Keloid Fibroblasts Resist Ceramide-Induced Apoptosis by Overexpression of Insulin-Like Growth Factor I Receptor

Hiroshi Ishihara, Hiroshi Yoshimoto, Masaki Fujioka, Ryuuichi Murakami, Akiyoshi Hirano, Tohru Fujii, Akira Ohtsuru,* Hiroyuki Namba,* and Shunichi Yamashita*
Department of Plastic and Reconstructive Surgery, *Department of Nature Medicine, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, Japan

Keloids are benign dermal tumors, characterized by overgrowth of lesions, invasiveness beyond the original boundary of the insult, and recurrence of lesions. The exact etiology is unknown, however. Our hypothesis is that keloids are acquired as a result of an abnormal or prolonged wound healing process, with persistent proliferation and extracellular matrix production of broblasts that should otherwise discontinue in normal wound healing. In this study, we examined the response of keloid broblasts to proapoptotic signaling. Cell-permeable ceramide, N-acetyl-D-sphingosine, induced apoptosis of dermal broblasts in a dose- and time-dependent manner, which was detected by phase contrast microscopy, uorescent microscopy, the TUNEL method, ow cytometric analysis, and WST-1 assay. In contrast, keloid broblasts resisted apoptosis induced by

N-acetyl-D-sphingosine (percent survival with 40 mM ceramide treatment for 12 h, normal versus keloid: 9.6% T 6.6% vs 66.8% T 5.5%). Western blotting analysis showed insulin-like growth factor I receptor overexpression in keloid broblasts, but not in normal broblasts. Exogenously added insulin-like growth factor I enhanced the resistance of keloid broblasts to ceramide-induced apoptosis. Wortmannin, a phosphatidylinositol 3 kinase inhibitor, suppressed the antiapoptotic action of insulin-like growth factor I in keloid broblasts. Our results suggest that keloid broblasts overexpressing insulin-like growth factor I receptor are resistant to apoptosis, thus allowing persistent proliferation and production of excessive extracellular matrix. Key words: programmed cell death/wound healing. J Invest Dermatol 115:10651071, 2000

eloids are characterized by proliferation of dermal broblasts, overproduction of extracellular matrix by dermal broblasts, invasiveness beyond the original boundary of the insult, and recurrence, and are classied as benign dermal tumors (Tredget et al, 1997). Although a benign disorder, surgical resection of keloids is contraindicated because of the likelihood of local invasion and recurrence. Accordingly various conservative therapies have been used such as steroid, tranilast, and pressure treatment, but denite and effective therapy has not yet been established. The normal wound healing process consists of three phases: inammation, proliferation, and remodeling. From the inammation phase to the proliferation phase, inammatory cells and small vessels as well as broblasts accumulate and proliferate. Fibroblasts produce extracellular matrix such as collagen and bronectin, and they form granulation tissue. In the late phase of wound healing, both cellularity and extracellular matrix decrease and the granulation tissue evolves into a mature scar. We speculated that inammation and proliferation phases are sustained in keloid
Manuscript received September 15, 1999; revised May 22, 2000; accepted for publication August 22, 2000. Reprint requests to: Dr. Hiroshi Ishihara, Department of Nature Medicine, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. Abbreviations: C2 ceramide, N-acetyl-D-sphingosine; PI, propidium iodide; PI3 kinase, phosphatidylinositol 3 kinase; TUNEL, terminaldeoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling.
0022-202X/00/$15.00

formation and keloid broblasts become more resistant to cell elimination by some unknown mechanism. In a previous study, we reported that keloids overexpress insulinlike growth factor I receptor (IGF-IR), and the IGF-I-IGF-IR signal mediates invasiveness of keloid broblasts (Yoshimoto et al, 1999). IGF-I is also a well-known major survival factor in the serum (O'Connor, 1998) and protects broblasts from apoptosis under a wide variety of conditions, including serum deprivation, Fas ligand, tumor necrosis factor a (Ortiz et al, 1997), chemotherapeutic drugs (Sell et al, 1995), and ceramide (Gerritsen et al, 1998). Ceramide is a lipid second messenger that is well known as a mediator of extracellular signals. It is formed by cell membrane sphingomyelin and inuences proapoptotic signal pathways induced by various stimuli such as Fas antigen (Tepper et al, 1995), tumor necrosis factor a (Kaipia et al, 1996), chemotherapeutic drugs (Jaffrezou et al, 1996), and radiation (Haimovitz, 1998), and can also induce apoptosis by itself. So we considered that ceramide might be one of the key mediators for apoptosis and chose it as the inducer in our experiments. In this study, we determined the expression of IGF-IR in keloid broblasts and investigated whether it inuences the response to proapoptotic signaling by N-acetyl-D-sphingosine (C2 ceramide).
MATERIALS AND METHODS Materials Keloid samples were obtained from three different Japanese patients after surgical excision. Three normal skin samples were obtained from three Japanese volunteers. Informed consent was obtained from each subject.

Copyright # 2000 by The Society for Investigative Dermatology, Inc.

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Reagents C2 ceramide and dihydro-C2-ceramide were purchased from Calbiochem (La Jolla, CA). They were dissolved in ethanol and used as 10 mM stock solution. Hoechst 33342 and propidium iodide (PI) were purchased from Sigma Chemical (St. Louis, MO). Human recombinant IGF-I was kindly provided by Fujisawa Pharmaceutical (Tokyo, Japan). Wortmannin was purchased from Wako Pure Chemical (Osaka, Japan) and dissolved in phosphate-buffered saline (PBS) as 100 mM stock solution. All other chemicals and reagents were of the highest purity available from commercial sources. Cell cultures Primary cultures of dermal broblasts were established as previously described (Arakawa et al, 1990). Explants were maintained in Dulbecco's modied Eagle's medium (DMEM) with 10% fetal bovine serum (Gibco BRL, Life Technologies), 100 U per ml penicillin, and 100 mg per ml streptomycin at 37C in a humidied incubator with 5% CO2. Fibroblasts at passages 37 were used in this study.

Western blot analysis for IGF-IR Expression of IGF-IR was also detected by western blotting. Cells were washed twice in ice-cold PBS before lysis in sodium dodecyl sulfate (SDS) sample buffer (62.5 mM TrisHCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue). The lysates were sonicated for 2 s, boiled for 5 min, and then microcentrifuged for 5 min. The protein concentration of the lysates was determined by the Bio-Rad assay. Proteins were separated on 7.5% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Hybond-p; Amersham, Arlington Heights, IL). The membranes were preincubated with a blocking buffer [1 Q Tris-buffered saline (TBS), 0.1% Tween-20, and 5% nonfat dry milk] overnight at 4C. They were then incubated with a primary antibody to IGF-IR (mouse monoclonal antibody for a subunits of human IGF-IR; Santa Cruz Biotechnology) with gentle agitation overnight at 4C. They were washed three times with TBS-T, and incubated with the secondary antibody (horseradish peroxidase conjugated goat antimouse IgG; ABC kit, Vector Laboratories). Proteins were detected by enhanced chemiluminescence (Pierce) using the protocol provided by the supplier. Induction and detection of cell death Dermal broblasts were preincubated for 48 h with serum-free medium. In the next step various concentrations of C2 ceramide were added and the broblasts were further incubated for an additional 12 h. The cells were viewed with a Nikon phase contrast microscope and then trypsinized and washed with PBS, xed with 2% glutaraldehyde, and stained with Hoechst 33342 (1 mM) and PI (1 mg per ml). Specimens were analyzed with an Olympus BH-2 uorescent microscope. Terminal-deoxynucleotidyl-transferase -mediated dUTP-biotin nick end labeling (TUNEL) method For the detection of apoptosis, the procedure is based upon the method described by Gavrieli et al (1992). To investigate the response of dermal broblasts to C2 ceramide, cultures reaching 80%90% conuence in the complete medium were starved for

Figure 1. Upregulation of IGF-I receptor expression in keloid broblasts compared with normal broblasts. Immunoblotting of IGF-IR a subunits. Total cell lysates were prepared from the three independent strains of normal and keloid broblasts, and equal amounts of proteins were subjected to 7.5% SDS-polyacrylamide gel electrophoresis followed by western blotting against IGF-IR a subunits. Lanes 13, normal broblasts; lanes 46, keloid broblasts.

Figure 2. Dose-dependent induction of cell death by C2 ceramide in normal and keloid broblasts. (a-f) Phase contrast microscopy of dermal broblasts cultured with DMEM containing various concentrations of C2 ceramide for 12 h. (a-c) Normal broblasts treated with 0 50 mM C2 ceramide; (d-f) keloid broblasts treated with 050 mM C2 ceramide; g, normal broblasts treated with ethanol only; (h) normal broblasts treated with 50 mM dihydro-C2-ceramide; (i) uorescent microscopy (Hoechst 33342) of normal broblasts cultured with DMEM containing 30 mM C2 ceramide for 12 h. Typical morphologic changes of apoptotic cells are observed among normal broblasts (arrow). Magnications: (ah) Q 40; (i) Q 200.

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Figure 3. Determination of apoptotic broblasts by the TUNEL method in vitro. (a, b) Normal broblasts treated with 30 and 50 mM C2 ceramide; (c, d) keloid broblasts treated with 30 and 50 mM C2 ceramide; (e) normal broblasts with no treatment; (f) normal broblasts treated with ethanol only; (g) normal broblasts treated with 50 mM dihydro-C2-ceramide. Typical apoptotic cells were observed in a concentration-dependent manner by C2 ceramide in normal broblasts, but only few TUNEL-positive cells were detected in keloid broblasts under the same conditions. Magnications: Q100.

48 h in serum-free DMEM. Then various concentrations of C2 ceramide were added and the cultures were further incubated for an additional 6 h. Apoptosis induction by C2 ceramide was detected in situ by the TUNEL method, using the Apoptosis in situ Detection Kit (Wako, Osaka, Japan) according to the manufacturer's protocol. Flow cytometry For ow cytometric analysis, cells (5 Q 105) were xed with 70% ethanol and washed with PBS. After preincubation with RNase A (0.1 mg per ml) at 37C, cells were stained with PI (25 mg per ml), and 0.1% bovine serum albumin was added. Fluorescence was measured by using a FACScan ow cytometer (Becton Dickinson, Mountain View, CA). Cell survival assay We also assessed cell survival by using a modied MTT assay, WST-1 assay, a colorimetric assay based on the cleavage of the tetrazolium salt WST-1 to a formazan dye by the mitochondrial dehydrogenase of viable cells. Fibroblasts suspended in DMEM containing 10% fetal bovine serum were seeded at 5 Q 103 cells per well into 96-well plates. The cells were preincubated for 24 h with serum-free medium at 37C. The medium was replaced with DMEM containing C2 ceramide (050 mM) with or without IGF-I (100 ng per ml) for 048 h at

37C. The ready-to-use WST-1 reagent was added to the cells during the last 2 h and incubated at 37C. Statistical analysis All values were expressed as mean T SD of triplicate samples and were representative of at least three experiments. Differences between groups were examined for statistical signicance using analysis of variance. A p-value less than 0.01 denoted the presence of a statistically signicant difference.

RESULTS Expression of IGF-IR on dermal broblasts In our previous study, we reported that keloids overexpress IGF-IR (Yoshimoto et al, 1999). In this study we used different strains of keloid and normal dermal broblasts, however, and therefore we rst checked the expression of IGF-IR in these cells. In western blotting, high expression of IGF-IR was detected in three keloid broblast strains (Fig 1, lanes 46) whereas only a relatively weak expression of IGFIR was present in three normal dermal broblast strains (Fig 1, lanes 13). These results conrmed our earlier ndings (Yoshimoto et al, 1999).

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Figure 4. DNA contents of normal and keloid broblasts after treatment with C2 ceramide. Cells were cultured with serum-free DMEM for 24 h and incubated with DMEM containing 25 mM C2 ceramide for 12 h. Following nuclear staining with PI, ow cytometric analysis was performed as described in Materials and Methods. (a, b) Normal broblasts; (c, d) keloid broblasts; (e, f) negative controls of normal broblasts. Note the increased population of apoptotic cells (48.61% T 8.82%) in normal broblasts after 12 h. In contrast, keloid broblasts underwent a G2 arrest and resisted apoptosis (7.76% T 3.48%).

C2-ceramide-induced apoptosis of dermal broblasts After 12 h of treatment, C2 ceramide induced a dose-dependent cell death of primary cultured human dermal broblasts (Fig 2a-c). To discriminate between apoptosis and necrosis, we examined the DNA fragmentation of C2-ceramide-treated dermal broblasts by uorescent microscopy analysis and the TUNEL method. In uorescent microscopy, typical apoptotic cells were easily observed in normal broblasts (Fig 2i), but necrotic cells were very rare. From this result, we considered that C2 ceramide mainly induced apoptosis but not necrosis in dermal broblasts within the range 1050 mM. In contrast, keloid broblasts were more resistant to C2ceramide-induced cell death compared with normal dermal broblasts and they maintained their original structure and viability (Fig 2d-f). In the TUNEL method, normal broblasts treated with C2 ceramide underwent apoptosis in a concentrationdependent manner, but only few TUNEL-positive cells were detected in keloid broblasts (Fig 3). FACScan analysis showed a high proportion of apoptotic cells among normal broblasts (48.61% T 8.82%) (Fig 4a, b). In contrast, keloid broblasts resisted apoptosis (7.76% T 3.48%) and underwent G2 arrest (Fig 4c, d). We also checked cell viability of dermal broblasts by WST-1 assay. In this assay, C2 ceramide induced loss of viability in normal dermal broblasts in a concentration- and time-dependent manner, but keloid broblasts resisted loss of viability compared with normal broblasts (Figs 5, 6).

IGF-I inhibits ceramide-induced apoptosis IGF-I is well known as a major survival factor in serum and can protect cells against apoptosis under a wide variety of conditions. In the next step, we examined the effects of IGF-I on normal and keloid broblasts treated with C2 ceramide. IGF-I (100 ng per ml) inhibited apoptosis of normal broblasts slightly, but the protective effects were more marked in keloid broblasts (Fig 7). Furthermore, the protective effect correlated with the level of IGFIR expression. Phosphatidylinositol 3 kinase (PI3-kinase) mediates the antiapoptotic signal of IGF-IR in keloid broblasts Finally, we investigated the mechanisms of antiapoptotic effects of IGF-IR in keloid broblasts. PI3-kinase and Akt are known to mediate the antiapoptotic signal of IGF-IR in broblasts (Kulik et al, 1997). In the next step, we used wortmannin as a PI3-kinase inhibitor and examined its effect on the antiapoptotic inhibitory action of IGF-I in keloid broblasts. Preincubation with 100 nM wortmannin blocked the antiapoptotic signal of IGF-IR (Fig 8). DISCUSSION Apoptosis is dened as programmed cell death. It is a genetically controlled process to eliminate cells in response to a variety of stimuli and provides protection against cancer, viral infections, cytotoxic drugs, and radiation as well as maintaining homeostasis

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Figure 5. Cell viability of normal and keloid broblasts following C2 ceramide treatment dose-response. Each of three primary culture cell lines of normal and keloid broblasts seeded at 5 Q 105 cells per well into 96-well plates were cultured with the indicated concentrations of C2 ceramide, and the cell viability was monitored by WST-1 assay as described in Materials and Methods. The dose-dependent effect of C2 ceramide on the cell viability of normal and keloid broblasts 12 h after incubation. Note that keloid broblasts were still resistant with the 40 mM concentration of C2 ceramide even though the same dose of C2 ceramide strongly abolished the cell viability of normal broblasts.

under physiologic conditions (Cuff et al, 1996; Blank et al, 1997; Dixon et al, 1997; Kinloch et al, 1999). Apoptosis also plays an important role in the wound healing process (Desmouliere et al, 1995), and this study provided clear evidence for the resistance of keloid broblasts to apoptosis, as evidenced by increased cell proliferation and decreased cell apoptosis. We also demonstrated that such properties correlated with overexpression of IGF-IR on keloid broblasts. To our knowledge, there are no previous reports on the role of IGF-IR in the apoptosis of keloid broblasts, except for one study on apoptosis in keloid tissues (Appleton et al, 1996). In contrast to our ndings, the above study showed increased apoptosis in keloid tissues by using in situ end labeling (the TUNEL method). The exact explanation of the different ndings is not clear at present. The number of TUNEL-positive cells relative to total cell number might be low, however, because of the markedly high cell density in keloid tissues. Also Appleton et al (1996) did not provide any functional studies using a cell culture system. In this study, western blotting analysis showed that the expression of IGF-IR on keloid broblasts was 1.42.9 times higher than that on normal broblasts. Furthermore, we also

demonstrated resistance of keloid broblasts to C2-ceramideinduced apoptosis compared with normal dermal broblasts. The addition of exogenous IGF-I enhanced the survival of keloid broblasts in a manner proportionate to IGF-IR expression. Comparison of cells derived from null mice of the IGF-IR gene with stable transfectants overexpressing the human IGF-IR demonstrated the importance of IGF-IR expression in resisting apoptosis (Nakamura et al, 1997). Although we did not examine the acquisition of resistance in normal skin broblasts by IGF-IR transfection, our results also suggest that the number of functional IGF-IR is important in the regulation of antiapoptotic signals. The antiapoptotic signal pathway of IGF-IR in broblasts and other cells has already been reported (Datta et al, 1997; Kulik et al, 1997). PI3-kinase is a major effector for signaling by IGF-IR (De Meyts et al, 1994; Carpenter and Cantley, 1996; Mendez et al, 1996; Myers and White, 1996). Furthermore, Akt is identied as the downstream component of survival signaling through PI3kinase (Kulik et al, 1997; Dudek et al, 1997; Kauffmann-Zeh et al, 1997; Kennedy et al, 1997; Khwaja et al, 1997), which is a growth factor that regulates serine/threonine kinase. Furthermore,

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Figure 6. Cell viability of normal and keloid broblasts following C2 ceramide treatment time course. Normal and keloid broblasts seeded at 5 Q 105 cells per well into 96-well plates were cultured with the indicated concentrations of C2 ceramide, and the cell viability was monitored by WST-1 assay in a similar method to Fig 5. The time course of cell viability in cells treated with 0 mM, 30 mM, and 40 mM C2 ceramide. The viability of normal broblasts decreased in a time-dependent manner, whereas the reduction rate of viability was small in keloid broblasts.

Figure 7. IGF-I rescues C2-ceramide-induced apoptosis of keloid broblasts but not of normal broblasts. Normal and keloid broblasts were cultured with DMEM containing various concentrations (050 mM) of C2 ceramide with or without 100 ng per ml IGF-I for 12 h, and the cell viability was studied by WST-1 assay. Incubation of keloid broblasts with IGF-I enhanced the resistance to C2-ceramide-induced apoptosis in keloid broblasts but not in normal broblasts.

cascades was investigated (Ladin et al, 1998; del Peso et al, 1997). This signal pathway has been recognized as a major antiapoptotic signal pathway for IGF-IR. Kulik et al reported the existence of a survival signaling pathway downstream of PI3-kinase, Akt/PKB, in broblasts overexpressing the IGF-IR (Kulik and Weber, 1998). In this study, wortmannin, a PI3-kinase inhibitor, completely blocked the antiapoptotic effect of IGF-I and IGF-IR in keloid broblasts. Thus, PI3-kinase may also play a major role in the antiapoptotic signal pathway in keloid broblasts. The underlying mechanisms for the increased number of IGFIR in keloid broblasts remain unclear at this stage. The expression of IGF-IR gene is modulated by a number of physiologic and pathologic factors, such as developmental stage, nutritional status, hormones, growth disorders, and malignancy (Werner et al, 1995). The tumor suppressor gene p53 is a regulatory factor of transcription of IGF-IR gene. Wild-type p53 suppresses IGF-IR promoter activity whereas mutant p53 stimulates IGF-IR gene transcription (Werner et al, 1996). Sead et al (1998) analyzed p53 gene mutation in keloids using polymerase-chain-reaction-based single-strand conformational polymorphism and DNA sequencing. They found mutations in exon 4 of p53 gene in all samples (codon 72, CGCCCC, arginineproline). We also checked this mutation of p53 gene; however, we could not nd such mutations in any of our keloid samples (data not shown). Viral protein interacts with p53 and changes the protein conformation to be similar to that of mutant p53. It is possible that changes in keloid p53 might occur without any genetic mutation. In conclusion, we demonstrated in this study that keloid broblasts overexpress IGF-IR, resist proapoptotic signals, and disturb the balance between cell death and proliferation in the lesions, eventually producing excessive extracellular matrix, resulting in the unique pathologic features of keloid tissues. Identication of the factors responsible for controlling the expression of IGF-IR and apoptotic signal in keloids may therefore allow the design of novel therapies for the treatment of keloids.
Human recombinant IGF-I was a kind gift from Fujisawa Pharmaceutical Co., Tokyo. This work was supported by Grants-in-Aid (10671682) from the Ministry of Education, Science, Sports and Culture, Japan.

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Figure 8. PI3-kinase is involved in IGF-I-mediated resistance to apoptosis in keloid broblasts. Keloid broblasts were cultured with DMEM containing 40 mM C2 ceramide with or without 100 ng per ml IGF-I, and the cell viability at the indicated time was monitored by WST-1 assay. Preincubation with 100 nM wortmannin for 30 min blocked IGF-Imediated resistance of keloid broblasts against C2-ceramide-induced apoptosis.

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