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Originally published In Press as doi:10.1074/jbc.M600294200 on March 8, 2006

J. Biol. Chem., Vol. 281, Issue 18, 12451-12457, May 5, 2006
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Cell Type-specific Differential Induction of the Human {gamma}-Fibrinogen Promoter by Interleukin-6*

Hai Ou Duan{ddagger}§ and Patricia J. Simpson-Haidaris{ddagger}§1

From the Departments of {ddagger}Medicine/Hematology-Oncology Division, §Pathology and Laboratory Medicine, and Microbiology and Immunology, University of Rochester School of Medicine and Dentistry Rochester, New York 14642

Received for publication, January 11, 2006 , and in revised form, March 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
During an acute phase response, interleukin-6 (IL-6) and glucocorticoids up-regulate expression of the three fibrinogen (FBG) genes (fga, fgb, and fgg) in liver and lung epithelium; however, little constitutive lung expression occurs. Recently, we showed that the magnitude of Stat3 binding to three IL-6 motifs on the human {gamma}FBG promoter correlates negatively with their functional activity in hepatocytes, although these cis-elements are critical for promoter activity. We determined the role of IL-6-receptor-gp130-Stat3 signaling in IL-6 activation of the {gamma}FBG promoter in liver and lung epithelial cells. Although IL-6 induced {gamma}FBG promoter activity ~30-fold in HepG2 cells, it was increased only 2-fold in lung A549 cells. Equivalent production of gp130 was demonstrated in both cell types by Western blotting; however, lower production of both IL-6-receptor and Stat3 explains, in part, reduced activity of the {gamma}FBG promoter in lung cells. Dexamethasone potentiated IL-6 induction of the {gamma}FBG promoter 2.3-fold in both HepG2 and A549 cells for a combined increase in promoter activity of 70-fold or 4.5-fold, respectively. Dexamethasone potentiation is likely due to the induction of IL-6-receptor expression as well as prolonged intensity and duration of Stat3 activation. By circumventing IL-6-receptor-gp130-coupled signaling with ectopic expression of the granulocyte colony-stimulating factor receptor (GCSFR)-gp130(133) chimeric receptor, overexpression of Stat3 induced {gamma}FBG promoter activity 30-fold in A549 cells. Together, the data suggest tissue-specific differences in IL-6-receptor-gp130-coupled signaling, thereby limiting the extent of Stat3 activation and {gamma}FBG expression during lung inflammation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fibrinogen (FBG)2 is best known for its hemostatic role in platelet aggregation and fibrin clot formation at the site of vessel injury. In addition, FBG plays an important role in homeostasis. Disrupters of homeostasis, such as infection, inflammation, tissue injury, or neoplasia, result in the activation of the acute phase response. During inflammation, both local and systemic responses occur resulting in activation of various cell types to produce proinflammatory cytokines including interleukin (IL)-1beta, tumor necrosis factor-{alpha}, and IL-6 (1). FBG is one of a number of acute phase proteins (APPs) up-regulated 2–10-fold during inflammation (2–4). Increased levels of plasma FBG augment the immune responses of the host to restore homeostasis (reviewed in Ref. 5). FBG serves as a substratum for epithelial (6, 7) and mesenchymal (8) cell migration during wound repair. Furthermore, FBG and fibrin degradation products stimulate fibroblast proliferation (9), leading to enhanced deposition of matrix collagen, and production of inflammatory cytokines (7).

Although the liver is the major site of plasma FBG production, epithelial cells from extrahepatic tissues, including respiratory epithelium, are capable of producing FBG (10–17). In addition to FBG, other APPs such as haptoglobin (18), annexin I (19), and lipopolysaccharide-binding protein (20) are up-regulated in the lung during inflammation. Little constitutive expression of the three FBG genes, fga, fbg, and fgg, occurs in lung cells; however, de novo synthesis is induced by IL-6 and glucocorticoids (GCs) (10, 17, 21). Lung-derived FBG is secreted from the basolateral surface of alveolar epithelial cells, where it is deposited into the extracellular matrix (22). IL-1beta down-regulates constitutive and IL-6-induced FBG production in liver and lung epithelial cells. However, GC treatment overcomes IL-1beta-mediated inhibition of fga, fbg, and fgg expression and FGB protein production in a lung epithelial cell type-specific manner (23).

Stat3 (signal transducer and activator of transcription 3; also known as acute phase response factor) is the major transcription factor mediating regulation of IL-6-responsive APP genes. GCs have a synergistic effect on IL-6-mediated up-regulation of type II APP genes including FBG (24). Recently, we defined a minimal promoter-enhancer region of the human fgg that is regulated by IL-6 and GCs in liver HepG2 epithelial cells (25). Three type II IL-6 response elements (IL-6REs) were identified and shown to be critical for transactivation of the {gamma}FBG promoter by IL-6; however, the magnitude of Stat3 binding to these elements correlates negatively with their functional activity (25).

Based on the aforementioned observations, we hypothesized that FBG production is regulated systemically (liver) and locally (lung) in a cell type-specific manner during inflammation. We investigated mechanisms that account, at least in part, for the different induction patterns of {gamma}FBG by IL-6 and GCs in lung A549 when compared with liver HepG2 epithelial cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Reagents, and Antibodies—HepG2 and A549 cells were grown as described previously (10). Recombinant human proteins, IL-6, soluble IL-6 receptor (sIL-6R), and granulocyte colony-stimulating factor (GCSF), were purchased from Research Diagnostics, Inc. (Flanders, NJ), and dexamethasone (DEX) was from Sigma. Mouse monoclonal antibody against total Stat3 (F-2)X and Tyr705-p-Stat3 (B-7), rabbit polyclonal antibody to Ser727-p-Stat3 (sc-8001-R), IL-6R{alpha} (C-20) and gp130 (H-255), and goat anti-actin (C-11) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antibody J88B, which specifically recognizes human {gamma}FBG, was produced as described (26).

Western Blot Analysis—Cells were grown under various conditions as described in the figure legends. After treatment, whole cell lysates were prepared as described (10). After determining the protein concentration by Bradford assay, equivalent amounts of protein from the cell lysates were reduced and resolved by SDS-10%-PAGE and transferred to nitrocellulose membranes; specific proteins were detected by immunoblotting as described previously (25). Blots were stripped and reprobed with antibody to actin to control for the protein load in each lane.

Luciferase Reporter Plasmid Construction—A human {gamma}FBG promoter construct in pGL2-basic vector containing 1359 bp of the 5'-flanking region of the FBG {gamma}-chain gene (pGL2–1359) was kindly provided by Dr. M. L. Tenchini (University of Milan, Italy) (27). The chimeric receptor, GCSFR-gp130(133), which is composed of the extracellular domain of human GCSF receptor (GCSFR) fused to the transmembrane and first 133 residues of the cytoplasmic domain of human gp130, and the rat wild type Stat3(WT) were generously provided by Dr. H. Baumann (Roswell Park Cancer Institute, Buffalo, NY) (28, 29). GCSFR-gp130(133) contains the tyrosine required for Stat3-specific gp130 signal transduction.

Transient Transfection and Luciferase Assays—Transfection was performed using SuperFect (Qiagen, Valencia, CA) according to the manufacturer's instructions. The cells were grown to 40–50% confluence in 35-mm culture dishes and transfected with 1 µg of total DNA including 2.5 ng of pRL-SV40 (Promega, Madison, WI) to control for transfection efficiency. The total amount of DNA was kept constant by adding pSG5 (Stratagene, La Jolla, CA). The amounts of reporter and expression vectors used in each experiment are indicated in the figure legends. After SuperFect-DNA precipitates were incubated with cells for 16–18 h, fresh medium with various concentrations of IL-6 ± DEX was added, and the cells were incubated another 24 h (25). Cell lysates were prepared, and the relative-fold luciferase activity was determined using the Dual-Luciferase reporter kit (Promega).

Electrophoretic Mobility Shift Assay (EMSA)—After the desired treatment, A549 nuclear extracts were prepared as described (25). The rat {alpha}2-macroglobulin ({alpha}2M) type II IL-6RE, which binds with high affinity to Stat3 (30), was used to monitor activation of Stat3 in A549 cells in response to various treatments over time. Double-stranded probes were end-labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase; DNA-protein binding reactions were performed as described (25) using 2 µg of nuclear extract and 20 fmol of labeled probe for each reaction.

Reverse Transcriptase (RT)-PCR—Total RNA was isolated using an RNeasy Mini Kit (Qiagen). Two µg of total RNA were reverse-transcribed with RETROscriptTM (Ambion, Austin, TX). One-tenth of the mRNA:cDNA heteroduplex was used in the PCR. Oligonucleotide primers used in this study are: Stat3 forward, 5'-ATTCGGGAAGTATTGTCG-3', Stat3 reverse, 5'-GCCTCAGTCGTATCTTTC-3'; IL-6R forward, 5'-GCTCCACGACTCTGGAAACTATTC-3', IL-6R reverse, 5'-GTTTTGCTGAACTTGCTCCCGAC-3'; gp130 forward, 5'-GGTACGAATGGCAGCATACACG-3', gp130 reverse, 5'-CTGGACTGGATTCATGCTGAC-3'. Glyceraldehyde-3-phosphate dehydrogenase primers were described previously (31). PCR amplification was performed through 35 cycles at 94 °C for 45 s, 60 °C for 30 s, and 72 °C for 1 min; final extension of PCR products was performed for 7 min at 72 °C. For semiquantitative RT-PCR, products were sampled every fifth cycle beginning at cycle 25 as described (31) to ensure that the PCR amplification had not reached plateau phase. Equal volumes of reaction mixture from each sample were loaded on a 1.7% agarose gel and resolved by electrophoresis. Images were digitally captured and band intensity analyzed using Kodak 1D image analysis software, version 3.5.3 (Eastman Kodak Co.).


Figure 1
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  		FIGURE 1.
Comparison of {gamma}FBG expression by IL-6 and DEX in HepG2 and A549 cells. A and B, transient transfection reporter gene assays were conducted using 750 ng of {gamma}FBG-Luc reporter construct (pGL-1359) and 2.5 ng pRL-SV40 as internal control for transfection efficiency. A, {gamma}FBG promoter activity in HepG2 (white bars) and A549 cells (black bars) in response to increasing concentrations of either IL-6 and DEX or a fixed concentration of IL-6 with increasing doses of DEX as indicated. The data were collected from six independent experiments with each cell type. B, because the differences in {gamma}FBG promoter activity in A549 cells is difficult to observe when plotted on the same scale (1–100) with the promoter activity in HepG2 cells, the {gamma}FBG promoter activity in A549 cells treated as described in panel A was plotted on a scale ranging from 1 to 6. C, Western blot assays of {gamma}FBG induction in both cell lines. Cells were grown to near confluence and then incubated in medium containing various concentrations of IL-6 ± DEX for 24 h followed by whole cell lysate preparation. Forty µg of whole cell lysates were loaded in each lane. A representative fluorograph from three separate experiments is shown.

 
Statistical Analysis—Data are expressed as the mean ± S.E. The experiments were repeated 3–6 times, and the differences between means were examined for statistically significance (p value <0.05) by two-way analysis of variance using StatViewTM (Abacus Concepts Inc., Beverly, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Transactivation Potential of the Human {gamma}FBG Promoter Is Significantly Reduced in Alveolar Epithelial Cells When Compared with Hepatocytes—In HepG2 cells, pGL2–1359 {gamma}FBG promoter activity increased in a dose-dependent and statistically significant manner reaching a maximal 30-fold induction with 50 ng/ml IL-6 (Fig. 1A). These results agree with those previously reported using the minimal 600-bp promoter-enhancer region of the human {gamma}FBG gene (25). In contrast, {gamma}FBG promoter activity reached only a 2-fold increase over basal levels in A549 cells, although a modest IL-6 dose-dependent increase in {gamma}FBG transactivation was observed (Fig. 1, A and B).

In the absence of a consensus glucocorticoid response element, DEX potentiates IL-6-induced {gamma}FBG promoter activity 2-fold in HepG2 cells (25). In the presence of a functional glucocorticoid-response element (27), increasing concentrations of DEX alone did not appreciably induce {gamma}FBG promoter activity driven by pGL2–1359 in either HepG2 or A549 cells (Fig. 1, A and B). In contrast, in the presence of 50 ng/ml IL-6, 0.1 µM DEX maximally potentiated by 2.3-fold the IL-6-induced luciferase expression driven by the human {gamma}FBG promoter in each cell type, resulting in a combined 70-fold induction in HepG2 cells (p < 0.001) and a 4.5-fold induction in A549 cells (p = 0.0013).


Figure 2
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  		FIGURE 2.
Trace amounts of Stat3beta mRNA were detected in both A549 and HepG2 cells by RT-PCR. A549 and HepG2 cells were treated with IL-6 (50 ng/ml) for 2, 4, 6, or 12 h, after which the cells were collected for total RNA extraction. The primers and conditions used for RT-PCR are described under "Experimental Procedures." The 311-bp Stat3{alpha} and the 261-bp Stat3beta bands are indicated with arrowheads. GAPDH, glyceralde-hyde-3-phosphate dehydrogenase.

 
Nonstimulated A549 cells constitutively produced ~1–2% the amount of {gamma}FBG protein when compared with the constitutive levels produced by HepG2 cells when compared with similar amounts of actin for each cell type (Fig. 1C). Furthermore, IL-6 induced a corresponding dose-dependent increase in {gamma}FBG protein, which was also potentiated ~2–3-fold by 0.1 µM DEX when compared with 50 ng/ml IL-6 alone in both cell types (Fig. 1C). However, two striking differences were noted. First, maximum potentiation of IL-6-mediated transactivation of the {gamma}FBG promoter was achieved at lower concentrations of DEX in A549 cells (0.001 µM) when compared with HepG2 cells (0.1 µM). Second, maximal potentiation of {gamma}FBG promoter activity occurred over a broader range of DEX concentrations (0.001–1.0 µM) in A549 than in HepG2 cells (Fig. 1, A and B). Together with our previous observations (10), these results demonstrate that lung and liver epithelial cells respond to IL-6 and DEX to produce increased levels of {gamma}FBG protein by transcriptional activation of the {gamma}FBG promoter. However, the transactivation potential of the human {gamma}FBG promoter is significantly reduced in lung when compared with liver epithelial cells.

Stat3beta mRNA Is Minimally Expressed in Lung and Liver Epithelial Cells—Two naturally occurring isoforms of Stat3 mRNA are produced by differential splicing of the primary transcript. The shorter form, Stat3beta, is missing the last 55 C-terminal residues of the longer form, Stat3{alpha}, which encode the transactivation domain and Ser727 phosphorylation site (32, 33). Instead, 7 unique residues in the C terminus of Stat3beta replace this region. Stat3beta retains its ability to bind to DNA but fails to effectively transactivate gene expression, thereby functioning as a dominant negative to Stat3 signaling. Therefore, to determine whether A549 cells express relatively higher proportions of steady state levels of Stat3beta when compared with Stat3{alpha} mRNA than that found in HepG2 cells, we performed RT-PCR using primer pairs spanning the Stat3 alternative splicing region. If differential mRNA splicing were responsible for the reduced transactivation of the {gamma}FBG promoter, we would expect to find a higher relative abundance of the 261-bp Stat3beta RT-PCR product when compared with the 311-bp Stat3{alpha} product in A549 when compared with HepG2 cells. RT-PCR results from both cell lines revealed little amplification of the Stat3beta fragment (261 bp) (Fig. 2). Although these results do not represent the relative abundance of Stat3 isoform mRNAs between cell types, they do demonstrate that both HepG2 and A549 cells express equivalent but very low steady state levels of Stat3beta relative to endogenous Stat3{alpha} mRNA. Stat3 mRNA expression was not modulated by IL-6 treatment over 12 h (Fig. 2). These results rule out the possibility that Stat3beta interferes with Stat3{alpha} transactivation to dampen down {gamma}FBG promoter activity in A549 cells.


Figure 3
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  		FIGURE 3.
Comparison of protein levels of IL-6R, gp130 and Stat3 in lung A549 and liver HepG2 cells. Forty µg of whole cell lysate from nonstimulated A549 and HepG2 cells were loaded in each lane; proteins were transferred onto nitrocellulose membrane for Western blotting.

 


Figure 4
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  		FIGURE 4.
DEX Up-regulates IL-6R mRNA and protein in A549 cells. Cells were treated with 0.1 µM DEX for different lengths of time then collected for total RNA extraction and whole cell lysate preparation. A, semiquantitative RT-PCR for human IL-6R and glyceral-dehyde-3-phosphate dehydrogenase (GAPDH). A, upper panel, representative RT-PCR result; lower panel, quantitation of three separate experiments from cell treatment, RNA isolation, and RT-PCR. B, Western blot analysis of whole cell lysates prepared at times indicated.

 
A549 Cells Express Constitutively Low Levels of IL-6R and Stat3 Protein When Compared with HepG2 Cells—It is well established that IL-6 signaling occurs by ligand activation of the high affinity IL-6R and homodimerization of the gp130 transmembrane spanning signal transduction molecule, leading to activation of Stat3 (24). To explore other possibilities for the low transactivation potential of the {gamma}FBG promoter in response to IL-6 in A549 cells, we tested whether the expression levels of some key components of the IL-6 signaling pathway were different in relationship to the amount of actin expressed in each of these cell lines. Western blotting indicated that these cells produce similar amounts of gp130 protein; however, constitutive levels of both IL-6R and Stat3 were reduced in A549 when compared with HepG2 cells (Fig. 3), suggesting cell type-specific differences in Stat3 signaling in response to IL-6.

DEX Induces IL-6R mRNA Expression and Protein Production—A previous study showed that DEX induces IL-6R mRNA expression and protein production in HepG2 cells (34). Therefore, to determine whether DEX treatment changes the expression of IL-6R-gp130-Stat3 signaling components in lung epithelial cells, we employed semiquantitative RT-PCR (Fig. 4A). DEX-induced expression of steady state levels of IL-6R mRNA 1.8-fold by 2 h (p < 0.05) peaked at 2.2-fold after 6 h (p = 0.003) and remained elevated by 2-fold at 12 h (p = 0.0112) after treatment (Fig. 4A). A similar increase in IL-6R protein production was also observed in response to DEX treatment, reaching peak production at 6 h and remaining elevated out to 24 h after treatment (Fig. 4B). The relative fold increase in IL-6R mRNA and protein in response to DEX treatment corresponds with the ~2.3-fold DEX potentiation of IL-6-mediated {gamma}FBG promoter activity (Fig. 1).


Figure 5
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  		FIGURE 5.
Phosphorylation status and nuclear translocation of Stat3 in response to IL-6, DEX, or both. A, a comparison of the activation profiles of Stat3 in both A549 and HepG2 cell lines by Western blot assay. Cells were treated with IL-6, DEX, or a combination of both for different periods of time, and then antibodies to Stat3-pTyr705 and Stat3-pSer727 were used to monitor the activation status of Stat3 protein in both cell lines. The numbers on the top of each panel indicate the period of time (in min) during which cells were incubated with the treatment. B, combined treatment with IL-6 and DEX leads to sustained binding of activated Stat3 to {alpha}2M type II IL-6RE. A549 cells were treated with 50 ng/ml IL-6, 0.1 µM DEX, or a combination of both for different lengths of time, and then cells were collected for preparation of nuclear extracts. The rat {alpha}2M type II IL-6RE was used as probe in the EMSA. NS, non-specific.

 
IL-6 + DEX Induces Elevated and Prolonged Stat3 Activation—A third possibility for reduced IL-6-mediated transactivation of the {gamma}FBG promoter in A549 cells was a difference in cell type-specific activation of Stat3. Therefore, we compared the activation profiles of Stat3 in both A549 and HepG2 cells using phospho-Stat3-specific antibodies for Tyr705 and Ser727 when compared with total Stat3 by Western blotting (Fig. 5). In response to 50 ng/ml IL-6, phosphorylation of Stat3-Tyr705 was both rapid and transient in both cell lines; peak Tyr705 phosphorylation was observed within 5 min and remained at or near peak levels for 30 min, after which it was barely discernible (Fig. 5A). In contrast, the kinetics of Ser727 phosphorylation was slower, reaching peak levels in both HepG2 and A549 cells after 120 min of IL-6 treatment and remaining active throughout the entire 4-h treatment. On the other hand, 0.1 µM DEX did not stimulate phosphorylation of Tyr705 in either cell type but instead induced phosphorylation of Ser727 in both HepG2 and A549 cells with similar kinetics observed in response to IL-6 treatment (Fig. 5A). Notably, when given both IL-6 + DEX, there was sustained phosphorylation of Ser727 as well as evidence of biphasic and prolonged phosphorylation of Tyr705 in both cell types, resulting in prolonged intensity and duration of Stat3 activation.

To determine whether IL-6 + DEX-sustained activation of Stat3 leads to prolonged nuclear localization, we tested the ability of Stat3 in A549 nuclear extracts to bind to type II IL-6REs in the human {gamma}FBG promoter and the rat {alpha}2M promoter by EMSA. Activated Stat3 from A549 cells did not bind to any of the three human {gamma}FBG type II IL-6REs (not shown); however, this was not surprising for three reasons. First, A549 cells produce lower amounts of Stat3 when compared with HepG2 cells relative to equivalent amounts of actin produced by these cells (Fig. 3). Second, activated Stat3 binds with low affinity to the type II IL-6REs in the {gamma}FBG promoter when compared with that in the {alpha}2M promoter in HepG2 cells (25). Third, the weak binding of Stat3 to the wild type {gamma}FBG IL-6REs is in accord with the general inability to demonstrate complex formation with wild type IL-6 promoter regions of several APP genes (25). Activated Stat3 can act as a transcriptional coactivator without direct association with its DNA binding motif (35). The combined effects of low affinity binding of Stat3 to the {gamma}FBG IL-6REs and reduced amounts of Stat3 precluded detection of Stat3-{gamma}FBG promoter DNA complexes from lung A549 cell nuclear extracts.


Figure 6
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  		FIGURE 6.
Overexpression of Stat3(WT) with cotransfection of chimeric receptor GCSFR-gp130(133) in A549 cells. In addition to 500 ng of {gamma}FBG luciferase reporter and 2.5 ng of pRL-SV40 control plasmid, A549 cells were transfected with 50 ng of the chimeric receptor, GCSFR-gp130(133), and different amounts of Stat3(WT) expression vector as indicated. A, cells treated with various concentrations of IL-6 for 24 h. B, cells treated with various concentrations of GCSF for 24 h. Cell lysates were prepared for detection of luciferase activity (n = 3).

 
EMSA using the {alpha}2M IL-6RE DNA probe revealed that IL-6 induced nuclear translocation and DNA-protein complex formation of activated Stat3 (Fig. 5B), which is consistent with IL-6 induction of rapid and transient phosphorylation of Stat3-Tyr705 (Fig. 5A). After 30–60 min, nuclear accumulation of IL-6-activated Stat3 returned to baseline. However, by 160–240 min, prolonged and sustained nuclear accumulation and {alpha}2M DNA-Stat3 complex formation occurred (Fig. 5B). This biphasic activation and nuclear accumulation of Stat3 likely reflects the slower kinetics and prolonged phosphorylation of Ser727 (Fig. 5A). The results shown in Fig. 5A demonstrated that DEX alone failed to stimulate phosphorylation of Stat3 Tyr705, indicating that DEX-mediated phosphorylation of Ser727 is not sufficient for nuclear translocation of Stat3 above baseline (Fig. 5B). Similarly, IL-6 + DEX induced rapid biphasic activation and nuclear localization of Stat3, leading to elevated and sustained DNA-protein complex formation with the {alpha}2M IL-6RE (Fig. 5B).

Ectopic Expression of GCSFR-gp130(133) and Overexpression of Stat3(WT) Restores the Transactivation Potential of the {gamma}FBG Promoter in A549 Cells—The result from Fig. 2 indicated that low expression of IL-6R and Stat3 might play an important role in the significantly reduced activation of the {gamma}FBG promoter in A549 cells in response to IL-6. Therefore, we tested whether overexpression of Stat3(WT) would reconstitute IL-6-mediated transactivation of the {gamma}FBG promoter in A549 cells to a similar degree observed in HepG2 cells (i.e. from 2- to ~30-fold, respectively). The overexpression of Stat3(WT) in A549 cells did not affect IL-6 induction of the {gamma}FBG promoter (Fig. 6A)(p = not significant), potentially due to the low endogenous levels of IL-6R. However, the addition of sIL-6R failed to restore the full transactivation potential of the {gamma}FBG promoter activity in response to IL-6 (not shown).

To bypass the need for endogenous IL-6R-gp130 signal transduction, we tested whether overexpression of the chimeric receptor, GCSFR-gp130(133), would overcome the low endogenous activation of the {gamma}FBG promoter in A549 cells. In cells expressing the chimeric receptor, dose-dependent GCSF activation of the {gamma}FBG promoter was observed, reaching a maximum 9-fold induction (Fig. 6B; p < 0.0001) when compared with the 2-fold activation observed in response to IL-6 in parental A549 cells (Figs. 1 and 6A). By circumventing the IL-6R, endogenous Stat3 has a higher potential to transactivate the {gamma}FBG promoter in A549 cells. Overexpression of both GCSFR-gp130(133) and Stat3(WT) induced {gamma}FBG promoter activity ~32-fold in response to 50 ng/ml GCSF (Fig. 6B; in the presence of 10 and 50 ng/ml GCSF and 225 or 450 ng/ml Stat3(WT), all p values <0.0005), which is equivalent to the ~30-fold induction achieved by IL-6 in HepG2 cells (Fig. 1A). Together, these results indicate that the reduced expression of both IL-6R and Stat3 in A549 cells likely plays a major role in limiting IL-6-induced transactivation of the {gamma}FBG promoter in a cell type-specific manner.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Stat family members participate in signaling pathways in response to a wide variety of cytokines and growth factors. Stat transcription factors are localized to the cytoplasm in latent form where, upon activation, they dimerize and translocate to the nucleus to activate transcription of target genes. Stat3 was initially identified as acute phase response factor for its role in induction of hepatic APP genes in response to IL-6 during systemic inflammation (36). However, Stat3 also plays important roles in regulation of cell proliferation, differentiation, and oncogenic transformation (37–39). Both Stat3 and GCs regulate expression of surfactant proteins, which are critical components for surfactant structure and function in lung homeostasis (40–43). Stat3 also functions as a survival factor to prevent apoptosis (44). Stat3-induced expression of surfactant protein B is required for remodeling of the alveolar epithelium following oxygen injury (41). In transgenic mice that overexpress IL-6 in a lung-specific manner, survival of animals in 100% oxygen-induced injury is markedly enhanced due to diminished alveolar-capillary protein leakage and epithelial cell injury. Furthermore, GCs enhance cell-cell tight junction formation to enhance barrier function and epithelial cell polarity (45, 46). Indeed, mothers at risk for pre-term delivery are treated with DEX to induce maturation of the lungs of the baby and promote surfactant protein production prior to birth (47).

This study focused on the regulation of human fgg gene expression in lung when compared with liver epithelial cells in response to IL-6 and GCs. We found that when compared with the HepG2 liver cell line, lung A549 cells exhibited a 15-fold lower activation of the human {gamma}FBG promoter in response to IL-6. Therefore, we tested several possibilities that could account for the cell type-specific differences in fgg gene expression. First, analysis of RT-PCR products obtained prior to the plateau phase of amplification showed that barely detectable levels of Stat3beta mRNA were expressed and that the relative abundance of Stat3beta to Stat3{alpha} mRNA was similar in HepG2 and A549 cells. Second, relative to actin, the expression of endogenous IL-6R and Stat3 protein was much lower in A549 than in HepG2 cells, whereas gp130 expression was similar in both cell types. Third, overexpression of Stat3(WT) in A549 cells failed to restore IL-6-induced {gamma}FBG promoter activity, suggesting that low expression of IL-6R contributes to the 15-fold lower activation of the {gamma}FBG promoter in A549 cells. The addition of sIL-6R, however, failed to significantly restore IL-6 activation of the {gamma}FBG promoter, indicating that low expression of IL-6R was not the sole cause of reduced IL-6-mediated transactivation of the {gamma}FBG promoter. Fourth, Western blotting taken together with EMSA showed that rapid and transient kinetics of Stat3 Tyr705 phosphorylation is required for nuclear translocation and binding of activated Stat3 to the {alpha}2M IL-6RE. Finally, when overexpression of a chimeric receptor, GCSFR-gp130(133), was used to bypass IL-6R-gp130-coupled signal transduction, {gamma}FBG promoter activity was induced only 9-fold. However, in combination with the chimeric receptor, increasing expression of Stat3(WT) restored {gamma}FBG promoter activity in A549 cells to match the ~30-fold activity achieved by IL-6 through the endogenous IL-6R-gp130 signaling pathway in HepG2 cells.

There is increasing evidence that demonstrates synergism between IL-6 and GCs in the synthesis of APPs (44, 48). DEX treatment stimulated IL-6R mRNA expression and protein production 2–3-fold in A549 cells, which could account for the similar-fold DEX potentiation of {gamma}FBG promoter activity in response to IL-6. Because compensation of low levels of endogenous IL-6R with sIL-6R failed to replicate the DEX effect, DEX-induced expression of IL-6R is not sufficient to explain DEX potentiation of IL-6-induced activation of the {gamma}FBG promoter in A549 cells. DEX alone promoted Ser727 phosphorylation of Stat3 in both HepG2 and A549 cells with a conspicuous absence of Tyr705 phosphorylation. These results indicate that GCs stimulate Stat3 serine phosphorylation in the absence of tyrosine phosphorylation in an IL-6-independent manner. EMSA revealed that Stat3 phosphorylated at Ser727 but not at Tyr705 was incapable of translocating to the nucleus. However, the combined treatment of IL-6 + DEX led to sustained activation of Stat3 by phosphorylation of both Tyr705 and Ser727; both tyrosine and serine phosphorylation of Stat3 is required for maximum transactivation of target genes (49). Furthermore, IL-6 + DEX induced phosphorylation of Stat3 at Tyr705 and Ser727 promotes nuclear accumulation of Stat3 as well as biphasic and sustained DNA-protein complex formation. Taken together, these results suggest that DEX induction of the high affinity IL-6R, as well as the elevated and sustained activation and nuclear accumulation of Stat3, accounts for the majority of DEX potentiation of type II IL-6RE-containing target gene expression.

In summary, the data reported here suggest that low constitutive expression of IL-6R and/or Stat3 in lung epithelium may be beneficial to lung homeostasis. Indeed, Stat3 activation occurs long before signs of acute lung injury are observed, suggesting that Stat3 plays a role in initiating local inflammatory responses (40). Furthermore, the results of this study show that lung alveolar A549 cells are more sensitive to DEX potentiation of IL-6-induced FBG expression when compared with HepG2 cells (0.001 µM when compared with 0.1 µM, respectively); GCs maintain lung epithelial cell tight junctions and cell polarity (45, 46). In that respiratory epithelium is subjected to daily insults from airborne contaminants, it is likely that IL-6 and GC modulation of Stat3 activation in lung is limited to restrict the intensity of a local (lung epithelial) inflammatory response to IL-6 and GCs while allowing a full-scale systemic (hepatic) response. FBG is secreted basolaterally and incorporated into the alveolar extracellular matrix in response to IL-6 and GCs only when tight junctions are maintained (17, 22, 50). Taken together, these observations suggest that limited activation of Stat3 by IL-6 and GCs and subsequent FBG production contributes to the maintenance of respiratory epithelial barrier integrity and lung homeostasis. Elucidation of the mechanisms regulating FBG expression in lung will enhance our understanding of signaling mechanisms by which acute phase response mediators and APPs function to regulate pulmonary inflammation.


   FOOTNOTES
 
* This work was supported by Grants R01-HL50616 and P01-HL30616 from NHLBI, National Institutes of Health. 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 Medicine/Hematology-Oncology Division, P.O. Box 610, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-275-8267; Fax: 585-473-4314; E-mail: pj_simpsonhaidaris{at}urmc.rochester.edu.

2 The abbreviations used are: FBG, fibrinogen; APP, acute phase protein; IL, interleukin; IL-6RE(s), IL-6 response element(s); sIL-6R, soluble IL-6 receptor; IL-6R, IL-6 receptor; GC, glucocorticoid; Stat3, signal transducer and activator of transcription; DEX, dexamethasone; GCSF, granulocyte colony-stimulating factor; GCSFR, GCSF receptor; EMSA, electrophoretic mobility shift assay; {alpha}2M, {alpha}2-macroglobulin; RT, reverse transcriptase; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Dr. M. L. Tenchini from the University of Milan and Dr. Heinz Baumann from the Roswell Park Cancer Institute for the generous gifts of plasmid constructs used in this report.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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T. Hou, S. Ray, and A. R. Brasier
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