Ann Dermatol.  2017 Aug;29(4):407-413. 10.5021/ad.2017.29.4.407.

Adiponectin Upregulates Filaggrin Expression via SIRT1-Mediated Signaling in Human Normal Keratinocytes

• Jin T
• Park KY
• Seo SJ

• KMID: 2388937
• DOI: http://doi.org/10.5021/ad.2017.29.4.407

Abstract

BACKGROUND
Filaggrin (FLG) is the major component of the epidermal granular layer and binds to and condenses the keratin cytoskeleton. FLG thus contributes to cell compaction and serves as a natural moisturizing factor by promoting unfolding and degradation into hygroscopic amino acids. Loss or downregulation of FLG has been shown to result in a weak stratum corneum, which causes water loss and increases the possibility of skin barrier-related seizure. Adiponectin (Acrp30) contributes to the functional recovery of somatic cells, including human normal epidermal keratinocytes (NHEKs).
OBJECTIVE
To investigate the effect of Acrp30 in FLG expression and identifying its signal transduction mechanism.
METHODS
Normal human keratinocytes were treated with Acrp30 and the levels of FLG were examined. Silent mating type information regulation 2 homolog (SIRT)-targeting siRNA and aryl hydrocarbon receptor nuclear translocator (ARNT)-targeting siRNA were used to identify the role of various signal transduction pathway components.
RESULTS
Acrp30 upregulated SIRT1 and ARNT expression in NHEKs, resulting in increased FLG expression. Treatment with both SIRT1-targeting siRNA and ARNT-targeting siRNA blocked Acrp30 stimulation and silenced FLG expression.
CONCLUSION
Adiponectin upregulates FLG expression through a SIRT1-mediated pathway. Our results suggest that Acrp30 is a promising agent for skin barrier permeability improvement.

Keyword

Adiponectin; ARNT; Filaggrin; Keratinocytes; SIRT1

MeSH Terms

Adiponectin*
Amino Acids
Aryl Hydrocarbon Receptor Nuclear Translocator
Cytoskeleton
Down-Regulation
Humans*
Keratinocytes*
Permeability
RNA, Small Interfering
Seizures
Signal Transduction
Skin
Water
Adiponectin
Amino Acids
Aryl Hydrocarbon Receptor Nuclear Translocator
RNA, Small Interfering
Water

Figure

• 

  Fig. 1 Adiponectin stimulation increases filaggrin (FLG) expression in a time-dependent and dose-dependent manner without affecting the viability. Human normal epidermal keratinocytes (NHEKs) were treated with Acrp30 for 24 h at the indicated concentrations or with 10 µg/ml Acrp30 at the indicated time lines. (A) The MTT assay was performed to determine NHEK viability after Acrp30 treatment. (B, C) Cell lysates were generated with described lysis buffer and silent mating type information regulation 2 homolog (SIRT1), aryl hydrocarbon receptor nuclear translocator (ARNT), and FLG expression level was analyzed by immunoblotting. Representative results are shown from 3 independent experiments. NT: no treatment, O.D: optical density.

• 

  Fig. 2 Silent mating type information regulation 2 homolog (SIRT1) inhibition blocks Acrp30-induced filaggrin (FLG) upregulation. Human normal epidermal keratinocytes were pretreated with 60 µM sirtinol for 4 h, after which they were treated with 10 µg/ml Acrp30 for 12 h (A) or 24 h (B). Cell lysates were generated with described lysis buffer and analyzed by immunoblotting. The protein amounts were quantified by comparison to β-actin. Immunoblotting was used to analyze the levels of SIRT1, aryl hydrocarbon receptor nuclear translocator (ARNT), and FLG. All blots shown are representative of 3 independent experiments. ***p<0.001 compared to the untreated group, ****p<0.0005 compared to the untreated group. NT: no treatment, ST: sirtinol.

• 

  Fig. 3 siRNA-mediated silencing of silent mating type information regulation 2 homolog (SIRT1) blocks Acrp30 stimulation and filaggrin (FLG) regulation. Human normal epidermal keratinocytes were transfected with 20 nM of SIRT1-targeting siRNA or scrambled siRNA and incubated for 48 h. Next, the transfected cells were treated with 10 µg/ml Acrp30 for 24 h. Cell lysates were generated with described lysis buffer and analyzed by immunoblotting. The protein amounts were quantified by comparison to β-actin. Immunoblotting was used to analyze the levels of SIRT1, aryl hydrocarbon receptor nuclear translocator (ARNT), and FLG. All blots shown are representative of 3 independent experiments. **p<0.05 compared to the untreated group,. ***p<0.001 compared to the untreated group, ****p <0.0005 compared to the untreated group.

• 

  Fig. 4 siRNA-mediated knockdown of aryl hydrocarbon receptor nuclear translocator (ARNT) blocks Acrp30 stimulation and filaggrin (FLG) regulation. Human normal epidermal keratinocytes were transfected with 20 nM of ARNT-targeting siRNA or scrambled siRNA and incubated for 48 h. Next, the transfected cells were treated with 10 µg/ml Acrp30 for 24 h. Cell lysates were generated with described lysis buffer and analyzed by immunoblotting. The protein amounts were quantified by comparison to β-actin. Immunoblotting was used to analyze the levels of silent mating type information regulation 2 homolog (SIRT1), ARNT, and FLG. All blots shown are representative results of 3 independent experiments. ***p<0.001 compared to the untreated group.

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