Byung-Chul Kim and Heung Tae Kim have contributed equally.
Fibroblasts from chronic wounds show altered TGF-β-signaling and decreased TGF-β Type II Receptor expression†
Article first published online: 31 MAR 2003
Copyright © 2003 Wiley-Liss, Inc.
Journal of Cellular Physiology
Volume 195, Issue 3, pages 331–336, June 2003
How to Cite
Kim, B.-C., Kim, H. T., Park, S. H., Cha, J.-S., Yufit, T., Kim, S.-J. and Falanga, V. (2003), Fibroblasts from chronic wounds show altered TGF-β-signaling and decreased TGF-β Type II Receptor expression. J. Cell. Physiol., 195: 331–336. doi: 10.1002/jcp.10301
- Issue published online: 14 APR 2003
- Article first published online: 31 MAR 2003
- Manuscript Accepted: 31 JAN 2003
- Manuscript Received: 27 JAN 2003
- NIH. Grant Numbers: AR42936, AR46557
- Wound Biotechnology Foundation
Chronic wounds are characterized by failure to heal in a defined time frame. However, the pathogenic steps leading from the etiological factors to failure to heal are unknown. Recently, increasing evidence suggests that resident cells in chronic wounds display a number of critical abnormalities, including senescence and unresponsiveness to the stimulatory action of transforming growth factor-β1 (TGF-β1). In this study, we have determined some of the mechanisms that might be responsible for unresponsiveness to TGF-β1. Using Northern analysis and affinity labeling, we show that venous ulcer fibroblasts have decreased TGF-β Type II receptor expression. This finding is not the result of genetic mutation, as shown by experiments with Type II receptor satellite instability. Decreased Type II receptor expression was accompanied by failure of ulcer fibroblasts to phosphorylate Smad 2, Smad 3, and p42/44 mitogen activating protein kinase (MAPK), and was associated with a slower proliferative rate in response to TGF-β1. We conclude that venous ulcer fibroblasts show decreased Type II receptor expression and display abnormalities in the downstream signaling pathway involving MAPK and the early Smad pathway. These findings suggest ways to address and treat the abnormal cellular phenotype of cells in chronic wounds. © 2003 Wiley-Liss, Inc.
Chronic wounds represent a difficult clinical problem, with considerable morbidity and substantial socio-economic costs. It is expected that the incidence and prevalence of chronic wounds will rise due to an increasingly elderly population with compromised cardiovascular status (Phillips et al., 2000). Therefore, a greater sense of urgency is developing for increasing the effectiveness of our clinical approach to chronic wounds. Recently, a number of advances have been made in how these wounds are cared for. Due in part to increased emphasis on evidence-based medicine, there has been greater standardization in treatment strategies and in what constitutes effective conventional therapy (Falanga et al., 1999; Falanga, 2000). Also, a number of new therapies, including the use of recombinant growth factors (Steed, 1995; Robson et al., 1998, 2001; Smiell et al., 1999) and bioengineered skin, (Gentzkow et al., 1996; Bowering, 1998; Falanga et al., 1998; Falanga and Sabolinski, 1999) have been tested successfully in clinical trials. However, in spite of this substantial progress in how chronic wounds are treated, there remains a great need for accelerating their healing and for preventing recurrences. Also, many chronic wounds heal extremely slowly or simply do not heal in spite of all appropriate therapies (Phillips et al., 2000; Falanga, 2001).
Further progress in the way we approach chronic wounds may require that we identify with greater accuracy the precise mechanisms underlying their pathogenesis and failure to heal. Indeed, except for the obvious etiological factors involved in the development of chronic wounds, i.e., venous or arterial insufficiency, pressure injury, etc., little is known of the steps leading to failure of tissue integrity and inability to heal in a timely fashion. In the case of venous ulcers, a number of hypotheses have been proposed. Some of these hypotheses have emphasized failure of the fibrinolytic system and the formation of pericapillary fibrin cuffs (Browse and Burnand, 1982; Van de Scheur and Falanga, 1997), while others have addressed the possibility of trapping of growth factors (Falanga and Eaglstein, 1993) and breakdown of extracellular matrix and cytokines by excessive amounts of tissue metalloproteinases (Trengove et al., 1999; Lobmann et al., 2002). More recently, however, a novel line of investigation has been centered on the hypothesis that chronic wounds fail to heal because their resident cells are senescent and/or have become unresponsive to the action of growth factors. There is increasing evidence in support of this hypothesis, much of it developed in the context of diabetic and venous ulcers. Fibroblasts cultured from diabetic ulcers show decreased mitogenic potential and differential responses to certain cytokines, either along or in combination (Loots et al., 1999; Loot et al., 2002). It has been shown that dermal fibroblasts cultured from venous ulcers are senescent (Stanley and Osler, 2001), show decreased proliferative potential (Stanley et al., 1997), and are unresponsive to stimulation by such critical growth factors as transforming growth factor-β1 (TGF-β1) (Hasan et al., 1997) and platelet-derived growth factor BB (PDGF-BB) (Agren et al., 1999). The reason for this unresponsiveness to growth factors is unclear, but it has been proposed that receptor expression may be at fault. In this study, we tested the hypothesis that venous ulcer fibroblasts have decreased receptor expression for TGF-β1. We show that, indeed, Type II receptor expression is markedly decreased in venous ulcer fibroblasts compared to normal control cells. Moreover, we provide evidence that the downregulation of Type II receptor expression is associated with failure in specific signal transduction pathways. We propose that this approach of identifying specific and detailed pathogenic abnormalities will ultimately lead to better treatments for chronic wounds.
PATIENTS AND METHODS
Patients with venous ulcers were enrolled in this study after signing an informed consent approved by the Institutional Review Board of Roger Williams Medical Center. Each patient was diagnosed as having venous ulcers on the basis of the following clinical and laboratory criteria: (1) a leg ulcer between the malleoli and the mid-calf; (2) presence of lipodermatosclerosis; (3) palpable pulses and an ankle brachial index (ABI) of 0.8 or greater; (4) shortened venous refilling time and evidence of venous incompetence by color duplex scanning. All the patients had venous ulcers that had been present for more than 6 months and which had not responded to conventional therapy with standard compression bandaging. At time of enrollment, the patients were being treated with a foam dressing and four-layer elastic compression bandages. Exclusion criteria included previous treatment with topical growth factors and any form of bioengineered skin, heavy exudate (requiring dressing changes more often than twice a week), histological signs of malignancy, and clinical findings of infection.
Biopsies for tissue culture
Excisional biopsies were performed from both the ulcers and the patient's own ipsilateral thigh (antero-medial aspect). For the thigh biopsy, a 6-mm punch biopsy was first taken on day 0, and this acute wound was then excised on day 3, at which time a biopsy was also taken of the patient's ulcer's edge. These two specimens, from the acute wound on the thigh and from the ulcer, were placed in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), plus penicillin and streptomycin. Dermal fibroblasts were cultured from both specimens and expanded in vitro.
Rabbit polyclonal anti-phospho-Smad 3 and anti-phospho-Smad 2 antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal anti-hemagglutinin (HA) epitope was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). DMEM, FBS, trypsin-EDTA, and penicillin–streptomycin were purchased from Life Technologies (Rockville, MD). Human TGF-β1 was a human recombinant form (R&D Systems, Inc., Minneapolis, MN).
Dermal fibroblasts were cultured from the biopsy specimens as previously described (Takagi et al., 1995) and were used in their first five in vitro passages. Cultures were established and expanded in T-75 flasks (Costar Co., Cambridge, MA) with DMEM and 10% FBS (GIBCO Laboratories, Grand Island, NY). Maintenance culture medium consisted of DMEM plus 10% FBS and antibiotics. Cell numbers were measured with a hemacytometer. Fibroblasts were grown and kept in standard conditions of 37°C in 5% CO2, 95% air.
For these experiments, fibroblasts were seeded at 50,000–100,000 per well in 6-well plates in DMEM plus 10% FBS. Cell counts were performed using a hemacytometer. Counts were done in quadruplicate for each experimental group. In these experiments, normal or ulcer fibroblasts were first seeded at a density of 5 × 104–1 × 105 per well in 6-well culture plates and allowed to attach for 24 h in DMEM plus 10% FBS. The following day, the medium was changed to DMEM plus 10% FBS with or without TGF-β1 (10 ng/ml). Cell counts were performed next day with a hemacytometer.
Amplification of TβRII
A 73-bp region of the TβRII gene (nucleotides 665–737) was amplified from 50 to 200 ng of genomic DNA with approximately 10 ng of 32P-end-labeled TA10-F1 primer (5′-CTTTATTCTGGAAGATGCTGC-3′) and 150 ng of reversed primer TA10-R1 (5′-GAAGAAAGTCTCACCAGG-3′) using 30 cycles 95°C for 30 sec, 55°C for 1 min, and 70°C for 1 min. The PCR products were separated by electrophoresis at 52°C on 6% polyacrylamide/7M urea gel and visualized by autoradiography. All assays were repeated in triplicate. Amplification, cloning, and sequencing of TβRII cDNA were performed as described (Markowitz et al., 1995).
SDS–PAGE and immunoblot analysis
Protein samples were heated at 95°C for 5 min and analyzed by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE). SDS–PAGE were performed on 12% acrylamide gels, followed by transfer to polyvinylidine difluoride (PVDF) membranes for 2 h at 100 V. The membrane was blocked for 2 h with 5% (w/v) nonfat dried milk. Blots were incubated overnight with primary antibody (1:100 dilution for Smad 3) and then for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody, prior to development using an enhanced chemiluminescence kit (Pierce Scientific, Clifton, NJ).
Whole-cell extracts were obtained in a 1% Triton X-100 lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM sodium chloride, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM β-glycerophosphate, 1 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Western blotting was performed using anti-phospho-p42/44 mitogen activating protein kinase (MAPK) (Thr202/Tyr204) (E10; Cell Signaling Technology, Inc., Beverly, MA), anti-phospho-SAPK/JNK (Thr183/Tyr185) (Cell Signaling Technology, Inc.), anti-phospho-p38 MAP Kinase (Thr180/Tyr182) (Cell Signaling Technology, Inc.), anti-phospho-Smad 3 and anti-phospho-Smad 2 (Upstate Biotechnology, Inc.). Protein samples were heated at 95°C for 5 min and analyzed by SDS–12% PAGE.
MAP kinase assay
Phosphorylated forms of ERKs, p38 MAP kinase and JNK were detected by Western blot analysis using phospho-specific antibodies. In brief, cells were harvested and resuspended in lysis buffer composed of 50 mM Tris, pH 8.0, 150 mM sodium chloride, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Cells were incubated on ice for 5 min and sonicated four times for 5 sec each on ice. After normalizing protein concentrations, samples were electrophoresed in 12% SDS–PAGE and transferred onto nitrocellulose membranes. Analyses were performed using Phospho-plus MAPK Antibody Kit. Phospho-plus p38 MAPK Antibody Kit and Phospho-plus SAPK/JNK (Thr183/Tyr185) Antibody Kit (New England Biolabs, Herts, UK) were used following protocols provided by the manufacturer.
RNA extraction and Northern analysis
Total cellular RNA from cells was isolated by extraction in guanidium isothiocyanate, using previously described methods (Chomczynski and Sacchi, 1987). The RNA was then separated for Northern analysis on 1% agarose gel containing 5% formaldehyde, and transferred to a nylon membrane (Onstic, Westborough, MA) in a gradient of 20× to 10× SSC. The following cDNA probes were used: a 1.5 kb EcoRI fragment cDNA from the original clone Hf677 for the α1(I) procollagen chain (Chu et al., 1985), and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was from the American Tissue Culture Collection (Rockville, MD). Human TGF-β Type II receptor cDNA was kindly provided by Dr. R. Weinberg (Massachusetts Institute of Technology, Cambridge, MA). Probes were labeled with 32P by random priming and used for Northern blot analysis. For RNA electrophoresis, 10 μg of total RNA was loaded into each lane, as measured by an absorbance at 260 nm. Confirmation of uniformity of RNA loading was obtained by staining the nylon blots with methylene blue. Northern hybridization was performed at 42°C in a solution containing 50% formamide, 6× SSC, 5× Denhardt's reagent, 0.5% SDS, salmon sperm DNA (Sigma Co., St. Louis, MO), and the labeled cDNA probe (2 × 108 cpm/mg). The blots were washed twice in 1× SSC with 0.1% SDS for 30 min at room temperature, followed by a wash at 68°C in 0.2× SSC with 0.1% SDS for 20 min. Autoradiography was generally carried out overnight at −70°C. Subsequently, the density of each RNA band was measured by 1D-densitometry scan analysis.
TGF-β1 cross-linking assay
Cells were plated at a density of 1 × 106 cells/well in 6-well dishes. Receptor–ligand binding was performed with 100 pM 125I-labeled TGF-β1 in the presence or absence of 100-fold molar excess of unlabeled TGF-β1. Bound proteins were cross-linked using 300 μM disuccinimidyl suberate, solubilized, and separated with gel electrophoresis (Cai et al., 1996).
We have previously shown that venous ulcer fibroblasts are unresponsive to a range of concentrations of TGF-β1, as measured by collagenous protein synthesis and Northern analysis for α1(I) procollagen (Hasan et al., 1997). This is confirmed in the representative Northern analysis for α1(I) procollagen shown in Figure 1A, which indicates a lack of responsiveness to TGF-β1 in two different strains of ulcer fibroblasts. Interestingly, and consistent with previously reported findings, baseline steady state levels of α1(I) procollagen were not lower than normal in ulcer fibroblasts. Proliferative response to TGF-β1 is also absent in venous ulcer fibroblasts, as shown in the representative example in Figure 1B. The lack of proliferative response to TGF-β1 in venous ulcer fibroblasts was observed in both 10% or 0.5% FBS. Thus, chronic wound fibroblasts are unable to respond properly to the action of TGF-β1, both in terms of synthetic activity and proliferation. Based on this lack of responsiveness, we have hypothesized that ulcer fibroblasts have decreased expression of TGF-β Type II receptors. Figure 2A shows a representative experiment where, by Northern analysis, the expression of TGF-β Type II receptors is markedly diminished in four sets of fibroblasts from ulcers and normal skin. To determine whether the expression of actual receptor protein is downregulated, we performed affinity labeling using 125I-TGF-β1. As shown by the representative experiment in Figure 2B, the Type II receptor protein is easily detectable in normal but not in ulcer fibroblasts. As expected, the addition of cold TGF-β1 in these experiments eliminates the band observed for normal fibroblasts in lane 1 of Figure 2B. Since the expression of Type II receptor was diminished in ulcer fibroblasts, as shown by Northern analysis and affinity labeling, the next question was whether a receptor mutation may have occurred in these fibroblast populations from venous ulcers. We determined this by testing for microsatellite instability of the Type II receptor (Markowitz et al., 1995). In the experiment shown in Figure 2C, where SNU-16 cells were used as control, lanes 4 and 5 show identical bands for normal and ulcer fibroblasts, respectively. There was no evidence of extra bands indicating receptor mutation. We next asked the question of whether downregulation of Type II receptor expression in venous ulcer fibroblasts has functional consequences for their TGF-β1 signal transduction. For this purpose, we first focused on the TGF-β1-induced phosphorylation of Smad 2 and Smad 3, critical proteins involved in the early phase of signal transduction after TGF-β1 receptor binding (Massague, 1998; Attisano and Wrana, 2002). Figure 3 indicates that the phosphorylation of both Smad 3 and Smad 2 in response to TGF-β1 is decreased more than threefold in ulcer fibroblasts. We then determined whether other TGF-β1 transduction pathways may be affected as well (Atfi et al., 1997; Moustakas et al., 2001; Yu et al., 2002). To do so, we first determined which pathways are operative in control fibroblasts in response to TGF-β1. As shown in Figure 4 with two representative strains of control fibroblasts, we tested for the phosphorylated forms of ERKs, p38 MAP kinase and JNK, which were sought by immunoblot analysis using phospho-specific antibodies. Normal control fibroblasts were cultured for the indicated times with TGF-β1 (10 ng/ml). In response to TGF-β1, no difference was found in the phosphorylation of p38 and JNK/SAPK. However, by 30 min, more than a fourfold increase was detected in the phosphorylation of the p42/44 MAPK. After this initial increase, phosphorylation of this protein decreased gradually over time, and was not detectable by 24 h. Since these results indicate that the p42/44 MAPK is appropriately phosphorylated in normal fibroblasts in response to TGF-β1, we therefore tested for the phosphorylation of this signaling molecule in ulcer fibroblasts. Indeed, as shown in Figure 5, appropriate phosphorylation of this kinase is specifically impaired in venous ulcer fibroblasts. These results indicate that, due to decreased TGF-β Type II receptor binding or, possibly, independent of that abnormality, signal transduction with the MAP kinase pathway is disrupted.
In this study, we show that fibroblasts cultured from a prototypic chronic wound, such as venous ulcers, have decreased expression of the TGF-β1 Type II receptor, as measured by both Northern analysis and affinity labeling, and that this is not due to a receptor mutation. The functional significance of these findings is evidenced by decreased phosphorylation of Smad 2, Smad 3, and p42/44 MAPK in response to TGF-β1. Taken together with previous data showing unresponsiveness of ulcer fibroblasts to TGF-β1 (Hasan et al., 1997), these findings strongly suggest that functionally important signal transduction abnormalities occur in venous ulcer fibroblasts.
Members of the TGF-β1 superfamily are a class of multifunctional peptides, which influence cellular development, and a variety of other crucial cellular functions (Massague, 1998). In recent years, the pathways involved in receptor binding of TGF-β1 and the subsequent steps in signal transduction are becoming increasingly recognized. TGF-β1 peptides bind to a family of transmembrane serine/threonine kinase receptors known as Type I and Type II. Upon ligand binding of the peptide to the Type II receptor, there is dimerization of both receptor types. Receptor Type II then phosphorylates the Type I receptor, which becomes activated and can propagate the signal (Massague, 1998). In the last few years, an entirely new class of signal transduction was found to be operative in the mechanism of action in TGF-βs. In addition to the conventional ERK pathway, the TGF-β receptor complex uses SMAD proteins, which comprise three classes of SMADs: pathway-restricted (SMAD 1, 2, 3, 5, and 8), common-mediator (SMAD 4), and those thought to be inhibitory (SMAD 6 and 7) (Attisano and Wrana, 2002). Once phosphorylated by the TGF-β receptor, the SMAD 2 protein is joined by the SMAD 4 protein. The SMAD 2 and SMAD 4 proteins then migrate across the nuclear membrane and activate the transcription machinery. This more unique pathway in TGF-β signaling operates alongside the ERK cascade (Atfi et al., 1997). For this reason, we evaluated both of these pathways in venous ulcer fibroblasts. We found that the decreased expression of TGF-β1 Type II receptors was accompanied by abnormalities in the downstream signaling pathway. Our results suggest that a decrease in Type II receptor expression may indeed have important consequences for certain signaling pathways. It has often been speculated that, given the relatively large number of receptors on the cell surface, a decrease in receptor number does not have important functional consequences. Our results indicate that this may not be the case. At this point, we cannot exclude that more than one abnormality, possibly several independent ones, exist in wound fibroblasts, and that these include receptor expression as well as phosphorylation of key signaling proteins. However, although speculative, it is possible that tissue integrity and, ultimately, ulcer recurrence might be due to decreased expression of TGF-β receptors and its consequences on signal transduction. Indeed, ulcer recurrence is possibly the greatest problem in these patients, and ways to address that would be highly beneficial.
It is not difficult to envision how phenotypically abnormal cells may accumulate in a chronic wound. Particularly in venous ulcers, failure to heal is accompanied by an active inflammatory process, with evidence of substantial proliferation of fibroblasts and keratinocytes. It might well be that these resident cells reach the limit of their proliferative lifespan and undergo changes similar to those described in this study. There is already evidence that fibroblasts from venous ulcers are indeed senescent (Stanley et al., 1997); more recently, a correlation was found between the in vitro decreased proliferative potential of venous ulcer fibroblasts and the failure of wounds to heal (Stanley and Osler, 2001). Evidence from studies of fibroblasts from diabetic ulcers have shown similar abnormalities (Loots et al., 1999; Loot et al., 2002). The microenvironment of the wound is likely to be another important determinant of cellular selection and phenotypic expression. Of particular relevance to our results is the observation that hypoxia, which is an established feature of chronic wounds, including venous ulcers, will downregulate the expression of the Type II receptor (Falanga et al., 1994).
Over the past several years, topically applied growth factors, including TGF-βs, have been tested extensively for their effectiveness in the treatment of chronic wounds (Steed, 1995; Smiell et al., 1999; Robson et al., 2001). The results have been promising but of rather modest magnitude. It has been speculated that topical application of these agents, especially in the harsh microenvironment of a chronic wound, is fraught with problems. There are questions about the appropriate topical dose and the delivery vehicle, penetration of the polypeptide into the ulcer bed and its breakdown by tissue metalloproteinases (Robson et al., 1998; Trengove et al., 1999; Lobmann et al., 2002). Also, single growth factors may not be enough (Robson et al., 2000; Smith et al., 2000a,b). However, another and perhaps more fundamental explanation for the relative lack of success of growth factors in the treatment of chronic wounds may reside in the phenotypic abnormalities of wound cells and their relative unresponsiveness to stimulatory signals.
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