Downregulation of [Beta]-Catenin and Transdifferentiation of Human Osteoblasts to Adipocytes Under Estrogen Deficiency

By Foo, Clara Frey, Soenke; Yang, Hong Hyun; Zellweger, Rene; Filgueira, Luis

Abstract Background and aim. Postmenopausal osteoporosis, caused by estrogen deficiency, is characterized by the structural deterioration of bone accompanied by an increase in bone marrow adipocytes. Transgenic animal models have shown that there is a reciprocal relationship between osteoblastogenesis and adipogenesis in vivo. The present study investigated whether the estrogen and the canonical Wnt signaling pathways are linked together and regulate the phenotype and function, differentiation and proliferation of human osteoblasts using an in vitro estrogen-deficiency model.

Methods. Human osteoblasts (hFOB 1.19) and fulvestrant, an estrogen receptor blocker, were used to mimic estrogen deficiency in vitro. Osteogenic and adipogenic differentiation was measured by using specific stains and microscopy, as well as by measuring the expression of bone cell-specific markers with reverse transcription polymerase chain reaction. Expression of estrogen receptor-alpha (ERalpha) and beta-catenin was detected in Western blots and by immunoprecipitation.

Results. The cells expressed the 46-kDa and the 77-kDa ERalpha isoforms and beta-catenin. Fulvestrant reduced expression of ERalpha and beta-catenin. beta-Catenin was co-immunoprecipitated with ERalpha, indicating that these two proteins form a new signaling complex and transcription factor. In addition, it induced intracellular lipid droplet accumulation and downregulation of bone cell markers, indicating adipocyte differentiation.

Keywords: Osteoblasts, adipocytes, estrogens, Wnt, beta-catenin

Introduction

Postmenopausal estrogen deficiency leads to osteoporosis, probably due to replacement of cancellous bone by bone marrow adipose tissue. More recent studies suggest that alterations in bone formation and adipogenesis during aging may be a consequence of preferred differentiation from osteoprogenitor cells into adipocytes and reduced differentiation into osteoblasts [1-3]. This association is further supported by the fact that bone marrow adipocytes share a common progenitor cell with bone-forming osteoblasts [4]. Furthermore, estrogen has been implicated in a reciprocal relationship between bone marrow adipocytes and osteoblasts. In addition, there is evidence that estrogen preferentially promotes the formation of osteoblasts instead of adipocytes [5].

Recent reports suggest that the canonical Wnt/beta-catenin signaling pathway is another mechanism that controls the development of precursor cells into osteoblasts or adipocytes [6]. There is evidence to suggest that the activation of Wnt signaling blocks preadipocyte differentiation and stimulates osteoblast precursor growth, differentiation and maturation via beta-catenin by downregulating the expression of adipogenic transcription factors [7,8]. A recent study demonstrated a relationship between the Wnt and estrogen signaling pathways through a functional interaction between estrogen receptor-alpha (ERalpha) and beta-catenin in Drosophila and also in cancer cell lines [9]. Therefore, the aim of the present study was to investigate whether estrogen and the canonical Wnt signaling pathway interact in human osteoblasts by using a human in vitro estrogen-deficiency model [10].

Methods

Cellular experiments

Human fetal osteoblasts (hFOB 1.19) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in 25 cm^sup 2^ culture flasks (Sarstedt, Nurnbrecht, Germany), using Dulbecco’s modified Eagle’s medium/F-12 medium, supplemented with 15% fetal calf serum and antibiotics (all from Invitrogen, Auckland, New Zealand). The cells were kept in culture at 34[degrees]C with 5% CO2 under humidified conditions [11]. These osteoblastic cells are at an early stage of differentiation as they express Stro-1, a stromal stem cell marker, but under optimal conditions they differentiate toward mature osteoblasts (data not shown).

For experiments, sub-confluent cultures were used and one flask was split into four new flasks or seeded into flat-bottomed 96-well plates (Sarstedt) at a density of 10 000 cells per well. Fulvestrant (kindly provided by Astra-Zeneca, Australia), dissolved in isopropyl alcohol, was added to the cell cultures at different concentrations (1 to 250 [mu]M) to mimic an estrogen-deficient cellular environment. Untreated or solvent (isopropyl alcohol)-treated cells were used as controls.

Cell proliferation experiments were done in quadruplet applying the CellTiter96(R) AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA).

All cellular experiments and corresponding measurement were done three to five times independently and showed consistent results.

Microscopic analysis

Osteoblasts cultured for 1 week under estrogen deficiency and control conditions were fixed with 1% paraformaldehyde in phosphate- buffered saline (PBS) and stained with Nile Red (1:1000 dilution in PBS for 10 min; Molecular Probes/Invitrogen) for detection of cytoplasmic lipid droplets, and nuclei were stained with DAPI (4′,6- diamidine-2′-phenylindole dihydrochloride, 1 mg/ml in PBS; Roche Diagnostics, Mannheim, Germany). Images were taken with a CoolSNAP ES digital camera (Photometrics, Tucson, AZ), a Nikon Eclipse 90i microscope and processed with V++ imaging software.

Quantitative real-time reverse transcription polymerase chain reaction

Osteoblasts were cultured for 1 week under estrogen deficiency and control conditions before total RNA was isolated by using Ultraspec (Biotecx, Houston, TX, USA). mRNA was reverse transcribed by using the Reverse Transcription System (Promega). Thereafter, DNA products were amplified and quantified using specific primers for beta-actin (forward: 5′-GCG AGA AGA TGA CCC AGA TCA TGTT-3′; reverse: 5′-GCT TCT CCT TAA TGT CAC GCA CGAT-3′), Runx2/cbfal (forward: 5′-TCT TCA CAA ATC CTC CCC-3′; reverse: 5′-TGG ATT AAA AGG ACT TGG-3′), osterix/ SP7 (forward: 5′-TCC TCC CTG CTT GAG GAG GA- 3′; reverse: 5′-AGT CCC GCA GAG GGC TAG AG-3′) and cathepsin K (forward: 5′-CCC GAA GGG AAA CAA GCA-3′; reverse: 5′-GCC TGT ACC TGT ACA GCA-3′) by using the iQ SYBR Green Supermix (Bio-Rad, Regents Park, NSW, Australia) and the Rotor-Gene 3000 cycler and corresponding software (Corbett Life Science, Mortlake, NSW, Australia).

Immunoprecipitation and Western blot analysis

Osteoblasts cultured for 1 to 3 days under estrogen deficiency and control conditions were lysed using either whole-cell lysate buffer (10% sucrose, 1% sodium dodecylsulfate, 5% 2-mercaptoethanol in 0.5 M Tris-HCl buffer, pH 6.8) or the Ne-Per Nuclear and Cytoplasmic Extract kit from Pierce (Rockford, IL). For immunoprecipitation and Western blot analysis, the lysates were processed according to the protocol recommended by Santa Cruz Biotechnology (Santa Cruz, CA, USA) using the following reagents: normal rabbit immunoglobulin G (IgG), protein A-agarose, rabbit polyclonal anti-estrogen receptor-alpha antibody (sc-543), mouse monoclonal anti-beta-catenin antibody (sc-7963 (all from Santa Cruz); horse radish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG (DAKO, Glostrup, Denmark); ECL-SuperSignal West Pico (Pierce). Gel documentation was done with the Kodak Image Station 2000 MM and data were analyzed with the corresponding software.

Statistical analysis

For the proliferation assays, mean and standard error of the mean were calculated for each treatment group and each dilution, and statistical significance was examined with the SPSS program (SPSS Inc., Chicago, IL, USA).

Results

Osteoblast differentiation and proliferation under estrogen deficiency

A human in vitro estrogen-deficiency model was established for this study using the well-characterized human fetal osteoblast cell line hFOB 1.19 that was cultured in the presence of fulvestrant, a pure estrogen antagonist.

After one week in culture with fulvestrant or under corresponding control conditions, the cells were fixed and stained with Nile Red, a fluorescent lipid stain, and with DAPI, a nuclear stain. Significant amounts of lipid droplets were detected in fulvestrant- treated cells, but not in the control cells, indicating that estrogen deficiency induces adipocyte differentiation of the cells (Figure 1). When applying increasing fulvestrant concentrations, an increased lipid accumulation was found. When cultured for a longer period of time, lipid accumulation in the cells increased and the lipid droplets became bigger in size and fewer in number, indicating differentiation of adipocytes from multilocular to monolocular cells (data not shown).

Differentiation markers of osteoblasts were measured using a quantitative reverse transcription polymerase chain reaction protocol. Thereby, decrease in mRNA expression of osteoblastic markers, including Runx2/cbf-al, osterix and cathepsin K, was documented (Table I).

Higher fulvestrant concentrations (above 15 [mu]M) significantly enhanced hFOB 1.19 proliferation, indicating an increased proliferation stimulus of the cells under severe estrogen deficiency (Figure 2).

Expression of estrogen receptor-ct by human osteoblasts

Expression of ERa by hFOB 1.19 cells was documented through Western blot analysis. For ERalpha, expression by MCF-7 cells (breast cancer cell line) was used as a comparison and positive control. Expression of the 46-kDa and the 77-kDa estrogen receptor isoforms were detected in whole-cell lysates, as well as in cytoplasmic and nuclear fractions of hFOB 1.19 cells (Figure 3). When the osteoblasts were treated with fulvestrant, expression of both ERa isoforms was downregulated in comparison with the control cells (Figures 4 and 5). Densitometric analysis indicated downregulation of ERa expression of between 20 and 50% in fulvestrant-treated cells, compared with control cells. Expression of beta-catenin in human osteoblasts

beta-Catenin was measured through Western blot analysis and was shown to be expressed in wholecell lysates as well as in cytoplasmic and nuclear fractions of hFOB 1.19 cells. beta-Catenin expression was significantly reduced in fulvestrant-treated cells in comparison with control cells (Figure 6). Densitometric analysis indicated that downregulation of beta-catenin in fulvestrant-treated cells was in the range of 75% as compared with control cells. In addition, beta- catenin was co-precipitated with ERa and the amount of precipitate also decreased in fulvestrant-treated cells in comparison with control cells (Figure 7).

Figure 1. Nile Red stain of human osteoblasts treated with increasing fulvestrant concentrations. Representative images of control cells (A) and fulvestrant-treated cells (B: 1 [mu]M, C: 10 [mu]M) cultured over 1 week, before being fixed and stained with Nile Red for lipid droplets (red) and DAPI for nuclei (blue). Scale bar =100 [mu]m.

Table I. Expression of osteoblast markers as measured with quantitative reverse transcription polymerase chain reaction (PCR).

Figure 2. Cell proliferation under estrogen deficiency. Osteoblasts were cultured over 5 days in the presence of different fulvestrant concentrations (3.9 to 250 [mu]M) or control conditions (untreated or solvent-treated). Each bar represents the mean, with the standard deviation shown by the vertical bar, of four wells of a representative experiment. Proliferation was measured with the CellTiter96 colorimetric assay, a indicates statistically significant differences.

Figure 3. Expression of estrogen receptor-alpha (ERalpha) isoforms in hFOB 1.19 osteoblasts. hFOB 1.19 osteoblasts or MCF-7 cells (an estrogen-responsive breast cancer cell line) were processed for Western blot detection of ERalpha by a specific polyclonal rabbit antibody. Detection of beta-actin with a specific mouse monoclonal antibody was used as positive control. Osteoblasts expressed the 77-kDa and 46-kDa isoforms, whereas MCF-7 cells additionally expressed the 66-kDa isoform (MWM, molecular weight marker: 98, 64 and 50 kDa).

Figure 4. Expression of the 46-kDa estrogen receptor-alpha (ERalpha) isoform in fulvestrant-treated and control cells. Osteoblasts were cultured for 3 days in the presence of fulvestrant or control conditions before being processed for detection of ERa expression through Western blot analysis.

Figure 5. Expression of the 77-kDa estrogen receptor-alpha (ERalpha) isoform in fulvestrant-treated and control cells. Osteoblasts were cultured for 3 days in the presence of fulvestrant or control conditions before being processed for detection of ERa expression through Western blot analysis.

Figure 6. beta-Catenin expression in fulvestrant-treated and control cells. Osteoblasts were cultured for 3 days in the presence of fulvestrant or control conditions before being processed for detection of beta-catenin expression through Western blot analysis.

Figure 7. Co-precipitation of beta-catenin through immunoprecitiation of estrogen receptor-alpha.

Discussion

There is a reciprocal relationship between differentiation of osteoblasts and bone marrow adipocytes, as both cell types derive from stromal stem cells [4,12,13]. Thereby, estrogen and Wnt signaling have been shown to play an essential role [4,14-16]. However, this is the first study to investigate whether the two signaling pathways may interact and converge in human osteoblasts. For that purpose, a human in vitro estrogen-deficiency model was established by using fulvestrant, a pure estrogen receptor blocker [17]. This approach has the advantage of not interfering with other steroid hormones or growth factors available in the cell culture medium, compared with charcoal treatment. Consequently, our model may be more specific toward deletion of the estrogen action on the cells, without deleting or decreasing the activity of other steroid hormones such as corticosteroids and androgens.

The hFOB 1.19 cell line was used throughout this study because this is one well-described cell line out of few commercially available human osteoblast cell lines. In addition, the cells are already committed toward osteoblast differentiation, but still at an early stage of differentiation as they still express Stro-1, a stromal stem cell marker [18,19]. Therefore, in comparison with mature osteoblasts, these cells may be more susceptible to be redirected toward other stromal lineages, including adipocytes [19,20].

This study indicates that estrogen deficiency preferentially induces adipocyte differentiation in osteoblastic cells. Using Nile Red stain and fluorescence microscopy, the results clearly indicate accumulation of lipid droplets in the estrogen-deficient cells. In addition, essential markers of bone-forming cells, such as Runx2/ cbfa1, osterix and cathepsin K, expressed in hFOB 1.19, were downregulated in cells cultured under estrogen deficiency. Furthermore, increased fulvestrant concentrations representing severe estrogen deficiency induced the resulting adipocytes to increase in number, as they undergo cell proliferation. Altogether, these findings indicate that postmenopausal estrogen deficiency may deviate osteoblasts at an early differentiation stage toward adipocytes, and additionally induce proliferation of the bone marrow adipocytes. This process may not only decrease recruitment of new, fully functional osteoblasts, but may also induce expansion of the yellow bone marrow, thus gradually replacing bone tissue.

ERalpha expression, modification and function in osteoblasts are still not well understood, including why there are different isoforms and what their exact functions are [21]. Most interestingly, this study reveals expression of the 46-kDa and the 77-kDa ERalpha isoforms in the human hFOB 1.19 osteoblast cell line, but no expression of the 66-kDa isoform nor expression of the estrogen receptor-beta (data not shown). Expression of the 46-kDa isoform in human osteoblasts has already been published, but so far the 77-kDa isoform has only been shown in MCF-7 breast cancer cells [22,23]. The 46-kDa isoform is an alternative splicing variant of the ERalpha gene product missing the activation domain AF-1, but still having the DNA-binding and ligand-binding domains and being able to dimerize after ligand binding [24]. Estrogen and fulvestrant certainly act on the 46-kDa isoform. Consequently, fulvestrant is expected to bind to the 46-kDa isoform and prevent dimerization, resulting in ubiquitination and degradation of the fulvestrant- receptor complex [25]. As a consequence, a decrease in the 46-kDa isoform in the cells is expected. Such a decrease was confirmed by this study. The 77-kDa isoform does have the DNA-binding domain but it does not have the ligand-binding domain, indicating that fulvestrant does not act on it. However, in parallel to the decrease of the 46-kDa isoform, there was also a decrease in the 77-kDa isoform which cannot be explained as a direct effect of fulvestrant, but must have other reasons. The reasons could be either a decreased stability of the 77-kDa isoform, without having formation of ERalpha46/ERalpha77 heterodimers, or downregulation at transcriptional level. Either of the two possibilities has to be clarified through future investigation.

Most important, in this study estrogen deficiency through the action of fulvestrant and the decrease of ERalpha also resulted in a decrease of beta-catenin. As beta-catenin was co-precipitated with ERalpha, this indicates that both signaling proteins interact with each other. For beta-catenin, that interaction is essential, as free cytoplasmic beta-catenin is easily ubiquitinated and degraded [16]. Consequently, forming a heterodimeric complex with ERa may prevent beta-catenin from being degraded and may enable it to move from the cytoplasm to the nucleus, where it can act as a transcription factor on the regulation of gene transcription. As beta-catenin is an important signaling molecule of the canonical Wnt signaling pathway and essential for Wnt-dependent osteoblast differentiation, downregulation of beta-catenin will result in deviation of osteoblast differentiation towards adipocytes [6,15]. However, beta- catenin may well also act independent of Wnt-related molecules but interact directly and independently with ERalpha. In that respect, bone morphogenetic protein-2 (BMP-2) has been shown to synergize with beta-catenin to promote osteoblast and block adipocyte differentiation in a murine mesenchymal stromal stem cell model [26]. Moreover, in vivo mouse models indicate that ERalpha, BMPs and Wnt signaling converge to regulate osteoblast differentiation [27].

Wnt signaling is a rather complex mechanism, as there is a multitude of extracellular molecules, including Wnts, secreted frizzled-related protein (sFRP), dickkopf (Dkk) and sclerostin, binding to the surface-membrane receptor complex composed of frizzled receptor and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) [28-30]. In addition to the beta-catenin- dependent pathway there are other beta-catenin-independent mechanisms, resulting in the Wnt signaling being rather complex [31]. This complexity increases even more when considering the interference of other signaling pathways, including ERalpha signaling [27]. However, future studies have to elucidate the detailed mechanisms of how Wnts, together with estrogens, orchestrate osteoblast differentiation. Acknowledgements

This study was supported by the SUVA Foundation, Switzerland.

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This paper was first published online on iFirst on 18 August 2007

CLARA FOO1, SOENKE FREY1’2, HONG HYUN YANG1’3, RENE ZELLWEGER2, &

LUIS FILGUEIRA1

1 School of Anatomy and Human Biology, The University of Western Australia, Perth, Australia, 2 Department of Orthopaedic

Surgery, Royal Perth Hospital, Perth, Australia, and 3 College of Veterinary Medicine, Chonbuk National University, Chonju,

South Korea

(Received 4 November 2006; revised 2 July 2007; accepted 3 July 2007)

Correspondence: L. Filgueira, School of Anatomy and Human Biology, University of Western Australia, M309, 35 Stirling Highway, Crawley, WA 6009, Australia. Tel: 61 8 64883907. Fax: 61 8 64881051. E-mail: lfilgueira@anhb.uwa.edu.au

Copyright Taylor & Francis Ltd. Sep 2007

(c) 2007 Gynecological Endocrinology. Provided by ProQuest Information and Learning. All rights Reserved.