Progesterone Receptor (PR) Inhibits Expression of Insulin-Like Growth Factor-Binding Protein-1 (IGFBP-1) in Human Endometrial Cell Line HEC-1B: Characterization of the Inhibitory Effect of PR on the Distal Promoter Region of the IGFBP-1 Gene
Jiaguo Gao and
Linda Tseng
Department of Obstetrics and Gynecology, School of Medicine,
State University of New York, Stony Brook, New York 11794
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ABSTRACT
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Progestin has been shown to have both stimulatory
and inhibitory effects on the expression of insulin-like growth factor
binding protein-1 (IGFBP-1) in human endometrial cells. In this study,
progestin was found to reduce levels of secreted IGFBP-1 and IGFBP-1
messenger RNA and IGFBP-1 promoter activity after stably transfecting a
progesterone receptor (PR; B form) expression vector into HEC-1B cells.
Deletion analysis of the IGFBP-1 promoter revealed that PR specifically
inhibited promoter activity derived from a 59-bp distal
BsaHI/RsaI fragment. It was concluded that PR
inhibited the promoter activity through protein-protein interactions
based on the facts that 1) no progesterone-responsive element was
revealed by a series block mutation in the
BsaHI/RsaI fragment; 2) PR bound by
the antiprogesterone ZK98299 inhibited IGFBP-1 promoter activity; 3) a
DNA-binding mutant of PR inhibited the IGFBP-1 promoter activity; and
4) in an in vivo competition assay, the DNA-binding domain
of PR did not release the inhibitory effect of intact PR. Analysis of
PR deletion mutants indicated that both transcriptional activation
domains of PR (TAF-1 and TAF-2) were involved in the inhibition of
IGFBP-1 expression. Thus, our data may explain the superinduction of
IGFBP-1 in human endometrial cells after progestin withdrawal or
progestin replacement with antiprogestin.
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INTRODUCTION
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Insulin-like growth factor-binding protein-1 (IGFBP-1), a member
of the family of IGFBPs (1, 2), is mainly expressed in the liver and
endometrium (3, 4). In vivo, the expression of IGFBP-1 in
human endometrium is minimum during the menstrual cycle. In
decidualized human endometrium, IGFBP-1 becomes the major secretory
protein during pregnancy (5, 6). Therefore, the expression of IGFBP-1
in the endometrium is correlated with serum levels of progesterone,
suggesting that the expression of IGFBP-1 is induced by
progesterone.
In long term primary cultures of human endometrial stromal cells,
levels of IGFBP-1 and IGFBP-1 mRNA and IGFBP-1 promoter activity are
induced by progestin (7, 8, 9, 10). However, in long term cultures,
expression of IGFBP-1 is further increased by withdrawing progestin
from the culture medium (7). Moreover, levels of IGFBP-1 mRNA and the
transcription rate of IGFBP-1 gene are transiently increased
(superinduction) by replacing progestin with antiprogestin (8). These
observations indicate that progestin has both stimulatory and
inhibitory effects on expression of the IGFBP-1 gene.
Progesterones effects are mediated via the nuclear progesterone
receptor (PR), which belongs to a superfamily of ligand-induced
transactivator (11). Upon activation by progesterone, the PR is able to
bind to a specific DNA sequence [progesterone-responsive element
(PRE)] in the promoter of responsive genes to activate gene
transcription (12). Like other trans-activators, PR consists
of a DNA-binding domain and transcriptional activation domains, one
located in the amino-terminal (TAF-1) and the other in the
carboxyl-terminal (TAF-2) (13, 14, 15). In humans, PR occurs as two forms,
PR-B and PR-A (16, 17). The amino acid sequences of PR-B and PR-A are
identical, except that PR-B contains an additional N-terminal fragment
of 164 amino acids.
To study the effects of PR on IGFBP-1 gene expression, we stably
transfected a PR (B form) expression vector into the human endometrial
cell line HEC-1B. We report that progestin reduced the expression of
endogenous IGFBP-1 in HEC-1B cells expressing PR. Furthermore, PR
specifically inhibited IGFBP-1 promoter activity derived from a distal
59-bp BsaHI/RsaI fragment (-2686 to -2628).
Finally, both activation domains of PR were shown to be involved in the
inhibitory effect through protein-protein interactions.
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RESULTS
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PR Inhibits the Expression of Endogenous IGFBP-1
Although HEC-1B cells express IGFBP-1 (18, 19), they lack
endogenous PR (20). To investigate the effects of PR on IGFBP-1
expression, an expression vector for PR-B was stably transfected into
HEC-1B cells to establish the cell line HEC-1B-PR. Expression of PR in
HEC-1B-PR cells was verified by histochemistry using PR antibody (data
not shown). In a transient transfection assay with a mouse mammary
tumor virus (MMTV) promoter construct (pMMTVCAT) that contains
PREs (21), medroxyprogesterone acetate (MPA) dramatically increased
chloramphenicol acetyltransferase (CAT) activity 90-fold in HEC-1B-PR
cells, but had no effect on CAT activity in parental HEC-1B cells
(Table 1
). These results demonstrated that PR were
expressed and activated by MPA in HEC-1B-PR cells.
When the IGFBP-1 promoter construct pBRPL was used in transfection
assays, MPA inhibited 77% of IGFBP-1 promoter activity in HEC-1B-PR
cells (Table 1
), but had no effect on promoter activity in parental
HEC-1B cells (Table 1
). To determine the effect of PR on the expression
of endogenous IGFBP-1, HEC-1B-PR and HEC-1B cells were cultured with or
without MPA for 48 h, and the level of IGFBP-1 was measured in
conditioned medium by enzyme-linked immunosorbent assay (ELISA). MPA
reduced the production of IGFBP-1 by 3-fold in HEC-1B-PR cells, but did
not affect the production of IGFBP-1 in HEC-1B cells (Fig. 1
). In parallel, IGFBP-1 mRNA levels were reduced
approximately 3-fold by MPA in HEC-1B-PR cells, but were unchanged by
MPA in parental cells (Fig. 2
). These results indicated
that ligand-activated PR inhibits the expression of IGFBP-1 in HEC-1B
cells.

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Figure 1. MPA Inhibits the Secretion of IGFBP-1 Protein in
HEC-1B-PR Cells
HEC-1B and HEC-1B-PR cells (1 x 106) were cultured in
60-mm dishes. Cells were treated with or without MPA in serum-free
medium for 48 h. The level of IGFBP-1 in the conditioned medium
was measured by ELISA as described in Materials and
Methods. Results are the mean ± SD calculated
from triplicate dishes.
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Figure 2. Administration of MPA Reduces the Level of
Endogenous IGFBP-1 mRNA in HEC-1B-PR Cells
Total RNAs were isolated from MPA-treated or untreated HEC-1B and
HEC-1B-PR cells as described in Materials and Methods.
The levels of endogenous IGFBP-1 mRNA were measured by nuclease
protection assay. The riboprobe was transcribed from the 370-bp
PstI/BamHI fragment of IGFBP-1
complementary DNA. 18S RNA probe was used as an internal
control.
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PR Specifically Inhibits the Promoter Activity Derived from the
59-bp BsaHI/RsaI fragment
Previously, we have shown that activated PR inhibits IGFBP-1
promoter activity derived from a 130-bp ClaI/XbaI
fragment (from -2732 to -2600 bp), which can enhance transcription
about 10-fold (20). To define DNA sequences mediating the effect of PR
in this ClaI/XbaI fragment, a series of IGFBP-1
promoter deletion constructs was cotransfected with the PR-B expression
vector (hPR1) into HEC-1B cells. As shown in Fig. 3
, MPA
inhibited 60% of CAT activity derived from IGFBP-1 promoter (from
-2732 to +68 bp, construct pCXPL). Consistent with previous results
(22), deletion of ClaI/BsaHI (-2730 to -2688
bp) increased CAT activity, but promoter activity was still repressed
by MPA (Fig. 3
, construct pBXPL). Deletion of the
RsaI/XbaI fragment (-2630 to -2600 bp) had no
effect on promoter activity, and promoter activity was repressed by MPA
(Fig. 3
, construct pCRPL). However, when the
BsaHI/RsaI fragment (-2688 to -2630 bp) that
mediates the activation (22) was missing from the promoter constructs,
MPA was no longer able to inhibit CAT activity (Fig. 3
, constructs
pRXPL, pCBPL, and pPL). These results indicate that ligand-activated PR
specifically inhibited activation derived from the
BsaHI/RsaI fragment.

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Figure 3. The Activation Derived from the
BsaHI/RsaI Fragment in the IGFBP-1 Promoter
Is Inhibited by PR
Restriction enzyme sites ClaI (-2732),
RsaI (-2630), and XbaI (-2600) were
used to make deletion constructs. The BsaHI (-2688) site
was created by oligo-directed mutagenesis, and this point mutation has
no effect on the promoter activity (22). Eight micrograms of IGFBP-1
promoter/CAT constructs were cotransfected with 1.5 µg hPR1 into
HEC-1B cells. After transfection, cells were treated with (+MPA) or
without (-MPA) MPA for 48 h in serum-free medium. Normalized CAT
activity derived from pCXPL (-MPA) was assigned a value of 100.
Results are the mean ± SD calculated from triplicate
dishes.
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When the BsaHI/RsaI fragment was 5'-linked to the
thymidine kinase (TK) promoter, it was able to enhance the activity of
TK promoter 15-fold in the absence of MPA (Fig. 4
, pBRTKCAT vs. pTKCAT). The enhanced activity of the TK
promoter was repressed 80% by activated PR as well (Fig. 4
, pBRTKCAT
plus hPR1, -MPA vs. +MPA). However, activated PR had no
effect on the TK promoter (Fig. 4
, pTKCAT plus hPR1, -MPA
vs. +MPA). These results indicate that the
BsaHI/RsaI fragment and the basal promoter are
sufficient to mediate the inhibitory effect of PR.

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Figure 4. The Activation Derived from the
BsaHI/RsaI Fragment Can Be Repressed by PR in
a Heterologous TK Promoter Construct
The BR fragment was 5'-linked to the TK promoter. Eight micrograms of
the promoter construct were cotransfected with 1.5 µg hPR1 or pSG5
(empty vector) into HEC-1B cells. After transfection, cells were
treated with (+MPA) or without (-MPA) MPA for 48 h in serum-free
medium. Normalized CAT activity derived from pBRTKCAT (-MPA) was
assigned a value of 100. Results are the mean ± SD
calculated from triplicate dishes.
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Repression of IGFBP-1 Promoter by PR Does not Involve DNA-PR
Interaction
Three activating cis-elements have been identified in
the BsaHI/RsaI fragment in HEC-1B cells (22)
(Fig. 5
). However, sequence analysis did not reveal a
consensus PRE. To identify potential PREs, a series of block mutants in
the BsaHI/RsaI fragment was tested for their
effects on PR function. Mutants 1 and 3, which have no effect on
promoter activity (22), did not affect the inhibitory effect of
activated PR (Fig. 5
, pBRm1PL and pBRm3PL, -MPA vs. +MPA).
The reduced activation from cis-element I mutant (pBRm2PL),
cis-element II mutants (pBRm4PL pBRm5PL, and pBRm6PL),
and cis-element III mutant (pBRm7PL) was still repressed by
activated PR (Fig. 5
, -MPA vs. +MPA). Even the activation
from the double mutated BsaHI/RsaI fragment
(pBRm2, 7PL and pBRm4, 6PL) was repressed by adding MPA (Fig. 5
, -MPA
vs. +MPA). These results indicate that there is no PRE in
the BsaHI/RsaI fragment, and therefore, that PR
does not repress the promoter activity via PR-promoter interaction.

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Figure 5. None of the Block Mutations through the
BsaHI/RsaI Fragment Eliminates the Repressive
Effect of PR Complex
Seven single block mutants were created by oligo-directed mutagenesis
as previously described (22). The double mutants were created by
recombination of the corresponding single mutant. I, II, and III
regions indicate cis-elements I, II, and III, respectively.
Eight micrograms of each construct were cotransfected with 1.5 µg
hPR1 into HEC-1B cells. Normalized CAT activity derived from pBRPL
(-MPA) was assigned a value of 100. Results are the mean ±
SD calculated from triplicate dishes.
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In the presence of PR, administration of ZK98299, whose complex
with PR does not bind to DNA (23), significantly reduced the IGFBP-1
promoter activity (Fig. 6
, pBRPL plus hPR1, control
vs. ZK98299). As a control, ZK98299 failed to activate the
MMTV promoter that contains functional PREs (Fig. 6
, pMMTVCAT plus
hPR1, control vs. ZK98299). When the PR mutant DBDcys that
contains the Cys587 to Ala587 DNA-binding
domain mutation (24) was cotransfected with pBRPL or pMMTVCAT, MPA was
able to inhibit IGFBP-1 promoter activity, but was unable to increase
CAT activity derived from pMMTVCAT (Table 2
). These
results provided additional evidences that PR represses promoter
activity via protein-protein interactions.

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Figure 6. PR Bound by Antiprogestin ZK98299 Retains Its
Ability to Repress the IGFBP-1 Promoter
Eight micrograms of construct pMMTVCAT or pBRPL were cotransfected with
1.5 µg hPR1 or pSG5 (empty vector) into HEC-1B cells. After
transfection, cells were treated with MPA, ZK98299, or ethanol
(control) for 48 h in serum-free medium. Normalized CAT activity
derived from pBRPL (pBRPL plus pSG5, control) was assigned a value of
100. Results are the mean ± SD calculated from
triplicate dishes.
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Repression of IGFBP-1 Promoter by PR Involves Both TAF-1 and TAF-2
Domains of PR
Deletion constructs of PR were used to locate the repressive
domains in PR. When the N-terminal that contains the activation domain
TAF-1 was deleted (15), administration of MPA reduced IGFBP-1 promoter
activity (Fig. 7
, hPR3). When the C-terminal of PR that
contains the activation domain TAF-2 and the progesterone-binding
domain was deleted (15), the repressive effect of PR became independent
of progestin (Fig. 7
, hPR5). Construct hPR5 contains about two thirds
of the hinge region of PR. When both activation domains were deleted
[hPR(C)] (25), no repressive effect was observed [Fig. 7
, hPR(C)].
These results indicated that both the N- and C-terminals of PR, which
contain TAF-1 and TAF-2, respectively, are involved in the repression
of IGFBP-1 promoter activity and that the DNA-binding domain of PR
alone is not sufficient to inhibit promoter activity.

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Figure 7. Both Transcriptional Activation Function Domains of
PR Are Involved in the Repression of IGFBP-1 Promoter
Eight micrograms of pBRPL were cotransfected with 1.5 µg PR
expression vectors into HEC-1B cells. hPR1 expresses the B form of PR
(amino acids 1933). hPR3, hPR5, and hPR(C) express truncated PR
containing amino acids 551933, 1673, and 556639, respectively.
TAF-1 and TAF-2 are transcriptional activation functions. The C domain
is the DNA-binding domain. After transfection, cells were treated with
MPA or without MPA for 48 h in serum-free medium. Normalized CAT
activity derived from pBRPL cotransfected with hPR1 (-MPA) was
assigned a value of 100. Results are the mean ± SD
calculated from triplicate dishes.
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The DNA-binding domain construct of PR, hPR(C), was also used in an
in vivo competition assay to study the effect of the
DNA-binding domain on promoter repression. MMTV promoter activity
enhanced by PR was inhibited 60% when hPR(C) was cotransfected with
the PR expression vector at a 12.5:1 ratio (Table 3
).
Under the same conditions, however, hPR(C) was not able to reverse the
inhibitory effect of PR on IGFBP-1 promoter activity (Table 3
).
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Table 3. Overexpression of DNA-Binding Domain of PR
Cannot Release the Inhibitory Effect of PR on IGFBP-1
Promoter
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DISCUSSION
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In the present study, we have shown that ligand-activated PR
reduced levels of secreted IGFBP-1 and IGFBP-1 mRNA and inhibited
IGFBP-1 promoter activity in HEC-1B-PR cells. We have also demonstrated
that the inhibitory effect of PR on promoter activity is specific to
the activation derived from the BsaHI/RsaI
fragment and did not involve PR-promoter interaction.
At present, it is unclear how the activated PR inhibits promoter
activity, but several potential explanations exist. Activation of
promoters by distal elements requires interactions between distal
activators and the basal transcription machinery. As PR can bind to the
basal transcription factor TFIID in a DNA binding-independent manner
(25), we tested whether PR binding to TFIID interfered with
interactions between distal activators and the basal transcriptional
machinery. Deletion analysis have shown that the DNA-binding domain of
PR [hPR(C)] is responsible and sufficient for TFIID binding (25).
However, hPR(C) was not able to either inhibit IGFBP-1 promoter
activity or reverse the inhibitory effect of intact PR in in
vivo competition assays. Therefore, this mechanism seems unlikely
to explain the observed repression of IGFBP-1 promoter.
Nuclear receptors have also been proposed to inhibit activation by
tethering to activator bound to DNA (26). Mutation analysis revealed
that activation from each of the cis-elements in the
BsaHI/RsaI fragment was inhibited by PR. We have
previously shown that a number of proteins bind to various
cis-elements in the BsaHI/RsaI
fragment (22). However, gel mobility shift assays with the
BsaHI/RsaI fragment and nuclear extracts from
HEC-1B-PR cells revealed that none of the specific complexes was
further retarded by activated PR (data not shown), suggesting that PR
did not bind to any of the proteins bound to these
cis-elements.
Therefore, we hypothesize that PR inhibits IGFBP-1 promoter activity by
competing with one or more coactivators that are needed for activation
derived from the BsaHI/RsaI fragment. It has been
shown that PR can bind to coactivator through its activation domain
(27). More experiments are needed to verify this hypothesis.
The inhibitory effect of PR on IGFBP-1 expression reported here may
explain the superinduction of IGFBP-1 observed in primary cultures
after progestin withdrawal or exposure to antiprogestin (7, 8), but
contradicts results showing that IGFBP-1 is induced by progestin in
primary culture cells. This discrepancy may be explained by the
hypothesis that progestin has a direct inhibitory and an indirect
stimulatory effect on IGFBP-1 expression (20). In primary cultures of
human endometrial cells, the induction of IGFBP-1 by progestin was
shown to be gradual, with maximum induction reached by approximately 25
days (7, 8). Therefore, it is likely that progestin initiates a
signaling pathway that ultimately leads to the activation of IGFBP-1,
instead of activating the promoter directly. PRL is another
decidual-specific protein whose expression pattern is similar to that
of IGFBP-1 in human endometrium (8, 28). Promoter analysis revealed
that progesterone activation of the PRL gene does not occur via direct
PR-promoter interactions (29).
In the present study, we have concentrated on the B form of the PR
because we were unable to select cell clones that express functional
PR-A. It has been reported that the B and A forms have either similar
or opposite effects on transcription (17, 30). However, when the
IGFBP-1 promoter (-3.6 kilobases to +68 bp) construct was
cotransfected with PR-A expression vector, administration of MPA did
not increase promoter activity (data not shown). Transfection data
suggested that PR-A also inhibited activation from the
BsaHI/RsaI fragment (data not shown), which
agrees with the finding that hPR3 (deletion of N-terminal) repressed
IGFBP-1 promoter activity.
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MATERIALS AND METHODS
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Establishment of the Cell Line HEC-1B-PR
HEC-1B cells (American Type Culture Collection, Rockville, MD)
were maintained in MEM in Earles salt solution supplemented with 10%
bovine serum. HEC-1B cells (1 x 106) were
cotransfected with 9 µg hPR1 (15) and 1 µg pSV2neo (31) by the
method of Chen and Okayama (32). Forty-eight hours after transfection,
cells were subcultured, and clones were selected with Geneticin (G418
sulfate; Life Technologies, Gaithersburg, MD) at a concentration of 400
µg/ml. Well separated clones were isolated and cultured with 200
µg/ml Geneticin. Expression of PR was verified by
immunohistochemistry as described previously (20). A construct
containing the mouse mammary virus promoter was transfected into viable
clones to test PR function.
Detection of IGFBP-1 Protein and mRNA
HEC-1B and HEC-1B-PR cells (1 x 106) were
seeded onto 60-mm culture dishes in MEM with 10% bovine serum. Sixteen
hours later, the cells were cultured in serum-free medium with or
without MPA for 48 h. Conditioned medium were collected, and cells
were lysed by an acid guanidinium thiocyanate solution.
Levels of IGFBP-1 in conditioned medium were measured by ELISA as
described previously (33), using IGFBP-1 (Upstate Biotechnology, Lake
Placid, NY) as a standard.
Total RNA was isolated in acid guanidinium thiocyanate solution
and further purified by CsCl centrifugation. A
PstI/BamHI fragment (from 512881 bp) of IGFBP-1
complementary DNA was cloned into pSP6/T719, and an antisense
riboprobe was made by transcription with SP6 RNA polymerase. Levels of
IGFBP-1 RNA were measured by solution hybridization/nuclease protection
assays using the RPAII kit (Ambion, Austin, TX). 18S RNA (Ambion) was
measured as an internal control.
Transient Transfection Assays
The deletion constructs of IGFBP-1 promoter, pCXPL, pBXPL,
pCRPL, pBRPL, pRXPL, and pPL, were described previously (22). Mutations
in the BsaHI/RsaI were generated by
oligo-directed mutagenesis as previously described (22). Plasmids hPR1,
hPR3, and hPR5 (15) were gifts from Pierre Chambon (Institute de Chimie
Biologique, Strasbourg, France). hPR(C) (25) was a gift from Ludger
Klein-Hitpass (Universitatsklinikum Essen, Germany), and DBDcys (24)
was a gift from K. B. Horwitz (University of Colorado, Denver, CO). All
plasmids were purified twice by CsCl centrifugation.
A total of 10 µg DNA including 0.5 µg pRSV-luc (34) were
transfected into 1 x 106 cells in a 60-mm dish as
described previously (20, 22, 32). CAT and luciferase assays were
carried out as previously described (20, 22). CAT activities were
normalized to luciferase activities.
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ACKNOWLEDGMENTS
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We thank Dr. John Bruno for his critical comments on the
manuscript.
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FOOTNOTES
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Address requests for reprints to: Dr. Jiaguo Gao, Department of Obstetrics and Gynecology, School of Medicine, State University of New York, Stony Brook, New York 11794.
This work was supported by NIH Grant HD-19247.
Received for publication December 23, 1996.
Revision received February 21, 1997.
Accepted for publication February 25, 1997.
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