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Endocrinology Vol. 143, No. 9 3681-3690
Copyright © 2002 by The Endocrine Society

ARTICLE

Stanniocalcin 1 Alters Muscle and Bone Structure and Function in Transgenic Mice

Ellen H. Filvaroff, Susan Guillet, Constance Zlot, Min Bao, Gladys Ingle, Hope Steinmetz, John Hoeffel, Stuart Bunting, Jed Ross, Richard A. D. Carano, Lyn Powell-Braxton, Graham F. Wagner, Renee Eckert, Mary E. Gerritsen and Dorothy M. French

Departments of Molecular Oncology (E.H.F., M.B.), Pathology (S.G., D.M.F.), Physiology (C.Z., G.I., H.S., J.H., S.B., J.R., R.A.D.C., L.P.-B., M.E.G.), and Safety Assessment (R.E.), Genentech Inc., South San Francisco, California 94080; and Department of Physiology (G.F.W.), Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada

Address all correspondence and requests for reprints to: Dr. Ellen Filvaroff, Department of Molecular Oncology, Genentech, Inc., MS 72B, 1 DNA Way, South San Francisco, California 94080. E-mail: filvarof{at}gene.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fish stanniocalcin (STC) inhibits uptake of calcium and stimulates phosphate reabsorption. To determine the role of the highly homologous mammalian protein, STC-1, we created and characterized transgenic mice that express STC-1 under control of a muscle-specific promoter. STC-1 transgenic mice were smaller than wild-type littermates and had normal growth plate cartilage morphology but increased cartilage matrix synthesis. In STC-1 mice, the rate of bone formation, but not bone mineralization, was decreased. Increased cortical bone thickness and changes in trabeculae number, density, and thickness in STC-1 mice indicated a concomitant suppression of osteoclast activity, which was supported by microcomputed tomography analyses and histochemistry. Skeletal muscles were disproportionately small and showed altered function and response to injury in STC-1 mice. Electron microscopy indicated that muscle mitochondria were dramatically enlarged in STC-1 mice. These changes in STC-1 mice could not be explained by deficits in blood vessel formation, as vascularity in organs and skeletal tissues was increased as was induction of vascularity in response to femoral artery ligation. Our results indicate that STC-1 can affect calcium homeostasis, bone and muscle mass and structure, and angiogenesis through effects on osteoblasts, osteoclasts, myoblasts/myocytes, and endothelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STANNIOCALCIN (STC), A SECRETED protein, was first discovered in fish, where it regulates calcium and phosphate homeostasis (1, 2, 3). More specifically, STC in fish inhibits uptake of calcium through the gills and intestines and stimulates phosphate reabsorption (1, 2, 3). The mammalian homolog of fish STC, human STC-1, shares approximately 80% amino acid similarity with fish STC (4, 5). As in fish, mammalian STC-1 controls phosphate transport across epithelia in the gut and kidney (6, 7). Mammalian STC-1 is expressed in many tissues (4, 5, 8). However, very little is known about mammalian STC-1 activity, and hypotheses about its function have been based largely on studies of the pattern of STC-1 expression during growth and development.

Recent data suggest that STC-1 plays a role in the nervous system. STC-1 is constitutively expressed in terminally differentiated neurons in the central nervous system (9), and culture of a human neural-crest-derived cell line, Paju, in hypercalcemic conditions induces STC-1 expression (10). STC-1 is up-regulated in neurons around infarcts in human and rat brains (10) and in hypoxic human glioblastoma cells (11). Because calcium mobilization and influx may mediate, at least in part, toxicity after ischemia, STC-1 may protect cells from hypoxic/ischemic insult (10). In fact, STC-1 can protect Paju cells after ischemic challenge and increase cellular uptake of inorganic phosphate (10). Expression of STC in the epithelium of the choroid plexus suggests a role for STC in the regulation of calcium and phosphate levels in cerebrospinal fluid and blood (12).

STC-1 has also been implicated in endothelial activation and angiogenesis. STC-1 is expressed at high levels in highly vascularized tissues such as the heart, kidney, and lung as well as the ovary, and it is up-regulated during gestation and lactation (4, 5, 13, 14). Consistent with its high, ubiquitous expression, STC-1 may also play a role in mesenchymal-epithelial interactions (15). STC-1 mRNA is highly up-regulated in endothelial cells undergoing differentiation into tube-like structures, and STC-1 mRNA is localized to blood vessels in tumors (16). In fact, STC-1 is expressed in 95% of cancer cell lines tested and in many types of tumors (17). Furthermore, STC-1 mRNA levels are higher in hepatocellular carcinoma and colorectal tumors than in normal tissues (17).

Based on its presence in chondrocytes and muscle cells, a role for STC-1 protein in chondrogenic and myogenic differentiation has been suggested (18). More specifically, STC-1 protein is present in developing bones and muscles of the mouse fetus. During bone development, STC-1 is found in chondroprogenitors, chondrocytes and osteoblasts (18, 19, 20). Just before ossification during intramembranous bone formation, STC-1 is present in the mesenchyme. During muscle development, STC-1 is found in heart myocardiocytes and at all stages of skeletal myoblast differentiation into myotubes (18). Recent data suggest that, as in other tissues (15), STC-1 signals between adjacent cell types in the skeleton (20).

In spite of a number of correlative studies, the exact function of STC-1 remains unknown. To explore the function of STC-1 in mammals, transgenic mice expressing human STC-1 under control of a muscle-specific promoter were generated. These transgenic mice expressed high levels of STC-1 in skeletal muscle and were significantly smaller than their wild-type (WT) littermates. Decreases in body weight were significant starting as early as 5 d following birth, and body weight differences were maintained throughout the life of the animals. Detailed analyses revealed substantial changes in the structure and function of the musculoskeletal system. Given the high, ubiquitous expression of STC-1 (4, 5, 8), our results provide insight into possible mechanism(s) of action of mammalian STC-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of STC-1 transgenic mice
All protocols were approved by the Institutional Animal Care and Use Committee. The gene for human STC-1 (hSTC-1) was cloned downstream of the rat myosin light chain 2 promoter sequence (21) and was followed by the splice donor/acceptor sites (between exons 4 and 5) of the human GH gene (22). Transgenic mice were made using this construct as per standard procedures (23). Pups were genotyped at 9 d of age by PCR of mouse tail DNA (QIAGEN, Santa Clarita, CA). Transgene expression was assessed in tissue (muscle, kidney, liver, intestine) biopsies taken at 4 wk of age by RT-PCR or Northern blot analysis (QIAGEN) of mRNA using hSTC-1 primers for exons 1–4.

All analyses were performed on adult mice except where noted in the text. Statistical analysis of differences between transgenic (STC-1) and WT mice was determined by a Student’s t test with a value of P < 0.05 considered to be statistically significant and P < 0.01 as highly significant.

Whole animal physiology
All animals were kept on a 12-h light cycle and fed standard rodent chow. Mice were weaned and single housed in microisolater cages at 3 wk of age. Six founders (three males, three females) were examined. Once the phenotype was determined to be consistent among founders, two founders with highest human STC-1 expression, as determined by Taqman analysis, were bred to establish lines. Data presented are from offspring from one founder line unless otherwise indicated. Body weights, evaluated every other day from 5–42 d of age for male and female mice (n = 10 for each group of transgenic or WT mice) were used to establish comprehensive growth curves. Organs—brain, heart, left kidney, right kidney, liver, lung, spleen, and thymus—were weighed at 14 wk of age.

Clinical chemistry was performed on serum samples taken at 50 d of age (n = 10 mice of each genotype). Pooled sera from male and female mice (n = 5/group) were sent to AniLytics, Inc. (Gaithersburg, MD) for analysis by immunoassay of the following hormones: calcitonin, thyroid hormone (T4), and GH. At 10 wk of age, founders were housed in metabolic chambers to assess food intake and oxygen consumption followed by a glucose tolerance test.

Whole body radiographs using a Faxitron x-ray (MX20, IDL software) were taken at 25-kV/120-sec exposures using Kodak X-Omat TL film (Eastman Kodak Co., Rochester, NY). Film was developed in a Kodak processor and scanned into Adobe Photoshop 6.0 (Adobe Systems Inc., San Jose, CA). Bone (femur and tibia) lengths were determined by manual measurements from planar x-rays.

Oxygen consumption was measured in a Columbus Instruments Oxymax open circuit calorimeter (Columbus, OH).

Histology
The following tissues were collected and fixed overnight in 10% neutral buffered formalin: skeletal muscle, ovary, testis, skin/mammary gland, lymph node, liver, intestines, pancreas, lungs, spleen, kidney, heart, aorta, thymus, urinary bladder, adrenal gland, pituitary gland, brain, and eye. After fixation, tissues were embedded in paraffin, sectioned at 5 µm, and stained with hemotoxylin and eosin or alcian blue (femurs). Femurs from a separate group of transgenic and WT mice were fixed in 70% ethanol, embedded in methyl-methacrylate, and stained using the Von Kossa method.

For osteoclast detection using histochemical staining for tartrate-resistant acid phosphatase (TRAP) activity, skulls were fixed in 2% paraformaldehyde overnight, equilibrated in methanol, and then TRAP stained using the leukocyte acid phosphatase kit (Sigma, St. Louis, MO).

Histomorphometry
For kinetic analyses, 6-wk-old STC-1 transgenic and WT mice were given ip injections of 2 mg/kg calcein, followed by similar injections 5 d later. Femur and tibia, harvested 2 d after the second injection, were dissected and fixed in 70% ethanol. Bones were embedded in the same orientation in methyl-methacrylate, sectioned at 10 µm, and viewed under UV light. Mineral apposition rate was determined following the recommended nomenclature (24).

Tissue processing for EM
Tissues were fixed in 1/2 Karnovsky’s (2% paraformaldehyde; 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4) and then washed in 0.1 M sodium cacodylate (pH 7.4), 2 x 15 min before being postfixed in 1% aqueous osmium tetroxide for 2 h at room temperature. Following washing in double-distilled H2O for 2 x 15 min, the samples were dehydrated through an EtOH series: 50%, 70%, 90%, for 15 min each, and then 100% 2 x 15 min, followed by propylene oxide for 2 x 15 min. Samples were infiltrated with Eponate 12 (Ted Pella, Redding, CA) first with 1:1 propylene oxide:Eponate 12 overnight, followed by 100% Eponate 12 for 8 h, and then transferred to fresh resin and polymerized in a 60 C oven overnight and ultrathin (80 nm) sections were cut. Sections were stained with uranyl acetate and lead citrate and observed on a Philips CM12 transmission electron microscope. Images were captured with a Gatan Retractable MultiScan Camera using Digital Micrograph software.

Measurement of cartilage matrix synthesis
Assays of cartilage matrix synthesis using patellae were performed essentially as previously described (25, 26). Briefly, patellae of mice were dissected away from surrounding soft tissue and incubated overnight in media [serum-free low glucose 50:50 DMEM:F12 media with 0.1% BSA, 100 U/ml penicillin/streptomycin (Life Technologies, Inc., Gaithersburg, MD), 2 mM L-glutamine, 1x GHT, 0.1 mM MEM sodium pyruvate (Life Technologies, Inc.), 20 µg/ml gentamicin (Life Technologies, Inc.), 1.25 mg/liter Amphotericin B, 10 µg/ml] with 35S-sulfur (30 µCi/ml). Patellae were then washed three times with PBS and fixed overnight in 10% formalin, followed by decalcification of the underlying bone with 5% formic acid. Cartilage, dissected away from the underlying bone, was dried, weighed, placed in 500 µl of a tissue solubilizer (Solvable, Packard Instrument Co., Meriden, CT), and incubated at 60 C for 1.5 h. Scintillation fluid designed for concentrated alkaline and salt solutions (HIONIC-fluor, Packard Instrument Co.) was added (10 ml) to each tube and mixed thoroughly and [35S] quantified using a scintillation counter. Levels of proteoglycan synthesis are reported as average cpm of patellae from at least five mice per group.

Microcomputed tomography (µCT)
To quantify trabecular and cortical structure, isolated femurs (n = 5/genotype) were imaged by a SCANCO Medical (Basserdorf, Switzerland) µCT40 microimaging system at 50 kV and 80 µAmp. Contiguous axial image slices were obtained with an isotropic voxel size of 16 µm. System calibration was performed with a hydroxyapatite phantom of known density (2.91 g/cm3). The femoral cortical bone volume, total volume, and density were determined by application of an automated image segmentation algorithm constructed from the Analyze (AnalyzeDirect Inc., Lenexa, KS) image analysis software libraries. The algorithm employs histogram and morphological filtering (27) to extract the cortex volume. A cortical bone threshold (0.77 g hydroxyapatite/cm3), determined by histogram analysis of the data, was applied to extract potential cortex voxels found within the image data. A series of morphological operations (erode, open, conditional dilate, and close) contained within Analyze, were then applied to link cortex voxels and remove voxels of similar density found within regions of trabecular bone. The performance of the algorithm was evaluated by comparison of automated and manual segmentation results for an independent group of mouse bone samples. The automated estimates of cortex volume (r2 = 0.94), total volume (r2 = 0.99), and density (r2 = 0.96) were highly correlated (P < 0.01) with manual estimates (data not shown). Morphometric analysis of the trabecular bone within the femur was performed with the SCANCO Medical (Basserdorf, Switzerland) µCT40 evaluation software. A volume of interest (VOI) was manually defined, dorsal to the patellar grove, that contained only secondary trabecular bone and marrow space. Cortical bone was excluded by placement of the VOI boundaries within the inner boundary of the cortical bone. Before image segmentation, a constrained three-dimensional (3D) Gaussian low-pass filter was applied to image data for noise suppression. A global threshold (0.49 g hydroxyapatite/cm3) was applied to extract a binarized trabecular structure from the VOI. The trabecular segmentation threshold was chosen by visual inspection of segmentation results from a representative subset of samples. Trabecular structural characteristics were quantified by direct 3D morphometric analysis (28). Morphometric analysis by µCT has been shown to correlate with those from histomorphometry (29). Degree of anisotropy (DA) was quantitated to indicate trabecular organization. DA of trabecular bone is defined as the ratio of the maximum mean intercept length (MIL) to the minimum MIL, where MIL is the mean length between marrow/trabecular bone interfaces along a given direction (30).

Muscle function analyses
A flow-dependent analysis of muscle function was performed using a modification of our previously developed method (31). Briefly, animals were anesthetized using 2.5% isoflurane in oxygen, and maintained at 37 C using a heat lamp. The right leg was immobilized and the third right metatarsal was sutured to a Biopac TSD 105A force transducer using 4-0 silk. Two Grass E2 platinum subdermal needle electrodes were placed through the gastrocnemius at a distance of 5 mm. A 5-V DC square wave with duration of 5 msec and an interval of 500 msec was passed through the electrodes using a Biopac STIM100C stimulator and STIMISOC isolation unit. The muscle was stimulated for 10 min, rested for 10 min, and stimulated for 10 min. Tension from the force transducer was collected and plotted using a Biopac UM100A (Biopac Systems, Inc., Santa Barbara, CA) with Acknowledge software.

Platelet-endothelial cell adhesion molecule (PECAM) measurements after femoral artery ligation
The femoral artery was isolated at the level of the inguinal ligament and ligated with 7-0 silk suture (Ethicon, Somerville, NJ). The wound was closed with 4-0 silk suture (Look) and a single 7.5-mm Michel wound clip (California Surgical, Hayward, CA). Animals recovered on a warm water heating pad until ambulatory. Anti-PECAM-1, i.e. rat antimouse PECAM-1, IgG2a (PharMingen Inc., San Diego, CA) clone MEC13.3, labeled with 125I (DuPont NEN Life Science Products, Boston, MA; NEZ-033A), specifically binds a membrane-bound molecule on endothelial cells of mouse vessels (32). A nonspecific isotype control antibody (rat antimouse CD35, IgG2a (PharMingen Inc.), clone 8C12) labeled with 131I (DuPont NEN Life Science Products, NEZ-035A) was used to correct for any nonspecific antibody binding, vascular leakage or any blood left in the tissue. All antibodies were iodinated using the iodogen method in a ratio of 1 µg of antibody to 1 µCi of either 125I or 131I (33).

To measure PECAM-1 binding, a mixture of 125I PECAM-1 monoclonal antibody (mAb) (10 µg) and 131I nonbinding mAb (equivalent to 500,000 cpm) was diluted with PBS to a volume of 200 µl. Starting radioactivity was counted in a 2-µl sample using a Wallac Wizard {gamma}-counter (model 1480, Perkin-Elmer, Gaithersburg, MD). Thirty micrograms of cold PECAM-1 mAb were added to the solution. The mixture was injected through the jugular vein catheter and allowed to circulate for 5 min (34) after which a blood sample was obtained from the carotid catheter to measure circulating radiolabeled antibody levels. The animal was then exsanguinated by perfusion with bicarbonate-buffered saline through the jugular catheter (6 ml) with simultaneous blood withdrawal from the carotid catheter. Bicarbonate-buffered saline was then perfused through the carotid catheter (15 ml) after severing the inferior vena cava at the thoracic level. Entire organs and muscles were collected, weighed, and the radioactivity was counted on the {gamma}-counter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Creation of STC transgenic mice
Two (of six) male founders with highest expression of hSTC-1 in muscle but not other tissues (data not shown) were used to establish independent lines. Because STC-1 is a secreted protein, systemic levels of STC-1 were determined in STC-1 transgenic mice (n = 30) and age-matched, wild-type (WT) mice (n = 33) using an RIA (35). Serum levels of STC-1 in trangenic mice ranged from 15–35 ng/ml with a mean value of 23.48 ± 0.78 ng/ml. In contrast, STC-1 levels in WT mice were just within assay detection limits (0.2 ng/ml), ranging from 0.3–0.8 ng/ml, with a mean value of 0.47 ± 0.02 ng/ml. No significant differences in STC-1 mRNA in muscle or serum STC-1 levels between male and female mice were detected. Thus, overexpression of STC-1 in muscle was associated with elevated systemic levels of STC-1 protein.

Growth retardation in STC transgenic mice
All six STC-1 founder mice were approximately 25% smaller than WT littermates (Fig. 1Go). Because growth retardation was identified in multiple founders (n = 6), the phenotype of the mice was not likely due to integration of the transgene at a locus required for normal growth. Comprehensive growth curves indicated that mice hemizygous for STC-1 were significantly smaller than WT littermates by 5 d of age for the males (Fig. 1AGo) and 14 d of age for the females (data not shown). STC-1 transgenic mice were viable and fertile. Blood pressure, as determined by carotid cannulation, was within normal limits.



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  		Figure 1. Size of STC transgenic (STC) mice relative to WT littermates. A, Growth curves of male mice (n = 10/group) from 5–42 d of age. B, X-ray analysis of the skeleton of representative male transgenic (STC) or WT littermate mice from a dorsolateral (left) or ventrodorsal (right) view.

 
Hormone and metabolic changes in STC-1 transgenic mice
Because STC is believed to play a role in calcium and phosphate homeostasis (6, 7), we measured levels of these minerals. Ionized calcium was significantly elevated in STC-1 transgenic female founders and offspring relative to nontransgenic littermates (11.07 mg/dl STC-1 mice vs. 9.95 mg/dl WT mice, P = 0.016). Although previous studies showed that STC can stimulate phosphate reabsorption by fish and rat renal proximal tubules (7, 36), serum phosphate levels were normal in STC-1 transgenic mice (8.93 mg/dl STC-1 mice vs. 8.89 mg/dl WT mice, P = 0.951). Renal function parameters including blood urea nitrogen and creatinine were also normal. Other serologic parameters including liver enzymes—alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and {gamma}-glutamyltransferase—as well as amylase, a pancreatic enzyme, and creatine kinase, a cytosolic enzyme with highest activity in skeletal muscle, were within normal range (data not shown). Serum hormones—calcitonin, IGF binding protein-1, T4, and GH—were not significantly altered in transgenic mice relative to WT mice (data not shown).

To further investigate metabolism in STC-1 mice, food intake and response to a glucose tolerance test were evaluated. At 10 wk of age, males ate 32.2% more (0.1800 ± 0.097 g/d·g body weight STC-1 vs. 0.1361 ± 0.0043 g/d·g body weight WT, P = 0.015), weighed 36% less (18.97 ± 1.10 g STC-1 vs. 29.67 ± 0.73 g WT P = 0.001), and had a 13.8% increase in oxygen consumption (8.925 ± 0.2546 ml/h·g0.75 STC-1 vs. 7.8406 ± 0.2546 ml/h·g0.75 WT P = 0.039). A glucose tolerance test further demonstrated metabolic differences in STC-1 transgenic mice relative to WT littermates. STC-1 transgenic mice had faster glucose clearance at 30, 45, 60, 90, and 120 min after glucose loading (data not shown). Serum-free fatty acids were normal (data not shown). Fat pad weights were decreased in female STC-1 transgenic mice relative to WT littermates even when calculated as percentage of total body weight (0.19 ± 0.09% STC-1 vs. 0.62 ± 0.18% WT, P = 0.03). Accordingly, STC-1 transgenic mice appeared leaner than their WT littermates upon dissection.

Skeletal changes STC-1 transgenic mice
As determined radiographically, the axial and appendicular skeleton was shorter in transgenics compared with WT mice (Fig. 1BGo). Transgenic mice had normal numbers of vertebrae (C7T13L6), but the average length of the vertebral column from the base of the atlas to the last sacral vertebra was shorter in STC-1 compared with WT mice (5.4 cm vs. 6.1 cm). Because longitudinal growth of bones is controlled by growth plate cartilage, histological examination of long bones was performed. In 1-d-old STC-1 transgenic mice, the structure of the femoral growth plate was histologically normal (data not shown). By 35 d of age, the growth plate of STC-1 mice was narrowed relative to WT littermates but retained normal organization and pattern of chondrocyte maturation relative to WT mice (Fig. 2AGo). Alcian blue staining of the growth plate showed slightly more cartilage matrix in STC-1 mice relative to WT mice.



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  		Figure 2. Cartilage phenotype. A, Alcian blue staining of the growth plate (brackets) of 35-d-old transgenic (STC) or WT mice. B, Patellar cartilage weights (left) and cartilage matrix synthesis corrected for patellar cartilage weight (right) in transgenic mice (STC) (n = 4), age-matched controls (WT) (n = 5) and weight-matched younger mice (y) (n = 5). *, P < 0.05; and **, P < 0.005 compared with age-matched control (WT) mice using a Student’s t test.

 
Because STC-1 transgenic mice were substantially smaller than age-matched littermates (Fig. 1Go), we tested the hypothesis that STC-1 mice were merely developmentally delayed by including weight-matched (younger) mice (Fig. 2BGo). Long bones of STC-1 transgenic mice were 74% (femur) and 94% (tibia) the length of those of age-matched control mice, and 78% (femur) and 97% (tibia) the length of those from weight-matched control mice (data not shown). Consistent with the smaller skeleton in STC-1 mice, patellar cartilage weight was 68% that of age-matched control mice, and 90% that of weight-matched control mice (Fig. 2BGo, left panel). When corrected for this decrease in patellar cartilage weight, cartilage matrix synthesis was increased by 45% in STC-1 mice relative to age-matched controls but was 51% less than that of younger, weight-matched controls (Fig. 2BGo, right panel). As indicated by both qualitative histochemistry and quantitative assays, cartilage matrix synthesis in STC-1 mice was enhanced relative to that of age-matched controls. However, STC-1 mice did not phenocopy younger, weight-matched control mice (Fig. 2BGo).

Radiographs of skull bones showed altered suture morphology and decreased cellular extravasation through the center of parietal bones suggesting decreased osteoclast activity in STC-1 mice relative to WT littermates (Fig. 3AGo). To determine whether such changes were due to alterations in bone-resorbing osteoclasts, we stained skulls for TRAP, an osteoclast marker. Skulls from STC-1 mice had a broader distribution of osteoclasts than did those from their WT littermates (Fig. 3BGo). These results are consistent with previous findings that STC affects osteoclast activity (37, 38, 39) and indicate that both intramembranous ossification (skulls) and endochondral ossification (long bones) were affected by STC-1 expression in muscle.



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  		Figure 3. The skull phenotype of STC-1 mice. A, Representative skull of an STC-1 transgenic or WT mouse. B, TRAP staining (arrows) for osteoclasts in parietal bones of two STC-1 transgenic mice (STC) and two WT mice.

 
To determine the quality of bone formed, we examined histological sections and three-dimensional structural organization by µCT of bones. Plastic embedded sections von Kossa stained for mineral did not demonstrate striking changes in mineralization of trabecular and cortical bone in STC-1 transgenic mice (data not shown). Quantitative µCT indicated that cortical bone volume and total volume (bone and bone marrow) were reduced in STC-1 mice relative to age-matched control mice (Fig. 4AGo) consistent with the smaller size of STC-1 mice. However, cortical bone volume per total volume (BV/TV) was increased in STC-1 transgenic mice relative to age-matched WT mice (P = 0.04) (Fig. 4BGo), suggesting increased relative cortical bone thickness in STC-1 mice.



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  		Figure 4. Analysis of bone mineralization and structure in transgenic mice. A, Volume of cortical bone (cortical volume) and of both bone and bone marrow (total volume). B, BV/TV for cortical bone and trabecular bone. C, Bone mineral density (BMD) of cortical bone and anisotropy of trabecular bone for STC-1 transgenic mice (STC) (n = 5), age-matched (WT) (n = 5) or younger, weight-matched control mice (y) (n = 4). *, P < 0.05; and **, P < 0.005 vs. age-matched (WT) control mice using a Student’s t test. D, µCT images of mouse femurs. Three-dimensional representations of trabecular bone from WT and STC-1 transgenic mice (STC) and younger (y) weight-matched control mice illustrating changes in trabecular anisotropy. E, Fluorescent micrographs of two representative sections of (top) trabecular and (bottom) cortical bone of femurs of 6-wk-old STC-1 (STC) and WT mice. Note the decrease in distance between the two labeled fronts of mineralization (arrows) in STC-1 mice (STC) relative to WT mice.

 
Despite the increase in serum calcium levels in STC-1 transgenic mice, bone mineral density of both cortical bone (Fig. 4CGo, left panel) and trabecular bone (data not shown) was not altered in STC-1 mice. However, the degree of anisotropy (DA) of trabecular bone (P = 0.014) was increased relative to age-matched control mice (Fig. 4Go, C, right panel, and D). This increase in DA indicated a higher directional dependence of trabecular organization corresponding to a more uniform alignment of trabeculae along the principal axis. Similarly, trabecular thickness was decreased (P = 0.04) in STC-1 transgenic mice (0.0516 ± 0.001) relative to WT littermates (0.0625 ± 0.004). Furthermore, although the absolute number of trabeculae was not altered (data not shown), STC-1 transgenic mice had significantly (P = 9.4 x 10-5) higher trabecular density (trabecular number/total volume) than their WT littermates (5.99 ± 0.28 STC-1 vs. 3.75 ± 0.14 WT mice). Interestingly, younger mice had increased trabecular number, thickness (data not shown) and trabecular bone volume fraction (BV/TV) (Fig. 4BGo) and decreased cortical bone volume fraction (BV/TV) (Fig. 4BGo) and decreased cortical bone mineral density (Fig. 4CGo, left panel). Thus, as was found in our cartilage assays, the skeleton of STC-1 mice did not phenocopy that of weight-matched, younger mice (Fig. 4Go, A–D), suggesting that the skeletal changes in STC-1 are not due merely to a developmental delay.

Given the changes in bone size and structure, we tested whether the bone-forming activity of osteoblasts was altered. No significant changes in expression of several genes—vitamin D receptor, osteopontin, osteocalcin, or bone morphogenetic protein-4—were found by quantitative real-time RT-PCR (Taqman) analysis of mRNA from bones of STC-1 and WT mice (data not shown). However, histomorphometric analysis revealed that the mineral apposition rate, a measure of osteoblast activity, was lower in both trabecular and cortical bone of STC-1 transgenic mice relative to WT littermates (1.04 µm/d STC-1 vs. 2.47 µm/d WT, P = 1.9 x 10 -13) (Fig. 4EGo). While the rate of matrix deposition was decreased, bone mineralization in STC-1 mice was normal (Fig. 4CGo, left panel). Taken together, our data suggest that both osteoblast and osteoclast activity were decreased in STC-1 transgenic mice.

Changes in muscle function and histology
Just as endogenous STC-1 protein is present in muscle tissue (18), expression of the STC-1 transgene was directed to muscle tissue by virtue of the myosin light chain promoter (21). All muscles of STC-1 transgenic mice weighed significantly less than those of age-matched control mice and approximated the weight of muscles from weight-matched, younger mice (Fig. 5AGo). Thus, unlike organs such as the kidney, which weighed more in STC-1 mice than in age-matched control mice when analyzed as percentage of body weight (Fig. 5AGo), muscles were disproportionately smaller in STC-1 transgenic mice relative to WT littermates. To further examine skeletal muscles, electron microscopy was performed on skeletal muscle from STC-1 and WT mice. Enlarged myocyte mitochondria, with normal structure and organized cristae, were found in STC-1 transgenic mice relative to age-matched WT mice (Fig. 5BGo).



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  		Figure 5. Muscle mass and function in transgenic mice. A, Weight (as percent body weight) of kidney, gastrocnemius muscle (Gastroc), tibialis anterialis (TA) muscle, quadriceps femoris (Thigh), Tibia, and Femur of STC-1 (stc), age-matched control (wt) and approximately weight-matched, younger (y) mice. *, P < 0.05; and **, P < 0.005 compared with age-matched control (wt) mice using a Student’s t test. B, EM photomicrographs of muscles from transgenic (STC) or WT mice showing enlarged mitochondria in transgenic mice. C, Maximal twitch response (left) and Stable twitch response (right) at d 0 and 7 d post ligation for STC-1 (TG) (circle, solid line), approximately weight-matched (wgt-match) (square, spotted line), and age-matched (age match) controls (diamond, dashed line).

 
To quantitate muscle function, the contractile profile of muscles in response to a repetitive pulsatile electrical stimulation was determined. In this system, the early peak in response corresponds to the maximal twitch, which then slowly decreases to a plateau called the stable twitch (Ross J., L. Powell-Braxton, and S. Bunting, unpublished observation). At baseline (d 0), the slight decrease in maximal twitch response in STC-1 mice relative to WT mice was not significant (Fig. 5CGo, left, d 0). However, the stable twitch response of STC-1 transgenic mice at d 0 was significantly (P = 0.039) less than that of younger, approximately weight-matched WT mice (Fig. 5CGo, right, d 0).

In addition to determining baseline muscle function, we also measured the ability of muscles to respond after femoral artery ligation. In WT mice (age- and weight-matched control mice), the maximal twitch response essentially returned to baseline (97–99%) at 7 d after femoral artery ligation (Fig. 5CGo, left, d 7, inset). In contrast, STC-1 mice had only an approximately 80% recovery in maximal twitch response at 7 d post ligation (Fig. 5CGo, left, d 7, inset). In terms of the stable twitch response, WT mice showed 82–85% recovery, whereas that of STC-1 mice was only 71% at 7 d post ligation (Fig. 5CGo, right, d 7, inset). Thus, as indicated by both the maximal and stable twitch responses, muscle from STC-1 did not recover appropriately at 7 d after femoral artery ligation.

Changes in vascularity in STC transgenic mice
The runted phenotype of STC-1 transgenic mice could in theory be due to a vascular defect, since changes in STC-1 expression have been associated with endothelial cell differentiation (16). To test this hypothesis, we measured baseline vascular density and the angiogenic response following femoral artery ligation (32, 33, 34). STC-1 mice had a significantly higher baseline vascular density in all tissues relative to that of age-matched control mice (Fig. 6AGo). Compared with younger, approximately weight-matched control mice, STC-1 mice exhibited a lower vascular density in all tissues examined (Fig. 6AGo). Thus, as with other parameters, STC-1 transgenic mice were not identical to younger, weight-matched control mice.



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  		Figure 6. Changes in vascularity in transgenic mice. Shown are measurements of PECAM, an indicator of new blood vessels in (A) Heart, kidney (L kid, R kid), Quadriceps femoris (Thigh), hamstring (Ham) muscle, femur (Fem), gastocnemius (Gast) muscle, tibialis anterialis (TA) muscle, Tibia, lower leg (Low), Foot, and Tail in STC-1 (stc), age-matched control (wt) and approximately weight-matched younger (y) mice (n = 6 for each set of mice). B, Ratios of PECAM measurements in the ligated (Left) vs. contralateral, unligated leg (Right) in STC-1 (stc), age-matched control (wt) and approximately weight-matched younger (y) mice (n = 8 mice/group). *, P < 0.05 for STC-1 compared with age-matched control (wt) mice using a Student’s t test.

 
Following femoral ligation, a pronounced ischemia in the lower limb of the ligated leg occurs, which is followed by a spontaneous angiogenic response. The proliferation of small blood vessels results in an increase in endothelial cell surface area, which occurs over the next 3–10 d with maximal increases in vascular density at 7 d (40). We therefore used this femoral artery ligation model to quantitate the angiogenic response to injury in STC-1 transgenic mice. Compared with age-matched WT control mice, induction of vascularity in the lower part of the left, ligated leg relative to the right (untreated) leg was significantly greater in STC-1 transgenic mice (P < 0.05) (Fig. 6BGo). Similarly, increases in vascularity in the upper leg (hamstring and femur) in response to ligation were more pronounced in STC-1 transgenic mice than in age-matched control mice. Interestingly, vascular density increases in STC-1 transgenic mice in response to femoral artery ligation were also higher than those of younger, approximately weight-matched control mice, even though baseline vascularity was higher in these younger mice (Fig. 6Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Overexpression of STC-1 in transgenic mice resulted in smaller mice with distinct changes in the musculoskeletal system. The skeleton of STC-1 mice was not identical to that of younger, weight-matched controls suggesting that the phenotype of STC-1 mice was not due to a developmental delay.

Expression studies have indicated that STC-1 may play a role in bone development and patterning (18, 19, 20). Our data indicate that overexpression of STC-1 results in increased cartilage matrix synthesis and decreased bone length. Given that endogenous STC-1 is expressed at the ends of bone primordia and growing limbs (20), it is tempting to speculate that endogenous STC-1 affects the length of the cartilage anlagen, which serves as a template for subsequent bone formation. STC-1 could also act later in development as a regulator of chondrocyte maturation, as high levels of STC-1 protein are found in chondrocytes moving from the proliferative to hypertrophic zone (18). Our finding of increased cartilage matrix synthesis in STC-1 transgenic mice suggests that STC-1 stimulates chondrocytes, consistent with hypotheses that chondrocytes are targets of endogenous STC-1 (18, 19, 20).

STC-1 protein is also found in fetal trabecular osteoblasts (20) and adult mouse osteoblasts (19), and STC can regulate activity of osteoblasts (39, 41) and osteoclasts (37, 38, 39). Kinetic analyses indicated that the rate of mineral deposition by osteoblasts, but not the degree of bone mineralization, was decreased in STC-1 transgenic mice. The increases in cortical bone thickness, trabecular density, and anisotropy in the face of decreased matrix deposition by osteoblasts suggests concomitant changes in osteoclastic resorption in STC-1 mice. This hypothesis was further supported by our findings of decreased extravasation and altered osteoclast distribution in parietal bones in the skull. Thus, the changes in cortical and trabecular bone remodeling are likely due to suppression of both osteoblast and osteoclast activity by STC-1. The presence of alterations in skulls and long bones of STC-1 mice implicates STC-1 in both intramembranous and endochondral bone formation, consistent with the distribution of endogenous STC-1 protein during growth and development (18, 19, 20).

STC-1 has recently been implicated in skeletal muscle development (18). Our findings indicate that STC-1 can regulate muscle mass and function. Muscles of STC-1 mice were smaller than age-matched control mice, and muscle mitochondria were dramatically enlarged. Our assay of muscle function measuring the contractile profile of the gastrocnemius muscle indicated that STC-1 muscle had impaired ability to recover following femoral artery ligation. Our finding that STC-1 transgenic mice had a robust angiogenic response following femoral artery ligation suggest that the deficit in muscle function observed after ligation was not due to a failure to stimulate new vessel growth.

Taken together, our results indicate that STC-1 can affect muscle mass and function, perhaps at least in part by regulating muscle metabolism through alterations in mitochondria. Recently, STC protein has been found to be localized to the outer mitochondrial membranes of putative nephron target cells in fish, suggesting that STC could have direct effects on mitochondrial function (42). The enlargement of mitochondrial might be related to the protective effects that mammalian STC has on post-ischemic neuronal cells, perhaps in the transport of calcium and/or phosphate (10). It is tempting to speculate that the alterations in body composition and mitochondria are key contributors to the increased food and oxygen consumption, and faster glucose clearance in STC-1 transgenic mice.

STC-1 may also regulate endothelial cell activity. STC-1 has been implicated in endothelial cell differentiation into tube-like structures in an in vitro model of angiogenesis (16). STC-1 mRNA is expressed at sites of pathological angiogenesis, i.e. in tumors (16, 17). To evaluate a potential role for STC-1 in angiogenesis, we employed the femoral artery ligation model which provides an in vivo model of spontaneous angiogenesis following lower limb ischemia. In our angiogenesis experiments, we normalized values to the weight of the tissue to adjust for differences in size. Measurements of baseline vascular density strongly suggested that STC-1 mice had significantly higher capillary density in organs (heart and kidney) and tissues (muscle and bone) compared with age-matched littermates. The ability of STC-1 mice to mount a spontaneous angiogenic response following an ischemic insult was significantly increased relative to age-matched WT mice. These changes could be due to direct effects of STC-1 on endothelial cells and their environment, consistent with previous studies showing up-regulation of STC-1 upon endothelial cell differentiation (16). The finding that STC-1 mice showed a larger increase in vascularity after femoral ligation than did younger, approximately weight-matched controls supports the conclusion that the phenotype of STC-1 mice is not due merely to a developmental delay.

Angiogenesis is also a critical component of organ and tissue growth. STC-1 mice exhibited a vascular density greater than age-matched control mice, suggesting that a perfusion deficit is unlikely to explain their runted phenotype. The lack of obvious changes in wound healing following the femoral ligation surgery supports our data indicating robust vascular responses in STC-1 transgenic mice. The dramatic decrease in body weight and size in STC-1 transgenic mice is likely due, at least in part, to effects of STC-1 on the musculoskeletal system.

While this manuscript was in preparation, analysis of transgenic mice overexpressing STC-1 by virtue of the metallothionein I minimal promoter indicated a role for STC-1 in growth and reproduction (43). Consistent with their data, and despite our use of a different promoter, our transgenic mice (which had 5–100 times lower serum levels of STC-1) were smaller and had higher serum calcium levels than WT mice. No changes in reproduction were observed with our mouse lines when we mated male transgenic mice with female WT mice, in accordance with their findings that reproductive function of male mice was not affected (43). The phenotype of our STC-1 transgenic mice similarly indicate a role for STC-1 in growth and calcium homeostasis and further identify and characterize the musculoskeletal changes induced by STC-1 overexpression.

In summary, our results indicate how STC-1 can affect muscle mass and function and bone size and structure. Future studies will help elucidate the relationship between STC-1 and other factors regulating development of the musculoskeletal system.


   Acknowledgments
 
The authors thank Sharon Erickson and her lab for generating and genotyping STC-1 mice, Patti Tobin and Robin E. Taylor for histology support, and Frank Peale for helpful discussions.


   Footnotes
 
Support from the Kidney Foundation of Canada and The Canadian Institutes of Health Research (to G.F.W.) is acknowledged.

Abbreviations: BV/TV, Bone volume per total volume; µCT, microcomputed tomography; DA, degree of anistropy; STC, stanniocalcin hSTC-1, human STC-1; mAb, monoclonal antibody; MIL, mean intercept length; PECAM, platelet-endothelial cell adhesion molecule; TRAP, tartrate-resistant acid phosphatase; VOI, volume of interest; WT, wild-type.

Received December 20, 2001.

Accepted for publication May 6, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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