proteins were present in a punctate pattern in the nucleus of cells after treatment with retinoic acid (Fig. 8, D and F, respectively). A low level of immunostaining for c-rel was detected in the cytoplasm of both undifferentiated and retinoic acid-treated cells (Fig. 8, G and H). Since no nuclear staining was evident and the cytoplasmic staining was near the limit of detection, it appears that functional c-rel is not present in P19 cells. The patterns of immunostaining in Fig. 8are at day 2 after treatment with retinoic acid; similar results were also seen at day 9 (results not shown).

Figure 8: Expression of NFkappaB/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 kappaB 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 kappaB sites during P19 cell differentiation. Both of these complexes have been shown previously to be involved in transcriptional activation through kappaB 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 NFkappaB/rel proteins bind the VCAM-1 gene promoter in P19 cells, antibodies to NFkappaB/rel proteins were included in gel retardation assays with VCAM-1 gene kappaB 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 kappaB sites, resulting in activation of the VCAM-1 gene during P19 cell differentiation.

Octamers from the VCAM-1 Gene Promoter Act as Negative Elements in Undifferentiated P19 Cells

The region of the VCAM-1 gene 5`-flanking region between position -2.1 kb and -288 bp contains multiple octamer consensus sequences. We have shown that these octamers act as silencers in endothelial cells (Iademarco et al., 1993). To determine if these octamers are responsible for the negative activity that prevents VCAM-1 gene expression in undifferentiated cells, we examined the activity of VCAM-1 gene octamers in the context of a heterologous promoter as described (Iademarco et al., 1993). Undifferentiated cells were transfected with pTA-ATF-CAT, which contains a TATA box and an ATF-binding site driving the CAT gene, and pTA-ATF-OCT-CAT and pTA-ATF-IgH-CAT, which contain a VCAM-1 gene octamer and a control octamer from the Ig heavy chain gene enhancer (IgH), respectively. The VCAM-1 gene octamer silenced transcription in undifferentiated P19 cells; however, the IgH octamer had no effect (Fig. 5B). In differentiated cells neither octamer had an effect on promoter activity. These results suggest that octamers in the VCAM-1 gene promoter are responsible for the negative activity of the region between position -2.1 kb and -288 bp in undifferentiated P19 cells, and they demonstrate that there is a difference in the activity of the VCAM-1 gene octamer and the IgH octamer. It should be emphasized that there are at least 10 consensus octamer-binding sites between position -2.1 kb and -288 bp making it difficult to assess the function of the octamers in the context of the VCAM-1 gene promoter directly by mutation analysis. However, the octamers do show the corresponding negative activity when they are placed on a heterologous promoter, suggesting that they are responsible for the negative activity of this region. Nevertheless, even though the octamers show the same activity as the -2.1 kb to -288 bp region during P19 cell differentiation, our experiments do not formally prove that the octamer sites are responsive for the negative activity of this region.

The Pattern of POU Protein Binding Appears to Control VCAM-1 Gene Octamer Activity during P19 Cell Differentiation

Next, we compared the VCAM-1 and IgH octamers in gel retardation assays with nuclear extracts from undifferentiated and differentiated P19 cells. Using nuclear extract from undifferentiated P19 cells, two minor complexes labeled ``1'' and ``2'' and a major complex labeled ``4'' were observed with the VCAM-1 gene octamer (Fig. 9A). Complexes of similar mobility were apparent with the IgH octamer; however, an additional major complex labeled ``3'' was also apparent. Competition assays with unlabeled octamers demonstrated that the IgH octamer is a higher affinity site than the VCAM-1 octamer: the IgH octamer competed for formation of each of the VCAM-1 and IgH octamer complexes more effectively than the unlabeled VCAM-1 octamer (Fig. 9B). As expected, competition with the VCAM-1 octamer had relatively little effect on formation of complex 3 with the IgH octamer. Complex 3 then appears to correlate with a lack of octamer activity in undifferentiated cells.

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 kappaB sites where nuclear protein binding increases either at or before day 2 (Fig. 7A). Therefore, the increase in NFkappaB/rel protein binding and the activation of kappaB 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.

Discussion

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 kappaB 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 NFkappaB/rel proteins could have a role in neural differentiation. Activation of the VCAM-1 gene kappaB 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 kappaB sites are constitutively active in the P19 cells. We found that three members of the NFkappaB/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 kappaB 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 kappaB 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 IkappaB binds to p65 retaining it in the cytoplasm, inflammatory cytokines cause disruption of this interaction allowing p65 to be translocated to the nucleus. Thus, IkappaB-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 kappaB 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.

FOOTNOTES

*
These studies were supported by Grants HL43418 and AR41908 from the National Institutes of Health (to D. C. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Institutes of Health Training Grant HL07317.

To whom correspondence should be addressed: Box 8052, Washington University School of Medicine, 660 S. Euclid, St. Louis, MO 63110. Tel.: 314-362-8989; Fax: 314-362-8987; Dean{at}telesphere.wustl.edu.

(^1)
The abbreviations used are: VCAM-1, vascular cell adhesion molecule-1; CNS, central nervous system; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase; ATF, activating transcription factor; GFAP, glial fibrillary protein; kb, kilobase(s); bp, base pair(s).

(^2)
M. Labow, personal communication.

ACKNOWLEDGEMENTS

We thank A. Frankfurter for TuJ1, A. Pearlman for RC2, S. Hockfield for Rat.401, and David Gottleib for P19 cells.

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