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Originally published In Press as doi:10.1074/jbc.M703597200 on June 7, 2007

J. Biol. Chem., Vol. 282, Issue 31, 22910-22920, August 3, 2007
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Differentiation of Human Circulating Fibrocytes as Mediated by Transforming Growth Factor-beta and Peroxisome Proliferator-activated Receptor {gamma}*

Kurt M. Hong{ddagger}§, John A. Belperio, Michael P. Keane, Marie D. Burdick||, and Robert M. Strieter||1

From the {ddagger}Center for Human Nutrition and the Departments of §Pathology and Laboratory Medicine and Medicine, Division of Pulmonary and Critical Care Medicine, David Geffen School of Medicine, UCLA, Los Angeles, California 90024 and the ||Department of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908

Received for publication, May 1, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fibrocytes are a distinct population of fibroblast-like progenitor cells in peripheral blood that have recently been shown to possess plasticity to differentiate along mesenchymal lineages, including commitment to myofibroblast and adipocyte cells. Here, we demonstrated that transforming growth factor (TGF) beta1 drives fibrocyte-to-myofibroblast differentiation through activating Smad2/3 and SAPK/JNK MAPK pathways, which in turn stimulates {alpha}-smooth muscle actin expression. We determined that SAPK/JNK signaling acts in a positive feedback loop to modulate Smad2/3 nuclear availability and Smad2/3-dependent transcription. Conversely, fibrocyte-to-adipocyte differentiation is driven by the peroxisome proliferator-activated receptor (PPAR) {gamma} agonist troglitazone, which is associated with cytoplasmic lipid accumulation and induction of aP2. Treatment with troglitazone also disrupted TGFbeta1-activated SAPK/JNK signaling, leading to decreased Smad2/3 transactivation activity and {alpha}-smooth muscle actin expression. Interestingly, TGFbeta1 was demonstrated to have reciprocal inhibition on fibrocyte differentiation to adipocytes. By activating SAPK/JNK signaling, which is normally suppressed during adipogenesis, PPAR{gamma}-dependent transactivation activity and induction of aP2 expression were disrupted. Taken together, within the context of the local microenvironmental niche, the delicate balance of PPAR{gamma} and TGFbeta1 activation drives the selection of an adipocyte or myofibroblast differentiation pathway through SAPK/JNK signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A large body of evidence now supports the existence of adult stem cells. Although they are predominantly found in the bone marrow, they have also been isolated from muscle, adipose tissue, connective tissue, and in peripheral circulation. Adult stem cells are characterized by their ability to differentiate along multiple lineages, giving rise to fully differentiated cells with distinct function. Multiple signaling networks orchestrate the development and differentiation of adult stem cells into functional mesenchymal, neuronal, and epithelial lineages.

We recently characterized the plasticity of an adult progenitor cell found in circulation, termed fibrocytes, which can differentiate into myofibroblast, osteoblast, and adipocyte lineages (1, 2). Fibrocytes are a distinct population of fibroblast-like cells in peripheral blood with a unique cell surface phenotype, expressing CD45RO, CXCR4, and collagen I (3, 4). They are distinct from tissue fibroblasts, monocyte/macrophages, T and B lymphocytes, dendritic cells, or their precursors, as well as epithelial and endothelial cells (5, 6). Known functions of fibrocytes include potent stimulation of T cells in antigen-specific immunity (7), wound healing following injury (4), as well as pathologic fibrosis in response to local inflammation (2).

The human transforming growth factor-beta isoforms constitute extracellular signaling molecules that have pleiotropic functions. Transforming growth factor-beta1 (TGFbeta)2 serves as a key fibrogenic mediator that initiates signaling by binding to type I and type II receptor kinases (TbetaR). This activates receptor-associated Smads such as Smad2 and Smad3, allowing them to complex with Smad4 for translocation to the nucleus. Alternatively, TbetaR can also signal through other pathways, the most prominent being mitogen-activated protein kinase (MAPK) (8, 9). Members of the MAPK family include ERK as well as two stress-activated protein kinases (SAPK): the c-Jun N-terminal kinase (JNK) and the p38 pathway. The mechanism whereby TGFbeta1 induces activation of MAPK pathways was recently elucidated and involved activation through the upstream mediators Ras, RhoA, and TGF-activated kinase 1 (TAK1) (10, 11).

Adipocyte differentiation is a complex process regulated by extracellular hormones and cytokines. Appropriate environmental exposure leads to up-regulation of specific transcription factors that serve as master regulators in the activation of adipocyte-specific genes, such as leptin and aP2 (12). One such transcription factor is peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). Following binding to natural ligands (i.e. 15-deoxy-PGJ2) or synthetic agonists (i.e. troligtazone), PPAR{gamma} becomes activated and forms a heterodimer with RXR{alpha} (13, 14). The complex then translocates to the nucleus and binds to specific PPAR response elements (PPREs) in the promoter region of its target genes, such as aP2, and contributes to differentiation (15).

In the present study, we examined the role of TGFbeta1 in activating distinct signaling pathways that lead to the stimulation of {alpha}-smooth muscle actin (SMA) expression during myofibroblast differentiation. Under different environmental cues, fibrocytes can differentiate into adipocytes, a process that involves activation of the PPAR{gamma} pathway leading to induction of aP2 expression. TGFbeta1 signaling in fibrocytes activates both Smad 2/3 and MAP kinases, specifically the ERK1/2 and SAPK/JNK pathways. PPAR{gamma} agonists can negatively regulate this process, through a PPAR{gamma}-independent pathway. A blockade of the SAPK/JNK pathway either by troglitazone or by chemical treatment (JNK inhibitor) inhibited TGFbeta1-induced {alpha}SMA expression. Conversely, we found that TGFbeta1 signaling suppressed PPAR{gamma} activity and aP2 expression. TGFbeta1-induced SAPK/JNK phosphorylation leads to downstream signaling that negatively affects the transactivation activity of PPAR{gamma}. Taken together, SAPK/JNK signaling affected the divergence of adipogenic and myofibrogenic differentiation of fibrocytes. This is important because it provides a therapeutic target whereby through the use of a synthetic PPAR{gamma} agonist we are able to block a key TGFbeta1-mediated pro-fibrotic effect, which has exciting implications for therapy of currently untreated fibrotic diseases.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Fibrocytes Isolation—Fibrocytes were harvested from peripheral blood mononuclear cells according to previously published methods (2, 3). Briefly, peripheral blood mononuclear cells were isolated from human leukopheresis packs by gradient centrifugation over Ficoll-Paque (Amersham Biosciences). Following culturing on fibronectin-coated flasks for 72 h in Dulbecco's modified Eagle's medium with 20% fetal bovine serum and 4% L-glutamine, non-adherent cells were removed. The adherent cells were supplemented with new media and remained incubated for 7-10 days. The cells were then gently detached from flasks by incubation with Accutase (Innovative Cell Technologies, San Diego, CA) for 10 min at 37 °C. This crude fibrocyte preparation was purified of contaminating monocytes/macrophages, T cells, and B cells by magnetic immunodepletion using anti-CD14; pan-T, anti-CD2; and pan-B, anti-CD19 Dynabeads, respectively (Dynal Inc., Brown Deer, WI).

Myofibroblast and Adipocyte Differentiation—For myofibroblast differentiation, enriched fibrocytes were treated with serum-depleted Dulbecco's modified Eagle's medium supplemented with 10 ng/ml TGFbeta1. Culture media was changed every 48-72 h. For adipocyte differentiation, fibrocytes were treated with PBM culture media (Cambrex, Charles City, IA) supplemented with 10 µM troglitazone. Culture media was changed every 48 h. All reagents with the exception of TGFbeta1 (R&D Systems, Minneapolis, MN) were purchased from Sigma.

Oil Red O Staining—Cells were stained with Oil Red O to assess for lipid accumulation, as previously described (16). Briefly, cells are washed with 1x phosphate-buffered saline, fixed in 10% formalin solution for 1 h at room temperature, rinsed twice with phosphate-buffered saline, followed by wash with 60% isopropyl alcohol for 5 min. Cells are then stained with Oil Red O solution for 90 min, followed by a gentle rinse with water. Cells are counterstained with hematoxylin for 3 min and washed with water before microscopic visualization.

Real Time RT-PCR—Total RNA was prepared using TRIzol (Invitrogen) as previously described (1, 2). For RT-PCR analysis, 2.0 µg of RNA was converted to cDNA utilizing random hexamer primers and reverse transcriptase from Maloney murine leukemia virus. Real time PCR was performed using a ABI Prism 7700 sequence detector and SDS analysis software (PE Applied Biosystems, Foster City, CA). The following specific oligonucleotide primers were used in experiments: {alpha}SMA, forward, 5'-CGGGCTTTGCTGGTGATG-3', reverse, 5'-CCCTCGATGGATGGGAAA-3'; aP2, forward, 5'-GGAAAATCAACCACCATAAAGA-3', reverse, 5'-GGAAGTGACGCCTTTCATGAC-3'.

Immunocytochemistry—Fibrocyte-derived myofibroblast or adipocyte cell monolayers were fixed in 4% paraformaldehyde for 2 h and then stained for {alpha}SMA or aP2 antibodies using the Vectastain ABC system (Vector Laboratories, Burlingame, CA). Briefly, cells were incubated with a 1:1 mixture of 3% hydrogen peroxide in methanol. Nonspecific binding sites were blocked with PowerBlock (Biogenex, San Ramon, CA) for 30 min, washed, and overlaid with either control or species-specific anti-human antibody. Slides were then rinsed and overlaid with secondary biotinylated goat anti-rabbit IgG and incubated for 30 min. After washing twice with phosphate-buffered saline, slides were overlaid with Vectastain ABC systems peroxidase-conjugated streptavidin and incubated for 30 min. 3,3'-Diaminobenzidine tetrahydrochloride reagent was used for chromogenic localization of antibody. After optimal color development, sections were immersed in sterile water, counterstained with Mayers hematoxylin, neutralized in 10% ammonia, and coverslipped with Permount solution after drying overnight.

Last, for immunostaining experiments that examined the role of TGFbeta1 in activating Smad2/3, we treated fibrocytes with 10 ng/ml TGFbeta1 for 60 min. The cells were stained with either appropriate isotype control or Smad2/3 antibody to monitor for nuclear localization of Smad2/3 proteins.

Immunoblot Analysis—Immunoblotting was performed based on 50 µg of protein from cellular nuclear/cytoplasmic fractions as previously described (1, 2). In brief, cell monolayer in flasks were washed with Hanks' balanced salt solution and then treated with lysis buffer, after which lysates were heated at 100 °C for 10 min and clarified by centrifugation. Equal amounts of protein (50 µg) were then loaded onto 10% gradient gels (Amersham Biosciences) and electrophoresed at 100 V until the dye-front reached the end of the gel. The gels were transferred to nitrocellulose membrane using the manufacturer's suggested protocol. The membranes are blocked using 5% (w/v) evaporated milk in Tris-buffered saline containing 0.001% (v/v) Tween 20. The membranes were probed with primary antibody overnight using antibodies against the targets listed: ERK1/2, pERK1/2, SAPK/JNK, pSAPK/JNK, p38 MAPK, pp38 MAPK (all from Cell Signaling Technology, Danver, MA), {alpha}SMA (R&D Systems), PPAR{gamma} (Upstate, Billerica, MA), aP2 (Hycult Biotechnology, Uden, Netherlands), and GAPDH. Appropriate horseradish peroxidase-conjugated secondary antibodies were used based on species-specific requirements; final chemiluminescence detection is based on the ECL+ kit (Amersham Biosciences) per the manufacturer's protocol.


Figure 1
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  		FIGURE 1.
Differentiation of human circulating fibrocytes to myofibroblasts and adipocytes. A, morphology of purified fibrocytes following in vitro expansion for 2 weeks. B, fibrocyte differentiation to myofibroblasts. Cultures of fibrocytes in chamber slides were treated with 10 ng/ml TGFbeta1 for 3 weeks (culture media changed every 48 h). The cells are stained with either {alpha}SMA or appropriate isotype control antibody. All slides were also counterstained with hematoxylin. C, fibrocyte differentiation to adipocytes. Cultures of fibrocytes in chamber slides were treated with 10 µM troglitazone for 3 weeks (culture media changed every 48 h). Fibrocyte-derived adipocytes transformed into cells of rounder morphology are shown in the top left panel. Adipogenesis is associated with intracellular lipid accumulation as evident by positive Oil Red O staining (top right panel). The cells are stained with either appropriate isotype control (bottom left panel) or aP2 (bottom right panel) antibodies. All slides are also counterstained with hematoxylin. Positively stained cells for {alpha}SMA and aP2 appear as reddish brown.

 
Vector Transfection—Fibrocytes were transfected using Amaxa Nucleofection TechnologyTM (Amaxa, Koeln, Germany). Cells were re-suspended in solution from a nucleofector kit, also available as part of the Amaxa cell optimization kit, following Amaxa guidelines for cell line transfection (see Amaxa literature). We have optimized this kit for use with fibrocytes. Briefly, 100 µl of 5 x 106 fibrocyte suspension mixed with 2 µg of cDNA was transferred to the provided cuvette and nucleofected with an Amaxa Nucleofector apparatus (Amaxa). Cells were transfected using the U023 pulsing parameter and immediately transferred into wells containing 37 °C pre-warmed serum-starved culture medium in 6-well plates. After transfection, cells were cultured from 8 h before treatment with appropriate parameters. PPRE (17) and Smad binding element (SBE) luciferase reporter gene plasmid as well as PPAR{gamma} overexpression vector (pCMV-PPAR{gamma}) were obtained from Panomics Inc. (Fremont, CA). PPRE (PPRE3X-Lux) and SBE (SBE3X-Lux) luciferase reporter constructs were used to monitor PPAR{gamma} and SMAD transactivation activities, respectively, with each vector containing multiple repeat-specific consensus binding sites. Twelve hours after transfection, fibrocytes were subjected to treatment based on specific experimental design. Cells were then harvested and luminescence was measured in a microplate luminometer.

RNA Interference—RNA duplex siRNA for JNK1 was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). A control scramble siRNA duplex was also used. 8 x 106 cells were plated into a 6-well plate with fresh media without antibiotics 24 h before testing. siRNA transfection was performed according to the manufacturer's protocol, except for modifications as noted below. Briefly, 160 pmol of siRNA was used per 100 µl of siRNA duplex medium. The cells were incubated for 3.5 h with siRNA duplex solution, followed by treatment in growth media containing 15% fetal calf serum. After siRNA transfection, the cells were incubated for three additional days at 37 °C before additional treatment and analysis. To assess and optimize transfection efficiency, GAPDH siRNA was used to examine appropriate GAPDH gene silencing in fibrocytes.

Statistical Analysis—Differences between groups were compared using either the Mann-Whitney U test if the data were not normally distributed, or the Student's t test if the observations were consistent with a sample from a normally distributed population. Data were analyzed on an IBM PC computer using GraphPad Prism 4 version 4.00 for Windows (GraphPad Software, San Diego CA). Results were determined to be statistically significant if p < 0.05 unless otherwise specified.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Fibrocytes Possess the Ability to Differentiate to Myofibroblasts and Adipocytes—Fibrocytes were extracted from peripheral blood mononuclear cells (Fig. 1A). After 10 days of expansion in vitro, contaminating T cells, B cells, and monocytes were removed using negative selection with magnetic beads (1, 2). The purified fibrocyte population expressed CXCR4, Col I, and CD45RO. These cells do not express myofibroblast marker {alpha}SMA or the mature adipocyte marker, adipocyte lipid-binding protein (aP2, also known as FABP4). To induce fibrocyte differentiation to myofibroblasts, the cells were cultured in media supplemented with TGFbeta1 (10 ng/ml) for 3 weeks. To evaluate the phenotype of fibrocytes following differentiation, we performed immunocytochemical staining for the mature myofibroblast protein {alpha}SMA. As compared with the isotype control, the majority of fibrocytes treated with TGFbeta1 markedly expressed cytoplasmic {alpha}SMA (Fig. 1B). No appreciable change in cell morphology was seen with myofibroblasts as compared with untreated fibrocytes. In cells maintained in media without TGFbeta1 supplementation, a small population of cells (~15%) underwent spontaneous differentiation to myofibroblast with associated expression of {alpha}SMA.


Figure 2
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  		FIGURE 2.
Dose and time-dependent stimulation of {alpha}SMA expression by TGFbeta1. A, fibrocytes were serum starved, then treated for 24 h with 0.3-100 ng/ml of TGFbeta1 or 10 ng/ml of TGFbeta1 for 1-48 h. For mRNA analysis, total RNA was extracted and reverse-transcribed into cDNA. cDNA was analyzed for mRNA expression of {alpha}SMA and GAPDH (for normalization) by real-time PCR. B, dose dependence and kinetics of TGFbeta1 induction of {alpha}SMA gene expression. All results are mean ± S.E. for relative {alpha}SMA mRNA levels from three independent experiments. C, treatment with 10 ng/ml TGFbeta1 and induction of {alpha}SMA protein expression. *, p ≤ 0.05 compared with control.

 
We previously demonstrated the ability of fibrocytes to undergo differentiation to adipocytes when exposed to cyclic treatment of adipogenic induction media containing indomethacin, dexomethasone, 3-isomethylxanthine, and insulin (1). An up-regulation of PPAR{gamma} was seen in fibrocyte-derived adipocytes. Here we demonstrate that treatment of fibrocytes with a synthetic PPAR{gamma} ligand, troglitazone (TGZ), induced similar fibrocyte to adipocyte differentiation. Following 21 days in culture, the cells were observed to accumulate lipids in intracellular vacuoles, as evidence by extensive Oil Red O Staining (Fig. 1C). Unlike differentiation to myofibroblasts, fibrocytes treated with TGZ transformed into cells of rounder morphology (Fig. 1C). To confirm differentiation into adipocytes, we performed immunocytochemical staining for the mature adipocyte marker aP2. As compared with isotype controls, fibrocyte-derived adipocytes stained extensively for aP2, localized in proximity to intracellular lipid vesicles (Fig. 1C). Whereas a small population of fibrocytes underwent spontaneous differentiation of {alpha}SMA expressing myofibroblasts, untreated cells did not express aP2 even after 6 weeks (data not shown). Not all treated cells underwent differentiation to adipocytes, as only ~45% of the cells committed to the adipose lineage. No increase in adipogenic differentiation was observed even with extended treatment.

TGFbeta1 Induces {alpha}SMA Expression in Fibrocytes and Promotes Its Differentiation to Myofibroblast—We next examined the role of TGFbeta1 in induction of {alpha}SMA expression. We assessed the kinetics and dose-dependence effect of TGFbeta1 activation in fibrocytes. Fibrocytes were treated with varying concentrations of TGFbeta1 (0.3-100 ng/ml) for different time periods (1-48 h). Semi-quantitative RT-PCR analysis showed that TGFbeta1, at concentrations of 10-100 ng/ml, was able to induce {alpha}SMA transcription (Fig. 2A). The maximal induction was observed with 10 ng/ml TGFbeta1, with {alpha}SMA mRNA level increasing 7-fold as compared with unstimulated cells. We also performed kinetic evaluation of {alpha}SMA expression using TGFbeta1 (10 ng/ml). The {alpha}SMA mRNA transcript was elevated within 8 h after treatment, with an increase of 4.1-fold (Fig. 2B). It was increased to a maximal of 7.3-fold by 24 h. Sustained increase in TGFbeta1-stimulated {alpha}SMA transcripts were seen even at 48 h.

To confirm our findings, we next looked at protein expression of {alpha}SMA following TGFbeta1 treatment. Fibrocytes were treated with TGFbeta1 (10 ng/ml) for 1-72 h, and protein whole cell extracts were obtained. When we analyzed for {alpha}SMA expression on Western blot, we observed a time-dependent increase in the expression of {alpha}SMA protein, with significant levels seen at 48 and 72 h following treatment (Fig. 2C). The augmented {alpha}SMA protein level was sustained up to 4 days following treatment.

Fibrocyte Differentiation to Adipocytes Is Dependent on Activation of PPAR{gamma} and Associated with an Induction of aP2 Expression—We had previously demonstrated that fibrocytes differentiate into adipocytes when cells were exposed to a permissive microenvironmental niche (1). In the current study, we examined in greater detail the role of PPAR{gamma} in mediating this process. PPAR{gamma} is well documented as a critical transcription factor in the regulation of adipogenic differentiation. Upon activation, PPAR{gamma} binds to common consensus PPRE sites on promoters of specific adipocyte genes in the nucleus, including that of aP2 (15, 18). aP2 is often used as a mature adipocyte marker because its expression parallels the degree of adipogenic differentiation. To activate PPAR{gamma} in fibrocytes, we treated the cells with troglitazone (10 µM) for varying lengths of time (1-72 h) and monitored the kinetics of aP2 transcript activation using semi-quantitative RT-PCR. When compared with untreated cells, an up-regulation of the aP2 mRNA transcript was seen within 12 h, with maximal expression of 13.8-fold at 24 h, and with a sustained response at 48 h (Fig. 3A).

To verify the role of PPAR{gamma} in aP2 induction in fibrocytes, we examined protein expression of PPAR{gamma} and aP2 following TGZ treatment. Both nuclear and cytoplasmic protein extracts were prepared for Western blot analysis. A low basal level of PPAR{gamma} was detected in unstimulated fibrocytes (Fig. 3B, lane 1). Following TGZ treatment, there was a significant time-dependent increase in accumulation of PPAR{gamma} in the nucleus of cells within 4 h (lane 3) and with maximal stimulation 24 h (lane 4). As for aP2, no basal expression was seen in untreated cells (Fig. 3B). In response to PPAR{gamma} activation, we observed a significant, but delayed, increase in aP2 level starting at 24 h (Fig. 3B). The protein level remained elevated at 72 h (lane 6). To corroborate this finding, we pretreated the cells with GW9662, a specific PPAR{gamma} antagonist. As shown in Fig. 3C, the ability of TGZ to induce aP2 expression was eliminated in the presence of GW9662. Taken together, these findings advocate that TGZ-mediated induction of aP2 in fibrocytes is dependent upon PPAR{gamma} activation.


Figure 3
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  		FIGURE 3.
Fibrocyte to adipocyte differentiation is regulated through a PPAR{gamma}-dependent mechanism, leading to activation of aP2 expression. A, time-dependent stimulation of aP2 expression by troglitazone. Fibrocytes were serum starved, then treated up to 48 h with 10 µM TGZ. Transcript analysis was performed using quantitative RT-PCR. B, nuclear translocation of PPAR{gamma} following TGZ treatment with an associated increase in cytoplasmic aP2 protein level. C, the PPAR{gamma} antagonist GW9662 inhibited the effect of TGZ in activation of aP2 expression. D, fibrocytes were transfected with a PPRE-luciferase reporter gene plasmid (PPRE3X-Lux). Increase in PPAR{gamma} transactivation activity was observed following treatment with TGZ (10 µM) or transfection with constitutive PPAR{gamma} overexpression vector. Luciferase assays were performed in duplicate and normalized to Renilla. Each bar represents the mean luciferase activity (as relative light units (RLU) per mg of protein ± S.E.).

 
As further verification, we examined the ability of TGZ to promote PPAR{gamma} transactivation activity in fibrocytes. We first transfected the cells with a luciferase reporter gene containing 3 PPREs (PPRE3x-Lux) (17), which was used as an index of PPAR{gamma} transactivation. They were then either treated with TGZ or were co-transfected with a construct encoding PPAR{gamma} under the constitutive cytomegalovirus promoter (pCMV-PPAR{gamma}). PPRE-driven luciferase activity was then monitored and normalized to Renilla luciferase. As shown in Fig. 3D, use of null vector (lane 2) did not result in spontaneous activation. Conversely, TGZ was able to induce a 5.9-fold increase in luciferase reporter activity (lane 6). Co-transfection with the pCMV-PPAR{gamma} expression construct also resulted in robust up-regulation (7.8-fold increase) of PPAR{gamma} transactivation in the absence of TGZ (lane 7). Taken together, these results strongly suggest the role of PPAR{gamma} as a key regulatory molecule in the activation of fibrocyte-to-adipocyte differentiation.

TGFbeta1-mediated Induction of {alpha}SMA Expression Is Negatively Regulated by Troglitazone via a PPAR{gamma}-independent Mechanism—We have now demonstrated the ability of fibrocytes to differentiate along two different lineages. This plasticity is dependent on the presence of specific growth factors such as TGFbeta1 for myofibroblast differentiation or PPAR{gamma} signaling for adipocyte differentiation. Here, we examined the effects of TGZ on TGFbeta1-mediated myofibroblast differentiation. Fibrocytes were treated with TGFbeta1 (1-100 ng/ml) in the presence or absence of TGZ (10-30 µM). Semi-quantitative RT-PCR analysis showed that TGZ, at a concentration of 30-100 µM, was able to suppress the induction of {alpha}SMA transcription activated by TGFbeta1 (Fig. 4, A, lanes 8 and 9; B, lane 7). Interestingly, whereas TGZ at 10 µM was sufficient to activate fibrocyte adipogenesis, a higher concentration was needed to effectively suppress the expression of {alpha}SMA.

We next tested whether the effects of TGZ in inhibiting {alpha}SMA was mediated through the activation of PPAR{gamma}. Although it is well documented that PPAR{gamma} ligands can influence transcription of genes in a PPAR-dependent manner, recent research has revealed that these drugs also elicit "nongenomic" PPAR-independent effects (19). For example, PPAR{gamma} ligands can rapidly induce phosphorylation of members of the MAPK family or phosphatidylinositol 3-kinase pathway (20). To examine this, we pretreated fibrocytes with the PPAR{gamma} antagonist GW9662 before treatment with TGFbeta1 or TGZ, either alone or in combination. As shown in Fig. 4B, GW9662 (3-10 µM) did not reverse the inhibitory effects of TGZ on {alpha}SMA transcript expression (lanes 8 and 9). Even higher concentrations of GW9662 did not improve on this effect (date not shown).

We also looked at the effect of TGZ on {alpha}SMA protein expression. Again, fibrocytes were pretreated with or without GW9662, then incubated in TGFbeta1 (10 µM) and/or TGZ (30 µM) for 72 h. The expression of {alpha}SMA was assessed by Western blot. Consistent with mRNA findings, treatment with TGZ inhibited TGFbeta1-induced {alpha}SMA expression (Fig. 4C, lane 5). The inhibitory effect of TGZ was not reversed by GW9662 (Fig. 4C, lane 6). Taken together, these findings suggest that, contrary to PPAR{gamma}-mediated induction of aP2, the effect of TGZ on {alpha}SMA expression is mediated by nongenomic PPAR{gamma}-independent effects.

TGFbeta1 Activates Regulatory Smads2/3 and MAPK Pathways—We now focused in detail on the molecular mechanisms of interaction between TGZ and TGFbeta1 during myofibroblast differentiation. To do this, we first dissected the exact mechanisms whereby TGFbeta1 signaling leads to {alpha}SMA expression. We examined the two signaling pathways, Smads and MAPK, which have been reported by others to be associated with TbetaR activation in other cells (8, 20, 21). First, we evaluated whether TGFbeta1 activates Smad2/3 in fibrocytes. The regulatory Smad2/3 have two major phosphorylation sites, at linker regions and at C-terminal regions (22). Treatment with TGFbeta1 (10 ng/ml) led to phosphorylation of Smad2/3 at the C-terminal SSXS region (Ser-465/467) but not at the linker region (data not shown). Phosphorylation of Smad2/3 was detected within 60 min and remained activated at 4 h (Fig. 5A).


Figure 4
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  		FIGURE 4.
TGFbeta1-mediated induction of{alpha}SMA expression is negatively regulated by troglitazone via a PPAR{gamma}-independent mechanism. A, fibrocytes were treated with TGFbeta1 in the presence or absence of TGZ. TGZ, at a concentration 30-100 µM, suppressed the induction of {alpha}SMA transcription activated by TGFbeta1. B, pretreatment of fibrocytes with the PPAR{gamma} antagonist GW9662 before TGFbeta1 or TGZ, either alone or in combination did not reverse the inhibitory effects of TGZ on {alpha}SMA transcript expression. C, treatment with TGZ inhibited TGFbeta1-induced {alpha}SMA protein expression and the inhibitory effect of TGZ was not reversed by GW9662.

 
We also confirmed nuclear localization of Smad2/3 proteins with immunocytochemical staining of fibrocytes following treatment with TGFbeta1. The cells were stained with either appropriate isotype control antibody (top left) or pSmad2/3 antibody (top right and bottom). Nuclear localization of Smad2/3 proteins was visualized following fibrocyte treatment with TGFbeta1 (10 ng/ml) for 60 min, which confirmed the presence of intranuclear Smad2/3 signaling.


Figure 5
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  		FIGURE 5.
TGFbeta1 activates both regulatory Smads and MAP kinase pathway, and troglitazone inhibition of TGFbeta1 signaling is partly regulated through suppression of the SAPK/JNK MAPK pathway. A, treatment with TGFbeta1 led to phosphorylation of Smad2/3 within 60 min as well as activation of both ERK1/2 and SAPK/JNK pathways, albeit at different kinetics. The total protein levels of total ERK and SAPK/JNK were not significantly affected by TGFbeta1 treatment. B, immunocytochemical staining of fibrocytes following treatment with TGFbeta1. The cells were stained with either appropriate isotype control (top left) or Smad2/3 (top right and bottom) antibody. Nuclear localization of regulatory Smad2/3 proteins was visualized following fibrocyte exposure to 10 ng/ml TGF-beta1 for 60 min.

 
TGFbeta1 Also Activates MAP Kinase Pathways—We next examined whether MAPK were also potential downstream effectors of TGFbeta1 receptor. As shown in Fig. 5A, ERK1/2 and SAPK/JNK activation were both observed following treatment with TGFbeta1. TGFbeta1, however, did not activate the SAPK/p38 MAPK pathway in fibrocytes (data not shown). Interestingly, the kinetics of activation of Smad2/3 and activation the MAPKs were different. SAPK/JNK phosphorylation occurred very early, detected within 5 min after treatment and resolved within 60 min (Fig. 5A). ERK1/2 were activated starting at 15 min of TGFbeta1 treatment, which peaked at 30 min and demonstrated persistent activation at 2 h. The total protein levels of total ERK and SAPK/JNK were not significantly affected by TGFbeta1 treatment.

We next investigated whether MAP kinases were intermediates in a TGFbeta1-initiated signaling pathway leading to transcriptional activation of {alpha}SMA. We have shown that TGFbeta1 induced a time-dependent activation of Smad2/3, kinetics of which lagged temporally behind ERK and SAPK/JNK activation. Considering the possibility that the MAPK pathway may influence Smad signaling, we evaluated whether a blockade of ERK1/2 or SAPK/JNK pathways altered activation of Smads or {alpha}SMA expression. We first pretreated the fibrocytes with PD98059 (20 µM), an ERK kinase (MEK1 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase)) inhibitor, prior to addition of TGFbeta1. We then extracted cytoplasmic and nuclear protein extracts for Western blot analysis. Whereas PD98059 pretreatment led to marked suppression of ERK phosphorylation (Fig. 6A, lane 6), there was no change in TGFbeta1-induced phosphorylation of Smad2/3 in either the cytoplasm or nucleus (lanes 2 and 6). There was also no difference in {alpha}SMA protein expression in cells pretreated with PD98059 (lanes 2 and 6), suggesting that the ERK1/2 MAPK pathway was not involved in TGF-beta-induced {alpha}SMA expression.


Figure 6
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  		FIGURE 6.
SAPK/JNK MAP kinases are intermediates in a TGFbeta1-initiated signaling pathway leading to transcriptional activation of {alpha}SMA. A, chemical blockade using the ERK1/2 inhibitor PD98059 did not affect TGFbeta1-induced phosphorylation of Smad2/3 or {alpha}SMA protein expression. Exposure to TGZ attenuated TGFbeta1-induced {alpha}SMA expression, an effect not reversed by PD98059 pretreatment. B, pretreatment using the SAPK/JNK inhibitor SP600125 markedly reduces the level of phosphorylated Smad2/3 in the nucleus and resulted in a significant loss in {alpha}SMA expression, an effect similarly seen with treatment using TGZ. C, siRNA knockdown of JNK1 leads to loss of JNK1 protein. Fibrocytes were treated with siRNA oligo specific for JNK1 and their effect on JNK1 protein levels were compared with that of a nonspecific siRNA oligo control or transfection reagent alone. Loss of JNK1 protein leads to reduced {alpha}SMA expression. D, transfection with a TGFbeta1 reporter construct (p3SBE-Lux). Unlike PD98059 pretreatment, use of a JNK inhibitor significantly inhibited TGFbeta1-driven luciferase activity in fibrocytes.

 
We next evaluated whether the JNK pathway may be implicated. Here, we pretreated the cells with a SAPK/JNK inhibitor, SP600125 (10 µM), prior to incubating cells with TGFbeta1. Unlike PD98059, use of SP600125 led to inhibition of the TGFbeta1-mediated stimulation of {alpha}SMA protein (Fig. 6B, lane 6). Whereas SAPK/JNK blockade did not influence the level of cytoplasmic pSmad2/3, it did markedly reduce the level of pSmad2/3 in the nucleus (Fig. 6B, lane 6). This was associated with significant loss in {alpha}SMA expression. These results suggest that whereas SAPK/JNK signaling did not affect the ability of TGFbeta1 to activate cytoplasmic Smad2/3, it did alter the level of activated Smad2/3 in the nucleus.

To rule out the possibility that importance of SAPK/JNK signaling is not due exclusively to a pharmacologic inhibitor, we used RNA interference to specifically knockdown endogenous JNK protein in fibrocytes. Fibrocytes were treated with siRNA oligo specific for JNK1 and the effect on JNK protein level was compared with that of a nonspecific siRNA oligo control or with transfection reagent alone. The specific knockdown of endogenous JNK protein using siRNA was confirmed with immunoblotting (Fig. 6C). Loss of SAPK/JNK protein again led to reduced expression of {alpha}SMA protein.

Lastly, we assessed the effect of JNK blockade on Smad transactivation activity. A TGFbeta1 reporter construct (SBE3X-Lux) containing a luciferase gene controlled by a TGFbeta1-inducible promoter (with SBE) was used to monitor TGFbeta1-induced changes in gene expression in fibrocytes. Transfection of SBE3X-Lux into the cells resulted in a strong induction (15.1-fold) of luciferase activity in response to TGFbeta1 (Fig. 6D, lane 4). We then performed experiments where we chemically blocked either ERK1/2 or SAPK/JNK pathways using PD98059 or SP600125, respectively, before stimulating the cells with TGFbeta1. TGFbeta1-driven luciferase activity was then monitored and normalized to Renilla luciferase. Use of null vector (lane 2) did not result in spontaneous activation. When pretreated with PD98059, there was no significant change in Smad-mediated promoter transactivation (lanes 4 and 7). Use of the JNK inhibitor significantly inhibited TGFbeta1-driven luciferase activity (lane 8). Thus, in concert with our earlier findings, these results suggest that SAPK/JNK signaling may be important in modulating Smad2/3-dependent transcription of pro-fibrotic genes, such as {alpha}SMA in fibrocytes.

Troglitazone Inhibition of TGFbeta1 Signaling Is Partly Regulated through Suppression of JNK MAP Kinase—With a better understanding of TGFbeta1 signaling, we re-examined our earlier inquiry into the precise mechanism whereby TGZ can inhibit {alpha}SMA expression. We showed that TGFbeta1 leads to ERK1/2 and SAPK/JNK activation in fibrocytes. Previous studies have shown that PPAR{gamma} ligands such as TGZ, beyond activating PPAR{gamma}, can also regulate activity of MAP kinases (19, 20). The activation of MAPK pathways by PPAR agonists have been shown to exert either anti-fibrotic or pro-fibrotic influence depending on the cellular context (20, 23-25). We have already shown that contrary to PPAR{gamma}-mediated induction of aP2 the effect of TGZ on {alpha}SMA expression was mediated by its PPAR{gamma}-independent actions. We now examine if this inhibition is mediated through MAP kinase signaling.

Interestingly, ERK1/2 was not only activated by TGFbeta1 (Fig. 6A, lane 2), but also by TGZ (lane 3). TGZ induced ERK phosphorylation without a change in total ERK1/2 level. TGFbeta1 and TGZ together induced a higher level of phosphorylated ERK1/2 than either treatment alone (lane 4). To determine whether TGZ inhibition of TGFbeta1-mediated {alpha}SMA expression involves ERK signaling, we performed pathway-specific inhibition studies using the ERK inhibitor PD98059 (20 µM). Pretreatment with PD98059 effectively decreased the level of pERK1/2 (lane 5-8). We suspect that if TGZ inhibition of {alpha}SMA is mediated through ERK activation, blockade of ERK signaling would abolish the inhibitory effect of TGZ treatment. This, however, was not observed. Whereas exposure of TGZ did result in loss of {alpha}SMA expression (lane 4), PD98059 pretreatment failed to reverse this effect (lane 7). Treatment with TGZ also resulted in a lower level of pSmads in the nucleus (lane 4), which also failed to reverse with chemical inhibition of ERK signaling (lane 7). Taken together, these results suggest that whereas TGZ activated ERK MAP kinase, this pathway was not involved in regulation of TGFbeta1-mediated {alpha}SMA expression.

We next looked at the SAPK/JNK pathway. We showed that TGFbeta1 activated the SAPK/JNK pathway (Fig. 6B, lane 2). To determine whether TGZ modulates its effect through regulation of TGFbeta1-activated SAPK/JNK signaling, we performed pathway-specific inhibition experiments using the JNK inhibitor SP600125. TGZ alone did not activate SAPK/JNK, unlike its ability to activate the ERK pathway (lane 3). Instead, treatment with TGZ interfered with the ability of TGFbeta1 to activate SAPK/JNK (lane 4). This outcome, mediated upstream of SAPK/JNK, is in effect similar to chemical blockade of SAPK/JNK (lane 6). Because abrogation of SAPK/JNK activity through chemical inhibition is associated with diminished Smad2/3 activation in the nucleus (lane 6), it was not surprising to see that TGZ treatment also provoked similar reduction in nuclear Smad activation (lane 4). Consequently, treatment with SAPK/JNK inhibitor or TGZ both resulted in loss of {alpha}SMA expression as stimulated by TGFbeta1 (lanes 4 and 6; as compared with lane 2). Taken together, the effect of TGZ is mediated through its repression of SAPK/JNK activation. Because full induction of {alpha}SMA requires cooperative TGFbeta1-mediated signaling through SAPK/JNK and Smad2/3, disruption of SAPK/JNK signaling may be one mechanism whereby TGZ exerts its important effects on expression of {alpha}SMA.

Fibrocyte Differentiation to Adipocyte Is Negatively Regulated by c-Jun N-terminal Kinase—We have demonstrated the ability of TGZ to initiate PPAR{gamma}-driven differentiation of fibrocytes to adipocytes. TGZ activation of PPAR{gamma}-independent signaling was also shown to negatively regulate the differentiation of fibrocytes to myofibroblasts. However, it remains unknown whether TGFbeta1 plays a reciprocal role in modulating fibrocyte commitment to adipogenesis. To examine this issue, fibrocytes were treated with TGZ, with or without TGFbeta1, for 48 h (i.e. the time point shown to induce maximal aP2 expression). TGFbeta1 treatment prevented cell rounding and lipid accumulation and conferred a more densely packed, spindly morphology to the cells (data not shown). In addition, as compared with TGZ alone, the presence of TGFbeta1 resulted in marked reduction in cytoplasmic aP2 expression (Fig. 7A, lane 4). No detectable level of aP2 protein was seen in either untreated cells or in cells treated with TGFbeta1 alone.

A number of previous studies have suggested that the PPAR{gamma} and TGFbeta1 pathways cross-talk to regulate adipogenesis of fat tissue preadipocytes. Whereas results have been conflicting, MAP kinases have been implicated to play an important role in mediating this downstream TGFbeta effect. To dissect the potential involvement of MAP kinases in regulation of fibrocyte adipogenesis, we again utilized our ability to chemically block each individual MAP kinase pathway. We inhibited ERK1/2 or SAPK/JNK pathways through pretreatment with either PD98059 or SP600125, respectively. With TGZ alone, we observed PPAR{gamma} translocation to the nucleus, followed by induction of aP2 expression (Fig. 7B, lane 2). Neither TGFbeta1 nor PD98059 alone induced spontaneous aP2 expression (lanes 3 and 5). When TGFbeta1 was added, induction of aP2 expression by TGZ was significantly repressed (lane 4). Blockade of ERK1/2 using PD98059 failed to reverse TGFbeta1-mediated inhibition of aP2 expression (lane 7). These findings suggest that the ERK1/2 MAP kinase pathway is not involved in this negative regulatory process.

We next examined whether the JNK pathway may be implicated. We targeted SAPK/JNK blockade using the inhibitor SP600125. Contrasting the effect with PD98059, SP600125 pretreatment led to the reversal of TGFbeta1-mediated inhibition of aP2 (Fig. 7C, lane 7). To confirm this finding, we again used RNA interference to specifically knockdown endogenous JNK protein in fibrocytes. Fibrocytes were treated with siRNA oligo specific for JNK1 or control and the effect of JNK protein on TGFbeta1 signaling was examined. Consistent with pharmacologic inhibition with SP600125, the knockdown of endogenous JNK protein resulted in abrogation of TGFbeta1 inhibition on aP2 expression (Fig. 7D).

Because induction of aP2 transcription is PPAR{gamma}-dependent and requires translocation of activated PPAR{gamma} into the nucleus, an increase in the level of nuclear PPAR{gamma} was noted following TGZ induction. Interestingly, despite repression of aP2 by TGFbeta1 or its reversal with JNK inhibition, no significant change in the level of nuclear PPAR{gamma} was seen with either treatment as compared with TGZ alone (Fig. 7C, lanes 4 and 6). These findings suggest that TGFbeta1-mediated activation of SAPK/JNK signaling does not impact PPAR{gamma} activation or nuclear translocation directly. Instead, SAPK/JNK activation may lead to downstream modulation of PPAR{gamma} DNA binding or transactivation activity.

To examine this premise, we assessed the effect of SAPK/JNK activation on PPAR{gamma} transactivation activity. We transfected cells with PPRE3x-Lux for measurement as index of PPAR{gamma} transactivation. The cells were then treated with TGZ, in the presence or absence of TGFbeta1. The measured PPRE-driven luciferase activity was monitored and normalized to Renilla luciferase. As expected, treatment with TGZ resulted in a strong induction of luciferase activity (Fig. 7E, lane 4). Addition of TGFbeta1 markedly attenuated this effect (lane 5). When the SAPK/JNK pathway was inhibited with SP600125, this led to a reversal of TGFbeta1-mediated inhibition of PPAR{gamma} transactivation activity (lane 8). Similar pretreatment with the ERK inhibitor PD98059 failed to reverse the negative regulatory effect by TGFbeta1. Altogether, these results suggest that whereas TGFbeta1 activates both ERK and SAPK/JNK signaling, only the SAPK/JNK pathway may be important in the regulatory ability of TGFbeta1 to dampen the fibrocyte adipogenic process.


Figure 7
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  		FIGURE 7.
Fibrocyte differentiation to adipocyte is negatively regulated by SAPK/JNK. A, TGFbeta1 treatment significantly reduced TGZ-induced aP2 expression. B, use of the ERK1/2 inhibitor PD98059 failed to reverse TGFbeta1-mediated inhibition of aP2 expression. C, contrasting the effect with PD98059, SP600125 pretreatment led to the reversal of TGFbeta1-mediated inhibition of aP2. D, siRNA knockdown of JNK1 leads to loss of JNK1 protein, resulting in abrogation of the TGFbeta1-mediated inhibition of TGZ signaling. As a result, TGZ-induced aP2 expression is restored. E, effect of SAPK/JNK activation on PPAR{gamma} transactivation activity. Fibrocytes were transfected with a PPRE-luciferase reporter gene plasmid for measurement as an index of PPAR{gamma} transactivation. TGZ resulted in a strong induction of luciferase activity, which was inhibited in the presence of TGFbeta1. Inhibition of SAPK/JNK pathway with SP600125 reversed this TGFbeta1-mediated inhibition, an effect not seen with ERK inhibitor PD98059.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrated that adult fibrocytes are circulating progenitors that possess the ability to differentiate along different mesenchymal lineages. We showed that TGFbeta1 was a key molecule that promotes the differentiation of fibrocyte toward myofibroblast lineage, whereas activation of PPAR{gamma} was critical for fibrocyte-to-adipocyte differentiation. Signaling pathways activated by TGFbeta1, including Smad2/3 and SAPK/JNK MAPK, collaborate to induce {alpha}SMA transcription, whereas TGZ-mediated PPAR{gamma} activation leads to intracellular lipid accumulation and induction of aP2 expression. Interestingly, treatment with TGZ negatively influenced TGFbeta1-stimulated {alpha}SMA expression. This is modulated through attenuation of the SAPK/JNK activity, leading to decreased Smad2/3 levels and transactivation activity. Conversely, TGFbeta1 was demonstrated to have reciprocal inhibition on the differentiation of fibrocytes to adipocytes. Through activation of SAPK/JNK MAP signaling, which is normally suppressed during adipogenesis, this results in the disruption of PPAR{gamma}-dependent induction of aP2 expression. Taken together, SAPK/JNK signaling seemed to affect the divergence of adipogenic and myofibrogenic lineages derived from fibrocyte progenitors, and ultimately, changed the commitment of cells directed into these lineages (Fig. 8).

In bone marrow-derived stem cells, the ability of TGFbeta1to regulate mesenchymal differentiation is well documented (26-28). The role of TGFbeta1 in fibrocyte lineage determination was previously unknown. Recent studies have demonstrated the pivotal role of fibrocytes in contributing to a number of patho-physiologic fibrotic processes. These include aberrant pulmonary fibrosis (2, 29), vascular intimal hyperplasia (30), as well as renal-related fibrotic diseases (31). We showed that TGFbeta1 leads to activation of both Smad2/3 and MAPK, with cross-talk between the two pathways.

Communication between MAPK and Smads have previously been reported for other cell lines (8, 31-34). Activation of MAPKs by TGFbeta1 was previously described to occur either with slow kinetics, possibly resulting from Smad-dependent transcription responses, or with rapid kinetics such as in fibrocytes. In the latter case, the rapid activation (5-30 min) of MAPK phosphorylation strongly suggests independence from Smad-driven transcription. Following activation, MAP kinases can modify Smad signaling by phosphorylation of Smad2/3, which may affect its capacity for nuclear translocation. Both ERK (35) and SAPK/JNK (22, 36) have been shown to phosphorylate the linker region of Smad2 and Smad3, which inhibits their nuclear translocation. In our study, inhibition of SAPK/JNK was associated with decreased levels of Smad2/3 in the nucleus as well as reduced transactivation activity. SAPK/JNK-mediated modification of Smad2/3 could have either altered their ability to form heterocomplex with Smad4 or impact their translocation into the nucleus.


Figure 8
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  		FIGURE 8.
Summary schematic demonstrating the different signaling pathways that participate in fibrocyte differentiation into either {alpha}SMA-expressing myofibroblasts or differentiation into aP2-expressing adipocytes. Signaling pathways activated by TGFbeta1, including Smad2/3 and SAPK/JNK MAPK, collaborate to induce {alpha}SMA transcription, whereas TGZ-mediated PPAR{gamma} activation leads to induction of aP2 expression. The cross-talk and complex balance of TGFbeta with PPAR{gamma} signaling, within the context of the local microenvironmental niche, drives the selection of defined differentiation pathways.

 
We next examined the mechanism whereby TGZ inhibits TGFbeta1 signaling in fibrocytes. Depending on cell type, PPAR{gamma} agonists can modulate MAPK signaling by different mechanisms. One simple possibility was that the agonist directly interfered with TGFbeta1 signaling, preventing cells from receiving profibrotic signals. In some studies, PPAR{gamma} agonists were found to inhibit Smad2/3 phosphorylation either through direct interaction with TGFbeta receptor or with native (non-phosphorylated) Smads. In our study, cytoplasmic phosphorylation of Smad2/3 was not affected by TGZ. Instead, a drop in level of activated Smads in the nucleus with decreased Smad transactivation activity was seen following TGZ exposure. Specifically, the inhibitory effect of TGZ on TGFbeta1 signaling was dependent on the ability of TGZ to repress SAPK/JNK activation. MAPKs, and in particular the p38 MAPK and SAPK/JNK, have been shown to be involved in the control of post-translational modification of Smads. This can occur through direct phosphorylation of R-Smads (i.e. at a different region than that by TGFbeta receptor), or alternatively, via sumoylation (by proteins of the PIAS family of E3 ligases) (37).

Whereas TGFbeta positively regulated myofibroblast differentiation, previous studies have suggested that TGFbeta can suppress adipocyte differentiation. TGFbeta1 has been shown to inhibit differentiation of tissue preadipocyte to adipocytes (38, 39). In addition, transgenic overexpression of TGFbeta1 in adipose tissue severely reduces adipose tissue masses, and results in the failure of adipocytes to differentiate (40). In fibrocytes, the addition of TGFbeta1 resulted in significant inhibition of adipogenic differentiation induced by TGZ. TGFbeta1 treatment resulted in strong inhibition of PPAR{gamma} trans-activation activity and decreased level of mature adipocyte marker aP2. We have already showed that TGFbeta1 activated both ERK1/2 and SAPK/JNK pathways. However, only SAPK/JNK activation reversed TGZ-induced PPAR{gamma} activity. In previous studies, a number of signaling effectors, including MAPK family members, have been shown to phosphorylate PPAR{gamma} (41-43). PPAR{gamma} phosphorylation can affect its activity, including reduction in the sensitivity of PPAR{gamma} to its cognate ligands or affect its translocation or transactivation activity. This inhibition of adipogenesis by SAPK/JNK has also been reported by others (44-46). Interestingly, Hirosumi and colleagues (45) reported abnormally elevated JNK activity in obese mice. Gene disruption of JNK1, a principal JNK isoform, alleviated dietary as well as genetic obesity, and protected the animals from the development of obesity-induced insulin resistance (45).

Lastly, although beyond the scope of this paper, a number of different mechanisms can modulate the effect of SAPK/JNK activation on PPAR{gamma} activity. SAPK/JNK MAPK phosphorylation of PPAR{gamma} can potentially alter PPAR{gamma} protein stability, ability of PPAR{gamma} to bind to DNA, or change in ability of PPAR{gamma} to interact with other transcription intermediary proteins (i.e. coactivator binding to PPAR{gamma}-RXR complex). Alternatively, a number of transcription factors are downstream substrates for SAPK/JNK MAPK, including c-Jun, which is the main component of AP-1 complexes. Activation of AP-1 can facilitate binding to specific AP-1 recognition sites and modulate transactivation of target genes, including those of the adipose differentiation program. This possibility warrants additional investigation because in a recent study, Fu and associates (47) demonstrated that introduction of dominant-negative AP1 led to reversal of TGFbeta1 suppression of PPAR{gamma} promoter activity.

In summary, the present study demonstrates that a complex signaling circuit involving PPAR{gamma}, Smads, and MAPK activation serve to modulate lineage plasticity of circulating fibrocytes. Within the context of the local microenvironmental niche, the delicate balance of PPAR{gamma} and TGFbeta1 activation drives the selection of defined differentiation pathways to converge on a specific gene program. When deregulated, changes in an individual signaling pathway may contribute to impaired differentiation and allow for the development of abnormal fibrosis or fat formation. We now have exciting molecular targets whereby pharmacologic attempts can be made to modulate signaling parameters that can ultimately regulate fibrocyte cell fate in early development toward or away from a specific mesenchymal lineage.


   FOOTNOTES
 
* 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: Hospital Dr., P. O. Box 800466, Charlottesville, VA 22908. Fax: 434-243-0399; E-mail: strieter{at}virginia.edu.

2 The abbreviations used are: TGFbeta1, transforming growth factor beta1; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; PPAR, peroxisome proliferator-activated receptor; JNK, c-Jun N-terminal kinase; SBE, Smad binding element; TGZ, troglitazone; PPRE, peroxisome proliferator-activated receptor response elements; MAPK, mitogen-activated protein kinase; {alpha}SMA, smooth muscle actin; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, small interfering RNA. Back



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 ABSTRACT
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 DISCUSSION
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