Effects of Chwn Salmon Stanniocalcin, a Calciwn
Regulating Honnone in Teleosts mul its Syntlretic
N-tenllinal Peptide Fraglllent on Bone Metabolisln

Yuji Yoshiko

1996

Effects of Chum Salmon Stanniocalcin, a Calcium
Regulating Hormone in Teleosts and its Synthetic
N-Terminal Peptide Fragment on Bone Metabolism

Ph. D. ThesisbyYuji Yoshiko

Hiroshima University School of Dentistry
Department of Oral Anatomy I

1996

ACKNOWLEDGMENTS
I would like to express my sincere gratitude to Professor Norihiko Maeda,
Department of Oral Anatomy I for his friendship, generous support, guidance andinterest throughout this study.
I am also very grateful for the generous support from and continued , fruitful
discussions with Professor Takashi Uchida, Department of Oral Anatomy II , andProfessor Yukio Kato, Department of Oral Biochemistry.
Many thanks are due to my coworkers, especially Dr. Yoshio Koide, associate
professor of Laboratory of Molecular Endocrinology, School of Fisheries Sciences,
Kitasato University, and Mr. Tetsuya Kosugi, Pharmaceutical Research Laboratories,
POLA Rand D Laboratories for their continuous guidances and invaluable supports .
My gratefulness is extended to Professor Hiroshi Kawauchi, Laboratory of
Molecular Endocrinology, School of Fisheries Sciences, Kitasato University, and Dr.
Hiromichi Sagitani, director of Pharmaceutical Research Laboratories, POLA Rand D
Laooratories, for their gainful encouragements.
My thanks are due to my colleagues in the Department of Oral Anatomy I.

ApPENDIX
This thesis is based on the following paper:

Yoshiko, Y., Kosugi, T. and Koide, Y. Effects of a synthetic N-terminal
fragment of stanniocalcin on the metabolism of mammalian bone in vitro.
Biochimica et Biophysica Acta, in press.

Effects of Chum Salmon Stanniocalcin, a Calcium
Regulating Hormone in Teleosts and its Synthetic
N-Terminal Peptide Fragment on Bone Metabolism

CON,TENTS
1. INTRODUCTION

2. MATERIALS AND METHODS

2.1. Preparation of chum salmon stanniocalcin (STC)
2.2. Intestinal Ca2 + influx of Atlantic cod2.3. Preparation of peptide fragments of STC
2.4. Amino acid sequence analysis
2.5. Cloning of cDNA encoding STC
2.6. Synthesis of N-terminal peptide fragment of STC (peptide N)
2.7 . Calvarial bone resorption assay
2.8. Generation of osteoclast-like cells
2.9. Production of cyclic AMP in osteoblast-like cells
2.10. Synthesis of collagen in cal varial bone
2.11. Statistical analysis

3. RESULTS

3.1. Isolation and characterization of STC
3.2. Amino acid and cDNA sequence analysis of STC
3.3. Synthesis of peptide N
3.4. Effects of STC and peptide N on the calvarial bone resorption
3.5. Effects of STC and peptide N on generation of osteoclast-like cells

3.6. Effects of peptide N on the production of cyclic AMP in osteoblast-like cells
3.7. Effects of peptide N on the synthesis of calvarial bone collagen

4. DISCUSSION

4.1. Chemical properties of STC
4.2. Effects of STC and peptide N on the metabolism of mammalian bone

5. SUMMARY

6. REFERENCES

1

1. INTRODUCTION
The predominant hypocalcemic hormone in bony fishes is stanniocalcin (STC),
previously referred to as hypocalcin or teleocalcin (e.g. Wagner et aZ., 1986, Lafeber
et aZ., 1988 and Wagner et aI., 1988). STC is synthesized and secreted by the

corpuscles of Stannius (CS), that are located on the mesonephric kidney (e.g. Wagner
et aI., 1986, Lafeber et aI., 1988, Wagner et aI., 1988 and Hirano, 1989) and firstdescribed by Stannius (1839). It has been proposed as the major active principle fromthe CS and it was purified from sockeye salmon (Wagner et aZ., 1986), Australian eel(Butkus et aZ., 1987), coho salmon (Wagner et at., 1988), and rainbow trout (Lafeber
et ai., 1988). It is a homodimeric glycoprotein with a molecular weight ranging from39 (sockeye salmon) to 54 kDa (rainbow trout) in the native form and from 18-32 kDa
under reducing conditions. The nucleotide sequences of STC cDNA from Australianeel (Butkus et aI., 1987) and coho salmon (Wagner et ai., 1992) have been elucidated.
However, the complete amino acid sequence of STC has not yet been determined,
except for the N-terminal region (Wagner et ai., 1986, 1988, Lafeber et aZ., 1988 andSundell etaZ., 1992). STC exhibits no similarities to calcitonin (CT) or to parathyroid
hormone (PTH) in terms of the amino acid sequence deduced from Australian eel
cDNA (Butkus et at., 1987). The target organs of STC appear to be the kidney (Lu andWagner, 1994), the intestine (Sundell et ai., 1992) and the gills (So and Fenwick,
1977, 1979, Milet et ai., 1979, Ma and Copp, 1982 and Fenwick, 1989), which play
an important role in calcium and potassium regulation in teleosts.
CT plays physiologically important roles in maintenance of calcium homeostasis
and it is a potent inhibitor of osteoclastic bone resorption (Chambers et at., 1983,
1985, 1986). In clinical studies, the specific activity of CT derived from bony fishes is2higher than that derived from mammals. However, the hypocalcemic effect is governed
by STC rather than CT in bony fishes. Therefore, the biological functions of STC havebeen noted in the comparative endocrinology and the phylogeny of calci unl
metabolism. Possible biological functions for components of the CS and STC in
mammals have been discussed, as has the possibility that the effects of STC are similar
to those of PTH (Milet et ai., 1979, Milet et ai., 1982, Lopez et ai., 1984, Lafeber elaI., 1986, Lafeber et al., 1989 and Stem et al., 1991). Serum levels of calcium andmagnesium are increased in rats after an intraperitoneal (i.p.) injection of an extract ofsilver eel CS (Milet et aI., 1982). A PTH-like molecule in silver eel plasma wasidentified from its cross-reactivity with an antiserum against bovine PTH (bPTH) andthe immunoreactive substance was no longer detected after removal of the CS (Lopezet al., 1984). The PTH-like substance was detected in all cells of the silver eel CS by

immunocytochemistry using an antiserum against bPTH (Lopez et al., 1984). In otherstudies, the administration (i. p.) to rats of an extract of sil ver eel CS resulted in theactivation of osteoclastic bone resorption at the periosteum of the femur (Milet et al.,1982). A bone resorption assay using organ cultures of fetal bones revealed thatextracts of silver eel CS (Milet et al., 1982) and of rainbow trout CS (Lafeber et aI.,1986), as well as purified STC from rainbow trout (Lafeber et ai., 1989), stimulate therelease of calcium in a manner that resembles the release of calcium in response tostimulation by bPTH. However, Ma and Copp (1982) have reported that pacificsalmon STC infusion has no significant effect on plasma calcium in rats, in contrast to
hypocalcemic effect of the protein in fish and young chicks. Moreover, pure STC from. coho salmon does not consistently stimulate bone resorption in a variety of rodent
bone-culture systems and that it has limited PTH-like activity, if any, on calcium
metabolism in a mammalian system (Stem et al., 1991).
Evidence was obtained for the existence of STC-like immunoreactivity in sera ofseveral vertebrates and in human kidney, but the corresponding gene was not been

3cloned (Wagner et al., 1995). However, Chang et al., (1995) was recently obtained ahuman cDNA eIone cooing for a protein highly homologous to fish STC. The mRNAis expressed in human tissues, wi th the highest levels in ovary, prostate and thyroid.
Human cell (SUSM-1 cells, immortalized liver fibroblasts) culture studies show thatthe mRNA is positively regulated by extracellular Ca2 + in the medium.
Some investigators have described the activities of fragments of the STC moleculein vivo (Butkus et al., 1989, Milliken et al., 1990, Verbost et al., 1993, Fenwick andVerbost., 1993 and Verbost and Fenwick, 1995). In fish, it has been found that an Nterminal peptide fragment of Australian eel (Milliken et aI., 1990, Verbost et al., 1993
and Verbost and Fenwick, 1995) and sockeye salmon (Milliken et aI., 1990) STC has
an inhibitory effect on whole-body calcium uptake, with the effect resembling that of
the intact molecule. There is no agreelnent about the function of the C-terminal region(Millikenetal., 1990, Verbost eta!., 1993, Fenwick and Verbost., 1993 and Verbost
and Fenwick, 1995). The actions of six peptide fragments of Australian eel STC havebeen tested in sheep, and plasma potassium levels were affected by either the Nterminal region or a mid- sequence fragment of the STC (Butkus et aI., 1989).
Identification of the active site of the STC molecule in mammalian systems helped
to clarify the actions of STC in mammals, as well as the relationship between STC andPTH. Therefore, we completely sequenced and characterized of STC from chumsalmon. We also characterized the effects of the native form of STC and its synthetic
N-terminal fragment, which includes the region that functions as the acti ve si te in fish,on the PTH-sensitive aspects of the metabolism of mammalian bone in vitro.

4

2. MATERIALS AND METHODS
2.1. Preparation of STC

The CS were collected from mature female chum salmon, Oncorhynchus keta, andSTC was prepared according to Sundell et al. (1992). The CS were homogenized andextracted with 35% ethanol-10% ammonium acetate, pH 8.0, containing 5 mM EDT A
and 1.5 mM phenylsulfonylfluoride at 4°C, then mixed into cold ethanol. The
precipitate was fractionated on a DE-52 column (Whatman, Kent, UK) and a Superdex
75 column (Pharmacia LKB, Uppsala, Sweden) and subsequently purified by reversed
phase high performance liquid chromatography (rpHPLC) on a TSK gel ODS-120T
column (Tosoh, Tokyo, Japan).
The protein peak in rpHPLC were subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) in a 12 % polyacrylamide gel at pH
8.8, essentially as described by Laemmli (1970) with and without 1 %~-mercaptoethanol and stained with Coomassie brilliant blue.

2.2. Intestinal Ca 2 + influx of Atlantic cod

To determine the effects of STC on the unidirectional intestinal Ca 2 + influx (jCa)of the Atlantic cod (Gadus morhua) , isolated intestinal preparation was used as
described in detail by Sundell and Bj6msson (1990). The intestine was perfusedthrough the coeliac artery with a cod Ringer's solution containing 0.1 % bovine serum
albumin (BSA~ Sigma, St Louis, MO). The outflow from the intestinal vein wascollected in fractions throughout the experiment. The intestinal lumen was perfusedwith a BIS based on ion levels measured in the intestinal fluid (Sundell and Bj6rnsson,

51990). 45CaCl 2 (specific activity 0.85 ± 0.06 MBq/!-lmol; Amersham International,
Buckinghamshire, UK) was added to the BIS. All perfusions were carried out for 3 h.
The first hour was to allow the 45Ca appearance rate to reach a steady state across theintestinal mucosa. During the second hour, the venous outflow was collected and thebasal JCa for each preparation calculated. During the third hour, purified protein or
vehicle was added to each preparation. JCa was calculated as the ratio of total
radioactivity of each fraction to the original radioactivity level of the tracer added to theperfusion-solution as well as for 6O-min periods with the flow linearly collected to thebody weight (Fig. 1).

2.3. Preparation of peptide fragments of STC
The STC was reduced and pyridyl-ethylated (RPE), according to Friedman et al.
(1970). STC dissolved in 1 M Tris-HCI buffer (pH 8.3) containing 6 M guanidineHCl and 2 mM EDT A was reduced with dithiothreitol and subsequently alkylated with
4-vinylpyridine. The protein solution was loaded onto a TSK gel ODS-120T column.
STC was also reduced with dithiothreitol and alkylated with iodoacetic acid in 2 N
NaOH according to Hirs (1967). The resulting reduced and carboxymethylated(RCM)-STC was recovered as described above. The RPE-protein was digested withtrypsin (T) and endoproteinase Asp-N (AN). The RCM-STC was digested with

Staphylococcus aureus V8 protease (SP). All fragment peptides were fractionated by
rpHPLC on a TSK gel ODS-120T column.

2.4. Amino acid sequence analysis
The amino acid composition of the STC and the fragment peptides was determinedusing an automated amino acid analyzer (Hitachi, Model L-8500, Tokyo) afterhydrolysis with 6 M HCI containing 0.6% phenol at 110°C (Muramoto et al., 1987).

6

Cysteine ·was determined as S-pyridylethyl cysteine. The amino acid sequence of the
C-terminal region was determined by measuring the amino acids released after
incubating RPE-STC with carboxypeptidase Y (CPY), according to Hayashi et al.
(1973).
Sequences were determined using a gas-phase automated amino acid sequencer(Shimadzu, Model PSQ-1, Tokyo) and 3-phenyl-2-thiohydantoin (pth)-amino acids
were identified by rpHPLC on a Wakosil-pth column eluted with pth-amino acids
mobile phase solvent (Wako Pure Chemical, Osaka, Japan).

2.5. Cloning of cDNA encoding STC
DNA was manipulated and transformed into E. coli according to Sambrook et al.
(1989) . The PCR primer was synthesized and DNA was sequenced using an
automated DNA synthesizer (Applied Biosystems, Model 381A, Foster City, CA) anda DNA sequencer (Applied Biosystems, Model 393A), respectively. To amplify the
DNA coding for the STC from the mRNA of CS, two degenerate primers weresynthesized (Fig. 2). Primer 1 corresponded to the N-terminal amino acid sequence of
the STC and contained an additional ATG, which is the initiation codon for expressionin E. coli. Primer 2 corresponded to the C-terminal amino acid sequence of STC andcontained an additional TAG codon with which to terminate translation.
CS was homogenized and mRNA was obtained using a Clontech kit (Takara,Siga, Japan). The cDNA was synthesized from 500 ng of mRNA using a Gene Amp
RNA PCR kit (Takara). DNA was amplified by 35 cycles of amplification (first 5'cycles: 95°C for 30 s, 35°C for 2 min, and 70 °c for 1 min, followed by 30 cycles:
95°C for 30 s, 40 °C for 2 min, and 70°C for 1 min). Amplified fragments wereligated to the pT7Blue T -vector (Novagen, Madison, WI) and transformed into E. coli

7

JM109 (Takara). Forty transformants were selected and the size of inserted DNA wasdetermined by digestion with restriction endonuc1eases.

2.6. Synthesis of the N -terminal peptide fragment of STC

A synthetic peptide of N-terminal fragment 1-20 of STC from chum salmon(peptide N) was prepared by solid-phase BOC chemistry (Merrifield, 1985) using anamino acid synthesizer (Applied Biosystems, Model 430A). The peptide was cleaved
from the resin with anhydrous hydrogen fluoride and the crude peptide was purified by
rpHPLC on a TSK gel ODS-120T column. The homogeneity of the preparation of thepurified peptide was confirmed with a gas-phase automated amino acid sequencer.

2.7. Calvarial bone-resorption assay

Pregnant Sprague-Dawley rats were injected subcutaneously with 45CaCl 2(Amersham International) at a dose of 3.7 MBq per rat on the 18th day of gestation.
On the following day, two calvariae from fetuses were selected randomly and cultured
in each well of a 24-well plate in Fitton-Jackson modified BGJb medium (Gibco BRL,
Gland Island, NY) containing 2 mg/ml BSA for 24 h. The bones were then transferred
to fresh medium that contained vehicle as a control, various concentrations of purified
STC or peptide N with or without the N-terminal fragment of human PTH (hPTHl-34;
Peninsula Laboratories, Belmont, CA) and incubated for an additional 2-4 days. The
extent of bone resorption was calculated as the release of 45Ca, expressed as thepercentage of original total radioactivity of the bones (sum of the radioactivity of themedium and the bones), as described (Raisz, 1965).

82.8. Generation of osteoclast-like cells

Our procedure was based on that described Takahashi et al. (1988) . Marrow cellsof the tibiae from 7 week-old ddY mice were collected, washed with minimu messential medium a medium (a-MEM~ Gibco BRL) and suspended in a-MEM
supplemented with 10% fetal calf serum (FCS; Gibco BRL) . The suspension was
divided among the wells of 24-well plates and incubated with test sampl es as
described above for 8 days. After culture period, adherent cells were rinsed withphosphate buffered saline (PBS), fixed with ethanol-acetone (50:50, vol/vol) for 1
min, and stained for tartrate-resistant acid phosphatase (TRAP) by incubating the cells
in O.lM sodium acetate buffer (pH 5.0) containing naphthol AS MX phosphate(Sigma) and red violet LB salt (Sigma) in the presence of 10 mM sodium tartrate.
TRAP positive cells containing three or more nuclei and having a podozome-likestructure at the peripheral edge of the cytoplasm were counted under a microscope.

2.9. Production of cyclic AMP in osteoblast-like cells

ROS 17/2.8-5 cells derived from a rat osteosarcoma (Riken Cell Bank, Ibaraki,
Japan) were grown in Ham's F-12 medium (Gibco BRL) supplemented with 100/0
FCS. The cells were subcultured at weekly intervals, then plated in 48-well plates.
When the cells were reached confluence, they were washed and incubated with thesame medium containing 0.3% BSA and 0.1 mM isobutylmethylxanthine (Sigma) as
described (Nakatani et ai., 1984). Test samples (except for STC) were added to thecells and incubated for a further 5 min. The reaction was stopped by aspirating the,medium then immediately adding ice-cold 65% ethanol. The plates were placed in aultrasonic bath to release cyclic AMP from the cells. The cyclic AMP was quantified bya cyclic AMP-specific enzyme immunoassay system (Amersham International).

92.10. Synthesis of collagen in calvarial bone

The synthesis of bone collagen was assayed as described (Dietrich et ai., 1976).
Calvariae obtained from 4-day-old ddY mice were cultured in modified BGJb medium
supplemented with 4 mg/ml BSA for 3 h. Three pieces of bone per flask were thentransferred randomly to flasks of fresh medium containing vehicle, peptide N or
hPTHl-34 and incubated on a shaker (60 oscillations/min) for 4 days. The medium
containing test samples was changed every day. The bones were pulse-labeled with L[2, 3 3 H]proline at 0.37 MBg/ml (999 MBg/mmol; Amersham International) during
the last 2 h of cuI ture. At the end of culture period, the bones were extracted, dried,weighed and homogenized. An aliguot of the homogenate was incubated with highly
purified bacterial collagenase (form III from Clostridium histolyticum; Advance
Biofactures, Lynbrook, NY). The amounts of radio labeled proline that had been
incorporated into collagenase-digestible (CDP) and non-collagen protein (NCP) were
determined by the method of Peterkofsky and Diegelmann (1971). The data areexpressed as dpm per microgram of dry weight of bones. The percent collagensynthesis was calculated after multiplying NCP by 5.4 to correct for the relative
abundance of proline in collagen and non-collagen protein (Peterkofsky, 1972).

2.11.

St~tisticalanalysis

Statistical evaluations were determined by analysis of variance (ANOV A) withmUltiple comparisons by the method of Fisher's protected least significant difference.
The significance of differences was accepted at P<O.05.

10

3. RESULTS
3.1. Isolation and characterization of STC

Figure 3 shows ion exchange chromatography of an ethanol extract of the CS
from chum salmon on a DE52 cellulose column. Figure 4 shows gel filtration of theDE6 fraction obtained from the DE52 column on a Superdex 75. The yields obtained
from each step in the extraction of 60g of the CS were as follows: DE-52 column,
fraction DE6, 184.1 mg; Superdex 75 column of fraction DE6, fraction G4, 2 mg.
When fraction 04 from Superdex 75 was applied to rpHPLC on TSK-gel ODS 120T
columns, one protein peak was eluted (Fig. 5).
In SDS-PAOE under nonreducing conditions, STC migrated with a molecularweight of 46.3 kDa (Fig. 6). In the presence of a reducing agent, the protein peakmigrated with a molecular weight of 23.4 kDa (Fig. 6).
The effects of STC on jCa, calculated over 60-min periods before and duringperfusion, are presented in Figure 7. Perfusion without STC resulted in no deference
from the basal flux, whereas the protein decreased jCa (2.0 nM STC, 0.89 ± 0.24~mol vs. control, l.02

± 0.23 ~mol; 10.0 nM STC, 0.97 ± 0.23 vs. control, 1.28 ±0.38 ~mol Ca 2 +/h/kg body weight). In the inset figure (Fig. 7), the jCa from the threeperfusion periods (third hour of perfusion) are normalized and expressed as apercentage of basal flux (second hour of perfusion) in each treatment group. This
,revealed a dose-related effect of the protein on the intestinal Ca2 + influx, in which 2.0
and 10 nM STC decreased the influx by 12.7 and 24.2%, respectively.

11

3.2. Amino acid and cDNA sequence of STC

RPE-STC eluted as a single peak on rpHPLC following the reduction and pyridylethylation (RPE) of intact STC and it had a single amino acid sequence that allowed theassignment of 24 residues. These and SDS-PAGE results described above, indicatethat the protein is a disulfide-linked homodimer.
To determine the complete amino acid sequence, peptide fragments were generated
with proteases (T, AN and SP). Tryptic digestion of RPE-STC yielded 14 fragments(T 1-T 13) by rpHPLC and rechromatography of T 13 (Fig. 8). The amino acidcomposition and yield of the pth-amino acids of the intact protein and fragments arelisted in Tables 1 and 2, respectively. Three AN fragments (AN33, AN26, and AN30)
and one of SP (SP44) are shown in these tables, although all the fragments wereanalyzed. T12 overlapped residues of 12-14 of the protein and extended 16 additional
residues up to position 40 with the exception of cycle 18 (residue 29). AN33
overlapped residues 39-40 of the protein and thus 47 addi tional residues were
determined up to position 87. T4, T2, T3, T9 and T7 confirmed the sequence of 5760, 61-64, 65-73, 74-79 and 81-86, respectively. T8 overlapped residue 87(methionine) of the protein and thus 11 additional residues were determined up to
position 98. SP44 overlapped residues 95-98 of the protein and extended 16 additional
residues up to position 114. AN26 overlapped residues 100-114 of the protein andextended 23 additional residues up to position 137. TI0 and T 11 confirmed thesequences of 99-106 and 107-125, respectively. T13B overlapped residues 126-137 of
the protein and extended 8 additional residues up to position 145. AN30 overlapped
residues 138-145 of the protein and extended 34 additional residues up to position
179. T5, T13A and Tl confirmed the sequences of 147-152, 153-167 and 168-179,
respectively. Tl provided the carboxyl-terminal sequence because it contained nolysine and arginine. Kinetic studies of CPY digestion confirmed that the carboxyterminus of the intact molecule was serine. The monomer consisted of 179 amino acids

12

including 11 half-Cys residues and one consensus N-glycosylation site at Asn-29 (Fig.
9). This Asn residue was confirmed by detecting Asp in the hydrolysate of the pthamino acid obtained at the 18th cycle of T12. Based on the sequence analysis, thecalculated molecular weight of the polypeptide was 19,327 which is lower than that
estimated by SDS-PAGE (23.4 kDa). This discrepancy can be accounted for by the
carbohydrate moiety of the molecule.
An analysis of forty transformants showed that one plasmid was suitable for
inserting DNA, deduced from the amino acid sequence. The plasmid pKDSTI had a
55-base pair insertion that encoded 179 amino acids, except for an additional first Met
and the primer region (Fig. 10). A comparison of the amino acid sequence deduced
from the cDNA with that of the protein revealed that total number of amino acids andthe positions of all the half-Cys residues were identical. However, the amino acidsequence deduced from the cDNA differed from that of the protein at two positions:Leu for Pro-160 and Pro for Ser-170.

3.3. Synthesis of N -terminal peptide fragment of STC

The synthetic peptide (peptide N)~ (Fig. 11) was eluted as a single peak after
rpHPLC, and the correct anticipated sequence of amino acid was determined by direct
sequencing of the amino acids.

3.4. Effects of STC and peptide N on the calvarial bone resorption

Because the reported PTH-like effects of STC are discrepant, we ini tially
examined the bone-resorptive activity of native form of STC and a synthetic peptide,
peptide N, from chum salmon on 4-day-cultured calvariae of fetal rats labeled with
45Ca. The effects of the samples from chum salmon were compared with those of
hPTHI-34. Although hfYfHI-34 (10- 7 -10- 10 M) increased the release of 45Ca into the13medium from calvariae in a dose-dependent manner (Fig. 12A), STC had no effect at
the concentrations we applied (10- 9 -10-13 M) (Fig. 12B). However, peptide N
inhibited the rate of release of 45Ca and the effect was significant at 10-1 L 10- 12 M
(Fig. 12B).
Figure 13 shows the time course of the effects of intact STC and peptide N on thecalvariae from fetal rats. In the control, the rate of release of 45Ca into the medium
increased with increase duration of culture, from the second to the fourth day. Whenthe bones were cultured with 10-11M peptide N, the rate of release was significantly
reduced on the fourth day (peptide N, 13.6 ± 1.60/0 vs. control, 19.1 ± 1.70/0 of 45Ca
released). There was no effect in the intact STC during the culture period.
Next, we examined the effects of intact STC and peptide N on PTH-stimulatedbone resorption. The rate of 45Ca release from calvariae cultured for 2 days in thepresence of hPTHl-34 (10- 8 M) was not affected by intact STC (Fig. 14). However,
peptide N inhibited the increased release of 45Ca in cultures exposed to hPTHl-34 atdose that was effective in the absence of the latter (Fig. 14). Peptide N inhibited
hPTHl-34-stimulated bone resorption by about 30.70/0 (hPTHl-34 plus peptide N,
14.1 % ± 1.9% vs. hPTHl-34 alone, 22.4 ± 3.4% of 45Ca released).

3.S. Effects of STC and peptide N on generation of osteoclast-like cells

The effect of peptide N on the formation of osteoclast-like cells is shown in Figure
15. These were few osteoclast-like cells in the original control culture after 8 days
(Figs. 15A, B). An incubation with hPTHl-34 (10-7 -10- 10 M) dose-dependently
increased the number of the cells (Figs. 15A, 16). The effect of peptide N wasapparent when cuI tures incubated in the presence and in the absence of hPTH 1-34
were compared. No effect of peptide N was detected at the tested concentrations (10-9 -14

10- 13 M) in the absence of hPTHl-34 (Fig. 15B), whereas the synthetic peptide
inhibited hPTH1-34-dependent increases in the number of osteoclast-like cells. This
increase was significantly reduced by peptide N (10-10_10-12 M) and the maximum
response was detected at 10- 11 M (hPTHl-34 plus peptide N, 12.8 ± 1.5 cells vs.
hPTHl-34 alone, 21.0 ± 2.5 cells per well); (Fig. 15C). Intact STC had no effectupon these systems with or without hPTHl-34 (Data not shown).

3.6. Effects of peptide N on the production of cyclic AMP in
osteoblast-like cells

Several lines of evidence indicate that one criterion of PTH potency is to stimulate
cyclic AMP formation. The effect of peptide N on the formation of cyclic AMP in
osteoblastic ROS17/2.8-5 cells was compared with that of hPTHl-34. Because there
was no effect of intact STC in PTH-sensitive systems, we examined the effects of
peptide N on this system. The addition of hPTHl-34 (10-7 -10-10 M) to the cells
increased cyclic AMP levels in a dose-dependent manner (Fig. 17A), whereas peptide
N (10- 9-10- 13 M) did not affect the levels of cyclic AMP (Fig. 17B). When peptide N
at various concentrations was added with 10-8 M hPTHl-34, the accumulation of
cyclic AMP in response to hPTHl-34 was significantly inhibited at 10- 11 -10-13 M(Fig. 17C). The maximum effect was seen at 10-11M (hPTHl-34 plus peptide N, 1.11

± 0.06 pmol vs. hPTHl-34 alone, 2.84 ± 0.49 pmol cyclic AMP per well).

3.7. Effects of peptide N on the synthesis of calvarial bone collagen

Table 3 summarizes the effects of hPTHl-34 and peptide N on the synthesis ofcollagen in calvariae of newborn mice. Human PTHl-34 (10-8 M) caused a significant
decrease in the rate of incorporation of pH]proline into CDP, while labeling of NCP

15

was increased (but not significantly). In bones incubated with peptide N, NCP labelingby proline was slightly increased (but not significantly). By contrast, the labeling of
CDP by proline was significantly increased by peptide N (10-11 -10-13 M), so that therelative level of collagen synthesis in the presence of the peptide appeared to be greaterthan that in the control. The effect of peptide N on the rate of incorporation ofpH]proline into CDP was maximal at 10- 12 M (control, 12.01 ± 0.57 dpm vs. peptide
N, 17.41 ± 1.31 dpmfl.,lg dry weight).

16

4. DISCUSSION
4.1. Chemical properties of chum salmon STC
Intact chum salmon STC migrated as one band on SDS-PAGE with a molecularweight of 46.3 kDa. This is comparable to reported STCs ranging from 39 kDa insockeye salmon (Wagner et a!., 1986) to about 54 kDa in the rainbow trout (Lafeber etai., 1988). After reduction with ~-mercaptoethanol, STC migrated as one 23 .4 kDaband, suggesting that STC is a homodimer. Further support for this notion was
obtained from rpHPLC after the reduction of intact STC. Thus, chum salmon STC is
likely consist of two identical polypeptide chains, as also proposed for coho salmon
STC (Wagner et a!., 1988) and rainbow trout STC (Lafeber et a!., 1988 and Filk et ai.,
1989, 1990). The purified STC from chum salmon represents a biological activity inthe intestine, which is proposed as one target organ of STC in marine teleosts (Sundell

et ai., 1992).
Chum salmon STC monomer, prepared from the intact STC by reduction andalkylation, consisted of 179 amino acid residues with 11 half Cys residues and one Nlinked glycosylation site at position 29. The complete amino acid sequence of chumsalmon STC was compared with those deduced from the cDNA of coho salmon(Wagner et al., 1992), Australian eel STC (Butkus et ai., 1987) and human (Chang etai., 1995); (Fig. 11). From residues 1-179, chum salmon STC was identical with coho
salmon STC except for the 3rd and 18th residues. It also had high sequence identitywith Australian eel STC (78%) and human (620/0). Moreover, all half-Cys residues
and an N-linked Asn of chum salmon STC were located in positions homologous tothose in the three deduced sequences.

17

The cDNA was cloned by RT -PCR by using two primers corresponding to the Nand C-teIininal sequences of the protein. However, the amino acid sequence deduced
from the cDNA differed from that of protein at two residues (positions 160 and 170).
Although it is possible that the cDNA is a mutant produced during the PCR process,this might be due to the occurrence of two genes encoding STC in chum salmon,
according to the tetraploid hypothesis of salmonid fishes. Although the cDNA obtai ned
here may have been a variant which is rarely or never expressed, the nu cleoti desequence of the cDNA was comparable with that of coho salmon STC cDNA (Fig.
10). Thus STC is well conserved in these salmonid fishes, at the gene and pro teinlevels.
It is notable that the chum salmon STC was 44, 52 and 35 residues shorter at the

C-terminal region than the sequences deduced from coho salmon , eel and human STC
cDNAs, respectively. The C-terminal residue of chum salmon STC is Ser, which is
not generally cleaved by proteolytic enzymes. There were also two dibasic pairs(Arg79-Arg80, and Lys167-His168) in the sequence of chum salmon STC, whichwere much more subject to enzymatic cleavage by endoproteinases than the Ser
residue. Thus, the possibility that the chum salmon STC obtained here was a productof proteolysis during purification, was negated.
The C-terminal region (180-223) of the deduced sequence of coho salmon STC,
which is absent in that of chum salmon STC has low sequence identity with Australian
eel and human STC , although the N-terminal to residue 179 has high identity. The
full length cDNA of chum salmon CS is required to clarify whether the STC is a
primary transitional product or a further post-transitional processing product.

18

4.2. Effects of STC and peptide N on the metabolism of mammalianbone
The PTH-like effects of STC have been mainly determined by bone resorption
assays in extracts of silver eel CS (Milet et al., 1979), rainbow trout CS (Lafeber etaI., 1986) and rainbow trout STC (Lafeber et al., 1989). However, coho (Stern et aI.,

1991) and chum salmon STC (this study) had no effect in that system. Moreover,
peptide N, which had high sequence identity among those bony fishes inhibited
hPTHl-34-stimulated bone resorption. The assay of formation of osteoclasts-like cells
from bone marrow cells seemed to support the lack of STC effects and the inhibition
by peptide N in the bone resorption assay. Evidence from a wide variety of studies
suggests that osteoclasts are derived from hemopoietic progenitor cells (Nijeweide etaI., 1986 and Mundy and Roodman, 1987). Osteoclast-like TRAP positive

multinucleated cells can be generated from mouse bone marrow cells in culture bystimulation with PTH (Takahashi et al., 1988). TRAP positive multinucleated cells
formed in response to osteotropic hormones satisfy most of the criteria for osteoclasts.
In this study, STC and peptide N did not induce the formation of such cells frommurine bone marrow cells, whereas peptide N alone inhibited the response to hPTH134 in a number of the cells. It is suggested that the differentiation of osteoclasts isregulated by stromal cells and various humoral factors (Takahashi et aI., 1988 and
Hattersley and Chambers, 1989), although the details remain uncertain. The
understanding the cell linage of osteoclasts may be an important clue to elucidating theeffect of STCl-20 on the formation of osteoclast-like cells.
Peptide N also decreased bone resorption in the absence of hPTHl-34, but anobvious effect was detected after a long culture period (4 days). This delay might havebeen due to the influence of non-PTH factors, such as endogenous prostaglandins(POs; Katz et aI., 1981 and Chambers et al., 1985). As Chambers et al. (1985)
pointed out, POs also exert bone-resorbing activities via increasing cyclic AMP levels

19

in osteoblasts, as does PTH. It seems that the effects of peptide N on the PTHsensitive systems, described above, represent an indirect, rather than a specific action
at the PTH receptor because the peptide did not have a pronounced effect on hPTH 134-dependent bone resorption and had only a slight effect on PTH-independent, PGrelated bone resorption (Katz et ai., 1981 and Chambers et ai., 1985).
It is apparent that cyclic AMP in osteoblasts is an important second messenger forPTH (e.g. Nakatani et ai., 1984 and Lerner et ai., 1989) in the PTH-dependent
activation of osteoclasts. PTH stimulates the accumulation of cyclic AMP in osteoblast
cell lines and osteoblasts from rat calvariae (e.g. Nakatani et ai., 1984 and Lerner elai., 1989). Lafeber etai. (1989) reported that rainbow trout STC resembled PTH in its
bone-resorbing effect, even though it did not stimulate the production of cyclic AMP.
They suggested that rainbow trout STC lacks a cyclic AMP-stimulating segment, since
fragments of PTH can induce bone resorption without increasing cyclic AMP levels. In
this study, peptide N did not stimulate the production of cyclic AMP in ROS 17/2.8-5
cells, as seen with native STC from trout (Lafeber et ai., 1989) and coho salmon(Stern el ai., 1991). However, peptide N reduced the production of cyclic AMP in
response to hPTHl-34 in ROS 17/2.8-5 cells. It is possible that the activities of
peptide N on the metabolism of mammalian bone are at least partly related to the
response of cyclic AMP-mediated signals.
PTH has bone-catabolic action and it may also have an anabolic effect (Dietrich elai., 1976, Linkhart et ai., 1989 and Canalis et ai., 1989). Since the effects of STC on
bone formation had not previously been reported, we examined the direct effects of
peptide N on the synthesis of bone collagen. Peptide N enhanced synthesis of bone
collagen specifically, whereas hPTHl-34 inhibited such synthesis, as r~ported(Dietrich el ai. 1976). Thus, we found that peptide Nand hPTHl-34 have dissimilareffects on the synthesis of bone collagen, as well as on bone resorption.

20

These effects of peptide N were apparently similar to those of CT (Holtrop et al.,
1974, Feidman et al., 1980, Baron and Vignery, 1981, Takahashi et al., 1988 andFarley et al., 1988). It is unlikely that peptide N binds to the CT receptor, because
there is no constitutional homology recognized between STC and CT (Butkus et aI. ,1987). STC decreases calcium levels in blood, although CT seem s of min or
importance as a calcium-regulating factor in fish (Feinblatt, 1986) . Moreover, the STC
receptor has not been cloned, even in fish . We will continue to examine thi s iss ue indetail. In addition, peptide N elicited biphasic concentration-dependence in our assay
systems. PGE 2 also biphasically affects bone formation (Raisz et aI., 1990), whi chmight have been due to the presence of two cell types. Different targets may respond to
peptide N, since it has diverse effects on mammalian systems.
It seems that STC and material from the CS show marked similarity, in term s of

bioactivity, to mammalian PTH in both mammalian (Milet et al., 1979, Lafeber et al. ,1986 and Lafeber et aI., 1989) and teleostean assay systems (Wendelaar Bonga et aI.,
1988). However, there is still some controversy as to whether STC is identical to a
PTH-like substance of the CS. In fact, PTH does not cross-react with antiserum
against STC secreted from primary cultures of rainbow trout CS cells (Gellersen et aI.,
1988). Two types of glandular cells have been described in the CS of several teleosts(Lopez, 1969, Krishnamurthy and Bern, 1969, Wendelaar Bonga and Greven, 1975,
Wendelaar Bonga et aI., 1976 and Olivereau and Olivereau, 1978). Type I cells aresupposed to produce a proteinaceous hypocalcemic hormone, while type II cells may
be engaged in the synthesis or metabolism of a hormone involved in the regulation of
sodium and potassium levels (Wendelaar Bonga et aI., 1976). Stem et al. (1991) havereported that purified STC from coho salmon has no effect on several types of fetalbone and by the same token, our observations indicated that chum salmon STC did notaffect the PTH-sensitive systems. On the other hand, calcium and phosphate release,
as well as lactate production in embryonic mouse cal variae are stimulated in a dose-21

depende~t manner by purified STC from rainbow trout (Lafeber et aI.,

1989). One

explanation for these differences is that the tertiary structure of the STC molecule reacts
to PTH-sensitive systems. Milliken et aI. (1990) have reported that the C-terminal
fragment of Australian eel STC (peptide W) enhances whole-body calcium uptake inthe trout fly. The work of Fenwick and Verbost (1993) suggests that the effect of
peptide W on calcium uptake in the trout and eel lies in the origin of the peptide. I t isindeed clear that the C-terminal end of STC differs among Australian eel (Butkus el aI.,
1987), coho salmon (Wagner et al., 1992), human (Chang et ai., 1995) and chumsalmon (this study). In our experiments moreover, the N-terminal peptide fragment of
STC did not have PTH-like activities in mammalian systems. Thus, the expression of
PTH-like activities of STC might be dependent on the amino acid sequence at the Cterminal end. One explanation for the various bioactivities of STC in mammals as wellas in te1eosts, is that there are several active sites in the entire STC molecule. There isevidence that STC is modified prior to or just after entering the blood circulation.
Several modified immunoreactive forms of the STC have been identified in trout(Wagner et al., 1991) and salmon (Wagner et al., 1992) plasma. Interestingly, eel,salmon and human STC molecule (Butkus etal., 1987, Wagner el al., 1992, Chang etal., 1995 and this study) possess a dibasic amino acid pair, Arg-Arg, which could bea key site for endopeptidase activity. Additional di- and tri-basic sequence domains exit
in STCs, that could account for the different truncated forms of STCs. Studies of theother peptide fragments of STC will clarify this issue.
For a long time, it had been widely assumed that STC is a specific hormone inbony fishes. However, Chang et al. (1995) was successful in cloning a human cDNA
coding for a protein highly homologous to STC. Northern blot analysis was carriedout on a variety of human tissues and the mRNA is expressed in the ovary, prostate,thyroid and many of other tissues. The results would be more consistent with aparacrine than an endocrine role and suggest that STC has important physiologicalIIfunctions in several organs including bone in human. It would be expected that thisstudy is available for elucidation of functions of STC in human and that a peptide
fragment from STC applys to developing agents which may be useful in the diseaseof bone loss and so on.

23

5. SUMMARY
Stanniocalcin (STC) from chum salmon (Oncorhynchus keta) was isolated byextracting the corpuscles of Stannius (CS) with ethanol-ammonium, followed by ionexchange chromatography, gel filtration and reversed phase high performance liquid
chromatography. STC migrated as a 46-kDa product under nonreducing conditions
and as a 23-kDa product under reducing conditions on sodium dodecylsulphatepolyacrylamide gel electrophoresis. The protein is likely to be a homodimer composedof two subunits of 23 kDa each. Purified STC decreased the intestinal calcium uptake
in a dose related manner in the Atlantic cod (Gadus morhua). A cDNA was cloned
from cDNAs of the CS by means of PCR using two primers corresponding to the Nand C-terminal amino acid sequence of STC. Sequence analysis of the protein and thecDNA revealed that chum salmon STC is a homodimer, and that the monomer consistsof 179 amino acids including 11 half-Cys residues and one N-linked glycosylation
site, which is 44, 52 and 35 residues smaller at the C-terminal region than thesequences deduced from coho salmon, Australian eel and human STC cDNA,
respecti vel y.
A synthetic peptide (peptide N) corresponding to the N-terminal amino acidresidues which had a high amino acid sequence identity wi th coho salmon andAustralian eel STC, was prepared from chum salmon. Its effects were compared withthose of intact STC on mammalian bone metabolism in vitro . Peptide N (10-10 _10- 11M) slightly decreased the rate of loss of radioactivity from fetal rat calvariae labeledwith 45Ca, both with and without stimulation by N-terminal peptide fragment of
human parathyroid hormone (hPTHI-34). Intact STC had no effect on the release of
45Ca from the calvariae. Peptide N (10-10_10- 12 M) also inhibited increases in the24number of tartrate-resistant acid phosphatase-positive multinucleated cells promoted by
hPTH 1-34 in cultures of murine hemopoietic cells, although intact STC had no effecL
on the number of the cells. The accumulation of cyclic AMP induced by hPTHl -34 in
ROS 17/2.8-5 cells was suppressed by peptide N (10-10-10-12 M). Peptide N (10-1L
10-13 M) increased the rate of incorporation of pH]proline into the collagenasedigestible protein of calvariae in newborn mice. These results indicated that the highly
conserved amino-terminal region of STC from teleosts has diverse effects on themetabolism of mammalian bone, causing a biphasic response. Such effects have notbeen observed with materials from the CS and intact STC and they also differ from theeffects of hPTH 1-34.

25

6. REFERENCES
Baron, R. and Vignery, A. (1981) Behavior of osteoclasts during a rapid change intheir number induced by high doses of parathyroid hormone or calcitonin in intact
rats. Metab. Bone Dis. Relat. Res. 2: 339-346.
Butkus, A., Roche, P.J., Fernley, R.T., Haralambidis, J., Penschow, J.D., Ryan,
G.B., Trahair, J.E., Tregear, G.W. and Coghlan, J.P. (1987) Purification andcloning of a corpuscles of Stannius protein from Anguilla australis. Mol. Cell.
Endocrinol. 54: 123-133.
Butkus, A., Yates, N.A., Copp, D.H., Milliken, C., McDougall, J.G., Roche, P.J.,
Tregear, G.W. and Coghlan, J.P. (1989) Processing and bioactivity of the
corpuscles of Stannius protein of the Australian eel. Fish Physiol. Biochem. 7:
359-365.
Canalis, E., Centrella, M., Burch, W. and McCarthy, T.L. (1989) Insulin-like growth
factor I mediates selective anabolic effects of parathyroid hormone in bone
cultures. J. Clin. Invest. 83: 60-65.
Chambers, T.J. and Moore, A. (1983) The sensitivity of isolated osteoclasts tomorphological transformation by calcitonin. 1. Clin. Endocrinol. Metab. 57: 819824.
Chambers, T.J., Chambers, J.C., Symonds, 1. and Darby, J.A. (1986) The effect ofhuman calcitonin on the cytoplasmic spreading of rat osteoclasts. J. Clin.
Endocrinol. Metab. 63: 1080-1085.
Chambers, T.J., McSheehy, P.M.1., Thomson, B.M. and Fuller, K. (1985) The
effect of calcium-regulating hormones and prostaglandins on bone resorption byosteoclasts disaggregated from neonatal rabbit bones. Endocrinology 116: 234-

26

239.
Chang, A.C.M, Janosi, J., Hulsbeek, M., De Jong, D., Jeffrey, K.J., Noble, J.R.
and Reddel, R.R. (1995) A novel human cDNA highly homologous to the fish
hormone stanniocalcin. Mol. Cell. Endocrinol. 112: 241-247.
Dietrich, J.W., Canalis, E.M., Maina, D.M. and Raisz, L.G. (1976) Hormonal
control of bone collagen synthesis in vitro: effects of parathyroid hormone andcalci tonin. Endocrinology 98: 943-949.
Farley, J.R., Tarbaux, N.M., Hall, S.L., Linkhart, T.A. and Baylink, D.J. (1988)
The anti-bone-resorptive agent calcitonin also acts in vitro to directly increase boneformation and bone cell proliferation. Endocrinology 123: 159-167.
Feinblatt, J.D. (1986) The comparative physiology of calcium regulation insubmammalian vertebrates. In: Lowenstein, O. (ed.) Advances in comparative
physiology and biochemistry, Vol. 8, Academic Press, New York, pp. 74-97.
Feldman, R.S., Krieger, N.S. and Tashjian A.H.Jr. (1980) Effects of parathyroid
hormone and calcitonin on osteoclast formation in vitro. Endocrinology 107:1137-1143.
Fenwick, J. C. (1989) Calcium exchange across fish gills. In: Pang PKT, Schreibman
M.P. (eds.) Vertebrate endocrinology: fundamentals and biomedical implications,vol. 3. Academic Press, San Diego, pp. 319-341.
Fenwick, J.C. and Verbost, P. (1993) A C-terminal fragment of the hormone
stanniocalcin is bioactive in eels. Gen. Compo Endocrinol. 91: 337-343.
Filk, G., Labedz, T., Lafeber, F.P.J.G., Wendelaar Bonga, S.E.and Pang, P.K.T.
(1989) Studies on teleost corpuscles of Stannius: physiological and biochemical
aspects of synthesis and release of hypocalcin in trout, goldfish and eel. Fish
Physiol. Biochem. 7: 343-349.
Filk, G., Labedz, T., Neelissen, J.A.M., Hanssen, R.G.J.M., Wendelaar Bonga,
S.E. and Pang P.K.T. (1990) Rainbow trout corpuscles of Stannius: stanniocalcin

27synthesis in vitro. Am. J. Physiol. 258: RI157-RII64.
Friedman, M., Krull, L.H. and Cavins, J.F. (1970) The chromatographi cdetermination of cysteine and cysteine residues in proteins as s-bcta-(4-pyridylethyl) cysteine. J. BioI. Chern. 245: 3868-3871.
Gellersen, B., Wagner, G.F., Copp, D.H. and Friesen, H.G. (1988) Development ofa primary culture system for rainbow trout corpuscles of Stannius andcharacterization of secreted teleocalcin. Endocrinology 123: 913 -921.
Hattereley, G. and Chambers, T.J. (1989) Generation of osteoclasts from hemopoietic
cells and a multipotential cell line in vitro. 1. Cell. Physiol. 140: 478-482.
Hirano, T . (1989) The corpuscles of Stannius. In; Pang, P.K.T. and Schreibman,
M.P. (eds.) Vertebrate endocrinology: fundamentals and biomedical implications,vol. 3. Academic Press, San Diego, pp. 139-169.
Hirs, C.H.W. (1967) Reduction and s-carboxymethylation of proteins. Methods
Enzymol. 11: 199-203.
Holtrop, M.E., Raisz, L.G. and Simmons, H.A. (1974) The effects of parathyroid
hormone, colchicine, and calcitonin on the ultrastructure and the activity ofosteoclasts in organ culture. J. Cell BioI. 60: 346-355.
Hyashi, R., Moore, S. and Stein, W.H. (1973) Carboxypeptidase from yeast. Large
scale preparation and the application to COOH-terminal analysis of peptides andproteins. 1. BioI. Chern. 248: 2296-2302.
Katz, J. M., Wilson, T., Skinner, S.J.M. and Gray, D.H. (1981) Bone resorption
and prostaglandin production by mouse calvaria in vitro: response to exogenous
prostaglandins and their precursor fatty acids. Prostaglandins 22: 537-551.
Krishnamurthy, V.G. and Bern, H.A. (1969) Correlative histologic study of the
corpuscles of Stannius and the juxtaglomerular cells of teleost fishes . Gen.
Compo Endocrinol. 13: 313-335.
Lafeber, F.P.J.G., Hanssen, R.G.J.M., Choy, Y.M., Flik, G., Herrmann-Erlee, M.

28

P.M., Pang, P.K.T. and Wendelaar Bonga, S.E. (1988) Identification of
hypoca1cin (teleoca1cin) isolated from trout Stannius corpuscles. Gen. Compo
Endocrinol. 69: 19-30.
Lafeber, F.P.J.G., Herrmann-Erlee, M.P.M., Frisk, G. and Wendelaar Bonga, S.E.
(1989) Rainbow trout hypoca1cin stimulates bone resorption in embryonic mouse
calvaria in a PTH-like fashion. 1. Exp. BioI. 143: 165-175.
Lafeber, F.P.J.G., Schaefer, H.LM.B., Herrmann-Erlee, M.P.M. and Wendelaar
Bonga, S.E. (1986) Parathyroid hormone-like effects of rainbow trout Stannius
products on bone resorption of embryonic mouse calvaria in vitro. Endocrinology
119: 2249-2255.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the headof the bacteriophage T4. Nature 227: 680-684.
Lerner, U.H., Ransjo, M., Sahlberg, K., Ljunggren, O. and Fredholm, B.B. (1989)
Forskolin sensitizes parathyroid hormone-induced cyclic AMP response, but not
the bone resorptive effect, in mouse calvarial bone. Bone and Mineral 5: 169-18l.
Linkhart, T.A. and Mohan, S. (1989) Parathyroid hormone stimulates release of
insulin-like growth factor-I (IGF-I) and IGF-II from neonatal mouse calvaria inorgan culture. Endocrinology 125: 1484-149l.
Lopez, E, Tisserrand-Jochem, E.M., Eyquem, A., Milet, C., Hillyard, C., Lallier, F.,
Vidal, B. and MacIntyre, 1. (1984) Immunocytochemical detection in eel
corpuscles of Stannius of mammalian parathyroid-like hormone. Gen. Compo
Endocrinol. 53: 28-36.
Lopez, E. (1969) Histophysiological study of the corpuscles of Stannius in salmosalar
L. during various stages of its life cycle. Gen. Compo Endocrinol. 12: 339-349.
Lu, M. and Wagner, G.F. (1994) Stanniocalcin stimulates phosphate reabsorption by
flounder renal proximal tubule in primary culture. Am. J. Physiol. 267: R1356R1362.

29

Ma, S.W.Y. and Copp, D.H. (1982) Role of the corpuscles of Stannius andteleocalcin in calcium regulation in the teleost. In: Oguro, C. and Pang, P.K.T.
(eds.) Comparative endocrinology of calcium regulation, Japan Scientific Societies
Press, Tokyo, pp. 173-179.
Merrifield, R. G. (1985) Solid phase synthesis. Angew. Chern. Int. Ed. Engl. 24:
799-892.
Milet, C., Hillyard, C.J., Martelly, E., Chartier, M.M., Girgis, G., Macintyre, I. and
Lopez, E. (1982) A parathyroid-like hormone from eel corpuscles of Stannius
which exhibits hypocalcemic action. In: Oguro, C. and Pang, P.K.T. (eds.)
Comparative endocrinology of calcium regulation, Japan Scientific Societies,
Tokyo, pp. 181-186.
Milet, C., Lopez, E., Chartier, M.M., Martelly, E., Lapier, F. and Vadil, B. (1979) A
new calcium regulating hormone from the corpuscles of Stannius. In: Macintyre,
I. and Szelke, M. (eds.) Molecular endocrinology, Elsevier Science Publishers,

Amsterdam, pp. 341-348.
Milet, C., Peignoux-Deville, 1. and Martelly, E. (1979) Gill calcium fluxes in the eel,Anguillaanguilla (L.). Effects of Stannius corpuscles and ultimobranchial bcx:ly.
Compo Biochem. Physiol. 63A: 63-70.
Milliken, C. E., Fargher, R.C., Butkus, A., McDnald, M. and Copp, D.H. (1990)
Effects of synthetic peptide fragments of teleocalcin (hypocalcin) on calcium
uptake in juvenile rainbow trout (SaZmo gairdneri). Gen. Compo Endocrinol. 77:
416-422.
Mundy, G.R. and Roodman, G.D. (1987) Osteoclast ontogeny and function. In:
Peck, W. A. (ed.) Bone and mineral research. Elsevier Science Publishers,
Amsterdam, Vol. 5, pp. 209-279.
Muramoto, K., Sunahara, S. and Kamiya, H. (1987) Measurement of tryptophan in
peptides by acid hydrolysis in the presence of phenol and its application to the

30amino acid sequence of a sea anemone toxin. Agric. BioI. Chern . 5 1: 1607-1616.
Nakatani~ Y., Tsunoi, M., Kurihara, N, Fujita, K. and Kumegawa, M. ( 1984) Effectsof parathyroid hormone on cAMP production and alkaline phosphatase ac ti vity inosteoblastic clone MC3T3-E1 cells. Biochem. Biophys. Res. Comn. 123: 894898.
Nijweide, P.l., Burger, E.H. and Feyen, 1.H.M. (1986) Cells of bone: proliferati on,differentiation, and hormonal regulation. Physiol. Rev. 66: 855-886.
Olivereau, M. and Olivereau, 1. (1978) Prolactin, hypercalcemia and corpuscles ofStannius in seawater eels. Cell Tiss. Res. 186: 81-96.
Peterkofsky, B. (1972) The effect of ascorbic acid on collagen polypeptide synthesis
and proline hydroxylation during the growth of cultured fibroblasts. Arch.
Biochem. Biophys. 152: 318-328.
Peterkofsky, B. and Diegelmann, R. (1971) Use of a mixture of proteinase-freecollagenases for the specific assay of radioactive collagen in the presence of otherproteins. Biochemistry 10: 988-994.
Raisz, L.G. (1965) Bone resorption in tissue culture. Factors influencing the response
to parathyroid hormone. 1. Chn. Invest. 44: 103-116.
Raisz, L.G. and Fall, P.M. (1990) Biphasic effects of prostaglandin E2 on boneformation in cultured fetal rat calvariae: interaction with cortisol. Endocrinology
126: 1654-1659.
Sambrook, l., Fritsch, E.F. and Maniatis, T. (1989) in molecular cloning, alaboratory manual, 2nd edn., Cold Spring Harbor Laboratory Press, New York.
So, Y.P. and Fenwick, l.C. (1977) Relationship between net 45calcium influx acrossa perfused isolated gill and the development of post-stanniectomy hypercalcemia.
1. Exp. Zool. 200: 259-264.
So, Y.P. and Fenwick, l.C. (1979) In vivo and in vitro effects of Stannius corpuscle
extract on the branchial uptake of 45Ca in stanniectomized North American eel

31(Anguilla rostrata). Gen. Compo Endocrinol. 37: 143-149.

Stannius; H. (1839) Ueber Nebennieren bei Knochenfischen. MUller's Arch.: 97-10l.
Stern, P.H., Schankar, G., Fangher, R.C., Copp, D.H., Milliken, C. E., Sato, K.J.,
Goitzman, D. and Herrmann-Erlee, M.P.M. (1991) Salmon stanniocalcin andbovine parathyroid hormone have dissimilar actions on mammalian bone. J. Bone
Mineral Res. 6: 1153-1159.
Sundell, K. and Bjbrnsson, B.T. (1990) Effects of vitamin 0 3 , 25(OH) vitamin 0 3 ,24, 25 (OH)2 vitamin 0 3 , and 1, 25 (OHh vitamin 0 3 on the in vitro intestinal
calcium absorption in the marine teleost, Atrantic cod (Gadus morhua). Gen.
Compo Endocrinol. 78: 74-79.
Sundell, K., Bjbrnsson, B.T., Itoh, H and Kawauchi, H. (1992) Chum salmon(Oncorhynchus keta) stanniocalcin inhibits in vitro intestinal calcium uptake in

Atlantic cod (Gadus morhua). J. Compo Physiol. 162B: 489-495.
Takahashi, N., Yamana, H., Yoshiki, Y., Roodman, G.D., Mundy, G.R., Jones,
S.J., Boyde, A. and Suda, T. (1988) Osteoclast-like cell formation and its
regulation by osteotrophic hormones in mouse bone marrow cuI tures.
Endocrinology 122: 1373-1382.
Verbost, P.M., Butkus, A., Atsma, W., Willems, P., Flik, G. and Wendelaar Bonga,
S.E. (1993) Studies on stanniocalcin: characterization of bioactive and antigenic
domains of the hormone. Mol. Cell. Endocrinol. 93: 11-16.
Verbost, P.M. and Fenwick, J.C. (1995) N-terminal and C-terminal fragments of thehormone stanniocalcin show differential effects in eels. Gen. Compo Endocrinol.
98: 185-192.
Wagner, G.F., Dimattia, G.E., Davie, J.R., Copp, D.H. and Friesen, H.G. (1992)
Molecular cloning and cDNA sequence analysis of coho salmon stanniocalcin.
Mol. Cell. Endocrinol. 90: 7-15.
Wagner, G.F., Fenwick, J.C., Park, C.M., Milliken, C., Copp, D.H. and Friesen,

32

H.G. (1988) Comparative biochemistry and physiology of teleocalcin fromsockeye and coho salmon. Gen. Compo Endocrinol. 72: 237-246.
Wagner, G.F., Guirandon, C.C., Milliken, C. and Copp, D.H. (1995)
Immunological and biological evidence for stanniocalcin-like hormone in humankidney. Proc. Natl. Acad. Sci. U.S.A. 92: 1871-1875.
Wagner, G.F., Hampong, M., Park, C.M. and Copp, D.H. (1986) Purification,
characterization, and bioassay of teleocalcin, a glycoprotein from salmon
corpuscles of Stannius. Gen. Compo Endocrinol. 63: 481-491.
Wagner, G.F., Milliken, C., Friesen, H.G. and Copp, D.H. (1991) Studies on theregulation and characterization of plasma stanniocalcin in rainbow trout. Mol.
Cell. Endocrinol. 79: 129-138.
Wendelaar Bonga, S.E. and Greven, l.A.A. (1975) A second cell type in Stannius
bodies of two euryhaline teleost species. Cell Tiss. Res. 159: 287-290.
Wendelaar Bonga, S.E., Greven, l.A.A. and Weenhuis, M. (1976) The relation
between ionic composition of the environment and secretary activity of theendocrine cell types of Stannius. Cell Tiss. Res. 175: 297-312.
Wendelaar Bonga, S.E., Pang, R.K. and Pang, P.K.T. (1986) Hypocalcemic effects
of bovine PTH(1-34) and Stannius corpuscle homogenates in teleost fish adapted
to low calcium water. 1. Exp. Zool. 240, 363-367.

t=n+60

L

JCa

t=n

(45Ca f · V)
45Ca sp

1000
X

Fig. 1. The formula for the unidirectional intestinal Ca 2 + influx (JCa) of
the Atlantic cod. JCa was calculated as the amount of 45Ca appearing ineach fraction of the vascular perfusate divided by the amount of
perfused 45Ca (sp. act. 0.85 ± 0.06 MBq/~mol, 45Casp ) JCa wasexpressed for each fraction as well as for 60-min periods with the flow
linearly correlated with body weight. The amount of 45Ca (dpm~ 45Ca f )in 1 ml of each fraction collected was assessed using a liquid
scintillation counter, and V was assessed by weighing. X is the bodyweight in grams.

Primer 1
MetPheSerProAsnSerPro
5'-TTCATATGTTCTCACCAAACCAGCCC-3'
C

Primer 25'-TTAGATCTAAGAGTTTGGCCCCTG-3'
G A A A A
T

Fig. 2. Nucleotide sequence of the synthetic primers. Primer 1
corresponds to the N-terminal amino acids of STC and contains anadditional ATO initiation codon as well as an Nde 1 site at its 5'-end.
Primer 2 corresponds to the C-terminal amino acids of STC andcontains an additional TAO termination codon as well as a BglII site
at its 5'-end. Ndel and BglII sites are shwon in italic.

0.05 M NI-I 4 HC0 3 0.1 M NH 4HC0 3

0.2 M NI-I 4HC0 3 1 M NH 4 IIC0 3

I

I

I

I
DE9

1.5
S

DE6

~

0
00
N

......
ro
~

1.0

C)

§
.D
~
0
U)
.D

~

DEI

0.5

o
I

o

I

I

I

I

II

50

100

150

200

255

Fraction number

Fig. 3. Ion exchange chromatography of an ethanol extract fron1 the CS
of chum salmon on a DE52 cellulose column (10 X 300 mm).

1.0

G4

S
~

0

~
~

ro

<l.)
()
~

ro

.e0

G2

r/:J

..0

~

0
I

I

I

0

60
Time (min)120

Fig. 4. Gel filtration of the DE6 fraction obtained from ion exchange
chromatography on a Superdex 75 column (16 X 600 mm).

0.57-----------

50
]

·S
·a

40 B
(])
o

~

o

o

Time (min)60

Fig. 5. rpHPLC of the 04 fraction obtained from gel fil tration on a
TSK-Oel ODS-120T column (0.46 X 25 cm) with a linear gradient ofacetonitrile in 0.1% TFA (dotted line).

SDS-PAGE
Marker+94000 - A-~
67000 - 43000 - -- 46300- . .
30000 - .-23400

20100 --

14400 -~

Fig. 6. Gel electrophoresis of chum salmon STC.
Proteins are stained with Coomassie blue. +, with and-, without reduction by ~-mercaptoethano1.

1
><

c~ 80
2

~
~

60

.D40
4-.
0

~

1.5

20
0

A

...c:

B

C

*

~

..D
bJ)

..!:o::

---+

1

N

cd

U

0

a 0.5
~

o

- L - - - - - L_ _ _

A

B

C

Fig. 7. Calcium uptake across the intestinal mucosa of in vitroperfused intestinal preparations from the Atlantic cod. For eachgroup of fish the basal influx was measured for 60 min (unfilledbars), followed by influx measurements during 60 min of treatment(filled bars). The treatment groups are: A, perfusion with cod
Ringer's solution only; B, perfusion with 2 nM STC and C,
perfusion with 10 nM STC. The inset figure presents the data
expressed as a percentage of the mean basal flux of each procedure.
Each value indicates the mean ± SE of results from 10 fishes.
Significantly different from the basal Ca2+ influx within group: *p<
0.05.

lilA

50~
0.5

T11

T8

T1

"--"

T12

'0
c
cd

0..

T13

o
H

0..

o

10~

T6

o

T10

Time (min)
60

T7

""

~

cd

T9

Q.)

90

"

,,'/

U

"

§

/

..0

"

H

o

'" "

C/)

..0

~

""

T4
T2 T3

'"

T5
//

5

o
I

o

I

I

105

15
Time (min)Fig. 8. rpHPLC of a trypsin digest of RPE-STC on a TSK gel ODS-120T
column (0.46 X 25 cm) with a linear gradient of isopropanol in 0.1 % TFA(dotted line). Inset: rechromatography of T13 on a TSK gel ODS -120T
column with a linear gradient of isopropanol in 0.01 M ammonium
acetate (pH 6.0).

10

20

H-PheSerProAsnSerProSer As pVa l AlaArgCys LeuAs nGl yAlaLeuAs pValGly(Intact=====================================================(T6------------------ - ------- - - - ) (T12 ================= ======
30
40
CysGlyThrPheAlaCysLeuGluAsnSerThrCysAspThrAspGlyMet HisAsp Ile============(AN 33============================== ======== == === ==================

50

60

CysGlnLeuPhePheHisThrAlaAlaThrPheAsnThrGlnGlyLysThr Pheva lLy s============================================== = =============-------------------- - ----------------------- --- ) (T4 === ===== )70
SO
GluSerLeuArgCysIleAlaAsnGlyValThrSerLysValPheGlnThr I leArgArg===================================== == = == ==================(T2========) (T3=======================) (T9============ == )90
100
CysGlyValPheGlnArgMetIleSerGluValGlnGluGluCysTyrSerArgLeuAsp==================--------------------------------- -----)(AN26(T7==============) (TS================================ ) (T10 ==(SP44 =============
110
120
IleCysGlyValAlaArgSerAsnProGluAlaIleGlyGluValValGlnValProAla=================) (T11===============================================================================)
130
140
HisPheProAsnArgTyrTyrSerThrLeuLeuGlnSerLeuLeuAlaCysAspGluGlu==================================================)(AN30==================) (T13B========================================
150
160
ThrValAlaValValArgAlaGlyLeuValAlaArgLeuGlyProAspMetGluThrLeu===========================================================================--) (T5==============) (T13A===================
170
179
PheGlnLeuLeuGlnAsnLysHisCysProGlnGlySerAsnGlnGlyProAsnSer-OH========================================================)====================) (T1============================== ==)(CPY)

Fig. 9. Proposed amino acid sequence of chum salmon STC monomer. The
double broken line represents residues that were determined by amino acidsequence analysis. T, AN, SP and CPY indicate fragment peptides preparedby digestion with trypsin, endoproteinase ASp-N, Staphylococcus aureus V8proteinase and carboxypeptidase Y, respectively. Asn29 is N-glycosylated.

coho salmon
203---------------------------------- 235
churn salmon TTTCATATGTTCTCACCAAhCAGCCCCTCGGATGTGGCTAGGTGTTTGAATGGCGCTCTA 60
7serAspValAlaArgCysLeuAsnGlyAlaLeu-C---------------------------------------------------------- 295
GACGTGGGATGTGGTACGTTTGCCTGCCTGGAGAATTCTACCTGTGACACTGATGGCATG 120
AspValGlycysGlyThrPheAlacysLeuGluAsnSerThrcysAspThrASpGlyMet--T--------------------------------------------------------- 355
CACGATATCTGTCAACTGTTCTTTCACACCGCAGCTACCTTTAACACACAGGGTAAGACA 180
HisAspIlecysGlnLeuPhePheHisThrAlaAlaThrpheAsnThrGlnGlyLysThr--------------------G--------------T------------------------ 415
TTTGTAAAGGAGAGTCTGAGATGTATTGCCAACGGCGTCACGTCTAAAGTCTTTCAGACC 240
PhevalLYSGluSerLeuArgcysIleAlaAsnGlyValThrserLysValPheGlnThr--------------A--------------------------------------------- 475
ATCAGGCGCTGTGGCGTCTTCCAGAGAATGATTTCTGAGGTCCAGGAGGAGTGTTACAGT 300
IleArgArgcysGlyValPheGlnArgMetIleSerGluValGlnGluGluCysTyrSer------------------------------------------------------------ 535
AGACTGGACATCTGTGGTGTGGCTCGCTCTAACCCTGAGGCCATTGGAGAGGTGGTGCAG 360
ArgLeuAspIlecysGlyvalAlaArgserAsnProGluAlaIleGlyGluValValGln
595
GTCCCTGCACACTTCCCCAACAGGTACTACAGCACTCTGCTCCAGTCCCTGCTAGCCTGT 420
valProAlaHisPheProAsnArgTyrTyrserThrLeULeUGlnSerLeuLeuAlaCys--------------------------------------------------G--------- 655
GATGAGGAGACAGTGGCTGTGGTCAGGGCAGGGCTTGTTGCTAGGCTGGGACCAGACATG 480
AspGluGluThrValAlaValValArgAlaGlyLeuValAlaArgLeuGlyProAspMet--------T------T--------------------C --------------536
GAAACTCCCAACCAGCTGCTGCAGAACAAACACTGCACCCAGGGTTCTAACCAGGGTCCA
GluThrELDPheGlnLeuLeuGlnAspLysHisCysserGlnGlyserAsn174
Leu
Pro

540

AATTCCTAGATCTAA 535

Fig. 10. Nucleotide and deduced amino acid sequences of the chumsalmon STC cDNA clone pKDST1 and comparison with coho salmon
STC cDNA. Underlines show the positions of synthetic oligonucleotide
primers for PCR. Double underlines show the positions of substitution
compared with the protein. The nucleotide sequence of coho salmon was
taken from Wagneretal. (1992). (-) represents bases identical with thoseof chum salmon STC.

10
40
20
30
FSPNSPSDVARCLNGALDVGCGTFACLENSTCDTDGMHDI--S--------------A-----------------------AS-------------Q---SA----D----N-----EVAAQNSAE-V----S--Q----A--------------Y--

1)
2)
3)
4)

Churn salmon
Coho salmon
Australian eel
Human

1)

50
60
70
100
90
80
CQLFFHTAATFNTQGKTFVKESLRCIANGVTSKVFQTIRRCGVFQRMISEVQEECYSRLD

2)------------------------------------------------------------

3)
4)-RS-L-G--K-D-----------K-----I-----L-----SS--K-----------K--KS-LYS--K-D----A------K-----------LA----ST-----A--------K-N

1)

110
120
130
140
150
160
ICGVARSNPEAIGEVVQVPAHFPNRYYSTLLQSLLACDEETVAVVRAGLVARLGPDMETL

2)------------------------------------------------------------

3)
4)

L-S--Q-----M---A---SQ--------------T---D--EQ------S--E-E-GVV-GI-KR-----T----L-NH-S----NR-VR---E---D--STI-DS-MEKIG-N-AS-

1)
2)
3)
4)

170
180
190
200
210
220
FQLLQNKHCPQGSNQGPNS-------------------APAGWRWPMGSPPSFKIQPSMRGRDPTHLFARKRSVEALER-----T-A--PSAAG-TGPVGAGGSWRCPWGPPCSRSSPTCAPGTPPTSLLRNARPAPNY-HI--TD--AQTHPRADFNRRRTNEPQKLKVLLRNLRGEEDSPSHIKRTSHESA

2)
3)

230
VME
HPPRLALMDCP

Fig. 11 . Comparison of the amino acid sequences of STCs. The amino acidsequences are taken from the Australian eel (Butkus et ai.,1987), coho
salmon (Wagner et aI., 1992) and human (Chang et al.,1995) . (-) represents
residues identical with those of chum salmon STC. The synthetic peptide
based on STC from chum salmon is underlined.

30

30
.....

25

l.....,.

0

~

20

"-"

ro

u~

\/) ;>-..
<:t .....

l.....,.

(A)

15

• .-1

o .~

~t)

10

Q)

.9

Q)

ro
.....

5

ro ro
........ -0

~

0

30

30
.....

10

9
8
hPfHI-34

7(-log M)

13

12

10

9

(B)

25

l.....,.

0

~
"-"
ro

20

\/) ;>-..

15u~<:t .....

c....... . .....

0 .::::Q) .....

C/l

0

ro ro
Q) 0
-;.0
Q) ro

0::: .....

10
5
0

11
STC

13

12

11
10
Peptide N

9Fig. 12. Effects of hPfHl-34, STC and peptide N on the release of 45Ca fromfetal rat calvariae. Two calvariae per well of a 24-well plate were cultured withvarious concentrations of hPfH 1-34 (A) for 2 days and STC or peptide N (B) for
4 days . Each value indicates the mean ± SE of results from 5 wells. Statisticalanalysis was performed in (B). Significantly different from control (-). *P<0.05.

(-log M)

30.-------------------------------~

T

.. *

----------------~

O~----------------------------------~

2

3
Time (days)

4

Fig. 13. Time course of the effects of STC and peptide N on therelease of 45Ca from fetal rat calvariae. Two calvariae per well of a
24-well plate were cultured with 10-11 M STC ( • ), 10-11 M
peptide N ( .) or vehicle ( 0 ) for 2-4 days. At the indicated times,
the release of 45Ca from the two bones was measured. Each val ue
indicates the mean ± SE of results from 5 wells. Significantly
different from control (vehicle) of the same time. *P<0.05.

30

"§
0
....,

25

t.-.

0

~

20

'-"

ro

u ~
If)

>-.

'\t .....
'-< . ......

o .::::<l.l .....
VJ

u
ro ro

.9
. . . . . "0

15
10

Q)
Q)

~

ro

5

I-.

0

13

12

11
STC

_13_ _1_2--,---_11-,--_10_ _9_ (-log M)
Peptide N
hPTHI-34

Fig. 14. Effects of STC and peptide N in the presence of hPTHI-34 on therelease of 45Ca [rom fetal rat calvariae. Two calvariae per well of a 24-wellplate were cultured wi th various concentrations of STC or Peptide N wi th 10-8
M hPTHI-34 for 2 days. Each value indicates the mean ± SE of results from 5
wells. Significantly different from hPTHI-34 only. *P<0.05.

25

(A)

ct· 2022
15
t:0 .Q)
~

~

.D

~ 10
(/J

S u 5
::l

Z

~ 0

9

hPTHI-34

8

7

12

11
Peptide N(-log M)

25

ct
22
t:0
~

~

.D

20
.- 15
Q)

~ 10
(/J

S u
Z

::l

Z

~

t:0

0

.-

Q)

9

(-log M)

15

-£ 10

Z

~

~

10

20

.D

~

8
13
hPTHI-34

25

ct

r2

5

S U
Z
::l

5
0

13

11
Peptide N
hPTHI-34
129(-log M)

Fig. 15. Effects of hPTHI-34 and/or peptide N on the formation of TRAP positive
multinucleated cells (TRAP+MNCs) from mouse bone marrow cells. Bone marrowcells (7 X lOS per well of a 24-well plate) were cultured with various concentrations
of hPTHI-34 (A), peptide N (B) or peptide N plus hPTHI-34 at 10-8 M (C). After
culture for 8 days, TRAP+MNCs were counted. Each value indicates the mean ± SEof results from 5 wells. Statistical analysis was performed in (B) and (C).
Significantly different from hPTHI-34 only. *P<O.05.

Fig. 16. TRAP positive multinucleated cell (arrow) .

4.r~--------------------------~

109hPTHl-34

87(-log M)

4~~--------------------------------------~

8---"'-"13'---_..;0.12=--_......:1..;0.1_----'1::;..:;:0:.-.-_.....:::..9_hPTHl-34
Peptide N(-log M)

4-r~--------------------------------~_1_3_ _ _12_ _ _1_1_ _1_0_ _ _9_ (-log M)
Peptide N
hPTHl-34

Fig. 17. Effects of hPTHl-34 and/or peptide N on the formation of cyclic AMP in
ROS 17/2.8-5 cells. ROS 17/2.8-5 cells in each well of a 48-well plate were stimulated
for 5 min with various concentrations of hPTHl-34 (A), peptide N (8) or peptide N
plus hPTHl-34 at 10-8 M (C). Levels of cyclic AMP were measured by an enzyme
immunoassay. Each value indicates the mean ± SE of results from 4 wells. Statisticalanalysis was performed in (8) and (C). Significantly different from hPTHl-34 only.
*P<0.05.

T2

1.1 (1)1.2 (1)1.2 (1)T3
1. 2( 1)
1. O( 1)
1. 2( 1)
1. 4 (1)
1. 1 (1)
1. 2( 1)
1. O( 1)

1. O( 1)T4

1.0( 1)1.2( 1)(4 )o. 8( 1)
1. O( 1)(9 )1. 0 (1)
1. 1 (1)
1. 8(2)
(4 )

57-60

0 . 5 (1)2.2(2)
1. 6 (2)2.0(2)

2.0(2)

T1

147-152( 6)

1. 0 (1)1.1 (1)

1. 0 (1)2.2 (2)
1. 3 (1)

T5

1-11

(11 )

2.1 (2)

1. 0 (1)0.8 (1)

1. 3 (1)
1. 2 (1)2.6(3)

1. 9 (2)

T6

T8

T9

1.6(1)

o. 9( 1)
1. 8(2)4.4 (4)

1.1 (1)1.2 (1)

1. 3( 1)
O. 9( 1)
1. O( 1)
O. 8( 1)T10

2.1 (2)T11

T12

1. 0 (1)1. 0 (1)3.6(3)
1. 0 (1)2.1 (2)3.0(3)

1.2 (2)

1. 3 (1)
1. 3 (1)
1. 3 (1)7.2 (7)4.2 (6)
1. 9( 1)
3.9(3 )
5.2(5)
3.5 (4)
1. 4 (1)0.8(1)
1. 2 (1)4.2 (4)
1.1 (1)
1. 3 (1)0.9(1)

1. 0 (1)0.6 (1)(45 )

0.8(1 )

1. O( 1)

(8 )

12-56

3. 8( 4)
1. O( 1)2.0(2)
1. O( 1)0.7(1 )

( 6)

99-106 107-125

1. 1 (1)1. 0 (1)1. 0 (1)0.7 (1)

(12 )

74-79

(19 )

4.3 (5)

(6 )

87-98

3.3 (3)81-86

0.7 (1)

1. O( 1)o. 9( 1)o. 9( 1)

T7

Table 1.
Amino acid composition of fragments and intact STC.
AA

Asp
Thr
Ser
Glu
Gly
Ala
Val
Met
Ile
Leu
Tyr
Phe
Lys
His
1/2Cys
Pro
(12 )

65-73

Arg

Total
61-64

o. 6( 1)position 168-179

SP44

PE-intact

AN33
AN26

AN30
T13B

3.8(3)
1. O( 1)2.0(2)
2.3(2)T13A

4.8(5 )
2.8(2 )
1. 9 (2)
6.7 (7)4.2(4)
3.6(3 )
2.8(4)
0.6(1)

18.5(18)
4.0(3)
1. 6( 2)10.4(12)4.7 (6)
1. 6( 2)12.7(13)
3. 9( 4)22.7(21)8.3(9)
2.2(2)
5.3(3)
1. 7 (2)13.7(13)
1. 8 (2)13.5(13)
3.7 (3)13.5(13)
4.5 (5)1.0(1)
2.9(3)(1 )
1.2(2)s 5.7(6)3.4 (4)14.8(15)
3.6(3)
1. O( 1)
2.5(3)
0.5(1 )
(1 )4.9(6) 10.2(10)3.9(4)
2.4(3)
4.0(4)
2 .1( 1)9.8(10)1.2 (2)4.5(5)
11.3(11)ND(2)3.3(4)
7. 9( 8)0.8(1 )
2.1 (2)
1.1 (1)

1. 7 (2)3.7 (5)
1. 2 (2)
1. 3 (1)2. O( 1)
1. 5 (1)
1. 2 (1)2.5(2)
1. 5 (1)0.9(3)

1. 5 (1)
1. 9 (2)
1. 8 (2)3.1 (3)3.6(4)
1. 7 (2)

1.1 (1)2.0 (2)0.9 (1)2.0(3)

4.2 (5)

1. 0 (1)0.8 (1)

1-179

(179)

(61)

(20)

(42 )

95-114

(38)

39-99

(21 )

3.1 (3)
1.3 (1)2.1 (3)4.3(4)
2.2 (2)3.9(4)
2.8 (4)o. 8( 1)
4.3(4)
1.1 (1)
1. O( 1)

1. 3( 1)
(15 )

153-167 126-146 100-137 138-179

1I2Cys was determined as pyridylethyl cysteine. Numbers in parenthesis were obtained from sequence analysis. ND, not determined.

Table 2.
Amino acid sequence of fragments and intact STC.
cycle

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

Tl
yield
H162.7
C267.5
P282.2
Q182.9G191.0
S42.4
N174.6
Q141.3
GI05.7
P123.3
N96.8
14.0

T2

T3

T4

T5

T7

T8

T9

TI0

yield

yield

yield

yield

yield

yield

yield

yield

M365.2 V292.7
I377.7 F289.9
S64.6 Q219.9
E267.6 T40.9
V269.8 I115.8
Q232.7 R21.1
E229.0E231. 2
C91.6
Y144.9S19.1
R24.4

L793.1
D421.1
I478.0C330.6
G235.6
V356.9A337.8R84.8

E259.3 C7.3
S37.3 I17.2
L92.7 A17.9
R6.3
N13.6
G5.9
V3.8
TO.5
SO.7
KO.5

T99.8 A417.1 C72.3
F337.8 G154.0 G67.7
V246.4 L189.1 V96.0
K175.0 V120.9 F75.7
A91.7 Q57.6
R14.5 R9.9

33

34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49

C, Detennined as PE-Cys and CM-Cys. ND, not detected. aNot recorded.

cyc~e

T11
yield

T13A
yield

T13B
yield

AN26
yield

1
2
3
4
5
6
7
S
9
10
11
12
13
14
15
16
17
lS
19
20
21
22
23
24
25
26
27
2S
29
30
31
32
33
34
35
36
37
3S
39
40
41
42
43
44
45
46
47
4S
49

S131.5 C249.2 L2S.9
N552.1 L274.2 G16.1
P427.6 N93.7 P21.4
E457.1 G74.9 P1S.4
A3S6.4 Al13.2 M17.0
I394.0 Ll14.7 E15.9
G245.S D66.7 T5.4
E314.9 VS9.S L1S.1
V332.2 G44.3 F17.1
V342.0 C62.5 Q13.4
Q275.6 G21.5 L13.6
V220.7 T14.0 L15.5
P206.9 F54.7 Q9.3
A1S9.4 A51.9 N11.3
H103.6 C45.S K4.S
FS9.9 L44.0
F60.9 E34.9
N5S.7 NND
R19.0 S5.3
T5.5
C25.3
D11. 0
T3.S
D4.6
G5.4
M9.5
H4.1
DS.5
I7.0

Y7.1
Y6.5
SO.9
T2.0
LS.1
L6.1
Q3.6
SO.5
L3.4
L3.5
A2.0
C1.5
D1.9
E1.5
ELl
TO.4
V1.2
AO.7
VO.S
VO.2

a D29S.6 C4S.24
D255.5 DI244.1 E327.1 I140.5 Y47.0
C2S6.9 E327.3 C231.2 SS.4
G121. 5 T62.7 Q276.6 R9.5
V224.3 V21S.4 L357.6 L32.1
A233.5 A196.2 F309.5 D17.1
R139.9 V1S0.1 F339.9 I1S.6
S34.3 V17S.4 H77.5 CS.1
N133.7 R11S.2 T63.7 G10.0
P99.7 A1S9.0 A279.3 V22.7
E125.2 G101.6 A291.4 A22.0
Al19.9 L153.4 T4l.S R12.2
I101. 9 V126.6 E136.5 S2.6
G75.0 A141 . 7 N105.S N12.3
ES9.3 R97.6 T2S.S P10.7
V93.6 L125.2 Q73.7 E9.7
V101. 4 G67.4 G69.6 A9.9
Q90.l P70.6 K104.l I7.2
V62.6 D51.5 T16.9 G6.7
P55.6 M92.1 F53.2 Q1.6
A7S.S E59.5 V53.4 C3.9
H39.2 T16.4 K66.6 G5.1
F66.4 L57.9 E2S.4 TO.9
F4.9
P49.0 F5S.5 S5.5
N59.S Q56.3 L2S.0
P39.S L44.2 R19.1
Y50.9 L45.6 C16.0
Y63.1 Q37.6 I24.4
N30.9 A29.0
S7.1
K36.9 N14.5
T5.2
L23.5 H21.3 G14.2
L30.9 c34.1 V15.6
Q14.7 P20.2 T3.l
S2.2
Q2S.4 S2.7
L10.S G10.6 K11.2
VS.2
L12.4 S2.6
A2.5
N14.1 F4.6
Q7.2
Q5.2
C1.4
G5.S
T1.6
I3.7
P6.7
N4.5
R2.9
S1.3
R2.3
Cl.S
G1.9
V1.7
F2.7
Ql.S
Rl. 0
M2.5

T12
yield

AN30
yield

AN33
yield

SP44
yield

Intact
yield
F22.3
S5.3
P14.3
N16.l
S3.6
P10.3
S3.0
D7.7
vlO.O
A12.9
R5.5
C9.2
L11.3
N3.9
G5.9
A6.9
L5.0
D4.1
V5.7
G4.S

7.38+0.67

NCP
5.81+1.20

27.60+0.24

17.14±2.17**

27.63±0.93

collagensynthesized (%)aTable 3.
Effects of hPTHl-34 and peptide N ·on the incorporation of pH]proline into collagen-digestible(CDP) and non-collagen protein (NCP) in calvariae of newborn mice.

Incorporation of [3H]proline
dry wt.)6.42±0.37

31.05+2.27(dpm/~g814.27+1.72

5.56+0.09

32.37±1.11*

CDP

9

13.59+0.96

5.55±0.36

32.20+0.30**-log (M)10

14.31+0.60*

6.77±0.47

31.44±0.76*

Treatment

11

17.41±1.31**

6.44+0.48

7.16+0.53***

12.01+0.57

PTH

12

16.09+0.58**

Control

STCI-20

13

Three calvariae per flask were cultured with hPTHl-34 at 10-8 M or with various
concentrations of peptide N for 4 days, then pulse-labeled with pH]proline for the last 2 hof culture. Each value indicates the mean ± SE of results from 5 flasks. aThe percentage ofcollagen synthesized was calculated correcting for the relative abundance of proline in
CDP vs. NCP as described in Materials and Methods. Significantly different from control:*P<O.05, **P<O.01 and ***P<O.OOl.