Ann Dermatol.  2018 Dec;30(6):645-652. 10.5021/ad.2018.30.6.645.

The Effect of Adiponectin on the Regulation of Filaggrin Expression in Normal Human Epidermal Keratinocytes

Affiliations
  • 1Department of Dermatology, Inje University Seoul Paik Hospital, Inje University College of Medicine, Seoul, Korea.
  • 2Department of Dermatology, Chung-Ang University Hospital, Chung-Ang University College of Medicine, Seoul, Korea. drseo@hanafos.com, kyky@caumc.or.kr
  • 3Department of Laboratory Medicine, Chung-Ang University Hospital, Chung-Ang University College of Medicine, Seoul, Korea.

Abstract

BACKGROUND
Adiponectin, an adipokine secreted from adipocytes, affects energy metabolism and also shows anti-diabetic and anti-inflammatory properties. Recent studies have reported that adiponectin plays a role in regulating skin inflammation.
OBJECTIVE
This study aimed to investigate the effect of adiponectin on the expression of filaggrin (FLG) in normal human epidermal keratinocytes (NHEKs).
METHODS
NHEKs were serum-starved for 6h before being treated with adiponectin. Afterward, cell viability was assessed by MTT assay. We also treated with calcium, interleukin (IL)-4, and IL-13 to provide positive and negative comparative controls, respectively. Gene mRNA expression was quantified using real time reverse transcription polymerase chain reaction, and protein expression was evaluated using Western blot. To evaluate the relationship among mitogen-activated protein kinases (MAPKs), activator protein 1 (AP-1), and FLG, we also treated cells with inhibitors for MAPKs JNK, p38, and ERK1/2.
RESULTS
FLG and FLG-2 mRNA expression in NHEKs significantly increased after treatment with 10 µg/ml adiponectin. Adiponectin also restored FLG and FLG-2 mRNA expression that was otherwise inhibited by treatment with IL-4 and IL-13. Adiponectin induced FLG expression via AP-1 and MAPK signaling.
CONCLUSION
Adiponectin positively regulated the expression of FLG and could be useful as a therapeutic agent to control diseases related to disrupted skin barrier function.

Keyword

Adiponectin; Cell differentiation; Filaggrin; Keratinocytes; Mitogen-activated protein kinases; Transcription factor AP-1

MeSH Terms

Adipocytes
Adipokines
Adiponectin*
Blotting, Western
Calcium
Cell Differentiation
Cell Survival
Energy Metabolism
Humans*
Inflammation
Interleukin-13
Interleukin-4
Interleukins
Keratinocytes*
Mitogen-Activated Protein Kinases
Polymerase Chain Reaction
Reverse Transcription
RNA, Messenger
Skin
Transcription Factor AP-1
Adipokines
Adiponectin
Calcium
Interleukin-13
Interleukin-4
Interleukins
Mitogen-Activated Protein Kinases
RNA, Messenger
Transcription Factor AP-1

Figure

  • Fig. 1 The effect of adiponectin on the differentiation of keratinocytes. (A) Keratinocyte cell viability after adiponectin treatment at 24 h, 48 h, and 72 h. Cells were serum-starved for 6 h before treatment with adiponectin (2.5, 5, 10, and 20 µg/ml). Cell viability was assessed by MTT assay and expressed as a percentage of the control (untreated) group. (B) Protein and mRNA expression of keratinocyte differentiation markers. Cells were treated with adiponectin (5, 10, and 20 µg/ml) for 72 h. Protein and mRNA expression was analyzed by Western blot and reverse transcription-polymerase chain reaction, respectively. Data are presented as the mean±standard deviation of three independent replicate experiments (n=3). NS: no significance, NC: normal control group, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, FLG, filaggrin. **p<0.005, control vs. adiponectin treatment group.

  • Fig. 2 Adiponectin induces filaggrin (FLG) and FLG-2 mRNA expression. The time dependent relative mRNA expression after adiponectin treated of (A) FLG and (B) FLG-2 was analyzed by real time reverse transcription-polymerase chain reaction. Data are represented in graphical form and show the fold change compared to normal control (NC) cells of the 1 h incubation group. The protective effect of adiponectin on (C) FLG and (D) FLG-2 mRNA expression under normally inhibitory treatment with Th2 cytokines interleukin (IL)-4 and IL-13. Calcium (1.2 mM) was used as a positive control for FLG expression. Data are presented in graphical form and show the fold change compared to NC cells. CypA expression levels were used as an internal control. Data are presented as the mean±standard deviation of three independent replicate experiments (n=3). (A, B) *p<0.05, **p<0.005, NC group vs. adiponectin-treated group. (C, D) *p<0.05; **p<0.005 vs. NC; #p<0.05, ##p<0.005 vs. IL-4- and IL-13-treated group.

  • Fig. 3 Adiponectin induced phosphorylation of mitogen-activated protein kinases (MAPKs) and activator protein 1. (A) The time-dependent phosphorylation of MAPKs was induced by adiponectin treatment (10 µg/ml). (B) Adiponectin and calcium (1.2 mM) differently phosphorylation of MAPKs proteins. Adiponectin regulated the phosphorylation of (C) Fra-1 and c-Jun protein expression. Protein and mRNA expression levels were analyzed by Western blot and reverse transcription-polymerase chain reaction, respectively. Data are presented in graphical form and show the fold change compared with sub-confluent normal control (NC) cells. Data are presented as the mean±standard deviation of three independent replicate experiments (n=3). *p<0.05, **p<0.005 vs. NC group; #p<0.05 vs. IL-4- and IL-13-treated group.

  • Fig. 4 Adiponectin regulation of filaggrin (FLG) mRNA expression depends on the activator protein 1 components c-Jun and fos related antigen-1 (Fra-1) and mitogen-activated protein kinase (MAPK) signaling. (A) MAPK inhibitors attenuated the phosphorylation of JNK/SAPK, ERK and p38. The relative (B) protein expression of Fra-1 and cJun and mRNA expression of (C) FLG and (D) FLG-2 after treatment with adiponectin and MAPK inhibitors. Normal human epidermal keratinocytes were treated with MAPK inhibitors SP600125, SB203580, and PD98059 before adiponectin treatment. Protein and mRNA expression levels were analyzed by Western blot and reverse transcription-polymerase chain reaction, respectively. Data are presented in graphical form and show the fold change compared with sub-confluent normal control (NC) cells. Data are presented as the mean±standard deviation of three independent replicate experiments (n=3). DMSO: used as vehicle, dimethyl sulfoxide, SP: SP600125, JNK inhibitor, SB: SB203580, p38 inhibitor, PD: PD98059, ERK inhibitor. *p<0.05, **p<0.005 vs. NC group; &p<0.05, &&p<0.005 vs. adiponectin-treated group.


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A Novel Effect of Lipids Extracted from Vernix Caseosa on Regulation of Filaggrin Expression in Human Epidermal Keratinocytes
Wu Qiao, Tinghan Jia, Hongjian Gu, Ruihua Guo, Ken Kaku, Wenhui Wu
Ann Dermatol. 2019;31(6):611-620.    doi: 10.5021/ad.2019.31.6.611.


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