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doi:10.1369/jhc.4C6561.2005
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Volume 53 (10): 1181-1187, 2005
Copyright ©The Histochemical Society, Inc.
RAPID COMMUNICATION |
Characterization of Osteocrin Expression in Human Bone
Cambridge University School of Clinical Medicine, Addenbrooke's Hospital, Cambridge, UK (SB,DCI,JEC), and Phenogene Therapeutics Inc., Montreal, Quebec, Canada (PM,GPT)
Correspondence to: Professor Juliet Compston, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Box 157, Cambridge, CB2 2QQ, UK. E-mail: jec1001{at}cam.ac.uk
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Summary |
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Osteocrin (Ostn), a bone-active molecule, has been shown in animals to be highly expressed in cells of the osteoblast lineage. We have characterized this protein in human cultured primary human osteoblasts, in developing human neonatal bone, and in iliac crest bone biopsies from adult women. In vivo, Ostn expression was localized in developing human neonatal rib bone, with intense immunoreactivity in osteoblasts on bone-forming surfaces, in newly incorporated osteocytes, and in some late hypertrophic chondrocytes. In adult bone, Ostn expression was specifically localized to osteoblasts and young osteocytes at bone-forming sites. In vitro, Ostn expression decreased time dependently (p<0.02) in osteoblasts cultured for 2, 3, and 6 days. Expression was further decreased in cultures containing 200 nM hydrocortisone by 1.5-, 2.3-, and 3.1-fold (p<0.05) at the same time points. In contrast, alkaline phosphatase expression increased with osteoblast differentiation (p<0.05). Low-dose estradiol decreased Ostn expression time dependently (p<0.05), whereas Ostn expression in cultures treated with high-dose estradiol was not significantly changed. These results demonstrate that Ostn is expressed in human skeletal tissue, particularly in osteoblasts in developing bone and at sites of bone remodeling, suggesting a role in bone formation. Thus, Ostn provides a marker of osteoblast lineage cells and appears to correlate with osteoblast activity. (J Histochem Cytochem 53:1181–1187, 2005)
Key Words: osteocrin • osteoblasts • osteocytes • human bone formation
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Introduction |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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OSTEOBLASTS secrete abundant amounts of non-collagenous matrix (Parfitt et al. 1996
) and play a key role in the maturation and mineralization of the bone matrix (Robey 1996). In addition to their role in bone formation, osteoblasts are directly and indirectly involved in regulating bone metabolism, secreting both autocrine and paracrine regulatory molecules. Such molecules can regulate osteoclastogenesis and/or osteoclast activity, such as receptor activator of NF kappa B ligand and osteoprotegerin (Suda et al. 1999; Bord et al. 2003) or act in an autocrine/paracrine fashion on the osteoblastic population such as growth factors (Khan et al. 2000). Recently, osteocrin (Ostn), a novel secreted protein, was identified during a screen of an embryonic mouse calvarial cDNA library using a viral-based signal-trap technology (Moffatt et al. 2002). The Ostn gene produces a 1280-bp mRNA encoding a mature protein of 103 aa with a molecular weight of 11.4 kDa. Ostn was shown to be highly expressed in cells of the osteoblastic lineage both in vitro and in vivo in rodents. To date, Ostn shows no strong homology to any known gene except for two conserved sequence motifs reminiscent of dibasic cleavage sites found in peptide hormone precursors, suggesting a putative regulatory role. Further, treatment of rat primary osteoblasts with Ostn resulted in inhibition of late-stage differentiation (Thomas et al. 2003). Thus, Ostn appears to represent a novel bone protein that can act as a soluble osteoblast regulator. To date, however, the presence of Ostn has not been investigated in human tissues. To demonstrate Ostn expression in human tissue and establish the potential role for Ostn in human skeletal disease, Ostn was immunolocalized in developing human neonatal rib bone and in iliac crest bone biopsies from postmenopausal women treated or not treated with estrogen therapy. Further, changes in Ostn expression with osteoblast differentiation were demonstrated in primary human osteoblasts treated with hydrocortisone and/or estrogen.
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Materials and Methods |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Cell Culture
Primary human osteoblasts were isolated from bone samples from three young female donors (4 months, 6 months, and 6 years) undergoing routine surgery. All bone samples were obtained with appropriate ethical approval. Cells were isolated by sequential enzymatic digestion as described previously (Meikle et al. 1992
). Briefly, the bone was cleaned and finely minced prior to digestion in trypsin (Difco, Becton Dickinson; Oxford, UK) and dispase (Roche Diagnostics; Mannheim, Germany). Osteoblasts were released by two collagenase (collagenase A; Roche Diagnostics) digestions and then grown to confluence in HAMS F12/ DMEM (Invitrogen Life Technologies; Paisley, UK) supplemented with 10% heat-inactivated FBS (Invitrogen) penicillin/streptomycin (Invitrogen) and ascorbic acid (100 mM; Wako, Alpha Labs, Eastleigh, UK). Cultures were incubated at 37C in a humidified chamber with 5% CO2. At confluence the cells were seeded into eight-well chamber slides (Nalge Nunc International; Naperville, IL) at 104 cells/well in McCoys 5A medium (Invitrogen) supplemented with 10% human serum (Labtech International; Lewes, UK). After 2-hr settling time, fresh medium was applied ±200 nM hydrocortisone (Sigma; Dorset, UK) to provide proliferating or differentiating conditions, respectively. Control cultures had medium containing hydrocortisone carrier (Sigma). Cells were cultured for 2, 3, and 6 days, after which time the osteoblast cell layer was assessed for alkaline phosphatase (ALP) activity or immunolocalized for Ostn expression. Similar cultures were treated with 17-ß estradiol (E2; Sigma) at a physiological concentration (low dose, 10–10 M) or a saturating concentration (high dose 10–8 M).
Bone Samples
Young Developing Human Bone
Neonatal ribs were collected postmortem from six infants (three males, two females, and one of unknown sex) born at full term (30–40 weeks) who had no evidence of growth retardation or skeletal abnormalities. These samples were obtained with informed parental consent after approval by the local Research Ethics Committee. The bone was fixed overnight in neutral-buffered formalin, decalcified in 14.5% buffered EDTA, washed in PBS, and paraffin-wax embedded. Sections were cut using a base sledge microtome. All sections were mounted on 3-aminopropyltriethoxy-silane (Sigma)-coated slides. Wax-embedded sections were cleared of paraffin in two changes of xylene (12 min) and rehydrated through descending concentrations of alcohol (100%, 70%, and 50%) to PBS for histology and immunolocalization.
Adult Bone
Nine-µm sections were obtained from transverse iliac crest bone biopsies from 14 postmenopausal women. Informed written consent was obtained from all women, and the study was approved by the local Ethics Committee. Ten of these women had undergone total abdominal hysterectomy and bilateral salpingo-oophorectomy for a benign indication and had received long-term estradiol therapy. Estradiol implants (100 mg) had been inserted approximately every 6 months, on demand, for at least 14 years, although in the last 2 years the dose had been reduced to an average of 50 mg every 6 months. Control samples were from four age-matched women who were under routine clinical investigation for osteoporosis, were in good health, and were not receiving estrogen therapy or other drugs affecting bone or mineral metabolism. Biopsies were decalcified and wax embedded as above.
Alkaline Phosphatase Assay
Chambers were removed from the culture slides at the end of each culture time point. Unfixed cells were assayed for ALP activity according to the method of Bradbeer et al. (1994). Briefly, cells were incubated in sodium barbitone buffer containing 
-naphthyl acid phosphatase (Sigma), magnesium chloride, and Fast Red (Sigma) for 3 min, after which time the color reaction was stopped by washing with distilled water and the slides coverslipped in aqueous mount. Extent of positive staining was quantified by image analysis. Cultures of dermal fibroblasts were used as negative controls.
Immunolocalization
At the end of the incubation period, the medium was removed and cells rinsed in PBS, pH 7.4, fixed with 4% paraformaldehyde for 5 min at room temperature, and immunostained using an indirect immunoperoxidase method as described previously (Bord et al. 2000). Briefly, following blocking steps and washes, Ostn primary antibody (rabbit polyclonal N-terminal fragment 1:10,000) (Thomas et al. 2003) was applied and incubated overnight at 4C. A biotinylated secondary antibody (horse anti-rabbit anti-mouse; Vector Laboratories Ltd, Peterborough, UK) at 3.5 µg/ml was added, and sites of antigenicity were amplified by avidin–biotin complex (Elite ABC substrate; Vector Laboratories). Signal was detected using DAB (Vector Laboratories). Cells were lightly counterstained with Gills Haematoxylin (1:50, 45 sec; Sigma) or methyl green (1:20, 30 sec; Vector Laboratories) and air dried prior to mounting. Specificity of antibody reaction was confirmed by substituting the primary antibody with preimmune serum (1:10,000), non-immune serum (2 µg/ml), and omission of primary and secondary antibodies. Cultures of dermal fibroblasts were used as negative controls.
Sections
Paraffin-wax-embedded sections were dewaxed and taken to PBS and then immunostained as above with Ostn. Preimmune serum was used to confirm specificity of staining. Sections of colon were used as negative control tissue.
Quantitation of Immunolocalization
Cells were examined by light microscopy with a Nikon E-800 fitted with a Basler digital camera (Nikon UK Ltd; Kingston-upon-Thames, UK). Extent of staining was measured using Lucia G image analysis (Nikon UK Ltd). Thresholds were set to detect positive staining with the entire eight-well chamber slide examined for each experiment. To standardize staining and measurements, all slides in each experiment were immunolocalized at the same time at precisely timed intervals. All slides for each antibody were measured at the same time with the same threshold parameters. Thus, it was possible to compare protein expression across osteoblast cell cultures. Results are shown as the mean of the experiments (±SD).
Statistical analysis was performed using the approximate test for unequal variance based on the t distribution (Armitage and Berry 1994).
RT-PCR Analysis
Human osteoblasts derived from bone from a 6-year-old female donor were collected into RNAlater after 4 days in culture (Ambion; Huntingdon, Cambs, UK) per the manufacturer's protocol. RNA was extracted from this preparation using Trizol with glycogen (5 µg/ml) as carrier according to the manufacturer's instructions. For RT-PCR, cDNAs were generated with Superscript II reverse transcriptase (Invitrogen, Burlington, Canada) and random hexamer priming, and PCR amplification was carried out with gene-specific primers using Taq DNA polymerase (Amersham; Little Chalfont, UK). To confirm Ostn expression was not an artifact, a "No RT" control was included in which the reverse transcriptase had been omitted during the cDNA generation step. Gene-specific primers and conditions were as follows: human Ostn forward 5'-GACTGGAGATTGGCAAGTGC-3' and reverse 5'-GCCTCTGGAATTTGAAAGCC-3' (annealing temperature = 56C, 40 cycles, product 393 bp), human Phex forward 5'-GCCTTCTGCAGACACTTG-3' and reverse 5'-GATGTAGCCCAGCCAGTC-3' (annealing temperature = 53C, 35 cycles, product 398 bp).
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Results |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Developing Bone
To investigate Ostn expression in developing human bone, Ostn immunohistolocalization was performed in neonatal rib bones. Marked Ostn expression was detected in a defined and restricted pattern. Positive immunoreactivity was seen in the growth plate in some late hypertrophic chondrocytes, but no expression was detected in resting, proliferating, or early hypertrophic chondrocytes. In cancellous bone, osteoblasts on forming surfaces were highly reactive for Ostn (Figure 1A) as well as osteocytes newly incorporated into the bone matrix, whereas osteocytes entombed deeper in the matrix showed no reactivity (Figure 1A). Interestingly, at sites where formation of new osteoid appeared complete, some osteoblasts, possibly in transition to osteocytes, stained positively (Figure 1B), whereas most adjacent surrounding osteoblasts were negative. All osteoclasts, defined by their large multinucleated morphology and location at sites of bone resorption, showed absence of staining (Figure 1C). Similar to cancellous bone, osteoblasts on both the periosteal and endosteal surfaces of the bony cortical collar, as well as newly incorporated osteocytes, showed positive Ostn immunoreactivity. No staining was seen in sections of colon stained for Ostn or in bone sections stained with preimmune serum.
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Adult Bone
To investigate expression in adult bone, sections of iliac crest bone from postmenopausal women were immunolocalized with Ostn. Positive immunoreactivity was mainly confined to sites of bone formation and was generally less intense than that seen in neonatal bone sections. Biopsy sections from postmenopausal women who had received long-term, high-dose estradiol and in bone sections from age-matched, non-estrogen-treated women were also compared. The distribution pattern of staining was generally the same in the two groups of women. Within cancellous bone, the staining was predominantly in osteoblasts and newly incorporated osteocytes at bone-forming sites. However, expression at these sites was more intense in the estrogen-treated group (Figure 1D) than in the non-treated group (Figure 1E). Sections localized with preimmune serum showed absence of staining (Figure 1F). In cortical bone, Ostn immunoreactivity was evident in osteoblasts and young osteocytes on both the periosteal and endosteal surfaces. As in cancellous bone, this expression was more intense in bone from the estrogen-treated women. Within the cortex, intense staining was seen in osteoblasts within Haversian systems and in newly incorporated osteocytes (Figure 1G), whereas expression was less intense in the bone from the non-estrogen-treated women (Figure 1H). Sections treated with the preimmune serum showed absence of staining (Figure 1I). Overall, the estrogen-treated group showed a noticeable increase in the extent and intensity of Ostn expression in both cancellous bone and remodeling sites in cortical bone.
Primary Human Osteoblasts
To further investigate the role of Ostn in human bone, expression patterns were characterized during primary osteoblast differentiation. Primary human osteoblasts from young female donors were cultured for 2, 3, and 6 days in proliferating and differentiating media. After 2 days in proliferating media, intense Ostn expression was localized in the cytoplasm of 40 ± 4.1% of osteoblasts, falling to 36.2 ± 3.2% after 3 days and only evident in 19.6 ± 3.1% of cells by day 6 (p<0.02) (Figures 1J and 1K, 2A). Osteoblasts cultured in the presence of 200 nM hydrocortisone, an osteoblast-differentiating factor, showed a 1.5-fold downregulation of Ostn expression after 2 days of culture. Ostn expression further decreased 2.3-fold and 3.1-fold after 3 days and 6 days in culture, respectively (all p<0.05) (Figure 1K, 2A). In contrast, as expected, ALP expression in the same cultures increased with osteoblast differentiation. In proliferating medium, 14.3 ± 2.7% of osteoblasts stained positively at 2 days for ALP, 24.7 ± 3.1% at 3 days, and 36.7 ± 3.4% at 6 days p<0.05) (Figure 2B). Further increases in ALP expression were seen in osteoblasts cultured in the presence of hydrocortisone with 27 ± 1.5%, 33 ± 3.8%, and 46.5 ± 6.0% cells staining positively at 2, 3, and 6 days, respectively (Figure 2B).
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To investigate the effects of estrogen on Ostn expression, osteoblasts were cultured in proliferating media with either low-dose or high-dose E2. Low-dose E2 significantly downregulated Ostn levels after 3 and 6 days in culture (p<0.02) compared with untreated controls (Figure 2C); 34.8 ± 2.1% of cells stained positively for Ostn at 2 days decreasing to 29.9 ± 2.5% and 9.6 ± 0.8% at 3 and 6 days, respectively. Ostn expression in cultures treated with high-dose E2 did not change compared with untreated cultures. There was no positive staining for Ostn or ALP in cultures of dermal fibroblasts. Cells immunolocalized with Ostn preimmune serum showed absence of staining (Figure 1L).
Expression of Ostn in human osteoblasts was also confirmed by RT-PCR (Figure 2D). The osteoblastic nature of the cell population was confirmed by expression of phosphate-regulating gene with homologies to endopeptidases on the X-chromosome (Phex), a protease that is predominantly expressed in osteoblast-lineage cells (Beck et al. 1997; Ruchon et al. 1998).
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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These in vitro and in vivo studies characterize, for the first time, the localization of Ostn in cultured human osteoblasts and in developing and adult human bone. Our findings also demonstrate an association of Ostn expression levels with osteoblast differentiation.
Ostn was originally identified in osteoblasts in mouse embryonic bone (Thomas et al. 2003). In young adult rodent bone, Ostn was associated with active osteoblasts on the endosteal surface. Temporally, peak expression of Ostn was observed just after birth with expression decreasing in older bones. Accordingly, Ostn expression was also downregulated in very mature rat primary osteoblasts (Thomas et al. 2003).
Our studies with human osteoblasts concur with results seen in animal osteoblasts. We have demonstrated downregulation of Ostn expression in differentiating human primary osteoblasts both with time in culture and with hydrocortisone treatment. This was in contrast to ALP, which increased, as expected, with differentiation (Lian et al. 2004). Upregulation of ALP expression is coupled to downregulation of proliferation with low levels in early proliferating osteoblasts, which increase rapidly with osteoblast differentiation and matrix maturation (Lian et al. 2004). Thus, Ostn shows the reverse pattern of expression to ALP in human osteoblasts. We have previously demonstrated that hydrocortisone at normal physiological concentrations increases the rate of cellular differentiation in cultured human osteoblasts (Ireland et al. 2004). Thus, the results reported here showing increasing ALP expression with differentiation and associated decreasing Ostn expression is consistent with Ostn as a marker of less-differentiated osteoblasts.
In vivo, our demonstration of Ostn expression at sites of bone formation in developing human neonatal bone supports the findings in rodent bone. Ostn expression was most prominent in young active osteoblasts, again suggesting Ostn as a marker of early osteoblast maturation. The pattern of Ostn expression in adult human bone was similar to that seen in developing human bone, although the intensity and extent of staining was less. To assess Ostn expression in adult human bone with known anabolic skeletal activity, we examined iliac crest bone biopsies from a cohort of women treated with high-dose estradiol and compared Ostn expression patterns to those in age-matched, untreated women. Sections from the estrogen-treated women showed more intense and extensive Ostn staining than those from the untreated women, particularly at sites of bone formation. Previously, we have demonstrated anabolic skeletal effects in this estrogen-treated group (Vedi et al. 1999; Bord et al. 2001), with increased osteoblastic activity.
In contrast, inhibitory effects of lower-dose estrogen were observed on osteoblasts in vitro, consistent with the recent evidence suggesting that estrogen-mediated regulation of osteoblast function is determined by the stage of differentiation, estrogen receptor isoform expression, and estrogen concentration (Waters et al. 2001; Ireland et al. 2002; Bord et al. 2004). Bone-derived cells from old donors contain a high proportion of mature cells, whereas young donor-derived osteoblasts have a greater proportion of immature proliferative cells (Marie 1994). To establish the effects of estrogen on Ostn expression, we have used young donor cells to be more representative of the total osteoprogenitor and osteoblastic cell population present in the bone marrow and bone of postmenopausal women. The downregulation of Ostn by estrogen is relatively slow and occurs only at the low dose. This suggests that estrogen does not have direct effects on Ostn via an estrogen-responsive element but rather acts through the secondary mechanism of altering osteoblast differentiation/activity. In vivo and in vitro differences according to differentiation in regulation of skeletal tissues and cells by steroid hormones such as vitamin D have been well documented (Haussler et al. 1998; Issa et al. 1998).
Although Ostn expression in our in vivo studies was confined mainly to osteoblasts and osteocytes, some late hypertrophic chondrocytes were also immunoreactive for Ostn. In mice and rats, Ostn is also expressed by a subset of mesenchymal tissues such as tendon, ligament, and muscle (Thomas G, unpublished data). A recent report identified Musclin, a muscle-derived secretory factor that is identical to Ostn, in mouse muscle (Nishizawa et al. 2004). However, in this study Ostn was not expressed in muscle cells adjacent to bone; thus, it remains to be established if this disparity is due to species, age, or tissue differences. This newly identified gene is likely to be more widely expressed than currently known. Within the bone marrow a small subset of cells stained positively for Ostn. It is currently not known if these are osteoblast precursors or a different cell type.
Interestingly, at some sites osteoblasts apparently at the point of incorporation into the bone matrix as osteocytes stained positively for Ostn, whereas surrounding osteoblasts showed no staining, possibly suggesting that Ostn could be a transdifferentiation factor. This immunoreactivity was maintained in these newly incorporated osteocytes but was lost as osteocytes became further embedded. This was in contrast to the majority of osteoblasts where expression was downregulated with differentiation, suggesting a retention of Ostn expression in a small subset of osteoblasts that could be involved in their transition to osteocytes. Currently, there is speculation about potential transdifferentiation factors. Recent evidence suggests that transforming growth factor ß, matrix metalloproteinase 14, and sclerostin (Winkler et al. 2003) may act as mediators in this process, either individually or in concert (Karsdal et al. 2002,2004).
In summary, these preliminary investigations demonstrate that Ostn is expressed in human osteoblasts and bone. It appears to be a novel osteoblast marker particularly prominent in developing bone and at sites of remodeling; the downregulation of Ostn by hydrocortisone suggests that synthesis may be linked to differentiation. Further studies investigating the association of Ostn expression with osteoblast activity in a wider age range of subjects and alongside other markers of the osteoblast phenotype may extend our knowledge of the role of Ostn in bone remodeling and establish whether Ostn is implicated in skeletal pathologies such as osteoporosis.
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Acknowledgments |
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The Wellcome Trust funds S.B. and D.C.I.
The authors are grateful to Mr. Per Hall and Mr. Rod Laing for supply of bone tissue.
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Footnotes |
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Received for publication October 26, 2004; accepted May 4, 2005
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