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From the
Received for publication, February 25, 2000, and in revised form, May 3, 2000
Although transforming growth factor- TGF- In the skeleton, TGF- Our laboratory has previously identified a TGF- TIEG has been shown to play a role in TGF- To gain information on the role of TIEG in the actions of TGF- Cell Culture and Northern Analyses
MG-63 human osteosarcoma cells were routinely maintained in
DMEM/F-12 (1:1) (Sigma) containing 10% (v/v) FBS. For Northern blot
analysis, the cells were grown to near confluency in 100-mm culture
dishes. Total RNA was isolated from these cells using the
guanidinium/cesium chloride method. Northern analyses were performed
using total RNA (8-15 µg) as described previously (10). The blots
were probed with 32P-labeled human TIEG cDNA, GAPDH
cDNA, and 18 S ribosomal RNA as a control. The Northern blots were
exposed to Kodak X-Omat AR film, and densitometry was determined using
the NIH Image 1.47 program.
Isolation of Stable Cell Lines with Vector Only or with TIEG
Overexpression Constructs
The 1.4-kilobase pair TIEG cDNA was cloned in-frame into the
multiple cloning site of a pEBV His (Invitrogen) expression vector, which was thereafter named pEBV His-TIEG. MG-63 cells were stably transfected with either 10 µg of pEBV His-TIEG DNA or pEBV-His control vector using an electroporation method. The transfected cells
were seeded onto 100-mm plates and allowed to grow for 48 h at
37 °C. The cells were then grown in selection medium containing hygromycin (100 µg/ml). The medium was replaced with selection medium
every 2-3 days until antibiotic-resistant colonies developed. The
colonies were then ring-cloned and propagated separately. The TIEG
expression clones were characterized by analyzing the expression of
TIEG mRNA and protein by Northern and immunoprecipitation, respectively.
Immunoprecipitation of TIEG Protein
The cells were seeded onto 100-mm plates and allowed to grow to
near confluency. Prior to labeling, the medium was removed, and the
cells were washed twice in methionine-free medium. The cells were
pre-incubated in methionine-free medium for 1 h. Following pre-incubation, 750 µCi of [35S]methionine (Amersham
Pharmacia Biotech) was added to each 100-mm plate, and the cells were
labeled for 2 h at 37 °C. To harvest the cells, they were
washed twice with phosphate-buffered saline and lysed in 1 ml of
radioimmune precipitation buffer containing 0.1% (v/v) Nonidet P-40,
0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.15 M
NaCl, and 5 mM Na/KPO4, pH 7.4. A protease
inhibitor mixture was added to give final concentrations of the
following protease inhibitors: 0.10 mg/ml phenylmethylsulfonyl
fluoride, 30 µl/ml of aprotinin, and 0.1 mM sodium
orthovanadate. The cells were sheared using a 20-guage needle,
incubated on ice for 20 min, and centrifuged at 14,000 rpm for 10 min
at 4 °C. The supernatant was precleared with protein A-Sepharose
beads. The precleared lysates were incubated with an equal amount of
radioactivity with 5 µg of affinity-purified TIEG polyclonal antibody
for 16 h at 4 °C. The immunocomplex was pelleted using protein
A-Sepharose beads. The beads were resuspended in SDS-sample buffer and
boiled, and the proteins were separated by 10% (w/v)
SDS-polyacrylamide gel electrophoresis. The gels were soaked in
Autofluor (National Diagnostics) and exposed to Kodak X-Omat AR film.
Densitometry was performed using the NIH Image 1.47 program.
Cell Proliferation
The cells were plated in 12-well plates at a density of 10,000 cells/cm2 in growth medium for 24 h. The cells were
then serum-starved for 24 h by replacing the medium with DMEM/F-12
(1:1) containing 0.25% (w/v) BSA, and the parent control MG-63 cells
were then treated with vehicle or 2 ng/ml TGF- Markers of the Osteoblast Phenotype
Alkaline Phosphatase Activity--
Alkaline phosphatase (AP)
activity was measured after 4 days as described previously by our
laboratory (16, 21, 22). Briefly, MG-63 cells were seeded onto 12-well
tissue culture plates at 5,000 cells/cm2, and after 24 h the medium was changed to DMEM/F-12 (1:1) containing 0.25% (w/v) BSA
with or without TGF- Osteocalcin Protein--
Osteocalcin protein was measured as
reported previously using the NovoCalcin immunoassay (Metra
Biosystems). Briefly, control and vector-transfected MG-63 cells,
TIEG-6 cells, and TIEG-7 cells were seeded onto 12-well tissue culture
plates at 40,000 cells/cm2. After 24 h, the medium was
replaced with DMEM/F-12 (1:1) containing 0.25% (w/v) BSA. The parent
MG-63 control cells were treated with or without TGF- RT-PCR--
RT-PCRs for osteocalcin and GAPDH were performed
from MG-63, TIEG-6, and TIEG-7 cells described earlier by Rickard
et al. (23). Amplification reactions were terminated during
the linear phase of amplification after 24 cycles (GAPDH) and 35 cycles
(OC). PCR products were visualized on 1.5% agarose gels stained with ethidium bromide.
Statistical Analysis--
Student's t test was used
for statistical analysis.
TGF- The effect of overexpressing TIEG protein on the MG-63 cell
proliferation was then examined. As shown in Fig.
3A, the parent MG-63 cells
display a 50% inhibition of cell proliferation when treated with
TGF-
Overexpression of a Nuclear Protein, TIEG, Mimics Transforming
Growth Factor-
Action in Human Osteoblast Cells*
§,§,,,, and
Department of Biochemistry and Molecular
Biology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota
55905 and the ¶ Division of Endocrine, Reproductive, and
Developmental Toxicology, Chemical Industry Institute of
Toxicology, Research Triangle Park, North Carolina 27709
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-)
is a growth factor with many known regulatory activities in many
different cell types, its intracellular signaling pathway is still not
fully understood. A TGF--inducible early gene (TIEG) was
discovered and shown by this laboratory to be a 3-zinc finger
transcription factor family member; its expression is rapidly induced
in cells treated with TGF-. To ascertain whether TIEG plays a major
role in the TGF- pathway, human osteosarcoma MG-63 cells were stably transfected either with an expression vector containing a TIEG cDNA
or with the vector alone. Clones that contain only the vector express
normal levels of TIEG mRNA and protein and display the same
patterns of gene expression and levels of cell proliferation as the
nontransfected, non-TGF--treated parental cells. However, transfected cells that overexpress TIEG mRNA and protein
(TIEG-6 and TIEG-7) display changes that mimic those of MG-63
cells treated with TGF-, i.e. increased alkaline
phosphatase activity, decreased levels of osteocalcin mRNA and
protein, and decreased cell proliferation. The degree of these changes
correlated with the level of TIEG expressed in the cell lines. TGF-
treatment of the overexpressed cells showed no added effects. These
findings and other published reports support a primary role of TIEG as
a transcription factor in the TGF- signaling pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 has been shown
to regulate many diverse tissue and cell processes in a multitude of
cell types (1, 2). The cellular processes affected by TGF- include
inhibiting proliferation, inducing cell differentiation and apoptosis,
and altering gene expression. These and additional properties have
resulted in the labeling of TGF- and members of its signaling
pathway as tumor suppressors (3, 4).
is involved in bone growth, cell
differentiation, and overall bone metabolism. In rodent and human OBs,
TGF- is involved in the regulation of 1) proliferation of OB cells,
which appears to occur at the G1 phase of the cell cycle; 2) the proliferation and differentiation of OB osteoprogenitor cells;
and 3) OB bone matrix production and mineralization (5-7). Interesting
studies by Derynck and co-workers (8, 9) using transgenic mice have
shown that TGF-2 overexpression causes a rapid,
age-dependent loss of bone mass, which resembles
osteoporosis. TGF- appears to directly increase OB activity and
differentiation, to uncouple OB and OCL activities, and to result in
bone loss.
-inducible early gene
(TIEG) in normal human OB cells (hFOB) in which protein is 1) rapidly
translocated from the cytoplasm to the nucleus and 2) rapidly induced
(as is its mRNA) by TGF- within 60 min after treatment in both
primary and immortalized hFOB in culture (10, 11). TIEG mRNA was
induced equally by all three isoforms of TGF-, which is not
surprising in view of the high binding affinity of the receptors for
all isoforms of TGF-. Our laboratory has reported that TIEG encodes
a 480-amino acid protein (72 kDa) and has a unique N-terminal end,
which distinguishes it from an early growth response-
(EGR-) gene. We have previously reported that these two
proteins are encoded in the same gene, but EGR- is transcribed at much lower levels than TIEG in all tissues (12, 13). The
zinc finger region of TIEG shows homology to the 3-zinc finger family
of transcription factors such as Sp-1, Wilm's tumor, BTEB, EGR-1, and
the Krüppel-like factors. The TIEG gene is localized on chromosome 8,q22.2, the same locus that contains genes related to
myeloma and osteopetrosis (11). TIEG protein has been identified in
many human tissues and cell types in addition to osteoblasts, including
certain cells in the breast, uterus, brain, pancreas, and muscle (11).
A mouse TIEG, termed mGIF for murine glial cell-derived neurotrophic
factor (GDNF)-inducible factor, has also been shown to be distributed
in several regions of mouse brain and is rapidly induced by GDNF in a
neuroblastoma cell line and in primary cultures of rat embryonic
cortical neurons (14, 15).
-induced inhibition of
cell proliferation and apoptosis in human osteoblast cells and
pancreatic carcinoma cells and more recently in epithelial and liver
cancer cells (16-19). The induction of apoptosis appears to be the
same for TGF- and TIEG when the latter is overexpressed (18,
19).
, we
developed and characterized two stably transfected human osteoblastic
MG-63 cell lines (TIEG-6 and TIEG-7), demonstrating that they
overexpress the TIEG protein by 200 and 300%, respectively. This paper
also describes the phenotypic properties of these two lines to show
that they mimic those of the TGF--treated parent osteosarcoma MG-63
compared with vector control cells or with untreated, nontransfected,
parent MG-63 cells.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. The TIEG-6 and
TIEG-7 cells were maintained in serum-containing medium. All cells were
incubated with 1 µCi/ml [3H]thymidine for 24 h;
[3H]thymidine incorporation was determined in the cells
by trichloroacetic acid precipitation (20, 21). The data are presented
as the percent of control values, i.e. those from the
untreated, nontransfected MG-63 cells.
(2 ng/ml) for the parent MG-63 control
cells. The data are presented as the percent of control values,
i.e. those from the untreated, nontransfected, parental
MG-63 cells or from the vector-transfected control cells.
(2 ng/ml).
After 72 h, the conditioned medium was collected, centrifuged to
remove all debris, dialyzed in 50 mM ammonium bicarbonate,
and analyzed for osteocalcin (21).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-treated and untreated MG-63 cells and vector control
cells, as well as stably transfected TIEG-6 and TIEG-7 cells, were analyzed for TIEG mRNA steady state levels by Northern blotting and
for TIEG protein levels by immunoprecipitation and SDS-gel electrophoresis. As reported previously in normal human osteoblast cells (10) and shown in Fig.
1A for osteosarcoma cells,
TGF- induces TIEG mRNA levels in parent MG-63 cells to a maximum
of 5-fold within 1 h post-treatment. Fig. 1B shows the
Northern blot of transfected cells wherein TIEG mRNA is increased
in TIEG-6 and TIEG-7 cell lines, with only background levels detected
in untreated, nontransfected, parental MG-63 cells or in
vector-transfected control cells. To estimate TIEG protein levels,
soluble protein was isolated from 35S-labeled cell extracts
and immunoprecipitated with anti-TIEG protein antisera. Fig.
2A shows that TGF-
treatment of the nontransfected MG-63 cells rapidly increases TIEG
protein levels by 1.8-fold. In Fig. 2B, TIEG protein levels
are shown to be 2- and 3-fold higher in TIEG-6- and TIEG-7-transfected
cells, respectively, compared with the untreated, nontransfected, MG-63
parent control cells.
View larger version (35K):
[in a new window]
Fig. 1.
Analyses of TIEG mRNA steady state levels
in MG-63, TIEG-6, and TIEG-7 cells. Northern blot analyses and
densitometry of the TIEG mRNA steady state levels are described
under "Experimental Procedures." Panel A represents the
Northern blots of TIEG mRNA levels in parent MG-63 cells treated
for various times. Panel B represents the Northern blots of
the expression of the transfected TIEG mRNA levels in untreated
parental MG-63 cells, vector control cells (Vect. Cont), and
TIEG-6 and TIEG-7 cells stably transfected with the TIEG expression
vector.
View larger version (31K):
[in a new window]
Fig. 2.
Analyses of TIEG protein levels in the MG-63,
TIEG-6, and TIEG-7 cells. MG-63, TIEG-6, and TIEG-7 cells were
labeled with [35S]methionine as described under
"Experimental Procedures." Panel A represents the
nontransfected parental MG-63 cells treated and untreated with TGF- ,
and Panel B shows the analyses of the parent MG-63 cells and
the transfected, overexpressing TIEG-6/TIEG-7 cells. The cell lysates
were immunoprecipitated using anti-TIEG polyclonal antibody 228, and
the samples were separated on a 10% (w/v) SDS-polyacrylamide gel as
described under "Experimental Procedures." The TIEG
immunoprecipitated protein is identified with an arrow.
Densitometry was performed on the original blots as described under
"Experimental Procedures." The gels represent examples of analyses
performed on three separate sets of cultures of each cell line.
. Similarly, Fig. 3B shows that the overexpressing TIEG-6 and TIEG-7 cells display a more than 50% decrease in the rate
of proliferation compared with the rates in the untreated, parent MG-63
cells, whereas the vector-transfected control cells show only a
moderate reduction. The effect of overexpressing TIEG in the MG-63
cells on the expression of bone markers for the OB phenotype was then
examined. As shown in Fig. 4A,
TGF--treated control MG-63 cells display a 2-fold induction of AP
activity. In Fig. 4B transfected TIEG-6 and TIEG-7 cells are
shown to contain ~1.4- and 1.8-fold enhanced AP activity,
respectively, compared with the AP levels in the untreated, parent
MG-63 cells or the vector control MG-63 cells. To determine whether
TGF--induced endogenous TIEG protein acts differently or in addition
to the overexpressed TIEG protein, we treated the TIEG-7 cells with
TGF- and assayed for AP levels. As shown in Fig. 4B,
although TGF- showed strong effects on the AP levels in the
nontransfected control or vector control MG-63 cells, no additional
TGF--induced changes were detected in TIEG-7 cells.
View larger version (18K):
[in a new window]
Fig. 3.
Analyses of the cell proliferation rate in
MG-63 and TIEG-6/7 cells. A and B, the cells
were labeled with [3H]thymidine, and all rates of
proliferation were determined as described under "Experimental
Procedures." In A, CONTROL represents untreated
cells, and TGF- represents treated parental MG-63 cells
incubated with 10 ng/ml TGF- for 24 h.
[3H]Thymidine was added subsequently for 24 h before
the rate of cell proliferation was determined. *, indicates
p < 0.01, significantly different from untreated
control. In B, MG-63 represents the cell
proliferation of untreated MG-63 cells, Vector Control
represents the rate in untreated vector control cells, and
TIEG-6 and TIEG-7 represent the rate in the TIEG
overexpressing cell lines. **, indicates p < 0.001, significantly different from MG-63 control. The mean and ± S.D.
for 3 separate experiments are illustrated.
View larger version (26K):
[in a new window]
Fig. 4.
Analyses of AP activity on the MG-63 cell
(A) and the TIEG-6/7 cells (B).
AP activity was measured as described under "Experimental
Procedures." A, CONTROL represents the activity
in untreated MG-63 cells, and TGF- represents the
activity in TGF--treated MG-63 cells. These cells were incubated
with 2 ng/ml TGF- for 4 days before the cells were harvested. The
data are presented as the % of control, i.e. values from
the untreated, nontransfected MG-63 cells and vector-transfected
control cells. The mean and S.D. from three separate experiments are
presented. **, indicates p < 0.001, significantly
different from untreated control. In B, MG-63
represents the activity in untreated MG-63 cell lines, Vector
Control represents the activities in untreated vector control cell
lines, and TIEG-6 and TIEG-7 represents the
activities in the TIEG-6 and TIEG-7 transfected cell lines,
respectively. Finally, TIEG-7 + TGF- represents values
from the TIEG-7 cells treated for 48 h with 2 ng/ml TGF-. *,
indicates p < 0.01, significantly different from the
MG-63 control. **, indicates p < 0.001, significantly
different from the MG-63 control.
Regarding the expression of the OC gene, the TGF- treatment of the
parent MG-63 cells results in a modest (25%) inhibition of OC
production (Fig. 5A).
Similarly, as shown in Fig. 5B, the TIEG-6 and TIEG-7 cells
display a significant decrease, i.e. 94 and 97%,
respectively, in the levels of OC production compared with the parent
MG-63 cells and the vector only control cells. To determine whether
TIEG overexpression actually regulates OC protein at the level of gene
expression, and as further support that TIEG overexpression mimics
TGF- action on osteoblast cells, we performed RT-PCR analysis of the
MG-63, TIEG-6, and TIEG-7 cells. As shown in Fig.
6, the TIEG-6 and TIEG-7 cells show
reduced OC mRNA steady state levels compared with MG-63 cells,
which correlates to the protein levels.
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|
Preliminary studies to identify the mechanistic pathway for TIEG
involved co-precipitation experiments using the anti-TIEG polyclonal
antibodies and anti-Smad 3 antibodies, under conditions similar to
those described above for measuring TIEG protein. The precipitated
proteins were analyzed by Western blotting using anti-Smad 3 antibody
on the samples precipitated by anti-TIEG antibody and vice
versa. These studies failed to detect any interactions between
TIEG and Smad 3.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
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The data presented in this paper support the hypothesis that TIEG
may serve an important role as a primary participant in the TGF-
signaling pathway and/or as an early response gene for TGF- in
osteoblast cells. The data show that cell proliferation is inhibited,
as are OC mRNA and protein levels, whereas the AP activity is
increased in TIEG-6 or TIEG-7 cells compared with the vector control
cells. These responses mimic the effects of TGF- in MG-63 cells.
Importantly, the overexpression displays a dose dependence in that the
effects are increased as the concentrations of TIEG protein increases
by 200 and 300% in stably transfected TIEG-6 and TIEG-7 cells,
respectively. It should also be noted that the concentrations of TIEG
protein in the TIEG-6 and TIEG-7 cells are lower, but more constant,
compared with the higher, but transient, induction of TIEG protein
levels in the TGF--treated (nontransfected) parental MG-63 cells.
Interestingly, TGF- treatments of TIEG-7 cells show no further
responses in the AP levels, supporting the idea that TIEG levels in the
transfected lines were saturating and that overexpressed TIEG in the
latter can fully substitute for endogenous TGF--induced TIEG protein.
The fact that TIEG mRNA and protein levels were previously shown to
be rapidly induced after TGF- treatment further supports a role for
TIEG as an early response gene in the TGF- signaling pathway (15,
16). TIEG has previously been implicated in tumor suppressor functions
by inhibiting cell proliferation and inducing apoptosis in pancreatic
carcinomas, hepatocarcinoma, and epithelial cells. In this regard, even
the transient overexpression of TIEG seems to mimic the actions of
TGF- (16-19). The data presented here support this function for
TIEG in osteosarcoma cells.
Previous studies (12, 13) from our laboratory have demonstrated that
the TIEG gene codes for two proteins (TIEG protein and
EGR-), which are members of a 3-zinc finger family of transcription factors. However, TIEG represents the predominant species (>95%) in
many tissues and cell types examined (12, 13). Interestingly, TIEG is a
cytonuclear protein in which the constitutive levels are rapidly
translocated to the nucleus following TGF- treatment. This
translocation is followed by a rapid induction of the TIEG gene expression (11, 16). In addition, the induction of TIEG mRNA
steady state levels in OB cells is specific for TGF- and not a
variety of other growth factors and cytokines (10). Treatments with
other cytokines, e.g. BMP-6, inteleukin-6, insulin like
growth factor-I and -II, tumor necrosis factor-, interleukin-1,
platelet-derived growth factor, and fibroblast growth factor, had no
effect on TIEG expression (10). Selected other TGF- family members,
e.g. BMP-2, BMP-4, and activin, as well as epidermal growth
factor, also showed some induction but only at much higher
concentrations than required for TGF- (11). Interestingly, other
members of the TGF- family regulate TIEG in other cell types,
e.g. GDNF rapidly induces TIEG levels in neuroblastoma cells
(14, 15). TIEG has been shown to be highly conserved and is homologous
with the previously reported mGIF in mouse neuroblastoma cells (14, 15). We have previously reported a tissue- and cell type-specific distribution of TIEG, with TIEG protein localized to specific cell
types in the cerebellum, pancreas, placenta, uterus, muscle, bone, bone
marrow, and breast epithelium (11, 15, 24). Because TGF- is known to
regulate cell functions in many of these tissues, it is probable that
TIEG is utilized by TGF-, or possibly some of its family members, in
these other cell types. These and other studies support a role for TIEG
in the TGF- signaling pathway as a possible tumor suppressor gene
(16-19).
Members of the TGF- superfamily appear to mediate their actions
through distinct receptors that have serine/threonine kinase activity
(25) and utilize signaling pathways involving either Smad proteins (1,
26), TGF--activated kinase 1 (TAK-1) (27), or possibly other, yet
undefined, pathways. After binding and activation by TGF- or BMPs,
the serine kinase membrane receptors phosphorylate receptor-regulated
Smads 1 and 5 in response to BMPs and Smads 2 and 3 for TGF- and
activin (25-29). The activated Smads complex with Smad 4 and
translocate to the nucleus, where the complex can interact with other
nuclear factors and modulate target gene expression. Smads 6 and 7 are
negative regulators of this pathway and function by binding and
inhibiting the actions of the type I TGF- receptor kinase domain or
the receptor-regulated Smads. It is unclear whether TIEG plays a role
in this signaling pathway.2
As a cytonuclear protein that is induced to rapidly translocate to the
nucleus in response to TGF- treatment, and as a TGF- rapidly
induced (early response) gene, TIEG could: 1) interact with Smads 2, 3, or 4, travel to the nucleus, and play a role as a
co-activator/repressor in transcriptional regulation; 2) play a direct
role in a unique and heretofore undefined signaling pathway of TGF-
family members; or finally 3) not play a signal pathway role but
function as an early response gene in which the protein product would
be involved in further mediating TGF- effects on late genes,
including those involved in maintaining cell
proliferation.2
If TIEG is a participant in the Smad pathway, the lack of any further
TGF- response on AP activity in the TIEG-7 cells suggests that the
Smads are already occupied by the overexpressed TIEG protein and
unavailable for further ligand-mediated activation of Smads. Regarding
the early response gene role, recent studies have reported that TIEG
represses TATA-containing and TATA-less promoters in reporter gene
assays (14) and contains repression domains that are known to repress
transcription in a heterologous GAL-4-based transcriptional assay (30).
The latter studies, combined with the results presented in this paper,
support the idea that TIEG acts as a transcription factor in the
TGF- action pathway. This view is supported by similar roles of
other members of the 3-zinc finger protein family members,
e.g. Sp-1, Krüppel, and EGR-1 factors.
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ACKNOWLEDGEMENTS |
|---|
The authors thank Larry Pederson and Kay Rasmussen for excellent technical assistance and Jacquelyn House for excellent clerical assistance.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health (NIH) Grant AR43627, the Howard Wagner Cancer research Fund, the Mayo Foundation, and NIH Training Grant Awards HD07108 (to G. G. R.) and CA09441 (to K. M. W.).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.
§ These authors contributed equally to this manuscript.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, 1601A Guggenheim Bldg., Mayo
Clinic, 200 First Street S.W., Rochester, MN 55905. Tel.: 507-284-2480; Fax: 507-284-2053; E-mail: spelsberg.thomas@mayo.edu.
Published, JBC Papers in Press, May 17, 2000, DOI 10.1074/jbc.C000135200
2
At present we are not sure whether TIEG
interacts only with the Smad binding elements or interacts directly
with the Smad proteins. Two lines of evidence suggest an interaction of
TIEG with Smads (S. A. Johnsen, M. Subramaniam, and T. C. Spelsberg, unpublished data). First, preliminary studies attempting a
co-transfection of the TIEG expression vector with the Smad binding
element reporter construct into AKR2B cells resulted in an enhanced
reporter response to TGF-. Second, when the cells were
co-transfected with Smads 3 and 4 expression vectors, TIEG
co-activation was further enhanced in the presence of TGF- treatment.
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ABBREVIATIONS |
|---|
The abbreviations used are:
TGF-, transforming growth factor-;
TIEG, TGF--inducible early gene;
OB, osteoblasts;
hFOB, human immortalized fetal osteoblasts;
AP, alkaline
phosphatase;
OC, osteocalcin;
OCL, osteoclasts;
TIEG-6/TIEG-7, MG-63
cell lines, stably transfected with the cDNA expression vector;
GDNF, glial cell-derived neurotrophic factor;
mGIF, murine
GDNF-inducible factor;
PCR, polymerase chain reaction;
RT-PCR, reverse
transcriptase-PCR;
DMEM, Dulbecco's modified Eagle's medium;
BSA, bovine serum albumin;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
BMP, bone morphogenetic protein;
EGR, early growth response.
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REFERENCES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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| 1. | Padgett, R. W., Savage, C., and Das, P. (1997) Cytokine Growth Factor Rev. 8, 1-9 |
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| 5. | Bonewald, L. F., and Dallas, S. L. (1994) J. Cell. Biochem. 55, 350-357 |
| 6. | Centrella, M., Horowitz, M., Wozney, J., and McCarthy, T. L. (1994) Endocr. Rev. 15, 27-39 |
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