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Am J Physiol Cell Physiol 294: C1332-C1341, 2008. First published April 9, 2008; doi:10.1152/ajpcell.90626.2007
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RECEPTORS AND SIGNAL TRANSDUCTION

Specific signals involved in the long-term maintenance of radiation-induced fibrogenic differentiation: a role for CCN2 and low concentration of TGF-β1

Valérie Haydont,1,2 Bruce L. Riser,3 Jocelyne Aigueperse,4 and Marie-Catherine Vozenin-Brotons1,2

1UPRES EA 27-10 Radiosensibilité des tumeurs et tissus sains, Institut de Radioprotection et de Sûreté Nucléaire/Institut Gustave Roussy, Villejuif; 2Laboratoire de Radiopathologie, SRBE/DRPH, Institut de Radioprotection et de Sûreté Nucléaire, Fontenay-aux-Roses, France; 3Rosalind Franklin University of Medicine and Science, North Chicago, and Baxter Healthcare, Renal Division, McGaw Park, Illinois; 4DRPH, Institut de Radioprotection et de Sûreté Nucléaire, Fontenay-aux-Roses, France

Submitted 13 December 2007 ; accepted in final form 14 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The fibrogenic differentiation of resident mesenchymal cells is a key parameter in the pathogenesis of radiation fibrosis and is triggered by the profibrotic growth factors transforming growth factor (TGF)-β1 and CCN2. TGF-β1 is considered the primary inducer of fibrogenic differentiation and is thought to control its long-term maintenance, whereas CCN2 is considered secondary effector of TGF-β1. Yet, in long-term established fibrosis like that associated with delayed radiation enteropathy, in situ TGF-β1 deposition is low, whereas CCN2 expression is high. To explore this apparent paradox, cell response to increasing doses of TGF-β1 was investigated in cells modeling initiation and maintenance of fibrosis, i.e., normal and fibrosis-derived smooth muscle cells, respectively. Activation of cell-specific signaling pathways by low TGF-β1 doses was demonstrated with a main activation of the Rho/ROCK pathway in fibrosis-derived cells, whereas the Smad pathway was mainly activated in normal cells. This leads to subsequent and cell-specific regulation of the CCN2 gene. These results suggested a specific profibrotic role of CCN2 in fibrosis-initiated cells. Furthermore, the modulation of CCN2 expression by itself and the combination of TGF-β1 and CCN2 was investigated in fibrosis-derived cells. In fibrosis-initiated cells CCN2 triggered its autoinduction; furthermore, low concentration of TGF-β1-potentiated CCN2 autoinduction. Our findings showed a differential requirement and action of TGF-β1 in the fibrogenic response of normal vs. fibrosis-derived cells. This study defines a novel Rho/ROCK but Smad3-independent mode of TGF-β signaling that may operate during the chronic stages of fibrosis and provides evidence of both specific and combinatorial roles of low TGF-β1 dose and CCN2.

fibrosis; CCN2/CTGF; transforming growth factor-β1; Rho; Smad

RADIATION FIBROSIS DISPLAYS the main structural and molecular hallmarks of fibrotic disease but is of known etiology. In the intestine, it is characterized by the fibrogenic differentiation of resident mesenchymal cells and accumulation of extracellular matrix (ECM) resulting from altered ECM remodeling (43, 47). This pathological accumulation of ECM causes bowel obstruction and is achieved by specialized smooth muscle cells (51) exhibiting an immature ({alpha}-smooth muscle actin expression and prominent stress fibers) (5, 24) and synthetic phenotype [expression of procollagen and connective tissue growth factor (CTGF, recently renamed CCN2)] (4).

Transforming growth factor (TGF)-β1 is today considered a primary inducer of tissue fibrosis including radiation-induced fibrosis (32). Its fibrogenic signal is mediated through two TGF-specific Ser/Thr kinase receptors, downstream activation of the Smad3/4 pathway and subsequent transactivation of various genes including the CTGF (34, 38) and ECM molecules (46). To date, most experimental approaches used to study fibrosis focused on the initiation steps of the fibrogenic process, in which a specific role for Smad3 (especially in radiation injury) has been demonstrated in mice lacking Smad3 (10). Although CCN2 is thought to be the main downstream fibrogenic effector of TGF-β1 because it controls both fibronectin and collagen gene expression (8, 39), its ability to trigger fibrogenic differentiation in normal cells is today still debated (3, 11, 17, 33, 37, 50). Although the initial cellular and molecular steps of fibrogenesis are now well described, remaining of great concern is the chronic and progressive nature and the fact that fibrosis is mostly diagnosed when the pathology is already established. In cancer patients treated by radiotherapy, fibrosis may occur many years after the treatment has been completed, and its development is today unpredictable. The chronic activation loops involved in the long-term maintenance of fibrosis have not been fully investigated, but several lines of evidence suggest that the mechanisms involved might be distinct from the pathways that trigger the initiation steps.

Indeed, the main parameter of chronic fibrosis, which is the production of ECM, is unaffected in Smad3 knockout mice, suggesting the existence of Smad3-independent mechanisms. In the same line in human delayed radiation enteropathy, TGF-β1 expression is low whereas the gene and protein levels of CCN2 are very high, suggesting occurrence of alternative stimulatory signals involved in CCN2 regulation during the chronic phase of the pathology (47). Similar observations have been made in scleroderma, in which CCN2 but not TGF-β1 expression correlated with the severity of the fibrosis (29, 40). This apparent paradox suggests that during the chronic phase of fibrotic disease the respective role of TGF-β1 and CCN2 might change. Grotendorst et al. (17) proposed that TGF-β1 was able to trigger a long-term cell response, in which its own presence was no longer required for sustained CCN2 expression. Here, we decided to investigate an alternative hypothesis in which CCN2 itself or/and in conjunction with low TGF-β1 doses could be able to control its own expression and subsequently the long-term fibrogenic differentiation.

Initiation and maintenance of fibrosis seemed to depend on the specific molecular pathway; thus the development of physiologically relevant in vitro models modeling these complex processes is critically required. Gabbiani and colleagues (13, 25, 26, 45) demonstrated that the differentiation of fibroblasts into myofibroblasts was the key cellular process in fibrosis initiation and maintenance in the skin, whereas in the intestine the pathological accumulation of ECM including collagen secretion is achieved by activated smooth muscle cells (15, 51). This activation of smooth muscle cells is characterized by a switch from a contractile to a secretory phenotype (35), which can be defined by using the VDA classification vimentin-desmin-{alpha}-smooth muscle actin (12). Therefore, we derived primary intestinal smooth muscle cells from normal ileum (N-SMC) and radiation enteropathy (RE-SMC). First, we investigated the fibrogenic signals mediated by TGF-β1 in N- and RE-SMC. Cell response to increasing doses of TGF-β1 showed activation of cell-specific signaling pathways by low TGF-β1 doses (below that typically used in vitro). A main activation of the Rho/ROCK pathway was obtained in fibrosis-derived cells, whereas the canonical Smad pathway was activated in normal cells and induced cell-specific regulation of the CCN2 gene. Then both CCN2 effect and combination of TGF-β1 and CCN2 were specifically investigated in fibrosis-derived cells. Results showed that CCN2 alone triggered its autoinduction in this cell type and that TGF-β1-enhanced CCN2 autoinduction. The results of this study showed that 1) distinct pathways controlled CCN2 expression during initiation vs. maintenance of fibrosis, 2) very low doses of TGF-β1 elicit potent profibrotic action in both normal and fibrosis-derived cells, 3) CCN2 drives its autoinduction in fibrosis-derived cells, and 4) low concentration of TGF-β1 enhanced CCN2 autoinduction in fibrosis-derived cells.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Growth factors and chemicals. Recombinant human (rh)CCN2 was produced in a baculoviral expression system as previously described (39) and used between 0.3 and 30 ng/ml. Pravastatin was a gift from Bristol-Myers Squibb and was used at 100 and 500 µM. TGF-β1 (R&D Systems, Abingdon, UK) was used between 0.01 and 10 ng/ml. Lysophosphatidic acid (LPA, 10 µM; Sigma-Aldrich, Saint-Quentin Fallavier, France) and Y-27632 (100 µM; Fisher-Bioblock, Illkirch, France) were used. The monoclonal mouse IgG1 antibody anti-Smad-4 (Santa Cruz, tebu-bio, France) was used at 1:100 for cell immunostaining or at 1:300 for Western blot detection and, respectively labeled with secondary antibody Alexa Fluor 488 conjugated anti-mouse IgG (1:250) (Molecular Probes, Eugene, OR) and peroxidase-labeled anti-mouse antibody (1:5,000) (Amersham Biosciences, Saclay, France). CCN2, phospho-Smad (pSmad)-2/3, and Smad-2/3 were detected by specific goat polyclonal antibodies (1:300) (Santa Cruz) and then by donkey anti-goat horseradish peroxidase-conjugated antibody (1:8,000) (Santa Cruz). ROCK I (1:250) (Becton Dickinson, Le Pont-De-Claix, France), lamin B (1:300) (Santa-Cruz), GAPDH (1:5,000) (Clone 6C5 Biodesign, Interchim, France), and {alpha}-smooth muscle actin (1:2,000) (Sigma, Clone 1A4 Sigma, Lyon, France) labeling were performed with a primary monoclonal mouse IgG1 antibody.

Cells and culture. Primary cells were isolated from six independent donors. N-SMC were isolated from normal terminal ileum in patients undergoing colonic surgery from distal colon cancer (n = 3; 62-, 65-, and 45-yr-old patients). RE-SMC were obtained after ileal resection due to intestinal obstruction subsequent to radiation fibrosis (n = 3; 48-, 60-, and 56-yr-old patients). Procurement of tissue samples received prior approval from our institution's Ethics Committee and was performed according to the French Medical Research Council guidelines. Prior microscopic observations confirmed the absence of malignancies in the samples. For primary smooth muscle cells isolation, muscularis propria was mechanically separated from the tissue by scraping. Then, muscularis propria was dilacerated and digested by enzymatic cocktail as previously described (4). By this method, fibroblast contamination is negligible according to studies by Graham et al. (14) and Brittingham et al. (5) and our laboratory's previous published experiences (4). Phenotypical characterization of the cells isolated from radiation enteropathy showed a positivity for tropomyosin, indicating that these cells are smooth muscle cells. At passage 4, RE-SMC and N-SMC were seeded at 3,500 cells/cm2 in SmGM2 medium (Clonetics, Cambrex, Emerainville, France). When 80% of confluence was reached, cells were starved for 24 h and then incubated in FCS-free medium for the various experiments.

Cell staining and confocal microscopy analysis. Direct microscopic analysis was performed after crystal violet staining (methanol 80%, formaldehyde 10%, crystal violet 1.25 g). Immunofluorescence was analyzed by laser scanning confocal microscopy (Zeiss LSM510). In these experiments, subconfluent cells were fixed in paraformaldehyde 4% and incubated with appropriate antibodies. Negative controls were performed to determine background signals. Nuclear staining was performed by 7-amino-actinomycin D (Sigma-Aldrich, Saint-Quentin Fallavier, France). Fluorescence quantification was performed by ImageJ software download on http://rsb.info.nih.gov/ij/.

FACS analysis. TGF-β1 receptor quantification was performed with Fluorokine kit from R&D and flow cytometer analysis (FACSCalibur, Becton-Dickinson).

Protein isolation and Western blot. For CCN2 analysis, supernatants were collected and cells were lysed in RIPA buffer. For Smad detection, nuclear and cytoplasmic protein extracts were isolated by the Schreiber method (41). Briefly, cells were scraped in PBS with protease cocktail inhibitor, and then cytoplasmic extract was collected by buffer A (10 mM HEPES pH = 8, 0.1 mM EGTA pH = 8, 0.01 M KCl, 1 mM DTT, 0.01 M NaF, 0.62% NP40, protease inhibitor). Nuclear protein extract were obtained after buffer B (20 mM HEPES pH = 8, 1 mM EGTA pH = 8, 400 mM NaCl, 1 mM DTT, 0.01 M NaF, protease inhibitor) addition. Protein concentrations were assessed by Bradford's assay (Bio-Rad, Marnes-la-Coquette, France). Proteins were detected by Western blotting. Briefly, 5–50 µg of proteins were separated by 12% SDS-PAGE and electrotransferred onto a 0.2-µm nitrocellulose membrane. The membrane was blocked in PBS-Tween with 5% of nonfat milk (Bio-Rad), incubated with appropriate primary antibody, washed, and probed with the peroxidase-labeled secondary antibody. Detection was achieved by enhanced chemiluminescence (ECL, Amersham).

mRNA expression analysis by quantitative RT-PCR. Total RNA was extracted from cells, quantified, and analyzed by real-time RT-PCR as already described (47). Primer sequences were, for CCN2, 5'-TCTGGGCAAACGTGTCTTC-3' (forward) and 5'-TGTGTGACGAGCCCAAGGA-3' (reverse); for TGF-β1, 5'-GCCTTTCCTGCTTCTCATGG-3' (forward) and 5'-TTCTCCGTGGAGCTGAAGCA-3' (reverse); for TGF-β1 receptor I (TRI), 5'-CAATGGGCTTAGTATTCTGGGAAA-3' (forward) and 5'-ATAAGGCAGTTGGTAATCTTCATGAA-3' (reverse); and for TGF-β1 receptor II (TRII), 5'-TGTCTGTGGATGACCTGGCTAA-3' (forward) and 5'-AAATTCATCCTGGATTCTAGGACTTCT-3' (reverse). For loading control, measurement of rRNA 18S was performed (Abyprism, Applied Biosystem, Courtaboeuf, France). Relative mRNA quantitation was performed by the comparative {Delta}{Delta}CT method, where CT is cycle threshold and {Delta}{Delta}CT is defined as the difference between the mean CT(CCN2, TGF-β1, TRI, TRII) and CT(18S).

Rho and Rho kinase activity assay. Active Rho proteins were isolated by pull-down assay according to the manufacturer's instruction (Pierce, Perbio, France). For Rho kinase assay, cells were washed in cold PBS before lysis by solution of Tris·HCl (10 mM, pH = 7.5), NaCl (100 mM), EGTA (1 mM), β-glycerophosphate (20 mM), Nonidet NP40 (0.5%), sodium orthovanadate (1 mM), inhibitor of protease (Roche). ROCK was immunoprecipitated: 500 µg of total protein lysis were incubated with 2 µg of specific ROCK I primary antibody (BD Biosciences) + 3% of BSA for 1 h at 4°C. Then, G protein-conjugated magnetic beads (Qiagen, Courtaboeuf, France) were added and incubated overnight at 4°C on rocket platform. Immunoprecipitate was washed threefold in lysis buffer and twofold more in kinase buffer [Tris·HCl 50 mM (pH = 7.5), EGTA 0.1 mM]. Beads were resuspended in ATP mix [MgCl2 75 mM, 500 µM of a nonradioactive ATP solution diluted in MOPS 20 mM (pH = 7.2), β-glycerophosphate 25 mM, EGTA 5 mM, sodium orthovanadate 1 mM, DTT 1 mM], 2 µg of histone H1 (Roche, Meylan, France) and 10 µCi of [{gamma}-32P]ATP were added. Then, kinase assay was performed for 15 min at 30°C. Enzymatic reaction was stopped by 6 M urea Laemmli buffer and boiled for 5 min at 100°C. [32P]histone H1 labeled were separated on 6 M urea 12% acrylamide gel and quantified by phosphorimager (Image Gauge software, FLA-3000, Fuji Ray test, Sèvres, France).

Statistical analysis. All values are reported as means ± SE. Data were analyzed by one-way ANOVA and the Student-Newman-Keuls test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Study of the TGF-β signaling machinery in normal vs. fibrosis-derived smooth muscle cells. RE-SMC exhibited a typical fibrosis-related morphology upon microscopic observation, with enlarged cellular bodies and numerous stress fibers containing a high density of {alpha}-smooth muscle actin compared with N-SMC (Fig. 1A). These primary cells retained their specific morphological and biochemical characteristics until passage 7 in culture and were therefore used in the present experiments at passage 4.


Figure 1
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  		Fig. 1. Expression and binding activity of transforming growth factor (TGF)-β1 receptors. A: primary smooth muscle cell lines were isolated from 3 individuals with healthy bowel (N-SMC) and 3 patients with radiation enteropathy (RE-SMC) and subsequently used. Cell morphology was observed after crystal violet staining and correlated with {alpha}-smooth actin ({alpha}-SmActin) expression. B: TGF-β receptor I and II (RI and RII) mRNA levels were determined by quantitative RT-PCR (QRT-PCR) and normalized to the amount of 18S rRNA. *P < 0.05 compared with N-SMC. C: cells were removed by trypsin treatment and conserved in culture medium for 7 h at 4°C on a rocket platform to enable receptor regeneration at the cell surface. Then, cells were incubated with biotinylated recombinant human (rh)TGF-β1. The binding was revealed by FITC-avidin conjugated staining and quantification was performed by flow cytometry. Data were expressed as percentages of positive cells and median values of fluorescence (arbitrary units) detected for N- and RE-SMC.

 
Quantitative RT-PCR (QRT-PCR) analysis showed that TRII mRNA level was higher in RE-SMC compared with N-SMC, whereas the level of TRI mRNA was not significantly different (Fig. 1B). FACS experiments showed that 98% of RE-SMC bound biotinylated rhTGF-β1, whereas only 77% of the N-SMC bound it. In addition, quantification of the amount of TGF-β1 bound at the cell surface showed that RE-SMC bound 2.4-fold (13 vs. 35 arbitrary units) more TGF-β1 than the N-SMC (Fig. 1C). The enhanced TGF-β1 binding activity measured in RE-SMC suggested increased sensitivity toward TGF-β1-induced fibrogenic stimuli and might help to explain the maintenance of fibrosis by low dose of TGF-β1 in situ.

Differential activation of Smad vs. Rho pathway by TGF-β1 in normal vs. fibrosis-derived smooth muscle cells. To investigate further the signaling pathways activated after TGF-β1 binding to its receptors and to explore the role of low doses of TGF-β1 on N-SMC and RE-SMC, activation of the canonical Smad pathway was first investigated by nucleocytoplasmic repartition of pSmad-2/3, Smad-2/3, and Smad-4. Differential nuclear isolation was checked with lamin B, a nucleus-specific marker.

Without exogenous TGF-β1 stimulation, the subcellular distribution of Smad-2/3 and Smad-4 proteins was similar in N- and RE-SMC, being mainly intracytoplasmic (Fig. 2A). After stimulation with low doses of TGF-β1 (0.01 ng/ml), pSmad-2/3 nuclear translocation occurred in both cell types (Fig. 2A). Interestingly, in RE-SMC, Smad-2/3 nuclear mobilization was weak because large amounts of Smad-2/3 remained in the cytoplasm, whereas in N-SMC Smad-2/3 nuclear import seemed more efficient. Similarly, Smad-4 nuclear recruitment was weaker in RE-SMC than in N-SMC. This is in accordance with our previous data obtained with high concentration of TGF-β1 (23) and indicates that Smad activation is weak in RE-SMC whatever TGF-β1 dose used and suggests activation of additional pathway to sustain fibrosis.


Figure 2
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  		Fig. 2. Differential activation of Smad vs. Rho pathway. A: N-SMC and RE-SMC were exposed to TGF-β1 for 45 min before nuclear and cytoplasmic protein extraction. Nuclear translocation of phosphorylated Smad-2/3, Smad-2/3, and Smad-4 was observed after SDS-PAGE (12%). Lamin B and GAPDH were used to check, respectively nucleo/cytoplasmic isolation and loading. Western blots are representative of 3 independent experiments. Quantifications were performed with ImageJ as follow: for nuclear extract, ratio = (integral of intensity for pSmad-2/3, Smad-2/3, or Smad-4/integral of intensity of lamin B) x 10; for cytoplasmic extract, ratio = (integral of intensity for pSmad-2/3, Smad-2/3, or Smad-4/integral of intensity of GAPDH) x 10. B: active Rho was studied by pull-down assay. N-SMC and RE-SMC were exposed to TGF-β1 at indicated concentrations and to lysophosphatidic acid (LPA) 10 µM for 45 min. After treatment, cells were washed in cold TBS buffer, and then 500 µg of protein extract were added to 400 µg of GST-Rotekin-RBD and 1 glutathione disc. Active Rho was immobilized on the spin column, before elution in Laemmli buffer. Total elution fraction was deposed on 12% SDS-PAGE and electrotransferred onto a 0.2-µm nitrocellulose membrane. The membrane was blocked in PBS-Tween with 3% of BSA, incubated with the primary antibody, washed, and probed with the peroxidase-labeled secondary antibody. Detection was achieved by enhanced chemiluminescence. Rho activity (Rho-GTP) was compared with Rho total level. Loading control was performed with GAPDH staining.

 
We have previously shown a Rho pathway activation in RE-SMC after stimulation with high TGF-β1 doses (4) and investigated in the present study whether low doses of TGF-β1 were able to switch on Rho cascade. As shown Fig. 2B, total Rho protein level was similar in N-SMC and RE-SMC and remained constant upon the incubations. Baseline level of active Rho (Rho-GTP) was similar in N- and RE-SMC, but, interestingly, a cell-specific effect appeared upon low doses of TGF-β1: in N-SMC low TGF-β1 doses (between 0.01 and 0.1 ng/ml) decreased Rho-GTP level whereas a slight activation was observed at higher doses. Conversely, important Rho activation occurred in RE-SMC incubated with low doses of TGF-β1 (from 0.01 ng/ml) that was similar to that obtained with 10 µM of LPA. Globally, these results show that the Rho pathway is highly activated by low doses of TGF-β1 in RE-SMC but not in N-SMC.

Differential CCN2 induction by TGF-β1 in normal vs. fibrosis-derived smooth muscle cells. To assess the functional relevance of these differential pathways to fibrosis development and maintenance, subsequent CCN2 response was investigated. As previously described (4, 23) high constitutive CCN2 mRNA and protein levels were observed in RE-SMC compared with N-SMC (x5; P < 0.05) (Fig. 3, A and C). In addition in RE-SMC, CCN2 always appeared as a double band, suggesting specific alteration of CCN2 glycosylation. A slight CCN2 protein induction was obtained after stimulation with the lowest TGF-β1 dose in both cell types (Fig. 3C), which was confirmed by QRT-PCR (x1.5 in N-SMC and x 1.3 in RE-SMC) (Fig. 3B). At higher TGF-β1 doses, the increase in CCN2 mRNA level was associated with a clear increase in CCN2 protein in both cell types. The magnitude of CCN2 induction was higher in N-SMC than in RE-SMC (x11.5 in N-SMC vs. x4.9 in RE-SMC) (Fig. 3B). However, the overall CCN2 level obtained after TGF-β1 stimulation remained always higher in RE-SMC than in N-SMC.


Figure 3
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  		Fig. 3. CCN2 induction by TGF-β1. A: Cells isolated from normal ileum (N-SMC) or late radiation enteropathy (RE-SMC) were exposed to increasing doses of TGF-β1 for 3 h. CCN2 mRNA expression level was subsequently analyzed by QRT-PCR. aP < 0.05 compared with unstimulated N-SMC. *P < 0.05 compared with unstimulated RE-SMC. B: ratios of relative mRNA level were calculated in N-SMC and RE-SMC as follows: relative mRNA in stimulated cell divided by relative mRNA in unstimulated cell. C: CCN2 expression level was analyzed after separation of protein extracts on SDS-PAGE 12%. Values are normalized to the level of GAPDH protein. D: N-SMC and RE-SMC were stimulated by increasing concentration of TGF-β1 for 3 h. Then, pravastatin was added to medium culture cell at 0, 100, and 500 µM for 6 h. Modulation of CCN2 expression was analyzed by Western blotting. E: CCN2 expression was observed by Western blot after cell treatment with TGF-β1 at indicated concentrations for 45 min before Y-27632 addition for 3 h more. Results were representative of 3 independent experiments.

 
These results along with the differential signaling pathway activation previously shown suggest that the RE-SMC require less stimulation to be maximally activated than the N-SMC or/and that a specific and more efficient pathway is activated in RE-SMC after stimulation with low doses of TGF-β1. A causal role for Rho activation in CCN2 stimulation by low doses of TGF-β1 was identified in RE-SMC by using a pharmacological inhibitor of Rho isoprenylation, pravastatin. Cells were preincubated for 3 h with TGF-β1 followed by a 6-h exposure to pravastatin. Pravastatin strongly inhibited CCN2 expression in RE-SMC incubated or not with low doses of TGF-β1 but was less efficient at higher doses (1 ng/ml) (Fig. 3D). Consistent with pull-down results, pravastatin inhibited TGF-β1-induced CCN2 expression in N-SMC (Fig. 3D). This inhibitory action of pravastatin was dose dependent, being most effective at 500 µM, corresponding to the ED50 of the drug (9).

These results encouraged us to examine the possible role of ROCK in CCN2 alteration induced by low doses of TGF-β1. Thus similar experiments were performed with the allosteric inhibitor of ROCK, Y-27632: cells were preincubated for 45 min with TGF-β1 followed by a 3-h exposure to Y-27632. This protocol repressed CCN2 expression level in N-SMC and RE-SMC (Fig. 3E). Lastly, we asked whether CCN2 alteration was mediated by the ROCK I. We found that baseline ROCK I activity was higher in RE-SMC than in N-SMC (4), but no significant modulation of ROCK I activity was found upon TGF-β1 stimulation (data not shown), suggesting that ROCK I was not the target of TGF-β1/Rho activation at low dose.

CCN2 autoregulation in fibrosis-derived smooth muscle cells. Next, we specifically questioned CCN2 action in fibrosis-derived cells, and interestingly we found that 3 and 30 ng/ml of CCN2 stimulate its own expression within 6 h as shown Fig. 4A. As control, the sensitivity of the Western blot assay was checked and proved to be too weak to detect 30 ng/ml of rhCCN2 (data not shown). This CCN2 stimulation was associated with an increase in Smad-4 nuclear fluorescence; first observed at 3 ng/ml of rhCCN2 and became statistically significant when RE-SMC were incubated with 30 ng/ml of CCN2 (P < 0.01) (Fig. 4B). Finally, as we have previously shown, a preferential activation of the Rho pathway in the fibrosis-derived cells, we investigated direct Rho activation by rhCCN2 incubation and found that an enhanced level of Rho-GTP formed upon rhCCN2 stimulation (Fig. 4C).


Figure 4
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  		Fig. 4. CCN2 autoinduction. A: RE-SMC were exposed for 6 h to increasing doses of CCN2 then cell were lysed. CCN2 expression was detected by Western blot. Values are normalized to the level of GAPDH protein. B: after 45 min stimulation with increasing doses of CCN2, RE-SMC were fixed, permeabilized, and stained with anti-Smad-4 antibody. Labeling was performed by an Alexa Fluor 488 antibody (green) and nucleus was colored by 7-amino-actinomycin D (blue). Immunofluorescences were analyzed by confocal microscopy and Smad-4 nuclear translocation was quantified by using ImageJ software by contouring nucleus area and calculation of pixel mean intensity. Quantification values represent the average of pixel mean intensity ± SE performed on 10 cells randomly chosen and representative of a global cell population response. *P < 0.05 compared with unstimulated RE-SMC. C: RE-SMC were stimulated for 45 min with increasing doses of rhCCN2. Then, cells were lysed and Rho pull-down assays were performed as previously described.

 
TGF-β1 enhanced CCN2 autoinduction in fibrosis-derived smooth muscle cells. To mimic as close as possible the physiological conditions of fibrotic tissue during radiation enteropathy, fibrosis-derived cells were coincubated with rhCCN2 and low concentration of TGF-β1. This coincubation enhanced CCN2 production in a dose-dependent manner, suggesting that low doses of TGF-β1 and CCN2 act in conjunction to sustain the long-term fibrogenic differentiation (Fig. 5A). Interestingly, TGF-β1 addition stimulates both Smad4 nuclear translocation and Rho activation, but Smad-4 nuclear fluorescence intensity remained unchanged when increasing doses of CCN2 were combined to 0.01 ng/ml of TGF-β1 (Fig. 5B), whereas Rho activation increased 1.5-fold (Fig. 5C).


Figure 5
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  		Fig. 5. Combination of CCN2 and low concentration of TGF-β1 enhanced CCN2 expression. A: RE-SMC were exposed for 6 h to increasing doses of CCN2 and TGF-β1. CCN2 expression was analyzed by Western blot. B and C: cells were stimulated for 45 min with increasing doses of CCN2 plus 0.01 ng/ml of TGF-β1. B: cells were fixed, permeabilized, and stained with anti-Smad-4 antibody and analyzed as previously described. *P < 0.05 compared with unstimulated RE-SMC. C: Rho pull-down assays were performed as previously described. A.U., arbitrary units.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Long-term persistence of the fibrogenic differentiation is a key parameter of fibrosis as it controls the accumulation of the ECM. If the fibrogenic process has been well characterized and is primarily mediated by the potent profibrotic growth factor TGF-β1, the molecular basis of chronic activation loops involved in the long-term maintenance of fibrosis have been poorly characterized. Using physiologically relevant primary human cells and biochemical approaches, characterization of the molecular pathways involved in the long-term maintenance of radiation-induced fibrogenic differentiation was the main goal of the present study. Our major findings include, first, the demonstration of a differential contribution between TGF-β1 and CCN2 in initiation vs. maintenance of fibrosis. The primary role of the Smad pathway in fibrogenesis is confirmed here, but the long-term maintenance of the fibrogenic differentiation depends preferentially on the Rho pathway. Secondly, we found that very low doses of TGF-β1 elicit potent profibrotic action both in normal and fibrosis-derived cells. Third, we determined that CCN2 is self-inducible in fibrosis-derived cells, suggesting that CCN2 alone is able to sustain fibrosis in "fibrosis-primed" cells. Last, we reported that enhancement of CCN2 stimulation by the combination of TGF-β1 and CCN2. The latter result extends the notion that complex growth factor cocktails and combinatorial pathways are likely required for the long-term maintenance of the fibrogenic differentiation and specific to it.

Although TGF-β1 is recognized as the key parameter in fibrosis generation and maintenance, its expression might be low in human fibrotic diseases that involve long-term evolution of the pathology such as late radiation enteropathy and scleroderma (30, 47). In these pathologies low TGF-β1 deposition is associated with strong accumulation of CCN2 (a TGF-β1 downstream effector) (2, 37). The molecular basis of this paradox and its functional significance are unclear and lead us to investigate two nonmutually exclusive hypotheses. In the first, a fibrogenic role for low-dose TGF-β1 within fibrotic tissues is proposed and was investigated in vitro in normal and pathological cells by using picograms per milliliter of rhTGF-β1. The second one is that CCN2 could be the key mediator of the maintenance of fibrosis and led us to specifically investigate CCN2 regulation by low doses of TGF-β1, rhCCN2, and a combination of both to mimic the in vivo situation.

The role of low-dose TGF-β1 in the fibrogenic differentiation is mentioned in the literature, particularly by C. Flanders and colleagues (10), but has never been specifically investigated. In normal cells, a dose-dependent biphasic cell response was observed in which low concentrations of TGF-β1 activated Smad but inhibited Rho, whereas high concentrations stimulated both pathways and triggered a high-magnitude (11x) stimulation of CCN2 production. This two-step activation process is likely to be involved in the tight control of CCN2 expression and could be one of the checkpoints leading to normal wound healing (Smad activation) or fibrosis development (Smad + Rho activation). This hypothesis requires further investigations and was not our main goal. In the present study, we aimed at investigating the regulation of CCN2 protein expression in fibrosis-primed cells. We found that CCN2 protein level was stimulated by low doses of TGF-β1 and largely depends on Rho pathway activation combined with slight activation of Smad. This preferential activation of the Rho pathway was obtained at all the concentrations of TGF-β1 used. In fibrosis-derived cells, high constitutive and inducible levels of CCN2 protein were measured that were always significantly above levels observed in normal cells. This finding helps to explain the high constitutive CCN2 expression previously reported in situ and supports a role for low TGF-β1 doses in the maintenance of fibrosis. Although CCN2 trans-activation by TGF-β has been initially shown to involve the Smad pathway (2, 7, 28), we and others have provided evidence that additional cascades might be more specifically involved in fibrotic conditions, including the Rho/ROCK pathway (4, 9, 18), MAPK p42-p44 (42), and JNK (36). These observations were extended to low doses of TGF-β1 and showed that CCN2 stimulation in fibrosis-derived cells correlated with both an elevation in TGF-β1 receptor number and activation of the Rho/ROCK cascade. Rho activation after TGF-β1 stimulation was shown by direct Rho-GTP analysis and by pharmacological inhibition approaches using pravastatin and Y-27632. Pravastatin action is not fully specific to Rho activation since it is known to target isoprenylated protein including ras or rac (19, 44). However, the strong efficacy of Y-27632, a specific inhibitor of the ROCK, in decreasing TGF-β1-induced CCN2 expression reported here and previously by Graness et al. (16) in fibroblasts confirmed the essential role of the Rho/ROCK pathway. Because ROCK I mRNA level was known to be altered in late radiation enteropathy-derived smooth muscle cells (4), we assessed its activity in the present study but found no modulation (data not shown), suggesting that ROCK I was not the downstream target of TGF-β1/Rho activation. In addition to Rho/ROCK activation, a weak role for Smad2/3/4 was also observed in RE-SMC that is not fully consistent with results reported by Leask et al. (31) in scleroderma-derived fibroblasts incubated with TGF-β1, showing that CCN2 induction was Smad independent. These discrepancies could account for differences between skin vs. intestinal cells. Another explanation is related to the approach used. Whereas Leask et al. mainly used a gene reporter strategy, we chose a direct biochemical approach to keep as close as possible to normal cell physiology and avoid transfection-based artifact. However, both studies concur to show that Smad pathway activation is not the key factor for the maintenance of CCN2 expression in fibrosis-derived human cells and underline the importance of truly representative cellular models to characterize complex pathological situations such as fibrosis.

The lack of adequate models has probably delayed the study of the mechanisms involved in the maintenance of fibrosis. The present study extends to intestinal smooth muscle cells derived from radiation enteropathy the observations reported in fibroblasts isolated from scleroderma biopsies (29, 40) in which CCN2 constitutive expression is higher in fibrosis-derived cells than in normal cells. This suggests that CCN2 high constitutive expression is of relevance in various fibrotic conditions. Few hypotheses have been proposed until now to explain this high constitutive expression. Several response elements located in CCN2 promoter might be involved, including Sp1 (27) and the stretch-responsive elements (2) whose action requires increased actin polymerization (6) and is associated with Rho/ROCK activation. Another interesting explanation could involve the paracrine action triggered by CCN2 itself. B. Riser's laboratory previously described CCN2 self-induction of mRNA in kidney mesangial cells from rats (39) and showed in the present study that CCN2 autoinduction was associated with Smad-4 nuclear translocation. Conversely, the combination of CCN2 with low TGF-β1 doses enhances CCN2 expression but no Smad-4 nuclear translocation. Direct activation of the Smad pathway by CCN2 has also been reported in mesangial cells by Wahab et al. (48). They showed that CCN2 alone was able to block the negative feedback induced by Smad-7, whereas a combination of TGF-β1 and CCN2 increased pSmad-2/3 nuclear translocation but they did not investigate CCN2 regulation in response to CCN2. Interestingly in our model, CCN2 self-induction was associated with Smad-4 nuclear translocation without TGF-β1 induction, suggesting that CCN2 could signal via the Smad pathway. The molecular mechanism responsible remains speculative. A CCN2-induced enhancement of the binding of endogenous TGF-β1 to its receptors has been proposed (1); however, a specific signal through a CCN2 receptor cannot be ruled out. Until recently Smad signaling was thought to be TGF-β-specific but Wang et al. (49) challenged this paradigm showing Smad3 activation by angiotensin-II. A similar mechanism could occur in our model and opens new research perspectives.

In conclusion, the present results challenge the paradigm in which high TGF-β1 levels are a prerequisite for the maintenance of fibrosis. Here, low TGF-β1 doses triggered specific-cell responses, suggesting that subtle regulation may take place in situ according to TGF-β1 concentration. In addition, precise sequential kinetics of TGF-β1 and CCN2 induction seem to be key parameters in the maintenance of fibrosis and might therefore be used to induce fibrolysis. The specific role played by Rho/ROCK indicates a possible target for therapeutic intervention, which has recently been proposed and validated in preclinical studies (20–22). This new strategy selectively targets phenotypically altered pathological cells within the fibrotic tissue without affecting new granulation tissue formation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
V. Haydont is a fellow of the Institut de Radioprotection et de Sureté Nucléaire et région Ile de France. This study was supported by the Comité de Radioprotection d'Electricité de France and Association de recherche sur le Cancer grant 3881.


   ACKNOWLEDGMENTS
 
The authors thank Prof. D. Mathé for suggestions and scientific advices and Dr. H. K. Lord for English edition of the manuscript; Drs. P. Lasser, M. Pocard (Surgery department Institut Gustave Roussy), and A. Lusinchi (Radiation therapy department Institut Gustave Roussy) for support; as well as Y. Lecluse and A. Jalil for technical assistance on flow cytometry and confocal microscopy analysis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. C. Vozenin-Brotons. Laboratoire UPRES EA 27-10, Radiosensibilité des tumeurs et tissus sains, PR1, 39, Rue Camille Desmoulins, 94805 Villejuif CEDEX, France (e-mail: vozenin{at}igr.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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