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A transcriptional factor decoy against AP-1 suppresses TGF-β1-induced type I collagen gene expression in cultured keloid fibroblasts
Journal of Dermatological Science (2005) 37, 49—51
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR A transcriptional factor decoy against AP-1 suppresses TGF-b1-induced type I collagen gene expression in cultured keloid fibroblasts To the Editor, During wound repair, type I collagen production and deposition in the extracellular matrix is required for the generation of normal physiological connective tissue but these events are regulated on several levels. Keloid occurs when this regulatory process is disrupted in some way (reviewed in Ref. [1]). TGFb proteins play an important role in all of these processes and transient TGF-b production may, therefore, promote wound healing and restoration of tissue architecture. However, persistent expression and/or activation of TGF-b may lead to keloid. Among the three types of TGF-b, TGF-b1 is a key mediator in the pathogenesis of keloid [2]. Several signaling pathways regulate type I collagen gene expression by TGF-b. For example, the Smad [3], AP-1 [4], and SP-1 [5] pathways, all of which involve AP-1-binding transcription factors, are involved in this process, indicating that AP-1 may play an important role in the regulation of collagen synthesis. AP-1 has been implicated in various gene regulatory activities, associated with cell proliferation and extracellular matrix production, by TGF-b [6]. Accordingly, we hypothesize that AP-1 plays a critical role in overproduction of type I collagen and keloid pathogenesis and that this may provide a therapeutic target for keloid treatment. Recently, decoy oligodeoxynucleotides (ODNs) have been used to inhibit the expression of specific genes. It has been reported that treatment with AP1 decoy ODN abolishes the expression of several genes, including plasminogen activator, TGF-b and endothelin-1 [7]. However, there are no previous reports of the effects of an AP-1 decoy ODN in skin fibroblasts or specifically in keloid fibroblasts. The aims of this study were to investigate whether AP-1
functions as a transcription factor in keloid formation and to determine the effect of decoy oligodeoxynucleotides to AP-1 on type I collagen gene expression in cultured TGF-b1-treated keloid fibroblasts. In this study, the transcriptional activity of the AP-1 binding element was examined via electrophoretic mobility shift assay (EMSA), and the effect of AP-1 decoy ODN on type I collagen synthesis was determined via northern blot analysis, and CAT assay. Primary cultures of normal human fibroblasts (n = 3, mean age: 33 years) and keloid fibroblasts (n = 3, mean age: 24 years) were established from left over adult skin following cosmetic surgery. We used the same AP-1 decoy ODN which was used in the previous study [8], though it was not performed to the same object. The sequences of circular dumbell AP-1 decoy ODN (CDODN) against AP-1 binding sites and mismatched AP-1 decoy ODN (MODN, as control) are as follows (note: consensus sequences are underlined): CDODN: 50 -GGATCCATGACTCAGAAGACGACACACGTCTTCTGAGTCAT-30 MODN: 50 -GGATCCAAATCTCAGAAGACGACACACGTCTTCTGAGATTT-30 CDODN and MODN were transfected to normal and keloid fibroblasts for 24 h prior to TGF-b1 treatment using Lipopectamine Plus1 (Gibco-BRL, USA). The transfected cells were further incubated in complete medium for 24 h and this was followed by EMSA, northern blot analysis, and CAT assay. Results are expressed as mean S.E.M. Variance analysis with a subsequent Student’s t-test was used to determine significant differences in unpaired comparisons. All experiments were performed at least three times. First, We performed EMSA to confirm the effect of TGF-b1 on DNA binding activity of AP-1. AP-1 binding activity of keloid fibroblasts was higher than those in normal fibroblasts (1.6 0.4 versus 2.8 0.2; Fig. 1A, lanes 1 and 4). Moreover, TGFb1 treatment (5 ng/ml) increased AP-1 binding activity in nuclear extracts of cultured keloid fibro-
0923-1811/$30.00 # 2004 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology. doi:10.1016/j.jdermsci.2004.09.008
50
Letter to the Editor
Fig. 1 (A) AP-1 decoy (CDODN) down regulates AP-1 binding activity in cultured normal and keloid fibroblasts treated with TGF-b1 and AP-1 decoy as analysed by gel mobility shift assay. Lane 1: normal skin fibroblasts; lane 2: normal + TGFb1; lane 3: normal + TGF-b1 + AP-1 decoy; lane 4: keloid; lane 5: keloid + TGF-b1; lane 6: keloid + TGF-b1 + AP-1 decoy; and lane 7: gel retardation assay with unlabeled AP-1 ODN (10). (B) In the mismatched decoy ODN (MODN) transfected keloid fibroblasts, the TGF-b1 induced AP-1 binding activity was not affected. Lane 1: keloid; lane 2: keloid + TGF-b1; and lane 3: keloid + TGF-b1 + MODN. The values are mean S.D. and expressed as fold as compared to untreated keloid groups. *Significantly different from untreated keloid fibroblast (p < 0.05). **Significantly different from only TGF-b1 treated group (p < 0.01). Values in parenthesis indicate fold difference.
blasts to 1.9- and 3.1-fold the level of normal skin fibroblasts and untreated keloids, respectively (Fig. 1A, lanes 2 and 5). To determine the effect of AP-1 decoy ODN transfection on TGF-b1-induced AP-1 binding activity, cultured keloid fibroblasts were transfected with AP-1 decoy ODN prior to treatment with TGF-b1 (5 ng/ml). In transfected cells, AP-1 decoy ODN treatment decreased AP-1 binding activity to 0.31-fold that of the TGF-b1treated keloid fibroblasts (Fig. 1A, lane 6). But, in the mismatched decoy ODN (MODN) transfected keloid fibroblasts, the TGF-b1 induced AP-1 binding activity was not affected. (Fig. 1B, lanes 2 and 3) In vitro competitive binding assays, an increase in concentration of unlabeled AP-1 decoy ODN was accompanied by a corresponding decrease in the intensity of the retarded band, which is indicative of the presence of an ODN-AP-1 protein complex
(Fig. 1A, lane 7). In northern blot analysis of cultured normal and keloid skin fibroblasts, [32P] labeled a1(I) procollagen and GAPDH cDNA probes specifically hybridized with each mRNA. Northern analysis with a cDNA probe for a1(I) procollagen following TGF-b1 treatment resulted in a signal that was increased (560 20 versus 1002 11, by approximately 92%) compared to the untreated keloid group, after normalization to GAPDH mRNA levels. Furthermore, the overexpression of procollagen was decreased (1002 11 versus 240 21) by AP-1 decoy treatment compared to the TGF-b1treated keloid group. This implies that AP-1 decoy treatment decreased TGF-b1-induced a1(I) procollagen mRNA levels by approximately 76% (Fig. 2). CAT analysis revealed that the degree of acetylation was 7.1% in the untreated keloid group, 12.3% in the TGF-b1-treated group, and 6.1% in the group
Fig. 2 Northern blot analysis of a1(I) procollagen in cultured normal and keloid fibroblasts treated with TGF-b1 and AP1 decoy. Lane 1: normal skin fibroblasts; lane 2: normal + TGF-b1; lane 3: normal + TGF-b1 + AP-1 decoy; lane 4: keloid; lane 5: keloid + TGF-b1; and lane 6: keloid + TGF-b1 + AP-1 decoy. The values are mean S.D. and expressed as densitometric absorbance unit, which are the percentage of the value of GAPDH. *Significantly different from untreated keloid fibroblast (p < 0.05). **Significantly different from only TGF-b1 treated group (p < 0.01). Values in parenthesis indicate fold difference.
Letter to the Editor
treated with both TGF-b1 and AP-1. In comparison to the untreated keloid group, procollagen promoter activity was increased (7.1 0.6 versus 12.3 1.3) in the TGF-b1-treated group and this increased activity was inhibited (12.3 1.3 versus 6.1 1.2) by AP-1 decoy treatment in comparison to the TGF-b1-treated keloid group. This implies that AP-1 decoy treatment decreased TGF-b1-induced a2(I) procollagen promoter activity by approximately 51% (figure not shown) Taken together, our data suggest that transfection of an AP-1 decoy ODN suppresses TGF-b1 induced type I collagen gene expression in cultured keloid fibroblasts. By extension, in vivo transfection of target cells with sufficient quantities of the AP-1 decoy ODN may be useful for keloid treatment by preventing expression of type I collagen induced by TGF-b1. Inhibition of AP-1 during treatment would minimize the likelihood of immune-related side effects. Thus, this AP-1 decoy strategy may be particularly suitable for short-term management of keloid formation. Further studies are required to prove that the blockade of transcriptional factor AP-1 by AP-1 decoy ODN is sufficient to block the progression of keloid in vivo. Despite these limitations, this transcription factor decoy strategy may provide a new tool for treating keloid.
References [1] Ghosh AK. Factors involved in the regulation of type I collagen gene expression; implication in fibrosis. Exp Biol Med 2002;227:301—14. [2] Lee TY, Chin GS, Kim WJ, Chau D, Gittes GK, Longaker MT. Expression of transforming growth factor beta 1, 2 and 3 proteins in keloids. Ann Plast Surg 1999;43:179—84. [3] Chen SJ, Yuan W, Mori Y, Levenson A, Trojanowska M, Varga J. Stimulation of type I collagen transcription in human skin fibroblasts by TGF-b; involvement of Smad3. J Invest Dermatol 1999;112:49—57. [4] Chung KY, Agarwal A, Uitto J, Mauviel A. An AP-1 binding sequence is essential for regulation of the human a2(I) collagen (COL1A2) promoter activity by transforming growth factor-b. J Biol Chem 1996;271:3272—8. [5] Greenwel P, Inagaki Y, Hu W, Walsh M, Ramirez F. Sp1 is required for the early response of a2(I) collagen to TGFb1. J Biol Chem 1997;272:19738—45.
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[6] Tang W, Yang L, Leng SX, Elias JA. Transforming growth factorbeta stimulates interleukin-11 transcription via complex activating protein-1-dependent pathways. J Biol Chem 1998;273: 5506—13. [7] Ahn JD, Morishita R, Kaneda Y, Lee KU, Park JY, Jeon YJ, Song HS, Lee IK. Transcription factor decoy for activator protein-1 (AP-1) inhibits high glucose and angiotensin II-induced type 1 plasminogen activator inhibitor (PAI-1) gene expression in cultured human vascular smooth muscle cells. Diabetologia 2001;44:713—20. [8] Ahn JD, Morishita R, Kaneda Y, Lee SJ, Kwon KY, Choi SY, et al. Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo. Circ Res 2002;90:1325—32.
Chang Wook Kima,c Seong Suhb,c Su Hang Sungc In Kyu Leec,d Kyu Suk Leea,c,* a
Department of Dermatology School of Medicine Keimyung University Dong-San Dong 194, Daegu, South Korea b
Department of Microbiology School of Medicine Keimyung University Dong-San Dong 194, Daegu, South Korea c
Institute for Medical Science School of Medicine Keimyung University, Dong-San Dong 194 Daegu, South Korea d
Department of Internal Medicine School of Medicine, Keimyung University Dong-San Dong 194, Daegu, South Korea *Corresponding author. Tel.: +82 53 250 7625 fax: +82 53 250 7626 E-mail address: [email protected] (K.S. Lee) 1 March 2004
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR A transcriptional factor decoy against AP-1 suppresses TGF-b1-induced type I collagen gene expression in cultured keloid fibroblasts To the Editor, During wound repair, type I collagen production and deposition in the extracellular matrix is required for the generation of normal physiological connective tissue but these events are regulated on several levels. Keloid occurs when this regulatory process is disrupted in some way (reviewed in Ref. [1]). TGFb proteins play an important role in all of these processes and transient TGF-b production may, therefore, promote wound healing and restoration of tissue architecture. However, persistent expression and/or activation of TGF-b may lead to keloid. Among the three types of TGF-b, TGF-b1 is a key mediator in the pathogenesis of keloid [2]. Several signaling pathways regulate type I collagen gene expression by TGF-b. For example, the Smad [3], AP-1 [4], and SP-1 [5] pathways, all of which involve AP-1-binding transcription factors, are involved in this process, indicating that AP-1 may play an important role in the regulation of collagen synthesis. AP-1 has been implicated in various gene regulatory activities, associated with cell proliferation and extracellular matrix production, by TGF-b [6]. Accordingly, we hypothesize that AP-1 plays a critical role in overproduction of type I collagen and keloid pathogenesis and that this may provide a therapeutic target for keloid treatment. Recently, decoy oligodeoxynucleotides (ODNs) have been used to inhibit the expression of specific genes. It has been reported that treatment with AP1 decoy ODN abolishes the expression of several genes, including plasminogen activator, TGF-b and endothelin-1 [7]. However, there are no previous reports of the effects of an AP-1 decoy ODN in skin fibroblasts or specifically in keloid fibroblasts. The aims of this study were to investigate whether AP-1
functions as a transcription factor in keloid formation and to determine the effect of decoy oligodeoxynucleotides to AP-1 on type I collagen gene expression in cultured TGF-b1-treated keloid fibroblasts. In this study, the transcriptional activity of the AP-1 binding element was examined via electrophoretic mobility shift assay (EMSA), and the effect of AP-1 decoy ODN on type I collagen synthesis was determined via northern blot analysis, and CAT assay. Primary cultures of normal human fibroblasts (n = 3, mean age: 33 years) and keloid fibroblasts (n = 3, mean age: 24 years) were established from left over adult skin following cosmetic surgery. We used the same AP-1 decoy ODN which was used in the previous study [8], though it was not performed to the same object. The sequences of circular dumbell AP-1 decoy ODN (CDODN) against AP-1 binding sites and mismatched AP-1 decoy ODN (MODN, as control) are as follows (note: consensus sequences are underlined): CDODN: 50 -GGATCCATGACTCAGAAGACGACACACGTCTTCTGAGTCAT-30 MODN: 50 -GGATCCAAATCTCAGAAGACGACACACGTCTTCTGAGATTT-30 CDODN and MODN were transfected to normal and keloid fibroblasts for 24 h prior to TGF-b1 treatment using Lipopectamine Plus1 (Gibco-BRL, USA). The transfected cells were further incubated in complete medium for 24 h and this was followed by EMSA, northern blot analysis, and CAT assay. Results are expressed as mean S.E.M. Variance analysis with a subsequent Student’s t-test was used to determine significant differences in unpaired comparisons. All experiments were performed at least three times. First, We performed EMSA to confirm the effect of TGF-b1 on DNA binding activity of AP-1. AP-1 binding activity of keloid fibroblasts was higher than those in normal fibroblasts (1.6 0.4 versus 2.8 0.2; Fig. 1A, lanes 1 and 4). Moreover, TGFb1 treatment (5 ng/ml) increased AP-1 binding activity in nuclear extracts of cultured keloid fibro-
0923-1811/$30.00 # 2004 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology. doi:10.1016/j.jdermsci.2004.09.008
50
Letter to the Editor
Fig. 1 (A) AP-1 decoy (CDODN) down regulates AP-1 binding activity in cultured normal and keloid fibroblasts treated with TGF-b1 and AP-1 decoy as analysed by gel mobility shift assay. Lane 1: normal skin fibroblasts; lane 2: normal + TGFb1; lane 3: normal + TGF-b1 + AP-1 decoy; lane 4: keloid; lane 5: keloid + TGF-b1; lane 6: keloid + TGF-b1 + AP-1 decoy; and lane 7: gel retardation assay with unlabeled AP-1 ODN (10). (B) In the mismatched decoy ODN (MODN) transfected keloid fibroblasts, the TGF-b1 induced AP-1 binding activity was not affected. Lane 1: keloid; lane 2: keloid + TGF-b1; and lane 3: keloid + TGF-b1 + MODN. The values are mean S.D. and expressed as fold as compared to untreated keloid groups. *Significantly different from untreated keloid fibroblast (p < 0.05). **Significantly different from only TGF-b1 treated group (p < 0.01). Values in parenthesis indicate fold difference.
blasts to 1.9- and 3.1-fold the level of normal skin fibroblasts and untreated keloids, respectively (Fig. 1A, lanes 2 and 5). To determine the effect of AP-1 decoy ODN transfection on TGF-b1-induced AP-1 binding activity, cultured keloid fibroblasts were transfected with AP-1 decoy ODN prior to treatment with TGF-b1 (5 ng/ml). In transfected cells, AP-1 decoy ODN treatment decreased AP-1 binding activity to 0.31-fold that of the TGF-b1treated keloid fibroblasts (Fig. 1A, lane 6). But, in the mismatched decoy ODN (MODN) transfected keloid fibroblasts, the TGF-b1 induced AP-1 binding activity was not affected. (Fig. 1B, lanes 2 and 3) In vitro competitive binding assays, an increase in concentration of unlabeled AP-1 decoy ODN was accompanied by a corresponding decrease in the intensity of the retarded band, which is indicative of the presence of an ODN-AP-1 protein complex
(Fig. 1A, lane 7). In northern blot analysis of cultured normal and keloid skin fibroblasts, [32P] labeled a1(I) procollagen and GAPDH cDNA probes specifically hybridized with each mRNA. Northern analysis with a cDNA probe for a1(I) procollagen following TGF-b1 treatment resulted in a signal that was increased (560 20 versus 1002 11, by approximately 92%) compared to the untreated keloid group, after normalization to GAPDH mRNA levels. Furthermore, the overexpression of procollagen was decreased (1002 11 versus 240 21) by AP-1 decoy treatment compared to the TGF-b1treated keloid group. This implies that AP-1 decoy treatment decreased TGF-b1-induced a1(I) procollagen mRNA levels by approximately 76% (Fig. 2). CAT analysis revealed that the degree of acetylation was 7.1% in the untreated keloid group, 12.3% in the TGF-b1-treated group, and 6.1% in the group
Fig. 2 Northern blot analysis of a1(I) procollagen in cultured normal and keloid fibroblasts treated with TGF-b1 and AP1 decoy. Lane 1: normal skin fibroblasts; lane 2: normal + TGF-b1; lane 3: normal + TGF-b1 + AP-1 decoy; lane 4: keloid; lane 5: keloid + TGF-b1; and lane 6: keloid + TGF-b1 + AP-1 decoy. The values are mean S.D. and expressed as densitometric absorbance unit, which are the percentage of the value of GAPDH. *Significantly different from untreated keloid fibroblast (p < 0.05). **Significantly different from only TGF-b1 treated group (p < 0.01). Values in parenthesis indicate fold difference.
Letter to the Editor
treated with both TGF-b1 and AP-1. In comparison to the untreated keloid group, procollagen promoter activity was increased (7.1 0.6 versus 12.3 1.3) in the TGF-b1-treated group and this increased activity was inhibited (12.3 1.3 versus 6.1 1.2) by AP-1 decoy treatment in comparison to the TGF-b1-treated keloid group. This implies that AP-1 decoy treatment decreased TGF-b1-induced a2(I) procollagen promoter activity by approximately 51% (figure not shown) Taken together, our data suggest that transfection of an AP-1 decoy ODN suppresses TGF-b1 induced type I collagen gene expression in cultured keloid fibroblasts. By extension, in vivo transfection of target cells with sufficient quantities of the AP-1 decoy ODN may be useful for keloid treatment by preventing expression of type I collagen induced by TGF-b1. Inhibition of AP-1 during treatment would minimize the likelihood of immune-related side effects. Thus, this AP-1 decoy strategy may be particularly suitable for short-term management of keloid formation. Further studies are required to prove that the blockade of transcriptional factor AP-1 by AP-1 decoy ODN is sufficient to block the progression of keloid in vivo. Despite these limitations, this transcription factor decoy strategy may provide a new tool for treating keloid.
References [1] Ghosh AK. Factors involved in the regulation of type I collagen gene expression; implication in fibrosis. Exp Biol Med 2002;227:301—14. [2] Lee TY, Chin GS, Kim WJ, Chau D, Gittes GK, Longaker MT. Expression of transforming growth factor beta 1, 2 and 3 proteins in keloids. Ann Plast Surg 1999;43:179—84. [3] Chen SJ, Yuan W, Mori Y, Levenson A, Trojanowska M, Varga J. Stimulation of type I collagen transcription in human skin fibroblasts by TGF-b; involvement of Smad3. J Invest Dermatol 1999;112:49—57. [4] Chung KY, Agarwal A, Uitto J, Mauviel A. An AP-1 binding sequence is essential for regulation of the human a2(I) collagen (COL1A2) promoter activity by transforming growth factor-b. J Biol Chem 1996;271:3272—8. [5] Greenwel P, Inagaki Y, Hu W, Walsh M, Ramirez F. Sp1 is required for the early response of a2(I) collagen to TGFb1. J Biol Chem 1997;272:19738—45.
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[6] Tang W, Yang L, Leng SX, Elias JA. Transforming growth factorbeta stimulates interleukin-11 transcription via complex activating protein-1-dependent pathways. J Biol Chem 1998;273: 5506—13. [7] Ahn JD, Morishita R, Kaneda Y, Lee KU, Park JY, Jeon YJ, Song HS, Lee IK. Transcription factor decoy for activator protein-1 (AP-1) inhibits high glucose and angiotensin II-induced type 1 plasminogen activator inhibitor (PAI-1) gene expression in cultured human vascular smooth muscle cells. Diabetologia 2001;44:713—20. [8] Ahn JD, Morishita R, Kaneda Y, Lee SJ, Kwon KY, Choi SY, et al. Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo. Circ Res 2002;90:1325—32.
Chang Wook Kima,c Seong Suhb,c Su Hang Sungc In Kyu Leec,d Kyu Suk Leea,c,* a
Department of Dermatology School of Medicine Keimyung University Dong-San Dong 194, Daegu, South Korea b
Department of Microbiology School of Medicine Keimyung University Dong-San Dong 194, Daegu, South Korea c
Institute for Medical Science School of Medicine Keimyung University, Dong-San Dong 194 Daegu, South Korea d
Department of Internal Medicine School of Medicine, Keimyung University Dong-San Dong 194, Daegu, South Korea *Corresponding author. Tel.: +82 53 250 7625 fax: +82 53 250 7626 E-mail address: [email protected] (K.S. Lee) 1 March 2004