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Endocrinology, doi:10.1210/en.2007-1219
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Endocrinology Vol. 149, No. 5 2403-2410
Copyright © 2008 by The Endocrine Society

The Murine Stanniocalcin 2 Gene Is a Negative Regulator of Postnatal Growth

Andy C.-M. Chang, Jeff Hook, Frances A. Lemckert, Michelle M. McDonald, Mai-Anh T. Nguyen, Edna C. Hardeman, David G. Little, Peter W. Gunning and Roger R. Reddel

Cancer Research Unit (A.C.-M.C., R.R.R.) and Muscle Development Unit (M.-A.T.N., E.C.H.), Children’s Medical Research Institute, and Oncology Research Unit (J.H., F.A.L., P.W.G.) and Department of Orthopedic Research and Biotechnology (M.M.M., D.G.L.), The Children’s Hospital, Westmead, New South Wales 2145, Australia; Faculty of Medicine (A.C.-M.C., E.C.H., D.G.L., P.W.G., R.R.R.), University of Sydney, New South Wales 2006, Australia

Address all correspondence and requests for reprints to: Roger Reddel, Children’s Medical Research Institute, 214 Hawkesbury Road, Westmead, New South Wales 2145, Australia. E-mail: rreddel{at}cmri.usyd.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stanniocalcin (STC), a secreted glycoprotein, was first studied in fish as a classical hormone with a role in regulating serum calcium levels. There are two closely related proteins in mammals, STC1 and STC2, with functions that are currently unclear. Both proteins are expressed in numerous mammalian tissues rather than being secreted from a specific endocrine gland. No phenotype has been detected yet in Stc1-null mice, and to investigate whether Stc2 could have compensated for the loss of Stc1, we have now generated Stc2–/– and Stc1–/– Stc2–/– mice. Although Stc1 is expressed in the ovary and lactating mouse mammary glands, like the Stc1–/– mice, the Stc1–/– Stc2–/– mice had no detected decrease in fertility, fecundity, or weight gain up until weaning. Serum calcium and phosphate levels were normal in Stc1–/– Stc2–/– mice, indicating it is unlikely that the mammalian stanniocalcins have a major physiological role in mineral homeostasis. Mice with Stc2 deleted were 10–15% larger and grew at a faster rate than wild-type mice from 4 wk onward, and the Stc1–/– Stc2–/– mice had a similar growth phenotype. This effect was not mediated through the GH/IGF-I axis. The results are consistent with STC2 being a negative regulator of postnatal growth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE STANNIOCALCINS (STCS), STC1 and STC2, are secreted glycoproteins that have been highly conserved from aquatic to terrestrial vertebrates. STC1 was first identified in fish as a hormone secreted by the corpuscles of Stannius, an endocrine organ unique to fish. It is secreted into the blood in response to elevated calcium and acts on the gills, kidneys and gut to regulate calcium absorption and phosphate excretion. In higher vertebrates, STC1 is expressed most highly in a variety of tissues, including the kidney, ovary, prostate, thyroid, and spleen (1, 2, 3, 4), and during mouse fetal development, the highest levels are detected in bone and muscle (5, 6, 7). On the basis of a number of studies, it has been proposed that STC1 has significant roles in metabolism, reproduction, and development and also in cancer (for reviews see Refs. 8, 9, 10, 11).

STC2 was identified by searching for related sequences in expressed sequence tag databases (12, 13, 14). Like STC1, mammalian STC2 is expressed in a wide variety of tissues. STC1 and STC2 both have predicted signal peptides, are secreted as phosphoproteins (15), and have moderately well-conserved primary amino acid sequences, especially at their N-terminal halves with spatial conservation of cysteine residues, suggesting that they might have similar biological functions. However, STC2 is larger (human STC2 is 55 amino acids longer than human STC1 protein) and has a histidine-rich C-terminal region capable of binding to metals like nickel (15, 16). Moreover, it has been shown that recombinant STC2 protein is unable to displace STC1 from its putative receptor (11).

The initial functional analyses of mammalian STC1 focused on determining whether its role in regulating blood calcium has been conserved. In vivo studies using recombinant STC1 protein injected into rats indicated that it could regulate blood Ca2+ by increasing renal reabsorption of phosphate and could reduce the flux of Ca2+ across rat and swine intestine and increase absorption of phosphate (2, 17, 18). Other studies suggested a role in the control of intracellular Ca2+ in endothelial cells, cardiomyocytes, and neuronal cells (19, 20). Transgenic mice that have increased STC1 expression have significant postnatal dwarfism, affecting multiple organs but no detectable abnormalities of systemic Ca2+ or phosphate levels (21, 22). Based on these and other expression studies, it has been proposed that mammalian STC1 may regulate intracellular Ca2+ pools rather than systemic Ca2+ levels.

To determine whether STC1 plays a crucial role during normal development and provide a model for studying the function of STC1 in vivo, we previously generated mice that were null for Stc1 (23). Surprisingly, these mice had no obvious phenotype, suggesting either that STC1 is not essential for normal growth or that STC2 can compensate for its absence. To distinguish between these possibilities, we have now generated Stc2-null mice and also double-Stc1/Stc2-null mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Targeting vector construction and generation of Stc2–/– mice
Genomic DNA encoding Stc2 was isolated from a mouse 129 SVJ{lambda} FIXII library (Stratagene, La Jolla, CA). Various inserts from positive {lambda}-isolates were then subcloned into vector pGEM5Zf+ (Promega, Madison, WI) for DNA sequencing. A 1.2-kb right targeting arm containing exon 3 was obtained by PCR of a {lambda}-clone with two primers A1440 (5'-ACCCAAGCTTGAAACAAACAAGCAAAAACA) and A1441 (5'-ACCCAAGCTTCTAGGGGCAGAGTGGAAAAT) containing a HindIII site (underlined) and inserted into the HindIII site of the vector ploxPneo-1 (23). A 2-kb left arm containing sequences upstream of exon 1 was obtained as a NheI-EcoRI fragment and inserted into the KpnI site of ploxPneo-1 after blunt ending. To enable negative selection of ES clones, a 2.8-kb pgk-herpesvirus thymidine kinase cassette (obtained from Dr. Frank Koentgen, Ozgene, Bentley DC, Western Australia, Australia) was inserted into the BamHI site. The final targeting construct, pAC521 (11.2 kb) was linearized with NotI and electroporated into R1 embryonic stem (ES) cells. The ES cells were subsequently cultured in knockout medium (Invitrogen, Carlsbad, CA) and selected using 300 µg G418 per milliliter and 1 µM ganciclovir (Roche, Castle Hill, Australia). Clones were isolated and grown to confluence in individual wells of 24-well tissue culture plates. Genomic DNA isolated from about 400 G418-resistant ES cell colonies was analyzed by Southern blot or PCR. One correctly targeted ES cell clone was injected into cultured BALB/c blastocysts followed by transfer into pseudopregnant foster mice. Chimeras obtained were subsequently mated to 129/SvJ females, and the resulting heterozygous animals were bred to generate wild-type, Stc2+/– and Stc2–/– animals for subsequent analyses.

Southern blot and PCR analyses
Genomic DNA isolated from ES cells or mouse tails were digested with various restriction enzymes, separated through a 0.8% agarose gel (Roche) with Tris-borate-EDTA buffer at pH 7.5, and transferred onto a BiodyneB membrane (Pall, Pensacola, FL) by capillary action in 0.4 M NaOH for 3–4 h. Two flanking DNA fragments, P1 and P2, outside the Stc2 targeting region, were used as hybridizing probes for genotyping. The probes were labeled with [{alpha}-32P]dCTP using the Gigaprime DNA labeling kit (GeneWorks Hindmarsh, South Australia, Australia). The P1 fragment was obtained by PCR from a region upstream of the Stc2 gene using primers A1283 (5'-CCAAAATGTACCCATCCACCC) and A1284 (5'-ACATCCACCCTAAGACTTGGAGG). The P2 fragment was generated by PCR from intron 3 of the Stc2 gene using primers A1281 (5'-TTTAATTGCTCCATCATCGT) and A1279 (5'-AACAGGGAATGGAGGGTTTC). To further confirm correct gene targeting, genomic DNA was PCR amplified using primer A1491 (5'-CCTACCGGTGGATGTGGAATGTG) from the Neo-cassette and A1492 (5'-AGTAGAGAAGGGGAAGGGGAGTC) from intron 3, outside the right targeting arm. All PCRs were carried out using the Expand high-fidelity system (Roche). After an initial denaturation step (94 C for 10 min), Taq polymerase was added and 35 cycles of PCR were then performed (94 C for 1 min, 63 C for 1 min, and 72 C for 2 min), ending at 72 C for 10 min.

mRNA expression and quantitation
For RT-PCR of Stc2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), primer pairs A1549 (exon 1; 5'-TGTGACCCTGGCTTTGGTGTTTG), A1550 (exon 4; 5'-CGTGGGAGGTCTCTGTATGTTGG), and A1505 (5'-ACCACAGTCCATGCCATCAC), A1506 (5'-TCCACCACCCTGTTGCTGTA) were used, respectively. To ascertain that no truncated Stc2 transcript was generated in the null cells, RT-PCR with primer pairs A1625 (exon 3, 5'-GCCCTGCGTCATAAATTTGG) and A1626 (exon 4, 5'-TCTGTTCACACTGAGCCTG) was used. One microgram of total RNA isolated from various tissues was treated with DNase I (Invitrogen), followed by reverse transcription (SuperScript III first-strand synthesis system; Invitrogen) according to the manufacturer’s instructions. Then 5 µl and 2 µl cDNA were used for PCR of Stc2 and GAPDH sequences, respectively. For Stc2 PCR, after an initial denaturation step (95 C for 10 min), Taq polymerase was added and the cDNA was amplified for 30 cycles (95 C for 50 sec, 60 C for 30 sec, and 72 C for 1 min). GAPDH cDNA was amplified for 20 cycles.

Hepatic IGF-I and acid labile subunit (ALS) mRNA levels were measured by quantitative real-time RT-PCR using published primers and conditions (24), with SYBR Green PCR master mix (Applied Biosystems, Warrington, UK) in a RotorGene-6000 thermal cycler (Corbett, Sydney, Australia). 18S rRNA was used as the reference gene for quantitation.

Generation of Stc1–/– Stc2–/– mice
We crossed the previously generated Stc1–/– mice (23) line L278nd (C57BL/6 background) with the Stc2–/– mice to generate Stc1+/–Stc2+/– double-heterozygous mice (mixed 129/SvJ/C57BL6). The double-heterozygous mice were then mated and the pups were genotyped by standard Southern blot analysis. To screen for Stc1 knockouts, genomic DNA was digested with NheI, Southern blotted, and probed with P3 as described previously (23).

To obtain sufficient numbers of Stc2–/– and Stc1–/– Stc2–/– mice for further phenotypic characterization, we bred Stc2–/– with Stc2–/– mice and Stc1–/– Stc2–/– with Stc1–/– Stc2–/– mice. This avoided the generation of large numbers of heterozygotes that would not have been analyzed. Because the Stc1–/– mice were previously shown to be grossly identical with wild-type mice, we did not include the former in most analyses to minimize animal usage.

All animal work was performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and was authorized by the institutional Animal Care and Ethics Committee.

Whole-animal muscle physiology
Three different types of muscle tests were carried out on 13-month-old male mice of wild-type, Stc2–/–, and Stc1–/– Stc2–/– genotypes. Six, 10, and nine mice were used, respectively.

Strength and fatigability tests.
The animals were placed with their forepaws on a metal rod covered in heat shrunk rubber with a diameter of 3 mm. The mice were required to pull themselves up to the top of the rod to pass the test. Muscle weakness was based on the average of passes of more than 15 attempts in a 3-min period (25).

Forelimb strength.
A computerized grip strength meter (Columbus Instruments, Columbus, OH) was used to measure the forelimb strength of a mouse holding a horizontal bar. When the animal was pulled away from the bar by its tail, a maximum force was registered when it released its grasp. The average force (newtons) was calculated from 10 measurements.

Open field test.
The Flex-Field photobeam activity system (San Diego Instruments, San Diego, CA) was used. This test involved placing a mouse in a 1 x 1 m open field box fitted with infrared sensors to monitor the number of ambulatory, rearing, and fine movements made over a 20 min period.

Histopathology, radiography, clinical chemistry, and body composition analysis
Mice were killed by CO2 inhalation, and necropsies were performed. Organs were fixed in 10% formalin, sectioned and stained, and examined by a veterinary pathologist. Blood chemistry determinations were carried out with a Vitros 5.1 device (OrthoClinical Diagnostics, Rochcester, NY) at the Children’s Hospital Westmead.

Eight-month-old male mice (n = 10–11) were measured for bone mineral content (milligrams) and bone mineral density (milligrams per square centimeter) using dual-energy x-ray absorptiometry (Lunar PIXImus2; General Electric Medical Systems, Bedford, UK) as described by the manufacturer. Skeletal radiography of whole animals was performed on a B7070 mammographic system (General Electric Medical Systems) after the animals were anesthetized with ip injections of ketamine/xylazine.

Major urinary protein (MUP) was analyzed by collecting urine from age-matched mice and separating the proteins in 2 µl urine by SDS-PAGE in a 12% gel. The gel was stained with Coomassie blue for visualization.

Total serum IGF-I levels were determined using a rat/mouse ELISA kit (Immunodiagnostic Systems, Boldon, UK) according to the manufacturer’s instructions. Each measurement was done in duplicate and samples were pretreated to remove interference from binding proteins.

Bone histology
Tibias from 3-, 5-, and 16-wk male and female mice were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 12 h and then decalcified in 0.34 M EDTA for 3 wk. Samples were then dehydrated with increasing concentrations of ethanol, infiltrated with xylene, and embedded in paraffin wax. Sections were cut at a thickness of 5 µm, stained with hematoxylin/eosin and safranin O, and counterstained with light green. Growth plate width, which is the distance separating the epiphyseal growth plate junction from the metaphyseal junction, was measured using an image-analysis system (BIOQUANT, Nashville, TN) coupled to a camera (QICAM, Scitech, Preston, Victoria, Australia).

Statistical analysis
All statistical analyses of data were performed with the unpaired Student’s t test, and for all experiments results were regarded as significant if P < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of Stc2 deleted mice
The Stc2 gene, like Stc1, contains four exons. Exon 1 encodes the 5' untranslated region and the first 50 amino acid residues of Stc2, exon 2 encodes 48 residues, exon 3 encodes 70 residues, and exon 4 encodes the last 128 residues plus the 3' untranslated region. The targeting construct for homologous recombination was designed to delete exons 1 and 2 (Fig. 1Go). After screening about 400 ES clones by Southern analysis or PCR, one ES clone (no. 144) was found to be targeted correctly. When genomic DNA of clone 144 was digested with restriction enzyme XbaI and hybridized with probe P1, a smaller band of 2.8 kb, indicative of correct targeting, was seen (Fig. 2AGo). To confirm this, clone 144 was digested with other restriction enzymes including EcoRI, HindIII or StuI, and hybridized with a different probe, P2. As predicted, smaller bands were seen with ES clone 144 but not incorrectly targeted clones such as no. 143 (Fig. 2BGo). The intensity of the smaller bands was weaker than the larger wild-type bands because the ES clone was not a pure clone at this stage. To further confirm that no. 144 is correctly targeted, PCR amplification of genomic DNA was done with the neomycin cassette-derived primer A1491 and A1492, which corresponds to genomic sequence outside the right targeting arm (Fig. 1Go). A product of 1.7 kb was obtained only with clone 144 and not with others such as no. 142 and 143 (Fig. 2CGo). Clone 144 was then used to generate Stc2 knockout mice.


Figure 1
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  		FIG. 1. Mouse Stc2 locus, targeting vector, and targeted allele. The upper line shows a map of the Stc2 locus with the four exons defined by shaded boxes. The XbaI (X), EcoRI (E), HindIII (H), StuI (S), and SacI (C) restriction sites are indicated. For clarity, not all sites for each restriction enzyme are shown. The 5' (P1) and 3' (P2) external probes used for Southern analyses are shown. The middle line represents the targeting plasmid. Neo, Neomycin phosphotransferase expression cassette; TK, herpesvirus thymidine kinase expression cassette. The bottom line shows the map of the correctly targeted Stc2locus. A1491 and A1492 represent the two PCR primers designed to confirm correct targeting. A1625 and A1626 are RT-PCR primers derived from exons 3 and 4, respectively.

 

Figure 2
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  		FIG. 2. Southern blot and PCR analysis of genomic DNA isolated from various ES clones. A, DNA was digested with XbaI and hybridized with radioactively labeled probe P1. A smaller 2.8-kb band indicative of correct targeting is seen with clone 144. B, DNA was digested with EcoRI, HindIII, or StuI and then hybridized with another probe P2. A faint smaller band is seen with each restriction digest of clone 144, indicating correct targeting. C, PCR of genomic DNA with primer A1491 and A1492 produced the 1.7-kb band expected only if the correct targeting event had occurred.

 
Analysis of offspring from Stc2 heterozygous breeding pairs
Mice that carried one copy of the deleted gene were interbred to generate pups that were +/+, +/–, and –/– for Stc2 as determined by Southern blot analysis of DNA extracted from tail snips (Fig. 3AGo). From a total of 187 pups born, 51 were +/+, 91 were+/–, and 45 were –/–. This is close to the predicted Mendelian ratios of 1:2:1 expected for nondeleterious alleles. Of the 45 null mice, 26 were female and 19 were male. Thus, the Stc2 knockout pups were no less viable than their wild-type littermates, and there was no obvious gender bias.


Figure 3
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  		FIG. 3. Analysis of wild-type (+/+), heterozygous (+/–), and homozygous Stc2 mutant (–/–) mice. A, Southern blot of tail DNA from pups digested with SacI and hybridized with probe P1. The predicted mt (mutant) and wt (wild-type) bands are indicated for each genotype. B, RT-PCR of Stc2 mRNA expression from total RNA isolated from kidney and heart of wild-type and Stc2–/– mice. RT-PCR of GAPDH mRNA was used as an internal control. Lower panel, Replicate samples amplified with primers specific for exon 3 (A1625) and exon 4 (A1626). No products were seen in Stc2–/– tissues indicating the absence of any truncated Stc2 transcripts.

 
To confirm gene deletion, RT-PCR analysis was performed on RNA isolated from heart and kidney, organs that are known to produce abundant Stc2 mRNA (12). An Stc2-specific band was seen in wild-type tissues as expected but not in Stc2–/– tissues, indicating that deletion had indeed occurred (Fig. 3BGo). To exclude the remote possibility that a truncated Stc2 transcript containing the remaining exons 3 and 4 could have been generated in the Stc2–/– tissues, RT-PCR using primer A1625 (exon 3) and A1626 (exon 4) was carried out. No band was seen in the Stc2–/– tissues (Fig. 3BGo). The integrity of the RNA samples was demonstrated by the detection of the housekeeping GAPDH transcript.

We monitored the postnatal body weight of these pups and found that Stc2–/– female and male mice were consistently heavier (about 15%) and larger than wild type. The difference became apparent only after weaning, which occurred at d 21 (Fig. 4Go). The males were weighed for 50 wk, and the difference in weight was maintained throughout this time. Some of the females from this cohort were used after 16 wk for breeding; hence, weighing was stopped at this time point. Female pups from another cohort were weighed for 44 wk, and at this point the average weight of wild-type was 27.03 ± 2.52 g (n = 9), and Stc2–/– was 32.29 ± 1.43 g (n = 7; P = 0.002), showing that weight differences were maintained. The weight of the Stc2+/– heterozygotes was intermediate between those for wild type and Stc2–/– at all time points examined (data not shown).


Figure 4
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  		FIG. 4. Postnatal body weight of wild-type (+/+) and homozygous Stc2 null (–/–) mice. The weights of female littermates were measured weekly for 7 wk and then at 2-wk intervals. Similar intervals were followed for males until the last three points, which were at wk 22, 26, and 50. Error bars indicate SD of the mean. The weight of +/+ and –/– mice was significantly different (P < 0.001) from wk 4 onward. Growth curves of heterozygotes were intermediate between those for +/+ and –/– and are not shown for clarity.

 
Generation of Stc1–/– Stc2–/– mice
Having observed that Stc1 deleted mice were essentially normal, we previously suggested that Stc2 may have compensated for the absence of Stc1 (23). We therefore crossed the Stc1–/– mice with Stc2–/– to generate Stc1+/–Stc2+/– double heterozygous mice. The double heterozygous mice were then mated and the pups were genotyped by standard Southern blot analysis (Fig. 5Go).


Figure 5
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  		FIG. 5. Southern blot analyses of pups generated from the breeding of Stc1+/–Stc2+/– mice. A, Tail DNA was digested with NheI and hybridized with Stc1 probe P3. B, Duplicate DNA samples were digested with SacI and hybridized with Stc2 probe P1. The predicted MT (mutant) and WT (wild type) bands are indicated for each genotype.

 
From a total of 127 pups, nine (three females and six males) were Stc1–/– Stc2–/– double knockouts, which is close to the ratio of 16:1 expected from Mendel’s second law (independent assortment), indicating that the double knockouts are not embryonic lethal. The double null mice were found to have growth rates that were similar to the Stc2–/– mice, showing that the loss of Stc1–/– did not exacerbate the Stc2–/– phenotype (data not shown).

Examination of body weight and histomorphology
The weight of female and male wild-type, Stc1–/–, Stc2–/–, and Stc1–/– Stc2–/– mice were compared at 4 months of age in two separate cohorts of mice born a month apart (Table 1Go). The weight of wild-type and Stc1–/– female and male mice was significantly lower than both the Stc2–/– and the Stc1–/– Stc2–/–, whereas the weight of the double Stc1–/– Stc2–/– was only slightly greater than the Stc2–/– mice. Mice were also weighed from the time of weaning until adulthood, and the growth rates of the Stc2–/– and the Stc1–/– Stc2–/– were both similar and greater than wild-type and Stc1–/– mice (Fig. 4Go and data not shown).


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  		TABLE 1. Body weight at age of 4 months for two cohorts of mice born a month apart

 
Histological examination by a veterinary pathologist of the lung, heart, liver, kidney, pancreas, spleen, thymus, ovary, skeletal muscle, and testis from Stc2–/– and Stc1–/– Stc2–/– mice did not reveal overt differences, compared with wild-type mice (data not shown).

Examination of bones
Because STC1 and STC2 have been suggested to have a role in bone development (5, 26, 27, 28), skeletal x-ray analyses on adult wild-type, Stc2–/– and the Stc1–/– Stc2–/– mice were performed. No skeletal abnormalities were seen (data not shown). Whole-body bone mineral content (wild type, 654.2 ± 50.1 mg; Stc2–/–, 674.0 ± 29.0; and Stc1–/– Stc2–/–, 702.0 ± 82.0) and density (wild type, 64.8 ± 3.4 mg/cm2; Stc2–/–, 67.0 ± 4.0; and Stc1–/– Stc2–/–, 67.0 ± 4.0) were measured using dual-energy x-ray absorptiometry, and no significant differences were seen.

To assess whether the larger overall size of the Stc2–/– and Stc1–/– Stc2–/– mice was due to an increase in gross appendicular growth, the tibias of 3-, 5-, and 16-wk old mice were collected. Analysis of the growth plate width did not reveal any significant differences between genotypes (data not shown). It has been reported that STC2 overexpressing transgenic mice exhibited delayed cranial suture formation (29). No difference in cranial patency was seen when Stc2–/– and Stc1–/– Stc2–/– mice were compared with wild-type mice 3 and 5 wk of age (data not shown).

Organ allometry
To determine the reason for the increased weight of Stc2–/– and the Stc1–/– Stc2–/– mice, we performed organ allometry studies on 6-month-old male mice (Table 2Go). The data showed that most of the increased body weight in the Stc2–/– and the Stc1–/– Stc2–/– mice resulted from an increase in the weight of the major organs. Interestingly, there was a decrease in the weight of the testes. Body length was also increased slightly for Stc2–/– (in other cohorts, the body length of both Stc2–/– and Stc1–/– Stc2–/– was greater than wild type). The wet organ weights were also calculated as a percentage of the body weight (Table 2Go). The normalized data revealed that some of the major organs of the Stc2–/– and Stc1–/– Stc2–/– mice were significantly larger, suggesting organomegaly. For reasons that are unclear, the brain weight of Stc1–/– Stc2–/– mice was significantly greater than in Stc2–/–, but the weights of other tissues in mice of these genotypes were similar.


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  		TABLE 2. Wet organ weights and organ-to-body weight percentage in 6-month-old male mice

 
Whole-animal muscle performance
In three different types of muscle physiological analyses, no consistent differences were seen between wild-type and Stc2–/– or Stc1–/– Stc2–/– mice. The analyses were as follows: a strength and fatigability test (wild type, 11.3 ± 4.9 passes per 15 attempts; Stc2–/–, 13.3 ± 3.0; and Stc1–/– Stc2–/–, 7.3 ± 6.0), forearm strength measurement (wild type, 1.53 ± 0.15 N; Stc2–/–, 1.49 ± 0.29; and Stc1–/– Stc2–/–, 1.46 ± 0.26), and open field activity were carried out (wild type, 1421 ± 471 movements per 20 min; Stc2–/–, 1897 ± 600; and Stc1–/– Stc2–/–, 1469 ± 666).

Reproductive performance
The ovary and testis have been reported to have high levels of Stc1 and Stc2 mRNA expression (12). To determine whether fertility was affected, six to seven breeding pairs of each genotype were set up for 7 wk. During that period the Stc1–/– Stc2–/– mice produced similar number of litters to those of wild-type or single Stc2 null animals, and the litters were of similar sizes (Table 3Go). This suggested that the complete removal of Stc2 or even of both Stc genes did not substantially affect the fertility and fecundity of female or male mice.


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  		TABLE 3. Progeny from different breeding pairs kept together for 7 wk

 
Clinical chemistry and endocrine measurements
Serum concentrations of ionized calcium and phosphate were very similar in the Stc2–/–, Stc1–/– Stc2–/– and wild-type mice. The mean alkaline phosphatase levels in Stc2–/– were 144% that of wild-type (P = 0.003). Other clinical chemistry measurements revealed no evidence of liver or renal disease (Table 4Go).


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  		TABLE 4. Clinical biochemistry from serum analysis of 8-month-old mice

 
Because the most obvious phenotype seen in these knockouts was growth enhancement, we decided to investigate whether the primary growth promoting trophic factors, GH and IGF-I, were affected. It has been shown that the production of MUP is dependent on a normal pulsatile GH secretion pattern and signaling pathway (30, 31). A comparison of MUP levels by SDS-PAGE between adult Stc2–/– mice, Stc1–/– Stc2–/– mice, and wild type did not show any differences, suggesting that GH signaling is not affected (data not shown).

We also investigated mRNA expression of GH-dependent genes such as IGF-I and ALS in the liver. The liver is the principal organ that produces circulating IGF-I and ALS, and ALS is required for the stability of IGF-I. Using quantitative realtime RT-PCR on duplicate samples of total RNA isolated from livers of 4-month-old male and female mice of wild-type (n = 9), Stc2–/– (n = 7), and Stc1–/– Stc2–/– (n = 6) genotypes, we found that the expression levels for both genes were similar for wild-type (IGF-I, 1.14 ± 0.26; ALS, 1.26 ± 0.29) and Stc2–/– mice (IGF-I, 1.13 ± 0.49; ALS, 1.28 ± 0.58). mRNA levels were slightly lower for Stc1–/– Stc2–/– mice (IGF-I, 0.49 ± 0.16; ALS, 0.52 ± 0.13).

Because circulating IGF-I plays a major role in mammalian growth and mediates most of the actions of GH, we also measured total serum IGF-I from nine six-month-old male mice of each genotype. No significant difference in circulating IGF-I was found in Stc2–/– (507 ± 144 ng/ml) and Stc1–/– Stc2–/– mice (576 ± 126 ng/ml), compared with wild type (580 ± 63 ng/ml). This is consistent with the MUP excretion, and the IGF-I and ALS mRNA expression analyses, which were essentially unchanged.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We generated a mouse line with the Stc2 gene deleted. The deletion was confirmed by Southern blot analyses using probes flanking both sides of the Stc2 gene. RT-PCR confirmed the absence of Stc2 transcripts in the Stc2–/– tissues. We also tried to detect endogenous Stc2 protein using Western blot and immunohistochemical analyses. However, no specific signals were detected with antibodies that were commercially available or generated in our own laboratory (data not shown).

The most obvious phenotype seen with the loss of Stc2 and of both Stc genes is that the mice were 15–20% heavier from the time of weaning onward. This was especially noticeable with male mice. The increased body weight in these mice resulted from an increase in the weight of most viscera.

The GH/IGF-I signaling pathway is a major determinant of postnatal growth, with IGF-I mediating most of the growth-promoting actions of GH (32). MUP levels and hepatic IGF-I and ALS gene expression levels were essentially unaltered, suggesting that GH is being expressed at normal levels and with a normal pulsatile pattern. This is supported by the finding that total circulating IGF-I levels in Stc2–/– mice, and Stc1–/– Stc2–/– mice, compared with wild type were also similar.

Our studies suggest that the negative effect of Stc2 on growth is independent of an intact GH/IGF-I signaling pathway. Similar conclusions were drawn from studies of transgenic mice that overexpressed human STC1 or STC2 (21, 22, 29).

In some regards, the phenotype of Stc2–/– mice is a milder, converse version of what has been reported for transgenic mice overexpressing human STC2, which exhibited substantial growth reduction resulting in a 45% decrease in size, compared with wild-type littermates (29), although in the transgenics, the altered growth occurred both pre- and postnatally. Stc2 mRNA was not detectable in mouse embryos from embryonic day 10.5 to embryonic day 18.5, so it is not surprising that the difference in growth of Stc2–/– mice appeared only postnatally (29). The transgenics had testicular organomegaly and a reduction in skeletal muscle mass, whereas the Stc2–/– mice had decreased testicular size and no significant difference in muscle size or strength. The authors of the transgenic study concluded that STC2 can act as a potent growth inhibitor, although the conclusion required the caveats that this was an overexpression study and that it was the human STC2 gene that was expressed. Our results suggest that the conclusion was nevertheless correct.

Stc1 expression has been reported in a variety of tissues during early mouse development, especially the skeletal and muscular tissues (6, 7, 33). Transgenic mice that overexpressed human STC1 were also dwarfed and had decreased bone length (21, 22). It was therefore unexpected to find that Stc1–/– mice were born without any overt abnormalities and grew at a rate that was indistinguishable from wild-type mice (23). Compensation by Stc2–/– was proposed as a possible explanation, but this now seems very unlikely because the Stc1–/– Stc2–/– mice had a similar phenotype to the Stc2–/– mice. If compensation occurs, it might be expected that tissues such as the heart, skeletal muscle, kidney, liver, testis, and ovary that were reported to express both genes (4, 12, 29, 34) would be most affected in the double knockout mice. Apart from the alterations in size, the gross and microscopic anatomy of these organs was indistinguishable from wild type, and we did not detect any abnormalities of function in activity levels, muscle strength, renal and hepatic function, and reproductive capacity.

The fecundity of Stc1–/– Stc2–/– mice was of particular interest. In mouse (and rat) uterus, expression of Stc1 and Stc2 was induced at implantation sites by the implanting blastocyst (35, 36). The ovary is the site of greatest Stc1 expression in adult mice, and the presence of a nursing litter results in increased Stc1 expression in lactating mice, suggesting that Stc1 is involved in regulation of ovarian function and lactation (3, 37). Moreover, Stc1 is expressed in mammary tissue (38, 39). However, wild-type, Stc2–/– and Stc1–/– Stc2–/– breeding pairs kept together for 7 wk produced similar numbers of litters and similar litter sizes. The Stc2–/– mice and Stc1–/– Stc2–/– mice did not have gross defects in reproductive ability (Table 3Go), and weight at weaning was unaffected. Although these data do not completely exclude the possibility, they do not suggest that deleting the Stc genes has had any effect on reproductive capacity and lactation.

STC1 in fish has a major role in calcium and phosphate homeostasis (40). Our finding that the absence of mouse Stc1 and/or Stc2 genes has no effect on serum calcium, and phosphate indicates it is unlikely that the mammalian stanniocalcins play this role under normal physiological conditions. This conclusion is consistent with results obtained with overexpression of STC1 or STC2 in transgenic mice (21, 29).

Our data indicate that under normal laboratory housing conditions, mice lacking Stc2 grew slightly heavier and larger than wild type, and the additional loss of Stc1 did not exacerbate the phenotype. However, it is clear from other studies that STC1 and STC2 production can be induced by stresses including inflammation, hypoxia, and exposure to xenotoxic agents that cause accumulation of misfolded proteins (41, 42, 43, 44). There are also many studies suggesting altered expression of STCs may have a role in cancer (8, 45, 46, 47, 48, 49). Additional aspects of the Stc2–/– and Stc1–/– Stc2–/– phenotype may emerge when the mice are subjected to an appropriate stressor. The availability of the Stc1–/–, Stc2–/– and Stc1–/– Stc2–/– mice now provides a model for investigating the role of these genes in cancer and stress responses.


   Acknowledgments
 
We thank Dr. Irma Villaflor and the other members of the Bioservices Unit of Children’s Medical Research Institute for their help in managing the animals and Ms. Elizabeth Collins for help with the manuscript preparation.


   Footnotes
 
This work was supported by project grants and research fellowships from the National Health and Medical Research Council (NHMRC; Australia) (to R.R.R. and P.W.G.). E.C.H. was supported by NHMRC Project Grant 321701.

Disclosure Statement: D.G.L. consults for Novartis. The other authors have no disclosures.

First Published Online February 7, 2008

Abbreviations: ALS, Acid labile subunit; ES, embryonic stem; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MUP, major urinary protein; STC, stanniocalcin.

Received September 4, 2007.

Accepted for publication January 28, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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