From the Department of Biochemistry, Biotechnology Research Institute, and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China
Received for publication, November 30, 2000, and in revised form, February 8, 2001
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ABSTRACT |
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Retinoic acid (RA), a derivative of vitamin A, is
essential for the normal patterning and neurogenesis during
development. RA treatment induces growth arrest and terminal
differentiation of a human embryonal carcinoma cell line (NT2) into
postmitotic central nervous system neurons. Using RNA fingerprinting by
arbitrarily primed polymerase chain reaction, we identified a novel
serine/threonine-rich protein, RA-regulated nuclear
matrix-associated protein (Ramp), that was
down-regulated during the RA-induced differentiation of NT2 cells.
Prominent mRNA expression of ramp could be detected in
adult placenta and testis as well as in all human fetal tissues
examined. The genomic clone of ramp has been mapped to the
telomere of chromosome arm 1q, corresponding to band 1q32.1-32.2.
Associated with the nuclear matrix of NT2 cells, Ramp translocates from
the interphase nucleus to the metaphase cytoplasm during mitosis.
During the late stage of cytokinesis, Ramp concentrates at the midzone
of the dividing daughter cells. The transcript expression of
ramp is closely correlated with the cell proliferation rate
of NT2 cells. Moreover, overexpression of Ramp induces a transient
increase in the proliferation rate of NT2 cells. Taken together, our
data suggest that Ramp plays a role in the proliferation of the human
embryonal carcinoma cells.
Retinoic acid (RA),1 a
derivative of vitamin A, serves as an important soluble factor that
mediates the differentiation of both the neuronal and hematopoietic
precursor cells. As a natural morphogen, RA specifies the axial
patterning during the embryonic development and affects neurogenesis
(1, 2). RA is a powerful differentiating agent and induces the
differentiation of many cell types such as epithelial cells and
mesenchyme cells (3, 4). It also induces the differentiation of cancer
cells such as teratocarcinoma and leukemia (5) and many
immortalized cell lines. Thus, RA has been used as an effective
therapeutic agent for the treatment of acute promyelocytic leukemia
(6).
The human embryonal carcinoma cell line, NTera2 cl. D1 (NT2), has been
extensively used as a model system to study growth and differentiation
as well as cancer therapy. RA treatment induces growth arrest and
terminal differentiation of NT2 cells into postmitotic central nervous
system neurons (7, 8). The RA-induced neuronal differentiation of NT2
cells is concomitant with the up-regulation of the homeotic genes, such
as Hox 2.1 and Hox 2.2. On the contrary, the expression of growth
factors, including transforming growth factor- We have employed the RNA fingerprinting by arbitrarily primed PCR
to identify candidate genes that are differentially regulated during
the RA-induced neuronal differentiation of the NT2 cells (20). Among
the candidate genes identified, clone 8.31 encoded a novel gene that
was down-regulated during the RA-induced neuronal differentiation of
NT2 cells. Here we report the cloning and characterization of this
novel gene, designated RA-regulated nuclear
matrix associated protein (Ramp). Ramp is a
serine/threonine-rich protein that is associated with the nuclear
matrix protein of the NT2 cells, and translocates from the nucleus to
the cytoplasm during mitosis and cytokinesis. Our data suggest that
Ramp plays a role in the cell proliferation of these human embryonal
carcinoma cells.
Cloning of Full-length cDNA of Ramp--
Full-length
cDNA of clone 8.31 (ramp) was obtained by screening an
expression cDNA library prepared from undifferentiated NT2 cells
(Stratagene) using the partial 8.31 cDNA fragment obtained by RNA
fingerprinting by arbitrarily primed PCR as probes. The cDNA
sequence encoding ramp has been submitted to GenBankTM
(accession number AF345896). Radioactive cDNA probes were prepared
using Megaprime DNA labeling system (Amersham Pharmacia Biotech) and hybridization was performed at 60 °C. Single phages were obtained and transformed into XLOLR bacterial cells (Stratagene) and the cDNA fragment was cloned into pBK-CMV mammalian expression vector by in vivo excision.
Cell Culture and Cell Proliferation Assays--
NT2 cells were
cultured as previously described (21). Cells were maintained in
Opti-MEM I reduced-serum medium (Life Technologies, Inc.) supplemented
with 5% fetal bovine serum (fetal bovine serum, Life Technologies,
Inc.). NT2 cells were differentiated with 10 µM
all-trans RA (t-RA; Sigma Chemical Co.) in
Dulbecco's modified Eagle's medium (high glucose formulation)
supplemented with 10% fetal bovine serum. Cell synchronization was
performed by treating NT2 cells with nocodazole (1 µM)
for 12 h, which arrested the cells at G2-M.
Transfection of NT2 cells was performed using FuGENE reagent (Roche
Molecular Biochemicals) in 96-well plates. Expression vectors (0.15 µg) plus 0.05 µg of the reference plasmid pCMV- Promoter Constructs and Alkaline Phosphatase Assays--
Genomic
DNA fragment (~2396 bp) containing the putative ramp
promoter and the first exon (2304-2354 bp) of ramp cDNA
(data not shown). The genomic DNA fragment was then fused to the 5'-end of the promoterless reporter secretory form of human placental alkaline
phosphatase (SEAP, CLONTECH). Deletion
mutant (Ramp RNA Preparation, RT-PCR, and Northern Blot Analysis--
Total
RNA was prepared using TRIzol reagent (Life Technologies, Inc.) or as
previously described (24). Northern blot analysis was performed as
previously described using ramp-specific cDNA probes at
60 °C (25).
Coupled in Vitro Transcription and Translation--
Two
micrograms of linearized plasmids were used for each coupled in
vitro transcription/translation reaction using the TnT-coupled reticulocyte lysate system (Promega). Antisense ramp
cDNA construct in the same expression vector was used as negative
control; luciferase gene was employed as positive control.
Chromosomal Localization of Ramp by FISH--
Genomic
DNA-encoding ramp was labeled with digoxigenin dUTP by nick
translation and was hybridized to normal metaphase chromosomes derived
from PHA-stimulated peripheral blood lymphocytes. After incubation with
fluorescein-conjugated anti-digoxigenin antibodies, the cells were
counterstained with DAPI. The location of ramp was labeled
in green (indicated by white arrows). A
biotin-labeled probe, which is specific for the heterochromatic region
of chromosome 1, was co-hybridized with the genomic ramp
clone. Chromosome 1-specific probes were detected by Texas avidin and
was labeled in red (indicated by gray arrows).
Antibodies and Immunofluorescent Analysis--
Cells were fixed
with 4% paraformaldehyde (Fluka) in phosphate-buffered saline (PBS)
and the immunofluorescent staining was performed. After blocking with
4% normal serum (Zymed Laboratories Inc.) in PBS
containing 0.25% Triton-X (Roche Molecular Biochemical), and 2%
bovine serum albumin (Sigma Chemical Co.), cells were incubated overnight at 4 °C with primary antibodies diluted in antibody diluent (4% normal serum, 0.25% Triton-X, and 2% bovine serum albumin). Incubation with secondary antibodies was performed at room
temperature for at least 2 h. Cells were visualized under a Zeiss
Axiophot fluorescence microscope. Polyclonal antibodies were generated
against peptides designed from the N and C termini of the translated
Ramp amino acid sequence. Monoclonal antibodies detecting Nuclear Matrix Preparation--
High salt isolation of nuclear
matrix was prepared according to He et al. (26). NT2 cells
were washed with ice-cold PBS and then extracted with cytoskeletal
(CSK) buffer (10 mM PIPES, pH 6.8, 100 mM NaCl,
300 mM sucrose, 3 mM MgCl2, 1 mM EDTA, protease inhibitors, 1 mM
phenylmethylsulfonyl fluoride, and 0.5% Triton-X) for 10 min at
4 °C. The cytoskeletal fraction was collected by centrifugation, and
the chromatin was solubilized by digestion with 1 mg/ml DNase I in CSK
buffer for 15 min at 37 °C. Ammonium sulfate was then added to a
final concentration of 0.25 M, and samples were collected
by centrifugation. High salt wash (2 M NaCl in CSK buffer)
was performed for 5 min at 4 °C. The final pellet (core nuclear
matrix) was resuspended in 8 M urea.
Western Blot Analysis--
Western blot analysis was performed
as previously described (13). Cells were washed twice with ice-cold PBS
followed by a wash with ice-cold PBS supplemented with 1 mM
sodium orthovanadate and protease inhibitor mixture (Complete, Roche
Molecular Biochemicals). Cells were then lysed with lysis buffer (50 mM Tris-Cl at pH 8, 150 mM NaCl, 1% Triton-X,
1× aprotinin; Sigma, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 1× Complete) at 4 °C for
15 min. Lysates were collected and centrifuged to remove cell debris.
Protein assays were performed with the Bio-Rad Protein Assay kit based
on the Bradford dye-binding procedure (Bio-Rad). Protein samples
(typically 40 µg) were separated by SDS-polyacrylamide gel
electrophoresis in 10% resolving gel using a Hoeffer minigel apparatus. Proteins were then electrotransferred to nitrocellulose membrane (Amersham Pharmacia Biotech). After blocking at room temperature for 1 h using TBS-Tween containing 5% non-fat milk, membranes were incubated overnight at 4 °C with primary antibodies in TBS-Tween containing 5% non-fat milk. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2500, Zymed Laboratories Inc.) for 1 h at room
temperature. After three washes with TBS-Tween, immunoreactive bands
were detected using an ECL kit (Amersham Pharmacia Biotech) and
visualized by x-ray film (Fuji).
Cloning of the Full-length Coding Sequence of Ramp--
The
cDNA encoding full-length 8.31 (ramp) was obtained by
screening a cDNA library prepared from the undifferentiated NT2 cells. Double-stranded sequencing by T7 DNA polymerase revealed that
the cDNA (~2831 bp) is novel in its gene identity. The full DNA
sequence and its deduced amino acid sequence are shown in Fig.
1A. The cDNA contains a
single open reading frame corresponding to a protein of 730 amino acids
(~79.4 kDa). The ATG codon preceded by a consensus Kozak sequence is
found at position 124, and the TAG codon is found at position 2314. Hydropathy analysis of the predicted amino acid sequence reveals that
the protein contains a high percentage of hydrophilic residues (Fig.
1B). Although a potential transmembrane domain cannot be
identified within the amino acid sequence, a putative nuclear
localization signal is located at amino acid positions 197-203. The
sequence is rich in serines and threonines (20.25% of all amino acids)
and in basic residues (13.04% arginines, lysines, and histidines).
There are five potential glycosylation sites at residues 190, 248, 289, 299, and 316, and three putative cAMP- and cGMP-dependent
kinase phosphorylation sites at residues 413-416, 482-485, and
688-691. Another interesting feature is the presence of a proline-rich motif that shares high homology with the SH3-binding consensus, PXXP, at residues 637-641. Moreover, there is a leucine
zipper located at 577 and two WD repeats at 113 and 231 of the cloned ramp cDNA.
To confirm that the full-length cDNA encoded a functional protein,
coupled in vitro transcription and translation was performed to estimate the molecular weight of the encoded protein. It was demonstrated that the cloned full-length ramp cDNA could
be translated into a protein of ~85 kDa (Fig. 1C). In
addition, a chimeric enhanced green fluorescence protein (EGFP)-tagged
Ramp protein (E-Ramp) was constructed by fusing the EGFP-encoding
cDNA to the N terminus of the full-length ramp cDNA.
The chimeric cDNA construct was transiently expressed in CHO cells
and total proteins were collected. Immunoblotting using the anti-Ramp
polyclonal antibodies revealed the expression of the EGFP-tagged Ramp
proteins (Fig. 1D). Two immunoreactive bands (~110 and
~180 kDa) were observed, the identity of which was confirmed by
immunoblotting with anti-EGFP antibodies. Because the Ramp protein
contains several putative N-glycosylation sites, it is
possible that the ~180-kDa immunoreactive represents the glycosylated protein.
Ramp Is Expressed in the Placenta and Testis--
To obtain clues
on the potential function(s) of ramp, we have examined the
expression profile of ramp in both adult and fetal human
tissues. Among the tissues examined, including brain, colon, heart,
kidney, liver, lung, skeletal muscle, placenta, small intestine, spleen, stomach, and testis, two transcripts (~5 and ~3 kb) of ramp were predominantly expressed in the placenta and testis
(Fig. 2A). Interestingly,
skeletal muscles also expressed low level of ramp (upon long
exposure of the autoradiogram).
The expression of ramp was observed in all the human fetal
tissues examined, which included brain, lung, liver, and kidney. All
fetal tissues expressed the ~5- and ~3-kb ramp
transcripts. Two extra transcripts of ~4 and ~2 kb were observed in
the messenger RNA prepared from the fetal lung (Fig. 2B).
However, similar prominent expression of ramp was not
observed in the corresponding adult tissues.
Based on abundant level of expression of ramp in fetal
liver, dot blot analysis was performed to examine the mRNA level in hematopoietic tissues (Fig. 2C). Although ramp
was detected in all hematopoietic tissues examined, mRNA level was
highest in thymus and bone marrow. A low level of
ramp transcript was detected in the spleen and lymph node,
and only a barely detectable level was observed in the peripheral leukocytes.
Chromosomal Localization of Ramp by Fluorescence in Situ
Hybridization--
DNA from a genomic clone of ramp was
labeled with digoxigenin dUTP by nick translation. Labeled probe was
combined with sheared human DNA and hybridized to normal metaphase
chromosomes derived from PHA-stimulated peripheral blood lymphocytes.
The initial experiment resulted in the specific labeling of the long
arm of a group A chromosome, which was believed to be chromosome 1 on the basis of size, morphology, and banding pattern (Fig.
3A). Thus, a biotin-labeled
probe, which was specific for the heterochromatic region of chromosome
1, was co-hybridized with the genomic clone of ramp. The
specific labeling of genomic fragment containing ramp was
labeled green (indicated by white arrows), and the
heterochromatin of the chromosome 1 labeled red (indicated by
gray arrows) (Fig. 3B). A total of 80 metaphase cells were analyzed with 76 exhibiting specific labeling.
Measurements of 10 specifically labeled chromosomes 1 demonstrated that
the genomic clone of ramp is located at a position that is
62% of the distance from the heterochromatic-euchromatic boundary of
the telomere of chromosome arm 1q, an area that corresponds to band
1q32.1-32.2. The result is schematized in Fig. 3C.
RA Treatment Down-regulated the Expression of Ramp in NT2
Cells--
To examine the regulation of ramp expression by
RA, the full-length ramp cDNA was used as a probe to
examine its change in expression profile upon treatment with RA for
0-28 days (Fig. 4A). Two
transcripts (~4.5 and ~3.5 kb) of ramp were detected in
NT2 cells. The transcript expression of ramp was transiently up-regulated after 1 day of RA treatment, followed by a decrease, reaching a low level of expression at day 28. Moreover, total proteins
were obtained from NT2 cells treated with t-RA for 6 or 12 days. An immunoprecipitation assay was performed using anti-Ramp (N
terminus) antibodies. The resulted immunoprecipitates were electrophoresed and immunoblotted with anti-Ramp (C terminus) antibodies. As depicted in Fig. 4B, undifferentiated NT2
cells expressed abundant level of Ramp proteins, which was repressed after 6 days of RA treatment. Interestingly, additional immunoreactive bands (~175 and ~180 kDa) were also detected by the anti-Ramp antibodies employed in the study.
Ramp Is Localized to the Nucleus and Is a Nuclear Matrix-associated
Protein--
To define the subcellular localization of Ramp protein,
we transiently transfected the ramp expression vector into
CHO cells. Transfected CHO cells were immunostained with anti-Ramp
polyclonal antibodies and counterstained with DAPI. It was observed
that the Ramp protein was localized to the nucleus (Fig.
5A). A chimeric Ramp was
constructed by fusing a 6X histidine tail to the C terminus of the
Ramp. After transient transfection into CHO cells, the expression of
Ramp was detected by double staining the transfected cells using the
anti-6X histidine antibodies and anti-Ramp antibodies. Ramp protein was
localized to the nuclear region of the transfected CHO cells, although
some expression of Ramp could also be detected in the cytoplasm (Fig.
5B). The nucleo-cytoplasmic trafficking of the transfected
Ramp observed in CHO cells raised the possibility that Ramp is
associated with the nuclear matrix.
To confirm that Ramp is a nuclear matrix-associated protein, we
examined whether Ramp was localized to the nuclear matrix proteins of
the NT2 cells. Nuclei were digested with DNase I and extracted with 2 M NaCl to enrich for the nuclear matrices and its
associated proteins. The nuclear matrix preparations were then
electrophoresed and immunoblotted with anti-Ramp antibodies. Ramp
proteins were detected in the crude nuclear preparation as well as the
DNase I-treated and NaCl-washed purified nuclear matrix proteins (Fig.
5C).
Ramp Redistributes during the Cell Cycle of the NT2 Cells--
The
subcellular distribution of Ramp was then examined throughout the cell
cycle of the unsynchronized NT2 cells. During the interphase, strong
Ramp immunoreactivity was detected mainly in the nuclei of the NT2
cells (Fig. 6A). Positive
signal, although less concentrated, could also be observed in the
cytoplasm. At pro-metaphase and metaphase, Ramp was excluded from the
condensed chromosomes, with the staining localized to the cytoplasmic
region. In telophase, the Ramp staining remained in the cytoplasmic
region of the daughter cells. It is interesting to observe that Ramp became concentrated at the midzone as the dividing NT2 cells progressed to late anaphase in cytokinesis. The subcellular localization of Ramp
during the cell cycle raises the possibility that Ramp is a nuclear
matrix-associated protein and actively participates in the cell
division of NT2 cells.
NT2 cells were then synchronized at G2-M phase by treatment
with nocodazole. At pro-metaphase, NT2 cells revealed a strong DNA
(DAPI) staining of the condensing chromosomes. Immunofluorescent staining using specific Ramp antibodies confirmed our previous observations with unsynchronized NT2 cells. Ramp proteins were localized to the cytoplasm and excluded from the chromatins (Fig. 6B). The G2-M-arrested NT2 cells were then
immunostained with control antibodies that detected the
tyrosine-phosphorylated cdc2 (Phospho-cdc2 Y15). Interestingly, the
subcellular distribution of phospho-cdc2 Y15 was similar to that of the
Ramp proteins. Thus, the localization of the Ramp proteins is
consistent with our hypothesis that Ramp actively participates in the
process of cell division.
RA Could Up-regulate the Transcriptional Expression of Ramp in NT2
Cells--
To evaluate the effect of RA on the expression of the
ramp gene, we cloned and sequenced a ~2639-bp genomic DNA
fragment containing the putative ramp promoter and the first
exon (2304-2354 bp) of ramp cDNA (data not shown). The
genomic DNA fragment was then fused to the 5'-end of the promoterless
reporter SEAP. The SV40 early and the acetylcholine receptor
As the first step to explore the possibility that Ramp might play a
role in the cell proliferation of the NT2 cells, we examined the effect
of short term RA treatment on the proliferation of NT2 cells. NT2 cells
were treated with t-RA (10 µM) for 1-7 days. The cell proliferation was quantitated by measuring the metabolic rate
of viable cells and by determining the total cell count of NT2 cells.
When NT2 cells were treated with t-RA for 1 day, there was a
significant increase in the proliferation of NT2 cells, both in terms
of metabolic activities (~12%, Fig. 7B), as well as in
total cell number (~18.9%, Fig. 7C). At day 2, significant difference between the RA treatment or the control solvent
treatment was not observed. This was followed by a sharp decrease in
the total metabolic rate at day 4 of RA treatment. To further confirm the effect of RA on the proliferation of NT2 cells, we examined the
rate of cell proliferation by monitoring the BrdUrd incorporation after
1-3 days of RA treatment. As shown in Fig. 7D, the rate of
BrdUrd incorporation was increased by more than 10% of the control
after 1 day of RA treatment. Similar down-regulation was observed after
day 2 of RA treatment. Thus, our data demonstrated a good correlation
between the expression of ramp and the cell proliferation
rate of NT2 cells.
Overexpression of Ramp Enhanced the Cell Proliferation of NT2
Cells--
To further evaluate the effect of ramp on the
cell proliferation, we transiently transfected the ramp
expression vector into the NT2 cells. Control
The effect of ramp on the proliferation of NT2 cells was also monitored
by the BrdUrd incorporation assays. We transiently transfected the
EGFP-tagged ramp (E-Ramp) expression vector into the NT2
cells and examine the rate of BrdUrd incorporation. Because the
expression of retinoid receptor (RXR In this paper, we report the identification and characterization
of a novel gene, designated ramp. This gene was initially identified as a candidate gene that was down-regulated during the
RA-induced neuronal differentiation of human NT2 cells. Cloning of the
full-length cDNA reveals that Ramp does not share any homology with
known proteins, and that it is a serine/threonine-rich protein expressed predominantly in adult placenta, testis, and hematopoietic tissues such as thymus and bone marrow. We demonstrate that Ramp associates with the nuclear matrix in the undifferentiated NT2 cells.
Furthermore, the distinct subcellular localization of Ramp during the
cell cycle, together with its capabilities to enhance the cell
proliferation, suggests a role for Ramp in the mitosis and cytokinesis
of the NT2 cells.
The deduced amino acid sequence of Ramp exhibits a number of
interesting features. Although Ramp contains a putative nuclear localization signal, prediction based upon the amino acid composition using PSORT server reveals that Ramp can both be a cytoplasmic and
nuclear protein. Another interesting feature is the proline-rich motif,
conforming to the SH3-binding consensus PXXP (27). The sequence TLPLPLRP shares high homology with the proline-rich domain of
yet another serine/threonine-rich protein, Sirm (28). In addition, Ramp
contains an LXXLL motif. The LXXLL motif is
demonstrated to be essential for the association of thyroid hormone
receptor-binding protein to the liganded thyroid hormone receptor and
retinoic acid receptor (29). It is well known that cellular
communication is made possible in part by the post-translational
modification of the signaling proteins. With serine and threonine
residues constituting more than 20% of the Ramp protein, Ramp may
serve different functions depending on the phosphorylation status. We have observed that Ramp proteins are phosphorylated at serine in
undifferentiated NT2 cells (data not shown). This suggests that
phosphorylation of Ramp may involve protein kinases, such as Akt, RSK1,
and p70S6K (30). However, its potential signaling kinases and
phosphatases remain to be identified. The presence of two WD
repeats in Ramp is also of interest. WD repeats were first identified
in the Subcellular localization of Ramp provides insight into the potential
functions of Ramp. Although low expression of Ramp is detected in the
cytoplasm, it is more condensed to the nuclei of the undifferentiated
NT2 cells. In actively dividing cells, as revealed by the DAPI
counterstain, higher expression of Ramp is observed and localized in
the cytoplasmic region of the NT2 cells. The subcellular distribution
of Ramp suggests its possible role as a nuclear matrix-associated
protein, which is the set of proteins that resists the nuclease
treatment, as well as the high salt treatment (26). In light of
the fact that the nuclear matrix-associated proteins normally
redistribute during the mitosis and cytokinesis (35), we have observed
that Ramp is concentrated at the midzone of the late telophase NT2
cells, consistent with a potential role of Ramp in cytokinesis. During
cytokinesis, daughter cells are cleaved in two by the constriction of
an actin-rich contractile ring, which encircles the equator of the
dividing cells. Myosin II present in the contractile ring is essential for the constriction of the furrow (36). In this midzone, the microtubule bundle between the chromosomes of the daughter cells and
assemble with other midzone-associated proteins, such as kinesin, and
inner centromere protein, to form the midbody (37). These structures
strongly affect the proper completion of cytokinesis, thus requiring an
accurate assembly of the midzone proteins. When NT2 cells are arrested
at the pro-metaphase (G2-M) by nocodazole, Ramp becomes
totally excluded from the condensed chromosomal DNA. During the
progression into mitosis, the phospho-cdc2 Y15 is activated by the
cdc25 phosphatase by dephosphorylating the cdc2 (38). Interestingly, we
have demonstrated that the subcellular distribution of Ramp is in close
association with the phospho-cdc2, which signifies the progression of
the NT2 cells into mitosis. The precise interplay between the cyclins,
Ramp, and the progression of the cell cycle, however, remains to be elucidated.
The functional role of nuclear matrix has been controversial. Until
recently, nuclear matrix-associated proteins, such as ATRX and AKAP95,
are demonstrated to be associated with an X-linked mental retardation
syndrome and anchors the protein kinase A during the mitosis,
respectively (39, 40). In this study, we have demonstrated a close
association of ramp expression with the rate of
proliferation of NT2 cells. The transient up-regulation of ramp observed at day 1 of RA treatment can be explained by
the up-regulation of cell proliferation rate during the first day of RA
treatment. Prolonged RA treatment reduced the NT2 cell proliferation rate, coincidental with the decrease in the expression of
ramp. This is further supported by the observation that the
proliferation rate of NT2 cells can be induced by the transient
overexpression of ramp. Although ramp was
originally identified in the human EC cells, its transcriptional
expression is also down-regulated in a number of RA-responsive leukemia
cell lines2.
The strong association of ramp with early embryonic
development is exemplified by the high expression of ramp in
the fetal tissues such as brain, lung, liver and kidney, but not in the adult tissues. Taken together with its restrictive expression pattern
in the adult tissues, it is possible that Ramp might play different
role during the embryogenesis, and in the functioning of specific adult
tissues. The differential roles of Ramp may be mediated by the presence
of different ramp isoforms, the expression of which can be
regulated during development.
The gene encoding Ramp was localized to the chromosome 1q32.1-32.2.
Chromosome 1q32 has been mapped to several genetic diseases including
the complement system malfunctioning, as well as the Usher disease,
which is characterized by a combined loss of both the hearing and
visual systems (41). Another gene located in this region is a
phosphoprotein known as Mitosin. Similar to Ramp, Mitosin is also
suggested to involve in the cell cycle progression (42). Moreover, the
Alzheimer's disease is also mapped to the region of 1q32, although the
exact position remains to be elucidated. Hence, it will be of great
interest to examine whether ramp is involved in any of these
genetic diseases.
In summary, we have cloned and characterized a novel gene,
ramp, which is regulated during the RA-induced neuronal
differentiation of NT2 cells. Ramp is a serine/threonine-rich protein
associated with the nuclear matrix of NT2 cells. While the
immunocytochemical study suggests its role in the cell cycle, promoter
analysis, overexpression studies and cell proliferation assays support
its role in the cell proliferation of NT2 cells. The mapping of
ramp to the chromosome 1q32.1-2 also suggests that it may
potentially be involved in genetic diseases such as the complement
malfunctioning and Usher disease.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and basic fibroblast
growth factor, and proto-oncogenes, such as c-myc and
N-myc, are down-regulated during the neuronal differentiation (9). These responses are mediated through two classes
of nuclear receptors, the RA receptors and retinoid X receptors
(10-12). Although it is well-documented that RA receptors and retinoid
X receptors play essential roles in RA-mediated events, including
primitive endodermal differentiation, cell proliferation, and
apoptosis, the molecular mechanisms underlying these diverse physiological actions have only recently been explored (13-16). Even
less is known about how different genes are regulated by RA during
these processes. The involvement of other signaling molecules, such as
the coordinated regulation of the neurotrophin receptors, may mediate
RA-induced central nervous system neuronal differentiation (17). Thus,
one major thrust has been the identification of genes that are
regulated during the RA-mediated cellular events (18, 19).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gal, expressing
-galactosidase as internal control for normalization, were
used. For cell proliferation assays, NT2 cells were transiently transfected with expression vectors containing wild type
ramp, 6X histidine-, or EGFP-tagged ramp.
Reference plasmid pCMV-
gal was co-transfected to serve as internal
control for normalizing the transfection efficiency. MTT assays and
-galactosidase assays were performed as previously described (Roche
Molecular Biochemicals) (22). BrdUrd incorporation assays were
performed using BrdUrd-based cell proliferation enzyme-linked
immunosorbent assay (Roche Molecular Biochemicals). Briefly, cells were
labeled with BrdUrd solution for 2 h at 37 °C. The cells
were then fixed and denatured using FixDenat solution. After incubation
with horseradish peroxidase-conjugated anti-BrdUrd
solution for 2 h, the cells were washed and incubated with
substrate solution. Absorbance was measured at 450 nm. All experiments were repeated at least three times.
, ~311 bp) was constructed by deleting a ~2080-bp
DNA fragment from the 5'-end of the putative promoter. For promoter
analysis, a putative ramp promoter was subcloned to
the 5'-region of the reporter gene SEAP. SV40 early and acetylcholine
receptor
subunit promoters (23) were used as control in the
experiment. NT2 cells transfected with promoter constructs were treated
with 10 µM t-RA in normal medium. After
24 h, transfected cells were lysed for luminescent SEAP and
-galactosidase assays (CLONTECH).
-tubulin
(Tub2.1) were obtained from Sigma Chemicals. Polyclonal antibodies
specific for EGFP (CLONTECH), 6× histidine (CLONTECH), phospho-cdc2 Y15 (New England BioLab),
and lamin A/C (Santa Cruz Biotechnology) were employed in the study.
Alexa568- or Alexa488-conjugated secondary antibodies were obtained
from Molecular Probes Inc.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cloning of full-length cDNA encoding
human ramp. A, deduced amino acid
sequence of human ramp cDNA. The putative nuclear
localization signal is boxed. The putative leucine zipper
and WD repeats are dotted and underlined,
respectively. B, hydrophobicity plot of the deduced amino
acid sequence of human ramp according to Kyte and Doolittle.
C, the ramp cDNA encoded a ~80-kDa protein.
Coupled in vitro transcription and translation using rabbit
reticulocyte extract demonstrated that full-length ramp
cDNA encoded a ~80-kDa protein (lane 2;
S*). Luciferase gene (Luc, lane 1) was
used as positive control. Antisense construct (AS,
lane 4) was used as negative control. Radioactive methionine
was not included in lane 3 (S), whereas no
cDNA was included in lane 5 ( ). D, CHO
cells were transfected either with the chimeric EGFP-tagged
ramp (E-Ramp) or the EGFP cDNA construct.
Total proteins were collected and immunoblotting was performed using
anti-Ramp antibodies (anti-Ramp). The protein blot was
stripped and reblotted with antibodies specific for the EGFP proteins
(anti-EGFP). Arrowheads depict the positions of
Ramp proteins; the arrow indicates the position of
EGFP.
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Fig. 2.
Expression profile of ramp
in human tissues. Northern blots of multiple tissues
(A, adult tissues; B, fetal tissues;
CLONTECH) were hybridized using the full-length
ramp cDNA as a probe. Size markers in kilobases are as
indicated. C, 2 µg of poly(A)+ RNAs from human
normal tissues (CLONTECH) was used in the dot blot
to examine the expression of ramp in various adult tissues
of hematopoietic origin, as well as fetal tissues.
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Fig. 3.
Chromosomal localization of
ramp by FISH. A, ramp
genomic DNA probes labeled with digoxigenin dUTP were hybridized to
normal metaphase chromosomes derived from PHA-stimulated peripheral
blood lymphocytes. Positive signals were detected by incubation with
fluorescent anti-digoxigenin antibodies and counterstained with DAPI.
Specific labeling of the long arm of chromosome 1 was observed based on
the size, morphology, and banding pattern. B, biotin-labeled
probe specific for the heterochromatic region of chromosome 1 was
co-hybridized with ramp. Heterochromatin was labeled with
Texas red avidin in red (indicated in the black and white figure by
gray arrows) and ramp in green (indicated by
white arrows). Measurements of 10 specifically labeled
chromosomes 1 demonstrated that ramp is located at a
position, which is 62% of the distance from the
heterochromatic-euchromatic boundary to the telomere of chromosome arm
1q, an area that corresponds to band 1q32.1-32.2. A total of 80 metaphase cells were analyzed with 76 exhibiting specific labeling.
C, two ideograms illustrating the chromosomal position of
ramp at 1q32.1-32.2. Both ideograms are from the
International System for Human Cytogenetic Nomenclature 1995.
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Fig. 4.
RA treatment down-regulated the expression of
Ramp in NT2 cells. A, Northern blot analysis of
ramp expression during the RA-induced neuronal
differentiation of NT2 cells. Total RNA (10 µg) prepared from NT2
cells treated with t-RA for 0-28 days was hybridized with
the full-length ramp cDNA at high stringency. The
position of the 28 S ribosomal RNAs is indicated, whereas
arrowheads depict the positions of the ramp
transcripts. B, total protein lysates were obtained from NT2
cells either treated with t-RA or control solvent for 6 or
12 days. Antibodies specific to the N terminus (N) were
added, and the immunoprecipitates were electrophoresed. Immunoblotting
was performed using anti-Ramp (C terminus specific; C)
antibodies.
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Fig. 5.
Ramp is localized to the nucleus and is
associated with the nuclear matrix of NT2 cells. A, CHO
cells were transiently transfected with ramp expression
vector. After 48-h incubation, the cells were immunostained with
anti-Ramp antibodies and Alexa568-conjugated secondary antibodies.
B, CHO cells were transiently transfected with Ramp-His
expression vectors. Transfected cells were immunostained with anti-Ramp
or anti-6Xhistidine antibodies as indicated. Nuclei were visualized by
counterstaining with DAPI. The scale bar represents 10 µm.
C, nuclear fractions (lane 1) were extracted from
NT2 cells, treated with DNase I (lane 2) and washed with 2 M NaCl. The remaining pellet representing the core nuclear
matrix (lane 3) was solubilized in 8 M urea.
Equal amounts of each fraction were subjected to separation by
SDS-polyacrylamide gel electrophoresis. Western blot analysis was
performed using specific antibodies for the Ramp proteins (upper
panel) and the lamin A/C (lower panel).
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Fig. 6.
Cell cycle redistribution of Ramp in NT2
cells. A, undifferentiated NT2 cells were fixed and
double-stained using specific anti-Ramp (Ramp) and
anti- -tubulin antibodies (Tubulin). Nuclei are visualized
by counterstaining with DAPI. Results of the Ramp staining are
indicated in the first column,
-tubulin in the second column, and
DAPI in the third column. Results obtained by superimposing the first
and second columns are indicated in the fourth column
(Combine) on the right. B, NT2 cells
were synchronized using nocodazole (1 µM), fixed, and
double-stained with anti-Ramp and anti-phospho-cdc2 Y15
antibodies as indicated. Results obtained by superimposing the
first and second columns are indicated in the fourth column on the
right. Nuclei were visualized by counterstaining with DAPI. The
scale bar represents 10 µm.
promoters (AChR
) (23) were used as control promoter reporter
plasmids in the study. Transient transfection into NT2 cells resulted
in the constitutive expression of SEAP, demonstrating that the putative
ramp promoter was transcriptionally active in
undifferentiated NT2 cells. The transfected NT2 cells were treated with
10 µM t-RA for 24 h, and the SEAP
expression was monitored. RA up-regulated the relative expression of
SEAP by ~40% (p < 0.005; Fig.
7A). This is consistent with
our previous observations that the expression of ramp was transiently up-regulated during the early stage of neuronal
differentiation. Deletion of the 2080-bp DNA fragment from the putative
ramp promoter (Ramp
) reduced the expression level of the
reporter SEAP as well as the induction by RA. Similarly, RA treatment
did not significantly affect the expression level of the control SV40
early and AChR
promoters.
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Fig. 7.
RA transiently up-regulated the
transcriptional expression of ramp promoter and the
cell proliferation of NT2 cells. A, Ramp promoter
vectors were constructed by subcloning the upstream genomic DNA
fragment of ramp to the 5'-end of the SEAP reporter gene.
Ramp deletion promoter vector (Ramp ) was constructed by deleting a
2080-bp DNA fragment from the 5'-end of the putative ramp
promoter. SV40 early and AChR
promoters were used as control in the
study. Promoter vectors were transiently transfected into the NT2
cells. The efficiency of transfection was normalized using a
-galactosidase expression vector in each transfection. The cells
were lysed after 24-h treatment with t-RA (10 µM). Alkaline phosphatase and
-galactosidase assays
were performed. Bars show mean ± S.E.;
n = 5. B, NT2 cells were treated with
t-RA (10 µM) for 1-7 days. MTT assays were
performed on days 1, 2, 4, and 7 as described. C, NT2 cells
were trypsinized and counted using a Coulter cell counter on the days
as indicated. D, NT2 cells were treated with t-RA
(10 µM) for 1-3 days. BrdUrd assays were performed as
described. For B to D, the mean absorbance
obtained using solvent control was designated as 100%. Results shown
represent the mean ± S.E. of a typical experiment;
n = 6. The mean is shown as: *, p < 0.005, unpaired Student's t test, compared with the
control.
-galactosidase
expression vector driven by the constitutively expressed CMV promoter
was used for normalizing the transfection efficiency. Rate of cell
proliferation was monitored by MTT assays 48 h after transfection.
Overexpression of ramp significantly up-regulated the
proliferation rate of the NT2 cells by ~10% (Fig.
8A). We could observe similar
up-regulation of cell proliferation rate (~14%) when the human
neuroblastoma SY5Y cells were transfected transiently with the wild
type ramp (Fig. 8B).
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Fig. 8.
Overexpression of ramp
increases the rate of cell proliferation. NT2 cells
(A, C) and SY5Y cells (B) were plated
on 96-well plates and then transiently transfected with ramp
expression vector as indicated. The transfection efficiency was
normalized by including -galactosidase expression vector in each
transfection. After incubation for 48 h, MTT (A,
B) and BrdUrd incorporation (C) assays were
performed to monitor the rate of cell proliferation.
-Galactosidase
assays were performed to normalize the transfection efficiency. Bars
show mean ± S.E. (n = 5). *, p < 0.005, unpaired Student's t test, compared with the
control.
) was not affected during the
early stage of RA treatment (13), EGFP-tagged RXR
(E-RXR) was used
as negative control in the experiment. Overexpression of E-Ramp
significantly up-regulated the NT2 cell proliferation rate by ~30%
whereas transient transfection of E-RXR did not significantly affect
the NT2 cell proliferation rate (Fig. 8C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of heterotrimeric GTP-binding proteins (G proteins)
(31). The role of WD repeats in facilitating protein-protein
interaction has been proposed (32). It is noteworthy that many of these
mammalian WD repeat-containing proteins, such as chromatin assembly
factor I, retinoblastoma-binding proteins, and Cdc20/fizzy, are
involved in cell cycle regulation (32-34). Our preliminary data on the
analysis of ramp promoter suggest that the transcriptional
expression of ramp in NT2 cells is up-regulated during the
early phase of RA treatment. Moreover, our data further demonstrate
that the essential regulatory elements are located in the 2102-bp DNA
fragment of the putative ramp promoter. However, the
detailed regulatory mechanism of the ramp expression still remains unknown and awaits to be determined.
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ACKNOWLEDGEMENTS |
---|
We thank Mr. Cheuk H. Lung for excellent technical assistance and we are grateful to Dr. Lei Yang and Prof. Donald C. Chang for helpful discussions.
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FOOTNOTES |
---|
* The study was supported by the Research Grants Council of Hong Kong (HKVST 529/94M), the Innovation and Technology Commission (AF/178/97) and the Hong Kong Jockey Club.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF345896.
A recipient of the Croucher Foundation Senior Research Fellowship.
To whom correspondence should be addressed: Department of Biochemistry,
Hong Kong University of Science and Technology, Clear Water Bay, Hong
Kong, China. Tel.: 852-2358-7304; Fax: 852-2358-1552; E-mail:
BOIP@UST.HK.
Published, JBC Papers in Press, March 5, 2001, DOI 10.1074/jbc.M010802200
2 Unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: RA, retinoic acid; NT2, human embryonal carcinoma cell line NTera2 cl. D1; PCR, polymerase chain reaction; Ramp, RA-regulated nuclear matrix-associated protein; CMV, cytomegalovirus; t-RA, all-trans RA; EGFP, enhanced green fluorescence protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; bp, base pair(s); FISH, fluorescence in situ hybridization; PHA, phytohemagglutinin; DAPI, 4',6-diamidino-2-phenyl-indole; CSK, cytoskeletal; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; E-Ramp, EGFP-tagged Ramp protein; CHO, Chinese hamster ovary; kb, kilobase(s); AChR, acetylcholine receptor; SEAP, secretory form of human placental alkaline phosphatase.
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