Regulation of CXCL12 expression by canonical Wnt signaling in bone marrow stromal cells

https://doi.org/10.1016/j.biocel.2011.01.021Get rights and content

Abstract

CXCL12 (stromal cell-derived factor-1, SDF-1), produced by stromal and endothelial cells including cells of the bone marrow, binds to its receptor CXCR4 and this axis regulates hematopoietic cell trafficking. Recently, osteoclast precursor cells were found to express CXCR4 and a potential role for the CXCL12–CXCR4 axis during osteoclast precursor cell recruitment/retention and development was proposed as a regulator of bone resorption. We examined the role of canonical Wnt signaling in regulating the expression of CXCL12 in bone marrow stromal cells. In mouse stromal ST2 cells, CXCL12 mRNA was expressed, while its expression was reduced in Wnt3a over-expressing ST2 (Wnt3a-ST2) cells or by treatment with lithium chloride (LiCl). Wnt3a decreased CXCL12 levels in culture supernatants from mouse bone marrow stromal cells. The culture supernatant from Wnt3a-ST2 cells also reduced migratory activity of bone marrow-derived cells in a Transwell migration assay. Silencing of glycogen synthase kinase-3β decreased CXCL12 expression, suggesting that the canonical Wnt signaling pathway regulates CXCL12 expression. In a transfection assay, LiCl down-regulated the activity of a reporter gene, a 1.8 kb fragment of the 5′-flanking region of the CXCL12 gene. These results show that canonical Wnt signaling regulates CXCL12 gene expression at the transcriptional level, and this is the first study linking chemokine expression to canonical Wnt signaling.

Introduction

The Wnt secreted proteins of molecular weight ∼40 kDa are a family of glycosylated-lipid-modified proteins which are powerful regulators of embryonic development, cell differentiation, and proliferation (Logan and Nusse, 2004, Sethi and Vidal-Puig, 2010). Two distinct Wnt signaling pathways, the β-catenin-dependent canonical pathway and the β-catenin-independent so-called “noncanonical” pathway, including the Wnt/planar cell polarity pathway and the Wnt/Ca2+ pathway, have been described. Two types of Wnt proteins have also been identified, one class of which comprises the canonical Wnts such as Wnt1 and Wnt3a. The other class is the “noncanonical” Wnts such as Wnt5a and Wnt11 which act independently of or inhibit β-catenin signaling (Logan and Nusse, 2004). According to the model of canonical Wnt action, in cells lacking a Wnt signal, glycogen synthase kinase (GSK)-3β phosphorylates β-catenin, inducing rapid degradation of β-catenin via the ubiquitin–proteasome pathway (MacDonald et al., 2009, Sethi and Vidal-Puig, 2010). Canonical Wnt signaling causes stabilization of β-catenin which then translocates to the nucleus, where it interacts with the transcription factors that regulate expression of several target genes including c-myc and osteoprotegerin (OPG) (MacDonald et al., 2009, Sato et al., 2009).

The Wnt signaling pathway has also been reported to be involved in regulation of bone formation (Krishnan et al., 2006, Liu et al., 2008, Tamura et al., 2010). Loss of function of β-catenin in osteoblasts leads to low bone mass caused by increased numbers of osteoclasts resulting in increased bone resorption, indicating enhanced osteoclastogenesis from hematopoietic progenitor cells (Glass et al., 2005). Previously, we have shown that canonical Wnt signaling induces OPG expression and analysis of the murine OPG gene promoter revealed that constitutively active forms of β-catenin regulate transcription of OPG via a promoter region containing two responsive sites (Sato et al., 2009). In addition, canonical Wnt signaling reduces receptor activator of NFkB ligand (RANKL) expression (Spencer et al., 2006, Sato et al., 2009). These observations suggest that canonical Wnt signaling regulates bone turnover via osteoclast formation from hematopoietic progenitor cells.

The Wnt signaling pathway plays a role in the regulation of hematopoiesis in bone marrow. Activation of canonical Wnt signaling enhances hematopoietic stem cell (HSC) self-renewal (Reya and Clevers, 2005, Staal and Luis, 2010). In contrast, mice with conditional inactivation of β-catenin show normal hematopoietic cell development and repopulating activity of HSC (Cobas et al., 2004, Staal and Luis, 2010). These controversial results indicate that the precise role of canonical Wnt signaling in bone marrow remains unclear. Recently Kim et al. (2009) reported that the population of canonical Wnt receptor molecules and β-catenin accumulation were predominantly enriched in the stromal rather than the hematopoietic compartment of bone marrow (Kim et al., 2009). Therefore, we investigated the role of canonical Wnt function in bone marrow stromal cells.

Hematopoiesis and mobilization of mature blood cells into the bloodstream are dependent on various types of chemokines present in the bone marrow. Chemokines are small molecular weight molecules that function as chemoattractants and serve as regulators of blood cell maturation, trafficking and homing (Schall and Bacon, 1994, Howard et al., 1996). CXCL12 (stromal derived factor: SDF-1α) and SDF-1β belong to the C-X-C chemokine family and were originally isolated from a murine bone marrow stromal cell line (Broxmeyer, 2008). Both molecules are derived by alternative splicing of the CXCL12 gene and share similar biological activities. CXCL12 is widely produced by stromal and endothelial cells including bone marrow, skeletal muscle, liver, brain and heart, and CXCL12 binds predominantly to a G-protein coupled transmembrane glycoprotein cell surface chemokine receptor, CXCR4, widely expressed by several types of tissue-committed stem cells including HSC or hematopoietic progenitor cells (Ratajczak et al., 2006). It has been reported that CXCL12–CXCR4 interactions mediate to maintain HSC niches on the endosteal surface of bone; the CXCL12–CXCR4 axis is involved in the homeostatic release of HSC from the bone marrow into the circulation, and also maintains the HSC pool in the bone marrow microenvironment, the so-called stem cell niche (Petit et al., 2002). In vitro, a variety of cytokines and signaling molecules such as interleukin-1β, platelet-derived growth factor-BB, transforming growth factor (TGF)-β1 and tumor necrosis factor-α stimulate CXCL12 expression and protein secretion (Jung et al., 2006). However, the regulation of CXCL12 production by Wnt signaling remains poorly understood.

In this report, we demonstrate that canonical Wnt signaling regulates CXCL12 production via the CXCL12 gene promoter in bone marrow stromal cells.

Section snippets

Cell cultures

Cells of the mouse stromal cell line ST2 (RIKEN Cell Bank, Tsukuba, Japan) were cultured in α-MEM containing 100 μg/ml of kanamycin (Meiji, Tokyo, Japan) and supplemented with 10% fetal bovine serum (FBS; SAFC Biosciences, Inc. Lenexa, KS) at 37 °C in 100-mm cell culture dishes (Corning, Corning, NY) in a humidified atmosphere of 5% CO2 in air. For osteoblastic differentiation of ST2 cells, the cells were cultured in α-MEM containing both 5 mM β-glycerophosphate and 100 μg/ml of ascorbic acid for 2

Wnt3a down-regulates CXCL12 expression in ST2 cells

To evaluate a potential role for canonical Wnt signaling in regulating CXCL12 expression of bone marrow stromal cells, we generated ST2 cell lines that expressed either Wnt3a (Wnt3a-ST2 cells), which stimulates canonical Wnt signaling, or non-canonical Wnt5a (Wnt5a-ST2 cells). As shown in Fig. 1A, while untransfected ST2 cells did not produce any detectable expression of Wnt3a or Wnt5a, Wnt3a-ST2 cells and Wnt5a-ST2 cells expressed high levels of Wnt3a and Wnt5a mRNA, respectively, as detected

Discussion

In bone marrow, CXCL12 appears to be constitutively expressed by stromal and endothelial cells. In the present study, we used the stromal cell line ST2, derived from mouse bone marrow, to examine CXCL12 expression. In normal culture, ST2 cells have the characteristics of preadipocytes and none of the features typical of the osteoblastic phenotype (Yamaguchi et al., 1996). However ST2 cells cultured with ascorbic acid exhibited characteristics typical of osteoblasts with the formation of

Acknowledgement

This study was supported in part by the Japan Ministry of Education, Culture, Sports, Science and Technology Grants-in-aid #22390346 (MT).

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