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Lea,25-Dihydroxyvitamin D3regulates the Transcription of Carbonic Anhydrase II Mrna in Avian Myelomonocytes

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Lea,25-Dihydroxyvitamin D3regulates the Transcription of Carbonic Anhydrase II Mrna in Avian Myelomonocytes Proc. Nati. Acad. Sci. USA Vol. 89, pp. 4688-4692, May 1992 Cell Biology lea,25-Dihydroxyvitamin D3 regulates the transcription of carbonic anhydrase II mRNA in avian myelomonocytes (myelomonocytes/gene 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 osteoclast, 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 osteoclasts, 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 osteopetrosis, renal tubular acidosis, 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 genes (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).
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