Korean J Physiol Pharmacol.  2019 Mar;23(2):113-120. 10.4196/kjpp.2019.23.2.113.

Mannosylerythritol lipids ameliorate ultraviolet A-induced aquaporin-3 downregulation by suppressing c-Jun N-terminal kinase phosphorylation in cultured human keratinocytes

• Bae IH
• Lee SH
• Oh S
• Choi H
• Marinho PA
• Yoo JW
• Ko JY
• Lee ES
• Lee TR
• Lee CS
• Kim DY

• KMID: 2438085
• DOI: http://doi.org/10.4196/kjpp.2019.23.2.113

Abstract

Mannosylerythritol lipids (MELs) are glycolipids and have several pharmacological efficacies. MELs also show skin-moisturizing efficacy through a yet-unknown underlying mechanism. Aquaporin-3 (AQP3) is a membrane protein that contributes to the water homeostasis of the epidermis, and decreased AQP3 expression following ultraviolet (UV)-irradiation of the skin is associated with reduced skin moisture. No previous study has examined whether the skin-moisturizing effect of MELs might act through the modulation of AQP3 expression. Here, we report for the first time that MELs ameliorate the UVA-induced downregulation of AQP3 in cultured human epidermal keratinocytes (HaCaT keratinocytes). Our results revealed that UVA irradiation decreases AQP3 expression at the protein and messenger RNA (mRNA) levels, but that MEL treatment significantly ameliorated these effects. Our mitogen-activated protein kinase inhibitor analysis revealed that phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38, mediates UVA-induced AQP3 downregulation, and that MEL treatment significantly suppressed the UVA-induced phosphorylation of JNK. To explore a possible mechanism, we tested whether MELs could regulate the expression of peroxidase proliferator-activated receptor gamma (PPAR-γ), which acts as a potent transcription factor for AQP3 expression. Interestingly, UVA irradiation significantly inhibited the mRNA expression of PPAR-γ in HaCaT keratinocytes, whereas a JNK inhibitor and MELs significantly rescued this effect. Taken together, these findings suggest that MELs ameliorate UVA-induced AQP3 downregulation in HaCaT keratinocytes by suppressing JNK activation to block the decrease of PPAR-γ. Collectively, our findings suggest that MELs can be used as a potential ingredient that modulates AQP3 expression to improve skin moisturization following UVA irradiation-induced damage.

Keyword

Aquaporin-3; JNK mitogen-activated protein kinases; Mannosylerythritol lipid; PPAR gamma; Ultraviolet

MeSH Terms

Down-Regulation*
Epidermis
Glycolipids
Homeostasis
Humans*
JNK Mitogen-Activated Protein Kinases*
Keratinocytes*
Membrane Proteins
Peroxidase
Phosphorylation*
Phosphotransferases
PPAR gamma
Protein Kinases
RNA, Messenger
Skin
Transcription Factors
Water
Glycolipids
JNK Mitogen-Activated Protein Kinases
Membrane Proteins
PPAR gamma
Peroxidase
Phosphotransferases
Protein Kinases
RNA, Messenger
Transcription Factors
Water

Figure

• 

  Fig. 1 AQP3 expression is downregulated by UVA irradiation. (A) HaCaT keratinocytes were irradiated with the indicated doses of UVA or left non-irradiated (control) and harvested after 24 h for Western blot analysis. (B) The protein level of AQP3 was quantified relative to that of GAPDH using the Image J software from National Institutes of Health. The results are presented as the mean expression level obtained from three independent experiments ± SD (**p < 0.01). (C) UVA 3 J/cm2-irradiated and non-irradiated control cells were harvested at the indicated time points, and qRT-PCR analysis of the AQP3 mRNA level was performed. The data were normalized with respect to the expression of GAPDH, and the results are presented as the mean expression ± SD (n = 3, **p < 0.01). (D) HaCaT keratinocytes were treated with the indicated doses of UVA and cell viability was determined using a CCK-8 kit (Dojindo, Kumamoto, Japan) 24 h after irradiation of indicated doses of UVA. The viability of cells treated with UVA 3 J/cm2 decreased to 90% without significance. AQP3, aquaporin-3; UVA, ultraviolet A; N.S., not significant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SD, standard deviation; qRT-PCR, quantitative real-time polymerase chain reaction; mRNA, messenger RNA.

• 

  Fig. 2 MELs ameliorate the UVA-induced downregulation of AQP3 expression in HaCaT keratinocytes. (A) A structure of MELs. Two fatty acids were attached to −OH groups on mannosylerythritol as an ester bond. The fatty acid contains mainly 6–18 carbon atoms and the major is 8 carbon atoms. (B) HaCaT keratinocytes were treated with the indicated concentrations of MELs for 24 h and cell viability was determined using a CCK-8 kit (Dojindo, Kumamoto, Japan). DMSO was used as the vehicle control. (C) Cells treated with UVA 3 J/cm2 and the indicated concentrations of MELs for 24 h were subjected to Western blot analysis of AQP3 proteins. (D) The protein levels of AQP3 were quantified relative to that of GAPDH using the Image J software. (E) The mRNA expression of AQP3 was analyzed by qRT-PCR. The results are presented as the mean expression level obtained from three independent experiments ± SD (**p < 0.01). MELs, mannosylerythritol lipids; UVA, ultraviolet A; AQP3, aquaporin-3; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; SD, standard deviation.

• 

  Fig. 3 Involvement of JNK phosphorylation in the UVA-induced downregulation of AQP3. (A) Cells were irradiated with UVA 3 J/cm2 and harvested at the indicated time points. Protein samples were subjected to Western blotting for phospho-JNK, total JNK, phospho-ERK, total ERK, phospho-p38, total p38, and GAPDH. (B) Cells pretreated with 100 nM SP600125 (a JNK inhibitor) for 1 h were irradiated with UVA 3 J/cm2 and harvested after 24 h. The protein level of AQP3 was analyzed by Western blotting and quantified relative to the level of GAPDH using the ImageJ software. (C) The mRNA expression of AQP3 was examined by qRT-PCR. (D) Cells pretreated with 10 µM PD98059 (an ERK inhibitor) or 5 µM SB203580 (a p38 inhibitor) for 1 h were irradiated with UVA 3 J/cm2 and harvested after 24 h. The protein level of AQP3 was analyzed by Western blotting and quantified relative to the level of GAPDH using the ImageJ software. Values represent the mean expression level ± SD (n = 3, *p < 0.05, **p < 0.01). JNK, c-Jun N-terminal kinase; UVA, ultraviolet A; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AQP3, aquaporin-3; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction; SD, standard deviation.

• 

  Fig. 4 The effects of MELs on JNK phosphorylation and PPAR-γ expression in UVA-irradiated HaCaT keratinocytes. (A) Cells pretreated with the indicated concentrations of MELs for 1 h were irradiated with UVA 3 J/cm2 and harvested after 15 min. The protein levels of phospho-JNK, total JNK, phospho-ERK, total ERK, phospho-p38, total p38, and GAPDH were evaluated by Western blot analysis. (B) Cells treated with UVA 3 J/cm2 and the indicated concentrations of MELs or a JNK inhibitor for 16 h were prepared for qRT-PCR analysis of PPAR-γ mRNA expression. Values represent the mean expression level obtained from three independent experiments ± SD (*p < 0.05, **p < 0.01). (C) Proposed model showing how MELs ameliorate the UVA-induced downregulation of AQP3. MELs, mannosylerythritol lipids; JNK, c-Jun N-terminal kinase; PPAR-γ, peroxidase proliferator-activated receptor gamma; UVA, ultraviolet A; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR, quantitative real-time polymerase chain reaction; SD, standard deviation.

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