Interleukin-6 Regulation of Transforming Growth Factor (TGF)-β Receptor Compartmentalization and Turnover Enhances TGF-β1 Signaling*

Transforming growth factor (TGF)-β1 is a key cytokine involved in the pathogenesis of fibrosis in many organs, whereas interleukin (IL)-6 plays an important role in the regulation of inflammation. Recent reports demonstrate interaction between the two cytokines in disease states. We have assessed the effect of IL-6 on TGF-β1 signaling and defined the mechanism by which this occurred. Stimulation of Smad-responsive promoter (SBE)4-Lux activity by TGF-β1 was significantly greater in the presence of IL-6 than that induced by TGF-β1 alone. Augmented TGF-β1 signaling following the addition of IL-6 appeared to be mediated through binding to the cognate IL-6 receptor, the presence of which was confirmed by fluorescence-activated cell sorting and Stat-specific signaling. TGF-β1 receptors internalize by both caveolin-1 (Cav-1) lipid raft and early endosome antigen 1 (EEA-1) non-lipid raft pathways, with non-lipid raft-associated internalization increasing TGF-β1 signaling. Affinity labeling of TGF-β1 receptors demonstrated that IL-6 stimulation resulted in increased partitioning of TGF-β receptors to the non-lipid raft fraction. There was no change in expression of Cav-1; however, following IL-6 stimulation, co-immunoprecipitation demonstrated decreased association of IL-6 receptor with Cav-1. Increased TGF-β1-dependent Smad signaling by IL-6 was significantly attenuated by inhibition of clathrin-mediated endocytosis and augmented by depletion of membrane cholesterol. These results indicate that IL-6 increased trafficking of TGF-β1 receptors to non-lipid raft-associated pools results in augmented TGF-β1 Smad signaling.

Transforming growth factor (TGF) 1 -␤1, which is the prototypic member of the TGF-␤ superfamily, exerts a broad range of biological activities. It has been implicated in the pathogenesis of renal fibrosis in both experimental and human disease (1)(2)(3)(4)(5). Furthermore, attenuation of its action has been postulated to be a target for therapeutic intervention in numerous disease models (2,3,6,7). Understanding the mechanisms that regu-late TGF-␤1-dependent responses is therefore an important goal. TGF-␤ receptors reside in both lipid raft and non-raft membrane domains (8). Alteration in the membrane compartmentalization of TGF-␤ receptors between these membrane domains has recently been identified as a mechanism that regulates TGF-␤1 signaling (8,9).
Interleukin (IL)-6 is a multifunctional cytokine produced by a variety of cells during infection, trauma, and immunological challenge and is known to play a central role in the regulation of host defense and inflammation (10). Dysregulation of IL-6 activity or of its signaling cascade has been implicated in the pathogenesis of a number of diseases including rheumatoid arthritis, breast carcinoma, hepatic cirrhosis, and inflammatory bowel disease. Dysregulated TGF-␤1 signaling has also been implicated in the pathogenesis of inflammatory bowel disease (11), and in this context, a recent report has highlighted cross-talk between TGF-␤ and IL-6 in intestinal epithelial cells, with TGF-␤ playing a role in the negative regulation of IL-6 signaling (12).
In the kidney, IL-6 is not expressed in the healthy proximal tubular epithelial cell. It has, however, been reported that expression of IL-6 is up-regulated in various forms of renal diseases and that IL-6 expression may be associated with glomerular and tubular injury (13)(14)(15). These studies suggest a potential role for IL-6 in modulating renal tubulo-interstitial injury, although the functional significance of altered IL-6 in proximal tubular epithelial cells is not clear.
The aim of the present study was to examine the potential interaction between IL-6 and TGF-␤1 signaling in proximal tubular cells. Our results demonstrate enhanced response to TGF-␤1 in the presence of IL-6. This effect was associated with activation of the IL-6 cognate receptor, decreased partitioning of TGF-␤ receptors to lipid raft-rich membrane fractions, and a marked reduction of TGF-␤ receptor turnover. Both increased IL-6 and TGF-␤1 may be associated with renal injury, and TGF-␤1 action is implicated in progressive renal interstitial fibrosis. These results suggest a synergistic effect of the two factors that may lead to an enhanced pro-fibrotic response within the kidney, driven by proximal tubular cell activation.
Cell Culture-HK-2 cells (human renal proximal tubular epithelial cells immortalized by transduction with human papilloma virus 16 E6/E7 genes; Ref. 16) were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 (Invitrogen) supplemented with 10% fetal calf serum (Biological Industries Ltd., Cumbernauld, United Kingdom), 2 M L-glutamine (Invitrogen), 20 mM HEPES buffer (Invitrogen), 5 g/ml insulin, 5 g/ml transferrin (Sigma), 40 ng/ml hydrocortisone (Sigma), and 5 ng/ml sodium selenite (Sigma). Cells were grown at 37°C in 5% CO 2 and 95% air. Fresh growth medium was added to the cells every 3-4 days until the cells reached confluence. Cells were grown to confluence and serum-deprived for 48 h prior to experimental manipulation. In all experiments, cells were stimulated with recombinant TGF-␤1 and/or IL-6 under serum-free conditions. In all aspects of cell biology that we have studied previously, HK-2 cells respond in an identical fashion to primary cultures of human proximal tubular cells (17)(18)(19)(20). HK-2 cells are therefore a good model from which general conclusions can be drawn in terms of proximal tubular cell biology.
Transient Transfection-The Smad-responsive promoter (SBE) 4 -Lux (21) was a gift from Aristidis Moustakas (Ludwig Institute for Cancer Research, Uppsala, Sweden). For transfection of the reporter construct, 1.3 ϫ 10 4 cells/well were seeded onto a 24-well plate (this density of cells produced a 70% confluence monolayer the following day). The next day, cells were transfected with 0.2 g of the Smad-responsive promoterluciferase construct using the mixed lipofection reagent FuGENE 6 at a ratio of 0.6 l of FuGENE 6 to 0.2 g of DNA in serum-free and insulin-free medium. Transfection efficiency was monitored by co-transfection with a ␤-galactosidase reporter plasmid. 24 h after transfection, cells were stimulated with either TGF-␤1 or the combination of TGF-␤1 and IL-6. Following lysis of the cells in Reporter Lysis Buffer, luciferase content was quantified by glow-type luminescence assay (Bright-Glo), and ␤-galactosidase activity was determined by commercial colorimetric assay (Promega). Luciferase activity was normalized to ␤-galactosidase activity.
Flow Cytometry-Cell surface expression of IL-6 receptor was assessed by fluorescence-activated cell-sorting analysis. Following detachment of HK-2 cell monolayers with trypsin/EDTA, the cells were incubated with R-phycoerythrin-conjugated mouse anti-human IL-6 receptor monoclonal antibody (BD Biosciences; 1:40 dilution) or mouse anti-human IgG antibody (BD Biosciences; 1:40 dilution) as a control at saturating concentrations in fluorescence-activated cell-sorting buffer (phosphate-buffered saline, 10 mM EDTA (Sigma-Aldrich), 15 mM sodium azide (Fisher Chemicals), and 5% bovine serum albumin (Sigma-Aldrich), pH 7.35) for 30 min at 4°C. After three washes in fluorescence-activated cell-sorting buffer, the data were collected using a BD Biosciences FACSCalibur and analyzed using CellQuest Pro TM software.
Immunoprecipitation and Immunoblotting/Western Analysis-Briefly confluent monolayers were washed once with cold phosphatebuffered saline (Invitrogen), scraped, and rinsed into 5 ml of cold phosphate-buffered saline. After centrifugation at 2500 rpm for 10 min, cell pellets were extracted in buffer (150 mM NaCl, 50 mM Tris-Cl, 0.01% NaN 3 , 2 mM EDTA, 1 mM sodium orthovanadate, 10 g/ml leupeptin, and 25 g/ml aprotinin) containing 1% Triton X-100 for 30 min on ice. Samples were centrifuged at 12,500 rpm for 30 min, and then the supernatant (Triton-soluble components including membrane and cytosolic fraction) was transferred to a separate tube and kept at Ϫ70°C until use.
Immunoprecipitation was performed by standard methodologies. Briefly, cell protein samples (200 g) were pre-cleared with 25 l of packed protein A-cross-linked 4% beaded agarose (Sigma) at 4°C for 30 min. The beads were removed by centrifugation (13,000 rpm, 10 min), and the supernatant was collected. Primary antibody (2 g/ml) was added to the cleared supernatant and incubated at 4°C with constant mixing overnight. The immune complex was captured by the addition of packed protein A-agarose beads (50 l) overnight at 4°C. Beads were washed with radioimmune precipitation assay buffer (50 mM Tris, 150 mM NaCl, 0.5% sodium deoxycholate, 10 mM MgCl 2 , 0.1% SDS, and 1% Triton X-100); 30 l of sample buffer was then added prior to boiling for 5 min. Separation of the beads was achieved by centrifugation (13,000 ϫ g for 10 min), and the supernatant was removed. Specificity of immunoprecipitation was confirmed by negative control reactions performed with either no primary antibody or IgG control.
Subsequently, samples were subjected to immunoblot/Western analysis. Briefly, equal amounts of sample were prepared in SDS sample buffer (2% SDS, 10% (v/v) glycerol, 60 mM Tris, and 0.05% (v/v) mercaptoethanol) and boiled for 5 min prior to loading onto 10% SDS-PAGE gels. Electrophoresis was carried out under reducing conditions according to the procedure of Laemmli (22). After electrophoresis, the separated proteins were transferred to a nitrocellulose membrane (Amersham Biosciences). The membrane was blocked with Tris-buffered saline containing 5% nonfat powdered milk for 1 h and then incubated with the primary antibody in Tris-buffered saline containing 5% nonfat powdered milk and 0.1% Tween 20 (Tris-buffered saline-Tween) overnight at 4°C. The blots were subsequently washed in Tris-buffered saline-Tween and then incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (Sigma) in Tris-buffered saline-Tween. Proteins were visualized using enhanced chemiluminescence (Amersham Biosciences) according to the manufacturer's instructions.
Electrophoretic Gel Mobility Shift Assay-Nuclear extracts were prepared according to the method of Andrews and Faller (23), and protein concentrations were determined using the Bio-Rad protein assay. Nuclear extracts (5 g) were incubated for 20 min at room temperature with 1.25 ng of 32 P-labeled double-stranded high-affinity sis-inducible element (hSIE) oligonucleotide corresponding to a consensus Stat-binding element, 5Ј-CGAGTGCATTTCCCGTAAATCTTGTCTACA-3Ј, in a 20-l binding reaction buffer and 1 g of poly(dI)/(dC) (Sigma-Aldrich). Complexes were resolved on nondenaturing 6% polyacrylamide gels, and separated proteins were visualized by autoradiography. In supershift experiments, 20 g/ml rabbit anti-human Stat1 or Stat3 antibody (Autogen Bioclear) was added and incubated for 20 min before addition of radiolabeled probe.
Affinity Labeling of TGF-␤1 Receptors and Assessment of Receptor Turnover-HK-2 cells were incubated with 250 pM 125 I-TGF-␤1 at 37°C for 1 h, and receptors were cross-linked to ligand with disuccinimidyl suberateas (Pierce) as described previously (24). Previous studies suggest that receptor cross-linking itself has no effect on receptor internalization (24). To assess endogenous receptor turnover, following affinity labeling and cross-linking, cells were incubated in serum-free medium at 37°C for up to 10 h. At each time point, cells were lysed and processed for SDS-PAGE, and endogenous receptors were visualized by autoradiography.
Detergent-free Purification of Lipid Raft-rich Membrane Fraction-HK-2 cells were grown to near confluence in 100-mm 2 dishes and affinitylabeled as described above. After two washes with ice-cold phosphatebuffered saline, two confluent dishes were scraped into 2 ml of 500 mM sodium carbonate, pH 11.0. Homogenization was carried out with the use of 10 strokes of a tight-fitting Dounce homogenizer, followed by three 10-s bursts of a tissue homogenizer (Powergen 125; Fisher Scientific), followed by three 20-s bursts of an ultrasonic disintegrator (Soniprep 150; Fisher Scientific) to disrupt cellular membranes (25). The homogenates were adjusted to 45% sucrose by addition of 2 ml of 90% sucrose prepared in MBS (2-(N-morpholino)ethanesulfonic acid-buffered saline, 25 mM 2-(Nmorpholino)ethanesulfonic acid, pH 6.5, and 0.15 M NaCl) and placed at the bottom of an ultracentrifuge tube. A discontinuous sucrose gradient (4 ml of 35% sucrose and 4 ml of 5% sucrose, both prepared in MBS) was formed above and centrifuged at 39,000 rpm for 16 -20 h in an SW40 TI rotor (Beckman Instruments). A light-scattering band was observed at the 3-35% sucrose interface. Twelve 1-ml fractions were collected from the top of the tubes, and a portion of each fraction was analyzed by SDS-PAGE. A 330-kDa band ( 125 I-TGF-␤⅐receptor complex) was detectable on autoradiography, and this was quantified by densitometry (Chemi Doc; Bio Rad). Data are expressed as percentage of the total 125 I-TGF-␤⅐receptor complex for that experiment in each fraction. This method does not allow comparative measurement of receptor expression under different experimental conditions but rather allows for the relative amount of receptor expressed in the lipid raft and non-raft fractions to be expressed as a percentage of the total receptor expressed in each individual experiment.

IL-6 Enhances TGF-␤1 (Smad-dependent)
Signaling-Activation of the Smad signaling pathway was examined using the (SBE) 4 -Lux reporter, which contains four repeats of the CA-GACA sequence identified as a Smad-binding element. Stimulation of HK-2 cells transiently transfected with TGF-␤1 led to a significant increase in luciferase activity of the reporter construct as described previously (26) (Fig. 1). Addition of IL-6 (0 -100 ng/ml) alone did not increase the signal above control values. In contrast, addition of IL-6 in the presence of TGF-␤1 led to a dose-dependent increase in luciferase activity that was significantly greater than that induced by TGF-␤1 alone (Fig.  1). This effect of IL-6 was statistically significant at IL-6 doses of Ն10 ng/ml.

HK-2 Cells Express Functional IL-6 Cognate
Receptor-Although expression of gp130 is found in almost all cell types, cellular distribution of IL-6 cognate receptor is more limited, with studies to date documenting that IL-6 cognate receptor expression is confined to hepatocytes and leukocyte subpopulations. Enhanced TGF-␤1-dependent Smad signaling seen following addition of IL-6 suggests that this is the result of binding of IL-6 directly to its cognate receptor and the resulting dimerization of gp130.
Flow cytometry was used to confirm the expression of IL-6 cognate receptor on the cell surface of HK-2 cells ( Fig. 2A). Functional integrity of the IL-6 signaling pathway was examined by gel shift assays performed with nuclear proteins and a Stat site-specific consensus probe (Fig. 2B). Stat activation was seen following addition of IL-6 ( Fig. 2B). We verified activation of both Stat1 and Stat3 following addition of IL-6 alone to HK-2 cells by supershift assay. Incorporation of an antibody to either Stat1 or Stat3 interfered with the formation of the probe⅐protein complexes because band intensities were reduced with inclusion of antibody, and an additional retarded band was seen in the presence of the Stat3 antibody (Fig. 2C).
Smad Protein Expression and Association-Previous studies have demonstrated cooperative binding of the Stat and Smad protein family members (27) and also demonstrated that TGF-␤1 activity may be modulated by Stat-dependent regulation of the inhibitory Smad protein Smad7 (28).
The association of Stat proteins with either Smad2 or Smad3 was examined by immunoprecipitation of either Stat1 or Stat3 and Western analysis of the Smad proteins (Fig. 3). Using this approach, we could demonstrate that Stat1 associates with both Smad2 and Smad3 but that this association is not affected by the presence of IL-6 or TGF-␤1. Similarly, Stat3 also associates with Smad2 and Smad3, but this is not modulated by either IL-6 or TGF-␤1.
It is also unlikely in our system that regulation of TGF-␤1 signaling is mediated by alterations in the expression of Smad proteins because stimulation of HK-2 cells with IL-6 and/or TGF-␤1 did not alter intracellular receptor-regulated Smad expression (Smad2 and Smad3) or expression of the inhibitory Smad (I-Smad) Smad7 (data not shown).
TGF-␤1 Receptor Compartmentalization-Previous studies have demonstrated co-localization of TGF-␤ receptors into both caveolin, cholesterol-and sphingolipid-rich membrane domains called lipid rafts (29) and EEA-1-containing compartments (8,9). We have demonstrated that altered TGF-␤1 function may be associated with trafficking of receptors between these two pools (9). To examine alterations in receptor partitioning, endogenous TGF-␤ receptors on HK-2 cells were affinity-labeled using 125 I-TGF-␤1. Subsequently, rafts were fractionated by sucrose density centrifugation (30). Following the addition of 125 I-TGF-␤1 (250 pM), receptors were found in both the raft and non-raft fractions, as we demonstrated previously (Fig. 4A). Scanning densitometry of the results of three separate experiments confirmed that following the addition of 125 I-TGF-␤1, 38% of the total TGF-␤ receptor partitioned into the non-raft fractions (Fig. 4B). Addition of 25 I-TGF-␤1 in the presence of IL-6 led to a significant increase in TGF-␤ receptor detected in the non-raft fraction. Under these conditions, 62% of total TGF-␤ receptor partitioned into the raft fractions (mean of n ϭ 3, p ϭ 0.02).
The sterol-binding antibiotic nystatin specifically interacts with cholesterol to sequester it in the membrane, thereby effectively reducing the ability of cholesterol to interact with and exert its effects on other membrane components (31,32). Disruption of cholesterol by pre-treatment of cells with 50 g/ml nystatin (29) at 37°C for 1 h led to an increase of TGF-␤ receptor partitioning into the non-raft fraction (Fig. 4A). The increase in trafficking of TGF-␤ receptor into the non-lipid raft membrane fraction was associated with a further increase in TGF-␤1-dependent Smad signaling following the addition of TGF-␤1 together with IL-6 (Fig. 5A). The effect of nystatin was dependent on cholesterol sequestration because it was prevented by addition of cholesterol (25 g/ml) together with nystatin to cells at 37°C for 1 h prior to addition of TGF-␤1 together with IL-6 ( Fig. 5A). Next we examined the effect of inhibition of clathrin-mediated endocytosis by K ϩ depletion, which prevents clathrin lattice assembly and has been shown to inhibit endosome-dependent TGF-␤1 signaling (8). Activation of the TGF-␤1 signaling pathway following addition of TGF-␤ or TGF-␤1 in combination with IL-6 was assessed by using the (SBE) 4 -Lux reporter (Fig. 5B). The augmentation of TGF-␤1-dependent Smad signaling by IL-6 was significantly attenuated when carried out in minimal, K ϩ -depleted medium (Fig. 5B). Cellular cholesterol disruption has been previously demonstrated to trigger shedding of membrane proteins (33). In our experimental system, addition of 50 g/ml nystatin at 37°C for 1 h to confluent monolayers of HK-2 cells did not induce shedding of either TGF-␤ type I receptor as assessed by Western analysis of cell culture supernatant or IL-6 as assessed by enzyme-linked immunosorbent assay (data not shown).
Growth factor signaling may be modulated by alteration of caveolin-1 expression, which is likely to influence trafficking of receptors between lipid and non-lipid membrane pools (34). Total caveolin-1 expression as assessed by Western analysis was unaltered following stimulation with either TGF-␤1 or TGF-␤1 ϩ IL-6 ( Fig. 6). In contrast, following stimulation with TGF-␤1 in the presence of IL-6, there was a decrease in the association of Cav-1 with the TGF-␤ receptor compared with addition of TGF-␤1 alone, as assessed by immunoprecipitation of TGF-␤ receptor type I and immunoblot analysis for Cav-1 (Fig. 6).
To evaluate the importance of the IL-6-dependent trafficking of TGF-␤ receptors between the lipid raft and non-raft-associ- FIG. 1. IL-6 increases TGF-␤1-mediated Smad signaling. HK-2 cells were transfected with a Smad-responsive promoter, (SBE) 4 -Lux, prior to stimulation with 0.1 ng/ml recombinant TGF-␤1 Ϯ increasing doses of recombinant IL-6 (0 -100 ng/ml), all under serum-free conditions. All stimuli were applied for 24 h; subsequently, luciferase content was quantified as described under "Materials and Methods," and the results were normalized for transfection efficiency (using ␤-galactosidase) expressed as the fold increase above the non-stimulated control. Data represent mean Ϯ S.D. (n ϭ 9). ated membrane fractions on receptor turnover, we followed cell surface receptors affinity-labeled with 125 I-TGF-␤1 (Fig. 7). In cells, addition of 125 I-TGF-␤1 (250 pM) alone demonstrated rapid decay of the receptor. In contrast, addition of 125 I-TGF-␤1 together with IL-6 stabilized the receptors. The observation that stabilization of the receptor complex was related to trafficking to the non-raft membrane fractions was further supported by the observation that cholesterol sequestration by the addition of nystatin also led to prolongation of receptor complex half-life. DISCUSSION Our interest in the regulation of TGF-␤1 function in the kidney stems from the overwhelming evidence that implicates this pro-fibrotic factor in the pathogenesis of renal fibrosis (1)(2)(3)(4)(5). TGF-␤1 also plays a role in fetal development, wound healing, and regulation of inflammatory processes (35,36). Furthermore, it has an anti-proliferative effect on normal epithelial cells and acts as tumor suppressor, yet it also functions as a promoter of cancer progression and metastasis at later stages of disease (37). It is also clear, therefore, that understanding the processes that modulate TGF-␤1 activity has a much wider implication than renal disease.
TGF-␤s elicit their signaling effects by binding mainly to three cell surface receptors: type I, type II, and type III. Type I and II receptors are serine/threonine kinases that form heteromeric complexes and are necessary for TGF-␤ signaling. These are initiated when the ligand induces assembly of a hetero-meric complex of type II and type I receptors. The receptor type II kinase then phosphorylates receptor type I on a conserved glycine/serine-rich domain. This activates the receptor type I kinase, which subsequently recognizes and phosphorylates members of the intracellular receptor-regulated Smad signal transduction pathway. For TGF-␤1, these include Smad2 and Smad3. This causes dissociation of the intracellular receptorregulated Smads from the receptor, stimulates the assembly of a heteromeric complex between the phosphorylated intracellular receptor-regulated Smad and the co-Smad Smad 4, and induces nuclear accumulation of this heteromeric Smad complex (reviewed in Ref. 38).
Endocytosis of cell surface receptors is an important regulatory event in signal transduction. TGF-␤1 receptors internalize into both caveolin-and EEA-1-positive vesicles and reside in both lipid raft and non-raft membrane domains (8). Clathrindependent internalization into the EEA1-positive endosome promotes TGF-␤1 signaling. In contrast, the lipid raft-caveolar internalization pathway contains Smad7-bound receptor and is required for receptor turnover. TGF-␤ receptor type II internalization is unaffected by ligand stimulation, suggesting that its role is not to regulate trafficking but to act to recruit the type I receptor and stabilize heterotetrameric receptor complexes during constitutive trafficking events. Subsequently, the activated type I receptor signals in the EEA-1-positive endosome by phosphorylating Smad2 or directing degradation through lipid raft pathways by binding Smad7⅐Smurf2 complexes. We have recently identified a novel mechanism by which this process may be regulated that is dependent on the interaction between distinct receptor populations on the cell surface. These studies demonstrate that co-localization of the hyaluronan receptor CD44 and TGF-␤ receptors facilitates modulation of both Smad and non-Smad-dependent TGF-␤1mediated events by hyaluronan (26). Engagement of CD44 by hyaluronan in proximal tubular epithelial cells (PTC) attenuates TGF-␤1 signaling by increasing trafficking of TGF-␤ receptors to non-signaling lipid raft associated pools (9).
In the current study we have demonstrated that, in addition to the negative regulation of TGF-␤1 receptor function described previously (9,26), an augmented response to TGF-␤1 may also occur as the result of alteration of membrane trafficking of the receptor complex between the lipid and non-lipidassociated membrane fraction. As with our previous observations, these effects were unrelated to the binding of the TGF-␤ receptor and its ligand but rather related to the engagement of another membrane receptor, in this case, the cognate IL-6 receptor, by its ligand, IL-6. The results demonstrate that an . Activation of IL-6 signaling was assessed by electrophoretic gel mobility assay. Growth-arrested cells were stimulated with 10 ng/ml IL-6. At the times indicated, nuclear extracts were prepared, and electrophoretic mobility shift analysis was carried out as described under "Materials and Methods" (B). Confirmation of specificity of electrophoretic mobility shift analyses was performed by supershift assay (C). Growth-arrested cells were stimulated with 10 ng/ml IL-6 for 5 min prior to preparation of nuclear extracts, which were incubated with 20 g/ml anti-Stat1 or -Stat3 antibody prior to addition of radiolabeled probe.

FIG. 3. Stat and Smad protein association is not influenced by IL-6 or TGF-␤1.
Growth-arrested HK-2 cells were stimulated with 0.1 ng/ml TGF-␤1 Ϯ 10 ng/ml IL-6, as indicated. After 24 h, cell lysates were prepared, and immunoprecipitation for either Stat1 or Stat3 was performed as indicated under "Materials and Methods." Subsequently, Smad2 or Smad3 expression in the precipitate was examined by Western analysis. One representative experiment of three repeated experiments is shown.
increase in the association of the TGF-␤ receptor complex with non-lipid raft membrane fractions is associated with reduced receptor turnover and decreased association of the receptor with Cav-1. This is consistent with the previously published hypothesis that targeting the receptor away from the Cav-1, lipid-associated membrane pool facilitates TGF-␤1 signaling and reduces TGF-␤1 receptor degradation (8).
It has been suggested that regulation of caveolin-1 may be a critical step in modulating cellular responses to growth factors. In support of this, recent studies have demonstrated that FIG. 4. Distribution of TGF-␤ receptors. A, TGF-␤ receptor distribution in lipid raft and non-raft fractions was analyzed in HK-2 cells affinity-labeled with 125 I-TGF-␤1 and subjected to sucrose gradient subcellular fractionation to separate lipid rafts from other cellular components. The effect of IL-6 on trafficking of TGF-␤ receptors was assessed by comparison of distribution in HK-2 cells exposed to either 250 pM radiolabeled TGF-␤1 alone (Control) or radiolabeled TGF-␤1 in the presence of 10 ng/ml IL-6. In all experiments, subcellular fractionation was performed 24 h after addition of the stimuli. Characterization of the contribution of lipid rafts was examined by pre-treatment of cells with 50 g/ml nystatin for 1 h at 37°C prior to affinity labeling of TGF-␤ receptors (Nystatin). An equal volume from each fraction was analyzed by SDS-PAGE electrophoresis followed by autoradiography. B, following scanning densitometry of autoradiographs, the distribution of TGF-␤ receptor into the raft (fractions 5 and 6) and non-raft fractions (fractions 7-10) was quantified, and the data from three separate experiments are expressed graphically.
FIG. 5. Attenuation of TGF-␤1 receptor signaling. A, sequestration of TGF-␤ receptor from lipid rafts. HK-2 cells were transiently transfected with the Smad-responsive promoter (SBE) 4 -lux prior to addition of 0.1 ng/ml TGF-␤1 Ϯ 10 ng/ml IL-6 as indicated for 24 h. The role of lipid rafts in Smad signaling was examined by pre-treatment of transfected cells with 50 g/ml nystatin at 37°C for 1 h prior to addition of TGF-␤1 together with IL-6. The effect of nystatin was dependent on chelation of cholesterol, as demonstrated by the addition of nystatin to cells at 37°C for 1 h prior to addition of TGF-␤1 together with IL-6 and cholesterol (25 g/ml). B, inhibition of endosomal internalization. HK-2 cells transfected with the reporter construct were incubated in medium (Dulbecco's modified Eagle's medium/Ham's F-12):water (1:1) for 5 min at 37°C, followed by incubation in minimal medium (K ϩ -depleted serum-free medium containing 20 mM HEPES, pH 7.5, 140 mM sodium chloride, 1 mM calcium chloride, 1 mM magnesium sulfate, and 5.5 mM glucose) or full medium containing 10 mM potassium chloride for 1 h at 37°C prior to stimulation with TGF-␤1 (0.1 ng/ml) Ϯ IL-6 (10 ng/ml) for 24 h. For both experimental protocols, luciferase content was quantified as described under "Materials and Methods," and the results were normalized for transfection efficiency (using ␤-galactosidase) expressed as the fold increase above the non-stimulated control. The data represent the mean Ϯ S.D. of six individual experiments. caveolin-1 suppresses mitogen-activated protein kinase activation and cell proliferation induced by basic fibroblast growth factor and platelet-derived growth factor in mesangial cells (34). Previous work has also demonstrated that ectopic expression of Cav-1 enhances TGF-␤ receptor turnover (8). Although caveolin-1 may facilitate receptor turnover, it is important to note that TGF-␤ receptor may still partition to lipid raft membrane fractions and that TGF-␤1 receptor turnover continues in the absence of Cav-1 (8). The data presented in our study support the notion that TGF-␤ receptor turnover may be regulated independently from that of Cav-1 because we have shown a decrease in receptor turnover in the absence of changes in caveolin-1 expression, and we propose a model whereby the interaction between distinct receptors at the cell surface orchestrates the trafficking of the receptors, which dictates receptor fate.
The receptor complex mediating the biological activities of IL-6 consists of two distinct membrane-bound glycoproteins, an 80-kDa cognate receptor subunit and a 130-kDa signal-transducing element. Expression of the transmembrane-spanning gp130 is found in almost all cell types. In contrast, cellular distribution of the cognate IL-6 receptor is predominantly confined to hepatocytes and leukocyte subpopulations (39). Signal transduction involves the activation of Janus-activated kinase tyrosine kinase family members, leading to the activation of transcription factors of the Stat family (40). In addition to the membrane-bound receptor, a soluble form of the IL-6 receptor has been identified. This soluble receptor binds to IL-6, and the soluble IL-6 receptor⅐IL-6 complex activates cells via an interaction with membrane-bound gp130; thus IL-6, in the presence of soluble IL-6 receptor, may be an agonist for cell types that express gp130 in the absence of the cognate receptor. In the current study, we have demonstrated that proximal tubular epithelial cells express cognate IL-6 receptor, and we have also demonstrated its functional integrity, as assessed by specific Stat activation, following addition of IL-6. Although these data confirm the functional integrity of IL-6 signaling via the cognate receptor in these cells, our data would seem to support the hypothesis that the major mechanism that regulates the level of TGF-␤1-dependent Smad signaling by IL-6 is upstream of Stat activation, and it is interesting to speculate that this may involve the modulation of the direct association of the TGF-␤ receptor complex and the cognate IL-6 receptor.
Previous studies have demonstrated an interaction between Stat activation and the TGF-␤1 signaling pathway. Inhibition of TGF-␤ Smad signaling by interferon-␥ involves interferon-␥ acting through Janus-activated kinase 1 and Stat1 to induce the expression of the inhibitory Smad, Smad7 (27). In our system, however, we were unable to demonstrate any change in Smad7 expression. Examples of Stat/Smad synergy also exist, with TGF-␤ and ciliary neurotrophic factor acting synergistically through the cooperation of Smad and Stat transcription factors to induce vasoactive intestinal gene expression (28). Although we were able to demonstrate interaction between Stat and Smad proteins, it is unlikely that this is the basis of augmented TGF-␤1 signaling, given that no changes in these interactions could be demonstrated in our experimental system. I-TGF-␤1 at 4°C followed by cross-linking. Following incubation in either serum-free medium alone, 10 ng/ml IL-6, or 50 g/ml nystatin as indicated at 37°C for the indicated times, cells were lysed and processed for SDS-PAGE, and endogenous receptors were visualized by autoradiography (A). Three separate experiments were performed, quantified by scanning densitometry of autoradiographs, and graphed as receptor quantity (percentage of time 0) versus time (B). Each point represents mean Ϯ S.D. छ, control; •, IL-6; f, nystatin; #, p Ͻ 0.05, nystatin versus control; *, p Ͻ 0.05, IL-6 versus control.
One of the emerging themes of TGF-␤ is its dual or multifunctionality. In carcinogenesis, TGF-␤1 plays a complex role because it has the potential to act as either a tumor suppressor or a pro-oncogenic pathway (41). Indeed, it can switch from a tumor suppressor to a pro-oncogenic factor during the course of carcinogenic progression in a single cell lineage (42). In the context of inflammatory bowel disease, TGF-␤ is a potent negative regulator of mucosal inflammation (43); however, many studies have demonstrated an up-regulation of intestinal TGF-␤ in a variety of models of colitis and patients with inflammatory bowel disease (44,45). Similarly, although TGF-␤1 has traditionally been thought of in the kidney as a pro-fibrotic and therefore damaging factor promoting progressive renal dysfunction, there is emerging evidence that it may be involved in the postmitotic remodeling phase of recovery following ischemic renal injury (46 -48). The regulation of TGF-␤ receptor trafficking that we have reported in the current study and in our previous studies (9,26), may provide one explanation for these seemingly contradictory effects of TGF-␤1 because even in the presence of TGF-␤1, independent regulation of TGF-␤ receptor trafficking may determine the net effect of TGF-␤1. Recent evidence has highlighted the importance of TGF-␤ in regulating T-cell phenotype during inflammation (49,50), whereas IL-6 signaling has been shown to play a pivotal role in regulating mononuclear cell recruitment (51). Understanding the impact of the coincidence of these two pathways in dictating cellular responses may provide further insights into the regulation of both inflammatory and fibrotic processes.