Human Osteocalcin and Bone Sialoprotein Mediating Osteomimicry of Prostate Cancer Cells: Role of cAMP-Dependent Protein Kinase A Signaling Pathway

Introduction

The progression of prostate cancer to androgen independence and bone metastasis is generally lethal. Death often results from bone and visceral organ metastasis (1). Despite the prevalence of prostate cancer metastasis to the skeleton, the molecular mechanisms of bone tropism are poorly understood. Previous studies suggest that prostate cancer cell adhesion, extravasation, migration, and interaction with bone cells are critical determinants that govern prostate cancer bone colonization (2–4) . Reports using clinical prostate cancer metastasis specimens (5–7) and experimental cell and animal models (8–10) found the bone-specific proteins osteocalcin and bone sialoprotein to be expressed in a heterogeneous manner by human prostate cancer specimens. We proposed that prostate cancer cells acquire osteomimetic or bone-like properties to improve their adhesion, proliferation, and survival in bone (11). This communication delineates the molecular mechanisms underlying the induction of human osteocalcin and bone sialoprotein promoter activities and their endogenous gene expression in the bone microenvironment by factors secreted from prostate cancer and bone stromal cells.

Osteocalcin (5-6 kDa) and bone sialoprotein (72-80 kDa) are synthesized and secreted by normal maturing osteoblasts. They are major noncollagenous bone matrix proteins, with osteocalcin comprising 1% to 2% of the total proteins in the skeleton (12). Osteocalcin binds with high affinity to hydroxyapatite crystals, the key mineral component of bone, and regulates bone crystal growth (13). Osteocalcin can also act as a chemoattractant in the recruitment of osteoblasts and osteoclasts, contributing to the dynamics of new bone formation and bone resorption (14). Ducy et al. (15) reported that osteocalcin-null mice exhibit increased bone formation without impaired bone resorption, suggesting a more complex interaction between recruited osteoblasts and osteoclasts and the participation of bone sialoprotein in this process. Bone sialoprotein is a highly sulfated, phosphorylated, and glycosylated protein that mediates cell attachment through a RGD motif to extracellular matrices (16). Due to its highly negatively charged characteristics, bone sialoprotein can sequester calcium ions while conserving polyglutamate regions, which have hydroxyapatite crystal nucleation potential (17). Through the RGD motif, bone sialoprotein mediates the attachment and activation of osteoclasts (18) and can facilitate attachment of normal bone or cancer cells to mineralized tissue surfaces (19, 20) . Through its binding to factor H, bone sialoprotein can protect cells from complement-mediated cell lysis, which may be important for cancer cell survival (21). In the absence of osteocalcin, bone sialoprotein could contribute to an overall metabolic shift toward new bone formation (22–24) . The published data suggest that osteocalcin and bone sialoprotein could complement each other by regulating the homeostasis of bone formation and bone resorption via controlled osteosclerotic and osteoclastogenic reactions.

The coupling of G proteins with a cyclic AMP (cAMP)–dependent protein kinase A (PKA) signaling pathway is triggered by a large number of ligand and receptor systems (25). Activation of this signaling pathway is associated with the control of cell growth and differentiation (26, 27) , ion channel conductivity (28), and gene transcription (29–31) . The cAMP-responsive element (CRE), a cis-element, interacts with a basic domain/leucine zipper motif contained within a transcription factor termed CRE-binding protein (CREB) through the cAMP-dependent PKA pathway (32). Studies in which both osteocalcin (30) and bone sialoprotein (33, 34) promoter activities were stimulated by the cAMP-dependent PKA pathway suggest the presence of the putative CRE site within these promoter regions that may interact with CREB to initiate osteocalcin and bone sialoprotein expression.

In this communication, we show that conditioned media collected from various human prostate cancer and bone stromal cell lines induce human osteocalcin and bone sialoprotein promoter activities and increase their steady-state levels of endogenous mRNA through a cAMP-dependent PKA pathway in human prostate cancer cells that targets CRE cis-elements within the human osteocalcin and bone sialoprotein promoter regions.

Materials and Methods

Reagents. Tissue culture medium and fetal bovine serum were obtained from Life Technologies, Inc. (Rockville, MD) and Sigma (St. Louis, MO), respectively. Reagents used for the study of cAMP-dependent signaling pathway, forskolin, dibutyryl cAMP (db cAMP), phorbol 12-myristate 13-acetate, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), were purchased from Alexis Biochemicals (San Diego, CA). Synthetic oligonucleotides were ordered from Invitrogen (Carlsbad, CA). Restriction enzymes, T4 DNA ligase and T4 polynucleotide kinase, were obtained from New England Biolabs (Beverly, MA). Radioactive nucleotides were purchased from Amersham Biosciences Corp. (Piscataway, NJ). Taq DNA polymerase was obtained from Roche Molecular Biochemicals (Indianapolis, IN). QuikChange site-directed mutagenesis kit was obtained from Stratagene (La Jolla, CA).

Cells, Cell Culture, and Conditioned Medium Collection. Human prostate cancer cell lines LNCaP, C4-2B, DU145, PC3, and ARCaP and human bone stromal cell lines MG63 (a human osteosarcoma cell line) and KeesII (a human normal osteoblast cell line) were cultured in T-medium (Life Technologies) supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin as described previously (35). The cells were maintained at 37°C in 5% CO₂. For conditioned medium collection, cells were cultured in T-medium with serum until 80% confluent. The cells were washed subsequently twice in PBS and incubated in T-medium without serum. After 2 days of additional incubation, conditioned media were collected, centrifuged, and stored at −20°C until use. The concentration of total proteins in conditioned medium was determined by the Bradford method using Coomassie plus protein reagent (Pierce, Rockford, IL).

Plasmid Construction. Genomic DNA was used in the PCR of the 0.8-kb human osteocalcin promoter inserting KpnI and XhoI sites, subsequently cloned into pGL3-Basic-luciferase reporter vector (Promega, Madison, WI) as described previously (9). The deletion constructs, ΔA [374 bp upstream of AP1/VDRE (AV) element], ΔTst-1 (POU-factor Tst-1/Oct-6 binding site), ΔCRE, and ΔIRF-1 (IFN regulatory factor-1 binding site) in human osteocalcin promoter/pGL3 were generated by recombinant PCR technique. The point mutation plasmids in CRE (−643 to −636 in human osteocalcin promoter, 5′-TGACCTCA-3′) were constructed with a QuikChange site-directed mutagenesis kit. The mutation constructs (see below) are the one-point substitution mutants, Mut1 (−642 G→T), Mut2 (−641 A→C), Mut3 (−640 C→A), Mut4 (−639 C→A), and Mut5 (−638 T→G); a two-point substitution mutant, Mut6 (−640 and −639 CC→AA); and a two-point deletion construct, Mut7 (−640 and −639 CC→XX). The 1.5-kb human bone sialoprotein promoter construct prepared in the pGL3-luciferase reporter vector has been described previously (36). The single deletion constructs ΔCRE1 (−79 to −72) and ΔCRE2 (−674 to −667) and a double deletion construct ΔCRE2/CRE1 in the human bone sialoprotein promoter/pGL3-luciferase plasmid were generated by recombinant PCR technique. All plasmid constructs were confirmed by DNA sequencing.

Transfection and Luciferase Activity Assay. Cells were trypsinized and seeded at a density of 1.5 × 10⁵ cells per well (LNCaP, C4-2B, DU145 and ARCaP), and 1.0 × 10⁵ cells per well (PC3 and MG63) in 12-well plates 24 hours before transfection. Plasmid DNAs were introduced into cells by complexing with a commercial reagent N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammonium methylsulfate (Roche Molecular Biochemicals) according to the manufacturer's protocol. Each transfection reaction contained 1.25 μg of tested DNA constructs and 0.25 μg of the transfection efficiency control cytomegalovirus promoter-driven β-galactosidase plasmid DNA. After 6 hours of transfection, DNA-liposome mixtures were replaced by fresh T-medium or conditioned medium. Transfected cells were harvested and lysed in 1× reporter lysis buffer (Promega) after 36 hours of additional incubation. Cell lysates were vortexed for 15 seconds and spun for 10 minutes. For luciferase activity assay, 20 μL of the lysate supernatant were mixed with 100 μL of the luciferase substrate (Promega) and detected by a luminometer (Monolight 3010 luminometer, PharMingen, San Diego, CA). For β-galactosidase activity assay, 100 μL of the supernatant were mixed with 100 μL of 2× β-galactosidase substrate [200 mmol/L sodium phosphate buffer (pH 7.3), 2 mmol/L MgCl₂, 100 mmol/L β-mercaptoethanol, 1.33 mg/mL ONPG] and incubated at 37°C for 30 minutes. β-galactosidase activity was detected by Microplate spectrophotometer (Molecular Devices Corp., Sunnyvale, CA) at 405 nm wavelength. Data were presented as normalized luciferase activity (means ± SD) defined as luciferase activity normalized to internal control β-galactosidase activity for transfection efficiency. All studies were done in three independent experiments with duplicate assays.

Reverse Transcription-PCR Analysis. Total RNA was isolated from confluent monolayers of cells using RNAZol B (Teltest, Inc., Friendswood, TX). Total RNA (5 μg) was used as template and random hexanucleotide primers (0.15 μg) were added for reverse transcription and amplification in a reaction volume of 20 μL according to the manufacturer's instruction (Invitrogen). After reverse transcription reaction, first-strand cDNA (3-5 μL) was used for PCR with a PTC-100 programmable thermal controller (MJ Research, Inc., Waltham, MA). The oligonucleotide primer sets used for PCR analysis of cDNA are human osteocalcin 5′-ACACTCCTCGCCCTATTG-3′ (forward) and 5′-GATGTGGTCAGCCAACTC-3′ (reverse), human bone sialoprotein 5′-GCATCGAAGAGTCAAAATAG-3′ (forward) and 5′-TTCTTCTCCATTGTCTTCTC-3′ (reverse), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 5′-ACCACAGTCCATGCCATCA-3′ (forward) and 5′-TCCACCACCCTGTTGCTGT-3′ (reverse). The thermal profile for human osteocalcin amplification is 30 cycles starting with denaturation of 1 minute at 94°C followed by 1 minute of annealing at 55°C and 1 minute for extension at 72°C. For human bone sialoprotein amplification, the thermal profile is 35 cycles starting with denaturation for 1 minute at 94°C followed by 30 seconds of annealing at 48°C and 40 seconds of extension at 72°C. The program for GAPDH amplification is 25 cycles starting with denaturation for 30 seconds at 94°C followed by 30 seconds of annealing at 60°C and 1 minute of extension at 72°C. The reverse transcription-PCR products were analyzed by 1.2% agarose gel electrophoresis. Quantity one-4.1.1 Gel Doc gel documentation software (Bio-Rad, Hercules, CA) or NIH image were used for quantification of human osteocalcin and bone sialoprotein mRNA expression normalized by GAPDH mRNA expression.

Immunohistochemical Staining. Human primary and bone metastatic prostate cancer tissue specimens were deparaffinized, treated with 3% H₂O₂, blocked with SuperBlock (Scytek Laboratories, Logan, UT), and reacted with antibodies against osteocalcin (OC 4-30, PanVera Corp., Madison, WI) or bone sialoprotein (kindly provided by Dr. J. Sodek, University of Toronto, Toronto, Ontario, Canada). The staining signals were amplified by biotinylated peroxidase-conjugated streptavidin system (Bio-Genex Laboratories, San Ramon, CA). Osteocalcin and bone sialoprotein were visualized in selective areas of the clinical tissue specimens where the immunohistochemical staining was revealed by the conjugated peroxidase reacted with AEC or 3,3′-diaminobenzidine as the substrate. Positive osteocalcin and bone sialoprotein are defined as >15% of the cell populations reacted positively with either anti-osteocalcin or anti–bone sialoprotein antibody.

Electrophoretic Mobility Shift Assay. A LNCaP-lineaged metastatic human prostate cancer cell line, C4-2B, and the human osteosarcoma cell line MG63 were plated in 15 cm diameter tissue culture dishes in T-medium (with 5% serum) until 80% confluent. Cells were then switched to 1-day complete serum-free condition and then treated with or without ARCaP conditioned medium (15 μg/mL) or forskolin (10⁻⁵ mol/L) for an additional 16 hours. Nuclear extracts were prepared from C4-2B and MG63 for electrophoretic mobility shift assay as described by Ausubel et al. (37). Briefly, cells were washed with cold PBS twice, harvested, and homogenized in hypotonic buffer [10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl₂, 10 mmol/L KCl, 0.5 mmol/L DTT, 0.2 mmol/L phenylmethylsulfonyl fluoride]. After centrifugation, the nuclear pellets were stirred and incubated for 30 minutes in high-salt buffer [20 mmol/L HEPES (pH 7.9), 25% glycerol, 1.5 mmol/L MgCl₂, 1.2 mol/L KCl, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 0.2 mmol/L phenylmethylsulfonyl fluoride] on ice. The nuclear pellets were centrifuged and the nuclear extract supernatants were dialyzed twice against dialysis buffer [20 mmol/L HEPES (pH 7.9), 20% glycerol, 100 mmol/L KCl, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 0.2 mmol/L phenylmethylsulfonyl fluoride] overnight. The concentration of nuclear proteins was determined by the Bradford method using Coomassie plus protein reagent. Synthetic oligonucleotides were purified by PAGE. Appropriate pairs were annealed by heating up to 95°C for 10 minutes and naturally cooled down to room temperature. The oligo sequences used as probes or competitors were wild-type CRE 5′-ACCAACCGGCTGACCTCATCTCCTGCC-3′ and Mut6 5′-ACCAACCGGCTGAAATCATCTCCTGCC-3′. The double-stranded probes were end labeled with [γ-³²P]ATP (3,000 Ci/mmol at 10 mCi/mL) using T4 polynucleotide kinase. Forty thousand counts per minute of the labeled probe and 10 μg of nuclear extracts were incubated with binding buffer containing 10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, 0.5 mmol/L EDTA, 0.5 mmol/L DTT, 4% glycerol, 1 mmol/L KCl, and 1 μg poly(deoxyinosinic-deoxycytidylic acid) (Amersham Pharmacia Biotech, Piscataway, NJ) at 30°C for 30 minutes. The samples were subjected to 6% nondenaturing PAGE in 1× TGE buffer [25 mmol/L Tris-HCl (pH 8.5), 188 mmol/L glycine, 1 mmol/L EDTA] at 35 mA and 200 V for 2 hours at room temperature. In competition experiments, unlabeled competitor oligos were preincubated with nuclear extracts for 30 minutes at room temperature before the addition of the probe. For the supershift experiment, 2 μg of anti-CREB antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-AML3/Runx2 antibody (Active Motif, Inc., Carlsbad, CA) were added to the nuclear extract reaction mixtures at room temperature for 30 minutes before the incubation of the probe. After electrophoresis, gels were dried by Gel Dryer (model 583, Bio-Rad) and exposed to BioMax film (Kodak, Rochester, NY).

Statistical Analysis. All statistical analyses were done using Microsoft Excel software. Significant differences were analyzed using Student's t test and two-tailed distribution.

Results

Human Osteocalcin and Bone Sialoprotein Expression in Tissue Specimens, Promoter Regulation in Human Prostate Cancer Cell Lines. Osteocalcin protein is prevalently expressed by both primary (85% positively stained) and metastatic (both lymph node and bone were 100% positively stained) human prostate cancer specimens (8). Likewise, bone sialoprotein protein is also expressed preferentially by malignant (89-100%) primary prostate cancer tissues (7). One common feature of osteocalcin and bone sialoprotein immunostaining in human prostate cancer tissues is the marked heterogeneities among prostate cancer cells in primary and bone metastatic specimens. Some cells stain strongly for osteocalcin and bone sialoprotein proteins ( Fig. 1A, bold arrows ) and others seem to be lightly stained or not stained at all ( Fig. 1A, arrowheads). Differential staining could either reflect intrinsic genetic variations among the prostate cancer cells or be an epiphenomenon of prostate cancer cell interaction with the microenvironment. We sought to evaluate the regulation of osteocalcin and bone sialoprotein expression in human prostate cancer and bone cells to define whether extrinsic factor(s) secreted by prostate cancer or bone cells could mediate osteocalcin and bone sialoprotein promoter activities and their respective steady-state levels of endogenous mRNA expression.

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Figure 1.

Immunohistochemical staining of osteocalcin (OC) and bone sialoprotein (BSP) in human primary and bone metastatic prostate cancer tissue specimens, and conditioned medium stimulates human osteocalcin and bone sialoprotein promoter activities and the steady-state levels of osteocalcin and bone sialoprotein mRNA expression in human prostate cancer cell lines. A, positive and strong osteocalcin and bone sialoprotein stains were detected in both primary and bone metastatic clinical prostate cancer specimens (bold arrows). Some areas of the prostate cancer cells were found only lightly or not stained at all (arrowheads). Osteomimicry was found to exist in prostate cancer cells when present in primary. Magnification, ×75. B, conditioned media (CM) were collected from human prostate cancer cell lines (LNCaP, C4-2B, DU145, PC3, and ARCaP), a normal human osteoblastic cell line (KeesII), and a human osteosarcoma cell line (MG63). The human osteocalcin promoter-reporter construct was cotransfected with cytomegalovirus promoter-driven β-galactosidase plasmid (for the correction of transfection efficiency as an internal control) into an androgen-independent and metastatic LNCaP cell subline, C4-2B. Conditioned medium induced human osteocalcin promoter activity in a dose-dependent manner (total protein concentration 0-15 μg/mL). C, ARCaP conditioned medium also stimulated human bone sialoprotein promoter activity in a dose-dependent manner (total protein concentration 0-15 μg/mL). D, human osteocalcin and bone sialoprotein promoter-reporter activities were determined in LNCaP, C4-2B, DU145, PC3, ARCaP, and MG63 cell lines in the presence or absence of ARCaP conditioned medium (15 μg/mL). Human osteocalcin and bone sialoprotein promoter activities were dramatically elevated by ARCaP conditioned medium in LNCaP and C4-2B cells. Fold induction was calculated from the promoter activities assayed in the presence or absence of conditioned medium. Columns, mean of three independent experiments with duplicate assays in each experiment; bars, SD. Significant differences of the fold inductions of human osteocalcin or human bone sialoprotein reporter activity were observed by the addition of ARCaP conditioned medium: **, P < 0.005. E, reverse transcription-PCR was done using total RNA isolated from LNCaP, C4-2B, PC3, and MG63 cells in the absence (-) or presence (+) of ARCaP conditioned medium (15 μg/mL of total protein) for a 12-hour incubation period. Expression of the housekeeping gene GAPDH was used as a loading control. Relative expression values of osteocalcin and bone sialoprotein mRNA, normalized by the amounts of GAPDH mRNA expression, were measured by Gel Doc gel documentation software. Fold induction represents the ratios of ARCaP conditioned medium–treated versus vehicle-treated control of each cell line.

To test the hypothesis that bone-specific protein expression by prostate cancer cells may be induced by factor(s) secreted from prostate cancer and bone stromal cells, we compared the effects of conditioned medium harvested from either prostate cancer or bone stromal cells on the expression of osteocalcin and bone sialoprotein in an androgen-independent C4-2B prostate cancer cell line of LNCaP lineage. A luciferase reporter construct with a 0.8-kb human osteocalcin promoter or a 1.5-kb human bone sialoprotein promoter was transfected into C4-2B cells, and the transfected cells were exposed to serum-free conditioned medium collected from various human prostate cancer cell lines with a gradient of malignant potentials [from LNCaP (the least malignant), C4-2B, DU145, and PC3 (cells with intermediate levels of aggressiveness) to ARCaP (the most malignant)], a normal nonmalignant osteoblast KeesII cell line or a malignant osteosarcoma MG63 cell line. As shown in Fig. 1B, conditioned medium stimulated human osteocalcin promoter activity in a concentration-dependent manner (0-15 μg/mL of total proteins in conditioned medium). Conditioned medium from LNCaP cells maximally stimulated human osteocalcin promoter activity by only 1.2 ± 0.1-fold, whereas conditioned medium collected from the most aggressive ARCaP prostate cancer cell line maximally enhanced the highest human osteocalcin promoter activity at 7.1 ± 0.3-fold. Conditioned medium from other cell lines, C4-2B, DU145, PC3, KeesII, and MG63, induced human osteocalcin promoter activity at intermediate levels (2.0 ± 0.2- to 5.1 ± 0.4-fold). These data suggest that the extent of stimulation of human osteocalcin promoter activity by conditioned medium correlated positively with the aggressiveness of the prostate cancer. In parallel with the induction of human osteocalcin promoter activity, ARCaP conditioned medium also up-regulated human bone sialoprotein promoter activity as much as 12-fold in a concentration-dependent manner in C4-2B cells ( Fig. 1C).

To determine further whether ARCaP conditioned medium is capable of stimulating human osteocalcin and bone sialoprotein promoter activities in a series of other human prostate cancer and bone stromal cell lines, we tested these promoter activities in LNCaP, DU145, PC3, ARCaP, and MG63 cells. As shown in Fig. 1D, both human osteocalcin and bone sialoprotein promoter activities were elevated by ARCaP conditioned medium in LNCaP and C4-2B but not in DU145, PC3, ARCaP, and MG63 cell lines.

We compared the effect of ARCaP conditioned medium in inducing human osteocalcin and bone sialoprotein promoter-reporter activities in LNCaP and C4-2B cells, and its effects on the steady-state levels of osteocalcin and bone sialoprotein mRNA, in several human prostate cancer cell lines by semiquantitative reverse transcription-PCR. Figure 1E shows that adding ARCaP conditioned medium (15 μg/mL) to LNCaP and C4-2B cells for a 12-hour period enhanced the steady-state levels of endogenous osteocalcin and bone sialoprotein mRNA expression by 4.8- and 5.9-fold and 4.5- and 7.8-fold (GAPDH as an internal control), respectively. In cells that already had high basal levels of osteocalcin and bone sialoprotein mRNA, such as PC3 and MG63 (38) cells, ARCaP conditioned medium did not enhance further osteocalcin and bone sialoprotein mRNA expression (0.9- to 1.3-fold induction, respectively). As with the promoter activity assay, the steady-state levels of endogenous osteocalcin and bone sialoprotein mRNA also did not show an increase in DU145 and ARCaP cell lines on exposure to ARCaP conditioned medium (data not shown).

CRE Is Responsible for Regulation of Conditioned Medium–Mediated Human Osteocalcin and Bone Sialoprotein Promoter Activities. Our previous data (9) showed that three cis-elements are critical for the regulation of human osteocalcin promoter activity: OSE1, OSE2, and AV. To determine whether these elements are important for ARCaP conditioned medium–activated human osteocalcin promoter activity, we assessed the activities of several human osteocalcin promoter deletion constructs, including single, double, or triple deletion constructs of OSE1, OSE2, and AV generated by the recombinant PCR method as described previously (9). Figure 2A shows that among the single deletion constructs, ΔAV did not seem to affect ARCaP conditioned medium–induced human osteocalcin promoter luciferase activity. In comparison, a slight drop of human osteocalcin promoter activity was observed on the deletion of OSE1 or OSE2. No further decrease in human osteocalcin promoter-luciferase activity induced by ARCaP conditioned medium was noted by deleting additional cis-elements, including the complete deletion of all three critical human osteocalcin regulatory elements, ΔAV, ΔOSE2, and ΔOSE1. These data suggest that regions outside of OSE1, OSE2, and AV must be responsible for human osteocalcin promoter activation by ARCaP conditioned medium.

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Figure 2.

CRE is responsible for the human osteocalcin and bone sialoprotein promoter activation induced by ARCaP conditioned medium. A, deletion analysis of human osteocalcin promoter. Three cis-elements, AV, OSE2, and OSE1 (9), are not critical for the human osteocalcin promoter activation regulated by ARCaP conditioned medium (basal luciferase activities, expressed as relative luciferase activity, in control human osteocalcin/Luc and ΔAV/OSE2/OSE1 were 1440 ± 58 and 1160 ± 200, respectively). ARCaP conditioned medium–mediated human osteocalcin promoter reporter activity was not affected by the elimination of these three cis-elements. B, deletion of CRE element abrogated the ARCaP conditioned medium–mediated activation of human osteocalcin promoter reporter activity. Region A (374 bp), upstream from AV element, contains three cis-elements, Tst-1 (−848 to −834), CRE (−643 to −636), and IRF-1 (−609 to −597). Deletion of region A (ΔA) mutant in human osteocalcin promoter dramatically decreased the conditioned medium–mediated activation of the promoter activity. Subsequently, ΔTst-1, ΔCRE, and ΔIRF-1 mutant constructs were generated from the human osteocalcin promoter using the recombinant PCR method. Only the ΔCRE construct abolished ARCaP conditioned medium–induced human osteocalcin promoter activity. Relative activities of various human osteocalcin mutation reporter constructs were determined in the presence or absence of ARCaP conditioned medium (minus ARCaP conditioned medium of the human osteocalcin/Luc promoter activity was designed as 1.0). *, P < 0.05; **, P < 0.005, significant difference of the relative luciferase activity. C, two putative CRE sites were cooperatively regulated in the human bone sialoprotein promoter activity by ARCaP conditioned medium. Single deletion of CRE1 (ΔCRE1, −79 to −72) or CRE2 (ΔCRE2, −674 to −667) in human bone sialoprotein promoter reduced partially the promoter activation; the double deletion ΔCRE2/CRE1 construct markedly decreased the ARCaP conditioned medium–induced human bone sialoprotein promoter activity (the human bone sialoprotein/Luc promoter activity was assigned as 1.0 in the absence of ARCaP conditioned medium). *, P < 0.05; **, P < 0.005. Columns, mean of three independent studies with duplicate assays in each experiment; bars, SD. D, point mutation constructs of the CRE site within human osteocalcin promoter were constructed using the QuikChange site-directed mutagenesis kit (see Materials and Methods). Relative activities of human osteocalcin mutation reporter constructs were determined and compared with the human osteocalcin/Luc construct (assigned as 1.0 without adding ARCaP conditioned medium). Columns, mean of three independent studies with duplicate assays in each experiment; bars, SD. *, P < 0.05; **, P < 0.005, significant difference from the relative luciferase activity of human osteocalcin/Luc.

To address this question, we generated three new deletion constructs with regions outside of the OSE1, OSE2, or AV element systematically deleted. These are designated ΔA (upstream of the AV element, 374 bp; Fig. 2B), ΔB (between AV and OSE2 site, 327 bp), and ΔC (between OSE2 and OSE1 site, 99 bp). As shown in Fig. 2B, a dramatic decrease in human osteocalcin promoter activity was observed only when region A was deleted. Minimal loss of ARCaP conditioned medium–induced human osteocalcin promoter luciferase activity was detected with deletion of region B or C (data not shown).

To find the specific cis-DNA element within region A that may be responsible for ARCaP conditioned medium–induced human osteocalcin promoter activity, we site-specifically deleted selected regions of A, ΔTst-1 (POU-factor Tst-1/Oct-6, −848 to −834), ΔCRE (−643 to −636), and ΔIRF-1 (−609 to −597), based on a computer database search, and tested the activity of these constructs in C4-2B cells exposed to either ARCaP conditioned medium or control medium. Figure 2B shows that only the ΔCRE construct exhibited a marked decreased in ARCaP conditioned medium–induced human osteocalcin promoter luciferase activity, suggesting that cAMP may mediate downstream signaling through CRE, regulating ARCaP conditioned medium–induced human osteocalcin promoter activity.

Then, we generated CRE deletion constructs of human bone sialoprotein promoter. Based on computer search, there are two putative CRE sites, CRE1 (−79 to −72) and CRE2 (−674 to −667), within human bone sialoprotein promoter. As shown in Fig. 2C, human bone sialoprotein promoter luciferase activity decreased partially in the two single deletion constructs of either CRE1 or CRE2 (designated as ΔCRE1 and ΔCRE2). However, human bone sialoprotein promoter activation was markedly reduced in the double-deleted ΔCRE2/CRE1 construct when exposed to ARCaP conditioned medium. This study shows that CREs are also important for the regulation of human bone sialoprotein promoter reporter activity enhanced by ARCaP conditioned medium.

To delineate the specific nucleotide(s) within the CRE of human osteocalcin promoter that may be responsible for ARCaP conditioned medium–regulated promoter activity, we introduced either one or two point mutations in CRE and examined ARCaP conditioned medium–induced human osteocalcin promoter luciferase activity in C4-2B cells. Only Mut3 (−640 C→A) and Mut4 (−639 C→A) greatly diminished the ARCaP conditioned medium–activated human osteocalcin promoter activity ( Fig. 2D). Other single base mutations, −642 G→T (Mut1), −641 A→C (Mut2), and −638 T→G (Mut5), failed to exert much influence on conditioned medium–mediated human osteocalcin promoter activity when assayed under the same conditions. Consistent with these results, double-base mutations at −640 and −639 CC→AA (Mut6) and deletion at this same region, CC→XX (Mut7), dramatically reduced ARCaP conditioned medium–induced human osteocalcin promoter activity. Although we did not perform similar mutational analysis of human bone sialoprotein promoter because of the structural identity between CREs among human osteocalcin and bone sialoprotein promoters (with the exception of a single base difference found in CRE1), it is highly likely that similar mutations in CRE1 and CRE2 will result in disruption of human bone sialoprotein promoter activity when assayed in prostate cancer cells. Taken together, the results show that two nucleotides, −640 (C) and −639 (C) within the CRE cis-element of human osteocalcin promoter, are cooperatively responsible for the ARCaP conditioned medium–mediated human osteocalcin promoter activation.

cAMP-Dependent PKA Signaling Pathway Is Essential for Mediating ARCaP Conditioned Medium–Activated Osteocalcin and Bone Sialoprotein Gene Expression in Human Prostate Cancer Cells. The cAMP-dependent PKA pathway has long been shown to mediate specific intracellular signaling events, including the transcription of specific genes via the CRE cis-element (25, 30, 31) . To determine whether ARCaP conditioned medium stimulation of human osteocalcin and bone sialoprotein promoter activities may be mediated through an activation of the PKA signaling pathway, we assessed the effects of PKA pathway activators, db cAMP and forskolin, on human osteocalcin and bone sialoprotein promoter activities in C4-2B cells and correlated this with endogenous mRNA expression in various human prostate cancer cell lines. The PKA pathway activators, db cAMP (10⁻⁶-10⁻³ mol/L) and forskolin (10⁻⁸-10⁻⁵ mol/L), stimulated human osteocalcin ( Fig. 3A ) and human bone sialoprotein ( Fig. 3B) promoter activities in a ligand concentration–dependent manner in C4-2B cells. These results were confirmed by an assessment of endogenous osteocalcin and bone sialoprotein mRNA expression after induction by a PKA activator, forskolin. Following forskolin treatment (10⁻⁶ mol/L), osteocalcin and bone sialoprotein mRNA expression in LNCaP and C4-2B but not in PC3 and MG63 were elevated ( Fig. 3C). In this study, the steady-state levels of osteocalcin mRNA were elevated by 5.2- and 7.8-fold, whereas the levels of bone sialoprotein mRNA were increased by 3.2- and 5.4-fold after forskolin stimulation in LNCaP and C4-2B cells, respectively. This result was in general agreement with the effect of ARCaP conditioned medium on endogenous osteocalcin and bone sialoprotein mRNA expression ( Fig. 1E), further supporting the involvement of PKA as the key downstream signaling pathway regulating soluble factor–mediated osteocalcin and bone sialoprotein gene expression in LNCaP and C4-2B human prostate cancer cells.

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Figure 3.

Human osteocalcin and bone sialoprotein promoter activities were stimulated by db cAMP and forskolin (FSK) in a dose-dependent manner. C4-2B cells were cotransfected with human (A) osteocalcin or (B) bone sialoprotein promoter plus cytomegalovirus/β-galactosidase plasmid (an internal control plasmid for transfection efficiency). Transient transfected cells were treated with different concentrations of db cAMP (10⁻⁶-10⁻³ mol/L, M), forskolin (10⁻⁸-10⁻⁵ mol/L, M), or ARCaP conditioned medium (15 μg/mL) for 16 hours. Human osteocalcin and bone sialoprotein promoter activities were induced by these pharmacologic reagents in a concentration-dependent manner through the activation of cAMP-dependent PKA pathway. Columns, mean fold induction of three separate studies with duplicate assays in each experiment; bars, SD. *, P < 0.05; **, P < 0.005, significant difference from control. C, reverse transcription-PCR was done using total RNA (5 μg) isolated from LNCaP, C4-2B, PC3, and MG63 cell lines in the absence (-) or presence (+) of forskolin (10⁻⁶ mol/L, M) exposure for 12 hours. Relative expression values of osteocalcin and bone sialoprotein mRNA, normalized by the amounts of GAPDH mRNA, were measured by Gel Doc gel documentation software. Fold induction represents the ratios of forskolin-treated versus untreated specimens from each cell line.

The involvement of the PKA pathway in mediating ARCaP conditioned medium activation of osteocalcin and bone sialoprotein expression was further confirmed using a selective inhibitor of PKA, H-89 (39). Forskolin-stimulated human osteocalcin and bone sialoprotein promoter activities were both inhibited by H-89 (10⁻⁸-10⁻⁶ mol/L) in a concentration-dependent manner ( Fig. 4A ). Consistent with this observation, H-89 also inhibited ARCaP conditioned medium– and db cAMP-mediated activation of human osteocalcin promoter activity in prostate cancer cells ( Fig. 4B). Interestingly, phorbol 12-myristate 13-acetate (PMA), an activator of the protein kinase C pathway, also induced human osteocalcin promoter activity to a lesser extent and such activation, as expected, was not blocked by H-89 ( Fig. 4B). In agreement with the above results from the human osteocalcin and bone sialoprotein promoter study, we confirmed that H-89 also inhibited the induction of endogenous osteocalcin and bone sialoprotein mRNA expression by ARCaP conditioned medium or forskolin in LNCaP and C4-2B cells (data not shown). We evaluated further whether the cAMP/PKA signaling pathway may be operative under induction by conditioned medium harvested from C4-2B, DU145, PC3, and MG63 cell lines. Figure 4C shows that H-89 (10⁻⁶ mol/L) nearly abolished conditioned medium–induced human osteocalcin promoter activity in all of conditioned medium harvested from prostate cancer and bone stromal cell lines. These data suggest that stimulation of bone-specific osteocalcin and bone sialoprotein gene expression in human prostate cancer cell lines by soluble factor(s) in prostate cancer or bone conditioned medium occurs primarily through the PKA signaling pathway involving cAMP as a mediator.

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Figure 4.

Effects of a selective inhibitor of PKA pathway H-89 on ARCaP conditioned medium–, db cAMP-, or forskolin-induced elevation of human osteocalcin (hOC) and bone sialoprotein (hBSP) promoter activities. A, C4-2B cells transfected with human osteocalcin or human bone sialoprotein promoter-reporter constructs were treated with various concentrations of H-89 (10⁻⁸-10⁻⁶ mol/L, M) for 2 hours and subsequently exposed to forskolin (10⁻⁶ mol/L, M) for an additional 16 hours. H-89 exerted a concentration-dependent inhibition of human osteocalcin and bone sialoprotein promoter-reporter activities induced by forskolin. Fold induction represents the folds of forskolin-treated reporter activities assayed in the presence or absence of H-89. *, P < 0.05; **, P < 0.005. B, H-89 inhibited ARCaP conditioned medium–, db cAMP-, or forskolin-induced human osteocalcin promoter activity but did not inhibit the promoter-reporter activity assayed under stimulation by phorbol 12-myristate 13-acetate (PMA). H-89 (10⁻⁶ mol/L, M) was added to human osteocalcin promoter-reporter transiently transfected C4-2B cells for 2 hours and then exposed to ARCaP conditioned medium (15 μg/mL), db cAMP (10⁻³ mol/L, M), forskolin (10⁻⁶ mol/L, M), or the protein kinase C pathway activator phorbol 12-myristate 13-acetate (PMA, 10⁻⁶ mol/L, M) for 16 hours. C, H-89 also abolished the human osteocalcin promoter activation induced by C4-2B, DU145, PC3, or MG63 conditioned medium. Columns, mean of three independent experiments with duplicate assays; bars, SD. *, P < 0.05; **, P < 0.005.

Evidence in Support of Nuclear CREB and Cis-Element, CRE, in the Regulation of Bone-Specific Gene Expression in Human Prostate Cancer Cells: Electrophoretic Mobility Shift Assay. To further establish a downstream link between the cAMP-dependent PKA signaling pathway and human osteocalcin and bone sialoprotein promoter activation in prostate cancer cells, we conducted electrophoretic mobility shift assay to compare the binding of a ³²P-labeled oligonucleotide CRE probe and nuclear factors extracted from C4-2B cells (an ARCaP conditioned medium–positive responder) and MG63 cells (an ARCaP conditioned medium–negative responder) that had been exposed previously to ARCaP conditioned medium (15 μg/mL) or forskolin (10⁻⁵ mol/L) for 16 hours in culture. Cells exposed to vehicle were used as controls. Nuclear factors extracted from either ARCaP conditioned medium– or forskolin-treated C4-2B cells strongly enhanced the specific CRE-nuclear protein complex formation ( Fig. 5A, lanes 3 and 5 ) in comparison with cells exposed to control medium ( Fig. 5A, lane 2). These DNA-protein complexes could be competed off by unlabeled specific CRE-oligo probe (lanes 4 and 6). However, no competition was observed with a mutant form of CRE-oligo probe, the Mut6-oligo (lane 9, two-point substitution; see Fig. 2D). Consistent with the biochemical data, H-89 was shown to abolish both ARCaP conditioned medium– and forskolin-induced CRE binding to the nuclear proteins extracted from C4-2B cells (lanes 7 and 8). Nuclear extracts from MG63 cells, in contrast, formed a low but detectable basal level of complexes with ³²P-labeled-CRE probe before and after treatment with ARCaP conditioned medium ( Fig. 5B, lanes 4 and 5). These complexes could be competed off by unlabeled-CRE probe (lane 6) but failed to be supershifted by anti-CREB-specific antibody (lane 7). As expected, nuclear extracts from C4-2B cells exposed to ARCaP conditioned medium did bind to CRE and these CRE-nuclear protein complexes can be supershifted by anti-CREB antibody (lane 2) but not by anti-Runx2 antibody in both cases (lanes 3 and 8). These data show that the trans-acting factor CREB may play a critical role in regulating bone-specific gene transcription through the cAMP/PKA pathway by ARCaP conditioned medium in human prostate cancer but not in bone stromal cells.

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Figure 5.

ARCaP conditioned medium and forskolin enhanced CREB and CRE binding through cAMP-dependent PKA signaling pathway in selective human prostate cancer but not in bone cells. A, C4-2B cells were exposed to ARCaP conditioned medium (CM, 15 μg/mL) or forskolin (F, 10⁻⁵ mol/L) for 16 hours; control cells were exposed to vehicles. Cells were harvested and nuclear extracts (NE) prepared. Electrophoretic mobility shift assay was done by incubating nuclear extracts and the ³²P-labeled CRE probe. Lanes 3 and 5, ARCaP conditioned medium and forskolin enhanced the formation of CRE-nuclear protein complexes. Lanes 4 and 6, complexes were competed off by unlabeled specific CRE-oligo probe. Lane 9, Mut6-oligo (the CRE mutant of two-point substitution; see Fig. 2D) did not compete with the nuclear proteins and ³²P-CRE-oligo complexes. Lanes 7 and 8, H-89 (10⁻⁶ mol/L) blocked both ARCaP conditioned medium– and forskolin-induced CRE binding to the nuclear factors extracted from C4-2B cells. B, C4-2B (lanes 1-3) and MG63 (lanes 4-8) cells were treated with ARCaP conditioned medium (15 μg/mL) or vehicle for 16 hours and nuclear extracts were prepared. Lanes 4 and 5, no or minimum changes of the binding complex formation when experiments were conducted using nuclear extracts from MG63 cells either exposed to ARCaP conditioned medium or untreated. Arrow, CRE and CREB complexes, which were supershifted by adding anti-CREB antibody to the nuclear extracts from C4-2B cells (lane 2) but not from MG63 cells (lane 7). Specificity of the supershift complex was confirmed by the lack of a supershift band when naive Runx2 antibody was used as a reagent (lanes 3 and 8).

Discussion

Prostate cancer expresses proteins normally restricted to bone, such as osteocalcin, bone sialoprotein, osteopontin, and osteonectin (6, 7, 11, 40–43) . We hypothesized that the osteomimetic property of prostate cancer cells results from transcription factor switching (9, 44) . Because of the potential importance of osteomimicry in enhancing cancer cell adhesion, invasion, and metastasis (6, 7, 11) , we designed studies to define how the expression of bone-specific proteins in prostate cancer cells is regulated. We chose to study osteocalcin and bone sialoprotein because of the prevalence of expression of these genes by prostate cancer cell lines (8, 45) and in clinical prostate cancer specimens (7, 8, 46) . Our data showed that osteocalcin and bone sialoprotein expression is stimulated in a selective manner in human prostate cancer but not in bone stromal cell lines. We observed that the induction of osteocalcin and bone sialoprotein expression in prostate cancer cell lines is mediated by paracrine/autocrine factors harvested from conditioned medium of prostate cancer and bone stromal cells (47). The selective nature of soluble factors serving as paracrine mediators in stimulating osteocalcin and bone sialoprotein expression in prostate cancer cell lines agrees with their immunohistochemical staining patterns that are generally heterogeneous (see Fig. 1A; refs. 7, 8, 46 ). Using the stimulatory response of human osteocalcin/human bone sialoprotein promoter reporter activities and the endogenous steady-state levels of osteocalcin/bone sialoprotein mRNA in C4-2B as the assay end points, we showed that the extent of human osteocalcin promoter activation by conditioned medium collected from prostate cancer and bone stromal cell lines correlated directly with the malignant potential of these cells in laboratory immune-compromised mice. We also observed that conditioned medium stimulated human osteocalcin and bone sialoprotein promoter activities in a dose-dependent manner and that these increased promoter reporter activities corresponded with the enhancement of the steady-state levels of endogenous osteocalcin and bone sialoprotein mRNA expressed in LNCaP and C4-2B cells. These results support the idea that osteocalcin and bone sialoprotein, once induced in prostate cancer cells, could facilitate the formation of hydroxyapatite complexes, leading to altered biological functions of prostate cancer cells (i.e., increased cell adhesion, migration and invasion, and recruitment of bone cells). Enhanced bone turnover could contribute to prostate cancer “seeding” in the skeleton (4, 11) .

A CRE cis-element, located upstream of the AV region, is responsible for mediating ARCaP conditioned medium activated human osteocalcin promoter activity and osteocalcin mRNA expression in human prostate cell lines ( Fig. 2B). This cis-element seems to be functional in LNCaP and C4-2B cells but remains silent in several other human prostate cancer (DU145, PC3, and ARCaP) and osteosarcoma (MG63) cell lines. This is supported by the lack of increased osteocalcin expression in these prostate cancer and osteosarcoma cell lines after exposure to ARCaP conditioned medium ( Fig. 1D and E). Further, nuclear extracts from prostate cancer and bone cells that failed to respond to PKA pathway activators showed a lack of CRE binding and supershift activity. In sharp contrast, however, an increased complex formation between cis-element CRE and trans-acting factor CREB and an expected supershift of this complex were observed in the responsive C4-2B cells on the addition of anti-CREB antibody ( Fig. 5A and B). The complex formation is likely to account for increased human osteocalcin promoter activity and enhanced endogenous osteocalcin mRNA expression in human prostate cancer cells in response to factor(s) from the tumor cell microenvironment. A similar mechanism regulating human bone sialoprotein promoter activity and mRNA expression was also observed in LNCaP and C4-2B cells after exposure to ARCaP conditioned medium, a result supported by additional studies using interfering pharmacologic agents that either stimulated (db cAMP and forskolin) or repressed (H-89) cAMP accumulation in target cells ( Figs. 1D and E, 2C, 3B and C, and 4A). Our results show for the first time that conditioned medium harvested from prostate cancer and bone stromal cells stimulated osteocalcin and bone sialoprotein expression primarily through a cAMP-dependent PKA signaling pathway in LNCaP and C4-2B human prostate cancer cells. This stimulation by conditioned medium lends further support to the observation that osteocalcin and bone sialoprotein are prevalently expressed by clinically localized and bone and lymph node metastatic human prostate cancer tissue specimens and can be the molecular basis of osteomimicry during disease progression.

Several growth factors (fibroblast growth factor-2 or basic fibroblast growth factor, insulin-like growth factor-I, and transforming growth factor-β) and hormones (glucocorticoids, estrogens, parathyroid hormone, and parathyroid hormone–related peptide) have been shown to regulate osteocalcin and bone sialoprotein expression in rodent, chick, or human cell lines (48–54) . Boudreaux and Towler (55) showed the induction of osteocalcin promoter activity by a growth factor, fibroblast growth factor-2, in MC3T3-E1 cells. Boguslawski et al. (30) observed that rat and human osteoblast-like cell lines stably transfected with osteocalcin promoter reporter construct responded to parathyroid hormone and growth factors (fibroblast growth factor-2 and insulin-like growth factor-I) via enhanced osteocalcin transcription mediated by a PKA-dependent pathway. A similar activation of bone sialoprotein promoter activity was documented for fibroblast growth factor-2 (54, 56) , parathyroid hormone (33, 53) , and prostaglandin E₂ (34) in which bone sialoprotein transcription was stimulated through a PKA-dependent pathway, although a protein kinase C–mediated pathway may play a minor role (33). The present study differs from previous reports in two important aspects. First, none of the earlier observations showed conclusively at the molecular level that the involvement of CRE elements within osteocalcin and bone sialoprotein promoters, as defined in the present study, conferred soluble factor–induced osteocalcin and bone sialoprotein expression by cancer or normal cells. Our results emphasized the roles of CRE cis-elements in osteomimicry by prostate cancer cells. Second, none of the previously reported factors tested in our assay system activated human osteocalcin and bone sialoprotein promoter activities in C4-2B cells (data not shown). These important distinctions suggest the following possibilities. (a) There are fundamental differences in terms of the soluble factors, downstream signaling network, and cis-elements within human osteocalcin and bone sialoprotein promoters that are responsible for mediating the osteomimetic properties of prostate cancer and bone cells. (b) At the molecular level, prostate cancer and bone cells may differ in their cell surface receptors and downstream cell signaling pathways responding to soluble factor(s) secreted by prostate cancer and bone stromal cells and their elicited activation of osteocalcin and bone sialoprotein expression. (c) With respect to the osteomimetic response of prostate cancer cells to soluble factors, prostate cancer cell lines clearly show heterogeneity. Because osteocalcin and bone sialoprotein expression in LNCaP, C4-2, and C4-2B cells are responsive to soluble factors and osteocalcin and bone sialoprotein are prevalently expressed in both primary and metastatic human prostate cancer tissues, we suggest that LNCaP and its derivative cell lines are superior models for the study of human prostate cancer progression. This conclusion is also supported by a large body of literature devoted to the study of the molecular mechanisms of androgen receptor and androgen-independent metastatic progression using this LNCaP prostate cancer progression model (57–59) . To further understand differences in responsiveness among prostate cancer and bone stromal cells to soluble factors in conditioned medium, we must isolate and characterize the responsible factors and evaluate cell surface or intracellular receptors coupling to cell signaling systems in prostate cancer and bone cells.

In summary, our data using human prostate cancer cell lines show dramatic cell background–dependent differences in responsiveness to ARCaP conditioned medium and the involvement of the cAMP-PKA signaling cascade in the induction of osteocalcin and bone sialoprotein gene expression in LNCaP and C4-2B cells. We established at the molecular level that a specific region of cis-element in human osteocalcin promoter, located between −643 and −636 (CRE), must be responsible for conferring cAMP regulation of human osteocalcin promoter activity in human prostate cancer cells. Likewise, other regions of CRE within human bone sialoprotein promoter, −79 to −72 (CRE1) and −674 to −667 (CRE2), must also be activated on exposure of human prostate cancer cells to cAMP mimetics and yet unidentified growth factor(s) in the conditioned medium of prostate cancer and bone stromal cells.

We propose that unknown soluble factors secreted by human prostate cancer or bone stromal cells could assume the key regulatory role in osteocalcin and bone sialoprotein expression in human prostate cancer ( Fig. 6 ). An ongoing study to identify and characterize the unknown soluble factors in our laboratory has revealed a small polypeptide (<30 kDa) with thermal sensitivity and ammonium sulfate–precipitable characteristics in conditioned medium, which seems exclusively responsible for the activation of human osteocalcin and bone sialoprotein promoter activities in prostate cancer cells. These results could have significant implications for understanding osteomimicry and targeting it therapeutically in human prostate cancer.

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Figure 6.

Proposed cAMP-dependent PKA signaling mechanism describing the regulation of human osteocalcin and bone sialoprotein promoter activities in human prostate cancer cells. An unknown soluble factor with a molecular mass of <30 kDa was proposed to be secreted by human prostate cancer and bone stromal cells. This putative factor is believed to interact with a cell surface receptor in prostate cancer cells and subsequently to activate adenylate cyclase (AC), resulting in a signal cascade through the PKA signaling pathway to enhance human osteocalcin and bone sialoprotein promoter activities. We propose the molecular basis for osteomimicry as follows: cAMP generated by ligand-receptor interaction promoted PKA activation and the activated PKA translocated into the cell nucleus and induced CREB phosphorylation, which interacted with CRE cis-elements in human osteocalcin (CRE) and human bone sialoprotein (CRE1 and CRE2) promoter regions and triggered marked downstream promoter activation and endogenous mRNA expression in human prostate cancer cells.