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Molecular and Cellular Biology, January 1999, p. 788-795, Vol. 19, No. 1
Department of Biological Chemistry,
Received 15 May 1998/Returned for modification 14 July
1998/Accepted 21 September 1998
The extended human acetylcholinesterase (AChE) promoter contains
many binding sites for osteogenic factors, including
1,25-(OH)2 vitamin D3 and 17 Osteogenesis in terrestrial
vertebrates involves the progeny of common mesenchymal progenitors
(3) that become committed to bone or cartilage lineages in
parallel pathways controlled by osteogenic hormones and growth factors
(9). These include 1,25-(OH)2 vitamin
D3 [1,25-(OH)2D3], which has been
found to be essential for endochondral ossification (6, 41).
Another osteogenic agent, 17 An intriguing candidate protein for such a role(s), with recently
established morphogenic capacities, is the acetylcholine-hydrolyzing enzyme acetylcholinesterase (acetylcholine acetyl hydrolase [AChE]; EC 3.1.1.7). AChE is expressed in both mesenchyme (26) and hematopoietic cells (28, 29). Antisense AChE suppression
attenuates erythropoiesis and induces stem cell expansion in primary
murine bone marrow cultures (39), and the chondrogenic
expression of AChE parallels the early development of rat lower limbs
(43) and embryonic chick limbs (1). Thus,
AChE fulfills at least some of the requirements for an
osteogenic/hematopoietic coordinator. This provides an incentive for
exploring genomic and transcriptional evidence to test whether it does
indeed function as such a coordinator.
In transgenic Xenopus embryos (35), transfected
rat gliomata (22), and cultured Xenopus
motoneurons (40), the morphogenic capacities of AChE were
limited to E6-AChE, translated from the alternative splicing E6-AChE
mRNA variant, 3' terminated with exon 6. Therefore, we examined E6-AChE
mRNA expression in human osteosarcoma cells, which are amenable to
controlled differentiation by 1,25-(OH)2D3 and
17 Thanatophoric dysplasia, like other severe forms of chondrodysplasia,
is frequently associated with point mutations in fibroblast growth
factor receptor 3 (13). Its diagnosis is based on
ultrasonography, X-ray findings, and pathologic changes in bone
structure. The lack of definitive therapy calls for studying the
molecular mechanisms which underlie the impaired chondrocyte
proliferation in affected embryos. Here, we report findings which
suggest involvement of AChE in osteo- and chondrogenesis, as well as
impairments of AChE gene expression in thanatophoric dysplasia.
Osteoblast cell culture.
Human osteosarcoma Saos-2 cells
were maintained in a fully humidified atmosphere at 37°C and 5%
CO2 in Ham's F-10 growth medium containing 10% (vol/vol)
fetal calf serum, 2 mM L-glutamine, 100 U of penicillin per
ml, 100 µg of streptomycin per ml, and 0.12% (wt/vol) bicarbonate
and were passaged once a week. 17 RT-PCR analyses.
Kinetic follow-up of reverse
transcription-PCR (RT-PCR) analyses was performed by using selective
primers for AChE mRNA, essentially as described previously
(28), with actin and glyceraldehyde 3-phosphate
dehydrogenase mRNA measurement (14) as a control. Plasmid
DNAs containing exons E2 to E6 and the pseudointron 14 region
(5) served as probes for blot hybridization. Densitometry calibration curves showing linearity and parallelism of intensity of
product with cycle number over the range encompassing the percent differences between treated and untreated cells were as previously described (21, 23).
Antisense oligonucleotide experiments.
2-O-methylated or
phosphorothioated 15- and 20-mer oligonucleotides, antisense
(designated by the prefix "AS") and inversely oriented sequences,
were targeted against the common sequence domain in human AChE mRNA and
were used as detailed elsewhere (15, 18, 29, 39), except for
the addition of a terminal 5'-hexa-ethylene glycol (HEG) group where
indicated. Oligonucleotides targeted against butyrylcholinesterase
(BChE) mRNA served as a control (18). Cell proliferation was
evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation
(19) or binding of methylene blue (33), both as
previously described.
In situ hybridization.
The following 5'-biotinylated,
2-O-methylated AChE cRNA probes complementary to 3' alternative human
AChE exons (5) were used: E6 (morphogenic form), (5402)
5'-CCGGGGGACGUCGGGGUGGGGUGGGGAUGGGCAGAGUCUGGGGCUCGUCU-3' (5352); E5 (hematopoietic form), (4457)
5'-AGGAAGAGGAGGAGAAGCUGGUGGAGGAGGAGGAGGGGCAGGGGGAGGCC-3' (4506); and I4 (readthrough form), (4397)
5'-CUAGGGGGAGAAGAGAGGGGUUACACUGGCGGGCUCCCACUC CCCUCCUC-3'
(4349). (Numbers denote nucleotide positions in the GenBank entry
[accession no. M55040].)
Immunohistochemistry, cytochemistry, and AChE catalytic activity
measurements.
For immunohistochemistry, cryocut sections were
incubated with human-specific anti-AChE monoclonal antibody 101-1 (primary antibody) (36) and biotinylated anti-mouse antibody
(secondary antibody) (Vectastain; Vector Laboratories, Burlingame,
Calif.); detection was carried out with 3,3'-diaminobenzidine and
urea-H2O2 as instructed by the manufacturer
(Sigma). Cytochemical staining for AChE activity on nonfixed cells
grown on glass slides and on cryocut sections, as well as determination
of hydrolysis rates of acetylthiocholine iodide in homogenates, was
performed essentially as detailed elsewhere (28) for the
noted time periods. Nuclear staining was done with
4',6-diamidino-2-phenylindole (DAPI).
Confocal laser scanning microscopy.
An MRC-1024 Bio-Rad
confocal microscope equipped with an inverted microscope and a 63×/1.4
oil immersion objective was used to scan the fast red precipitate used
for detection during in situ hybridization of Saos-2 cells. The fast
red was excited at 488 nm, and emission was measured with a 580df32
filter. A section was scanned every 0.54 µm, and a three-dimensional
projection was created from all sections.
AChE gene expression in cultured Saos-2 cells.
AChE mRNA
transcripts were first detected in cultured Saos-2 cells by
high-resolution in situ hybridization using alternative splicing-specific probes for the various AChE mRNA transcripts. An exon
6-derived probe revealed efficient production of the morphogenic E6-AChE mRNA, terminated with exon E6 (40). Smaller amounts of E5-AChE mRNA 3' terminated with exon E5, which encodes the erythrocyte AChE, and negligible levels of the readthrough I4-AChE mRNA
form including pseudointron I4 (22) were also detected (Fig.
1A). Similar to the situation in cells of
hematopoietic origin (21, 28), the major detectable
transcript was that encoding E6-AChE. Especially prolonged (72-h)
cytochemical staining of nonfixed Saos-2 cells revealed accumulation of
acetylthiocholine hydrolysis products that were suppressed by the
AChE-specific inhibitor BW 284C51 but resistant to the BChE-specific
inhibitor tetraisopropylpyrophosphoramide (iso-OMPA) (Fig. 1B). We
conclude that the AChE gene is expressed in Saos-2 cells to yield its
catalytically and morphogenically active E6-AChE form, with
considerably smaller amounts of its other variants.
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Human Osteogenesis Involves Differentiation-Dependent Increases
in the Morphogenically Active 3' Alternative Splicing Variant of
Acetylcholinesterase
ABSTRACT
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Abstract
Introduction
Materials and methods
Results
Discussion
References
-estradiol. In
differentiating osteosarcoma Saos-2 cells, both of these factors
enhanced transcription of the AChE mRNA variant 3' terminated with exon
6 (E6-AChE mRNA), which encodes the catalytically and morphogenically
active E6-AChE isoform. In contrast, antisense oligodeoxynucleotide
suppression of E6-AChE mRNA expression increased Saos-2 proliferation
in a dose- and sequence-dependent manner. The antisense mechanism of action was most likely mediated by mRNA destruction or translational arrest, as cytochemical staining revealed reduction in AChE gene expression. In vivo, we found that E6-AChE mRNA levels rose following midgestation in normally differentiating, postproliferative fetal chondrocytes but not in the osteogenically impaired chondrocytes of
dwarf fetuses with thanatophoric dysplasia. Taken together, these
findings suggest morphogenic involvement of E6-AChE in the proliferation-differentiation balance characteristic of human osteogenesis.
INTRODUCTION
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Abstract
Introduction
Materials and methods
Results
Discussion
References
-estradiol, interacts with high-affinity binding sites in normal human osteoblast-like and osteosarcoma cells
(16, 24). This suggests osteoblast mediation for the action
of estrogen on bone. The resultant skeletal tissue supports hematopoiesis through the function of bone marrow stromal cells, which
share a common progenitor with the osteogenic lineages. These complex
interrelationships predict the existence of common coordinator proteins
with control functions in the proliferation-differentiation balance
characteristic of both osteogenic and hematopoietic processes.
-estradiol, and tested the outcome of antisense AChE suppression
on cell proliferation. In parallel, we searched for E6-AChE mRNA in
vivo in developing chondrocytes from normal fetuses and from dwarf
bones of proliferation-impaired fetuses with thanatophoric dysplasia
(8). This inherited skeletal disorder is one of the most
severe forms of chondrodysplasia, causing dwarfism and abnormal body
proportions that lead to late prenatal or early postnatal death. While
data on its frequency are not yet available, chondrodysplastic
disorders are believed to occur with a frequency as high as 1:10,000.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References
-Estradiol and
1,25-(OH)2D3 (Sigma Chemical Co., St. Louis,
Mo.) dissolved in ethanol were diluted into growth medium (1:5,000
[vol/vol]) before use. For antisense manipulations, 20,000 cells/well, plated in 96-well flat-bottomed plates, were washed twice
with phosphate-buffered saline and maintained in RPMI 1640 medium
without phenol red (which exerts an estrogenic effect on osteoblasts
[17]) and with a serum substitute, Biogro-1
(Biological Industries Co., Beit-Haemek, Israel). Oligonucleotides were
added without carrier at the noted doses for 12 h of incubation at
37°C and 5% CO2, followed by two washes in
phosphate-buffered saline.
RESULTS
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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FIG. 1.
AChE gene expression in Saos-2 osteosarcoma cells. (A)
AChE mRNA alternative splicing variants. Presented are
three-dimensional projections created from confocal scanned sections of
Saos-2 cells subjected to in situ hybridization with 5'-biotinylated
AChE cRNA probes selective for the morphogenic E6-AChE mRNA variant
(E6), the hematopoietic E5-AChE mRNA variant encoding
glycophospholipid-anchored AChE (E5), and the readthrough form
including pseudointron I4 (I4). Detection was carried out by forming
alkaline phosphatase-streptavidin conjugates producing fast red
precipitates, which were excited at 488 nm. Note that E6 is the
predominant AChE mRNA transcript in Saos-2 cells. (B) Cytochemical
staining. Saos-2 cells were subjected to cytochemical staining of AChE
catalytic activity in the presence of 10 
5 M iso-OMPA
(ISO) or BW 284C51 (BW), selective inhibitors of BChE and AChE,
respectively. Nuclear staining was done with DAPI. Note the selective
depiction of brown precipitates of AChE but not BChE reaction
products.
AChE transcription in Saos-2 cells is up-regulated by
17-estradiol and 1,25-(OH)2D3.
Kinetic
follow-up of RT-PCR amplification (21, 23) using Saos-2 RNA
yielded a logarithmic increase in a 482-bp fragment derived from
E6-AChE mRNA, as expected, up to cycle 33. Calculations based on actin
mRNA levels from the same preparations revealed 106 E6-AChE
mRNA molecules per 200 ng of total Saos-2 RNA. For average yields of
20 ± 5 µg of total RNA from 3 × 106 Saos-2
cells, this indicates fewer than 50 AChE mRNA molecules per cell, at
the same order of magnitude as hematopoietic DAMI cells (75 molecules/cell [28]) but at much lower concentrations than brain cells (104 molecules/cell
[21]). Physiologically relevant concentrations of the
osteogenic agent 17-estradiol (109 M
[32]) increased E6-AChE mRNA levels in Saos-2 cells
3.3-fold within 24 h (Fig. 2A). A
more prominent effect was caused by
1,25-(OH)2D3, which increased E6-AChE mRNA
levels in a dose-dependent manner. Increases of 6.7- and 12.3-fold for
1012 and 107 M
1,25-(OH)2D3 (Fig. 2A), respectively,
demonstrated an association between AChE transcription and
1,25-(OH)2D3-induced osteogenesis.
|
Upstream AChE sequences include a plethora of binding sites for osteogenic factors. The MatInspector (34) and FindPatterns programs of the University of Wisconsin software package were used to search for consensus motifs known to bind transcription factors important for osteoblast differentiation and bone remodeling. The recently sequenced 30-kb region upstream from the human AChE gene (accession no. AF002993) was found to include five clusters of such sequences, bearing 100% identity to the core consensus motifs. Of these, the most distal are located ca. 18 and 17 kb upstream from the transcription start site (Fig. 2B), a domain demonstrated to be active in regulating AChE expression (37). The osteogenic binding motifs include vitamin D receptor binding elements (VDREs) arranged in pairs or, in one case, in a triplet as found in the vitamin D-regulated promoter of the c-fos gene (10). Several of these VDREs are positioned near CAAT boxes which may act synergistically with them, as was shown for factors of the CTF/NF-1 family (30). Other elements include half-palindromic sequences capable of activating transcription by binding the estrogen receptor (42), in line with the recently demonstrated glucocorticoid effect on AChE gene expression (7). Recognition motifs were also found for the vitamin D-regulated repressor HES-1 (31) and for the brain development and bone-remodeling factor Krox-20/24 (27). Six elements for the fetus-specific core binding factor A1 (Cbfa1), essential for osteoblast development and bone formation in the mouse (25) (three of which are depicted in Fig. 2A), are characteristic of bone-specific genes (e.g., osteocalcin and osteopontin genes). In contrast, no Cbfa1 motif was found in the human liver-specific promoters for aldolase B (accession no. D00175), liver-type phosphofructokinase (accession no. X80853), and serum albumin (accession no. M12523 and J04457). Together with our RT-PCR data, this finding suggests that the binding elements in the AChE upstream sequences do indeed reflect functionally significant osteogenic regulation of AChE gene expression.
Antisense suppression of AChE gene expression enhances proliferation of Saos-2 cells. Since osteogenic differentiation is often associated with arrest of proliferation, we tested the effect of loss of AChE function on Saos-2 proliferation. To this end, two different 2-O-methylated AChE-targeted antisense oligonucleotides (AS1 and AS3 [18]) were added to Saos-2 cultures, and cell numbers were measured after a 12-h incubation in the presence of BrdU (see Materials and Methods). Both AS1 and AS3, at doses of 0.2 to 200 nM, suppressed the cytochemical staining capacity of treated cells under conditions where an irrelevant AS-BChE oligomer (18) had no effect on AChE activity (Fig. 3A). Dose-dependent increases in cell proliferation up to 25% above the level for untreated cells, reflecting enhanced BrdU incorporation, were observed for both AS1 and AS3 but not for AS-BChE. A phosphorothioated AS-AChE oligonucleotide, targeted against the initiator AUG domain in the AChE gene (39), was also used. This oligomer, with and without a HEG protecting group, was also found to be effective in enhancing proliferation (25 and 40% increases in methylene blue binding above control levels for untreated cells, respectively). In contrast, inverse and sense oligonucleotides were both ineffective in these tests (data not shown). The differences in proliferation that were induced by the three AS-AChE oligomers are considered significant in osteoblast cultures (17), indicating sequence dependence of the observed effects and suggesting functional significance for osteoblastic AChE in inhibiting proliferation.
|
Developmental regulation of AChE mRNA expression in human fetal
chondrocytes.
Cytochemical staining of AChE activity disclosed no
catalytic activity of AChE in bones from aborted human fetuses at 18 weeks' gestation, even under prolonged incubation conditions in which developing osteoblastic cells were positive. Also, immunohistochemistry staining for AChE revealed an exceedingly weak signal (data not shown).
However, high-resolution in situ hybridization analyses demonstrated
AChE mRNA transcripts in the cytoplasm of developing perichondria and
mature chondrocytes within developing fetal axial bones. The three
alternative forms of AChE mRNA, 3' terminated with E5, E6, and I4, were
all detected by fast red staining in bone cells throughout gestational
weeks 9 to 25 (Fig. 4A), whereas control
sections incubated with no probe revealed no staining. Image analysis
of E5-AChE mRNA expression patterns showed relatively constant levels
during the entire gestational period examined (Fig. 4B). In contrast,
the level of E6-AChE mRNA labeling, which was lower than that of
E5-AChE mRNA during the early gestational period, increased
considerably (ANOVA, P 
0.009) during advanced pregnancy, becoming significantly higher than that of E5-AChE mRNA by
25 weeks. In line with the analysis in Saos-2 cells, I4-AChE mRNA
labeling was minimal in the bone tissues examined. This demonstrated developmentally regulated splicing choices for AChE mRNA in
chondrocytes in vivo, with the morphogenic E6-AChE mRNA isoform
appearing relatively late in fetal development.
|
Attenuated E6-AChE mRNA production in thanatophoric bone
dysplasia.
To study the association between normal osteogenic
differentiation and AChE expression, we tested AChE mRNA labeling in
femur bones from two unrelated fetuses with thanatophoric bone
dysplasia. E5-AChE mRNA transcripts displayed expression levels much
lower than those in normal bone, and E6-AChE mRNA was drastically
suppressed (ANOVA, P 0.05), demonstrating
consistently deficient E4-E6 splicing in addition to attenuated AChE
transcription (Fig. 4). It is noteworthy that the hematopoietic organs
expressed AChE mRNA with similar efficiencies in normal fetuses and
fetuses with thanatophoric bone dysplasia (data not shown), reflecting
bone-specific attenuation of AChE expression associated with bone dysplasia.
0.01), for
both E5- and E6-AChE mRNAs. This difference was directly associated
with the differentiation-related decrease in chondrocyte density
(ANOVA, P 0.04). In bones from fetuses with
thanatophoric bone dysplasia, considerably lower levels were observed
for E5-AChE mRNA expression (ANOVA, P 0.01), although with parallel patterns of differentiation-related increases (Fig. 4). In contrast, E6-AChE mRNA remained absent in both zones of
the dysplastic bone (Fig. 4 and 5). This absence of E6-AChE mRNA
coincided with deficient chondrocyte condensation in the dysplastic
bone (ANOVA, P 0.04). Taken together, these findings suggest a suppressive role for the E6-AChE protein in chondrocyte proliferation.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
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By combining in vitro and in vivo approaches, we have demonstrated
that the human AChE gene is expressed in osteoblasts and chondrocytes
in a manner dependent both on their state of proliferation and
differentiation and on the presence of binding sites for osteogenic factors such as 17-estradiol and
1,25-(OH)2D3, which were found in the extended
AChE promoter. In cultured tumor osteoblasts, E6-AChE mRNA expression
increases with differentiation, whereas its suppression increases the
proliferation rate. In the already committed, differentiated fetal
chondrocytes, stable in vivo expression of E5-AChE mRNA persists
through most of the gestation period. However, E6-AChE mRNA levels
increase with differentiation, in parallel with decreases in cell
density and after gestational week 20. In bones of fetuses with
thanatophoric dysplasia, in which chondrocyte condensation is impaired,
E5-AChE mRNA expression is drastically down-regulated. Its levels
decrease further together with cell density and in a manner parallel to
that observed in normal fetal bones. In contrast, E6-AChE mRNA levels
did not increase in the dysplastic bones, suggesting that a threshold
stage of development is required for such increases. The weak
immunodetection and failure to observe catalytically active AChE in in
vivo chondrocytes may be due to the low-level expression of the AChE
gene in these cells, virtually at the limit of detection, as also seen
in Saos-2 cells. That this protein nevertheless exerts a morphogenic
effect further supports its in vivo function as a signaling factor, a role which could be fulfilled by a very small number of molecules.
Several arguments suggest that the osteogenic expression of E6-AChE
mRNA is functionally significant: (i) the abundance of binding motifs
for osteogenic factors in the extended AChE promoter; (ii) the
increased expression under the influence of 17-estradiol and
1,25-(OH)2D3; (iii) the direct correlation
between E6-AChE mRNA expression and chondrocyte differentiation, itself
inversely correlated with chondrogenic proliferation; and (iv) the
proliferation-enhancing effect of antisense AChE suppression, which
corroborates the inverse correlation with cell density. The deficient
AChE mRNA expression in dysplastic bones demonstrates early induction
during osteogenesis as well as dependence on those osteogenic agents
missing in bone dysplasia and thus impairing chondrocyte condensation.
The paucity of E6-AChE mRNA in dysplastic bones may, therefore, reflect
deficiencies in transcription factors, splicing proteins such as SF2
and ASF, which were found to decrease in association with diminished
splicing of E6-AChE mRNA in human megakaryocytes (28), or
cell surface signals essential for E6-AChE production. Recently
accumulated data on the cell-cell interaction capacities of AChE and
its catalytically inactive homologs (4, 11, 12, 20)
tentatively provide a mechanism for the morphogenic activities of
cholinesterasic domains. Finally, the apparently normal hematopoietic
expression of AChE in fetuses with thanatophoric dysplasia points to a
clear distinction between the osteogenic and hematopoietic control
processes governing AChE production.
Splicing choices of AChE mRNA transcripts were notably different in cultured osteoblasts, where E6-AChE mRNA predominated, from those in in vivo chondrocytes, where E6-AChE mRNA levels increased only late in gestation, following a long period when E5-AChE mRNA transcripts predominated. This difference may be related to the different growth conditions, cell type properties, or differentiation states involved. In addition, the predominance of E6-AChE may be attributed to its distinct morphogenic properties, so that E6 splicing deficiencies may reflect insufficient development of the dysplastic chondrocytes which are unprepared for the next phase in the cell-cell interaction activities particular to this AChE variant.
Antisense suppression of AChE production enhanced osteoblast proliferation in a dose- and sequence-dependent manner. The possibility of triple helix involvement had been excluded for all of the oligonucleotides used (15, 18). The suppressed cytochemical labeling points to mRNA destruction and/or interference with translation as the most likely mechanism(s) for functioning of these antisense oligonucleotides, which further suggests that interference with E6-AChE production impairs the proliferation-differentiation balance in osteoblasts. This argument is strengthened by the inverse in vivo relationship between AChE mRNA levels and chondrocyte density. The function implied by these studies for the AChE protein is, therefore, an association with the cell-cell signaling that arrests proliferation in committed, differentiating bone cells.
Having a developmental osteogenic function may imply involvement of
AChE in the replacement of osteoblasts for maintenance of both fetal
and adult bones. In this respect, it would be of interest to
investigate the effect of antisense AChE agents on osteogenesis in
fetuses of chondrodysplastic animal models. The association of bone
defects (e.g., osteoporosis) with Alzheimer's disease, in which
peripheral AChE levels are reduced (2), further requires an
examination of AChE mRNA levels following 17-estradiol and
1,25-(OH)2D3 treatment in vivo. Finally,
17-estradiol and 1,25-(OH)2D3 treatments
should be examined for secondary effects on hematopoiesis through the
predicted increase in bone marrow-supportive stromal cells. The
expression of E6-AChE in bone therefore has both basic and applied
significance, due to the morphogenic capacities of this intriguing protein.
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ACKNOWLEDGMENTS |
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We are grateful to A. Cohen (Milford, Mass.) for a gift of 2-O-methyl antisense oligomers, to Letizia Shraiber for assistance with pathological analysis, to Avi Orr-Urteger and Joseph Weissman for genetic and scientific counseling, and to N. Melamed-Book for confocal imaging.
This research was supported by grants to H.S. from the German-Israeli Fund (grant I-0512-206.01/96) and Ester/Neuroscience, Ltd. D.G. was the recipient of a Sourasky Medical Center postdoctoral fellowship.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. Phone: 972-2-6585109. Fax: 972-2-6520258. E-mail: Soreq{at}shum.huji.ac.il.
Present address: Department of Human and Molecular Genetics, Baylor
College of Medicine, Houston, TX 77030.
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REFERENCES |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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| 1. | Alber, R., O. Sporns, T. Weikert, E. Willbold, and P. G. Layer. 1994. Cholinesterases and peanut agglutinin binding related to cell proliferation and axonal growth in embryonic chick limbs. Anat. Embryol. (Berlin) 190:429-438[Medline]. |
| 2. | Appleyard, M. E., and B. McDonald. 1991. Reduced adrenal gland acetylcholinesterase activity in Alzheimer's disease. Lancet 338:1085-1086[Medline]. (Letter.) |
| 3. | Aubin, J. E., F. Liu, L. Malaval, and K. Gupta. 1995. Osteoblast and chondroblast differentiation. Bone 17:77S-83S[Medline]. |
| 4. | Auld, V. J., R. D. Fetter, K. Broadie, and C. S. Goodman. 1995. Gliotactin, a novel transmembrane protein on peripheral glia, is required to form the blood-nerve barrier in Drosophila. Cell 81:757-767[Medline]. |
| 5. |
Ben Aziz-Aloya, R.,
S. Seidman,
R. Timberg,
M. Sternfeld,
H. Zakut, and H. Soreq.
1993.
Expression of a human acetylcholinesterase promoter-reporter construct in developing neuromuscular junctions of Xenopus embryos.
Proc. Natl. Acad. Sci. USA
90:2471-2475 |
| 6. | Boskey, A. L. 1981. Current concepts of the physiology and biochemistry of calcification. Clin. Orthop. 157:225-257. |
| 7. | Brank, M., K. Zajc-Kreft, S. Kreft, R. Komel, and Z. Grubic. 1998. Biogenesis of acetylcholinesterase is impaired, although its mRNA level remains normal, in the glucocorticoid-treated rat skeletal muscle. Eur. J. Biochem. 251:374-381[Medline]. |
| 8. | Brenner, R. F., A. Nerlich, R. Terinde, and P. Bartmann. 1996. In vitro studies on clonal growth of chondrocytes in thanatophoric dysplasia. Am. J. Med. Genet. 63:401-405[Medline]. |
| 9. | Bruder, S. P., D. J. Fink, and A. I. Caplan. 1994. Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J. Cell. Biochem. 56:283-294[Medline]. |
| 10. |
Candeliere, G. A.,
P. W. Jurutka,
M. R. Haussler, and R. St-Arnaud.
1996.
A composite element binding the vitamin D receptor, retinoid X receptor  , and a member of the CTF/NF-1 family of transcription factors mediates the vitamin D responsiveness of the c-fos promoter.
Mol. Cell. Biol.
16:584-592[Abstract].
|
| 11. | Darboux, I., Y. Barthalay, M. Piovant, and R. Hipeau-Jacquotte. 1996. The structure-function relationships in Drosophila neurotactin show that cholinesterasic domains may have adhesive properties. EMBO J. 15:4835-4843[Medline]. |
| 12. | de la Escalera, S., E. O. Bockamp, F. Moya, M. Piovant, and F. Jimenez. 1990. Characterization and gene cloning of neurotactin, a Drosophila transmembrane protein related to cholinesterases. EMBO J. 9:3593-3601[Medline]. |
| 13. |
Delezoide, A. L.,
C. Lasselin-Benoist,
L. Legeai-Mallet,
P. Brice,
V. Senee,
A. Yayon,
A. Munnich,
M. Vekemans, and J. Bonaventure.
1997.
Abnormal FGFR 3 expression in cartilage of thanatophoric dysplasia fetuses.
Hum. Mol. Genet.
6:1899-1906 |
| 14. | Dukas, K., P. Sarfati, N. Vaysse, and L. Pradayrol. 1993. Quantitation of changes in the expression of multiple genes by simultaneous polymerase chain reaction. Anal. Biochem. 215:66-72[Medline]. |
| 15. | Ehrlich, G., D. Patinkin, D. Ginzberg, H. Zakut, F. Eckstein, and H. Soreq. 1994. Use of partially phosphorothioated "antisense" oligonucleotides for sequence-dependent modulation of hematopoiesis in culture. Antisense Res. Dev. 4:173-183[Medline]. |
| 16. |
Eriksen, E. F.,
D. S. Colvard,
N. J. Berg,
M. L. Graham,
K. G. Mann,
T. C. Spelsberg, and B. L. Riggs.
1988.
Evidence of estrogen receptors in normal human osteoblast-like cells.
Science
241:84-86 |
| 17. |
Ernst, M.,
C. Schmid, and E. R. Froesch.
1988.
Enhanced osteoblast proliferation and collagen gene expression by estradiol.
Proc. Natl. Acad. Sci. USA
85:2307-2310 |
| 18. | Grifman, M., and H. Soreq. 1997. Differentiation intensifies the susceptibility of pheochromocytoma cells to antisense oligodeoxynucleotide-dependent suppression of acetylcholinesterase activity. Antisense Nucleic Acid Drug Dev. 7:351-359[Medline]. |
| 19. | Huong, P. L., A. H. Kolk, T. A. Eggelte, C. P. Verstijnen, H. Gilis, and J. T. Hendriks. 1991. Measurement of antigen specific lymphocyte proliferation using 5-bromo-deoxyuridine incorporation. An easy and low cost alternative to radioactive thymidine incorporation. J. Immunol. Methods 140:243-248[Medline]. |
| 20. |
Ichtchenko, K.,
T. Nguyen, and T. C. Sudhof.
1996.
Structures, alternative splicing, and neurexin binding of multiple neuroligins.
J. Biol. Chem.
271:2676-2682 |
| 21. | Karpel, R., R. Ben Aziz-Aloya, M. Sternfeld, G. Ehrlich, D. Ginzberg, P. Tarroni, F. Clementi, H. Zakut, and H. Soreq. 1994. Expression of three alternative acetylcholinesterase messenger RNAs in human tumor cell lines of different tissue origins. Exp. Cell Res. 210:268-277[Medline]. |
| 22. | Karpel, R., M. Sternfeld, D. Ginzberg, E. Guhl, A. Graessmann, and H. Soreq. 1996. Overexpression of alternative human acetylcholinesterase forms modulates process extensions in cultured glioma cells. J. Neurochem. 66:114-123[Medline]. |
| 23. | Kaufer, D., A. Friedman, S. Seidman, and H. Soreq. 1998. Acute stress facilitates long-lasting changes in cholinergic gene expression. Nature 393:373-377[Medline]. |
| 24. |
Komm, B. S.,
C. M. Terpening,
D. J. Benz,
K. A. Graeme,
A. Gallegos,
M. Kore,
G. L. Greene,
B. W. O'Malley, and M. R. Hausler.
1988.
Estrogen binding, receptor mRNA, and biologic response in osteoblast-like osteosarcoma cells.
Science
241:81-84 |
| 25. | Komori, T., H. Yagi, S. Nomura, A. Yamaguchi, K. Sasaki, K. Deguchi, Y. Shimizu, R. T. Bronson, Y. H. Gao, M. Inada, M. Sato, R. Okamoto, Y. Kitamura, S. Yoshiki, and T. Kishimoto. 1997. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturation arrest of osteoblasts. Cell 89:755-764[Medline]. |
| 26. | Layer, P. G., and E. Willbold. 1995. Novel functions of cholinesterases in development, physiology and disease. Prog. Histochem. Cytochem. 29:1-94[Medline]. |
| 27. |
Lemaire, P.,
C. Vesque,
J. Schmitt,
H. Stunnenberg,
R. Frank, and P. Charnay.
1990.
The serum-inducible mouse gene Krox-24 encodes a sequence-specific transcriptional activator.
Mol. Cell. Biol.
10:3456-3467 |
| 28. |
Lev-Lehman, E.,
V. Deutsch,
A. Eldor, and H. Soreq.
1997.
Immature human megakaryocytes produce nuclear-associated acetylcholinesterase.
Blood
89:3644-3653 |
| 29. | Lev-Lehman, E., D. Ginzberg, G. Hornreich, G. Ehrlich, A. Meshorer, F. Eckstein, H. Soreq, and H. Zakut. 1994. Antisense inhibition of acetylcholinesterase gene expression causes transient hematopoietic alterations in vivo. Gene Ther. 1:127-135[Medline]. |
| 30. |
Liu, M., and L. P. Freedman.
1994.
Transcriptional synergism between the vitamin D3 receptor and other nonreceptor transcription factors.
Mol. Endocrinol.
8:1593-1604 |
| 31. | Matsue, M., R. Kageyama, D. T. Denhardt, and M. Noda. 1997. Helix-loop-helix-type transcription factor (HES-1) is expressed in osteoblastic cells, suppressed by 1,25(OH)2 vitamin D3, and modulates 1,25(OH)2 vitamin D3 enhancement of osteopontin gene expression. Bone 20:329-334[Medline]. |
| 32. | Moore, J. W., G. M. G. Clark, O. Takatani, Y. Wakabayashi, J. L. Hayward, and R. D. Bulbrook. 1983. Distribution of 17 beta-estradiol in the sera of normal British and Japanese women. J. Natl. Cancer Inst. 71:749-755. |
| 33. |
Oliver, M. H.,
N. K. Harrison,
J. E. Bishop,
P. J. Cole, and G. J. Laurent.
1989.
A rapid and convenient assay for counting cells cultured in microwell plates: application for assessment of growth factors.
J. Cell Sci.
92:513-518 |
| 34. |
Quandt, K.,
K. Frech,
H. Karas,
E. Wingender, and T. Werner.
1995.
MatInd and MatInspector new fast and versatile tools for detection of consensus matches in nucleotide sequence data.
Nucleic Acids Res.
23:4878-4884 |
| 35. | Seidman, S., M. Sternfeld, R. Ben Aziz-Aloya, R. Timberg, D. Kaufer-Nachum, and H. Soreq. 1995. Synaptic and epidermal accumulations of human acetylcholinesterase are encoded by alternative 3'-terminal exons. Mol. Cell. Biol. 15:2993-3002[Abstract]. |
| 36. | Seidman, S., R. Ben Aziz-Aloya, R. Timberg, Y. Loewenstein, B. Velan, A. Shafferman, J. Liao, B. Norgaard-Pedersen, U. Brodbeck, and H. Soreq. 1994. Overexpressed monomeric human acetylcholinesterase induces subtle ultrastructural modifications in developing neuromuscular junctions of Xenopus laevis embryos. J. Neurochem. 62:1670-1681[Medline]. |
| 37. | Shapira, M., M. Korner, L. Bosgraaf, I. Tur-Kaspa, and H. Soreq. The human ACHE locus includes a polymorphic enhancer domain 17 kb upstream from the transcription start site. In D. M. Quinn, B. P. Doctor, and P. Taylor (ed.), Cholinesterases and related proteins, in press. Plenum Press, New York, N.Y. |
| 38. | Sillence, D. O., W. A. Horton, and D. L. Rimoin. 1979. Morphologic studies in skeletal dysplasias. Am. J. Pathol. 96:813-859[Abstract]. |
| 39. |
Soreq, H.,
D. Patinkin,
E. Lev-Lehman,
M. Grifman,
D. Ginzberg,
F. Eckstein, and H. Zakut.
1994.
Antisense oligonucleotide inhibition of acetylcholinesterase gene expression induces progenitor cell expansion and suppresses hematopoietic apoptosis ex vivo.
Proc. Natl. Acad. Sci. USA
91:7907-7911 |
| 40. |
Sternfeld, M.,
G. Ming,
H. Song,
K. Sela,
R. Timberg,
M. Poo, and H. Soreq.
1998.
Acetylcholinesterase enhances neurite growth and synapse development through alternative contributions of its hydrolytic capacity, core protein, and variable C termini.
J. Neurosci.
18:1240-1249 |
| 41. | Sylvia, V. L., Z. Schwartz, L. Schuman, R. T. Morgan, S. Mackey, R. Gomez, and B. D. Boyan. 1993. Maturation-dependent regulation of protein kinase C activity by vitamin D3 metabolites in chondrocyte culture. J. Cell. Physiol. 157:271-278[Medline]. |
| 42. | Tora, L., M. P. Gaub, S. Madar, A. Dierich, M. Bellard, and P. Chambon. 1988. Cell-specific activity of a GGTCA half-palindromic oestrogen-responsive element in the chicken ovalbumin gene promoter. EMBO J. 7:3771-3778[Medline]. |
| 43. | Umezu, Y., N. Nagata, Y. Doi, H. Furukawa, T. Sagara, T. Hayashida, H. Ogata, and S. Fujimoto. 1993. Cytochemical and immunocytochemical demonstration of acetylcholinesterase of the prenatal rat lower limb. Arch. Histol. Cytol. 56:217-224[Medline]. |
Molecular and Cellular Biology, January 1999, p. 788-795, Vol. 19, No. 1
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