Human and Murine Osteocalcin Gene Expression: Conserved Tissue Restricted Expression and Divergent Responses to 1,25-Dihydroxyvitamin D3 in Vivo

Natalie A. Sims, Christopher P. White, Kate L. Sunn, Gethin P. Thomas, Melanie L. Drummond, Nigel A. Morrison, John A. Eisman and Edith M. Gardiner

Bone and Mineral Research Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales, Australia


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human and murine osteocalcin genes demonstrate similar cell-specific expression patterns despite significant differences in gene locus organization and sequence variations in cis-acting regulatory elements. To investigate whether differences in these regulatory regions result in an altered response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in vivo, we compared the response of the endogenous mouse osteocalcin gene to a bacterial reporter gene directed by flanking regions of the human osteocalcin gene in transgenic mice. Transgene expression colocalized with endogenous osteocalcin expression in serial sections, being detected in osteoblasts, osteocytes and hypertrophic chondrocytes. In calvarial cell culture lysates from transgenic and nontransgenic mice, the endogenous mouse osteocalcin gene did not respond to 1,25-(OH)2D3 treatment. Despite this, transgene activity was significantly increased in the same cells. Similarly, Northern blots of total cellular RNA and in situ hybridization studies of transgenic animals demonstrated a maximal increase in transgene expression at 6 h after 1,25-(OH)2D3 injection (23.6 ± 3.6-fold) with a return to levels equivalent to uninjected animals by 24 h (1.2 ± 0.1-fold). This increase in transgene expression was also observed at 6 h after 1,25-(OH)2D3 treatment in animals on a low calcium diet (25.2 ± 7.7-fold) as well as in transgenic mice fed a vitamin D-deficient diet containing strontium chloride to block endogenous 1,25-(OH)2D3 production (7.5 ± 0.9-fold). In contrast to the increased transgene expression levels, neither endogenous mouse osteocalcin mRNA levels nor serum osteocalcin levels were significantly altered after 1,25-(OH)2D3 injection in transgenic or nontransgenic mice, regardless of dietary manipulations, supporting evidence for different mechanisms regulating the response of human and mouse osteocalcin genes to 1,25-(OH)2D3. Although the cis- and trans-acting mechanisms directing cell-specific gene expression appear to be conserved in the mouse and human osteocalcin genes, responsiveness to 1,25-(OH)2D3 is not. The mouse osteocalcin genes do not respond to 1,25-(OH)2D3 treatment, but the human osteocalcin-directed transgene is markedly up-regulated under the same conditions and in the same cells. The divergent responses of these homologous genes to 1,25-(OH)2D3 are therefore likely to be due to differences in mouse and human osteocalcin-regulatory sequences rather than to variation in the complement of trans-acting factors present in mouse osteoblastic cells. Increased understanding of these murine-human differences in osteocalcin regulation may shed light on the function of osteocalcin and its regulation by vitamin D in bone physiology.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Osteocalcin is the most abundant noncollagenous protein expressed in bone (1, 2), with its expression limited specifically to cells of the osteoblast lineage, including mature osteoblasts, osteocytes, and hypertrophic chondrocytes (3, 4, 5, 6). There is also some evidence for low levels of osteocalcin expression in megakaryocytes, platelets (7), and brain (8). Despite its well characterized specificity of expression, the precise function of osteocalcin in bone remodeling is not clear. The location of osteocalcin at bone-forming surfaces (9, 10) and the increased bone mineralization observed in osteocalcin gene knockout mice (11) and warfarin-treated rats (12, 13) supports a role for osteocalcin in suppression of bone mineralization. Alternatively, osteocalcin has been suggested to increase bone resorption through osteoclast recruitment (10, 14).

Serum levels of osteocalcin increase in humans (15) and rats (16) after injection with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], and increased osteocalcin mRNA expression occurs in rat femurs 6 h after 1,25-(OH)2D3 treatment in vivo (17). Expression of osteocalcin mRNA and protein is stimulated by 1,25-(OH)2D3 in rat osteoblastic cell lines (18, 19, 20, 21, 22) and in human cultured primary osteoblasts (23, 24), as well as in transfection studies of the human (25, 26) and rat (27, 28, 29) osteocalcin genes.

Stimulation of gene transcription by 1,25-(OH)2D3 depends on binding of vitamin D receptor/retinoid X receptor (VDR/RXR) heterodimers or VDR homodimers to specific vitamin D-responsive elements (VDREs) within the human and rat osteocalcin gene promoters (30, 31, 32). The organizations of the human, rat, and mouse osteocalcin genes differ, however, with some rat and all mouse strains studied, having a cluster of osteocalcin-related genes rather than a single-copy gene as described in the human (18, 33, 34, 35). Despite these differences, the same pattern of tissue-specific expression is shared by the three species, and the promoters of the two mouse osteocalcin genes exhibit the same modular organization described in the rat and human genes, including a VDRE consensus sequence at a similar distance upstream of the transcription start site (33). Cis-acting elements thought to direct tissue-specific and hormone-responsive osteocalcin gene transcription differ, however, between species (33, 34), and trans-acting factors that bind to these regions are also likely to vary (34, 36, 37), leading to potential variation in transcriptional control mechanisms. Supporting this concept, recent studies have verified such a species-related difference, showing the mouse osteocalcin gene to be down-regulated rather than induced by 1,25-(OH)2D3 in vivo and in vitro (38, 39). In each of these studies, the inhibitory effect was demonstrated on the endogenous mouse osteocalcin gene, and direct comparisons of rat and mouse osteocalcin-regulatory activities were made in transfected rat and mouse cell lines. In the present study, basal tissue-specific and 1,25-(OH)2D3-responsive regulation by the mouse and human osteocalcin promoters were directly compared in transgenic mice carrying a reporter transgene in an osteoblast-targeting vector based on 5'- and 3'-sequences from the human osteocalcin gene locus.

The human regulatory sequences conferred a spatially restricted transgene expression pattern virtually identical to that of the endogenous osteocalcin gene, confirming that the cis- and trans-acting mechanisms directing the restricted pattern of osteocalcin gene expression are conserved across species. However, although the human osteocalcin-directed transgene was up-regulated by 1,25-(OH)2D3 treatment, endogenous mouse osteocalcin gene expression was not induced by 1,25-(OH)2D3 treatment in transgenic or nontransgenic mice, a confirmation of previous reports (38, 39). Clearly, the vitamin D responsiveness of the osteocalcin gene does differ between species, and this variation is not due simply to differences in the cellular milieu in human and murine bone.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transgene Expression Analyses
In the two sublines of OSCAT8 transgenic mice, transgene expression was detectable only in RNA preparations from bone tissues (Fig. 1AGo), with no detectable expression in any other tissue, including brain or kidney, in either subline and no expression detectable in any nontransgenic tissues. Quantification of transgene expression normalized to the 18S RNA signal indicated that transgene expression in bones from the high copy number animals (OST2) was 8-fold higher than in low copy number (OST1) animals. As the OST2 line carries approximately 60 transgene copies compared with the two copies in OST1 mice, the OSCAT8 construct therefore does not confer copy number-independent transgene expression.



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Figure 1. Restriction of CAT Expression to Bone in Two Mouse Sublines Carrying the OSCAT Transgene

A, Northern blot analysis of transgene expression in transgenic (OST1, OST2) and nontransgenic (FVB/N) mice. Bone (femur and calvaria), brain, liver, kidney, muscle, heart, and lung were collected from sublines carrying low (OST1) and high (OST2) numbers of copies of the OSCAT transgene and from FVB/N mice. Total RNA was analyzed by Northern blots probed for CAT (upper panel) and 18S RNAs (lower panel). The doublet of CAT expression in the OST2 bone may be due to alternative mRNA processing in the SV40 sequence included in the OSCAT8 construct (70) or may result from retardation of the mRNA by the 18S ribosomal RNA. B, CAT enzyme activity in tissues from transgenic and nontransgenic mice. Black bars represent activity from OST2; white bars indicate activity from OST1; and stippled bars depict activity from FVB/N tissues. Each assay was performed in triplicate, and the results are the mean ± SEM of two to six experiments.

 
Measurement of chloramphenicol acetyl transferase (CAT) enzyme activity in protein extracts from transgenic tissues was consistent with the Northern blot results. In both OST1 and OST2 animals, substantial transgene activity was detected in calvarial and femoral extracts but not in extracts from other tissues, with greater activity in calvarial preparations (Fig. 1BGo). CAT activity levels in nonosseous tissues were negligible and did not differ between OST and wild type mice. As in the RNA analysis, greater transgene activity was seen in mice with higher transgene copy number; activities in calvarial and femoral extracts were 8- and 2-fold higher, respectively, in OST2 than in OST1 mice.

In femoral, tibial, calvarial, and vertebral sections from 8-week-old OST2 mice, CAT protein was immunohistochemically detected in cuboidal and flattened osteoblasts, osteocytes, and hypertrophic chondrocytes (femurs shown in Fig. 2Go, A-D). Positively stained osteocytes were located close to bone-forming surfaces. Osteoblastic transgene expression was detected on trabecular bone surfaces and along endosteal and periosteal surfaces of cortical bone. Trabecular CAT protein stained surface in the femoral metaphysis was 27% of the trabecular bone surface. CAT protein was not detectable by immunohistochemistry in OST1 mice or in bones from nontransgenic mice (Fig. 2EGo).



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Figure 2. Cellular Localization of Transgene Expression by Immunohistochemistry

In femurs of 8-week-old transgenic mice, CAT was detected: A, in both cuboidal and flattened osteoblasts and osteocytes in trabeculae; B, in cuboidal osteoblasts and osteocytes on endosteal surfaces, but not in the marrow; C, in osteoblasts on the periosteal surface; and D, in hypertrophic chondrocytes at the base of active chondrocyte stacks in the growth plate. E, No CAT-specific staining was detected in nontransgenic FVB/N bones by immunohistochemistry. Magnification: 400x. b, Bone; c, chondrocyte; m, marrow; ob, osteoblast; oy, osteocyte; hc, hypertrophic chondrocyte.

 
The pattern of transgene expression detected by in situ hybridization reflected that observed by immunohistochemistry (Fig. 3Go, A and B), with CAT mRNA detected in flattened and cuboidal osteoblasts on both trabecular and cortical surfaces, in osteocytes and in hypertrophic chondrocytes. Few positively stained osteocytes, all of them immediately adjacent to positively stained osteoblastic surfaces, were detected by in situ hybridization. No specific transgene signal was detected by immunohistochemistry or in situ hybridization in bones from nontransgenic animals (Fig. 3Go, E and F). Sections hybridized to sense strand probes or to antisense probes, but without anti-digoxigenin antibody, were similarly devoid of positive signal (Fig. 3Go, C and D). Transgene expression followed osteocalcin expression at the single-cell level, shown by in situ hybridization on serial sections (Fig. 3Go, G and H). In no area did CAT expression overlap regions of acid phosphatase activity in the same or adjacent sections (Fig. 3IGo). Osteocalcin mRNA was detectable in megakaryocytes of both transgenic and nontransgenic mice by in situ hybridization in some sections only, but transgene mRNA expression was not detected in megakaryocytes by this technique, even after 1,25-(OH)2D3 treatment.



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Figure 3. Transgene Expression Pattern in Femurs of OST2 Mice

Transgene expression pattern by CAT immunohistochemistry (A) and in situ hybridization (B) on sequential sections of femurs of 8-week-old OST2 mice. Transgene expression was detected in osteoblasts on the endosteal surface of the cortical bone (c) and on trabecular surfaces (t) by both staining techniques. No specific transgene staining was detected in marrow (m). C, No CAT antibody, negative control for panel A; D, sense probe, negative control for panel B; E, nontransgenic (FVB/N) femoral section immunohistochemically stained for CAT protein; F, FVB/N femoral section hybridized to antisense CAT probe. G-I, Colocalization of transgene and osteocalcin expression by in situ hybridization in serial OST2 femoral sections hybridized to CAT (G) and osteocalcin (H) antisense probes. The CAT transgene and endogenous osteocalcin gene were expressed in endosteal osteoblasts (ob) and osteocytes (oy) close to the bone-forming surfaces. Variation in intensity of staining of osteocytes in panels G and H may result from unequal partitioning of the major portion of the osteocyte cell bodies in serial sections. I, Adjacent section stained for osteoclasts (oc) by tartrate-resistant acid phosphatase activity, with hematoxylin counterstain. Magnification: 200x (A-F); 400x (G-I).

 
1,25(OH)2D3 Response
Osteoblastic cultures from OST2 transgenic calvariae responded to 1,25-(OH)2D3 treatment with a significant increase in transgene activity (P = 0.003 compared with vehicle-treated OST2 cultures). In contrast to this elevation of transgene expression by 1,25-(OH)2D3, there was a slight but nonsignificant decrease in endogenous osteocalcin released into cell culture supernatants in both OST2 (P = 0.08) and FVB/N (P = 0.13) cultures (Fig. 4Go).



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Figure 4. Increase of CAT Activity, but Not Osteocalcin Release, in Primary Cell Cultures from Transgenic Mice Treated with 1,25-(OH)2D3

Calvarial osteoblastic cell cultures from transgenic (OST2) and nontransgenic (FVB/N) mice were treated with 10-8 M 1,25-(OH)2D3 for 5 days. CAT enzymatic activity was significantly increased in the OST2 culture lysates after 1,25-(OH)2D3 treatment. In the same OST2 cultures, and in parallel FVB/N control cultures, osteocalcin protein content in the culture supernatants, corrected for DNA content, was slightly decreased, but this decrease did not reach statistical significance. *, P < 0.005. Values shown are the mean ± SEM of eight OST2 and five FVB/N lysates.

 
Consistent with calvarial culture results, a significant increase in transgene mRNA levels after 1,25-(OH)2D3 treatment of OST1 and OST2 mice fed the high calcium (HighCa) diet was detected by Northern blot. Transgene mRNA levels in bones reached maximal increases of similar magnitude in both low (20.5 ± 9.7) and high (17.0 ± 2.5) copy number mice at 6 h after 1,25-(OH)2D3 treatment. In contrast, the endogenous osteocalcin mRNA level was not significantly changed by 1,25-(OH)2D3 treatment in either transgenic line or in nontransgenic FVB/N mice (Fig. 5Go, A and B). Baseline osteocalcin mRNA levels were lower in uninjected OST1 and OST2 mice than in FVB/N mice, with the following OC/18S mRNA ratios: FVB/N, 1.7± 0.3; OST1, 0.3 ± 0.2; OST2, 0.5 ± 0.3, P = 0.045.



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Figure 5. Increased Transgene Expression in OST1 and OST2 Sublines Treated with 1,25-(OH)2D3 with Minimal Effect on Osteocalcin (OC) Expression in Transgenic or Nontransgenic Mice

A and B, OST1, OST2, and FVB/N mice were fed the high calcium diet and given a single intraperitoneal injection of 1,25-(OH)2D3. Tissues were collected at 0, 6, or 24 h after treatment for Northern blot analysis of total RNA from bone (Bo), brain (Br), and kidney (Ki). A, A representative Northern blot analysis of tissues from mice injected with 1,25-(OH)2D3 (+) or vehicle (-). Filter was probed sequentially for CAT transgene, osteocalcin, and 18S transcripts. B, Phosphorimage analysis of Northern blot signals. The treated/control ratios of CAT and osteocalcin signals were quantitated and calculated as described in Materials and Methods. (Values are mean ± SEM; *, P < 0.05 vs. uninjected control value.) This experiment was repeated three times. C–E, OST2 and FVB/N mice were fed the low calcium or SrCl2 diet for 10 days before injection with 1,25-(OH)2D3 or vehicle. Quantification of Northern blot analyses of CAT (C) and osteocalcin (D) expression in OST2 and osteocalcin (E) expression in FVB/N mice at the indicated times after injection. Data are expressed as the ratios of normalized signals of treated mice to uninjected mice, as described in Materials and Methods [mean ± SEM; *, P < 0.05 vs. vehicle value at the same time point and vs. uninjected value (To)]. This experiment was repeated five times for each diet.

 
The SrCl2 diet significantly reduced serum osteocalcin levels in both transgenic and nontransgenic mice (P = 0.004). Levels in untreated FVB/N mice fed the SrCl2 diet were 93.5 ± 11.7 compared with 125.7 ± 8.6 in low calcium-fed (LowCa) mice. In OST2 mice, levels were 78.6 ± 7.8 on SrCl2 and 103.8 ± 11.2 on LowCa. The lower level of serum osteocalcin in OST2 mice compared with FVB/N was also statistically significant (P = 0.048). This reduction in serum osteocalcin in OST2 mice and the reduction in osteocalcin mRNA levels in OST1 and OST2 mice described above may reflect competition for transcriptional regulatory factors by the large number (40) of integrated OSCAT8 transgene copies. Treatment of the mice with 1,25-(OH)2D3 did not significantly alter serum osteocalcin levels at any time after hormone injection, regardless of dietary manipulations or transgene status.

Similar CAT responses to 1,25-(OH)2D3 treatment were seen in mice fed SrCl2 and LowCa diets. Transgene mRNA levels were maximally increased 6 h after 1,25-(OH)2D3 treatment in both dietary groups, remained significantly elevated at 9 h after treatment, and had returned to uninjected control levels by 12 h (Fig. 5CGo). The increase in transgene expression at 6 h in SrCl2-treated OST2 mice (6.6 ± 0.9-fold higher than uninjected values) was considerably lower than that observed in mice on the LowCa or HighCa diets treated with 1,25-(OH)2D3 (23.6 ± 3.6- and 25.2 ± 7.7-fold, respectively). As previously noted for animals fed the HighCa diet, osteocalcin mRNA levels in both transgenic and nontransgenic mice fed LowCa and SrCl2 diets were not altered by 1,25-(OH)2D3 treatment (Fig. 5Go, D and E).

Cellular responses to 1,25-(OH)2D3 treatment were examined histologically. In situ hybridization analyses indicated that in femoral sections from all OST2 mice, the spatial distribution of transgene expression was unchanged in hormone-treated animals, with strong staining in trabecular and endosteal osteoblasts, in osteocytes close to bone-forming surfaces and in hypertrophic chondrocytes (Fig. 6AGo). With reduced CAT probe concentration, only the most strongly transgene-expressing cells were positively stained (Fig. 6BGo, 0-h sample).



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Figure 6. Increased Transgene, but Not Osteocalcin, Expression in 1,25-(OH)2D3-Treated OST2 and Nontransgenic FVB/N Mice

Animals were treated with a single injection of 1,25-(OH)2D3 or vehicle, and in situ hybridization was carried out on paraffin-embedded distal femurs. Three experiments were performed in mice fed the high calcium diet and five were performed in mice fed the low calcium or SrCl2 diet, and five sections of each bone sample were hybridized with CAT and osteocalcin probes. Representative sections for each time point were selected for this figure. A, CAT in situ hybridization, showing staining in trabecular and endosteal osteoblasts, in osteocytes close to bone-forming surfaces and in hypertrophic chondrocytes. B, CAT in situ hybridization using a low concentration of probe (1.5 ng/slide) in uninjected (0 h) and at 3, 6, 9, and 12 h post 1,25-(OH)2D3 treatment. The number of cells expressing a high level of transgene was increased at 3, 6, and 9 h after treatment, with a maximum after 6 h. Note the low number of stained cells in uninjected animals compared with panel A. C, CAT expression in vehicle-treated animals was unchanged by 1,25-(OH)2D3 treatment (6 h posttreatment shown). D, In situ hybridization for endogenous osteocalcin expression, showing staining in the same cells as the transgene (panel A). E, Expression of the endogenous mouse osteocalcin gene using low levels of probe (0.75 ng/slide) in 1,25-(OH)2D3-treated animals. Endogenous mouse osteocalcin expression was not increased by 1,25-(OH)2D3 treatment, with no alteration in the number of positive cells or in staining intensity. F, Expression of endogenous mouse osteocalcin mRNA in vehicle-treated animals was unchanged (6 h posttreatment shown).

 
At 6 h after 1,25-(OH)2D3 injection, the number of cells positive for transgene mRNA expression using the low probe concentration was dramatically increased in all three dietary groups. Transgene expression was also increased at 3 and 9 h after treatment, but the maximal level of staining consistently occurred at 6 h. Intense staining was seen in the same cell types and along the same surfaces that stained positive using high CAT probe concentration in uninjected animals (compare Fig. 6AGo with 6B, 6-h sample). At 12 (Fig. 6BGo) and 24 (not shown) h after injection, the number of strongly positive cells had returned to levels observed in uninjected controls (Fig. 6AGo) or in vehicle- treated animals (Fig. 6CGo). In OST1 mice (even using the high probe concentration) transgene expression, which was below the level of detection in uninjected and vehicle-treated mice, was apparent at 6 h after 1,25-(OH)2D3 treatment in osteoblasts in the primary spongiosa and in some hypertrophic chondrocytes and was again undetectable at 24 h after treatment (not shown).

The distribution of transgene expression mirrored osteocalcin expression detected by in situ hybridization at the high probe concentration (Fig. 6DGo). Consistent with the Northern blot results, expression of the endogenous mouse osteocalcin mRNA was not increased by 1,25-(OH)2D3 treatment, with no alteration in the number of positive cells or staining intensity, regardless of transgene status or dietary manipulation (Fig. 6Go, E and F).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The osteoblast-targeting vector, OSCAT8, with 5'- and 3'-flanking sequences from the human osteocalcin gene, directed reporter gene expression specifically to osteoblastic lineages in transgenic mice. The levels of transgene mRNA and transgene activity were high in bone, and levels in nonosseous tissues were comparable to nontransgenic levels. Brain and kidney tissue extracts yielded no detectable CAT activity or RNA in the OSCAT8 mice. The histological pattern of transgene expression closely followed osteocalcin expression and was comparable to in situ hybridization patterns of osteocalcin expression in rat femurs (5, 6, 41). Transgene and osteocalcin expression were detected in osteoblasts, osteocytes, and hypertrophic chondrocytes in the present study. Although some osteocalcin expression was detectable in megakaryocytes (7), transgene expression was not detected in bone marrow megakaryocytes in this study, even after 1,25-(OH)2D3 treatment. It is not clear whether the previously reported low levels of osteocalcin gene expression in megakaryocytes (7) and brain, intestine, and kidney (8) are derived from the osteocalcin genes or from the osteocalcin-related genes described in mice and rats. If osteocalcin expression does occur in human megakaryocytes, data from the present study indicate either that such expression is directed by regions of the osteocalcin promoter not included in the 5'- and 3'-sequences in the OSCAT8 construct or that the human sequences present in the transgene do not function in murine megakaryocytes.

The pattern of transgene expression seen in the OST sublines is similar to those reported in previous studies utilizing varying extents of 5'-flanking sequences from the human and rat osteocalcin genes, with the exception that all previous reports have demonstrated ectopic transgene expression in kidney and/or brain (42, 43, 44). Although the present study suggests a regulatory role for the 3'-flanking sequence, this possibility cannot be confirmed because there has been no direct comparison of constructs with and without this 3'-sequence.

In situ hybridization analysis demonstrated that after 1,25-(OH)2D3 treatment, the spatial pattern of the increased transgene expression was unchanged. Before hormone treatment, although there was extensive transgene expression in trabecular and endosteal osteoblasts as well as in osteocytes and hypertrophic chondrocytes, only a few cells of each type exhibited a high level of transgene expression. After 1,25-(OH)2D3 treatment, the number of trabecular and cortical osteocytes and hypertrophic chondrocytes expressing high levels of the transgene was increased, supporting a function for vitamin D receptors in hypertrophic chondrocytes (45, 46) and suggesting a presence of functional receptors in osteocytes. 1,25-(OH)2D3 regulation of collagen gene products has been described in hypertrophic chondrocytes (40, 47), but regulation of a noncollagenous protein in this cell type has not previously been described.

Whereas the basal transgene expression pattern closely followed endogenous osteocalcin gene expression in bone cells, the hormone responsiveness of the human-derived transgene differed from that of the endogenous gene. In both primary cell culture and whole animal studies, expression of the human osteocalcin-directed transgene was markedly increased by 1,25-(OH)2D3 treatment. In the same experiments and in nontransgenic controls, however, hormone treatment did not significantly alter levels of mouse osteocalcin protein in vitro, osteocalcin mRNA levels in vivo, or serum osteocalcin levels over the time course studied, indicating a species-related difference in osteocalcin promoter responses to 1,25-(OH)2D3 treatment.

Other studies have also indicated a minimal response of the mouse osteocalcin gene to 1,25-(OH)2D3 treatment. Whereas rat osteocalcin gene expression increased in response to 1,25-(OH)2D3 treatment, in three of four murine osteoblast-like osteosarcoma cell lines, expression of the mouse gene was unchanged after 1,25-(OH)2D3 treatment, and primary mouse calvarial osteoblast cultures had only a weak positive response (18). Similarly, a recent study demonstrated that a human osteocalcin locus transgene, including 4 kb of upstream sequence, the coding region, and 15 kb of downstream sequence, stably incorporated in the mouse genome, responded to 1,25-(OH)2D3 treatment with increased serum levels of human osteocalcin but no change in endogenous mouse osteocalcin protein (48), as demonstrated in the present study.

Other recent studies have reported a decrease in mouse osteocalcin expression after 1,25-(OH)2D3 treatment. For example, osteocalcin mRNA levels in mouse calvaria were reduced 6-fold after 1,25-(OH)2D3 treatment in vivo, and osteocalcin mRNA expression was halved in primary mouse osteoblast cultures treated with 1,25-(OH)2D3 (38). Furthermore, gel mobility shift analysis indicated that the VDRE-like sequence in the mouse osteocalcin genes does not mediate this response to 1,25-(OH)2D3 (38); rather, indirect regulation via decreased activity of the OSF2 transcription factor was proposed (34). In another study, 1,25-(OH)2D3 caused a decrease in osteocalcin release into culture supernatant of the murine osteoblast-like cell line MC3T3-E1, whereas in the same cell line, a stably transfected rat osteocalcin promoter construct responded positively to 1,25-(OH)2D3 treatment. Moreover, in transient transfections, activity of a reporter construct bearing the mouse osteocalcin VDRE fused to a minimal TATA-containing promoter was repressed by 1,25-(OH)2D3 treatment, whereas the activity of an analogous construct bearing the rat VDRE sequence was stimulated by the hormone treatment (39). Contrary to the previous report, however, this latter study also demonstrated that the mouse osteocalcin VDRE is bound by a complex containing VDR and RXR{alpha} in gel mobility shift assays, a difference from the previously cited work that may stem from variation in probe length in the two studies. Despite this difference, the common finding in those published studies, that 1,25-(OH)2D3 reduces mouse osteocalcin expression to a significant extent, differs from those of the present study. Whether these differences relate to duration of hormone treatment, extent of mineralization of the primary osteoblast cultures at the time of treatment, and/or variation in response between mouse strains is not yet known, but the influence of these factors clearly requires further investigation.

It is possible that the mouse osteocalcin gene shows no positive response to 1,25-(OH)2D3 treatment because the endogenous gene is maximally stimulated in the untreated state. This interpretation would be consistent with the findings of Clemens and colleagues (48) who reported higher serum levels of mouse osteocalcin than human osteocalcin in their transgenic mouse model. However, even though inhibition of vitamin D metabolism reduced serum osteocalcin levels in the present study, 1,25-(OH)2D3 still did not induce endogenous osteocalcin transcription. Interestingly, the transgene response to 1,25-(OH)2D3 was reduced in vitamin D-deficient mice. Since 1,25-(OH)2D3 may increase VDR transcription (49) or stabilize VDR protein (50) in rat osteosarcoma cells, it may follow that VDR is less stable in vitamin D deficiency, attenuating any transcriptional response to the hormone.

Although osteocalcin and VDR do not appear to be required for embryonic development in the mouse (11, 51), they clearly play important roles in postnatal bone physiology. In mice lacking functional osteocalcin genes, bone density is increased with apparently elevated osteoblast activity, suggesting a role for osteocalcin in limiting bone formation in the mouse (11). Similarly, growth retardation and widening of the growth plate are observed in VDR knockout mice, although serum osteocalcin levels are not altered (51). These alterations in bone structure are similar to those seen in vitamin D- deficient states in human (52, 53) and rat (54, 55, 56). Thus, although the interaction between 1,25-(OH)2D3 and bone is important for postnatal bone growth, the present study suggests VDRE-mediated up-regulation of osteocalcin is not required for this process in the mouse. By extension, 1,25-(OH)2D3 regulation of osteocalcin expression may not be involved in bone growth in humans. Alternatively, the physiological role of VDRE-mediated osteocalcin regulation may differ between species.

Given the murine-human difference in osteocalcin regulation by 1,25-(OH)2D3 described here, it is important that an appropriate model be used to study the physiological regulation of the human osteocalcin gene. The OSCAT8 transgenic mouse may represent such a model, utilizing 5'- and 3'-human osteocalcin-flanking regions to direct transgene expression and mimic the human osteocalcin gene in both expression pattern and 1,25-(OH)2D3 regulation. In conjunction with other reports, the present study provides strong evidence for a species-related difference in the effect of 1,25-(OH)2D3 on VDRE-mediated regulation of osteocalcin transcription in the mouse and human, with a 1,25-(OH)2D3-stimulated increase in the human osteocalcin-directed transgene without a concomitant increase in endogenous mouse osteocalcin transcription. This species difference in 1,25-(OH)2D3 responsiveness relates to sequence variations in osteocalcin gene-regulatory regions rather than inherent differences in the bone cellular milieu. Insights gained from further investigation of these species-related differences may shed light on the function of osteocalcin and its regulation by vitamin D in bone physiology.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DNA Constructs and Probes
The pOSCAT8 construct contained the CAT reporter gene flanked by 5'- and 3'-regions of the human osteocalcin gene in natural position and orientation. The 3.8 kb of continuous 5'-flanking sequence, including a known VDRE (25, 26), was generated by inserting a 3.5-kb HindIII-SacI fragment of genomic DNA (26) into the SacI site at position -344 of the human osteocalcin gene in the pOSCAT1 construct (26). A 3.5-kb BamHI-HindIII fragment from the human osteocalcin gene including 108 bp of exon 4 and 3.4 kb of 3'-untranslated and flanking DNA was inserted downstream of the SV40 polyadenylation signal.

Southern blots were probed with a CAT-specific DNA probe (1.8 kb BglII-BamHI fragment from the pCAT-promoter vector (Promega, Madison, WI) or with a probe specific for the human osteocalcin 3'-sequence (1.1 kb KpnI fragment from pOSCAT8 containing human osteocalcin 3'-flanking DNA). DNA slot blots were probed for the mouse neurofilament gene (57) and subsequently with the human osteocalcin 3'-probe. Northern blots were probed with random primed CAT-specific DNA probe or osteocalcin-specific probe and subsequently with a 32P-kinased oligonucleotide probe for 18S ribosomal RNA (5'-CGGCATGTATTAGCTCTAGAATTACCACAG-3').

For in situ hybridization, riboprobes were generated from linearized pOC918 construct containing a rat osteocalcin cDNA insert (a gift from Dr. S. E. Harris). For the CAT probes, a 1.7-kb HindIII fragment of the pCAT-promoter vector was subcloned into pGEM-II to form pGEMCAT6, which was linearized and transcribed to generate the antisense strand CAT riboprobe. A reverse orientation construct (pGEMCAT2) was similarly transcribed to generate the sense strand riboprobe.

Generation of Transgenic Mice and Genomic DNA Analysis
Transgenic mice were generated by pronuclear injection of the purified HindIII insert from pOSCAT8 into inbred FVB/N embryos (58) and screened by Southern blot analysis of genomic DNA prepared from tail biopsy. Upon back-crossing the founder animal to FVB/N, two transgene insertions segregated. Inbred sublines (OST1 and OST2) with stable transgene transmission patterns were generated by back-crossing to FVB/N mice for two further generations before interbreeding hemizygous siblings. Eight-week-old hemizygous transgenic animals were bred from homozygous sires and nontransgenic FVB/N dams for all studies. Nontransgenic FVB/N mice were specifically bred as age-matched controls for these experiments. All experimental animals and mice used to generate transgenic lines were bred at the Garvan Institute in studies approved by the Institute Animal Experimentation Ethics Committee.

Quantitative slot blot analysis was performed using 5 µg DNA and probed with the mouse neurofilament probe and quantitated on a Molecular Dynamics 445 SI PhosphorImager (Sunnyvale, CA). The filter was stripped then reprobed with the transgene-specific 3'-osteocalcin DNA probe and quantitated. Transgene copy number was calculated by normalization to the neurofilament signal. In these two sublines of OSCAT8 transgenic mice, quantification of the number of incorporated copies of the transgene showed low and high copy number insertions, with OST1 and OST2 containing 2 and 60 transgene copies per haploid genome, respectively.

Primary Bone Cell Isolation and Culture
Osteoblast-like cells were derived from 3- to 6-day-old FVB/N and OST2 mice as follows (59). Frontal and parietal bones were removed and washed twice in PBS, and adherent soft tissue and sutures were dissected away. Cleaned calvariae were transferred to culture medium containing 30 mM HEPES. Bones were washed again and minced into normal culture media ({alpha}-MEM) containing 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 40 µg/ml gentomycin, 10 mM ß-glycerophosphate, 50 µg/ml ascorbic acid, 20 mM HEPES, 2 mM glutamine. Calvariae were washed in PBS and transferred to 1 ml digest mix per five calvaria [1 mg/ml collagenase (Boehringer-Mannheim, Mannheim, Germany), 0.05% trypsin, 0.02% EDTA in PBS] and stirred vigorously for 30 min at 37 C. Five sequential digests were carried out; the last three were washed in PBS and resuspended in fresh culture medium, pooled, aspirated repeatedly through 19- and 21-gauge needles, and filtered through a 200-µm polyester filter (Spectrum Industries, Houston, TX) to a single cell suspension. Cells were seeded at a density of 104 cells per cm2 into six-well plates and cultured in a humid atmosphere with 5% CO2 at 37 C. Medium was changed after 3 days and every 2–3 days thereafter. Mineralizing cultures were treated with 10-8 M 1,25-(OH)2D3 from day 29 and stopped on day 35; untreated controls were stopped on day 35.

DNA, CAT, and Protein Assays
Cells were lysed in 500 µl 0.1% Triton-X100, 0.5 mM MgCl2 in 20 mM Tris, pH 10, and stored at -20 C until assayed. The fluorometric DNA assay was adapted from Rao and Otto (60) and Downs and Wilfinger (61). Duplicate samples of 50 µl lysate were mixed with 200 ml of 1 µg/ml bisbenzimide in water in clear polystyrene 96-well plates, allowed to stand for 5 min, then read on a Fluoroskan II (Labsystems, Helsinki, Finland) with excitation and emission settings at 355 nm and 460 nm, respectively. DNA values were calculated using DeltaSoft analytical software (Biometallics, Princeton, NJ).

For the CAT and protein assays the same cell lysates were freeze-thawed three times to further release cellular proteins. A 1-h 65 C incubation to inactivate nonspecific acetyl transferases was carried out. CAT was assayed as described by Morrison et al. (26) based on the nonchromatographic assay of Sleigh (62). Protein was assayed using the Bio-Rad (Hercules, CA) protein assay based on the colorimetric method of Bradford (63).

Osteocalcin levels in the culture medium were determined by RIA using the method of Gundberg et al. (64) except sample sizes assayed were 50 µl instead of 5 µl. The concentration of primary antibody was altered accordingly to maintain the same total assay volume. Primary antibody and osteocalcin standards were kindly provided by Dr. C. Gundberg. Iodinated osteocalcin was purchased from Biomedical Technologies, Inc (Stoughton, MA). Donkey anti-goat IgG secondary antibody was purchased from Sigma (St. Louis, MO).

1,25(OH)2D3 Treatment in Vivo
Animals were maintained on standard laboratory chow (Barastoc, Ridley Agri Products, Victoria, Australia) containing 0.94% calcium, 0.81% phosphorus, and 1800 IU vitamin D3/kg and supplied with water ad libitum. Ten days before treatment, at 6.5 weeks of age, animals were allocated one of three diets: standard laboratory chow (HighCa); semisynthetic AIN-93 diet (65) containing 0.1% calcium, 0.27% phosphorus, and 1800 IU vitamin D3/kg (LowCa); or vitamin D metabolism-inhibiting diet containing 0.1% calcium, 0.27% phosphorus, no vitamin D3, and 0.8% strontium chloride (SrCl2) added to AIN-93. At 8 weeks of age, animals were given a single intraperitoneal injection of 1,25-(OH)2D3 in 10% argon-saturated isopropanol/90% propylene glycol at a dose rate of 2 µg/kg body weight or an equivalent volume of vehicle (0.1 ml). At various times after injection, animals were bled by cardiac puncture under methoxyflurane anesthesia (Metofane, Pittman-Moore, Mundelein, IL) and killed by cervical dislocation. Femurs, tibias, calvariae, brain, kidney, muscle, heart, lung, spleen, and liver were collected at the time of death and from uninjected mice at the time of injection. One femur, taken for in situ hybridization, was immediately fixed in 4% paraformaldehyde/PBS at 4 C overnight. Other tissues collected were snap-frozen in liquid nitrogen and stored at -70 C until RNA preparation. Serum was prepared from blood and stored at -70 C before serum osteocalcin measurement (64).

Initial experiments, repeated three times, included OST1, OST2, and FVB/N animals fed only the HighCa diet. Tissues were collected at the time of injection or at 6 or 24 h after injection. In subsequent comparisons of OST2 and FVB/N mice fed the LowCa and SrCl2 diets, tissues were collected at the time of injection or at 3, 6, 9, 12, or 24 h after injection. These experiments were repeated five times. For all experiments, at each repetition, single animals were injected with hormone or vehicle for each time point. Each experiment was analyzed once by Northern blot.

Preparation and Analysis of RNA
Tissues were homogenized at 3000-5000 rpm for 15 sec in 4.5 ml guanidine thiocyanate buffer (4 M guanidinium isothiocyanate, 0.5% sarkosyl, and 0.1 M ß-mercaptoethanol) using a Polytron (Brinkman, Westbury, NJ). Total cellular RNA was isolated from homogenates by acid phenol-chloroform extraction (66) followed by cesium chloride gradient centrifugation (67). RNA (15 µg) was analyzed by Northern blot analysis on a nylon filter (HybondN+, Amersham, Buckinghamshire, UK) and probed with the random primed {alpha}-32P-CTP-labeled CAT probe followed, after stripping, by the human osteocalcin probe, then the {gamma}-[32P]ATP 18S oligonucleotide probe. For each probe, radioactive counts bound to the filter were quantitated by phosphorimaging. For each sample, CAT and osteocalcin values were normalized to the corresponding 18S signal. Treated/control ratios were calculated by dividing the normalized CAT and osteocalcin values for bone samples from 1,25-(OH)2D3-treated mice by the corresponding normalized CAT and osteocalcin values for bones from vehicle-treated mice at the same time points. For the treated/T0 ratios, the normalized CAT and osteocalcin values for the 1,25-(OH)2D3-treated mice were divided by normalized CAT and osteocalcin values from uninjected mice.

Histology
Skull, vertebrae, tibia, and femur samples were fixed in 4% paraformaldehyde/PBS at 4 C. Skulls were bisected sagittally during fixation. Femurs were trimmed and the distal third of each was used for analysis. Specimens were decalcified for 4 days in 15% EDTA/0.5% paraformaldehyde/PBS at 4 C and embedded in paraffin. Sections (5 µm) were taken onto chrome alum-coated slides, dewaxed in Histoclear (National Diagnostics, Atlanta, GA), and rehydrated in graded ethanols followed by PBS before staining.

For immunohistochemistry, sections were blocked using 5% normal goat serum and incubated for 1 h at room temperature with a rabbit polyclonal anti-CAT antibody (5 Prime -> 3 Prime Inc., Paoli, PA) diluted 1:100 in PBS containing 2% normal goat serum. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide, followed by a 30-min incubation with 0.3% biotinylated goat anti-rabbit IgG and an avidin-biotinylated peroxidase complex (Vector Laboratories, Burlingame, CA). Peroxidase was detected with a diaminobenzidine staining kit (Vector Laboratories). For quantification of immunohistochemical CAT staining, six femoral sections at 10-µm intervals were analyzed from each of five 8-week-old transgenic mice. CAT surface as a percentage of total trabecular bone surface was determined in the metaphysis of each section using the Osteomeasure system (Osteometrics Inc., Decatur, GA).

In situ hybridization was modified from Zhou and colleagues (68). Rat osteocalcin and CAT antisense and sense probes were generated as described above. Sections were incubated with 0.2 M HCl for 20 min at room temperature, followed by digestion with 1 µg/ml proteinase K in 0.1 M Tris buffer (pH 8.0) for 45 min at 37 C, followed by 2 mg/ml glycine in PBS for 5 min. The tissues were refixed in 4% paraformol saline for 15 min before acetylation in 0.25% acetic anhydride/0.1 M triethanolamine (pH 8.0) for 10 min and rinsed in PBS between all pretreatments. Slides were stained for 1 h at room temperature with Fast Red TR (Sigma) to eliminate endogenous alkaline phosphatase staining and washed for 20 min in absolute ethanol before rehydration in PBS. Prehybridization was performed at room temperature for 30 min, followed by 2 h at 37 C in hybridization buffer: 50% formamide, 5x NaCl-sodium citrate (SSC), 2% block reagent (Boehringer Mannheim) 0.1% N-lauroyl-sarcosine, and 0.02% SDS. Digoxigenin-labeled probes were applied in hybridization buffer at concentrations described below before incubation for 16–18 h at 42 C in a humidified chamber. Slides were washed in 2x SSC, then 1x SSC, and finally 0.1x SSC, each for 30 min at 37 C. Hybridized probe was detected using the alkaline phosphatase-coupled anti-digoxigenin antibody method (Boehringer Mannheim) with the addition of 1.2 mg/ml levamisole (Sigma) in the final staining solution.

Osteocalcin and CAT mRNA transcripts were initially detected using 7.5 ng and 15 ng probe/slide, respectively, for in situ hybridization. However, with these excess probe concentrations, the transgene signal was so strong that it was not possible to detect the increase in transgene RNA that had been documented using Northern blot analysis. Therefore, reduced probe concentrations were empirically chosen based on hybridization to control sections of uninjected OST2 femurs until a minimal level of osteocalcin and CAT mRNA staining was observed, thus allowing detection of even small changes in expression. Final concentrations used for 1,25-(OH)2D3 treatment experiments were 0.75 ng/slide and 1.5 ng/slide for osteocalcin and CAT, respectively.

Femoral sections were also stained for tartrate-resistant acid phosphatase using a method modified from Parkinson et al. (69) by inclusion of tartaric acid in the staining and prestaining buffers with a hematoxylin counterstain. Some femoral sections stained for CAT by immunohistochemistry were also stained for tartrate-resistant acid phosphatase before immunohistochemical staining. Sections were photographed using a Zeiss Axiophot microscope (Carl Zeiss, Thornwood, NY) with differential interference contrast optics at all magnifications shown.

Statistical Analysis
Treatment responses of mRNA expression and serum osteocalcin were compared by one, two, and three-way ANOVA followed by Tukey’s post-hoc test. In all analyses, P < 0.05 was considered significant. Histomorphometric measurements are expressed as the mean ± SEM from five femoral sections from each of six animals.


    ACKNOWLEDGMENTS
 
The authors thank A. Bourne and R. Enriquez for excellent technical assistance, and the director and staff (Dr. J. Ferguson, P. Gregory, T. Chaplin, L. Peters) of the Garvan Institute Biological Testing Facility for consistently high quality animal care and breeding. We also thank Dr. S. E. Harris (University of Texas Health Sciences Center, San Antonio, TX) for the rat osteocalcin cDNA clone, Dr. C. Gundberg (Yale University, New Haven, CT) for mouse osteocalcin antibody, standards, and RIA method, E. Vasac (Centre for Immunology, St. Vincent’s Hospital, Sydney, Australia) for sharing the Fast Red TR technique, and Dr. K. W. Ng (St. Vincent’s Hospital, Melbourne, Australia) for the use of the Osteomeasure Image Analysis System.


    FOOTNOTES
 
Address requests for reprints to: Edith M. Gardiner, Bone and Mineral Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, New South Wales 2010, Australia.

This work was supported by NIH Grant 1RO1-AR43421 and by funding from Aza Research Pty. Ltd.

Received for publication May 14, 1997. Revision received July 22, 1997. Accepted for publication July 23, 1997.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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