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ORIGINAL ARTICLE

Green Tea Extract and ( )-Epigallocatechin-3-Gallate
Inhibit Mast Cell-Stimulated Type I Collagen
Expression in Keloid Fibroblasts via Blocking
PI-3K/Akt Signaling Pathways
Qunzhou Zhang1, A. Paul Kelly2, Lina Wang3, Samuel W. French4, Xudong Tang1, Hai S. Duong1,
Diana V. Messadi5 and Anh D. Le1,6

Keloid, a chronic fibro-proliferative disease, exhibits distinctive histological features characterized by
an abundant extracellular matrix stroma, a local infiltration of inflammatory cells including mast cells (MCs),
and a milieu of enriched cytokines. Previous studies have demonstrated that co-culture with MCs stimulate type
I collagen synthesis in fibroblasts, but the signaling mechanisms remain largely unknown. In this study, we
investigated the signaling pathways involved in MC-stimulated type I collagen synthesis and the effects of green
tea extract (GTE) and its major catechin, ( )-epigallocatechin-3-gallate (EGCG), on collagen homeostasis in
keloid fibroblasts. Our results showed that MCs significantly stimulated type I collagen expression in keloid
fibroblasts, and the upregulation of type I collagen was significantly attenuated by blockade of phospha-
tidylinositol-3-kinase (PI-3K), mammalian target of rapamycin (mTOR), and p38 MAPK signaling pathways, but
not by blockade of ERK1/2 pathway. Furthermore, GTE and EGCG dramatically inhibited type I collagen
production possibly by interfering with the PI-3K/Akt/mTOR signaling pathway. Our findings suggest that
interaction between MCs and keloid fibroblasts may contribute to excessive collagen accumulation in keloids
and imply a therapeutic potential of green tea for the intervention and prevention of keloids and other fibrotic
diseases.
Journal of Investigative Dermatology (2006) 126, 2607–2613. doi:10.1038/sj.jid.5700472; published online 13 July 2006

INTRODUCTION neous regression as observed in hypertrophic scars (Ehrlich
Keloids are fibrous overgrowth induced by cutaneous injury et al., 1994). Histologically, keloids and hypertrophic scars
(Tuan and Nichter, 1998; Niessen et al., 1999). Clinically, differ from normal skin and normal scars by their rich
keloids behave like benign dermal fibro-proliferative tumors vasculature (Rockwell et al., 1989; Lee et al., 2004), a high
as they continue to grow and extend beyond the confines of density of mesenchymal cells, a thickened epidermal cell
the original wound margins, without evidence of sponta- layer, an increased infiltration of inflammatory cells including
lymphocytes, mast cells (MCs), and macrophages (Amadeu
1
Center for Craniofacial Molecular Biology, University of Southern California,
et al., 2003), and an abundant deposition of extracellular
School of Dentistry, Los Angeles, California, USA; 2Division of Dermatology, matrix (ECM) (Niessen et al., 1999).
Department of Medicine, Charles R. Drew University of Medicine and Human type I collagen, the major component of ECM in
Science, Los Angeles, California, USA; 3Department of Pathology, Kenneth skin, bone, and ligament, comprises of two a1 (I) chains and
Norris Jr. Cancer Center, University of Southern California Keck School
of Medicine, Los Angeles, California, USA; 4Department of Pathology, one a2 (I) chain, which are derived from pro-COL1A1 and
Harbor-UCLA Medical Center, Torrance, California, USA; 5Department pro-COL1A2 genes, respectively (Ghosh, 2002). Abnormality in
of Oral Biology and Medicine, University of California, Los Angeles, collagen synthesis leading to an imbalance in ECM metabolism
California, USA and 6Department of Surgical, Therapeutic and
has been recognized as an essential factor in the pathogenesis
Bioengineering Sciences, University of Southern California, School
of Dentistry, Los Angeles, California, USA of several fibrotic diseases including keloids (Niessen et al.,
Correspondence: Dr Anh D. Le, Department of Surgical, Therapeutic and 1999; Myllyharju and Kivirikko, 2001). However, the mole-
Bioengineering Sciences, School of Dentistry, Center for Craniofacial cular mechanisms underlying the overproduction of type I
Molecular Biology, University of Southern California, Los Angeles, California collagen in keloids still remain largely unknown.
90033, USA. E-mail: anhle@usc.edu
MCs are tissue dwelling cells containing prominent
Abbreviations: ECM, extracellular matrix; EGCG, ( )-epigallocatechin-3-
cytoplasmic granules. MC hyperplasia and activation have
gallate; GTE, green tea extract; MC, mast cell; mTOR, mammalian target of
rapamycin; PI-3K, phosphatidylinositol-3-kinase been implicated in the pathogenesis of several chronic
Received 8 March 2006; revised 19 May 2006; accepted 30 May 2006; inflammatory fibrotic diseases such as aberrant wound
published online 13 July 2006 healing, idiopathic lung fibrosis, scleroderma, liver fibrosis,

& 2006 The Society for Investigative Dermatology www.jidonline.org 2607

. against type I collagen (Col-I).1/1 0... HMC-1 cells stimulated type I collagen expression in fibroblasts further understanding of the molecular mechanisms under. Nigrovic and Lee. 2003. Puxeddu 0/1 0.1/1 0. Volume 126 . In addition. Q Zhang et al. We reported for the first time that both GTE and EGCG significantly suppressed MC-stimulated type I collagen expression in keloid fibroblasts. harbors several polyphenolic components known NSK KF as catechins. and directly co-cultured with increasing number of HMC-1 cells in a six-well plate helped to further delineate the targets of therapeutic under normal culturing condition for 24 hours. (c) Dual-color immunofluorescent staining of type I collagen (Green) in keloid fibroblasts MCs stimulate type I collagen expression in keloid and c-kit on MCs (Red).. several recent studies have shown that polyphenols from green tea significantly improved the quality of wound healing and scar formation in a rat model (Kapoor et al. Representative results from three numbers of HMC-1 cells (a human leukemic mast cell independent experiments are shown. 1987. Huttunen et al. 1986. Our results also indicated that the inhibitory effects of GTE and EGCG on type I collagen expression in keloid fibroblasts appeared to be mediated via the phosphatidylinositol-3-kinase (PI-3K)/Akt/ mTOR signaling pathways. derived from keloids and their corresponding peripheral normal skins. and such increase was Most recently. Bar: 200 mM. and dramatically KF KF+HMC-1 attenuated experimental cholestasis-induced liver fibrosis in rats (Zhong et al. (b) Densitometric analysis of the results of (a). and c NSK NSK+HMC-1 ( )-epicatechin. 2005). which may correlate with a prolonged inflammatory state. Zaveri. and a HMC-1/NSK HMC-1/KF rheumatoid arthritis (Benoist and Mathis.. GTE and EGCG Inhibit MC-Stimulated Type I Collagen I Synthesis inflammatory bowel diseases such as Crohn’s disease. 2005. Smith et al. cardiovascular. 2006). and PI-3K/Akt signaling pathways (Zhang et al. 1 Green tea. Co-cultured cells (cell density ratio of 1:1) were fibroblasts in vitro fixed in cold methanol:acetone (1/1) and stained with mouse monoclonal To investigate the effects of MCs on type I collagen antitype I collagen antibody and rabbit polyclonal anti-c-kit antibody expression in keloid fibroblasts. Previous studies Col-l have shown that MCs undergo significant qualitative and -Actin quantitative changes during abnormal wound healing. (a) Keloid fibroblasts (KF) or normal skin fibroblasts (NSK) (1  105/well) were lying the antifibrogenic effects of GTE and EGCG. 2003). remarkably suppressed both collagen production and collagenase activity in hepatic stellate cells (Nakamuta et al.5/1 1/1 0/1 0. line) under the condition where direct cell–cell contact is allowed. and neurological diseases (Shimizu and Weinstein. 2005).. derived from dried leaves of the plant Camelia 0 0/1 0. we explored whether GTE and EGCG had any effect on type I collagen synthesis in keloid fibroblasts co-cultured with human MCs. 2608 Journal of Investigative Dermatology (2006).. A wide range of pharmacological effects of green tea and its major catechins has been reported with promising therapeutic potentials in cancer. The relative density ratio of the two bands of type I collagen to b-actin in RESULTS fibroblasts (FB) cultured alone was arbitrarily set as 1. 2000. 2002. Lee and Vijayasingam. 2006). 1999. including ( )-epigallocatechin-3-gallate (EGCG). 2 1995). an b 7 increase in fibroblast proliferation. 2002). and an aberrant collagen FB FB+HMC-1 6 production (Artuc et al. ( )-epicatechin gallate..5/1 1/1 0/1 0.. 2004)..1/1 0. ( )-epigallocatechin.5/1 1/1 et al. keloid fibroblasts and HMC-1 cells under the condition of Similar results were obtained by immunofluorescence studies direct cell–cell contact led to the activation of both ERK1/2 (Figure 1c). a fixed density of normal followed by Alexa Fluors568 conjugated goat anti-rabbit IgG and Alexa skin or keloid fibroblasts was co-cultured with different Fluors488 conjugated goat anti-mouse IgG. Equal amount of cell lysates intervention and prevention of keloids and other fibrotic (100 mg) was subjected to Western blot analysis with a specific antibody diseases.0. thus implicating an 4 important role of MCs and their mediators in keloid formation 3 (Craig et al. In this study.1/1 0. Such changes in MCs have also been observed 5 in keloids and hypertrophic scars.5/1 1/1 sinensis. These unique findings provided Figure 1. we have demonstrated that co-culture of dependent on the number of HMC-1 cells (Figure 1a and b). Our results showed that co-culture with HMC-1 MCs stimulate type I collagen expression in keloid fibroblasts cells led to a substantial increase in type I collagen synthesis through activation PI-3K/Akt and p38 MAPK signaling pathways in both normal and keloid fibroblasts. Abe Relative density et al.

The results indicated 8 HMC-1 (1/1) – + + + + + + + Wortmannin (nM) – – 10 50 100 – – – 6 Rapamycin (nM) – – – – – 5 10 50 4 CoI-I 2 0 1/4 1/2 1 2 3 4 6h -Actin p-ERK1/2 0 T-ERK1/2 e f 10 KF + + + + + + + + Relative density p-Akt 8 HMC-1 (1/1) – + + + + + + + U0126 (M) – – 5 10 50 – – – 6 T-Akt SB203580 (M) – – – – – 1 5 10 4 p-p38 CoI-I 2 -Actin -Actin 0 p-4E-Bp Figure 3..05) (Figure 3c–f). or SB203580. The results represent three experiments are shown. Activation of multiple signaling pathways in the co-cultured keloid of cell lysates (100 mg) were subjected to Western blot analysis with specific fibroblasts and HMC-1 cells. Similar dose-dependent reduction in HMC-1-stimulated type I findings were obtained with wortmannin. different concentrations of GTE or EGCG for 1 hour followed Our results showed that pretreatment of keloid fibroblasts by co-culture with the same cell density of HMC-1 cells for with LY294002. 2005).05) PD98059 (M) – – – – – 5 10 50 4 (Figure 3a. and f) Densitometric the same density of HMC-1 cells for different time intervals. keloid fibroblasts were pretreated with as determined by trypan blue exclusion (data not shown). and e) p-p70 Keloid fibroblasts (KF) were pretreated with different concentrations of various -Actin inhibitors followed by direct co-culture with the same density of HMC-1 cells under normal culturing condition for 24 hours. implying the GTE and EGCG suppressed HMC-1-stimulated type I collagen involvement of several important signaling pathways.05) (Figure 3a and b). c.05) structurally different inhibitor of PI-3K (Po0. tion of type I collagen expression in keloid fibroblasts. respectively. The relative density ratio amounts of cell lysates were subjected to Western blot analysis with various of the two bands of type I collagen (Col-I) to b-actin in keloid fibroblast (KF) specific antibodies. expression in keloid fibroblasts by interfering with PI-3K/Ak Next. (b. To rule out the possibility that the Col-I 2 suppression of MCs stimulated type I collagen synthesis was -Actin 0 secondary to drug toxicity. with maximal activity at 1–2 hours tions indicated that both PI-3K/Akt/mTOR and p38 MAPK (Figure 2). Taken together. and e). www. To rule specific inhibitor of p38 MAPK.05) (Figure 3c (Figure 4a and b).jidonline. or SB203580 at maximal concentra. Likewise. we also observed a time-dependent signaling pathways are involved in MC-stimulated upregula- increase in the phosphorylated levels of p38 MAPK. herein we demonstrated a time-dependent that mere exposure to these inhibitors had no obvious effects increase in both phosphorylated ERK1/2 and Akt levels in on type I collagen levels in keloid fibroblasts in the absence the co-cultured keloid fibroblasts and HMC-1 cells under of HMC-1 cells (Figure S1). Representative results from three independent cultured alone was arbitrarily set as 1. a cells (Figure 4c. dramatically additional 24 hours under normal culturing conditions. eukaryotic initiation factors (eIFs) binding protein (p-4E-BP)- 1 and p70S6K1 in the co-culture (Figure 2). also led to a dose-dependent inhibition of type-I collagen expression stimulated by HMC-1 cells (Po0. the right panel vs the middle panel). c. Cell viability of both fibroblasts and gallate (EGCG) had any effects on MC-stimulated type I HMC-1 cells was more than 95% at all concentrations tested collagen expression. Immunofluorescence studies also illu- and d). we ask whether these activated signaling pathways signaling pathway are involved in MC-induced upregulation of type I collagen Previous studies have shown that green tea and its major expression. a specific inhibitor of collagen signals in keloid fibroblasts stimulated by HMC-1 mammalian target of rapamycin (mTOR).0. e. pretreatment of keloid fibroblasts with strated that treatment with GTE significantly attenuated type I different concentrations of rapamycin.. but also possess antifibrogenic activity in kinase inhibitors for 1 hour followed by co-culture with the some animal models (Nakamuta et al. rapamycin. blocking ERK1/2 a b10 signaling pathway by pretreatment of keloid fibroblasts with KF + + + + + + + + Relative density 8 PD98059 or U0126 had no obvious inhibitory effects on HMC-1 (1/1) – + + + + + + + 6 LY294002 (M) – – 5 10 50 – – – HMC-1-stimulated type I collagen expression (P40. d. To explore same cell density of HMC-1 cells for 24 hours under normal whether green tea extract (GTE) and ( )-epigallocatechin-3- culturing conditions. However. and f). a specific inhibitor of PI-3K. keloid fibroblasts were exposed to LY294002. Equal amounts Figure 2. Our decreased HMC-1-stimulated type collagen expression in a results showed that pretreatment with GTE or EGCG led to a dose-dependent manner (Po0. independent experiments and expressed as the mean7SD. Involvement of PI-3k/Akt/mTOR and p38 MAPK pathways in s6k MC-stimulated type I collagen production in keloid fibroblasts. these observa- normal conditions. 2005). (a. Keloid fibroblasts at about 80% confluence were catechins not only have inhibitory effects on MC activation pretreated with different concentrations of various protein (Li et al. an alternative and collagen protein production in keloid fibroblasts (Po0. c d10 KF + + + + + + + + Relative density tions for 24 hours in the absence of MCs. Meanwhile. Q Zhang et al. GTE and EGCG Inhibit MC-Stimulated Type I Collagen I Synthesis Consistently. and equal analysis of results from (a.org 2609 . Keloid fibroblasts were directly co-cultured with antibody against type I collagen (Col-I).

Our results showed no obvious EGCG under normal conditions for 24 hours. Equal amounts of cell lysates (100 mg) were subjected to Western blot analysis with specific antibody against type I Next. p38 MAPK (c).05. The relative density the inhibitory effects of GTE and EGCG on HMC-1-stimulated ratio of the two bands of type I collagen (Col-I) to b-actin in KF cultured alone type I collagen expression in keloid fibroblasts. GTE and EGCG Inhibit MC-Stimulated Type I Collagen I Synthesis a KF + + + + + + + + a b 5 HMC-1 – + + + + + + + KF + + + + + + + + + Relative density GTE (g/ml) – – 20 40 80 – – – HMC-1 – + + + + + + + + 4 GTE (g/ml) – – 20 40 80 – – – – EGCG (M) – – – – – 25 50 100 3 EGCG (M) – – – – – 25 50 100 – CoI-I PD98059 (M) – – – – – – – – 50 2 p-ERK1/2 1 -Actin T-ERK1/2 0 4 b c d 3 KF + + + + + + + + + 2.05. GTE and EGCG inhibited MC-stimulated type I collagen Data presented are representative of results from three independent production in keloid fibroblasts. 4E-BP. we explored the signaling mechanisms underlying collagen. The relative density ratio of the p-ERK1/2 or p-Akt to (g/ml) (M) (g/ml) (M) total (T-) ERK1/2 or total (T-) Akt in KF cultured alone was arbitrarily set as 1. Q Zhang et al. EGCG only led to a moderate decrease in the phosphorylated followed by Alexa Fluors568 conjugated goat anti-rabbit IgG and Alexa ERK1/2 levels. n 20 40 80 10 25 50 0 n 20 40 80 10 25 50 0 Co 10 Co 10 p70S6K. (b and d) Densitometric analysis of GTE EGCG GTE EGCG results from (a) and (c). On the other treated with various concentrations of GTE or EGCG for 24 hours under normal culturing conditions and cell viability was assayed using MTT method. Volume 126 . hand. keloid fibroblasts were MTT assay was performed to determine the cell viability exposed to increased concentrations of LY294002. Figure 4. (b) Densitometric analysis of results from (a). (Figure 4). respectively. followed by direct co-culture with the same cell 40 40 number of HMC-1 cells under normal culturing condition for 1 hour. (c) Dual-color immunofluorescent staining of type I collagen (Green) in KF and c-kit on HMC-1 cells (Red). Keloid fibroblasts (KF) 80 80 were pretreated with different concentrations of GTE or EGCG. The co-cultured cells were ERK1/2 and p38 MAPK. and expressed as the mean7SD. treatment The percentage of viable cells represented the mean7SD from three replicate with GTE or EGCG resulted in a dose-dependent reduction in experiments. these data suggest that fibroblasts and HMC-1 cells (Figure 4d). similar to the inhibitory effects of LY294002.0. No obvious changes in the levels of these phosphorylated signaling changes in cell viability were observed in both keloid components (Figure S2). Equal 20 20 amounts of cell lysates were subjected to Western blot analysis with specific 0 0 antibody against the phosphorylated form of ERK1/2 (a).5 Relative density Relative density 3 HMC-1 – + + + + + + + + 2 GTE (g/ml) – – 20 40 80 – – – – EGCG (M) – – – – – 25 50 100 – 1. (a) Keloid fibroblasts (KF) were pretreated experiments. Effects of GTE and EGCG on the activation of signaling pathways Percentage of viable cells (%) Percentage of viable cells (%) 120 120 100 100 in the co-cultured keloid fibroblasts and HMC-1 cells. Keloid fibroblasts pretreatment of keloid fibroblasts with PD98059 (50 mM) and (KF) were pretreated with 80 mg/ml GTE for 1 hour followed by co-cultured SB203580 (10 mM) significantly inhibited the activation of with HMC-1 (cell density ratio of 1:1) for 16 hours. However. Akt. (d) KF or HMC-1 ells were activation of p38 MAPK (P40. PD98059. was arbitrarily set as 1. with different concentrations of GTE or EGCG for 1 hour followed by direct co-culture with the same number of HMC-1 cells under normal culturing condition for 24 hours. GTE and EGCG inhibited MC-stimulated type I collagen 2610 Journal of Investigative Dermatology (2006). As expected. Bar: 200 mM. but had no obvious inhibitory effects on the Fluors488 conjugated goat anti-mouse IgG. exposure of keloid fibroblasts to GTE or antibody against type I collagen and rabbit polyclonal antibody against c-kit. or various 60 60 kinase inhibitors for 1 hour. and 4E-BP1 (e).5 2 SB203580 (M) – – – – – – – – 10 1 p38 MAPK 0. p70S6K in the co-cultured keloid fibroblasts and HMC-1 cells (Figure 5e). respectively (Po0. To rule out drug toxicity. The results represent three independent experiments and the increased levels of phosphorylated Akt. following treatment with different concentrations of GTE and and SB203580 for 1 hour. Figure 5a fixed in cold methanol:acetone (1:1) and stained with mouse monoclonal and b).0.5 1 -Actin 0 0 e KF + + + + + + + + + HMC-1 – + + + + + + + + c KF KF/HMC-1 KF/HMC-1+GTE GTE (g/ml) – – 20 40 80 – – – – EGCG (M) – – – – – 25 50 100 – LY294002 (M) – – – – – – – – 50 p-AKt T-AKt p-4E-BP -Actin S6k p70 -Actin d KF HMC-1 Figure 5. Figure 5a–d). Collectively. which paralleled their inhibitory effects on out the possibility that the inhibitory effect of GTE and EGCG HMC-1-stimulated upregulation of type I collagen expression on type I collagen expression was due to cellular toxicity.

2005). 2004). Several studies have shown that excessive collagen deposition. 2004. MO) that co-culture with HMC-1 cells upregulated both phos. Fibroblasts were maintained in DMEM (Gibco. our group has reported the activation of tion of the PI-3k/Akt/mTOR signaling pathways in keloid ERK1/2 and PI-3K/Akt signaling pathways in the co-cultured fibroblasts. The purity and components of GTE were previously regulatory components of protein translational machinery described (Lu et al. our results indicated that the University of Southern California and were conducted with strict treatment with GTE or EGCG suppressed the phosphorylated adherence to the Declaration of Helsinki Principles.. (Eugene. we have demonstrated for the first MCs substantially stimulated type I collagen synthesis in time that both GTE and EGCG significantly inhibited MC- keloid fibroblasts (Figure 1). are characterized by Nigrovic and Lee. The understanding of the molecular mechanisms of keloid current study has extended our previous observation and pathogenesis and identify a therapeutic potential of green found that co-culture with HMC-1 cells activated not only tea for the intervention and prevention of keloids and other ERK1/2 and PI-3K/Akt signaling pathways but also p38 MAPK fibrotic diseases. CA) been reported to possess various pharmacological activities and Rockland Immunochemicals (Gilbertsville. further studies are required to explore whether the PI-3K/Akt signaling pathways. LY294002. Provo. Li IgG and Alexa Fluors488 conjugated goat anti-mouse IgG were et al. 2005). CA). pathway (Figure 2). with a broad variety of health benefits (Yang et al. respectively. Primary cultures levels of Akt.. Asano et al. All studies have been approved by the Institutional Review Board of blasts (Figure 4). GTE and EGCG Inhibit MC-Stimulated Type I Collagen I Synthesis expression in keloid fibroblasts mainly by interfering with the However.. et al. This finding is consistent with previous report that co-culture of MC and lung fibroblast with direct MATERIALS AND METHODS cell–cell interaction led to the activation of p38 MAPKs Reagents (Fitzgerald et al. 2003. In this study. PI-3K/Akt/mTOR. both PI-3K/Akt/mTOR and p38 MAPK signaling pathways. 2004. and (La Jolla. but had no of human dermal fibroblasts were isolated by enzymatic digestion of obvious inhibitory effects on p38 MAPK in the co-cultured keloid tissues obtained from patients at King Drew Medical Center keloid fibroblasts and HMC-1 cells (Figure 5). Rabbit polyclonal antibodies against human c-kit and type I GTE and its major polyphenolic components have long collagen were from OncogeneTM Research Products (San Diego. Mouse monoclonal antibodies against human type 2005). In addition. or both.. 2005).. 2000. chronic fibroproliferative disease. inhibitors used. All reagents used were several experimental models (Zhong et al. SB203580.. notoriously prone to co-culture with MCs promote type I collagen synthesis in recurrence. Antibodies for phosphorylated Mr 70. two important solutions. CA).. activity by inhibiting MC-stimulated type I collagen produc. wortman- that are downstream targets of PI-3K/Akt/mTOR signaling nin. Cells tion mainly via the PI-3K/Akt/mTOR signaling pathways. we have demonstrated for the first time I collagen and b-actin were obtained from Sigma. 2004. while treatment Biolabs Inc. stimulated type I collagen expression by suppressing activa- Most recently. OR). 2005).. PD98059. UT) and EGCG (Sigma. our results also indicated GTE (Pharmanex Inc.. the present study has demonstrated that MCs conditions via their ability to respond to a composite range of can stimulate type I collagen production via activating stimuli. 2000. MA). 2002). IL).. MCs have been attributed to several patho-physiological In summary. 2004. and. Alexa Fluors568 conjugated rabbit anti-goat tea can inhibit MC activation (Yamamoto et al. Burd and Consistently. Ihn et al.. modality has proven successful (Kelly. and ERK1/2. our findings demonstrated that co-culture with Huang. St Louis. and release of a wide array of biologically active multiple signaling pathways in keloid fibroblasts. 4E-BP-1. no single fibroblasts (Abe et al. Nakamuta et al. and rapamycin were purchased from Calbiochem pathways. 2006). Ribatti et al. 1998. (Beverly. p38 MAPK were from New England collagen production in keloid fibroblasts. Cell viability p38 MAPK signaling pathways have been described in the using trypan blue exclusion was determined for both keloid upregulation of type I collagen expression in dermal fibroblasts and HMC-1 cells at the highest concentrations of fibroblasts (Lim et al.. Rockville. MD) supplemented with 10% fetal bovine serum.jidonline. Mouse mono- that treatment with specific inhibitors of PI-3K.000 with specific inhibitors of ERK1/2 had no obvious inhibitory ribosomal protein S6 kinase 1 (p70S6K) (Thr421/Ser424) and effects (Figure 3). All keloidal tissues were from untreated. 2002). p70S6K1. These findings suggest that MCs stimulate eukaryotic initiation factor 4E (eIF)-binding protein 1 (4E-BP1) type I collagen expression in keloid fibroblasts via activating (Ser65/Thr70) were from Santa Cruz Biotechnology (Santa Cruz. 2003). and affect ECM metabolism/remodeling in from Molecular Probes Inc. a mediators (Mekori and Metcalfe. Garbuzenko et al. from passage 2 through 8 were used for experiments and routinely www. Our unique findings have provided further keloid fibroblasts and HMC-1 cells (Zhang et al.. These results (Zhang et al. Herein. 2003. Kapoor analytical grade. Horseradish peroxidase conjugated secondary antibodies were from Recent studies have shown that some components of green PIERCE (Rockford. 2005).. All inhibitors were dissolved in DMSO. we reported that GTE and EGCG had strong inhibitory effects on Cell origin and cell culture MC-stimulated type I collagen production in keloid fibro.. In this study. The involvement of ERK1/2.org 2611 . U0126. inhibitory effects of GTE and EGCG on MC-stimulated type I collagen expression were due to their direct effects either on DISCUSSION the activation of MCs or keloid fibroblasts. Despite several treatment approaches. PA). suggest that GTE or EGCG harbor potential antifibrogenic primary lesions. Moreover. Keloids. mTOR and clonal antibodies against human total or phosphorylated ERK1/2 p38 MAPK significantly inhibited MC-stimulated type I (Thr202/Tyr204) or Akt (Ser473). were dissolved in distilled water and stored at 801C as stock phorylated 4E-BP and p70S6K (Figure 2).. Q Zhang et al..

Hall I.000. set from these three independent experiments is presented where a human MC leukemia cell line (a generous gift of Dr JH Butterfield. and phosphorylated ERK1/2 and Akt Effect of activated human mast cells and mast cell-derived mediators on levels. followed by a horse. Exp Dermatol 9:258–65 stained red. Rochester. Hall HK. 200 mM Na3VO4. Tamaki K (2005) Increased phosphorylation and activation antibodies in the absence of primary antibodies were used as of mtogen-activated protein kinase p38 in scleroderma fibroblasts.000. One representative data 17:212–8 2612 Journal of Investigative Dermatology (2006). small Keloid fibroblasts were seeded on four-well Lab-TeksII Chamber intestine. Type I collagen Alterations in mast cells showing tryptase and chymase activity in expression appeared green and c-kit-positive HMC-1 cells were epithelialization and chronic wounds. Mathis D (2002) Mast cells in autoimmune disease. IL) (1  104) and monoclonal antibody against tryptase. Krishnaswamy G (2004) Human lung 24 hours. Am J Pathol 145:105–13 direct cell–cell contact. cells were solubilized in lysis buffer (50 mM Tris-HCl. 0. DeBlois G. 5 mM EDTA. 200 mM Amadeu T. CONFLICT OF INTEREST The authors state no conflict of interest. Diegelmann RF.2 mmol/l a-thioglycerol (Sigma. human type I collagen antibody (1:100) and a rabbit polyclonal anti. J Immunol 172:7123–35 radish peroxidase conjugated secondary antibodies (1:2. Clin Exp Allerg 32:237–46 temperature for 1 hour with Alexa Fluors568 conjugated goat anti- Ghosh AK (2002) Factors involved in the regulation of type I collagen gene rabbit IgG (1:2. and visualized using an enhanced chemilu- 420:875–8 minescent (ECL) detection.5 mg/ml). 2 mmol/l L-glutamine. J Invest Dermatol 125:247–55 negative control. and Alexa Fluors488 conjugated expression: implication in fibrosis. All cultures were maintained at 371C. Nature (Pierce. Phosphatidylinositol 3-kinase is involved in a2 (I) collagen gene expression I noral and scleroderma fibroblasts. Pickholtz D. Dermatol Ther carried out for at least three separate runs. Lee SA. MO). Am J Respir Cell Mol Biol 30:585–93 and incubated at 41C overnight with a mouse monoclonal anti.. Horsmanheimo M. Ishikawa O (2002) To determine type I collagen. 0. and lung by an immunoperoxidase technique using a murine SlideTM System (Nalge Nunc Int. the membranes were Asano Y. purposes. St Louis. Maquart F-X et al. and 20% O2. Amersham Pharmacia). Mimura Y. Nagler A. (2002) Human mast cells stimulate fibroblast proliferation. Yokoyama Y. Henz BM (1999) Mast cells on 7. antibiotics. Cell lysates were prepared for Abe M. EGCG. Kurosawa M. The fixed cells were washed. contact. innocent bystanders? Exp Dermatol 8:1–16 After blocking with TBS/5% skim milk. incubated at room synthesis and lattice contraction: a direct role for mast cells in skin fibrosis. 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