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[Cancer Research 60, 3470-3476, July 1, 2000]
© 2000 American Association for Cancer Research

Endocrinology

A Plasmin-derived Hexapeptide from the Carboxyl End of Osteocalcin Counteracts Oxytocin-mediated Growth of Inhibition of Osteosarcoma Cells1

J. F. Novak2, M. B. Judkins, M. I. Chernin, P. Cassoni, G. Bussolati, J. A. Nitche and S. K. Nishimoto

Department of Biology, Bucknell University, Lewisburg, Pennsylvania 17837 [J. F. N., M. B. J., M. I. C., J. A. N.]; Department of Biomedical Sciences and Oncology, University of Turin, Turin, Italy 10126 [P. C., G. B.]; and Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163 [S. K. N.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 OS Cells Synthesize and...
 OTRs of OS Cells...
 DISCUSSION
 REFERENCES
 
We have previously described the presence of the functional plasminogen activator system on the surfaces of bone neoplastic cells and the fact that plasmin specifically cleaves bone matrix protein osteocalcin (OC). The cleavage of OC to NH2-midterminal (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44) and COOH-terminal RFYGPV hexapeptide (44 45 46 47 48 49) proceeds with detachment of both products from bone mineral. Because the sequence of OC-derived hexapeptide (HP) is nearly identical to the E2 region of the oxytocin receptor (OTR), we set out to ascertain whether the HP interferes with the osteosarcoma (OS)-associated oxytocin (OT) system. We documented the presence and functional activity of OTRs in several OS cells by means of (a) OT-mediated inhibition of OS growth; (b) expression of OTR mRNA by means of reverse transcription-PCR; (c) immunofluorescence staining with IF3 monoclonal antibody specific for human OTR; and (d) saturation binding and Scatchard analysis of OT binding to the receptors of isolated membranes or intact OS cells. Although we could not demonstrate direct binding of HP to OT, the presence of HP in cultures of OS cells antagonizes the inhibitory effect of OT on these cells. Additionally, in competitive binding assays, the HP effectively competes with binding of OT to its cognate receptors. The results indicate the existence of an OTR/OT system in tumor cells of bone origin. Suggested plasminogen activator-OC-OTR/OT interactions may have an effect on the regulation of cell proliferation within the bone tissue as well as properties of the extracellular matrix surrounding the tumor foci in the bone.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 OS Cells Synthesize and...
 OTRs of OS Cells...
 DISCUSSION
 REFERENCES
 
The mechanism of bone metastasis from extraskeletal tumors as well as the progression of primary bone tumors in situ through the bone tissue is an active area of research (1 , 2) . The PA3 system has been implicated in extracellular matrix degradation, facilitation of tumor proliferation, local invasion, and metastasis (3) . Plasminogen, a substrate of PAs, may be readily available in the bone marrow/bone interface via bone marrow sinusoids that exhibit large fenestrations that facilitate the exchange of plasma proteins. Once in the interstitial tissue space, the plasminogen may be retained by high-capacity/low-affinity receptors present on osteoblast cells (4) . The conformation of the cell-bound plasminogen renders this molecule amenable to cleavage and activation by PAs (5) , resulting in a plasmin that remains bound to the same receptor with an increased affinity (6) . Plasmin is a serine protease acting on extracellular matrix proteins or activating latent matrix metalloproteases or growth factors (7 , 8) . A bone-specific effect of plasmin could be mediated by the bone protein OC. OC is a highly conserved protein produced predominantly by osteoblasts late in the mineralization process. The precise function of OC in bone metabolism is not known. A relationship between serum OC and bone formation led to the hypothesis that OC is a marker for enhanced bone activity (9 , 10) .

We found that plasmin can cleave OC at a single site within its COOH end. The cleavage creates a NH2-midterminal 1–43 peptide and a short COOH-terminal 44–49 HP (RFYGPV) (11) . The NH2-midterminal peptide is the most abundant fragment in serum (12) . Plasmin cleaves OC both in solution and when bound to hydroxyapatite. When treated with plasmin, both OC cleavage products detach from the hydroxyapatite. We hypothesize that the plasmin-mediated lysis of the free and hydroxyapatite-bound OC could be responsible for the abundant NH2-midterminal peptide and COOH-terminal HP in serum. If so, plasmin cleavage of OC could play a role in OC metabolism as well as bone homeostasis. The COOH-terminal pentapeptide was previously shown to function as a cellular chemoattractant (13) . The objective of this investigation was to ascertain whether the COOH-terminal HP RFYGPV produced by plasmin cleavage of OC has biological activity. The COOH-terminal RRFYGPV sequence, a plasmin cleavage site in OC, is evolutionarily conserved in tetrapod vertebrates from frog to man (14, 15) . A BLASTP4 search for proteins including the sequence ... R-F-Y-G-P-V... matched the OTR in which the RFYGPD sequence constitutes a part of the highly conserved second extracellular E2 region (Fig. 1)Citation . The E2 region is essential for OT binding (16) . Here we present evidence for the interaction between plasmin action, the OC HP, and OTR in modulating the growth of OS cells.



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  		Fig. 1. Schematic representation of OTR and its common sequence with the COOH-terminal of OC. The four extracellular regions labeled E1 through E4 are composed of the NH2-terminal and three loops between the seven transmembrane domains. The binding of OT to the receptor requires intact E1 and E2 regions for high-affinity binding as indicated by the arrow. The similarity between the OTR E2 and COOH-terminal OC sequences has been found with BLASTP program.

 

   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 OS Cells Synthesize and...
 OTRs of OS Cells...
 DISCUSSION
 REFERENCES
 
Materials.
OC HP (NH2-Arg-Phe-Tyr-Gly-Pro-Val-COOH) was synthesized by Bio-Synthesis, Inc. (Lewisville, TX). OT acetate was purchased from Sigma (St. Louis, MO). The peptides were reconstituted in PBS (pH 8) and stored at -80°C. [3H]OT was obtained from New England Nuclear (Boston, MA); [tyrosyl-2,6-3H]OT, 43.5 Ci/mmol].

Cells.
Human OS cell lines U2OS and MG-63 were purchased from American Type Culture Collection (Manassas, VA). Established human OS cell lines OS9 and OS15 were generously provided by Dr. Nicola Baldini (Istituti Ortopedici Rizzoli, Bologna, Italy). OS9 cells were established from bone metastasis, whereas OS15 cells are from primary osteoblastic OS. MCF-7 human breast carcinoma cell line was provided by Dr. David Beidler (University of Michigan, Ann Arbor, MI). The cells were maintained under the following conditions: {alpha}-MEM/F12 (1:1 mixture of MEM and F-12 nutrient mixture) media were used for MG-63 and U2OS cells; Iscove’s modified essential medium was used for maintaining OS9, OS15, and MCF-7 cells. All media (Life Technologies, Inc., Grand Island, NY) were enriched with 10% heat-inactivated fetal bovine serum (PAA Laboratories, Inc., Parker Ford, PA), 1 mg/ml streptomycin, and 1000 units/ml penicillin (Sigma). All cells were maintained at 37°C in a humidified 5% CO2 atmosphere.

Cell Growth Studies.
U2OS and MG-63 cells were seeded into 6-well tissue culture plates (2.5 x 104 cells/well; Corning, Cambridge, MA) using the serum-rich media as described above (2 ml/well). After 16 h, the media were replaced with fresh media containing appropriate peptide concentrations. The control and experimental media were replaced every 48 h until the time of harvest [120 h (U2OS cells) and 144 h (MG-63 cells) after peptide treatment]. At the end of culture periods, the cells were harvested by trypsinization and counted with a hemocytometer. Each condition was repeated with eight individual samples, and statistical analysis was performed with SPSS.

RNA Extraction.
Total RNA was extracted by the method described by Chomczynski and Sacchi (17) using 4 M guanidinium thiocyanate, 0.75 M sodium citrate (pH 7), 10% sarcosyl, and 0.1 M 2-mercaptoethanol. All solutions were prepared in diethylpyrocarbonate-treated water.

RT-PCR.
The first-strand cDNA synthesis was performed with 1 µg of total RNA using Moloney murine leukemia virus reverse transcriptase provided with Amplimer Sets for RT-PCR (Clontech, Palo Alto, CA). Oligo(dT)18 primer (20 µM) was added to the RNA preparation, and the samples were denatured at 70°C for 2 min and quenched on ice. To complete cDNA synthesis, the following components were added to each reaction: 5x reaction buffer (4 µl); deoxynucleotide triphosphate mix (10 mM each); recombinant RNAse inhibitor (20 units); and Maloney murine leukemia virus reverse transcriptase (200 units). The reaction was incubated at 42°C for 1 h and then heated at 94°C for 5 min to stop cDNA synthesis and destroy DNase activity. The following PCR protocol was revised from that provided along with Advatage RT-for-PCR kit (Clontech). Each reaction contained the following reagents (volume/final concentration): 10x buffer KCl-Tris-HCl (5 µl/Tris-HCl, 10 mM; KCl, 50 mM); MgCl2 (3 µl/1.5 mM); deoxynucleotide triphosphate mix (1 µl/0.2 mM each); cDNA [5 µl; (1:20)]; primers (1.25 µl of 5', 1.25 µl of 3'; 12.5 pM); and AmpliTaq DNA polymerase (0.4 µl/2.0 units). Oligonucleotide primers (OTR-sense, 5'-CCTTCATCGTGTGCTGGACG-3'; OTR-antisense, 5'-CTAGGAGCAGAGCACTTATG-3') were synthesized by Genosys Biotechnologies, Inc. (Woodlands, TX). These primers were designed to amplify a 391-bp fragment of the OTR cDNA and modeled after those used by Ito et al. (18) . A PvuII digest of the PCR product produced the expected 118- and 273-bp fragments. Target mRNA was amplified using the following temperatures and intervals: critical denaturing (94°C, 1 min), annealing (59°C, 1 min), and strand elongation (72°C, 2 min) temperatures for 30 cycles in a thermometer (ERICOMP Single Block System). PCR reaction products were stored at 4°C until analyzed by gel electrophoresis.

Immunofluorescence and Flow Cytometry.
The MG-63 OS cells were grown on glass coverslips to subconfluent density. The cells were washed with PBS, fixed in paraformaldehyde for 5 min at room temperature, and incubated for 30 min with a primary monoclonal antibody (MAb) to human OTR, clone IF3 (19) , diluted to 1:20 in PBS. Fluorescein-labeled secondary antiserum Sera-Lab Ltd., Sussex, England) diluted 1:10 in PBS was used for 30 min at room temperature.

The MG-63 cells (5 x 106 cells ml) were resuspended in PBS and fixed in paraformaldehyde for 30 min at 4°C with anti-OTR MAb IF3. After two washes, the cells were incubated at 4°C for 30 min with fluorescein-labeled secondary antiserum. Fluorescence was analyzed by means of a FACSort (Becton Dickson, San Jose, CA).

Isolation of Plasma Membrane.
Membranes were isolated from cells collected by rubber policeman into a cell collection buffer [250 mM sucrose, 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA, and 1 mM phenylmethylfulfonyl fluoride]. The cells were collected by centrifugation and homogenized in a homogenization buffer [50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA, and protease inhibitors mixture (Sigma)] using a glass/Teflon Elvehjem homogenizer (five cycles each 10 s using a Wheaton Instruments overhead stirrer). Large debris was removed by centrifugation (1,500 x g, 10 min, 4°C), and the supernatant was centrifuged for 30 min at 160,000 x g. The crude membrane preparation was resuspended in binding buffer (see below), and its protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA).

Radioligand Binding Assays.
The binding to the membrane receptors was conducted with isolated membranes and intact cells. The intact cells, when used for binding studies, were scraped into a Locke’s solution [154 mM NaCl, 5.6 mM KCl, 5.6 mM glucose, 5 mM 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid, 40 mg/liter gentamycin, 200,000 units/liter penicillin, 2 g/liter BSA, 1 mM MgCl2, and 2.3 mM CaCl2] containing 1 mM phenylmethyl sulfonyl fluoride, collected by centrifugation, and enumerated by counting in a hemocytometer. The cells were resuspended into binding buffer or Locke’s solution without Ca2+ and Mg2+ ions.

The saturation binding was conducted with increasing concentrations of [3H]OT in the presence or absence of 1 µM unlabeled OT at 4°C for 4 h. The binding was conducted in duplicate or triplicate assays. The membranes or cells were retained on the Whatman GF/B filters (Clifton, NJ). The filters were presoaked in the binding buffer for 1 h, and filtration was conducted with Locke’s solution without divalent ions. The filters were placed into scintillation vials, and radioactivity was determined with a Packard scintillation counter fitted with the quenching correction curves. The saturation binding data were determined using the Prism (GraphPad Software, Inc., San Diego, CA) nonlinear least square analysis program.

Competition Binding Assays.
Competition binding studies were conducted with one concentration of [3H]OT and increasing concentrations of OC HP with and without the presence of 1 µM OT. The incubations and analyses were performed as described above, and IC50 values were derived from nonlinear least square analysis.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
OS Cells Synthesize and...
 OTRs of OS Cells...
 DISCUSSION
 REFERENCES
 
Growth of OS Cells Is Modulated by OT and the OC COOH-terminal HP
OT Inhibits OS Cell Growth.
Previously, we found that the growth of cells derived from neuroectodermal and mammary tumors is inhibited by OT (20 , 21) . To determine the growth effect of OT and that of OC COOH-terminal HP, the 2.5 x 104 OS cells were seeded into 6-well plates and treated with peptides as described in "Materials and Methods." Fig. 2Citation shows the effect of OT on the growth of two osteoblast-like OS cell lines. OT significantly depresses the cell number when present in the complete medium at 10-7 M (MG-63, Fig 2ACitation , P < 0.02; U2OS, Fig. 2BCitation , P < 0.001). The inhibition of growth was concentration dependent because OT at 10-8 M had only a minor effect on the growth of the cells. The growth inhibition became significant after more than 100 h of growth in the presence of OT. Preliminary tests for hormone-induced apoptosis did not indicate evidence of DNA ladder formation. A similar OT-mediated inhibition of growth was observed for OS15 and OS9 OS cell lines. Thus, OT inhibits all tested OS cell lines (MG-63, U2OS, OS9, and OS15) in the presence of 10% heat-inactivated FCS.



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  		Fig. 2. OT inhibits OS cell proliferation. Osteoblast-like OS cell lines MG-63 and U2OS exhibit a dose-dependent decrease in cell number when grown in the presence of OT. The asterisk indicates P < 0.02 (t test for paired samples), the double asterisk indicates P < 0.001; n = 8 for all data. A and C, MG-63 mean cell number after 144-h incubation with OT at 10-8 and 10-7 M (dark bars, A), or with 10-9 and 10-6 M OC COOH-terminal HP (hatched bars, C) compared to control cells cultured in control medium ({square}). B and D, U2OS mean cell number after 120-h incubation with OT (), B) or OC COOH-terminal HP (hatched bars, D) compared to the control cells cultured in control medium. Error bars, SD.

 
The OT-mediated Growth Inhibition Is Reversed by OC COOH-terminal HP.
Realization that the COOH-terminal peptide of OC matches the essential and conserved sequence of the E2 region of the OTR led us to experiments that tested the effects of HP on the growth of OS cells. Fig. 2, C and DCitation , shows that the HP alone at 10-6 M to 10-9 M has a somewhat promoting but not significant effect on OS cell growth.

Fig. 3Citation shows that the simultaneous addition of OT and OC HP at equimolar concentrations negates the growth-inhibitory activity of OT alone for MG-63 cells. Addition of oxytocin at 10-7 M decreased cell number compared to control cultures in full media (P < 0.002), but the presence of both the HP and OT at 10-7 M recovered control levels of cells. Identical responses were seen for U2OS cells (data not shown). Lower doses of HP 10-8 and 10-9 M did not reverse the growth-inhibitory activity of OT (data not shown).



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  		Fig. 3. OC COOH-terminal HP blocks the growth-inhibitory effect of OT. MG-63 OS cells were cultured in control medium (Control), medium containing 10-7 M OT, or medium with 10-7 OT and 10-7 M OC COOH-terminal HP (OT + HX); n = 8 for all conditions. The asterisk indicates that the OT-treated cultures were significantly different (P < 0.02) from control or OT + HP-treated cells. Error bars, SD. Note that 10-7 M HP alone has no effect on cell number, as indicated by the experiment in Fig. 2Citation .

 

   OS Cells Synthesize and Express OTR
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 OS Cells Synthesize and...
OTRs of OS Cells...
 DISCUSSION
 REFERENCES
 
RT-PCR Evidence That OS Cells Express OTR mRNA.
Growth inhibition of OS cell lines by OT implies the presence of OTRs in OS cells. Synthesis of OTR mRNA was verified by PCR amplification of the expected 391-bp product from cDNA mixtures reverse-transcribed from OS cell mRNA. RT-PCR was conducted with 10 µg of total RNA and oligonucleotide primers specific for OTR cDNA sequences (18) . Fig. 4Citation shows that MG-63 and U2OS OS cells express the OTR, as does the breast carcinoma cell line MCF-7 used here as a positive control (21) . The OS15 and OS9 OS cells also express the OTR (data not shown). In parallel experiments, we found that HT29 colon cancer cells do not express OTR, and these cells were therefore used as a negative control.



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  		Fig. 4. Proof of OTR gene expression in OS cells by RT-PCR. Isolated mRNA from OS cell lines MG-63 and U2OS or MCF-7 breast carcinoma cells produced the expected 391-bp PCR product after reverse transcription using oligo(dT) primers and amplification by intron-spanning OTR-specific PCR primers. The internal positive controls were PCR amplification of the OTR plasmid (data not shown) and RT-PCR or mRNA and primers provided with the kit (Clontech), which resulted in a band of 900 bp as expected. The negative control was RT-PCR of HT29 colon carcinoma cells. The left lane shows molecular weight markers and the estimated size (bp). RT-PCR of OS mRNAs was performed as described in the "Materials and Methods." Two µl of the PCR reactions were mixed with glycerol-bromophenol blue containing sample buffer, loaded onto 2% agarose gel, electrophoresed, and visualized by UV fluorescence after ethidium bromide staining.

 
OTRs Are Present on Cell Surface of OS Cells.
The presence of OTR on cell membranes of the OS cells was studied by means of the OTR-specific IF3 MAb, followed by visualization via fluorescence secondary antibody. Fig. 5Citation shows that OTR is localized on the cell surfaces and exhibits either a uniformly stained pattern all along the cell periphery or manifests itself as bright heavily stained spots. MCF-7 cells that were previously shown to express OTR were used as a positive control. A replacement of IF3 with a normal mouse serum and the use of IF3 with HT29 cells colon carcinoma cells that do not express OTR constituted negative controls.



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  		Fig. 5. Immunofluorescence of the OTR. Immunofluorescence microscopy was conduced as described in the "Materials and Methods" using IF3 MAb. The antibody staining revealed the presence of OTR on the surfaces of the majority of MG63 OS cells.

 
The fluorescence intensity and the percentage of OTR-positive cells were also determined by fluorescence-activated cell-sorting analyses. As reported in Fig. 6Citation , the large majority of MG-63 cells (~90%) showed a strong reactivity to OTR antibodies. The results were identical in unfixed (data not shown) and paraformaldehyde-fixed cells. An identical pattern was observed in U2OS cells (data not shown).



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  		Fig. 6. One-parameter flow-cytometric analysis of the OTR reactivity with MG-63 cells. Anti-OTR IF3 MAb shows a strong reaction with the majority (~90%) of MG-63 OS cells. C-axis, fluorescence intensity/cell; y-axis, number of cells registered/channel. A total of 20,000 of cells was analyzed.

 

   OTRs of OS Cells Exhibit Saturation Binding that Can Be Inhibited by OC COOH-terminal HP
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 OS Cells Synthesize and...
 OTRs of OS Cells...
DISCUSSION
 REFERENCES
 
Intact OS Cells or Cell Membranes Exhibit Saturation Binding of [3H]OT Ligand.
Fig. 7Citation shows that [3H]OT binding to intact U2OS cells is saturable with a calculated Kd of 7.1 x 10-8 M by Scatchard analysis. Fig. 8Citation shows that [3H]OT binds to the membranes isolated from MG-63 cells in a saturation manner (Kd = 1.5 x 10-8 M). Indicated Kd values are significantly lower than the values reported for OT binding to endogenous OTR in OT target tissues (22) .



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  		Fig. 7. Specific binding of OT to isolated U2OS cells. Increasing concentrations of [3H]OT added to intact U2OS cells exhibited saturation binding. Nonspecific binding estimated in the presence of a 1000-fold excess of unlabeled OT was subtracted from total binding as described in "Materials and Methods." Scatchard analysis (inset) reveals a calculated Kd of 7.1 x 10-8 M.

 


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  		Fig. 8. Specific binding of OT by isolated cell membranes of MG-63 OS cells. Increasing concentrations of [3H]OT added to isolated membranes of MG-63 cells exhibited saturation binding. Nonspecific binding estimated in the presence of 1000-fold excess of unlabeled OT was subtracted from total binding as described in "Materials and Methods." Calculations from Scatchard analysis yield a Kd of 1.5 x 10-8 M.

 
The COOH-terminal HP of OC Inhibits Binding of OT to Cell Membrane Receptors.
Fig. 9Citation shows that when increasing concentrations of the OC COOH-terminal HP are present, there is a dose-dependent inhibition of [3H]OT binding by isolated MG-63 cell membranes. The calculated IC50 for inhibition is 1.5 x 10-7 M. There was no demonstrable binding of iodinated HP to either the cell membranes or OT (data not shown). It is possible that iodination of the tyrosine abrogated binding because the tyrosine is a conserved feature of both the HP and the OTR E2 domain sequence RFYGPD.



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  		Fig. 9. The OC COOH-terminal HP inhibits binding of OT to cell membrane receptors. Increasing concentrations of OC COOH-terminal HP inhibit binding of OT to MG-63 cell membranes. At 1.5 x 10-7 M HP, one half of the binding of OT to cell membranes was blocked (IC50).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 OS Cells Synthesize and...
 OTRs of OS Cells...
 DISCUSSION
REFERENCES
 
Until recently, the OT/OTR system has been studied exclusively in the context of its functions in traditional target tissues and cells: epithelial cells in the breast-smooth muscle of the uterus; and hypothalamic neurons of the brain (23) . Consequently, milk ejection, initiation of parturition, and behavioral effects are well known physiological processes controlled in part by OT. Recently, however, the OTR has been found in a number of primary tumor cells of both epithelial [breast and endometrium (19 , 24) ] and neural origin [neuroblastoma and glioblastoma (25) ]. Here we report on the expression of the OTR by OS cells, a tumor cell of mesenchymal origin. The OTR has been localized on OS cell membranes by immunofluorescence, and OT displays saturation binding to both isolated membranes and intact cells. The finding of OT-mediated inhibition of cell growth suggests that the OTR is connected to the growth-regulatory pathways. The functionality of the OT/OTR system in other tumor cells has been previously ascertained because OT inhibited proliferation of some breast cancer cell lines (20) but promoted their growth under different cell culture conditions (26) . The differences in responses are likely to depend on the cell subclone, its steroid responsivity, specific experimental conditions, and the presence of appropriate ligands in their surroundings. The steroid hormone progesterone has been found recently to bind the OTR and to decrease OT binding (27) . Other conditions may change the affinities and/or specificity of ligands for their receptors, including changes in the amino acid sequences and interactions between different receptor subunits, neighboring molecules at the cell membrane, or the presence of similar ligands (28) . Thus, both biological and experimental conditions may pertain to the OT/OTR system in OS cells (Kd = 15 nM, membranes; 71 nM, intact cells) and may explain the difference in Kd values obtained with target tissues [~2–3 nM (29) ] and those with model cells [0.6 nM (30) ].

OC is known to affect several processes that impact on bone remodeling. This is supported by its ability to bind osteopontin (31) , collagen type I (32) , and bone mineral with high affinity and stereospecificity (33) . In addition, OC inhibits cross-linking of osteopontin by transglutaminase (34) . OC was demonstrated to alter adhesive, chemotactic, and gene expression properties of giant tumor cells, a neoplastic counterpart of osteoclasts (35) . Thus, the plasmin-mediated lysis of OC may involve the release of OC not only from the hydroxyapatite (11) but also from other components of the extracellular matrix. The action of cell-bound plasmin on extracellular proteins has specific consequences in neural tissues. Deprivation of cell attachment substrates via plasmin digestion of laminin in normal neural tissues leads to anoikis of neurons (36) . Plasmin cleavage of OC and its loss from the bone mineral should lead to profound changes in the extracellular matrix surrounding bone cells and should therefore affect bone remodeling. Both bone density and cortical bone thickness are increased in OC-null mice (37) , suggesting that either the intact OC or one of its fragments may be a negative regulator of bone formation. Plasmin cleavage of the conserved site within OC and subsequent release of active peptides with novel properties would constitute proteolytic activation of OC, not unlike that of other vitamin K-dependent proteins involved in blood homeostasis (38) . Thus, in addition to the effect of intact OC, released OC peptides, a COOH-terminal HP (11) and NH2-mid peptide (39) , could also have new biological activities. This may be substantiated by the fact that the plasmin-mediated cleavage occurs at a highly evolutionary conserved RR site of the OC COOH-terminal sequence. The similarity between the COOH-terminal OC HP NH2-RFYGPV-COOH and E2 region of OTR-RFYGPD- might extend to the sixth position valine because both COOH-terminal valine and aspartic acid have negatively charged carboxylate groups. Our results support the interaction between HP, the OTR, and OT. The OC-derived HP reverses the growth-inhibitory effect of OT, a phenomenon with potential significance for in situ regulatory bone mechanisms. This presumably could occur via binding to the OT itself, a possibility that we could not confirm by any of the following methods: gel chromatography; native gel polyacrylamide electrophoresis; and capillary electrophoresis (results not shown). These negative data are reminiscent of the recent report that peptides representing various extracellular regions of the vasopressin receptor interfere with vasopressin binding without measurable binding to the ligand itself (40) . We conclude that the interference of HP must occur at the site of the receptor because HP competes for OT binding with IC50 of 1.7 x 10-7 M.

The enhanced expression of a cell surface plasmin system is one of the hallmarks of the neoplastic and metastatic states (41) and a predictor of poor prognosis in mammary and other tumors (42) . We have shown that such a system is also active on OS cells (4 , 43) . The production of COOH-terminal OC by a cell surface plasmin system in bone may have relevance to the in situ development of OS and the ability of other tumors to metastasize into bone. We suggest (Fig. 10)Citation that the presence of HP could enhance tumor growth by relieving OT-mediated growth inhibition of OS cells. By virtue of the nearly exclusive occurrence of OC in skeletal tissues, the plasmin-OC OT system would be specific for the bone environment and could therefore determine the success of OS cells or metastatic bone tumors to produce localized growth in skeletal tissues. Interactions among the OTR/OT system, OC, and steroid hormones may also reflect on the possible role of these mechanisms in the development of bone metabolic diseases such as osteoporosis. Similar interactions could also occur in normal or metabolic disease-affected bone tissue where all three components of the PA-OT/OTR system were already documented: (a) hormonally regulated PA system (44) ; (b) presence of OC fragments (12) resembling those derived by plasmin (11) ; and (c) high-affinity OTRs in normal osteoblasts (45) .



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  		Fig. 10. The model depicting interrelations among the PA, OC-derived active HP and OT system in bone-derived cells. The model starts at the upper left, with an osteoblast (OB), OS cell (OS), or metastatic bone tumor cell (CA) with PA (uPA) and plasminogen (Plg) bound to its surface. The activated plasmin (Pl) that results from the activation of plasminogen remains bound to the surface of the cell. Plasmin cleaves OC, which causes detachment of OC from the bone matrix and the release of OC NH2 midterminal fragment (N-mid) and COOH-terminal HP (HP). The HP can block the inhibitory effect of circulating OT to its receptor (OTR). The presence of both HP and OT in the bone environment enhances cell growth of tumor cells within bone or may modulate remodeling processes in the normal bone environment (inset).

 
In conclusion, we have shown that OS cells express the OTR and that the binding of this receptor by its ligand results in the inhibition of growth. Our results with osteoblast-like cells in culture add to a growing list of tumor systems that express endogenous OTR and in which the OT/OTR systems have been shown to modulate either the growth or properties of the tumor cells. The participation of the OT/OTR system in a number of novel physiological mechanisms has been documented (46 , 47) , and in addition, normal human osteoblasts (45) and endothelial cells (48) have also been recently shown to express OTRs. These results suggest that OT possesses much broader physiological significance for both females and males than previously thought. The present findings may be of particular significance for understanding primary bone tumors and those neoplasms with predilection for metastatic growth in the bone. It may be of importance that OS cells can express OC in both pre- and postproliferative stages of growth (49) , whereas the normal osteoblasts do so only during mineralization phase (50) . Nearly without exception, the tumors and particularly their metastatic variants are known to express a cell surface plasmin system. We suggest that the ability to invade bone may be due in part to release of the OC COOH-terminal peptide that in turn acts as a potent antagonist of OT binding to the OTR. We plan to evaluate the proposed sequence of events in a suitable in vivo model of bone neoplastic disease. It could be of therapeutic value to identify compounds that could interfere with degradation of OC. Alternatively, potent ligands for OTRs and strong competitors for OC HP could also lead to the inhibition of tumor growth within the bone environment.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Preliminary results were reported at "Towards the Eradication of Osteosarcoma Metastases" meeting held in Oslo, August 1998, and the 90th AACR Meeting, Philadelphia, March 1999. Back

2 To whom requests for reprints should be addressed, at Department of Biology, Bucknell University, Lewisburg, PA 17837. Phone: (570) 577-1286; Fax: (570) 577-3537; E-mail: novak{at}bucknell.edu Back

3 The abbreviations used are: PA, plasminogen activator; OC, osteocalcin; HP, hexapeptide; OT, oxytocin; OTR, oxytocin receptor; OS, osteosarcoma; RT-PCR, reverse transcription-PCR; MAb, monoclonal antibody. Back

4 www.ncbi.nlm.nih.gov/BLAST/. Back

Received 12/ 3/99. Accepted 5/ 3/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 OS Cells Synthesize and...
 OTRs of OS Cells...
 DISCUSSION
 REFERENCES
 

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