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Molecular and Cellular Biology, September 2005, p. 8139-8149, Vol. 25, No. 18
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.18.8139-8149.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

RNA-Binding Protein HuR Is Required for Stabilization of SLC11A1 mRNA and SLC11A1 Protein Expression

Yong Zhong Xu,1 Sergio Di Marco,1,2 Imed Gallouzi,2 Marek Rola-Pleszczynski,3 and Danuta Radzioch1*

McGill University Health Centre, McGill University, Department of Experimental Medicine and Department of Human Genetics, Montreal, QC, Canada,1 McGill University, Department of Biochemistry, Montreal, QC, Canada,2 University of Sherbrooke, Department of Pediatrics, Sherbrooke, QC, Canada3

Received 7 March 2005/ Returned for modification 15 April 2005/ Accepted 30 June 2005


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ABSTRACT
 
The solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1) gene is associated with infectious and autoimmune diseases and plays an important role in macrophage activation. Human SLC11A1 mRNA contains an AU-rich element (ARE) within the 3' untranslated region; however, its role in the regulation of SLC11A1 gene expression has not been elucidated. Here we analyze the expression of SLC11A1 in human monocytes and HL-60 cells and then use HL-60 cells as a model to determine whether RNA-binding protein HuR is associated with the ARE and involved in SLC11A1 mRNA turnover. Our results demonstrate a binding of HuR to the SLC11A1 ARE in phorbol myristate acetate (PMA)-differentiated cells dramatically increased compared to that in undifferentiated cells. Interestingly, PMA-induced accumulation of cytoplasmic HuR occurs in parallel with an increase in the binding of HuR to SLC11A1 ARE and with an increase in the SLC11A1 mRNA level. This suggests that HuR's cytoplasmic localization plays an important role in the regulation of SLC11A1 expression. We also observe that down-regulation of HuR expression by RNA interference (RNAi) results in a decrease in SLC11A1 expression which can be restored by the addition of recombinant HuR protein to the RNAi-treated cells. Finally, we show that HuR overexpression in HL-60 cells significantly increases the SLC11A1 mRNA stability. Taken together, our data demonstrate that HuR is a key mediator of posttranscriptional regulation and expression of the SLC11A1 gene.


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INTRODUCTION
 
In mouse, natural resistance to infection with unrelated intracellular parasites such as Salmonella enterica serovar Typhimurium, Leishmania donovani, and various Mycobacterium species, including Mycobacterium bovis and Mycobacterium lepraemurium, is controlled by the solute carrier family 11 member 1 (Slc11a1) gene, also known as Ity/Lsh/BCG or Nramp1 (6, 8, 44, 52, 56). In addition to restricting the growth of intracellular pathogens, the murine Slc11a1 gene product was also shown to have many pleiotropic effects on macrophage activation, including regulation of the chemokine KC as well as several cytokines, such as interleukin-1ß (IL-1ß) and tumor necrosis factor-{alpha} (TNF-{alpha}); induction of nitric oxide (NO) release; major histocompatibility complex class II molecule expression; and oxidative burst (7, 27). The human homologue (SLC11A1) gene has also been reported to be involved in susceptibility to infectious diseases, such as tuberculosis, leprosy, and human immunodeficiency virus infection, as well as to autoimmune diseases, such as rheumatoid arthritis, juvenile rheumatoid arthritis, sarcoidosis, and Crohn's diseases (7, 19). A large number of additional disorders with either known or suspected infectious etiologies have also been investigated for possible contributions of the SLC11A1 gene to disease risk. In most examples, further studies are needed before firm conclusions can be reached.

The murine Slc11a1 gene is expressed in monocytes/macrophages (28). Biochemical studies have shown that Slc11a1 is an integral membrane protein with a molecular mass of 90 to 110 kDa which is extensively glycosylated and phosphorylated in macrophages (4, 57). Interestingly, the expression of the Slc11a1 gene in murine macrophages cells is up-regulated by bacterial lipopolysaccharide, interferon {gamma}, granulocyte/macrophage colony-stimulating factor, and inflammatory stimuli (10, 28). In human, SLC11A1 is expressed not only in monocytes/macrophages but also in polymorphonuclear neutrophils (11, 45). SLC11A1 gene expression is undetectable in transformed human cell lines from either erythroid or lymphoid T or B lineages as well as in the progenitors of the monocyte/macrophage pathway (KG1, U937, THP) and the promyelocytic leukemia cell line HL-60. It can, however, be strongly induced in these cells when they are differentiated toward either the monocyte/macrophage or the granulocyte pathway (11). So far, little is known about how the SLC1A1 gene is regulated by the above-mentioned stimuli. In differentiated HL-60 cells, the induction of SLC11A1 protein expression correlates with a high level of SLC11A1 mRNA, suggesting that SLC11A1 expression may be controlled primarily at the level of transcription and/or mRNA stability (45).

A recent survey of the 3' untranslated regions (3'UTR) of the human mRNA database showed that the SLC11A1 message contains four AUUUA repeats, a typical AU-rich element (ARE). This sequence is homologous to the destabilizing sequence found in many short-lived mRNAs such as oncogene, cytokine, and growth hormone mRNAs (5, 9) and is involved in the regulation of the mRNA turnover of these genes. The role of this motif in the regulation of SLC11A1 gene expression has not yet been elucidated. In general, mRNAs that contain AREs are rapidly degraded in unstimulated cells, but specific growth or stress conditions can modify the fate of specific messages. For example, IL-1{alpha} has been shown to enhance the stability of a variety of cytokine and chemokine mRNAs that otherwise exhibit short half-lives, and this depends, at least in part, upon the presence of ARE motifs in the 3'UTRs (30, 35). Similarly, eotaxin mRNA is stabilized following treatment with TNF-{alpha} and IL-4 (2), and treatment with phorbol esters, calcium ionophores, or interleukins increases the stability of lymphokine mRNA in lymphoid cells (31, 51, 64). Furthermore, multiple studies have shown that a specific mRNA/protein complex is formed or increased in response to extracellular stimulation, leading to changes in the half-lives of the target mRNAs (14, 48, 58, 65). In the case of SLC11A1 mRNA, the possible implication of the ARE in its turnover comes from the fact that the murine counterpart does not contain any ARE and seems to be very stable with a long half-life (33). Indeed, this observation suggests that the presence of AUUUA repeats in the SLC11A1 3'UTR could indicate a posttranscriptional regulation affecting the stability and/or the mRNA export levels.

The AREs are known to bind RNA-binding proteins that positively or negatively regulate ARE-containing mRNA turnover (5, 9, 12, 38, 46, 47). A number of proteins that bind to 3'UTRs, in particular to AREs, have been identified (1, 25, 62). Among these proteins, AUF1 (also called heterogeneous nuclear ribonucleoprotein D [hnRNP D]) and HuR are well studied for their influence on mRNA turnover. AUF1 has been shown to promote mRNA degradation in most cases, while HuR stabilizes mRNA (9, 34). HuR is a member of the embryonic lethal abnormal vision family of mRNA-binding proteins. It is a 36-kDa protein, containing three RNA recognition motifs, which binds with high affinity to AREs and thus stabilizes specific ARE-containing mRNAs (43). Unlike the other three embryonic lethal abnormal vision proteins (Hel-N1, HuC, and HuD), which are developmentally regulated and expressed uniquely in the nervous system, HuR is ubiquitously expressed in all cell types (26). Although it is localized mainly in the nucleus, HuR can shuttle between the nucleus and cytoplasm by virtue of its shuttling signal, HNS (HuR nucleocytoplasmic shuttling domain), which is located in the hinge region between its second and third RNA recognition motifs. It is postulated that HuR binds to specific mRNAs in the nucleus, transporting them to the cytoplasm and protecting them from rapid degradation (3, 9). Studies have demonstrated that HuR's cytoplasmic localization increases under conditions of stress, such as heat shock (23), UV irradiation (59), amino acid starvation (65), chronic ethanol exposure (39), and T-cell receptor signaling (50). Furthermore, it was suggested that while HuR associates with its mRNA targets, thus stabilizing them (21), it serves as an adaptor for their rapid export to the cytoplasm (24). Thus, it is possible that HuR, through the (AUUUA)4 element, regulates the posttranscriptional events of the SLC11A1 gene, leading to its rapid expression upon extracellular stimulation.

To define the molecular mechanisms of the posttranscriptional regulation of SLC11A1 genes as well as the role of its AREs, we chose the human promyelocytic leukemia HL-60 cell line as a model. We observed that the increased expression of the SLC11A1 mRNA in phorbol myristate acetate (PMA)-differentiated HL-60 cells is in part due to its stabilization by the RNA-binding protein HuR. We present evidence supporting the implication of the above-described AREs in this process as mediators of HuR's association with SLC11A1 mRNA turnover.


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MATERIALS AND METHODS
 
Plasmid constructions. Vectors used for synthesis of RNA probes in electrophoretic mobility shift assays were constructed as follows. For the SLC11A1 3'UTR probe, human genomic DNA prepared from U937 cells was first amplified by PCR using the primers 5'-CCATCAACCTCTACTTCGTGGTCAGCTATC-3' and 5'-CTTGGGTTCCTTATGACCTGGTGCCTGA-3'. The PCR product was then amplified again using another set of primers: 5'-ATAAGAATGCGGCCGCCCACACCAGGGCCTGGCT –3' and 5'-GCTCTAGAGGTGGCTCACGCCTGTAATT-3'. The final PCR product (spanning nucleotides 1829 to 2540 of SLC11A1 3'UTR; NCBI GenBank accession number D50402) was gel purified and modified using the A-tailing procedure and then ligated into PGEM-T easy vector (Promega, Madison, WI). This construct was termed PGEM-T-3'UTR. For the 40-nucleotide ARE probe, the synthesized oligonucleotide (corresponding to positions 2248 to 2287 of SLC11A1 3'UTR) was cloned into PGEM-T easy vector and termed PGEM-T-ARE. The plasmids Gateway-AP-HuR-GST and Gateway-GST-AP, for expression of the recombinant proteins, and the pcDNA3.0-HuR-Flag plasmid, for overexpression of the recombinant human HuR in mammalian cells, were constructed as described previously (21, 55).

Protein expression and purification. The fusion proteins AP-HuR-GST and GST-HuR were expressed and purified as previously described (55). Briefly, Escherichia coli BL21(DE3) cells were transformed with the plasmid Gateway-AP-HuR-GST or Gateway-GST-AP, grown in LB medium (containing 50 µg/ml ampicillin) until the optical density at 600 nm was approximately equal to 0.6, and then induced with 0.3 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) for 4 h. After induction, cell pellets were washed with lysis buffer (50 mM Tris, pH 8.0, 200 mM NaCl, and 1 mM EDTA) and resuspended in lysis buffer containing 100 µg/ml lysosome for 15 min, followed by the addition of dithiothreitol, protease inhibitors (Roche, Laval, QC, Canada), and sarkosyl (1.5% final concentration) and then sonication. After sonication, the lysate was centrifuged and the supernatant was transferred to a new tube, a procedure followed by the addition of 10% Triton X-100 to a final concentration of 3%. The lysate was incubated with glutathione-Sepharose 4B (Amersham Biosciences, Baie d'Urfé, QC, Canada), and the recombinant proteins were eluted from the beads with glutathione elution buffer and dialyzed against phosphate-buffered saline (PBS) for the use described below.

Preparation of monocytes and cell culture. Human venous blood samples from healthy, medication-free volunteers were collected on citrate/dextrose/adenine. The peripheral blood mononuclear leukocytes were enriched by dextran sedimentation, layered over a Ficoll-Hypaque cushion, and centrifuged at 400 x g for 20 min. Mononuclear leukocytes were then collected at the interface, washed twice with PBS, resuspended in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 ng/ml streptomycin and allowed to adhere to plastic petri dishes coated with defibrinized autologous serum for 1 h at 37°C. Nonadherent cells were removed by rinsing with PBS, and adherent cells (>90% monocytes) were detached with EDTA (0.01 M) in RPMI 1640. Mononuclear cells were further purified with anti-CD14 monoclonal antibody-coated microbeads by using MACS CD14 isolation kits from Miltenyi Biotec (Auburn, CA). Both monocytes and HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin. For PMA treatment, the cells were seeded in six-well tissue culture plates at densities of 1 x 106 (monocytes) or 4 x 105 (HL-60) cells/ml and treated with 10 ng/ml PMA (Sigma, Oakville, ON, Canada).

Western blot analysis. After the appropriate treatment, total cell extracts as well as nuclear and cytoplasmic fractions were prepared as described previously (17, 20). Total cellular, cytoplasmic, and nuclear lysates were resolved on 4 to 12% Bis-Tris gel (Invitrogen, Burlington, ON, Canada) and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 5% milk in TBS-T (Tris-buffered saline-0.1% Tween 20) overnight, a procedure followed by incubation with primary antibody at room temperature for 1 h. A monoclonal antibody against HuR, 19F12, was used at a 1:3,000 dilution. A monoclonal antibody against SLC11A1 (Santa Cruz) was used at a 1:500 dilution. A monoclonal antibody against ß-actin (Sigma, Saint Louis, Missouri) was used at a 1:5,000 dilution. A polyclonal antibody against ß-tubulin (Santa Cruz) was used at a 1:1,000 dilution, and a polyclonal antibody against HDAC1 (Santa Cruz) was used at a 1: 1,000 dilution. After washing, membranes were further incubated with appropriate secondary antibodies coupled to horseradish peroxidase and developed using enhanced chemiluminescence detection reagents.

Northern blot analysis. Total cellular RNA was extracted by using TRIzol reagent (Invitrogen, Burlington, ON, Canada). Equal amounts (30 µg/lane) of RNA were denatured in formamide/formaldehyde (65°C, 10 min), separated on 1.2% agarose/formaldehyde gels, and then transferred to Hybond-N+ membranes (Amersham Biosciences, Baie d'Urfé, QC, Canada) and hybridized with a specific cDNA probe. To analyze SLC11A1 RNA, a fragment of SLC11A1 cDNA spanning positions 444 to 815 of the open reading frame (GenBank accession number D50402) was used. The PCR-amplified and gel-purified SLC11A1 and G3PDH cDNAs were labeled with [{alpha}-32P]dCTP by using the nick translation system (Invitrogen, Burlington, ON, Canada). The probe for the 18S rRNA was obtained by in vitro transcription of the pTRI RNA 18S antisense control template (Ambion, Austin, TX). After hybridization, membranes were washed and then exposed for 2 to 4 days to Bioflex MRI films (Clonex, Markham, ON, Canada).

Immunofluorescence. HL-60 cells were seeded on four-well culture slides (Becton Dickinson Labware, Franklin Lakes, NJ) and treated with PMA. Untreated HL-60 cells were collected by centrifugation at 500 x g for 5 min and placed on culture slides. Both treated and untreated cells were fixed for 15 min in PBS containing 3.7% paraformaldehyde and 0.18% Triton-X. After soaking in blocking buffer (PBS containing 1% goat serum) for 30 min, slides were then incubated with the primary antibodies (mouse anti-HuR [1:500] and rabbit anti-G3BP [Ras-GTPase-activating protein SH3 domain-binding protein] [1:1,000]) in blocking buffer for 1 h at room temperature. Following washes with blocking buffer, samples were incubated with a mixture of fluorescein isothiocyanate- and tetramethyl rhodamine isothiocyanate-labeled secondary antibodies, both at 1:500 dilutions in blocking buffer for 1 h. Finally, cells were stained with 1:20,000 DAPI (4'6'-diamidino-2-phenylindole) (1 mg/ml) for 5 min. After washing with blocking buffer, slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and visualized with a Zeiss Axiovision 3.1 microscope using a 63x oil objective, and an Axiocam HR (Zeiss) digital camera was used for photography.

RNA-binding assay and supershift assay. Plasmid PGEM-T-3'UTR was linearized with the restriction endonuclease XbaI (New England Biolabs, Beverly, MA) and PGEM-T-ARE with SpeI (New England Biolabs, Beverly, MA) to generate the DNA template for in vitro transcription. RNA probes were prepared by using the SP6/T7 transcription kit according to the manufacturer's instruction (Roche, Laval, QC, Canada). Briefly, a 20-µl transcription reaction mixture containing 1 µg of template, 2.5 mM ATP, GTP, and CTP, 200 µM UTP, 5 µl [{alpha}-32P]UTP, and 10 units of T7 RNA polymerase was incubated for 30 min at 37°C. Template DNA was removed by DNase I treatment, and the radiolabeled probe was purified through spin columns. The RNA-binding assay and the HuR supershift assay were performed as described previously (17).

Transfection of HL-60 cells. The electrotransfection of HL-60 cells has been described previously (36). Twenty µg of plasmid pcDNA3.0-HuR-Flag or pcDNA3.0-Flag was added to a 0.4-cm electroporation cuvette containing 0.5 ml of HL-60 cells (1 x 107 cells/ml) in RPMI 1640 medium supplemented with 20% fetal calf serum. Electroporation was performed at a setting of 240-V pulse and 960-µF capacitance by using a Bio-Rad gene pulser (Bio-Rad Laboratories, Hercules, CA). Cells were cultured in RPMI 1640 medium containing 20% FBS and allowed to recover for 3 days, a process followed by G418 (Geneticin, Invitrogen, Burlington, ON, Canada) selection for 10 days at a final concentration of 1.5 mg/ml. Clonal sublines were selected by plating on semisolid methyl cellulose Iscove's medium (Stem Cell Technologies, Paisley, Scotland) containing 1.5 mg/ml G418. The stably transfected colonies were grown in RPMI 1640 medium supplemented with 1 mg/ml G418 and screened for expression of the HuR-Flag recombinant protein.

Immunoprecipitation of mRNP complex and RT-PCR. To investigate the interaction of the SLC11A1 mRNA and HuR protein, a combination of immunoprecipitation and reverse transcription (RT)-PCR analysis was used as previously described (54). HL-60 cells treated with PMA for different periods of time were lysed in 1 ml polysome lysis buffer (10 mM KCl, 5 mM MgCl2, 10 mM HEPES [pH 7.0], 0.5% NP-40, 1 mM dithiothreitol, 100 units/ml RNase inhibitor, 0.2% vanadyl ribonucleoside complex, and protease inhibitors). Immunoglobulin G1 (IgG1) (Sigma, St. Louis, Missouri), anti-HuR antibody (3A2)- and anti-Flag antibody (Sigma, St. Louis, Missouri)-coated protein A beads were used to immunoprecipitate and separate mRNA-HuR complexes. RNA was extracted using TRIzol reagent. RT was performed using a SuperScript first-strand synthesis system for RT-PCR (Invitrogen, Burlington, ON, Canada), and the produced template was amplified by PCR using the sense primer 5'-TCAAACTTCTCTGGGTGCTGCTCT-3' and the antisense primer 5'-ACTCATAGCCAAAGGTCAAGGCCA-3'. PCR products (372 bp) were resolved on ethidium bromide-stained 1.5% agarose gel.

Protein labeling. AP-HuR-GST was conjugated with Alexa Fluor 594 using the Alexa Fluor 594 monoclonal antibody labeling kit (Molecular Probes, Eugene, OR) following the manufacturer's instructions. Briefly, 1 mg/ml AP-HuR-GST protein solution was prepared by adding 0.1 M sodium bicarbonate buffer to the lyophilized protein powder. One hundred µl of the protein solution was transferred to the vial of reactive dye, gently mixed, and incubated at room temperature for 1 h. The labeled protein was then purified through a spin column.

RNA interference experiment and protein transfection. The transfection of small interfering RNA (siRNA) into HL-60 cells was performed in six-well plates by using the Lipofectamine 2000 and Lipofectamine Plus reagents (Invitrogen, Burlington, ON, Canada) according to the manufacturer's instructions. The day before transfection, cells were split as necessary to achieve log growth by transfection time. On the day of transfection, the medium was removed and the cells were resuspended in fresh growth medium without antibiotics to a final density of 1 x 106 cells/ml. The siRNA duplexes used in the experiment are as follows. HuSi-1 (Dharmacon, Lafayette, CO) (5'-AAGCCUGUUCAGCAGCAUUGG-3') targets to position 111 to 131 of nucleotides relative to the start codon of HuR mRNA, and HuSi-C (Dharmacon, Lafayette, CO) (5'-AAGCC AAUUCAUCAGCAAUGG-3') is a mutant version of HuSi-1 (underlined nucleotides), which was used as a negative control. siRNA duplex (0.12 µM) was added to each well of cells seeded on six-well plates. Four h after transfection, PMA was added to the cells at a final concentration of 10 ng/ml. Twenty-four h later, the medium was changed with fresh growth medium containing PMA. Seventy-two h posttransfection, total cellular extracts were prepared. For protein transfection, recombinant AP-conjugated proteins were added to the HL-60 cells 4 h after siRNA treatment at concentrations of 30 or 50 nM, and then the HL-60 cells were induced to differentiation with PMA 6 h later. Twenty-four h after siRNA treatment, fresh medium containing PMA was added, and the transfected cells were kept in culture for another 48 h.


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RESULTS
 
Effect of PMA and M-CSF treatment on the expression of SLC11A1 in human monocytes and HL-60 cells. To test the effect of PMA on the expression of SLC11A1, human monocytes were treated with the compound for 72 h and examined for their SLC11A1 protein levels. As shown in Fig. 1A and B, freshly isolated monocytes (Med, 0 h) did not express any detectable SLC11A1 protein. Even after 72 h of culturing (Med, 72 h) of monocytes, the SLC11A1 protein was barely detectable. However, when the monocytes were differentiated into macrophages by using PMA, the expression of SLC11A1 protein was dramatically increased. The increased expression of SLC11A1 protein was also observed when monocytes were differentiated by using macrophage colony-stimulating factor (M-CSF). Western blot analysis showed a band of 90- to 110-kDa electrophoretic mobility. As illustrated in Fig. 1C, we have also tested SLC11A1 protein expression in the HL-60 cell line with and without PMA treatment to assess whether this cell line can faithfully reproduce the modulation in SLC11A1 protein expression during the course of differentiation from promyelocytic cells to macrophages. As expected, differentiated HL-60 cells became adherent to plastic and spread in a manner characteristic of macrophages and they also lost their proliferation potential, as previously described by Harris and Ralph (29). Similarly, as we observed in monocytes, SLC11A1 protein expression was induced by the PMA treatment and was undetectable in protein extracts from untreated HL-60 cells. The SLC11A1 protein detected in the extracts from PMA-treated HL-60 cells showed an electrophoretic mobility the same as that observed in PMA-treated monocytes (Fig. 1A to C).



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  		FIG. 1. PMA and M-CSF induce the expression of SLC11A1 in human monocytes and HL-60 cells. Human monocytes and HL-60 cells were either untreated (Med) or treated with PMA (10 ng/ml) or M-CSF (30 ng/ml). Total protein extracts were prepared by lysis of cells in radioimmunoprecipitation buffer (0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% NP-40 in 1x PBS). Total RNA was isolated by using TRIzol reagent. (A) SLC11A1 protein expression in human monocytes was assessed using Western blot analysis (20 µg of total protein extract/lane). The protein extracts were prepared from fresh monocytes (0 h; lanes 1 and 4), from monocytes cultured for 72 h (lanes 2 and 5), and from monocytes treated with PMA for 72 h (lanes 3 and 6). The protein extracts isolated from untreated and PMA-treated monocytes purified from the blood of volunteer 1 are shown in lanes 1 to 3, and those from volunteer 2 are shown in lanes 4 to 6. (B) Monocytes isolated from volunteer 3 were untreated or else treated with PMA or M-CSF for 72 h. The effect of PMA and M-CSF on SLC11A1 protein expression was assessed (20 µg of total protein/lane). (C) Twenty µg of each protein sample was subjected to Western blot analysis to assess SLC11A1 protein expression in HL-60 cells. (D) Thirty µg of total RNA from HL-60 cells was separated by electrophoresis in denaturing formaldehyde gel and transferred to Hybond-N+ membrane. Blots were probed with cDNA probes corresponding to SLC11A1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Days of PMA treatment [PMA (d)] are indicated at the tops of panels C and D. (E) Schematic representations of SLC11A1 cDNA and probes used to study mRNA stability. 3'UTR and ARE probes spanning nucleotides 1829 to 2540 and 2248 to 2287 of human SLC11A1 mRNA, respectively, are shown.

To determine whether the induction of SLC11A1 protein expression reflects an increase in the mRNA level, total RNA from HL-60 cells treated and untreated with PMA was detected by Northern blotting. As shown in Fig. 1D, SLC11A1 mRNA levels were increased upon treatment with PMA. Overall, these results indicated that PMA-induced differentiation of promyelocytic cells towards macrophage lineage leads to an induction of SLC11A1 mRNA and subsequently to the induction of SLC11A1 protein expression.

PMA treatment, which induces SLC11A1 expression in HL-60 cells, increases HuR's level in the cytoplasm. The above results, as well as the fact that the SLC11A1 message presents a typical ARE in its 3'UTR (Fig. 1E), suggest that the PMA-induced expression of SLC11A1 mRNA in HL-60 cells could be due, in part, to the increase in its stability. Furthermore, a repeat of AUUUA sequence was shown to mediate the association of ARE-containing messages with the stabilizing factor HuR (9). Based on this information, we performed experiments to determine whether HuR, through the (AUUUA)4 elements, regulates the posttranscriptional events of the SLC11A1 gene, leading to its rapid expression during the differentiation of HL-60 cells. Since HuR's cytoplasmic localization is associated with the nuclear export and stabilizing of its mRNA targets (9), it was of particular interest to first investigate the subcellular localization of HuR under conditions in which SLC11A1 is induced. Western blot analysis using whole-cell lysates revealed no obvious increase in total HuR expression during PMA treatment of HL-60 cells (Fig. 2A), so we hypothesized that PMA could affect the cellular localization of HuR. Indeed, when subcellular fractions from untreated HL-60 cells were analyzed, no HuR protein was detected in the cytoplasm. Following treatment of HL-60 cells with PMA for 48 h, HuR could readily be detected in the cytoplasm. (Fig. 2B). Western blotting of the same membranes to detect nucleus- and cytoplasm-specific markers (histone deacetylase 1 and ß-tubulin, respectively) verified that the nuclear protein did not leak into the cytoplasmic fractions during cell treatment or fractionation and further monitored the equal loading and transfer of the samples (Fig. 2B). These results demonstrated that the observed cytoplasmic accumulation of HuR in response to PMA treatment was not due to an overall increase of HuR expression but likely resulted from an elevated export of nuclear HuR, as previously reported (22, 32, 39). These observations were further confirmed by immunofluorescence experiments. In untreated cells, we observed that HuR is exclusively nuclear (Fig. 2C, control panel); however in PMA-treated HL-60 cells, HuR was also detectable in the cytoplasm (Fig. 2C, PMA-treated panel). These data confirmed the biochemical fractionation data (Fig. 2B) and strongly suggest the possibility that HuR escorts the SLC11A1 mRNA from the nucleus to the cytoplasm, protecting it from the decay machinery.



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  		FIG. 2. Effect of PMA treatment on the subcellular localization of HuR. (A) HuR protein levels in total lysate (20 µg) prepared from untreated HL-60 cells (–) and HL-60 cells treated with PMA (10 ng/ml) (+) for 1, 2, and 4 days [PMA(d)] were monitored with Western blot analysis. (B) HuR protein levels in cytoplasm fractions (40 µg) and nuclear fractions (10 µg) prepared from both untreated HL-60 cells and HL-60 cells treated with PMA (10 ng/ml) were monitored using Western blot analysis. Expressions of the cytoplasmic marker ß-tubulin and the nuclear marker HDAC1 were also monitored. (C) Immunofluorescence detection of HuR in HL-60 cells either untreated (control) or treated with PMA for 4 days. Left, HuR immunofluorescence; middle, G3BP immunofluorescence used to indicate cytoplasmic localization; right, DAPI staining used to localize nuclei. Pictures are representative of three separate experiments. Magnification, x63.

HuR specifically binds to the SLC11A1 3'UTR-ARE following treatment with PMA. The stabilization effect of HuR on its mRNA targets was always associated with direct binding through specific cis-acting elements located in the 3'UTR. We already found a potential ARE in the 3'UTR of SLC11A1, which prompt us to verify the existence of SLC11A1 mRNA-HuR complex in HL-60 cells before and after PMA treatment. Indeed, evidence for the in vivo association of endogenous SLC11A1 mRNA and HuR in the cells was obtained through immunoprecipitation of endogenous or Flag-tagged HuR under conditions that preserved its association with target mRNAs by using a previously described method (54). For the analysis of Flag-tagged HuR binding to SLC11A1 mRNA in vivo, plasmid pcDNA3.0-HuR-Flag was transfected into HL-60 cells, and stable transfectants of HL-60 cells that constitutively overexpress HuR were selected. As a control, HL-60 cells stably transfected with the empty expression plasmid pcDNA3.0-Flag were also generated. The relative amounts of endogenous HuR and Flag-tagged HuR were analyzed by Western blotting using anti-HuR and anti-Flag antibody, respectively (Fig. 3A). The level of HuR in the transfected cells increased by ~3.5-fold over that in nontransfected cells (Fig. 3A). The blot was stripped and probed with an anti-Flag monoclonal antibody, yielding a band at 37 kDa only in extracts from the cells transfected with pcDNA3.0-HuR-Flag (Fig. 3A). Total extracts prepared from untreated and PMA-treated HL-60 cells and cells stably transfected with the plasmid pcDNA3.0-HuR-Flag to overexpress HuR (Fig. 3A) were incubated with IgG1, anti-HuR antibody, and anti-Flag antibody, respectively. The precipitated RNAs were isolated and analyzed using primers specific for SLC11A1 mRNAs (Fig. 3B). RT-PCR analysis revealed the absence of detectable SLC11A1 mRNA in complexes immunoprecipitated with all three antibodies when the extracts of untreated HL-60 cells were used (Fig. 3B). However, after PMA treatment, endogenous SLC11A1 mRNA was present when cells were immunoprecipitated using anti-HuR or anti-Flag antibody but not when a nonspecific isotype-matched antibody (IgG1) was used (Fig. 3B).



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  		FIG. 3. HuR specifically binds to the SLC11A1 3'UTR ARE, and this binding increases after PMA treatment. (A) Western blot analysis of endogenous and Flag-tagged HuR expression. Total-cell extracts from wild-type HL-60 cells (lane 1), cells stably transfected with the empty expression plasmid pcDNA3.0-Flag (lane 2), and HuR expression plasmid pcDNA3.0-HuR-Flag (lane 3) were prepared and resolved using 4 to 12% Bis-Tris gel and immunoblotted using anti-HuR (top) or anti-Flag (middle) antibody. (B) Total-cell extracts were prepared from wild-type HL-60 cells (lanes 1 to 5) and stably transfected HL-60 cells (lanes 6 to 8) either untreated or treated with PMA (10 ng/ml) for 2 and 4 days [PMA(d)]. Protein-RNA complexes were immunoprecipitated using anti-HuR antibody, anti-Flag antibody, or IgG1. SLC11A1 mRNAs present in the complexes were isolated and detected by using RT-PCR. PCR products were analyzed using 1.5% agarose gels and stained with ethidium bromide. (C) HL-60 cells were treated or not with 10 ng/ml PMA for 2 and 4 days. Cytoplasmic (C) and nuclear (N) extracts were prepared and incubated with {alpha}-32P-labeled RNA probes containing 712-bp SLC11A1 3'UTR. Reaction mixtures were then resolved by electrophoresis on native polyacrylamide gel. (D) Cytoplasmic and nuclear extracts from HL-60 cells treated or untreated with 10 ng/ml PMA were incubated with {alpha}-32P-labeled RNA probes containing 40-bp ARE in the SLC11A1 3'UTR. Reaction mixtures were then resolved by electrophoresis on a native polyacrylamide gel. (E) Cytoplasmic extracts [Extract(C)] from HL-60 cells treated or untreated with 10 ng/ml PMA were incubated with {alpha}-32P-labeled RNA probes for 40-bp SLC11A1 ARE. Complexes containing HuR were shifted using the monoclonal anti-HuR antibody 19F2 and anti-p38 kinase antibody. Ab, antibody.

The SLC11A1 gene was induced to express at both the mRNA and protein levels in the HL-60 cells treated with PMA. Under the same stimulations, we also observed that primarily nuclear HuR accumulates in the cytoplasm and HuR binds to endogenous SLC11A1 mRNA. This data implies that the increase of SLC11A1 mRNA is causally related to an increase in cytoplasmic HuR levels. To confirm this, RNA-binding assays were first performed on nuclear and cytoplasmic extracts from untreated and PMA-treated HL-60 cells by using the following two {alpha}-32P-labeled RNA probes: the full-length SLC11A1 3'UTR probe (720 nucleotides in length, spanning nucleotides 1829 to 2540) and the SLC11A1-ARE probe, which contains an AU-rich element (40 nucleotides in length, comprising positions 2248 to 2287) (Fig. 1E). As shown in Fig. 3C and D, multiple protein-RNA complexes were formed when the nuclear or cytoplasmic extracts were incubated with either of the two labeled probes. The full-length SLC11A1-3'UTR and the SLC11A1-ARE probes display almost identical patterns of complex formation with one of the complexes (complex 1), which is dramatically increased following PMA treatment. To determine whether HuR is involved in complex 1, gel supershift assays were also performed using an antibody specific to HuR to supershift the protein-RNA complexes formed between the {alpha}-32P-labeled ARE probe and the proteins present in the cytoplasmic extracts. As shown in Fig. 3E, the inclusion of antibodies specific to HuR resulted in a depletion of complex 1 and a strongly supershifted band, demonstrating that HuR is one of the components of this complex and that the PMA-induced redistribution of HuR from nuclei to cytoplasm is associated with the increased SLC11A1 mRNA-binding activity. No supershifting was observed when the antibody specific to p38 kinase (Fig. 3E) or the anti-FXR antibody was used in the experiment (data not shown). Similar results were generated using nuclear extracts (data not shown).

HuR knockdown inhibits SLC11A1 expression, which can be recovered by recombinant HuR transfection. To test whether HuR was essential for the induction of SLC11A1 gene expression, we knocked down HuR by using a small RNA interference experiment. As shown in Fig. 4A, the transfection of specific HuR siRNAs into HL-60 cells led to a substantial down-regulation of HuR protein levels (almost 32% ± 4% of the amount of control), with no effect on actin expression. Transfection using a control HuR siRNA had no effect on either HuR or actin levels (Fig. 4A). Interestingly, SLC11A1 gene expression was significantly inhibited in HuR knockdown cells in terms of both protein and mRNA levels (Fig. 4A and B, respectively). Statistical analysis of the results using the Mann-Whitney rank sum test gave a P value of 0.008. These results documented a causal relationship between the level of HuR protein and that of SLC11A1 gene expression.



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  		FIG. 4. SLC11A1 expression is inhibited and can be restored in HuR knockdown cells. (A) HL-60 cells were transiently transfected with siRNA targets HuR (HuSi-1), control siRNA (HuSi-C, a mutant of HuSi-1), or mock (transfection reagents only). Four h after transfection, HL-60 cells were treated with PMA (10 ng/ml) for 68 h. Protein extracts of mock-transfected HL-60 cells, as well as those of HuSi-C- and HuSi-1-transfected HL-60 cells, were prepared and analyzed by Western blotting with antibodies specific to SLC11A1, HuR, and ß-actin. (B) HL-60 cells were transfected with siRNA and then treated with PMA as described above. Forty-eight and 72 h after PMA treatment, total RNA from HL-60 cells transfected with HuSi-1, HuSi-C, or mock were isolated, and Northern blot analysis was performed to determine SLC11A1 mRNA levels. (C) Schematic representations of fusion proteins AP-HuR-GST and GST-HuR (a control) used to rescue HuR siRNA-treated HL-60 cells. (D) The Alexa Fluor 594-labeled AP-HuR-GST was added to HL-60 cells 4 h after transfection with HuSi-1. Six h after the addition of labeled AP-HuR-GST, the cells were washed, fixed (4% paraformaldehyde; 15 min), and visualized with a Zeiss Axiovision 3.1 microscope. (E) HL-60 cells were transfected with HuSi-1. The recombinant protein AP-HuR-GST was added to cells 4 h posttransfection, and total protein extracts were prepared 6 h after the addition of AP-HuR-GST recombinant protein. Western blot analysis was performed to detect the absorbance of AP-HuR-GST by HL-60 cells. (F) HL-60 cells were transfected with HuSi-1 or HuSi-C, and 4 h after transfection, recombinant proteins GST-AP and AP-HuR-GST were added to the cells. Six h later, cells were treated with PMA (10 ng/ml). Seventy-two h after HuR siRNA treatment, total protein extracts were prepared. Western blot analysis was performed using antibodies specific to SLC11A1 and ß-actin.

To further confirm that the observed down-regulation of SLC11A1 gene expression was due specifically to the HuR knockdown, AP-HuR-GST and GST-AP (negative control) recombinant proteins were prepared (Fig. 4C) and transfected into HuR siRNA-treated HL-60 cells. These AP-conjugated proteins were added to the HL-60 cells 4 h after siRNA treatment. As shown in Fig. 4D and E, the AP-HuR-GST protein was effectively taken up by the cells. Six h after the addition of recombinant proteins, the HL-60 cells were treated with PMA and allowed to differentiate. After 72 h of the HuR siRNA treatment, we observed that addition of AP-HuR-GST, but not of GST-AP, was required to induce a normal level of SLC11A1 expression in HuR siRNA-treated cells (Fig. 4E). The expression of ß-actin protein was not affected by the addition of either of the recombinant proteins (Fig. 4E).

HuR overexpression stabilizes PMA-induced SLC11A1 mRNA. The studies described above demonstrate that HuR is critical for PMA-induced SLC11A1 expression; however, they do not explain the molecular mechanism responsible for the HuR-mediated augmentation of SLC11A1 gene expression. In order to determine the role of HuR in SLC11A1 gene expression, we performed actinomycin D chase experiments using HL-60 cells transfected with the plasmid pcDNA3-HuR-Flag or a control plasmid, pcDNA3.0-Flag (Fig. 5A). We determined that after PMA treatment for 48 h, the half-lives of SLC11A1 mRNA were similar in both pcDNA3.0-Flag-transfected cells (mean, 4.7 ± 0.3 h) and wild-type cells (mean, 4.1 ± 0.4 h). However, the half-life of SLC11A1 mRNA in pcDNA3-HuR-Flag-transfected cells was much longer (mean, 8.8 ± 0.4 h) than those observed in cells transfected with control vector or in wild-type cells (Fig. 5A and B). When using the t test, there is a significant difference in the mRNA half-lives for wild-type and HuR-overexpressing cell lines (P < 0.001). These results clearly demonstrated that HuR stabilizes mRNA, and therefore it plays an important role in the regulation of SLC11A1 gene expression.



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  		FIG. 5. Overexpression of HuR stabilizes PMA-induced SLC11A1 mRNA. (A) HL-60 cells stably transfected with either plasmid pcDNA3.0-HuR-Flag or pcDAN3.0-Flag, as well as wild-type HL-60 cells, were treated with PMA for 3 days, a procedure followed by the addition of actinomycin D (Act D) (2 µg/ml), and RNA was isolated 0, 1, 2, 4, 6, and 8 h later. Northern blotting was performed by using SLC11A1 and 18S rRNA cDNA probes. (B) SLC11A1 mRNA decay curves derived from a densitometry analysis of the Northern blot hybridization. The half-life of SLC11A1 mRNA in pcDNA3-HuR-Flag-transfected cells was much longer (mean, 8.8 ± 0.4 h) than those observed in pcDNA3.0-Flag-transfected cells (mean, 4.7 ± 0.3 h) and in wild-type cells (mean, 4.1 ± 0.4 h). There was a significant difference between the half-life of SLC11A1 mRNA in the wild type and that in HuR-overexpressing cell lines (t test; P < 0.001).


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DISCUSSION
 
The SLC11A1 gene plays an important role in macrophage activation and is linked to susceptibility to some infectious and autoimmune diseases (7, 19). Human SLC11A1 protein is expressed in monocytes, macrophages, and polymorphonuclear neutrophils. Here we found that following the treatment of human monocytes with PMA for 72 h, the expression of SLC11A1 protein was dramatically increased in monocytes derived from tested healthy medication-free volunteers. SLC11A1 protein was detectable, albeit at a very low level, in the monocytes cultured for 72 h in the absence of PMA from one of the two volunteers but was undetectable in monocytes isolated from the other volunteer. This difference in the basal SLC11A1 protein expression levels of the two tested volunteers might reflect individual variability. HL-60 cells were shown to be a useful model in the study of the regulation of SLC11A1 gene expression during experimentally induced granulocytic and monocytic differentiation. Previous work (11, 45) and our present study have shown that both SLC11A1 mRNA and protein are undetectable in untreated HL-60 cells. However, when the cells are induced with PMA to become macrophage-like, both steady-state mRNA and protein for SLC11A1 are expressed (Fig. 1B and C). So far, the molecular mechanisms involved in regulating SLC11A1 gene expression are not well characterized. The present study provides evidence that during PMA-induced differentiation of HL-60 cells towards the monocytic/macrophage pathway, the RNA-binding protein HuR shuttles from nucleus to cytoplasm, binds to the ARE within the 3'UTR of human SLC11A1 mRNA, and plays an important role in regulating its stability. To our knowledge, this is the first report demonstrating that HuR is essential for the regulation of SLC11A1 mRNA stability and the induction of SLC11A1 gene expression in human macrophages.

So far, little is known about the mechanism by which HuR shuttles between the nucleus and cytoplasm after induction by stimuli such as heat shock (23), UV irradiation (59), and amino acid starvation (65). Although protein kinase C, as well as several protein kinases involved in the mitogen-activated protein kinase pathway, have been implicated in regulating mRNA turnover (13, 41, 42, 60, 61, 63), a previous study showed that none of them affects the subcellular localization of HuR, and the cytoplasmic HuR level is regulated only by the activity of AMP-activated kinase, an enzyme involved in response to metabolic stresses (58). In this study, we report that the levels of cytoplasmic HuR are potently influenced by the treatment of cells with PMA. Although HL-60 cells express substantial levels of the HuR protein, we did not detect any HuR in the cytoplasm of untreated cells. Differentiation of HL-60 cells for at least 2 days with PMA resulted in the appearance of HuR in the cytoplasm, and its level in the cytoplasm increased over time. A number of cell signaling pathways can be activated by PMA treatment, but no report has shown whether PMA treatment has an effect on AMP-activated kinase activity. Therefore, the mechanisms whereby PMA regulates the translocation of HuR from nuclei to cytoplasm remain to be elucidated.

Here we found, both in vitro and in vivo, that HuR shuttles from the nucleus to the cytoplasm and binds to the 3'ARE-SLC11A1 mRNA during PMA treatment. There exists an association between the level of cytoplasmic HuR and the ability of cytoplasmic extracts to form the HuR-containing RNA-protein complex 1. Therefore, the increased cytoplasmic HuR during PMA treatment most likely contributes to the increased binding between HuR and SLC11A1 mRNA. We propose that PMA-induced SLC11A1 expression may be partly due to increased SLC11A1 stability mediated by the binding of HuR to the ARE. However, increased SLC11A1-mRNA stability may not be exclusively dependent on cytoplasmic HuR levels. The basal HuR abundance in the nuclear extracts from untreated cells is almost the same as that in PMA-treated cells, but the level of complex 1 formed between the 3'UTR/ARE and nuclear extracts from untreated cells is much lower than that from PMA-treated cells. It is possible that PMA treatment could cause covalent modification of HuR (methylation or phosphorylation of HuR) or changes in the levels of other proteins that bind to the SLC11A1 mRNA along with HuR and affect the formation of HuR-containing complex 1. Previously published studies have shown that lipopolysaccharide stimulation increased the methylation of HuR by coactivator-associated arginine methyltransferase as well as the formation of HuR-containing complexes on tumor necrosis factor {alpha} mRNA in macrophages (37, 39), but it is unclear whether there is a correlation between the methylation of HuR and its ability to stabilize labile mRNA.

It is well established that HuR binds to ARE-containing mRNAs and stabilizes them (9), and here we have revealed an interaction between HuR and SLC11A1 mRNA; hence, we hypothesized that the knockdown of HuR in HL-60 cells could result in the inhibition of PMA-induced SLC11A1 gene expression. Our data confirmed that the disruption of endogenous HuR levels by using small RNA interference led to the concomitant decrease of PMA-induced SLC11A1 expression in terms of both protein and mRNA levels. The causal relationship between the HuR protein and SLC11A1 gene expression was further supported by the restoration of SLC11A1 gene expression by using recombinant HuR protein in HuR siRNA-treated HL-60 cells. Overall, our data imply that an appropriate level of HuR expression is required for the stabilization of SLC11A1 mRNA and the induction of SLC11A1 expression.

To further explore the biological consequences of HuR binding to the SLC11A1-ARE, we analyzed the effect of HuR overexpression on SLC11A1 mRNA stability. Actinomycin D chase experiments performed on stably transfected HL-60 cells overexpressing HuR provide us with interesting observations. In untreated HuR-overexpressing HL-60 cells, no SLC11A1 mRNA could be detected by Northern blot analysis (data not shown), as observed similarly in untreated wild-type HL-60 cells. Therefore, HuR on its own is unable to activate the human SLC11A1 promoter unless HL-60 cells are differentiated with PMA. HuR overexpression did increase SLC11A1 mRNA stability, suggesting that HuR participates in the PMA-induced SLC11A1 expression and that this mechanism takes place at the posttranscriptional level. Several studies have shown that overexpression of HuR is associated with increased cyclooxygenase 2 expression in tumor tissues such as those from colon cancers, ovarian carcinomas, and breast carcinomas (15, 16, 18). Therefore, the overexpression of HuR may be part of a regulatory pathway to control the mRNA stability of several important target genes.

Since HuR is involved in the stabilization of a number of short-lived mRNAs containing ARE, PMA-induced changes in HuR may also contribute to the increased expression of other genes during the differentiation of monocytes. Immunoprecipitation of mRNP complex with HuR antibody combined with RT-PCR analysis revealed that HuR binds to the TNF-{alpha} mRNA but not to phospholipid scramblase 1 (RLSCR1) mRNA during the differentiation of HL-60 cells (data not shown). Both TNF-{alpha} and RLSCR1 gene expression were shown to increase during monocyte differentiation (66). Since TNF-{alpha} mRNA contains ARE, whereas RLSCR1 mRNA is devoid of ARE, these findings are suggestive of the importance of ARE as a target sequence for HuR protein binding.

It seems clear that SLC11A1 polymorphisms are associated with susceptibility to infectious and autoimmune diseases in human (7, 40, 53). In the promoter region of SLC11A1, seven different alleles (alleles 1 to 7) of the polymorphism containing a functional Z-DNA-forming repeat have been reported for different populations. Interestingly, a study showed that the polymorphic repeat acts as a functional polymorphism, influencing SLC11A1 gene expression. The high-promoter-activity-level sequence, allele 3, shows an association with susceptibility to autoimmune diseases, whereas the low-promoter-activity-level sequence, allele 2, is associated with susceptibility to infectious diseases (49). Therefore, host susceptibility to a disease may be linked to differential protein expression resulting from a genetic variant. In the present study, our results demonstrate that the ARE present in the 3'UTR of SLC11A1 mRNA has an important biological function in posttranscriptional regulation and indicate that HuR, which binds to the ARE, is essential for PMA-induced SLC11A1 expression. Therefore, further studies on the mechanism of HuR regulation will contribute to a better understanding of the regulation of SLC11A1 gene expression and disease susceptibility and may help us to develop new ways for treating infectious and autoimmune diseases.


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ACKNOWLEDGMENTS
 
We are grateful to Rachid Mazroui for his help with the fluorescence microscopy analysis, to James Garnon for many helpful technical suggestions, to Maryse Thivierge for monocyte preparation, and to Marie-Linda Boghdady and Dominigue Marion for a critical review of the text.

This work was supported by Canadian Institute of Health Research Grant FRN #36337 (6033; D.R.) and Strategic Training Centre in Infectious Diseases and Autoimmunity CIHR Grant (Y.Z.X.)


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FOOTNOTES
 
* Corresponding author. Mailing address: McGill University, Departments of Experimental Medicine and Human Genetics, 1650 Cedar Avenue, L11-218, Montreal, QC, Canada H3G 1A4. Phone: (514) 934-1934, ext. 44517. Fax: (514) 934-8260. E-mail: danuta.radzioch{at}muhc.mcgill.ca. Back


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Molecular and Cellular Biology, September 2005, p. 8139-8149, Vol. 25, No. 18
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.18.8139-8149.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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