We developed a gene gun method for the transfer of human agouti signalling protein (ASP) cDNA to alter rat skin colour in vivo. Human ASP cDNA was cloned into a modified cytomegalovirus plasmid and delivered to the skin of Long–Evans rats by gene gun bombardment. Skin pigmentation, body weight and blood sugar of ASP cDNA-transfected rats were recorded against the control group, which were injected with plasmids encoding for green fluorescent protein. The treated skin showed lighter skin colour after 3 days of ASP gene transfection. This depigmentation effect was most prominent on day 14 and the skin gradually returned to its original pigmentation by day 28. Successful transfection of ASP gene in skin and hair follicles, as well as downregulation of melanocortin-1 receptor (MC1R) and tyrosinase expression upon treatment, was confirmed using immunohistochemistry and Western blot analysis. Body weight and blood sugar in the treated rats did not show statistically significant differences as compared to control groups. These observations demonstrate that gene transfer using the gene gun method can induce high cutaneous ASP production and facilitate a switch from dark to fair colour without systemic pleiotropic effects. Such a colour switch may be that ASP is acting in a paracrine fashion. In addition, this study verifies that ASP exerts its functions by acting as an independent ligand that downregulates the melanocyte MC1R and tyrosinase protein in an in vivo system. Our result offers new, interesting insights about the effect of ASP on pigmentation, providing a novel approach to study the molecular mechanisms underlying skin melanogenesis.
Skin pigmentation is an ideal phenotypic model to understand quantitative traits because it is easily measured and its study has important implications in medical and evolutionary studies. The regulation of human and animal pigmentation is complex and its mechanism remains to be further explored. Genetics plays a vital role at the molecular level in determining skin colour of mice, which is largely determined by melanocytes that can produce black/brown eumelanin or red/yellow phaeomelanin. Mammalian skin pigmentation and hair colour also depend on the relative amounts of the two types of melanin in skin and hair follicles. The switch between the synthesis of eumelanin and phaeomelanin is regulated by agouti signaling protein (ASP) and by α-melanocyte stimulating hormone (α-MSH), respectively.1 ASP acts as a competitive inhibitor of α-MSH in binding to the melanocortin-1 receptor (MC1R) on melanocytes.2, 3 ASP binding results in MC1R desensitization, consequently reducing cAMP and tyrosinase concentrations, as well as tyrosinase-related proteins 1 (TYRP1) and dopachrome tautomerase (DCT) activity.4 Thus, transient expression of the ASP gene in mouse hair follicles during the early hair growth cycle will initiate phaeomelanogenesis and, following α-MSH expression, switches on eumelanogenesis, giving rise to the characteristic subapical yellow bands in individual hairs of wild-type agouti mice.5, 6 Meanwhile, in addition to the yellow coat colour, several pleiotropic effects, such as obesity and hyperglycaemia, were presented in ASP transgenic mice or the ASP gene dominant mutations mouse model.
The human and mouse agouti genes are highly homologous; however, the role of ASP in human subjects has not yet been extensively investigated. In vitro studies have shown that human ASP acts in a similar fashion to mouse agouti protein, in that it downregulates melanogenic genes controlling eumelanin synthesis in human melanocytes, and its in vivo expression in transgenic mice gives rise to yellow coat colours.7, 8, 9, 10 Thus, it appears that the ASP plays an important role in the regulation of mice and human skin pigmentation and hair colour.
There is an increasing focus on gene therapy as a potential treatment for a number of specific disorders. To date, viral vectors have been widely used in most gene therapy trials. However, this often requires the construction of complex recombinant viruses and expensive viral production facilities. Furthermore, the relatively high cytotoxicity and immunogenicity of viral vectors hinder their potential use in clinical situations.11 The easy accessibility and visualization of the skin makes it an attractive target for gene therapy, and also serves as an ideal model for the application of ballistic gene transfer techniques.12 Bombarding DNA-coated microscopic gold particles into skin via a gene gun has shown to result in high concentrations of local transgenic expression, and thus has been a useful tool in genetic vaccination, cancer gene therapy and pain control.13, 14, 15 In this study, we report a technique of gene gun-mediated delivery of human ASP cDNA into the skin of Long–Evans (LE) rats to achieve skin depigmentation. Neither the feeding behaviour nor body weight, serum inflammatory marker interleukin-1β nor tumour necrosis factor-α, were changed compared to the control group. Our results show that local cutaneous transfer of ASP plasmids using gene gun technology can effectively alter rat skin colour without systemic pleiotropic effect.
Effects of ASP-cDNA gene gun injection on local skin depigmentation
To achieve depigmentation in vivo, the Helios gene gun was used to bombard the pigmented area on the dorsum of LE rats with pCMV-hASP, pCMV-hGFP or pCMV constructs (n=5–9 rats per group). We found that skin bombarded with gold particles containing pCMV-hASP started skin depigmentation from the third day and the effect was most prominent on day 14, but gradually returned to original pigmentation by day 28. The results of depigmentation at the site of ASP-cDNA gene gun injection for each time point are shown in Figure 1. No depigmentation was attained on skin bombarded with gold particles containing pCMV-hGFP or pCMV.
Time course of gene gun injection on local skin pigmentation
The alteration in skin pigmentation was quantified objectively using a Mexameter MX18 (Courage + Khazaka Electronic, GmbH, Koln, Germany). The dorsal skin colour of LE rats was measured at baseline and at days 1, 3, 7, 14, 21 and 28 after gene gun injection. ASP-cDNA gene gun-injected skin showed statistically significant (P<0.05) decreases in pigmentation when compared with the other control groups at days 3, 7, 14 and 21 (Figure 2).
Effect of ASP-cDNA gene gun injection on ASP, MC1R, tyrosinase and α-tubulin of skin
Skin biopsies were taken at each time point and the concentrations of ASP in skin, measured by Western blot analysis, are shown in Figure 3. Specimens taken at day 7 demonstrated the greatest ASP expression. There was also a significant increase in concentrations of ASP in the ASP-cDNA gene gun-treated animals on days 3 and 14 when compared with the GFP-treated group, but there was no significant difference on day 28. Importantly, there were significant differences between the ASP-treated and GFP-treated animals in the concentrations of MC1R and tyrosinase on days 3, 7 and 14 after gene gun injection. MC1R and tyrosinase concentrations were decreased associated with ASP overexpression in ASP-treated LE rats on days 3, 7 and 14.
Immunohistochemistry of ASP and tyrosinase expression after gene gun injection
ASP production in gene gun-treated skin tissue was further confirmed by immunohistochemistry. Cells expressing tyrosinase stained green and those expressing ASP stained red: the nuclei were stained blue for ease of identification. Before gene gun bombardment, tyrosinase was shown in the epidermis and in hair follicles (Figure 4a). In the skin samples bombarded with ASPcDNA, ASP was detected in the epidermis and in hair follicles on days 3, 7, 14 and 21 (Figures 4b–e). Tyrosinase expression was suppressed on day 3, 7 and 14 and gradually returned to the original level on day 28 (Figure 4f). In contrast, sections taken from the GFP-expressing controls or from control animals from which the primary antibody was omitted displayed no immunoreactivity (data not shown). Moreover, haematoxylin–eosin (HE) staining showed no abnormal inflammatory cell infiltration or skin necrosis after gene gun injection (Figures 5a–d). Fontana–Masson stain also demonstrated decreased melanin content in the epidermis on day 14 after ASP gene delivery (Figure 5e, f).
Body weight, blood sugar and cytokine measurements
All rats were weighed regularly up to 4 weeks after treatment. The treatment did not significantly influence weight gain as analysed by ANOVA with multiple measurements (P>0.05). No differences in blood glucose concentrations were found between the groups (data not shown). In addition, interleukin-1β and tumour necrosis factor-α concentration in sampled blood specimens did not vary significantly (data not shown).
Skin pigmentation results from the production and distribution of melanin in the epidermis, and is the major physiological defence against solar irradiation. Melanin is synthesized in epidermal melanocytes and then transferred into neighbouring keratinocytes via melanocyte dendrites. In mammals, melanogenesis is stimulated by ultraviolet irradiation, α-MSH and by cyclic adenosine monophosphate (cAMP)-elevating agents such as forskolin and isobutylmethylxanthine. The mechanism involves upregulating tyrosinase, which is the rate-limiting enzyme in melanogenesis by converting tyrosine to dopaquinone.16 Most studies have proposed that ASP acts by antagonizing the action of α-MSH at MC1R, resulting in decreased cell tyrosinase concentrations leading to suppression of eumelaninogenesis.6, 9 In this study, depigmentation in the rats started as early as day 3 after the pCMV-hASP gene was delivered to their skin using the gene gun (Figure 1). The effect was most prominent on day 14, but gradually returned to original pigmentation by day 28. Similarly, Western blot analysis also demonstrated that the expression of ASP protein was most prominent on days 3, 7 and 14, exhibiting an inverse relationship with tyrosinase expression (Figure 3). In addition to skin colour fading, newly grown hairs in the treated region faded simultaneously (figure not shown). Thus, the pCMV-hASP plasmid can be transfected to skin via the gene gun, and the expressed ASP gene acts on the melanocytes of both the skin and the hair follicles.
In this study, we also focused on a novel signalling mechanism in which ASP exerts its function by binding to and inducing the downregulation of MC1R. Decreased MC1R concentrations associated with ASP overexpression were demonstrated by Western blot analysis in ASP-treated LE rats on days 3, 7 and 14 after gene gun injection (Figure 3). Most studies have proposed that ASP acts as an antagonist of MC1R, causing MC1R desensitization and consequent reduction of tyrosinase activity.4, 17 However, Scott et al18 observed that ASP plays a role in the regulation of skin pigmentation through downregulation of MC1R mRNA transcription in cultured human melanocytes. Recently, microphthalmia transcription factor (MITF), which belong to the basic helix–loop–helix family, was found to regulate the gene for MC1R through its promoter region in cultured cells.19 Thus, the inhibitory effect of ASP on MC1R mRNA concentrations in a melanoma cell line might be mediated by inhibition of MITF expression.20 It is therefore possible that ASP exerts its effects on mammalian melanocytes not only by binding to MC1R, but also by activating another signal transduction pathway to reduce MC1R expression.
The success of gene therapy is largely dependent on the development of vectors that can deliver, and the efficient expression of the therapeutic gene in specific cell populations. Promoter-based gene expression was developed for cutaneous gene therapy to regulate the expression of introduced genes limited to the epidermis.21 High concentrations of transcription products in both the dermis and epidermis have been achieved using cytomegalovirus (CMV) promoters.22 We found that gene gun administration of pCMV-hASP caused the local skin melanin index of rats to decrease by 70% (Figure 2). The production of ASP on days 3, 7, 14 and 21 in the skin of LE rats in areas that were bombarded with pCMV-hASP plasmid was determined by immunohistochemistry (Figure 4b–e). These results demonstrate a positive correlation in the relative activities measured by immunohistochemical staining and a Mexameter. Therefore, the continuous production of ASP protein by keratinocytes, which have a lifespan of 19–34 days,23 is one of the important factors to keep the skin depigmented for up to 4 weeks long. However, it is possible that the transfected keratinocytes are secreting the ASP protein temporally, and partial activity of ASP is retained as a result of incomplete degradation.
Dominant mutations of the mouse ASP gene or the production of transgenic mice that ubiquitously express ASP from the human β-actin promoter will lead to a pleiotropic ‘yellow obesity’ syndrome.24 Yellowing is the result of chronic binding of ASP to MC1R, and the obese, hyperglycaemic phenotype may be due to the interaction between ASP and MC4R. However, in a transgenic mice model using the melanocyte-specific TYRP1 gene promoter and keratinocyte-specific keratin-14 gene promoter to limit ASP gene expression in mouse skin, the overexpressed agouti protein enables the fading of mouse coat colour phenotype, while leaving the characteristics of obesity and hyperglycaemia unaffected.7, 10 This indicates that ASP expressed in skin does not circulate as an endocrine or growth factor. Therefore, the diverse effects of ASP may depend upon the tissue of origin, possibly acting as a paracrine factor in a specific tissue, such as fat or brain, thus leading to the pleiotropic phenotypes. In our study, obesity, hyperglycaemia and changes in feeding behaviour were not recorded after pCMV-hASP gene delivery into rat skin. Thus, this approach can result in high cutaneous ASP concentrations without inducing systemic pleiotropic effects.
The gene gun offers several advantages over other in vivo gene delivery systems.25 First, the procedure requires minimal manipulation of the target tissue. Second, it is relatively easy to prepare compared with a viral delivery system. Third, this approach is a cell receptor-independent delivery system that allows DNA to penetrate directly through cell membranes into the cytoplasm or even the nuclei while bypassing the endosome/lysosome system, thus avoiding enzymatic degradation. Lastly, the advantage of the gene gun approach is the ease with which both the duration and amount of protein produced in vivo can be controlled. Moreover, unlike viral vectors, DNA plasmids induce no detectable vector-specific immune response and can be administered repeatedly. Neither interleukin-1β nor tumour necrosis factor-α, which are serum markers of inflammatory response, were elevated compared with the control group (data not shown). The skin biopsy specimens also demonstrated little inflammatory cell infiltration. The gene gun-mediated delivery system has thus proved to be a highly efficient and effective means of delivering the ASP gene to the epidermis with subsequent alteration of skin pigmentation.
Materials and methods
Male LE rats, 10–12 weeks old, (300–350 g; National Laboratory Animal Center, Taiwan), were used in this study. The rat is born with pigmented eyes, and a white flank coat with a black hood on the head and back of the neck, with a line down the back. Clipping the dorsal part of the hair, the shaved skin is presented as brownish with a variable irregular blackish patch individually. The rats were housed two per cage in chip-lined metal cages in a central animal care facility on a 12–12 h light–dark cycle, and fed on rat chow and water ad libitum and their dorsal hair was clipped daily. The ethic guidelines for all experiments with conscious animals were obeyed and the protocols were approved by the health guidelines of the Institutional Animal Care and Use Committee.
Human ASP cDNA
The human ASP construct (pCMV-hASP) was kindly provided by Dr GS Barsh (Department of Pediatrics and Genetics, Stanford University School of Medicine, CA). The full-length coding region of the ASP gene with an expected length of 540 bp was amplified by PCR using specific upstream and downstream oligonucleotides. The amplified fragment was cloned into the pUC18 vector and white colonies (lacZ disruption) were selected. The plasmid DNA was purified using Mini Plasmid DNA Preparation Kits (Qiagen, Valencia, CA, USA), digested with restriction enzymes, and separated electrophoretically on agarose gels. Plasmids with the correct insert were verified by DNA sequencing.
Gold particle-mediated gene transfer by gene gun bombardment
Gold particle-mediated gene transfer was applied into rat skin using gene gun bombardment. Plasmid DNA was precipitated on gold particles (1.5–2.0 μm) in the presence of 50 mM spermidine and 0.5 M CaCl2 at a final concentration of 3.75 μg DNA/mg of gold particle. The Versatile Helios Gene Gun System (Bio-Rad Laboratories, Hercules, CA, USA) was used for rapid and direct transfer of 10 pulses of DNA-coated gold microcarriers into a range of targets in vivo. Each pulse of helium expels the DNA-coated gold beads from a single 0.5-in (12.7 mm) segment of gold bead-coated tubing, and results in the delivery of 0.5 mg gold and 1.875 μg plasmid DNA per pulse. Group A (n=9) was injected with the pCMV-hASP plasmid in the pigmented back area. Group B (n=7), serving as the control group, received an injection of plasmid encoding green fluorescent protein (GFP). Group C (n=5) was injected with the pCMV vector only.
Measurement of skin colour
A Mexameter MX18 (Courage+Khazaka Electronic GmbH, Koln, Germany) was used to objectively quantify changes in melanin index. Melanin was measured at wavelengths of 660 and 880 nm. The higher the values, the greater the amount of melanin present. The dorsal skin colour was measured at baseline and on days 3, 7, 14, 21 and 28 after gene gun injection.
Blood glucose and body weight measurements
Animals were weighed, and 0.1 ml of blood was collected from the femoral vein under anaesthesia at baseline and on days 3, 7, 14, 21 and 28 after receiving experimental or control treatments. Blood sugar concentrations were measured using a LifeScan One-Touch blood glucose monitor (LifeScan, Milpitas, CA, USA).
Skin protein extraction and Western blot analysis
Skin tissue biopsies (∼0.5 g) were removed from the injected skin regions on days 0, 3, 7, 14, 21 and 28 after treatment. Skin tissue samples were kept frozen on dry ice and stored at −70°C. The tissue was defrosted, weighed and then homogenized using a polytron tissue homogenizer (1 min) in phosphate-buffered saline (PBS) containing 34 mg/l bacitracin, Complete™ protease inhibitors (Boehringer Mannheim, Indianapolis, IN, USA) and phosphatase inhibitors (1 mM sodium vanadate; 20 mM sodium fluoride). Samples were centrifuged (3000 g, 15 min at 4°C), and an aliquot (1 ml) was removed, lyophilized and stored at −20°C until further processing. Western blot analysis was performed on RIPA lysates (20 μg per lane), which were electrophoresed in 10% sodium dodecyl sulphate—polyacrylamide electrophoresis gels, and proteins were analysed by enhanced chemiluminescence Western blotting (Amersham, Arlington Heights, IL, USA). Antibodies used were monoclonal antibodies against tyrosinase (Cell Marque Corporation, Austin, Texas) and MC1R (Chemicon International Inc., Temecula, CA, USA). A polyclonal antibody against ASP (aPEP16; diluted 1:500) was raised in rabbits using the synthetic aPEP16 peptide kindly provided by Dr VJ Hearing (National Cancer Institute, National Institutes of Health, MD, USA). An antibody against α-tubulin was used as a loading control.
Skin sections of 10 μm were cryostatically made and fixed in acetone. Sections were rinsed in 0.1 M PBS for 20 min and blocked with 2% normal goat serum for 2 h at room temperature. The samples were then incubated with primary antibodies (1:500 dilution) against ASP (CN Biosciences Inc., San Diego, CA, USA) and tyrosinase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in 10% normal goat serum and 0.3% Triton-X 100 octylphenoxypolyethoxyethanol (Sigma, St Louis, MO, USA) for 20 h at 4°C. After washing with PBS with 0.3% Triton-X three times, the sections were then incubated with fluorescence conjugated secondary antibodies (Alexa fluor™ 488, 594, Molecular Probe, Eugene, OR, USA) for 1 h. The slides were then sealed with prolong antifade kit (Molecular Probe, Eugene, OR, USA) after drying and subjected to image study. To record the images, slides were examined with a fluorescence microscope (Leica, Germany) coupled to a CCD colour video camera and an image-analysing system (Meta-Morph, Universal Imaging Corporation, PA, USA). Control sections were also stained in a similar way without the primary antibody to ensure the specific binding of the antibody to the protein. The tissue processed in the absence of primary antibody had little immunostaining. Three sections per skin stained with HE for microscopic examination.
Data are expressed as means±s.e.m. (standard error of mean). One-way analysis of variance with post hoc analysis was used to detect differences among groups. Differences were deemed significant at P<0.05.
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This work was performed with the support of the Industrial Technology Research Institute of Taiwan and the National Science Council Grant NMRP 92-2314-B-182A-202 and Genome Project. We thank Dr VJ Hearing (National Cancer Institute, National Institutes of Health, MD, USA) for the polyclonal antibody (aPEP16) and Dr GS Barsh (Department of Pediatrics and Genetics, Stanford University School of Medicine, CA, USA) for the ASP plasmid.
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Yang, CH., Shen, SC., Lee, J. et al. Seeing the gene therapy: application of gene gun technique to transfect and decolour pigmented rat skin with human agouti signalling protein cDNA. Gene Ther 11, 1033–1039 (2004). https://doi.org/10.1038/sj.gt.3302264
- gene gun
- agouti signalling protein
- melanocortin-1 receptor
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