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Originally published In Press as doi:10.1074/jbc.M800902200 on February 4, 2008

J. Biol. Chem., Vol. 283, Issue 16, 10461-10469, April 18, 2008
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AMP-activated Protein Kinase Inhibits Transforming Growth Factor-β-induced Smad3-dependent Transcription and Myofibroblast Transdifferentiation*

Rangnath Mishra{ddagger}, Barbara L. Cool§, Keith R. Laderoute, Marc Foretz||**, Benoit Viollet||**, and Michael S. Simonson{ddagger}1

From the {ddagger}Division of Nephrology and Hypertension, Department of Medicine, Case Western Reserve University and University Hospitals Case Medical Center, Cleveland Ohio 44106, the §Department of Metabolic Disease Research, Abbott Laboratories, Abbott Park, Illinois 60064, SRI International, Menlo Park, California 94025, ||Institut Cochin, Universite Paris Descartes, CNRS (UMR 8104), 75014 Paris, France, and **INSERM, U567, 75014 Paris, France

Received for publication, February 4, 2008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In wound healing, myofibroblast transdifferentiation (MFT) is a metaplastic change in phenotype producing profibrotic effector cells that secrete and remodel the extracellular matrix. Unlike pathways that induce MFT, the molecular mechanisms that negatively regulate MFT are poorly understood. Here, we report that AMP-activated protein kinase (AMPK) blocks MFT in response to transforming growth factor-β (TGFβ). Pharmacological activation of AMPK inhibited TGFβ-induced secretion of extracellular matrix proteins collagen types I and IV and fibronectin. AMPK activation also prevented induction of the myofibroblast phenotype markers {alpha}-smooth muscle actin and the ED-A fibronectin splice variant. AMPK activators did not prevent MFT in cells transduced with an adenovirus expressing dominant negative, kinase-dead AMPK{alpha}2. Moreover, AMPK activators did not inhibit MFT induction in AMPK{alpha}1,2–/– fibroblasts, demonstrating a requirement for AMPK{alpha} expression. Adenoviral transduction of constitutively active AMPK{alpha}2 was sufficient to prevent TGFβ-induced collagen I, {alpha}-smooth muscle actin, and ED-A fibronectin. AMPK did not reduce TGFβ-stimulated Smad3 COOH-terminal phosphorylation and nuclear translocation, which are necessary for MFT. However, AMPK activation inhibited TGFβ-induced transcription driven by Smad3-binding cis-elements. Consistent with a role for AMPK in transcriptional regulation, nuclear translocation of AMPK{alpha}2 correlated with the appearance of active AMPK{alpha} in the nucleus. Collectively, these results demonstrate that AMPK inhibits TGFβ-induced transcription downstream of Smad3 COOH-terminal phosphorylation and nuclear translocation. Furthermore, activation of AMPK is sufficient to negatively regulate MFT in vitro.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Myofibroblast transdifferentiation (MFT)2 is a fundamental cellular program activated in embryonic development, tumorstroma interactions, wound repair, and fibrosis in the lung, heart, liver, and kidney (1–4). Myofibroblasts are mesenchymal cells that display phenotypic markers of both muscle and nonmuscle cells. At sites of tissue injury, MFT produces profibrotic effector cells that assemble a fibrotic lesion (1, 3, 4). MFT is induced in an autocrine or paracrine fashion by cytokines, growth factors, and mechanical forces such as shear stress (3, 5).

The temporal sequence of MFT in wound healing is not entirely clear, but it involves early de novo expression of the ED-A fibronectin (ED-A FN) splice variant and formation of a proto-myofibroblast (1, 6). The transition of proto-myofibroblasts into myofibroblasts is marked by abundant de novo expression of {alpha}-smooth muscle actin ({alpha}SMA) (7). Stress fibers rich in {alpha}SMA develop and link to focal adhesions at the plasma membrane (1, 2). Finally, the mature myofibroblasts secrete a fibrillar collagen- and fibronectin-rich extracellular matrix that is remodeled into a fibrotic lesion (3, 8).

AMP-activated protein kinase (AMPK) was initially characterized as a protein activated by nutrient and bioenergetic stress that raises intracellular AMP and lowers ATP (9–11). AMPK belongs to the evolutionarily conserved SNF1 (sucrose non-fermentor) family of serine/threonine kinases that regulate energy homeostasis in eukaryotes (12, 13). The kinase exists as a heterotrimer with a catalytic {alpha}-subunit and regulatory β- and {gamma}-subunits. AMPK is highly sensitive to small increments in AMP and to elevations in the AMP/ATP ratio (12, 14). Stimulation of AMPK also requires phosphorylation of a critical threonine residue (Thr-172) in the activation loop of the {alpha}-subunit. At least two upstream AMPK kinases have been identified, including the tumor suppressor LKB1 and the Ca2+-calmodulin-dependent protein kinase kinases {alpha} and β (11, 15, 16). In cells, AMPK activation slows metabolic reactions that consume ATP and stimulates reactions that produce ATP, thereby restoring the AMP/ATP ratio and the normal cellular energy state (12). Recently it has become clear that AMPK can be activated independent of changes in the AMP/ATP ratio (15–17), implying that AMPK might have cellular functions unrelated to its role as a nutrient stress defense pathway.

In this study we asked whether MFT is negatively regulated by AMPK. Numerous stimuli of MFT have been identified, such as endothelin-1 and transforming growth factor-β (TGFβ), but negative regulation of MFT has not been extensively characterized. Here, we report that AMPK activation blocks Smad3-dependent transcription and MFT in response to TGFβ.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Reagents—We purified and characterized recombinant human adiponectin from the supernatant of 293-T cells (generous gift from Philipp Scherer, Albert Einstein College of Medicine) stably transfected with the pFM1 vector containing an Acrp30 gene exactly as described (18). Adiponectin from a mammalian tissue culture system was used because bacteria cannot properly process the hydroxylprolineated collagenous domain of adiponectin (18). Recombinant human TGFβ1 was from R&D, and AICAR was from Sigma. A-769662 has been previously described (19). Antibodies were as follows: affinity-purified rabbit anti-human collagen type I (Biodesign International); collagen IV and fibronectin (Rockland); {alpha}SMA (Epitomics); mouse monoclonal ED-A FN and β-actin (Sigma). All other antibodies were from Cell Signaling.

Cell Culture and Induction of MFT—Human primary mesangial cells (HMC) were from Cambrex Bioscience Inc. (Walkersville, MD) and were characterized and cultured as previously reported (20, 21). Human primary mesangial cells were used in passages 4–10. Where indicated we also studied MFT in murine embryonic fibroblasts null for AMPK{alpha}1,{alpha}2 (22). To induce MFT, cells in 60 mm plates at ~80% confluence were made quiescent for 24 h in DMEM with 0.5% FBS, then stimulated with 0.5 ng/ml TGFβ-1. The media contained 50 µg/ml β-aminopropionitrile to minimize cross-linking. Twelve, 24 or 48 h after adding TGFβ, the supernatant was collected and the monolayer was solubilized in a 5 M guanidine-0.1 M Tris buffer (pH 8.6) with protease inhibitors as described (23).

Adenoviral Transduction of Human Primary Mesangial Cells with Dominant Negative and Constitutively Active AMPK{alpha}2—Replication-defective adenoviral vectors encoding dominant negative AMPK{alpha}2 (Ad.dnAMPK{alpha}2), constitutively active AMPK{alpha}2 (Ad.caAMPK{alpha}2) and control adenovirus (Ad.trGFP) have been described (24–26). Adenovirus expressing a dominant negative mutant of AMPK {alpha}2 was provided by Dr. Ken Walsh (25, 27). All viral constructs were amplified in 293 cells and purified by using an Adeno-XTM virus mini-purification kit from Clontech Inc. (Mountain View, CA). Viral titers were determined as plaque forming units. For transduction, human primary mesangial cells were incubated with adenovirus at a multiplicity of infection of 50 for 12 h. The optimal multiplicity of adenoviral infection was determined using a GFP-encoding adenovirus. Under these conditions, the transduction efficiency was >90%.


Figure 1
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  		FIGURE 1.
The AMPK activators AICAR and adiponectin block TGFβ-stimulated collagen I secretion. A, time course of TGFβ-induced collagen I secretion with and without AMPK activation by AICAR and adiponectin (Adipo). Quiescent cells were preincubated for 30 min with 0.5 mM AICAR or 20 µg/ml adiponectin before adding 0.5 ng/ml TGFβ for 12, 24, and 48 h. Collagen I secretion in the supernatant was measured by ELISA. *, p < 0.05 versus time 0. B, cells were preincubated with 100 µM A-769662 or vehicle (0.01% Me2SO) for 30 min before addition of TGFβ for 48 h and measurement of secreted collagen I by ELISA. *, p < 0.05 versus untreated with vehicle. Data are mean ± S.D. for three experiments in duplicate.

 
Direct ELISA for Collagens, Fibronectins, and {alpha}SMA—ELISA for secreted collagen I, collagen IV, and FN in the medium was as described (24, 28). We also used ELISA to measure cell-associated ED-A FN and {alpha}SMA (29). 100 µl of media or cell extracts were absorbed to Nunc Maxisorp 96-well plates overnight at 4 °C. After washing three times with phosphate-buffered saline/0.1%Tween, nonspecific binding was blocked in 1.0% bovine serum albumin, 100 mM phosphate buffer, pH 8.2. The wells were then incubated with a primary antibody, the appropriate affinity-purified and biotin-conjugated goat anti-IgG, and a horseradish peroxidase-conjugated streptavidin. A solution of 3,5,2',5'-tetramethylbenzidine was added followed by quenching with acid and an absorbance reading at 450 nm in a SpectraMax 190 (Molecular Devices). All values were in the linear range and were normalized for cell number.

Immunocytochemical and Immunofluorescent Analysis of ED-A FN and PSMAD3—For immunocytochemistry, cells were fixed in 2% paraformaldehyde for 20 min, incubated with 0.6% hydrogen peroxide for 30 min at room temperature, and permeabilized with 0.1% Triton X-100 in TBS for 15 min. After blocking with 3.0% bovine serum albumin in TBS-T for 1 h, primary antibody (1:500) was added overnight at 4 °C. The primary antibody was detected using the ImmPress Universal Antibody detection system (Vector Laboratories) exactly as described by the manufacturer. The monolayer was mounted in Vectamount (Vector Laboratories). A similar protocol without hydrogen peroxide pretreatment was used for immunofluorescent staining with detection by a fluorescein isothiocyanate-conjugated secondary antibody, counterstaining with 4',6-diamidino-2-phenylindole and mounting in Vectashield. The PSmad3 antibody recognizes Smad3 phosphorylated at Ser-423/425. Images were acquired with a SPOT RT camera (Diagnostic Instruments).


Figure 2
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  		FIGURE 2.
AMPK activators prevent collagen IV and fibronectin secretion in cells stimulated with TGFβ. Quiescent cells were stimulated for 48 h with 0.5 ng/ml TGFβ plus or minus 0.5 mM AICAR, 20 µg/ml adiponectin, or 100 µM A-769662. Media were collected and analyzed by ELISA for collagen IV (A) and fibronectin (B) secretion. In A and B, *, p < 0.05 versus untreated control. Data are mean ± S.D. for three independent experiments in duplicate.

 
Measurement of Smad3-directed Transcriptional Activity—We employed mink lung cells stably transfected with a minimal promoter from the plasminogen activator inhibitor-1 (PAI-1) gene linked to luciferase (30). Cells were also transiently transfected (31) with pSBE4-Luc (46), kindly provided by Dr. Bert Vogelstein, to measure Smad3 transactivation. After treatment with TGFβ and AMPK activators for 24 h, cytosolic luciferase activity was measured as described (31).

Subcellular Fractionation—Cells were homogenized at 4 °C in 0.25 M sucrose containing 10 mM Tris-Cl (pH 8.0), 1 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol with protease inhibitors (Roche Applied Science). The cell lysate was centrifuged at 3000 x g for 10 min at 4 °C, and the resulting pellet and supernatant were collected as the nuclear and cytoplasmic (post nuclear supernatant) fractions.


Figure 3
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  		FIGURE 3.
In cells treated with TGFβ, AMPK activators inhibit induction of the MFT markers {alpha}SMA and ED-A FN. A, TGFβ was added to mesangial cells for 48 h with and without the AMPK activators (preincubated for 30 min) AICAR (0.5 mM), adiponectin (20 µg/ml), or A-769662 (100 µM). {alpha}SMA was measured in cell lysates by ELISA. B, the ED-A FN splice variant was assessed qualitatively by immunocytochemistry in quiescent cells and in cells stimulated with TGFβ (C) or TGFβ plus A-769662 (D). E, to control for nonspecific binding, cells stimulated with TGFβ were stained with a monoclonal isotype control antibody. F, cells were treated as described, and secreted ED-A FN was measured by ELISA in the cell media. Data in A and F are mean ± S.D. for three experiments. In A and F, *, p < 0.05 versus untreated control.

 
Data Analysis and Statistics—Data are mean ± S.D. from 3–4 independent experiments. Statistical significance was calculated by unpaired Student's t test or by analysis of variance with Bonferroni multiple correction using InStat (GraphPad) (asterisks represent p < 0.05 in the figures).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
AMPK Activators Inhibit MFT in Cells Treated with TGFβ—To study MFT in response to TGFβ, we used human renal mesangial cells, an in vitro model of MFT (32–34). In cultured mesangial cells, TGFβ induces the myofibroblast phenotype with expression of collagen type I (24, 35), ED-A FN (36–38), and {alpha}SMA (35). We asked whether activators of AMPK prevent induction of MFT marker proteins by TGFβ. AICAR (5-aminoimidazole-4-carboxamide riboside) is a cell-permeable adenosine analog phosphorylated to form 5-aminoimidazole-4-carboxamide-1-d-ribofuranosyl 5'-monophosphate, an AMP mimetic. Recombinant human adiponectin is also a potent stimulus of AMPK (12).


Figure 4
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  		FIGURE 4.
Dominant negative AMPK{alpha}2 reverses inhibition of TGFβ-stimulated MFT marker proteins by AMPK activators. A, schematic illustration of the AMPK{alpha}2 dominant negative mutant with an inactivating mutation in the kinase domain. Quiescent cells were transduced for 24 h with adenoviral vectors expressing dnAMPK{alpha}2 (Ad.dnAMPK) or GFP (Ad.trGFP). Transduced cells were treated with 0.5 ng/ml TGFβ after a 30-min preincubation with the AMPK activators A-769662 (100 µM) and AICAR (0.5 mM). After 48 h collagen I in the media (B), {alpha}SMA in cell lysates (C), and secreted ED-A FN (D) were measured, and the results were normalized to the untreated cells transduced with Ad.trGFP. *, p < 0.05 versus untreated with Ad.trGFP. Data are mean ± S.D. of three experiments.

 
AICAR and adiponectin prevented TGFβ-induced secretion of matrix proteins that are induced in MFT. TGFβ stimulated collagen 1 secretion at 24 and 48 h, but preincubation with AICAR or adiponectin blocked stimulation by TGFβ (Fig. 1A). Cool and coworkers (19) recently described a small molecule, direct activator of AMPK{alpha}, the thienopyridone derivative A-769662. Similar to the effect of AICAR and adiponectin, A-769662 prevented induction of collagen I secretion in cells exposed to TGFβ (Fig. 1B). In mesangial MFT, TGFβ also induces collagen IV and fibronectin (39). We observed that the AMPK activators AICAR, adiponectin, and A-769662 blocked the 2.3-fold induction of collagen IV by TGFβ (Fig. 2A). Similarly, the AMPK activators prevented the 2.1-fold induction of fibronectin secretion by TGFβ (Fig. 2B). Collectively, these results suggest that AMPK prevents TGFβ-stimulated induction of matrix proteins induced in MFT.

To directly determine whether AMPK blocks MFT, we added AMPK activators to cells exposed to TGFβ. We then measured induction of {alpha}SMA and the ED-A FN fibronectin splice variant, well established markers of the MFT phenotype. AICAR, adiponectin, and A-769662 blocked the 2.1-fold increment in {alpha}SMA in cells exposed to TGFβ (Fig. 3A). Similarly, the AMPK activators prevented induction of ED-A FN by TGFβ. Quiescent mesangial cells expressed minimal ED-A FN by immunocytochemistry and ELISA, but 48 h after TGFβ abundant ED-A FN extracellular fibrils were detected (Fig. 3, B–F), similar to induction of ED-A FN in other cellular models of MFT (6). A-769662 blocked the 2.6-fold increase of ED-A FN by TGFβ (Fig. 3, C, D, and F). AICAR and adiponectin also inhibited ED-A FN expression (Fig. 3F). Taken together, these results suggest that AMPK negatively regulates MFT.

Genetic Evidence That AMPK{alpha} Blocks TGFβ-stimulated MFT—To further investigate negative regulation of MFT by AMPK, we asked whether a dominant negative, kinase-dead AMPK{alpha} mutant that antagonizes AMPK signaling (40) prevents inhibition of MFT by AMPK activators. Adenovirus transduction with dnAMPK{alpha}2 prevented inhibition of TGFβ-stimulated collagen I secretion by AICAR, adiponectin, and A-769662 (Fig. 4, A and B). Transduction with an adenoviral vector expressing GFP had no effect on basal or TGFβ-stimulated collagen I secretion, demonstrating that the effect of dnAMPK{alpha}2 was not a nonspecific consequence of adenoviral gene transfer. Moreover, AICAR blocked expression of collagen I in cells transduced with Ad.trGFP. In cells transduced with Ad.dnAMPK{alpha}2, the 2.3-fold induction of {alpha}SMA by TGFβ was not inhibited by AICAR, adiponectin and A-769662 (Fig. 4C). Similarly, AMPK activators did not block the 2.7-fold elevated secretion of ED-A FN in cells transduced with Ad.dnAMPK{alpha}2 (Fig. 4D). Basal and stimulated levels of {alpha}SMA or ED-A FN were not altered by adenoviral transduction with Ad.trGFP. These results with dnAMPK{alpha}2 support the hypothesis that AMPK signaling blocks MFT.

Experiments in murine embryonic fibroblasts lacking AMPK{alpha}1 and AMPK{alpha}2 also demonstrated that inhibition of TGFβ-stimulated MFT by the activators specifically requires AMPK. We confirmed that basal or activated AMPK{alpha} protein was absent in AMPK –/–{alpha} fibroblasts (data not shown). TGFβ-induced collagen I secretion was similar in AMPK –/–{alpha} compared with AMPK +/+{alpha} fibroblasts (1.9-compared with 1.8-fold stimulation, Fig. 5A), demonstrating intact TGFβ signaling in the null fibroblasts. A-769662 blocked TGFβ-stimulated collagen I secretion in AMPK +/+{alpha} cells but not in AMPK –/–{alpha} cells (Fig. 5A), suggesting a requirement for AMPK. AICAR was similarly ineffective in the AMPK –/–{alpha} fibroblasts but was inhibitory in AMPK +/+{alpha} cells (Fig. 5A). These results demonstrate that AMPK is required for inhibition of TGFβ-stimulated collagen I by A-769662 and AICAR.


Figure 5
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  		FIGURE 5.
AMPK activators do not inhibit MFT in murine embryonic fibroblasts null for AMPK{alpha}. Quiescent AMPK –/–{alpha} fibroblasts lacking both {alpha}1 and {alpha}2 subunits and AMPK +/+{alpha} wild-type controls were stimulated for 48 h with 0.5 ng/ml TGFβ with and without 100 µM A-769662 or 0.5 mM AICAR. ELISA was then used to measure collagen I secretion (A), {alpha}SMA accumulation (B), and ED-A FN in the media (C). *, p < 0.05 versus untreated control in respective cell type. Data are mean ± S.D. for three experiments in duplicate.

 
Similarly, in AMPK –/–{alpha} null fibroblasts A-769662 and AICAR did not inhibit expression of the MFT markers {alpha}SMA and ED-A FN. Induction of {alpha}SMA by TGFβ was similar in AMPK +/+{alpha} or AMPK –/–{alpha} cells, demonstrating that the signals linking TGFβ receptor activation to induction of {alpha}SMA are unaffected in the null fibroblasts (Fig. 5B). However, the 2.5-fold induction of {alpha}SMA in AMPK +/+{alpha} cells treated with TGFβ was not inhibited by A-769662 or AICAR in AMPK –/–{alpha} null fibroblasts (Fig. 5B). A-769662 and AICAR also failed to block ED-A FN secretion in AMPK –/–{alpha} fibroblasts exposed to TGFβ (Fig. 5C). Collectively, these experiments in AMPK –/–{alpha} fibroblasts provide strong evidence that AMPK negatively regulates TGFβ-stimulated MFT.

We next asked whether AMPK activity is sufficient to block TGFβ-stimulated MFT. An AMPK{alpha}2 mutant consisting of only the kinase domain and lacking the auto-inhibitory and β/{gamma} interaction domains (Fig. 6A) is constitutively active (caAMPK{alpha}2), even in the absence of AMPK activators (26). We expressed caAMPK{alpha}2 by means of an adenoviral gene transfer vector. Transduction of Ad.caAMPK{alpha}2 had no effect on basal collagen I secretion, but it blocked TGFβ-stimulated expression (Fig. 6B). Neither basal nor TGFβ-stimulated collagen I secretion were affected by transduction with Ad.trGFP (Fig. 6B). caAMPK{alpha}2 also blocked induction of {alpha}SMA and ED-A FN protein by TGFβ (Fig. 6, C and D). Transduction with Ad.trGFP had no effect on induction of {alpha}SMA and ED-A FN. Taken together, these results with dn.AMPK{alpha}2, AMPK –/–{alpha} null fibroblasts, and ca.AMPK{alpha}2 demonstrate that AMPK{alpha} is sufficient to prevent MFT in cells exposed to TGFβ.

AMPK Blocks Smad3-mediated Transcription without Inhibiting Smad3 Phosphorylation or Nuclear Translocation—To determine the mechanism underlying inhibition of MFT by AMPK, we studied signals downstream of TGFβ receptor activation. Because Smad3 is essential for TGFβ-stimulated MFT (41–44), we asked whether AMPK blocks Smad3 phosphorylation or nuclear translocation.

Immunofluorescent analysis showed rapid TGFβ-stimulated COOH-terminal activating phosphorylation of Smad3 and nuclear localization. PSmad3 was nearly undetectable in the cytosol or nucleus of unstimulated cells, but after 30 min of TGFβ 83% of cell nuclei were positive for PSmad3 (Fig. 7, A and B). Two hours after TGFβ, nuclear levels of PSmad3 started to modestly decline. The AMPK activators AICAR and A-769622 did not reduce the staining intensity or nuclear localization of PSmad3 in response to TGFβ (Fig. 7, A and B). An unexpected finding was that AMPK activators alone transiently increased PSmad3 levels and translocation, albeit much less intensely than with TGFβ alone or TGFβ plus the AMPK activators (Fig. 7B). These results demonstrate that AMPK does not inhibit Smad3 phosphorylation at the activating epitope. Moreover, AMPK does not inhibit nuclear translocation of PSmad3.

Because AMPK did not block phosphorylation or nuclear translocation of Smad3, we investigated whether AMPK inhibited Smad3-mediated transcription downstream of the nuclear localization step. To that end we employed reporter cells with three Smad3-sensisitive cis-elements in the context of a heterologous human PAI-1 promoter driving transcription of luciferase (30, 45). TGFβ increased luciferase reporter activity 24.7-fold (Fig. 8A), and the increase in Smad3-dependent transcription was blocked by a TGFβ-neutralizing antisera, demonstrating specificity for TGFβ (data not shown). Both AICAR and A-769662 blocked the increase in luciferase activity in cells exposed to TGFβ (Fig. 8A). Activation of Smad3-dependent transcription was not inhibited by transduction with Ad.trGFP but was blocked by the dominant negative AMPK{alpha}2 mutant (Fig. 8A). We confirmed that AMPK blocked Samd3-dependent transcription in mesangial cells transfected with pSBE4-Luc (Fig. 8B), which is transactivated only by Smad3 and not by other R-Smad proteins (46). Collectively, these data suggest that AMPK inhibits Smad3-mediated transcription by a mechanism downstream of TGFβ-stimulated Smad3 phosphorylation and nuclear translocation.


Figure 6
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  		FIGURE 6.
A constitutively active AMPK{alpha}2 mutant suffices to prevent induction of MFT marker proteins by TGFβ. A, schematic diagram of the truncated AMPK{alpha}2 mutant with constitutively active (caAMPK{alpha}2). Quiescent cells were transduced for 24 h with adenoviral vectors expressing constitutively active AMPK{alpha}2 (Ad.caAMPK) or GFP (Ad.trGFP). TGFβ (0.5 ng/ml) or medium alone was added for an additional 48 h and collagen I (B), {alpha}SMA (C), and ED-A FN (D) were assessed by ELISA. *, p < 0.05 versus untreated with Ad.trGFP. Data are mean ± S.D. from n = 3 experiments.

 
We also asked whether active AMPK was present in mesangial cell nuclei, consistent with a role for AMPK in transcriptional regulation. Because it is well established that AMPK{alpha}1 does not translocate to the nucleus (47, 50), we assessed translocation of AMPK{alpha}2 in subcellular fractions. The AMPK{alpha}2 subunit was barely detectable in nuclei of quiescent cells, but the kinase rapidly translocated from cytosol to the nucleus when cells were stimulated with AICAR (Fig. 9). Nuclear translocation of AMPK{alpha}2 correlated with the appearance of P172ThrAMPK{alpha} (active kinase) in the nucleus. AICAR-stimulated nuclear localization and activation of AMPK{alpha}2 was unaffected by addition of TGFβ, and addition of TGFβ alone weakly elevated P172ThrAMPK{alpha} at 1 h, which presumably reflected phosphorylation of AMPK{alpha} by the TGFβ-activated protein kinase (49, 50). However, we note that the level of AMPK{alpha} activation by TGFβ was of insufficient magnitude and duration to inhibit MFT. These results demonstrate that AMPK{alpha}2 can translocate to the nucleus in an activated form in mesangial cells and support a role for AMPK{alpha} in regulation of Smad3-dependent transcription.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
MFT, a metaplastic change in cell phenotype, is essential for the development of profibrotic effector cells in the wound healing response and is induced by extracellular cues such as growth factors, cytokines, and mechanical stress. Yet the molecular mechanisms that negatively regulate MFT are poorly understood. In this study, we demonstrate that AMPK is sufficient to repress TGFβ induction MFT of mesangial cells in vitro.

AMPK as a Negative Regulator of MFT—In eukaryotes AMPK has an evolutionarily conserved role regulating the catabolic and anabolic metabolism of glucose and fatty acids at the cellular and whole body levels (10–12). Recent studies have revealed novel functions for AMPK, including regulation of a cell cycle checkpoint (51), the decision to enter autophagy or apoptosis (52, 53), and cell fate decisions in development (54). Our experiments show that two mechanistically different pharmacological activators of AMPK block induction of myofibroblast marker proteins in response to TGFβ. A caveat for interpreting results with AICAR is that 5-aminoimidazole-4-carboxamide-1-d-ribofuranosyl 5'-monophosphate can weakly stimulate glycogen phosphorylase (55, 56) and inhibit fructose-1,6-bisphosphatase (57). Unlike AICAR, however, A-769662 does not affect glycogen phosphorylase or fructose-1,6-bisphosphatase activity (19). A-769662 does not alter the activity of a broad spectrum of proteins in a high-throughput screen (19), suggesting that A-769662 is selective for AMPK. The finding that AICAR and A-769662 failed to repress MFT marker proteins in AMPK{alpha}1,2 null fibroblasts treated with TGFβ suggests that the pharmacological agonists were specific for AMPK. Regarding the role of MFT in fibrosis, an important phenotypic change in mesangial cells undergoing MFT is secretion of the fibrillar collagen type 1, which is not secreted by normal mesangial cells in vivo but is robustly induced in glomerulosclerosis. Excessive secretion of fibronectin and the basement membrane protein collagen IV is also observed in mesangial-myofibroblasts in glomerulosclerosis in vivo. It is therefore significant that the AMPK activators blocked induction of collagen I, collagen IV, and fibronectin in mesangial cells.

The notion that AMPK negatively regulates TGFβ-stimulated MFT was also demonstrated in this study using three genetic approaches. First, mutation of lysine 45 to arginine in the AMPK{alpha}2 kinase domain results in a dominant negative mutant that antagonizes AMPK signaling (40). Adenoviral transduction of this AMPK{alpha}2 mutant prevented repression of collagen I, {alpha}SMA, and ED-A FN in mesangial cells stimulated with TGFβ. Second, a constitutively active mutant of AMPK{alpha}2, which stimulates AMPK signaling (26), prevented induction of the MFT marker proteins. These findings also suggest that AMPK activity is sufficient in mesangial cells to block the MFT transition in response to TGFβ and concurs with our results with AICAR and A-769662. Finally, AMPK activators were unable to block MFT in murine embryonic fibroblasts null for AMPK{alpha} subunits. Double knockouts of AMPK{alpha}1 and AMPK{alpha}2 are embryonic lethal (22), thus mesangial cells lacking both AMPK{alpha} subunits could not be prepared. These results in murine embryonic fibroblasts are also important, because they suggest that the negative effect of AMPK on MFT is not restricted to glomerular mesangial cells and that AMPK might be a more general inhibitor of MFT in other cellular contexts.


Figure 7
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  		FIGURE 7.
AMPK activation does not alter the activating phosphorylation and nuclear translocation of Smad3. A, quiescent mesangial cells on glass coverslips were stimulated for 30 min with 0.5 ng/ml TGFβ alone or TGFβ plus 0.5 mM AICAR or 100 µM A-769662. PhosphoSer-423/425 Smad3 (top row) and 4',6-diamidino-2-phenylindole nuclear staining (bottom row) in fixed cells were detected by immunofluorescence. B, in the time course studies cells were treated for the times indicated and analyzed for PSmad3 by immunofluorescence, and cells with nuclear PSmad3 were counted and expressed as a percentage of the 4',6-diamidino-2-phenylindole-stained cells. Data are mean ± S.D. from two experiments.

 
A Smad3-based Mechanism for Inhibition of MFT by AMPK—As a window into potential mechanisms that underlie negative regulation of MFT by AMPK, we asked whether AMPK inhibits transcriptional regulation by Smad3. Numerous signal transduction pathways have been associated with MFT in vitro and in vivo, but most studies have shown that Smad3 is required for MFT in response to TGFβ (see Refs. 41–44 for review). The activated TGFβ receptor kinase phosphorylates the COOH-terminal domain of regulatory Smads (Smad2/3), which then form heterodimers or trimers with Smad4 and translocate to the nucleus. Smad complexes in the nucleus bind to Smad-binding elements and activate or repress gene transcription. In our experiments COOH-terminal stimulatory phosphorylation of Smad3 was unaffected by the AMPK activators. Moreover, nuclear translocation of PSmad3 was not altered by AMPK, suggesting that AMPK does not block the early steps in Smad3 signaling.

Inhibition of Smad3-stimulated signaling by AMPK occurs downstream of nuclear translocation and involves transcriptional regulation. AMPK blocked TGFβ-stimulated transactivation of a luciferase reporter construct (pSBE4-Luc) that is highly selective for Smad3. AMPK also inhibited activation of the human PAI-1 promoter, an informative model of Smad3-dependent transcription (45, 58–60). Stimulation of the human PAI-1 promoter by TGFβ has been previously shown to require Smad3 and three Smad3-binding cis-elements (45, 58–60). In addition, PAI-1 gene induction is relevant to the study of MFT and glomerular fibrosis: several laboratories have documented that PAI-1 is potently induced in MFT and that it contributes to the increase in fibrosis that results from MFT (61–63). TGFβ also induces transcription and expression of PAI-1 in human mesangial cells (64), and genetic depletion of PAI-1 augments mesangial matrix protein accumulation and glomerular fibrosis in an experimental model of chronic kidney disease (65). Our results demonstrate that AMPK prevents transcriptional activation of the PAI-1 promoter by TGFβ. Inhibition of Smad3-dependent activation of the PAI-1 promoter by the AMPK activators was antagonized by adenovirus transduction of the dnAMPK{alpha}2 mutant, confirming that repression by AICAR and A-769662 required AMPK activity. Translocation of active AMPK{alpha}2 to the mesangial cell nucleus, which has previously been demonstrated in other cells types (47, 48), provides further support for the notion that AMPK regulates Smad3-directed gene expression at the level of transcription. Whether AMPK prevents regulated recruitment of Smad3 to the PAI-1 promoter, or instead regulates Smad3-binding co-activators or co-repressors, remains to be determined.


Figure 8
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  		FIGURE 8.
AMPK inhibits Smad3-directed transcription of a luciferase reporter gene. A, schematic (top) of the stably transfected luciferase reporter with three upstream Smad3-responsive cis-elements from the human PAI-1 promoter. Quiescent cells were treated with 0.5 ng/ml TGFβ for 48 h in the presence and absence of 0.5 mM AICAR, 100 µM A-769662, or vehicle. B, mesangial cells were transiently transfected with pSBE4-Luc (schematic), and after being made quiescent for 24 h the cells were treated with AICAR to stimulate AMPK as in A. In some experiments cells were infected with adenoviral vectors for 24 h before transfection with pSBE4-Luc. In A and B luciferase was measured in cell lysates and expressed as -fold increase compared with untreated cells. *, p < 0.05 versus untreated control. Data are mean ± S.D. for n = 3 experiments.

 
In the wound healing response of kidney, heart, liver, and lung, MFT is a common phenotypic transition associated with reorganization of the extracellular matrix, development of fibrosis and loss of functional tissue. Thus regulators of MFT represent a potential approach for limiting fibrotic tissue injury in vivo (3, 4, 66). With the present observations, we define AMPK as a new negative regulator of MFT and speculate that AMPK activation in vivo might modulate the wound healing response and fibrosis.


Figure 9
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  		FIGURE 9.
Nuclear translocation of AMPK{alpha}2 and localization of active AMPK{alpha}. Quiescent mesangial cells were stimulated for 1 or 2 h with 0.5 mM AICAR, 0.5 ng/ml TGFβ, or AICAR plus TGFβ. Subcellular cytosolic and nuclear fractions were prepared by centrifugation and analyzed by Western blotting for the indicated proteins. The blots were reprobed with β-actin or lamin-A to confirm equal protein loading and to assess the relative purity of the cytosolic and nuclear fractions. Results are representative of three independent experiments.

 

   FOOTNOTES
 
* This work was supported by grants from the Rosenberg Foundation of the Centers for Dialysis Care (Cleveland, OH) and from the EXGENESIS Integrated Project (Grant LSHM-CT-2004-005272) funded by the European Commission (to B. V.). 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, Division of Nephrology, Biomedical Research Bldg., Rm. 427, Case Western Reserve University, 2109 Adelbert Rd., Cleveland, OH 44106. Tel.: 216-368-1251; Fax: 216-368-1249; E-mail: mss5{at}po.cwru.edu.

2 The abbreviations used are: MFT, myofibroblast transdifferentiation; AMPK, AMP-activated protein kinase; TGFβ, transforming growth factor β; AICAR, 5-aminoimidazole-4-carboxamide riboside; {alpha}SMA, {alpha}-smooth muscle actin; PAI-1, plasminogen activator inhibitor-1; FN, fibronectin; ELISA, enzyme-linked immunosorbent assay; dn, dominant negative; Ad, adenovirus; GFP, green fluorescent protein; ca, constitutively active. Back


   ACKNOWLEDGMENTS
 
Mink lung epithelial cells stably transfected with the PAI-1 promoter were generously provided by Dr. Daniel Rifkin. We thank David Nethery and Dr. Jeff Kern for help with immunofluorescent microscopy.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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