ARTICLE |
Correspondence to: Klaus Klaushofer, Ludwig Boltzmann Inst. of Osteology, Fourth Medical Dept., Hanusch-Hospital, Heinrich Collinstr. 30, A-1140 Vienna, Austria.
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Summary |
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Osteocalcin (OC), a bone-specific protein, is a marker of late osteoblastic differentiation. Its expression is influenced by various growth factors and hormones. We investigated the effect of 1,25-dihydroxy vitamin D3 (D3) and tri-iodothyronine (T3) on OC expression in osteoblast-like MC3T3-E1 cells. A heterologous OC green fluorescence protein (GFP) fusion vector was established and expressed to study possible effects on protein transport. Immunostaining of endogenous OC revealed a significant increase in the percentage of positive cells after D3 and T3 treatment. This was consistent for MC3T3-E1 cells as well as nonosteogenic NIH-3T3 and mammary carcinoma cells, but not for neuroblastoma cells. The perinuclear immunostaining corresponded to the NBD C6 ceramide Golgi staining. Conversely, we found a strong induction of OC in MC3T3-E1 cells at the mRNA and protein levels only with T3 and not with D3. OC mRNA and protein expression was not detected in NIH fibroblasts. OC GFP transfection experiments indicate rapid transport and secretion of OC, because OC GFP was not found to be accumulated at intracellular compartments after hormone treatment. We conclude that the strong perinuclear immunostaining does not represent OC but a protein immunologically related to OC, as indicated by preabsorption experiments. The expression of this OC epitope-sharing protein is regulated by both D3 and T3 in the osteoblastic MC3T3-E1 and in nonosteogenic cells. (J Histochem Cytochem 46:477486, 1998)
Key Words: osteocalcin, immunohistochemistry, green fluorescent protein, 1,25-dihydroxy vitamin D3, tri-iodothyronine, MC3T3-E1 cells
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Introduction |
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1,25-Dihydroxy vitamin D3 (D3) and tri-iodothyronine (T3) exert their diverse effects on normal bone growth and development primarily through nuclear receptor-mediated processes (
Osteocalcin (OC) is a small (5.25.9 kD) protein that comprises 1020% of the noncollagenous organic material of the extracellular bone matrix (-carboxyglutamic acid)-dependent binding of Ca2+. OC-deficient mice develop a phenotype characterized by higher bone mass without impaired bone resorption (
The effects of D3 and T3 on OC mRNA and protein expression have been studied in various chicken, mouse, rat, and human osteo-derived cell lines, indicating that the hormonal regulation is species- and differentiation-dependent (
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Materials and Methods |
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Cell Culture
MC3T3-E1 mouse calvaria-derived osteoblast-like cells (Dr. Kumegawa; Sakado, Japan), NIH-3T3 mouse fibroblasts (ATCC; Rockville, MD), I34 mouse mammary carcinoma cells, and NB1 mouse neuroblastoma cells (Institute of Cancer Research and Tumor Biology; University of Vienna, Vienna, Austria) were cultured in MEM supplemented with 4.5 g/liter glucose, 5% fetal calf serum (FCS), and 30 µg/ml gentamicin (regular medium). The cells were kept in humidified air under 5% CO2 at 37C. They were subcultured twice a week using 0.001% pronase E and 0.02% EDTA in PBS. For the experiments, the cells were seeded at a density of 50,000/cm2. Continuous treatment with T3 (10-7 M; Sigma, Diesenhofen, Germany) or D3 (10-8 M; Hoffmann LaRoche, Vienna, Austria) was started 24 hr after seeding. Medium was changed every third day.
Immunostaining of Endogenous OC
Cells were cultured on sterile glass coverslips placed in the culture dishes. At the respective time points (24 hr, and 3 and 8 days of culture) cells were rinsed with PBS, fixed with 4% paraformaldehyde (15 min), and permeabilized with methanol (5 min, -20C). After several rinses with PBS, cells were incubated with goat anti-mouse osteocalcin antiserum (RIA grade; BTI, Stoughton, MA; 1:100 in 3% BSA, 1 hr), followed by rabbit anti-goat IgG (Nordic Immunology; Tilburg, The Netherlands; 1:100 in 3% BSA, 45 min) and donkey anti-rabbit IgG conjugated with Cy3 (Accurate, Westbury, NY; 1:500 in 3% BSA, 45 min). According to the supplier, this OC antiserum is specific for the intact molecule, with the recognition site at the carboxyl terminal. The epitope is further along the molecule than the common Gla domain. In addition, for some experiments cell nuclei were counterstained with PicoGreen (Molecular Probes, Leiden, Netherlands; 1:1000 in A. dest., 45 min) after treatment with RNase A (Boehringer, Mannheim, Germany; 0.1 mg/ml in A. dest., 37C, 20 min). Coverslips were mounted on glass slides in Vectashield (Vector Laboratories; San Diego, CA) and examined using a confocal laser scanning microscope (Leica TCS4D).
Percentages of immunopositive cells were determined by evaluating at least 300 cells for each time point and treatment. The specificity of the immune reaction was assessed by staining with nonimmune goat serum (BTI; 1:100) or by omitting the OC antiserum. Preabsorption of the OC antiserum with purified mouse OC (BTI) was performed with 5 µg, 500 ng, 50 ng, and 5 ng OC per ml of 1:100 diluted antiserum, overnight at 4C.
Golgi Staining with NBD C6 Ceramide
MC3T3-E1 and NIH-3T3 cells grown on glass coverslips were incubated with NBD C6 ceramide (Molecular Probes, Leiden, The Netherlands; 5 nM in 1% DF-BSA, 6C, 30 min), a vital stain for the Golgi apparatus. The cells were treated as described in
Western Immunoblotting
Denaturing SDS-PAGE of 20 µg protein extract/lane or 100 ng mouse OC standard (BTI) was performed on 12% or 15% gels, respectively, according to the method of
For determining the binding capacity of the OC antiserum to denatured OC, we applied 50 ng OC (BTI; diluted in 0.1% BSA) as well as 50 ng SDS- and/or heat-denatured OC on a nitrocellulose and/or PVDF membrane using a slot-blot filtration apparatus.
Radioimmunoassay
MC3T3-E1 and NIH-3T3 cells were seeded in 96-well culture plates and cultured as described above. At Days 3 and 4, respectively, the cell culture media were removed and kept at -20C until assayed by a competitive liquid-phase RIA (BTI). According to the supplier's description of the RIA, there is no crossreaction with bovine OC.
Northern Blotting
RNA electrophoresis, transfer, and hybridization were performed as described previously (
Construction of OC GFP Fusion Plasmid
The recombinant mouse OC-enhanced green fluorescence protein (GFP) fusion construct was obtained by fusing the mouse OC cDNA (
Transfection Experiments
MC3T3-E1 cells were seeded on coverslips placed in culture dishes and were grown overnight in regular medium. The cells were transfected with 1 µg OC GFP fusion vector and 5 µg of transfection reagent DOSPER (Boehringer) in 700 µl MEM for 6 hr, and this was continued for a further 18 hr after addition of 2.5 ml regular medium. Then the medium was completely replaced by regular medium with or without T3 (10-6 to 10-9 M) or D3 (10-7 to 10-10 M). A temperature block (15C for 1 or 2 hr; cf.
Statistical Analysis
ANOVA (Statview 4.0; post hoc test, Scheffe) was used for statistical analysis.
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Results |
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Immunostaining of Endogenous OC
MC3T3-E1 Mouse Osteoblast-like Cells.
After 3 days of culture, cells reached a subconfluent monolayer. In controls, only few cells exhibited positive immunostaining (Figure 1A). In contrast, the majority of T3- and D3-treated cells exhibited intense perinuclear staining (Figure 1B and Figure 1C). Three days of treatment with T3 and D3 resulted in a highly significant increase in the percentage of positive cells (cf. Figure 4; Co 16 ± 7%, T3 69 ± 5%, D3 82 ± 3%).
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After 8 days of culture the percentage of immunopositive cells remained constant compared with Day 3 (cf. Figure 4; Co 17 ± 6%, T3 78 ± 5%, and D3 85 ± 3%). However, an increase in the intensity of the immunostaining in a distinct juxtanuclear region could be observed (Figure 1DF), especially after treatment with D3 (Figure 1F). The tubulo-vesicular structure of the staining appeared consistent with an accumulation of the antigen in the Golgi region. This is supported by comparison with the NBD C6 ceramide Golgi staining (Figure 1GI). In general, the stained Golgi stacks appeared to become enlarged with culture time. However, enlarged Golgi complexes were more abundant in cultures treated with T3 and D3. In D3-treated cultures, many large binucleate cells could be observed, showing extensive strong immunostaining mostly located between the two nuclei (Figure 2A). A similar localization was observed after NBD C6 ceramide Golgi staining (Figure 2D). Such conspicuous binucleate figures were found exclusively in D3-treated and, less commonly, in T3-treated MC3T3-E1 cells.
Preabsorption of the OC antiserum with purified OC (5 µg) resulted in complete inhibition of immunostaining (Figure 2B) comparable to the negative staining when nonimmune goat serum was used instead of the OC antiserum (Figure 2C). The perinuclear immunostaining gradually reappeared in response to increasing dilutions of the OC protein used for the preabsorption experiment (500 ng, 50 ng, and 5 ng OC), indicating the specificity of the antiserum for OC.
To evaluate whether both hormones also stimulate the immunostaining at the single-cell level in actively growing MC3T3-E1 cells, cells were seeded and immediately treated with T3 or D3. Only 24 hr (Day 1) after seeding, many single cells without contact with neighboring cells showed positive perinuclear and vesicular staining in the cytoplasm (Figure 2E and Figure 2F), predominately in hormone-treated but also in control cells.
NIH-3T3 Mouse Fibroblasts, I34 Mouse Mammary Carcinoma Cells, and NB1 Mouse Neuroblastoma Cells. Surprisingly, some nonosteoblastic cell lines also showed similar positive results when stained with the OC antiserum.
NIH-3T3 Cells. After 3 days of culture, many control as well as T3- and D3-treated cells showed perinuclear immunostaining as well as immunoreactive vesicles throughout the cytoplasm (Figure 3AC). Again, immunostained regions corresponded to NBD C6 ceramide-positive Golgi elements (data not shown). However, in contrast to the results with MC3T3-E1 cells, a higher percentage of untreated NIH-3T3 cells were immunopositive (cf. Figure 4; Co 75 ± 5%, T3 88 ± 2%, D3 95 ± 1%).
I34 mammary carcinoma cells also showed a similar positive immunostaining pattern after 3 days of culture (Figure 3e), but the stimulatory effects of D3 or T3 did not reach significance (cf. Figure 4; Co 23 ± 5%, T3 32 ± 3%, D3 42 ± 12%).
Neither untreated nor hormone-treated NB1 neuroblastoma cells showed any immunohistochemical reaction with the OC antiserum (Figure 3F).
Western Immunoblot
The specificity of the OC antiserum was tested by immunoblotting under denaturing conditions. Three distinct bands of approximately 14, 18, and 40 kD were detected in MC3T3-E1 (Figure 5A) and in NIH-3T3 protein extracts (data not shown), none of them having the appropriate molecular weight of osteocalcin (5.8 kD). The OC antiserum did not react with 100 ng mouse OC standard after SDS-PAGE (Figure 5A) but immunoprecipitated native iodinated mouse OC (data not shown). Slot-blots with native mouse OC and denatured OC revealed that the OC antiserum failed to bind to denatured OC, whereas strong binding to native osteocalcin could be observed (Figure 5B).
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Northern Blot and RIA
Expression of OC was studied at both the mRNA and the protein level in MC3T3-E1 and NIH-3T3 cells. No OC mRNA was detected in untreated or D3-treated cultures. T3 significantly increased OC mRNA levels in the osteoblastic but not in the fibroblastic cells (Figure 6).
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Measurement of the OC protein by RIA in the cell culture media exactly reflected the mRNA data (Table 1).
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Transfection Experiments with OC GFP
By recombinant DNA techniques, we generated an OC GFP fusion protein. This fusion protein consisted of the OC prepropeptide and propeptide with specific recognition sites for signal peptidases and maturases in the secretory transport process (Figure 7). The correct recombination of the OC GFP was confirmed by sequencing and its expression at the mRNA level was proved by Northern blotting. The expression of the construct was not influenced by D3 or T3 treatment. The "functional integrity" of the fusion protein at the secretory pathway was demonstrated by incubating transiently transfected MC3T3-E1 cells for 1 or 2 hr at 15C. This arrest of protein transport due to low temperature resulted in strong perinuclear staining (Figure 8A). Release of the transport block led to a vesicular staining pattern (Figure 8B and Figure 8C) and the complete disappearance of fluorescent OC GFP from the cells after appropriate incubation at 37C. Transfection of the pEGFP-N1 vector into MC3T3-E1 cells without the prepropeptide of OC resulted in uniform expression of GFP in the cytoplasm and nuclei (Figure 8D).
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Different doses of T3 and D3 were used to study possible inhibitory effects on OC GFP transport and secretion. None of the hormones altered transport of OC GFP in comparison to untreated controls; 99.8% of the investigated cells did not show any accumulation of OC GFP in the Golgi complex (Figure 8E and Figure 8F).
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Discussion |
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Osteocalcin expression is a characteristic of the mature osteoblast phenotype (
Treatment with D3 and T3 resulted in a highly significant increase in the percentage of immunopositive MC3T3-E1 cells that showed strong perinuclear staining. A comparable localization of OC has been shown previously (
At this point we speculated that the contradiction of apparent immunolocalization of intracellular OC in the absence of detectable mRNA or protein secretion after treatment with D3 might be based on transport-inhibitory phenomena. To test this possibility, we initiated experiments with GFP (
We established an OC GFP fusion vector and transiently transfected this fusion vector into MC3T3-E1 cells treated with or without D3 and T3. To demonstrate its functional integrity, the fusion protein was visualized along the secretory pathway by exposing the cells to low temperature (15C), which resulted in a simple and reversible arrest of intracellular protein transport at the Golgi level (
The discrepancies between OC immunohistochemistry and the mRNA and RIA data, as well as the OC GFP transfection results found in MC3T3-E1 cells, suggest that the OC antiserum reacts with one or more immunogenic epitopes that are not solely specific to OC. Therefore, we tested it in nonosteogenic cells, i.e., NIH-3T3 mouse fibroblasts and I34 mouse mammary carcinoma cells. Unexpectedly, we found positive immunostaining identical to that of the osteoblastic cells. However, we did not detect OC mRNA or OC protein (RIA) in NIH-3T3 fibroblasts, a result that is in agreement with earlier reports showing the inability of skin fibroblasts to synthesize OC (
Some evidence exists in the literature that OC antisera may recognize epitopes on proteins other than OC (
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Acknowledgments |
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We thank Prof Dr M.P.M. Erlee for critical reading of the manuscript.
Received for publication June 6, 1997; accepted October 22, 1997.
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