Download the wnt antagonist sfrp1 is expressed in epiphyseal cartilage and

THE WNT ANTAGONIST SFRP1 IS EXPRESSED IN EPIPHYSEAL CARTILAGE AND FUNCTIONS AS A
NEGATIVE REGULATOR OF CHONDROCYTE MATURATION
*Rich, L; **Lengner, C; **Gaur, T; ** Trevant, B; ***Bodine, P; ***Komm, B; *Ayers, D; **Stein, G S; +**Lian, J B
*Department of Orthopedic Surgery, +**Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, MA
[email protected]; [email protected]
INTRODUCTION:
The Wnt family of at least 22 cysteine rich secreted glycoproteins
participates in key developmental processes including axis determination
in early embryos, tissue induction and regulation of cellular
differentiation. A number of Wnts are expressed in the developing
skeleton having distinct roles in formation of cartilage and bone (1-4).
Wnt signaling is activated by binding of Wnt proteins to a Frizzled
receptor complex and is highly regulated by Wnt antagonists during
limb skeletogenesis (2). A small family of secreted frizzled-related
proteins (sFRPs) has been identified. These proteins possess a cysteine
rich domain and thus can interact with the frizzled receptor to block Wnt
binding to its receptors and signaling. Recently, a knock-in mutation of
SFRP-1 in the mouse identified its importance as a negative regulator of
bone formation. In the absence of SFRP-1, trabecular bone mass was
significantly greater than WT in mice over 6 month of age due to a delay
in age-related bone loss. Since previous studies identified SFRP-1 by in
situ hybridizations in the early embryo to be expressed in many
developing tissues, we addressed sFRP-1 expression and regulation of
the Wnt pathway during chondrogenesis in vivo and in an ex vivo model
of chondrocyte differentiation.
METHODS:
To investigate the regulatory role of Wnt antagonist sFRP-1 and
Wnt signaling in chondrocytes, we examined an SFRP-1 knock-in
mutant mouse model in which the lac Z reporter gene was inserted in
exon 1 resulting in an inactive Sfrp-1 allele (sFRP1lz/lz) (5). Thus -gal
staining of embryos and tissue sections revealed expression of the sFRP1 gene in its native locus. Whole embryos characterization was
performed by soft xrays, Alizarin red/Alcian blue and histological
analysis of frozen sections. We examined the effects of sFRP-1 on
chondrocyte differentiation using a novel ex-vivo model in which
mouse embryo fibroblasts (MEFs) were induced to a chondrocyte
phenotype by plating in micro-mass and treating with 100ng/ml BMP2
(5). Cells are harvested at 0, 3, 6, 9 and 12 days after inducing
chondrogenesis for preparation of total cellular RNA. Expression of
cartilage phenotypic genes was determined by RT-PCR.
RESULTS:
During embryogenesis, the sFRP1lz/lz mouse developed a normal
appearing skeleton based on Alizarin red and Alcian blue staining of
skeletal elements. SFRP-1 expression was detected in skeletal tissues
initially in association with the developing scapula and the epiphysis of
developing limbs. At later stages (E 16.5-birth), sFRP-1 was expressed
in vertebrae and growth plate and -gal activity remained in the
epiphyses after birth. In histological sections, a pattern of robust sFRP-1
expression was found in articular cartilage defining the limits of the joint
surface, indicating that sFRP-1 may play a role in formation of
cartilaginous structure of the limb. To address a functional role for
sFRP-1 in regulating chondrogenesis, mouse embryo fibroblasts were
prepared at E 12 from Wt and sFRP-1lz/lz embryos and induced to
undergo chondrocytic differentiation with BMP2 and high density
culture. WT MEFs exhibit a sequential expression of chondrogenic
genes in response to BMP2 treatment, including an induction of the prochondrogenic transcription factor Sox9 and the cartilaginous ECM
protein Collagen Type II by 3 days of culture. Between 8 and 12 days of
culture, there is an induction of genes associated with the hypertrophic
phenotype, including Collagen Type X, Alkaline Phosphatase, and
Indian Hedgehog. In this system, sFRP-1 expression is induced in
response to high density culture throughout the time course. Upon
addition of BMP2 to these cultures, sFRP-1 expression is suppressed,
suggesting a role for sFRP-1 in limiting the rate of chondrocyte
maturation. Upon chondrogenic differentiation of sFRP-1lz/lz MEFs, we
observed an earlier onset of chondrogenesis in the presence of BMP2,
and surprisingly found that Collagen Type II gene expression was
induced in the absence of both BMP2 and sFRP-1. These results suggest
that BMP2 signaling is acting upstream of sFRP-1 in order to suppress
the potentially anti-chondrogenic activity of this Wnt antagonist.
DISCUSSION:
Specific Wnt family members have been identified as critical for
chondrogenic pattern formation in the developing embryo (1-4). For
example, Wnt14 plays a critical role in initiating joint formation (7).
Several in vitro studies have also shown that specific Wnts can either
promote or inhibit chondrogenesis and influenced by the stage of
chondrogenesis (1). In addition, BMP2, an inducer of mesenchymal cell
recruitment into the chondrogenic lineage, regulates specific Wnts and
frizzled receptors (8,9). Thus regulation of Wnt signaling is critical for
regulation of chondrocyte differentiation. Our findings of the sFRP-1
Wnt antagonist highly expressed by chondrocytes in the epiphyses,
underscores the importance of regulating Wnt signal for progression of
chondrogenesis. We have provided direct evidence from analysis of the
SFRP-1 knockout mouse that SFRP-1 may (i) contribute to
specification of the articular surface for joint formation based on its
expression pattern; and (ii) regulate the induction of chondrocyte
differentiation as cells lacking sFRP-1 proceed into chondrogenesis in
the absence of the BMP2 signals. Lastly, our data suggests that BMP2
signaling pathways regulate the anti-chondrogenic activity of sFRP-1.
Taken together, these findings also suggest a potential therapeutic
approach for promoting chondrogenesis by blocking activity of sFRP-1,
a secreted soluble Wnt antagonist factor.
REFERENCES:
1.
Church V, Nohno T,Linker C, Marcelle C, Francis-West P. 2002. J
Cell Sci, 115:4809-18.
2.
Hartmann C, Tabin CJ. 2000. Development, 127:3141-59.
3.
Kawakami Y, Wada N, Nishimatsu SI, Ishikawa T, Noji S, Nohno
T. 1999. Dev Growth Differ, 41:29-40.
4.
Rudnicki JA, Brown AM. 1997. Dev Biol, 185:104-118.
5.
Bodine PV, Zhao W, Kharode YP, Bex FJ, Lambert AJ, Goad MB,
Gaur T, Stein GS, Lian JB, Komm BS. 2004. Mol Endocrinol,
18:1222-37
6.
Lengner CJ, Lepper C, van Wijnen AJ, Stein JL, Stein GS, and
Lian JB. 2004. J Cell Physiol, in press
7.
Hartmann 2001 Hartmann C, Tabin CJ. 2001. Cell 104:341-351
8.
Fischer L, Boland G, Tuan RS. 2002. J Cell Biochem, 84:816-831.
9.
Chimal-Monroy J, Montero JA, Ganan Y, Macias D, GarciaPorrero JA, Hurle JM. 2002. Dev Dyn, 224:314-320.
10. Enomoto-Iwamoto M, Kitagaki J, Koyama E, Tamamura Y, Wu C,
Kanatani N, Koike T, Okada H, Komori T, Yoneda T, Bodine PV,
Zhao W, Kharode YP, Bex FJ, Lambert AJ, Goad MB, Gaur T,
Stein GS, Lian JB, Komm BS. 2004. Mol Endocrinol 18:12221237.
AFFILIATED INSTITUTIONS FOR CO-AUTHORS:
*** Women’s Health Research Institute, Wyeth Research, Collegeville,
PA
51st Annual Meeting of the Orthopaedic Research Society
Poster No: 1438