Gene array analysis of Wnt-regulated genes in C3H10T1/2 cells
Introduction
Wnt proteins constitute a family of highly conserved secreted glycoproteins that have diverse roles in development and disease. Wnt signaling regulates cell differentiation, cell polarity, cell migration, and cell fate [29], [34]. Inappropriate activation of the canonical Wnt signaling pathway is associated with a high frequency of tumors in specific tissues such as prostate [6] and colon [30]. To date, nineteen different Wnt genes have been identified in the mouse and human genomes (http://www.stanford.edu/rnusse/wntwindow.htlm).
As currently understood, secreted Wnt proteins bind to receptors of the frizzled seven-transmembrane-span family and the low-density lipoprotein (LDL) receptor-related proteins 5 and 6 (LRP5 and LRP6) at the cell surface. In the cell, the Wnt signal is transduced to β-catenin via several different relay proteins leading to the stabilization of cytosolic β-catenin. In the absence of Wnt signaling, β-catenin is phosphorylated by glycogen synthase kinase 3β (GSK3β) and casein kinase I (CKI) [11]. This triggers its ubiquitination by β-transducin repeats-containing proteins (beta-TrCP) and subsequent degradation by the 26S proteasome. In the presence of Wnt, disheveled (dsh) and GBP/Frat block β-catenin degradation resulting in accumulation of cytoplasmic β-catenin. Stabilized β-catenin enters the cell nucleus where it associates with, and activates members of the T-cell transcription factor/lymphoid-enhancer binding factor (TCF/LEF) family, driving transcription of Wnt target genes [26], [27], [56]. In the absence of Wnts, certain TCFs repress transcription by interacting with the co-repressors groucho and C-terminal binding protein (CtBP). The Wnt pathway involving β-catenin is referred to as the canonical Wnt pathway.
Recently, a role for the Wnt/β-catenin signaling pathway has been demonstrated in bone formation where it appears to be an important regulator of bone accrual during growth. Loss of function mutations of the Wnt co-receptor, LRP5, leads to low bone mass accompanied by fractures causing Osteoporosis Pseudoglioma Syndrome (OPPG) in humans and in animal models [13], [22]. LRP5 gain-of-function mutations in humans result in a high bone mass (HBM) trait and fracture resistance [3], [24]. Additional diseases caused by LRP5 gain-of-function mutations in humans include Van Buchem disease type II, autosomal dominant osteosclerosis, endosteal hyperostosis, and osteopetrosis type I [52]. Furthermore, the Wnt pathway has been shown to regulate osteoblast differentiation and function both in vivo and in vitro [36]. Osteoblasts are the fully differentiated skeletal cells responsible for the production of bone matrix. Osteoblasts arise from mesenchymal stem cells which are pluripotential in nature and capable of giving rise to a number of committed and restricted cell lineages including osteoblast, chondroblast, adipoblast, fibroblast, and myoblast lines (for a review, see Triffitt JT, in Principles of Bone Biology [49]). We have recently demonstrated that a number of Wnt proteins are capable of inducing the osteoblast marker (ALP) and inhibiting several adipogenesis markers (PPARγ, CEBP/2α, and aP2) in mesenchymal cells [13], [36]. Moreover, we have also demonstrated that integrity of Wnt signaling is necessary for mineralization in the osteoblastic cell line MC3T3-E1. Nonetheless, the precise mechanism of Wnt action on osteoblast differentiation remains unclear as gene expression of osteoblast characteristic markers such as Runx2, collagen type I, or osteocalcin is unchanged in mesenchymal C3H10T1/2 cells treated with Wnt3a [36]. In order to better understand the gene expression changes that promote osteoblast differentiation in response to Wnt signaling, we have analyzed gene expression profiles induced by Wnt3a in C3H10T1/2 cells using the Affymetrix Murine Genome U74v2 Set encompassing approximately 36,000 full-length genes and EST clusters.
Section snippets
Cell culture
The C3H10T1/2 cell line (obtained from ATCC) was cultured in α-MEM supplemented with 10% heat-inactivated fetal calf serum at 37°C and 5% CO2. For transient transfection, cells were plated at 2 × 104/cm2, and after 24 h, the culture medium was changed to 2% fetal calf serum. Transfections were carried out as described below. Murine L-cells and Wnt3a producing L-cells (obtained from ATCC) were cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum at 37°C and 5% CO2. G418 at
Microarray analysis of Wnt3a-treated C3H10T1/2 cells
C3H10T1/2 is a pluripotent mesenchymal stem cell line that can be manipulated to differentiate into adipocytes, myoblasts, chondrocytes, and osteoblasts [37], [46], [48], [57]. We have previously reported that Wnt proteins capable of stabilizing β-catenin induce the expression of the osteoblast marker alkaline phosphatase (ALP) in C3H10T1/2 cells [36]. In contrast, Wnt3a was shown to inhibit the expression of adipocyte markers such as aP2 and PPARγ2 in the same cells [36]. To further understand
Discussion
The complete set of target genes of the Wnt/β-catenin pathway in mesenchymal cells has not been defined. C3H10T1/2 is a pluripotent mesenchymal cell line that possesses the ability to differentiate into osteoblasts, chondrocytes, as well as adipocytes, depending on culture conditions or treatments. In the present study, we performed microarray analysis of genes regulated in C3H10T1/2 cells stimulated by Wnt3a. Although this data set may provide a global view of genetic program driven by Wnt3a
Acknowledgment
We would like to thank Dr. JM Le Moullec for providing OPG-luciferase and mutated OPG-luciferase reporter constructs.
References (59)
- et al.
A twist code determines the onset of osteoblast differentiation
Dev. Cell
(2004) - et al.
Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription
Chem. Biol.
(2000) - et al.
Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells
Bone
(2001) - et al.
LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development
Cell
(2001) - et al.
CD44 expression in fetal rat bone: in vivo and in vitro analysis
Exp. Cell Res.
(1996) Lithium and GSK-3: one inhibitor, two inhibitory actions, multiple outcomes
Trends Pharmacol. Sci.
(2003)- et al.
Activation of beta-catenin-LEF/TCF signal pathway in chondrocytes stimulates ectopic endochondral ossification
Osteoarthritis Cartilage
(2003) - et al.
A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait
Am. J. Hum. Genet.
(2002) - et al.
Decorin modulates matrix mineralization in vitro
Biochem. Biophys. Res. Commun.
(2003) - et al.
WNTs modulate cell fate and behavior during vertebrate development
Trends Genet.
(1997)
WNT targets: repression and activation
Trends Genet.
Glycogen synthase kinase-3 inhibition by lithium and beryllium suggests the presence of two magnesium binding sites
Biochem. Biophys. Res. Commun.
Expression of galectin-3 in skeletal tissues is controlled by Runx2
J. Biol. Chem.
Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine
Cell
Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor
Cell
Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF
Cell
Six novel missense mutations in the LDL receptor-related protein 5 (LRP5) gene in different conditions with an increased bone density
Am. J. Hum. Genet.
Expression of CD44 in Apc and Tcf mutant mice implies regulation by the WNT pathway
Am. J. Pathol.
Protein kinase C is a mediator of the synthesis and secretion of osteoprotegerin in osteoblast-like cells
Biochem. Biophys. Res. Commun.
ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology; implication for Coffin–Lowry Syndrome
Cell
High bone mass in mice expressing a mutant LRP5 gene
J. Bone Miner. Res.
High bone density due to a mutation in LDL-receptor-related protein 5
N. Engl. J. Med.
Osteoclast differentiation and activation
Nature
Bone morphogenetic proteins, their antagonists, and the skeleton
Endocr. Rev.
Detection and analysis of beta-catenin mutations in prostate cancer
Prostate
Wnt regulation of chondrocyte differentiation
J. Cell Sci.
Wnt signaling during BMP-2 stimulation of mesenchymal chondrogenesis
J. Cell. Biochem.
Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling
J. Cell Biol.
In vivo inhibition of osteoblastic metalloproteinases leads to increased trabecular bone mass
J. Bone Miner. Res.
Cited by (144)
Bone formation and bone repair: The roles and crosstalk of osteoinductive signaling pathways
2022, Process BiochemistryThe cytokine interleukin-11 crucially links bone formation, remodeling and resorption
2021, Cytokine and Growth Factor ReviewsCD8<sup>+</sup> T lymphocytes enhance the anabolic effect of intermittent parathyroid hormone on cementoblasts
2019, International ImmunopharmacologyIron overload as a high risk factor for microgravity-induced bone loss
2019, Acta AstronauticaThe role of magnesium ions in bone regeneration involves the canonical Wnt signaling pathway
2019, Acta Biomaterialia
- 1
Equally contributed.