Negative Regulation of Myofibroblast Differentiation by PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10)
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《美国呼吸和危急护理医学》
Division of Pulmonary and Critical Care Medicine and Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan
University Health Network, Ontario Cancer Institute, University of Toronto, Toronto, Ontario, Canada
Division of Cardiology, Duke University Medical Center, Durham, North Carolina
ABSTRACT
Rationale: Myofibroblasts are primary effector cells in idiopathic pulmonary fibrosis (IPF). Defining mechanisms of myofibroblast differentiation may be critical to the development of novel therapeutic agents.
Objective: To show that myofibroblast differentiation is regulated by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) activity in vivo, and to identify a potential mechanism by which this occurs.
Methods: We used tissue sections of surgical lung biopsies from patients with IPF to localize expression of PTEN and –smooth muscle actin (-SMA). We used cell culture of pten–/– and wild-type fibroblasts, as well as adenoviral strategies and pharmacologic inhibitors, to determine the mechanism by which PTEN inhibits -SMA, fibroblast proliferation, and collagen production.
Results: In human lung specimens of IPF, myofibroblasts within fibroblastic foci demonstrated diminished PTEN expression. Furthermore, inhibition of PTEN in mice worsened bleomycin-induced fibrosis. In pten–/– fibroblasts, and in normal fibroblasts in which PTEN was inhibited, -SMA, proliferation, and collagen production was upregulated. Addition of transforming growth factor- to wild-type cells, but not pten–/– cells, resulted in increased -SMA expression in a time-dependent fashion. In pten–/– cells, reconstitution of PTEN decreased -SMA expression, proliferation, and collagen production, whereas overexpression of PTEN in wild-type cells inhibited transforming growth factor-–induced myofibroblast differentiation. It was observed that both the protein and lipid phosphatase actions of PTEN were capable of modulating the myofibroblast phenotype.
Conclusions: The results indicate that in IPF, myofibroblasts have diminished PTEN expression. Inhibition of PTEN in vivo promotes fibrosis, and PTEN inhibits myofibroblast differentiation in vitro.
Key Words: fibrosis myofibroblast phosphatase PTEN smooth muscle actin
–Smooth muscle actin (-SMA) expression in fibroblasts, increased proliferative capacity, and increased generation and secretion of the extracellular matrix (ECM) proteins collagen and fibronectin are key hallmarks of myofibroblast differentiation in fibrotic disorders (1, 2). In idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia (UIP), persistence of myofibroblasts is believed to contribute to the development of fibrosis. It has been established that myofibroblast differentiation occurs through combinatorial signals involving transforming growth factor-1 (TGF-) and integrin signaling (3). Conversely, mechanisms that inhibit or reverse myofibroblast differentiation are less well defined. Currently, no effective therapies exist to quell ongoing fibrosis in IPF/UIP. Thus, further investigation into the mechanisms that regulate myofibroblast differentiation may yield better insights into potential therapeutic options for patients with IPF/UIP.
The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a dual-specificity protein and lipid phosphatase that uses phosphatidylinositol-3,4,5-trisphosphate as a physiologic substrate (4). Altered PTEN protein expression or activity has increasingly been implicated in the pathogenesis of nonmalignant diseases characterized by tissue destruction and remodeling, such as rheumatoid arthritis (5), asthma (6), and pulmonary fibrosis (7), thereby highlighting the importance of PTEN in physiologic regulation of nontransformed cellular phenotypes. Through its lipid phosphatase activity, PTEN inhibits cell migration (8), promotes cellular apoptosis (9), and inhibits cell growth (10). In addition, PTEN possesses protein phosphatase activity, and can act through the dephosphorylation of focal adhesion kinase (FAK) and Src homology 2–containing protein (11). Interestingly, pten expression is markedly decreased by TGF- (12). We have previously observed diminished PTEN expression in fibroblasts isolated from lungs of patients with pulmonary fibrosis (7), and have hypothesized that this may account for the increased migratory capacity of these cells (7, 13).
Here, we show that in lung biopsy samples of patients with IPF/UIP, only fibroblasts that demonstrate a distinct inhibition or loss of PTEN correlate with the expression of -SMA. We further show that pharmacologic inhibition of PTEN induces lung fibrosis in mice. Importantly, we show that inhibition of PTEN activity is both necessary and sufficient to induce myofibroblast differentiation. Finally, we demonstrate the novel finding that PTEN overexpression suppresses -SMA expression, proliferation, and collagen production in myofibroblasts, a process that can occur via either lipid or protein phosphatase activity. Our data suggest that inhibition of PTEN expression in fibroblasts may contribute to the pathogenesis of fibrotic lung disease. Some of the results of this study have been previously reported in the form of an abstract (14).
METHODS
See online supplement for additional details.
Cell Culture and Reagents
C57Bl/6 embryonic mouse fibroblasts and National Institutes of Health 3T3 murine fibroblasts were from the American Type Culture Collection (Rockville, MD). Embryonic mouse fibroblasts lacking both pten alleles (pten–/–) were described previously (9). The FAK/Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), and its inactive control, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3), were from EMD Biosciences (San Diego, CA). The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 was from Cell Signaling Technologies (Beverly, MA). Porcine TGF-1 was from R&D Systems (Minneapolis, MN). Bisperoxo(pyridine-2-carboxyl)oxovanadate (bpV[pic]) was from EMD Biosciences.
Antibodies
Antibodies to phospho-Smad2, total Smad2, and Smad7 were from Cell Signaling Technologies. -SMA antibody (clone 1A4) was from Dako Corporation (Carpinteria, CA). -Tubulin antibody was from Upstate (Waltham, MA). Anti-human PTEN (clone 6H2.1) was from Cascade Biosciences (Winchester, MA). Neutralizing antibody against TGF- 1, 2, 3 was from R&D Systems.
Intratracheal Bleomycin Administration
Animals were anesthetized with pentobarbital. The trachea was exposed, and sterile bleomycin sulfate (0.05 U/mouse) or sterile phosphate-buffered saline (PBS) alone (50 μl) was delivered via a 26-gauge needle inserted directly into the trachea.
Collagen Measurements
Total lung collagen measurements were determined 21 d after bleomycin instillation. Total lung collagen and secreted collagen from cultured cells were determined using the Sircol collagen dye binding assay (Accurate Chemical Corporation, Westbury, NY) as previously described (15) following the manufacturer's instructions.
Adenovirus Construction
Adenovirus containing cDNA constructs encoding wild-type PTEN (Ad-PTEN) or the empty virus control (Ad-EV) were prepared as previously described (16).
Immunofluorescent Staining
Cells affixed to glass slides were permeabilized, blocked, and incubated with anti–-SMA antibodies (1:1,000) for 1 h. Cells were washed and then incubated with fluorescein isothiocyanate (FITC)–conjugated secondary antibody for 1 h in the dark. Cells were again washed, mounted with 4',6-diamidino-2-phenylindole–containing mounting media (Vector Labs, Burlingame, CA), and viewed under an epifluorescent microscope (Nikon Eclipse 50i; Nikon Inc., Melville, NY).
Immunohistochemistry
Immunohistochemistry was performed on 3- to 5-μm sections of formalin-fixed, paraffin-embedded surgical lung biopsy specimens obtained from patients undergoing surgical lung biopsy for diagnosis of idiopathic interstitial pneumonia. Prior written informed consent was obtained from all subjects in accordance with the University of Michigan Institutional Review Board. Rehydrated tissue sections were incubated with primary antibody, followed by biotinylated secondary antibody, and stained using the Vectastain ABC Universal kit (Vector Labs). Color development was achieved using 3,3'-diaminobenzidine, and sections were counterstained with hematoxylin. For immunofluorescent staining, the method of Mason and colleagues (17) was followed.
Fibroblast Proliferation Assay
Fibroblasts were plated in 96-well plates and serum-starved for 24 h. Media were changed, including indicated reagents, and cells cultured for 24 h. 3H-thymidine was added for the last 16 h of culture, and radioactive counts per minute were assessed using a scintillation counter.
Semiquantitative Real-Time Reverse Transcriptase–Polymerase Chain Reaction
Semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) used primers and probes for -SMA as previously described (7, 18), and glyceraldehyde phosphate dehydrogenase was used to normalize results. Relative gene expression was calculated using the comparative cycle–threshold method (19).
Western Blot Analysis
Western blot analysis under reducing conditions was performed on equal concentrations of protein from whole-cell lysates and immunoprecipitated proteins, as previously described (7).
Statistical Analyses
Statistical analyses were performed using GraphPad InStat 3.05 (San Diego, CA). Differences between groups were evaluated using Student's t test. For multiple comparisons, one-way analysis of variance with Bonferroni's post-test analysis was used. Data were considered significant at a p value less than 0.05. Results were plotted using GraphPad Prism 3.02. Densitometry of visualized bands on Western blot was performed using Image J software (version 1.31; National Institutes of Health, Bethesda, MD).
RESULTS
Decreased PTEN Expression in Fibroblasts of Fibrotic Lesions Correlates with Increased -SMA Expression
We have previously shown that PTEN expression is decreased in lung fibroblasts from patients with fibroproliferative disease compared with normal lung fibroblasts (7). To determine whether decreased PTEN expression was a general feature of lung fibroblasts or was localized to -SMA–expressing myofibroblasts, we performed immunohistochemical analysis of surgical lung biopsy specimens from 10 patients with UIP, the histologic pattern associated with IPF (20). Consecutive sections were stained for -SMA and PTEN and compared. A representative sample of fibroblastic foci, shown in Figure 1A, demonstrates that -SMA expression is observed in spindle-shaped fibroblasts where PTEN staining is diminished or lost, thus indicating that -SMA and PTEN expression may be inversely related (Figure 1A). Similar findings were observed in all other cases evaluated (data not shown). To confirm that PTEN was not being expressed in the same cell where -SMA was being expressed, we employed triple immunofluorescent staining (17) on specimens from 10 patients with pulmonary fibrosis. Figure 1B shows a separate section from the same patient in Figure 1A stained for -SMA (Cy3, red) and PTEN (FITC, green). We observed that Cy3-positive myofibroblasts do not demonstrate significant FITC staining. 4',6-Diamidino-2-phenylindole (blue) staining identifies nuclei. Similar results were observed in all other samples (data not shown).
Inhibition of PTEN Activity in Fibroblasts Results in Myofibroblast Differentiation
To determine whether a cause-and-effect relationship exists between PTEN inhibition and myofibroblast differentiation, we initiated our studies by examining fibroblasts isolated from the embryos of pten-deficient transgenic mice (pten–/–) compared with wild-type murine embryonic fibroblasts. These cells were defined as fibroblasts based on morphology under a light microscope, as well as by the expression of fibroblast-specific protein-1 and vimentin, and have been previously characterized (9). To determine -SMA expression, cultured pten–/– and wild-type fibroblasts were assessed by immunoblotting. We observed that -SMA was expressed in both pten–/– and wild-type fibroblasts under serum-free, quiescent conditions, although to a significantly greater degree in pten–/– fibroblasts (p < 0.0001). To ensure equal protein loading, membranes were stripped and reprobed for -tubulin (Figure 2A). Immunofluorescent staining for -SMA in serum-deprived wild-type and pten–/– cells confirmed this increase in -SMA, and demonstrated that all pten–/– cells expressed -SMA (Figure 2B). To investigate whether inhibition of PTEN activity is sufficient to promote -SMA gene and protein expression, PTEN activity was inhibited in wild-type fibroblasts by the PTEN inhibitor dipotassium bpV(pic) (21). Treatment of cells with bpV(pic) does not alter protein levels of PTEN (21), but inhibits PTEN activity, as demonstrated by increasing levels of phosphorylated Akt at serine-473 (21). When we inhibited PTEN activity by treating wild-type cells with 200 nM bpV(pic) for 24 h, we observed a roughly fourfold increase in -SMA expression. This increase in -SMA was accompanied by a threefold increase in levels of phosphorylated Akt at serine-473 (Figure 2C).
We next investigated the possibility that PTEN affected other myofibroblast behaviors, namely proliferation and collagen secretion. To achieve this, pten–/– and wild-type cells were assessed for basal proliferation rates and collagen secretion. Using a 3H-thymidine incorporation assay, we determined that pten–/– fibroblasts proliferated roughly 60 times faster than wild-type cells (Figure 3A). Similarly, cultures of pten–/– and wild-type cells were assessed for collagen production. Whole-cell lysates (to evaluate cell-associated collagen) and culture supernatants (to evaluate secreted collagen) were assessed for collagens I–V by Sircol assay. Although we did not observe significant amounts of soluble collagens in supernatants (not shown), we did observe significant elevations in cell- and matrix-associated collagens (Figure 3B, left panel).
Overexpression of PTEN Inhibits Myofibroblast Differentiation
To show that PTEN could inhibit myofibroblast differentiation, we next infected pten–/– cells with an adenoviral construct encoding either full-length, functional Ad-PTEN, or Ad-EV, and assessed for -SMA expression, proliferation, and collagen production. We observed a resultant down-regulation of proliferation (Figure 3A, right panel), collagen production (Figure 3B, right panel), and -SMA expression (Figure 3C) in cells infected with Ad-PTEN as compared with Ad-EV. Collectively, these data indicate that suppression of PTEN is sufficient to promote myofibroblast differentiation, and that reconstitution of PTEN into pten–/– cells will reverse the phenotype.
Increased -SMA Expression in pten–/– Fibroblasts Is Not Dependent on TGF- Signaling
TGF- is a multifunctional cytokine that is a potent inducer of myofibroblast differentiation (22) and repressor of pten transcription (12). It has been well documented that autocrine release of TGF- accounts, at least in part, for ongoing ECM secretion and myofibroblast differentiation in numerous model systems (23–26). Given that pten–/– cells possess a predominant myofibroblast phenotype, it was important to determine whether this was mediated by autocrine signaling by endogenous TGF-. To address this, wild-type and pten–/– cells were cultured until subconfluent and extensively washed. Cells were then serum-starved and treated for 24 h with either control antibody or an antibody to TGF- that is capable of neutralizing the activity of the three isoforms of TGF-. Cells were then lysed and assayed for -SMA expression by Western blot. As shown in Figure 4A, pten–/– cells expressed higher amounts of -SMA than wild-types after 24 h of serum starvation (compare lanes 1 and 4, Figure 4A), confirming our initial observation. After the addition of neutralizing antibody to TGF- or control antibody for 24 h, there was no decrease in -SMA expression in wild-type or pten–/– cells (Figure 4A), suggesting that autocrine TGF- is not a significant contributor to the constitutive myofibroblast phenotype seen in pten–/– cells.
TGF- signaling predominantly occurs through the Smad pathway, where receptor–ligand interaction results in rapid phosphorylation and nuclear translocation of Smad2 and Smad3 (27, 28). Our data indicate that autocrine TGF- signaling did not play a significant role in -SMA expression in pten–/– fibroblasts. However, it was necessary to exclude the possibility that constitutive Smad2 or Smad3 activity accounted for the differences in -SMA expression between pten–/– and wild-type fibroblasts. To do this, pten–/– and wild-type fibroblasts were plated until subconfluent, serum-starved overnight, and treated with exogenous TGF- (2 ng/ml) for 1 h. Cells were then lysed and assayed for Smad2 and Smad3 phosphorylation by Western blot. After serum deprivation, no phosphorylation of Smad2 could be observed in either wild-type or pten–/– cells (Figure 4B). However, treatment of both cells with TGF- for 1 h resulted in robust Smad2 phosphorylation (Figure 4B, top panel). Blots were stripped and probed for total Smad2 to ensure equal loading. Similar results were obtained for Smad3 (data not shown). This finding implies that basal Smad2 and Smad3 phosphorylation in pten–/– fibroblasts does not account for the increased constitutive -SMA expression observed, but that both cells are equally capable of responding to an exogenous TGF- challenge. Furthermore, these data suggest that surface TGF- receptor expression and activity is equivalent between the two cell lines.
Regulation of Smad2/3 nuclear translocation occurs in part via the activity of Smad7. To determine whether dysregulation of Smad signaling could account for the constitutive myofibroblast phenotype observed in pten–/– cells, we assessed the levels of Smad7 in pten–/– and wild-type cells by Western blot. Unexpectedly, we observed that Smad7 levels were increased in pten–/– cells compared with wild-type cells (Figure 4B, bottom panel). This interesting observation suggests that another possible action of PTEN is to inhibit Smad7 expression. Because Smad7 is a regulatory Smad, our data would appear to support the theory that PTEN inhibition or loss in nonmalignant cells disrupts normal cellular homeostatic mechanisms, promoting aberrant behaviors.
TGF- Markedly Induces -SMA Expression in Wild-Type Fibroblasts, but Only Minimal Further Increases in pten–/– Fibroblasts
Our data indicate that pten–/– fibroblasts express -SMA independent of TGF- stimulation, but that these cells are capable of responding to exogenous TGF- through the Smad signaling pathway. Smad proteins have been shown to mediate TGF-–induced -SMA expression (29); we therefore asked whether the addition of TGF- to pten–/– fibroblasts would further increase -SMA expression. To test this, wild-type and pten–/– fibroblasts were growth-arrested for 24 h in serum-free media, washed with PBS, and then cultured in serum-free media in the absence or presence of TGF- (2 ng/ml) for 24 h. Cell lysates from two separately grown cultures of each cell type were harvested and assessed by Western blot for -SMA expression. Figure 5A shows that wild-type fibroblasts significantly increased -SMA expression 24 h after TGF- exposure (p < 0.02), whereas -SMA expression in pten–/– fibroblasts was not substantially altered. This finding appears to be in agreement with our data demonstrating increased levels of Smad7 in pten–/– cells.
Because -SMA is elevated constitutively in pten–/– fibroblasts as compared with wild-type fibroblasts, we next assessed whether this was due to increased gene expression. After isolating RNA from quiescent, serum-deprived pten–/– and wild-type fibroblasts, we used semiquantitative RT-PCR for -SMA. We found that -SMA gene expression was 1.8-fold greater in pten–/– cells compared with wild-type cells (p < 0.0001; Figure 5B), which correlated with increased -SMA promoter activity (data not shown). In aggregate, these data establish that loss of PTEN expression resulted in increased -SMA gene transcription and protein expression. Moreover, these findings imply that -SMA expression induced by inhibition of PTEN occurs independent of Smad signaling.
Inhibition of PTEN Activity Is Necessary for TGF-–induced -SMA Expression
Because our data demonstrate that inhibition of PTEN activity was sufficient to induce -SMA expression, we questioned whether it was also necessary to promote -SMA expression in fibroblasts after TGF- stimulation. To test this, we induced myofibroblast differentiation in normal fibroblasts by treating with TGF- (2 ng/ml) for 24 h while concomitantly infecting with Ad-PTEN or Ad-EV. Cell lysates were collected and evaluated by Western blot for -SMA. As shown in Figure 6, TGF-–induced -SMA expression in fibroblasts was attenuated in the presence of Ad-PTEN, but not in the presence of Ad-EV. Together, these data indicate that suppression of PTEN expression is both necessary and sufficient for -SMA expression.
-SMA Inhibition by PTEN Can Be Accomplished through Both Lipid- and Protein-Phosphatase Activity
Because PTEN is a dual-specificity phosphatase, we next sought to determine whether the lipid- or protein-phosphatase activity was responsible for -SMA regulation. Thus, serum-starved wild-type fibroblasts were induced to express -SMA, either through TGF- treatment (2 ng/ml) or bpV(pic) treatment (200 nM), in the presence or absence of the FAK/Src-specific inhibitor PP2 (or its negative control, PP3; 10 μM), or the PI3K-specific inhibitors LY294002 (50 μM) or wortmannin (50 nM), for 24 h. Untreated serum-free cells were assessed as a negative control. Whole-cell lysates were prepared and proteins separated by electrophoresis. Immunoblotting for -SMA revealed the expected increase in -SMA expression after treatment with TGF-. Consistent with our previous findings, bpV(pic) also induced -SMA expression, although to a lesser extent than TGF- (Figure 7). Importantly, we observed that, in the presence of PP2, but not PP3, TGF-–induced and bpV(pic)-induced -SMA expression was attenuated, indicating that FAK and/or Src phosphorylation is necessary for -SMA expression and that PTEN is capable of dephosphorylating one or both of these proteins (Figure 7), consistent with previous data (30). Similarly, we observed that, in the presence of LY294002 or wortmannin, TGF-–induced and bpV(pic)-induced -SMA expression was completely inhibited, providing evidence that unopposed PI3K activity after PTEN inhibition also contributes to -SMA expression (Figure 7). Together, these data signify that PTEN is capable, through multiple signaling pathways, of inhibiting -SMA expression during myofibroblast differentiation.
PTEN Inhibition Worsens Experimental Pulmonary Fibrosis and Is Associated with Greater Numbers of -SMA–expressing Cells
Our aggregate data indicate that inhibition of PTEN is both necessary and sufficient to induce myofibroblast differentiation in vitro, a process that may also occur in vivo in UIP/IPF. To determine whether inhibition of PTEN would have similar effects in experimental pulmonary fibrosis, we used a murine bleomycin model. On Day 14 after intratracheal injection of bleomycin, a time point corresponding to resolution of the inflammatory phase and progression of the fibrotic phase, animals were treated with daily intraperitoneal injections of bpV(pic) (5 mM diluted in PBS) (31), or PBS as a control. Mice were killed 21 d after bleomycin and lungs were assessed for total collagen and presence of -SMA–expressing cells. Bleomycin-treated animals demonstrated a significant increase in total lung collagen compared with control animals (Figure 8A). As predicted, bleomycin-exposed animals treated with bpV(pic) had substantially more lung collagen when compared with both control and bleomycin-exposed animals (Figure 8A). Histologically, we found that both collagen deposition (as evidenced by trichrome staining) and -SMA expression (as evidenced by immunostaining) paralleled the total lung collagen findings (Figure 8B), indicating that PTEN inhibition in vivo results in increased -SMA–expressing cells and worse fibrosis.
DISCUSSION
In this article, we describe the novel finding that PTEN negatively regulates myofibroblast differentiation in fibroblasts, and that inhibition of PTEN allows for increased myofibroblast differentiation both in vitro and in vivo. We base this conclusion on our results showing that both pten–/– fibroblasts and fibroblasts in which PTEN is inhibited demonstrate increased expression of -SMA, increased proliferation, and increased production of collagen. In addition, we show that PTEN suppression is necessary for TGF-–mediated myofibroblast differentiation, and is sufficient to induce myofibroblast differentiation in wild-type cells. Importantly, we confirm the in vivo relevance of the association between PTEN and myofibroblast differentiation both in human IPF/UIP and in bleomycin-induced murine lung fibrosis, thereby identifying PTEN as a physiologic regulator of the myofibroblast phenotype. Mechanistically, our data suggest that suppression of PTEN promotes -SMA expression via unopposed signaling through the PI3K and/or FAK/Src pathways, resulting in increased -SMA gene expression and transcription.
Smad2/3 activation and nuclear localization following TGF- exposure results in myofibroblast differentiation and -SMA transcription (22, 28). Inhibition of Smad7, which binds and prevents nuclear translocation of the Smad2/3 complex, might also allow for increased -SMA transcription after TGF- stimulation. Somewhat unexpectedly, we observed that Smad7 levels were increased in pten–/– cells compared with wild-type cells. Although this may appear antithetical to our hypothesis, it is possible that elevated Smad7 levels in pten–/– cells may account for our observation of the relative inability of TGF- to promote -SMA expression further in pten–/– cells. This could be interpreted as a compensatory mechanism to prevent further -SMA induction by external stimuli. Alternatively, Smad7 increases in pten–/– cells may indicate that Smad7 is also negatively regulated by PTEN. Regardless, our data describe a novel, non–Smad-dependent pathway of -SMA expression that is tightly regulated by PTEN. As such, the data presented here support a new paradigm in the concept of fibroblast activation. Whereas previous investigation has focused on direct stimulus-induced activation, our study reveals that loss of negative regulatory elements, independent of Smad activation, can also result in myofibroblast differentiation. Thus, our data provide one possible explanation for the development of fibrotic disease in the absence of an identified inciting injury, and extend our current understanding of myofibroblast differentiation.
Fibroproliferative disorders are characterized by unrelenting, progressive ECM secretion and remodeling by -SMA–expressing myofibroblasts, which ultimately causes organ dysfunction (32). Heterogeneity in subpopulations of fibroblasts derived from patients with fibroproliferative diseases suggests selective clonal expansion (33) that, although not malignant per se, is reminiscent of malignant cells. Features of myofibroblasts in fibroproliferative disorders include a relative resistance to apoptosis (34), increased migration/invasion of tissue (7), and increased proliferation (35)—all characteristics of malignant cells. It has been well established that PTEN negatively regulates cell survival (9) and fibroblast proliferation (36). We have previously shown that PTEN is suppressed in lung fibroblasts isolated from patients with IPF, which may account for increased migratory/invasive behavior (7). We therefore propose that phenotypic behavior of myofibroblasts in fibroproliferative disorders is similar to that of malignant cells in cancers in which PTEN is mutated, deleted, or otherwise inactivated. Thus, it is likely that other features of myofibroblasts not evaluated here, such as contractility or secretion of other ECM proteins, may be orchestrated through the activity of PTEN. Further investigation into this area will likely shed more light on the spectrum of phenotypic behaviors modulated by PTEN.
To assess whether PTEN could be involved in the regulation of myofibroblast differentiation, we employed complementary techniques to inhibit or overexpress PTEN. Using pten–/– cells, we found a marked increase in -SMA expression, proliferation, and collagen secretion compared with wild-type cells. Interestingly, a clear difference was observed in the magnitude of effect among -SMA expression, proliferation, and collagen production in pten–/– cells. Whereas -SMA expression was 3-times greater, proliferation was 60-times greater in pten–/– cells compared with wild-type cells. The reason for this is not immediately apparent, but likely reflects the preferential impact of PTEN on intracellular signaling pathways that regulate proliferation compared with those that regulate protein synthesis.
Confirmatory experiments with PTEN inhibition using bpV(pic) in wild-type cells resulted in similar phenotypic findings. At high doses, bisperoxyvanadium compounds are potent, nonspecific phosphatase inhibitors (37). However, we chose to use bpV(pic) at a dose of 200 nM, a dose that inhibits PTEN but not PTP-1B or PTP- (21); thus, our findings of increased myofibroblast differentiation are likely due to specific PTEN inhibition rather than nonspecific phosphatase inhibition. Hence, our data are consistent with our hypothesis that PTEN negatively regulates the myofibroblast phenotype. It is important to note that our studies demonstrate a correlation between decreased PTEN activity and myofibroblast differentiation. Whether this is due to increased numbers of myofibroblasts (an effect of proliferation) or increased amounts of -SMA and collagen per cell (an effect of protein synthesis) is not yet clear. Data presented in Figures 2B and 8B would lend support to the former possibility, but the contribution of increased -SMA and collagen produced per cell cannot be discounted.
To verify that PTEN plays an active role in suppressing myofibroblast differentiation, we used adenoviral infection strategies. These experiments revealed that in pten–/– cells, reconstitution of PTEN resulted in inhibition of the myofibroblast phenotype. Furthermore, in wild-type cells stimulated with TGF-, overexpression of PTEN prevented the expected increase in -SMA, whereas empty virus had no such effect. Importantly, under both experimental conditions, -SMA expression was not completely abrogated. This is not unexpected. Indeed, we observed a slight increase in -SMA transcription and protein in pten–/– cells treated with TGF- (Figure 5), substantiating our hypothesis that a separate, PTEN-independent pathway also promotes -SMA expression. Given our findings that loss of PTEN induces -SMA in a Smad-independent fashion, it is possible that the further increase in -SMA in pten–/– cells after TGF- reflects the contribution of the Smad pathway. Alternatively, small increases in -SMA expression seen after TGF- in pten–/– cells may simply reflect a near-maximal basal expression and/or transcription of -SMA beyond which further increases are unattainable.
Phosphoinositol-3-phosphates generated by the activity of PI3Ks are the primary physiologic target of PTEN, although FAK and Src homology 2–containing protein have been shown to be dephosphorylated as well. One of the best-studied actions of the PI3K product phosphatidylinositol-3,4,5-trisphosphate is to recruit Akt to the plasma membrane through binding to its SH3 domain, thereby facilitating the phosphorylation and activation of Akt. As such, it is conceivable that -SMA expression might be induced as a result of PI3K-mediated Akt activity, and our data bolster this hypothesis. It is intriguing to note that induction of -SMA expression after PTEN inhibition was also abrogated using PP2, a tyrosine kinase inhibitor specific for FAK and the Src-family kinases. Previous data have shown that FAK activity is necessary for -SMA expression in fibroblasts (38), which is consistent with our findings. However, we cannot exclude the possibility that inhibition of Src kinases also plays a role. Indeed, Src kinases have been shown to phosphorylate PTEN, although the effects of tyrosine phosphorylation on PTEN activity are contradictory (39, 40). The ability of PTEN to inhibit -SMA expression via antagonizing either the PI3K or FAK/Src pathways (or both) is not surprising, and demonstrates the multiple means by which cellular phenotypes are regulated.
Our previous studies have shown a stable suppression of PTEN at both the gene and the protein level in primary lung fibroblasts isolated from patients with pulmonary fibrosis, at least through 5 to 7 passages ex vivo (7). Given the heterogeneity of fibroblast cultures derived from primary tissues, it was unclear whether cultured fibroblasts expressing low levels of PTEN were actually representative of myofibroblasts within fibroblastic foci. Immunohistochemical analysis now conclusively demonstrates that suppression of PTEN in fibroblasts is a marker of a myofibroblast phenotype, as demonstrated by colocalization of -SMA in vivo. To address whether similar findings were observed in a murine model of pulmonary fibrosis, we used bpV(pic) to inhibit PTEN activity. As predicted by our hypothesis, this had the effect of worsening fibrosis and increasing numbers of -SMA–expressing cells.
It is unclear how stable suppression of pten in fibrotic-lung fibroblasts occurs. TGF- is a potent inhibitor of pten (12), and is found in abundance in fibrotic tissue (41); yet we observed no difference in -SMA expression in our studies using neutralizing antibody to TGF-, suggesting that this is an unlikely mechanism for prolonged pten suppression in our model system. Other investigations have revealed that length of the 5'-untranslated region may regulate the constitutive expression of pten (42), as can the tumor suppressor p53 (43), and the transcription factors nuclear factor-B and early growth response–1 (44, 45). Furthermore, recent studies have shown that epigenetic modifications of pten may result in inactivation of the gene in a number of cancers, including endometrial (46) and breast (47). Currently, it is unclear how pten may be stably inhibited in mesenchymal cells; nonetheless, studies designed to address these questions will clearly be of importance.
In conclusion, we have established that the tumor suppressor PTEN is a central negative regulator of myofibroblast differentiation. Furthermore, we have shown that this in vitro mechanism has in vivo relevance to human fibroproliferative disease and experimental pulmonary fibrosis. Finally, we have demonstrated that strategies designed to enhance PTEN expression or activity within cells can inhibit further fibroblast proliferation and collagen secretion, and may be a viable approach to the treatment of these diseases. Indeed, we have recently demonstrated that prostaglandin E2 inhibits fibroblast migration via augmenting PTEN activity (48), thus highlighting the feasibility of this concept. Future work will be required to more precisely define the regulation of PTEN in fibroblasts.
Acknowledgments
The authors thank Dr. David Y. Mason (Hematology Department, John Radcliffe Hospital, Oxford, UK) for technical assistance with triple fluorescence immunohistochemistry. The authors are similarly indebted to Dr. Bethany B. Moore (University of Michigan) for assistance with the 3H-thymidine incorporation assay, and to Dr. Douglas Arenberg for critical review of the manuscript.
FOOTNOTES
Supported by National Institutes of Health grants HL070990 (to E.S.W.) and HL52285 (to S.H.P.) and Specialized Center of Research (SCOR) grant HL56402 (to C.M.H., K.R.F., F.J.M., and G.B.T.).
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200507-1058OC on September 22, 2005
Conflict of Interest Statement: E.S.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.G.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. B.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.H.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. V.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.W.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.R.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.J.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.D.K. has a patent for PTEN gene delivery that is pending. G.B.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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University Health Network, Ontario Cancer Institute, University of Toronto, Toronto, Ontario, Canada
Division of Cardiology, Duke University Medical Center, Durham, North Carolina
ABSTRACT
Rationale: Myofibroblasts are primary effector cells in idiopathic pulmonary fibrosis (IPF). Defining mechanisms of myofibroblast differentiation may be critical to the development of novel therapeutic agents.
Objective: To show that myofibroblast differentiation is regulated by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) activity in vivo, and to identify a potential mechanism by which this occurs.
Methods: We used tissue sections of surgical lung biopsies from patients with IPF to localize expression of PTEN and –smooth muscle actin (-SMA). We used cell culture of pten–/– and wild-type fibroblasts, as well as adenoviral strategies and pharmacologic inhibitors, to determine the mechanism by which PTEN inhibits -SMA, fibroblast proliferation, and collagen production.
Results: In human lung specimens of IPF, myofibroblasts within fibroblastic foci demonstrated diminished PTEN expression. Furthermore, inhibition of PTEN in mice worsened bleomycin-induced fibrosis. In pten–/– fibroblasts, and in normal fibroblasts in which PTEN was inhibited, -SMA, proliferation, and collagen production was upregulated. Addition of transforming growth factor- to wild-type cells, but not pten–/– cells, resulted in increased -SMA expression in a time-dependent fashion. In pten–/– cells, reconstitution of PTEN decreased -SMA expression, proliferation, and collagen production, whereas overexpression of PTEN in wild-type cells inhibited transforming growth factor-–induced myofibroblast differentiation. It was observed that both the protein and lipid phosphatase actions of PTEN were capable of modulating the myofibroblast phenotype.
Conclusions: The results indicate that in IPF, myofibroblasts have diminished PTEN expression. Inhibition of PTEN in vivo promotes fibrosis, and PTEN inhibits myofibroblast differentiation in vitro.
Key Words: fibrosis myofibroblast phosphatase PTEN smooth muscle actin
–Smooth muscle actin (-SMA) expression in fibroblasts, increased proliferative capacity, and increased generation and secretion of the extracellular matrix (ECM) proteins collagen and fibronectin are key hallmarks of myofibroblast differentiation in fibrotic disorders (1, 2). In idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia (UIP), persistence of myofibroblasts is believed to contribute to the development of fibrosis. It has been established that myofibroblast differentiation occurs through combinatorial signals involving transforming growth factor-1 (TGF-) and integrin signaling (3). Conversely, mechanisms that inhibit or reverse myofibroblast differentiation are less well defined. Currently, no effective therapies exist to quell ongoing fibrosis in IPF/UIP. Thus, further investigation into the mechanisms that regulate myofibroblast differentiation may yield better insights into potential therapeutic options for patients with IPF/UIP.
The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a dual-specificity protein and lipid phosphatase that uses phosphatidylinositol-3,4,5-trisphosphate as a physiologic substrate (4). Altered PTEN protein expression or activity has increasingly been implicated in the pathogenesis of nonmalignant diseases characterized by tissue destruction and remodeling, such as rheumatoid arthritis (5), asthma (6), and pulmonary fibrosis (7), thereby highlighting the importance of PTEN in physiologic regulation of nontransformed cellular phenotypes. Through its lipid phosphatase activity, PTEN inhibits cell migration (8), promotes cellular apoptosis (9), and inhibits cell growth (10). In addition, PTEN possesses protein phosphatase activity, and can act through the dephosphorylation of focal adhesion kinase (FAK) and Src homology 2–containing protein (11). Interestingly, pten expression is markedly decreased by TGF- (12). We have previously observed diminished PTEN expression in fibroblasts isolated from lungs of patients with pulmonary fibrosis (7), and have hypothesized that this may account for the increased migratory capacity of these cells (7, 13).
Here, we show that in lung biopsy samples of patients with IPF/UIP, only fibroblasts that demonstrate a distinct inhibition or loss of PTEN correlate with the expression of -SMA. We further show that pharmacologic inhibition of PTEN induces lung fibrosis in mice. Importantly, we show that inhibition of PTEN activity is both necessary and sufficient to induce myofibroblast differentiation. Finally, we demonstrate the novel finding that PTEN overexpression suppresses -SMA expression, proliferation, and collagen production in myofibroblasts, a process that can occur via either lipid or protein phosphatase activity. Our data suggest that inhibition of PTEN expression in fibroblasts may contribute to the pathogenesis of fibrotic lung disease. Some of the results of this study have been previously reported in the form of an abstract (14).
METHODS
See online supplement for additional details.
Cell Culture and Reagents
C57Bl/6 embryonic mouse fibroblasts and National Institutes of Health 3T3 murine fibroblasts were from the American Type Culture Collection (Rockville, MD). Embryonic mouse fibroblasts lacking both pten alleles (pten–/–) were described previously (9). The FAK/Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), and its inactive control, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3), were from EMD Biosciences (San Diego, CA). The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 was from Cell Signaling Technologies (Beverly, MA). Porcine TGF-1 was from R&D Systems (Minneapolis, MN). Bisperoxo(pyridine-2-carboxyl)oxovanadate (bpV[pic]) was from EMD Biosciences.
Antibodies
Antibodies to phospho-Smad2, total Smad2, and Smad7 were from Cell Signaling Technologies. -SMA antibody (clone 1A4) was from Dako Corporation (Carpinteria, CA). -Tubulin antibody was from Upstate (Waltham, MA). Anti-human PTEN (clone 6H2.1) was from Cascade Biosciences (Winchester, MA). Neutralizing antibody against TGF- 1, 2, 3 was from R&D Systems.
Intratracheal Bleomycin Administration
Animals were anesthetized with pentobarbital. The trachea was exposed, and sterile bleomycin sulfate (0.05 U/mouse) or sterile phosphate-buffered saline (PBS) alone (50 μl) was delivered via a 26-gauge needle inserted directly into the trachea.
Collagen Measurements
Total lung collagen measurements were determined 21 d after bleomycin instillation. Total lung collagen and secreted collagen from cultured cells were determined using the Sircol collagen dye binding assay (Accurate Chemical Corporation, Westbury, NY) as previously described (15) following the manufacturer's instructions.
Adenovirus Construction
Adenovirus containing cDNA constructs encoding wild-type PTEN (Ad-PTEN) or the empty virus control (Ad-EV) were prepared as previously described (16).
Immunofluorescent Staining
Cells affixed to glass slides were permeabilized, blocked, and incubated with anti–-SMA antibodies (1:1,000) for 1 h. Cells were washed and then incubated with fluorescein isothiocyanate (FITC)–conjugated secondary antibody for 1 h in the dark. Cells were again washed, mounted with 4',6-diamidino-2-phenylindole–containing mounting media (Vector Labs, Burlingame, CA), and viewed under an epifluorescent microscope (Nikon Eclipse 50i; Nikon Inc., Melville, NY).
Immunohistochemistry
Immunohistochemistry was performed on 3- to 5-μm sections of formalin-fixed, paraffin-embedded surgical lung biopsy specimens obtained from patients undergoing surgical lung biopsy for diagnosis of idiopathic interstitial pneumonia. Prior written informed consent was obtained from all subjects in accordance with the University of Michigan Institutional Review Board. Rehydrated tissue sections were incubated with primary antibody, followed by biotinylated secondary antibody, and stained using the Vectastain ABC Universal kit (Vector Labs). Color development was achieved using 3,3'-diaminobenzidine, and sections were counterstained with hematoxylin. For immunofluorescent staining, the method of Mason and colleagues (17) was followed.
Fibroblast Proliferation Assay
Fibroblasts were plated in 96-well plates and serum-starved for 24 h. Media were changed, including indicated reagents, and cells cultured for 24 h. 3H-thymidine was added for the last 16 h of culture, and radioactive counts per minute were assessed using a scintillation counter.
Semiquantitative Real-Time Reverse Transcriptase–Polymerase Chain Reaction
Semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) used primers and probes for -SMA as previously described (7, 18), and glyceraldehyde phosphate dehydrogenase was used to normalize results. Relative gene expression was calculated using the comparative cycle–threshold method (19).
Western Blot Analysis
Western blot analysis under reducing conditions was performed on equal concentrations of protein from whole-cell lysates and immunoprecipitated proteins, as previously described (7).
Statistical Analyses
Statistical analyses were performed using GraphPad InStat 3.05 (San Diego, CA). Differences between groups were evaluated using Student's t test. For multiple comparisons, one-way analysis of variance with Bonferroni's post-test analysis was used. Data were considered significant at a p value less than 0.05. Results were plotted using GraphPad Prism 3.02. Densitometry of visualized bands on Western blot was performed using Image J software (version 1.31; National Institutes of Health, Bethesda, MD).
RESULTS
Decreased PTEN Expression in Fibroblasts of Fibrotic Lesions Correlates with Increased -SMA Expression
We have previously shown that PTEN expression is decreased in lung fibroblasts from patients with fibroproliferative disease compared with normal lung fibroblasts (7). To determine whether decreased PTEN expression was a general feature of lung fibroblasts or was localized to -SMA–expressing myofibroblasts, we performed immunohistochemical analysis of surgical lung biopsy specimens from 10 patients with UIP, the histologic pattern associated with IPF (20). Consecutive sections were stained for -SMA and PTEN and compared. A representative sample of fibroblastic foci, shown in Figure 1A, demonstrates that -SMA expression is observed in spindle-shaped fibroblasts where PTEN staining is diminished or lost, thus indicating that -SMA and PTEN expression may be inversely related (Figure 1A). Similar findings were observed in all other cases evaluated (data not shown). To confirm that PTEN was not being expressed in the same cell where -SMA was being expressed, we employed triple immunofluorescent staining (17) on specimens from 10 patients with pulmonary fibrosis. Figure 1B shows a separate section from the same patient in Figure 1A stained for -SMA (Cy3, red) and PTEN (FITC, green). We observed that Cy3-positive myofibroblasts do not demonstrate significant FITC staining. 4',6-Diamidino-2-phenylindole (blue) staining identifies nuclei. Similar results were observed in all other samples (data not shown).
Inhibition of PTEN Activity in Fibroblasts Results in Myofibroblast Differentiation
To determine whether a cause-and-effect relationship exists between PTEN inhibition and myofibroblast differentiation, we initiated our studies by examining fibroblasts isolated from the embryos of pten-deficient transgenic mice (pten–/–) compared with wild-type murine embryonic fibroblasts. These cells were defined as fibroblasts based on morphology under a light microscope, as well as by the expression of fibroblast-specific protein-1 and vimentin, and have been previously characterized (9). To determine -SMA expression, cultured pten–/– and wild-type fibroblasts were assessed by immunoblotting. We observed that -SMA was expressed in both pten–/– and wild-type fibroblasts under serum-free, quiescent conditions, although to a significantly greater degree in pten–/– fibroblasts (p < 0.0001). To ensure equal protein loading, membranes were stripped and reprobed for -tubulin (Figure 2A). Immunofluorescent staining for -SMA in serum-deprived wild-type and pten–/– cells confirmed this increase in -SMA, and demonstrated that all pten–/– cells expressed -SMA (Figure 2B). To investigate whether inhibition of PTEN activity is sufficient to promote -SMA gene and protein expression, PTEN activity was inhibited in wild-type fibroblasts by the PTEN inhibitor dipotassium bpV(pic) (21). Treatment of cells with bpV(pic) does not alter protein levels of PTEN (21), but inhibits PTEN activity, as demonstrated by increasing levels of phosphorylated Akt at serine-473 (21). When we inhibited PTEN activity by treating wild-type cells with 200 nM bpV(pic) for 24 h, we observed a roughly fourfold increase in -SMA expression. This increase in -SMA was accompanied by a threefold increase in levels of phosphorylated Akt at serine-473 (Figure 2C).
We next investigated the possibility that PTEN affected other myofibroblast behaviors, namely proliferation and collagen secretion. To achieve this, pten–/– and wild-type cells were assessed for basal proliferation rates and collagen secretion. Using a 3H-thymidine incorporation assay, we determined that pten–/– fibroblasts proliferated roughly 60 times faster than wild-type cells (Figure 3A). Similarly, cultures of pten–/– and wild-type cells were assessed for collagen production. Whole-cell lysates (to evaluate cell-associated collagen) and culture supernatants (to evaluate secreted collagen) were assessed for collagens I–V by Sircol assay. Although we did not observe significant amounts of soluble collagens in supernatants (not shown), we did observe significant elevations in cell- and matrix-associated collagens (Figure 3B, left panel).
Overexpression of PTEN Inhibits Myofibroblast Differentiation
To show that PTEN could inhibit myofibroblast differentiation, we next infected pten–/– cells with an adenoviral construct encoding either full-length, functional Ad-PTEN, or Ad-EV, and assessed for -SMA expression, proliferation, and collagen production. We observed a resultant down-regulation of proliferation (Figure 3A, right panel), collagen production (Figure 3B, right panel), and -SMA expression (Figure 3C) in cells infected with Ad-PTEN as compared with Ad-EV. Collectively, these data indicate that suppression of PTEN is sufficient to promote myofibroblast differentiation, and that reconstitution of PTEN into pten–/– cells will reverse the phenotype.
Increased -SMA Expression in pten–/– Fibroblasts Is Not Dependent on TGF- Signaling
TGF- is a multifunctional cytokine that is a potent inducer of myofibroblast differentiation (22) and repressor of pten transcription (12). It has been well documented that autocrine release of TGF- accounts, at least in part, for ongoing ECM secretion and myofibroblast differentiation in numerous model systems (23–26). Given that pten–/– cells possess a predominant myofibroblast phenotype, it was important to determine whether this was mediated by autocrine signaling by endogenous TGF-. To address this, wild-type and pten–/– cells were cultured until subconfluent and extensively washed. Cells were then serum-starved and treated for 24 h with either control antibody or an antibody to TGF- that is capable of neutralizing the activity of the three isoforms of TGF-. Cells were then lysed and assayed for -SMA expression by Western blot. As shown in Figure 4A, pten–/– cells expressed higher amounts of -SMA than wild-types after 24 h of serum starvation (compare lanes 1 and 4, Figure 4A), confirming our initial observation. After the addition of neutralizing antibody to TGF- or control antibody for 24 h, there was no decrease in -SMA expression in wild-type or pten–/– cells (Figure 4A), suggesting that autocrine TGF- is not a significant contributor to the constitutive myofibroblast phenotype seen in pten–/– cells.
TGF- signaling predominantly occurs through the Smad pathway, where receptor–ligand interaction results in rapid phosphorylation and nuclear translocation of Smad2 and Smad3 (27, 28). Our data indicate that autocrine TGF- signaling did not play a significant role in -SMA expression in pten–/– fibroblasts. However, it was necessary to exclude the possibility that constitutive Smad2 or Smad3 activity accounted for the differences in -SMA expression between pten–/– and wild-type fibroblasts. To do this, pten–/– and wild-type fibroblasts were plated until subconfluent, serum-starved overnight, and treated with exogenous TGF- (2 ng/ml) for 1 h. Cells were then lysed and assayed for Smad2 and Smad3 phosphorylation by Western blot. After serum deprivation, no phosphorylation of Smad2 could be observed in either wild-type or pten–/– cells (Figure 4B). However, treatment of both cells with TGF- for 1 h resulted in robust Smad2 phosphorylation (Figure 4B, top panel). Blots were stripped and probed for total Smad2 to ensure equal loading. Similar results were obtained for Smad3 (data not shown). This finding implies that basal Smad2 and Smad3 phosphorylation in pten–/– fibroblasts does not account for the increased constitutive -SMA expression observed, but that both cells are equally capable of responding to an exogenous TGF- challenge. Furthermore, these data suggest that surface TGF- receptor expression and activity is equivalent between the two cell lines.
Regulation of Smad2/3 nuclear translocation occurs in part via the activity of Smad7. To determine whether dysregulation of Smad signaling could account for the constitutive myofibroblast phenotype observed in pten–/– cells, we assessed the levels of Smad7 in pten–/– and wild-type cells by Western blot. Unexpectedly, we observed that Smad7 levels were increased in pten–/– cells compared with wild-type cells (Figure 4B, bottom panel). This interesting observation suggests that another possible action of PTEN is to inhibit Smad7 expression. Because Smad7 is a regulatory Smad, our data would appear to support the theory that PTEN inhibition or loss in nonmalignant cells disrupts normal cellular homeostatic mechanisms, promoting aberrant behaviors.
TGF- Markedly Induces -SMA Expression in Wild-Type Fibroblasts, but Only Minimal Further Increases in pten–/– Fibroblasts
Our data indicate that pten–/– fibroblasts express -SMA independent of TGF- stimulation, but that these cells are capable of responding to exogenous TGF- through the Smad signaling pathway. Smad proteins have been shown to mediate TGF-–induced -SMA expression (29); we therefore asked whether the addition of TGF- to pten–/– fibroblasts would further increase -SMA expression. To test this, wild-type and pten–/– fibroblasts were growth-arrested for 24 h in serum-free media, washed with PBS, and then cultured in serum-free media in the absence or presence of TGF- (2 ng/ml) for 24 h. Cell lysates from two separately grown cultures of each cell type were harvested and assessed by Western blot for -SMA expression. Figure 5A shows that wild-type fibroblasts significantly increased -SMA expression 24 h after TGF- exposure (p < 0.02), whereas -SMA expression in pten–/– fibroblasts was not substantially altered. This finding appears to be in agreement with our data demonstrating increased levels of Smad7 in pten–/– cells.
Because -SMA is elevated constitutively in pten–/– fibroblasts as compared with wild-type fibroblasts, we next assessed whether this was due to increased gene expression. After isolating RNA from quiescent, serum-deprived pten–/– and wild-type fibroblasts, we used semiquantitative RT-PCR for -SMA. We found that -SMA gene expression was 1.8-fold greater in pten–/– cells compared with wild-type cells (p < 0.0001; Figure 5B), which correlated with increased -SMA promoter activity (data not shown). In aggregate, these data establish that loss of PTEN expression resulted in increased -SMA gene transcription and protein expression. Moreover, these findings imply that -SMA expression induced by inhibition of PTEN occurs independent of Smad signaling.
Inhibition of PTEN Activity Is Necessary for TGF-–induced -SMA Expression
Because our data demonstrate that inhibition of PTEN activity was sufficient to induce -SMA expression, we questioned whether it was also necessary to promote -SMA expression in fibroblasts after TGF- stimulation. To test this, we induced myofibroblast differentiation in normal fibroblasts by treating with TGF- (2 ng/ml) for 24 h while concomitantly infecting with Ad-PTEN or Ad-EV. Cell lysates were collected and evaluated by Western blot for -SMA. As shown in Figure 6, TGF-–induced -SMA expression in fibroblasts was attenuated in the presence of Ad-PTEN, but not in the presence of Ad-EV. Together, these data indicate that suppression of PTEN expression is both necessary and sufficient for -SMA expression.
-SMA Inhibition by PTEN Can Be Accomplished through Both Lipid- and Protein-Phosphatase Activity
Because PTEN is a dual-specificity phosphatase, we next sought to determine whether the lipid- or protein-phosphatase activity was responsible for -SMA regulation. Thus, serum-starved wild-type fibroblasts were induced to express -SMA, either through TGF- treatment (2 ng/ml) or bpV(pic) treatment (200 nM), in the presence or absence of the FAK/Src-specific inhibitor PP2 (or its negative control, PP3; 10 μM), or the PI3K-specific inhibitors LY294002 (50 μM) or wortmannin (50 nM), for 24 h. Untreated serum-free cells were assessed as a negative control. Whole-cell lysates were prepared and proteins separated by electrophoresis. Immunoblotting for -SMA revealed the expected increase in -SMA expression after treatment with TGF-. Consistent with our previous findings, bpV(pic) also induced -SMA expression, although to a lesser extent than TGF- (Figure 7). Importantly, we observed that, in the presence of PP2, but not PP3, TGF-–induced and bpV(pic)-induced -SMA expression was attenuated, indicating that FAK and/or Src phosphorylation is necessary for -SMA expression and that PTEN is capable of dephosphorylating one or both of these proteins (Figure 7), consistent with previous data (30). Similarly, we observed that, in the presence of LY294002 or wortmannin, TGF-–induced and bpV(pic)-induced -SMA expression was completely inhibited, providing evidence that unopposed PI3K activity after PTEN inhibition also contributes to -SMA expression (Figure 7). Together, these data signify that PTEN is capable, through multiple signaling pathways, of inhibiting -SMA expression during myofibroblast differentiation.
PTEN Inhibition Worsens Experimental Pulmonary Fibrosis and Is Associated with Greater Numbers of -SMA–expressing Cells
Our aggregate data indicate that inhibition of PTEN is both necessary and sufficient to induce myofibroblast differentiation in vitro, a process that may also occur in vivo in UIP/IPF. To determine whether inhibition of PTEN would have similar effects in experimental pulmonary fibrosis, we used a murine bleomycin model. On Day 14 after intratracheal injection of bleomycin, a time point corresponding to resolution of the inflammatory phase and progression of the fibrotic phase, animals were treated with daily intraperitoneal injections of bpV(pic) (5 mM diluted in PBS) (31), or PBS as a control. Mice were killed 21 d after bleomycin and lungs were assessed for total collagen and presence of -SMA–expressing cells. Bleomycin-treated animals demonstrated a significant increase in total lung collagen compared with control animals (Figure 8A). As predicted, bleomycin-exposed animals treated with bpV(pic) had substantially more lung collagen when compared with both control and bleomycin-exposed animals (Figure 8A). Histologically, we found that both collagen deposition (as evidenced by trichrome staining) and -SMA expression (as evidenced by immunostaining) paralleled the total lung collagen findings (Figure 8B), indicating that PTEN inhibition in vivo results in increased -SMA–expressing cells and worse fibrosis.
DISCUSSION
In this article, we describe the novel finding that PTEN negatively regulates myofibroblast differentiation in fibroblasts, and that inhibition of PTEN allows for increased myofibroblast differentiation both in vitro and in vivo. We base this conclusion on our results showing that both pten–/– fibroblasts and fibroblasts in which PTEN is inhibited demonstrate increased expression of -SMA, increased proliferation, and increased production of collagen. In addition, we show that PTEN suppression is necessary for TGF-–mediated myofibroblast differentiation, and is sufficient to induce myofibroblast differentiation in wild-type cells. Importantly, we confirm the in vivo relevance of the association between PTEN and myofibroblast differentiation both in human IPF/UIP and in bleomycin-induced murine lung fibrosis, thereby identifying PTEN as a physiologic regulator of the myofibroblast phenotype. Mechanistically, our data suggest that suppression of PTEN promotes -SMA expression via unopposed signaling through the PI3K and/or FAK/Src pathways, resulting in increased -SMA gene expression and transcription.
Smad2/3 activation and nuclear localization following TGF- exposure results in myofibroblast differentiation and -SMA transcription (22, 28). Inhibition of Smad7, which binds and prevents nuclear translocation of the Smad2/3 complex, might also allow for increased -SMA transcription after TGF- stimulation. Somewhat unexpectedly, we observed that Smad7 levels were increased in pten–/– cells compared with wild-type cells. Although this may appear antithetical to our hypothesis, it is possible that elevated Smad7 levels in pten–/– cells may account for our observation of the relative inability of TGF- to promote -SMA expression further in pten–/– cells. This could be interpreted as a compensatory mechanism to prevent further -SMA induction by external stimuli. Alternatively, Smad7 increases in pten–/– cells may indicate that Smad7 is also negatively regulated by PTEN. Regardless, our data describe a novel, non–Smad-dependent pathway of -SMA expression that is tightly regulated by PTEN. As such, the data presented here support a new paradigm in the concept of fibroblast activation. Whereas previous investigation has focused on direct stimulus-induced activation, our study reveals that loss of negative regulatory elements, independent of Smad activation, can also result in myofibroblast differentiation. Thus, our data provide one possible explanation for the development of fibrotic disease in the absence of an identified inciting injury, and extend our current understanding of myofibroblast differentiation.
Fibroproliferative disorders are characterized by unrelenting, progressive ECM secretion and remodeling by -SMA–expressing myofibroblasts, which ultimately causes organ dysfunction (32). Heterogeneity in subpopulations of fibroblasts derived from patients with fibroproliferative diseases suggests selective clonal expansion (33) that, although not malignant per se, is reminiscent of malignant cells. Features of myofibroblasts in fibroproliferative disorders include a relative resistance to apoptosis (34), increased migration/invasion of tissue (7), and increased proliferation (35)—all characteristics of malignant cells. It has been well established that PTEN negatively regulates cell survival (9) and fibroblast proliferation (36). We have previously shown that PTEN is suppressed in lung fibroblasts isolated from patients with IPF, which may account for increased migratory/invasive behavior (7). We therefore propose that phenotypic behavior of myofibroblasts in fibroproliferative disorders is similar to that of malignant cells in cancers in which PTEN is mutated, deleted, or otherwise inactivated. Thus, it is likely that other features of myofibroblasts not evaluated here, such as contractility or secretion of other ECM proteins, may be orchestrated through the activity of PTEN. Further investigation into this area will likely shed more light on the spectrum of phenotypic behaviors modulated by PTEN.
To assess whether PTEN could be involved in the regulation of myofibroblast differentiation, we employed complementary techniques to inhibit or overexpress PTEN. Using pten–/– cells, we found a marked increase in -SMA expression, proliferation, and collagen secretion compared with wild-type cells. Interestingly, a clear difference was observed in the magnitude of effect among -SMA expression, proliferation, and collagen production in pten–/– cells. Whereas -SMA expression was 3-times greater, proliferation was 60-times greater in pten–/– cells compared with wild-type cells. The reason for this is not immediately apparent, but likely reflects the preferential impact of PTEN on intracellular signaling pathways that regulate proliferation compared with those that regulate protein synthesis.
Confirmatory experiments with PTEN inhibition using bpV(pic) in wild-type cells resulted in similar phenotypic findings. At high doses, bisperoxyvanadium compounds are potent, nonspecific phosphatase inhibitors (37). However, we chose to use bpV(pic) at a dose of 200 nM, a dose that inhibits PTEN but not PTP-1B or PTP- (21); thus, our findings of increased myofibroblast differentiation are likely due to specific PTEN inhibition rather than nonspecific phosphatase inhibition. Hence, our data are consistent with our hypothesis that PTEN negatively regulates the myofibroblast phenotype. It is important to note that our studies demonstrate a correlation between decreased PTEN activity and myofibroblast differentiation. Whether this is due to increased numbers of myofibroblasts (an effect of proliferation) or increased amounts of -SMA and collagen per cell (an effect of protein synthesis) is not yet clear. Data presented in Figures 2B and 8B would lend support to the former possibility, but the contribution of increased -SMA and collagen produced per cell cannot be discounted.
To verify that PTEN plays an active role in suppressing myofibroblast differentiation, we used adenoviral infection strategies. These experiments revealed that in pten–/– cells, reconstitution of PTEN resulted in inhibition of the myofibroblast phenotype. Furthermore, in wild-type cells stimulated with TGF-, overexpression of PTEN prevented the expected increase in -SMA, whereas empty virus had no such effect. Importantly, under both experimental conditions, -SMA expression was not completely abrogated. This is not unexpected. Indeed, we observed a slight increase in -SMA transcription and protein in pten–/– cells treated with TGF- (Figure 5), substantiating our hypothesis that a separate, PTEN-independent pathway also promotes -SMA expression. Given our findings that loss of PTEN induces -SMA in a Smad-independent fashion, it is possible that the further increase in -SMA in pten–/– cells after TGF- reflects the contribution of the Smad pathway. Alternatively, small increases in -SMA expression seen after TGF- in pten–/– cells may simply reflect a near-maximal basal expression and/or transcription of -SMA beyond which further increases are unattainable.
Phosphoinositol-3-phosphates generated by the activity of PI3Ks are the primary physiologic target of PTEN, although FAK and Src homology 2–containing protein have been shown to be dephosphorylated as well. One of the best-studied actions of the PI3K product phosphatidylinositol-3,4,5-trisphosphate is to recruit Akt to the plasma membrane through binding to its SH3 domain, thereby facilitating the phosphorylation and activation of Akt. As such, it is conceivable that -SMA expression might be induced as a result of PI3K-mediated Akt activity, and our data bolster this hypothesis. It is intriguing to note that induction of -SMA expression after PTEN inhibition was also abrogated using PP2, a tyrosine kinase inhibitor specific for FAK and the Src-family kinases. Previous data have shown that FAK activity is necessary for -SMA expression in fibroblasts (38), which is consistent with our findings. However, we cannot exclude the possibility that inhibition of Src kinases also plays a role. Indeed, Src kinases have been shown to phosphorylate PTEN, although the effects of tyrosine phosphorylation on PTEN activity are contradictory (39, 40). The ability of PTEN to inhibit -SMA expression via antagonizing either the PI3K or FAK/Src pathways (or both) is not surprising, and demonstrates the multiple means by which cellular phenotypes are regulated.
Our previous studies have shown a stable suppression of PTEN at both the gene and the protein level in primary lung fibroblasts isolated from patients with pulmonary fibrosis, at least through 5 to 7 passages ex vivo (7). Given the heterogeneity of fibroblast cultures derived from primary tissues, it was unclear whether cultured fibroblasts expressing low levels of PTEN were actually representative of myofibroblasts within fibroblastic foci. Immunohistochemical analysis now conclusively demonstrates that suppression of PTEN in fibroblasts is a marker of a myofibroblast phenotype, as demonstrated by colocalization of -SMA in vivo. To address whether similar findings were observed in a murine model of pulmonary fibrosis, we used bpV(pic) to inhibit PTEN activity. As predicted by our hypothesis, this had the effect of worsening fibrosis and increasing numbers of -SMA–expressing cells.
It is unclear how stable suppression of pten in fibrotic-lung fibroblasts occurs. TGF- is a potent inhibitor of pten (12), and is found in abundance in fibrotic tissue (41); yet we observed no difference in -SMA expression in our studies using neutralizing antibody to TGF-, suggesting that this is an unlikely mechanism for prolonged pten suppression in our model system. Other investigations have revealed that length of the 5'-untranslated region may regulate the constitutive expression of pten (42), as can the tumor suppressor p53 (43), and the transcription factors nuclear factor-B and early growth response–1 (44, 45). Furthermore, recent studies have shown that epigenetic modifications of pten may result in inactivation of the gene in a number of cancers, including endometrial (46) and breast (47). Currently, it is unclear how pten may be stably inhibited in mesenchymal cells; nonetheless, studies designed to address these questions will clearly be of importance.
In conclusion, we have established that the tumor suppressor PTEN is a central negative regulator of myofibroblast differentiation. Furthermore, we have shown that this in vitro mechanism has in vivo relevance to human fibroproliferative disease and experimental pulmonary fibrosis. Finally, we have demonstrated that strategies designed to enhance PTEN expression or activity within cells can inhibit further fibroblast proliferation and collagen secretion, and may be a viable approach to the treatment of these diseases. Indeed, we have recently demonstrated that prostaglandin E2 inhibits fibroblast migration via augmenting PTEN activity (48), thus highlighting the feasibility of this concept. Future work will be required to more precisely define the regulation of PTEN in fibroblasts.
Acknowledgments
The authors thank Dr. David Y. Mason (Hematology Department, John Radcliffe Hospital, Oxford, UK) for technical assistance with triple fluorescence immunohistochemistry. The authors are similarly indebted to Dr. Bethany B. Moore (University of Michigan) for assistance with the 3H-thymidine incorporation assay, and to Dr. Douglas Arenberg for critical review of the manuscript.
FOOTNOTES
Supported by National Institutes of Health grants HL070990 (to E.S.W.) and HL52285 (to S.H.P.) and Specialized Center of Research (SCOR) grant HL56402 (to C.M.H., K.R.F., F.J.M., and G.B.T.).
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200507-1058OC on September 22, 2005
Conflict of Interest Statement: E.S.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.G.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. B.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.H.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. V.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.W.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.R.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.J.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.D.K. has a patent for PTEN gene delivery that is pending. G.B.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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