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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Mol Endocrinol. Author manuscript; available in PMC 2007 October 10.
Published in final edited form as:
PMCID: PMC2013302
NIHMSID: NIHMS23423

DICKKOPF HOMOLOG 1 MEDIATES ENDOTHELIN-1-STIMULATED NEW BONE FORMATION*

Abstract

Tumor-produced endothelin-1 (ET-1) stimulates osteoblasts to form new bone and is an important mediator of osteoblastic bone metastasis. The anabolic actions of ET-1 in osteoblasts were investigated by gene microarray analyses of murine neonatal calvarial organ cultures. Targets of ET-1 action were validated by real-time RT PCR in murine primary osteoblast cultures. Interleukin 6, interleukin 11, the CCN family members Cyr61 and connective tissue growth factor, inhibin beta-A, serum/glucocorticoid regulated kinase, RANKL, snail homolog 1, tissue inhibitor of metalloproteinase 3 and TG-interacting factor transcripts were increased by ET-1. ET-1 decreased the transcript for the Wnt signaling pathway inhibitor, dickkopf homolog 1 (Dkk1). Calvarial organ cultures treated with ET-1 had lower concentrations of DKK1 protein in conditioned media than control cultures. High DKK1 concentrations in bone marrow suppress bone formation in multiple myeloma. We hypothesized that the converse occurs in osteoblastic bone metastasis, where ET-1 stimulates osteoblast activity by reducing autocrine production of DKK1. Recombinant DKK1 blocked ET-1-mediated osteoblast proliferation and new bone formation in calvarial organ cultures, while a DKK1-neutralizing antibody increased osteoblast numbers and new bone formation. ET-1 directed nuclear translocation of β-catenin in osteoblasts indicating activation of the Wnt signaling pathway. The data suggest that ET-1 increases osteoblast proliferation and new bone formation by activating the Wnt signaling pathway through suppression of the Wnt pathway inhibitor DKK1.

Keywords: endothelin-1, dickkopf homolog 1, osteoblast, Wnt signaling pathway

INTRODUCTION

Endothelin-1 (ET-1) is a 21-amino acid peptide first identified as a potent vasoconstrictor and since found to have many actions (1), including regulation of blood pressure, renal sodium excretion, cardiac remodeling and nociception. Mice carrying a homozygous deletion of the ET-1 gene have craniofacial and cardiovascular defects and die shortly after birth from respiratory failure (2). ET-1 acts through a pair of G protein-coupled receptors, ETAR and ETBR (3, 4). ET-1 activity is primarily via ETAR during development, since the knockout of the ETAR, but not the ETBR, phenocopies the knockout of the ligand (5).

ET-1 is also a key mediator of osteoblastic bone metastases, which are characteristic of breast and prostate cancers (6, 7) The human breast cancer cell lines ZR-75-1, MCF-7 and T47D produce osteoblastic bone lesions when inoculated into the left cardiac ventricle of nude mice. These cell lines also abundantly secrete ET-1, and ET-1 is a potent stimulator of osteoblast activity and new bone formation in organ cultures of murine calvariae. The bone anabolic response to ET-1 in this assay was blocked by the selective ETAR antagonist ABT-627 but not by inhibition of ETBR. Oral ABT-627 prevented development of osteoblastic bone lesions due to ZR-75-1 cancer cells in nude mice but had no effect on these cells grown as mammary tumors. The drug had no effect on growth of ET-1-negative MDA-MB-231 breast cancer cells either in bone or the mammary fat pad (6, 7).

ET-1 is also important in prostate cancer bone metastasis. Prostate epithelial cells secrete large amounts of ET-1 (8, 9). ET-1 is secreted by a majority of prostate cancer cell lines (7). In men with advanced prostate cancer, plasma ET-1 concentrations are increased compared with men who have local disease or age-matched controls (7). ETAR antagonists reduce the progression of bone metastasis (10, 11) and decrease markers of bone turnover in men with advanced prostate cancer (12).

The data support a central role for ET-1 in the development of bone metastases, in which the tumorsecreted ligand stimulates osteoblasts via the ETAR. Preliminary evidence from our group also suggests that the endothelin axis has a role in normal bone development. Oral administration of an ETAR antagonist or targeted deletion of ETAR in the osteoblast results in reduced bone mineral density and trabecular bone volume (13, 14).

The molecular mechanisms by which ETAR activation stimulates bone responses were unknown. We have now identified downstream targets of ET-1 by gene microarray analysis, including several secreted factors that could serve as paracrine regulators of bone formation. One of the targets identified was dickkopf homolog 1 (DKK1), a secreted inhibitor of the Wnt signaling pathway. In the studies presented here, we show that ET-1 regulates DKK1 message and protein secretion in osteoblasts and that this inhibitor of the Wnt signaling pathway can mediate the anabolic responses of osteoblasts to ET-1.

Experimental Animals

Animal experimentation described followed the guidelines of The University of Virginia Animal Care and Use Committee.

RESULTS

Identification of ET-1 targets

Osteoblasts form new bone through a spatially and temporally complex process that is not accurately reproduced by cell lines. To preserve the physiologically relevant responses of bone to ET-1 treatment we carried out gene microarray analysis on calvarial organ cultures (15). ET-1 treatment of these cultures increases osteoblast numbers and new bone formation in five to seven days (6). Calvariae were treated in duplicate with and without 100 nM ET-1 for six and twenty-four hours, and four and seven days. Total RNA was extracted and hybridized to mouse Affymetrix gene chips containing 22,626 or 12,422 probe sets. Relatively few gene transcripts were changed by ET-1 treatment (Table 1). Changes in mRNA were considered significant if the fold change in signal intensity was > 1.5 or < -1.5, p values ≤ 0.05 and absolute signal intensity >100.

Table 1
Number of genes regulated by ET-1 by gene microarray analysis

The greatest changes were seen at twenty-four hours: dChip identified 130 genes and DMT identified 69 that were significantly changed by ET-1, representing 0.57% and 0.30% of the entire data set respectively. A complete list of genes can be found in Supplemental Data. Table 2 contains an abbreviated list of transcripts regulated by ET-1 at the four treatment time points as determined by dChip analyses. Gene transcripts in Table 2 were selected based on potential function in osteoblasts and include secreted cytokines, extracellular matrix proteins, receptors, signaling molecules, transcription factors, and components of the cytoskeleton.

Table 2
Targets of ET-1 in osteoblasts

Real-time RT PCR validation of microarray data

Nineteen genes identified from the dChip and DMT microarray analyses as regulated by ET-1 were chosen for further study on the basis of being known targets of ET-1 or potentially active in osteoblast biology. Fifteen candidate genes identified by dChip are indicated in bold in Table 2 [CCAAT/enhancer binding protein, delta (Cebpd), connective tissue growth factor (Ctgf), cysteine-rich protein 61 (Cyr61), dickkopf homolog 1 (Dkk1), distal-less homeobox 3 (Dlx3), distal-less homeobox 5 (Dlx5), inhibin beta-A (Inhba), inhibin beta-B (Inhbb), interleukin 6 (Il6), interleukin 11 (Il11), secreted frizzled-related protein 1 (Sfrp1), snail homolog 1 (Snai1), tissue inhibitor of metalloproteinase 3 (Timp3), tumor necrosis factor ligand superfamily member 11 (Rankl), and Wnt5a]. Four additional genes, serum/glucocorticoid regulated kinase (Sgk), TG interacting factor (Tgif), TGF-β1 induced transcript 4 (Tsc22), and twist homolog 2 (Twist2) were identified by DMT analysis.

Two days postconfluence, murine primary osteoblast cultures were serum-starved for 24 hours and treated with 100 nM ET-1 for 1, 6, 24, and 48 hours. Total RNA was extracted and analyzed by real-time RT-PCR. Transcripts for Ctgf, Cyr61, Dkk1, Dlx5, Inhba, Il6, Il11, Timp3, Rankl, Sgk, Snai1 and Tgif were significantly changed by ET-1 treatment (Figure 1). Trends towards changed mRNA concentrations with ET-1 treatment for Dlx3 and Inhbb did not reach statistical significance. ET-1 regulation of Cebpd, Sfrp1, Tsc22, Twist2, and Wnt5a mRNA in primary osteoblasts was not seen (data not shown). Since the original array data were obtained with whole calvariae, the changes seen for these five genes may occur in cell types of the calvariae other than osteoblasts.

Fig 1
Real-time RT PCR of primary osteoblast cultures treated with ET-1. Mouse primary osteoblast cultures were treated with or without 100 nM ET-1 in quadruplicate for 1, 3, 6, 24 and 48 hours. Total RNA was isolated and real-time RT PCR performed. Fold mRNA ...

IL-6 and DKK1 secretion in response to ET-1

We tested whether changes in mRNAs for the secreted factors, Il6 and Dkk1, were reflected at the protein level in response to ET-1. Mouse neonatal calvariae were treated with and without ET-1. Conditioned media were collected and replaced with fresh media at two, four and six days, and analyzed for IL-6 and DKK1 protein by ELISA. ET-1 significantly increased IL-6 and decreased DKK1 secretion into osteoblast-conditioned media at days two, four and six (Figure 2), in agreement with the changes observed at the mRNA level.

Fig 2
ET-1 increases IL-6 and DKK1 secretion from calvariae. Calvariae were treated with or without 100 nM ET-1 for six days. Media were collected at days two, four, and six days with replacement of fresh media. IL-6 and DKK1 protein concentrations were measured ...

Regulation of osteoblast function by IL-6 and DKK1

Interleukin 6 has multiple effects on both osteoclast and osteoblasts (16, 17). However, the addition of an IL-6-neutralizing antibody to calvarial organ cultures had no effect on ET-1-induced new bone formation (Figure 3). The role of DKK1 as a candidate mediator of the anabolic effects of ET-1 on bone was then examined. This secreted factor binds and sequesters low density lipoprotein receptor-related proteins 5 (LRP5) and 6 (LRP6), cofactors in Wnt ligand-mediated signaling, resulting in the inhibition of Wnt signaling (18, 19). In agreement with other reports (20), both LRP5 and 6 were expressed in murine primary osteoblast cultures by real-time RT PCR (data not shown). We next examined whether the transmembrane protein Kremen was regulated by ET-1. Kremen associates with the DKK1-LRP complex and could potentially modulate the negative effects of DKK1 on Wnt signaling (21). However, ET-1 did not alter Kremen mRNA concentrations in calvarial organ or primary osteoblast cultures (Figure 1).

Fig 3
IL-6 does not mediate the osteoblast-stimulatory action of ET-1. Murine neonatal calvarial organ cultures were treated singly with 100 nM ET-1, 1 μg/ml goat anti-mouse IL-6 neutralizing antibody (αIL-6), 1 μg/ml goat IgG control ...

High concentrations of DKK1 protein are found in the bone marrow of patients with multiple myeloma and have been strongly correlated with osteolytic bone disease. Moreover, multiple myeloma patients’ serum can block osteoblast differentiation in vitro, and this effect is inhibited by a neutralizing antibody to DKK1 (22). DKK1 may contribute to the suppressed bone formation that is characteristic of this malignancy (22). Based on the observations, we hypothesized that an opposite response occurs in osteoblastic metastases, where decreased DKK1 (due to ET-1) results in enhanced Wnt signaling and increased bone formation

The contribution of DKK1 to ET-1-stimulated osteoblast activity was examined in organ cultures. Seven days of ET-1 treatment resulted in a robust increase in new bone formation compared to control (Figure 4). No statistically significant increase in osteoblast numbers was detected. DKK1 alone did not suppress basal new bone formation or change osteoblast numbers. However, DKK1 effectively blocked ET-1-stimulated new bone formation. The concentration (50 ng/ml) of DKK1 selected in these experiments is similar to the concentration of DKK1 found in bone marrow plasma of myeloma patients (22).

Fig 4
DKK1 blocks ET-1-stimulated new bone formation but not basal osteoblast activity. Murine neonatal calvarial organ cultures were treated with 50 ng/ml DKK1, 100 nM ET-1, or both ET-1 and DKK1 (ET+DKK1) in quadruplicate for seven days and compared to control ...

When calvarial organ cultures were treated with a DKK1-neutralizing antibody, there was a significant increase in new bone formation compared to matched control antibody (Figure 5), demonstrating the presence of endogenous DKK1 protein in the organ cultures at concentrations sufficient to regulate basal bone formation.

Fig 5
DKK1 neutralizing antibody stimulates new bone formation. Murine neonatal calvarial organ cultures were treated with 100 nM ET-1, 0.1 μg/ml antibody against human DKK1 (αDKK1), or 0.1 μg/ml goat IgG control antibody for seven days ...

ET-1 activates Wnt signaling

Immunohistochemistry was performed on control and ET-1 treated calvarial organ culture and examined for nuclear translocation of β-catenin, an indication of Wnt pathway activation. In control cultures, β-catenin was located on the cell-surface of the osteoblasts (Figure 6A). However, treatment with ET-1 led to nuclear staining especially in active osteoblasts at the mineralizing bone surface (Figure 6B).

Fig 6
ET-1 activates Wnt signaling in osteoblasts. Murine calvarial organ cultures were treated with (B) and without (A) 100 nM ET-1 for seven days. H&E staining was performed. Immunohistochemistry was performed on adjacent sections using an anti-β-catenin ...

DISCUSSION

ET-1 is a mediator of osteoblastic bone metastasis and a potent osteoblast-activating peptide (6). To elucidate the mechanisms of ET-1 action in osteoblasts, we performed gene microarray analyses of neonatal mouse calvariae. Although the developing calvaria is a complex organ with multiple cell types, the gene microarray yielded a surprisingly small set of gene transcripts significantly affected by ET-1. When the array data were analyzed in detail, less than 0.6% of the probed gene transcripts were significantly changed at 24 hours by ET-1. We further narrowed the candidate genes by two criteria: a) potential relevance of the encoded products to the osteoblastic phenotype and b) validation of changed expression in primary osteoblast cultures (Table 2). The utility of this empirical approach was upheld by comparison of our gene set with those identified in independent microarray studies of cell lines induced to undergo osteoblastic differentiation in vitro. The gene expression profile from our study was compared to microarray analyses of the targets of BMP-2, TGF-β, activin A and PTH in osteoblast-like cell lines (23-26). When the ET-1-responsive factors in Table 2 were compared to the mRNAs changed in these other studies, consistent matches were observed with the secreted factors Ctgf, Cyr61, and Timp3 and the transcription factors Snai1, Tgif, and Tsc22 (Table 3). The bone-anabolic actions of ET-1, BMP-2, TGF-β, activin A and PTH may share common transcriptional activation pathways and the common secretion of osteoblast-activating factors. The previous microarray studies did not identify Il6, Il11, Rankl, or Dkk1. We selected 19 gene targets of ET-1 for further analysis based on significance when analyzed with two different software packages (dChip and DMT) and potential for activity in bone. Fourteen of the selected genes were confirmed to be significantly regulated by ET-1 in primary osteoblasts cultures by real-time RT PCR analysis.

Table 3
Common targets of ET-1, activin, BMP-2, TGF-β and PTH in osteoblasts

We focused on the possible bone anabolic effects of ET-1 via Wnt pathway activation and in particular the ET-1-mediated decrease in mRNA and protein secretion of DKK1. DKK1 is a secreted factor that sequesters low-density lipoprotein-related proteins-5 (LRP5) and -6 (LRP6), essential cofactors of the frizzled receptors, resulting in the inhibition of canonical Wnt signaling (18, 19). Wnt signaling is important in osteoblast development and bone remodeling. Activating mutations of LRP5 cause high bone mass (27, 28), and loss-of-function mutations result in osteoporosis-pseudoglioma syndrome (29). Mice with osteoblasttargeted deletions of LRP5 or 6 display limb defects and decreased bone mineral density, with compound mutants more severely affected (30). Transgenic mice overexpressing Dkk1 in osteoblasts exhibit skeletal dysplasia and low bone mass (31, 32). Conversely, a Dkk1 neutralizing antibody increases bone mass in rats (33). Similarly, mice containing a targeted knock-out of the soluble Wnt antagonist Sfrp1 have increased bone mass (34).

Activation of the Wnt signaling pathway is a critical step in steering uncommitted mesenchymal precursor cells from a chondrocytes to an osteoblast fate and subsequent steps in osteoblastogenesis (35-38). DKK1 may play a central role in this process. DKK1 is expressed in mesenchymal stem cells and promotes proliferation at the expense of differentiation (39, 40). This fact fits well with data presented here which suggests that ET-1 downregulates DKK1, which shifts the balance toward Wnt signaling pathway activation and osteoblast differentiation. Further evidence of Wnt signaling activation is nuclear β-catenin translocation in ET-1-treated osteoblasts. Interestingly, we did not identify any of the secreted Wnt proteins that activate canonical Wnt signaling as targets of ET-1 (ET-1-target WNT5A activates the non-canonical Wnt pathway). This suggests that the osteoblast is primarily under inhibitory control by basally secreted DKK1 and that suppression of DKK1 secretion by ET-1 increases osteoblast activity and new bone formation. This is supported by our finding that a DKK1-neutralizing antibody alone has osteoblast anabolic effects.

DKK1 may contribute to the pathogenesis of multiple myeloma bone disease, which is characterized by strong osteolytic resorption with suppressed osteoblastic responses. Dkk1 transcripts were higher in afflicted patients’ plasma cells compared to control plasma cells (22). Protein levels were also higher in bone marrow plasma and in peripheral blood of these study patients. While increased DKK1 may inhibit osteoblast activity in multiple myeloma, decreased DKK1 may enhance osteoblast activity in certain solid tumor metastases. Hall et al (41) recently described two models of prostate cancer bone metastasis, in which the amount of DKK1 produced by the cancer cells controls the phenotype of the bone lesions. In an osteolytic tumor model, decreasing tumor DKK1 secretion with siRNA changed the bone responses to a net osteoblastic one, while DKK1 overexpression converted an osteoblastic model to an osteolytic phenotype. Although not mutually exclusive, these models of DKK1 action in bone metastasis are distinct from our hypothesized mechanism in that regulation of bone metastasis osteoblastogenesis is through DKK1 secretion by the osteoblast and not the tumor cell.

The molecular mechanism by which ET-1 regulates DKK1 expression and secretion is unknown. The Dkk1 promoter contains nine putative binding sites for TCF/LEF transcription factors, the targets of the Wnt signaling pathway, and the promoter itself is responsive to canonical Wnt signaling in kidney cells (42). A similar negative feedback loop may also regulate Dkk1 expression in osteoblasts. Another clue from the data presented here is the temporal changes in mRNA concentrations with ET-1 treatment. Dkk1 declined after six hours of treatment with ET-1, while Il6, Cyr61, Ctgf, Sgk and Tgif increased by one hour with ET-1 treatment (Figure 2). The latter two genes encode a signaling kinase and a transcription factor, which could provide the intermediary regulatory pathway from the ETAR to DKK1 in osteoblasts. SGK is activated by ET-1 in endothelial cells (43). Similarly, Snai1 expression, as well as β-catenin, is increased by ET-1 in ovarian cancer cells (44). Dlx5 in osteoblasts was decreased by ET-1, is a target of BMP-4 and is expressed in early osteoblastogenesis (45). Dlx3 is a target of ETAR activation during mouse craniofacial development (46). The decrease in Dlx3 and Dlx5 transcription factors may reflect decreasing numbers of immature osteoblasts upon ET-1 stimulation of osteoblast differentiation.

In addition to DKK1, our experiments identified other secreted, osteoblast-regulatory factors. Two structurally similar members of the CCN family of secreted proteins, CYR61 and CTGF, were upregulated by ET-1. These gene products are expressed in the developing skeleton, stimulate bone formation and may have a role in bone metastasis progression (47, 48). CTGF is increased in breast cancer cell subclones that metastasize selectively to bone, and its targeted overexpression contributes to specific breast cancer metastases to bone (49). CYR61 expression correlates with tumor stage and lymph node status in patient breast cancer samples (50). Both CYR61 (51) and CTGF (52) can activate Wnt signaling, although such actions have not been studied in mammalian bone. These two factors are also upregulated by ET-1 in bladder epithelium (53), and bladder cancer metastasis to lung is blocked by the ETAR antagonist, ABT627 (54), although a role in this system for Wnt signaling or DKK1 has not been reported. Inhibin beta-A and beta-B were also targets of ET-1 in osteoblasts. Dimers of these proteins yield activin, which enhances osteoblastogenesis (55). Little is known about how TIMP3 might contribute to osteoblast function (56).

Several of the mRNAs identified in Table 2 encode secreted factors with the potential to regulate normal and pathologic bone remodeling. Il6 mRNA was markedly increased in both calvarial organ and primary osteoblast cultures by ET-1 treatment. Il6 is known to be regulated by ET-1 (57). IL-6 protein secretion was also increased by ET-1 (Figure 2), although our data with IL-6-neutralizing antisera (Figure 3) suggest that IL-6 does not substantially mediate osteoblastic responses to ET-1. Il11 was also increased by ET-1 treatment. In lung fibroblasts ET-1 stimulates IL-11 secretion by activation of MAPK kinase downstream of ETAR (58). Rankl mRNA was increased at 1 and 3 hours (Figure 1). Rankl transcription is negatively regulated by the canonical Wnt pathway (59); so the return of Rankl mRNA to baseline by 6 hours may represent increased Wnt signaling at longer times in ET-1-treated osteoblasts. IL-6, IL-11 and RANKL are potent osteoclast-activating agents (60-62). The increase of these transcripts suggests that ET-1 may increase osteoclast activity, as well as stimulating a net osteoblastic response. Such a role is consistent with the observation that prostate cancer patients treated with the ETAR antagonist atrasentan (ABT-627) have decreased markers of bone resorption (12). These markers are typically very high in patients with osteoblastic bone metastases due to prostate cancer (63), although circulating IL-6 concentrations do not correlate with the markers of resorption (64).

The development of bone metastasis involves complex and cooperative signals between bone and cancer cells. Cancer cells not only proliferate in bone but are able to coax osteoblasts and osteoclasts to produce factors that further stimulate cancer cell growth within the bone microenvironment. Tumor production of ET-1 stimulates osteoblast proliferation and new bone formation. We have shown that the anabolic actions of ET-1 on calvarial organ cultures are mediated by the Wnt signaling pathway inhibitor DKK1. This factor has autocrine effects on the osteoblast, but DKK1 and other ET-1 regulated factors may also have local effects within normal bone and the metastasis microenvironment to promote the progression of bone metastasis. These results, plus those of Tian et al (22) in multiple myeloma and those presented here, suggest that the amount of DKK1 present in the bone microenvironment could be a central regulator of the balance of bone formation to destruction in response to tumor cells. Disruption of Wnt signaling by DKK1 upsets the dynamic equilibrium between these two processes. Therapy aimed at blocking the pathological effects of dysregulated DKK1 actions could provide a novel avenue for treatment of cancer effects on the skeleton.

Materials and Methods

Animals and cell preparation

Animal care followed the guidelines of The University of Virginia Animal Care and Use Committee. Timed-pregnant ICR Swiss female mice were obtained from Harlan. Four-day-old pups were euthanized by CO2 inhalation. Calvariae were excised, cut along the sagital suture, and each half placed on a stainless steel grid in a 12-well tissue culture plate containing 1 ml BGJ media/0.1% BSA/100 I.U./ml penicillin/100 μg/ml streptomycin (15). Calvariae remained in culture for one day before treatment. Cultures were treated with synthetic ET-1 (American Peptide), recombinant mouse DKK1 (R&D Systems), goat anti-human DKK1 polyclonal antibody (R&D Systems), goat anti-mouse IL-6 polyclonal antibody (R&D Systems), or control IgG antibody (R&D Systems)

For primary osteoblast cultures, calvariae were washed in PBS and then placed in PBS (2 ml per mouse litter) containing 0.1% collagenase (Wako Pure Chemical Industries) and 0.2% dispase (Roche Applied Science). Calvariae were agitated at 37°C for seven minutes to release cells. The first extraction was discarded and the process was repeated three times. Osteoblasts isolated in the last three extractions were combined (65). Cells were plated at a density of 106 cells/ml in αMEM/10% fetal calf serum/100 I.U./ml penicillin/100 μg/ml streptomycin. Experimental treatments were begun 48 hours after the cells reached confluence.

RNA extraction and real-time RT PCR

For microarray analysis, pairs of whole calvariae were homogenized and RNA extracted using Trizol (Invitrogen) according to the manufacturer’s directions. RNA was further purified using an RNeasy Kit (Qiagen). For real-time RT PCR experiments, adherent primary osteoblasts in cultures were washed in ice-cold PBS and RNA extracted using an RNeasy Kit. A DNase digestion step was included during RNA purification.

First-strand cDNA was prepared using 1.2 μg total RNA, 2 mM dNTPs, 0.5 μg oligo (dT)12-18 (Invitrogen), 10 mM DTT, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 200 units SuperScript II reverse transcriptase (Invitrogen) in 20 μl reactions according to the manufacturer’s instructions.

PCR reactions were performed using 1.0 μl of cDNA template, 10 mM Tris, 1.5 mM MgCl2, 50 mM KCl, 0.5 μM each primer, 1 mM dNTPs, 1:75,000 dilution SYBR Green (Molecular Probes), 10 nM fluorescein and 1 U Taq polymerase (Roche Applied Science) with a BioRad MyIQ Single-Color Real-Time PCR Detection System. Changes in mRNA concentration were determined by subtracting the Ct (threshold cycle) of the study gene from the Ct of GAPDH (Δ=Ctgene-CtGAPDH). The mean of Δcontrol was subtracted from each of the ΔET-1 reactions (mean Δcontrol - ΔET-1 = ε). The fold difference was calculated as 2ε.

Mouse gene-specific primers were designed using the Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) using the mRNA target sequences associated with the GenBank files listed in Table 2. The forward (F) and reverse (R) primer sequences were: Cebpd: F- atc gct gca gct tcc tat gt, R- aga act gca gag ggc aaa ga; Ctgf: F- gta acc ggg gag gga aat ta, R- gct tta tca cct gca cag ca; Cyr61: F- gca cct cga gag aag gac ac; R- caa acc cac tct tca cag ca; Dkk1: F- tat gag ggc ggg aac aag ta, R- acg gag cct tct tgt cct tt; Dlx3: F- cag acg gcg agg agt tct ac, R- ctt tgg ttt gga ccc tca aa; Dlx5: F- tct cag gaa tcg cca act tt, R- ctg gtg act gtg gcg agt ta; Gapdh: F- tgt tcc tac ccc caa tgt gt, R- tgt gag gga gat gct cag tg; Il6: F- ccg gag agg aga ctt cac ag, R- gga aat tgg ggt agg aag ga; Il11: F- ctg tgg gga cat gaa ctg tg, R- aag ctg caa aga tcc caa tg; Inhba: F- atc atc acc ttt gcc gag tc, R- ccc ttt aag ccc att tcc tc; Inhbb: F- cga gat cat cag ctt tgc ag, R- tcc acc ttc ttc tcc acc ac; Kremen: F- agc tgt gcc ctg aag gta ga, R- tga gat gat gac ccc cta gc; LRP5: F- ggt cac ctg gac ttc gtc at, R- tcc agc gtg tag tgt gaa gc; LRP6: F- ggg cag aca cag gaa caa at, R- tgc gtt cac ttc cat cca ta; Rankl: F- tgt act ttc gag cgc aga tg, R- ccc aca atg tgt tgc agt tc; Sfrp1: F- gct caa caa gaa ctg cca ca, R- cgt tct tca gga aca gca ca; Sgk: F- ggg tgc caa gga tga ctt ta, R- cag aaa agg cac att gca ga; Snai1: F- gag gac agt ggc aaa agc tc, R- tcg gat gtg cat ctt cag ag; Tgif: F- ttg aat tcc aac cca gaa gc, R- agg gag gtt tgg gag aca ct; Timp3: F- ttt cca act ggg gat ctc tg, R- caa gct tcc agc caa act tc; Tsc22: F- ttc tcg ctt tct ccc cag ta, R- tag gaa gga caa gcc aca cc; Twist2: F- acc agt gag gaa gag ctg ga, R- tga ggg cac aga agt cac tg;Wnt5a: F- ctg gca gga ctt tct caa gg, R- gcg gcg cta tca tac ttc tc.

Microarray analysis

Microarray analysis of Affymetrix chips was performed by the University of Virginia Biomolecular Research Facility essentially as previously described (66). Briefly, cRNA was prepared from 8 ug of total RNA. The 430A 2.0 chip representing 22,626 probe sets was utilized for the 6 and 24 hour ET-1-treated groups and the U74Av2 chip representing 12,422 probe sets was utilized for the 4 and 7 day ET-1-treated groups. After washing in a fluidic station, the arrays were scanned with a 2.5 micron resolution HP Microarray Scanner (Hewlett Packard). The scanned image file was analyzed using the Affymetrix Microarray Analysis Suite 5.0 (MAS 5.0, Affymetrix, Santa Clara, CA) and the dChip software (67, 68). The detection of a particular gene, called ‘present’, ‘absent’, or ‘marginal’, was made using the nonparametric Wilcoxon ranked score algorithm in MAS 5.0, those detection calls were later imported into and utilized by the dChip program. Scatter plots were also generated using this software to inspect the reproducibility of the replicates as well as the degree of changes of the samples under comparison. Quantitation of the genes was obtained using the dChip, which applied the PM/MM model-based approach to derive the probe sensitivity index and expression index. The two indices were used in a linear regression to quantify a particular gene. When certain probes or transcripts deviated from the model to a set extent, they were excluded from the quantitation process. Normalization of the arrays was performed using the invariant set approach. Comparative analysis of the samples using dChip generated fold changes and Student t-test p-values. Usually the significance criterion matrix consisted of: 1) p<= 0.05; 2) fold change >= 1.5; and 3) signal differential >=Min (100, 10× 10 % mean signal intensity of absent probe sets). Hierarchical clustering of the genes was performed after an appropriate filtration of the data. Alternatively, data were analyzed with Affymetrix Data Mining Tool (DMT), which was a statistical tool package for MAS 5 generated data. The same criterion matrix applied to DMT to determine the significance of the changes.

Histologic methods and analysis

Calvariae were fixed in 10% buffered formalin for 24 hours and decalcified with 10% EDTA for 48 hours. Tissue samples were then processed, paraffin-embedded, sectioned and stained with H&E/Orange-G staining. New bone area and osteoblast number were analyzed using the MetaMorph imaging analysis system (Universal Imaging Corporation). Histomorphometric analysis of calvariae was performed in quadruplicate using a 20X objective lens. New bone area and osteoblast number was determined from a calvarial microscopic field that measured 0.57 mm in length.

Interleukin 6 and DKK1 ELISA

Mouse calvariae were harvested and treated with or without 100 nM ET-1 in quadruplicate. Media were collected and replaced with fresh media on days two, four and six. Mouse interleukin 6 ELISA analysis was performed using a Quantikine mouse interleukin 6 immunoassay (R&D Systems). Mouse DKK1 ELISA was performed as described in Tian, et al. (22).

Immunohistochemistry

Paraffin-embedded murine calvarial sections were deparafinized and hydrated. Sections were treated with 0.3% hydrogen peroxide for 30 minutes and stained using a Vectastain Elite ABC kit (Vector Laboratories) according to the manufacturer’s directions. A rabbit anti-β-catenin primary antibody (Chemicon International) was used at a concentration of 1:250.

Statistical analyses

Analyses were performed using Graph Pad Prism 4.00 software. Comparisons of two groups were performed with an unpaired t test, using a one-tailed analysis for confirmation of gene microarray data; otherwise a two-tailed analysis was used. Comparisons of three or more groups were performed using one-way ANOVA; post-test analyses were performed using Tukey multiple comparison testing.

ACKNOWLEDGEMENTS

This work was supported by grants from the National Institutes of Health (CA69158, DK067333 and CA40035 to T.A.G.; AR52295 to G.A.C.; and CA55819 to J.D.S.), Gerald D. Aurbach Endowment (T.A.G.), the Mellon Institute and Cancer Center of The University of Virginia (T.A.G.) and the Endocrine Fellows Foundation (G.A.C.).

Footnotes

*This is an un-copyedited author manuscript copyrighted by The Endocrine Society. This may not be duplicated or reproduced, other than for personal use or within the rule of ‘Fair Use of Copyrighted Materials’ (section 107, Title 17, U.S. Code) without permission of the copyright owner, The Endocrine Society. From the time of acceptance following peer review, the full text of this manuscript is made freely available by The Endocrine Society at http://www.endojournals.org/. The final copy edited article can be found at http://www.endojournals.org/. The Endocrine Society disclaims any responsibility or liability for errors or omissions in this version of the manuscript or in any version derived from it by the National Institutes of Health or other parties. The citation of this article must include the following information: author(s), article title, journal title, year of publication and DOI.

Disclosure Statement: G.A.C., K.S.M., Y.B., O.W.S., L.J.S., J.W.F., J.M.C., and T.A.G. having nothing to declare. J.D.S. consults for and has received lecture fees from Novartis and Millennium.

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