Adiponectin Suppresses UVB-Induced Premature Senescence and hBD2 Overexpression in Human Keratinocytes

<div><p>Recent studies have revealed that adiponectin can suppress cellular inflammatory signaling pathways. This study aimed to elucidate the effect of adiponectin on the unregulated production of hBD2 in UVB-induced premature senescent keratinocytes. We constructed an <i>in vitro</i> model of premature senescent keratinocytes through repeated exposure to low energy UVB. After repeated low energy UVB exposure, there was significant generation of reactive oxygen species (ROS) and induction of senescence-associated markers, including senescence associated beta-galactosidase activity and expression of p16^INK4a and histone H2AX. In addition, the present clinical study showed higher expression of hBD2 in sun-exposed skin of elderly group, and the overexpression of hBD2 was observed by c-Fos activation <i>in vitro</i>. Adiponectin has the ability to scavenge ROS and consequently inhibit MAPKs and SA-markers in UVB-exposed keratinocytes. An inhibitor study demonstrated that adiponectin downregulated hBD2 mRNA expression through suppression of the AP-1 transcription factor components c-Fos via inactivation of p38 MAPK. Collectively, the dysregulated production of hBD2 by the induction of oxidative stress was attenuated by adiponectin through the suppression of p38 and JNK/SAPK MAPK signaling in UVB-mediated premature senescent inducible conditions. These results suggest the feasibility of adiponectin as an anti-photoaging and anti-inflammatory agent in the skin.</p></div

Repeated low energy UVB irradiation-induced increase of SA markers and hBD2 expression in NHEK.

<p>NHEK were treated with repeated UVB radiation exposures (5 mJ/cm²) at scheduled exposure times (0 to 7 times) and the time interval between exposures was 30 min. (a) Viability, (b) induction of hBD2 expression, (c) β-gal activity and (d) SA-protein markers at 64 h after each repeated exposure to UVB on NHEK. The degree of cell senescence was quantified as the percentage of SA-β-gal positive cells and expressed as a percentage of the 0 exposure group. hBD2 expression was measured using whole cell lysates by ELISA assay. Data are presented as the mean ± SEM of three independent experiments (n = 3). *, <i>p</i><0.05, control <i>vs</i>. UVB treatment group. ns, no significance.</p

Adiponectin Suppresses UVB-Induced Premature Senescence and hBD2 Overexpression in Human Keratinocytes - Fig 4

<p><b>The protective effect of adiponectin pre-treatment on repeated UVB exposure induced (a) SA-β-gal activity, (b) SA-markers, and (c) hBD2 expression.</b> NHEK pre-treatment of adiponectin for 24 h, and then treated with 6 repeated UVB exposures. After 64 h, the degree of cell senescence was quantified as the percentage of SA-β-gal positive cells and expressed as a percentage of NC cells. Protein expression levels were analyzed by Western blot. hBD2 expression was measured using the whole cell lysates by ELISA assays (representative fluorescence images of hBD2 expression levels in the presence or absence of adiponectin (10 μg/ml), showed as photographs). Data are presented as the mean ± SEM of three independent experiments (n = 3). *, <i>p</i><0.05; **, <i>p</i><0.005, <i>vs</i>. NC. #, <i>p</i><0.05, <i>vs</i>. UVB treated group.</p

ROS scavenging effect of adiponectin in UVB exposed NHEK.

<p>UVB exposure was repeated 6 times and the time interval between exposures was 30 min. Intracellular ROS levels were measured by detecting the fluorescence intensity of the oxidant-sensitive florescent probe DCFH-DA. The florescence intensity was recorded in the presence or absence of adiponectin (10 μg/ml) at 485 nm/ 535 nm. Normalizing of the ROS florescence intensity was calculated using the PI fluorescence measurement intensity ratio. Data are presented as the mean ± SEM of three independent experiments (n = 3). *, <i>p</i><0.05; **, <i>p</i><0.005 <i>vs</i>. NC. #, <i>p</i><0.05, <i>vs</i>. UVB treated group. ns, no significance.</p

The inhibitory effect of adiponectin on hBD2 expression signaling molecules on repeated UVB exposed NHEK.

<p>(a) The time dependent phosphorylation of MAPKs induced by repeated UVB exposure. Adiponectin attenuated the phosphorylation of (b) JNK/SAPK, ERK and p38 MAPK phosphorylation (the relative expression levels were quantified and presented in graphical form) and (c) c-Fos and c-Jun protein expression. The UVB induced upregulation of hBD2 was suppressed through (d) the AP-1 components c-Fos and c-Jun. NHEK were treated with both SP600125 and SB203580 inhibitors before UVB exposure. Protein expression levels were analyzed by Western blot. And the relative (e) mRNA expression of c-Fos, c-Jun and hBD2 are represented in graphical form (fold change compared with NC cells). Data are presented as the mean ± SEM of three independent experiments (n = 3). *, <i>p</i><0.05, <i>vs</i>. NC. #, <i>p</i><0.05, <i>vs</i>. UVB treated group.</p

hBD2 expression of human-derived corneocytes affected by UV exposure and aging.

<p>Concentrations of hBD2 were determined by ELISA assay. The hBD2 expression of (a) hBD2 expression of total corneocytes between the elderly and young adult groups (n = 42). (b) All groups (elderly adult dorsal hand (Elderly-H), elderly adult abdomen (Elderly-A), young adult dorsal hand (Young-H), and young adult abdomen (Young-A)), elderly adult (c) dorsal hand and (d) abdomen (elderly male n = 10 and female n = 11). In both elderly and young adult groups n = 21. Data are presented as the mean. <i>p</i> values for the differences between groups were calculated with the Mann-Whitney test (without outliners).</p

Effect of sRAGE in Rat Aorta Vascular Smooth Muscle Cells.

<p>Western blot analysis showing expression of A, RAGE inflammation receptor protein, ICAM-1, MCP-1, and TNF-α; and B, Quantitative data from panel A. Expression of RAGE, ICAM-1, MCP-1, TNF-α, and β-actin were detected by western blot. Lane 1, control; lane 2, AngII-treated for 24 hours (100 nM); lane 3, HMGB1 treated for 15 minutes (1 µg/ml); lane 4, AngII plus HMGB1; lane 5, AngII plus HMGB1 and sRAGE (0.5 µg/ml); lane 6, AngII plus HMGB1 and sRAGE (1 µg/ml); lane 7, AngII plus HMGB1 and sRAGE (2 µg/ml); lane 8, treatment with sRAGE alone at 2 µg/ml. The optical density is expressed in arbitrary units normalized against a control. Data in histograms represent mean ± SD from 3 experiments. *<i>p</i><0.01 vs control, ^†<i>p</i><0.01 vs lane 4, ^#<i>p</i><0.01 vs lane 4, ns = non-significant.</p

Survival and Development of Atherosclerotic Lesions in Apo E KO mice with AngII-induced atherosclerosis.

<p>A. Survival graph of Angiotensin II-induced atherosclerosis in Apo E KO mice with saline injection, AngII-infused, and AngII with sRAGE-treated groups. B. Immunohistochemistry sections of aorta (left =  ascending aorta, right =  infrarenal abdominal aorta) staining for inflammation markers (RAGE, ICAM-1, VCAM-1, and MCP-1) from Apo E KO mice saline injection and AngII-induced atherosclerotic Apo E KO mice (magnification: x10 and below RAGE data: x20). C. Decreased intensity in atherosclerotic areas of sRAGE-treated AngII-induced Apo E KO mice compared to AngII-induced Apo E KO mice (n = 7). The positive stain is expressed in arbitrary units normalized against a control. *<i>p</i><0.01, Scale bar = 200 µm.</p

Effects of sRAGE on mRNA Levels in Aorta of AngII-induced Apo E KO mice.

<p>A. Reverse transcription PCR analysis of RAGE, ICAM-1 and MCP-1 gene expression. Data in histograms represent mean ± SD from 3 experiments. Lane 1, saline injection; lane 2, infusion of AngII; lane 3, infusion of AngII with 0.5 µg/day of sRAGE; lane 4, infusion of AngII with 1 µg/day of sRAGE; lane 5, infusion of AngII with 2 µg/day of sRAGE. B. RAGE, ICAM-1 and MCP-1 mRNA expression level were determined by Real-Time PCR. Results are means ± SD from 3 experiments each performed in duplicate. *<i>p</i><0.05, **<i>p</i><0.01, *** <i>p</i><0.001 and ns = non-significant.</p

Effect of sRAGE in Apo E KO mice on Expression of Cytokines and Adhesion Molecules.

<p>A-B. Apo E KO mice infused with AngII were treated with various concentrations of sRAGE. Expressions of RAGE, ICAM-1 and MCP-1 were detected by western blot. The optical density is expressed in arbitrary units normalized against β-actin control. Data in histograms represent mean ± SD from individual 3 experiments. Lane 1, saline injection; lane 2, infusion of AngII; lane 3, infusion of AngII with 0.5 µg/day of sRAGE; lane 4, infusion of AngII with 1 µg/day of sRAGE; lane 5, infusion of AngII with 2 µg/day of sRAGE, *<i>p</i><0.01.</p