Elsevier

Bone

Volume 36, Issue 4, April 2005, Pages 585-598
Bone

Gene array analysis of Wnt-regulated genes in C3H10T1/2 cells

https://doi.org/10.1016/j.bone.2005.01.007Get rights and content

Abstract

Wnt/β-catenin signaling is involved in a large variety of modeling and remodeling processes including cell polarity, cell differentiation, and cell migration. Recently, a role of the Wnt pathway in bone biology has been demonstrated. However, the precise mechanism by which Wnt proteins regulate bone formation still remains to be elucidated. We have previously shown that the Wnt pathway mediates induction of alkaline phosphatase, an osteoblast differentiation marker, in the pluripotent mesenchymal cells C3H10T1/2. In the present study, we performed a genome-wide expression analysis using Affymetrix oligonucleotide chips to determine the Wnt3a-induced gene expression profile in C3H10T1/2 cells. The expression profiles of 447 Wnt3a-regulated genes, classified into distinct functional families, are presented here. Our data reveal that Wnt3a regulates several genes that are involved in osteoblast and adipocyte differentiation. Importantly, Wnt3a induces the expression of osteoprotegerin by a β-catenin dependent mechanism indicating that the Wnt pathway may also affect osteoclastogenesis. Through the analysis of our expression profiling data, we have established a TaqMan panel as a tool to rapidly compare the expression profiles of a specific set of genes induced by distinct stimuli acting in the Wnt/β-catenin pathway. Using the TaqMan panel, we have compared the gene expression profiles induced by Wnt1, Wnt2, and Wnt3a in C3H10T1/2 cells, and also by two different GSK-3β inhibitors: LiCl and SB216773. Our data show that Wnt1 and Wnt3a act in a similar manner, distinct from Wnt2. Finally, we found that LiCl and SB216773 displayed different profiles in the TaqMan panel evidencing their distinct inhibitory action toward GSK-3β. Overall, data presented herein will aid further understanding of the involvement of the Wnt signaling pathway in its regulation of osteoblast and adipocyte differentiation and function and, in addition, will enhance current knowledge of the Wnt signaling pathway itself.

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.

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