Proc. Nati. Acad. Sci. USA Vol. 89, pp. 4688-4692, May 1992 Cell Biology lea,25-Dihydroxyvitamin D3 regulates the transcription of II mRNA in avian myelomonocytes (myelomonocytes/ regulation/mRNA stability) ABDERRAHIM LOMRI AND ROLAND BARON Departments of Cell Biology and Orthopedics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510 Communicated by Robert W. Berliner, February 3, 1992

ABSTRACT Carbonic anhydrase II (CAII) is highly ex- possibility that the promoter region contained vitamin D-re- pressed in the , where it is involved in the process of sponsive elements (VDRE) (8, 11, 12), no change in CAII extracellular acidification required for bone resorption. We mRNA levels were observed before 24 hr in the human have previously shown that la,25-dihydroxyvitamin D3 promonocytic cell line HL-60 (7). The present study was, [1,25(OH)2D3], a steroid hormone that regulates the differen- therefore, done to determine whether 1,25(OH)2D3 increases tiation of macrophages and , induces the expression CAII mRNA by acting at the transcriptional and/or post- of CAII mRNA and protein in avian bone marrow cells. To transcriptional levels and whether this increase required the determine whether this regulation occurred at the gene level, transcription and translation of other gene products. we have studied the effects of 1,25(OH)2D3 on CAII expression Using a transformed myelomonocytic avian cell line in a transformed myelomonocytic avian cell line (BM2). As (BM2), we found that the regulation of CAII levels by observed in nontransformed cells, 1,25(OH)2D3 markedly in- 1,25(OH)2D3 in mononuclear phagocytes occurs mostly via creased CAII biosynthesis and mRNA levels. The increase in an increase in gene transcription. This effect is independent CAII mRNA was detected as early as 3 hr after adding the of de novo protein synthesis, thereby suggesting that the hormone (1.9-fold) and reached 4.7-fold by 48 hr. These effects effect does not require the synthesis of other gene products. were completely blocked by actinomycin D, and nuclear run-on analysis confirmed that 1,25(OH)2D3 increased the rate ofCAII MATERIALS AND METHODS gene transcription. In contrast, induction of CAII mRNA expression was not affected by inhibition of protein synthesis Materials. Dulbecco's modified Eagle's medium (BT-88), with cycloheximide, and no significant changes in mRNA calf serum, chicken serum, folic acid, tryptose phosphate, stability were seen. Thus, 1,25(0H)213 modulates CAM gene and gentamycin were purchased from GIBCO BRL. Cyclo- expression at the transcriptional level, and this effect does not heximide (CHX), actinomycin D, lipopolysaccharide, phor- require de novo synthesis of other gene products. These results bol 12-myristate 13-acetate (PMA), and 5,6 dichloro-1-,3-D- suggest that activation of the CAll gene occurs early in the ribofuranosylbenzimidazole (DRB) were from Sigma. Re- differentiation events triggered by vitamin D3 in myelomono- striction endonucleases were from Boehringer Mannheim. cytic cells. 1,25(OH)2D3 was provided by M. Uskokovic, Hoffman-La Roche. Radioisotopes were purchased from Amersham. Cell Culture and Cell Activation. BM2 cells, a line of Carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) is a chicken myeloblasts transformed with the avian myeloblas- zinc metalloenzyme involved in the reversible hydration of tosis virus (clone C3A) (13), were cultured in BT-88 medium CO2. The carbonic anhydrase isoform II (CAII) is highly containing folic acid at 160 ,ug/ml, 10% tryptose phosphate, expressed in cells where acid-base regulation is a primary 5% heat-inactivated calf serum, 5% heat-inactivated chicken function-i.e., gastric parietal cells, salivary glands, renal serum, and gentamycin at 50 jig/ml. The BM2 cells are tubular cells (1), and the osteoclast in bone (2, 3). In all these nonadherent and are induced to differentiate into adherent cells, the role of CAII is to generate H+ for acid extrusion by myelomonocytic cells by treatment with lipopolysaccharide the ATP-driven proton pumps, and HCO- is usually extruded (10 ,ug/ml for 24 hr) (14). The adherent cells were passed via bicarbonate/chloride exchangers. The importance of every 2 days up to 8 weeks. For all experiments the cells were CAII in the function of osteoclasts is best demonstrated by plated at 105 cells per cm2 in BT-88 medium/serum for 48 hr. the fact that mutation ofthe CAII-encoding gene is associated Then fresh serum-free medium containing 0.5% bovine serum with , , and cerebral cal- albumin was added to the cells for 36 hr. At that time the BM2 cification (4). Furthermore, inhibition of CAII in vitro in- cells were differentiated to nondividing macrophages by duces a decrease in bone resorption (5), and CAII activity and stimulation with PMA at 250 ng/ml for 12 hr. Incubation of levels of expression can be regulated by calciotropic and cells with 10-8 M 1,25(OH)2D3 was for 3, 6, 12, 24, or 48 hr. thyroid hormones (6-9). This regulation includes la,25- Actinomycin D (5 ,ug/ml), CHX (10 ,ug/ml), and DRB (25 dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of ,ug/ml) were used as indicated. the steroid hormone vitamin D, which regulates CAII mRNA Immunoblot Analysis. BM2 cells were cultured with or and protein levels in human and avian mononuclear phago- without 10-8 M 1,25(OH)2D3 for 24 hr, rinsed twice with cytes (7, 8). The action of steroid hormones involves their phosphate-buffered saline (PBS), lysed in urea buffer (10 mM high-affinity binding to specific receptors in nuclei, and the Tris'HCl, pH 7.4/6 M urea), and centrifuged at 3000 x g for regulation of specific gene transcription is mediated by bind- 5 min at 4°C to remove cellular debris. Ten micrograms of ing of the hormone-receptor complex to steroid-responsive protein per lane was analyzed by electrophoresis through elements present in the promoter regions of the affected 10% SDS/PAGE (15). The proteins were transferred to (10). Although analysis of the CAII gene suggested the Abbreviations: 1,25(OH)2D3, la,25-dihydroxyvitamin D3; CAII, car- The publication costs of this article were defrayed in part by page charge bonic anhydrase type II; PMA, phorbol 12-myristate 13-acetate; payment. This article must therefore be hereby marked "advertisement" DRB, 5,6-dichloro-1-,8-D-ribofuranosylbenzimidazole; CHX, cyclo- in accordance with 18 U.S.C. §1734 solely to indicate this fact. heximide; VDRE, vitamin D-responsive elements.

4688 Downloaded by guest on September 26, 2021 Cell Biology: Lomri and Baron Proc. Natl. Acad. Sci. USA 89 (1992) 4689

nitrocellulose filters (16) and incubated with anti-CAII mono- PMA + + clonal antibody [7C6-1 (17) diluted 1:5] in PBS/0.5% bovine 1,25(OH)2D3 - + serum albumin/0.1% Tween 20 for 1 hr at 370C. The mem- branes were washed and treated with peroxidase-labeled goat KDa anti-mouse IgG antibody (Cappel Laboratories) for 30 min at 370C. After being washed, the membranes were incubated in 97 - 2 M Tris HCl, pH 7.6/0.03% H202/1.4 mM 3.3'-diaminoben- 66 - zidine. 45 - RNA Preparation and Analysis. Total RNA was isolated by CAII using the guanidium isothiocyanate method (18). Ten micro- 29 - - 4- grams of RNA per lane was separated on 1.2% formalde- hyde/agarose gels and transferred to nylon filters (Hybond- N+, Amersham) in 0.05 M NaOH for 3 hr. Filters were prehybridized 1 hr at 420C in buffer containing 50%o (vol/vol) FIG. 1. Effects of 1,25(OH)2D3 on CAII biosynthesis. BM2 cells formamide, 5x standard saline citrate (SSC), 5x Denhardt's were induced with PMA (250 ng/ml) for 12 hr and then cultured in solution, 0.5% SDS, and boiled salmon sperm DNA at 20 serum-free medium without (left lane) or with (right lane) 10-8 M 1,25(OH)2D3 for 24 hr. Total cell lysates (10 ;tg per lane) were pug/ml. Blots were probed with a 1.2-kilobase (kb) cDNA separated on 10%6 SDS/PAGE, and the nitrocellulose transfers were probe encoding the chicken CAII (19). Inserts were purified treated with the monoclonal antibody to CAII (7C6) as culture by electrophoresis and labeled with [a-32P]dCTP by random supernatants diluted 1:5 in buffer. The 29- to 30-kDa band corre- priming (20). Hybridization was carried out in the prehybrid- sponding to CAII is clearly enhanced with vitamin D3 (right lane and ization solution containing 106 cpm of radiolabeled probe per arrow). ml. Membranes were washed twice at room temperature in 2x SSC/0.1% SDS for 15 min, then once at 60°C in lx marrow cells (8) or in the PMA-treated transformed human SSC/0.1% SDS and in 0.1 x SSC/0.1% SDS for 30 min each. promonocytic cell line HL-60 (7). However, addition of Membranes were exposed to x-ray film at -70°C with an 1,25(OH)2D3 (10-8 M) in serum-free medium for 24 hr in- intensifying screen. For reprobing, the blots were washed duced an increase in CAlI levels, as detected by immunoblot once in boiling 0.5% SDS for 15 min. analysis (Fig. 1). BM2 cells in which differentiation was not In Vitro Transcription Assay. Nuclear run-on assays were induced with PMA showed undetectable amounts of CAII, done according to published methods (21) with some modi- even when cultured in the presence of 1,25(OH)2D3 (data not fications. Cell monolayers were washed with PBS, then shown). scraped in PBS, and pelleted by low-speed centrifugation. To examine whether the addition of 1,25(OH)2D3 affected The cells were suspended in 10 mM Tris-HCl, pH 7.4/10 mM the CAll mRNA level in BM2 cells, RNA was isolated from NaCl/3 mM MgCl2/0.5% Nonidet P-40 and disrupted by cells stimulated for 3, 6, 12, 24, or 48 hr with 1,25(OH)2D3 and repeated pipetting through a Pasteur pipet. After centrifuga- examined by Northern (RNA) blot hybridization, using as a tion, the nuclei (2-3 x 107) were suspended in 200 ,ul of a probe a cDNA encoding the chicken CAII (1.2 kb) (19). As transcription buffer containing 40%(vol/vol) glycerol, 10 mM shown in Fig. 2, treatment of BM2 cells with PMA alone Tris-HCl (pH 7.4), 5 mM MgCl2, 100 mM KCl, 0.1 mM resulted in an increase in mRNA for both CAII and LEP100, EDTA, 2 mM dithiothreitol, 2 mM ATP, GTP, CTP each, 100 a lysosomal membrane protein (22) that, unlike actin (8), did units of RNasin, and 125 1ACi of [a-32P]UTP (3000 Ci/mmol; not change its level of mRNA or protein expression in 1 Ci = 37 GBq) and incubated at 30°C for 20 min. Transcripts response to 1,25(OH)2D3 in nontransformed avian bone mar- were isolated by adding 500 ,ul of a solution containing 4 M row cells (8). The PMA effects peaked at 12 hr and decreased guanidium isothiocyanate, 25 mM sodium citrate (pH 7.0), thereafter. However, the addition of 1,25(OH)2D3 to PMA- 0.5% Sarkosyl, 0.1 M 2-mercaptoethanol plus 5 pLI of yeast treated cells increased CAII mRNA levels above the PMA- tRNA (10 mg/ml), 70 .ul of 2 M sodium acetate (pH 4.0), 700 treated levels (Fig. 2A), with a significant increase being seen pu1 ofphenol, and 140 ,ul of chloroform/isoamylalcohol, 49:1. as early as 3 hr (1.9-fold + 0.5, P < 0.05). Although the levels After vigorous mixing, the solution was placed on ice for 15 of LEP100 mRNA were slightly increased by the addition of min and then centrifuged for 10 min at 10,000 x g at 4°C. The 1,25(OH)2D3 in this culture system, the relative abundance of RNA was precipitated once with 1 vol of isopropanol and CAII mRNA was determined by densitometric analysis of recovered by centrifugation. The pellet was washed with 80o autoradiographs, normalized to the expression level of (vol/vol) ethanol and dissolved in sterile 10mM Tris-HCl, pH LEP100 (Fig. 2B, Inset) and the changes expressed as a ratio 7.0/1 mM EDTA/0.1% SDS. At this point, each sample was of treated/control. As shown on Fig. 2B, there was an applied to a spin column packed with Sephadex G-50 (Boeh- increase in the relative levels of CAll mRNA with time (R = ringer Mannheim) to remove the unincorporated [a-32P]UTP. 0.81, P < 0.001) with a transient peak after 6 hr (2.8-fold + This procedure yields labeled RNA with a total activity of 0.7, P < 0.01) and an increase thereafter to reach values of 1-2 x 107 cpm/assay. Equal numbers of counts (107 cpm per >4-fold (4.7 + 1.2, P < 0.001) after 48 hr (Fig. 2B), confirm- reaction) from 1,25(OH)2D3-treated and control BM2 cells ing in BM2 cells our results in normal bone marrow cells (8). were hybridized to denatured cDNAs applied to a nylon To determine the role of transcriptional changes in the membrane (Hybond-N+). Denatured DNAs included increase in CAII mRNA, BM2 cells were cultured in the pBR322 vector (1.0 ,ug), the chicken CAII cDNA (1.2 kb), the absence (Fig. 3, lanes a and b) or presence (Fig. 3, lanes c and human a-actin cDNA (1.0 kb), and the chicken LEP100 d) ofactinomycin D. Treatment ofthe cells with actinomycin cDNA (1.63 kb) (0.05 ,ug each). Hybridization conditions D completely blocked the ability of 1,25(OH)2D3 to increase were as described in ref. 21, and the membranes were CAlI mRNA (Fig. 3, lane d), thereby demonstrating that the processed as described above. effects of 1,25(OH)2D3 on CAll levels require transcription. Here again, these effects were specific for CAll because the levels of LEP100 mRNA were not affected by 1,25(OH)2D3 RESULTS or by actinomycin D (Fig. 3, lane d). Cells ofthe avian myelomonocytic cell line BM2 treated with To further address whether the effects of 1,25(OH)2D3 PMA for 24 hr to induce their differentiation expressed only reflected control at the transcriptional level, we also per- low levels of CAII (Fig. 1), consistent with previous obser- formed nuclear run-on assays. BM2 cells were cultured with vations made in the absence ofPMA in nontransformed avian or without 10-8 M 1,25(OH)2D3 for 12 hr, and their nuclei Downloaded by guest on September 26, 2021 4690 Cell Biology: Lomri and Baron Proc. Natl. Acad. Sci. USA 89 (1992)

\ IFi III et(11' .4 12 24 48 'I 1h I'MA 12N1A.1 - - + . ._ - 1,.2501 'I) ; I 2 (M 1X ) I _ . It _ WinoXmcI; in ( cIoheximidc

C 'AII "**'' b CA I .-1 - ob 60

1. i: .'Ich".9wu m 'II.I, of) 0 w Om6SW e@amam(i 6060m40 n. IEi1it lhlr 3 6' 3 6 B -a Control FIG. 3. Effects of actinomycin D and CHX on 1,25(OH)2D3- _. 1,25(OH)2D3 induced accumulation of CAII mRNA. PMA-pretreated BM2 cells were incubated with the vehicle (lanes a), 10-8 M 1,25(OH)2D3 (lanes b), actinomycin D (5 .g/ml) (lanes c), or both (lanes d) for 3 or 6 hr. a In lanes e and f CHX (10 gg/ml) was added for 3 or 6 hr. In lanes g 0 BM2 cells were preincubated with CHX at 10 j&g/ml and then treated in its presence with 1,25(OH)2D3 at time indicated below. Total RNA was isolated and subjected to Northern blot analysis (10 ,ug per lane), 5- 0 12 24 36 48 hybridized with the CAII probe (upper bands), stripped, and rehy- Time (hr) bridized with the LEP100 probe (lower bands). As previously O. observed, 1,25(OH)2D3 induced an increase in CAII mRNA above go the effect of PMA, and LEP100 levels were not affected (lanes a and b). Actinomycin D completely blocked the effects of PMA and 1,25(OH)2D3 (lanes c and d), but CHX did not (lanes e-). decrease differed for the two mRNA species. LEP100 mRNA o 12 24 36 48 was relatively stable, with a decrease of only 30-40% after 12 Time (hr) hr (Fig. SC). In contrast, CAII mRNA was rapidly degraded, with only 9.1 ± 2.3% of the message remaining after 12 hr in FIG. 2. Time course of the effects of 1,25(OH)2D3 on CA11 control cells (Fig. SB). Although the relative amount of CAII mRNA accumulation. PMA-pretreated BM2 cells were cultured in serum-free medium with or without 10-8 M 1,25(OH)2D3 for the message was higher in the presence of 1,25(OH)2D3 over the indicated periods (3-48 hr). (A) Northern blot analysis. Total RNA course of the experiment, with 26.3 ± 7.3% remaining after was extracted at the time indicated, loaded (10 gg per lane) and 12 hr (P < 0.02), this was mostly due to an early (< 3 hr) effect electrophoresed on agarose gels, blotted to Hybond-N' membranes, and hybridized with 32P-labeled chicken CA11 cDNA probe (1.2 kb). .AX I'M A 4 .. After being stripped, the same blots were rehybridized with the 1,2510Ei1w12D LEP100 (1.63 kb) probe, used as a control for loading and specificity. 1,.a (B) Quantitative densitometric analysis of the Northern blots (mean pBR1322 0. ± SD from three separate experiments). Relative changes in CAll mRNA (1.7 kb) levels were quantified by densitometric scanning, (All ON v normalized to LEP100 mRNA (2.2 kb) (Inset), and expressed as a (t-actin, i ratio (treated/control). The Inset shows that 1,25(OH)2D3 signifi- " S- cantly increased (P < 0.0001) CAll mRNA levels above PMA. When ILEPIOO im. expressed as the ratio of treated/control, CAHl mRNA increased significantly with time (r = 0.81, P < 0.001); a significant increase occurred as early as after 3 hr (1.9-fold, P < 0.05), a first peak occurred at 6 hr (P < 0.01), and a 4.7-fold increase occurred by 48 hr (P < 0.001). a were isolated. Run-on transcripts were labeled in vitro with I_ v ExpL 2 [a-32P]UTP and hybridized to cDNA. As shown in Fig. 4A, -m ._ hybridization to vector DNA (pBR322) was negligible, and ,C.j * Expt. I transcription of LEP100 or a-actin, used as internal controls, 1; 6 was not affected or slightly decreased by the hormone (ratio a oftreated/control from two separate experiments: 0.65-0.78 and 0.91-0.91, respectively). In contrast, and although CAII transcripts were present in nuclei from PMA-stimulated cells Control 1,25(OH)2D3 (controls), 1,25(OH)2D3 increased the absolute rate of tran- FIG. 4. Nuclear run-on assays of the effects of 1,25(OH)2D3 on scription of the CAII gene by 2.5- to 3.5-fold (Fig. 4B). transcriptional activity of the genes encoding CA11, a-actin, and Elevation of steady-state mRNA levels could result not LEP100. PMA-pretreated BM2 cells were cultured in serum-free only from an increased rate of transcription but also from medium with or without 10-8 M 1,25(OH)2D3 for 12 hr. 32P-labeled enhanced mRNA stability. To estimate CAII mRNA stabil- RNA isolated from nuclei was hybridized to cDNA probes immo- ity, control BM2 cells and cells preincubated with bilized on filters. (A) Hybridization. pBR 322 plasmid DNA was used 1,25(OH)2D3 for 24 hr were treated with the mRNA-specific as a negative control, and a-actin and LEP100 cDNAs were used as for or 12 and mRNA positive controls. Although the transcripts for actin and LEP100 are transcription inhibitor DRB (23) 3, 6, hr, not affected by the treatment, the levels of CA11 transcripts are was extracted. Fig. 5A shows the progressive decrease in increased. (B) Quantitative densitometric analysis. Autoradio- CAII mRNA as assessed by Northern hybridization with the graphic signals from two separate experiments (Expt.) were quan- chicken CAII cDNA probe. Although CAII and LEP100 titated by scanning densitometry, normalized to LEP100, and plot- transcripts progressively decreased in both control (lanes a) ted; the results show a 3-fold increase in CA11 transcriptional rates and 1,25(OH)2D3-treated (lanes b) cells, the kinetics of this with 1,25(OH)2D3. Downloaded by guest on September 26, 2021 Cell Biology: Lomri and Baron Proc. Natl. Acad. Sci. USA 89 (1992) 4691

A i h DISCUSSION

I'AMA t + + + + t+ + 1,254(011 i IDI - - - - + + + +t Expression of the CAII-encoding gene is required for the D)RB - + + I differentiation of myelomonocytic cells into the osteoclast phenotype, this enzyme being highly expressed in the fully differentiated osteoclast (24, 25) and its inhibition leading to (AII *VW a decrease in bone resorption both in vitro and in vivo (5, 26). We and others (27, 28) have previously shown that 1,25(OH)2D3, a potent regulator of osteoclast and macro- phage differentiation, can induce a marked increase in CAII IjXli A d9 mRNA and protein expression in myelomonocytic cells after a 24-hr exposure (7, 8). In HL-60 cells, where earlier time 0 3 1 2 3 6 2 lime thri 6 {I (.w 1 points have been tested, no increase was seen after a 3-hr exposure to the hormone (7), raising the question of whether B these regulatory effects were at the transcriptional or post- transcriptional level and whether the induction of CAII by C 100,25, ( M t2D.1 Control 1,25(OH)2D3 required the transcription and translation of R \,,1},~~~~~1)11 other gene products that would be activated earlier. 12 1) In the present study, we have analyzed the effects of l, _()I 1)<--a.v - O 1,25(OH)2D3 on the transcriptional level of CAII in a trans- formed avian myelomonocytic cell line [BM2, C3A (13)] at

([.( O ; the presence or absence of 1 early time points and in inhibitors v 1 I {} I 1Fiffc ll -)el arie1 lrlr of transcription or protein synthesis. Treatment of the cells with phorbol esters, as required for their differentiation (14), FIG. 5. Effects of 1,25(OH)2D3 on stability of the CA11 and LEP100 mRNA. (A) Northern blot analysis. PMA-pretreated BM2 induced a nonspecific increase in mRNA levels. 1,25(OH)2D3 cells were cultured for 24 hr in the absence (lanes a) or presence was, however, found to markedly and specifically increase (lanes b) of 10-8 M 1,25(OH)2D3. The transcription inhibitor DRB (25 the level of CAII mRNA in BM2 cells as early as 3 hr after ,ug/ml) was added to the culture medium, and RNA was extracted hormone addition (1.9-fold) and to increase the level 4.7-fold after 0-12 hr. Total RNA was isolated and subjected to Northern blot after 48 hr. All effects of the hormone were inhibited by analysis (10 gig/lane), hybridized with the CAII probe (upper bands), actinomycin D, suggesting that the increase in CAII level was stripped, and rehybridized with the LEP100 probe (lower bands). mostly from enhanced gene transcription. Furthermore, in Degradation of mRNA is visible as a function of time. (B and C) vitro transcription of the CAII gene in nuclear run-on assays Levels of CAII and LEP100 mRNA were quantitated by densito- also demonstrated a 2.5- to 3.5-fold increase in CAII tran- metric scanning with 10lo representing their respective levels at scripts with 1,25(OH)2D3. CAII mRNA was rapidly degraded time of inhibitor addition (time 0). CAII mRNA (B), in contrast to and addition induced only a LEP100 mRNA (C), was rapidly degraded (50%6 of the CAII mRNA without 1,25(OH)2D3, hormone was degraded after 8 hr without vitamin D3). Although 1,25(OH)2D3 small increase in CAII mRNA amounts but did not alter slightly increased CAII mRNA amounts over the duration of the significantly the rate of CAII mRNA degradation. Finally, experiment (for 12 hr, mean SD of three separate experiments; for inhibition of protein synthesis by CHX did not prevent, and other points, average from two separate experiments), the slopes of indeed amplified, the increase in CAII mRNA level after the degradation curves were identical between treated and untreated treatment with 1,25(OH)2D3, showing that the hormone does cells, suggesting a lack of significant stabilization of CA11 mRNA by not require the de novo translation of other gene products to the hormone. The higher amount of CAll mRNA seen after 3 hr with mediate its effects on CAII mRNA levels. The increase in DRB probably reflects continued and higher transcription after CAII mRNA in the presence of CHX probably reflects exposure to 1,25(OH)2D3. inhibition of the synthesis of degradative enzymes or of of the hormone, and the slopes of the degradation curves repressors of gene expression. were identical between treated and untreated cells from 3 to Taken together, these results suggest that the effects of 12 hr. We interpret these results to suggest that the increased 1,25(OH)2D3 on CAII expression are both rapid and direct, characterizing the CAII-encoding gene as one of the early transcription rate induced by 1,25(OH)2D3 was maintained genes involved in the vitamin D3 response of myelomono- for a short period of time after adding the transcription cytic cells, leading to their differentiation into the osteoclast inhibitor, leading to a small accumulation ofmRNA, followed phenotype. by degradation at similar rates in treated and untreated cells. It is known that steroid hormones can regulate mRNA We concluded that the stability of both CAII and LEP100 levels via gene transcription and/or mRNA stability (10, 29). mRNA was not affected by treatment with 1,25(OH)2D3. In the case of 1,25(OH)2D3, several genes other than CAII Finally, to determine whether the induction of CAII have been reported to be affected by the hormone at the mRNA by 1,25(OH)2D3 requires the de novo synthesis of transcriptional and, in some cases, posttranscriptional levels. other gene products, we tested the effects of the protein Interestingly, both 9-kDa calbindin-D- (30) and osteocalcin- synthesis inhibitor CHX (Fig. 3, lanes e-g) on the responses encoding gene expression (11, 12, 31) are up-regulated by of CAll and LEP100 mRNA to 1,25(OH)2D3. The addition of 1,25(OH)2D3, both at the transcriptional level and via an CHX in the presence of PMA induced a 2-fold increase in increase in mRNA stability, when our data would suggest CAll mRNA (Fig. 3, lane e) with no change in the levels of that CAII gene expression is mostly regulated at the tran- LEP100 mRNA (compare lanes a and e). However, when the scriptional level. On the other hand, in instances where cells were treated with 1,25(OH)2D3 in the presence of CHX 1,25(OH)2D3 down-regulates the expression of some genes, (lane f), the level of CAII mRNA was 1.5-fold higher than in as reported for c-myc (32), parathyroid hormone (33, 34), PMA plus CHX-treated cells (lane e) and than in parathyroid hormone-related peptide (35), and a 1-collagen 1,25(OH)2D3-treated cells (lane b), indicating that the hor- gene transcription (36), this down-regulation occurs without mone-induced effect does not require ongoing protein syn- alterations in mRNA stability. Furthermore, in all these thesis. A similar result was obtained when the cells were instances and as reported here for CAII mRNA, inhibition of incubated with CHX for 1 hr before the addition of protein synthesis by CHX did not block the effects of 1,25(OH)2D3 (lane g). 1,25(OH)2D3, indicating that the responses did not necessi- Downloaded by guest on September 26, 2021 4692 Cell Biology: Lomri and Baron Proc. Nad. Acad Sci. USA 89 (1992) tate the de novo synthesis ofother gene products. Instead, we 6. Silverton, S. F., Dodgson, S. J., Fallon, M. D. & Forster, R. E. that it induced an increase in CAII mRNA level, as (1987) Am. J. Physiol. 253, 670-674. found 7. Shapiro, L. H., Venta, P. J., Yu, Y. L. & Tashian, R. E. (1989) reported for these other gene products and for differentiation- FEBS Lett. 249, 307-310. related protooncogenes in HL-60 cells (37-39). 8. Billecocq, A., Emanuel-Rettig, J., Levenson, R. & Baron, R. (1990) 1,25(OH)2D3 induces the concomitant expression of sev- Proc. Nat!. Acad. Sci. USA 87, 6470-6474. eral other osteoclast genes in addition to CAII. The Na',K+- 9. Zenke, M., Mufioz, A., Sap, J., Vennstrom, B. & Beug, H. (1990) Cell 61, 1035-1049. ATPase and the vitronectin receptor (40), the tartrate- 10. Beato, M. (1989) Cell 56, 335-344. resistant acid phosphatase (27), the calcitonin receptor (41, 11. Morrison, N. A., Shine, J., Fragonas, J. C., Verkest, V., McMen- 42), and the vacuolar proton pump (43) are all induced by emy, M. L. & Eisman, J. A. (1989) Science 246, 1158-1161. 1,25(OH)2D3 in bone marrow cultures or in vivo. Although it 12. Kerner, S. A., Scott, R. A. & Pike, J. W. (1989) Proc. Natl. Acad. Sci. USA 86, 4455-4460. is possible that some of these genes are activated early and 13. Gazzolo, L., Moscovici, C., Moscovici, M. G. & Samarut, J. (1979) lead to a cascade of events and osteoclast differentiation, the Cell 16, 627-638. initial responses have not yet been characterized. Some 14. Symonds, G., Klempnauer, K. H., Evans, G. I. & Bishop, J. M. genes may be directly activated and some indirectly, requir- (1984) Mol. Cell. Biol. 4, 2587-2593. and translation of the products of these 15. Laemmli, U. K. (1970) Nature (London) 227, 680-685. ing the transcription 16. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. early genes. The fact that the increase in CAII gene tran- Sci. USA 76, 4350-4359. scriptional activity induced by 1,25(OH)2D3 in BM2 cells is 17. Linser, P. J., Perkins, M. S., Fitch, F. W. & Moscona, A. A. (1984) rapid (<3 hr) and does not require the de novo translation of Biochem. Biophys. Res. Commun. 125, 690-697. other gene products strongly suggests that activation of this 18. Chomczynski, P. & Sacchi, N. (1987)Anal. Biochem. 162,156-159. gene represents one of the early events in 1,25(OH)2D3- 19. Yoshihara, C. M., Federspiel, M. & Dodgson, J. B. (1984) Ann. N. Y. Acad. Sci. 429, 332-334. induced osteoclast differentiation. 20. Feinberg, A. P. & Vogelstein, B. (1984) Anal. Biochem. 137, In this respect it is noteworthy that the avian CARl gene 266-267. promoter contains sequences [GGTGA(G)TGAAC and 21. Greenberg, M. E. & Ziff, E. B. (1984) Nature (London) 311, 433- GGTGA(T)TCAAC) strongly reminiscent of the vitamin 438. D-responsive elements (VDRE) identified in rat and human 22. Fambrough, D. M., Takeyasu, K., Lippincott-Schwartz, J. & Sie- osteocalcin promoters (11, 12). Like the rat VDRE, the gel, N. R. (1988) J. Cell Biol. 106, 61-67. 23. Noda, M., Yoon, K. & Rodan, G. A. (1988) J. Biol. Chem. 263, sequences identified in the chicken CAII promoter include 18574-18577. AP-1-like palindromic binding sites (TGAITCA and 24. Gay, C. V. & Mueller, W. J. (1974) Science 183, 432-434. TGAQTGA instead of TGA(CTCA) when the AP-1 site and 25. Vaananen, H. K. & Parvinen, E. K. (1983) Histochemistry 78, the VDRE are juxtaposed in the human promoter (12). AP-1 481-485. represents a distinct class of regulatory factor with a con- 26. Hott, M. & Marie, P. J. (1989) J. Dev. Physiol. 12, 277-281. genes c-jun and 27. Roodman, G. D., Ibbotson, K. J., MacDonald, B. R., Kuehl, T. J. sensus binding site for the early-response & Mundy, G. R. (1985) Proc. Nat!. Acad. Sci. USA 82,8213-8217. c-fos heterodimers. Such binding of AP-1 on the VDRE has 28. Takahashi, N., Mundy, G. R., Kuehl, T. J. & Roodman, G. D. been reported to interfere with responses to vitamin D3, (1987) J. Bone Min. Res. 2, 311-317. suppressing the induction of the osteocalcin gene by both 29. Vannice, J. L., Taylor, J. M. & Ringold, G. M. (1984) Proc. Nat!. vitamin D3 and retinoic acid (44). In contrast, and although Acad. Sci. USA 81, 4241-4245. the AP-1 site also mediates the transcriptional response to 30. Dupret, J. M., Brun, P., Perret, C., Lomri, N., Thomasset, M. & Cuisinier-Gleizes, P. (1987) J. Biol. Chem. 262, 16533-16557. phorbol ester tumor promoters (45-48), PMA did not prevent 31. Demay, M. B., Gerardi, J. M., Deluca, H. F. & Kronenberg, H. M. the 1,25(OH)2D3-mediated increase in CAII mRNA levels in (1990) Proc. Nat!. Acad. Sci. USA 87, 369-373. BM2 cells. Because these motifs are only partially identical 32. Simpson, R. U., Hsu, T., Begley, D. A., Mitchell, B. S. & Aliza- to the identified VDRE, in particular, because they may lack deh, B. N. (1987) J. Biol. Chem. 262, 4104-4108. repetitive elements (49), it will be ofinterest to transfect cells 33. Silver, J., Naveh-Many, T., Mayer, H., Schmelzer, J. H. & Pop- with the chicken CAII promoter upstream of the chloram- ovtzer, M. M. (1986) J. Clin. Invest. 78, 1296-1301. 34. Russell, J., Lettieri, D. & Sherwood, L. M. (1986) Endocrinology phenicol acetyltransferase reporter gene to better define the 119, 2864-2866. DNA sequences constituting the chicken CAII VDRE and 35. Ikeda, K., Lu, C., Weir, E. C., Mangin, M. & Broadus, A. E. (1990) compare them with other responsive elements. J. Biol. Chem. 265, 5398-5402. In conclusion, our results suggest that vitamin D3 increases 36. Harrison, J. R., Petersen, D. N., Lichtler, A. C., Mador, A. T., CAII expression in myelomonocytic cells mostly via an Rowe, D. W. & Kream, B. E. (1989) Endocrinology 125, 327-333. is detected as 37. Brelvi, Z. S. & Studzinski, G. P. (1986) J. CellBiol. 102, 2234-2243. increase in CAII gene transcription. This effect 38. Dani, C., Blanchard, J. M., Piechaczyk, M., El Sabuty, S., Marty, early as 3 hr after treatment of the cells with 1,25(OH)2D3, L. & Jeanteur, P. (1984) Proc. Natl. Acad. Sci. USA 81, 7047-7050. making activation of the CAII gene an early event in the 39. Kelly, K., Cochran, B. H., Stiles, C. D. & Leder, P. 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