| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Department of Orthopaedics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
Address all correspondence and requests for reprints to: Regis J. O’Keefe, M.D., Box 665, Department of Orthopaedics, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, New York 14642. E-mail: regis_okeefe{at}urmc.rochester.edu
 |
Abstract |
|---|
 Top Abstract IntroductionTGF-{beta} signals through...Materials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionTGF-{beta} signals through...Materials and MethodsResultsDiscussionReferences |
|---|
Although TGF-ß is highly expressed in the growth plate (8), its effects on terminal differentiation, interaction with other growth factors, and role in endochondral ossification are less clear. In contrast to BMPs, recent data demonstrate that TGF-ß may inhibit terminal differentiation (9, 10, 11); we and others have shown that it regulates the expression of PTHrP (11, 12). These findings suggest that TGF-ß may be one of the critical factors involved in endochondral ossification in the growth plate and during reparative processes. Thus, investigation of TGF-ß-regulated signaling events may provide clues to the mechanisms involved in chondrocyte differentiation and define other potential targets to regulate this process.
|
TGF-ß signals through multiple distinct and overlapping pathways |
|---|
|
TopAbstractIntroductionTGF-{beta} signals through...Materials and MethodsResultsDiscussionReferences |
|---|
However, in addition to the classical Smad signaling paradigm, TGF-ß also activates other signaling pathways. TGF-ß has been shown to stimulate mitogen-activated protein (MAP) kinase signaling pathways directly and through an associated molecule called TAK (TGF-ß-activated kinase) (23, 24). In articular chondrocytes, TGF-ß stimulates TIMP expression through activation of protein kinase C (25), whereas TGF-ß has been shown to induce PKC activity in immature rat sternal chondrocytes (26). Thus, TGF-ß effects on chondrocyte maturation could be regulated through one or more different pathways.
The present study focuses on the role of Smad2 and 3 in mediating TGF-ß1 signaling in upper sternal chondrocytes and defines their role as regulators of the rate of chondrocyte maturation during endochondral ossification. Our results show that both Smad2 and 3 mediate TGF-ß1 signaling in chondrocytes and identify Smad3 as a more potent mediator of TGF-ß1 effects on chondrocyte maturation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionTGF-{beta} signals through...Materials and MethodsResultsDiscussionReferences |
|---|
c) of human Smad 1–3 were a gift from Dr.
Rik Derynck (27), and subcloned into the mammalian
expression vector pCMX (28) and into the replication
competent avian sarcoma retrovirus RCASBP(A) (29).
Wild-type Smad1–3 and dominant negative Smad1–3c sequences were
verified following subcloning using automated sequencing. The TGF-ß
responsive p3TP-Lux reporter construct (30) was a gift
from Dr. Joan Massagué.
Cell culture
Chondrocytes were isolated from the cephalic portion (upper 1/3)
of sterna from 14-day chick embryos by digestion for 4 h at 37 C,
5% CO2 in HBSS containing collagenase (450 U/ml,
Sigma St. Louis, MO) and trypsin (2.5%,
Sigma, St. Louis, MO). The cells were resuspended in DMEM
(Life Technologies, Inc., Grand Island, NY) with 10%
NuSerum IV (Collaborative Biomedical Products, Bedford, MA) and 100
U/ml Penicillin/Streptomycin (Life Technologies, Inc.)
then plated at 2.5 sterna per 100-mm plate. After 6 days, chondrocytes
were harvested, counted, and replated in six-well plates for
transfection experiments and alkaline phosphatase experiments or 60 mm
dishes for Northern analysis. This is a well characterized primary
culture model of spontaneous differentiation in which chondrocytes are
responsive to growth factors and other signaling molecules (2, 31, 32, 33, 34).
Transient transfection and luciferase assay
Upper sternal chondrocytes, cultured at 30–40% confluence in
six-well plates, were transfected on day 2 after plating using the
transfection reagent Superfect (QIAGEN, Santa Clarita, CA)
according to the manufacturer’s guidelines. Individual experiments
were internally controlled for amount of total plasmid DNA, with equal
amounts of p3TP-Lux reporter, control SV40-renilla plasmid for
normalization of transfection efficiency, and Smad construct and/or
vector control DNA for Smad cotransfection experiments. For experiments
in Fig. 2A
, 1.0 µg/well of reporter was used, whereas experiments in
Fig. 2, B–D, used 0.5 µg of reporter cotransfected with 1 µg total
of Smad construct and/or vector control DNA per well. Following
transfection, chondrocytes were placed in media containing DMEM,
penicillin/streptomycin, and 10% NuSerum IV. After 12 h,
chondrocytes were incubated for 6 h in serum-free media
(containing DMEM, hyaluronidase 4 U/ml, penicillin/streptomycin, and
supplemented with 10 pM triiodothyronine
(Sigma, St. Louis, MO), 60 ng/ml insulin, and 1
mM cysteine (Sigma), followed by addition of
0.1–10 ng/ml of TGF-ß1 (Calbiochem, La Jolla, CA) or 50
ng/ml BMP-2 (gift from the Genetics Institute) to selected
treatment groups. Eighteen hours later, chondrocytes were harvested and
assayed for luciferase activity using the Promega Corp.
dual luciferase assay system, as previously described
(35). Renilla luciferase values were used to normalize
each sample for transfection efficiency. Data presented is the mean of
triplicate samples, and error bars represent
SEM. Statistical analysis was performed using
one-way ANOVA.
|
|
c, and 3c in upper
sternal chondrocytesc and 3c inserts. Cells were passaged three times and
replated in 100-mm dishes. Upon confluence, media was changed to DMEM
containing 10% NuSerum IV and penicillin/streptomycin. Viral
supernatants were collected at 24 h intervals for 3 days. At the
time of secondary plating, upper sternal chondrocytes were incubated
with a 1 to 1 mixture of fresh viral supernatant and chondrocyte
plating medium. After 48 h, fresh ascorbate-containing medium was
added to the cultures with TGF-ß1 [1 ng/ml (Calbiochem,
La Jolla, CA)] added to selected treatment groups. Media and growth
factors were replenished at 48-h intervals.
Western blot
Retrovirally infected 7-day confluent cultures of third passaged
chicken embryonic fibroblasts or infected 9-day chicken upper sternal
chondrocytes were washed with cold PBS and lysed on ice in Golden lysis
buffer (GLB) (36) supplemented with protease inhibitor
cocktail tablets (Roche Molecular Biochemicals), 1
mM sodium orthovanadate, 1 mM EGTA, 1
mM NaF, 1 µM microcysteine. Insoluble
material was removed by centrifugation at 12,000 x g.
The protein concentration of the soluble material was estimated using
Coomassie Plus Protein Assay kit (Pierce Chemical Co.,
Rockford, IL). One hundred micrograms of each extract was separated by
SDS-PAGE. After transfer to a nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH), the blots were probed with the an
anti-Flag antibody (Sigma) at a concentration of 0.4
µg/ml. Horseradish peroxidase-conjugated goat antimouse polyclonal
antibodies (Bio-Rad Laboratories, Inc.) were used as
secondary antibody. The immune complexes were detected using ECL+Plus
(Amersham Pharmacia Biotech, Arlington Heights, IL) with
X-OMAT AR film (Kodak, Rochester, NY).
Northern analysis
For RNA analysis of TGF-ß1 effects on chondrocyte maturation,
cells were treated 24 h following plating with TGF-ß1 [10, 5,
3,0.5,0.3,0.1 ng/ml (Calbiochem, La Jolla, CA)] in the
presence of ascorbate and harvested at intervals spanning 12–96 h
following growth factor addition. For experiments studying effects of
Smad2, 3, 2c, and 3c on chondrocyte maturation, chondrocytes
infected with RCASBP(A) constructs were harvested 7d following growth
factor addition for Northern analysis of collagen type X
(colx) expression. RNA extraction was performed using the
Rneasy Mini Kit (QIAGEN, Santa Clarita, CA) according to
manufacturer’s instructions. RNA samples were denatured by heating,
electrophoresed on agarose gels, and transferred to a nylon membrane
(NEN Life Science Products, Boston, MA). Levels of
colx messenger RNA (mRNA) were assessed by hybridizing to a
32P-labeled chick colx oligonucleotide
probe, as previously described (2). All Northern blots
were subsequently hybridized to a 32P-labeled
chick GAPDH cDNA probe for loading controls, as previously described
(37). The ratios of colx to GAPDH
band intensities were determined with the use of NIH Image
1.61/ppc.
Assay for alkaline phosphatase activity
Sternal chondrocytes were treated with TGF-ß1 (10, 5, 3, 1,
0.5, 0.3, 0.1 ng/ml) for 3 days in the presence of ascorbate. Sternal
chondrocytes infected with RCASBP(A) constructs were treated with
TGF-ß1 in the presence of ascorbate for 5 days. Alkaline phosphatase
activity was determined as described previously (2) and
normalized for protein concentration. Data presented is the mean of
triplicate samples, and error bars represent SEM.
Statistical analysis was performed using one-way ANOVA.
Immunofluorescence
Chrondrocytes cultured in LAB-TEK chambers were transiently
transfected with wild-type Smad2, and 3. Twelve hours following
transfection, cells were serum starved for 16 h. Chondrocytes were
then treated with TGF-ß1 5ng/ml or BMP-2 50 ng/ml for 40 min or
remained untreated. Cells were fixed with 4% paraformaldehyde in PBS.
After 10 min incubation with 0.1% Triton/PBS, cells were blocked with
5% donkey serum/0.5% BSA/PBS and incubated overnight at 4 C with
anti-Flag antibody (1 µg/ml, Sigma) and washed with PBS,
followed by incubation with fluorescein isothiocyanate-conjugated
antimouse IgG antibody (1:1000, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 30 min. The cells were
then washed with PBS, mounted, and visualized by fluorescence confocal
microscopy (Leica Corp., 400x magnification) as
previously described (38, 39).
|
Results |
|---|
|
TopAbstractIntroductionTGF-{beta} signals through...Materials and MethodsResultsDiscussionReferences |
|---|
), with maximal
effects achieved at a concentration of 3 ng/ml (Fig. 1A, lane 4). We
subsequently examined the effect of TGF-ß1 on the development of
colx expression in upper sternal chondrocytes treated
between 12 and 96 h at a concentration of 3 ng/ml. As previously
shown (2, 40), ascorbate-treated control cultures
progressively differentiated and increased expression of
colx (Fig. 1B, lanes 1–5). In contrast, TGF-ß1 slowed the
rate of chondrocyte differentiation and inhibited colx
expression compared with control cultures. These effects were greatest
in cultures receiving the longest duration of exposure to TGF-ß1
(Fig. 1B, lanes 6–10). After 96 h in culture with TGF-ß1,
colx expression was nearly completely suppressed, with
effects similar to that previously observed with PTHrP
(41).
Similar to its effects on colx expression, TGF-ß1 also
suppressed alkaline phosphatase activity, another important marker of
chondrocyte differentiation. The inhibition of alkaline phosphatase
activity was dose dependent, with maximal effects also occurring at a
concentration of 3 ng/ml in cultures treated for 72 h (Fig. 1C).
Thus, TGF-ß1 is a potent inhibitor of chondrocyte
differentiation.
TGF-ß1 signals through Smad2 and 3 in chondrocytes
Smad activation was evaluated to determine a potential role for
these downstream targets as effectors of TGF-ß1 signaling in
chondrocytes. Upper sternal chondrocytes were transiently transfected
with the TGF-ß responsive p3TP-Lux reporter, which has been shown to
be transcriptionally activated by Smad3 (30, 42). TGF-ß1
caused a dose-dependent induction of luciferase activity that was
significant by 0.1 ng/ml (P 
0.005) and maximal
between 3 and 5ng/ml (Fig. 2A). In
contrast, BMP-2 (50 ng/ml) did not stimulate luciferase activity,
consistent with the selectivity of this reporter for TGF-ß1 mediated
signals in sternal chondrocytes, and suggesting a role for Smad3 in
mediating TGF-ß1 effects in chondrocytes.
To further define the role of Smad3 in the TGF-ß1 signaling pathway,
p3TP-Lux transfected cells were cotransfected with wild-type or
dominant negative Smad3, or a control vector (Fig. 2B). In the
controls, TGF-ß1 caused approximately a 6-fold increase in luciferase
activity. Transfection with wild-type Smad3 markedly stimulated
p3TP-Lux reporter activity even in the absence of TGF-ß1, thus
mimicking the effects we observed in the TGF-ß1-treated controls.
Furthermore, wild-type Smad3 potentiated TGF-ß1 effects, doubling the
response to TGF-ß1 (Fig. 2B).
To confirm the role of Smad3 as a downstream effector of TGF-ß1
signaling, chondrocytes were transfected with the dominant negative
Smad3 signaling molecule. Transfection with a C-terminally truncated
dominant negative Smad3 (Smad3c) inhibited both basal p3TP-Lux
luciferase activity and diminished the stimulatory effects of TGF-ß1
in chondrocytes, suggesting that Smad3 is essential for TGF-ß1 signal
transduction in chondrocytes.
In contrast to Smad3, Smad2 and Smad2c had minimal effects on
TGF-ß1 stimulated p3TP-Lux activity (Fig. 2C). These findings are
consistent with the structural differences between Smad2 and 3 that
make the p3TP-Lux reporter unresponsive to Smad2 (42) but
do not exclude Smad2 as a potential mediator of TGF-ß1 signaling in
chondrocytes.
We also investigated the effect of Smad1, which has been described as a
mediator of BMP signaling, on the TGF-ß1-induced activation of
p3TP-Lux (Fig. 2D). Smad1 overexpression in chondrocytes did not
activate p3TP-Lux either in control or TGF-ß1 treated cultures, and
in fact, inhibited TGF-ß1 stimulated luciferase activity. We also
found that the Smad1c construct did not alter luciferase activity.
The results obtained with Smad1 suggest that BMP-specific Smad
molecules may inhibit TGF-ß1 mediated signaling events.
To further establish the pathway-specific activation of both Smad2 and
3 by TGF-ß1, we transfected upper sternal chondrocytes with
constructs expressing Flag-tagged Smad2 and 3 (Fig. 3). In control cultures, Smad2 and 3
localized to the cytoplasm, as detected by immunofluorescent staining
(Fig. 3, A and D). Treatment with TGF-ß1 resulted in the nuclear
localization of Smad2 and 3 (B and E). In contrast, following treatment
with BMP-2, Smad2 and 3 remained in the cytoplasm (C and F). Thus,
Smad2 and 3 are selectively activated by TGF-ß1 in sternal
chondrocytes.
|
). Subsequent experiments were
performed to examine the functional effects of overexpression of the
various Smad constructs on the maturational markers, type X
collagen, and alkaline phosphatase activity.
|
shows a representative experiment, whereas Fig. 4C
demonstrates the mean relative expression of type X collagen in three
independent experiments from retrovirally infected cultures. The
experiments demonstrate that both Smad2 and Smad3 regulate type X
collagen expression in chondrocytes and potentiate the effects of
TGF-ß1. The basal level of colx was reduced in cultures
infected with the Smad3 viral expression vector, and colx
expression was completely inhibited following the addition of TGF-ß1
(lanes 7–8 vs. 1–2). Similar, but less pronounced effects
were observed in cultures infected with the Smad2 viral expression
vector (lanes 3–4 vs. 1–2). In contrast, both dominant
negative Smad2 and 3 slightly increased basal colx mRNA
levels and blocked the inhibitory effect of TGF-ß1 on colx
(lanes 5–6, 9–10 vs. 1–2). Consistent with the effects of
the wild-type constructs, Smad3c was more potent than Smad2c.
Uninfected chondrocytes had similar levels of colx
expression as the control virus infected cultures, indicating that
viral infection did not in itself appreciably affect maturation (lanes
1 and 11, Fig. 4B).
Additional experiments were performed examining the effect of Smad2 and
3 on alkaline phosphatase activity in virally infected upper sternal
chondrocyte cultures. Similar to the inhibitory effects observed with
TGF-ß1, both Smad2 and 3 down-regulated alkaline phosphatase activity
and the effect was further enhanced in the presence of TGF-ß1 (Fig. 4D). Collectively, these findings suggest that both Smad2 and 3 play
important roles as mediators of TGF-ß1 effects on chondrocyte
differentiation.
|
Discussion |
|---|
|
TopAbstractIntroductionTGF-{beta} signals through...Materials and MethodsResultsDiscussionReferences |
|---|
Prior studies have supported a role for TGF-ß as an important regulator of chondrocyte differentiation. TGF-ß has been shown to inhibit the expression of markers of a differentiated chondrocyte phenotype in a number of different cell culture systems, including rat epiphyseal chondrocytes (46), chicken growth plate chondrocytes (47), and chick cephalic sternal chondrocytes (9). Furthermore, TGF-ß inhibits chondrocyte differentiation in embryonic mouse metatarsal organ cultures (11), whereas chondrocytes undergo maturation at an accelerated rate in the growth plate of mutant mice expressing a dominant negative TGF-ß receptor (10). Our findings suggest that Smad2 and 3 signaling is the molecular basis for the inhibitory effect of TGF-ß on chondrocyte differentiation. In contrast, the overexpression of the BMPspecific signaling molecule, Smad1, inhibited activation of the TGF-ß responsive reporter. This suggests that the previously reported BMP-mediated stimulation of chondrocyte maturation may be due in part to interference with TGF-ß signaling, as has been described in other cell types (18).
The PTHrP/Ihh signaling pathway has also been shown to regulate the rate of chondrocyte differentiation in the growth plate (5). Recent data suggests that TGF-ß may exert its effects on differentiation, in part, by increasing the expression of PTHrP. We have recently demonstrated that TGF-ß induces the expression of PTHrP in cultured chick epiphyseal cells (12). Similarly, it has been shown that TGF-ß induces the expression of PTHrP in the peri-articular region of embryonic metatarsal organ cultures, and TGF-ß’s inhibition of chondrocyte maturation was dependent on this effect (11). As effectors of TGF-ß mediated inhibition of chondrocyte maturation, Smad2 and 3 may act to induce downstream targets such as PTHrP, thus integrating TGF-ß in the PTHrP/Ihh signaling loop regulating chondrocyte development.
TGF-ß has been shown to regulate the expression of target genes through other, associated signaling pathways, such as the mitogen-activated protein kinase family (MAP kinase) cascade. TGF-ß activates p44/42 MAP Kinase (ERK) in a variety of cells, including articular chondrocytes (24, 48), as well as c-Jun N-terminal kinase (JNK) and p38 MAP kinase (49, 50). Recently, Erk kinases have been shown to inhibit Smad1, 2, and 3 signaling by phosphorylating these molecules within their linker domain and thereby preventing nuclear translocation (23, 51). In contrast, Smads have been reported to undergo activating phosphorylation at the linker domain by JNK (52, 53). Therefore, TGF-ß not only activates Smad signaling, but also parallel pathways with both independent actions as well as modulatory effects on Smad-mediated signal transduction. Future studies investigating the effects of MAP kinase signaling cascades on chondrocyte maturation as well as their interaction with Smads will shed light on the intricate mechanisms regulating chondrocyte physiology.
In conclusion, here we demonstrate that both Smad2 and 3 are key mediators of chondrocyte maturation. Prior work suggests that TGF-ß is upstream of other important regulators of chondrogenesis like PTHrP. Thus, it is critical that future work identifies the downstream targets directly regulated by Smad signaling molecules. As the signaling mechanisms regulated by TGF-ß and their interactions with other growth factors and signaling pathways are defined, a clear picture of the events involved in the regulation of chondrocyte maturation during endochondral bone formation will emerge.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received April 14, 2000.
|
References |
|---|
|
TopAbstractIntroductionTGF-{beta} signals through...Materials and MethodsResultsDiscussionReferences |
|---|
B
. Mol Cell Biol 16:3554–3559[Abstract]
B-mediated inhibition of
apoptosis is required for encephalomyocarditis virus virulence: a
mechanism of resistance in p50 knockout mice. J Virology 72:5654–5660This article has been cited by other articles:
 |
|
 |
|
  D. Y. DAO, X. YANG, D. CHEN, M. ZUSCIK, and R. J. O'KEEFE Axin1 and Axin2 Are Regulated by TGF- and Mediate Cross-talk between TGF- and Wnt Signaling Pathways Ann. N.Y. Acad. Sci., November 1, 2007; 1116(1): 82 - 99. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-F. Dong, D. Y. Soung, Y. Chang, M. Enomoto-Iwamoto, M. Paris, R. J. O'Keefe, E. M. Schwarz, and H. Drissi Transforming Growth Factor-{beta} and Wnt Signals Regulate Chondrocyte Differentiation through Twist1 in a Stage-Specific Manner Mol. Endocrinol., November 1, 2007; 21(11): 2805 - 2820. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Zheng, H.-J. Baek, G. Karsenty, and M. J. Justice Filamin B represses chondrocyte hypertrophy in a Runx2/Smad3-dependent manner J. Cell Biol., October 3, 2007; 178(1): 121 - 128. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Frazier, R. Thomas, M. Scicchitano, R. Mirabile, R. Boyce, D. Zimmerman, E. Grygielko, J. Nold, A.-C. DeGouville, S. Huet, et al. Inhibition of ALK5 Signaling Induces Physeal Dysplasia in Rats Toxicol Pathol, February 1, 2007; 35(2): 284 - 295. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T.-F. Li, D. Chen, Q. Wu, M. Chen, T.-j. Sheu, E. M. Schwarz, H. Drissi, M. Zuscik, and R. J. O'Keefe Transforming Growth Factor-beta Stimulates Cyclin D1 Expression through Activation of beta-Catenin Signaling in Chondrocytes J. Biol. Chem., July 28, 2006; 281(30): 21296 - 21304. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Furumatsu, M. Tsuda, N. Taniguchi, Y. Tajima, and H. Asahara Smad3 Induces Chondrogenesis through the Activation of SOX9 via CREB-binding Protein/p300 Recruitment J. Biol. Chem., March 4, 2005; 280(9): 8343 - 8350. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Scheijen, M. Bronk, T. van der Meer, and R. Bernards Constitutive E2F1 Overexpression Delays Endochondral Bone Formation by Inhibiting Chondrocyte Differentiation Mol. Cell. Biol., May 15, 2003; 23(10): 3656 - 3668. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. T. Ballock and R. J. O'Keefe The Biology of the Growth Plate J. Bone Joint Surg. Am., March 31, 2003; 85(4): 715 - 726. [Full Text] [PDF]
|
|
|
|
|
|
M. Shen, E. Yoshida, W. Yan, T. Kawamoto, K. Suardita, Y. Koyano, K. Fujimoto, M. Noshiro, and Y. Kato Basic Helix-loop-helix Protein DEC1 Promotes Chondrocyte Differentiation at the Early and Terminal Stages J. Biol. Chem., December 13, 2002; 277(51): 50112 - 50120. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
