(The) biological impacts of blue light on skin cells : mechanisms and implications

다국어 초록 (Multilingual Abstract)

Circadian rhythm refers to an endogenous oscillation with a cycle of approximately 24 hours, playing a critical role in maintaining skin homeostasis. In this study, the biological influences of blue light on the interplay between the skin’s circadian rhythm and skin physiology were investigated. Exposure to blue light disrupted the circadian rhythm in human keratinocyte by inducing ER stress and altering the expression of core clock genes. Chromatin immunoprecipitation assays revealed that blue light suppressed the transcription of clock components through activation of one of UPR sensors, ATF6, which binds CRE in clock gene promoter. Blue light-induced upregulation of PER2 stabilized p53 protein levels by inhibiting MDM2-mediated degradation, resulting in p53 activation, p21 upregulation, and G2/M cell cycle arrest. In addition, since AhR promoter contains E-box elements, BMAL1 alteration in response to blue light exposure affected AhR rhythmicity and its nuclear translocation. Blue light also activated AP-1 and NF-κB signaling pathways, which are involved in skin inflammation and elasticity. Collectively, these findings demonstrate that blue light-induced circadian dysregulation accelerates skin aging, implying the essential role of the circadian clock in skin physiology. These suggest that targeting circadian regulation via ATF6 may offer a novel therapeutic strategy for managing blue light-induced skin aging.

다국어 초록 (Multilingual Abstract)

Blue light, a high energy component of visible light spectrum (380-500 nm), has been reported to have various biological impacts on skin. While it has long been associated with phototoxic effects, recent studies have demonstrated its therapeutic potential in treating several skin disorders. However, our understanding of the biological effects of blue light remains limited and largely based on phenotypic observations, raising concerns regarding its long-term safety and highlighting the need to delineate the underlying molecular mechanisms. This study aims to elucidate the molecular mechanisms of blue light-induced skin responses and to identify target molecules that may enhance the safety and efficacy of blue light therapy.
This study explored the effects of blue light on hyperpigmentation, anti-cancer responses and circadian rhythm regulation across various skin cell types. A key focus was the role of TRPV1, a non-selective cation channel expressed in skin cells, in mediating stress responses triggered by disruptions in cellular homeostasis, particularly fluctuations in intracellular calcium levels.
Notably, TRPV1 was identified as a downstream effector of OPN3. Blue light activated TRPV1 and upregulated its expressions via OPN3, inducing a calcium influx. Elevated intracellular calcium levels not only stimulated melanogenesis but also inhibited melanosome degradation, contributing to pigment accumulation. Furthermore, blue light activated TRPV1 localized on ER membrane, eliciting ER stress and triggering UPR. In melanoma cells, excessive ER stress attenuated IRE1α signaling, disrupted Ca2+ homeostasis and ultimately exerted anti-tumor effects. In keratinocytes, blue light-induced ER stress modulated circadian rhythm via ATF6 upregulation, potentially accelerating skin aging.
Collectively, these findings are expected to provide comprehensive insights into dermatologic responses to blue light and support the development of safter, more targeted and mechanism-based approaches in blue light therapy.

다국어 초록 (Multilingual Abstract)

Blue light, a high-energy radiation in the visible light spectrum, was recently reported to induce skin pigmentation. In this study, the involvement of TRPV1-mediated signaling along with OPN3 in blue light-induced melanogenesis, as well as its signaling pathway was investigated. Operating downstream target of OPN3 in blue light-induced melanogenesis, blue light activated TRPV1 and upregulated its expression, resulting in calcium influx. Intracellular Ca2+ induced activation of CaMKII and MAPK. It also downregulated clusterin expression, leading to the nuclear translocation of PAX3, ultimately affecting melanin synthesis. In addition, blue light interfered with autophagy-mediated regulation of melanosomes by decreasing not only the interaction between CLU and LC3B but the expression of ATF family. These findings demonstrate that the pigmenting effects of blue light are mediated by CaMKII- and MAPK-mediated signaling, as well as CLU-dependent inhibition of autophagy through OPN3-TRPV1-calcium influx, suggesting a new signaling pathway by which blue light regulates melanocyte biology. Furthermore, these results suggest that TRPV1 and CLU could be potential therapeutic targets for blue light-induced pigmentation due to prolonged exposure to blue light.

다국어 초록 (Multilingual Abstract)

Blue light has recently been reported to have various biological impacts on skin. In this study, whether disruption of intracellular Ca2+ homeostasis induced by blue light irradiation could lead to DNA damage and subsequent cell cycle arrest in mouse skin cancer cell were investigated. Here, I found blue light has stronger cytotoxic effects on melanoma cells than normal melanocytes. Blue light increased Ca2+ level in cytosol through activation of TRPV1 both on plasma membrane and ER membrane. In addition, while IRE1α, one of UPR branches, dimerized in response to ER stress, blue light inhibited its dimerization and promoted opening of IP3R, a calcium channel on ER. Loss of control of IP3R due to blue light increased the cytosolic calcium level, leading to mitochondrial damage through MCU. Finally, DNA damage evoked by mitochondrial Ca2+ overload induced G2/M phase cell cycle arrest through activating ATM-CHK1/2. Furthermore, blue light irradiation activated apoptosis by regulation Bax/Bcl-2 proteins. Collectively, these findings demonstrated that blue light exerts anti-cancer activity on melanoma cells by disrupting Ca2+ homeostasis mediated through activation of two kinds of cation/calcium channel, TRPV1 and IP3R.

국문 초록 (Abstract)

청색광은 가시광선 중 높은 에너지를 가지는 영역 (380-500 nm)으로, 피부에 다양한 생물학적 영향을 미치는 것으로 보고되고 있다. 일반적으로 청색광의 세포 독성 효과가 보고되고 있지만, 최근 연구에서는 여러 피부 질환의 치료에 있어 가능성도 제시되고 있다. 그러나 청색광의 생물학적 효과에 대한 연구는 아직 제한적이며 대부분 현상 관찰 수준에 머물러 있어, 청색과의 장기적 사용 시 안정성에 대한 우려와 함께 그 분자적 기전을 규명할 필요성이 강조되고 있다. 본 연구는 청색광에 의해 유도되는 피부 반응의 분자적 기전을 규명하고, 청색광 치료의 안전성과 유효성을 향상시킬 수 있는 표적 분자를 발굴하는 것을 목적으로 한다.
본 연구는 다양한 피부 세포에서 청색광이 피부 색소침착, 항암 효과, 그리고 생체 리듬 조절에 미치는 영향을 분석하였다. 특히, 피부 세포에서 발현되는 비선택적 양이온 채널인 TRPV1의 역할에 주목하였으며, TRPV1이 세포 내 칼슘 농도의 변화 등 항상성 교란으로 유도되는 스트레스 반응에 관여하는 것을 확인하였다.
TRPV1 은 OPN3의 하위 신호전달 인자로 확인되었으며, 청색광에 의해 활성화되고 발현이 증가하면서 세포 내 칼슘 유입을 유도하였다. 세포 내 칼슘 이온의 농도가 증가하면서 멜라닌 색소 생성이 촉진되는 동시에 멜라노좀의 분해가 억제되어 색소 침착이 유발되었다. 나아가, 청색광은 소포체에 존재하는 TRPV1을 활성화시켜 소포체 스트레스와 함께 미접힘 단백질 반응을 유도하였다. 흑색종 세포에서는 이로 인한 과도한 소포체 스트레스가 IRE1α 신호를 억제하고 칼슘 항상성을 붕괴시켜 항암 효과를 나타냈다. 각질형성세포에서는 ATF6 경로를 통해 유도된 소포체 스트레스가 피부 생체리듬을 조절함으로써 피부 노화를 가속화할 가능성이 제기되었다.
본 연구 결과는 청색광에 대한 피부 반응의 분자적 기전을 통합적으로 이해하는데 기여하며, 향후 보다 안전하고 표적화 된 기전 기반의 피부 치료 전략 개발에 기여할 것으로 기대된다.

목차 (Table of Contents)

• Chapter 1. Literature Review 1
• 1.1. Introduction 2
• 1.1.1. What is blue light 3
• 1.1.2. The source of blue light 3
• 1.2. Research on blue light and the eye 7
• 1.2.1. Visual response 7
• 1.2.2. Non-visual response 8
• 1.3. How does the skin sense blue light 10
• 1.3.1. Opsin 11
• 1.3.2. Porphyrin 12
• 1.3.3. Flavin 12
• 1.3.4. Nitrosated proteins 12
• 1.3.5. TRPV1 13
• 1.4. Skin biology and blue light 16
• 1.4.1. Oxidative stress 16
• 1.4.2. Skin barrier damage 17
• 1.4.3. Inflammation 17
• 1.4.4. Skin aging 18
• 1.4.5. Reactive nitrogen species overproduction 18
• 1.4.6. Hyperpigmentation 19
• 1.5. Blue light therapy in skin disorder 21
• 1.5.1. Acne vulgaris 21
• 1.5.2. Psoriasis Vulgaris 22
• 1.5.3. Atopic Dermatitis 23
• 1.5.4. Photoaging 23
• 1.6. Conclusion 25
• Chapter 2. Blue Light induced Pigmentation is Mediated by Both Melanogenesis Activation and Autophagy Inhibition through OPN3-TRPV1 27
• Abstract 28
• 2.1. Introduction 29
• 2.2. Materials & Methods 31
• 2.2.1. Materials 31
• 2.2.2. Cell culture 31
• 2.2.3. Blue light stimulation 32
• 2.2.4. Fluo-4 NW calcium assay 32
• 2.2.5. Cell viability assay 33
• 2.2.6. Melanin content assay 33
• 2.2.7. Cellular tyrosinase assay 33
• 2.2.8. RNA extraction and PCR 34
• 2.2.9. Western blot analysis 35
• 2.2.10. Immunofluorescence 35
• 2.2.11. Chromatin immunoprecipitation (ChIP) assay 36
• 2.2.12. Measurement of autophagic flux 36
• 2.2.13. Lentiviral infection 37
• 2.2.14. Statistical analysis 38
• 2.3. Results 39
• 2.3.1. Blue light increases melanin synthesis by regulating MITF 39
• 2.3.2. Blue light activates TRPV1-calcium influx signaling, which results in CREB/p38/ERK pathway through CaMKⅡ activation 42
• 2.3.3. Blue light activates TRPV1-mediated signaling through OPN3 46
• 2.3.4. TRPV1-dependent calcium influx downregulates CLU expression, leading to the nuclear translocation of PAX3 52
• 2.3.5. Blue light decreases the interaction between CLU and LC3B, resulting in the interference with melanosome degradation 56
• 2.4. Discussion 60
• Chapter 3. Anti-melanoma activity of blue light irradiation is mediated by disruption of intracellular Ca2+ homeostasis through TRPV1-IP3R axis 64
• Abstract 65
• 3.1. Introduction 66
• 3.2. Materials & Methods 68
• 3.2.1. Antibodies 68
• 3.2.2. Cell culture 68
• 3.2.3. Blue light stimulation 69
• 3.2.4. Measurement of calcium levels 69
• 3.2.5. MitoSOX 70
• 3.2.6. Cell Counting Kit-8 (CCK-8) assay 70
• 3.2.7. Cell Titer Glo assay 70
• 3.2.8. Clonogenic assay 71
• 3.2.9. 5-Ethynyl-2-deoxyuridine (EdU) incorporation assay 71
• 3.2.10. Cell cycle and Apoptosis assay 72
• 3.2.11. Luciferase reporter and β-Galactosidase activity assays 72
• 3.2.12. RNA extraction and PCR 73
• 3.2.13. Western blot analysis 75
• 3.2.14. Cytochrome C release assay 75
• 3.2.15. Immunofluorescence 76
• 3.2.16. JC-1 assay 76
• 3.2.17. Chromatin immunoprecipitation (ChIP) assay 77
• 3.2.18. Proximal ligation assay (PLA assay) 78
• 3.2.19. siRNA transfection 78
• 3.2.20. Statistical analysis 79
• 3.3. Results 80
• 3.3.1. Blue light suppressed proliferation and promoted apoptosis in melanoma cells 80
• 3.3.2. Inhibition of cell growth was mediated through delay in G2/M phase followed by DNA damage, especially double strand break 83
• 3.3.3. Blue light disrupted intracellular Ca2+ homeostasis through activation of TRPV1 on ER membrane 87
• 3.3.4. Loss of control of IP3R increased the cytosolic Ca2+ level, leading to mitochondrial damage through MCU 94
• 3.3.5. TRPV1 mediated blue light-induced ER stress and cell viability 98
• 3.3.6. CLU acted as a positive regulator of NF-κB signaling pathway by interacting with IκBα 101
• 3.4. Discussion 105
• Chapter 4. Blue Light Disrupts the Circadian Rhythm Through ATF6, Leading to Premature Aging in Human Skin 109
• Abstract 110
• 4.1. Introduction 111
• 4.2. Materials & Methods 114
• 4.2.1. Antibodies 114
• 4.2.2. Cell culture 114
• 4.2.3. Blue light stimulation 115
• 4.2.4. Cell synchronization 115
• 4.2.5. Cell Titer Glo assay 115
• 4.2.6. 5-Ethynyl-2-deoxyuridine (EdU) incorporation assay 115
• 4.2.7. RNA extraction and PCR 116
• 4.2.8. Western blot analysis 118
• 4.2.9. Luciferase reporter and β-Galactosidase activity assays 119
• 4.2.10. Chromatin immunoprecipitation (ChIP) assay 119
• 4.2.11. Senescence β-Galactosidase assay 120
• 4.2.12. Statistical analysis 121
• 4.3. Results 122
• 4.3.1. Blue light-induced ER stress impaired expressions of clock genes in human keratinocyte 122
• 4.3.2. Blue light disrupted circadian oscillation of clock genes through ATF6 126
• 4.3.3. Blue light inhibited the degradation of p53, maintaining its stability and leading to p21 upregulation 130
• 4.3.4. Disruption of circadian rhythm downregulated proliferation through cell cycle arrest 133
• 4.3.5. Blue light altered AhR oscillation and its activity in a clock-dependent manner 136
• 4.3.6. Blue light exposure accelerated cellular senescence 139
• 4.4. Discussion 144
• References 146
• 논문요약 172