THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1983 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 6 , Issue of February 25, pp. 3072-3077,1389 Printed in U. S A .

Isolation and Characterization of the Rat d ( 1 ) Collagen Promoter REGULATION BY 1,25-DIHYDROXYVITAMIN D*

(Received for publication, June 27, 1988)

Alexander Lichtler, Mary Louise Stover, Jane Angilly, Barbara KreamSQ, and David W. Rowell From the Departments of Pediatrics and $Medicine, University of Connecticut Health Center, Farmington, Connecticut 06032

Our previous work demonstrated that the inhibition of type I collagen synthesis by 1,25-dihydroxyvitamin D ( 1,25-(OH)2D3) in fetal rat calvaria and cultured rat osteosarcoma cells is accompanied by equivalent re- duction in steady state levels of al(1) and a2(I) collagen mRNA. To pursue the mechanism for this effect, we isolated and sequenced a 3.6-kilobase DNA fragment that contained the promoter for the rat al(1) collagen gene. This promoter fragment was fused to the chlor- amphenicol acetyltransferase gene and was introduced into ROS 17 /23 cells by calcium phosphate co-precip- itation. Expression of this construct was diminished by 1,25-(0H)zD3 to the same degree as the endogenous collagen gene in both transient expression assays and in permanently selected bone cells. However, a fibro- blast cell line did not show a similar reduction in the activity of the transgene or the endogenous collagen gene. These experiments indicate that the al(1) pro- moter contains cis-active elements which are regulated by the 1,25-(OH)zD3 receptor in ROS 17/2.8 cells.

The synthesis of type I collagen in bone cells, in contrast to a wide variety of other type I collagen-producing cells, appears to be highly regulated. For example, the calcitrophic hormones 1,25-dihydroxyvitamin D3 (1,25-(OH)zD3)’ and parathyroid hormone (PTH) decrease type I collagen synthe- sis in osteoblasts of fetal rat calvaria (1-4) and in cultured osteosarcoma cells (5) yet have no effect on collagen produc- tion in the periosteal fibroblastic cells of calvaria (1, 4). The question is: what are the mechanisms which allow this single copy gene to be transcriptionally active in a wide variety of cell types yet be regulated in a characteristic manner in a particular cell type? Presumably trans-acting factors, which are either unique to particular cell types or are present in

* This work was supported by National Institutes of Health Grants AR-29983 and AR-29850. A portion of this work was presented at the Ninth Annual Meeting of the American Society for Bone and Mineral Research, June 6-9, 1987, Indianapolis, IN. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted

504464. to the GenBankTM/EMBL Data Bank with accession number(s)

§ Recipient of Research Career Development Award AR-01017. ll Recipient of Research Career Development Award HD-00330.

The abbreviations used are: 1,25-(OH)2D3, 1,25-dihydroxyvitamin D; PTH, parathyroid hormone: CAT, chloramphenicol acetyltrans- ferase; CAT 3M, DNA fragment containing the chloramphenicol acetyltransferase gene; Col Cat 3.6, plasmid construct containing the rat cul(1) promoter driving CAT; pAZ1003, plasmid construct contain- ing the mouse cu2(I) promoter driving CAT; pSV2 CAT, plasmid construct containing the SV40 early promoter driving CAT; bp, base pair(s): kb, kilobase(s).

varying amounts, are able to interact with the collagen genes and influence their expression in a specific manner. This hypothesis implies that these genes contain many cis-acting regulatory domains whose function is dependent on its cellular environment.

Type I collagen promoters have been isolated from human (6, 71, mouse (8), and chicken (9, 10). Domains in the 5’- flanking sequences have been identified which control the constitutive expression of the promoter in fibroblasts ( l l ) , enhance expression by transforming growth factor+ (12), and down-regulate expression in virally transformed fibroblasts (13). In transgenic animals it has been shown that 5’-up- stream sequences contain sufficient information for prefer- ential expression in tendons and presumably other tissues rich in collagen (14). In vitro these sequences do not appear to fully account for tissue-specific expression; instead enhan- cer-like sequences have recently been identified within the first intron of both type I genes which are active in fibroblasts (15-17). In vivo or in vitro models necessary to analyze the differential expression of type 1 collagen in more highly dif- ferentiated or hormone-regulated cells have yet to be explored.

In this paper we describe the isolation and characterization of the al(1) rat collagen promoter. This species was selected because of the availability of well characterized cultured bone cell lines for studying regulation of type I collagen by calci- trophic hormones. The data indicate that the al(1) promoter carries the information necessary for regulation by 1,25- (OH)& in bone but not fibroblast-derived cell lines.

MATERIALS AND METHODS

Cell Culture-ROS 17/2 and 17/2.8 are clonal cell lines derived from a chemically induced rat osteosarcoma (18). These cells respond to 1,25-(OH)& and PTH with a reduction in type I collagen produc- tion (5). EL2 cells are a spontaneously immortalized rat fibroblast line with growth and transformation properties similar to 3T3 cells (19). Both cell lines were maintained in Dulbecco’s minimum essential medium supplemented with nonessential amino acids, 100 microun- its/ml penicillin, 100 lg/ml streptomycin, 4 mM glutamine, and 10% fetal calf serum in a humidified atmosphere of 5% CO,. When cells were incubated with hormone, the medium was changed to Ham’s F- 12 containing 2% fetal calf serum and 50 pg/ml ascorbic acid. Ethanol was used to dissolve the 1,25-(OH)2D3, and its concentration in test medium did not exceed 0.01%.

Bacteria and Plasmids-Bacteria-containing plasmids were grown in LB medium containing the appropriate antibiotic. Plasmids were prepared by the alkaline lysis method of Birnboim and Doly (20) as modified by Ish-Horowitz (21) and purified by Sephacryl S-1000 chromatography (Pharmacia LKB Biotechnology Inc.). Plasmids ob- tained from other investigators include: p13-3, the mouse cul(1) pro- moter extending from -350 to +850 bp (22), prepared by Dr. R. Jaenish, Whitehead Institute, Cambridge, MA; pAZ1003, a 2.6-kb fragment encoding -2000 bp of the promoter through +54 bp of the 5”untranslated region of the mouse a2(I) gene driving the chloram- phenicol acetyltransferase (CAT) gene (14), prepared by Dr. A. Schmidt in Dr. B. decrombrugghe’s laboratory at the National Cancer Institute, National Institutes of Health pSV2CAT (23) and pSV2Neo

3072

Regulation of the Rat a l (0 Collagen Promoter 3073

(24), both containing the SV40 enhancer and promoter driving CAT and the neomycin resistance gene, respectively, obtained from Dr. B. Howard, National Cancer Institute; pY3, the hygromycin resistance gene from Dr. H. Diggelmann (25); pCAT3M containing the chlor- amphenicol acetyltransferase gene within convenient restriction sites (26) was prepared by Dr. L. Laimins while in Dr. G. Khoury's laboratory, National Cancer Institute.

Isolation of the Rat Promoter-The partial Hue111 rat genomic library made by Bonner and co-workers (27) was screened (28) using p13-3, a subclone containing the murine al(1) promoter (22). Limited DNA sequencing (29) from both directions of a Xba site within the EcoRI fragment that hybridized to the screening probe showed greater than 90% homology to the screening murine probe, thus confirming and orienting the presence of the rat al(1) promoter within the insert.

DNA Sequencing Strategy-The 3.6-kb Xba fragment containing sequences that extend from the untranslated region of the mRNA into the 5"flanking region was shotgun-cloned (30, 31) into the HincII site of M13mpll and transfected into JMlOl cells. Approxi- mately 100 isolates containing inserts of various sizes from each strand were sequenced to give sufficient overlap to construct the entire sequence. Sequence alignment and analysis utilized IBI soft- ware and gel reader.

Construction of Col Cat 3.6-The chloramphenicol acetyltransfer- ase gene plus the SV40 small T antigen splice and polyadenylation signals are contained within the XbaIIBamHI fragment of the plas- mid CAT 3M (26). This fragment was cloned into the Xba/Bam site of pUC12. The 3.6-kb Xba collagen fragment was then ligated into the 5' side of the CAT gene. Because there is a BamHI site just internal to the 5'-Xba site of the collagen 3.6-kh fragment, the collagen promoter/CAT cassette can be subsequently excised as a 4.6- kb BarnHI fragment (see Fig. 3).

DNA Transfection-The property of the ROS 17/2 cell which makes transient expression of transfected genes difficult is their dependence on a high cell density for optimal collagen expression and regulation by 1,25-(OH)2D3.' Thus it was necessary to transfect the cell just prior to confluency to obtain efficient expression of the plasmid yet maintain hormonal responsiveness for the 48 h subse- quent to the transfection. Variability in responsivity of these cells to this hormone that occurred between separate experiments probably is related to differences in cell density. ROS cells were plated a t 2-4 X lo4 cells/cm2 in 60-mm Petri dishes 1-2 days prior to transfection because it usually required 24 hours for the cells to resume their polygonal shape after passaging.

Cells were transfected (24) with 5 pg of DNA. Six hours after addition of the DNA, the cells were shocked with 1.5 ml of 15% glycerol in 1 X HBS for 1 min. This step was not tolerated by the EL2 cells. The cells were incubated in 4 ml of Ham's F-12 medium containing 2% fetal calf serum and either lo-' M 1,25-(OH)2D3 (test) or ethanol (control). Twenty-four hours after transfection, 5 gCi/ml [5-'H]proline was added directly to the culture medium from the stock solution. Fifteen to 24 h later the medium was removed and assayed for the incorporation of [3H]proline into collagen and non- collagen proteins (5,32). The cell layer was extracted for CAT activity (23).

Cells containing the integrated Col Cat 3.6 gene were prepared by co-transfection of the collagen plasmid with the pSV2-Neo gene. Forty-eight hours after the transfection, the cells were passaged into three 60-mm dishes and placed under selection with 400 pg/ml G418. Approximately 20 clones from a specific transfection were isolated with cloning rings, expanded, and assayed for CAT activity. For these assays, cells were plated into 60-mm dishes, grown to confluency, and then placed in Ham's F-12 or Dulbecco's minimal essential medium containing lo-* M 1,25-(OH)*D$ or vehicle. Subsequent labeling of the cells with [3H]proline and extraction of the cell layer for CAT activity was done as described for the transient transfection assays.

Assay of CATActiuity-In most cases, one or two 60-mm plates of cells were transfected per assay point. Aliquots of cell extract con- taining 50-75 pg of protein were analyzed for CAT activity using a modification of the method of Gorman et al. (23). The assay was carried out in the presence of 4 mM acetyl-CoA, 0.46 M Tris, pH 7.8, and 2.5 pl of the stock ['4C]chloramphenicol in a final reaction volume of 150 pl of which 50 g1 was the cell extract. The reaction was carried out at 37 "C overnight with the addition of another 4 mM acetyl-coA after 6-8 h of the incubation. Radiolabeled products were separated on a thin layer silica gel plate (Baker-flex) and visualized by autora- diography with Kodak X-Omat film at room temperature for 6-24 h.

' B. Kream, unpublished data.

Quantitation of t,he spot intensities was done by scintillation spec- trometry of the unmodified chloramphenicol and its acetylated deriv- atives.

RESULTS

Isolation of Promoter-Initial screening of the rat Hue111 genomic library yielded eight positive isolates that hybridized with the murine al(1) collagen promoter probe. One clone, H- 3, upon further characterization was shown to contain the al(1) promoter. The insert was 13 kb and had two internal EcoRI sites and four Xba sites (Fig. 1A). The initial data suggested that this insert contained the rat collagen gene, the 8-kb EcoRI fragment hybridized to the al(1) collagen mRNA on a Northern gel (data not shown). Three XbaI fragments, 3.6, 3.1, and 3.3 kb, were present within the insert. The 3.6- and 3.1-kb fragments hybridized with the initial screening plasmid, and the 3.6-kb fragment contained a 300-bp BglII/ Xba fragment which was also present in the screening plas- mid. Preliminary DNA sequencing from both directions of the Xba site that separated the 3.6- and 3.1-kb fragments identified and localized the direction of the collagen promoter and the 5"untranslated domain of the gene by comparison with the sequence surrounding the corresponding Xba site o f the murine promoter.

The 3.6-kb fragment was subcloned into pUCl2, mapped as indicated in Fig. 1B, and then shotgun-subcloned into the HincII site of M13mpll for complete DNA sequencing. 3600 bases were sequenced from the universal primer of the sub- clones and the data are given in Fig. 2. The fragment contains the presumptive mRNA start site at +1 and continues to +115. The palindromic sequence which has been found in the 5'-untranslated region of other collagen genes (33) has been interrupted by the Xba restriction site. The TATA box is located a t -28 bases from the mRNA start site whereas a reverse CAT box is located at -100 bases.

Potentially interesting structures within the promoter in- clude a GC-rich 26-member (with one mismatch) interrupted palindrome (AGGGGGGAGGGGG ... CCTCCTCCCCCCT) located between -194 and -142. The downstream portion of the palindrome is part of a 35-bp stretch of pyrimidines located from -172 to -138. Two other large indirect repeats (16 bases with only one mismatch) are present at a great distance from the RNA start site. One is located a t -3109 through -3092 and is complementary to a sequence at -2584 through -2568; the second begins at -3312 to -3296 and is complementary to a sequence at -923 through -904.

One GC-rich direct repeat (TGGGGGCCGGGC) flanks the reverse CAT box from -128 through -116 and -98 through -86. Three other direct repeats of 12 or more bases are found a t more widely separated distances: two are pyrimidine-rich and are located at -2519 and -530 (TCTCCTCCCTCT) and a t -3469 and -1569 (TTTTTTTTTCTTT); the other is located at -3164 and -1382 (GCCACCCACACA). A com- puter search through the 3600 bases for the core glucocorticoid binding sequence (TGTTCT (34)) revealed the reverse com- plement (AGAACA) at -649. However, a palindromic se- quence characteristic of many glucocorticoid-responsive genes was not present (35).

Transient Expression of Col Cat 3.6-The plasmid Col Cat 3.6 contains 3.6 kb of upstream oll(1) collagen promoter and extends to +115 of the 5"untranslated region of the mRNA. This collagen RNA transcript contains two nonauthentic AUG start sites. Both are terminated with in-phase stop codons, and the latter one is included within the Xba cloning site. This collagen promoter fragment generates a mRNA with an unt,ranslated leader sequence joined to CAT mRNA which provides the utilized translation start codon (see Fig.

3074 Regulation of the Rat al(I) Collagen Promoter

FIG. 1. Map of the X phage insert containing the al(1) promoter. A , the entire insert relative to the short and long arms of the X phage is illustrated. The transcribed region of the gene is darkened. B, the 3.6 Xba subclone con- taining the 5"flanking domain and the RNA transcription start site. This frag- ment was cloned upstream of the CAT gene to yield Col Cat 3.6. Restriction site abbreviations used: E, EcoRI; X, XbaI; H, HindIII; P, PuuII; B, BamHI.

FIG. 2. Sequence of a 3.6-kb Xba fragment containing the rat al(1) promoter. The sequence from -3517 to -1020 is given in small type while the remainder from -1020 to +115 is in larger type. The transcribed sequence is shown in italics and includes the two AUG codons and their termination co- dons. The glucocorticoid consensus se- quence at -649 is boxed as is the CAT at -100 and TATA box at -28. The pyrimidine-rich sequence upstream from the CAT box at -138 to -172 is boxed, and the indirect repeat which is included within the pyrimidine-rich sequence is indicated by bold inverse arrows. The direct repeat that flanks the CAT box is shown by thin direct arrows.

8.4Kb

A.

-3 Kb - 1 .qKb Ol(b

(Subcloned with CAT B. t I I I tn

C.

-3600 -3500 -3400

-3200 -3300

-3100 -3000 -2900 -2800 -2700 -2603 -2500 -2400 -230C -2200 -2100 -2000 -1900 -1800 -1700 -1600 -1500

-1300 -1400

-1200 -ilK

-1000

-900

-800

-700

-600

-500

-400

-300

-200

-99

+1

15

0

3). Preliminary experiments revealed that the promoter was active for 24-48 h after transfection of ROS 17/2 cells. Opti- mization of transfection demonstrated that a llh-2-min glyc- erol shock greatly increased the measurable CAT activity extracted from ROS 17/2 cells (data not shown). Further preliminary studies were necessary to find the optimal con- centration of cells that would efficiently express the CAT gene yet also be sufficiently confluent to show responsiveness to 1,25-(OH)2D3. The sequence of transfection and hormone addition is also an important variable because addition of 1,25-(OH)zD3 12-24 h prior to the transfection results in down-regulation of all chimeric promoter constructs.

Fig. 4 illustrates an experiment in which the activity of Col Cat 3.6 is contrasted with pAZ1003, the mouse a2(I) promoter, and pSV2 CAT. The percent conversion of the input chlor- amphenicol to its acetylated forms and the percent collagen synthesis in the media are given. Despite some variability, Col Cat 3.6 consistently demonstrates more activity than

pAZ1003, and the activity of both is significantly reduced when the cells are treated with 1,25-(OH)2D3. In contrast pSV2 CAT does not show similar down-regulation in the presence of the hormone. The activity of the endogenous collagen gene was reduced by approximately 50% by 1,25- (OH)2D3 in all cases. There was some variability in the effect of 1,25-(OH),D3 between individual experiments and occa- sionally within individual assay points of a given experiment. Pooling the results of six separate experiments (Fig. 5) , in which the hormone inhibited endogenous collagen gene activ- ity, showed that the activity of Go1 Cat 3.6 fell from 19.0 k 17.7 to 5.0 f 4.6% CAT conversion ( p < 0.01) whereas pAZ1003 fell from 5.7 f 2.6 to 3.2 +- 1.2% ( p < 0.04). In contrast pSV2 CAT did not change significantly. The differ- ence in base-line expression of CAT activity from Col Cat 3.6 was approximately twice that of pAZ1003, but this difference failed to reach statistical significance ( p < 0.06).

The EL2 rodent fibroblasts did not show 1,25-(OH)~D3

Regulation of the Rat d ( I ) Collagen Promoter 3075 (-943 RatAlpha l(1) pVvll Collagen Promoter

Tlsnscription St& Site

Col Cat 3.6 (9.0 Kb)

sa1

Hind 111 PStl

FIG. 3. Map of Col Cat 3.6. The promoter fragment from rat type I collagen (open bar) is ligated into the Xba site of a CAT plasmid cloned into pUC12. The CAT enzyme contains the t antigen splice and polyadenylation signals. The polylinker sites flanking the pUCl2 plasmid as well as the transcription and translation start sites of the promoter and CAT gene are indicated.

Co SVPCAT ColCAT3.8 ~AZ1003 Std

C D C D C D C D C C D D

% CAT 0.0 96.8 6.7 4.8 6.4 4.1 8.1 2.5 9.3 3.8 19.7 13.4 16.7 17.0

yo COll 125.7 14.8125.9 15.5124.8 14.1 124.8 14.21 24.9 14.0124.713.61

FIG. 4. Effect of 1,25-(0H)*Ds on transfected promoters in ROS 17/2 cells. Plasmids containing the SV40 (pSVZCAT), crl(1) (Col Cat 3.6), or n2(I) (pAZ1003) promoters were transfected into ROS 17/2 cells in the presence or absence of 10 r n ~ 1,25-(OH)&. Twenty-four hours after the glycerol shock, 5 pCi/ml [3H]proline was added to the culture medium. Eighteen hours later the medium was taken for collagen analysis and the cells for CAT activity. The percent conversion of the chloramphenicol (% CAT) and percent collagen accumulation in the culture medium (% Coll) are given beneath each lane for control ( C ) and 1,25-(OH)2D3 @)-treated cells. The standard CAT enzyme spot was obtained from 0.5 unit of activity while lane Co is the background CAT activity.

regulation of the transfected plasmids or of the endogenous collagen gene (data not shown). Basal CAT expression and collagen synthesis in these cells was approximately 20% of the levels present in ROS cells, although Col Cat 3.6 was still a stronger promoter than pAZ1003 in these cells.

Permanently Selected ROS Cell Lines-Col Cat 3.6 and pAZ1003 were co-transfected with pSV2Neo or pY3 to gen- erate permanent cell lines so that the effect of 1,25-(OH)2D3 could be analyzed under conditions of higher cell density than could be used with the transient transfection protocol. After selection in G418 or hygromycin, individual clones of cells

eo T

H -20 L

Alpha l(1) Alpha 2(1) SV 40

Sld. Deviation: 17.7 4.69 2.65 1.23 5.75 3.25 21.5 19.0

9.7 10.4 Observations: 13 13 1-statistic: Degrees of Freedom: 24

2.74 2.25 0.56 12

Significance: 0.01 0.04 0.57

Mean: 19.0 5.07

7 7 10 10

18

FIG. 5. Summary of transient expression assays of pro- moters in ROS 17/2 cells. The data from six separate transfections, in which a control (pSV2CAT) and test (Col Cat 3.6 or pAZ1003) promoter constructs were incubated with control ( C ) and 1,25- (OH)*Dn (D)-containing media, were pooled and analyzed statisti- cally. The mean ? 2 S.D. is shown graphically, and the calculated values and Student's t test results are given beneath the graph.

were subcultivated, and those colonies demonstrating high basal CAT activity were subsequently tested for hormone responsiveness. Fig. 6A shows that certain colonies expressing Col Cat 3.6 responded to the hormone by a reduction in the activities of the endogenous collagen gene and the transfected gene (colonies H.3 and G.l). In other colonies, either the transfected gene (H.l) or both the transfected and endogenous gene (G.3) failed to respond to 1,25-(OH)2D3. Less variability was observed in the colonies that had been transfected with pAZ1003. In Fig. 6B it can be seen that although there is some variation in the activity of the CAT gene under basal conditions, all of the cell lines when incubated with 1,25- (OH)ZDS showed a significant decrease in the activity of the transfected cu2(I) promoter to a level that was proportional to the fall in activity of the endogenous gene. Also note that the expression of Col Cat 3.6 was in general 2-fold greater than pAZ1003 in these permanently selected cells (Fig. 6, A uersus B ) . There was no inhibition of either the transfected gene or the endogenous gene by 1,25-(OH)2D3 in the permanently selected EL2 cells (Fig. 6C). The relatively higher expression of Col Cat 3.6 over pAZ1003 was also observed in the perma- nently transfected EL2 fibroblasts (data not shown).

DISCUSSION

We undertook the cloning and characterization of the rat cul(1) collagen promoter because many models of type I col- lagen regulation have been developed in this species. In the case of calcitrophic hormone regulation of type I collagen synthesis in bone cells, the availability of ROS cells provided an in uitro model to analyze 1,25-(OH)*D3 regulation of the collagen promoter. These cells have been extensively charac- terized in terms of their regulation of alkaline phosphatase, a bone-specific protein, by PTH and 1,25-(OH)& (36,37). The cells contain 1,25-(OH)& receptors (38) and respond to this hormone by induction of bone gla protein (39), another bone- specific gene product. We have previously shown that, al- though the collagen synthesized by the ROS cells is not exclusively type I nor do the cells have the high rate of collagen synthesis found in primary cultures of osteoblasts or rat calvaria, there was a reduction of type I collagen synthesis by

3076 Regulation of the Rat d ( I ) Collagen Promoter A. Col Cat 3.6 in ROS 1712.8

CO H.3 G.1 SId G.3 H.1 Std

75.6 33 7 43 0 39.2 30 3 78.

88 16.2 " I r 16.8 " I 8 2

B. pAZlOO3 in ROS 1712.8 Co G.l G.2 G.3 H.l "" ~~ .~ H.2

C. Col Cat 3.6 in EL2 H.l H.2 H.3 Std

C D 8.9 2.6

13.3 6 7

* * - 0 0

T a m C D I C D I C Dl"- % CAT 3 1 2 7 2 0 1.3 3.7 3 2 76.9

X Coll 4.9 8.3 2.9 3.2 3.7 3.6

FIG. 6. Expression and regulation of promoters in perma- nently selected 17/2.8 cells. ROS and EL2 cells were co-trans- fected with the test plasmid and pSV2Neo or pY3 and placed under G418 or hygromycin selection. Resistant cell lines were tested for base-line CAT activity, and those showing the highest expression were tested for regulation by 1,25-(OH)2D3. After 24 h of exposure to the hormone, the cells were labeled with [3H]proline. The following day the medium was taken for analysis of percent collagen synthesis and the cell layer for CAT activity. Panel A, Col Cat 3.6 in ROS 17/ 2.8; panel B, pAZ1003 in ROS 17/2.8; panel C, Col Cat 3.6 in EL2. The percent conversion of the chloramphenicol (% CAT) and percent collagen accumulation in the culture medium (% Coll) are given beneath each lane for control (C ) and 1,25-(OH)*D3 (D)-treated cells. The G and H labeling refer to (3418 or hygromycin selection, respec- tively.

1,25-(OH)& similar to that found in rat calvaria (5). The rat al(1) promoter has a high degree of homology

(>go%) with the published sequence of the human and murine al(1) promoter (6, 22). It has organizational differences with the a2(I) promoter (11). For example, the al(1) promoter has the TATA sequence at -27 bp and a reverse CCAAT box at -100 which is flanked by a GC-rich direct repeat. The a2(I) promoter has a CCAAT sequence at -82 bp and is not flanked

by the direct repeats. Both promoters have a long pyrimidine- rich domain immediately upstream of the CCAAT box that demonstrates S1 nuclease and DNase hypersensitivity (40, 41). However, the al(1) promoter contains a 13-member pur- ine-rich sequence (AGGGGGGAGGGGG) just upstream to this region of DNA hypersensitivity which is complementary to a region within the pyrimidine sequence. A similar purine- rich sequence is not present in the a2(I) promoter domain.

Our previous work with ROS 17/2 cells has shown a reduc- tion in the d (1) and a2(I) mRNA content that is proportional to the fall in total collagen and percent collagen synthesis (5). Recently Harrison et ~ 1 . ~ has utilized nuclear run-on assays in 1,25-(OH)zD3-treated ROS cells and found a similar fold reduction in transcriptional activity and collagen synthesis. Thus the results of the present transfection studies are con- sistent with a mechanism of transcriptional regulation of the al(1) and a2(I) promoter by 1,25-(OH)& in ROS cells. Furthermore, the cis element responsible for this hormone response is contained within the transfected promoter se- quences. A similar degree of inhibition in response to 1,25- (OH)& has been found in the 5'-flanking sequences of the PTH gene (43). Since a putative down-regulatory sequence for the PTH gene is not yet evident, the promoter that we have isolated should be valuable in identifying the sequences that respond to this hormone.

The activity of the transfected collagen promoters and the endogenous genes were equally inhibited by 1,25-(OH)2D3 in the ROS 17/2.8 cells but not the fibroblastic cells. Vitamin D receptors have been demonstrated in most fibroblast cell lines (44) although the receptor number was not specifically tested in EL2 cells. Thus it is possible that the 1,25-(OH)&- regulated expression of collagen within osteoblasts is related to trans-acting factors associated with the osteoblastic phe- notype. An alternative is that fibroblastic cells express levels of 1,25-(OH)2D3 receptor which are below a threshold level necessary to produce a detectable change in transcription of the collagen gene. The ability of the ROS 17/2 and 17/2.8 cells to express collagen in a manner distinct from other collagen-producing cells provides a model to finely dissect the regulatory domains of the al(1) gene and the trans-acting factors within bone cells that mediated the bone-specific effects of various hormones and growth factors. Furthermore, these cells can provide a model to identify and contrast regulatory elements of the al(1) and cr2(I) promoters.

The al(1) promoter consistently expressed CAT activity greater than a2(I) in both the ROS cells and EL2 fibroblasts. Judgment of the relative activity of these two promoters and whether it underlies the basis for the 2:1 ratio of al(1) mRNA and a2(I) mRNA in type I collagen-producingcells will require transfection experiments that utilize constructs from the same species, with similar size and containing the recently de- scribed enhancer elements within the first intron (15-17).

It is likely that defining the major regulatory units within type I collagen that confer bone-specific regulation will be a very complex task. Besides the bone-directed action of 1,25- (OH)ZDB and PTH, other hormones that regulate type I collagen in bone but not fibroblasts include insulin (45) and insulin-like growth factor (46). Another example of bone- specific regulation of type I collagen production was recently observed in a patient with Ehlers-Danlos syndrome in whom the a2(I) chain was not synthesized by dermal fibroblasts (47). Deficient a2(I) chain synthesis in skin and presumably bone cells has been observed in a patient with severe osteo- genesis imperfecta (48). Since the Ehlers-Danlos case did not

J. H. Harrison, D. N. Petersen, A. Lichtler, A. T. Mador, D. W. Rowe, and B. E. Kream, manuscript submitted for publication.

Regulation of the Rat a

have bone disease, it is probable that the a2(I) chain was being synthesized in bone cells but not the dermal fibroblasts. Clearly the 4 1 ) gene was not being expressed equally in both tissues in these clinical examples.

There are many potential sites for regulation of these two promoters. Besides the potential regulating elements within the 5'-flanking sequences described in this report, the role of the tissue-enhancing domains located within the first intron of the collagen al(1) and a2(I) genes (15-17) needs to be compared in different cell types. DNase-hypersensitive sites have also been mapped to the 3'-flanking domain in the human al(1) gene (42) suggesting that important regulatory sequences may also exist in this region of the gene. Presump- tive translational control signals located within the 5'-un- translated region of the collagen mRNA (33) will need eval- uation in various cell types that synthesize type I collagen. Thus it appears that ROS 17/2 and 17/2.8 cells may provide an in uitro cell system that can be used to identify cis-acting domains within type I collagen genes that respond to tissue- specific and hormone-directed signals.

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2. Genovese, C., Rowe, D., and Kream, B. (1984) Biochemistry 2 3 ,

3. Kream, B. E., Rowe, D. W., Gworek, S. C., and Raisz, L. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,5654-5658

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