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

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 in vitro 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 p16INK4a 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 in vitro. 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.


Introduction
As the skin is the most exposed tissue to environmental stress, repeated exposure to environmental stimuli include ultraviolet (UV) light leads to an enhanced inflammatory state and even aging of skin and a reduction of barrier function [1,2]. An essential component of the cutaneous defensive barrier, human beta defensin 2 (hBD2) is a cysteine-rich cationic 41 amino acid antimicrobial peptide, which is induced in cutaneous infection and inflammatory dermatitis [3]. It plays a role not only in cellular immune reactions and antimicrobial cytotoxic activities against bacteria, fungi, and viruses but also as a sacrificial sunscreens in UV exposed tissue [4,5]. Moreover, as a chemoattractant for immature dendritic cells, memory helper T cells, and p38 (SB203580) were purchased from Calbiochem (San Diego, CA, USA). Other chemicals and reagents used in this study were of analytical grade.

Repeated UVB irradiation
For UVB exposure studies, we use two TL 20 W/12 RS lamps (Philips, Eindhoven, Netherlands), emitting UVB peaking at 313 nm that were placed 15 cm above the cell culture flasks. The emitted radiation was checked at the bottom of each flask using a UV-meter (Waldmann, Villingen-Schwenningen, Germany). For repeated exposures to UVB, keratinocytes were seeded in flasks 2 days before and the cells were exposed to UVB 5 mJ/cm 2 in a serum free medium without flask cover. This exposure was repeated 6 times and the time interval between exposures was 30 min. Normal control (NC) cells were submitted to the same conditions but with no exposure to UVB.
Cell culture and viability assay NHEK were maintained in a 5% CO 2 humidified atmosphere at 37 in EpiLife supplemented with calcium. The cells were used at the second to third passages. Before the experiment, the cells were conditioned 4 to 6 h in serum-free medium. Cell viability was determined by MTT reduction assays, as described by Hansen, Nielsen [39]. In brief, the cells were incubated for at least 24 h in 96-well plates and pre-treated with 10 μg/mL concentrations of adiponectin for 24 h. Then, the cells were treated with repeated UVB exposures at room temperature. After the culture supernatants were removed, the resulting purple formazan was dissolved with dimethyl sulfoxide (DMSO) [40]. Absorbance values were read at 562 nm using a microplate spectrophotometer, SpectraMax340pc (Molecular Devices, Sunnyvale, CA, USA). Cell viability was calculated relative to the absorbance of the UVB untreated NC group.
Assay for senescence-associated beta-galactosidase (SA-β-gal) activity SA-β-gal activity was determined as described by Debacq-Chainiaux et al. [41]. In brief, the cells were washed twice with phosphate-buffered saline (PBS) and fixed with 2% formaldehyde/0.2% glutaraldehyde at room temperature for 10 min. After two additional washes with PBS, 1 ml of staining solution (40 mM citric acid/Na phosphate buffer, 5 mM K 4 [Fe (CN) 6 ] 3 ÁH 2 O, 5 mM K 3 [Fe(CN) 6 ], 150 mM sodium chloride, 2 mM magnesium chloride and 1 mg/mL X-gal in distilled water, pH 6.0) were added to the cells, and incubated overnight at 37°C. The degree of cell senescence was quantified as the percentage of SA-β-gal positive cells and expressed as a percentage of the NC.

Western blot analysis
Standard procedures were used for Western blotting. Briefly, the cells were lysed in 1% Triton-X radioimmunoprecipitation assay (RIPA) buffer for 1 min. The remaining cell debris was removed by centrifugation. The protein concentrations of the cell lysates were determined using the bicinchoninic acid assay (BCA) method. Cell lysates containing equal amounts of protein (~35 μg of total protein) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% bovine serum albumin (BSA) and then incubated with desired primary and secondary antibodies. Protein expression was detected using the EzWestLumi plus system (ATTO, Tokyo, Japan), according to the manufacturer's instructions. Blots were visualized using a ChemiDoc TM XRS image analyzer (Bio-Rad, Hercules, CA, USA), and the protein expression levels were quantified using ImageJ software and normalized to GAPDH and β-actin.

Intracellular ROS assay
Intracellular ROS levels were measured by detecting fluorescence intensity of the oxidant-sensitive 2',7'-dichloro-dihydro-fluorescein diacetate (DCFH-DA). In brief, DCFH-DA diffuses into cells and is de-acetylated by cellular esterases to non-fluorescent DCFH, which is rapidly oxidized to highly fluorescent 2',7'-dichlorodihydrofluorescein (DCF) by ROS. Human keratinocytes grown in fluorescence microtiter 96-well black plates were pre-incubated for at least 24 h and incubated with adiponectin (10 μg/ml) for an additional 24 h. The conditioned cells were washed once with PBS, labeled with 20 uM DCFH-DA in serum free media, and incubated for 30 min in the dark at 37°C. Then, UVB exposure was repeated 6 times and the time interval between exposures was 30 min. After the cells were washed once with PBS, intensity of the fluorescence signal was detected with an excitation wavelength of 485 nm and emission wavelength of 535 nm using a SpectraMax i3x microplate reader (Molecular Devices). The relative induction of ROS was normalized by subsequent staining and measurement of PI (propidium iodide) fluorescence.

RNA extraction and quantitative reverse transcription (RT)-PCR analysis
Total RNA was extracted from keratinocytes using TRIzol1 reagent (Welgene, Seoul, Korea) as reported in the manufacturer's manual. The cDNA synthesized from mRNA was then incubated for an additional 1 h at 42°C. Single-stranded cDNA was amplified by PCR with specific primers for genes encodinghBD2, c-Jun, c-Fos, and GAPDH. Quantification of mRNA was performed using a real-time thermal cycler (Bio-Rad) using SYBR premix Ex Taq (Takara, Shiga, Japan), as reported in the manufacturer's manual. All reactions were carried out in duplicate and the results were analyzed via the 2 -ΔΔCT method and data were normalized to GAPDH.

Quantification of human beta defensin 2 from human-derived corneocytes
The Institutional Review Board (IRB) of the Chung-Ang University hospital (Seoul, Korea) (IRB No. C2015210) approved the protocol for the collection of human-derived corneocytes. Forty-two healthy individuals were recruited, 21 individuals from 20 to 29 years of age (young adults) and 21 individuals from 64 to 81 years of age (elderly adults). All participants had no autoimmune diseases, cancer, diabetes, or dermatitis (either atopic, psoriasis, eczema, contact, or seborrheic) and did not use immunosuppressive drugs for at least 6 months. To obtain human skin corneocytes, 10 sheets of D-squame discs (a diameter of 22 mm, CuDerm Corp., Dallas, TX, USA) were applied to both the abdomen (sun protected) and dorsal hand (sun exposed) areas. The acquired D-squame discs were stored in a 20 ml glass bottle in a -80°C deep freezer until use. Proteins were extracted from the acquired D-squame discs using the Qproteome Mammalian Protein Prep kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The concentration of acquired proteins was determined by Bradford assay. The expression level of hBD2 in the corneocytes was determined with a human beta defensin 2 ELISA kit (Peprotech, Cedarlane, Burlington, ON, Canada). The concentration of beta defensin 2 total protein was determined by comparing the optical density (O.D.) of the samples to the standard curve. The measurements were carried out in duplicate.

Statistical analysis
All in vitro data are presented as the mean ± standard error (SE). The mean values were calculated based on data from at least three independent experiments that were conducted on separate days using freshly prepared reagents. Data were analyzed using the paired t-test. Clinical characteristics data were analyzed using non-parametric multiple comparison Kruskal-Wallis tests with a post hoc Mann-Whitney test to identify differences between groups (n = 21). The significance of differences was defined at a P value < 0.05 and the statistical analysis was performed using SPSS (PASW Statistics 18 software).

High expression of hBD2 in photo-aged skin
It has been reported that hBD2 expression is dependent on UV exposure and aging [15,42]. Therefore, the relationship of cutaneous hBD2 expression between the elderly (64 to 81 years) and young adults (20 to 29 years) was explored in human skin corneocytes collected from sunexposed (dorsal hand) and unexposed (abdomen) skin. The IRB of the Chung-Ang University hospital (Seoul, Korea) (IRB No. C2015210) approved the protocol for the collection of human-derived corneocytes. Forty-two healthy individuals were recruited, 21 individuals from 20 to 29 years of age (young adults) and 21 individuals from 64 to 81 years of age (elderly adults). All participants were screened based on medical history and the intake of drugs. As expected, a statistical difference between the elderly and young adults was observed ( Fig 1A). Also, the elderly and young adults groups were divided into a sun-exposed aged skin (-H) and unexposed skin (-A), respectively, the elderly-H showed higher hBD2 expression than the young-A (p = 0.002), and the difference of young-A compared to young-H showed statistical significance (p = 0.001). Interestingly, no significance was seen in between the elderly-A and -H, but elderly male adults had higher hBD2 levels than females (Fig 1B and 1C). The young adult group had no statistically significant sexual differences (data not shown).

Induction of senescence-associated markers by repeated low energy UVB exposure
Over the last years, studies on aging suggested that p16 INK4a is a robust biomarker of intrinsic cellular aging in skin, and is especially involved in UVB-induced senescence [43]. In addition, the histone H2AX has been identified as a component in DNA repair mechanisms and is regarded as a marker to analyze the impact of UVB exposure on the epidermis and is also an indicator of oxidative cell stress in senescent epidermal cells [44,45]. To determine the optimal condition for the induction of premature senescence, NHEKs were repeatedly irradiated with UVB (5 mJ/cm 2 ) with scheduled exposure times (2 to 7 times) with a 30 min time interval between exposures. At 64 h after the last UVB exposure, accumulation of p16 INK4a and phosphorylated H2AX protein reached a maximal level after 5 and 7 repeated UVB exposures, respectively ( Fig 2D). Also, the percentage of positive cells for SA-β-gal activity increased under the same conditions ( Fig 2C). However, cell viability at 64 h after a series of 7 exposures showed sub-cytotoxic stress conditions in the NHEK (Fig 2A). Therefore, a total of 6 exposures to UVB at 5 mJ/cm 2 were selected as the optimal premature senescence inducible condition.
The intracellular ROS scavenging effect of adiponectin in repeated low energy UVB-exposed NHEK The dose of adiponectin used in this study was based on the report by Tomizawa, Hattori [46] who showed that pre-incubation with 10 μg/ml adiponectin for 24 h has the maximal ability to Fig 1. hBD2 expression of human-derived corneocytes affected by UV exposure and aging. 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. p values for the differences between groups were calculated with the Mann-Whitney test (without outliners).
doi:10.1371/journal.pone.0161247.g001 suppress pro-inflammatory molecules. To analyze the intracellular ROS scavenging effect of adiponectin, NHEK were exposed with repeated low energy UVB (5 mJ/cm 2 ) followed by treatment with 10 μg/ml adiponectin for 24 h. Intracellular ROS levels were measured by detecting the fluorescence intensity of the oxidant-sensitive probe DCFH-DA. As shown in Fig 3, intracellular ROS levels were markedly increased after repeated low energy UVB irradiation exposures ( ÃÃ , p = 0.005) and adiponectin significantly reduced UVB induced intracellular ROS levels (#, p = 0.011) compared to the control group untreated with adiponectin.
Effect of adiponectin on the expression of senescence associated markers and hBD2 in premature senescent NHEK NHEK were prepared as described earlier. In premature senescent NHEK (Fig 4A and 4B), adiponectin evidently inhibited SA-β-gal activity (#, p = 0.033) and p16 INK4a and phosphorylated H2AX protein expression. Moreover, Fig 2B shows an exposure frequency-dependent induction of hBD2 expression with a significant difference compared with NC ( Ã , p = 0.023) on premature senescence conditioned NHEK. Indeed, the representative fluorescence images show the morphological changes of premature senescent NHEK and overexpression of hBD2 ( Fig 4C). Adiponectin has a statistically significant ability to reduce hBD2 overexpression (#, p = 0.039). Adiponectin attenuates p38 and JNK/SAPK MAPK signaling in repeated low energy UVB exposed NHEK Western blot analysis was used to assess the effect of adiponectin on MAPK signaling pathway molecules. NHEK were prepared as described earlier and protein lysates were harvested at 10 min (ERK1/2) and 30 min (p38 and JNK/SAPK) after a series of 6 UVB exposures. As shown in Fig 5A, repeated UVB exposures induced the phosphorylation of p38 ( Ã , p = 0.018) and JNK/SAPK ( Ã , p = 0.05) while ERK1/2 protein was constitutively expressed. The UVB-induced phosphorylation levels of p38 and JNK/SAPK were inhibited in the adiponectin pre-treatment groups (Fig 5B).  factor AP-1, were phosphorylated at 64 h after 6 UVB exposures. To determine whether the c-Fos and c-Jun (serine 63) expression was affected by p38 and JNK/SAPK signaling, the p38 and JNK inhibitors SB203580 (10 μM) and SP600125 (125 nM), respectively, were used. The inhibitors were applied to the cells 1 h before the first UVB exposure and again after the third exposure. Fig 5D shows a reduction of c-Fos and c-Jun phosphorylation by the p38 and JNK/SAPK inhibitors, respectively. Moreover, the phosphorylation of the p38 and c-Fos were most effectively attenuated by applying the SB203580 with adiponectin. Likewise, the induction of hBD2 mRNA expression was observed with the same trend as c-Fos mRNA expression, which was markedly reduced by p38 inhibitor treatment with adiponectin at 30 min after last UVB exposure (Fig 5E). In contrast with c-Fos expression, the c-Jun expression was not parelle with JNK/ SAPK expression.

Discussion
As the first immune barrier component against environmental stress, hBD2 plays a role in cellular immune reactions and provides a link between the innate and adaptive responses against cutaneous infection as a chemotactic factor [3,8]. However, its uncontrolled expression caused by inflammatory responses leads to immune disorders. Overexpression of hBD2 was found in elderly individuals and UV exposed condition [2,[14][15][16]. Therefore, the present study explored the relation between photoaging and hBD2 expression. For the investigate the hypothesis, we checked the hBD2 expression on the human derived corneocytes and constructed photoaging in vitro model.
There was no significant difference between dorsal hand and abdomen skin in elderly group represented that whether they exposed to sun light or not their hBD2 expression ascended with increasing age and these same trends already revealed in skin fluid and peripheral blood mononuclear cells (PBMCs) [2,14]. Meanwhile, the possible reasons why elderly adults' dorsal skin has higher hBD2 expression relative to young adults' abdomen skin could be due to an accumulation of UV exposure. Interestingly, unlike the young adult group, the elderly adult group showed a statistical difference between genders (Fig 1C and 1D). Based on a recent survey, these results could be explained by the difference in the using frequency of sunscreen and moisturizers (sunscreen (male: female = 51: 96.5), moisturizers (male: female = 27.5: 74.5)). Its cumulative effects throughout the life span might make a difference (Foundation of Korea Cosmetic Industry Institute, 2012). To prove this, a premature senescent NHEK in vitro model was constructed by a series of repeated low energy UVB exposures. At one or two exposures to low energy UVB, keratinocytes were able to completely repair the UVB-damaged DNA and continue normal cellular functions. However, increased repeated UVB exposures induced acute oxidative stress in the keratinocytes and impacted the increase of H2AX phosphorylation involved in DNA repair intermediates and accumulation of the tumor suppressor p16 INK4a , which both have been implicated in cellular senescence. The repeated UVB exposures also showed a high level of SA-β-gal activity (Fig 2). Indeed, morphological changes and overexpression of hBD2 were also observed in senescent keratinocytes. Moreover, the result of clinical research supports the correlation between photoaging and hBD2 expression (Fig 1A).
Recent reports showed the effect of adiponectin secreted from adipose tissue against the inflammatory response and cutaneous disease [35][36][37][38]. In the current study, our results show that adiponectin decreases intracellular ROS generation resulting from UVB irradiation. UV irradiation affects calcium influx in keratinocytes, and consequently induces the generation of intracellular ROS [47]. UVB irradiation-induced ROS generation mediated a variety of cellular responses including activation of the MAPK cascade and the induction of DNA damage, resulting in hBD2 overexpression and cellular senescence, respectively. Therefore, as the ROS scavenging effect of adiponectin could result in the prevention of DNA damage mediated by oxidative stress, this novel approach of adiponectin treatment in UV damaged keratinocytes has the possibility to delay cell senescence. Also, all of the in vitro results showed that p38 and JNK/SAPK MAPKs that are linked to AP-1 expression were markedly suppressed by adiponectin. It means that the excessive hBD2 expression was attenuated by ROS scavenging which down-regulated of hBD2 trascription in accordance with the decreased of AP-1 expression. And the inhibitor study elucidated c-Fos mRNA expression via p38 activation was the most closely associated with hBD2 mRNA expression.
In conclusion, the present study demonstrates that the effect of adiponectin on scavenging ROS and inhibiting SA-markers in UVB-exposed keratinocytes. The unregulated 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 NHEK. All these results provide a possibility of adiponectin for anti-photoaging and antiinflammatory agent in the skin.