- Open Access
Autocrine transforming growth factor β signaling regulates extracellular signal-regulated kinase 1/2 phosphorylation via modulation of protein phosphatase 2A expression in scleroderma fibroblasts
© Samuel et al; licensee BioMed Central Ltd. 2010
- Received: 7 July 2010
- Accepted: 6 December 2010
- Published: 6 December 2010
During scleroderma (SSc) pathogenesis, fibroblasts acquire an activated phenotype characterized by enhanced production of extracellular matrix (ECM) and constitutive activation of several major signaling pathways including extracellular signal-related kinase (ERK1/2). Several studies have addressed the role of ERK1/2 in SSc fibrosis however the mechanism of its prolonged activation in SSc fibroblasts is still unknown. Protein phosphatase 2A (PP2A) is a key serine threonine phosphatase responsible for dephosphorylation of a wide array of signaling molecules. Recently published microarray data from cultured SSc fibroblasts suggests that the catalytic subunit (C-subunit) of PP2A is downregulated in SSc. In this study we examined the role and regulation of PP2A in SSc fibroblasts in the context of ERK1/2 phosphorylation and matrix production.
We show for the first time that PP2A mRNA and protein expression are significantly reduced in SSc fibroblasts and correlate with an increase in ERK1/2 phosphorylation and collagen expression. Furthermore, transforming growth factor β (TGFβ), a major profibrotic cytokine implicated in SSc fibrosis, downregulates PP2A expression in healthy fibroblasts. PP2A-specific small interfering RNA (siRNA) was utilized to confirm the role of PP2A in ERK1/2 dephosphorylation in dermal fibroblasts. Accordingly, blockade of autocrine TGFβ signaling in SSc fibroblasts using soluble recombinant TGFβ receptor II (SRII) restored PP2A levels and decreased ERK1/2 phosphorylation and collagen expression. In addition, we observed that inhibition of ERK1/2 in SSc fibroblasts increased PP2A expression suggesting that ERK1/2 phosphorylation also contributes to maintaining low levels of PP2A, leading to an even further amplification of ERK1/2 phosphorylation.
Taken together, these studies suggest that decreased PP2A levels in SSc is a result of constitutively activated autocrine TGFβ signaling and could contribute to enhanced phosphorylation of ERK1/2 and matrix production in SSc fibroblasts.
- Dermal Fibroblast
- Okadaic Acid
- Sphingosine Kinase
- PP2A Activity
- PP2A Expression
Scleroderma (SSc) is an autoimmune connective tissue disease characterized by excess production and deposition of extracellular matrix proteins leading to fibrosis of the tissue. During this process, normal fibroblasts become 'activated' and acquire a fibrotic phenotype. Transforming growth factor β (TGFβ) is a major profibrotic cytokine that plays important roles in a variety of physiological processes including cell proliferation, differentiation and survival. Although the mechanism of SSc fibrosis is not fully understood, there is strong evidence to suggest that TGFβ is central to the development and maintenance of the SSc phenotype [1–3]. Normal healthy dermal fibroblasts treated with TGFβ reproduce characteristics of SSc fibroblasts, further supporting the notion that TGFβ is a major mediator of SSc fibrosis .
During tissue injury, rapid release of TGFβ attracts inflammatory cells and fibroblasts to the site of injury, resulting in extracellular matrix production/remodeling and myofibroblast differentiation . In normal tissue, following the injury response, coordinated apoptosis of fibroblasts and myofibroblasts prevents scarring and excessive fibrosis . Published data suggests that normal and SSc dermal fibroblasts in culture secrete similar levels of TGFβ ligand [7, 8] However, there is evidence of increased TGFβ signaling in SSc fibroblasts when compared to normal fibroblasts. Several studies have shown elevated levels of TGFβ receptors in SSc fibroblasts, which contribute to an autocrine TGFβ signaling cascade that is maintained in culture even in the absence of exogenous ligand [9–11]. The chronic activation of the TGFβ pathway in SSc produces fibroblasts with constitutively activated Akt and ERK1/2 pathways that are resistant to apoptosis [12–14]. The ERK1/2 pathway regulates numerous cellular processes and more recently has also been implicated in the process of fibrosis. Several papers have reported the function of the activated ERK1/2 pathway in fibrosis. For example, it has been demonstrated that the ERK1/2 pathway is required for Smad1 phosphorylation in response to overexpression of TGFβRI and for subsequent upregulation of connective tissue growth factor (CCN2) and other profibrotic genes . Activation of mitogen-activated protein kinase kinase 1(MEK1)/ERK1/2 pathway was also shown to be a primary mechanism responsible for the TGFβ-induced upregulation of early growth response factor 1 (Egr-1) . In addition, Chen et al. recently reported that activation of the ERK1/2 pathway contributes to the enhanced fibrosis and contractile ability of scleroderma fibroblasts . The ERK1/2 pathway also induces up regulation of αvβ3 integrin, which contributes to the autocrine TGFβ signaling in scleroderma fibroblasts . However, although constitutively phosphorylated ERK1/2 may play important roles in SSc pathogenesis, the mechanism of prolonged activation of this pathway is largely unknown.
Protein phosphatase 2A (PP2A) is a member of the PPP family and one of the most abundant serine-threonine phosphatases, accounting for a substantial part of the total phosphatase activity. PP2A plays an important role in signal transduction pathways, regulation of cell cycle and transcriptional and translational regulation . PP2A has a complex structure, comprising of three subunits: the catalytic (C), regulatory (B) and structural subunit (A). The catalytic subunit (C) and structural subunit (A) have two isoforms: α and β. The regulatory subunit (B) consists of four families with many isoforms that confer specificity of location and function . The phosphatase activity of PP2A is present in the C-subunit and its effects include dephosphorylation of various transcription factors and protein kinases including MEK, ERK1/2, Akt, and sphingosine kinase (SK) [19–21]. Recently published microarray data from cultured early passage SSc fibroblasts suggests that the β isoform of the catalytic subunit of PP2A is downregulated in SSc . Based on the evidence of constitutive activation of ERK1/2 pathways in SSc fibroblasts and recent microarray data suggesting that PP2A may also be altered in SSc, we wished to further study the mechanism and significance of dysregulated PP2A in SSc fibroblasts.
TGFβ stimulates prolonged phosphorylation of ERK1/2 in dermal fibroblasts
PP2A expression is decreased upon treatment with TGFβ
Since PP2A has been previously described as a major ERK1/2 phosphatase we next sought to determine whether TGFβ could also be involved in the regulation of PP2A expression in dermal fibroblasts. Confluent dermal fibroblasts were serum starved and then treated with TGFβ for different time periods. The mRNA levels of α and β isoforms of the PP2A catalytic subunit were analyzed by quantitative reverse transcription (qRT)-PCR. As shown in Figure 1b, the mRNA levels of PP2A were decreased as early as 6 h after TGFβ addition and the lower levels persisted up to 48 h. Treatment of cells with TGFβ affected both catalytic subunit isoforms, but the β isoform showed a greater overall decrease. To further validate the effects of TGFβ on PP2A gene expression we measured the protein levels of PP2A (Figure 1c,d) after 24 h of TGFβ treatment. PP2A protein levels were decreased at 24 h, correlating with and confirming the mRNA data. These observations show that TGFβ negatively regulates PP2A expression, suggesting that PP2A may be involved in TGFβ-mediated ERK1/2 phosphorylation.
PP2A inhibition contributes to increased ERK1/2 phosphorylation
PP2A expression is decreased and correlates with increased ERK1/2 phosphorylation in SSc dermal fibroblasts
Autocrine TGFβ signaling regulates PP2A expression in SSc fibroblasts
Blockade of ERK1/2 phosphorylation increases PP2A expression in SSc fibroblasts
PP2A is a negative regulator of collagen expression
In this study we have demonstrated that TGFβ is a negative regulator of PP2A. We found decreased expression of both the α and β isoform of the catalytic subunit of PP2A after TGFβ stimulation. To our knowledge this is the first report to demonstrate that TGFβ is a negative regulator of PP2A gene expression. In accordance with previously published studies our data also confirms that TGFβ treatment activates prolonged ERK1/2 phosphorylation. This study provides new evidence for the contribution of PP2A to the pathogenesis of SSc. Analysis of fibroblasts cultured from SSc skin biopsies shows decreased protein levels of the PP2A catalytic subunit, with downregulation of both α and β isoforms at the mRNA levels, reproducing the effects of TGFβ in normal dermal fibroblasts. These data validate a previous gene array study, which showed decreased levels of the β isoform in cultured SSc fibroblasts. However, downregulation of the α isoform, which is the most abundant isoform in vivo, has not been described in SSc fibroblasts. Previous reports indicated that an autocrine TGFβ signaling pathway contributes to the SSc phenotype . We hypothesized that PP2A downregulation in SSc could be the result of constitutive TGFβ signaling. This hypothesis was supported by our data showing that recombinant soluble TGFβ receptor II, an antagonist of TGFβ signaling, was able to block the downregulation of PP2A and to reverse the constitutive phosphorylation of ERK1/2 in cultured SSc fibroblasts. This suggests that autocrine TGFβ signaling in SSc induces prolonged ERK1/2 phosphorylation, possibly via modulation of PP2A expression. Furthermore, in our study we observed that activated ERK1/2 can suppress PP2A expression in SSc fibroblasts but not in normal control fibroblasts. This suggested the presence of a self-sustained signaling loop between PP2A and ERK1/2 in SSc fibroblasts, whereby increased ERK1/2 phosphorylation in response to TGFβ downregulates PP2A expression and in turn results in a further increase in ERK1/2 phosphorylation. ERK1/2 phosphorylation has been previously implicated in fibrosis [12, 26, 27]. In this study, we observe that PP2A is also involved in regulation of collagen. The modest increase in collagen upon PP2A blockade suggests that the collagen production in SSc fibroblasts is a cumulative result of many dysregulated pathways present in SSc fibroblasts.
Reversible protein phosphorylation plays a central role in the regulation of vast majority of the biological processes. This process is tightly controlled by the protein kinases and phosphatases that together regulate the levels of cellular phosphoproteins. The balance between the activities of kinases and phosphatases is often disrupted during pathological conditions including neurodegenerative diseases and cancer [18, 28]. Persistent downregulation of PP2A in SSc fibroblasts strongly suggests that this pathway is involved is the pathogenesis of SSc. It is noteworthy that the study of Tan and colleagues , who first reported on the aberrant expression of PP2A, was performed using fibroblasts from uninvolved skin. This suggests that this defect is present in the early stages of the disease. The constitutive activation of the ERK1/2 pathway in SSc may play a critical role in the development and maintenance of fibrosis and the activated status of explanted SSc fibroblasts. In addition to its role as a major ERK1/2 phosphatase, PP2A has been also implicated in the regulation of sphingosine kinase (SK), a profibrogenic sphingolipid enzyme induced by TGFβ [20, 29]. SK catalyzes the conversion of sphingosine to sphingosine1 phosphate, which mimics some of the profibrotic effects of TGFβ [30, 31]. Additionally, SK is a major prosurvival molecule and may also indirectly contribute to fibrosis by inducing resistance to apoptosis in activated fibroblasts . Further experiments using animal models of PP2A knockout or transgenic mice would be essential to study and dissect the pathways involved in PP2A downregulation in vivo and its role in fibrosis. However there are several limitations to this approach considering the vast number of subunits and splice variants present for this molecule as well as the numerous substrates and methods of posttranslational regulation. Several experimental mouse models have been generated including the PP2AC knockout mouse and transgenic models of various other PP2A subunits . The PP2Acα knockout mouse is embryonic lethal and results in degeneration of the embryo and lack of formation of the mesoderm. Interestingly, in these embryos, the two highly homologous catalytic subunits are found in different subcellular locations, the Cα in the plasma membrane and Cβ in the cytosol, making it unlikely that Cβ can compensate for Cα in these mice . However, since these mice are embryonic lethal, a tissue-specific knockout of PP2Acα in fibroblasts would provide key insights into the role of PP2A in fibrosis.
In conclusion, this study describes a novel role for TGFβ in the regulation of PP2A gene expression. While our study focused on ERK1/2, PP2A dephosphorylates numerous signaling molecules, many of them with a potential role in fibrosis, and it is likely that such global downregulation of PP2A activity would modulate additional cellular pathways. We also show that SSc fibroblasts have decreased levels of PP2A and that this could be restored by blockade of autocrine TGFβ signaling, suggesting that negative regulation of the PP2A catalytic subunit gene expression may be a physiological mechanism by which sustained ERK1/2 phosphorylation occurs in SSc. This study highlights an unanticipated regulatory function for TGFβ in modulating PP2A activity and provides support for an essential role of PP2A in the pathogenesis of SSc. Further studies are necessary to gain insight into the role of PP2A and ERK1/2 activation in the modulation of ECM components in SSc fibroblasts.
The following antibodies were used: anti-PP2A (Upstate, Temecula, CA, USA), anti-phospho-ERK1/2, anti-ERK1/2, anti-Akt (Cell Signaling, Beverly, MA, USA), anti-phospho-Akt, Ser 473 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal β actin (Sigma Aldrich, St Louis, MO, USA), anti-type 1 collagen (Southern Biotech, Birmingham, AL, USA).
Recombinant human TGFβ1 was obtained from R&D Systems (Minneapolis, MN, USA). OA was purchased from Sigma Aldrich. Tissue culture reagents, Dulbecco's modified Eagle medium (DMEM) and 100× antibiotic antimycotic solution (penicillin streptomycin and amphotericin B) were obtained from Gibco BRL (Grand Island, NY, USA) and fetal bovine serum was purchased from HyClone (Logan, UT, USA). Enhanced chemiluminescence reagent and bovine serum albumin (BSA) protein assay reagent were obtained from Pierce (Rockford, IL, USA). TriReagent was purchased from the Molecular Research Center (Cincinnati, OH, USA). Primers were purchased from Operon (Huntsville, AL, USA). SMARTpool siRNA against PP2A C-subunit was purchased from Dharmacon RNA Technologies (Lafayette, CO, USA) and Hiperfect siRNA transfection reagent from Qiagen (Germantown, MD, USA).
Human dermal fibroblast cultures were established from biopsy specimens obtained from the dorsal forearms of SSc patients with diffuse cutaneous disease and from age, race and gender matched healthy donors, upon informed consent and in compliance with the Institutional Review Board. Dermal fibroblasts were cultured from the biopsy specimens as described previously . Normal and SSc skin fibroblasts were cultured in DMEM supplemented with 10% FBS and 1% antibiotic antimycotic solution. For experiments cells were pretreated with serum-free media for 24 h. Cells were treated with TGFβ, 5 ng/ml.
Total RNA was isolated from dermal fibroblasts using TriReagent (Molecular Research Center) according to the manufacturer's instructions. RNA (2 μg) was reverse transcribed in a 20-μl reaction using random primers and Transcriptor First Strand synthesis kit (Roche Applied Sciences Indianapolis, IN. Quantitative (q)PCR was carried out using IQ SYBR Green mixture (Bio-Rad, Hercules, CA) on an iCycler PCR machine (Bio-Rad) using 1 μl of cDNA in triplicate with β actin as the internal control. The primers used are as follows. PP2A C-subunit α isoform: forward, 5'-GCACTTGATCGCCTACAAGA-3' and reverse, 5'-GAAATATCTTGCCCAAAGGTGT-3'. PP2A C-subunit β isoform: forward, 5'-TTCTTGTAGCATTAAAGGTGCGT-3' and reverse, 5'-CATTCCCATACTTCGCAGACA-3'.
Whole cell protein extracts were prepared according to the manufacturer's recommendations (Pierce). Immunoblotting was performed as previously described .
SMARTpool siRNA directed against human PP2A catalytic subunit was purchased from Dharmacon RNA Technologies. Negative-control siRNA was purchased from Qiagen (Chatsworth, CA, USA) and Hiperfect transfection reagent (Qiagen) was used for transfection of dermal fibroblasts according to the manufacturer's recommendations.
This study was supported by NIH grant AR-44883.
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