ARTICLE |
Correspondence to: Yuji Yoshiko, Dept. of Anatomy, Faculty of Dentistry, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan. E-mail: yyuji@hiroshima-u.ac.jp
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Summary |
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STC1, a mammalian homologue of stanniocalcin (STC) which plays a major role in calcium/phosphate homeostasis in fish, has been recently isolated. We have characterized the spatiotemporal distribution of STC1 mRNA and protein during mouse embryonic development generally and osteogenesis specifically. Northern blotting analysis of whole embryos showed that STC1 mRNA is highly and differentially expressed during embryogenesis. By in situ hybridization, STC1 mRNA was detected early in mesenchymal condensations and was then found to be highly expressed in perichondrial cells, periosteal cells, and then osteoblasts during endochondral bone formation. In bones forming by intramembranous ossification, STC1 mRNA was not detected until osteogenic cells appeared. The cellular distribution of STC1 protein closely corresponded to that of its mRNA, but the protein was also detected in hypertrophic chondrocytes. In the MC3T3-E1 osteogenic cell model, STC1 protein and mRNA were detectable throughout proliferation and differentiation stages but levels were relatively higher late during nodule formation/mineralization phases. For comparison, STC1 mRNA was also found in epithelial cells of both embryonic and adult intestine that had not previously been described among tissues responsive to calcium/phosphate transport. These results suggest that STC1 is expressed in a time- and cell-specific manner and may play an autocrine/paracrine role during osteoblast development and bone formation. (J Histochem Cytochem 50:483491, 2002)
Key Words: STC1 protein, STC1 mRNA, cellular distribution, embryonic mouse osteogenesis, MC3T3-E1 cells
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Introduction |
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STANNIOCALCIN (STC) is a glycoprotein hormone produced in the corpuscles of Stannius (CS) (
Similarly to the activity of fish STC, human STC1 recombinant protein (rhSTC1) is able to regulate calcium uptake in the gill (
In this study we sought to describe in detail the spatiotemporal distribution of STC1 mRNA and protein in embryonic mouse osteogenesis and in an in vitro mouse model of osteogenesis, the MC3T3-E1 cell line. Our data indicate that STC1 is already expressed at the stage of mesenchymal condensation in the developing embryo but becomes increasingly restricted to osteoblast lineage cells and mature osteoblasts as development advances. Taken together with the fact that STC1 is also distributed specifically in epithelial cells of intestine and kidney, our data support the view that STC1 may be an autocrine/paracrine factor in tissues responsive to calcium/phosphate transport.
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Materials and Methods |
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Animals
Timed-pregnant or other ddY mice of appropriate ages for this study were purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan). Mice were housed and handled according to protocols approved by Hiroshima University Research Facilities of Laboratory Animal Science.
Cell Culture
MC3T3-E1, a clonal mouse normal osteoblastic cell line, was obtained from the Riken Cell Bank (Tsukuba, Japan). Cells were grown at 37C in Dulbecco's modified Eagles medium containing 10% fetal calf serum (Upstate Biotechnology; Lake Placid, NY) and antibiotics in a humidified 5% CO2 atmosphere. For the developmental sequence study, the medium was supplemented with 50 µg/ml ascorbic acid and changed every second or third day until mineralized nodules had developed. In some cases, 10 mM ß-glycerophosphate was added to cultures for 2 days before culture termination to induce matrix mineralization. To detect mineralization, cultures were fixed in neutral buffered formalin for 15 min and incubated in 2.5% silver nitrate solution (von Kossa staining).
Western Blotting Analysis
Cells were rinsed with PBS on ice, suspended in 0.5% IGEPAL CA-630 (Sigma; St Louis, MO), 150 mM NaCl, 5 mM EDTA, Tris-HCl, pH 7.3, including 1 mM phenylmethylsulfonyl fluoride, and incubated for 2 hr at 4C on a rotating platform. After centrifugation, an aliquot of the supernatant (50 µg total protein) was mixed with 2 x SDS reducing buffer and boiled for 5 min. Standard SDS-PAGE procedures were used with a Mini-PROTEAN II cell system (Bio-Rad; Hercules, CA) and 15% acrylamide gels. Thirty ng of bacterial recombinant human STC1 (rhSTC1) was loaded as a positive control. Protein blotting from the gels to nitrocellulose membranes (Hybond ECL; Amersham Pharmacia Biotech, Poole, UK) was performed using a TRANS-BLOT SD (Bio-Rad). The membranes were blocked at room temperature (RT) for 2 hr with 0.1% Tween-20, 0.1 M NaCl, 0.1 M Tris-HCl, pH 7.5 (TTBS), including 0.2% casein. Membranes were incubated overnight at 4C with mouse anti-rhSTC1 monoclonal antibody (anti-rhSTC1, 0.5 µg/ml), followed by a Vectastain ABC kit (Vector Lab; Burlingame, CA). Briefly, the membranes were incubated with biotinylated secondary antibody (horse anti-mouse IgG, 1:500) at 4C overnight and then avidinbiotinylated horseradish peroxidase (HRP) macromolacular complex (ABC reagents, 1:200) at RT for 30 min. Each incubation step was followed by three washes (15 min) with TTBS. Chemiluminescence detection was carried out with luminol visualization solution (0.4 mg/ml luminol, 0.1 mg/ml p-iodophenol, 0.015% H2O2 in 50 mM Tris-HCl, pH 7.5). The uniformity of protein loading was assessed by rabbit anti-actin antibody (1:1000; Miles, Elkhart, IN) and HRP-conjugated goat anti-rabbit IgG antibody for secondary antibody (1:3000, Bio-Rad). The specificity of anti-rhSTC1 was demonstrated by preabsorption of anti-rhSTC1 with rhSTC1 (100 µg/ml) and by normal mouse IgG (Santa Cruz Biotech; Santa Cruz, CA) instead of the anti-rhSTC1.
Northern Blotting Analysis
Digoxygenin (DIG)-11-UTP-labeled single-stranded RNA probes were prepared using a DIG labeling kit (Roche Diagnostics; Mannheim, Germany) according to the manufacturer's instructions. Full-length (1.2-kb) human STC1, a 0.6-kb fragment of mouse alkaline phosphatase (ALP), a 0.47-kb fragment of mouse osteocalcin (OCN), and a 0.5-kb fragment of mouse Type I () collagen (Coll
I) cDNAs were used to generate antisense and sense probes.
Whole embryos at known developmental ages (E10.5, E12.5, E14.5, and E16.5; embryonic days post coitum), several adult tissues, and MC3T3-E1 cells were subjected to total RNA extraction by acidguanidinium thiocyanatephenolchloroform extraction (
Tissue Preparation
For immunohistochemistry, 7-mm frozen sections from embryonic mouse tissues (littermates served for total RNA preparation) were stored at -70C until use. For ISH, the tissues were immersion-fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4C. The specimens were then dehydrated in a graded series of ethanol, defatted in chloroform, and embedded in paraffin. Sections of 6 µm were placed on 3-aminopropyltriethoxysilane-treated slides and stored at 4C.
Immunohistochemistry
Frozen sections were air-dried, fixed in PLP solution (10 mM NaIO4, 75 mM lysine, 37.5 mM phosphate buffer, pH 7.4, containing 2% paraformaldehyde) at RT for 10 min. To block nonspecific staining, the sections were incubated with Dako Protein Block (Carpinteria, CA) at RT for 2 hr. The sections were incubated with anti-rhSTC1 (5 µg/ml) at 4C overnight and then with biotinylated secondary antibody (1:1000) at RT for 2 hr, followed by incubation with 0.3% H2O2 in methanol at RT for 30 min. To make the avidinbiotin complex, ABC reagents (1:100; Vector) as described above were applied for 30 min at RT. Each incubation step was followed by two washes (15 min) with PBS. Staining was developed with a Vector VIP substrate kit (Vector). The sections were counterstained with methyl green and observed under a light microscope. As negative control, normal mouse IgG (Santa Cruz Biotech) was used in place of anti-rhSTC1.
In Situ Hybridization
ISH analysis was carried out as previously described (
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Results |
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STC1 mRNA Transcripts Are Differentially Regulated During Embryogenesis
Because high levels of STC1 mRNA have been detected in whole developing mouse embryos (
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STC1 Is Expressed in Specific Cell Types During Embryonic Osteogenesis
To elucidate the spatiotemporal profile of STC1 mRNA expression during embryonic mouse osteogenesis, ISH was performed; sections of representative examples of bones forming by endochondral or intramembranous ossification are shown in Fig 2. For bones forming by endochondral ossification, STC1 mRNA was expressed in the mesenchymal condensations (e.g., developing vertebral column at E10.5; Fig 2A) but, as cartilage development progressed, STC1 mRNA was highly expressed in perichondrial cell layers surrounding cartilage primordia (e.g., developing ribs at E12.5; Fig 2B). Perichondrial/periosteal cells of growing but still cartilaginous bones also expressed high levels of STC1 mRNA (e.g., occipital bone at E14.5; Fig 2C). However, when ossification of bones was clearly under way, intense STC1 mRNA hybridization signal was seen in osteoblasts and the associated periosteal cells (e.g., occipital bone at E16.5; Fig 2D). Notably, chondroblasts and cells of other tissues surrounding osteogenic cells expressed low/undetectable levels of STC1 mRNA (Fig 2A2D). With respect to bones forming by intramembranous ossification, such as the calvaria, little to no STC1 mRNA was detectable in condensed mesenchyme at E12.5 (Fig 2E). However, both osteoblasts and periosteal cells expressed high levels of STC1 mRNA in the developing calvaria at E16.5 (Fig 2F). For comparison, STC1 mRNA was also highly expressed throughout the epithelium of the E16.5 intestine (Fig 3A). However, the signal was restricted to cells at the base of the villi and crypt regions but was undetectable in cells from the middle to the top of the villi in adult intestine (Fig 3B). No signal was detected in any tissues with the sense probe or by hybridization with the antisense probe after pretreatment with RNase.
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Immunohistochemistry showed that the STC1 protein largely co-localized with STC1 mRNA (Fig 4). For example, diffuse staining of STC1 protein was seen in the perichondrium of the developing endochondral sphenoid bone at E12.5 (Fig 4A). Osteoblasts and associated periosteal cells of the intramembranous frontal (Fig 4B) and parietal (Fig 4C) bones stained intensely for STC1 protein at E16.5. In E16.5 metatarsals (endochondral bones), hypertrophic chondrocytes but not other chondrocytes and osteoblasts labeled for STC1 protein (Fig 4D). When anti-rhSTC1 antibodies were replaced with normal mouse IgGs, no labeling over background was seen (e.g., metatarsals; Fig 4G).
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STC1 Is Upregulated During MC3T3-E1 Cell Development
We previously demonstrated that STC1 mRNA is expressed in several osteoblastic cell lines, including the mouse calvaria-derived line, MC3T3-E1 (
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Expression of STC1 mRNA in differentiating MC3T3-E1 cell cultures was compared to that of several well-established osteoblast markers, including Coll1, ALP, and OCN (Fig 6). As expected, Coll
1 was detectable throughout the culture time but levels were highest at earlier times, whereas ALP mRNA was detected and upregulated from Day 13 and OCN mRNA from Day 18 as bone nodules formed and mineralized. STC1 mRNA was detectable from the earliest time analyzed (Day 4) but increased and remained high during late differentiation and mineralization phases (from Day 18).
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Discussion |
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We previously demonstrated that STC1 mRNA is expressed in osteoblasts of developing mouse long bone, calvaria, and several osteoblastic cell lines (
Northern blotting analysis revealed that STC1 mRNA is highly expressed in extracts from whole mouse embryos and in various adult tissues. In contrast to an earlier analysis in which steady-state levels of a 4-kb transcript were detected in whole embryos (
It has been observed that both STC1 protein and mRNA expression are ubiquitous in mouse embryonic tissues but, as shown in Fig 2 and Fig 4, both STC1 protein and mRNA are higher in osteoblastic cells than in surrounding musculoskeletal tissue cell types as well as in developing brain, skeletal muscle, and skin. The high and specific localization of STC1 in osteoblast lineage cells during embryonic osteogenesis suggests a significant role for STC1 in bone development and is in partial agreement with an earlier study in which the protein was found to be abundant in the musculoskeletal system in fetal mice (
STC1 protein was also located in hypertrophic chondrocytes but the mRNA was not, a discrepancy shown previously in kidney, in which STC1 protein is detected exclusively in the segments that do not express the mRNA (
In contrast to a recent immunohistochemical study of E15.5 embryos in which STC1 protein was not detected in intestine (
In summary, we conclude that STC1 is expressed in specific cell types in tissues responsive to calcium/phosphate transport including intestinal epithelial cells in embryonic and adult tissues. Notably, we have demonstrated that STC1 expression is present as early as the mesenchymal condensations and subsequently is high in cells committed to the osteoblast lineage, especially the mature osteoblasts, during embryonic mouse osteogenesis. Our results suggest that osteoblasts may be a rich source of and a target for action of STC1 during both endochondral and intramembranous bone formation.
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Acknowledgments |
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Supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan and by CIHR (MT-12390 to JEA) of Canada.
We are grateful to Dr Roger R. Reddel of Children's Medical Research Institute (NSW, Australia) for providing us with the recombinant plasmid pAC 143 including human STC cDNA, and to Akira Igarashi and Dr Shoichi Takano of BML Inc. (Saitama, Japan) for providing us with rhSTC1 and anti-rhSTC1. We also thank Aoi Son and Usha Bhargava for technical assistance.
Received for publication June 11, 2001; accepted October 10, 2001.
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