BAPTA-AM | Ckit Signal

Solar light induces expression of acetylcholinesterase in skin keratinocytes: Signalling mediated by activator protein 1 transcription factor

Qiyun Wu a,b, Panzhu Bai b, Yingjie Xia b, Queenie W.S. Lai b, Maggie S.S. Guo b, Kun Dai b, Zhongyu Zheng b, Christine S.J. Ling b, Tina T.X. Dong a,b, Rongbiao Pi c, Karl W.K. Tsim a,b,*

A B S T R A C T

Acetylcholinesterase (AChE) hydrolyses acetylcholine to choline and acetate, playing an important role in terminating the neurotransmission in brain and muscle. Recently, the non-neuronal functions of AChE have been proposed in different tissues, in which there are various factors to regulate the expression of AChE. In mammalian skin, AChE was identified in melanocytes and keratinocytes. Our previous study has indicated that AChE in keratinocyte affects the process of solar light-induced skin pigmentation; however, the expression of AChE in keratinocytes in responding to sunlight remains unknown. Here, we provided several lines of evidence to support a notion that AChE could be upregulated at transcriptional and translational levels in keratinocytes when exposed to solar light. The light-mediated AChE expression was triggered by Ca2+, supported by an induction of Ca2+ ionophore A23187 and a blockage by Ca2+ chelator BAPTA-AM. In addition, this increase on AChE transcriptional expression was eliminated by mutagenesis on the activating protein 1 (AP1) site in

Keywords:
Acetylcholinesterase
Keratinocyte
Solar light
Calcium AP1

1. Introduction

Neurotransmitter is a type of chemical that transmits signal in a synapse. Acetylcholine (ACh) is a neurotransmitter in the cholinergic synapse, which plays an important role in signal transduction in nervous system and neuromuscular junction (NMJ) (Blokland et al., 1995; Tiwari et al., 2013). In cholinergic synapse, ACh is loaded into synaptic vesicles by the vesicular acetylcholine transporter (VAChT) in pre-synaptic cells. The action potential depolarizes the membrane of pre-synaptic cells to cause the Ca2+ influx, resulting in the release of ACh that subsequently binds to the acetylcholine receptor (AChR) (Tiwari et al., 2013). Finally, the enzyme acetylcholinesterase (AChE) hydrolyses ACh into acetate and choline to terminate this signal transmission (Rosenberry et al., 1975; Massouli´e et al., 1982, 1993).
The regulation of AChE is varied in different cells, which has been studied extensively for years. In myogenesis, the regulation of AChE is theorised to be mediated by a cAMP/PKA-dependent signalling (Choi et al., 2000; Siow et al., 2002). Muscle paralysis leads to a decrease in AChE transcript at the post-synaptic sarcoplasm (Michel et al., 1994). During myotube formation, AChE is upregulated at the NMJ (Tsim et al., 1992, 2006; Leung et al., 2009). Similar in neuronal cells, the expression of AChE in muscle is regulated by activating the cAMP signalling cascade (Wan et al., 2000). In the process of neuronal differentiation of pluripotent stem cells, the stabilization of mRNA could result in an increase of AChE mRNA and protein (Coleman et al., 1996).
AChE in different tissues is proposed to have various biological functions. The existence of AChE in non-neural tissues and the diversity of molecular structure of AChE suggest the possibility of non-neuronal and/or non-cholinergic functions of AChE (Jbilo et al., 1994; Soreq et al., 2001; Mor et al., 2008; Luk et al., 2012; Xu et al., 2017, 2018). For example, AChE is expressed in bone tissue, osteoblast and osteoblast-like cell lines. During bone remodelling processes in both in vitro and in vivo models, the cholinergic signal induces bone formation (Eimar et al., 2013). The expression of AChE in osteoblast is found to be regulated by Wnt/β-catenin signalling pathway during osteoblastic differentiation, suggesting its function in bone formation (Xu et al., 2017). In mouse skin, the highly diluted ACh has been shown to promote the wound repair in vivo, which suggested a role of cholinergic system in wound healing (Uberti et al., 2018). In TF-1 erythroblast cells, the manipulation of AChE expression led to the change of GATA-1, α and β globin expressions, suggesting a regulatory role of AChE during erythropoiesis (Luk et al., 2012; Xu et al., 2018). The presence of low AChE activity in skin epidermis is evident in patients suffering from vitiligo (Iyengar et al., 1989), implying possible functional role of AChE in skin pigmentation (Schallreuter et al., 1988, 2005). Besides, ACh inhibits the long-term hypoxia-induced apoptosis in mouse stem cells, suggesting its protective and trophic effects (Kim et al., 2008).
The cholinergic system in skin has been shown to play a critical role in skin pigmentation (Wu et al., 2018, 2020). In skin, ACh was released from keratinocyte when stimulated by solar light, which then inhibited melanin production in melanocyte to attenuate skin pigmentation, as induced by ultraviolet. Thus, the amount of AChE, or application of AChE inhibitor, could regulate the skin pigmentation. Here, we further explored the proposed notion of non-neuronal function of AChE in skin, and we aimed to reveal the regulation of AChE in keratinocyte. The expression of AChE was significantly upregulated by solar light through Ca2+ signalling, mediated by the transcriptional factor activating protein 1 (AP1) in the ACHE gene. These findings indicate that the exposure of solar light could regulate the expression of cholinergic protein, suggesting its potential role in skin.

2. Materials and methods

2.1. Chemicals

BAPTA-AM, A23187 and CaCl2 were purchased from Sigma-Aldrich (St Louis, MO). All cell culture reagents were from Thermo Fisher Scientific (Waltham, MA).

2.2. Cell culture

Immortalized human epidermal keratinocyte line, HaCaT cells, was purchased from AddexBio (San Diego, CA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin (10,000 U and 10,000 μg/mL) in a humidified atmosphere with 5% CO2 at 37 ◦C. Primary murine epidermal keratinocytes were obtained as described previously (Li et al., 2017) and cultured in EpiLife™ Medium with 60 μM calcium (Thermo Fisher Scientific) with 1% (v/v) penicillin/streptomycin (10,000 U and 10,000 μg/mL) in a humidified atmosphere with 5% CO2 at 37 ◦C.

2.3. Animals

Animals were obtained from Animal and Plant Care Facility of Hong Kong University of Science and Technology and performed according to the guidelines of Department of Health, The Government of Hong Kong SAR. The experimental procedures had been reviewed and approved by Animal Ethics Committee at the University (Reference No. (15–50) in DH/SHS/8/2/2 Pt.2). Housing was at a constant temperature (21 ◦C) and humidity (60%), under a fixed 12 h light/dark cycle and free access to food and water.

2.4. Solar light exposure

HaCaT cells, seeded onto 35-mm culture dishes, were exposed to solar light generated by small area solar light simulator machine (Newport Corporation, Irvine, CA) for 2, 5, 10 and 20 min. Each 1− s experimental irradiance contained UVA (0.5 mW/m2), UVB (50 mW/ m2), and UVC (50 mW/m2). Afterwards, cells were incubated overnight under 5% CO2 at 37 ◦C and then for following experiments.

2.5. qRT-PCR

Total RNA was extracted using RNAzol@RT reagent (Molecular Research Center, Cincinnati, OH). Briefly, cells were incubated in RNAzol@RT at room temperature. Then, total RNA was precipitated in 75% ethanol (v/v) by centrifugation at 12,000 g for 10 min. The RNA pellet was washed by 75% ethanol and dissolved in RNAse-free water. The RNA quality was determined according to the ratio (~2.0) of absorbance at 260 nm and 280 nm by NanoDrop™ (Thermo Fisher Scientific). Three μg RNA samples were applied for reverse transcription using First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), in accord to the manufacturer’s protocol. The sequences of specific primers are as follows: sense 5’ -CAC CGA TAC TCT GGA CGA GG-3′, antisense 5’ -TCC TGC TTG CTA TAG TGG TCG-3′ for human ACHE; sense 5′-ACA ACT TTG GTA TCG TGG AAG G-3′, antisense 5′-GCC ATC ACG CCA CAG TTT C-3′ for human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Amplification was performed for 45 cycles. Each cycle consisted of denaturation at 95 ◦C for 30 s, annealing at 55 ◦C for 30 s, and extension at 72 ◦C for 20 s, performed on Roche Lightcycler 480 System (Roche, Basel, Switzerland).

2.6. Frozen skin section

The dorsal skin was collected from C57BL/6 male mice (3-week-old) after sacrificed. Skin tissue was fixed with 4% PFA overnight at room temperature then perfused with sucrose solution. Afterwards, the skin was embedded in OCT and frozen at − 80 ◦C overnight, followed by sectioning to 10 μm with Thermo CryoStar NX 70 Cryostat (Thermo Fisher Scientific). The section was preserved at − 20 ◦C for following experiments.

2.7. Immuno-fluorescent staining

Tissue sections or cultured cells were fixed with 4% paraformaldehyde for 30 min. Samples were incubated with 1% BSA with 0.2% Triton X-100 for 1 h. Cultures were incubated with primary antibodies, anti-cytokeratin at 1:100 (Abcam Ltd, Cambridge, UK), anti- AChE at 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA), at 4 ◦C overnight, followed with Alexa 488/647-conjugated antibodies (Abcam Ltd). Samples were mounted with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) and then subjected to immunohistochemistry analysis by a Zeiss Laser Scanning Confocal Microscope.

2.8. Sucrose density gradient and Ellman assays

Continuous 5–20% sucrose gradients in lysis buffer containing 10 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.2% Triton X-100, and 150 mM NaCl was prepared in 12-mL polyallomer ultracentrifugation tubes. Two hundred μL (1 μg/μL) cell lysates mixed with sedimentation markers, including alkaline phosphatase (6.1 S) and β-gal (16 S), were loaded onto the gradients followed by centrifugation at 38,000 rpm in SW 41 Ti Rotor (Beckman, Indianapolis, IN) at 4 ◦C for 16 h. Approximately 48 fractions were collected for determination of AChE activity by Ellman assay: 0.1 mM tetra-isopropylpyrophosphoramide (iso-OMPA; pre-treated for 5 min, an inhibitor of butyrylcholinesterase activity), 625 μM acetylthiocholine (ATCh), and 0.5 mM 5,5-dithiobis-2-nitrobenzoic acid (Sigma-Aldrich) in 80 mM Na2HPO4, pH 7.4 were added to 30 μL of each fraction. The mixture was incubated at room temperature for 30 min, and AChE activities were measured at 405 nm using Multiskan™ FC Microplate Photometer. AChE forms were determined by summation of the enzymatic activities corresponding to the peaks of sedimentation profile.

2.9. SDS-PAGE and western blot analysis

Cells were lysed in whole cell lysis buffer and shaken for 30 min at 4 ◦C, followed by centrifugation at 12,000 g at 4 ◦C for 10 min. The aliquots, normalized to 40 μg of protein, were applied to 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels (SDS-PAGE) and then transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk powder in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h. After blocking, the membranes were incubated at 4 ◦C overnight with specific primary antibodies, including anti-IVL at 1:200 (Abcam Ltd), anti-AChE at 1:200, and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) at 1:500,000 (Sigma-Aldrich), followed by incubated with horseradish peroxidase (HRP) secondary antibodies (Sigma-Aldrich) at 25 ◦C for 1 h. The immune-reactive proteins were detected using enhanced chemiluminescence (ECL) western blotting detection kit (Thermo Fisher Scientific). The intensities of the bands were quantified using ChemiDoc Imaging System (Bio-Rad).

2.10. Measurement of intracellular Ca2+

HaCaT cells were loaded with calcium indicator by incubation with 2 μM fluo-4 AM (Thermo Fisher Scientific) at 37 ◦C for 30 min. Then, cells were exposed to solar light generated by small area solar light simulator machine or treated with drugs. The intracellular Ca2+ was observed using Zeiss Laser Scanning Confocal Microscopy. The intensity of the fluorescent signal was quantified using ImageJ software (NIH Image).

2.11. Luciferase assay

The DNA construct of human pAChE-Luc and pAChEΔAP1-Luc were described previously (Siow et al., 2002; Zhang et al., 2008). The DNA construct of AP1 sequences tagged with a luciferase gene (pAP1-Luc) was from BD Biosciences (Pal Alto, CA). Transient transfection was performed using jetPRIME® reagent (Polyplus Transfection). Luciferase assay was performed using Pierce™ Firefly Luciferase Glow Assay Kit (Thermo Fisher Scientific). In brief, cells were lysed by 100 mM potassium phosphate buffer (pH 7.8), 0.2% Triton X-100 and 1 mM DTT and agitated for 30 min at 4 ◦C. Afterwards, cells were centrifuged at 16,000 g for 10 min at 4 ◦C 50 μL of cell lysate was used for assay. The luminescent reaction was quantified in a GloMax® 96 Microplate Luminometer (Thermo Fisher Scientific), and the activity was expressed as fold of control group.

2.12. Protein assay

Protein concentration was measured by Bradford protein assay dye from Bio-Rad (Bio-Rad Laboratories, Hercules, CA). Briefly, the protein sample was collected after cell was lysed. Then, the aliquot of sample, or different dose of bovine serum albumin, was mixed with the assay dye by shaking for 5 min, followed by measurement at 595 nm using Multiskan™ FC Microplate Photometer (Thermo Fisher Scientific). The protein concentration was calculated according to a standard curve.

2.13. Statistics analysis

Comparison of the means for untreated control cells and treated cells were analysed using one-way ANOVA and Student’s t-test. Significant values were represented as *p < 0.05, **p < 0.01, ***p < 0.001.

3. Result

3.1. Solar light increases AChE expression in keratinocytes

To investigate the regulation of AChE in keratinocyte, the expression and localization of AChE in keratinocyte and skin were recognized by immunostaining with specific antibody. In mouse skin cross sections, AChE was observed to be widely distributed in both epidermis and dermis layers (Fig. 1A). The negative control of AChE staining, the mouse skin underwent staining without anti-AChE antibody, was shown in Sup. Fig. 1. In parallel, AChE expression was evident in cultures of HaCaT cell and primary mouse epidermal keratinocyte (MEK) (Fig. 1B). By sucrose density gradient analysis, the molecular forms of AChE in HaCaT cells and MEKs were identified. The G1/G2 and G4 forms of AChE were expressed in HaCaT cells and MEKs; while the G4 tetramer was dominant form in both cell types (Fig. 1C).
In skin, solar light triggers ACh release from keratinocytes by affecting Ca2+ mobilization, which substance also influences neighbouring melanocytes by inhibiting the melanin production. Thus, the AChE inhibitor could enhance the ACh-induced inhibition on melanin production (Wu et al., 2020). To determine the role of solar light in regulating the expression of AChE, cultured keratinocytes were exposed to solar light generated by a solar light simulator containing UVA (365 nm, 0.5 mW/m2), UVB (311 nm, 50 mW/m2) and UVC (254 nm, 50 mW/m2) for 0–20 min. After the short exposure to light, the culture was incubated further overnight before Western blot and qPCR assays. The keratinocyte differentiation marker, involucrin (~68 kDa), was increased due to solar light exposure, suggesting the differentiation of keratinocytes (Fig. 2A). In parallel, AChE expression was upregulated by solar light exposure in a time-dependent manner, in which the translational expression of AChE was increased by 60% more than that of control group, after 10 min of the treatment (Fig. 2A). To investigate the alternation in AChE form, the sucrose density gradient analysis was performed. The activities of G1/G2 and G4 forms of AChE were all enhanced by solar light exposure in a time-dependent manner (Fig. 2B). In addition, the qPCR results indicated that solar light increased the amount of AChE mRNA, robustly (Fig. 2C). 3.2. Solar light promotes transcription of AChE
In keratinocytes, solar light significantly triggers Ca2+ mobilization that leads to the release of ACh (Wu et al., 2020). Here, Fluo-4 AM was used to label Ca2+ in the culture. The solar light induced the Ca2+ mobilization in keratinocytes, which was fully blocked by a Ca2+ chelator BAPTA-AM (Fig. 3A). The calcium ionophore A23187 was adopted as a positive control. To further investigate the mechanism on the solar light-induced upregulation of AChE, a luciferase reporting system was used to detect the activity of ACHE promoter. The DNA construct containing human ACHE promoter region and gene encoding luciferase (pAChE-Luc) was transfected onto cultured HaCaT cells (Siow et al., 2002). After exposure to solar light for 0–20 min, the ACHE promoter activity was increased, significantly (Fig. 3B). The 20-min exposure upregulated the activity to ~4 folds of control. However, this increase of ACHE promoter activity was eliminated by the pre-treatment with BAPTA-AM (Fig. 3C), suggesting this promotion was regulated by Ca2+. To confirm the effect of Ca2+ in AChE expression, Ca2+ ionophore A23187 and CaCl2 were applied onto HaCaT cells, respectively. The luciferase assay showed that the activity of ACHE promoter was stimulated by either A23187 or CaCl2, and this increase was blocked by pre-treatment with BAPTA-AM (Fig. 3C).
To reveal the role of Ca2+ in triggering ACHE gene transcription, Ca2+ signalling, mediated by solar light, was probed. An AP1 site was identified in human ACHE promoter (Fig. 4A): this transcription factor has been shown to play a role in Ca2+-mediated AChE regulation (Zhang et al., 2008). To investigate the role of the AP1 site in solar light-induced ACHE promoter activity, site-directed mutagenesis was performed on the AP1 site of the promoter, as to generate a mutated construct, i.e. pAChEΔAP1-Luc. In the transfected cultures, the construct pAChE-Luc, as expected, demonstrated a robust activation upon sun light stimulation in a time-dependent manner. In contrast, the stimulation was fully abolished in pAChEΔAP1-Luc transfected cultures (Fig. 4B). Besides, the DNA construct containing four repeats of AP1 site sequences tagged with luciferase reporting gene, i.e. pAP1-Luc, was transfected into cultured keratinocytes. The solar light-induced activity of pAP1-Luc was revealed, which was fully blocked by BAPTA-AM (Fig. 4C). These results suggest that solar light promotes AChE expression through Ca2+ signalling via AP1 site of the ACHE promoter.

4. Discussion

Sunlight is a portion of electromagnetic radiation emitted by sun, containing infrared, visible, and UV light (Setlow et al., 1974). The impact of sunlight to human could be both positive and negative. Skin pigmentation is one of the most common impacts in exposing sunlight. The mechanism of the UV-induced DNA damage in skin has been well studied. However, whether the cholinergic system involving in sunlight-induced skin pigmentation still remains unclear. Previous studies have revealed that ACh is released from skin under exposure to sunlight. The release of ACh could be increased from 205 ± 58 to 349 ± 122 pmol in skin (Schlereth et al., 2006). A complete set of cholinergic molecules has been identified in skin keratinocytes and/or melanocytes (Wu et al., 2018, 2020). The specific interaction of keratinocyte and melanocyte is considered as forming a “skin synapse”. Keratinocyte, as pre-synaptic, expresses ChAT to synthesize ACh, and which is released from keratinocyte, as triggered by sunlight. Melanocyte, as post-synaptic, reacts to ACh challenge by having expression of mAChR. AChE, located in both keratinocyte and melanocyte, is able to terminate the transmission of ACh. In this report, the sunlight-induced Ca2+ mobilization was identified as an impulse to trigger the release of ACh (Wu et al., 2020), as well as regulating AChE expression in skin keratinocyte. On the other hand, the expression of ChAT was significantly increased after exposure to sunlight (unpublished): this enzyme expression was highly possible to accelerate the generation of ACh in keratinocytes in acting on melanogenesis.
In mammals, AChE is encoded by ACHE gene that contains seven exons, and the different transcriptional variants are generated by alternative splicing on 3’ region. AChE exists as monomer or oligomer in mammals and associated with anchoring subunits. Based on the quaternary structure, the forms of AChE can be divided into globular (G) and asymmetric (A) forms. Through post-translational modifications, AChE forms soluble G1 and G2: G2 is anchored via GPI to erythrocyte membrane (Low et al., 1986; Eichler et al., 1992; Luk et al., 2012). The G4 AChE is anchored with PRiMA in brain and muscle (Massouli´e et al., 1993; Xie et al., 2010; Chen et al., 2011). The asymmetric forms of AChE are associated with ColQ subunits, which can form one, two or three catalytic tetramers, namely A4, A8, A12, respectively (Bon et al., 1978; Tsim et al., 1988). Here, G1/G2 and G4 forms of AChE were found in cultured keratinocytes. Besides, the assemblies of AChE forms were all upregulated by solar light in keratinocytes.
The regulation of AChE in cell development of different tissues has been extensively studied for years. In myogenesis of muscle, the regulation of AChE has been proposed to be mediated by a cAMP/PKA- dependent signalling (Choi et al., 2000; Siow et al., 2002). Similar in neuronal cells, the expression of AChE was shown to be activated by cAMP signalling cascade (Wan et al., 2000). In cAMP-treated muscle or neuron, the transcriptional rate of ACHE gene is regulated by the transcription factor CREB (Choi et al., 2000). In erythrocyte, the glycosylation of AChE and its transcription are regulated during erythrogenesis (Luk et al., 2012). The transcriptional factor GATA-1 was shown to be an activator of transcription of ACHE gene (Xu et al., 2018). In development of bone, Runx2 binds to ACHE promoter via Wnt/β-catenin signalling pathway to promote AChE expression during osteoblastic differentiation (Xu et al., 2018). In our previous findings, AChE was downregulated by cAMP signalling during melanogenesis in melanocytes and melanoma cells (Wu et al., 2018). In melanocytes and melanoma cells, the downregulation of AChE was mediated by phosphorylated CREB via cAMP signalling. On the other hand, the E-box site in ACHE promoter between Exon 1 and Exon 2 was the element bound by MITF to promote transcription of ACHE gene. However, the suppressive effect from phosphorylated CREB is much stronger than the promoting effect triggered by MITF, which therefore leads to a reduction of AChE expression during melanogenesis (Wu et al., 2018).
The AP1-mediated up regulation of AChE in keratinocyte is different to the regulation of cAMP in melanocytes in suppressing the expression of AChE (Wu et al., 2018). We believe this specific regulation should be applied to all mammalian species: because AP1 sequence is conserved in ACHE genes of mammals. However, the purpose of this up-regulation of AChE in keratinocyte under sunlight remains unknown. In our hypothesis, sunlight containing UV irradiation may cause differentiation of keratinocyte and initiate BAPTA-AM the apoptosis process. Indeed, the expression of AChE was shown to be changed during cellular differentiation and apoptosis (Jiang et al., 2008; Xiang et al., 2008; Zhang et al., 2012; Ye et al., 2015). The current work is based solely on HaCaT cells, and therefore confirmation is needed in primary epidermal keratinocytes in the future. In addition, questions, such as which component or what specific wavelength, affecting the expression of cholinergic system are yet to be addressed.

5. Conclusion

In this study, we have revealed both transcriptional and translational levels of AChE in skin keratinocytes were upregulated by exposure to solar light. Solar light stimulated the Ca2+ mobilization in keratinocytes and then triggered the AP1 binding to AP1 site in the ACHE promoter region. Based on our previous work, this study further elucidated that the solar light affects non-neuronal cholinergic system in skin, subsequently, to influence the dermatological functions.

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