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1,25-Dihydroxyvitamin D3 inhibition of Col1a1 promoter expression in calvariae from neonatal transgenic mice
Biochimica et Biophysica Acta 1398 (1998) 285^293
1,25-Dihydroxyvitamin D3 inhibition of Col1a1 promoter expression in calvariae from neonatal transgenic mice Antonio Bedalov 1;a , Roberto Salvatori 2;a , Milan Dodig a , Belinda Kapural a , Dubravko Pavlin 3;a , Barbara E. Kream b , Stephen H. Clark c;d , Charles O. Woody e , David W. Rowe a , Alexander C. Lichtler a; * a c
Department of Pediatrics, MC1515, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030, USA b Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030, USA Division of Rheumatic Diseases, Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030, USA d Department of Veterans A¡airs Medical Center, Newington, CT 06111, USA e Department of Animal Science University of Connecticut, Storrs, CT 06268, USA Received 25 March 1998; accepted 9 April 1998
Abstract We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to 33518, 32297, 31997, 31794, 31763, and 31719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2 D3 had a dosedependent inhibitory effect on the expression of the 33518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2 D3 , suggesting that the sequences required for 1,25(OH)2 D3 inhibition are downstream of 31719 bp. The inhibitory effect of 1,25(OH)2 D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2 D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2 D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1,25(OH)2 D3 responsive element downstream of 31719 bp. The inhibitory effect does not require new protein synthesis. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Collagen; Calcium regulating hormone; Bone; Osteoblast; Transcription
* Corresponding author. Fax: +1 (860) 679-1047; E-mail: [email protected] 1 Permanent address: University of Zagreb, School of Medicine, Salata 3b, 41000 Zagreb, Croatia. 2 Present address: Division of Endocrinology, Johns Hopkins University, Baltimore, MD 21287, USA. 3 Present address: Departments of Orthodontics and Cellular and Structural Biology, The University of Texas Health Science Center, San Antonio, TX 78284, USA. 0167-4781 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 8 ) 0 0 0 7 9 - 7
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1. Introduction The seco-steroid hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3 ) is an important regulator of calcium homeostasis. Decreased serum calcium increases 1,25-(OH)2 D3 production, which increases intestinal calcium absorption and stimulates bone resorption [1]. 1,25-(OH)2 D3 inhibits collagen synthesis in many bone culture systems, including cultured intact fetal and neonatal rat and mouse [2^4], primary cultures of osteoblast-enriched calvarial cells [5] and clonal osteoblastic cell lines [6]. Previous work from our laboratory demonstrated that the inhibition of collagen synthesis by 1,25-(OH)2 D3 is accompanied by a decrease in procollagen mRNA levels in fetal rat calvariae and in the rat osteoblastic cell line ROS 17/2 [3,7]. We also showed that 1,25(OH)2 D3 inhibits Col1a1 and Col1a2 transcription in ROS 17/2.8 cells [8], and decreases the expression of a Col1a1 promoter-chloramphenicol acetyltransferase (CAT) construct extending to 33518 bp (ColCAT3.6) in transiently transfected ROS 17/2.8 cells [9]. More recent studies using stably transfected ROS 17/2.8 cells showed that a ColCAT construct extending to 32.3 kb, and a construct containing an internal deletion from 32256 to 32216 bp were both inhibited by 1,25-(OH)2 D3 , however a construct extending to 31670 bp was not inhibited. This suggested that a DNA sequence necessary for the 1,25(OH)2 D3 response may be located in the region between 32216 and 31670 bp [10]. We have previously observed that there are signi¢cant di¡erences in the regions of the Col1A1 promoter which are responsible for maintaining high basal activity in transgenic mouse calvariae and osteoblast-like cell lines including ROS 17/2.8 [11^13]. This raised the possibility that di¡erent regulatory elements are involved in 1,25-(OH)2 D3 regulation of the Col1a1 gene in vivo and in vitro. In addition, we have found that the response of the Col1a1 gene to 1,25-(OH)2 D3 treatment in ROS 17/2.8 cells is not as consistent as the response in cultured calvariae. We have generated a series of transgenic mice containing deletions of the Col1a1 promoter [12]. Therefore, in this study we have used transgenic mouse calvariae containing these deletions to analyze the 1,25-(OH)2 D3 responsive region in the Col1a1 promoter. Calvariae obtained from these mice were cul-
tured in the presence and absence of 1,25-(OH)2 D3 . We show that 1,25-(OH)2 D3 decreases CAT activity and transgene mRNA levels in organ cultures of calvariae from transgenic mice and that these e¡ects do not require protein synthesis. We also show that the proximal 1719 bp of the 5P £anking region of the Col1a1 promoter are su¤cient to confer 1,25(OH)2 D3 inhibition. 2. Materials and methods 2.1. Materials Crystalline 1,25-(OH)2 D3 was a generous gift of Dr. Milan Uskokovic of Ho¡man-LaRoche (Nutley, NJ) and was diluted in ethanol to give a 10 mM stock solution. BGJb medium was purchased from Gibco (Grand Island, NY). 2.2. Transgenic mouse lines The construction of ColCAT3.6, ColCAT2.3, ColCAT1997, ColCAT1794, ColCAT1764, ColCAT1719 and ColCAT1.7 (Fig. 1), containing portions of the rat Col1a1 promoter fused to the CAT reporter gene, and production of transgenic mice with these constructs, have been previously reported [12]. Transgenic animals were initially identi¢ed via dot-blot analysis of DNA using a CAT-speci¢c probe. Subsequent generations were identi¢ed by analyzing CAT activity in tail fragments. All animal experiments were conducted in accordance with the highest possible standards of humane animal care, as outlined in the NIH Guide for the Care and Use of Laboratory Animals. 2.3. Calvarial organ cultures Frontal and parietal bones were dissected from 6^ 8-day-old mice and cut along the sagittal suture. Each hemicalvaria was cultured in a 35 mm tissue culture well (Falcon) containing 2 ml of BGJb medium with 1 mg/ml bovine serum albumin, 100 Wg/ml ascorbic acid and 1 mM proline. 1,25-(OH)2 D3 or ethanol vehicle was added and the culture dishes were placed on a rocking platform in an incubator at 37³C in 5% CO2 /95% air. After 24 h, fresh me-
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dium containing 1,25-(OH)2 D3 or vehicle was added. After culture for the times speci¢ed, hemicalvariae were washed twice in cold phosphate-bu¡ered saline (PBS) and either analyzed for CAT activity or utilized for total RNA extraction. 2.4. Measurement of CAT activity After washing with PBS, each hemicalvaria was put in an Eppendorf tube containing 250 mM TrisHCl (pH 7.5), and 0.5% Triton X-100. Hemicalvariae were then subjected to three cycles of freezing in dry ice and thawing at 37³C. The extracts were then heated at 65³C for 15 min to inactivate endogenous deacetylases. Tubes were then centrifuged for 1 min and the supernatants were collected. This method is e¡ective in releasing about 90% of CAT activity from each hemicalvaria [14]. Protein content in the extracts was measured using the BCA colorimetric assay (Pierce, Rockford, IL) [15]. CAT activity was measured using the £uor di¡usion method [16]. Different amounts of the extracts (depending on the line examined) were mixed with 0.2 ml of a solution containing 0.1 M Tris-HCl pH 7.8, 1 mM chloramphenicol and 0.2 mCi of [3 H]acetyl coenzyme A (200 mCi/mmol) obtained from New England Nuclear (Boston, MA) in a scintillation vial and covered with 4 ml of toluene-based scintillation £uid. The vials were incubated at room temperature and counted at 30 min intervals. Background counts (mean of two vials containing no tissue extract) were subtracted from measured counts and the results plotted against time. CAT activity is expressed as the slope of this line. 2.5. RNA isolation and Northern blot analysis Total RNA was extracted from calvariae using TRI Reagent [17]. Brie£y, six hemicalvariae were pooled and homogenized in TRI Reagent using a polytron (Brinkmann, Lucerne, Switzerland), and RNA extracted following the manufacturer's instructions. 10 Wg of RNA was denatured and fractionated using a 1% agarose gel containing 1.1 M formaldehyde, transferred to nylon membrane and hybridized at 42³C in 50% formaldehyde by standard methods. The CAT probe was a 1.4 kb EcoRI fragment of ColCAT3.6 containing CAT and SV40 sequences.
Fig. 1. Col1a1-CAT transgenes used to generate transgenic mice. The parental construct, ColCAT3.6, contains 3518 bp of rat Col1a1 5P £anking DNA (solid horizontal line), 116 bp of transcribed Col1a1 DNA (black bar), and the SV40 small t antigen splice (shaded bar and short horizontal line) and polyadenylation site (PA) derived from SV2CAT [9,35]. Deletions were produced using restriction sites shown, or Bal-31 digestion. The numbers to the left indicate the distance from the transcription start site.
The rat Col1a1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probes have been described [7,18]. Probes were labeled with [K-32 P]deoxy-CTP (3000 Ci/mmol) (New England Nuclear, Boston, MA) using random primer oligonucleotides [19]. Speci¢c RNA levels were quantitated using a PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA). 2.6. In vivo experiments To assess the e¡ect of in vivo 1,25-(OH)2 D3 treatment on Col1a1 expression, 7-day-old mice were given a single subcutaneous injection of 1,25-(OH)2 D3 (in 65% PBS/35% ethanol) or vehicle at two di¡erent doses: 0.16 or 1.6 ng/g body weight, as previously reported [3]. Each group consisted of six mice. After 16 or 48 h, animals were killed. Calvariae and tail tendons were excised, rinsed in PBS, and analyzed for CAT activity. 2.7. Statistical analysis The results of CAT assays were analyzed by unpaired Student's t-test and di¡erences were considered statistically signi¢cant at P 6 0.05.
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3. Results 3.1. Time course of 1,25-(OH)2 D3 e¡ect on CAT activity To determine the time course of action of 1,25(OH)2 D3 on transgene activity in vitro, we treated transgenic mouse calvariae containing ColCAT2.3 (line 8) for 24 and 48 h with 10 nM 1,25-(OH)2 D3 . CAT activity was not signi¢cantly decreased at 24 h but was inhibited by approximately 50% at 48 h (Fig. 2). This level of suppression is consistent with the previously published level of inhibition of the endogenous Col1a1 gene [3,7] by 1,25-(OH)2 D3 in rat calvariae. 3.2. Dose response for the e¡ect of 1,25-(OH)2 D3 on CAT activity We treated calvariae from a ColCAT3.6 line (line 18) for 48 h with 1,25-(OH)2 D3 ranging from 0.1 nM to 10 nM. CAT activity was signi¢cantly reduced in the treated calvariae in a dose-dependent fashion: 39% inhibition at 0.1 nM, 50% inhibition at 1 nM and 59% inhibition at 10 nM (Fig. 3). 3.3. E¡ect of 1,25-(OH)2 D3 on Col1a1 promoter deletion constructs To localize a 1,25-(OH)2 D3 -responsive region in
Fig. 2. Time course of the e¡ect of 10 nM 1,25-(OH)2 D3 on CAT activity in calvariae from line 8 (ColCAT2.3) transgenic mice. Calvariae were excised from 6-day-old mice. One hemicalvaria was cultured in control medium while the other hemicalvaria was treated with 1,25-(OH)2 D3 for 24 or 48 h. CAT activity was then measured as described in Section 2. Each value represents the mean þ S.E.M. of six hemicalvariae. Asterisk indicates values signi¢cantly di¡erent from control (P 6 0.01).
the Col1a1 promoter, we analyzed deletion constructs of ColCAT3.6 terminating at 32296, 31997, 31796, 31764, and 31719 bp. All lines showed signi¢cant inhibition after 48 h of treatment with 10 nm 1,25-(OH)2 D3 (Table 1). These data suggest that the site of action of 1,25-(OH)2 D3 on the Col1a1 promoter in transgenic mouse calvariae is located in the proximal 1719 bp. Since the 31670
Table 1 E¡ect of 1,25(OH)2 D3 on CAT activity in 48 h cultures of transgenic calvariae carrying fragments of the COL1A1 promoter linked to CAT Construct ColCAT3.6 ColCAT2.3 ColCAT1997 ColCAT1794 ColCAT1763 ColCAT1719
Line 14 18 5 8 53 54 60 61 286 312
CAT (cpm/h/Wg protein)
Inhibition (%)
Control
1,25(OH)2 D3 10 nM
11441 þ 2537 6505 þ 1292 5504 þ 370 16634 þ 1972 3677 þ 278 8367 þ 803 1914 þ 275 5771 þ 979 3410 þ 403 3645 þ 477
3913 þ 587* 1330 þ 152* 2402 þ 482* 4915 þ 544* 1257 þ 77* 4892 þ 351* 905 þ 126* 3480 þ 264* 1145 þ 155* 1233 þ 266*
66 80 56 70 66 42 53 40 66 66
Calvariae were cultured for 48 h in the presence or absence of 10 nM 1,25(OH)2 D3 . Extracts were prepared from individual calvariae and assayed for CAT activity as described in Section 2. Each value is the mean þ S.E.M. for CAT activity in six to eight calvariae. *Signi¢cant e¡ect of 1,25(OH)2 D3 , P 6 0.05.
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Fig. 3. E¡ect of 48 h treatment with di¡erent concentrations of 1,25-(OH)2 D3 on CAT activity in calvariae from line 18 (ColCAT3.6) transgenic mice. Each value represents the mean þ S.E.M. of six hemicalvariae. Asterisk indicates values signi¢cantly di¡erent from control (P 6 0.01).
construct did not have detectable CAT activity in calvariae [11], the e¡ect of 1,25-(OH)2 D3 on this construct could not be tested. 3.4. E¡ect of cycloheximide on 1,25-(OH)2 D3 inhibition of the Col1a1 promoter and time course of inhibition of CAT mRNA levels We next determined whether the inhibitory 1,25(OH)2 D3 e¡ect on transgene expression was dependent on new protein synthesis by measuring the e¡ect of 1,25-(OH)2 D3 on CAT mRNA levels in the presence or absence of cycloheximide (CHX), a protein synthesis inhibitor. Transgenic calvariae were pretreated with CHX or vehicle for 1 h and then treated with 10 nM 1,25-(OH)2 D3 or vehicle for 3, 6, 12 or 24 h longer. RNA was extracted, fractionated on a denaturing agarose gel and hybridized to CAT and 18S ribosomal RNA speci¢c probes (Fig. 4A). The CAT-speci¢c hybridization was normalized to the 18S signal, and the normalized CAT signal from 1,25-(OH)2 D3 -treated calvariae was expressed as the percent of the normalized control CAT signal (Fig. 4B). In the non-CHX-treated cultures, CAT mRNA levels from 1,25-(OH)2 D3 -treated cultures were somewhat decreased relative to control cultures by 3 h, and were further decreased to 53% of control by 24 h. CHX increased CAT mRNA levels, and decreased the inhibitory e¡ect of 1,25-(OH)2 D3 at 3 h and 6 h. However, by 12 and 24 h the inhibition
Fig. 4. (A) Northern blot analysis of the time course of the effect of treatment with 1,25-(OH)2 D3 on CAT mRNA in the presence or absence of 3 Wg/ml cycloheximide (CHX). (B) Graphic representation of quanti¢ed mRNA levels from A. CAT-speci¢c signals were normalized to 18S RNA to control for variation in sample loading. At each time point, the experimental value is expressed as a percentage of the control value. (C) E¡ect of 1,25-(OH)2 D3 on Col1a1 and GAPDH mRNA. CON, control calvarial cultures. VD, calvarial cultures treated with 1,25-(OH)2 D3 for 24 h. 28S and 18S indicate ethidium bromide-stained ribosomal RNA used as a loading control.
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of CAT mRNA levels by 1,25-(OH)2 D3 was not affected by CHX. The presence of two CAT-speci¢c RNA species is due to aberrant splicing of the SV40 small t antigen intron which is present in the ColCAT vectors [14,20]. The ratio of the two species was not changed by 1,25-(OH)2 D3 or cycloheximide treatment. To demonstrate that 1,25-(OH)2 D3 did not have a generalized e¡ect on protein synthesis in calvariae, we hybridized RNA from control and 24-h 1,25-(OH)2 D3 -treated calvarial cultures with probes
for Col1a1 and GAPDH (Fig. 4C). Although the Col1a1 mRNA levels were inhibited by about 50%, the levels of GAPDH mRNA were not signi¢cantly a¡ected. 3.5. E¡ect of in vivo 1,25-(OH)2 D3 treatment of transgenic mice To determine whether the 1,25-(OH)2 D3 e¡ect on the Col1a1 transgene could be observed in vivo, 7day-old transgenic mice (ColCAT2.3, line 8) were treated with 0.16 or 1.6 ng 1,25-(OH)2 D3 /g body weight for 16 or 48 h (Fig. 5A). 1,25-(OH)2 D3 at 0.16 ng/g had little e¡ect on the CAT activity in calvariae. However, 1,25-(OH)2 D3 at 1.6 ng/g caused a 40% inhibition after 48 h. CAT activity did not decline signi¢cantly after 1,25-(OH)2 D3 treatment in extracts from tail tendons (Fig. 5B), suggesting that the 1,25-(OH)2 D3 action on the Col1a1 transgene in bone does not re£ect a generalized inhibitory e¡ect on transgene expression. 4. Discussion
Fig. 5. (A) E¡ect of in vivo 1,25(OH)2 D3 treatment on CAT activity in calvariae. 8-day-old transgenic mice (ColCAT2.3, line 8) were injected subcutaneously with two di¡erent doses of 1,25(OH)2 D3 : 0.16 ng/g body weight or 1.6 ng/g body weight. Calvariae were excised after 16 or 48 h. CAT activity was assayed as described in Section 2. Asterisk indicates values signi¢cantly di¡erent from control (P 6 0.01). (B) E¡ect of 48 h in vivo 1,25(OH)2 D3 treatment on CAT activity in tendon. Tendons from the 48 h treated animals used in A were excised and assayed for CAT activity. Double asterisk indicates values not signi¢cantly di¡erent from control (P = 0.121).
We studied the e¡ect of 1,25-(OH)2 D3 on the expression of Col1a1 transgenes in calvariae to determine whether the transgene responds to this hormone in the same way as the endogenous gene and the same transgenes in transfected ROS 17/2.8 cells. Moreover, the generation of transgenic mice with constructs containing progressive deletions of the Col1a1 promoter gave us the opportunity to identify regulatory regions of the gene which mediate the inhibitory e¡ect of 1,25-(OH)2 D3 on collagen gene transcription. Previous experiments [11] showed that ColCAT3.6 and ColCAT2.3 are expressed equally well in transgenic mice. Therefore, mouse lines containing either construct were used for dose-response and time course experiments. 1,25-(OH)2 D3 had a dose-dependent inhibitory e¡ect on transgene activity. The maximal inhibitory e¡ect of 10 nM 1,25-(OH)2 D3 on transgene activity in calvariae was similar to the effect on the endogenous Col1a1 gene in calvariae [3,7] and on the endogenous gene and the ColCAT transgene in osteoblastic cells [10] (about 50%). Inhibition of CAT activity in organ cultures was
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not statistically signi¢cant at 24 h and was maximal at 48 h. In a previous study we observed a 50% reduction of Col1a1 mRNA by 1,25-(OH)2 D3 in fetal rat calvariae after 24 h [21]. Since the half-life of the CAT protein is about 16 h [22], we expected that maximal repression of ColCAT expression as assessed by measuring CAT activity would be observed after longer treatment times. We analyzed several deletion constructs of ColCAT between 32297 (ColCAT2.3) and 31670 (ColCAT1.7): ColCAT1997, ColCAT1794, ColCAT1763 and ColCAT1719. All of these construct were expressed at similar levels in organ cultures of calvariae, and all were inhibited by 1,25-(OH)2 D3 to approximately the same degree. Since our constructs include 116 bp of sequence downstream of the transcription initiation site, these results suggest that a 1,25-(OH)2 D3 responsive element is present in the proximal 1834 bp of the Col1a1 promoter. Unfortunately, ColCAT1.7, which extends to 31670 bp, has no detectable activity; therefore it was not possible to determine whether this construct responds to 1,25(OH)2 D3 . Since experiments using transfected ROS 17/2.8 cells indicate that ColCAT1.7 (which has low but detectable activity in these cells) does not respond to 1,25-(OH)2 D3 [10], combining these two experiments suggests the possibility that the response element may be located in the 49 bp region between 31719 and 31670 bp, although there may be di¡erences in the regulation of the gene in cultured cells and transgenic mice. It is also possible that a 1,25(OH)2 D3 -responsive element is located downstream of 31670 and that it interacts with a region necessary for high basal activity in osteoblasts which is between 31683 and 31677 bp [23]. It has been shown that 1,25-(OH)2 D3 mediates many of its e¡ects by initially binding to a speci¢c cytoplasmic receptor (VDR) [24]. Cytoplasmic receptors have been identi¢ed in 1,25-(OH)2 D3 -responsive tissues, including bone [25,26]. The receptor has been shown to interact with speci¢c DNA sequences (VDREs) and mediate positive and negative 1,25(OH)2 D3 responses in several promoters [27]. This mechanism does not require new protein synthesis. To determine whether new protein synthesis is required for 1,25-(OH)2 D3 inhibition of the Col1a1 promoter, we examined the e¡ect of the protein synthesis inhibitor CHX on the ability of 1,25-(OH)2 D3
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to inhibit CAT mRNA. Because the half-life of the CAT mRNA is much shorter than the CAT protein or the Col1a1 mRNA (D. Breault, personal communication), this allowed us to study the rate of decrease in gene expression at earlier time points. CHX itself increased the steady-state transgene mRNA levels. This e¡ect was probably due to increased mRNA stability, as reported for other mRNAs, such as c-jun [28], tumor necrosis factor K [29] and insulin-like growth factor binding protein [30]. 1,25-(OH)2 D3 treatment without CHX caused a more rapid reduction in CAT mRNA levels than was seen in the CHX-treated calvariae (Fig. 4A,B). Increased stability of the CAT mRNA may explain the delayed response of the CHX-treated cultures to 1,25-(OH)2 D3 . The relative degree of reduction of CAT mRNA at 12 and 24 h was unchanged by the pre-treatment with CHX, suggesting that the 1,25(OH)2 D3 e¡ect on the Col1a1 transgene in calvariae does not require new protein synthesis. Studies on genes which are positively regulated by 1,25-(OH)2 D3 showed that the typical VDRE consists of two loosely conserved half-sites separated by 3 bp, which binds a heterodimer of the VDR with the RXR receptor [27]. Negative 1,25-(OH)2 D3 response elements (nVDRE) have been described for the human [31] and chicken [32] parathyroid hormone (PTH) genes, and the rat PTH-related peptide gene [33]. Although an initial study suggested that the human PTH nVDRE contained only one copy of a motif similar to those found in positive VDREs, [31], a more recent study indicated that the human PTH nVDRE has two half-sites which bind the VDR-RXR heterodimer [34]. Taken with other studies on 1,25-(OH)2 D3 inhibited promoters [32,33], this suggests that nVDREs are similar to positive VDREs. We performed a computer search of the region downstream of 31719 bp of the Col1a1 promoter using all reported VDR binding sites and derived consensus sites. We found a match with a 1,25(OH)2 D3 response consensus derived by Demay et al. [35] between +20 and +30 bp. Preliminary results indicate that this site does not bind the 1,25-(OH)2 D3 receptor (M.S. Kronenberg, personal communication), so it is unlikely to mediate repression by 1,25-(OH)2 D3 . Several other partial matches with 1,25-(OH)2 D3 receptor binding sequences were dis-
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covered in di¡erent areas of the promoter downstream of 31670. These sequences are candidate sites of action of 1,25-(OH)2 D3 on the Col1a1 promoter. Alternatively, the activated 1,25-(OH)2 D3 receptor may exert an inhibitory e¡ect on the Col1a1 promoter by interacting with trans-activating factors present in the bone cells, as described for glucocorticoid action on the collagenase gene [36]. Glucocorticoids inhibit basal and induced transcription of collagenase by interfering with the AP-1 complex. This mechanism does not require new protein synthesis and depends on the hormone receptor, but does not require its direct binding to DNA. The results we have obtained on 1,25-(OH)2 D3 action on Col1a1 promoter in transgenic mouse calvariae are consistent with this model. A third possibility is that 1,25-(OH)2 D3 may inhibit Col1a1 transcription through the protein kinase C pathway, based on the observation that 1,25(OH)2 D3 can signal through this pathway [37,38]) and our observation that phorbol esters inhibit Col1a1 transcription in osteoblasts [39]. Our in vivo experiments show that 1,25-(OH)2 D3 is able to inhibit Col1a1 activity in calvariae of intact transgenic animals. The fact that the activity of Col1a1 in tail tendons was not inhibited indicates that the action of 1,25-(OH)2 D3 on the Col1a1 gene in calvariae is not the result of a generalized inhibition of this promoter in all tissues. Kesterson et al. [40] have shown that the human osteocalcin gene in transgenic mouse calvariae is induced by 3 days of treatment with doses of 1,25-(OH)2 D3 similar to those used in our experiments, indicating that the 1,25-(OH)2 D3 inhibition of Col1a1 is not due to generalized inhibition of gene expression in calvariae. These results support our conclusion that calvarial organ cultures are a physiologically appropriate model to study the 1,25-(OH)2 D3 regulation of the Col1a1 promoter. Acknowledgements This work was supported by the following grants from the National Institutes of Health: AR29983 (A.C.L.), AR38933 (B.E.K., S.H.S., and A.C.L.) and AR29850 (B.E.K.), and grants from NASA and the American Heart Association, AHA 92015860 (D.W.R.). Dr. Clark was supported in
part by the Medical Research Service, Department of Veterans A¡airs. References [1] L.G. Raisz, B.E. Kream, Regulation of bone formation, New Engl. J. Med. 309 (1983) 83^89. [2] L. Raisz, D.M. Maina, S.G. Gworek, J.W. Deitrich, E.M. Canalis, Hormonal control of bone collagen synthesis in vitro: inhibitory e¡ect of 1-hydroxylated vitamin D metabolites, Endocrinology 102 (1978) 731^735. [3] D.W. Rowe, B.E. Kream, Regulation of collagen synthesis in fetal rat calvaria by 1,25-dihydroxyvitamin D3 , J. Biol. Chem. 257 (1982) 8009^8015. [4] F.R. Bringhurst, J.R. Pots, E¡ects of vitamin D metabolites and analogs on bone collagen synthesis in vitro, Calcif. Tissue Int. 34 (1982) 103^110. [5] G.L. Wong, R.A. Lubin, D.V. Cohn, 1,25-Dihydroxycholecalciferol and parathormone: e¡ects on isolated osteoclastlike and osteoblast-like cells, Science 197 (1977) 663^665. [6] B.E. Kream, D. Rowe, M.D. Smith, V. Maher, R. Majeska, Hormonal regulation of collagen synthesis in a clonal rat osteosarcoma cell line, Endocrinology 119 (1986) 1922^1928. [7] C. Genovese, D. Rowe, B. Kream, Construction of DNA sequences complementary to rat K1 and K2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D, Biochemistry 23 (1984) 6210^6216. [8] J.R. Harrison, D.N. Petersen, A.C. Lichtler, A.T. Mador, D.W. Rowe, B.E. Kream, 1,25-Dihydroxyvitamin D3 inhibits transcription of type I collagen genes in the rat osteosarcoma cell line ROS 17/2.8, Endocrinology 125 (1989) 327^ 333. [9] A. Lichtler, M.L. Stover, J. Angilly, B. Kream, D.W. Rowe, Isolation and characterization of the rat K1(I) collagen promoter. Regulation by 1,25-dihydroxyvitamin D, J. Biol. Chem. 264 (1989) 3072^3077. [10] D. Pavlin, A. Bedalov, M.S. Kronenberg, B.E. Kream, D.W. Rowe, C.L. Smith, J.W. Pike, A.C. Lichtler, Analysis of regulatory regions in the COl1A1 gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression in osteoblastic cells, J. Cell Biochem. 56 (1994) 490^501. [11] Z. Bogdanovic, A. Bedalov, P.H. Krebsbach, D. Pavlin, C.O. Woody, S.H. Clark, H.F. Thomas, D.W. Rowe, B.E. Kream, A.C. Lichtler, Upstream regulatory elements necessary for expression of the rat COL1A1 promoter in transgenic mice, J. Bone Miner. Res. 9 (1994) 285^291. [12] A. Bedalov, R. Salvatori, M. Dodig, M.S. Kronenberg, B. Kapural, Z. Bogdanovic, B.E. Kream, C.O. Woody, S.H. Clark, K. Mack, D.W. Rowe, A.C. Lichtler, Regulation of COL1A1 expression in type I collagen producing tissues: Identi¢cation of a 49 base pair region which is required for transgene expression in bone of transgenic mice, J. Bone Miner. Res. 10 (1995) 1443^1451. [13] D. Pavlin, A.C. Lichtler, A. Bedalov, B.E. Kream, J.R. Har-
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BBAEXP 93119 1-7-98
1,25-Dihydroxyvitamin D3 inhibition of Col1a1 promoter expression in calvariae from neonatal transgenic mice Antonio Bedalov 1;a , Roberto Salvatori 2;a , Milan Dodig a , Belinda Kapural a , Dubravko Pavlin 3;a , Barbara E. Kream b , Stephen H. Clark c;d , Charles O. Woody e , David W. Rowe a , Alexander C. Lichtler a; * a c
Department of Pediatrics, MC1515, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030, USA b Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030, USA Division of Rheumatic Diseases, Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030, USA d Department of Veterans A¡airs Medical Center, Newington, CT 06111, USA e Department of Animal Science University of Connecticut, Storrs, CT 06268, USA Received 25 March 1998; accepted 9 April 1998
Abstract We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to 33518, 32297, 31997, 31794, 31763, and 31719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2 D3 had a dosedependent inhibitory effect on the expression of the 33518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2 D3 , suggesting that the sequences required for 1,25(OH)2 D3 inhibition are downstream of 31719 bp. The inhibitory effect of 1,25(OH)2 D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2 D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2 D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1,25(OH)2 D3 responsive element downstream of 31719 bp. The inhibitory effect does not require new protein synthesis. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Collagen; Calcium regulating hormone; Bone; Osteoblast; Transcription
* Corresponding author. Fax: +1 (860) 679-1047; E-mail: [email protected] 1 Permanent address: University of Zagreb, School of Medicine, Salata 3b, 41000 Zagreb, Croatia. 2 Present address: Division of Endocrinology, Johns Hopkins University, Baltimore, MD 21287, USA. 3 Present address: Departments of Orthodontics and Cellular and Structural Biology, The University of Texas Health Science Center, San Antonio, TX 78284, USA. 0167-4781 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 8 ) 0 0 0 7 9 - 7
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1. Introduction The seco-steroid hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3 ) is an important regulator of calcium homeostasis. Decreased serum calcium increases 1,25-(OH)2 D3 production, which increases intestinal calcium absorption and stimulates bone resorption [1]. 1,25-(OH)2 D3 inhibits collagen synthesis in many bone culture systems, including cultured intact fetal and neonatal rat and mouse [2^4], primary cultures of osteoblast-enriched calvarial cells [5] and clonal osteoblastic cell lines [6]. Previous work from our laboratory demonstrated that the inhibition of collagen synthesis by 1,25-(OH)2 D3 is accompanied by a decrease in procollagen mRNA levels in fetal rat calvariae and in the rat osteoblastic cell line ROS 17/2 [3,7]. We also showed that 1,25(OH)2 D3 inhibits Col1a1 and Col1a2 transcription in ROS 17/2.8 cells [8], and decreases the expression of a Col1a1 promoter-chloramphenicol acetyltransferase (CAT) construct extending to 33518 bp (ColCAT3.6) in transiently transfected ROS 17/2.8 cells [9]. More recent studies using stably transfected ROS 17/2.8 cells showed that a ColCAT construct extending to 32.3 kb, and a construct containing an internal deletion from 32256 to 32216 bp were both inhibited by 1,25-(OH)2 D3 , however a construct extending to 31670 bp was not inhibited. This suggested that a DNA sequence necessary for the 1,25(OH)2 D3 response may be located in the region between 32216 and 31670 bp [10]. We have previously observed that there are signi¢cant di¡erences in the regions of the Col1A1 promoter which are responsible for maintaining high basal activity in transgenic mouse calvariae and osteoblast-like cell lines including ROS 17/2.8 [11^13]. This raised the possibility that di¡erent regulatory elements are involved in 1,25-(OH)2 D3 regulation of the Col1a1 gene in vivo and in vitro. In addition, we have found that the response of the Col1a1 gene to 1,25-(OH)2 D3 treatment in ROS 17/2.8 cells is not as consistent as the response in cultured calvariae. We have generated a series of transgenic mice containing deletions of the Col1a1 promoter [12]. Therefore, in this study we have used transgenic mouse calvariae containing these deletions to analyze the 1,25-(OH)2 D3 responsive region in the Col1a1 promoter. Calvariae obtained from these mice were cul-
tured in the presence and absence of 1,25-(OH)2 D3 . We show that 1,25-(OH)2 D3 decreases CAT activity and transgene mRNA levels in organ cultures of calvariae from transgenic mice and that these e¡ects do not require protein synthesis. We also show that the proximal 1719 bp of the 5P £anking region of the Col1a1 promoter are su¤cient to confer 1,25(OH)2 D3 inhibition. 2. Materials and methods 2.1. Materials Crystalline 1,25-(OH)2 D3 was a generous gift of Dr. Milan Uskokovic of Ho¡man-LaRoche (Nutley, NJ) and was diluted in ethanol to give a 10 mM stock solution. BGJb medium was purchased from Gibco (Grand Island, NY). 2.2. Transgenic mouse lines The construction of ColCAT3.6, ColCAT2.3, ColCAT1997, ColCAT1794, ColCAT1764, ColCAT1719 and ColCAT1.7 (Fig. 1), containing portions of the rat Col1a1 promoter fused to the CAT reporter gene, and production of transgenic mice with these constructs, have been previously reported [12]. Transgenic animals were initially identi¢ed via dot-blot analysis of DNA using a CAT-speci¢c probe. Subsequent generations were identi¢ed by analyzing CAT activity in tail fragments. All animal experiments were conducted in accordance with the highest possible standards of humane animal care, as outlined in the NIH Guide for the Care and Use of Laboratory Animals. 2.3. Calvarial organ cultures Frontal and parietal bones were dissected from 6^ 8-day-old mice and cut along the sagittal suture. Each hemicalvaria was cultured in a 35 mm tissue culture well (Falcon) containing 2 ml of BGJb medium with 1 mg/ml bovine serum albumin, 100 Wg/ml ascorbic acid and 1 mM proline. 1,25-(OH)2 D3 or ethanol vehicle was added and the culture dishes were placed on a rocking platform in an incubator at 37³C in 5% CO2 /95% air. After 24 h, fresh me-
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dium containing 1,25-(OH)2 D3 or vehicle was added. After culture for the times speci¢ed, hemicalvariae were washed twice in cold phosphate-bu¡ered saline (PBS) and either analyzed for CAT activity or utilized for total RNA extraction. 2.4. Measurement of CAT activity After washing with PBS, each hemicalvaria was put in an Eppendorf tube containing 250 mM TrisHCl (pH 7.5), and 0.5% Triton X-100. Hemicalvariae were then subjected to three cycles of freezing in dry ice and thawing at 37³C. The extracts were then heated at 65³C for 15 min to inactivate endogenous deacetylases. Tubes were then centrifuged for 1 min and the supernatants were collected. This method is e¡ective in releasing about 90% of CAT activity from each hemicalvaria [14]. Protein content in the extracts was measured using the BCA colorimetric assay (Pierce, Rockford, IL) [15]. CAT activity was measured using the £uor di¡usion method [16]. Different amounts of the extracts (depending on the line examined) were mixed with 0.2 ml of a solution containing 0.1 M Tris-HCl pH 7.8, 1 mM chloramphenicol and 0.2 mCi of [3 H]acetyl coenzyme A (200 mCi/mmol) obtained from New England Nuclear (Boston, MA) in a scintillation vial and covered with 4 ml of toluene-based scintillation £uid. The vials were incubated at room temperature and counted at 30 min intervals. Background counts (mean of two vials containing no tissue extract) were subtracted from measured counts and the results plotted against time. CAT activity is expressed as the slope of this line. 2.5. RNA isolation and Northern blot analysis Total RNA was extracted from calvariae using TRI Reagent [17]. Brie£y, six hemicalvariae were pooled and homogenized in TRI Reagent using a polytron (Brinkmann, Lucerne, Switzerland), and RNA extracted following the manufacturer's instructions. 10 Wg of RNA was denatured and fractionated using a 1% agarose gel containing 1.1 M formaldehyde, transferred to nylon membrane and hybridized at 42³C in 50% formaldehyde by standard methods. The CAT probe was a 1.4 kb EcoRI fragment of ColCAT3.6 containing CAT and SV40 sequences.
Fig. 1. Col1a1-CAT transgenes used to generate transgenic mice. The parental construct, ColCAT3.6, contains 3518 bp of rat Col1a1 5P £anking DNA (solid horizontal line), 116 bp of transcribed Col1a1 DNA (black bar), and the SV40 small t antigen splice (shaded bar and short horizontal line) and polyadenylation site (PA) derived from SV2CAT [9,35]. Deletions were produced using restriction sites shown, or Bal-31 digestion. The numbers to the left indicate the distance from the transcription start site.
The rat Col1a1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probes have been described [7,18]. Probes were labeled with [K-32 P]deoxy-CTP (3000 Ci/mmol) (New England Nuclear, Boston, MA) using random primer oligonucleotides [19]. Speci¢c RNA levels were quantitated using a PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA). 2.6. In vivo experiments To assess the e¡ect of in vivo 1,25-(OH)2 D3 treatment on Col1a1 expression, 7-day-old mice were given a single subcutaneous injection of 1,25-(OH)2 D3 (in 65% PBS/35% ethanol) or vehicle at two di¡erent doses: 0.16 or 1.6 ng/g body weight, as previously reported [3]. Each group consisted of six mice. After 16 or 48 h, animals were killed. Calvariae and tail tendons were excised, rinsed in PBS, and analyzed for CAT activity. 2.7. Statistical analysis The results of CAT assays were analyzed by unpaired Student's t-test and di¡erences were considered statistically signi¢cant at P 6 0.05.
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3. Results 3.1. Time course of 1,25-(OH)2 D3 e¡ect on CAT activity To determine the time course of action of 1,25(OH)2 D3 on transgene activity in vitro, we treated transgenic mouse calvariae containing ColCAT2.3 (line 8) for 24 and 48 h with 10 nM 1,25-(OH)2 D3 . CAT activity was not signi¢cantly decreased at 24 h but was inhibited by approximately 50% at 48 h (Fig. 2). This level of suppression is consistent with the previously published level of inhibition of the endogenous Col1a1 gene [3,7] by 1,25-(OH)2 D3 in rat calvariae. 3.2. Dose response for the e¡ect of 1,25-(OH)2 D3 on CAT activity We treated calvariae from a ColCAT3.6 line (line 18) for 48 h with 1,25-(OH)2 D3 ranging from 0.1 nM to 10 nM. CAT activity was signi¢cantly reduced in the treated calvariae in a dose-dependent fashion: 39% inhibition at 0.1 nM, 50% inhibition at 1 nM and 59% inhibition at 10 nM (Fig. 3). 3.3. E¡ect of 1,25-(OH)2 D3 on Col1a1 promoter deletion constructs To localize a 1,25-(OH)2 D3 -responsive region in
Fig. 2. Time course of the e¡ect of 10 nM 1,25-(OH)2 D3 on CAT activity in calvariae from line 8 (ColCAT2.3) transgenic mice. Calvariae were excised from 6-day-old mice. One hemicalvaria was cultured in control medium while the other hemicalvaria was treated with 1,25-(OH)2 D3 for 24 or 48 h. CAT activity was then measured as described in Section 2. Each value represents the mean þ S.E.M. of six hemicalvariae. Asterisk indicates values signi¢cantly di¡erent from control (P 6 0.01).
the Col1a1 promoter, we analyzed deletion constructs of ColCAT3.6 terminating at 32296, 31997, 31796, 31764, and 31719 bp. All lines showed signi¢cant inhibition after 48 h of treatment with 10 nm 1,25-(OH)2 D3 (Table 1). These data suggest that the site of action of 1,25-(OH)2 D3 on the Col1a1 promoter in transgenic mouse calvariae is located in the proximal 1719 bp. Since the 31670
Table 1 E¡ect of 1,25(OH)2 D3 on CAT activity in 48 h cultures of transgenic calvariae carrying fragments of the COL1A1 promoter linked to CAT Construct ColCAT3.6 ColCAT2.3 ColCAT1997 ColCAT1794 ColCAT1763 ColCAT1719
Line 14 18 5 8 53 54 60 61 286 312
CAT (cpm/h/Wg protein)
Inhibition (%)
Control
1,25(OH)2 D3 10 nM
11441 þ 2537 6505 þ 1292 5504 þ 370 16634 þ 1972 3677 þ 278 8367 þ 803 1914 þ 275 5771 þ 979 3410 þ 403 3645 þ 477
3913 þ 587* 1330 þ 152* 2402 þ 482* 4915 þ 544* 1257 þ 77* 4892 þ 351* 905 þ 126* 3480 þ 264* 1145 þ 155* 1233 þ 266*
66 80 56 70 66 42 53 40 66 66
Calvariae were cultured for 48 h in the presence or absence of 10 nM 1,25(OH)2 D3 . Extracts were prepared from individual calvariae and assayed for CAT activity as described in Section 2. Each value is the mean þ S.E.M. for CAT activity in six to eight calvariae. *Signi¢cant e¡ect of 1,25(OH)2 D3 , P 6 0.05.
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Fig. 3. E¡ect of 48 h treatment with di¡erent concentrations of 1,25-(OH)2 D3 on CAT activity in calvariae from line 18 (ColCAT3.6) transgenic mice. Each value represents the mean þ S.E.M. of six hemicalvariae. Asterisk indicates values signi¢cantly di¡erent from control (P 6 0.01).
construct did not have detectable CAT activity in calvariae [11], the e¡ect of 1,25-(OH)2 D3 on this construct could not be tested. 3.4. E¡ect of cycloheximide on 1,25-(OH)2 D3 inhibition of the Col1a1 promoter and time course of inhibition of CAT mRNA levels We next determined whether the inhibitory 1,25(OH)2 D3 e¡ect on transgene expression was dependent on new protein synthesis by measuring the e¡ect of 1,25-(OH)2 D3 on CAT mRNA levels in the presence or absence of cycloheximide (CHX), a protein synthesis inhibitor. Transgenic calvariae were pretreated with CHX or vehicle for 1 h and then treated with 10 nM 1,25-(OH)2 D3 or vehicle for 3, 6, 12 or 24 h longer. RNA was extracted, fractionated on a denaturing agarose gel and hybridized to CAT and 18S ribosomal RNA speci¢c probes (Fig. 4A). The CAT-speci¢c hybridization was normalized to the 18S signal, and the normalized CAT signal from 1,25-(OH)2 D3 -treated calvariae was expressed as the percent of the normalized control CAT signal (Fig. 4B). In the non-CHX-treated cultures, CAT mRNA levels from 1,25-(OH)2 D3 -treated cultures were somewhat decreased relative to control cultures by 3 h, and were further decreased to 53% of control by 24 h. CHX increased CAT mRNA levels, and decreased the inhibitory e¡ect of 1,25-(OH)2 D3 at 3 h and 6 h. However, by 12 and 24 h the inhibition
Fig. 4. (A) Northern blot analysis of the time course of the effect of treatment with 1,25-(OH)2 D3 on CAT mRNA in the presence or absence of 3 Wg/ml cycloheximide (CHX). (B) Graphic representation of quanti¢ed mRNA levels from A. CAT-speci¢c signals were normalized to 18S RNA to control for variation in sample loading. At each time point, the experimental value is expressed as a percentage of the control value. (C) E¡ect of 1,25-(OH)2 D3 on Col1a1 and GAPDH mRNA. CON, control calvarial cultures. VD, calvarial cultures treated with 1,25-(OH)2 D3 for 24 h. 28S and 18S indicate ethidium bromide-stained ribosomal RNA used as a loading control.
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of CAT mRNA levels by 1,25-(OH)2 D3 was not affected by CHX. The presence of two CAT-speci¢c RNA species is due to aberrant splicing of the SV40 small t antigen intron which is present in the ColCAT vectors [14,20]. The ratio of the two species was not changed by 1,25-(OH)2 D3 or cycloheximide treatment. To demonstrate that 1,25-(OH)2 D3 did not have a generalized e¡ect on protein synthesis in calvariae, we hybridized RNA from control and 24-h 1,25-(OH)2 D3 -treated calvarial cultures with probes
for Col1a1 and GAPDH (Fig. 4C). Although the Col1a1 mRNA levels were inhibited by about 50%, the levels of GAPDH mRNA were not signi¢cantly a¡ected. 3.5. E¡ect of in vivo 1,25-(OH)2 D3 treatment of transgenic mice To determine whether the 1,25-(OH)2 D3 e¡ect on the Col1a1 transgene could be observed in vivo, 7day-old transgenic mice (ColCAT2.3, line 8) were treated with 0.16 or 1.6 ng 1,25-(OH)2 D3 /g body weight for 16 or 48 h (Fig. 5A). 1,25-(OH)2 D3 at 0.16 ng/g had little e¡ect on the CAT activity in calvariae. However, 1,25-(OH)2 D3 at 1.6 ng/g caused a 40% inhibition after 48 h. CAT activity did not decline signi¢cantly after 1,25-(OH)2 D3 treatment in extracts from tail tendons (Fig. 5B), suggesting that the 1,25-(OH)2 D3 action on the Col1a1 transgene in bone does not re£ect a generalized inhibitory e¡ect on transgene expression. 4. Discussion
Fig. 5. (A) E¡ect of in vivo 1,25(OH)2 D3 treatment on CAT activity in calvariae. 8-day-old transgenic mice (ColCAT2.3, line 8) were injected subcutaneously with two di¡erent doses of 1,25(OH)2 D3 : 0.16 ng/g body weight or 1.6 ng/g body weight. Calvariae were excised after 16 or 48 h. CAT activity was assayed as described in Section 2. Asterisk indicates values signi¢cantly di¡erent from control (P 6 0.01). (B) E¡ect of 48 h in vivo 1,25(OH)2 D3 treatment on CAT activity in tendon. Tendons from the 48 h treated animals used in A were excised and assayed for CAT activity. Double asterisk indicates values not signi¢cantly di¡erent from control (P = 0.121).
We studied the e¡ect of 1,25-(OH)2 D3 on the expression of Col1a1 transgenes in calvariae to determine whether the transgene responds to this hormone in the same way as the endogenous gene and the same transgenes in transfected ROS 17/2.8 cells. Moreover, the generation of transgenic mice with constructs containing progressive deletions of the Col1a1 promoter gave us the opportunity to identify regulatory regions of the gene which mediate the inhibitory e¡ect of 1,25-(OH)2 D3 on collagen gene transcription. Previous experiments [11] showed that ColCAT3.6 and ColCAT2.3 are expressed equally well in transgenic mice. Therefore, mouse lines containing either construct were used for dose-response and time course experiments. 1,25-(OH)2 D3 had a dose-dependent inhibitory e¡ect on transgene activity. The maximal inhibitory e¡ect of 10 nM 1,25-(OH)2 D3 on transgene activity in calvariae was similar to the effect on the endogenous Col1a1 gene in calvariae [3,7] and on the endogenous gene and the ColCAT transgene in osteoblastic cells [10] (about 50%). Inhibition of CAT activity in organ cultures was
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not statistically signi¢cant at 24 h and was maximal at 48 h. In a previous study we observed a 50% reduction of Col1a1 mRNA by 1,25-(OH)2 D3 in fetal rat calvariae after 24 h [21]. Since the half-life of the CAT protein is about 16 h [22], we expected that maximal repression of ColCAT expression as assessed by measuring CAT activity would be observed after longer treatment times. We analyzed several deletion constructs of ColCAT between 32297 (ColCAT2.3) and 31670 (ColCAT1.7): ColCAT1997, ColCAT1794, ColCAT1763 and ColCAT1719. All of these construct were expressed at similar levels in organ cultures of calvariae, and all were inhibited by 1,25-(OH)2 D3 to approximately the same degree. Since our constructs include 116 bp of sequence downstream of the transcription initiation site, these results suggest that a 1,25-(OH)2 D3 responsive element is present in the proximal 1834 bp of the Col1a1 promoter. Unfortunately, ColCAT1.7, which extends to 31670 bp, has no detectable activity; therefore it was not possible to determine whether this construct responds to 1,25(OH)2 D3 . Since experiments using transfected ROS 17/2.8 cells indicate that ColCAT1.7 (which has low but detectable activity in these cells) does not respond to 1,25-(OH)2 D3 [10], combining these two experiments suggests the possibility that the response element may be located in the 49 bp region between 31719 and 31670 bp, although there may be di¡erences in the regulation of the gene in cultured cells and transgenic mice. It is also possible that a 1,25(OH)2 D3 -responsive element is located downstream of 31670 and that it interacts with a region necessary for high basal activity in osteoblasts which is between 31683 and 31677 bp [23]. It has been shown that 1,25-(OH)2 D3 mediates many of its e¡ects by initially binding to a speci¢c cytoplasmic receptor (VDR) [24]. Cytoplasmic receptors have been identi¢ed in 1,25-(OH)2 D3 -responsive tissues, including bone [25,26]. The receptor has been shown to interact with speci¢c DNA sequences (VDREs) and mediate positive and negative 1,25(OH)2 D3 responses in several promoters [27]. This mechanism does not require new protein synthesis. To determine whether new protein synthesis is required for 1,25-(OH)2 D3 inhibition of the Col1a1 promoter, we examined the e¡ect of the protein synthesis inhibitor CHX on the ability of 1,25-(OH)2 D3
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to inhibit CAT mRNA. Because the half-life of the CAT mRNA is much shorter than the CAT protein or the Col1a1 mRNA (D. Breault, personal communication), this allowed us to study the rate of decrease in gene expression at earlier time points. CHX itself increased the steady-state transgene mRNA levels. This e¡ect was probably due to increased mRNA stability, as reported for other mRNAs, such as c-jun [28], tumor necrosis factor K [29] and insulin-like growth factor binding protein [30]. 1,25-(OH)2 D3 treatment without CHX caused a more rapid reduction in CAT mRNA levels than was seen in the CHX-treated calvariae (Fig. 4A,B). Increased stability of the CAT mRNA may explain the delayed response of the CHX-treated cultures to 1,25-(OH)2 D3 . The relative degree of reduction of CAT mRNA at 12 and 24 h was unchanged by the pre-treatment with CHX, suggesting that the 1,25(OH)2 D3 e¡ect on the Col1a1 transgene in calvariae does not require new protein synthesis. Studies on genes which are positively regulated by 1,25-(OH)2 D3 showed that the typical VDRE consists of two loosely conserved half-sites separated by 3 bp, which binds a heterodimer of the VDR with the RXR receptor [27]. Negative 1,25-(OH)2 D3 response elements (nVDRE) have been described for the human [31] and chicken [32] parathyroid hormone (PTH) genes, and the rat PTH-related peptide gene [33]. Although an initial study suggested that the human PTH nVDRE contained only one copy of a motif similar to those found in positive VDREs, [31], a more recent study indicated that the human PTH nVDRE has two half-sites which bind the VDR-RXR heterodimer [34]. Taken with other studies on 1,25-(OH)2 D3 inhibited promoters [32,33], this suggests that nVDREs are similar to positive VDREs. We performed a computer search of the region downstream of 31719 bp of the Col1a1 promoter using all reported VDR binding sites and derived consensus sites. We found a match with a 1,25(OH)2 D3 response consensus derived by Demay et al. [35] between +20 and +30 bp. Preliminary results indicate that this site does not bind the 1,25-(OH)2 D3 receptor (M.S. Kronenberg, personal communication), so it is unlikely to mediate repression by 1,25-(OH)2 D3 . Several other partial matches with 1,25-(OH)2 D3 receptor binding sequences were dis-
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covered in di¡erent areas of the promoter downstream of 31670. These sequences are candidate sites of action of 1,25-(OH)2 D3 on the Col1a1 promoter. Alternatively, the activated 1,25-(OH)2 D3 receptor may exert an inhibitory e¡ect on the Col1a1 promoter by interacting with trans-activating factors present in the bone cells, as described for glucocorticoid action on the collagenase gene [36]. Glucocorticoids inhibit basal and induced transcription of collagenase by interfering with the AP-1 complex. This mechanism does not require new protein synthesis and depends on the hormone receptor, but does not require its direct binding to DNA. The results we have obtained on 1,25-(OH)2 D3 action on Col1a1 promoter in transgenic mouse calvariae are consistent with this model. A third possibility is that 1,25-(OH)2 D3 may inhibit Col1a1 transcription through the protein kinase C pathway, based on the observation that 1,25(OH)2 D3 can signal through this pathway [37,38]) and our observation that phorbol esters inhibit Col1a1 transcription in osteoblasts [39]. Our in vivo experiments show that 1,25-(OH)2 D3 is able to inhibit Col1a1 activity in calvariae of intact transgenic animals. The fact that the activity of Col1a1 in tail tendons was not inhibited indicates that the action of 1,25-(OH)2 D3 on the Col1a1 gene in calvariae is not the result of a generalized inhibition of this promoter in all tissues. Kesterson et al. [40] have shown that the human osteocalcin gene in transgenic mouse calvariae is induced by 3 days of treatment with doses of 1,25-(OH)2 D3 similar to those used in our experiments, indicating that the 1,25-(OH)2 D3 inhibition of Col1a1 is not due to generalized inhibition of gene expression in calvariae. These results support our conclusion that calvarial organ cultures are a physiologically appropriate model to study the 1,25-(OH)2 D3 regulation of the Col1a1 promoter. Acknowledgements This work was supported by the following grants from the National Institutes of Health: AR29983 (A.C.L.), AR38933 (B.E.K., S.H.S., and A.C.L.) and AR29850 (B.E.K.), and grants from NASA and the American Heart Association, AHA 92015860 (D.W.R.). Dr. Clark was supported in
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