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TGF-ß down-regulates IL-6 signaling in intestinal epithelial cells: critical role of SMAD-2¹

Division of Digestive Diseases, Emory University, Atlanta, Georgia, USA

²Correspondence: Division of Digestive Diseases, Room 201-F, 615 Michael St., Whitehead Research Bldg., Emory University, Atlanta, GA 30322, USA. E-mail: ssitar2{at}emory.edu

SPECIFIC AIMS

The aim of the present study is to determine whether TGF-ß modulates IL-6-mediated signaling in intestinal epithelial cells especially with regard to STAT3 phosphorylation and ICAM-1, an adhesion molecule induced by IL-6 that plays an important role in inflammation.

PRINCIPAL FINDINGS

1. TGF-ß1 receptor type II is present predominantly at the basolateral membrane of intestinal epithelial cells
Model intestinal epithelial cell line Caco2-BBE was assessed for expression of TGF-ß receptor type II (TßRII). Plasma membrane proteins were labeled by biotinylation of each membrane domain (apical and basolateral). Western blot revealed the presence of TßRII predominantly at the basolateral membrane. These finding were confirmed by confocal microscopy.

2. TGF-ß1 induces Smad2 phosphorylation and nuclear translocation of Smad4 complex
Smad activation by receptor-mediated phosphorylation is a central event in TGF-ß signal transduction. We therefore analyzed by Western blot Smad2 phosphorylation in the Caco2-BBE cells after TGF-ß1 stimulation for 30 min, 1, 2, and 4 h. Phospho-Smad2 was detectable at 30 min, peaked at 2 h, then declined 4 h after TGF-ß1 stimulation (Fig. 1 A, upper panel). However, there was no change in total Smad2 at any time (Fig. 1A , middle panel). In contrast, although immunoreactive Smad4 showed no significant change at 30 min, 1 h, and 2 h, there was a significant decrease in Smad4 4 h after TGF-ß1 stimulation (Fig. 1A , lower panel). Using immunoprecipitation, we demonstrate that the interaction between Smad2 and Smad4 was significantly enhanced upon stimulation with TGF-ß1 for 2 h (twofold higher than control cells as determined by scanning densitometry) (Fig. 1B ). We next examined the subcellular localization of Smad4 by immunofluorescence in the presence and absence of TGF-ß1. Immunofluorescence staining of Smad4 in unstimulated monolayer was detectable in the periphery of cells, consistent with its presence in the cytosol (Fig. 1C ; a–d). TGF-ß1 (2 h) -stimulated cells exhibited a predominantly nuclear SMAD4-specific staining compared with unstimulated cells (Fig. 1C ; e–h) following the kinetics of p-Smad2 accumulation. These data demonstrate that TGF-ß1 signals effectively in intestinal epithelial cells.

View larger version (69K): [in this window] [in a new window]   Figure 1. TGF-ß1 induces Smad signaling pathway. A) Time course of Smad phosphorylation in Caco2-BBE cells. Monolayers were treated basolaterally with 10 ng/mL TGF-ß1 and whole cell lysates were prepared and analyzed by Western blot with antibodies against phospho-Smad2 (upper panel), total Smad2 (middle panel), and Smad4 (lower panel). Data shown are representative of 4 independent experiments. B) Smad2 and Smad4 form a complex upon TGF-ß stimulation. Whole cell lysates prepared from cells treated with basolateral TGF-ß1 (10 ng/mL) were immunoprecipitated with anti-Smad4 or SMAD2 antibody, followed by Western blot with Smad2 or SMAD4 antibody, respectively. Representative of 3 separate experiments. C) Smad4 is translocated to nucleus upon TGF-ß stimulation. Filter-grown Caco2-BBE monolayers were stimulated with basolateral TGF-ß1 (10 ng/mL) for 2 h. Monolayers were fixed, permeabilized, stained with rhodamine/phalloidin (actin, red), then incubated with rabbit anti-Smad4 antibody followed by incubation with To-Pro3-iodide to stain cellular nuclei. Monolayers were visualized with secondary antibody coupled to fluorescein isothiocyanate using Zeiss 510 confocal microscope. En-face (xy) Sections of the confluent monolayer containing representative stained cells are shown. a, e) Immunoreactive Smad 4 (green); c, g) nuclear staining (blue) of sections corresponding to a and e, respectively. x40.

3. TGF-ß down-regulates IL-6-induced tyrosine phosphorylation of STAT1 and STAT3 and nuclear translocation of activated STAT3
To determine the mechanism by which TGF-ß inhibits IL-6-induced gene expression, the effect of TGF-ß1 on IL-6 signaling pathway was examined. IL-6 stimulation is associated with tyrosine phosphorylation and activation of transcription factors STAT1 and STAT3, so we examined the effect of TGF-ß1 on IL-6-induced tyrosine phosphorylation of STAT1 and STAT3 proteins. Phospho-STAT-1and -3 were barely detectable in control and TGF-ß1-treated monolayers. As expected, stimulation of Caco2-BBE cells with IL-6 resulted in tyrosine phosphorylation of STAT-1 and -3 at 5 and 30 min. Pretreatment of monolayers with TGF-ß1 for 4 h resulted in a significant inhibition of IL-6-induced phospho-STAT1 and STAT3 at 5 and 30 min (Fig. 2 A). TGF-ß1 pretreatment, however, did not alter expression of total STAT-1 or -3 (Fig. 2A ), indicating modulation of the IL-6/STAT signal transduction pathway by TGF-ß1. We further characterized the negative regulation of IL-6/STAT3 signaling by TGF-ß. We examined the effects of TGF-ß1 pretreatment on subcellular localization of STAT3 by immunoblot and immunofluorescence analysis. IL-6 treatment of Caco2 BBE cells induced nuclear translocation of activated STAT3 (Fig. 2B, C ; e–h); pretreatment with TGF-ß1 for 4 h reversed the effect of IL-6 (Fig. 2B, C ; i–l), indicating a functional interaction between TGF-ß and IL-6 signaling cascades.

View larger version (44K): [in this window] [in a new window]   Figure 2. TGF-ß1 inhibits IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. A) Caco2-BBE cells were treated with IL-6 alone or pretreated with basolateral TGF-ß1 for 4 h, then treated with vehicle or incubated with basolateral IL-6 for 5 and 30 min. Cells were harvested and Western blot was performed using an anti-p-STAT3 (top panel) or anti-p-STAT1 (lower panel) antibody. The blots were stripped and reprobed with anti-STAT1 or STAT3 antibody. Representative data from 3 individual experiments using 2 samples/condition are shown. B) TGF-ß inhibits IL-6-induced nuclear translocation of activated STAT3: Caco2-BBE cells were treated with vehicle (lane 1) or 100 ng/mL basolateral IL-6 (30 min, lane 2) or pretreated with basolateral TGF-ß1 (10 ng/mL) for 4 h, then incubated with 100 ng/mL IL-6 for 30 min. Nuclear extracts were prepared and Western blot was performed using anti-p-STAT3 (tyr) antibody. C) Filter-grown Caco2-BBE monolayers were stimulated with IL-6 alone for 30 min or pretreated with basolateral TGF-ß1 for 4 h, then incubated with IL-6 for 30 min. Monolayers were fixed, permeabilized, and stained with rhodamine/phalloidin, then incubated overnight with anti-p-STAT3 (tyr) antibody at 4°C. Monolayers were incubated with To-Pro3-iodide to stain cellular nuclei and visualized with secondary antibody coupled to fluorescein isothiocyanate using Zeiss 510 confocal microscope. En-face (xy) sections of the confluent monolayer containing representative stained cells are shown. x60

TGF-ß inhibits IL-6-mediated ICAM-1 induction
We next investigated the consequences of TGF-ß1 pretreatment on ICAM-1 expression, a downstream effector of IL-6 signaling. IL-6 induced a significant increase in ICAM-1 expression that was maximal 8 h after IL-6 stimulation. Pretreatment with TGF-ß1 resulted in 80% decrease in IL-6-induced ICAM-1 expression. To support this finding, we examined whether TGF-ß1 inhibits the induction of ICAM-1 promoter by IL-6. IL-6 induced a fivefold increase in relative luciferase activity compared with cells stimulated with vehicle alone. Pretreatment with TGF-ß1 abolished the increase in luciferase activity induced by IL-6. Because induction of ICAM-1 by IL-6 is dependent on the JAK-STAT signaling pathway, the result shows that blocking IL-6 signaling via STATs blocks IL-6 activation of a STAT-dependent gene.

4. Smad2 is required for TGF-ß1-mediated inhibition of ICAM-1
To establish the involvement of Smad2 in the TGF-ß-mediated inhibition of ICAM-1 expression, we studied the effect of dominant-negative (DN) Smad2 mutant (Smad2C) overexpression on IL-6-induced ICAM-1 expression. The DN SMAD2 lacked the last four amino acids, including the COOH-terminal serines, and thus cannot be phosphorylated in response to TGF-ß. Cells were transfected with either vector alone or with DN Smad2C-Flag plasmid. 48 h after transfection, cells were stimulated with IL-6 alone or pretreated with TGF-ß1 for 4 h, then incubated with IL-6. Cells were harvested and subjected to Western blot with an anti-ICAM-1 antibody. While TGF-ß1 was able to inhibit ICAM-1 expression induced by IL-6 in vector transfected cells, overexpression of Smad2C suppressed TGF-ß1-mediated inhibition of IL-6-induced ICAM-1 expression. To further confirm the role of Smad2, we studied the effect of exogenous DN Smad2 expression on TGF-ß1-mediated inhibition of ICAM-1-Luc reporter gene response to IL-6. Normalized luciferase activity indicated that overexpression of DN Smad2 efficiently blocked the TGF-ß1 inhibition of IL-6-induced ICAM-1-Luc activity. These results argue that by signaling through Smad2, TGF-ß1 inhibits the induction of ICAM-1 in response to IL-6. Mutating the phosphorylation sites within the intracellular signal transducer Smad2 abrogates the temporal restriction of Smad2 to accumulate in the nucleus and constitutes a mechanism that leads to loss of TGF-ß1-mediated inhibition of ICAM-1 expression.

CONCLUSIONS AND SIGNIFICANCE

We demonstrate here that antagonistic signals between TGF-ß and IL-6 involves cross-talk between TGF-ß/Smad and JAK/STAT signaling pathways in intestinal cell model system. We show that TGF-ß1 exerts its inhibitory action in this system by down-regulating IL-6-induced phosphorylation of STAT3 and further blocks the expression of ICAM-1, a downstream effector of IL-6 signaling. Because ICAM-1 gene expression by IL-6 is regulated by STATs, our results identify inhibition of STAT activation by TGF-ß1 as one molecular mechanism underlying suppression of IL-6-induced gene expression by TGF-ß1 and demonstrate that a block in signal transduction was effectively translated into a suppression of gene activation. The functional requirement for cellular Smads in mediating TGF-ß1 suppression of IL-6-induced ICAM-1 was confirmed using dominant negative Smad2. These results identify a novel level of cytokine cross-talk in intestinal epithelial cells and advance the concept that cytokine antagonism at the level of signal transduction contributes to the balance of cytokine activity, and thus to pathogenesis of inflammatory diseases. Modulation of cytokine signaling to block the proinflammatory actions of pleiotropic cytokines such as IL-6 or to potentiate the actions of anti-inflammatory cytokines may be a promising therapeutic approach in diseases such as rheumatoid arthritis and IBD.

View larger version (59K): [in this window] [in a new window]   Figure 3. Schematic representation of the cross-talk between TGF-ß and IL-6 signaling pathway. The IL-6 signaling pathway is depicted on the right. In unstimulated cells, signaling molecules such as JAKs and STATs are inactive. After ligand (IL-6) binding, receptor aggregation occurs; receptor-associated JAKs are brought together, which allows cross phosphorylation (P) and activation. The active JAKs tyrosine phosphorylate the receptor and STAT-3 is recruited through interaction with the tyrosine phosphorylated residues in the IL-6 receptor. STAT-3 in turn is phosphorylated, dimerized, and translocates to the nucleus to activate the transcription of IL-6 response genes such as ICAM-1. The TGF-ß signaling pathway is shown on the left. Upon ligand binding, the TGF-ß receptor subunits TßRI and TßRII aggregate and trigger phosphorylation of receptor-regulated Smad2. Phosphorylated Smad-2 (P) forms a hetero-oligomeric complex with a common partner (Co-Smad), Smad4. The Smad complex is then translocated to the nucleus, where they regulate transcription of target genes. The broken arrow depicts inhibition of IL-6-induced STAT-3 by TGF-ß, likely via the Smad pathway.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1211fje; doi: 10.1096/fj.02-1211fje

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