- Email: [email protected]
Changes in osteocalcin response to 1,25-dihydroxyvitamin D3 stimulation and basal vitamin D receptor expression in human osteoblastic cells according to donor age and skeletal origin
Bone Vol. 29, No. 1 July 2001:35– 41
Changes in Osteocalcin Response to 1,25-Dihydroxyvitamin D3 Stimulation and Basal Vitamin D Receptor Expression in Human Osteoblastic Cells According to Donor Age and Skeletal Origin P. MARTI´NEZ,1 I. MORENO,1 F. DE MIGUEL,2 V. VILA,3 P. ESBRIT,2 and M. E. MARTI´NEZ1 1
Biochemistry Division, Hospital La Paz, Madrid, Spain Bone and Mineral Metabolism Laboratory, Research Unit, Fundacio´n Jime´nez Dı´az, Madrid, Spain 3 Centro de Biologı´a Molecular, Consejo Superior de Investigaciones Cientı´ficas, Madrid, Spain 2
Key Words: Human osteoblastic (hOB) cells; Skeletal site; Aging; 1,25-Dihydroxyvitamin D3; [1,25(OH)2D3]; Osteocalcin; Vitamin D receptor (VDR).
Age-related osteopenia is known to occur differently throughout the skeleton. In the present study, we examine the influence of donor age (<50 and >50 years), and bone structure (cortical vs. trabecular) on osteocalcin and vitamin D receptor (VDR) expression in primary cultures of human osteoblastic cells (hOB) cells. Cells were isolated from trabecular bone samples obtained from donors undergoing either knee (mainly trabecular) (n ⴝ 22; 4 <50 years, 18 >50 years) or hip (mainly cortical) (n ⴝ 16; 6 <50 years, 10 >50 years) arthroplasty. Pooling the results from knee and hip hOB cell cultures, we found that secreted osteocalcin was higher in hOB cells from the younger donors, compared with that in older donors, and peaked after stimulation with 10ⴚ6–10ⴚ8 mol/L 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In cells from the latter donors, this secretion was maximal after 10ⴚ6 mol/L 1,25(OH)2D3 treatment. On the other hand, using reverse transcription followed by polymerase chain reaction, baseline osteocalcin mRNA was found to be lower in hOB cells from the older donors than in those from younger donors. After treatment with 10ⴚ6–10ⴚ8 mol/L 1,25(OH)2D3, osteocalcin mRNA increased over baseline in all groups of hOB cells studied. In age-matched cultures, both basal and 10ⴚ6–10ⴚ8 mol/L 1,25(OH)2D3-stimulated osteocalcin mRNA showed similar values in hOB cells from both skeletal sites in younger donors. However, in the older donors, baseline as well as 10ⴚ8 mol/L 1,25(OH)2D3-stimulated osteocalcin mRNA were higher in knee hOB cells than in hip hOB cells. Furthermore, baseline VDR mRNA expression was also higher in the former cells than in the latter cells in the older group. Considering the influence of donor age at each skeletal site of origin, we found lower baseline osteocalcin and VDR mRNA levels in hip hOB cells in the older group than in the younger group. Our findings indicate that the response of osteocalcin secretion and its mRNA to physiological doses of 1,25(OH)2D3 decreases with aging and is associated with decreased VDR mRNA expression in hOB cells from mainly cortical bone. (Bone 29:35– 41; 2001) © 2001 by Elsevier Science Inc. All rights reserved.
Introduction Bone loss with aging is a well-known event, although the underlying mechanisms of age-related osteopenia are still unclear.39 Histological findings have shown a sharp decrease in bone-forming surfaces compared with bone-resorbing areas with aging,37 which is thought to reflect an osteoblastic inability to reconstitute resorbed bone.24 Thus, age-associated bone loss might be explained, at least in part, by a decrease in osteoblastic cell proliferation, which could be related to an increased osteoblastic maturation.31 The process of aging is known to follow a different pattern throughout the skeleton.5,11,36 Therefore, bone loss occurs mainly in the hip in older subjects, whereas it occurs in the lumbar area in postmenopausal women.11,36 Histomorphometric studies have shown differences in bone remodeling depending on skeletal sites,36 and it has been hypothesized that these differences are associated with variations in osteoblastic differentiation at different skeletal sites. Treatment with vitamin D metabolites has been shown to increase bone mineral density, depending on the subject’s age and bone type.12 We previously found that cultured human osteoblastic cells from trabecular knee areas appear to be less differentiated than those from trabecular bone of the hip.31 Other studies in human osteoblastic cell populations have also demonstrated differences in cell phenotype, cell response to calciotropic hormones, and androgen receptor expression, depending on the skeletal site of origin.25,26,32 These differences could be related to the variable trabecular/ cortical bone ratio at different skeletal sites throughout the skeleton, and that it is higher at the knee compared with the hip.16 Osteoblastic differentiation is a multistep development process, characterized by the ordered expression of genes related to bone growth and differentiation. The osteocalcin gene is one of the marker genes for the progression of osteoblastic differentiation.42 However, the specific role of osteocalcin in bone metabolism remains unclear. Osteocalcin is thought to be a key factor for bone mineralization and bone resorption by enhancing the recruitment and differentiation of osteoclast precursors.13,23 This
Address for correspondence and reprints: Dr. M. E. Martı´nez, Hospital La Paz C/Paseo de la Castellana 261, 28046 Madrid, Spain. E-mail: [email protected] © 2001 by Elsevier Science Inc. All rights reserved.
35
8756-3282/01/$20.00 PII S8756-3282(01)00479-3
36
P. Martinez et al. Differences in osteocalcin response to 1,25(OH)2D3
is consistent with a possible role for osteocalcin in the coupling of bone formation and resorption.42 On the other hand, knockout mice used for study of the osteocalcin gene showed increased bone formation and unaltered bone resorption.18 In osteoblast, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] regulates cell proliferation and differentiation through the vitamin D receptor (VDR).33,41 Thus, osteocalcin gene expression is regulated by 1,25(OH)2D3 through its binding to the VDR, followed by translocation of the 1,25(OH)2D3/VDR complex to the nucleus, where it interacts with vitamin D response elements in the osteocalcin gene.14,28 After stimulation with 1,25(OH)2D3, an increase in osteocalcin mRNA and protein has been reported in human bone,9,40,43 and in rat and chicken osteoblastic cells.22,33 However, this metabolite decreases osteocalcin gene expression in mouse osteoblastic cells.9 Thus, variation in vitamin D regulation of the osteocalcin gene seems to occur among different species.9 An age-associated decline in the total content of osteocalcin in human bone has previously been reported.6 Furthermore, transgenic mice overexpressing the rat osteocalcin promoter in bone have shown a decrease of this promoter activity in osteoblastic cells with aging.20 Interestingly, it is well known that vitamin D deficiency occurs with aging. Because VDR protein levels appear to be regulated by 1,25(OH)2D3,38 vitamin D deficiency might explain the decreased VDR-dependent osteocalcin synthesis by osteoblasts. However, the relationship of this feature to putative osteocalcin changes in different bone types with aging is presently unknown. We recently found differences in the response of osteocalcin to 10⫺8 mol/L 1,25(OH)2D3 in osteoblastic (hOB) cells from human trabecular bone, depending on donor age and skeletal origin.32 In this regard, a diminished response of another vitamin D-dependent protein, calbindin D, to 1,25(OH)2D3 has recently been reported to occur with aging in the rat duodenum.2 The present study evaluates the possible changes in osteocalcin mRNA and its protein secretion after stimulation with different 1,25(OH)2D3 concentrations in hOB cells, as they relate to donor age and skeletal site of origin. We also assessed VDR mRNA changes as a function of skeletal site and donor age because the stimulating effects of 1,25(OH)2D3 on osteocalcin are mediated through this receptor in these cells. Thus, this DR content could provide a way to assess these cells’ ability to respond or not to l,25(OH)2D3-stimulation. Materials and Methods Subjects Osteoblastic cells were isolated from human trabecular bone fragments from 38 osteoarthritic donors ⬍70 years of age (15 men and 23 women), who were undergoing knee or hip replacement surgery. Twenty-two of these bone samples were obtained from subjects during knee arthroplastia. Four of these subjects were ⬍50 years of age (48 ⫾ 1 years), whereas 18 were ⬎50 years of age (64 ⫾ 5 years). The other 16 bone specimens were obtained from subjects during hip arthroplastia. Six of these patients were aged ⬍50 years of age (46 ⫾ 4 years), and 10 were from donors ⬎50 years of age (67 ⫾ 2 years). These subjects had no clinical symptoms or history of bone metabolic disorders. The study was approved by the local ethics commitee. Cell Culture Trabecular bone fragments from either the knee or hip, which would otherwise have been discarded, were cultured as previously described.31,32 Briefly, the samples were minced into
Bone Vol. 29, No. 1 July 2001:35– 41
pieces 0.3– 0.5 cm in diameter, and then washed thoroughly with phosphate-buffered saline to remove adherent bone marrow cells. The fragments were cultured in Dulbecco’s modified Eagle medium containing 4.5 g/L glucose, 15% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 g/mL streptomycin, in a humidified atmosphere with 5% CO2 at 37°C. Confluent cells were subcultured at a density of 104 cells/cm2, and again grown to confluence. These hOB cells display various osteoblastic features, such as alkaline phosphatase and type I collagen secretion, and 1,25(OH)2D3-stimulated osteocalcin secretion.31,32 Cells grown as just described were cultured for 72 h in FBS-free medium with 1 g/L glucose, 10 nmol/L vitamin K, 50 g/mL ascorbic acid, and 0.1% bovine serum albumin (BSA), in the presence or absence of different concentrations of 1,25(OH)2D3 (kindly provided by Roche, Basel, Switzerland). After this time period, the medium was removed and immediately frozen at ⫺20°C. The cells were washed in saline and frozen at ⫺80°C, until total RNA extraction. Osteocalcin Assay Osteocalcin in the cell-conditioned medium was measured by an immunoradiometric assay (Nichols, San Juan Capistrano, CA), using specific antibodies against intact human osteocalcin. The sensitivity of this assay is 0.05 ng/mL. Intra- and interassay variation coefficients were ⬍10%. Osteocalcin secretion was expressed as a ratio over the cell protein concentration, as measured by the Bicinchoninic Acid (BCA) protein assay (Pierce, Rockford, IL), using BSA as standard. Cell protein was extracted from the organic phase obtained following the Trizol method (Gibco, Grand Island, NY) for RNA extraction, according to the manufacturer’s instructions. Osteocalcin in the cell extracts was measured by the aforementioned immunoradiometric assay. Osteocalcin and VDR mRNA Total RNA was extracted from the aqueous phase obtained by the Trizol method, following the manufacturer’s instructions. About 12 g of total RNA was isolated from each 25 cm2 flask containing confluent hOB cells. Osteocalcin and VDR mRNA levels were assayed by reverse transcription followed by semiquantitative polymerase chain reaction (RT-PCR), using conditions providing submaximal amplification.15 Total RNA (0.1–1 ng for osteocalcin, 5 ng for VDR) was added to a reaction mixture containing: 1 mmol/L MgSO4; 0.2 mmol/L dinitrophenolphosphate (dNTP); 1 U of Myeloblastosis Virus Avian reverse transcriptase; 1 U of thermostable DNA polymerase from Thermus flavus (Access RT-PCR System, Promega, Madison, WI); 1 mol/L of specific primers 5⬘-CATGAGAGCCCTCACACTCC-3⬘ (sense) and 5⬘-CAGCAGAGCGACACCCTAGACC-3⬘ (antisense) (human osteocalcin), corresponding to nucleotides 18 –37 and 315–336, respectively, in human osteocalcin (Gene Bank Accession No. X 51699); and 5⬘-GGAAGTGCAGAGGAAGCGGGAGATG-3⬘ (sense) and 5⬘-AGAGCTGGGACAGCTCTAGGGTCAC-3⬘ (antisense) (human VDR), corresponding to nucleotides 344 – 368 and 699 –723 nucleotides, respectively, in the human VDR gene (Gene Bank Accession No. X 67482). Using these primers, PCR amplification yielded 319 bp and 380 bp products, respectively. The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was coamplified, using specific primers for the human gene, as a constitutive control.21 Total RNA and the primers were preincubated for 5 min at 65°C. The reaction mixture (10 L) was then incubated for 45 min at 48°C, and 2 min at 95°C, followed by 35 cycles of 1 min
Bone Vol. 29, No. 1 July 2001:35– 41
P. Martinez et al. Differences in osteocalcin response to 1,25(OH)2D3
37
at 95°C, 1 min at 60°C, and 2 min at 68°C, with a final extension of 7 min at 68°C. The PCR products were separated on 2% agarose gels. Bands were visualized by ethidium bromide staining and quantified by densitometric scanning (1D Manager, Madrid, Spain). Osteocalcin and VDR densitometric values were normalized against those of the corresponding GAPDH product. The identity of the PCR products was confirmed by sequencing with a dye-terminator cycle-sequencing kit (Perkin-Elmer, Norwalk, CT), using Taq FS-DNA polymerase. Sequences were resolved on an ABI-Prism 377 automatic sequencer. Statistical Analysis All data are expressed as mean ⫾ SE. Differences between values from basal and 1,25(OH)2D3-treated cell cultures were analyzed by the nonparametric paired Wilcoxon test. Differences between the values of two different groups of hOB cell cultures were analyzed by the unpaired Mann–Whitney U-test. Results Time Course of Osteocalcin Secretion in Human Osteoblastic Cells Preliminary time-course experiments using eight hOB cell cultures, four from the hip and four from the knee, were obtained from ⬍50- or ⬎50-year-old donors. We found that baseline osteocalcin secretion increased during the time period studied: 2.7 ⫾ 1 ng/mg protein at 24 h; 5.7 ⫾ 2.6 at 48 h; and 15 ⫾ 3 at 72 h (p ⬍ 0.05 between values at 48 h and 72 h, and that at 24 h). A similar time-course pattern for osteocalcin secretion was found after treating these cell cultures with 1,25(OH)2D3. Thus, osteocalcin secretion values were 8.3 ⫾ 1.8 at 24 h, 46.3 ⫾ 6.2 at 48 h and 74.4 ⫾ 5.1 at 72 h after stimulation with 10⫺8 mol/L 1,25(OH)2D3, or 22.4 ⫾ 11.0 at 24 h, 89.7 ⫾ 29.1 at 48 h, and 115.4 ⫾ 31.8 at 72 h after stimulation with 10⫺6 mol/L 1,25(OH)2D3 [p ⬍ 0.05 between values at 48 h and 72 h, and that at 24 h for each 1,25(OH)2D3 concentration]. These results indicate that the levels of osteocalcin secretion after 1,25(OH)2D3 stimulation were higher at 72 h in these cell cultures, and therefore further studies on osteocalcin secretion were carried out during this time period. Dose-dependent Effect of 1,25(OH)2D3 on Osteocalcin Secretion in Human Osteoblastic Cells We found that treatment with 10⫺4–10⫺10 mol/L 1,25(OH)2D3 induced a biphasic response of osteocalcin secretion in all groups of hOB cells studied. Thus, secreted osteocalcin increased dose dependently between 10⫺6 and 10⫺10 mol/L, but decreased thereafter at 10⫺4 mol/L 1,25(OH)2D3 in these cells (Figure 1). Influence of Donor Age and Skeletal Site of Origin on 1,25(OH)2D3-stimulated Osteocalcin Secretion in Human Osteoblastic Cells ⫺6
Figure 1. Secreted osteocalcin response to different 1,25(OH)2D3 concentrations: 10⫺10 mol/L (Vit. D-10); 10⫺8 mol/L (Vit. D-8); 10⫺6 mol/L (Vit. D-6); and 10⫺4 mol/L (Vit. D-4), in hOB cells from either different skeletal site of origin (hip or knee) (A) or donor age (⬍50 years and ⬎50 years) (B). Osteocalcin secretion was measured in the medium conditioned by hOB cells isolated from bone explants from adult donors, before (basal) and after stimulation with different 1,25(OH)2D3 concentrations for 72 h. Results are mean ⫾ SE. *p ⬍ 0.05 vs. basal condition; # p ⬍ 0.05 between Vit. D-8 and Vit. D-6 stimulation; ap ⬍ 0.05, between ⬍50 year- and ⬎50-year-old donors, after stimulation with Vit. D-8.
these cells in the younger group (Table 1). On the other hand, cell osteocalcin content was significantly higher after treatment with 10⫺6 mol/L 1,25(OH)2D3, compared with 10⫺8 mol/L in these cells in the older group (Table 1). We also considered the influence of skeletal site of origin on 1,25(OH)2D3-stimulated osteocalcin secretion. We found that this secretion reached a maximum value after 10⫺6–10⫺8 mol/L 1,25(OH)2D3 treatment in knee hOB cells (Figure 1B). In conTable 1. Osteocalcin cell content in human osteoblastic (hOB) cells
⫺8
We found maximum osteocalcin secretion after 10 –10 mol/L 1,25(OH)2D3 treatment in the younger group (Figure 1A). However, in the older group, this maximum was reached after 10⫺6 mol/L 1,25(OH)2D3 stimulation (Figure 1A). Furthermore, cell osteocalcin content, assessed in some of these hOB cell cultures from both age groups, was also found to be significantly increased after both 10⫺8 mol/L and 10⫺6 mol/L 1,25(OH)2D3 treatment, compared with the respective baseline value (Table 1). Furthermore, we found that this content was similar after stimulation with either 10⫺6 or 10⫺8 mol/L 1,25(OH)2D3 in
⬍50 years ⬎50 years
Basal
1,25(OH)2D3 (10⫺8 mol/L)
1,25(OH)2D3 (10⫺6 mol/L)
0.7 ⫾ 0.2 0.3 ⫾ 0.2
8.0 ⫾ 6.1a 2.8 ⫾ 1.5a,b
12.8 ⫾ 7.2a 5.0 ⫾ 2.3a
Cell osteocalcin protein (ng/mg protein) after 10⫺8 or 10⫺6 mol/L 1,25(OH)2D3 treatment in hOB cells in relation to donor age (⬍50 years and ⬎50 years). Osteocalcin was measured in hOB cell extracts before (baseline) and after 1,25(OH)2D3 stimulation for 72 h. Results are mean ⫾ SE (n ⫽ 6). a p ⬍ 0.05 vs. baseline; bp ⬍ 0.05 between 10⫺8 and 10⫺6 mol/L 1,25(OH)2D3 treatment.
38
P. Martinez et al. Differences in osteocalcin response to 1,25(OH)2D3
Bone Vol. 29, No. 1 July 2001:35– 41
Figure 2. Effect of 1,25(OH)2D3 on osteocalcin mRNA in hOB cells, in relation to donor age (⬍50 years and ⬎50 years) and skeletal site of origin (knee or hip). Osteocalain mRNA expression was measured by semiquantitative RT-PCR in hOB cells before (basal) and after stimulation with 10⫺8 mol/L (Vit. D-8) or 10⫺6 mol/L (Vit. D-6) 1,25(OH)2D3 for 72 h. Results are mean ⫾ SE. *p ⬍ 0.05, **p ⬍ 0.001 vs. basal condition; # p ⬍ 0.05, between vit. D-8 and vit. D-6 stimulation; bp ⬍ 0.05, between hip and knee in age-matched donors; ap ⬍ 0.001, and cp ⬍ 0.05, between ⬍50 years and ⬎50 years old donors at the knee and the hip, respectively. Inset: Representative gel corresponding to a hip hOB cell culture in the older group. Cells were treated with or without vit. D-8 and vit. D-6 for 72 h.
trast, the concentration of 1,25(OH)2D3 that induced maximal osteocalcin secretion was shifted to 10⫺6 mol/L in hip hOB cells (Figure 1B). Influence of Donor Age on Osteocalcin and Vitamin D Receptor Depending on Skeletal Site of Origin in Human Osteoblastic Cells We further analyzed the influence of donor age, as it relates to the skeletal site of origin, on osteocalcin mRNA and protein secretion in hOB cells. We found that baseline osteocalcin mRNA was significantly higher in hOB cells in the younger group than in the older group, at both skeletal sites (Figure 2). Treatment with either 10⫺6 or 10⫺8 mol/L 1,25(OH)2D3 induced a significant and similar increase over baseline in osteocalcin mRNA in knee hOB cells in both age groups studied (Figure 2). However, in hip hOB cells from the older group, osteocalcin mRNA expression increased to a higher value after treatment with 10⫺6 mol/L 1,25(OH)2D3 than with 10⫺8 mol/L 1,25(OH)2D3 (Figure 2 and inset). Furthermore, baseline VDR mRNA expression was similar in knee hOB cells in both age groups studied (Figure 3), but was higher in hip hOB cells from the younger donors than in these cells in the older group (Figure 3). Osteocalcin secretion also increased to a similar value after both 10⫺6 and 10⫺8 mol/L 1,25(OH)2D3 at both skeletal sites in the younger group (Table 2). However, in older donors, this secretion was significantly higher after treatment with 10⫺6
mol/L 1,25(OH)2D3 than with 10⫺8 mol/L of this metabolite at both skeletal sites (Table 2). Influence of Skeletal Site of Origin on Age-associated Changes in Osteocalcin and Vitamin D Receptor in Human Osteoblastic Cells The influence of skeletal site on 1,25(OH)2D3-stimulated osteocalcin was then evaluated in age-matched donors. In the older group, baseline and 10⫺8 mol/L 1,25(OH)2D3-stimulated osteocalcin mRNA levels were higher in knee hOB cells than in these cells from the hip, but the levels were similar after 10⫺6 mol/L 1,25(OH)2D3 stimulation at both skeletal sites (Figure 2). No significant differences were found in baseline levels of both osteocalcin and VDR mRNA in hOB cells at both skeletal sites in the younger group (Figures 2 and 3). However, VDR mRNA expression was significantly higher in knee hOB cells than in these cells from the hip in the older donors (Figure 3). Osteocalcin secretion was also significantly higher in knee hOB cells than in these cells from the hip after either 10⫺6 or 10⫺8 mol/L 1,25(OH)2D3 stimulation, but only in the older group (Table 2). Discussion The present study extends our previous studies31,32 by evaluating the influence of age and skeletal site of origin (hip or knee) on osteocalcin, protein, and mRNA in response to different concen-
Figure 3. Differences in basal VDR mRNA levels in relation to donor age (⬍50 years and ⬎50 years) and skeletal site of origin (hip or knee). VDR mRNA levels were measured by semiquantitative RT-PCR in hOB cells after 72 h in serum-free medium. Results are mean ⫾ SE. *p ⬍ 0.001, between subjects ⬍50 years and those ⬎50 years, at the hip; **p ⬍ 0.05, between hip and knee from age-matched donors.
Bone Vol. 29, No. 1 July 2001:35– 41
P. Martinez et al. Differences in osteocalcin response to 1,25(OH)2D3
Table 2. Osteocalcin secretion in human osteoblastic (hOB) cell cultures
Basal Knee ⬍ 50 ⬎ 50 Hip ⬍ 50 ⬎ 50
1,25(OH)2D3 (10⫺8 mol/L)
years years
7.3 ⫾ 3.0 5.2 ⫾ 2.1
43.8 ⫾ 15.2a 60.4 ⫾ 10.6a
years years
8.0 ⫾ 4.2 3.1 ⫾ 1.0
55.2 ⫾ 19.4a 27.1 ⫾ 9.0a,c
1,25(OH)2D3 (10⫺6 mol/L) 99.0 ⫾ 31.2a 120.3 ⫾ 23.0a,b 88.5 ⫾ 19.0a 55.2 ⫾ 18.0a,b,c
Osteocalcin secretion (ng/mg protein) at baseline and after 10⫺8 or 10⫺6 mol/L 1,25(OH)2D3 stimulation in hOB cells in relation to donor age (⬍50 years and ⬎50 years) and skeletal site of origin (hip or knee). Osteocalcin secretion was measured in the cell-conditioned medium before (basal) and after stimulation with 1,25(OH)2D3 for 72 h. Results are mean ⫾ SE (n ⫽ 4 –18). a p ⬍ 0.05 vs. basal; bp ⬍ 0.05 between 10⫺8 and 10⫺6 mol/L 1,25(OH)2D3 treatment; cp ⬍ 0.05 between different skeletal sites of origin (hip or knee) in age-matched donors.
trations of 1,25(OH)2D3 in hOB cells. We also assessed baseline VDR mRNA expression in these cells. We used hOB cells isolated from explants of trabecular bone at each skeletal site because this appears to be a potent tool for addressing issues related to skeletal pathology and aging.8,30 Our findings show that osteocalcin secretion, and also cell osteocalcin protein content, was similar after stimulation with 10⫺6–10⫺8 mol/L 1,25(OH)2D3 in these cells in younger donors (⬍50 years). Human OB stimulation with a higher 1,25(OH)2D3 concentration was found to decrease osteocalcin secretion in both age groups studied. These results differ somewhat from those of Beresford et al.,3 who found maximal osteocalcin response after stimulation with 10⫺7–10⫺9 mol/L 1,25(OH)2D3, decreasing at 10⫺6 mol/L of this metabolite, in human-derived osteoblastic cell cultures. However, in their study, only two subjects, aged 2 and 17 years, were the source of osteoblastic cells, whereas our donors were older and more numerous. In addition, we found that peak osteocalcin secretion, and its cell protein content, shifted to a higher concentration (10⫺6 mol/L) of 1,25(OH)2D3 at both skeletal sites in the older group compared with those in the younger group. Thus, the present findings, and those of Beresford et al.,3 are consistent with the hypothesis that higher 1,25(OH)2D3 concentrations are needed to induce maximal production of osteocalcin by osteoblasts in aged human subjects.19,35,45 Moreover, a decrease in circulating osteocalcin, as well as its content in both cortical and trabecular bone, occurs with age.6,44 We also found that baseline osteocalcin mRNA was higher in hOB cells in younger donors than in older donors at both skeletal sites. Our findings are consistent with a recent report by Frenkel et al.,20 showing that rat osteocalcin promoter activity overexpressed in the bone of mice decreases with aging. These results are also in agreement with Katzburg et al.27 who showed a decrease in osteocalcin secretion with age between pre- and postmenopausal women from human bone-derived cell cultures. A previous report showed differences in bone osteocalcin content, according to differences in trabecular bone composition at various skeletal sites.1 We therefore assessed the osteocalcin response to 1,25(OH)2D3 in hOB cells from explants of trabecular bone at the knee or hip, with the latter showing a higher cortical/trabecular ratio16 in age-matched donors. We found that both osteocalcin secretion and gene expression levels were similar after stimulation with 10⫺6–10⫺8 mol/L of 1,25(OH)2D3 in knee or hip hOB cells in the younger group, but were higher after 10⫺6 mol/L of 1,25(OH)2D3 than after 10⫺8 mol/L in hip hOB
39
cells of older donors. Thus, hOB cells from the hip, a predominantly cortical bone, require a higher 1,25(OH)2D3 concentration to reach an osteocalcin response similar to that observed in hOB cells from the knee, which is mainly trabecular bone. These findings are consistent with those of a clinical study indicating that age-related bone loss was higher in predominantly cortical bone than in predominantly trabecular bone in vitamin D-deficient subjects,4 and that vitamin D supplements decreased the relative risk of fracture mainly in cortical bone in aging subjects.7 In the present study, we found lower baseline osteocalcin mRNA levels in hOB cells from each skeletal site in older donors. Our findings also show that the increment of increase in osteocalcin mRNA after of 1,25(OH)2D3 stimulation was inversely related to its baseline mRNA, as previously shown to occur in other osteoblastic cell types at various developmental stages of the osteoblast phenotype.33,34 Taken together, the aforementioned findings suggest that a less efficient 1,25(OH)2D3-induced response of osteocalcin mRNA occurs with aging in a predominantly cortical type of bone. Other investigators also described a decrease in the number of osteogenic progenitors with aging in a predominantly cortical type of bone.17,29 Our previous studies have shown that hOB cells from the knee, and in younger subjects, display less differentiated features, which could be related to the presence of a higher content of osteoprogenitor cells in these cultures, compared with those in cells from the hip, and in older subjects.31,32 In this study, knee hOB cells, and those in the younger donors, showed higher osteocalcin mRNA levels. Frenkel et al.,20 using transgenic mice overexpressing the rat osteocalcin promoter in bone, found that the activity of this promoter remains absent in osteoprogenitor cells isolated from bone stroma until they begin to differentiate into osteoblasts. Therefore, although the different types of bone used in this study to obtain various hOB cell cultures originally had a variable content of stromal cells, these cells appear to have been differentiated into osteoblastic cells in our experimental setting. Consistent with our osteocalcin results, we observed a decrease in baseline VDR mRNA with aging in the more differentiated hOB cells from the hip. This is also consistent with previous findings in orofacial bone showing that VDR expression, using in situ hybridation, increases with osteoblast differentiation.10 These results could be explained by differences in VDR gene expression levels in the different bone types used in both studies. Also in agreement with the osteocalcin results described herein, in the older donors, we found lower VDR mRNA levels in hip hOB cells than in those from the knee. Thus, VDR expression appears to differ depending on bone type, which is consistent with previous findings for androgen receptors in other hOB cell populations.25 In summary, we demonstrated that the more differentiated hOB cells from the hip (predominantly cortical bone) in older subjects show a lower response of osteocalcin gene expression and osteocalcin secretion to 1,25(OH)2D3. Our findings also support the hypothesis that age-related cortical bone loss in vitamin D deficiency occurs in association with decreased VDR gene expression.
Acknowledgments: This work was supported by grants from the Spanish Fondo de Investigacio´n Sanitaria (FIS 97/0307 and 00/0125) and FAES (Madrid, Spain). The authors thank the medical staff of the Orthopaedic Department, Hospital La Paz, for providing us with the bone samples. We also thank Mark S. Davis for proofreading the manuscript.
40
P. Martinez et al. Differences in osteocalcin response to 1,25(OH)2D3
Bone Vol. 29, No. 1 July 2001:35– 41
References 1. Aerssens, J., Boonen, S., Joly, J., and Dequeker, J. Variations in trabecular bone composition with anatomical site and age: Potential implications for bone quality assessment. J Endocrinol 155:411– 421; 1997. 2. Armbrecht, H. J., Boltz, M. A., Christakos, S., and Bruns, M. E. Capacity of 1,25-dihydroxyvitamin D to stimulate expression of calbindin D changes with age in rats Arch Biochem Biophys 352:159 –164; 1998. 3. Beresford, I. N., Gallagher, J. A., Poser, J. W., and Russell, R. G. G. Production of osteocalcin by human bone cells in vitro. Effects of 1,25(OH)2D3, 24,25(OH)2D3, parathyroid hormone, and glucocorticoids. Metab Bone Dis Rel Res 5:229 –234; 1984. 4. Bettica, P., Bevilacqua, M., Vago, T., and Norbiato, G. High prevalence of hypovitaminosis D among free-living postmenopausal women referred to an osteoporosis outpatient clinic in northern Italy for initial screening. Osteopor Int 9:226 –229; 1999. 5. Bonjour, J. P., Theintz, G., Buchs, B., Slosman, D., and Rizzoli R. Critical years and stages of puberty for spinal and femoral bone mass accumulation during adolescence. J Clin Endocrinol Metab 73:555–563; 1991. 6. Boonen, S., Aerssens, J., Dequeker, J., Nicholson, P., Chen, X., Lowet, G., Verbeker, G., and Bouillon, R. Age-associated decline in human femoral neck cortical and trabecular content of insulin-like growth factor I: Potential implications for age-related (type II) osteoporotic fracture ocurrence. Calcif Tissue Int 61:173–178; 1997. 7. Chapuy, M. C., Arlot, M., Duboeut, F., Brun, J., Crouzet, B., Arnaud, S., Delmas, P. D., and Meunier, P. J. Vitamin D3 and calcium to prevent hip fracture in elderly women. N Engl J Med 327:1637–1642, 1992. 8. Chavassieux, P. M., Chenu, C., Valentin-Opran, A., Merle, B., Delmas, P. D., Hartmann, D. J., Saez, S., and Meunier, P. J. Influence of experimental conditions on osteoblast activity in human primary bone cell cultures. J Bone Miner Res 5:337–343; 1990. 9. Clemens, T. L., Hui, T. Maeda, S., Kesterson, R. A., Demayo, F., Pike, J. W., and Gundberg, C. A. Analysis of osteocalcin expression in transgenic mice reveals a species difference in vitamin D regulation of mouse and human osteocalcin genes. J Bone Miner Res 12:1570 –1576; 1997. 10. Davideau, J. L., Papagerakis, P., Hotton, D., Lezot, F., and Berdal, A. In situ investigation of vitamin D receptor, alkaline phosphatase, and osteocalcin gene expression in oro-facial mineralized tissues. Endocrinology 137:3577–3585; 1996. 11. Davis, J. W., Ross, P. D., and Wasnich, R. D. Evidence for both generalized and regional low bone mass among elderly women. J Bone Miner Res 9:305–309; 1994. 12. Dawson-Hughes, B., Dallal, G. E., Krall, E. A., Harris, S., Sokoll, L. J., and Falconer, G. Effect of vitamin D supplementation on wintertime and overall bone loss in healthly postmenopausal women. Ann Intern Med 115:505–512; 1991. 13. Defranco, D. J., Glowacki, J., Cox, K. A., and Lian, J. B. Normal bone particles are preferentially resorbed in the presence of osteocalcin-deficient bone particles in vivo. Calcif Tissue Int 49:43–50; 1991. 14. Demay, M. B., Gerardi, J. M., DeLuca, H. F., and Kronenberg, H. M. DNA sequences in the rat osteocalcin gene that bind the 1,25-dihydroxyvitamin D3 receptor and confer responsiveness to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 87:369 –373; 1990. 15. De Miguel, F., Martinez-Ferna´ndez, P., Guille´n, C., Valin, A., Rodrigo, A., Martı´nez, M. E., and Esbrit, P. Parathyroid hormone-related protein (107–139) stimulates interleukin-6 expression in human osteoblastic cells. J Am Soc Nephrol 10:796 – 803; 1999. 16. Dempster, D. W. Bone remodeling. In: Coe, F. L. and Favus, M. J., Eds. Disorders of Bone and Mineral Metabolism. New York: Raven; 1992; 355– 380. 17. D’Ippolito, G., Schiller, P. C., Ricordi, C., Roos, B. A., and Howard, G. A. Age-related osteogenic potential of mesenchymal stromal cells from human vertebral bone marrow. J Bone Miner Res 14:1115–1122; 1999. 18. Ducy, P., Desbois, C., Boyce, B., Pinero, C., Story, B., Dunstan, C., Smith, E., Bonadio, J., Golstein, S., Gundberg, C., Bradley, A., and Karsenty, G. Increased bone formation in osteocalcin-deficient mice. Nature 382:448 – 452; 1996. 19. Evans, C. E., Charles, S. B., Galasko, B., and Ward, C. Effect of donor age on the growth in vitro of cells obtained from human trabecular bone. J Orthop Res 8:234 –237; 1990. 20. Frenkel, B., Capparelli, C., Van Auken, M., Baran, D., Bryan, J., Stein, J. L., Stein, G. S., and Lian, J. B. Activity of the osteocalcin promoter in skeletal sites of transgenic mice and during osteoblast differentiation in bone marrow-
21.
22.
23.
24.
25.
26.
27.
28.
29.
30. 31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
derived stromal cell cultures: Effects of age and sex. Endocrinology 138:2109 – 2116; 1997. Garcia-Ocan˜a, A., Go´mez-Casero, E., Pen˜aranda, C., Sarasa, J. L., and Esbrit, P. Cyclosporine increases renal parathyroid hormone-related protein expression in vivo in the rat. Transplantation 65:860 – 863; 1998. Gerstenfeld, L. C., Chipman, S. D., Glowacki, J., and Lian, J. B. Expression of differentiated function by mineralizing cultures of chicken osteoblast. Dev Biol 122:49 – 60; 1987. Glowacki, J. and Lian, J. B. Impaired recruitment and differentiation of osteoclast progenitors by osteocalcin-depleted bone implants. Cell Differ 21: 247–254; 1987. Hock, J. M., Onyia, J., and Bidwell, J. Comparisons of in vivo and in vitro models of the response of osteoblasts to hormonal regulation with aging. Calcif Tissue Int 56: (Suppl.):S44 –S47; 1995. Kasperk, C., Helmboldt, A., Bo¨rcso¨, I., Heuthe, S., Cloos, O., Niethard, F., and Ziegler, R. Skeletal site-dependent expression of the androgen receptor in human osteoblastic cell populations. Calcif Tissue Int 61:464 – 473; 1997. Kasperk, C., Wergedal, J., Stong, D., Farley, J., Wangerin, K., Gropp, H., Ziegler, R., and Baylink, D. J. Human bone cell phenotypes differ depending on their skeletal site of origin. J Clin Endocrinol Metab 80:2511–2517; 1995. Katzburg, S., Lieberherr, M., Ornoy, A., Klein, B. Y., Hendel, D., and Somjen, D. Isolation and hormonal responsiveness of primary cultures of human bone-derived cells: Gender and age differences. Bone 25:667– 673; 1999. Kerner, S. A., Scott, R. A., and Pike, J. N. Sequence elements in the human osteocalcin gene confer basal activation and inducible response to hormonal vitamin D3 Proc Natl Acad Sci USA 86:4455– 4459; 1989. Majors, A. K., Boehm, A., Nitto, H., Midura, R. J., and Muschler, G. F. Characterization of human bone marrow stromal cells with respect to osteoblastic differentiation. J Orthop Res 15:546 –557; 1997. Marie, P. J. Human endosteal osteoblastic cells: Relationship with bone formation. Calcif Tissue Int 56(Suppl.):S13–S16; 1995. Martinez, M. E., Del Campo, M. T., Medina, S., Sa´nchez, M., Sa´nchezCabezudo, M. J., Esbrit, P., Martinez, P., Moreno, I., Rodrigo, A., Garce´s, M. V., and Munuera, L. Influence of skeletal site of origin and donor age on osteoblastic cell growth and differentiation. Calcif Tissue Int 64:280 –286; 1999. Martı´nez, M. E., Medina, S., Sa´nchez, M., Del Campo, M. T., Esbrit, P., Rodrigo, A., Martı´nez, P., Sa´nchez-Cabezudo, M. J., Moreno, I., Garce´s, M. V., Munuera, L. Influence of skeletal site of origin and donor age on 1,25(OH)2D3induced response of various osteoblastic markers in human osteoblastic cells. Bone 24:203–209; 1999. Owen, T. A., Aronow, M. S., Barone, L. M., Bettencourt, B., Stein, G. S., and Lian, J. B. Pleiotropic effects of vitamin D on osteoblast gene expression are related to the proliferative and differentiated state of the bone cell phenotype: Dependency upon basal levels of gene expression, duration of exposure, and bone matrix competency in normal rat osteoblast cultures. Endocrinology 128:1496 –1504; 1991. Owen, T. A., Bortell, R., Shalhoub, V., Heinrichs, A., Stein, J. L., Stein, G. S., and Lian, J. B. Postproliferative transcription of the rat osteocalcin gene is reflected by vitamin D-responsive developmental modifications in protein– DNA interactions at basal and enhancer promoter elements. Proc Natl Acad Sci USA 90:1503–1507; 1993. Pleilschifter, J., Diel, I., Pilz, U., Brunotte, K., Naumann, A., and Ziegler, R. Mitogenic responsiveness of human cells in vitro to hormones and growth factors decreases with age. J Bone Miner Res 8:707–717; 1993. Podenphant, J. and Engel, U. Regional variations in histomorphometric bone dynamics from the skeleton of an osteoporotic woman. Calcif Tissue Int 40:184 –188; 1987. Roholl, P. J. M., Blauw, E., Zurcher, C., Dermans, J. A. M. A., and Theuns, H. M. Evidence for a diminished maturation of preosteoblasts into osteoblasts during aging in rats: An ultrastructural analysis. J Bone Miner Res 9:355–366; 1994. Santiso-Mere, D., Teruki, S., Hilliard, G. M. IV, Wesley Pike, J., and McDonell, D. P. Positive regulation of the vitamin D receptor by its cognate ligand in heterologous expression systems. Mol Endocrinol 7:833– 839; 1993. Sherman, S., Gehron Rabey, P., and Hadley, E. C. Aging and the human skeleton: Recommendations for research. Calcif Tissue Int 56(Suppl.):S3–S4; 1995. Sims, N. D., White, C. P., Sunn, K. L., Thomas, G. P., Drummond, M. L., Morrison, N. D., Eisman, J. A., and Gardiner, E. M. Human and murine osteocalcin gene expression: Conserved tissue restricted expression and divergent responses to 1,25-dihydroxyvitamin D3 in vivo. Mol Endocrinol 11:1695– 1708; 1997.
Bone Vol. 29, No. 1 July 2001:35– 41 41. Skjodt, H., Gallagher, J. A., Beresford, J. N., Couch, M., Poser, J. W., and Russel, R. G. G. Vitamin D metabolites regulate osteocalcin synthesis and proliferation of human bone cells in vitro. J Endocrinol 105:391–396; 1985. 42. Stein, G. S. and Lian, J. B. Molecular mechanisms mediating proliferation/ differentiation: Interrelationships during progressive development of the osteoblast phenotype. Endocrine Rev 14:424 – 442; 1993. 43. Subramaniam, M., Colvard, D., Keeting, P. E., Rasmussen, K., Riggs, B. L., and Spelsberg, T. C. Glucocorticoid regulation of alkaline phosphatase, osteocalcin, and proto-oncogenes in normal human osteoblast-like cells. J Cell Biochem 50:411– 424; 1992. 44. Vanderschueren, D., Gevers, G., Raymaekers, G., Devos, P., and Dequeker, J. Sex and age-related changes in bone and serum osteocalcin. Calcif Tissue Int 46:179 –182; 1990.
P. Martinez et al. Differences in osteocalcin response to 1,25(OH)2D3
41
45. Wong, M. M., Rao, L. G., Ly, H., Hamilton, L., Ish-Shalom, S., Sturtridge, W., Tong, J., McBroom, R. G., and Murray, T. M. In vitro study of osteoblastic cells from patients with idiopathic osteoporosis and comparison with cells from nonosteoporosis controls. Osteopor Int 4:21–31; 1994.
Date Received: November 30, 1999 Date Revised: June 1, 2000 Date Accepted: March 6, 2001
Changes in Osteocalcin Response to 1,25-Dihydroxyvitamin D3 Stimulation and Basal Vitamin D Receptor Expression in Human Osteoblastic Cells According to Donor Age and Skeletal Origin P. MARTI´NEZ,1 I. MORENO,1 F. DE MIGUEL,2 V. VILA,3 P. ESBRIT,2 and M. E. MARTI´NEZ1 1
Biochemistry Division, Hospital La Paz, Madrid, Spain Bone and Mineral Metabolism Laboratory, Research Unit, Fundacio´n Jime´nez Dı´az, Madrid, Spain 3 Centro de Biologı´a Molecular, Consejo Superior de Investigaciones Cientı´ficas, Madrid, Spain 2
Key Words: Human osteoblastic (hOB) cells; Skeletal site; Aging; 1,25-Dihydroxyvitamin D3; [1,25(OH)2D3]; Osteocalcin; Vitamin D receptor (VDR).
Age-related osteopenia is known to occur differently throughout the skeleton. In the present study, we examine the influence of donor age (<50 and >50 years), and bone structure (cortical vs. trabecular) on osteocalcin and vitamin D receptor (VDR) expression in primary cultures of human osteoblastic cells (hOB) cells. Cells were isolated from trabecular bone samples obtained from donors undergoing either knee (mainly trabecular) (n ⴝ 22; 4 <50 years, 18 >50 years) or hip (mainly cortical) (n ⴝ 16; 6 <50 years, 10 >50 years) arthroplasty. Pooling the results from knee and hip hOB cell cultures, we found that secreted osteocalcin was higher in hOB cells from the younger donors, compared with that in older donors, and peaked after stimulation with 10ⴚ6–10ⴚ8 mol/L 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In cells from the latter donors, this secretion was maximal after 10ⴚ6 mol/L 1,25(OH)2D3 treatment. On the other hand, using reverse transcription followed by polymerase chain reaction, baseline osteocalcin mRNA was found to be lower in hOB cells from the older donors than in those from younger donors. After treatment with 10ⴚ6–10ⴚ8 mol/L 1,25(OH)2D3, osteocalcin mRNA increased over baseline in all groups of hOB cells studied. In age-matched cultures, both basal and 10ⴚ6–10ⴚ8 mol/L 1,25(OH)2D3-stimulated osteocalcin mRNA showed similar values in hOB cells from both skeletal sites in younger donors. However, in the older donors, baseline as well as 10ⴚ8 mol/L 1,25(OH)2D3-stimulated osteocalcin mRNA were higher in knee hOB cells than in hip hOB cells. Furthermore, baseline VDR mRNA expression was also higher in the former cells than in the latter cells in the older group. Considering the influence of donor age at each skeletal site of origin, we found lower baseline osteocalcin and VDR mRNA levels in hip hOB cells in the older group than in the younger group. Our findings indicate that the response of osteocalcin secretion and its mRNA to physiological doses of 1,25(OH)2D3 decreases with aging and is associated with decreased VDR mRNA expression in hOB cells from mainly cortical bone. (Bone 29:35– 41; 2001) © 2001 by Elsevier Science Inc. All rights reserved.
Introduction Bone loss with aging is a well-known event, although the underlying mechanisms of age-related osteopenia are still unclear.39 Histological findings have shown a sharp decrease in bone-forming surfaces compared with bone-resorbing areas with aging,37 which is thought to reflect an osteoblastic inability to reconstitute resorbed bone.24 Thus, age-associated bone loss might be explained, at least in part, by a decrease in osteoblastic cell proliferation, which could be related to an increased osteoblastic maturation.31 The process of aging is known to follow a different pattern throughout the skeleton.5,11,36 Therefore, bone loss occurs mainly in the hip in older subjects, whereas it occurs in the lumbar area in postmenopausal women.11,36 Histomorphometric studies have shown differences in bone remodeling depending on skeletal sites,36 and it has been hypothesized that these differences are associated with variations in osteoblastic differentiation at different skeletal sites. Treatment with vitamin D metabolites has been shown to increase bone mineral density, depending on the subject’s age and bone type.12 We previously found that cultured human osteoblastic cells from trabecular knee areas appear to be less differentiated than those from trabecular bone of the hip.31 Other studies in human osteoblastic cell populations have also demonstrated differences in cell phenotype, cell response to calciotropic hormones, and androgen receptor expression, depending on the skeletal site of origin.25,26,32 These differences could be related to the variable trabecular/ cortical bone ratio at different skeletal sites throughout the skeleton, and that it is higher at the knee compared with the hip.16 Osteoblastic differentiation is a multistep development process, characterized by the ordered expression of genes related to bone growth and differentiation. The osteocalcin gene is one of the marker genes for the progression of osteoblastic differentiation.42 However, the specific role of osteocalcin in bone metabolism remains unclear. Osteocalcin is thought to be a key factor for bone mineralization and bone resorption by enhancing the recruitment and differentiation of osteoclast precursors.13,23 This
Address for correspondence and reprints: Dr. M. E. Martı´nez, Hospital La Paz C/Paseo de la Castellana 261, 28046 Madrid, Spain. E-mail: [email protected] © 2001 by Elsevier Science Inc. All rights reserved.
35
8756-3282/01/$20.00 PII S8756-3282(01)00479-3
36
P. Martinez et al. Differences in osteocalcin response to 1,25(OH)2D3
is consistent with a possible role for osteocalcin in the coupling of bone formation and resorption.42 On the other hand, knockout mice used for study of the osteocalcin gene showed increased bone formation and unaltered bone resorption.18 In osteoblast, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] regulates cell proliferation and differentiation through the vitamin D receptor (VDR).33,41 Thus, osteocalcin gene expression is regulated by 1,25(OH)2D3 through its binding to the VDR, followed by translocation of the 1,25(OH)2D3/VDR complex to the nucleus, where it interacts with vitamin D response elements in the osteocalcin gene.14,28 After stimulation with 1,25(OH)2D3, an increase in osteocalcin mRNA and protein has been reported in human bone,9,40,43 and in rat and chicken osteoblastic cells.22,33 However, this metabolite decreases osteocalcin gene expression in mouse osteoblastic cells.9 Thus, variation in vitamin D regulation of the osteocalcin gene seems to occur among different species.9 An age-associated decline in the total content of osteocalcin in human bone has previously been reported.6 Furthermore, transgenic mice overexpressing the rat osteocalcin promoter in bone have shown a decrease of this promoter activity in osteoblastic cells with aging.20 Interestingly, it is well known that vitamin D deficiency occurs with aging. Because VDR protein levels appear to be regulated by 1,25(OH)2D3,38 vitamin D deficiency might explain the decreased VDR-dependent osteocalcin synthesis by osteoblasts. However, the relationship of this feature to putative osteocalcin changes in different bone types with aging is presently unknown. We recently found differences in the response of osteocalcin to 10⫺8 mol/L 1,25(OH)2D3 in osteoblastic (hOB) cells from human trabecular bone, depending on donor age and skeletal origin.32 In this regard, a diminished response of another vitamin D-dependent protein, calbindin D, to 1,25(OH)2D3 has recently been reported to occur with aging in the rat duodenum.2 The present study evaluates the possible changes in osteocalcin mRNA and its protein secretion after stimulation with different 1,25(OH)2D3 concentrations in hOB cells, as they relate to donor age and skeletal site of origin. We also assessed VDR mRNA changes as a function of skeletal site and donor age because the stimulating effects of 1,25(OH)2D3 on osteocalcin are mediated through this receptor in these cells. Thus, this DR content could provide a way to assess these cells’ ability to respond or not to l,25(OH)2D3-stimulation. Materials and Methods Subjects Osteoblastic cells were isolated from human trabecular bone fragments from 38 osteoarthritic donors ⬍70 years of age (15 men and 23 women), who were undergoing knee or hip replacement surgery. Twenty-two of these bone samples were obtained from subjects during knee arthroplastia. Four of these subjects were ⬍50 years of age (48 ⫾ 1 years), whereas 18 were ⬎50 years of age (64 ⫾ 5 years). The other 16 bone specimens were obtained from subjects during hip arthroplastia. Six of these patients were aged ⬍50 years of age (46 ⫾ 4 years), and 10 were from donors ⬎50 years of age (67 ⫾ 2 years). These subjects had no clinical symptoms or history of bone metabolic disorders. The study was approved by the local ethics commitee. Cell Culture Trabecular bone fragments from either the knee or hip, which would otherwise have been discarded, were cultured as previously described.31,32 Briefly, the samples were minced into
Bone Vol. 29, No. 1 July 2001:35– 41
pieces 0.3– 0.5 cm in diameter, and then washed thoroughly with phosphate-buffered saline to remove adherent bone marrow cells. The fragments were cultured in Dulbecco’s modified Eagle medium containing 4.5 g/L glucose, 15% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 g/mL streptomycin, in a humidified atmosphere with 5% CO2 at 37°C. Confluent cells were subcultured at a density of 104 cells/cm2, and again grown to confluence. These hOB cells display various osteoblastic features, such as alkaline phosphatase and type I collagen secretion, and 1,25(OH)2D3-stimulated osteocalcin secretion.31,32 Cells grown as just described were cultured for 72 h in FBS-free medium with 1 g/L glucose, 10 nmol/L vitamin K, 50 g/mL ascorbic acid, and 0.1% bovine serum albumin (BSA), in the presence or absence of different concentrations of 1,25(OH)2D3 (kindly provided by Roche, Basel, Switzerland). After this time period, the medium was removed and immediately frozen at ⫺20°C. The cells were washed in saline and frozen at ⫺80°C, until total RNA extraction. Osteocalcin Assay Osteocalcin in the cell-conditioned medium was measured by an immunoradiometric assay (Nichols, San Juan Capistrano, CA), using specific antibodies against intact human osteocalcin. The sensitivity of this assay is 0.05 ng/mL. Intra- and interassay variation coefficients were ⬍10%. Osteocalcin secretion was expressed as a ratio over the cell protein concentration, as measured by the Bicinchoninic Acid (BCA) protein assay (Pierce, Rockford, IL), using BSA as standard. Cell protein was extracted from the organic phase obtained following the Trizol method (Gibco, Grand Island, NY) for RNA extraction, according to the manufacturer’s instructions. Osteocalcin in the cell extracts was measured by the aforementioned immunoradiometric assay. Osteocalcin and VDR mRNA Total RNA was extracted from the aqueous phase obtained by the Trizol method, following the manufacturer’s instructions. About 12 g of total RNA was isolated from each 25 cm2 flask containing confluent hOB cells. Osteocalcin and VDR mRNA levels were assayed by reverse transcription followed by semiquantitative polymerase chain reaction (RT-PCR), using conditions providing submaximal amplification.15 Total RNA (0.1–1 ng for osteocalcin, 5 ng for VDR) was added to a reaction mixture containing: 1 mmol/L MgSO4; 0.2 mmol/L dinitrophenolphosphate (dNTP); 1 U of Myeloblastosis Virus Avian reverse transcriptase; 1 U of thermostable DNA polymerase from Thermus flavus (Access RT-PCR System, Promega, Madison, WI); 1 mol/L of specific primers 5⬘-CATGAGAGCCCTCACACTCC-3⬘ (sense) and 5⬘-CAGCAGAGCGACACCCTAGACC-3⬘ (antisense) (human osteocalcin), corresponding to nucleotides 18 –37 and 315–336, respectively, in human osteocalcin (Gene Bank Accession No. X 51699); and 5⬘-GGAAGTGCAGAGGAAGCGGGAGATG-3⬘ (sense) and 5⬘-AGAGCTGGGACAGCTCTAGGGTCAC-3⬘ (antisense) (human VDR), corresponding to nucleotides 344 – 368 and 699 –723 nucleotides, respectively, in the human VDR gene (Gene Bank Accession No. X 67482). Using these primers, PCR amplification yielded 319 bp and 380 bp products, respectively. The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was coamplified, using specific primers for the human gene, as a constitutive control.21 Total RNA and the primers were preincubated for 5 min at 65°C. The reaction mixture (10 L) was then incubated for 45 min at 48°C, and 2 min at 95°C, followed by 35 cycles of 1 min
Bone Vol. 29, No. 1 July 2001:35– 41
P. Martinez et al. Differences in osteocalcin response to 1,25(OH)2D3
37
at 95°C, 1 min at 60°C, and 2 min at 68°C, with a final extension of 7 min at 68°C. The PCR products were separated on 2% agarose gels. Bands were visualized by ethidium bromide staining and quantified by densitometric scanning (1D Manager, Madrid, Spain). Osteocalcin and VDR densitometric values were normalized against those of the corresponding GAPDH product. The identity of the PCR products was confirmed by sequencing with a dye-terminator cycle-sequencing kit (Perkin-Elmer, Norwalk, CT), using Taq FS-DNA polymerase. Sequences were resolved on an ABI-Prism 377 automatic sequencer. Statistical Analysis All data are expressed as mean ⫾ SE. Differences between values from basal and 1,25(OH)2D3-treated cell cultures were analyzed by the nonparametric paired Wilcoxon test. Differences between the values of two different groups of hOB cell cultures were analyzed by the unpaired Mann–Whitney U-test. Results Time Course of Osteocalcin Secretion in Human Osteoblastic Cells Preliminary time-course experiments using eight hOB cell cultures, four from the hip and four from the knee, were obtained from ⬍50- or ⬎50-year-old donors. We found that baseline osteocalcin secretion increased during the time period studied: 2.7 ⫾ 1 ng/mg protein at 24 h; 5.7 ⫾ 2.6 at 48 h; and 15 ⫾ 3 at 72 h (p ⬍ 0.05 between values at 48 h and 72 h, and that at 24 h). A similar time-course pattern for osteocalcin secretion was found after treating these cell cultures with 1,25(OH)2D3. Thus, osteocalcin secretion values were 8.3 ⫾ 1.8 at 24 h, 46.3 ⫾ 6.2 at 48 h and 74.4 ⫾ 5.1 at 72 h after stimulation with 10⫺8 mol/L 1,25(OH)2D3, or 22.4 ⫾ 11.0 at 24 h, 89.7 ⫾ 29.1 at 48 h, and 115.4 ⫾ 31.8 at 72 h after stimulation with 10⫺6 mol/L 1,25(OH)2D3 [p ⬍ 0.05 between values at 48 h and 72 h, and that at 24 h for each 1,25(OH)2D3 concentration]. These results indicate that the levels of osteocalcin secretion after 1,25(OH)2D3 stimulation were higher at 72 h in these cell cultures, and therefore further studies on osteocalcin secretion were carried out during this time period. Dose-dependent Effect of 1,25(OH)2D3 on Osteocalcin Secretion in Human Osteoblastic Cells We found that treatment with 10⫺4–10⫺10 mol/L 1,25(OH)2D3 induced a biphasic response of osteocalcin secretion in all groups of hOB cells studied. Thus, secreted osteocalcin increased dose dependently between 10⫺6 and 10⫺10 mol/L, but decreased thereafter at 10⫺4 mol/L 1,25(OH)2D3 in these cells (Figure 1). Influence of Donor Age and Skeletal Site of Origin on 1,25(OH)2D3-stimulated Osteocalcin Secretion in Human Osteoblastic Cells ⫺6
Figure 1. Secreted osteocalcin response to different 1,25(OH)2D3 concentrations: 10⫺10 mol/L (Vit. D-10); 10⫺8 mol/L (Vit. D-8); 10⫺6 mol/L (Vit. D-6); and 10⫺4 mol/L (Vit. D-4), in hOB cells from either different skeletal site of origin (hip or knee) (A) or donor age (⬍50 years and ⬎50 years) (B). Osteocalcin secretion was measured in the medium conditioned by hOB cells isolated from bone explants from adult donors, before (basal) and after stimulation with different 1,25(OH)2D3 concentrations for 72 h. Results are mean ⫾ SE. *p ⬍ 0.05 vs. basal condition; # p ⬍ 0.05 between Vit. D-8 and Vit. D-6 stimulation; ap ⬍ 0.05, between ⬍50 year- and ⬎50-year-old donors, after stimulation with Vit. D-8.
these cells in the younger group (Table 1). On the other hand, cell osteocalcin content was significantly higher after treatment with 10⫺6 mol/L 1,25(OH)2D3, compared with 10⫺8 mol/L in these cells in the older group (Table 1). We also considered the influence of skeletal site of origin on 1,25(OH)2D3-stimulated osteocalcin secretion. We found that this secretion reached a maximum value after 10⫺6–10⫺8 mol/L 1,25(OH)2D3 treatment in knee hOB cells (Figure 1B). In conTable 1. Osteocalcin cell content in human osteoblastic (hOB) cells
⫺8
We found maximum osteocalcin secretion after 10 –10 mol/L 1,25(OH)2D3 treatment in the younger group (Figure 1A). However, in the older group, this maximum was reached after 10⫺6 mol/L 1,25(OH)2D3 stimulation (Figure 1A). Furthermore, cell osteocalcin content, assessed in some of these hOB cell cultures from both age groups, was also found to be significantly increased after both 10⫺8 mol/L and 10⫺6 mol/L 1,25(OH)2D3 treatment, compared with the respective baseline value (Table 1). Furthermore, we found that this content was similar after stimulation with either 10⫺6 or 10⫺8 mol/L 1,25(OH)2D3 in
⬍50 years ⬎50 years
Basal
1,25(OH)2D3 (10⫺8 mol/L)
1,25(OH)2D3 (10⫺6 mol/L)
0.7 ⫾ 0.2 0.3 ⫾ 0.2
8.0 ⫾ 6.1a 2.8 ⫾ 1.5a,b
12.8 ⫾ 7.2a 5.0 ⫾ 2.3a
Cell osteocalcin protein (ng/mg protein) after 10⫺8 or 10⫺6 mol/L 1,25(OH)2D3 treatment in hOB cells in relation to donor age (⬍50 years and ⬎50 years). Osteocalcin was measured in hOB cell extracts before (baseline) and after 1,25(OH)2D3 stimulation for 72 h. Results are mean ⫾ SE (n ⫽ 6). a p ⬍ 0.05 vs. baseline; bp ⬍ 0.05 between 10⫺8 and 10⫺6 mol/L 1,25(OH)2D3 treatment.
38
P. Martinez et al. Differences in osteocalcin response to 1,25(OH)2D3
Bone Vol. 29, No. 1 July 2001:35– 41
Figure 2. Effect of 1,25(OH)2D3 on osteocalcin mRNA in hOB cells, in relation to donor age (⬍50 years and ⬎50 years) and skeletal site of origin (knee or hip). Osteocalain mRNA expression was measured by semiquantitative RT-PCR in hOB cells before (basal) and after stimulation with 10⫺8 mol/L (Vit. D-8) or 10⫺6 mol/L (Vit. D-6) 1,25(OH)2D3 for 72 h. Results are mean ⫾ SE. *p ⬍ 0.05, **p ⬍ 0.001 vs. basal condition; # p ⬍ 0.05, between vit. D-8 and vit. D-6 stimulation; bp ⬍ 0.05, between hip and knee in age-matched donors; ap ⬍ 0.001, and cp ⬍ 0.05, between ⬍50 years and ⬎50 years old donors at the knee and the hip, respectively. Inset: Representative gel corresponding to a hip hOB cell culture in the older group. Cells were treated with or without vit. D-8 and vit. D-6 for 72 h.
trast, the concentration of 1,25(OH)2D3 that induced maximal osteocalcin secretion was shifted to 10⫺6 mol/L in hip hOB cells (Figure 1B). Influence of Donor Age on Osteocalcin and Vitamin D Receptor Depending on Skeletal Site of Origin in Human Osteoblastic Cells We further analyzed the influence of donor age, as it relates to the skeletal site of origin, on osteocalcin mRNA and protein secretion in hOB cells. We found that baseline osteocalcin mRNA was significantly higher in hOB cells in the younger group than in the older group, at both skeletal sites (Figure 2). Treatment with either 10⫺6 or 10⫺8 mol/L 1,25(OH)2D3 induced a significant and similar increase over baseline in osteocalcin mRNA in knee hOB cells in both age groups studied (Figure 2). However, in hip hOB cells from the older group, osteocalcin mRNA expression increased to a higher value after treatment with 10⫺6 mol/L 1,25(OH)2D3 than with 10⫺8 mol/L 1,25(OH)2D3 (Figure 2 and inset). Furthermore, baseline VDR mRNA expression was similar in knee hOB cells in both age groups studied (Figure 3), but was higher in hip hOB cells from the younger donors than in these cells in the older group (Figure 3). Osteocalcin secretion also increased to a similar value after both 10⫺6 and 10⫺8 mol/L 1,25(OH)2D3 at both skeletal sites in the younger group (Table 2). However, in older donors, this secretion was significantly higher after treatment with 10⫺6
mol/L 1,25(OH)2D3 than with 10⫺8 mol/L of this metabolite at both skeletal sites (Table 2). Influence of Skeletal Site of Origin on Age-associated Changes in Osteocalcin and Vitamin D Receptor in Human Osteoblastic Cells The influence of skeletal site on 1,25(OH)2D3-stimulated osteocalcin was then evaluated in age-matched donors. In the older group, baseline and 10⫺8 mol/L 1,25(OH)2D3-stimulated osteocalcin mRNA levels were higher in knee hOB cells than in these cells from the hip, but the levels were similar after 10⫺6 mol/L 1,25(OH)2D3 stimulation at both skeletal sites (Figure 2). No significant differences were found in baseline levels of both osteocalcin and VDR mRNA in hOB cells at both skeletal sites in the younger group (Figures 2 and 3). However, VDR mRNA expression was significantly higher in knee hOB cells than in these cells from the hip in the older donors (Figure 3). Osteocalcin secretion was also significantly higher in knee hOB cells than in these cells from the hip after either 10⫺6 or 10⫺8 mol/L 1,25(OH)2D3 stimulation, but only in the older group (Table 2). Discussion The present study extends our previous studies31,32 by evaluating the influence of age and skeletal site of origin (hip or knee) on osteocalcin, protein, and mRNA in response to different concen-
Figure 3. Differences in basal VDR mRNA levels in relation to donor age (⬍50 years and ⬎50 years) and skeletal site of origin (hip or knee). VDR mRNA levels were measured by semiquantitative RT-PCR in hOB cells after 72 h in serum-free medium. Results are mean ⫾ SE. *p ⬍ 0.001, between subjects ⬍50 years and those ⬎50 years, at the hip; **p ⬍ 0.05, between hip and knee from age-matched donors.
Bone Vol. 29, No. 1 July 2001:35– 41
P. Martinez et al. Differences in osteocalcin response to 1,25(OH)2D3
Table 2. Osteocalcin secretion in human osteoblastic (hOB) cell cultures
Basal Knee ⬍ 50 ⬎ 50 Hip ⬍ 50 ⬎ 50
1,25(OH)2D3 (10⫺8 mol/L)
years years
7.3 ⫾ 3.0 5.2 ⫾ 2.1
43.8 ⫾ 15.2a 60.4 ⫾ 10.6a
years years
8.0 ⫾ 4.2 3.1 ⫾ 1.0
55.2 ⫾ 19.4a 27.1 ⫾ 9.0a,c
1,25(OH)2D3 (10⫺6 mol/L) 99.0 ⫾ 31.2a 120.3 ⫾ 23.0a,b 88.5 ⫾ 19.0a 55.2 ⫾ 18.0a,b,c
Osteocalcin secretion (ng/mg protein) at baseline and after 10⫺8 or 10⫺6 mol/L 1,25(OH)2D3 stimulation in hOB cells in relation to donor age (⬍50 years and ⬎50 years) and skeletal site of origin (hip or knee). Osteocalcin secretion was measured in the cell-conditioned medium before (basal) and after stimulation with 1,25(OH)2D3 for 72 h. Results are mean ⫾ SE (n ⫽ 4 –18). a p ⬍ 0.05 vs. basal; bp ⬍ 0.05 between 10⫺8 and 10⫺6 mol/L 1,25(OH)2D3 treatment; cp ⬍ 0.05 between different skeletal sites of origin (hip or knee) in age-matched donors.
trations of 1,25(OH)2D3 in hOB cells. We also assessed baseline VDR mRNA expression in these cells. We used hOB cells isolated from explants of trabecular bone at each skeletal site because this appears to be a potent tool for addressing issues related to skeletal pathology and aging.8,30 Our findings show that osteocalcin secretion, and also cell osteocalcin protein content, was similar after stimulation with 10⫺6–10⫺8 mol/L 1,25(OH)2D3 in these cells in younger donors (⬍50 years). Human OB stimulation with a higher 1,25(OH)2D3 concentration was found to decrease osteocalcin secretion in both age groups studied. These results differ somewhat from those of Beresford et al.,3 who found maximal osteocalcin response after stimulation with 10⫺7–10⫺9 mol/L 1,25(OH)2D3, decreasing at 10⫺6 mol/L of this metabolite, in human-derived osteoblastic cell cultures. However, in their study, only two subjects, aged 2 and 17 years, were the source of osteoblastic cells, whereas our donors were older and more numerous. In addition, we found that peak osteocalcin secretion, and its cell protein content, shifted to a higher concentration (10⫺6 mol/L) of 1,25(OH)2D3 at both skeletal sites in the older group compared with those in the younger group. Thus, the present findings, and those of Beresford et al.,3 are consistent with the hypothesis that higher 1,25(OH)2D3 concentrations are needed to induce maximal production of osteocalcin by osteoblasts in aged human subjects.19,35,45 Moreover, a decrease in circulating osteocalcin, as well as its content in both cortical and trabecular bone, occurs with age.6,44 We also found that baseline osteocalcin mRNA was higher in hOB cells in younger donors than in older donors at both skeletal sites. Our findings are consistent with a recent report by Frenkel et al.,20 showing that rat osteocalcin promoter activity overexpressed in the bone of mice decreases with aging. These results are also in agreement with Katzburg et al.27 who showed a decrease in osteocalcin secretion with age between pre- and postmenopausal women from human bone-derived cell cultures. A previous report showed differences in bone osteocalcin content, according to differences in trabecular bone composition at various skeletal sites.1 We therefore assessed the osteocalcin response to 1,25(OH)2D3 in hOB cells from explants of trabecular bone at the knee or hip, with the latter showing a higher cortical/trabecular ratio16 in age-matched donors. We found that both osteocalcin secretion and gene expression levels were similar after stimulation with 10⫺6–10⫺8 mol/L of 1,25(OH)2D3 in knee or hip hOB cells in the younger group, but were higher after 10⫺6 mol/L of 1,25(OH)2D3 than after 10⫺8 mol/L in hip hOB
39
cells of older donors. Thus, hOB cells from the hip, a predominantly cortical bone, require a higher 1,25(OH)2D3 concentration to reach an osteocalcin response similar to that observed in hOB cells from the knee, which is mainly trabecular bone. These findings are consistent with those of a clinical study indicating that age-related bone loss was higher in predominantly cortical bone than in predominantly trabecular bone in vitamin D-deficient subjects,4 and that vitamin D supplements decreased the relative risk of fracture mainly in cortical bone in aging subjects.7 In the present study, we found lower baseline osteocalcin mRNA levels in hOB cells from each skeletal site in older donors. Our findings also show that the increment of increase in osteocalcin mRNA after of 1,25(OH)2D3 stimulation was inversely related to its baseline mRNA, as previously shown to occur in other osteoblastic cell types at various developmental stages of the osteoblast phenotype.33,34 Taken together, the aforementioned findings suggest that a less efficient 1,25(OH)2D3-induced response of osteocalcin mRNA occurs with aging in a predominantly cortical type of bone. Other investigators also described a decrease in the number of osteogenic progenitors with aging in a predominantly cortical type of bone.17,29 Our previous studies have shown that hOB cells from the knee, and in younger subjects, display less differentiated features, which could be related to the presence of a higher content of osteoprogenitor cells in these cultures, compared with those in cells from the hip, and in older subjects.31,32 In this study, knee hOB cells, and those in the younger donors, showed higher osteocalcin mRNA levels. Frenkel et al.,20 using transgenic mice overexpressing the rat osteocalcin promoter in bone, found that the activity of this promoter remains absent in osteoprogenitor cells isolated from bone stroma until they begin to differentiate into osteoblasts. Therefore, although the different types of bone used in this study to obtain various hOB cell cultures originally had a variable content of stromal cells, these cells appear to have been differentiated into osteoblastic cells in our experimental setting. Consistent with our osteocalcin results, we observed a decrease in baseline VDR mRNA with aging in the more differentiated hOB cells from the hip. This is also consistent with previous findings in orofacial bone showing that VDR expression, using in situ hybridation, increases with osteoblast differentiation.10 These results could be explained by differences in VDR gene expression levels in the different bone types used in both studies. Also in agreement with the osteocalcin results described herein, in the older donors, we found lower VDR mRNA levels in hip hOB cells than in those from the knee. Thus, VDR expression appears to differ depending on bone type, which is consistent with previous findings for androgen receptors in other hOB cell populations.25 In summary, we demonstrated that the more differentiated hOB cells from the hip (predominantly cortical bone) in older subjects show a lower response of osteocalcin gene expression and osteocalcin secretion to 1,25(OH)2D3. Our findings also support the hypothesis that age-related cortical bone loss in vitamin D deficiency occurs in association with decreased VDR gene expression.
Acknowledgments: This work was supported by grants from the Spanish Fondo de Investigacio´n Sanitaria (FIS 97/0307 and 00/0125) and FAES (Madrid, Spain). The authors thank the medical staff of the Orthopaedic Department, Hospital La Paz, for providing us with the bone samples. We also thank Mark S. Davis for proofreading the manuscript.
40
P. Martinez et al. Differences in osteocalcin response to 1,25(OH)2D3
Bone Vol. 29, No. 1 July 2001:35– 41
References 1. Aerssens, J., Boonen, S., Joly, J., and Dequeker, J. Variations in trabecular bone composition with anatomical site and age: Potential implications for bone quality assessment. J Endocrinol 155:411– 421; 1997. 2. Armbrecht, H. J., Boltz, M. A., Christakos, S., and Bruns, M. E. Capacity of 1,25-dihydroxyvitamin D to stimulate expression of calbindin D changes with age in rats Arch Biochem Biophys 352:159 –164; 1998. 3. Beresford, I. N., Gallagher, J. A., Poser, J. W., and Russell, R. G. G. Production of osteocalcin by human bone cells in vitro. Effects of 1,25(OH)2D3, 24,25(OH)2D3, parathyroid hormone, and glucocorticoids. Metab Bone Dis Rel Res 5:229 –234; 1984. 4. Bettica, P., Bevilacqua, M., Vago, T., and Norbiato, G. High prevalence of hypovitaminosis D among free-living postmenopausal women referred to an osteoporosis outpatient clinic in northern Italy for initial screening. Osteopor Int 9:226 –229; 1999. 5. Bonjour, J. P., Theintz, G., Buchs, B., Slosman, D., and Rizzoli R. Critical years and stages of puberty for spinal and femoral bone mass accumulation during adolescence. J Clin Endocrinol Metab 73:555–563; 1991. 6. Boonen, S., Aerssens, J., Dequeker, J., Nicholson, P., Chen, X., Lowet, G., Verbeker, G., and Bouillon, R. Age-associated decline in human femoral neck cortical and trabecular content of insulin-like growth factor I: Potential implications for age-related (type II) osteoporotic fracture ocurrence. Calcif Tissue Int 61:173–178; 1997. 7. Chapuy, M. C., Arlot, M., Duboeut, F., Brun, J., Crouzet, B., Arnaud, S., Delmas, P. D., and Meunier, P. J. Vitamin D3 and calcium to prevent hip fracture in elderly women. N Engl J Med 327:1637–1642, 1992. 8. Chavassieux, P. M., Chenu, C., Valentin-Opran, A., Merle, B., Delmas, P. D., Hartmann, D. J., Saez, S., and Meunier, P. J. Influence of experimental conditions on osteoblast activity in human primary bone cell cultures. J Bone Miner Res 5:337–343; 1990. 9. Clemens, T. L., Hui, T. Maeda, S., Kesterson, R. A., Demayo, F., Pike, J. W., and Gundberg, C. A. Analysis of osteocalcin expression in transgenic mice reveals a species difference in vitamin D regulation of mouse and human osteocalcin genes. J Bone Miner Res 12:1570 –1576; 1997. 10. Davideau, J. L., Papagerakis, P., Hotton, D., Lezot, F., and Berdal, A. In situ investigation of vitamin D receptor, alkaline phosphatase, and osteocalcin gene expression in oro-facial mineralized tissues. Endocrinology 137:3577–3585; 1996. 11. Davis, J. W., Ross, P. D., and Wasnich, R. D. Evidence for both generalized and regional low bone mass among elderly women. J Bone Miner Res 9:305–309; 1994. 12. Dawson-Hughes, B., Dallal, G. E., Krall, E. A., Harris, S., Sokoll, L. J., and Falconer, G. Effect of vitamin D supplementation on wintertime and overall bone loss in healthly postmenopausal women. Ann Intern Med 115:505–512; 1991. 13. Defranco, D. J., Glowacki, J., Cox, K. A., and Lian, J. B. Normal bone particles are preferentially resorbed in the presence of osteocalcin-deficient bone particles in vivo. Calcif Tissue Int 49:43–50; 1991. 14. Demay, M. B., Gerardi, J. M., DeLuca, H. F., and Kronenberg, H. M. DNA sequences in the rat osteocalcin gene that bind the 1,25-dihydroxyvitamin D3 receptor and confer responsiveness to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 87:369 –373; 1990. 15. De Miguel, F., Martinez-Ferna´ndez, P., Guille´n, C., Valin, A., Rodrigo, A., Martı´nez, M. E., and Esbrit, P. Parathyroid hormone-related protein (107–139) stimulates interleukin-6 expression in human osteoblastic cells. J Am Soc Nephrol 10:796 – 803; 1999. 16. Dempster, D. W. Bone remodeling. In: Coe, F. L. and Favus, M. J., Eds. Disorders of Bone and Mineral Metabolism. New York: Raven; 1992; 355– 380. 17. D’Ippolito, G., Schiller, P. C., Ricordi, C., Roos, B. A., and Howard, G. A. Age-related osteogenic potential of mesenchymal stromal cells from human vertebral bone marrow. J Bone Miner Res 14:1115–1122; 1999. 18. Ducy, P., Desbois, C., Boyce, B., Pinero, C., Story, B., Dunstan, C., Smith, E., Bonadio, J., Golstein, S., Gundberg, C., Bradley, A., and Karsenty, G. Increased bone formation in osteocalcin-deficient mice. Nature 382:448 – 452; 1996. 19. Evans, C. E., Charles, S. B., Galasko, B., and Ward, C. Effect of donor age on the growth in vitro of cells obtained from human trabecular bone. J Orthop Res 8:234 –237; 1990. 20. Frenkel, B., Capparelli, C., Van Auken, M., Baran, D., Bryan, J., Stein, J. L., Stein, G. S., and Lian, J. B. Activity of the osteocalcin promoter in skeletal sites of transgenic mice and during osteoblast differentiation in bone marrow-
21.
22.
23.
24.
25.
26.
27.
28.
29.
30. 31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
derived stromal cell cultures: Effects of age and sex. Endocrinology 138:2109 – 2116; 1997. Garcia-Ocan˜a, A., Go´mez-Casero, E., Pen˜aranda, C., Sarasa, J. L., and Esbrit, P. Cyclosporine increases renal parathyroid hormone-related protein expression in vivo in the rat. Transplantation 65:860 – 863; 1998. Gerstenfeld, L. C., Chipman, S. D., Glowacki, J., and Lian, J. B. Expression of differentiated function by mineralizing cultures of chicken osteoblast. Dev Biol 122:49 – 60; 1987. Glowacki, J. and Lian, J. B. Impaired recruitment and differentiation of osteoclast progenitors by osteocalcin-depleted bone implants. Cell Differ 21: 247–254; 1987. Hock, J. M., Onyia, J., and Bidwell, J. Comparisons of in vivo and in vitro models of the response of osteoblasts to hormonal regulation with aging. Calcif Tissue Int 56: (Suppl.):S44 –S47; 1995. Kasperk, C., Helmboldt, A., Bo¨rcso¨, I., Heuthe, S., Cloos, O., Niethard, F., and Ziegler, R. Skeletal site-dependent expression of the androgen receptor in human osteoblastic cell populations. Calcif Tissue Int 61:464 – 473; 1997. Kasperk, C., Wergedal, J., Stong, D., Farley, J., Wangerin, K., Gropp, H., Ziegler, R., and Baylink, D. J. Human bone cell phenotypes differ depending on their skeletal site of origin. J Clin Endocrinol Metab 80:2511–2517; 1995. Katzburg, S., Lieberherr, M., Ornoy, A., Klein, B. Y., Hendel, D., and Somjen, D. Isolation and hormonal responsiveness of primary cultures of human bone-derived cells: Gender and age differences. Bone 25:667– 673; 1999. Kerner, S. A., Scott, R. A., and Pike, J. N. Sequence elements in the human osteocalcin gene confer basal activation and inducible response to hormonal vitamin D3 Proc Natl Acad Sci USA 86:4455– 4459; 1989. Majors, A. K., Boehm, A., Nitto, H., Midura, R. J., and Muschler, G. F. Characterization of human bone marrow stromal cells with respect to osteoblastic differentiation. J Orthop Res 15:546 –557; 1997. Marie, P. J. Human endosteal osteoblastic cells: Relationship with bone formation. Calcif Tissue Int 56(Suppl.):S13–S16; 1995. Martinez, M. E., Del Campo, M. T., Medina, S., Sa´nchez, M., Sa´nchezCabezudo, M. J., Esbrit, P., Martinez, P., Moreno, I., Rodrigo, A., Garce´s, M. V., and Munuera, L. Influence of skeletal site of origin and donor age on osteoblastic cell growth and differentiation. Calcif Tissue Int 64:280 –286; 1999. Martı´nez, M. E., Medina, S., Sa´nchez, M., Del Campo, M. T., Esbrit, P., Rodrigo, A., Martı´nez, P., Sa´nchez-Cabezudo, M. J., Moreno, I., Garce´s, M. V., Munuera, L. Influence of skeletal site of origin and donor age on 1,25(OH)2D3induced response of various osteoblastic markers in human osteoblastic cells. Bone 24:203–209; 1999. Owen, T. A., Aronow, M. S., Barone, L. M., Bettencourt, B., Stein, G. S., and Lian, J. B. Pleiotropic effects of vitamin D on osteoblast gene expression are related to the proliferative and differentiated state of the bone cell phenotype: Dependency upon basal levels of gene expression, duration of exposure, and bone matrix competency in normal rat osteoblast cultures. Endocrinology 128:1496 –1504; 1991. Owen, T. A., Bortell, R., Shalhoub, V., Heinrichs, A., Stein, J. L., Stein, G. S., and Lian, J. B. Postproliferative transcription of the rat osteocalcin gene is reflected by vitamin D-responsive developmental modifications in protein– DNA interactions at basal and enhancer promoter elements. Proc Natl Acad Sci USA 90:1503–1507; 1993. Pleilschifter, J., Diel, I., Pilz, U., Brunotte, K., Naumann, A., and Ziegler, R. Mitogenic responsiveness of human cells in vitro to hormones and growth factors decreases with age. J Bone Miner Res 8:707–717; 1993. Podenphant, J. and Engel, U. Regional variations in histomorphometric bone dynamics from the skeleton of an osteoporotic woman. Calcif Tissue Int 40:184 –188; 1987. Roholl, P. J. M., Blauw, E., Zurcher, C., Dermans, J. A. M. A., and Theuns, H. M. Evidence for a diminished maturation of preosteoblasts into osteoblasts during aging in rats: An ultrastructural analysis. J Bone Miner Res 9:355–366; 1994. Santiso-Mere, D., Teruki, S., Hilliard, G. M. IV, Wesley Pike, J., and McDonell, D. P. Positive regulation of the vitamin D receptor by its cognate ligand in heterologous expression systems. Mol Endocrinol 7:833– 839; 1993. Sherman, S., Gehron Rabey, P., and Hadley, E. C. Aging and the human skeleton: Recommendations for research. Calcif Tissue Int 56(Suppl.):S3–S4; 1995. Sims, N. D., White, C. P., Sunn, K. L., Thomas, G. P., Drummond, M. L., Morrison, N. D., Eisman, J. A., and Gardiner, E. M. Human and murine osteocalcin gene expression: Conserved tissue restricted expression and divergent responses to 1,25-dihydroxyvitamin D3 in vivo. Mol Endocrinol 11:1695– 1708; 1997.
Bone Vol. 29, No. 1 July 2001:35– 41 41. Skjodt, H., Gallagher, J. A., Beresford, J. N., Couch, M., Poser, J. W., and Russel, R. G. G. Vitamin D metabolites regulate osteocalcin synthesis and proliferation of human bone cells in vitro. J Endocrinol 105:391–396; 1985. 42. Stein, G. S. and Lian, J. B. Molecular mechanisms mediating proliferation/ differentiation: Interrelationships during progressive development of the osteoblast phenotype. Endocrine Rev 14:424 – 442; 1993. 43. Subramaniam, M., Colvard, D., Keeting, P. E., Rasmussen, K., Riggs, B. L., and Spelsberg, T. C. Glucocorticoid regulation of alkaline phosphatase, osteocalcin, and proto-oncogenes in normal human osteoblast-like cells. J Cell Biochem 50:411– 424; 1992. 44. Vanderschueren, D., Gevers, G., Raymaekers, G., Devos, P., and Dequeker, J. Sex and age-related changes in bone and serum osteocalcin. Calcif Tissue Int 46:179 –182; 1990.
P. Martinez et al. Differences in osteocalcin response to 1,25(OH)2D3
41
45. Wong, M. M., Rao, L. G., Ly, H., Hamilton, L., Ish-Shalom, S., Sturtridge, W., Tong, J., McBroom, R. G., and Murray, T. M. In vitro study of osteoblastic cells from patients with idiopathic osteoporosis and comparison with cells from nonosteoporosis controls. Osteopor Int 4:21–31; 1994.
Date Received: November 30, 1999 Date Revised: June 1, 2000 Date Accepted: March 6, 2001