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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 18, 2001 as doi:10.1096/fj.00-0784fje. |
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Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
2Correspondence: Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, Thomas Jefferson University, 1015 Walnut St., Room 501. Philadelphia, PA 19107, USA. E-mail: Rocky.S.Tuan{at}mail.tju.edu
SPECIFIC AIM
In this study, we have examined the mechanism of action of the Wnt family members of signaling molecules in the regulation of limb mesenchymal chondrogenesis. Using micromass cultures of chick limb bud mesenchymal cells in vitro, we analyzed the effects of retrovirally mediated mis-expression of Wnt-5a, the primary member of the proposed Wnt-5a class expressed in limb mesenchyme, and Wnt-7a, a proposed Wnt-1 class member expressed in the dorsal ectoderm of the limb bud, on the early events of chondrogenesis, i.e., cellular condensation and differentiation, particularly with respect to N-cadherin-related activities.
PRINCIPAL FINDINGS
1. Mis-expression of Wnt-7a, not Wnt-5a, inhibits
chondrogenesis in vitro
Chondrogenesis is inhibited in day 3 micromass cultures
transfected with RCAS-Wnt-7a or RCAS-Wnt-5a/RCAS-Wnt-7a, but not with
RCAS-Wnt-5a alone, based on Alcian blue staining, collagen type II
immunohistochemistry, and metabolic
[35S]sulfate incorporation.
2. Effect of Wnt mis-expression on chondrogenesis does not
involve cellular condensation and the formation of precartilage
aggregates
Binding of peanut agglutinin lectin, a characteristic of
condensing precartilage cells, is similar in all three groups of
transfected cultures—RCAS vector only, RCAS-Wnt-5a, and
RCAS-Wnt-7a—at 24 and 48 h, suggesting that Wnt-7a is unlikely to
affect the cellular condensation phase of the chondrogenic program, but
instead probably acts at the ‘overt differentiation’ stage.
3. Wnt-7a mis-expression affects N-cadherin expression
In control micromass cultures transfected with RCAS empty
vector (Fig. 1A
, D
, G
, J
, M
, P
), N-cadherin is immunodetected by 12 h
in aggregating and/or condensing regions of the culture, whereas cells
surrounding the mesenchymal aggregates are negative (Fig. 1A
); N-cadherin staining intensity increases as a function
of culture time and is maximal around 24–36 h (Fig. 1D
, G
),
with N-cadherin positive cells eventually adding to the developing
precartilage nodules (Fig. 1M
). By 36 h (Fig. 1G
), cartilaginous nodules appear and N-cadherin expression
is down-regulated, starting from the mostly differentiated central
regions of the culture, whereas the peripheral areas remain N-cadherin
positive. Finally, by 48 h (Fig. 1J
),
N-cadherin expression is almost completely down-regulated except for
the very peripheral edges of the culture, representing less mature
cells. In individual nodules (Fig. 1P
), a similar pattern of
N-cadherin down-regulation was also observed in the centrally located
chondrocytes, whereas perinodular cells remained positive. However, in
RCAS-Wnt-7a transfected cultures, N-cadherin expression within the
cellular aggregates is delayed at 12 h (Fig. 1C
),
becoming more diffusely oriented but localized to the condensation
areas throughout the culture at 24 h (Fig. 1F
), and followed by persistent, high levels of
expression at 36 h (Fig. 1I
) and 48 h
(Fig. 1L
, R
). On the other hand, RCAS-Wnt-5a cultures showed
N-cadherin expression patterns very similar to the control cultures at
all time points (Fig. 1B
, E
, H
, K
, N
, Q
). Western and
Northern blot analyses confirmed these results.
|
4. Effect of Wnt-5a and Wnt-7a mis-expression on collagen
type II promoter activity and Sox9 gene expression
Cultures were cotransfected with the RCAS-Wnt constructs
and a Col2
1 promoter-luciferase construct. Col21 promoter
activity was low in all three cotransfection groups on day 1. Upon
initiation of chondrogenic differentiation on day 2 and during
progression of chondrogenesis on day 3, Col21 promoter activity
gradually increased in RCAS empty vector cotransfected control cultures
and RCAS-Wnt-5a cotransfected cultures, whereas that in RCAS-Wnt-7a
cotransfected cultures remained persistently low. Northern analysis of
Sox9 gene expression, a regulator of Col21 promoter, showed
comparable expression in all culture groups.
5. Effect of Wnt-5a and Wnt-7a mis-expression on
ß-catenin/N-cadherin cell adhesion complex stabilization and
phosphorylation
ß-Catenin, a major component of the Wnt signaling
pathway, accumulates in the cytoplasm when the Wnt signaling pathway is
active and can participate in the N-cadherin dependent cell–cell
adhesion and/or activation of nuclear target gene transcription.
Although total ß-catenin protein levels are comparable on culture
days 1, 2, and 3 in all three culture groups (Fig. 2A
), the gradual down-regulation of the high levels of
ß-catenin-bound N-cadherin present at the condensation phase seen in
RCAS and RCAS-Wnt-5a cultures is absent in RCAS-Wnt-7a cultures,
which showed a persistent level of ß-catenin-bound N-cadherin even on
days 2 and 3 (Fig. 2B
), indicating stabilization of the
ß-catenin/N-cadherin cell adhesion complex at the plasma membrane.
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The serine/threonine phosphorylation state of the cadherins and their
associated cytoplasmic proteins, the catenins, was also altered upon
Wnt-7a mis-expression. In both RCAS and RCAS-Wnt-5a cultures,
N-cadherin is constitutively phosphorylated on mainly serine residues
on days 1 and 2, i.e., at the time of active N-cadherin-dependent cell
adhesion during cellular condensation (Fig. 2C
). On day 3,
as chondrogenic differentiation proceeds, the level of N-cadherin
serine phosphorylation decreased, indicative of the turnover of
N-cadherin and N-cadherin-dependent cell adhesion complex (Fig. 2C
). In Wnt-7a cultures, N-cadherin showed a gradual
increase in threonine phosphorylation accompanying serine
phosphorylation on days 1 and 2 (Fig. 2D
); a high level of
serine/threonine phosphorylation persisted even on day 3, suggesting
inhibition of N-cadherin turnover in Wnt-7a transfected cultures (Fig. 2C
, D
).
ß-Catenin serine/threonine phosphorylation, on the other hand,
was high in RCAS and RCAS-Wnt-5a cultures on day 1 and decreased on
days 2 and 3 (Fig. 2E
), consistent with N-cadherin-mediated
cell–cell adhesion being stabilized through ß-catenin during
condensation and subsequently down-regulated during overt chondrogenic
differentiation. However, Wnt-7a cultures showed a gradual increase in
ß-catenin serine/threonine phosphorylation throughout the 3-day
culture period (Fig. 2E
).
6. Wnt-7a mis-expression stimulates limb mesenchymal cell
proliferation
BrdU labeling showed that cell proliferation in Wnt-7a
cultures was strongly stimulated at all time points when compared with
the RCAS empty vector control cultures.
CONCLUSIONS AND SIGNIFICANCE
The Wnt family of secreted glycoproteins has been suggested to consist of two major functional groups: the Wnt-1 class and the Wnt-5a class. Our results showed that Wnt-5a, the primary member of the Wnt-5a class, and Wnt-7a, a proposed Wnt-1 class member, act as functionally divergent Wnt family members in the regulation of limb mesenchymal chondrogenesis. Mis-expression of Wnt-7a, but not Wnt-5a, strongly inhibits the chondrogenesis of primary chick limb mesenchymal cells in vitro. Wnt-7a inhibition of chondrogenesis is unlikely to involve the condensation step of the chondrogenic program, but instead probably influences the cellular differentiation phase by targeting the expression of N-cadherin and the prolonged stabilization of N-cadherin-mediated cell–cell adhesion. In addition, Wnt-7a mis-expression stimulates limb mesenchymal cell proliferation in vitro, possibly contributing to its chondro-inhibitory effect.
It has been previously suggested that the chondro-inhibitory effect of ectopically expressed Wnt-1 on limb chondrogenesis in vivo and in vitro is due to the mimicking of the activity of another Wnt family member, such as Wnt-5a, that is normally expressed in the limb mesenchyme or, more specifically, in the progress zone, where the mesenchymal cells are kept in a highly proliferative state. Recent in vivo studies have shown that Wnt-5a mis-expression delays the maturation of chondrocytes and collagen type X expression, a marker for hypertrophic chondrocytes, whereas mesenchymal condensation and chondrocyte differentiation actually proceed normally. Our results here on the early phase of chondrogenesis, i.e., the cellular condensation and chondrogenic differentiation events, thus agree with the literature. Furthermore, the late Wnt-5a effect on chondrocyte maturation is unlikely to account completely for the inhibitory effect of exogenous Wnt-1 expression upon early chondrogenic differentiation, although both result in a similar abnormal phenotype of the long bones in vivo, particularly in view of the proposed functional diversity between Wnt-1 and Wnt-5a.
An alternative explanation for the chondro-inhibitory effect of Wnt-1 is that it mimics the activity of a Wnt family member, such as Wnt-7a, that is normally expressed in the dorsal ectoderm of the developing limb, i.e., outside the region of chondrogenesis, and known to interact with the underlying mesenchyme. We thus hypothesize that Wnt-7a acts in vivo as a dorsal ectodermal factor to limit the chondrogenic activity within the limb mesenchyme, in addition to its known involvement in the dorsal-ventral and anterior-posterior limb patterning.
How do Wnt molecules such as Wnt-7a affect mesenchymal chondrogenesis?
One potential mechanism is suggested by the consequences of Wnt
signaling on cell adhesion. During overt chondrogenic differentiation,
the expression of cell adhesion molecules, such as N-cadherin and
N-CAM, is down-regulated and the abundant cell–cell contacts acquired
during the condensation phase, including gap junctions and adherens
junctions, are lost. Wnt-1 class signaling in other vertebrate cell
systems has been shown to induce stabilization of ß- and/or
-catenin, and in some cases leads to increased cell–cell adhesion;
Wnt-1 expression also causes increased gap junction permeability in
Xenopus embryos, possibly as a consequence of
increased adhesion. Therefore, Wnt-7a mis-expression in chondrogenic
cells might prevent the down-regulation of cell–cell adhesion and gap
junction communication required for cells to progress to differentiate
into chondrocytes. The results from N-cadherin immunohistochemistry
(Fig. 1)
and Western blot analysis support this hypothesis.
During mesenchymal chondrogenic differentiation, levels of N-cadherin
mRNA and protein decrease in parallel, whereas the balance
between serine/threonine and tyrosine phosphorylation for both
N-cadherin and ß-catenin regulates the stability and proteolytic
turnover of N-cadherin. Tyrosine phosphorylation of ß-catenin and the
switch from serine/threonine phosphorylation to tyrosine
phosphorylation of N-cadherin are correlated with loss of N-cadherin
function. In addition to the persistent high level of N-cadherin mRNA,
Wnt-7a transfected cultures also showed a persistent high level of
serine/threonine phosphorylation of both ß-catenin and N-cadherin
(Fig. 2C
, D
, E
), suggesting altered balance in the
phosphorylation of these molecules in favor of serine/threonine, which
should subsequently lead to the stabilization of N-cadherin-mediated
cell–cell adhesion.
In conclusion, we have demonstrated here that retrovirally mediated
Wnt-7a mis-expression in chick limb bud micromass cultures targets the
regulation of N-cadherin gene expression and prolongs the stabilization
of the ß-catenin/N-cadherin complex and cell–cell adhesion,
thereby inhibiting chondrogenic differentiation, a process dependent
not only on initial cell–cell interactions, but also their progressive
down-regulation. Stimulation of limb mesenchymal cell proliferation by
mis-expressed Wnt-7a during the course of early condensation and
chondrogenic differentiation may also contribute to the inhibition of
overt chondrogenic differentiation. The observed difference between
Wnt-5a signaling and Wnt-7a signaling, i.e., lack of effect of Wnt-5a
mis-expression on mesenchymal chondrogenesis, may be a result of
differential interaction with specific Frizzled receptor homologues
(Fig. 3
).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0784fje ; to cite this
article, use FASEB J. (April 18, 2001) 10.1096/fj.00-0784fje 
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