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Originally published In Press as doi:10.1074/jbc.M602445200 on August 24, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31212-31221, October 20, 2006
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Interferon-{gamma}-inducible Protein (IP)-10 mRNA Stabilized by RNA-binding Proteins in Monocytes Treated with S100b*

Narkunaraja Shanmugam{ddagger}, Richard M. Ransohoff§, and Rama Natarajan{ddagger}1

From the {ddagger}Gonda Diabetes Research Center, Beckman Research Institute of City of Hope, Duarte, California 91010 and the §Lerner Research Institute, Cleveland, Ohio 44195

Received for publication, March 15, 2006 , and in revised form, August 23, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines mediate the recruitment and activation of blood monocyte/macrophages and lymphocytes to sites of inflammation. Expression of the chemokine IP-10 (interferon-{gamma}-inducible protein) has been documented in several inflammatory and autoimmune disorders including type 1 diabetes. However, the mechanism of its expression in monocytes or its functional role in diabetes is not known. Advanced glycation end products acting via their receptor, RAGE, play major roles in diabetic complications. In this study, we observed for the first time that S100b, an inflammatory protein as well as a specific RAGE ligand, significantly increased IP-10 mRNA and protein levels in THP-1 monocytes as well as peripheral blood monocytes. Promoter luciferase assays showed that IP-10 mRNA accumulation by S100b was not via increased transcription. On the other hand, S100b significantly increased IP-10 mRNA half-life and stability. This appeared to be mediated by S100b-induced binding of specific RNA-binding protein(s) to a 3'-untranslated region-responsive region of the IP-10 mRNA. Our results demonstrate for the first time that diabetic stimuli such as RAGE ligands can induce inflammatory gene expression in monocytes via increased message stability.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Proinflammatory chemokines and cytokines secreted by peripheral blood mononuclear cells have been implicated in the pathogenesis of diabetes and its complications because they can play key roles in atherosclerosis and pancreatic islet beta-cell dysfunction. Chemokines constitute a superfamily of chemoattractant cytokines (<20 kDa) that contain between two and four highly conserved NH2-terminal cysteine amino acid residues. They are involved in the recruitment and activation of neutrophils, monocyte/macrophages, and lymphocytes to sites of inflammation (1). The CXC family of chemokines, in which the first two cysteines are separated by another amino acid residue, is chemotactic for neutrophils and T cells. The CC chemokine family (including macrophage inflammatory protein-1, macrophage chemoattractant protein-1, and RANTES (regulated on activation normal T cell expressed and secreted)) in which the first two cysteine residues are juxtaposed is chemotactic for monocytes and subpopulations of T cells. IP-10 (interferon-{gamma}-inducible protein-10), a member of the CXC chemokine superfamily, is a highly inducible chemoattractant for activated T cells but has pleiotropic activities, such as stimulation of monocytes and NK cells, bone marrow progenitor cell maturation, and modulation of adhesion molecule expression after stimulation with IFN-{gamma}2 (1–3). Elevated serum IP-10 levels have been shown in diabetes (4). However, it is not known whether monocyte IP-10 is altered under diabetic conditions. Studies with inflammatory cells such as monocytes demonstrate that simulated diabetic conditions in vitro, such as high glucose culture or treatment with advanced glycation end products (AGEs), can induce the expression of inflammatory cytokines and chemokines via activation of specific signaling pathways and transcription factors such as NF-{kappa}B (5–9). This could result in increased monocyte activation, migration, and adhesion to the endothelium, key events in the pathogenesis of diabetic vascular disease. In the present study, we evaluated the hypothesis that proinflammatory RAGE ligands can increase IP-10 levels in monocytes. We also report novel new mechanisms regulating IP-10 mRNA accumulation under these conditions.

AGEs are products of nonenzymatic glycation/oxidation of proteins/lipids that accumulate during natural aging and are also greatly augmented in disorders such as diabetes, renal failure, and Alzheimer disease (10–12). Their formation is related to circulating high glucose concentrations in diabetes. Several receptors for AGEs have been identified (12–16). The well studied cell surface AGE receptor, namely RAGE, is a multiligand member of the immunoglobulin superfamily (12–16). Ligands for RAGE include AGEs, EN-RAGE, the S100/calgranulin family of proteins, amphoterin, and amyloid-{alpha}-peptide (14, 15). They exert their effects in various cells including vascular cells, blood mononuclear phagocytes, and lymphocytes (15, 16) and now serve as valuable tools to study RAGE signaling.

Interaction of these ligands with RAGE can induce oxidant stress, production of growth factors and cytokines, chronic inflammatory responses, and vascular dysfunction associated with diabetic complications (15, 16). Blockade of RAGE can suppress the inflammatory response in murine models and reduce vascular hyperpermeability and accelerated atherosclerosis observed in diabetic apoE null mice (16–18). RAGE activation induces inflammatory genes via key signaling pathways and transcription factors such as mitogen-activated protein kinases, JAK-STAT (Janus kinases and signal transducers and activators of transcription) pathway, and NF-{kappa}B (5, 6, 9, 11, 12, 19–22). However, it is not known whether AGEs and RAGE ligands lead to IP-10 expression. Furthermore, there has been no report showing that RAGE activation can lead to gene expression via mRNA stability. In the present study, we demonstrate for the first time that S100b, a specific RAGE ligand that is augmented in diseases such as diabetes and Alzheimer disease, can induce potent increases in IP-10 mRNA accumulation in monocytes and that this occurs via increased stability of IP-10 mRNA rather than increased transcription.

In addition to transcriptional control, mRNA stability is a key cellular mechanism for rapid and additional tight regulation of mRNA levels (23, 24), which ultimately reflects in increased protein levels and thus modulates several biological processes. mRNA stability is a consequence of not only cis-acting sequences but also trans-acting factors that bind directly or indirectly to cis-acting elements and promote the deadenylation and degradation of mRNA. The role of mRNA stability in mediating pathologic gene expression under diabetic conditions is not known.

A major objective of the current study was to gain insights into the mechanisms of AGE-induced mRNA accumulation of the chemokine IP-10 in monocytes. We found that IP-10 mRNA accumulation induced by the RAGE ligand S100b is mediated by mRNA-binding proteins that interact with a sequence in the 3'-untranslated region (UTR) of IP-10 mRNA. Recent studies have shown that high glucose, or AGE-RAGE interaction in monocytes triggered by RAGE ligands, can induce the expression of inflammatory genes such as tumor necrosis factor {alpha} and cyclooxygenase-2 via increased transcription (5, 6, 9, 20, 21). Our current results demonstrate that S100b can also induce inflammatory chemokines via increased mRNA stability, thus indicating for the first time that diseases such as diabetes that are associated with elevated AGEs and S100b may promote stabilization of the mRNAs of pathologic genes via novel post-transcriptional regulatory mechanisms. In the context of monocytes in a diabetic environment, this can trigger an amplifying inflammatory loop, islet dysfunction as well as accelerated vascular complications.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Actinomycin D, cycloheximide, and S100b protein (bovine brain) were obtained from Calbiochem; heparin was from Sigma-Aldrich. [{alpha}-32P]UTP (3000 Ci/mmol) was from New England Nuclear (Boston, MA). RT-PCR and SYBR green quantitative PCR reagents were from Applied Biosystems (Foster City, CA), and MAXIscrip T7 kit and Quantum RNA 18 S internal standards were from Ambion Inc. (Austin, TX). Ficoll-Paque Plus were from Amersham Biosciences. Luciferase assay system was from Promega, Inc. (Madison, WI), and human IP-10/CXCL10 immunoassay kit was from R & D Systems (Minneapolis, MN).

Cell Culture and Treatments—Human THP-1 monocytic cells were obtained from American Type Culture Collection or from the National Cell Culture Center (Minneapolis, MN) and subsequently cultured as described (20) in RPMI 1640 medium supplemented with 10% fetal calf serum and either 5.5 mM D-glucose (normal glucose, NG) or treated with 5–10 µg/ml of S100b protein. In some experiments, THP-1 cells were pretreated with inhibitors as indicated. They were then incubated alone in NG medium or with S100b for various time periods.

Isolation of Peripheral Blood Monocytes (PBMC)—Blood collections for PBMC isolations from normal healthy volunteers or those with documented type 1 or type 2 diabetes were performed with an approved institutional review board protocol as described earlier (20). The mean HbA1C of the patients was 7.76 ± 0.284 9 (range, 7.1–8.9). Duration of diabetes varied from 7 to 45 years for both type 1 and type 2 diabetic subjects. Type 2 diabetes was identified based on nonautoimmunity as far as mechanism, regardless of insulin use. All of the patients were on insulin and/or oral agents. Isolated monocytes were collected and washed in fresh RPMI medium. About 1 x 106 cells in 6-well plates were treated with S100b for 4 h, and the total RNA was isolated as described below.

RNA Preparation and Relative RT-PCR—THP-1 cells (2 x 106/sample) in 4 ml of medium containing 5.5 mM (NG) with or without 6.5 µg/ml S100b were cultured in duplicate in 6-well dishes for different time intervals. Total RNA was isolated, and multiplex PCRs were performed with gene-specific primers paired with Quantum RNA 18 S internal standards as described (20, 21). The 5' and 3' primers for human IP-10 were 5'-CACCAAATCAGCTGCTACTA-3' (187bp) or 5'-GGAAGATGGGAAAGGTGAGG-3' (450bp) and antisense primer 5'-TGAAAAAGAAGGGTGAGAAGAG-3'. The 5' and 3' primers for firefly luciferase mRNA were 5'-ACGGATTACCAGGGATTTCAGTC-3' and 5'-AGGCTCCTCAGAAACAGCTCTTC-3'. PCR products were fractionated on 2.5% agarose gels and photographed using AlphaImager 2000 documentation and analysis system. DNA bands corresponding to amplified products and 18 S RNA were quantified with QuantityOne software (Bio-Rad). The results are expressed as fold stimulation over NG after normalizing with paired 18 S RNA levels. The relative RT-PCRs were performed for 32 cycles for IP-10 based on initial optimization experiments where we determined that linear increase occurred between 25 and 38 cycles. For 18 S, we used 30 cycles based on optimization experiments showing linear increase between 20 and 33 cycles.

Real Time PCR—Real time quantitative PCR was performed on the Applied Biosystems 7300 real time PCR system (Foster City, CA). The human IP-10 5' and 3' primers for real time PCR were 5'-GCCTCTAGACTGAGAATTCTGATAAACCC-3' and 5'-CACCAAATCAGCTGCTACTA-3'. Primers for control GAPDH were 5'-GGTGAAGGTCGGAGTCAACG-3' and 5'-CACCATTCTCGCTCCTGGAAGATGGTG-3'. The 5' and 3' primers for luciferase mRNA amplification were 5'-TCTCTGGCATGCGAGAATCT-3' and 5'-ACGGATTACCAGGGATTTCAGTC-3'. Each sample was run in triplicate. The relative RNA amount was calculated and normalized with the internal control, GAPDH, according to the method described by Jing et al. (25).

IP-10 Enzyme-linked Immunosorbent Assay—THP-1 cells (5 x 105 cells/ml) were incubated in 6-well tissue culture plates in RPMI 1640 medium with 0.2% bovine serum albumin. The cells were treated with or without S100b for 8 h. The supernatant conditioned medium was then harvested and assayed for IP-10 levels using a specific Quantikine kit. Medium alone without cells was incubated under the same conditions and used as blank control.

Preparations of S130 Extract Nuclear and Cytoplasmic Proteins—THP-1 cells (6.5 x 105 cells/ml) were resuspended in adequate volume of RPMI 1640 medium and treated with 6.5 µg/ml of S100b for 1 h. The cells were harvested by 1500 x g centrifugation and then resuspended at a concentration of 1.5 x 106 cells/ml of buffer A (10 mM Tris, pH 7.5, 1 mM potassium acetate, 1.5 mM magnesium acetate, 2 mM dithiothreitol). The cells were lysed with 25 strokes of a type B Dounce homogenizer and pelleted with a 15-min 4,000 x g centrifugation. The supernatant was layered over buffer A containing 30% (w/v) sucrose and centrifuged at 130,000 x g for 1 h. The resulting supernatant (S130) was removed without disturbing the S130-sucrose interface, supplemented with glycerol to a final concentration of 10% (v/v), and frozen at –70 °C in aliquots. For the preparation of nuclear proteins, THP-1 cells (5 x 108 cells) were treated without (NG) or with S100b (6.5 µg/ml) protein for 1 h. In some experiments, the cells were pretreated with actinomycin D (1–10 µg/ml) or cycloheximde (0.5–5 µg/ml) for 1 h and then treated with or without S100b. They were then centrifuged at 1400 rpm, and the supernatants were discarded and washed twice with cold Hanks' salt solution. The cells were resuspended in 10–20 ml of cytoplasmic extraction buffer (10 mM Tris-Cl, pH 7.9, 60 mM KCl, 1 mM EDTA, and 1 mM dithiothreitol), kept on ice for 15 min, and centrifuged at 1400 rpm for 15 min. Lysis buffer (cytoplasmic extraction buffer containing 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1x protease mixture) was added to the pellets (50x packed cell volume), kept on ice for 10 min, and then centrifuged to collect the nuclear pellets. The nuclear pellets were washed twice with ice-cold cytoplasmic extraction buffer containing protease inhibitors but no detergents. The supernatants were pooled and saved (cytoplasm protein). Washed nuclear pellets were resuspended in 2 ml of nuclear extraction buffer (20 mM Tris-Cl, pH 8.0, 0.4 M NaCl, 1.5 mM MgCl2, 1.5 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1x protease inhibitor mixture, 25% glycerol) on ice for 10 min and centrifuged at 7000 rpm for 15 min. The supernatants were collected and snap frozen on dry ice before storage at –70 °C.

Construction of Recombinant Plasmids—The template for the IP-10 3'-UTR was RT-PCR-amplified from THP-1 cells using 5'-TTGAGTTATAATTACTTTAT-3' (antisense) and 5'-TGAAAAGAAGGGTGAGAAGAG-3' (sense) primers and cloned into pGEM vector using pGEM-T vector system I (Promega) to yield pIP10-UTR. The orientation of the fragment was confirmed by restriction enzyme map analysis. The luciferase reporter expression constructs were prepared in the vector pcDNA3.1/Zeo (+) containing the luciferase cDNA from pGL3-Basic (Promega) cloned into the HindIII and XbaI sites to yield pZeo/Luc. Incorporation of the IP-10 3'-UTR was accomplished by PCR amplification of the IP-10 3'-UTR using XbaI-tailed primers (forward primer 5'-TAGTCTAGATGAAAAAGAAGGGTGAGAAGAG-3' and reverse primer 5'-GCCTCTAGAGAGTTATAATTACTTTATTAACC-3') and inserting it adjacent to the luciferase coding region to yield pZeo/Luc+IP-10 3'-UTR. PCR-amplified antisense UTR DNA fragment was cloned in to the 3' end of the luciferase gene to yield pZeo/Luc+IP-10 3'-UTR antisense.

DNA Transfections and Luciferase Assays—THP-1 cells plated in 6-well plates (1.2 x 106/well) were transfected with 1 µg of the indicated plasmids, pGL, pGLT, pIkB (mut), pISRE3 (mut), pZeo-Luc+IP-10 3'-UTR, or pZeo-Luc+IP-10 3'-UTR antisense and the control pGL3-Luc plasmid (Promega) using Lipofectamine 2000 or Amaxa Nucleofector (Amaxa Biosystem) in RPMI medium with serum according to manufacturer's protocols. Following an overnight recovery period, the transfected cells were cultured in either control medium alone containing 5.5 mM glucose (NG) or with 6.5 µg/ml S100b. The cells were then washed with phosphate-buffered saline, lysed with 100 µl of lysis buffer, and stored overnight at –70 °C. The samples were thawed and brought to room temperature, and 20 µl of each lysate was used to determine luciferase activity according to the manufacturer's instructions.

Measurement of mRNA Stability—THP-1 cells were treated 4 h with or without S100b. Then, after S100b treatment, cell transcription was stopped by treating with actinomycin D (1 µg/ml). 5 x 105 cells/ml were collected at the indicated time intervals with RNA-STAT60 added and stored at –70 °C until needed. IP-10 mRNA levels were measured by RT-PCR. The relative RNA levels were quantified after normalizing with internal control 18 S mRNA. For luciferase mRNA stability, luciferase reporter constructs were transiently expressed into THP-1 cells. The transcription of luciferase was stopped by adding actinomycin D (10 µg/ml) after 4 h of S100b treatment. 5 x 106 cells/ml were collected at the indicated time intervals with RNA-STAT60 added and stored at –70 °C until needed. Total RNA was isolated, and luciferase mRNA levels were quantified by real time PCR. Each sample was run in triplicate. The relative RNA amount was calculated and normalized with the internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), according to the method described by Jing et al. (25).

In Vitro Transcription and Preparation of 3'-UTR Deletion Constructs—pIP10-UTR was linearized with SpeI enzyme. RNA transcript was generated using a MAXIscripT7 kit. The full-length UTR RNA was uniformly labeled by including [{alpha}-32P]UTP. Deletions of 3'-UTR RNA were prepared by linearizing pIP10-UTR with following restriction enzymes, ScaI (730 nt), EcoRI, (453 nt), and PvuII (255 nt). IP-10 180-nt UTR was obtained by digesting pIP-10-UTR with ScaI enzyme and then with NcoI. The NcoI site was filled in with Klenow enzyme and then religated using T4-DNA ligase to yield pIP10–180nt. Plasmid pIP10–180nt was linearized with SpeI, and 180nt RNA was prepared by in vitro transcription using a MAXIscripT7 kit.

Protein Preparation, UV Cross-linking, and Electromobility Shift Assays (EMSAs)—400 µl of S130 or nuclear extracts from of NG- and S100b-treated samples were treated with 400 units/ml of micrococcal nuclease in buffer containing 50 mM glycine, pH 9.2, and 5 mM of CaCl2 and incubated at 30 °C for 30 min. After incubation, 5 mM of EGTA was added and centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatants were centrifuged again. 400 µl of samples were treated with 40 µg/ml of yeast tRNA, 400 units of RNAsin and beta-mercaptoethanol to 1% final and incubated on ice for 15 min. UV cross-linking was carried out in a 20-µl reaction volume with 32P-labeled RNA (10 pmol) and incubated with corresponding extracts for 15 min at room temperature in a buffer containing 10 mM Tris-Cl (pH 7.5), 100 mM potassium acetate, 2 mM magnesium acetate, 1 mM dithiothreitol, 10 mM creatine phosphate, 1 mM ATP, and 0.1 mM spermine. To the reaction mix 30 µg/ml of heparin was added and incubated for a further 10 min to reduce nonspecific binding. The samples were subsequently transferred to ice and covalently cross-linked by UV irradiation for 10 min with UV Stratalinker 1800 equipment. Following UV cross-linking, the RNA body was cleaved with RNase buffer containing 10 mM Tris (pH 7.5), 1% mixture of protein inhibitors, 100 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml RNase A, and RNase U2 for 30 min at 37 °C. The proteins were resolved by 12% SDS-PAGE and visualized by autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Ligation of RAGE Increases the Accumulation of IP-10 mRNA in THP-1 Monocytic Cells—We first examined whether a RAGE ligand such as S100b can increase the expression of IP-10 in cultured monocytes. THP-1 monocytes were treated with or without S100b protein for various time intervals from 1 to 24 h as shown in Fig. 1A. RNA extracted from these experiments was subjected to relative multiplex RT-PCR analyses in which specific primers for human IP-10 were paired with 18 S rRNA primers as internal standard. S100b-induced changes in IP-10 gene levels (lower band) were evaluated as fold over control NG samples after normalizing to 18 S rRNA internal control (upper band). The results show that S100b treatment markedly increased IP-10 mRNA accumulation, peaking around 4 h and declining by 24 h (Fig. 1A).

Next we analyzed the dose-response effect of S100b. THP-1 cells were treated with various concentrations (2.5, 5, 7.5, or 10 µg/ml) of S100b (Fig. 1B). Peak accumulation of IP-10 mRNA was noticed at around 5 µg/ml of S100b. The bar graph in Fig. 1C shows that the stimulatory effects of S100b on IP-10 mRNA were significant at these doses.

In addition, we performed quantitative PCR, and the bar graph in Fig. 1D shows that S100b treatment for 4 h leads to significant increase in IP-10 mRNA levels as quantified by real time quantitative PCR.

To determine whether S100b acts through the RAGE receptor, THP-1 cells were pretreated with a specific anti-RAGE antibody (generous gift from Dr. David Stern and Dr. Ann Marie Schmidt, Columbia University, New York) for 1 h prior to S100b. This led to complete blockade of S100b-induced IP-10 mRNA (Fig. 1E, fourth lane), confirming that S100b-induced IP-10 mRNA accumulation is via RAGE activation.

In the next step, we evaluated whether IP-10 protein levels were also regulated by S100b in THP-1 cells. IP-10 levels in culture supernatants were quantified by enzyme-linked immunosorbent assay. S100b (6.5 µg/ml) treatment for 8 h led to a significant 4-fold increase in IP-10 levels relative to untreated control cells (136 ± 31 pg/ml versus 28 ± 11 pg/ml, p < 0.001).


Figure 1
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  		FIGURE 1.
Analyses of S100b-induced IP-10 mRNA accumulation. Relative RT-PCRs were performed with total RNA isolated from THP-1 cells cultured in NG (5.5 mM) medium with or without S100b (6.5 µg/ml) for 1–24 h, using IP-10 gene-specific primers. 18 S RNA primers were included in the same reaction as internal controls. PCR products were analyzed on 2.5% agarose gels. A, agarose gel of RT-PCR products from S100b treated (+) or untreated (–) THP-1 cells. B, dose-dependent effects of S100b. Total RNA samples isolated from THP-1 cells treated for 4 h with 0, 2.5, 5, 7.5, or 10 µg/ml S100b were used to perform multiplex relative RT-PCRs with IP-10 and 18 S RNA primers. C, bar graph quantification of IP-10 mRNA levels. *, p < 0.001. The values are normalized to 18 S and shown as the means ± S.E. fold induction from three to six independent experiments. D, bar graph shows IP-10 mRNA levels quantified by real time qPCR at 4 h after S100b treatment. *, p < 0.001. The values are normalized to GAPDH and the means ± S.E. of three independent experiments. E, anti-RAGE antibody (Ab) blocks S100b-induced IP-10 mRNA accumulation. THP-1 cells were pretreated with 70 µg/ml of anti-RAGE antibody for 1 h followed by 6.5 µg/ml S100b for 4 h.

 
S100b Induces IP-10 mRNA Accumulation in PBMC—Because THP-1 cells may not fully represent the phenotype of blood monocytes, we next examined whether S100b can also induce IP-10 mRNA accumulation in PBMC isolated from normal healthy adult donors. These PBMC were treated with S100b for 4 h, total RNA was isolated, and IP-10 mRNA levels were analyzed by RT-PCR as described for Fig. 1. Fig. 2 (A and B) shows that S100b induced significant IP-10 mRNA accumulation in PBMC to a similar extent as in THP-1 cells.


Figure 2
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  		FIGURE 2.
Analysis of IP-10 mRNA levels in primary human PBMC. A, 2 x 105 isolated PBMC from normal healthy adult donors in 4 ml of RPMI medium were stimulated with S100b (6. 5 µg/ml) for 4 h. IP-10 mRNA levels were measured by RT-PCR as described in Fig. 1. Representative gel in A shows stimulatory effect of S100b on IP-10 mRNA accumulation in PBMC. B, fold induction of IP-10 mRNA by S100b from three to four experiments each from four separate donors. The values are normalized to 18 S and shown as the means ± S.E. *, p < 0.01. C and D, monocytes from type 1 diabetic subjects show elevated IP-10 mRNA levels relative to nondiabetic. PBMC were isolated from four type 1 and three type 2 diabetic patients (T1D and T2D, respectively) or normal subjects (N). 2 x 105 isolated monocytes were directly processed for total RNA isolation. C and D show RT-PCR analyses of IP-10 mRNA levels from PBMC from type 1 and type 2 diabetic patients. E, bar graph showing the fold increase in IP-10 mRNA levels. *, p < 0.01.

 
IP-10 mRNA Levels Are Elevated in PBMC Isolated from Diabetic Subjects Relative to Normal Controls—To further determine the in vivo relevance of the in vitro data, we next examined whether blood monocytes from diabetic patients had increased levels of IP-10. We therefore isolated PBMC from patients with documented type 1 or type 2 diabetes and analyzed IP-10 mRNA levels relative to monocytes from normal healthy control volunteers. Fig. 2C shows that PBMC from four type 1 diabetic patients had markedly elevated levels of IP-10 mRNA relative to the normal controls. PMBC from three type 2 diabetic patients also showed some increases (Fig. 2D). The bar graph quantitation in Fig. 2E shows a significant 2–3-fold increase of IP-10 mRNA levels in PBMC from type 1 diabetic patients, whereas the increase in type 2 diabetic patients was not statistically significant, at least with this limited number of patients.

S100b Treatment Increases IP-10 mRNA Levels via mRNA Stabilization—Time course analyses showed that S100b-induced accumulation of IP-10 mRNA is evident by 2 h, peaking at 4–8 h and then declining to control levels by 24 h (Fig. 1A). To determine whether S100b-induced IP-10 mRNA accumulation at 4 h is due to increase in transcription, THP-1 cells were pretreated for 1 h with actinomycin D (1 µg/ml, inhibitor of transcription) or with cycloheximide (5 µg/ml, inhibitor of de novo protein synthesis) and then treated with S100b for 4 h. RT-PCR analyses in Fig. 3A shows that at 4 h, in actinomycin D-treated cells, there was a slight decrease in basal IP-10 mRNA, but S100b could still increase IP-10 mRNA accumulation. On the other hand, cycloheximide was clearly inhibitory. These interesting results indicate that increased IP-10 accumulation by S100b at 4 h is most likely not via increased transcription but instead may be due to increases in mRNA stability. Furthermore, new protein synthesis appears to be also involved. This was further confirmed by additional experiments shown below.


Figure 3
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  		FIGURE 3.
S100b treatment increases IP-10 mRNA levels by increasing RNA stability. THP-1 cells were pretreated with actinomycin D (1 µg/ml, Act-D) or cycloheximide (0. 5 µg/ml, Cyclo) and then stimulated with or without S100b for 4 h. A, representative gel of RT-PCR products. B, fold induction of IP-10 mRNA as mean ± S.E. from three independent experiments. *, p < 0.001; **, p < 0.01. C, effect of S100b on IP-10 promoter activity in THP-1 cells. THP-1 cells were transfected with either a control plasmid containing the promoterless luciferase gene (pGL3-Luc) or plasmids containing the luciferase gene under the control of human IP-10 promoter (–960/+97) (pGL) sequences or deletion (–435/+97) (pTGL) and mutant promoter (pkB2-mut and pISRE3-mut) as indicated. After a 24-h recovery period, the cells were treated with S100b for 4 h and lysed, and then luciferase activity was determined with a luminometer. The results show that S100b had no effect on IP-10 promoter activity. The values shown are the means ± S.E. of three independent experiments.

 
S100b Treatment Does Not Activate Transcription from the IP-10 Promoter—Our data show that after actinomycin D treatment, S100b could still increase IP-10 mRNA accumulation (Fig. 3, A and B), suggesting an absence of transcription mechanisms for S100b-induced effects. To further determine whether S100b does or does not increase IP-10 transcription, we transfected THP-1 cells with deletion and mutant promoter reporter constructs pGL, pTGL, pkB-2 (mut), and pISRE-3 (mut) that expresses the firefly luciferase (Luc) gene under the control of human IP-10 gene promoter segments (Fig. 3C). S100b-stimulated (4 h) THP-1 cells transfected with these plasmids showed no significant elevation in Luc activity with any of the constructs (Fig. 3C). However, basal activity was reduced when the distal NF-{kappa}B site was mutated (pkB-2, mut), suggesting that NF-{kappa}B may be necessary for basal IP-10 transcription (26). These results further confirm that S100b-induced IP-10 mRNA accumulation is not via increased transcription but could be via increased mRNA stability.


Figure 4
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  		FIGURE 4.
S100b treatment enhances IP-10 mRNA half-life. The cells were treated with or without S100b for 4 h and then with actinomycin D (Act D), and RNA was harvested at the indicated time points. The levels of IP-10 mRNA in S100b-treated THP-1 cells (open squares) and untreated control cells (diamonds) were determined using RT-PCR and expressed as a percentage of the mRNA level present at various time points. A and B, show representative blots (upper blot without S100b and lower blot with S100b). C, quantitation of data from three independent experiments, The results show that IP-10 mRNA has a shorter half-life in untreated relative to S100b-treated THP-1 cells.

 
IP-10 mRNA Has a Shorter Half-life in Control Cells Relative to S100b-treated THP-1 Cells; Involvement of a 3'-UTR Regulatory Region—To further determine whether S100b treatment can increase IP-10 mRNA stability, we treated THP-1 cells with actinomycin D at 4 h after S100b and IP-10 mRNA levels subsequently analyzed at various time intervals thereafter. Fig. 4A shows that, in control and actinomycin-treated cells, IP-10 mRNA decays quickly and is completely gone by 60 min. On the other hand, Fig. 4B shows that in S100b- and actinomycin-treated cells, the destabilization is much slower, and significant message levels are present even beyond 60 min. This is quantitatively depicted in Fig. 4C.

IP-10 3'-UTR Is Responsible for S100b-mediated Stabilization of IP-10 mRNA—We fused the IP-10 3'-UTR RNA (910 nt, 263–1172) into a plasmid-containing Luc reporter at the 3' end (pZeo/Luc+IP-10). THP-1 cells transfected with this plasmid showed a significant 3-fold (p < 0.005) increase in Luc activity compared with control plasmid pZeo/Luc when treated for 4 h with S100b (Fig. 5A). This result further supports a role for increased mRNA stability by S100b and indicates that the IP-10 3'-UTR region has a potential S100b-responsive regulatory element.


Figure 5
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  		FIGURE 5.
The IP-10 3'-UTR is responsible for S100b-mediated stabilization of IP-10 mRNA. A, a 910-nucleotide full-length IP-10 3'-UTR region was fused to the 3' end of the luciferase reporter gene. THP-1 cells were transfected either with this human IP-10 3'-UTR-luciferase construct (pZeo/Luc+IP-10) or control plasmid (pZeo/Luc). After transfection, the cells were treated with or without S100b. Luciferase activities were measured in cellular extracts 4 h later. The results are expressed as fold induction of luciferase activity by S100b relative to control and shown as the means ± S.E. of three independent experiments. *, p < 0.001 versus control. B, in addition to pZeo/Luc+IP-10 and pZeo/Luc, full-length antisense IP-10 3'-UTR region was also transfected, and luciferase activities were measured in cellular extracts 4 h later. The results are expressed as relative luciferase units (RLU) and shown as the means ± S.E. of three independent experiments. *, p < 0.002 versus untreated control. C, luciferase mRNA levels also analyzed from the same samples by RT-PCR with 18 S as internal control.

 
To derive additional support for the above data that S100b can stabilize IP-10 mRNA, we measured luciferase mRNA stability in these transiently transfected THP-1 cells. As shown in Fig. 5, luciferase mRNA levels (Fig. 5C, first and second lanes) and its activities (Fig. 5B, first and second bars) are not changed when plasmid-containing luciferase gene alone is used in both S100b-treated and control THP-1 cells. On the contrary, the presence of the IP-10 3'-UTR within this same construct transfection with plasmid containing IP-10 3'-UTR fused to 3' end of the luciferase gene (pZeo/Luc+IP-10), luciferase mRNA levels (Fig. 5C, third lane), and its activity (Fig. 5B, third bar) were dramatically reduced, thus showing the marked destabilizing effect of the 3'-UTR. Treatment of S100b could significantly reverse this, and thus both luciferase mRNA levels and activity were significantly increased (fourth lane and fourth bar). Specificity of the effect of the 3'-UTR was confirmed by testing the construct cloned in the antisense direction (pZeo-IP-UTR-antisense), which also did not respond to S100b (Fig. 5, C, fifth and sixth lane for Luc mRNA, and B, fifth and sixth bars for Luc activity). These results further support the notion that IP-10 UTR mediates S100b-induced IP-10 mRNA accumulation in THP-1 cells. Because our luciferase constructs are not under the control of the IP-10 promoter, these results further support the negative data on transcription in Fig. 3C and rule out the potential presence of required elements outside the short IP-10 promoter segment studied therein.


Figure 6
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  		FIGURE 6.
Measurement of luciferase mRNA stability. After transfection of pZeo/Luc+IP-10, pZeo/Luc+IP-10 (antisense) and pZeo/Luc THP-1 cells were treated with or without S100b for 4 h. At the end of the S100b treatment (0 min), actinomycin D (10 µg/ml) was added to each sample, and aliquots of THP-1 cells were collected at 10-, 30-, 60-, and 120-min time intervals. Luciferase mRNA levels were quantified by real time PCR with GAPDH as internal control. Luciferase mRNA levels at 0 min were considered as 100%. Percentage of mRNA remaining after actinomycin D treatment at indicated times were plotted against time. The values shown are the means ± S.E. of three independent experiments. Filled circles (•) and filled squares ({blacksquare}) represent pZeo/Luc transfected cells. Open circles ({circ}) and open squares ({square}) represent pZeo/Luc+IP-10 transfected cells. The solid line and broken line represent S100b-treated and untreated, respectively.

 
To further ascertain the involvement of IP-10 3'-UTR in mRNA stability, we also checked stability of luciferase mRNA. THP-1 cells were transfected with pZeo/Luc, pZeo/Luc+IP-10, and pZeo/Luc+IP10UTR (antisense). Transfected cells were stimulated with S100b for 4 h, at which time cell transcription was completely blocked by adding excess actinomycin D (10 µg/ml). Aliquots of cells were collected at indicated time periods (Fig. 6). Luciferase mRNA levels were quantified by real time RT-PCR. As shown in Fig. 6, luciferase mRNA fused with IP-10 3'-UTR showed a much shorter half-life in control cells (open squares, broken line) than the S100b-treated cells (open circles, solid line). No change in luciferase mRNA levels was observed in cells transfected with luciferase without UTR (filled circles, filled square) or the negative control (luciferase fused with antisense IP-10 UTR) (data not shown) This suggests that the IP-10 3'-UTR contains S100b-specific regulatory elements that can mediate IP-10 mRNA stabilization.


Figure 7
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  		FIGURE 7.
A nuclear protein(s) from S100b-treated THP-1 cells interacts with the IP-10 3[prime]-UTR. A, 15 µg of S130 extract or nuclear or cytoplasmic proteins were incubated with a 32P-labeled in vitro transcribed IP-10 3'-UTR. The complexes were UV cross-linked, and excess RNA was removed by RNase T1 and RNase A treatment. The samples were analyzed on 12% SDS-PAGE. The first, third, fifth, and eighth lanes represent total S130, nuclear, and cytoplasmic proteins from control cells; the second, fourth, and sixth lanes represent total, nuclear, and cytoplasmic proteins from S100b-treated cells, respectively. The seventh lane is 32P-labeled in vitro transcribed IP-10 3'-UTR-RNA only. The arrow shows the specific complex formed at the 3'-UTR. The eighth and ninth lanes on the right from samples treated with proteinase K before UV cross-linking show that the complex formed is due to protein-RNA interaction. B, EMSA performed with nuclear protein from actinomycin D- or cycloheximide-treated cells. First lane, RNA only. The second, fourth, and sixth lanes represent control, whereas the third, fifth, and eighth lanes represent S100b-treated.

 
A Nuclear Protein(s) from S100b-treated THP-1 Cells Interacts with the IP-10 3'-UTR—Our previous results suggest that S100b increases IP-10 accumulation in THP-1 cells via increased mRNA stability. We next evaluated whether trans-acting factor(s) bind to the 3'-UTR and thereby stabilize IP-10 mRNA. To examine the ability of the IP-10 mRNA 3'-UTR to interact with potential regulatory proteins, UV cross-linking and EMSAs were performed. 32P-Labeled in vitro transcribed IP-10 3'-UTR was incubated with total extracts (S130 fraction) from THP-1 cells treated with or without S100b, and the formation of RNA-protein complexes analyzed by SDS-PAGE. UV cross-linking and EMSA showed stable RNA-protein complex formation with total cell extracts from S100b-treated THP-1 cells but not in control untreated cell extracts (Fig. 7A, second and first lanes, respectively). Although this RNA-protein complex formation was noted in total extracts, we wanted to further determine the cellular localization of proteins involved in this complex. We therefore isolated nuclear and cytoplasmic protein extracts from both S100b-treated and untreated THP-1 cells. UV cross-linking experiments showed the formation of an S100b-specific RNA-protein complex in the nuclear (Fig. 7B, fourth lane) but not cytoplasmic extract (Fig. 7B, sixth lane). The last lane is 32P-labeled in vitro transcribed IP-10 3'-UTR-RNA only. These results indicate that IP-10 mRNA is stabilized by a RNA-protein complex formed in the nuclear fractions of S100b-treated cells. Pretreatment of the RNA-protein complex with proteinase K before UV cross-linking (Fig. 7A, right panel, Prot.K) results in the loss of specific RNA-protein complex formation in S100b-treated cells, thus confirming that the complex formed is indeed from RNA-protein interactions.


Figure 8
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  		FIGURE 8.
Specificity of IP-10-UTR and nuclear protein complex formation. A, five picomoles of IP-10 3'-UTR were allowed to interact with 5, 10, 15, or 30 µg of nuclear extract from S100b treated THP-1cells. Increasing concentrations of protein gradually augmented the RNA-protein complex formation (indicated by the arrow). Fifth lane, control NG extract; second through fifth lanes, 5–30 µg of nuclear protein from S100b-treated cells; sixth lane, RNA only. B, competition experiment with cold sense RNA in which nuclear protein extracts from S100b-treated cells were incubated with a 32P-labeled in vitro transcribed oligonucleotide containing the IP-10 3'-UTR and a 5–20 fold excess of unlabeled cold oligonucleotide containing IP-10 3'-UTR. First lane, RNA only; second lane, control extract; third lane, S100b-treated nuclear extract; fourth lane, same as third lane with 5-fold cold IP-10 3'-UTR RNA; fifth lane, 10-fold; sixth lane, 20-fold. C, competition experiment with cold antisense IP-10 3'-UTR RNA. First lane, control; second lane, S100b without competitor; third lane, X1; fourth lane, X5; fifth lane, X10; sixth lane, X20.

 


Figure 9
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  		FIGURE 9.
Identification of the minimal S100b-responsive region of IP-10 3'-UTR. A, 15 µg of nuclear proteins were incubated with a 32P-labeled in vitro transcribed full-length (910 nt) IP-10 3'-UTR or various deletions (730, 453, and 255 nt). First lane, IP-10 3'-UTR RNA only. The second, fourth, sixth, and eighth lanes are with control nuclear extracts; the third, fifth, seventh, and ninth lanes are with nuclear extracts from S100b-treated cells. The arrow shows the protein-RNA complex formed in S100b-treated sample. B, confirmation of the minimal 180-nt UTR region needed for protein-RNA complex formation. Nuclear proteins were incubated with 32P-labeled in vitro transcribed full-length (910 nt) or a 180-nt deletion construct of IP-10 3'-UTR (731–910) or nonspecific RNA (pGEM-MC). Complex formation is indicated by the arrow. + and – indicate with and without S100b, respectively.

 
Our data in Fig. 3 (A and B) showed that after pretreatment with actinomycin D, S100b could still increase IP-10 mRNA accumulation, whereas cycloheximide completely blocked the effects of S100b, suggesting increased RNA stability and potential involvement of new protein synthesis. To test this further, we performed EMSAs with nuclear proteins prepared from THP-1 cells that were pretreated with actinomycin D (10 µg/ml) or cycloheximide (5 µg/ml) before S100b treatment. As shown in Fig. 7B, Actinomycin-treated samples still showed strong S100b-specific RNP complex formation (sixth and seventh lanes) but not cycloheximide-treated samples (fourth and fifth lanes). The first lane is 32P-labeled in vitro transcribed IP-10 3'-UTR RNA only. These data suggest that new protein synthesis is involved in this complex formation and needed for IP-10 mRNA stability. Furthermore, the data suggest that this new protein is synthesized from pre-existing mRNA in response to S100b and not via new transcription. These results indicate that S100b can also promote regulation at the translational level.

Formation of RNA-Protein Complex at the IP-10 3'-UTR Is Specific—UV cross-linking experiments showed formation of a stable RNA-protein complex when S100b-treated nuclear extracts interact with IP-10 3'-UTR RNA. To determine whether this is specific for IP-10 mRNA, we treated increasing amounts of S100b nuclear protein extracts with 5 pmol 32P-labeled IP-10 3'-UTR (910 nt). The autoradiograph in Fig. 8A shows that this increases the intensity of RNA-protein complex formation in a dose-dependent manner (the NG lane represents 15 µg of control protein from untreated cells in NG). We next performed competitive experiments using limited concentration (5 pmol) of 32P-labeled IP-10 3'-UTR transcripts and 5–20-fold excess of unlabeled cold IP-10 3'-UTR transcripts. Fig. 8B shows that the unlabeled transcripts compete effectively for protein binding to labeled IP-10 3'-UTR with almost total inhibition of complex formation at 20-fold molar excess of cold RNA. Specificity was further confirmed by data in Fig. 8C showing that, on the other hand, 20 molar excess of cold antisense IP-10 3'-UTR does not inhibit complex formation (Fig. 8C). These results confirm that the RNA-protein complex formation in the S100b-treated nuclear extracts is specific to the IP-10 3'-UTR RNA.

Identification of the Minimal IP-10 3'-UTR Region for RNA-Protein Complex Formation—For this, we cloned various deletions of IP-10 3'-UTR, namely, 3'-UTR-del-1 (730 nt, 263/992), 3'-UTR-del-2 (453 nt, 263/715), and 3'-UTR-del-3 (255 nt, 263/517) into the pGEM vector as described under Experimental Procedures" and shown in Fig. 9A (bottom panel). These 32P-labeled transcripts (full 910-nt 3'-UTR, del-1, del-2, and del-3) were incubated with nuclear extracts from THP-1cells with or without S100b treatment, and the generation of RNA-protein complexes was analyzed by EMSA. Fig. 9A shows the representative autoradiograph of the EMSA. The full IP-10 3'-UTR RNA (910 nt) yielded an RNA-protein complex (Fig. 9A, third lane) with S100b-treated samples, but none of the other deletions were effective, thus suggesting that the minimal 180-nt region from 992 to 1172 (which is absent even in del-1 construct) was responsible for the RNA-protein complex formation with the IP-10 3'-UTR. To confirm this further, the 32P-labeled transcript of IP-10 180 nt (nt 992–1172) was incubated with nuclear extracts from THP-1 cells treated with or without S100b. Fig. 9B shows that, similar to the 910-nt transcript full IP-10 3'-UTR, the minimal 180-nt IP-10 did indeed form a similar stable RNA-protein complex as anticipated (Fig. 9B, third and fifth lanes). 32P-Labeled transcripts of the multiple cloning site of the pGEM vector was used as a negative control unrelated RNA (last two lanes). These new results suggest for the first time that diabetic stimuli such as S100b can induce the accumulation of inflammatory chemokines such as IP-10 at the mRNA stability level by synthesizing and/or mobilizing specific RNA-binding proteins to bind to the specific 3'-UTR response elements of these genes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
IP-10, a key chemokine involved in T cell recruitment, (2, 3, 26, 27) has been reported to mediate chronic inflammatory and immune response. Diabetes is associated with significantly accelerated rates of inflammation, atherosclerosis, and hypertension. Increased circulating levels of IP-10 have been reported in patients with diabetes (4). However, the cells involved or the molecular pathways mediating IP-10 induction under diabetic conditions have not been previously described. Because AGEs accumulate under diabetic conditions and contribute to the progression of diabetic complications, we hypothesized that activation of RAGE by a specific ligand, S100b, in inflammatory cells can mimic the diabetic state and induce IP-10 expression. This could also unravel additional pro-inflammatory consequences of diabetic stimuli. In the present studies, we demonstrated for the first time that treatment of monocytes with S100b can significantly increase IP-10 mRNA and protein expression via the RAGE receptor. S100b-induced IP-10 mRNA accumulation was blocked by cycloheximide but not by actinomycin D. Furthermore, S100b did not increase transcription from the IP-10 promoter. These results suggested that IP-10 mRNA induction by S100b is not mediated by increased transcription but instead by increased mRNA stability and also involves new protein synthesis. This is interesting because IP-10 is normally regulated by transcriptional mechanisms (26) and induced by cytokines such as IFN-{gamma}. THP-1 cells treated with S100b showed no clear induction of IFN-{gamma} mRNA in THP-1 cells, and furthermore, pretreatment of THP-1 cells with a neutralizing antibody to IFN-{gamma} did not block S100b-induced IP-10 mRNA accumulation.3 This suggests that diabetic stimuli such as RAGE ligands can induce IP-10 mRNA in an IFN-{gamma}-independent manner.

Importantly, S100b also increased IP-10 mRNA accumulation in normal PBMC from nondiabetic volunteers. Furthermore, we observed significantly higher levels of IP-10 mRNA accumulation in monocytes isolated from type 1 diabetic patients relative to monocytes from nondiabetic subjects. These data support the in vivo and pathological relevance of our in vitro findings.

Because S100b-induced IP-10 mRNA accumulation was not transcriptionally regulated, we speculated that increased mRNA stability might be involved. This was also supported by mRNA decay assays showing prolonged IP-10 mRNA stability in S100b-treated cells relative to control. Two recent reports in other cells showed for the first time that IP-10 mRNA may be regulated by post-transcriptional mechanisms in response to a viral oncoprotein (27) or interferon {gamma} (28). The latter report indicated that IP-10 transcript in the murine cells studied were stabilized in a MyD88-dependent manner and required the presence of a short adenine-uridine-rich element (ARE) in the 3'-untranslated region of the transcript (29).

Interestingly, however, our sequence analysis of the human IP-10 3'-UTR did not reveal any typical classic ARE regions but only one atypical short AUUUA region unlike mouse IP-10 3'-UTR where two short AUUUA regions are present (29). AREs are among the most common and largely studied cis-acting elements that govern mRNA stability (24, 30). They have been implicated in mRNA stabilization of cyclooxygenase-2, another inflammatory gene (31). There are, however, also other non-ARE cis-acting sequences in certain mRNAs, including those of c-Myc, histone, {alpha}-globlin, hormones, and cyclic nucleotides that are also implicated in stabilizing mRNAs.

We obtained several lines of evidence to support the concept of IP-10 transcript stability in response to S100b. S100b could increase IP-10 mRNA accumulation even after pretreatment with actinomycin. In the presence of actinomycin D under basal conditions, there was a decrease in IP-10 mRNA levels. IP-10 normally transcribed in the cell under basal conditions is expected to be degraded rapidly because of endogenous destabilizing mechanisms. Our data suggest that S100b prevents this by promoting mRNA stability and increasing mRNA accumulation. We envision that actinomycin can block the basal IP-10 transcription. Thus the net effect on the increase in IP-10 mRNA levels in response to S100b in actinomycin D-pretreated samples is due both to a balance between S100b-induced stability and basal transcription. On the other hand, in the mRNA decay experiment in Fig. 4, actinomycin treatment was done after S100b to prevent any further basal IP-10 transcription, and here we observed that S100b can promote IP-10 mRNA accumulation by delaying/inhibiting its degradation or destabilization.

Additional support for this is derived from our data presented in Fig. 5 (B and C). The PCR blot of Luc mRNA in Fig. 5C shows that when we fuse IP-10 3'-UTR to luciferase gene, there is a dramatic decrease (destabilization) in luciferase mRNA levels, and this is restored by S100b treatment, which stabilizes the luciferase mRNA (third and fourth lanes). On the other hand, this kind of regulation is not seen when we used the control construct with IP-10 3'-UTR in the antisense orientation (fifth and sixth lanes). The corresponding luciferase activity data in Fig. 5B shows a similar supportive trend. Because the luciferase constructs are not controlled by the IP-10 promoter, these data further confirm the absence of transcription mechanisms. Further support that the IP-10-UTR plays a main role in IP-10 mRNA stability was obtained by measuring luciferase mRNA levels after actinomycin pretreatment (Fig. 6). These data confirm our aforementioned observation that, after blocking basal transcription with actinomycin, the presence of the IP-10 3'-UTR leads to rapid destabilization of the remaining Luc mRNA, but S100b prevents this and increases its half-life.

Our novel results clearly demonstrated the presence of S100b-specific positive regulatory cis-acting elements in the IP-10 3'-UTR because there was a 3-fold increase in Luc activity in S100b-treated cells when transfected with IP-10 UTR-Luc constructs. UV cross-linking and EMSA assays using IP-10-UTR RNA revealed RNA-protein complex formation with nuclear proteins in S100b-treated cells, but not with cytoplasmic protein fractions, confirming that the transacting factor is nuclear in origin. Furthermore, using deletion constructs we were able to narrow down the S100b-specific protein interaction region in the IP-10 UTR to a 180-nt segment that does not contain the AUUUA region. This suggests that the 3' end 180-nt segment of the RNA encompasses novel cis-acting elements sufficient to form the protein complex.

We noted that cycloheximide blocked S100b-induced IP-10 mRNA accumulation, suggesting that new protein synthesis may be needed to regulate mRNA stability. EMSAs with cycloheximide-treated THP-1 cells showed that S100b-induced RNA protein complex is abrogated in these treated cells. Thus S100b treatment may induce new protein synthesis from preexisting mRNA or other unknown post-translational mechanisms. This potential additional role in translational regulation will be the focus of future studies. The current data suggest that S100b can induce a protein by as yet unknown mechanisms, and this protein may be involved directly or indirectly in RNP complex formation leading to IP-10 mRNA stabilization.

The mechanisms whereby S100b and RAGE ligation lead to changes in target gene expression are not fully understood. Previous studies (5, 20) along with our current observations suggest that S100b may induce gene expression through a combination of transcriptional, post-transcriptional and message stabilization mechanisms. Changes in message stabilization occur through specific interaction with RNA-binding proteins. It is possible that the synthesis of novel nuclear proteins by RAGE ligation and diabetic conditions can greatly stabilize and increase the expression of inflammatory genes such as IP-10 by subsequently binding to their 3'-UTR regions. Future studies will be aimed at determining the identity and characterization of mechanisms of action of S100b-specific RNA-binding proteins that stabilize IP-10 mRNA and possibly other key mRNAs. Furthermore, this could uncover key proteins in blood cells that regulate inflammatory genes by augmenting message stability. Taken together we have shown for the first time that increased mRNA stability is a key mechanism for the increased expression of monocyte inflammatory genes such as IP-10 during RAGE activation associated with diseases such as diabetes and Alzheimer disease.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants RO1 DK065073 (to R. N.) and RO1 NS32151 (to R. M. R.), funds from the Juvenile Diabetes Research Foundation (to R. N.), and in part by General Clinical Research Center Grant NCRR MO1RR00043 from the National Center for Research Resources (awarded to the City of Hope). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Diabetes, Beckman Research Institute of City of Hope, 1500 East Duarte Rd., Duarte, CA 91010. Tel.: 626-256-4673 (ext. 62289); Fax: 626-301-8136; E-mail: rnatarajan{at}coh.org.

2 The abbreviations used are: IFN, interferon; AGE, advanced glycation end product(s); RAGE, receptor of AGE; UTR, untranslated region; PBMC, peripheral blood monocytes; RT, reverse transcription; NG, normal glucose; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; nt, nucleotide(s); EMSA, electromobility shift assay; Luc, luciferase; ARE, adenine-uridine-rich element; IP, interferon-{gamma}-inducible protein. Back

3 N. Shanmugam and R. Natarajan, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. I. Gaw-Gonzalo, Dr. M. Al-Sayed, A. Geva, and also the staff of the General Clinical Research Center for all of the help.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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