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The Journal of Immunology, 2003, 171: 6780-6787.
Copyright © 2003 by The American Association of Immunologists

Hyaluronic Acid or TNF-{alpha} Plus Fibronectin Triggers Granulocyte Macrophage-Colony-Stimulating Factor mRNA Stabilization in Eosinophils Yet Engages Differential Intracellular Pathways and mRNA Binding Proteins1

Stéphane Esnault and James S. Malter2

Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, WI 53792


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Eosinophils (Eos) accumulate in airways and lung parenchyma of active asthmatics. GM-CSF is a potent inhibitor of Eos apoptosis both in vitro and in vivo and is produced by activated fibroblasts, mast cells, T lymphocytes as well as Eos. Cytokine release by Eos is preceded by GM-CSF mRNA stabilization induced by TNF-{alpha} plus fibronectin. Hyaluronic acid (HA) is a major extracellular matrix proteoglycan, which also accumulates in the lung during asthma exacerbations. In this study we have analyzed the effects of HA on Eos survival and GM-CSF expression. We demonstrate that like TNF-{alpha} plus fibronectin, HA stabilizes GM-CSF mRNA, increases GM-CSF secretion, and prolongs in vitro Eos survival. GM-CSF mRNA stabilization accounts for most of the observed GM-CSF mRNA accumulation and protein production. Unlike TNF-{alpha} plus fibronectin, GM-CSF mRNA stabilization induction by HA requires continuous extracellular signal-regulated kinase phosphorylation. Finally, to identify potential protein regulators responsible for GM-CSF mRNA stabilization, immunoprecipitation-RT-PCR studies revealed increased GM-CSF mRNA associated with YB-1, HuR, and heterogeneous nuclear ribonucleoprotein (hnRNP) C after TNF-{alpha} plus fibronectin but only hnRNP C after HA. Thus, our data suggest that both TNF-{alpha} plus fibronectin and HA, which are relevant physiological effectors in asthma, contributes to long-term Eos survival in vivo by enhancing GM-CSF production through two different posttranscriptional regulatory pathways involving extracellular signal-regulated kinase phosphorylation and RNA binding proteins YB-1, HuR, and hnRNP C.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A predominant feature of asthma is the accumulation of eosinophils (Eos)3 in the lung parenchyma and airways (1). The pulmonary persistence of Eos likely reflects the anti-apoptotic effects of GM-CSF at the site of inflammation. We and others have demonstrated that Eos are a significant source of GM-CSF (2, 3, 4) and thus capable of supporting their own survival by autocrine or paracrine mechanisms (5, 6, 7). Eos produce GM-CSF after stimulation with a variety of agonists including fibronectin via interactions with integrins {alpha}4{beta}1 and {alpha}4{beta}7 (8), mAbs against CD40, CD9, or CD32, and cytokines including TNF-{alpha} or IL-15 (5, 7, 9, 10, 11, 12). We have recently demonstrated that the combination of TNF-{alpha} and fibronectin phosphorylates extracellular signal-regulated kinase (Erk) leading to GM-CSF mRNA stabilization, GM-CSF secretion and GM-CSF-dependent Eos survival (5, 13).

However, given the large number of potential activators encountered by Eos as they migrate from the peripheral blood into the lung, the list mentioned is likely incomplete. One intriguing candidate mediator is hyaluronic acid (HA), a major extracellular matrix (ECM) proteoglycan. HA is a glycosaminoglycan polymer consisting of repeating sugar residues D-glucuronic acid and N-acetyl-D-glucosamine. HA exists in both a high molecular mass form (1–6 x 106 Da) and a polydisperse lower molecular mass form (0.3–0.5 x 106 Da), the latter predominating under inflammatory conditions (14). Beside its classical functions as an ECM component (matrix structure, water balance, lubrication, and others), HA can activate B and T lymphocytes (15), induce intracellular protein phosphorylation (16), and enhance the surface expression of molecules involved in Ag presentation (17). Low molecular mass HA induces macrophages to express numerous cytokines and chemokines as well as inducible NO synthase (18, 19). Concentrations of HA as high as 4 mg/ml are present in human tissues (20), including the lung (21). HA levels were increased in bronchoalveolar lavage fluid from healthy donors after histamine inhalation (22). In 1978, Sahu and Lynn (23) reported that HA was the only glycosaminoglycan present in the pulmonary secretions of patients with asthma and has been localized by immunohistochemistry to the submucosa and around the smooth muscle bundles in the airways of severe asthma patients (24).

CD44, which is a receptor for HA, is expressed by human Eos. CD44 was present on resting Eos and up-regulated after in vitro activation with IL-5 or in vivo on hypodense Eos (25). CD44 mRNA has also been detected in resting Eos by gene chip expression analysis. In these studies, expression was dramatically increased after activation with IL-5 or GM-CSF.4Recently, low molecular mass HA prolonged Eos survival, through a GM-CSF-dependent mechanism, which was partially inhibited by a blocking anti-CD44 mAb (26).

Many cytokines and proto-oncogenes are regulated at the posttranscriptional level. We have shown that GM-CSF mRNA stability in PBMC or Eos was dependent on the AU-rich elements (AREs) in the 3'-untranslated region (UTR) (27). AREs are found in many rapidly degraded cytokine and proto-oncogene messages and mediate both rapid decay in quiescent cells and stability in activated cells (28). Several ARE-binding proteins have been identified that destabilize (tristetraprolin) or stabilize (HuR, YB-1) ARE-containing mRNAs (29, 30, 31, 32). It has been proposed that cytokine mRNA stabilization likely occurs when sequence specific binding proteins interact with, and possibly mask the AREs from cellular ribonucleases (33, 34).

The heterogeneous nuclear ribonucleoproteins (hnRNP) are nucleic acid binding proteins involved in various aspects of mRNA regulation. Among them the most abundant, hnRNP C1 and C2 proteins form stable tetramers with RNA (35) and contain both an RNA recognition motif and a unique basic ZIP-like RNA binding domain (36). In a previous study, hnRNP C extracted from PHA-activated human lymphocyte cytoplasmic lysates bound in vitro to AU-rich RNA (37). However, the mRNA binding activity of proteins like hnRNP C in vivo or in asthma remains to be explored.

In this study we have analyzed the contribution and mechanism of GM-CSF posttranscriptional regulation in Eos following HA activation. We show that GM-CSF mRNA accumulation in response to HA is a consequence of GM-CSF mRNA stabilization, with obligatory signals through Erk1/2. Erk activation, which is an early event in TNF-{alpha} plus fibronectin-activated Eos, needs to be maintained continuously in HA-activated cells to sustain GM-CSF mRNA stabilization and accumulation. CD44 blocking Ab partially inhibits HA-induced increases in GM-CSF mRNA steady-state levels, suggesting alternative pathways must exist. Of note, HA-increased stabilization does not involve YB-1 as we have shown previously in TNF-{alpha} plus fibronectin-activated Eos but rather enhanced hnRNP C interactions with GM-CSF mRNA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

HA from human umbilical cord was purchased from ICN Pharmaceuticals (Costa Mesa, CA). Recombinant human TNF-{alpha} was purchased from R&D Systems (Minneapolis, MN). Human cellular fibronectin was purchased from Sigma-Aldrich (St. Louis, MO). Human plasma fibronectin-coated 96-well tissue culture plates were bought from BD Biosciences (Bedford, MA). The blocking anti-CD44 Ab (clone 5F12) and the isotype control monoclonal IgG1 were respectively provided by Lab Vision (Fremont, CA) and by BD Biosciences. The blocking anti-Toll-like receptor (TLR)-4 Ab (clone HTA125) was obtained from eBioscience (San Diego, CA). The anti-GM-CSF polyclonal Ab was purchased from R&D Systems. The anti-TatYB-1 is a rabbit polyclonal raised against recombinant TatYB-1 fusion protein (32) and purified with the ImmunoPure G IgG Purification kit (Pierce, Rockford, IL), anti-TatYB-1 was biotinylated using the FluoReporter MiniBiotin-XX Protein Labeling kit and visualized with streptavidin-HRP conjugated secondary (Molecular Probes, Eugene, OR). The anti-HuR mAb and the anti-hnRNP C1 and C2 were, respectively, purchased from Molecular Probes and Santa Cruz Biotechnology, Santa Cruz, CA. Anti-rabbit HRP-conjugated secondary Ab and the ECL Western blotting detection system (Amersham, Pharmacia, Piscataway NJ) were used to detect primary Abs. The ERK inhibitor (PD98059) was supplied by New England Biolabs (Beverly, MA). The inhibitors were dissolved in DMSO and added to cell cultures 15 min before activation or after as indicated (see Fig. 7).



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  		FIGURE 7. Erk activation is continuously required for GM-CSF mRNA accumulation. A, GM-CSF or {beta}-actin RT-PCR was performed with resting (R) or low molecular mass HA-treated (100 µg/ml) Eos. In some cases, the specific Erk1/2 inhibitor (PD98059) was added 15 min before (PDb) or after (PDa) HA from 0 to 4 h. The specific inhibitors SB203508 or SB202190 were added 15 min before HA. Positive control (GM-CSF mRNA in vitro synthesized) and negative control (no cDNA) for RT-PCR are shown. B, The same experiment was performed but Eos were activated with TNF-{alpha} plus fibronectin (T+F) instead of HA. Results shown are representative of three experiments with three different donors.

 
Subjects and Eos preparation

Peripheral blood was obtained by venipuncture from patients with allergic rhinitis or asthma without current symptoms. All subjects have a clinical record at University of Wisconsin Hospital and an established and relatively constant count of Eos per cubic millimeter of blood. Normal blood Eos in sufficient numbers from normal donors were not available for these studies.

All informed consent was acquired according to a protocol approved by the University of Wisconsin Human Subjects Committee.

Peripheral blood Eos were purified using a negative immunomagnetic procedure as previously described (38). Briefly, heparinized whole blood was centrifuged (700 x g, 20 min) over a Percoll density gradient (density 1.090 g/ml; Pharmacia Biotech, Piscataway, NJ) to separate mononuclear cells from granulocytes. After removal of the mononuclear cell band, RBC were lysed by twice incubating (for 30 s) in sterile deionized water. The remaining white blood cells were incubated with anti-CD16-conjugated immunomagnetic microbeads and collected by exposure to a magnetic field (AutoMac system; Miltenyi Biotec, Auburn, CA). The cells in the eluent were stained (Diff Quik; Baxter, Miami, FL), and 400 cells were examined microscopically. The cells were used only if >99% were Eos. If few contaminating mononuclear cells were observed, the cell population underwent a second separation by adding anti-CD14 mAb-conjugated magnetic beads. After isolation, Eos were maintained in RPMI 1640 medium, 10% FCS, and 50 µg/ml gentamicin (all from Life Technologies, Grand Island, NY), at 37°C in a 5% CO2 environment.

Eos were activated with low molecular mass HA (100 µg/ml) in 96-well tissue culture plates (BD Biosciences, Meylan, France) or with TNF-{alpha} (10 ng/ml) plus 20 µg/ml soluble cellular fibronectin in 96-well tissue fibronectin-coated plates (BD Biosciences).

Eos survival

Eos (1 x 106 cells/ml) were cultured in 96-well tissue culture plates. Peripheral blood Eos viability was assessed by trypan blue exclusion on a hemocytometer (39). The percentage of peripheral blood Eos survival was determined by the following: survival (%) = (number of viable cells at 96 h)/(number of viable cells at 0 h) x 100.

Where used, neutralizing anti-GM-CSF (5 µg/ml; R&D Systems) was added at the initiation of culture.

RT-PCR

RT-PCR for GM-CSF was performed as previously described (5). Briefly, after 5 h of activation, 1 x 106 Eos/ml were pelleted, lysed in TriReagent (Molecular Research Center, Cincinnati, OH) and total RNA isolated as described by the manufacturer. RT-PCR was performed using the manufacturer’s protocol (Promega, Madison, WI). Primers for {beta}-actin mRNA were complementary to nucleotides 227–246 and 429–410, whereas those for GM-CSF mRNA corresponded to nucleotides 241–260 and 438–421. Thirty cycles were performed for {beta}-actin or GM-CSF. Because the signals obtained in an ethidium bromide gel were generally weak for the GM-CSF PCR products, Southern blotting was performed using a radioactively labeled GM-CSF cDNA probe as previously described (5).

mRNA transfection

Particle-mediated gene transfer of expression vectors or in vitro transcribed mRNAs into cultured cells was performed using the Accell Gene-Gun (PowderJect Vaccines, Madison, WI), as previously described (27, 40). Briefly, mRNAs in aqueous solution were precipitated at -20°C for 1 h with 1 volume of 2-propanol and 0.10 volume of 5 M ammonium acetate onto 1 µm gold beads at a concentration of 5 µg of mRNA per milligram of gold beads. The input mRNA of 80–95% was typically loaded onto the beads. Successive transfections of 2 x 106 cells were pooled and washed twice in culture medium to remove any extracellular mRNAs. The transfected Eos were placed in culture at 1 x 107 cells/ml.

Northern blotting

At indicated times, cells were pelleted, lysed in TriReagent (Molecular Research Center) and total RNA was quantitatively isolated and analyzed by Northern blotting with a radioactively labeled, cDNA GM-CSF or actin probe as previously described (27). GM-CSF mRNA signals were normalized to those for actin mRNA to accommodate any differences in the extraction, gel loading, and transfer of total RNA. After stringent washing at 50°C for 5 min with 0.1x SSC, 0.1% SDS, the blots were quantitated by PhosphorImager (model 445SI; Molecular Dynamics, Sunnyvale, CA).

Immunoprecipitation

As already described (32), resting Eos or Eos activated with TNF-{alpha} plus fibronectin or HA were snap frozen at -80°C and cell pellets were dissolved in 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 200 µg/ml Pefabloc, protease inhibitor mixture P8340 (Sigma-Aldrich), 1 mM DTT, 1 U recombinant RNasin/µl (Promega), 1% Triton X-100, and 0.1% SDS and 1% Nonidet P-40 by passing them through a 29-gauge needle. Cleared lysate was made by centrifugation at 12,000 x g for 3 min, and 12 µg of Ab was added for 2 h with rocking at 4°C. Protein G-agarose beads (Sigma-Aldrich) were added and incubation was continued overnight. Pellets were washed five times with lysis buffer (without detergent) and the last wash was split with 40% dissolved in TriReagent (Molecular Research Center) and 60% dissolved in SDS-PAGE loading buffer. RNA was isolated according to manufacturer’s recommendation. RT reactions were primed with oligo(dT) primers (Life Technologies) and PCR/Southern blotting was as previously described.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HA increases GM-CSF mRNA steady-state level and stability in Eos

After purification, human peripheral blood Eos were incubated with 100 µg/ml HA, which was the optimum concentration based on preliminary dose-response experiments (data not shown), as well as prior studies (41, 42). As shown in Fig. 1A, 5 h incubation with low molecular mass HA enhanced GM-CSF mRNA accumulation from 2- to 20-fold over that expressed in resting Eos. In a previous study (26), high molecular mass HA had a lesser effect on Eos. We incubated Eos with either high or low molecular mass HA and measured GM-CSF RNA accumulation by RT-PCR. As shown in Fig. 1B, the low molecular mass HA reproducibly enhanced GM-CSF mRNA accumulation nearly 100% over high molecular mass HA. As we had previously observed that peripheral blood Eos treated with TNF-{alpha} plus fibronectin or Eos from bronchoalveolar lavage stabilized GM-CSF mRNA (5), we evaluated whether HA had a similar effect. Control or Eos treated for 5 h with low molecular mass HA were transfected with exogenous in vitro transcribed GM-CSF mRNA by particle-mediated gene transfer and its decay measured by Northern blot. Specific mRNA could not be detected in the absence of transfection with GM-CSF mRNA loaded beads (Ref. 27 and data not shown). As shown in Fig. 2, GM-CSF mRNA decay was extremely rapid in untreated cells (t1/2 {approx} 10 min), which slowed by >2-fold after HA activation. Multiple donors showed similar data, which is presented as a mean t1/2 in Table I. This augmentation of GM-CSF mRNA stability is quite similar to our previous observations with peripheral blood Eos activated with TNF-{alpha} plus fibronectin or Eos from bronchoalveolar lavage after segmental challenge (5).



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  		FIGURE 1. GM-CSF mRNA expression is increased in activated Eos. GM-CSF and {beta}-actin RT-PCR were performed with total RNA extracted from resting Eos (R) or those activated with low molecular mass HA or high molecular mass HA (100 µg/ml) for 5 h. A and B, Southern blots (upper panels) using 32P-labeled GM-CSF cDNA probes, whereas the bottom panels are ethidium bromide stained agarose gels of actin RT-PCR. Positive (+) RT-PCR control (GM-CSF mRNA synthesized in vitro) and negative (-) control (no cDNA included) are shown. A, Represents numerous experiments and (B) is representative of three different donors.

 


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  		FIGURE 2. GM-CSF mRNA is stabilized in peripheral blood Eos activated by low molecular mass HA. Resting peripheral blood Eos (resting) or HA-activated Eos were transfected with GM-CSF mRNA. At the indicated time points, equal numbers of cells were harvested and total RNA was quantitatively isolated and Northern blotted with 32P-labeled GM-CSF or {beta}-actin cDNA probes. Signals were visualized using a PhosphorImager. This experiment is representative of three experiments with three different donors.

 

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  		Table I. GM-CSF mRNA half-life time is doubled after low molecular mass HA activationa

 
HA increases GM-CSF mRNA steady-state level partially via CD44

There is compelling evidence that CD44 is the main cellular receptor for HA. To elucidate the signaling pathways between HA binding and GM-CSF mRNA stabilization, we first sought to confirm HA-mediated signaling required CD44 function by blocking this receptor with an mAb. Fig. 3 shows that the mAb anti-CD44 inhibited >60% HA-induced GM-CSF accumulation whereas the control IgG1 had a modest effect (p < 0.032). Abs to TLR-4 (10 µg/ml), as TLR-4 has been proposed as a potential receptor for HA (43), had similar inhibitory effects as control IgG1 (34.4 ± 6.2%). Control IgG1 mediated decreases in GM-CSF mRNA, which suggests receptors for Fc{gamma}R can influence Eos function. Recently, it was reported that Fc{gamma}R ligation with IgG totally repressed low molecular mass HA-induced IL-12 production by macrophages (44).



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  		FIGURE 3. Low molecular mass HA induces GM-CSF accumulation partially via CD44. GM-CSF and {beta}-actin RT-PCR were performed with total RNA extracted from Eos activated with HA (100 µg/ml) for 5 h, which had been pretreated with blocking anti-CD44 (10 µg/ml) (CD44) or control IgG1 (10 µg/ml) (IgG1) for 60 min. Southern blots using a 32P-labeled GM-CSF cDNA probe or ethidium bromide stained agarose gels of actin RT-PCR were performed. The signals obtained from three donors were quantified, normalized with {beta}-actin, and the percentage of inhibition of GM-CSF accumulation after anti-CD44 (CD44) or IgG1 treatments were calculated. The paired t test was performed and the two groups were found significantly different (p < 0.032).

 
HA-prolonged Eos survival is GM-CSF dependent

Eos survival as measured by trypan blue exclusion at 4 days in culture was 9% for resting Eos that increased to 30% after HA activation (p < 0.001, Fig. 4A). HA-prolonged survival was completely inhibited by anti-GM-CSF Ab (p = 0.002, Fig. 4A). Isotype polyclonal IgG control or anti-IL-5 also had no effect on HA-induced survival activity (data not shown). Therefore, HA-mediated increased Eos survival was entirely GM-CSF dependent. Despite the fact that high molecular mass HA had modest effects on Eos function and anti-CD44 Ab largely blocked low molecular mass HA effects, LPS contamination was a potential confounding variable. Thus, we treated Eos with LPS as high as 200 ng/ml but observed no change in Eos survival from control (Fig. 4B). Therefore, the data support a specific interaction between low molecular mass HA and CD44, which triggers GM-CSF mRNA stabilization, GM-CSF secretion, and enhanced Eos survival.



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  		FIGURE 4. Eos survival is prolonged by low molecular mass HA via a GM-CSF dependent mechanism. Cell viability was determined by trypan blue exclusion after 4 days in culture with or without HA or LPS (2, 20, or 200 ng/ml). A, Eos were activated with 100 µg/ml HA or vehicle alone (control). Neutralizing anti-GM-CSF mAb was added at the beginning of the culture. Each value represents the mean of three different donors ± SEM. For statistical analysis, the one-way ANOVA was used. B, Eos survival was assessed after 4 days of incubation with HA or LPS. Each value is representative of two experiments with two different donors.

 
HnRNP C binds to GM-CSF mRNA in activated Eos

In a previous study, we demonstrated that YB-1 bound and stabilized GM-CSF mRNA in TNF-{alpha} plus fibronectin Eos (32). As shown in Fig. 5AI and 5BI, HA-induced GM-CSF mRNA accumulation was generally slightly less than what we observed with TNF-{alpha} plus fibronectin. However despite many attempts we were unable to immunoprecipitate GM-CSF mRNA with anti-YB-1 Abs in HA-activated Eos whereas the YB-1/GM-CSF mRNA interaction was always observed in TNF-{alpha} plus fibronectin-activated Eos (Fig. 5AII). HuR, which has been established as a major AU-rich mRNA binding protein (30), showed the same interaction profile as YB-1 even though a faint GM-CSF signal from HA-activated Eos was occasionally detectable (Fig. 5BII). Unexpectedly, hnRNP C bound to GM-CSF mRNA in HA-activated Eos (Fig. 6A). This interaction was even more striking after HA or TNF-{alpha} plus fibronectin-activated Eos obtained from another donor (Fig. 6B). These data suggest that HA or TNF-{alpha} plus fibronectin signaling activates hnRNP C to interact with GM-CSF mRNA.



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  		FIGURE 5. GM-CSF mRNA does not interact with YB-1 or HuR protein in low molecular mass HA-activated Eos. For each donor (AI, BI), GM-CSF mRNA steady-state levels in resting (Rest) or after 5 h incubations with either HA or TNF-{alpha} plus fibronectin (T+F) were determined by RT-PCR as described in Materials and Methods. For each condition, whole cell lysates underwent immunoprecipitation with either anti-YB-1 (AII) or anti-HuR (BII) Ab. Western blots (WB) were conducted with part of the immunoprecipitate pellet and the Abs shown (anti-YB-1 or anti-HuR). A positive control (C+) was included for each Western blot. The positive control for YB-1 and HuR were prepared from an Eos-like cell line (AML14.3D10) lysates as previously described. The other part of the immunoprecipitate pellet was used for GM-CSF RT-PCR and Southern blot with a radiolabeled GM-CSF cDNA probe (SB).

 


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  		FIGURE 6. GM-CSF mRNA interacts with hnRNP C in low molecular mass HA and TNF-{alpha} plus fibronectin-activated Eos. A, Lysates from resting (Rest) or HA-activated Eos were immunoprecipitated with polyclonal anti-hnRNP C. The upper panel shows a Western blot performed with the same Ab as for immunoprecipitation. The lower panel is a Southern blot with radiolabeled GM-CSF cDNA probes after GM-CSF RT-PCR of part of the immunoprecipitate pellet. B, This experiment was repeated with another donor after TNF-{alpha} plus fibronectin activation. The PhosphorImager signals obtained after RT-PCR and Southern blot are presented.

 
Erk phosphorylation is a continuous and necessary condition for HA-induced GM-CSF mRNA accumulation

We have demonstrated in a recent report that Erk phosphorylation was necessary and sufficient for GM-CSF mRNA stabilization after TNF-{alpha} plus fibronectin activation treatment (27). Based on Western blot, phosphorylation reached a maximum at 30 min after activation, which was prevented by pretreatment of Eos with PD98059 (27). HA also induced Erk phosphorylation, which was typically detectable from 30 min to at least 60 min after activation but showed some variations in kinetics from donor to donor (data not shown). Based on these results, PD98059 was added to Eos cultures 15 min before HA, which completely prevented GM-CSF mRNA accumulation (Fig. 7A). SB203580 or SB202190, respective inhibitors of p38 or p38 and JNK (27, 45) had a small effect on HA-induced GM-CSF mRNA expression (Fig. 7A). We observed similar data with these inhibitors on TNF-{alpha} plus fibronectin-treated Eos (27). Overall, Fig. 7A suggests that although all the mitogen-activating protein kinases (MAPK) are involved, Erk1/2 activation is predominant for GM-CSF mRNA accumulation. We next asked whether transitory or continuous Erk activation was necessary to maintain GM-CSF mRNA stability and accumulation. Thus PD98059 was added at various times after HA or TNF-{alpha} plus fibronectin, as seen in Fig. 7A. Erk inhibition 4 h after HA (1 h before cell harvest and Northern blotting) prevented GM-CSF mRNA accumulation. After TNF-{alpha} plus fibronectin, GM-CSF mRNA accumulation was inhibited only when PD98059 was added before but had no significant effects when added after activation (Fig. 7B). This suggests that TNF-{alpha} plus fibronectin induces Erk activation, which need only be brief to up-regulate GM-CSF mRNA accumulation. HA, conversely, must continuously signal through Erk. These data suggest signaling divergence downstream from the MAPK cascade, despite affecting GM-CSF mRNA decay to an equivalent degree.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we show that GM-CSF mRNA stabilization and GM-CSF-dependent prolonged Eos survival are tightly correlated with hnRNP C binding to GM-CSF mRNA after either HA or TNF-{alpha} plus fibronectin activation. These results implicate hnRNP C as a major GM-CSF mRNA binding protein in activated Eos. Previously, RNA mobility shift assays demonstrated hnRNP C bound in vitro to 3'-UTR AREs in cytokine mRNAs. HnRNP C binding activity to GM-CSF mRNA increased after PHA-mediated activation of human PBMC (37) or to IL-2 mRNA in cycling MLA 144 cells (46). Moreover, in the latter cell line, hnRNP C binding activity was closely correlated with IL-2 mRNA stabilization. Our laboratory has previously demonstrated in a cell-free system that hnRNP C stabilized the amyloid precursor protein mRNA (47) by binding to an uridine rich, 29 base element in the 3'-UTR (48). TNF-{alpha} plus fibronectin or HA-mediated effects presumably include altered hnRNP C phosphorylation given MAPK activation. This is consistent with a prior study showing hnRNP C binding activity was enhanced by dephosphorylation in activated cells (49). The fact that YB-1 or HuR does not immunoprecipitate with GM-CSF mRNA after HA treatment implies the existence of multiple and non-overlapping pathways leading to GM-CSF mRNA stabilization, accumulation, and presumably translation. However given the complexity of the 40S ribonucleoprotein complex formed by hnRNP proteins A, B, and C and many auxiliary proteins (50), it is possible Eos activation with HA but not with TNF-{alpha} plus fibronectin induces conformational changes resulting in a loss of epitopes of HuR and YB-1. Immunoprecipitation with anti-hnRNP C Ab followed by immunoblotting with anti-HuR or YB-1 should resolve this issue. Besides GM-CSF, other ARE-containing mRNAs may be stabilized following HA activation. However, as additional regulatory domains located in the 5'-UTR participate in the regulation of ARE-containing IL-2 mRNA (51), the decay rate of additional cytokine mRNAs after HA must be addressed individually.

Only a few physiologically relevant activators have been identified, which increase Eos survival by inducing autocrine GM-CSF production. In this study, we demonstrate that the abundant ECM molecule, HA, induces GM-CSF secretion after causing GM-CSF mRNA stabilization. The >2-fold increase in GM-CSF mRNA t1/2 (22 min ± 6.25, Table I) is nearly identical to that observed after in vitro treatment of resting Eos with ionomycin or TNF-{alpha} plus fibronectin or Eos derived from bronchoalveolar lavage 48 h after in vivo allergen challenge (5). These data suggest that there may be a maximal stabilization of GM-CSF mRNA, which can be achieved by multiple agonists through distinct signaling cascades and effector mechanisms. Although a 2-fold increase in t1/2 may seem modest, GM-CSF secretion was increased by 10–20-fold under such conditions in PBMC (40). Consistent with enhanced GM-CSF secretion, in vitro, Eos survival was reproducibly increased by 3–4-fold. The amount of GM-CSF produced likely depends on the duration of stabilization, which in turn depends on HA-mediated Erk activation. Of note, HA in human sera has a t1/2 of only a few minutes (52). Once initiated, signaling may be terminated by HA-CD44 internalization, which has been observed in macrophages (53) and during lung inflammation (54). Internalized HA is then broken down in an endosomal-lysosomal pathway.

We cannot rule out the possibility that part of the in vitro, GM-CSF-dependent prolongation of survival following HA activation reflects the release of stored GM-CSF (55). The release of preformed cytokines from Eos is typically described as an early and transitory event (4, 56). However, Eos survival requires prolonged rather than episodic exposure to GM-CSF (6). A hallmark of our data is that Erk signaling must be continuous to maintain GM-CSF mRNA stabilization and by inference, GM-CSF production. This suggests that after degranulation, other cellular regulatory events such as mRNA stabilization are elicited to maintain GM-CSF production. Indeed, shortly after Eos stimulation with IFN-{gamma}, confocal microscopy revealed increased IL-6 staining in granules (57). Although the mechanism for this effect is unclear, new translation of preexisting and possibly stabilized mRNA is a possible explanation.

Based on our results, CD44 seems to be the main receptor involved in HA signaling and GM-CSF production. There is precedent for the induction of proinflammatory mediators by the HA-CD44 interaction. Ligation of CD44 with HA on macrophages induces the production of IL-12 (58), monocyte chemoattractant protein-1 (59), and inducible NO synthase (19). Recently, GM-CSF and IL-6 were up-regulated in bone marrow macrophages by a mAb directed against CD44 v4 and v6 (60). However, CD44 is unlikely to be the only receptor involved in HA-Eos interactions as blocking Ab prevented only 60% of GM-CSF mRNA accumulation. Other HA-binding proteins have been described, including TLR-4 (43), receptor for HA-mediated motility (RHAMM) (61), and TNF-stimulated gene (TSG)-6 (62). However, as previously mentioned in Results, a blocking mAb anti-TLR-4 had no greater effect than the control IgG. We did not try to block RHAMM because coding mRNA was never detected in resting or in IL-5 or GM-CSF activated Eos by microarrays.4 Finally, a poorly characterized macrophage receptor for HA has been suggested, which may participate in signaling (63). It is worth noting that CD44 also has affinity for additional ligands, including fibronectin (64). Low molecular mass HA present in the ECM interacts with CD44 on endothelial cells resulting in the up-regulation of VCAM-1 (65). Thus HA-CD44 ligature in Eos as well as other inflammatory cells in asthma can have pleiotropic effects.

In addition to TNF-{alpha} plus fibronectin (5), HA enhanced GM-CSF mRNA stability in Eos. In a previous report, we demonstrated that Erk activation, which has been implicated in many intracellular signaling cascades in Eos (66, 67, 68), was necessary and sufficient to induce GM-CSF mRNA stability (13). P38 had an intermediate effect whereas JNK, which has been described as constitutively activated in Eos (13, 68), did not participate. By using a specific Erk inhibitor (PD98059), we demonstrate that Erk is necessary for GM-CSF mRNA stability in HA-activated Eos. Most of the studies analyzing the role of MAPK on mRNA stability have used transformed cell lines (Jurkat, HeLa, THP-1, fibroblast-like or mast cell-like) as models, and have generally implicated p38 or JNK as critical kinases (51, 69, 70, 71, 72, 73, 74). A recent study showed IL-1-mediated cyclooxygenase-2 mRNA stability in an intestinal myofibroblast cell line was p38 rather than Erk dependent (75). Erk has, however, been involved in macrophage inflammatory protein-2 or nucleolin mRNA stability in fresh rat peritoneal neutrophils or human PBMC, respectively (76, 77). Whether mRNA regulation in primary cells or Eos specifically depends on ERK phosphorylation whereas mRNA regulation in cell lines depends on p38 and JNK remains unknown. We also show that Erk phosphorylation was continuously required for GM-CSF mRNA stabilization with the resumption of rapid decay after Erk inhibition. Continuous exposure to ionomycin was necessary to maintain IL-3 mRNA stabilization (78). Activated Erk has been physically linked to cytoskeletal-protein complexes after RHAMM-HA interactions, which triggers cell motility (79). Thus, it is tempting to speculate a similar role for Erk as a complex partner for GM-CSF mRNA binding proteins important in preventing RNase action. Interestingly, HA activation with subsequent Erk phosphorylation affected GM-CSF production through different downstream pathways than did TNF-{alpha} plus fibronectin. The former required continuous Erk activation and culminated in hnRNP C, whereas the latter was pulsatile, and induced HuR and YB-1 along with hnRNP C as ultimate effectors. As the degree of GM-CSF mRNA stabilization was equivalent after either agonist, the physiologic rational for this partial redundancy is unclear.


   Acknowledgments
 
We thank members of the University of Wisconsin, Specialized Center of Research-Asthma Research Group for stimulating ideas and in particular, Julie B. Sedgwick for providing eosinophils.


   Footnotes
 
1 This work was supported by National Institutes of Health P50HL56396 (to J.S.M.). Back

2 Address correspondence and reprint requests to Dr. James S. Malter, Waisman Center for Developmental Disabilities, 1500 Highland Avenue, R509T, Madison, WI 53705. E-mail address: jsmalter{at}facstaff.wisc.edu Back

3 Abbreviations used in this paper: Eos, eosinophils; ECM, extracellular matrix; Erk, extracellular signal-regulated kinase; TLR, Toll-like receptor; ARE, AU-rich element; HA, hyaluronic acid; RHAMM, receptor for HA-mediated motility; hnRNP, heterogeneous nuclear ribonucleoprotein; UTR, untranslated terminal region; JNK, c-jun NH2-terminal kinase; MAPK, mitogen-activating protein kinase. Back

4 M. E. Bates, L. Y. Liu, S. Esnault, J. B. Sedgwick, B. A. Stout, E. Fonkem, V. Kung, E. A. B. Kelly, D. M. Bates, J. S Malter, et al. Expression of IL-5- and GM-CSF-responsive genes in blood and airway eosinophils. Submitted for publication. Back

Received for publication March 18, 2003. Accepted for publication September 26, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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YB-1 Coordinates Vascular Smooth Muscle {alpha}-Actin Gene Activation by Transforming Growth Factor {beta}1 and Thrombin during Differentiation of Human Pulmonary Myofibroblasts
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R. Pullmann Jr., M. Juhaszova, I. L. de Silanes, T. Kawai, K. Mazan-Mamczarz, M. K. Halushka, and M. Gorospe
Enhanced Proliferation of Cultured Human Vascular Smooth Muscle Cells Linked to Increased Function of RNA-binding Protein HuR
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D. E. Sullivan, M. Ferris, D. Pociask, and A. R. Brody
Tumor Necrosis Factor-{alpha} Induces Transforming Growth Factor-{beta}1 Expression in Lung Fibroblasts Through the Extracellular Signal-Regulated Kinase Pathway
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Q. Peng, D. Lai, T. T.-B. Nguyen, V. Chan, T. Matsuda, and S. J. Hirst
Multiple {beta}1 Integrins Mediate Enhancement of Human Airway Smooth Muscle Cytokine Secretion by Fibronectin and Type I Collagen
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