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Endocrinology Vol. 144, No. 9 4134-4143
Copyright © 2003 by The Endocrine Society

Stanniocalcin 1 Stimulates Osteoblast Differentiation in Rat Calvaria Cell Cultures

Yuji Yoshiko, Norihiko Maeda and Jane E. Aubin

Department of Oral Growth and Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences (Y.Y., N.M.), Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan; and Department of Molecular and Medical Genetics, Faculty of Medicine, University of Toronto (Y.Y., J.E.A.), Toronto, Ontario, Canada M5S 1A8

Address all correspondence and requests for reprints to: Jane E. Aubin, Ph.D., Department of Molecular and Medical Genetics, Faculty of Medicine, University of Toronto, Room 6230, Medical Sciences Building, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: jane.aubin{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stanniocalcin 1 (STC1) is a mammalian homolog of STC, the fish calcium/phosphate-regulating polypeptide whose functions are only beginning to be elucidated. Recently, we demonstrated that STC1 stimulates, in an autocrine/paracrine fashion, bone mineralization by increasing phosphate uptake in osteoblasts apparently via the functional activity of the sodium-dependent phosphate transporter, Pit1. We have now assessed the role of STC1 on osteoblast development in fetal rat calvaria (RC) cell cultures. STC1 mRNA and protein were differentially expressed over the time course of cultures, and dexamethasone, a potent stimulator of differentiation in this model, shifted peak STC1 expression levels to earlier times. Overexpression [recombinant human (rh) STC1] and underexpression (antisense oligonucleotides) of STC1 accelerated and retarded, respectively, osteogenic development as well as osteopontin and osteocalcin mRNA expression in mature osteoblast cultures, but not osteoprogenitor cell cultures. Dexamethasone shifted the effective doses required for these effects to higher and lower concentrations of antisense oligonucleotides and rhSTC1, respectively. Concomitantly, rhSTC1 increased both sodium-dependent phosphate uptake and Pit1 gene expression in nodule formation stages, but not in primitive progenitor stages of RC cell cultures. Thus, STC1 accelerates osteoblast development in an autocrine/paracrine manner in the RC cell culture model.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GENES ENCODING the homolog of fish stanniocalcin (STC), STC1/stc1 (1, 2, 3) and STC2/stc2 (4, 5, 6), have been isolated in human, mouse, and other mammals. Many aspects of STC family structure and function are not yet known, but the predicted human and mouse STC1 proteins are closely related to each other (98% amino acid sequence similarity) and share about 80% amino acid sequence similarity with their fish counterpart, whereas human STC2 is much less closely related. The glycosylation consensus sequence, Asn-Ser-Thr, is also conserved in the STC family, and STC1 is more similar than STC2 to fish STC with respect to the number and location of Cys residues. STCs are characterized as secreted phosphoglycoproteins, and STC1 and STC2 may be phosphorylated by protein kinase C and an ecto-protein kinase, respectively (7). Although STC receptor(s) has not yet been cloned, binding assays using an STC1-alkaline phosphatase (ALP) fusion protein suggest that STC1 may enter cells through a specific receptor(s) on the plasma membrane and then shuttle to the mitochondria (8).

In fish, an elevated level of calcium in plasma is a major stimulus for STC secretion. STC regulates calcium/phosphate (Pi) homeostasis by acting on gills to reduce calcium uptake (9), on gut to inhibit intestinal calcium absorption (10), and on kidney to increase Pi reabsorption (11). The close structural similarities between STC1 and fish STC make it tempting to hypothesize that STC1 may also regulate mammalian calcium/Pi homeostasis. STCs are expressed in a variety of adult and fetal tissues (1, 2, 3, 4, 5, 6, 12, 13, 14). The specific cellular distribution of STC1 protein and/or mRNA has been established in kidney (15, 16, 17, 18), intestine (14), and bone (14, 19, 20), all tissues known to be involved in calcium/Pi homeostasis. Recombinant human STC1 (rhSTC1) has been shown to inhibit not only gill calcium transport (3), but also rat renal Pi excretion, consistent with the actions of fish STC in both tissues (3, 21). rhSTC1 also decreases calcium absorption, which is coupled to an increase in Pi absorption, in swine and rat duodenal tissues (22). Although STC1 itself does not bind or sequester calcium, an analysis of Pi uptake in rat renal tubular brush-border membrane vesicles has suggested that the sodium-dependent Pi (NaPi) transporter(s) may be a target of STC1 activity (21). The importance of Pi handling by STC1 is also suggested by results in brain (23), where STC1 has been implicated in a protective effect of Pi uptake in cerebral neurons otherwise subject to hypoxic/ischemic damage. Taken together, these observations suggest that STC1 plays a significant role in Pi-mediating cellular and/or tissue-specific processes, including calcium/Pi metabolism.

Consistent with the proposed role of STC1 in calcium/Pi metabolism, we recently found that the polypeptide plays a role in bone mineralization via a functional relationship with the type III NaPi transporter (Pit1) in osteoblasts (24). These latter results have helped to clarify the long-standing issue of the role of Pi in bone mineralization. In addition, however, a series of studies demonstrating regulation of Pi handling by osteotropic factors also suggests the possibility that Pi contributes to osteogenic development (25, 26, 27, 28, 29, 30). In support of this hypothesis, increased extracellular Pi levels are linked to the induction of osteopontin (OPN) mRNA (31) and nuclear export of Runx2/Cbfa1 (32), an essential transcription factor for osteoblast differentiation and bone formation, respectively. We therefore sought to determine whether STC1 might functionally participate in osteoblast development. To do this, we first assessed the expression profile of STC1 in the fetal rat calvaria (RC) cell osteoblast differentiation model and then determined the consequences on the developmental sequence of under- or overexpressing the protein using an antisense oligonucleotide strategy and recombinant protein, respectively.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Animal use and procedures were approved by the University of Toronto animal care committee and Hiroshima University Research Facilities of Laboratory Animal Science. Tweny-one-day-old fetal Wistar RC cells were isolated by sequential collagenase digestion, which resulted in five cell fractions as previously described (33). Briefly, calvariae were dissected free from loosely adherent connective tissues, minced, and sequentially digested in collagenase (type I; Sigma-Aldrich Corp., St. Louis, MO) solution. Cells from the last four of five digestion fractions were separately grown in {alpha}MEM supplemented with 10% fetal bovine serum (Cansera, Etobicoke, Canada) and antibiotics. After 24 h, cells were trypsinized, pooled, and grown in 24-well plates or 35-mm dishes (0.3 x 104 cells/cm2) in the same medium supplemented additionally with 50 µg/ml ascorbic acid and with or without 10 nM dexamethasone (DEX). To determine matrix mineralization, 10 mM ß-glycerophosphate (ßGP) was added to cultures for 2 d before culture termination. To obtain single cell colonies, RC cells were plated at limiting dilution (~500 cells/in 100-mm dish) in DEX-containing medium until some mineralized colonies had appeared (~21 d of cultures). All cultures were maintained at 37 C in a humidified atmosphere with 5% CO2, and medium was changed every second or third day.

RNA extraction, Northern blotting, and RT-PCR
Total RNA was isolated from cells with TRIzol reagent (Invitrogen Canada, Inc., Burlington, Canada) according to the manufacturer’s directions. Twenty micrograms of total RNA were electrophoresed on 1% agarose-17% formaldehyde gels and transferred onto positively charged nylon membranes (Hybond-XL, Amersham Pharmacia Biotech, Baie d’Urfé, Canada). The RNA was cross-linked to the membranes with UV light, and then hybridization and washing were carried out according to the manufacturer’s standard protocol (Amersham Pharmacia Biotech). The following rat cDNA probes were labeled with [{alpha}-32P]deoxy-CTP using a Multiprime DNA labeling system (Amersham Pharmacia Biotech). Bone sialoprotein (BSP; pBSP1) and osteocalcin (OCN; pOC9) cDNAs were isolated and subcloned from cDNA libraries made from RC cells and ROS17/2.8 cells (34). The insert of pBSP1 is from nucleotides 15–1823 of rat BSP mRNA; pOC9 contains an insert from nucleotides 153–480 of rat OCN mRNA. Rat ALP and OPN cDNAs were provided by Dr. G. A. Rodan (Merck, Sharp, and Dohme, West Point, PA) and Dr. B. Mukherjee (McGill University, Montréal, Canada), respectively. Rat STC1 and the housekeeping gene ribosomal protein L32 (35) cDNA fragments were generated by RT-PCR from RC cells as described below.

Total RNA (2 µg) was reverse transcribed by ReverTra Ace (Toyobo, Osaka, Japan) or SuperScript II (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s instructions. In single cell-derived colonies, cDNA was directly synthesized from the total RNA recovered from each colony, with glycogen (5 µM, final concentration) used as carrier for ethanol precipitation. The sequences of primers for rat STC1, ALP, BSP, OPN, OCN, Pit1, Pit2, and L32 were designed using Primer Picking (primer 3; Table 1Go). cDNAs were amplified with Qiagen Taq polymerase (Qiagen, Mississauga, Canada) using the GeneAmp PCR system 2400 thermal cycler (PE Applied Biosystems, Foster City, CA). PCR consisted of 16–37 cycles with denaturing at 94 C for 30 sec, annealing at 56 C for 30 sec, and extension at 72 C for 30 sec. Each product was subcloned into the pCRII TOPO TA-cloning vector (Invitrogen Canada, Inc.) and sequenced. L32 was used as the internal control.


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  		TABLE 1. Primer sequences for STC1, Pits, and osteoblast markers

 
Western blotting
Cells were lysed with RIPA buffer [1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 158 mM NaCl, 10 mM Tris (pH 7.2), 1 mM EGTA, 100 µg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride, and 1 mM Na3VO4]. After centrifugation (15,000 rpm for 10 min), the supernatant (30 µg protein) was subjected to SDS-PAGE on 15% gels under reducing conditions and electroblotted onto nitrocellulose membranes (Hybond-ECL, Amersham Pharmacia Biotech). The membranes were treated with TTBS (0.1% Tween 20 and 0.1 M NaCl in 0.1 M Tris-HCl, pH 7.5) containing 0.2% casein for 2 h and with mouse anti-rhSTC1 monoclonal antibody (anti-rhSTC1, 0.5 µg/ml; provided by Dr. S. Takano and A. Igarashi; BML, Saitama, Japan) overnight at 4 C. The membranes were then incubated with horseradish peroxidase-conjugated goat antimouse IgG antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by chemiluminescence detection (0.4 mg/ml Luminol, 0.1 mg/ml 4-iodophenol, and 0.015% H2O2 in 50 mM Tris-HCl, pH 7.5). Immunoblots incubated with anti-rhSTC1 preabsorbed with rhSTC1 (5 µg/ml) or normal mouse IgG (Santa Cruz Biotechnology, Inc.) served as negative controls; 30 ng rhSTC1 were used as the positive control. Each incubation step was followed by three washes (three times, 15 min each time) with TTBS. The reproducibility of protein loading was determined by immunoblotting with rabbit antiactin antibody (1:1000; Miles, Elkhart, IN) and horseradish peroxidase-conjugated goat antirabbit IgG as secondary antibody (Bio-Rad Laboratories, Hercules, CA).

Immunocytochemistry
Cells were fixed in periodate-lysine-paraformaldehyde solution (10 mM NaIO4, 75 mM lysine, and 37.5 mM Pi buffer containing 2% paraformaldehyde) for 10 min. After freezing and thawing, the cells were incubated with Protein Block Reagent (DAKO, Carpinteria, CA) and then anti-rhSTC1 (5 µg/ml in PBS containing 1.5% normal horse serum) overnight at 4 C. They were then incubated with biotinylated secondary antibody (1:1000) for 3 h, followed by avidin-biotin complex (1:200; Vector Laboratories, Inc., Burlingame, CA) for 30 min. Each incubation step was followed by two washes (15 min each) with PBS. As a negative control, normal mouse IgG (Santa Cruz Biotechnology, Inc.) replaced anti-rhSTC1 in the primary incubation.

Oligonucleotide treatment
All oligonucleotides were purchased from Invitrogen or Sawady (Tokyo, Japan) and were purified by reverse phase HPLC. The oligonucleotides were 22 nucleotides in length, and the last three nucleotides at both the 5' and 3' ends had their internucleotidic linkages phosphorothioated. These chimeric oligonucleotides with the partially phosphorothioated modification were used to prevent both nuclease hydrolysis and nonspecific and nonantisense effects as previously described (36). We designed antisense (AS) oligonucleotides so as to target the AUG initiation region of rat STC1 mRNA. Inverted (INV) and scrambled (SCR) sequences were used as controls. The sequences of the oligonucleotides were as follows: AS, 5'-TCACTGCTGAGTTTTGGAGCAT3'; INV, 5'-TACGAGGTTTTGAGTCGTCACT-3'; and SCR, 5'-TATGGTCTGTGTCGAACTCTAG-3'. To block STC1 protein expression, AS oligonucleotides were added to cultures every day during the culture periods outlined in Fig. 1Go.



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  		FIG. 1. Outline of differentiation and treatment protocols during typical time windows of RC cell cultures. Cells (0.3 x 104 cells/cm2) reach confluence at approximately d 6 and subsequently initiate nodule formation. To estimate the effects of various treatments during different developmental stages, treatments spanned times from subconfluence (d 5) to culture termination, namely from early osteogenic differentiation to late maturation stages (A), during exponential proliferation/primitive progenitor stages (B), or during osteoblast and bone nodule maturation stages (C) as indicated. To determine matrix mineralization, ßGP is added to cultures for 2 d before culture termination.

 
rhSTC1 treatment
rhSTC1 protein was prepared using the QIAexpress system (Qiagen) according to the manufacturer’s instructions. The open reading frame of hSTC1 without the first Met was amplified by PCR and cloned into the bacterial expression vector pQE-30 that contained an N-terminal 6xHis tag. The His-tagged rhSTC1, purified using Ni-NTA (Qiagen) and gel chromatography, was added to the cultures every day in a schedule similar to that used for the AS oligonucleotide experiment, as shown in Fig. 1Go. The purity of rhSTC1 was confirmed by electrophoresis and Western blotting as described above.

Cell growth assay
To determine the effect of oligonucleotides or rhSTC1 on cell proliferation, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide thiazolyl blue colorimetric assay was carried out in the presence or absence of oligonucleotides or rhSTC1 while primitive osteoprogenitor cells were proliferating (from d 2–6). These results were also supplemented by electronically counting (Coulter counter, Hialeah, FL) the number of cells over the same culture period.

ALP/von Kossa staining
For histochemical analysis of osteoblastic nodules (33), cultures were fixed in neutral buffered formalin and treated with naphthol AS MX/red violet Luria broth in 0.1 M Tris-HCl (pH 8.3) for ALP activity. Mineralized nodules were confirmed by further incubation with 2.5% silver nitrate solution.

NaPi transport assay
NaPi transport in RC cells was determined as previously described (37). Briefly, confluent cell monolayers (d 6) or nodule-forming multilayers (d 12) in 24-well plates were washed with fresh serum-free medium containing 0.1% BSA, maintained, and preincubated with or without rhSTC1. Before the Pi uptake assay, the cells were washed three times with 500 µl 150 mM choline chloride, 1.8 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES buffered with Tris-HCl (pH 7.4) (washing buffer). NaPi transport was measured in 200 µl washing buffer with (150 mM NaCl instead of choline chloride) or without Na+ and containing 0.1 mM KH2PO4 including 1 µCi/ml (32) Pi (Amersham Pharmacia Biotech) at 37 C. After the specified time intervals, the cells were washed three times with 500 µl ice-cold washing buffer, followed by solubilization in 0.2 N NaOH. Pi uptake was determined by liquid scintillation counting. The raw data (in counts per minute) were transformed to nanomoles per milligram of protein.

Statistical analysis
Data are expressed as the mean ± SD. Statistical differences were evaluated by ANOVA and post hoc Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
STC1 is differentially expressed during osteoblast development in RC cell cultures
To determine the expression pattern of STC1 over the entire proliferation-differentiation-maturation sequence of osteogenic cells in RC cell populations, cells were grown with or without DEX, a stimulator of differentiation in this model, and subjected to Northern blot analysis (Fig. 2AGo). Under conditions both with and without DEX (DEX+/-) growth conditions, STC1 mRNA was detected at very low levels in exponentially growing cells, but its expression was clearly up-regulated when cells reached confluence (d 6). For comparison, the osteoblast markers ALP, a relatively early marker of osteoblast differentiation; OPN, which peaks during both proliferation and again during late differentiation stages; and BSP and OCN, later markers of osteoblast differentiation, are also shown. In cultures without DEX, STC1 mRNA reached maximal levels on d 11, as nodules began developing and before the time (d 15) when BSP and OCN mRNAs were significantly up-regulated, and then decreased. As expected, earlier and more abundant osteoblast differentiation occurred in DEX-containing cultures. Concomitantly, STC1 mRNA expression also peaked earlier (d 6; onset of nodule formation) and then decreased gradually as late osteoblast differentiation markers increased.



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  		FIG. 2. Developmental regulation of STC1 mRNA expression in RC cell cultures. A, Northern blot analysis in RC cell populations cultured with or without DEX. Twenty micrograms of total RNA, obtained on the days indicated, were electrophoresed, blotted, and probed with probes specific for STC1 and osteoblast markers (ALP, OPN, BSP, and OCN). L32 was used as internal control. B, PCR analysis in single cell-derived colonies from RC cell cultures grown at limiting dilution. Each colony was classified on the basis of morphology into one of two main categories: primitive osteoprogenitors and/or fibroblastic cells (CFU-F; F) are monolayers (m) without mineralization (-) and without BSP and OCN expression; discrete CFU-Os (O) are characterized by nodule-forming multilayers (n) comprising cuboidal cells and expressing all osteoblast markers examined. CFU-O colonies were further categorized by how far nodule formation (n~3n) and matrix mineralization (+~3+) had progressed. The type III NaPi transporters, Pit1 and Pit2, are also shown. L32 was used to determine the relative abundance.

 
Because RC cell populations comprise a heterogeneous mixture of cell types, we next wished to confirm STC1 mRNA expression by osteoblast lineage cells; to do this, we analyzed discrete osteogenic colony-forming units (CFU-Os). Fifteen single cell-derived colonies were randomly selected from 5 culture dishes (100 mm) under a phase contrast microscope; of these, 11 definitive CFU-Os and 4 colonies comprising more primitive osteoprogenitors and/or fibroblastic cells (CFU-Fs) were identified morphologically and molecularly as shown in Fig. 2BGo. By RT-PCR, STC1 mRNA was found to be expressed in 14 of the colonies; coexpression profiles of the osteoblast markers and morphological phenotypes confirmed that STC1 was expressed in all CFU-Os and 3 of the CFU-Fs. Notably, however, STC1 mRNA was consistently found to be relatively more highly expressed in maturing osteoblastic colonies coexpressing ALP, BSP, and OCN compared with fibroblastic colonies or colonies with more mature osteoblastic/osteocytic cells and mineralized matrix. Expression of the type III NaPi transporter, Pit1, was detected in all colonies, whereas Pit2 was detected in only 6; STC1-positive colonies all coexpressed Pit1, but only some of them coexpressed Pit2.

Cell extracts from RC cultures, corresponding to those used for Northern blot analysis, were evaluated by Western blotting for STC1 protein expression with monoclonal antibodies against rhSTC (Fig. 3AGo). Similar to previous results with rhSTC1 (reported to be 25 kDa) (14), a single band of approximately 28 kDa was detected in extracts from both DEX+/- cultures. Consistent with the Northern blot results, STC1 protein expression increased as osteoblasts developed and bone nodules formed; however, protein levels remained relatively high even in mature osteoblast cultures. Immunocytochemistry confirmed that STC1 staining is widely distributed in RC cell cultures, but intense staining is restricted to some of the cuboidal cells associated with bone nodules that coexpressed ALP (Fig. 3Go, B–E).



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  		FIG. 3. Developmental regulation of STC1 protein levels in RC cell cultures. A, Cell lysates in parallel cultures to those used for Northern blotting were assessed by Western blot analysis. A single band (~28 kDa) was seen in RC cell extracts corresponding well to the band (~25 kDa) of rhSTC1 recognized by the anti-rhSTC1 monoclonal antibody. Actin was used as internal control. B–E, Immunocytochemical detection of STC1 in differentiated RC cells on d 18 of cultures without DEX. Low magnification (B) and high magnification (B) view of a nodule and surrounding RC cells. D, Double staining (dark red) with anti-rhSTC1 (brown) and ALP activity (light red). E, Negative control (normal mouse IgG was used as a substitute for anti-rhSTC1).

 
Decreasing or increasing STC1 protein levels retards or accelerates, respectively, differentiation of bone nodule-forming osteoblasts
We next investigated a functional role for STC1 by using AS oligonucleotides to block STC1 expression. Given the expression of STC1 over the time course of cultures, we first treated RC cells daily with AS oligonucleotides throughout nodule formation/maturation stages (Fig. 1Go). Western blotting confirmed that AS oligonucleotides, but neither of the two control oligonucleotides (INV and SCR oligonucleotides), decreased STC1 protein levels (Fig. 4AGo). Treatment with AS, but not control, oligonucleotides also significantly decreased the number of ALP-positive (ALP+) nodules formed (Fig. 4Go, B and C) and levels of mRNA expression of all osteoblast markers examined (Fig. 4EGo). These effects of AS oligonucleotides were observed in both DEX+/- conditions, but the treatment was particularly effective without DEX, i.e. 75% reduction in ALP+ nodules formed in cultures without DEX vs. 55% reduction with DEX (Fig. 4CGo). We next examined whether the effect of AS oligonucleotides was seen during two other well established developmental time windows in cultures without DEX. Treatment with AS oligonucleotides earlier, during proliferation/primitive progenitor stages (Fig. 1Go), resulted in no detectable change in either osteoprogenitor cell growth (not shown) or subsequent formation of ALP+ nodules (Fig. 4GGo). Similarly, AS oligonucleotide treatment later, during late nodule maturation stages, had no significant effect on the number of ALP+ nodules formed (Fig. 4GGo), but did result in a large and selective decrease in OPN and OCN gene expression (Fig. 4FGo), an effect not seen at either early (Fig. 4DGo) or late (not shown) time points in cells treated during proliferation stages.



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  		FIG. 4. Effect of AS STC1 oligonucleotides on osteoblast development in RC cell cultures. Cells were cultured with or without oligonucleotides in the presence or absence of DEX. Oligonucleotides were added to cultures every day during nodule formation/early maturation stages (A–C and E), progenitor proliferation stages (D and G), or nodule late maturation stages (F and G). A, A decrease in STC1 protein expression by AS oligonucleotides. Cell lysates from d 18 DEX- cultures were subjected to Western blot analysis. B, Photographs of ALP+ nodules formed on d 18 with 3.0 µM AS or control (INV) oligonucleotides in DEX- cultures. C, Number of ALP+ nodules formed in d 18 cultures with or without DEX. The data are presented as the mean ± SD. *, P < 0.05; **, P < 0.01 (compared with control samples; n = 4). D–F, RT-PCR analysis of osteoblast markers (ALP, OPN, BSP, and OCN) and L32 mRNA expression in the absence of DEX. Total RNA was extracted from d 6 (d) and d 18 (E and F). G, Number of ALP+ nodules formed in d 18 DEX- cultures (n = 4).

 
To further confirm that STC1 plays a functional role in osteogenic differentiation, RC cell cultures were treated with rhSTC1 during the same developmental time windows as those used for AS oligonucleotide studies (Fig. 1Go). When we treated cells continuously during nodule formation/maturation stages, the number of ALP+ nodules increased dose-dependently in both DEX+/- conditions, and DEX markedly shifted the effective concentration of rhSTC1 to lower concentrations (maximal effective dose shifted from 20 to 0.2 ng/ml; Fig. 5AGo). The increased formation of ALP+ nodules was reflected in the increased expression of all osteoblast markers tested; a representative example at 20 ng/ml rhSTC1 without DEX is shown in Fig. 5CGo. Consistent with the results obtained in the AS studies, treatment with rhSTC1 during primitive progenitor stages resulted in no change in osteoprogenitor cell growth, subsequent ALP+ nodule formation (not shown), or osteoblast marker expression (Fig. 5BGo). In contrast, and again consistent with the results from AS treatments, treatment with rhSTC1 during nodule maturation stages increased OPN and OCN mRNA levels (Fig. 5DGo), but resulted in no other detectable osteoblast phenotypic changes. Pit1 mRNA levels, however, were also up-regulated when we treated cells either throughout nodule formation/maturation or only during nodule maturation stages (Fig. 5Go, B–D). We also confirmed that treatment of cultures with rhSTC1 (20 ng/ml) in combination with STC1 AS oligonucleotides (3.0 µM) throughout nodule formation/maturation stages rescued the down-regulation of STC1 caused by AS treatment, i.e. significantly increased the number of ALP+ nodules formed compared with treatment with AS oligonucleotides alone (Fig. 5EGo). In addition, we confirmed that the ALP+ nodules that formed also mineralized when cultures were further incubated with ßGP (not shown).



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  		FIG. 5. Effect of rhSTC1 on osteoblast development in RC cell cultures. Cells were cultured for 18 d with or without rhSTC1 in the presence or absence of DEX. rhSTC1 was added to cultures every day either throughout nodule formation/maturation stages (A, C, and E), or during progenitor proliferation stages (B) or nodule maturation stages (D). A, Number of ALP+ nodules formed with and without DEX. The data are presented as the mean ± SD. *, P < 0.05; **, P < 0.01 (compared with control samples; n = 4). B–D, PCR analysis of osteoblast marker (ALP, OPN, BSP, and OCN), Pit1 and L32 mRNA expression in cultures treated with (+) or without (-) 20 ng/ml rhSTC1 in the absence of DEX. Total RNA was extracted from d 18 cultures. E, Number of ALP+ nodules formed with (+) or without (-) rhSTC1 (20 ng/ml) in combination with (+) or without (-) STC1 AS oligonucleotides (3.0 µM) in d 18 DEX- cultures. *, P < 0.05 compared with AS oligonucleotide-treated samples (AS; n = 4).

 
rhSTC1 stimulates NaPi transport in nodule-forming osteogenic cells
Based on the effects of over- and underexpression of STC1 during nodule formation/maturation stages in RC cell cultures, we next assessed Pi uptake at two different times in cultures without DEX, i.e. in confluent monolayers (d 6, nonnodular/primitive progenitor cell populations) and in multilayers (d 12; nodule-forming osteogenic cell-rich populations not yet mineralizing matrix). Pi accumulated time-dependently in multilayer cultures; we confirmed that Na-independent Pi uptake accounted for less than 10% of the total Pi uptake at the times examined, except during the first 2 min, and NaPi uptake increased linearly with incubation time up to 20 min (Fig. 6AGo). rhSTC1 increased Pi uptake dose dependently (Fig. 6BGo) after 18- and 26-h treatment, but not after 3-h treatment (Fig. 6CGo). Notably, the rhSTC1 effect was observed in multilayer, but not in monolayer, cultures (Fig. 6DGo), consistent with its effect on Pit1 gene expression in cultures treated in parallel to those used for NaPi uptake studies (Fig. 6EGo).



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  		FIG. 6. Effect of rhSTC1 on NaPi transport in RC cell cultures. Cells were cultured in standard differentiation medium without DEX; when cells reached confluence (monolayers, d 6) or the nodule-forming multilayer stage (d 12), cultures were changed to serum-free medium and pretreated with and without rhSTC1, and Pi uptake was determined. A, Pi uptake by multilayer cultures of RC cells as a function of time. Pi uptake was measured in the presence of sodium ({bullet}) or choline ({circ}). B, Dose-dependent effects of rhSTC1 on NaPi transport in multilayer cultures after 18 h treatment. C, The time dependency of 20 ng/ml rhSTC1 on NaPi transport in multilayer cultures. D, Comparison of monolayer and multilayer cultures with ({blacksquare}) and without ({square}) 20 ng/ml rhSTC1 treatment for 18 h. An increase in NaPi uptake was seen in multilayer cultures treated with rhSTC1, but not in cultures without rhSTC1 treatment or in monolayer cultures. The numerical data are presented as the mean ± SD. *, P < 0.05 (compared with control samples; n = 4). E, RT-PCR analysis of Pit1 and L32 mRNA expression shown in D.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
STC1 is differentially expressed during osteoblast development, with high levels in maturing osteogenic cells in both total RC cell populations and discrete single cell-derived osteoblast colonies (CFU-O). Modification of STC1 expression levels by treatment with either STC1 AS oligonucleotides (retardation effect) or rhSTC1 (acceleration effect) throughout nodule formation/maturation stages alters osteogenic differentiation in the RC cell model; these treatments during nodule maturation stages also have effects (decreased by STC1 AS oligonucleotides and increased by rhSTC1) on OPN and OCN mRNA levels. DEX, concomitant with its stimulatory effect on differentiation in this model, shifts peak levels of STC1 mRNA/protein expression to earlier times of culture, shifting effective doses of the reagents above those required for measurable developmental perturbations. Moreover, rhSTC1 increases NaPi transport during the nodule formation stage, but not during osteoprogenitor cell proliferation stages; a corresponding increase in Pit1 gene expression by STC1 was also observed in the former, but not in the latter, time window. These data suggest that STC1 acts in an autocrine/paracrine fashion on osteogenic cells, possibly via its stimulatory effect on NaPi uptake in the RC cell culture model.

These data taken together with our previous studies in vivo and in vitro (24) suggest that STC1 stimulates bone formation at two different stages, namely during osteoblast differentiation and later during matrix mineralization. This appears to be at least in part via the ability of the polypeptide to increase NaPi transport, consistent with its postulated role in kidney and intestine. Although it is not yet certain how Pi affects bone formation (see below), not surprisingly based on their structural similarity, STC1 regulates Pi transport in mammals in a manner similar to STC regulation of renal and intestinal Pi transport in fish (3, 22), and, in turn, rhSTC1 mimics the fish hormone in gill calcium transport assays (3). However, although there are interesting parallels between STC in fish and calcitonin in mammals with respect to stimulus-secretion coupling and function (38), the biological activity of fish STC is known to resemble that of PTH on serum calcium levels in rats (39) and in organ cultures of fetal rat calvariae (40, 41), results that together seem to be in conflict. On the other hand, transgenic mice overexpressing STC1 in muscle via the myosin light chain 2 promoter show pleiotropic effects in many tissues, e.g. cartilage, bone, muscle, and endothelial cells. In bone, several morphometric parameters, such as osteoblast and osteoclast activities and the rate of mineral deposition, were affected (positively and negatively) in an age- and bone site-dependent manner (42). Given these diverse effects and the multiple tissues and cell types affected, it is difficult to ascribe the bone phenotype in these models to effects on osteoblasts, and it remains important to study direct bone effects of the protein in simpler models. Our data may be helpful in this regard, and despite the presence of STC1 in several tissues and cell types, the uniqueness of the bone formation and mineralization pathways make these important biological processes in which to further assess the mechanisms of action of STC1 in mammals.

The reasons for the discrepancy apparent in STC1 mRNA and protein expression in late maturation stages of RC cultures and for the relatively slow and small effect of the protein on NaPi uptake activity are not yet known. However, as mentioned in the introduction, some STC1 binding sites appear to be localized not only in the plasma membrane, but also the mitochondrial membrane of nephron cells and hepatocytes (8), suggesting that STC1 protein levels may differ from mRNA levels due to protein sequestration at multiple sites. In addition, it is thought that intracellular Pi levels in osteoblastic cells must be rigorously regulated, because changes in Pi dose-dependently lead to two adverse effects, i.e. the induction of apoptosis (43) and changes in developmentally regulated gene expression (31, 32) (see also below). Therefore, it seems reasonable to expect that Pi uptake in osteogenic cells is regulated to an optimum level and timing during normal bone formation, consistent with the developmental time-specific effects of STC1 reported here.

The two type III NaPi transporters (Pit1 and Pit2) were originally discovered as a class of retroviral receptors (44, 45, 46) and have been identified in several osteogenic cell models (25, 30, 44, 47, 48, 49, 50). It is notable, however, that STC1 and Pit1, but not Pit2, are coexpressed at high levels in primary RC cell populations and single CFU-Os. STC1 and Pit1 coexpression at the tissue level has been reported in several other tissues examined to date, including thymus, marrow, lung, liver, heart, muscle, kidney, and brain (1, 2, 3, 12, 14, 44). However, coexpression of STC1 and Pit1 at the cellular level has been reported to date only in kidney (18, 51) and in hypertrophic chondrocytes, the latter in a region where matrix mineralization begins in endochondral ossification (14, 48). Spleen is an exception in which STC1 is expressed, but Pits appear not to be. One possible explanation for this exception is that several STC1 mRNA transcripts exist and are differentially expressed among tissues. For example, a 1.4-kb band is abundant in spleen, but not the 4-kb band that is highly expressed in other tissues such as kidney, intestine, and osteoblasts, suggesting that each STC1 transcript may have tissue-specific functions (2, 19).

To date, NaPi transport and its regulation by osteotropic factors have been determined in monolayer cultures of immortalized or osteosarcoma-derived osteoblastic cell cultures that do not express late markers of osteoblast differentiation, form nodules, or mineralize matrices (25, 26, 27, 28, 29, 30). Our NaPi transport assays suggest, however, that NaPi transport and the stimulatory effects of exogenously added rhSTC1 on Pi transport are much higher in relatively mature osteoblasts than in more primitive progenitors present at earlier times in the development process, consistent with the endogenous STC1 expression profile and over- and underexpression results. To augment the results with STC1 that we present here, we recently assessed the role of Pit1 and NaPi transport during osteogenic lineage progression in the RC cell culture model; our results suggest that Pi handling via Pit1 is indispensable for osteoblast development in vitro (52) (Yoshiko, Y., N. Maeda, and J. E. Aubin, in preparation), consistent with the current STC1 data. Although more remains to be done, the results presented here by multiple kinds of analyses suggest that STC1 may act during osteogenic differentiation/nodule formation/maturation stages rather than early primitive progenitor stages. Such an effective time window may help to explain our observations in the DEX-treated RC cells: DEX shifted peak STC1 expression, changed the effective doses of rhSTC1, and decreased the magnitude of the rhSTC1 effect, but did not increase STC1 mRNA levels; these observations suggest that the DEX effects reflect its effects on differentiation and how much/when osteogenic cells express STC1, rather than a direct regulatory activity of DEX on STC1 expression. However, this remains to be confirmed.

Although our current data do not directly address whether Pi contributes to osteogenic development, there is growing evidence that Pi plays a role in bone formation. Among osteotropic factors that have been described to regulate NaPi transport in several osteogenic cell lines (25, 26, 27, 28, 29, 30), notable are IGF-I in the SaOS-2 osteoblastic cell line and TGFß in the ATDC5 chondrocytic cell line, both of which stimulated NaPi transport as well as Pit1 mRNA expression, effects not blocked by cycloheximide. Together with our recent results showing the relationship between STC1 and Pit1 (24), these data suggest that the NaPi transport system, under the control of multiple regulatory pathways in osteogenic cells, is involved in osteoblast development. Pi has generally not been thought to act as a specific signal for induction and/or modulation of gene and/or protein expression, except for Pits (44, 47, 53). However, a recent study indicates that the expression of OPN, one of the noncollagenous bone matrix proteins, is strongly regulated at the RNA level in response to elevated extracellular Pi in the mouse osteoblastic line, MC3T3-E1, an effect dependent on the function of the NaPi transport system(s) (31). The role of OPN in bone development is not yet clear. Some data indicate that OPN facilitates osteoblast attachment to extracellular matrix, which is considered an essential part of the process of osteoblast development (54, 55). However, mice in which OPN is ablated appear to develop bone normally, although they manifest a number of anomalies with resorption (56). Pi supplementation has been found to induce nuclear export of Runx 2/Cbfa1 in several clonal bone and chondrocyte cell lines (MC3T3-E1, ATDC5, and osteocytic MLO-Y4) (32). We also showed that STC1 regulates the expression of OCN mRNA, one of the most abundant of the noncollagenous bone matrix proteins. This is consistent with our related study in which expression of not only OPN, but also OCN, was found to be positively correlated at the RNA levels with Pi transport in the RC cell model (52). The function of OCN, suggested by the analysis of genetically engineered OCN-deficient mice, is inhibition of bone formation without changes in osteoblast or osteoclast number (57). Further analysis of these mice has provided evidence that OCN is also required at least in part to stimulate bone mineral maturation (58). In any case, these observations suggest that some of the bone matrix proteins act downstream of the STC1-Pi pathway during bone formation. Although our own and other data suggest a clear functional separation of the two processes of osteoblast differentiation and matrix mineralization, they do appear to share a requirement for Pi (24, 31, 32, 52).


   Acknowledgments
 
We thank U. Bhargava for her skillful technical assistance. Also special thanks to Dr. S. Takano and A. Igarashi (BML, Inc., Saitama, Japan) for their kind gifts of rhSTC1 and anti-rhSTC1 monoclonal antibody.


   Footnotes
 
This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports. and Culture of Japan (13771074, to Y.Y.) and an operating grant from the Canadian Institutes of Health Research (MT-12390, to J.E.A.).

Abbreviations: ALP, Alkaline phosphatase; AS, antisense; BSP, bone sialoprotein; CFU-F, fibroblastic colony-forming units; CFU-O, osteogenic colony-forming units; DEX, dexamethasone; ßGP, ß-glycerophosphate; INV, inverted; NaPi, sodium-dependent phosphate; OCN, osteocalcin; OPN, osteopontin; Pi, phosphate; RC, rat calvaria; rh, recombinant human; SCR, scrambled; STC1, stanniocalcin 1.

Received January 28, 2003.

Accepted for publication May 30, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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