Therapeutic evaluation of HIV transduction basic domain-conjugated superoxide dismutase solution on suppressive effects of the formation of peroxynitrite and expression of COX-2 in murine skin
© Liu et al. 2016
Received: 16 May 2015
Accepted: 12 January 2016
Published: 20 January 2016
Homeostasis of reactive oxygen species (ROS) in the skin is regulated by antioxidant defenses. The inflammatory states of skin diseases which range from acute rashes to chronic conditions are related to the level of ROS. The involvement of superoxide dismutase (SOD) in restoring the antioxidant capacity can then neutralize the inflammatory response.
We found that denatured Tat-SOD formulated in an aqueous medium could be delivered into mouse skin and the penetration signals of Tat-SOD were detected in the epidermis and dermis. According to immunohistochemical staining, Tat-SOD successfully suppressed inflammation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), the expression of sodium nitroferricyanide (SNP)-induced cyclooxygenase-2 (COX-2), and the production of nitrotyrosine proteins. In nerve growth factor (NGF) induced differentiated PC12 pheochromocytoma cells, we demonstrated that the denatured Tat-SOD regained its antioxidant activity and effectively protected PC12 cells from DNA fragmentation induced by paraquat. Using a luciferase reporter assay, the data was shown Tat-SOD protected PC12 cells from ROS damage, through suppression of COX-2 or nuclear factor-κB (NF-κB) activity occurred at the transcriptional level.
We showed that Tat-SOD inhibited SNP-induced COX-2 expression similarly to celecoxib and prevented the formation of peroxynitrite as 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The results suggest that denatured Tat-SOD solution may perform potential protein therapy for patients suffering from disorders related to ROS.
In physiological surroundings, the skin is constantly exposed to free radicals generated from external and internal circumstances. The buildup of reactive oxygen species (ROS) in the skin is determined by numerous antioxidant defense reactions, including both enzymatic and non-enzymatic mechanisms that either prevent the formation of ROS or scavenge those ROS generated. However, inflammatory conditions which overwhelm the antioxidant defense system can cause abnormal ROS levels that raise oxidative stress to pathological levels, resulting in deterioration of cells into diseased states, ranging from acute rashes with itching and redness, chronic syndromes like dermatitis (eczema) to psoriasis and even burn trauma, by way of oxidative modifications that alter biomolecular functions and structure and dysregulate cell signaling that results in aberrant cytokine release . For example, acute sunburn induced by UV-radiation opens up vulnerable skin to attack by excessive ROS due to depletion of antioxidant defenses [2–4]. Psoriasis, a chronic inflammatory skin disease, was also suggested to involve unregulated ROS levels, and its etiology is related to dysregulation of inflammatory pathways, like nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK) and JAK-STAT [5–8]. Burn injuries, a posttraumatic inflammatory condition, are thought to involve oxygen restoration which promotes superoxide production to exacerbate ischemic tissue injuries by increasing lipid peroxidation products, conjugated dienes, or malondialdehyde. Meanwhile, burns induce the upregulation of inducible nitric oxide (NO) synthase (iNOS), thus increasing the production of NO for peripheral vasodilatation, activation of the transcription factor NF-κB, and responses by numerous cytokines. NO highly reacts with superoxide anions to produce peroxynitrite, a risky mediator of cell and tissue injury [9–11]. Reducing oxidative stress by producing homeostasis through regulating ROS levels can then neutralize the inflammatory response [12–14].
Since the skin consists principally of a layer of vascular connective tissue (namely, the corium or cutis vera) and an external covering of epithelium tissue (namely, the epidermis or cuticle) with the peripheral endings of many sensory nerves, the sudoriferous, sebaceous glands and the hair follicles, it has limited excretory and absorbing powers. Some injuries that may affect all layers of the skin to cause significant cell damages by the subsequent inflammatory response. Competent therapeutic approaches to restore the antioxidant capacity in the skin can be achieved by preventing the formation of ROS, strengthening endogenous antioxidant enzymatic activity, and scavenging ROS using pharmacological or dietary supplements. Superoxide dismutase (SOD, EC 126.96.36.199) catalyzes the dismutation of superoxide and is considered a therapeutic protein for treating human inflammatory diseases. In several countries, SOD (orgotein) is locally injected for relief of sports injuries, osteoarthritis, and several inflammatory symptoms [15, 16]. Among available formulations of SOD, a liposomal-encapsulated native SOD injection was effective in treating systemic inflammatory diseases and skin ulcer lesions, especially those from wounds and burns [9, 17, 18]. Topical application of native Cu,Zn-SOD was also effective against skin lesions, antipruritic activity, and burns with skin transplantation [19, 20]. However, native SOD formulated by dissolving it in a vehicle of petrolatum or water did not retain its activity for long periods [9, 21, 22].
Several in vivo studies by intraperitoneal injection or oral administration of Tat-SOD indicated that the Tat-SOD has been observed the distribution in rats and mice, including liver, kidney, heart and brain. Topical application on mice ears of Tat-SOD has been found to suppress TPA-induced expression of proinflammatory cytokines and enzymes regulated by reducing the activation of NF-κB and MAPK . Increase of intracellular SOD activity resulting in decreasing the ROS induced damage was also reported in mammalian cells [24–27]. We previously constructed a human Cu,Zn-SOD (SOD1) fused with a nine-amino-acid (RKKRRQRRR) transactivator of transcription domain (Tat 49-57) of human immunodeficiency virus (HIV) type 1 in a bacterial vector to produce a Tat-SOD recombinant protein. The exogenous Tat-SOD, a novel modified form of SOD, exhibited direct cellular transduction into undifferentiated PC12 pheochromocytoma cells and a cellular protective function against oxidative stress from paraquat-induced cell death . In the other study, we found that Tat-SOD is conjugated to mesoporous silica nanoparticles (MSNs) to form MSN-Tat-SOD. The data showed that MSN-Tat-SOD effectively reduces cell apoptosis in HeLa cells through an endosomal escape action .
The aim of the present study was to develop an effective therapy using the denatured Tat-SOD solution. We first demonstrated that topical application of the denatured Tat-SOD protein delivered in vivo into murine skin and the data showed that Tat-SOD exhibited multiple effects of suppressing TPA-induced inflammation, SNP-induced cyclooxygenase-2 (COX-2), and nitrotyrosine production. Then, we also demonstrated Tat-SOD protein in vitro in differentiated PC12 cells suggested increasing intracellular SOD activity to protect neuronal cells from the ROS induced damage. The suppression of COX-2 or NF-κB expression by Tat-SOD was attributable to inhibition at the transcriptional level. The results suggest that denatured Tat-SOD solution may perform potential protein therapy for patients suffering from disorders related to ROS.
Isopropyl-L-D-thiogalactopyranoside (IPTG) and a luciferase assay kit were purchased from Promega (Madison, WI). Sodium nitroferricyanide (SNP), 12-O-tetradecanoylphorbol-13-acetate (TPA), indomethacin (Indo), and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) were purchased from Sigma (St. Louis, MO). Ni2+-nitrilotriacetic acid (NTA) Sepharose superflow was purchased from Qiagen (Valencia, CA). Lastly, celecoxib (CELEBREX™) was from Phamacia (Northumberland, UK).
Preparation of denatured recombinant Tat-SOD fusion proteins
Expression and purification of recombinant Tat-SOD fusion proteins were according to previous study [28, 30]. The recombinant Tat-SOD fusion proteins were purified under denaturing condition. To denature Tat-SOD fusion proteins, harvested cells were disrupted by sonication in a phosphate buffer (300 mM NaCl, and 50 mM NaH2PO4, pH 8) containing 8 M urea. After centrifugation, supernatants containing Tat-SOD fusion proteins were collected and purified by Ni2+-NTA Sepharose metal affinity resins according to the manufacturer’s instructions (Qiagen, Valencia, CA, USA). The purified denature-form proteins were concentrated and the salts were removed using Amicon® centrion (10 kDa MWCO,Millipore, Bedford, MA, USA). The concentrated and desalted proteins were estimated by the Bradford procedure using bovine serum albumin as a standard. Finally, the purified denatured proteins were dissolved in PBS containing 20 % glycerol and then aliquoted and stored at -80 °C.
Preparation of quantum dots conjugated with Tat-SOD
CdSe/ZnS quantum dots (560 nm; Nano Ocean Tech, Springdale, AR) were dissolved in chloroform and mixed with an equal volume of hydrating solution (distilled water: ethanol = 1: 1, 10 mg/mL sodium thioglycolate, and 2 μL ammonia). After the mixture was stirred for 1 h, the water layer was washed with the same volume of acetone and centrifuged at 5000 rpm. The pellet was dried and suspended in phosphate-buffered saline (PBS). The quantity of quantum dots was determined by a spectrophotometric method. The calculation followed Beer’s rule with a coefficient factor of 1.7 × 105. Equal moles of quantum dots and denatured Tat-SOD were mixed and incubated for 1 h before cell transduction.
Cell lines and culture conditions
PC12 cells, from a rat pheochromocytoma cell line, were obtained from American Type Culture Collection (Manassas, NJ) and cultured as recommended at 37 °C in Dulbecco’s modified Eagle medium (DMEM; GIBCO, Life Technologies Inc.) supplemented with 5 % heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT), 10 % horse serum, penicillin G (100 U/mL), streptomycin (100 μg/mL), and L-glutamine (2 mM) in a humidified atmosphere of 5 % CO2. Other reagents were purchased from HyClone.
Western blot analysis
PC12 cell lysates were separated on 10 % sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were then electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated 1 h at room temperature in blocking buffer (1× Tris-buffered saline-0.1 % Tween 20, and 5 % w/v nonfat dry milk), and then incubated with a polyclonal anti-human Cu,Zn-SOD antibody for 16 h at 4 °C. The membrane was extensively washed and incubated with a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G antibody (diluted 1:3000; Jackson, West Grove, PA) for 1 h at room temperature. Immunoreactive bands were visualized with an enhanced chemiluminescence substrate kit (NEN, Boston, MA) according to the manufacturer’s protocol.
Immunocytochemical staining for Tat-SOD expression
PC12 cells were fixed with 4 % paraformaldehyde, and permeabilized with 0.1 % Triton X-100 in PBS. After being washed with PBS, cells were blocked in blocking buffer for 1 h and then incubated with a polyclonal anti-human Cu,Zn-SOD antibody for 16 h at 4 °C. Cells were extensively washed and visualized with an anti-rabbit Cy3-labeled secondary antibody at a 1: 1000 dilution for 2 h, and nuclei were counterstained with DAPI (a DNA marker) for 5 min. Cells were analyzed with an Olympus IX70-FLA inverted fluorescence microscope. Images were taken using the SPOT system (Diagnostic Instruments, Sterling Heights, MI).
DNA fragmentation analysis
Cells were lysed for 3 h at 50 °C in lysis buffer (50 mM Tris–HCl, 10 mM EDTA, 0.5 % N-lauryl-sarcosine, and 0.5 mg/mL proteinase K), and then supplemented with an RNase solution in lysis buffer for 3 h at 50 °C. DNA was precipitated with phenol/chloroform (1: 1). DNA samples were then separated on a 1 % agarose gel, and visualized under UV illumination with staining.
Determination of SOD activity
SOD activities of cell lysates were measured according to a method of Marklund . The assay mixture, containing 500 μL buffer (50 mM Tris-cacodylic acid buffer at pH 8.4, 1 mM diethylenetriamine pentaacetic acid, and 1 mM phosphate buffer at pH 7.0), 50 μL of sample, 50 μL of pyrogallol, and 400 μL of water, was measured at an optical density of 420 nm. The increase in absorbance was measured for 3 min. SOD-specific activity was expressed as units per milligram (U/mg) of protein.
Inductively coupled plasma atomic emission spectroscopy (ICP-AES)
The plasma technology used with mass spectrometry (ICP-MS) for simultaneous trace element determination (Zn, Cu, Hg, Cd, and As) in recombinant proteins was carried out as the most suitable method . The water was Milli-Q grade (>18 MΩ). Equipment parameters, such as the torch position, voltages of the ionic lenses, and also the voltage of the detector and nebulization systems, were optimized and followed the manual instructions: power of 1.5 kW; plasma flow of 15 L/min; and auxiliary flow of 1.5 L/min. For calibration, a copper standard (1000 ppm, Merck, Darmstadt, Germany) was diluted with 0.15 % HNO3 to 0 ~ 50 ppb for use.
Circular dichroism (CD) measurements
Proteins for the CD analysis were dissolved in PBS to a concentration of 2.5 μg/μL. CD spectra were measured with a Jasco J-715 spectropolarimeter, and CONTIN software was used to analyze the data. The mean residue ellipticity was estimated from the mean residue weight, which was calculated from the primary structure .
Luciferase assay for COX-2 and NF-κB promoter activities
PC12 cells were transfected with a COX-2 or NF-κB luciferase reporter plasmid using Lipofectamine 2000 (Life Technologies Inc.) according to the manufacturer’s recommendations. At 24 h after transfection, cells were treated with Tat-SOD (0, 0.5, 1.0, 1.5, 2.0 μM), indomethacin (20 μg/mL), or PTIO (50 mM) for 2 h before the addition of TPA (50 ng/mL) or SNP (50 mM) for 4 h. Both firefly luciferase (FL) and Renilla luciferase (RL) activities were measured using a dual luciferase reporter assay kit (Promega Co.). The FL activity was normalized to the RL activity.
All experimental procedures were designed to minimize the total number of animals used and animal suffering during sacrifice of animal through cervical dislocation. All rules and regulations were followed during experimentation on animals and were approved by the local Animal Care Committee. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Taipei Medical University. (protocol #LAC-2013-0293). Six or seven-week-old female ICR mice were used as the experimental animals and purchased from the Laboratory Animal Center (National Taiwan University, Taipei, Taiwan). Animals were maintained in a room temperature and humidity-controlled facility with a 12-h light/dark cycle. A day before the experiments, the dorsal side skin was shaved using electric clippers. All samples in 0.05 mM CuCl2 and ZnCl2 were diluted with acetone and applied using an adjustable pipette (1 cm in diameter). The indicated doses of vehicle (acetone, 200 μL/site), Tat-SOD, celecoxib, or PTIO were topically administered to the shaven back of a mouse for 2 h, and then it was further treated with TPA (10 nmol/200 μL/site)  or SNP (20 μmol/200 μL/site) for 4 h. Finally, mice were sacrificed by cervical dislocation, and skin samples (1 cm in diameter) were obtained from the central dorsum of the mice for further experiments.
Immunohistochemical (IHC) staining of COX-2 and nitrotyrosine protein in mouse skin
Sections (4 μm) of formalin-fixed, paraffin-embedded tissue were collected onto silcanized glass slides and deparaffinized. For antigen retrieval, sections were boiled in 10 mM citrate buffer (pH 6.0) for 10 min. Each section was treated with 3 % hydrogen peroxide in methanol for 15 min and incubated with 2 % normal goat serum for 30 min. Sections were incubated with a primary polyclonal anti-COX-2 (Cayman, Ann Arbor, MI) or polyclonal anti-nitrotyrosine (Upstate, Lake Placid, NY) antibody. The section was developed using DAB (HPR EnVisionTM system, Dako, Glostrup, Denmark) and counterstained with Mayer’s hematoxylin.
Histological analysis of mouse skin
Skin samples were fixed with 4 % paraformaldehyde and then histologically stained with hematoxylin and eosin (H&E). Images were captured using an Olympus DP-70 camera on a Nikon ECLIPSE E800 microscope (at an original magnification of × 200).
Experiments were repeated at least 3 times with similar results. Statistical significance was determined using Student’s t-test as indicated in the legend. P-values are reported to 4 decimal points, and results are expressed as mean ± standard error of the mean (sem). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Preparation of denatured recombinant Tat-SOD
Tat-SOD attenuated SNP- and TPA-induced stress via cellular regulation of COX-2 and NF-κB in murine skin
Characterization of exogenous Tat-SOD function in vitro
In addition to evaluating the antioxidant ability, PC12 cells transduced with the Tat-SOD protein were then treated with paraquat (70 mM), an intracellular superoxide anion generator. Results (Fig. 5c) showed that the addition of paraquat induced abundant DNA fragmentation via cell apoptosis, and the Tat-SOD protein prevented this fragmentation, demonstrating that Tat-SOD effectively protected PC12 cells against attack by superoxide anions. Even though NGF protects neuronal cells from superoxide anion damage , ROS-mediated apoptotic damage has also found in the differentiated cells and could be diminished by the Tat-SOD protein.
Oxidative stress is defined as an impaired balance between ROS production and antioxidant defenses and is involved in many diseases . Removing ROS can be an effective precautionary measure against pathogenesis by exogenous SOD. SOD1, an oligomeric protein with Cu2+ and Zn2+, has a two-domain structure: one domain contains α-helices and the second is composed of both α-helices and β-sheets . Metal-binding sites are located between two domains with side chains of histidine and aspartate. In this paper, we demonstrated that the addition of heavy metals caused the loss of zinc, resulting in conformational changes of α-helices and β-sheets. It also implied that if the formulation was combined with protective chelating agents in the ointment, such as Na2EDTA or diethyldithiocarbamate, this would reduce the binding of Cu2+ and Zn2+ in SOD molecules . Improper vehicles such as large quantities of water or vaselinum album also caused rapid instability of the native SOD. Therefore, the strategy of using denatured protein makes preparation and formulation much easier and simpler by avoiding the risk of deactivating the native form and loss of ligand metals. The intracellular delivery of macromolecules still remains problematic because of the bioavailability restriction imposed by their intrinsic poor stability, membrane permeability, and endosomal trapping/degradation. Some experiments demonstrated the efficacy of oral SOD supplements or administration by subcutaneous, intravenous, intraperitoneal, or intramuscular injections. However, all these routes have limitations with transducing a protein into cells or the circulation, and almost all uses of native SOD could be still unnecessarily true . The design of Tat-SOD contains a Tat domain (RKKRRQRRR) at the NH2-terminus . Using cell-penetrating peptides to carry the peptides into cells was demonstrated to bypass the barrier in animal and culture models . Over 50 proteins of different sizes were reported in human or murine cell types in vitro [42–45]. We herein illustrated that the Tat-SOD penetrated into murine cells which was independent of receptors and transporters , and via an endosome escape mechanism .
In the present work, we assessed the effects of TPA on inducing inflammation in mouse skin. Our results of H&E histostaining agreed with TPA-induced vascular congestion and neutrophil accumulation in the perivascular region observed as a primary inflammatory reaction. Tat-SOD effectively attenuated cutaneous inflammation, suggesting that Tat-SOD affords a high therapeutic benefit in skin disorders, such as neutrophil dermatose and cutaneous inflammation . Similar to TPA, SNP-generated oxidative stress induced expressions of iNOS and COX-2 and stimulated NF-κB migration associated with the degradation of IκBα . Our results showed that topical administration of Tat-SOD inhibited SNP-induced COX-2 expression as well as did celecoxib. Celecoxib, a selective COX-2 inhibitor, is widely used to treat rheumatoid arthritis, psoriatic arthritis, adenomatous polyposis, and osteoarthritis [49, 50]. Compared to celecoxib, Tat-SOD exhibited a similar potency in dose-dependently inhibiting COX-2 expression through a different mechanism. For SNP-induced upregulation of iNOS, the presence of NO produced by reacting with superoxide anions leads to the formation of peroxynitrites, which immediately interact with tyrosine residues of proteins to form nitrotyrosines at a rate of 6.7×109 M-1 s. PITO serves for a stable NO scavenger without affecting NOS activity and significantly inhibits NO biological actions. In our study, Tat-SOD scavenged superoxide anions rather than NO, to attenuate the formation of nitrotyrosines. This suggests that applying Tat-SOD without blocking NO’s biological activities can prevent peroxynitrite-related diseases, such as sunburn erythema, contact hypersensitivity, and poly (ADP-ribose) polymerase-mediated diseases .
In conclusion, we developed an effective therapeutic application using the denatured Tat-SOD solution, a newly modified form of SOD, which could overcome the instability of native SOD formulations and side effects of anti-inflammatory agents. Tat-SOD was efficiently delivered in vivo with restoration of biological activity in mammalian tissues. Topical administration of Tat-SOD solution exhibited a dual effect of suppressing the formation of peroxynitrite and the expression of COX-2 in murine skin. This success lays the groundwork for eventual transduction of therapeutic proteins into patients with ROS-mediated diseases.
This work was supported by the National Science Council of Taiwan (NSC97-2113-M038-003). We also thank the Proteomic Core facility of Genomic Research Center at Academia Sinica (Taipei, Taiwan) for the protein structure analysis. We thank Ms. Ysanne Chen for her helpful English correction.
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