Ann Dermatol.  2015 Oct;27(5):507-516. 10.5021/ad.2015.27.5.507.

Oxidative Damage and Nuclear Factor Erythroid 2-Related Factor 2 Protein Expression in Normal Skin and Keloid Tissue

• Lee YJ
• Kwon SB
• Kim CH
• Cho HD
• Nam HS
• Lee SH
• Lee MW
• Nam DH
• Choi CY
• Cho MK

• KMID: 2171450
• DOI: http://doi.org/10.5021/ad.2015.27.5.507

Abstract

BACKGROUND
Reactive oxygen species (ROS) play an important role in the induction of apoptosis under pathological conditions. Recently, a significant increase in ROS production and disrupted apoptosis mechanisms in keloids have been reported. Nuclear factor erythroid 2-related factor 2 (Nrf2) represents one of the most important cellular defense mechanisms against oxidative stress and is implicated in the regulation of apoptosis. Recently, it has been reported that Nrf2 upregulates Bcl-2, an anti-apoptotic protein.
OBJECTIVE
To compare Nrf2 protein expression in normal skin tissues to keloid tissues.
METHODS
ROS generation in keloid tissues was evaluated with OxyBlot analysis. Western blotting and/or immunohistochemical staining approaches were used to study expression of Nrf2 or Bcl-2 in keloid and normal skin tissues. Cellular fractionation was performed to examine subcellular distribution of Nrf2. Transfection of fibroblasts with Nrf2-specific small interfering RNA (siRNA) was conducted to understand the relationship between Nrf2 expression and apoptosis induction.
RESULTS
Protein oxidation, a marker of oxidative stress, is increased in keloid tissues. Western blot analysis clearly showed that Nrf2 and Bcl-2 are downregulated in keloid tissues. Immunohistochemical staining of Nrf2 confirmed the results of the western blot analysis. Transfection of fibroblasts with the Nrf2-specific siRNA results in increased apoptosis and decreased cell viability.
CONCLUSION
Collectively, our data indicate that Nrf2 expression is downregulated in keloid tissues, and that Nrf2 is involved in the development of apoptosis in Nrf2 siRNA-transfected fibroblasts. We propose that a defective antioxidant system and apoptotic dysregulation may participate in keloid pathogenesis.

Keyword

Apoptosis; BCL2; Keloid; NF-E2-related factor 2; Reactive oxygen species

MeSH Terms

Apoptosis
Blotting, Western
Cell Survival
Defense Mechanisms
Fibroblasts
Keloid*
NF-E2-Related Factor 2
Oxidative Stress
Reactive Oxygen Species
RNA, Small Interfering
Skin*
Transfection
NF-E2-Related Factor 2
RNA, Small Interfering
Reactive Oxygen Species

Figure

• 

  Fig. 1 Measurements of relative values for dinitrophenylhydrazine (DNP) derivatives in the oxyblot analysis of normal skin and keloid tissues. (A) The OxyBlot Protein Oxidation Detection kit (S7150; Millipore, Billerica, MA, USA) was used to detect overall carbonyl groups introduced into the protein side chains by oxidative modification. (B) The median values of normal skin and keloid tissues were measured by the Mann-Whitney test (n=8, *p<0.05).

• 

  Fig. 2 Nuclear factor erythroid 2-related factor 2 (Nrf2) protein expression in the nucleus and cytoplasm of normal skin and keloid tissues. (A) Nrf2 was seen in the nuclear (N) and the cytoplasmic (C) fraction of normal skin tissues and keloid tissues. β-actin (a control for cytoplasmic fractionation) and Lamin A/C (a control for nuclear fractionation) were detected by western blotting in the fractions. (B) Western blot analysis. Nrf2 expression was low in keloid tissues. β-actin used as a loading control. The human fibroblasts served as a positive control for Nrf2 expression. (C) The median values of normal skin and keloid tissues were measured by the Mann-Whitney test (n=8, *p<0.05).

• 

  Fig. 3 Representative immunohistochemistry staining for nuclear factor erythroid 2-related factor 2 protein expression in (A, B) paraffin-embedded normal skin tissue and (C, D) keloid tissue. (A) Strongly positive staining in keratinocyte of epidermis (×100). (B) Strongly positive staining in nucleus and cytoplasm of keratinocyte of epidermis (×400). (C) Weakly positive staining in keratinocyte of epidermis (×100). (D) Weakly positive staining in nucleus and cytoplasm of keratinocyte of epidermis (×400).

• 

  Fig. 4 Confirmation of cell viability in human fibroblast after transfection with nuclear factor erythroid 2-related factor 2 (Nrf2)-specific small interfering (siRNA). (A) Cells were transfected with 10 nM of Nrf2-targeting siRNA (siNrf2) or Stealth RNAi control (siC) for 24 h and 48 h, after which the levels of Nrf2 were measured by Western blot analysis. (B) Phase contrast images of cells treated with Nrf2-siRNA and control cells. (C) Annexin V-binding assay. 7-Aminoactinomycin D (7-AAD), phycoerythrin (Annexin V-PE). (D) Effects of Nrf2-siRNA silencing on proliferation of the fibroblasts.

• 

  Fig. 5 Bcl-2 expression in normal skin and keloid tissues. (A) Western blot analysis. Bcl-2 expression was low in keloid tissues. β-actin used as a loading control. The G361 cell line served as a positive control for Bcl-2 expression. (B) The median values of normal skin and keloid tissues were measured by the Mann-Whitney test (n=8, *p<0.05).

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