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Endocrinology, doi:10.1210/en.2003-0862
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Endocrinology Vol. 145, No. 4 1730-1738
Copyright © 2004 by The Endocrine Society

Interaction between 3' Untranslated Region of Calcitonin Receptor Messenger Ribonucleic Acid (RNA) and Adenylate/Uridylate (AU)-Rich Element Binding Proteins (AU-Rich RNA-Binding Factor 1 and Hu Antigen R)

Shigemitsu Yasuda, Seiki Wada, Yukitomo Arao, Masakazu Kogawa, Fujio Kayama and Shigehiro Katayama

Fourth Department of Internal Medicine (S.Y., S.W., M.K., S.K.), Saitama Medical School, Saitama 350-0495, Japan; and Division of Environmental Immunology and Toxicology (Y.A., F.K.), Department of Health Science, Jichi Medical School, Tochigi 329-0498, Japan

Address all correspondence and requests for reprints to: Seiki Wada, M.D., Ph.D., Department of Clinical Science, Faculty of Pharmaceutical Science, Josai International University, 1 Gumyo, Togane, Chiba 283-8355, Japan. E-mail: wadas{at}saitama-med.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our previous results in mouse osteoclasts suggested that calcitonin (CT) alters CT receptor (CTR) mRNA stability. The CTR mRNA transcript contains several adenylate/uridylate (AU)-rich destabilizing elements in the 3' untranslated region (3'UTR). When the 3'UTR of mouse CTR mRNA was labeled by [{alpha}-32P]-uridine 5-triphosphate, interactions were observed between the transcript and several cell extracts, including those from the osteoclast progenitor monocyte/macrophage cell line, RAW 264.7. The molecular masses of the interacting proteins ranged from approximately 35 to 50 kDa, similar to AU-rich RNA-binding factor 1 (AUF1) and Hu antigen R (HuR). Radiolabeled 3'UTR transcripts bound with a 40-kDa protein, which could be extracted from cells transfected with AUF1 p40. To confirm the binding specificity, a pSG5 vector construct, containing the AUF1 p40 with an hemagglutinin tag, was transiently transfected into NIH3T3 cells. The extracts were incubated with poly(A)-added CTR3'UTR. The reaction mixture was immunoprecipitated using an antihemagglutinin antibody and precipitated mRNA species were extracted and reverse transcribed using oligo-dT primers. It was found that PCR primers specific for the 3'UTR of CTR mRNA sequence generated a PCR signal. No signal was observed when mutated AUF1 p40 was transfected. In a manner similar to the AUF1 binding, HuR was also found to bind to the 3'UTR. Specific binding of AUF1 p40 and HuR was also found with RNA extracted from mouse osteoclasts. Treatment of osteoclasts with CT did not significantly affect the expression of AUF1 but decreased the levels of HuR and its mRNA. The role of CTR3'UTR in mRNA stability was further tested by expressing luciferase reporter constructs that did, or did not, contain the CTR3'UTR, under the control of the tetracycline-regulatory system. The results showed that the addition of 3'UTR considerably shortened the mRNA half-life of the luciferase reporter gene. These results suggest that AUF1 p40, HuR, and the 3'UTR of the CTR mRNA transcript could be involved in posttranscriptional regulation of CTR mRNA expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BECAUSE CALCITONIN (CT) directly inhibits osteoclastic bone resorption, it has been used widely to treat metabolic bone disorders, such as Paget’s disease of bone, malignancy-associated hypercalcemia, and osteoporosis. It is well known, however, that continuous treatment with CT causes a diminution of its inhibitory actions on bone resorption (1). We and other investigators (2, 3, 4, 5) have studied the cellular mechanisms for this, using mouse and human osteoclasts in vitro. The results indicated that desensitization to CT was closely associated with down-regulation of the CT receptor (CTR). This down-regulation was due to not only the internalization or phosphorylation of the receptor but also reduced cell surface receptor concentration as a result of inhibition of de novo CTR synthesis (2, 4, 5). It was also found that the CT-induced reduction of CTR mRNA expression could not be accounted for by decreased transcription of the CTR gene (3), although another report indicated that decreased transcription rate could be involved in the homologous down-regulation of the CTR in osteoclasts (6). The CTR belongs to the seven-transmembrane G protein-coupled receptor (7TMGCR) superfamily. The mechanism of desensitization and down-regulation of another member of this receptor class, the ß2-adrenergic receptor (AR), has been studied extensively. It has been shown that the ligand-induced down-regulation of ß2-AR correlated well with the reduction of its mRNA (7).

Recent investigations have revealed that changes in mRNA half-life influence cell growth, differentiation, and cellular responses (8, 9, 10). Steady-state mRNA levels can fluctuate many fold after a change in mRNA half-life with little change in transcription rate (11). Degradation and stabilization of mRNA are now recognized as regulated processes that provide powerful means for the control of gene expression. Notable examples include the mRNA of various cytokines, i.e. granulocyte macrophage colony-stimulating factor and IL-3, and the oncogenes c-myc and c-fos (12, 13, 14). Cognate sequences in the 3'-untranslated region (UTR) of mRNAs have been shown to function as signals for rapid mRNA degradation. These include adenylate/uridylate (AU)-rich elements (ARE), where the core pentamer AUUUA, or the larger UUAUUUA(U/A)(U/A) motif, can function as potent mRNA destabilizing elements (15, 16). These motifs were also critically important in the regulation of certain 7TMGCR mRNAs, i.e. in the case of the thrombin receptor (17). In the case of the ß2-AR, an ARE in the 3'UTR was shown to bind with several cytosolic factors of approximate molecular size 35 kDa (18).

The AU-rich RNA-binding factor 1 (AUF1), or heterogeneous nuclear ribonucleoprotein (RNP), which consists of four protein isoforms (45, 42, 40, and 37 kDa), is an example of an RNA-binding protein that can influence mRNA stability. AUF1 isoforms p40 and p37 were purified from cytoplasmic extracts of K562 human erythroleukemia cells by monitoring their binding to the 3'UTR of c-myc mRNA (19). ARE-binding proteins can be nuclear or cytoplasmic and can shuttle between both compartments. It has been shown that ARE-mediated binding may trigger mRNA decay, but some other studies indicate that AUF1 may have a role in stabilizing mRNA (20). Hu antigen R (HuR) is the human homologue of the Xenopus gene embryonic lethal abnormal vision family, which was recently identified as a factor involved in mRNA degradation and stabilization (21). Although a complete understanding of the underlying mechanism awaits further experiments, protein-mRNA binding is likely to be widely important in ARE-directed mRNA modulation (22).

In this report, we examined a potential role for AUF1 p40 and HuR in altering the stability of CTR mRNA. Specific binding of AUF1 p40 and HuR to the 3'UTR of the CTR mRNA was found using the technique of immunoprecipitation and reverse transcribed PCR amplification with specific oligonucleotides for the 3'UTR of CTR mRNA. Mutated AUF1 p40 failed to bind with the sequence. Treatment with salmon CT (sCT) did not affect expression of AUF1 but decreased the levels of HuR. The functional importance of the CTR3'UTR in destabilizing mRNA stability was shown, although more precise roles of AUF1, HuR, and the 3'UTR of the CTR mRNA remain to be explored in the CT bone target cell, the osteoclast.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
DNA encoding mouse CTR3'UTR (nucleotides 2056–3707; GenBank accession no. NM-007588) was obtained by PCR amplification from a mouse brain cDNA library (Takara Bio Inc., Otsu, Japan) and ligated into the pGEM3Zf+ vector (Promega Corp, Madison, WI). First, PCR was preformed using the T3 primer at the 3' end of pGEM3Zf+ and CTR2013 (5'-AAT GAT CCC CAT GAA CGT C-3'). The amplified products were further purified by nested PCR using CTR 2116-EcoRI (5'-CAA GAA TTC CAT GTG TTT CAA GTG ATT CCC-3') and CTR3684-BamHI (5'-CAA GGA TCC ATA CAT GTA CAC AGC ATA AGC GTT-3'). The cloned DNA derived from 3'UTR of CTR mRNA was inserted into EcoRI and BamHI sites of pGEM3Zf+ vector (pGEM CTR3'UTR), such that transcription with T7 RNA polymerase yielded sense strand RNA. The identities and orientations of the inserts were verified by restriction enzyme digestion and sequencing.

The plasmids pSG5-HA-AUF1p45, pSG5-HA-AUF1p42, pSG5-HA-AUF1p40, pSG5-HA-AUF1p37 (23), and pSG-HA-HuR were used to examine the binding between these molecules and the 3'UTR of CTR mRNA. A cloned CTR 3'UTR fragment was ligated into BamHI and SmaI sites of the pSP64poly(A) vector [pSP64poly(A)-CTR3'UTR] to examine the binding between 3'UTR of CTR mRNA and AUF1 p40. A fragment containing five AUUUA repeats (5'-GGA TCC TTA TTT ATT TAT TTA TTT ATT TAG AT-3') was cloned into the EcoRI and BglII sites of the pSG5 vector (pSG5-AU5).

The mutant AUF1 p40, containing an altered RNA binding domain (RBD) was constructed using the site-directed mutagenesis system (Stratagene, La Jolla, CA). In the first step, using the mutagenic primers 1 (5'-TCA AGG GGT GCT GGC GCT GTG CTA TTT-3' and 5'-AAA TAG CAC AGC GCC AGC ACC CCT TGA-3'), the template plasmid (pSG5-HA-AUF1p40) was amplified by Pfu Turbo DNA polymerase (Stratagene), and then the template plasmid was digested by DpnI. mutated plasmid (pSG5-HA-AUF1p40RBD1mut) was transformed into Escherichia coli (XL-1 Blue) and amplified. In the second step, using the mutagenic primers 2 (5'-GG CGT GGG GCT TGC GCT ATT ACC TTT AAG G-3' and 5'-C CTT AAA GGT AAT AGC GCA AGC CCC ACG CC-3'), the template plasmid (pSG5-HA-AUF1p40RBD1mut) was amplified by Pfu Turbo DNA polymerase, and then the template plasmid was digested by DpnI. Mutated plasmid (pSG5-HA-AUF1p40RBD1,2mut) was transformed into E. coli (XL-1 Blue) and amplified. Mutation of the target element was confirmed by sequencing.

The tetracycline (Tet)-regulated expression vector, which contains the luciferase gene fused to the CTR3'UTR, was constructed to study the effects of the CTR3'UTR on mRNA half-life. A pGL3-basic vector containing the Tet-responsive element (TRE) was digested by XbaI and then blunted. The CTR3'UTR fragment was excised from pGEM CTR 3'UTR vector by EcoRI and BamHI and then blunted. The blunted CTR3'UTR fragment was inserted into the blunted XbaI site of pGL3-TRE vector (pGL3-TRE-CTR3'). The direction of the insert was verified by restriction enzyme digestion and sequencing.

Cell culture and preparation of cellular extracts
Mouse osteoclasts were prepared as described by Akatsu et al. (24). Briefly, primary osteoblasts prepared from newborn mouse calvaria (5 x 105 cells/dish) and bone marrow cells from male mice (5 x 106 cells/dish) were cocultured on dishes coated with type I collagen gel in {alpha}MEM containing 10% fetal bovine serum (FBS) with 10-8 M 1,25(OH)2D3 for 7–8 d. The culture media were replaced with fresh media every 3–4 d. At the end of the culture, dishes were treated with collagenase (0.2%), and intercellular attachment was gently disrupted by syringing in 21-gauge needles. Equal aliquots of the cells were subcultured into the dishes, and cells other than osteoclasts were removed by the treatment of 0.25% trypsin for 5 min. In these cultures, tartrate-resistant acid phosphatase-positive osteoclasts were predominant, comprising approximately 95% of the whole cell population. Osteoclasts were also prepared from bone marrow cells or RAW 264.7 cells by treatment with 20 ng/ml soluble receptor activator for nuclear factor {kappa}B ligand (sRANKL) and 100 ng/ml macrophage-colony stimulating factor (M-CSF) for 7 d. RAW 264.7 and MC3T3E1 cells were cultured in {alpha}MEM containing 10% FBS. NIH3T3 and MCF7 cells were cultured in DMEM containing 10% FBS.

Cell extracts were prepared from mouse osteoclasts, RAW 264.7, NIH3T3, and MC3T3E1 cells by lysis in an extraction buffer containing 0.2% Nonidet P-40, 40 mM KCl, 10 mM HEPES (pH 7.9), 3 mM MgCl2, 1 mM dithiothreitol (DTT), 5% glycerol, 8 ng/ml aprotinin, 2 ng/ml leupeptin, and 0.5 mM phenylmethyl sulfonyl fluoride (PMSF). The mixture was sonicated for 25 sec. After centrifugation (4000 x g for 2 min), aliquots of the extracts were used as a source of cellular proteins and stored at -80 C until experimental use. In the experiments of heparin-agarose purification, the diluted extract (2.5 mg/dl) was loaded onto a heparin-agarose column (Sigma-Aldrich, St. Louis, MO) equilibrated with a buffer [12 mM HEPES-KOH (pH 7.9), 60 mM KCl, 0.12 mM EDTA, 0.3 mM DTT, 0.3 mM PMSF, and 12% glycerol], as described previously (25). The loading was performed at 0.1 ml/min; the flow-through fraction was collected and reloaded onto the column. The process was repeated five times. The column was then washed with five volumes of the buffer and additionally with five volumes of a buffer containing 200 mM KCl. The heparin-bound proteins were eluted with buffer containing 0.5 M KCl and used for the RNA-protein binding experiments. Animal handling and treatment were performed according to protocols provided by the Animal Research Committee of Saitama Medical School. Approval for the procedures described herein was granted by Saitama Medical School ethics committee, and the approval number is 000016.

UV cross-linking assay
Plasmid pGEM CTR3'UTR was linearized by EcoRI and was used as a template for in vitro transcription with T7 RNA polymerase of Riboprobe System-T7 (Promega). The transcript was labeled by [{alpha}-32P]-uridine 5-triphosphate (UTP) (800 Ci/mmol) through incubation with 0.2 µg DNA template, RNA polymerase, and reaction buffer containing 100 mM DTT; 25 U/µl RNasin; 2.5 mM ATP, GTP, and CTP; and 0.2 mM UTP at 37 C for 1 h. Five-microgram cell extracts from each of osteoclasts, RAW 264.7, NIH3T3, and MC3T3E1 cells were incubated with 4.0 fmol of the labeled transcript (a specific activity of approximately 1.0 x 105 cpm/µg) in a buffer containing 10 mM HEPES (pH 7.6), 50 mM KCl, 50 mM DTT, 3 mM MgCl2, and 5% glycerol at 37 C for 20 min. The volume of each reaction was 20 µl. One unit per microliter RNase A (Toyobo Co. Ltd., Osaka, Japan) was added to cleave unbound RNA. The reaction mixtures were exposed to UV irradiation (254 nm, 250 mJ/cm2) and resolved by 10% SDS-PAGE. Gels were dried and then signals were identified using FMBIO II fluoroimager (Takara Bio Inc.).

RNA extraction and PCR amplification of reverse transcribed RNA
Total RNA was extracted by the guanidine thiocyanate-phenol chloroform method, as described previously (5). First-strand cDNA was synthesized from 2.5 µg total RNA by incubation for 1 h at 42 C using AMV reverse transcriptase (Promega) with random hexanucleotides (for semiquantitative RT-PCR experiments) or oligo dT (for RNA-protein binding experiments). The cDNA mixture (2.5 µl) was submitted to PCR to amplify the target gene sequence. The reaction mixture contained 50 pmol of each specific primer for AUF1 mRNA (nucleotides 250–271 and 1085–1111; GenBank accession no. AB046615), HuR mRNA (nucleotides 119–139 and 1079–1096; GenBank accession no. NM-001419), RPS2 (nucleotides 58–79 and 641–667; GenBank accession no. U92700), or the 3'UTR of the CTR mRNA (nucleotides 2058–2083 and 3678–3707; GenBank accession no. U18542); 25 mM deoxy (d)-ATP, dGTP, dCTP, and dTTP (Pharmacia, Uppsala, Sweden); 2 µl of a 10-fold concentrated reaction buffer (Roche Molecular Biochemicals, Indianapolis, IN); 1 U Taq DNA polymerase (Roche Molecular Biochemicals); and sterile distilled water (26). Amplification was performed by a DNA Thermal Cycler 480 (PerkinElmer, Norwalk, CT), with cycles of denaturation at 96 C for 30 sec, annealing for 50 sec at 66 C (AUF1), 60 C (RPS2 and 3'UTR of CTR), and 66 C (HuR), and extension was performed at 72 C for 4 min. Preliminary experiments were preformed to ensure that the number of cycles employed was within the exponential phase of the amplification curve for semiquantitative experiments. PCR products were resolved on a 1.0% (wt/vol) agarose gel. Gels were stained with ethidium bromide, and the intensity of signals was quantified using the FMBIO II fluoroimager (Takara Bio Inc.).

Western blot analysis
Protein expression was examined by Western blot analysis using cell extracts of osteoclasts, RAW 264.7, NIH3T3, and MC3T3E1 cells. The extracts were separated by 10% SDS-PAGE and were electrically blotted (15 V, overnight) to Hybond-C membrane (Amersham Bioscience, Arlington Heights, IN). The blotted membrane was washed with PBS containing 5% nonfat milk (4 C, 1 h), incubated with 300 ng/ml rabbit polyclonal anti-AUF1 antibody (Upstate, Lake Placid, NY) or mouse monoclonal anti-HuR antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 C overnight, washed three times by PBS with 0.05% Tween 20, and then treated with antirabbit IgG coupled horseradish peroxidase (HRP) for AUF1 and antimouse IgG-coupled HRP for HuR. Immunoreactive signals were detected with ECL Western blotting analysis system (Amersham Bioscience) and compared with standard molecular mass markers (Bio-Rad Laboratories, Hercules, CA).

Preparation of hemagglutinin (HA)-tagged AUF1 and HuR proteins and the mutant of AUF1 p40
The plasmids pSG5-HA-AUF1 isoforms (p45, p42, p40, or p37), pSG-HA-HuR, or pSG5-HA-AUF1p40RBD1,2mut were transfected into NIH3T3 cells to investigate the binding between AUF1/HuR and the 3'UTR of the CTR mRNA. NIH3T3 cells were transfected at 50–70% confluence in 100-mm petri dishes with a total of 20 µg DNA, using a calcium phosphate coprecipitation technique (26). After 24 h, the cells were harvested and proteins were extracted.

Binding of HA-tagged AUF1 p40 and HuR with 3'UTR of CTR mRNA
Plasmid pSP64poly(A)-CTR3'UTR was used as a template for in vitro transcription by SP6 RNA polymerase. The transcribed product was purified by Oligotex-dT30 (Roche Co., Basel, Switzerland). Binding between the poly(A)-added CTR3'UTR RNA and cell extracts containing expressed HA-tagged AUF1 p40, HuR, or AUF1 p40RBD1,2mut was examined as follows. The HA-tagged AUF1 p40, HuR, or AUF1 p40RBD1,2mut enriched cell extracts (10 µg) were incubated with 0.5 µg purified poly(A)-added CTR3'UTR RNA in a buffer containing 10 mM HEPES (pH 7.6), 50 mM KCl, 50 mM DTT, 3 mM MgCl2, 5% glycerol, and 5 µg tRNA at 4 C for 30 min. Anti-HA tag antibody (MBL, Nagoya, Japan) was added to the reactive solution and incubated for 1 h at 4 C and then combined with 60 µl protein G resin (Amersham Bioscience). The reaction mixture was centrifuged and thoroughly washed with incubation buffer. RNA was extracted from the mixture by the guanidine thiocyanate-phenol chloroform method and reverse transcribed (RT) with 1 nmol oligo-dT (18 mer). Using a pair of specific primers for 3'UTR of CTR cDNA, the RT products were amplified by PCR as described above. Aliquots (10 µl) of the PCR products were resolved in 1% (wt/vol) agarose gels and stained by ethidium bromide and viewed by the FMBIO II fluoroimager (Takara Bio Inc.).

Measuring mRNA half-life
MCF7 cells were transfected with a total amount of 2 µg DNA at 90–95% confluence in 35-mm petri dishes, using LipofectAMINE2000 (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol. TRE containing pGL3-basic vector (Clontech, Palo Alto, CA) was inserted into the XbaI site with (pGL3-TRE-CTR3') or without (pGL3-TRE) the cDNA fragment encoding the 3'UTR in CTR mRNA. Cells were transfected with 0.4 µg pGL3-TRE or pGL3-TRE-CTR3' plasmids, 0.8 µg pTet-off (Clontech) for tetracycline-mediated transcription suppressor expression plasmid, and 0.4 µg pCAT3 control plasmid (Promega) for normalization of transfection efficiency. pBluescript (Stratagene) was used as a carrier to adjust the total amount of DNA. Eighteen hours later, the cells were treated by doxycycline (DOX) (1 µg/µl) to suppress de novo transcription of the luciferase reporter gene. Cells were harvested at 2, 4, 6, or 8 h after treatment with DOX. Total RNA was extracted and reverse transcribed, as described above. PCR was performed using specific primer sets for luciferase (5'-AAG GCT ATG AAG AGA TAC GCC CTG G-3' and 5'-TGT CAA TCA AGG CGT TGG TCG CTT C-3') and CAT (5'-GGA TAT ACC ACC GTT GAT ATA TCC CAA TGG-3' and 5'-TGC CAC TCA TCG CAG TAC TGT TGT AAT TC-3'). The PCR was run for 19 cycles (one cycle was at 96 C for 30 sec, 60 C for 50 sec, and 72 C 4 min) using TAKARA Taq. Aliquots (10 µl) of PCR products were resolved in 2% (wt/vol) agarose gels, stained with ethidium bromide, and viewed with a FMBIO II fluoroimager (Takara Bio Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Binding between the 3'UTR of CTR mRNA and cellular extracts of mouse osteoclasts, RAW 264.7, NIH3T3, and MC3T3E1 cells
Because our previous study suggested the possible involvement of posttranscriptional regulation in CTR mRNA expression (3), we first examined cellular factors binding to the 3'UTR in CTR mRNA. The cDNA corresponding to the 3'UTR of CTR mRNA was subcloned from a mouse brain cDNA library using nested PCR, as described in Materials and Methods. Cellular proteins were extracted from mouse osteoclasts, RAW 264.7, NIH3T3, and MC3T3E1 cells. Each extract was incubated at 37 C with the 32P-labeled 3'UTR of CTR mRNA and then treated by RNase to cleave unbound RNA. RNA-protein complexes were fixed by UV irradiation. The RNA-protein complexes were not obtained using osteoclast extracts. However, several proteins that interacted with the 3'UTR of CTR mRNA were present in extracts of RAW 264.7, NIH3T3, and MC3T3E1 cells (Fig. 1Go). In particular, a 40-kDa binding protein was prominent in RAW 264.7 cells, a mouse macrophage osteoclast precursor cell. When osteoclasts were prepared from RAW 264.7 cells by treatment with sRANKL and M-CSF, the protein-RNA interaction was not observed, as was the case for mouse osteoclasts derived in a coculture system or by treatment of bone marrow cultures with sRANKL and M-CSF (Fig. 1Go, Exp. 2). Purification of cell extracts with heparin agarose, or heat inactivation (95 C, 10 min) of osteoclast extracts, did not reveal significant interaction of the molecules.



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  		FIG. 1. Binding between 3'UTR of CTR mRNA and cellular extracts from various types of cells. The cloned 3'UTR of mouse CTR cDNA was inserted into pBluescript II SK. The plasmid was linearized by EcoRI and used as template for in vitro transcription. Mouse osteoclasts prepared by a coculture system, RAW 264.7, NIH3T3, and MC3T3E1 cells were extracted with a buffer containing 0.2% Nonidet P-40, 40 mM KCl, 10 mM HEPES (pH 7.9), 3 mM MgCl2, 1 mM DTT, 5% glycerol, 8 ng/ml aprotinin, 2 ng/ml leupeptin, and 0.5 nM PMSF. Each cell extract was incubated with the [{alpha}-32P]-UTP-labeled 3'UTR of CTR mRNA, and then treated by RNase to cleave unbound RNA. RNA-protein complexes were fixed by UV irradiation. Each mixture was resolved by 10% SDS-PAGE. Gels were dried and the signals were compared with standard molecular mass markers. The results were reproduced in two independent experiments. In the experiment 2 (Exp 2), osteoclasts were also prepared from RAW 264.7 cells and bone marrow (BM) cells by treatment with sRANKL and M-CSF. To eliminate or reduce the nucleases or enrich the binding proteins, the extract of osteoclasts was heat inactivated (95 C, 10 min) or purified with heparin-agarose gel, as reported in Materials and Methods. OCL, Osteoclasts.

 
Binding between the 3'UTR of mouse CTR mRNA and AUF1 isoforms
As shown in Fig. 1Go, molecular masses of the interacting proteins ranged from approximately 35 to 50 kDa, similar to the size of AUF1 isoforms. We then studied whether AUF1 could bind to the 3'UTR of CTR mRNA. HA-tagged AUF1 isoforms (p45, p42, p40, and p37) were extracted from NIH3T3 cells transfected with the respective plasmid constructs, as described in Materials and Methods. We have found that AU5, which contains five repeats of AUUUA, could bind with the isoforms of AUF1 (Arao, Y., unpublished data). Extracts enriched for HA-tagged AUF1 isoform proteins were incubated with the 3'UTR of CTR mRNA or AU5, labeled with [{alpha}-32P]-UTP. Binding between the 3'UTR of CTR mRNA and the extracts of either AUF1 p42- or p37-transfected cells was not seen (data not shown). In contrast, both the 45- and 40-kDa proteins bound with the labeled AU5 transcripts, as shown by binding activity in the extracts of cells transfected with AUF1 p45 or p40 but not cells transfected with vector alone (Fig. 2AGo). Because 45-kDa proteins interacted with the 3'UTR of CTR mRNA in each cell extract, it seemed that an endogenously expressed factor, other than AUF1 p45, might bind with the CTR 3'UTR. Therefore, we could not verify the binding specificity to the AUF1 p45 to CTR 3'UTR. On the other hand, binding between AUF1 p40-enriched cell extracts and the CTR 3'UTR was verified: the specific binding of AUF1 p40 with the protein extracts could be competed off with the unlabeled transcript of AU5 (Fig. 2CGo).



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  		FIG. 2. Binding between 3'UTR of mouse CTR mRNA and AUF 1 isoforms p45 and p40. pSG5 vectors containing AUF1 isoform of either p40 or p45 cDNAs were individually transfected into NIH3T3 cells using lipofectAMINE 2000, as described in Materials and Methods. AUF1 isoform proteins-enriched cell extracts were incubated with 3'UTR of CTR mRNA or AU5 (containing five repeats of AUUUA), which were labeled by [{alpha}-32P]-UTP. The mixtures, containing labeled AU5 or 3'UTR of CTR mRNA with protein-enriched extracts, were treated with RNase to cleave unbound RNA. Each reaction mixture was cross-linked by UV irradiation and resolved by 10% SDS-PAGE. Gels were dried and then exposed to x-ray film. Binding complexes were compared with molecular mass markers. Representative binding between the extracts and AU5 (A) and the extracts and 3'UTR of CTR mRNA (B) is shown. Incubation with the unlabeled AU5 transcript (~10 times of the amounts of CTR 3'UTR) revealed a decrease of the binding between AUF1 p40 and 3'UTR of CTR mRNA (C). EV, Empty vector.

 
Binding of AUF1 p40 and HuR to the 3'UTR of the CTR mRNA
To examine the binding specificity between the 3'UTR of CTR mRNA and AUF1 p40, HA-tagged AUF1 p40, the mutant (AUF1 p40RBD1,2mut) or HA-tagged HuR proteins were extracted from NIH3T3 cells, which were transfected with the plasmids pSG5-HA-AUF1p40, pSG5-HA-AUF1p40RBD1,2mut, or pSG5-HA-HuR, respectively. AUF1 p40RBD1,2mut protein was the result of six phenylalanine to alanine substitutions at positions of 111, 113, 116, 196, 198, and 201, in the RNP particle consensus sequence 1 motif of RBD 1 and 2 (Fig. 3AGo), and this mutant protein was unable to bind with the ARE. HA-tagged AUF1 p40, HA-tagged AUF1 p40RBD1,2mut, or HA-tagged HuR protein-enriched extracts were individually incubated with the poly(A)-added 3'UTR of CTR mRNA. The mixtures were immunoprecipitated with an anti-HA tag antibody. After thoroughly washing, poly(A)-added RNAs were extracted and then reverse transcribed using an oligo dT primer. The PCR-amplified product, obtained using primers specific for the 3'UTR in CTR mRNA, was identical to the CTR sequence. A strong PCR signal was obtained when the binding protein was wild-type AUF1 but not with the mutant protein (Fig. 3CGo). This result clearly shows that AUF1 p40 directly binds with the 3'UTR of CTR mRNA. The binding of HuR to the 3'UTR of the CTR mRNA was also verified using this system, although the binding was observed as a rather faint signal in comparison with that of AUF1 (Fig. 3CGo). Equal amounts of cellular proteins were verified by Western blotting in this experiment (Fig. 3BGo). The binding of AUF1 and HuR to the 3'UTR of the CTR mRNA was also found using RNA extracts from mouse osteoclasts (Fig. 3DGo).



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  		FIG. 3. Binding of AUF1 p40 and HuR to the 3'UTR of the CTR mRNA. HA-tagged AUF1 p40, the mutant (AUF1p40RBD1,2mut), or HA-tagged HuR proteins were extracted from NIH3T3 cells, which were transfected with the plasmid pSG5-HA-AUF1p40, pSG5-HA-AUF1p40RBD1,2mut, or pSG5-HA-HuR. AUF1p40RBD1,2mut protein was phenylalanine to alanine substituted at positions of 111, 113, 116, 196, 198, 201 for alanine, in the RNP1 motif (A). HA-tagged AUF1 p40, AUF1p40RBD1,2mut, and HuR protein-enriched extracts were individually incubated with the poly(A)-added 3'UTR of CTR mRNA in a buffer containing 10 mM HEPES (pH 7.6), 50 mM KCl, 50 mM DTT, 3 mM MgCl2, 5% glycerol, and 5 µg tRNA for 30 min at 4 C. Equal amounts of cellular proteins were verified by Western blotting in this experiment (B). The mixtures were immunoprecipitated with anti-HA tag antibody and then recovered by protein G resin. After thoroughly washing, poly(A)-added RNAs that complexed with HA-tagged protein were extracted and then reverse transcribed with oligo dT. Using specific primers for 3'UTR of mouse CTR cDNA, the RT products were amplified by PCR. Aliquots of the products were resolved in 1% (wt/vol) agarose gels, stained with ethidium bromide, and compared with standard molecular mass marker (C). The binding of AUF1 and HuR to the 3'UTR was also studied using the RNA extracts of mouse osteoclasts prepared by a coculture system (D).

 
Expression of AUF1 isoforms and HuR, and CT effects on AUF1 and HuR expression in mouse osteoclasts
Expression of AUF1 and HuR in RAW 264.7 cells and mouse osteoclasts was investigated by Western blot analysis using specific antibodies against AUF1 and HuR. Cellular proteins were extracted from the cells and resolved by 10% SDS-PAGE. The identified signals were compatible with the four AUF1 isoforms and HuR when compared with standard molecular mass markers (Fig. 4AGo). Furthermore, the expression of AUF1 mRNA isoforms and HuR was examined by RT-PCR analysis, as described in Materials and Methods. Expression of mRNA corresponding to the four types of AUF1 isoforms and HuR was also found in RAW 264.7 cells and osteoclasts (Fig. 4BGo).



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  		FIG. 4. Expression of AUF 1 and HuR proteins and their mRNAs in RAW 264.7 cells and osteoclasts. A, Mouse osteoclasts were prepared by coculture of mouse primary osteoblasts and bone marrow cells in the presence of 10-8 M 1,25 dihydroxyvitamin D3 and 10-6 M prostaglandin E2. Cellular proteins were extracted from osteoclasts and RAW 264.7 cells, resolved by 10% SDS-PAGE, and electrically blotted to Hybond-C membrane. The membrane was incubated with anti-AUF1 or anti-HuR antibodies at 4 C overnight and then treated with antirabbit IgG antibody-coupled HRP (for AUF1) or antimouse IgG antibody coupled HRP (for HuR). Immunoreactive signals were detected by ECL Western blotting analysis system and compared with standard molecular mass markers. B, Total RNA was extracted by the guanidine thiocyanate-phenol chloroform method, as described in Materials and Methods. Reverse-transcribed RNA was subjected to PCR with specific primers, designed to recognize all isoforms of AUF1 mRNA or HuR mRNA. PCR products were resolved on a 2.0% (wt/vol) agarose gel. RAW, RAW 264.7 cells.

 
Because we have shown previously that treatment with CT-induced down-regulation of CTR through a decrease of CTR mRNA expression (2), possible involvement of AUF1 and HuR in this process was examined in mouse osteoclasts. The effect of CT on the expression levels of AUF1 proteins and mRNA was examined by Western blotting and semiquantitative RT-PCR. When osteoclasts were treated with sCT (10-9 M) for the times indicated, treatment with sCT in osteoclasts did not markedly alter the mRNA levels (Fig. 5AGo) or protein levels of AUF1 (Fig. 5CGo). Because decreased levels of HuR were recently shown to be associated with destabilization of some mRNA species, we studied the effect of CT on the protein and mRNA levels of HuR in osteoclasts. The expression of HuR protein, together with its mRNA, decreased dramatically after treatment with CT in mouse osteoclasts (Fig. 5Go, B and C).



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  		FIG. 5. Effects of CT on expression of AUF1 and HuR mRNAs (A, B) and their proteins (C) in osteoclasts. Mouse osteoclasts were prepared by coculture of mouse primary osteoblasts and bone marrow cells in the presence of 10-8 M 1,25 dihydroxyvitamin D3. Osteoclasts were treated with sCT (10-9 M) for the times indicated. Total RNA was extracted, reverse transcribed with random hexamers, and then subjected to PCR with specific primers, designed to recognize all isoforms of AUF1 mRNA (A) or HuR mRNA (B). PCR products were resolved on a 2.0% (wt/vol) agarose gel. The intensity of signals was compared with those of RPS2 (ribosomal protein S2). After treatment with sCT (10-9 M) for the times indicated, extracted osteoclast proteins were resolved by 10% SDS-PAGE and then electrically blotted to Hybond-C membrane. The membrane was incubated with anti-AUF1 or anti-HuR antibodies at 4 C overnight and then treated with antirabbit IgG antibody-coupled HRP (for AUF1) or antimouse IgG antibody-coupled HRP (for HuR). Immunoreactive signals of AUF1 and HuR were detected by ECL Western blotting analysis system and compared with standard molecular mass markers (C).

 
The effects of 3'UTR in CTR mRNA on mRNA half-life
To investigate the functional role of the 3'UTR of CTR mRNA, we employed a transcriptional pulsing strategy, based on the Tet-regulatory system, to monitor the influence of CTR 3'UTR on mRNA turnover rates. The expression plasmids for Tet-controlled suppressor and Tet-regulated reporter (luciferase) genes (pGL3-TRE-CTR3' or pGL3-TRE) were transfected into MCF7 cells, as described in Materials and Methods. Eighteen hours later, the cells were treated with DOX (1 µg/µl) to shut off the transcription of reporter genes and the rate of clearance of the two transcripts [luciferase (Luc) or Luc-CTR3'UTR] was monitored by RT-PCR. As shown in Fig. 6Go, the amount of CTR3'UTR/Luc mRNA at each time after DOX treatment was lower than Luc mRNA alone. The half-lives of Luc and CTR3'UTR fused Luc mRNAs were estimated as 4 and 2 h, respectively. This result suggested that the 3'UTR of CTR mRNA was able to destabilize mRNA and to reduce its half-life.



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  		FIG. 6. The effects of 3'UTR in CTR mRNA on mRNA half-life. The expression plasmids for Tet-controlled suppressor and Tet-regulated reporter genes (pGL3-TRE-CTR3' or pGL3-TRE) were transfected into MCF7 cells. Cells were transfected with 0.4 µg pGL3-TRE or pGL3-TRE-CTR3' plasmids, 0.8 µg pTet-off plasmid, and 0.4 µg pCAT3 control plasmid for normalization of transfection efficiency. Eighteen hours later, the cells were treated with DOX (1 µg/µl) to shut off the transcription of reporter genes and the rate of clearance of the two transcripts [Luc without CTR3'UTR (-) or Luc with CTR3'UTR (+)] was monitored by RT-PCR, as described in Materials and Methods. Transfection efficiency was confirmed by the amount of CAT transcript. Aliquots (10 µl) of PCR products were resolved on 2% (wt/vol) agarose gels stained with ethidium bromide and viewed by the FMBIO II fluoroimager (A). The levels of amplified fragments (Luc and CAT) were estimated by the quantification program of the FMBIO II fluoroimaging system. Results were normalized to CAT level in each lane. The level of Luc without CTR3'UTR mRNA is shown as closed circle, the level of Luc with CTR3'UTR mRNA is shown as open circle (B). Similar results were obtained from three independent experiments, of which representative data are shown.

 
Because MCF7 cells express CTR along with reasonable amounts of AUF1 p40 and HuR, we next examined the effects of CT in this system. Although transfection of 3'UTR shortened mRNA half-life, treatment with CT did not significantly alter the mRNA half-lives in this system. These results, however, were consistent with our previous observation, in which we found that the effects of CT were cell type specific. Thus, the prolonged decrease of CTR mRNA after CT treatment is observed only in osteoclasts (2, 5) and was not observed in CTR-bearing T47D (2), MCF7 cells, or CTR-transfected COS7 cells (Wada, S., private communication).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Steady-state mRNA levels are shown to change markedly by alteration of mRNA half-life, not solely by modulation of transcription rate. Cellular factors binding to AREs in the 3'UTR of mRNAs have been shown to affect mRNA stabilization, and play essential roles in changing cell function (27). The addition of an ARE to normally stable mRNAs such as ß-globin mRNA renders them unstable (28), and the deletion of the sequence from oncogene mRNAs such as c-myc or c-fos results in stabilization (29, 30). Turnover of mRNA is also important in the process of homologous down-regulation of 7TMGCR (17, 18), in which rapid ligand-induced receptor mRNA decay has been observed. Although the precise mechanism of mRNA degradation has not been fully elucidated, a number of investigations indicate that nuclear and/or cellular proteins (trans-acting factors) are involved in regulating steady-state mRNA levels (19, 23, 25). AUF1 has been studied in detail with respect to the manner in which it selectively recognizes AREs and facilitates mRNA degradation (19). However, some other studies have also shown that AUF1 might have a role in stabilizing mRNAs (20). Because AUF1 belongs to a posttranscriptional machinery complex, a possible explanation to reconcile the apparent discrepancy between these actions might be an altered requirement for the complex, together with other unidentified endogenous mRNA degrading factors. It is unlikely that a single pathway or mechanism can account for all mRNA processing (22).

In this study, we examined a potential role of AUF1 and HuR in the process of modulating CTR mRNA stability. As shown in Fig 1Go, there was interaction between the transcripts of the 3'UTR and cellular factors; a strong signal was noted around 40 kDa in the extracts of RAW 264.7 macrophages. Although several signals at approximately 70–100 kDa were observed, it is possible that mRNA binding factors assembled into dimers or trimers in vitro, as found in the study of Pende et al. (31). It was found that 3'UTR of CTR mRNA could bind with AUF1 p40 but not with p45, p42, or p37. Although a 45-kDa protein was noted to bind to the 3'UTR, it was possible that additional binding protein(s), besides AUF1 p45, constituted a complex with the 3'UTR. We therefore studied in more detail the binding between AUF1 p40 and 3'UTR of CTR mRNA. The specificity of binding between the two molecules was confirmed by combining the techniques of immunoprecipitation and RT-PCR (Fig. 3Go). Mutated AUF1 p40, with substitution of several amino acids at mRNA recognition motifs, was unable to bind to the 3'UTR. Although binding between the 3'UTR and extracts of osteoclasts was not amenable to study, expression of AUF1 protein and mRNA was found in osteoclasts and an osteoclast precursor RAW 264.7 macrophage. Treatment with sCT did not significantly affect the expression of AUF1 in osteoclasts, although interestingly, the expression of HuR and its mRNA was decreased in a time-dependent manner (Fig. 5Go). HuR has been shown to prevent degradation of AU-rich mRNAs by inhibiting endonuclease digestion (21, 32). Chen et al. (33) reported that recombinant HuR and AUF1 showed a significant overlap in ARE binding and indicated that these two molecules not only cannot discriminate different classes of ARE but may also compete with each other. It is, therefore, plausible that HuR displaces AUF1 in the nucleus, leading to the stabilization of its target mRNA (33).

Many studies have indicated that embryonic lethal abnormal vision family proteins, including HuR, up-regulate gene expression by stabilizing ARE-containing mRNAs. The mRNA turnover rate could be affected by the abundance, or RNA-binding properties, of ARE-binding factors (34). A decrease of HuR was observed after treatment with sCT in osteoclasts. This decrease, however, might not necessarily relate to the accelerated decay of CTR mRNA. The rapid loss of CTR mRNA seemed much faster than the changes of HuR protein levels, although the amounts of RNA-binding proteins needed for these processes are not known (34). The rapid loss of CTR mRNA might involve phosphorylation of HuR and/or other RNA binding proteins. This possibility is supported by recent findings that ARE-binding proteins can be posttranslationally modified or phosphorylated in response to diverse stimuli (35, 36, 37). For example, reversible phosphorylation of AUF1 was shown to constitute a critical mechanism for regulating mRNA turnover rates (35, 36). It is also reported that activation of AMP-dependent protein kinase induced nuclear retention of HuR, which inhibited the ability of HuR to stabilize ARE-containing transcripts in the cytoplasm (37). Expression of HuR and binding with some target mRNAs were recently shown to be altered by protein kinase A activation (38), which is also consistent with CTR regulation in osteoclasts because we have shown that the homologous down-regulation of CTR by CT is modulated by activation of protein kinase A.

Several investigators have successfully shown that RNA EMSA, using anti-AUF1 antibody, can determine the binding specificity of certain mRNAs. However, we were unable to show by this technique the specific binding by AUF1 to CTR mRNA in cells of the osteoclast lineage or to obtain direct evidence that cytosolic factors were involved in the reduction of CTR mRNA, in circumstances in which we have shown previously that the effect of CT on CTR down-regulation was highly specific for the osteoclasts (2, 3). Osteoclasts are multinuclear giant cells with bone-resorbing activity and originate from monocyte-macrophage lineage of hematopoietic cells. They possess unique characteristics of dense actin band (ring) formation at peripheral bone resorbing surfaces and a ruffled border in the center and secrete protons and proteolytic enzymes (e.g. lysosomal cysteine proteinases and matrix metalloproteinases) beneath the cells. It has been reported that characteristics of this unique membrane domain differ from any other known plasma membrane, and Baron et al. (39) indicated that the ruffled border is a specialized form of lysosomal membranes. These distinctive features could provide an explanation why apparent interactions between cellular extracts of osteoclasts and the transcript of 3'UTR in CTR mRNA could not be studied by RNA EMSA. Sirenko et al. (34) previously reported that, although the involvement of AUF1 in mRNA destabilization in monocytes was strongly suspected, it was not possible to prove this mechanism in this cell type. It seems possible that certain enzymes (including endonucleases) or other factors in mature macrophage-like cells may provide difficulties in the exploration of protein-RNA interaction; similarly, mature osteoclasts may possess unique characteristics, unlike, for example, those of the epithelial origin.

Although our present technique was unable to identify the factors associated with CTR mRNA in osteoclasts, it is possible that the factors involved in these nuclear and/or cytoplasmic processes are found in other cell types. Because RAW 264.7 cells represent osteoclast precursors and can differentiate into osteoclasts by treatment with sRANKL, the strong signal indicating interaction with the 3'UTR of CTR mRNA suggests that multiple factors bind to CTR mRNA. It has been shown that the number of AUUUA possibly affects the binding of AUF1 or other ARE-binding factors. Because the 3'UTR of mouse CTR mRNA possesses four AUUUA, located at a distance from each other, the strong signal found for binding of AU5 and AUF1 p40 with 3'UTR (Fig. 2Go) is likely to be a function of the number of AUUUA motifs and the distance between them. The binding, however, could be also altered by the number of other uridine-rich sequences along with the three dimensional conformation of the molecules. It will be interesting to investigate the different binding characteristics between AU5 and the 3'UTR of the CTR mRNA on AUF1 in a future study.

There is ample evidence that the control of mRNA degradation and translatability is linked to alteration of the cytoskeleton (34, 40, 41). It was reported that as much as 90% of mRNA transcripts are bound to cytoskeletal components, including actin and microfilaments, which are the principal sites for mRNA anchoring. Untranslated mRNA could be transmitted as a posttranscriptional complex to neighboring intermediate filaments, or cytoskeletal components from the nucleus, to the correct localization in the cytoplasm for subsequent translation (40). An adherence-dependent inactivation of the AUF1 complexes was also reported by Sirenko et al. (41), in experiments in which electroinjection of glutathione-S-transferase-AUF1 substantially altered cytoskeletal organization and stability of transcript. A prominent effect of CT in osteoclasts is a rapid induction of cellular contraction together with immediate disruption of polarization and actin ring formation (42). This effect of CT is mediated by activation of PKA signaling in mouse osteoclasts. We have also shown previously that cAMP-mediated pathway was crucial for homologous down-regulation of CTR and decreased expression of CTR mRNA (43). Activation of MAPK was shown to be related to alterations in the availability of mRNA degradation factors (44), and CT was recently shown to induce ERK and p38 MAPK in kidney cells (45). Rapid cytoskeletal changes accompany cellular adherence to matrix components, highlighted by the appearance of lamellipodia and the absence of focal adhesions. These intracellular events can be linked to the ubiquitin proteasome degrading network, and it would be intriguing to investigate further whether AUF1 promotes ARE-mediated mRNA decay in a ubiquitin-proteasome-dependent manner (46). Integrin engagement during cellular adhesion initiates transcription of numerous immediate early-response genes (47), which suggests that changes in cytoskeletal organization are likely to play an important role in unique responsiveness to ligands in various types of cells. It is also possible that CT-induced changes of cytoskeletal organization, along with destabilization of CTR mRNA in osteoclasts, may be an event involving the whole cell, including peripheral actin, microtubules, and intermediate filaments. As such, it seems likely that mRNA tethered to the cytoskeleton is involved in CT action in osteoclasts.

In summary, we have shown in this study potential roles for AUF1 p40 and HuR in modulating CTR mRNA stability. Specific binding of these molecules to the 3'UTR in the CTR gene was verified, but the elucidation of the exact roles of AUF1 p40 and HuR requires further examination in cells of the osteoclast lineage. Changes in cytoskeletal organization are also likely to play an important role in the unique responsiveness of CT in osteoclasts.


   Acknowledgments
 
We acknowledge helpful discussions and suggestion with Drs. Masahito Matsumoto, Shinji Kitahama, and Makoto Iitaka (Saitama Medical School, Saitama, Japan). We also thank Ms. Atsumi Kikuchi (Jichi Medical School) and Ms. Keiko Nagatani (Saitama Medical School) for their experimental assistance and Professor David M. Findlay (Adelaide University, Adelaide, Australia) for valuable comments on the manuscript.


   Footnotes
 
This work was supported by grants from Japan Osteoporosis Foundation, Research Society for Metabolic Bone Diseases, Daiwa Securities Health Foundation, and Japan Research Foundation for Clinical Pharmacology. F.K. was granted by Core Research for Evolutional Science and Technology and Japan Science and Technology Corp.

Abbreviations: AR, Adrenergic receptor; ARE, AU-rich element; AU, adenylate/uridylate; AUF1, AU-rich RNA-binding factor 1; CT, calcitonin; CTR, CT receptor; DOX, doxycycline; DTT, dithiothreitol; FBS, fetal bovine serum; HA, hemagglutinin; HRP, horseradish peroxidase; HuR, Hu antigen R; Luc, luciferase; M-CSF, macrophage-colony stimulating factor; PMSF, phenylmethyl sulfonyl fluoride; RBD, RNA binding domain; RNP, ribonucleoprotein; RT, reverse transcription; sCT, salmon CT; sRANKL, soluble receptor activator for nuclear factor {kappa}B ligand; Tet, tetracycline; 7TMGCRseven-transmembrane G protein-coupled receptor; TRE, Tet-responsive element; UTP, uridine 5-triphosphate; UTR, untranslated region.

Received July 11, 2003.

Accepted for publication December 31, 2003.


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 Discussion
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