Original Contribution Journal of Cosmetic Dermatology, 12, 287--295
Protective effects of skin permeable epidermal and fibroblast growth factor against ultraviolet-induced skin damage and human skin wrinkles Jae Jin An, PhD,1,2,† Won Sik Eum, PhD,2,† Hyuck Se Kwon, MS,3 Jae Sook Koh, PhD,4 Soo Yun Lee, BS,4 Ji Hwoon Baek, PhD,4 Yong-Jun Cho, MD,5 Dae Won Kim, PhD,2 Kyu Huyng Han, PhD,2 Jinseu Park, PhD,2 Sang Ho Jang, PhD,1 & Soo Young Choi, PhD2 1
Bioceltran co., Ltd., Chuncheon, Gangwon-do, South Korea Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chuncheon, Korea 3 Adbiotech, co., Ltd., Chuncheon, Gangwon-do, Korea 4 Dermapro Skin Research Center, Seoul, South Korea 5 Department of Neurosurgery, Hallym University Medical Center, Chuncheon, Korea 2
Summary
Background Epidermal and fibroblast growth factor (EGF and FGF1) proteins play an important role in the regeneration and proliferation of skin cells. EGF and FGF1 have considerable potential as possible therapeutic or cosmetic agents for the treatment of skin damage including wrinkles. Objectives Using protein transduction domains (PTD), we investigated whether PTDEGF and FGF1 transduced into skin cells and tissue. Transduced proteins showed protective effects in a UV-induced skin damage model as well as against skin wrinkles. Methods Transduced PTD-EGF and FGF1 proteins were detected by immunofluorescence and immunohistochemistry. The effects of PTD-EGF and FGF1 were examined by WST assay, Western blotting, immunohistochemistry, and skin wrinkle parameters. Results The PTD-EGF and FGF1 increased cell proliferation and collagen type 1 alpha 1 protein accumulation in skin tissue. Also, PTD-EGF and FGF1 inhibited UV-induced skin damage. Furthermore, topical application of PTD-EGF and FGF1 contained ampoules which were considered to improve the wrinkle parameters of human skin. Conclusion These results show that PTD-EGF and FGF1 can be a potential therapeutic or cosmetic agent for skin damaged and injury including wrinkles and aging. Keywords: PTD-EGF and FGF1, skin permeation, wrinkle, UV, protein therapy
Correspondence: Dr. S Y Choi, Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea. E-mail: [email protected] Dr. S H Jang, Adbiotech, co., LTD. Geodudanji 1 gil 39, Dongnae-myeon, Chuncheon, Gangwon-do, Korea. E-mail: [email protected] †These authors contributed equally to this work. Accepted for publication September 5, 2013
© 2013 Wiley Periodicals, Inc.
Introduction Skin is continuously exposed to many hazardous environmental agents including ultraviolet (UV) light. The consequences of UV exposure are implicated in skin aging and cell death. Skin aging and damage are well characterized by loss of epidermal polarity and
287
PTD-EGF and FGF1 ameliorate skin damage
. J J An et al.
reduction in collagen, which can result in skin wrinkling, dryness, and cancer.1–4 Growth factors are implicated in the processes and suggested as therapeutic agents for skin healing. Among the many growth factors, epidermal growth factor (EGF) is a single polypeptide composed of 53 amino acids with three disulfide bonds, which promotes epithelial cell division and proliferation.5–7 Fibroblast growth factor-1 (FGF1), also called acidic fibroblast growth factor, is a member of the FGF family and is known to regulate cellular proliferation and survival. FGF1 is expressed in hair follicles and regulates hair growth.8–10 Although these growth factors are considered to be good agents for the prevention and treatment of skin damage including wrinkles, their inability to enter cells and tissues limits their therapeutic and cosmetic applications. Many studies have demonstrated that the delivery of exogenous full-length proteins into living cells via protein transduction, facilitated by protein transduction domains (PTDs), can be used as a successful tool for the therapeutic application of proteins.11–14 Studies by this research team have demonstrated both in vitro and in vivo the therapeutic effects of fusion proteins against a variety of diseases including those afflicting skin.15–19 In this study, we demonstrated that PTD-EGF and FGF1 proteins can be directly transduced into fibroblast cells and skin tissue. In addition, topical application of PTD-EGF and FGF1 proteins to mice markedly inhibited UV-induced skin damage and improved human skin wrinkles. Therefore, we suggest that PTD-EGF and FGF1 proteins may act as a potential therapeutic or cosmetic agent for skin injury including wrinkles.
Materials and methods Cell culture and materials
CCD-986sk cells, human fibroblast cells, were purchased from the American Type Culture Collection (ATCC; USA). The CCD-986sk cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS) and antibiotics (100 lg/mL streptomycin, 100 U/mL penicillin) at 37 °C under humidified conditions of 95% air and 5% CO2. Ni2+-nitrilotriacetic acid Sepharose superflow was purchased from Qiagen (Valencia, CA, USA). Antibodies against EGF, actin, and an His probe were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and FGF1 and phsopho-Erk1/2 were purchased from Cell signal Technology (Cell signal, MA, USA), and collagen
288
type 1 alpha 1, phosphor-JNK and phosphor-p38 were purchased from Abcam (Cambridge, UK). Human EGF and FGF1 cDNA were isolated using the polymerase chain reaction (PCR) technique. All other chemicals and reagents were of the highest analytical grade available. Expression and purification of PTD-EGF and FGF1 proteins
A PTD expression vector, PEP-1, was prepared in our laboratory as described previously.15 The cDNA sequences for human EGF and FGF1 were PCR-amplified using the following sense and antisense primers: EGF sense primers, 5′-CTCGAGATGGCAGAACCGCAGCCCCCGTCC-3′; EGF antisense primer, 5′-GGATCCCTACATTAAGGTTG CCATTTTGTT-3′; FGF1 sense primers, 5′-CTCGAGGTT TAATCTGCCTCCA-3′; and FGF1 antisense primer, 5′GGATCCTTAATCAGAAGAGAC-3′. The resulting PCR products were subcloned in a TA cloning vector and ligated into the PTD expression vector in frame with six histidine open-reading frames to generate the expression vector, and were cloned into Escherichia coli DH5a cells. The recombinant PTD-EGF and FGF1 plasmids were transformed into E. coli BL21 cells and induced with 0.5 mM IPTG at 37 °C for 3–4 h. Harvested cells were lysed by sonication at 4 °C in a lysis buffer (1% Triton X-100, 1 mg/mL lysozyme in sodium carbonate) three times. The isolated inclusion body was dissolved in solubilization buffer (50 mM sodium carbonate 8 M urea pH 9.6) containing 1 mM cysteine, stirred for 1 h and 5 mM cysteine, stirred for 24 h at room temperature. The supernatants were loaded onto a CM-sepharose column (Sigma-Aldrich, St. Louis, MO, USA), and the recombinant PTD-EGF and FGF1 were purified using a Ni2+-nitrilotriacetic acid Sepharose affinity column and PD-10 column chromatography (Amersham, Piscataway, NJ, USA). To remove endotoxins, purified PTD-EGF and FGF1 were treated using a Detoxi-GelTM Endotoxin Removing Gel (Pierce, Rockford, IL, USA). Endotoxin levels for PTD-EGF and FGF1 were below the detection limit (
Protective effects of skin permeable epidermal and fibroblast growth factor against ultraviolet-induced skin damage and human skin wrinkles Jae Jin An, PhD,1,2,† Won Sik Eum, PhD,2,† Hyuck Se Kwon, MS,3 Jae Sook Koh, PhD,4 Soo Yun Lee, BS,4 Ji Hwoon Baek, PhD,4 Yong-Jun Cho, MD,5 Dae Won Kim, PhD,2 Kyu Huyng Han, PhD,2 Jinseu Park, PhD,2 Sang Ho Jang, PhD,1 & Soo Young Choi, PhD2 1
Bioceltran co., Ltd., Chuncheon, Gangwon-do, South Korea Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chuncheon, Korea 3 Adbiotech, co., Ltd., Chuncheon, Gangwon-do, Korea 4 Dermapro Skin Research Center, Seoul, South Korea 5 Department of Neurosurgery, Hallym University Medical Center, Chuncheon, Korea 2
Summary
Background Epidermal and fibroblast growth factor (EGF and FGF1) proteins play an important role in the regeneration and proliferation of skin cells. EGF and FGF1 have considerable potential as possible therapeutic or cosmetic agents for the treatment of skin damage including wrinkles. Objectives Using protein transduction domains (PTD), we investigated whether PTDEGF and FGF1 transduced into skin cells and tissue. Transduced proteins showed protective effects in a UV-induced skin damage model as well as against skin wrinkles. Methods Transduced PTD-EGF and FGF1 proteins were detected by immunofluorescence and immunohistochemistry. The effects of PTD-EGF and FGF1 were examined by WST assay, Western blotting, immunohistochemistry, and skin wrinkle parameters. Results The PTD-EGF and FGF1 increased cell proliferation and collagen type 1 alpha 1 protein accumulation in skin tissue. Also, PTD-EGF and FGF1 inhibited UV-induced skin damage. Furthermore, topical application of PTD-EGF and FGF1 contained ampoules which were considered to improve the wrinkle parameters of human skin. Conclusion These results show that PTD-EGF and FGF1 can be a potential therapeutic or cosmetic agent for skin damaged and injury including wrinkles and aging. Keywords: PTD-EGF and FGF1, skin permeation, wrinkle, UV, protein therapy
Correspondence: Dr. S Y Choi, Department of Biomedical Science and Research Institute of Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea. E-mail: [email protected] Dr. S H Jang, Adbiotech, co., LTD. Geodudanji 1 gil 39, Dongnae-myeon, Chuncheon, Gangwon-do, Korea. E-mail: [email protected] †These authors contributed equally to this work. Accepted for publication September 5, 2013
© 2013 Wiley Periodicals, Inc.
Introduction Skin is continuously exposed to many hazardous environmental agents including ultraviolet (UV) light. The consequences of UV exposure are implicated in skin aging and cell death. Skin aging and damage are well characterized by loss of epidermal polarity and
287
PTD-EGF and FGF1 ameliorate skin damage
. J J An et al.
reduction in collagen, which can result in skin wrinkling, dryness, and cancer.1–4 Growth factors are implicated in the processes and suggested as therapeutic agents for skin healing. Among the many growth factors, epidermal growth factor (EGF) is a single polypeptide composed of 53 amino acids with three disulfide bonds, which promotes epithelial cell division and proliferation.5–7 Fibroblast growth factor-1 (FGF1), also called acidic fibroblast growth factor, is a member of the FGF family and is known to regulate cellular proliferation and survival. FGF1 is expressed in hair follicles and regulates hair growth.8–10 Although these growth factors are considered to be good agents for the prevention and treatment of skin damage including wrinkles, their inability to enter cells and tissues limits their therapeutic and cosmetic applications. Many studies have demonstrated that the delivery of exogenous full-length proteins into living cells via protein transduction, facilitated by protein transduction domains (PTDs), can be used as a successful tool for the therapeutic application of proteins.11–14 Studies by this research team have demonstrated both in vitro and in vivo the therapeutic effects of fusion proteins against a variety of diseases including those afflicting skin.15–19 In this study, we demonstrated that PTD-EGF and FGF1 proteins can be directly transduced into fibroblast cells and skin tissue. In addition, topical application of PTD-EGF and FGF1 proteins to mice markedly inhibited UV-induced skin damage and improved human skin wrinkles. Therefore, we suggest that PTD-EGF and FGF1 proteins may act as a potential therapeutic or cosmetic agent for skin injury including wrinkles.
Materials and methods Cell culture and materials
CCD-986sk cells, human fibroblast cells, were purchased from the American Type Culture Collection (ATCC; USA). The CCD-986sk cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS) and antibiotics (100 lg/mL streptomycin, 100 U/mL penicillin) at 37 °C under humidified conditions of 95% air and 5% CO2. Ni2+-nitrilotriacetic acid Sepharose superflow was purchased from Qiagen (Valencia, CA, USA). Antibodies against EGF, actin, and an His probe were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and FGF1 and phsopho-Erk1/2 were purchased from Cell signal Technology (Cell signal, MA, USA), and collagen
288
type 1 alpha 1, phosphor-JNK and phosphor-p38 were purchased from Abcam (Cambridge, UK). Human EGF and FGF1 cDNA were isolated using the polymerase chain reaction (PCR) technique. All other chemicals and reagents were of the highest analytical grade available. Expression and purification of PTD-EGF and FGF1 proteins
A PTD expression vector, PEP-1, was prepared in our laboratory as described previously.15 The cDNA sequences for human EGF and FGF1 were PCR-amplified using the following sense and antisense primers: EGF sense primers, 5′-CTCGAGATGGCAGAACCGCAGCCCCCGTCC-3′; EGF antisense primer, 5′-GGATCCCTACATTAAGGTTG CCATTTTGTT-3′; FGF1 sense primers, 5′-CTCGAGGTT TAATCTGCCTCCA-3′; and FGF1 antisense primer, 5′GGATCCTTAATCAGAAGAGAC-3′. The resulting PCR products were subcloned in a TA cloning vector and ligated into the PTD expression vector in frame with six histidine open-reading frames to generate the expression vector, and were cloned into Escherichia coli DH5a cells. The recombinant PTD-EGF and FGF1 plasmids were transformed into E. coli BL21 cells and induced with 0.5 mM IPTG at 37 °C for 3–4 h. Harvested cells were lysed by sonication at 4 °C in a lysis buffer (1% Triton X-100, 1 mg/mL lysozyme in sodium carbonate) three times. The isolated inclusion body was dissolved in solubilization buffer (50 mM sodium carbonate 8 M urea pH 9.6) containing 1 mM cysteine, stirred for 1 h and 5 mM cysteine, stirred for 24 h at room temperature. The supernatants were loaded onto a CM-sepharose column (Sigma-Aldrich, St. Louis, MO, USA), and the recombinant PTD-EGF and FGF1 were purified using a Ni2+-nitrilotriacetic acid Sepharose affinity column and PD-10 column chromatography (Amersham, Piscataway, NJ, USA). To remove endotoxins, purified PTD-EGF and FGF1 were treated using a Detoxi-GelTM Endotoxin Removing Gel (Pierce, Rockford, IL, USA). Endotoxin levels for PTD-EGF and FGF1 were below the detection limit (
