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(Investigative Ophthalmology and Visual Science. 2002;43:41-46.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

TGFß1-Dependent Contraction of Fibroblasts Is Mediated by the PDGF{alpha} Receptor

Yasushi Ikuno and Andrius Kazlauskas

From the Department of Ophthalmology, The Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Contraction of fibroblasts and the resultant tractional force is a contributing factor to fibrotic diseases of the eye, such as proliferative vitreoretinopathy (PVR). Transforming growth factor (TGF)-ß is abundant in the eye, and is one of the growth factors thought to contribute to the development of PVR. A second is platelet-derived growth factor (PDGF). In the current study, the relationship between TGFß1 and PDGF was investigated at the level of cellular contraction.

METHODS. To study cellular contraction, an in vitro type I collagen gel contraction assay was used with a panel of fibroblast lines that expressed the PDGF{alpha} receptor ({alpha}PDGFR) or PDGFß receptor (ßPDGFR) or no PDGFRs. The agents tested included rabbit vitreous, TGFß1, and PDGF.

RESULTS. Vitreous promoted cellular contraction, and approximately 60% of this activity was eliminated by preincubation of the vitreous with neutralizing TGFß antibodies. The {alpha}PDGFR-expressing cells responded better than cells expressing the ßPDGFR or no PDGFRs. Both of the PDGFR-expressing cell lines contracted in response to PDGF, whereas the best response to TGFß1 was observed with cells expressing the {alpha}PDGFR. Finally, TGFß1 promoted the tyrosine phosphorylation of both of the PDGFRs, and the {alpha}PDGFR was more strongly phosphorylated than the ßPDGFR.

CONCLUSIONS. The results show that the vitreous promotes cellular contraction, that TGFß is the major factor responsible, and that at least a portion of the TGFß-dependent contraction proceeds through the {alpha}PDGFR—that is, indirectly. Therefore, the {alpha}PDGFR is responsible for mediating cellular contraction of multiple growth factors: TGFß and members of the PDGF family.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Proliferative vitreoretinopathy (PVR) is characterized by the formation of a membrane in front of the retina that is composed of extracellular matrix (ECM) and cells. Contraction of the epiretinal membrane results in tractional retinal detachment (TRD).1 2 Once the retina loses its functional contact with the underlying layer of retinal pigment epithelial (RPE) cells, it is irreversibly damaged by apoptosis of the photoreceptors.3 4 PVR occurs in up to 10% of patients who undergo surgery to reattach the retina.5 Anatomic correction is achieved in 60% to 80% of the patients who require additional surgery.1

Contraction of the epiretinal membrane is likely to involve integrin-dependent interactions between the cells and the ECM. Evidence supporting this idea includes the findings that administration of the Arg-Gly-Asp (RGD)-containing peptide, which interferes with cellular attachment to the ECM, prevents PVR.6 7 8 Furthermore, the typical PVR membrane is mainly composed of collagens I, II, and III.9 Such ECM components bind to cells through integrins, such as {alpha}2ß1, that are expressed by mesenchymal cells and induced by PDGF and other growth factors.10 11 12 Thus, understanding cellular contraction is likely to provide insight into the pathogenesis of PVR and also to identify new approaches for treatment.

As mentioned, growth factors appear to be important contributors to PVR. Transforming growth factor (TGF)-ß and platelet-derived growth factor (PDGF) have been most strongly implicated, and interleukin (IL)-6, fibroblast growth factor, and hepatocyte growth factor may also contribute.13 14 15 16 17 18 19 20 TGFß is present in the vitreous under normal conditions and is upregulated in PVR.16 PDGF is present in the vitreous of patients with PVR,15 and PDGF receptors (PDGFRs) are detected in PVR membranes excised from humans.20 Furthermore, cells unable to respond to PDGF induce PVR poorly in a rabbit model of the disease, and re-expression of the PDGF{alpha} receptor ({alpha}PDGFR) markedly elevates the PVR potential of these cells.13 Similarly, inhibiting the endogenous PDGFR by expressing a dominant negative PDGFR mutant suppresses the PVR potential of rabbit conjunctival fibroblasts.17

The receptors for PDGF and TGFß are from fundamentally different classes of growth factor receptors. The receptor for TGFß is a ubiquitously expressed transmembrane protein that encodes a serine-threonine kinase within the cytoplasmic domain. Binding of TGFß to its receptor results in the phosphorylation of the Smad family of transcription factors.21 As a result of phosphorylation, the Smads move from the cytoplasm into the nucleus, where they regulate gene expression.21 In contrast, the receptors for PDGF are tyrosine kinase–encoding receptors and trigger cellular responses, primarily through signaling cascades that involve SH2 domain–containing proteins.22 23 TGFß is able to indirectly activate the PDGFR by promoting the synthesis and secretion of PDGF.24 25 26

In this study we focused on the relationship between TGFß1 and PDGF in cell contraction. We report that TGFß1 is the major agent in the vitreous responsible for initiating cell contraction, and this response appears to proceed through the {alpha}PDGFR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
F cells are a simian virus (SV)40–immortalized line of mouse embryo fibroblasts derived from mice nullizygous for both the {alpha}- and ßPDGFRs. They were generously provided by Michelle Tallquist and Philippe Soriano (Fred Hutchinson Cancer Research Center, Seattle, WA). The F{alpha} and Fß cells express only one of the PDGFRs, the {alpha}- or ßPDGFR, respectively.13 The FCX2 cells are F cells infected with an empty expression vector. The generation, characterization, and maintenance of these cell lines have been described.13 Normal growth conditions were Dulbecco’s modified Eagle’s medium (DMEM) with high glucose and 10% fetal bovine serum (FBS). The serum concentration was reduced to 1% when the cells were serum starved.

Immunoprecipitation and Immunoblot Analysis
Cells were grown to 80% confluence, incubated in DMEM containing 1% FBS for 20 hours, and exposed at 37°C for 5 minutes to 50 ng/mL PDGF-BB or left unstimulated. After treatment, the cells were washed twice with H/S (20 mM HEPES [pH 7.4] and 150 mM NaCl) and then lysed in EB (10 mM Tris-HCl [pH 7.4], 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 1% Triton X-100, 0.1% BSA, 20 µg/mL aprotinin, 2 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride [PMSF]). Lysates were centrifuged for 15 minutes at 13,000g, the pellet was discarded, and the soluble fraction was used as the total cell lysate. The protein concentration was measured with a protein assay kit (Pierce, Rockford, IL) according to the manufacturer’s instructions.

Receptors were immunoprecipitated from the soluble fraction with the 27P or 30A antibody.27 28 Both are rabbit polyclonal antibodies that recognize the carboxyl terminus of the {alpha}- or ßPDGFR, respectively. They were made against a glutathione-S- transferase fusion protein encoding the entire C terminus of the human {alpha}PDGFR (amino acids 951-1089) or ßPDGFR (amino acids 958-1106). The antibodies are monospecific—that is, the PDGFR is the predominant species recognized in total cell lysates. Immune complexes were bound to formalin-fixed membranes of Staphylococcus aureus, spun through an EB sucrose gradient, and washed twice with EB and then with PAN (10 mM piperazine-N,N'-bis (2-ethanesulfonic acid) [PIPES; pH 7.0] 100 mM NaCl, and 1% aprotinin) with 0.5% Nonidet P (NP)-40, and finally again with PAN.

Receptor immunoprecipitates from 1.0 x 106 cells were resolved in 7.5% SDS-PAGE gel under reducing conditions. Proteins were transferred onto membranes (Immobilon; Millipore, Bedford, MA), and the membranes were blocked (10 mM Tris-HCl [pH 7.5], 1.5 M Tris base, 150 mM NaCl, 10 mg/mL BSA, 10 mg/mL ovalbumin, and 0.05% Tween 20; Block) for anti-phosphotyrosine blot analysis. The membranes were blocked (10 mM Tris-HCl [pH 7.5], 1.5 M Tris base, 150 mM NaCl, 10 mg/mL nonfat dry milk, and 0.05% Tween 20; Blotto) for other antibodies. Membranes were incubated with primary antibodies for 1 hour at room temperature and washed five times (150 mM NaCl, 10 mM Tris-HCl [pH 7.5], and 1.5 mM Tris base; Western Rinse solution). Afterward, they were incubated with secondary antibody for 1 hour at room temperature, washed five times with Western Rinse, and visualized using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Piscataway, NJ).

Reagents and Antibodies
Recombinant human TGFß1, PDGF-BB, neutralizing anti-pan-TGFß antibody, anti-PDGF antibody, and control affinity-purified goat or rabbit IgG were purchased from R&D Systems (Minneapolis, MN). Anti-TGFß antibody neutralizes TGFß1, -ß2, -ß3, and -ß5, and the anti-PDGF antibody neutralizes PDGF-AA, -AB, and -BB.

The 27P (anti-{alpha}PDGFR), 80.8 (anti-{alpha}PDGFR), 69.3 (anti-Ras GTP activating protein; RasGAP), and 30A (anti-ßPDGFR) are rabbit crude antisera and have been characterized.27 28 29 4G10 and PY20 are mouse monoclonal anti-phosphotyrosine antibodies, purchased from Upstate Biotechnology, Inc. (Lake Placid, NY) and Transduction Laboratories (Lexington, KY), respectively. For Western blot analysis the following dilutions were used for each of the primary antibodies: anti-{alpha}PDGFR, a 1:1 mixture of the 27P and 80.8 antibodies, 1:1000; anti-ßPDGFR, 1:5000; anti-phosphotyrosine, 4G10:PY20 (1:1), 1:5000; and 69.3, 1:4000. Secondary antibodies were horseradish peroxidase–conjugated donkey anti-rabbit (catalog no. NA934; Amersham Pharmacia Biotech) or sheep anti-mouse (catalog no. NA931; Amersham Pharmacia Biotech) whole antibodies diluted 1:5000.

Collagen I Contraction Assay
The contraction assay was as previously described,30 with slight modifications. Cells were suspended in 1.5 mg/mL neutralized collagen I (Cohesion Vitrogen 100; Invitrogen, Palo Alto, CA) at a density of 106 cells/mL, and were transferred into a 24-well plate (Falcon, Franklin Lakes, NJ) that had been preincubated with a solution of phosphate-buffered saline (PBS) and 5 mg/mL BSA overnight. The gel was solidified by incubating at 37°C for 90 minutes, and then the well was flooded with DMEM and 5 mg/mL BSA, supplemented with buffer or the agent to be tested. The gels were incubated at 37°C with 5% CO2. The initial gel diameter was 15 mm. The medium was replaced every 24 hours, and the gel diameter was measured after 24, 48, and 72 hours. The extent of contraction was calculated by subtracting the diameter of the well at a given time point from the initial diameter (15 mm). Each experimental condition was assayed in triplicate, and at least three independent experiments were performed.

Rabbit Vitreous Extraction
Vitreous was collected from freshly isolated normal rabbit eyes by first removing the anterior segment (cornea, iris, and lens), and then the vitreous was squeezed out of the remaining posterior portion of the eye. The extracted vitreous was resuspended in PBS containing 5 mg/mL BSA. The samples were centrifuged at 2500g for 10 minutes at 4°C, and the resultant supernatant was aliquoted and frozen at -70°C until use. Vitreous prepared in this way could include trace amounts of retinal and/or choroidal materials.

Statistic Analysis
An unpaired t-test was performed to detect statistically significant differences between experimental conditions in the contraction assay. In all cases, P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of TGFß in Vitreous on Fibroblast Contraction
We tested the possibility that vitreous promotes cellular contraction. The vitreous was excised from healthy rabbits and added to the culture medium of F{alpha} cells seeded in collagen type I gels. The diameter of the gels was measured at the start of the experiment and 48 hours later. As shown in Figure 1A , vitreous promoted contraction in a dose-dependent manner.



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  		Figure 1. Contraction of cells triggered by vitreous was dependent on TGFß. (A) Contraction of the cells was dependent on the dose of vitreous added. Vitreous was obtained from normal rabbit eyes. DMEM supplemented with 5 mg/mL BSA and the indicated amount of vitreous was added to cultures of F{alpha} cells plated in a collagen type I gel. F{alpha} cells are mouse embryo fibroblasts that express the {alpha}PDGFR. The gel diameter was measured at the start of the experiment and after 48 hours, and the extent of contraction was calculated by subtracting the two values. The data are representative of three independent experiments. (B) TGFß was one of the biologically active agents of the vitreous. Anti-TGFß or control IgG (100 µg/mL) was added to DMEM supplemented with vitreous (20%) or TGFß1 (10 ng/mL), and the collagen gel contraction assay was performed. The data are representative data of three independent experiments. Each experimental condition was assayed in triplicate. The data are the mean ± SD. **P < 0.01, compared with the control (control IgG with 20% vitreous).

 
To identify the agents within vitreous that were responsible for this cellular response we focused on TGFß, which is present in vitreous and stimulates contraction of collagen type I gels containing fibroblast or RPE cells.31 32 When vitreous was pretreated with 100 µg/mL of neutralizing TGFß antibody, approximately 60% of the contraction activity disappeared (Fig. 1B) . This dose of neutralizing antibody was sufficient to neutralize at least 10 ng/mL TGFß1 (data not shown). In contrast, the same amount of a control IgG had no effect on contraction stimulated by the vitreous (Fig. 1B) .

Response to Vitreous of Cells That Express the {alpha}PDGFR
We have observed that cells expressing the {alpha}PDGFR induced PVR in a rabbit model of the disease better than cells that express the ßPDGFR or no PDGFRs.13 We related these PVR findings to in vitro contraction by testing the in vitro contraction response of the cell lines used in the PVR studies. As shown in Figure 2 , vitreous triggered contraction in cells expressing the {alpha}PDGFR (F{alpha}) more potently than in cells expressing the ßPDGFR (Fß) or no PDGFRs (FCX2).



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  		Figure 2. Expression of the {alpha}PDGFR potentiated vitreal-dependent contraction. The collagen gel assay was used to monitor the response of F cells devoid of PDGFRs (FCX2) or F cells expressing either the ßPDGFR (Fß) or {alpha}PDGFR (F{alpha}). DMEM containing 5 mg/mL BSA was supplemented with buffer (-) or vitreous (+) to a final concentration of 20% and added to the indicated cell lines. Contraction was scored at the 48-hour time point. F{alpha} cells responded significantly better than either FCX2 (P < 0.01) or Fß cells (P < 0.01). Fß responded slightly better than FCX2, but the difference was not significant.

 
Relationship between TGFß1 and PDGF
In light of the fact that PDGF promotes contraction of fibroblasts,33 a likely explanation for the results shown in Figure 2 is that the vitreous contains PDGF family members that activate the {alpha}PDGFR but not the ßPDGFR. However, pretreating the vitreous with a neutralizing PDGF antibody had no effect on the contractile response, even though the same dose (100 µg/mL) of this antibody completely blocked contraction induced by 10 ng/mL of PDGF-AA or -BB (data not shown).

Because TGFß was a major contributor to the contraction activity in vitreous, we tested whether contraction induced by purified TGFß1 was influenced by expression of PDGFRs. Indeed, we observed an even more pronounced dependence on expression of PDGFRs when using purified TGFß1 than with vitreous (Fig. 3D) . TGFß1 triggered robust contraction of F{alpha} cells, whereas the response was modest (Fß) or undetectable (FCX2) in the other cell lines (Fig. 3D) . All three cell types responded comparably to FBS (Fig. 3B) , indicating that all cell lines had the capacity to contract under these experimental conditions. Furthermore, both of the PDGFR-positive cell lines contracted to a comparable extent after stimulation with PDGF-BB (Fig. 3C) , indicating that each of the receptors are capable of triggering this response.



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  		Figure 3. TGFß1-dependent contraction was greatly potentiated by expression of the {alpha}PDGFR. Cells expressing no PDGFRs (FCX2), the {alpha}PDGFR (F{alpha}), or the ßPDGFR (Fß) were subjected to the collagen gel contraction assay in the presence of buffer (A), 10% FBS (B), 50 ng/mL PDGF-BB (C), or 1 ng/mL TGFß1 (D). The gel’s diameter was measured after 24, 48, or 72 hours, and the media were replaced every day. All cells responded similarly to buffer or serum, and both of the PDGFR-expressing cells contracted when PDGF was added to the media. Cells expressing the ßPDGFR or no PDGFRs responded poorly to TGFß1, whereas those expressing the {alpha}PDGFR contracted robustly. Each experimental condition was assayed in triplicate. The data are the mean ± SD. The data are representative of three independent experiments. *P < 0.05; **P < 0.01, compared with FCX2 cells; {dagger}P < 0.05, compared with the Fß cells.

 
We next investigated why TGFß1 promoted contraction of cells expressing the {alpha}PDGFR but not the ßPDGFR. This phenomenon did not appear to be due to a differential ability of the two PDGFRs to trigger cell contraction, because both the {alpha}- and ßPDGFRs drove this response when the receptors were directly activated with PDGF (Fig. 3C) . Consequently, we tested whether TGFß1 could activate either of the two PDGFRs. To this end, F{alpha} or Fß cells were stimulated with TGFß1 and then immunoprecipitated and subjected to an anti-phosphotyrosine Western blot analysis. TGFß1 triggered tyrosine phosphorylation of the {alpha}PDGFR (Fig. 4A) . By comparison to the PDGF-dependent response, TGFß1-induced tyrosine phosphorylation of the {alpha}PDGFR was much slower and less intense. This may reflect the fact that TGFß1 does not directly activate the {alpha}PDGFR, but probably functions by promoting the synthesis and secretion of PDGF.24 In contrast to the {alpha}PDGFR, the ßPDGFR was very modestly tyrosine phosphorylated in TGFß1-treated cells at any of the time points tested (Fig. 4B) .



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  		Figure 4. TGFß1 induced tyrosine phosphorylation of the PDGFRs. Cells expressing the {alpha}- or ßPDGFR were serum starved overnight and then left unstimulated (0) or stimulated with 50 ng/mL PDGF-BB or 1 ng/mL TGFß1 for the indicated times. The cells were lysed, and the {alpha}PDGFR (A) or ßPDGFR (B) was immunoprecipitated and subjected to anti-phosphotyrosine Western blot analysis (top panels). The membranes were stripped and reprobed with an anti-{alpha}PDGFR antibody (A; bottom) or an anti-ßPDGFR antibody (B; bottom). The {alpha}PDGFR was tyrosine phosphorylated in response to TGFß1 to a much greater extent than the ßPDGFR. The 210- to 140-kDa region (A) and 250- to 160-kDa region (B) of the blots are shown; bar on the right indicates the position of the 205-kDa molecular mass marker.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A novel finding in this study is that vitreous promoted cellular contraction and that TGFß was the growth factor responsible for most of the activity. Furthermore, TGFß1-induced contraction was dependent on expression of the {alpha}PDGFR. One question that arises from these experiments is which cell type(s) is the source of TGFß in vitreous? A likely candidate is the hyalocytes, because these cells reside in the cortex of the vitreous. These cells are of the monocyte-macrophage lineage and are capable of secreting TGFß.34 35 An additional question is how is the TGFß activated. TGFß is typically secreted in a latent form and must undergo activation.36 We did not intentionally activate the TGFß in the vitreous, and thus it either existed in the activated state in the vitreous of healthy rabbits or underwent activation during preparation or within the assay itself.

A second question that is brought to light by our findings is how does TGFß1 make use of the {alpha}PDGFR? The receptors for PDGF are not activated by ligands outside the PDGF family,37 38 and hence it is highly improbable that TGFß1 directly activates the receptor. A more likely scenario is that TGFß1 stimulates the synthesis and secretion of a PDGF family member. Numerous groups have shown that TGFß indeed has this capability.24 25 26 39 40 41 However, we were not able to block TGFß1-dependent contraction using neutralizing antibodies to PDGF (Ikuno and Kazlauskas, unpublished observations, 2000). A caveat of this approach is that, although the antibody used in our experiments blocked all three of the traditional PDGF isoforms (PDGF-AA, -BB, and -AB), its reactivity toward the newly discovered PDGF-CC and PDGF-DD42 43 44 isoforms is unknown. Additional studies, and most probably the development of new reagents, are needed to assess the possible role of new PDGF family members in TGFß1-dependent cellular contraction.

We also considered the possibility of a relationship between TGFß1 and the {alpha}PDGFR at the level of signal relay. In this scenario, TGFß1 would activate the {alpha}PDGFR intracellularly. We generated cells expressing an {alpha}PDGFR that is missing most of the extracellular domain and thus is unable to bind ligand. These cells failed to contract when exposed to TGFß1, and this truncated {alpha}PDGFR was not tyrosine phosphorylated in TGFß1-treated cells (Ikuno and Kazlauskas, unpublished observations, 2000). Thus, we were not able to demonstrate intracellular cross talk between TGFß1 and the {alpha}PDGFR.

One of the key events in PVR is the contraction of the epiretinal membrane and consequent retinal detachment. Our findings suggest that {alpha}PDGFR is an important contributor to this step of the disease, because this receptor is required for the contraction induced by several different growth factors. The use of cell lines that individually express the two receptor for PDGF was critical for this discovery. This is because most cell lines, including the more relevant RPE cell line, express both PDGFRs,45 and none of the PDGF ligands specifically activates the ßPDGFR.42 43 44 46 47 Consequently, it is not possible to assess the relative contribution of each of the PDGFRs in cells that have not been modified. Additional studies are needed to determine whether the {alpha}PDGFR is particularly important for PVR other animal models and in the clinical setting.

There are a number of differences between the clinical disease and the rabbit model of PVR that we have used13 17 that are relevant to the findings described herein. RPE cells, not fibroblasts, are the major cell type found in epiretinal membrane isolated from patients with PVR.1 2 Our preliminary studies indicate that cultured RPE cells contract after exposure to vitreous or TGFß1 (Ikuno and Kazlauskas, unpublished observations, 2000), and thus the RPE and fibroblasts are similar in this regard. Additional studies are needed to determine whether the {alpha}PDGFR is the primary mediator of TGFß1-dependent contraction in the RPE cells, as it is in fibroblasts.

An additional potentially critical difference between the rabbit model and human disease is the vascularity of the retina. Circulating platelets contain relatively high levels of TGFß48 and thus may serve as a source of TGFß in the vascular human retina. In contrast, the rabbit retina is avascular, and TGFß therefore does not come from this source. Given the potential involvement of TGFß in PVR, this difference in source of TGFß may influence susceptibility and/or progression of PVR.

The in vitro contraction assay may be a simple screen for identifying compounds that have the potential to inhibit fibrotic diseases such as PVR. This is because of the good correlation between in vitro contraction of cells (Figs. 2 3) and their in vivo PVR potential in a rabbit model of the disease.13 Finally, because the {alpha}PDGFR is a mediator of cellular responses of several different growth factors, it may be a particularly relevant target for strategies to prevent PVR.


   Acknowledgements
 
The authors thank the members of Kazlauskas Laboratory for constructive discussions.


   Footnotes
 
Supported by The Schepens Eye Research Institute Ocular Gene Therapy Program and National Institutes of Health Grant EY-012509 (AK).

Submitted for publication February 21, 2001; revised September 28, 2001; accepted October 2, 2001.

Commercial relationships policy: N.

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

Corresponding author: Andrius Kazlauskas, The Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114; kazlauskas{at}vision.eri.harvard.edu.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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