Keloids are abnormal wound responses that are caused by hyperproliferative growth of connective tissue during the healing process. Recent research findings introduced the roles of reactive oxygen species (ROS) in the process of keloid formation. ROS induces oxidative stress and promotes the activities of oxidative damage-inducible genes. Aldo-keto reductase 1C3 (AKR1C3) prevents destructive ROS toxicity by detoxification of reactive carbonyl species. Thus, this study aimed to compare the expression of AKR1C3 in both normal and keloid skin
Six specimens of normal skin and six specimens of keloid tissues from human subjects were used to evaluate the expression of AKR1C3 by immunofluorescent staining of tissues and western blotting.
By western blotting, it was confirmed that the amount of AKR1C3 protein is significantly reduced in keloid tissues compared to normal tissues. Weak expression of AKR1C3 was also found in keloid tissues by immunofluorescent staining.
This study confirmed that the expression of AKR1C3 protein participates in ROS metabolism and plays a part in the downregulation of human keloid formation. To the best of our knowledge, this is the first work that reveals that AKR1C3 can affect the formation of keloids.
Keloids are among the most noticeable types of scars and may occur as a result of abnormal wound healing. They are formed by deposition of extracellular matrix components and proliferation of fibroblasts [
There have been many ongoing keloid-related studies because keloids cause serious functional and aesthetic problems. However, there are no uniformly successful treatments for keloid scars. Excision and primary repair frequently result in recurrence.
It was recently reported that keloid formation is associated with reactive oxygen species (ROS) [
ROS, including superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl anion (OH-), can oxidize and modify intracellular molecules. ROS production is induced either directly by oxidants/ oxidases or by a decrease in antioxidants and related upregulation of oxidative damage-inducible genes [
Lee et al. [
Aldo-keto reductases (AKRs) are engaged in metabolizing aldehydes that are byproducts of oxidative stress. AKRs are known to generate nicotinamide adenine dinucleotide phosphate (NAPDH)-dependent enzymes from several oxidoreductase families and reduce carbonyl groups and ketones of proteins [
This study examined the expression of AKR1C3 in both normal and keloid skin. To determine the correlation between AKR1C3 and keloid formation, the expression of AKR1C3 in normal and keloid skin tissues was analyzed using western blot and immunochemistry.
This research protocol, including the use of tissue samples, was reviewed and approved by the Institutional Review Board (IRB) of Seoul Soonchunhyang University Hospital. A total of six normal skin tissue samples and six keloid skin samples were collected from patients who underwent plastic surgery between December 2012 and February 2013 in the Departments of Plastic and Reconstructive Surgery at Soonchunhyang University Hospital in Korea. Six normal skin tissues were obtained from female patients who had breast reconstruction using a latissimus dorsi flap. Six keloid tissues were obtained from patients who had a surgical operation. The obtained tissue specimens were frozen in liquid nitrogen immediately after the resection and kept at -80°C until use. All tissues were used in conventional pathological confirmation and immunohistochemistry for a definite diagnosis. The keloid tissues were fixed with formalin and embedded in paraffin for immunohistochemical studies.
Tissue samples were homogenized in whole-cell enzyme-linked immunosorbent assay (ELISA) buffer. The tissue suspension was rotated for 10 minutes at 4°C. The supernatants were collected and kept at -70°C until use. Proteins extracted from tissues were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using NuPAGE 4–12% Bis-Tris gels and transferred to an Immobilon-P membrane. The membrane was blocked with 5% bovine serum albumin (BSA) in a tris-buffered saline with Tween 20 (TBST) solution, and then reacted with the primary mouse antibody diluted to a 1:1,000 concentration in TBST buffer. The membrane was subsequently washed four times (10 minutes, 10 minutes, 15 minutes, and 15 minutes) and then stored at 4°C for 16 hours. Proteins on the membrane were detected using an enhanced chemiluminescence solution kit. The membranes were stripped and reblotted with anti-actin antibody.
Paraffin sections (4 µm) were deparaffinized in xylene, rehydrated in 10 mM citrate buffer (pH 6.0), and then heated in a microwave oven for 15 minutes to restore antigens. To suppress endogenous peroxidase within tissues, the tissues were treated with 3% peroxide for 5 minutes and then with blocking solution for 30 minutes. The slides were then incubated with primary mouse anti-AKR1C3 antibody (catalog #A6229; Sigma, St. Louis, MO, USA) in a damp environment for 1 hour. Tissues were stained with 3,3ʹ-diaminobenzidine (ScyTek, Logan, UT, USA). The quantitative analysis of the comparative expression between keloid and normal skin tissues as expressed by western blotting was made with the TINA software version 2.10e (Raytest Isotopenmessgeraete, Straubenhardt, Germany) software program.
The data from the Raytest TINA program were evaluated by SPSS 17.0 (IBM Corp., Armonk, NY, USA). Our team used the nonparametric Mann-Whitney test to compare the keloid and normal skin groups. If a P-value was less than 0.05, it was considered highly statistically significant.
In the immunohistochemical study, AKR1C3 was weakly expressed in keloid skin tissues but rather strongly expressed in normal skin tissues (
According to western blotting results, AKR1C3 protein was expressed in both keloid and normal skin tissues. However, AKR1C3 protein was expressed at relatively lower levels in keloid skin tissues compared with normal skin tissues (
There are several reports that AKR1C3 is associated with ROS [
In this study, we measured AKR1C3 in keloid and normal skin tissues by western blotting and immunohistochemistry. We found that keloid skin tissues have lower AKR1C3 protein levels than do normal skin tissues.
Many biochemical processes are involved in cellular oxidationreduction reactions. Proteins are one of the major targets for oxidative stress [
ROS act as polyunsaturated fatty acids to generate lipid peroxides, known as RCS, including 4-hydroxy-2-nonenal (4-HNE) [
As mentioned above, a previous study reported that RCS are more highly expressed in keloid tissues than in normal skin using the OxyBlot kit [
There are carbonyl-reducing enzymes such as antidiuretic hormone (ADH), aldehyde dehydrogenase (ALDH), glutathione Stransferases (GSTs), and AKRs that lower the level of RCS via detoxification. The AKR superfamily consists of more than 100 members. In humans, 10 enzymes fall in the following subfamilies: AKR1A, AKR1B, AKR1C, AKR1D, and AKR7A, which are expressed in the tissues of the liver, brain, and kidney [
The AKR superfamily is characterized as monomeric, cytosolic, NADPH-dependent oxidoreductases that convert carbonyl groups into alcohols [
The physiological functions of AKR1Cs remain unclear, but members of AKR1C3 are considered major players in catalyzing the reductive detoxification of RCS and ketones, which are produced as a result of oxidative stress. The decrease in AKR1C3 that occurs under conditions of increased oxidative stress involved in the pathogenesis of keloid formation is one of the defense mechanisms against ROS toxicity [
Our study demonstrated that only a small amount of AKR1C3 is expressed in keloids by western blot analysis. Thus, keloid skin tissues may not be able to defend against oxidative stresses, such as ROS. Increased fibroblast proliferation in keloid skin is related to the increase in ROS generation and downregulation of detoxification enzymes such as AKR1C3, which account for the excessive proliferation of keloids. Accordingly, it is concluded that the decreased expression of AKR1C3 is associated with skin keloids in the altered metabolic state of increased ROS generation. Further studies are needed to understand the exact mechanism of signal transduction and modulation of AKR1C3 in keloids.
This work was supported in part by the Soonchunhyang University Research Fund.
No potential conflict of interest relevant to this article was reported.
Immunohistochemical study. (A) Aldo-keto reductase 1C3 (AKR1C3) in normal skin (immunochemical stain, ×20). (B) AKR1C3 in keloid tissue (Immunochemical stain, ×100).
Western blot analysis. Aldo-keto reductase 1C3 (AKR1C3) in normal (N) and keloid (K, diabetic) skin tissue.
Relative protein expression of Aldo-keto reductase 1C3 (AKR 1C3). The Raytest TINA software was used to assess protein expression in normal (SD=0.083) and keloid (SD=0.190) skin (P < 0.05).