자외선 조사에 의한 인체 각질형성세포 세포고사 방어인자에 관한 연구

박수홍,박준홍,이종석,황규왕,김계정 순천향의학연구소 1998 Journal of Soonchunhyang Medical Science Vol.4 No.2

Back ground: Human skin is continuously exposed to UV irradiation. Ultraviolet irradiation of human skin cause sunburn cell which is relevant to the apoptosis of keratinocytes. In the epidermis, apoptosis inducing factors and anti-apoptotic factors probably exist to maintain the integrity of keratinocytes. Objective: The purpose of this study was to investigate the exsistence of apoptosis inducing factors and anti-apoptotic defence factors by evaluation of UV induced apoptosis in cultured human keratinocyte and other keratinocyte cell lines(A-431 cells, KB cells) and its susceptibility of UV induced apoptosis in various conditions. Method: In this study, the percentages of apoptosis, necrosis and cell viability of irradiated human keratinocytes and othe keratinocyte cell lines by MMT assay and AO/EO stain. The percentages of those were measured before UVB irradiation and 8, 24, 48 hours after UVB irradiation. Also, the same evaluations were performed with irradiated human keratinocytes cultured without growth factor and with enough growth factors, both results were compared with each other. And the effect of cycloheximide, a protein synthesis inhibitor was evaluated. Also that of aurintricarboxylic acid(ATA), an inhibitor of endonucleases which play an important role in inducing apoptosis of human keratinocytes was evaluated. Result: Human keratinocytes and other keratinocyte cell lines(A-431 cells, KB cells) were cultured in vitro developed maximal apoptosis 48 hours after irradiation, keratinocytes were more resistant to UV induced apoptosis than the others. The withdrawal of growth factors from keratinocyte and addition of cycloheximide decreased the cell survival rate following UV irradiation and increased the induction of apoptosis. And ATA inhibited UV induced apoptosis. Conclusion: These results indicate that human keratinocytes have both anti-apoptotic factors and apoptosis inducing factors to maintain the homeostasis. And survival signals mediated through growth factors or cellular proteins are responsible for the resistance to apoptosis observed in keratinocytes in vitro.

표피 분화와 피부장벽

오정숙,장경자 대한피부미용학회 2015 대한피부미용학회지 Vol.13 No.6

The skin is a organ that represents an immune response while performing an essential barrier function that prevent water loss from the body inside and external factors from breaking inside. Stratum corneum generated by normal differentiation on healthy skin perform a skin barrier function, which is the most important role of epidermis. The principal cell of epidermal cell is keratinocyte, which is divided into 4 layers according to the level of differentiation, and various structural proteins are expressed. Corneodesmosome is expressed on the upper part of stratum spinosum and granular layer, which is stored in lamellar body and secreted outside the cells in stratum corneum, connecting one keratinocyte to another. Lipids that were contained in lamellar body of epidermal keratinocytes on the boundary of strata cornea and granular layer are secreted between keratinocytes, forming lipid cell envelope among keratinocytes. If epidermal keratinocytes reach the granular layer, they synthesize keratohyalin granule, which contains profilaggrin and loricrin. The skin barrier is formed by epidermal differentiation, acting as an epidermal permeability barrier as a “brick and mortar" structure of corneocyte and surrounding intercellular lipid layer. The typical ingredients that form stratum corneum are corneodesmosome that is a protein structure connecting keratinocytes, cornified protein envelope that surrounds keratinocytes, and cornified lipid envelope. Cornified envelope, which is a protein envelope on the outside of keratinocytes, and cornified lipid envelope, which exists on the outside of the cornified envelope, are connected in a covalent bond. Thus, it forms a combination necessary to the barrier function of stratum corneum, thereby performing the role of a support for precise array of lipids among keratinocytes. If differentiation of epidermal keratinocytes is abnormal, it will cause defects to the skin barrier function and dehydrate the skin, consequently resulting in chronic skin diseases such as atopy.

The Association of Treponema denticola with Human Gingival Keratinocytes

Camargo, Paulo M.,Wolinsky, Lawrence E.,Park, No-Hee Korean Academy of Oral Biology and the UCLA Dental 1996 International Journal of Oral Biology Vol.21 No.1

The adhesion of Treponema denticola to human gingival kerationcytes was quantified amd examined with electron microscopy. This study was undertaken as an initial step in defining the potential role of oral treponemes in the initiation and progression of periodontal disease. The adhesion of ^35S-labeled T. denticola to gingival keratinocytes was very similar to the adhesion to human gingival fibroblasts. Heat-treatment of T. denticola resulted in a significant reduction in bacterial adhesion to the keratinocytes. Treatment of T. denticola cells with proteinase K prior to incubation with keratinocytes resulted in a dose-dependent adhesion reduction (41% at 0.5 mg/ml of proteinase K). Treatment of the keratinocytes with neuraminidase prior to incubation with T. denticola led to a 29% increase in bacterial adhesion to gingival keratinocytes over control. Exposure of T. denticola to 50 mM D-galactose, 50 mM methyl-β-D-galactopyranoside and 50mM D-N-acetyl galactosamine reduced subsequent T. denticola adhesion to gingival keratinocytes by up to 70%. The adhesion of T. denticola was significantly inhibited by heat-inactivated rabbit serum. Electron microscopic examination revealed a "body"-oriented localization of T. denticola at the intercellular junctions of the gingival keratinocyte monolayer. No T. denticola cells were seen penetrating the cell membranes or within the cytoplasm of the keratinocytes. The results suggest that T. denticola adhesion to human gingival keratinocytes may be mediated through carbohydrate-protein interactions localized at the intercellular junctional region of the gingival keratinocyte monolayers.

Induction of Interleukin-22 (IL-22) production in CD4+ T Cells by IL-17A Secreted from CpG-Stimulated Keratinocytes

( Zheng Jun Li ),( Dae Kyoung Choi ),( Kyung Cheol Sohn ),( Seul Ki Lim ),( Myung Im ),( Young Lee ),( Young Joon Seo ),( Chang Deok Kim ),( Jeung Hoon Lee ) 대한피부과학회 2016 Annals of Dermatology Vol.28 No.5

Background: Interleukin-17A (IL-17A) is mainly secreted from Th17 cells that are activated by various stimuli including CpG oligodeoxynucleotide, a Toll-like receptor 9 (TLR9) ligand. Recently, it has been demonstrated that keratinocytes play an important role in the pathogenesis of psoriasis. Objective: To investigate the potential role of keratinocytes, we examined whether TLR9 ligand CpG induces IL-17A expression in keratinocytes. Methods: We used HaCaT keratinocytes as a model system, and determined CpG-induced IL-17A using enzyme-linked immunosorbent assay and Western blot. Results: When HaCaT keratinocytes were treated with CpG, the expression of several cytokines including IL-17A, tumor necrosis factor-α and CCL20 was markedly increased. Treatment with nuclear factor (NF)-κB inhibitor significantly blocked the CpG-induced IL-17A production, indicating that CpG induced IL-17A expression through the NF-κB signaling pathway. In addition, IL-17A secreted from keratinocytes stimulated the CD4+ T cells, resulting in strong induction of IL-22 production. Conclusion: Since IL-22 is an important mediator for psoriatic inflammation, our data suggest that keratinocytes can participate in the pathogenesis of psoriasis via the TLR9-dependent IL-17A production. (Ann Dermatol 28(5) 579∼585, 2016)

각질형성세포의 염증 반응 및 분화에 미치는 Activating Transcription Factor 3의 영향

김경민,김도연,신정민,홍동균,정경은,서영준,이영,김창덕 대한피부과학회 2022 대한피부과학회지 Vol.60 No.10

Background: Pathogenesis of psoriasis is related to dysregulated keratinocyte function and immune responses. Geneticbackground is one of the most important factors in disease pathogenesis. However, psoriasis-associated genes havenot yet been fully identified. Activating transcription factor 3 (ATF3) is a member of the cyclic adenosine monophosphateresponsive element-binding protein family of transcription factors, which may regulate epidermal keratinocytes. Objective: We aimed to evaluate the effects of ATF3 on inflammation and differentiation of keratinocytes. Methods: We evaluated the expression of ATF3 in polyinosinic:polycytidylic acid (poly[I:C])-treated keratinocytes. Subsequently,we compared ATF3 levels in psoriatic and normal skin using immunohistochemical staining. To illustrate therole of ATF3, we generated ATF3-overexpressing keratinocytes and ATF3-knockdown keratinocytes using a recombinantadenovirus. We investigated inflammation and differentiation of keratinocytes by measuring the mRNA levelsof inflammatory cytokines and differentiation markers. Results: Treatment of keratinocytes with poly(I:C) increased ATF3 expression in a time-dependent manner. Immunohistochemicalstaining showed that ATF3 expression was increased in the epidermis of psoriatic tissues. WhenATF3 was overexpressed in keratinocytes using a recombinant adenovirus, poly(I:C)-induced inflammation was reduced. Conversely, ATF3 knockdown increased poly(I:C)-induced inflammation. Thus, ATF3 overexpression inhibited keratinocytedifferentiation, while ATF3 knockdown promoted it. Conclusion: ATF3 may be involved in the pathogenesis of psoriasis by influencing the inflammatory response anddifferentiation of keratinocytes.

Flavonoid myricetin inhibits TNF-α-stimulated production of inflammatory mediators by suppressing the Akt, mTOR and NF-κB pathways in human keratinocytes

Lee, D.H.,Lee, C.S. North-Holland ; Elsevier Science Ltd 2016 european journal of pharmacology Vol.784 No.-

<P>Flavonoid myricetin has been shown to exhibit anti-inflammatory and anti-oxidant effects. Nevertheless, the effect of myricetin on the TNF-alpha-stimulated production of inflammatory mediators in keratinocytes has not been studied. Using human keratinocytes, we examined the effect of myricetin on the TNF-alpha-stimulated production of inflammatory mediators in relation to the Akt, mTOR and NF-kappa B pathways, which regulate the transcription genes involved in immune and inflammatory responses. TNF-alpha-stimulated production of the inflammatory mediators and reactive oxygen species in keratinocytes, and activation of the Akt, mTOR and NF-kappa B pathways in HaCaT cells and primary keratinocytes. Myricetin, Akt inhibitor, Bay 11-7085 (an inhibitor of NF-kappa B activation), rapamycin (mTOR inhibitor) and N-acetylcysteine attenuated TNF-alpha-induced activation of Akt, mTOR and NF-kappa B. Myricetin and N-acetylcysteine attenuated the TNF-alpha-stimulated production of cytokines and chemokines, and production of reactive oxygen species in keratinocytes. The results show that myricetin may reduce TNF-alpha-stimulated inflammatory mediator production in keratinocytes by suppressing the activation of the Akt, mTOR and NF-kappa B pathways. The effect of myricetin appears to be associated with inhibition of the production of reactive oxygen species. Further, myricetin appears to attenuate the proinflammatory mediator-induced inflammatory skin diseases. (C) 2016 Elsevier B.V. All rights reserved.</P>

Keratinocytes-Derived Reactive Oxygen Species Play an Active Role to Induce Type 2 Inflammation of the Skin: A Pathogenic Role of Reactive Oxygen Species at the Early Phase of Atopic Dermatitis

( Da-in Choi ),( Jun-hyeong Park ),( Jee-young Choi ),( Meishan Piao ),( Min-song Suh ),( Jee-bum Lee ),( Sook-jung Yun ),( Seung-chul Lee ) 대한피부과학회 2021 Annals of Dermatology Vol.33 No.1

Background: Atopic dermatitis (AD) is characterized by chronic, relapsing skin inflammation (eczema) with itchy sensation. Keratinocytes, which are located at the outermost part of our body, are supposed to play important roles at the early phase of type 2 inflammation including AD pathogenesis. Objective: The purpose of this study was to evaluate whether keratinocytes-derived reactive oxygen species (ROS) could be produced by the allergens or non-allergens, and the keratinocytes- derived ROS could modulate a set of biomarkers for type 2 inflammation of the skin. Methods: Normal human epidermal keratinocytes (NHEKs) were treated with an allergen of house dust mites (HDM) or a non-allergen of compound 48/80 (C48/80). Then, biomarkers for type 2 inflammation of the skin including those for neurogenic inflammation were checked by reverse transcriptase-polymerase chain reaction and western immunoblot experiments. Results: HDM or C48/80 was found to upregulate expression levels of our tested biomarkers, including type 2 T helper-driving pathway (KLK5, PAR2, and NFκB), epithelial-cell-derived cytokines (thymic stromal lymphopoietin, interleukin [IL]-25, IL-33), and neurogenic inflammation (NGF, CGRP). The HDMor C-48/80-induced expression levels of the biomarkers could be blocked by an antioxidant treatment with 5 mM N-acetyl- cysteine. In contrast, pro-oxidant treatment with 1 mM H₂O₂ could upregulate expression levels of the tested biomarkers in NHEKs. Conclusion: Our results reveal that keratinocytes- derived ROS, irrespective to their origins from allergens or non-allergens, have a potential to induce type 2 inflammation of AD skin. (Ann Dermatol 33(1) 26∼36, 2021)

배양한 각질형성세포의 증식과 분화에 미치는 Calcipotriol ( MC 903 ) 의 효과

이동윤 ( Dong Youn Lee ),조광현 ( Kwang Hyun Cho ),민경원 ( Kyong Won Minn ),손영숙 ( Young Sook Son ) 대한피부과학회 1996 대한피부과학회지 Vol.34 No.6

Background : Calcipotriol(MC903), a new vitamin D, analogue, has been reported to be effective in the treatment of patients with psoriasis. Objective : The purpose of this study is to examine the effects of calcipotriol on proliferation and differentiation of the keratinocytes in monolayer cultures and three-dimensional cultures. Methods. Using moriolayer cultures, we examined morphological changes of keratinocytes and performed (3H)thymidine incorporation after calcipotriol was added into the medium. Using three dimensional cultures, we performed two experiments. one with cultures treated with calcipotriol immediately after the keratinocytes had been exposed to the air and another set of cultures treated with calcipotriol after three dimensional morphogenesis of the kerat.inocytes. We examined morphological changes of keraitinocytes and performed a immunohistochemical study for proliferation differentiation markers. Results : In monolayer cultures, at calcipotriol concentrations of 10 'M 10 'M, keratinocytes be- came larger, more irregular, and flattened in a dose-dependent manner. At 10-9 M-10-6 M, [3Hl thymidine incorporatiorn was decreased dose-dependently as compared to the control culture. In the first experiment using three-dimensional cultures, at 10 M-10 'M, total epidermal layers were thinned. This was assnciated with thinnings of nucleat.ed and horny layers in a dose-dependent manner. In the seconci experiment using three-dimensional cultures, at 10 M-10 M, nucleated layers were thinned in a dose dependent manner, but the horny layer was slightly thickened, as compared to the control culture. Immunohistochemical studies showed a reduction of differentiation markers such as keratin 1, involucrin, filaggrin, loricrin consistent with a thinning of nucleated layers in the epidermal architecture in both experiments. In the basal layer, at 10 M-10 'M, PCNA-positive cells were and BrdU-positive cells were decreased dose-dependently as compared to the control culture. Conclusions : In this study, we demonstrated that at 10-9M - 10-6M calcipotriol inhibited keratinocytes proliferation and stimulated keratinocytes differentiation in a dose-dependent manner. (Kor J Dermatol 1996;34(6): 942-952)

Inhibition of Poly(I:C)-Induced Inflammation by Salvianolic Acid A in Skin Keratinocytes

( Qing-ling Zhang ),( Ri-hua Jiang ),( Xue Mei Li ),( Jung-woo Ko ),( Chang Deok Kim ),( Ming Ji Zhu ),( Jeung-hoon Lee ) 대한피부과학회 2019 Annals of Dermatology Vol.31 No.3

Background: Skin keratinocytes participate actively in inducing immune responses when external pathogens are introduced, thereby contributing to elimination of pathogens. However, in condition where the excessive inflammation is occurred, chronic skin disease such as psoriasis can be provoked. Objective: We tried to screen the putative therapeutics for inflammatory skin disease, and found that salvianolic acid A (SAA) has an inhibitory effect on keratinocyte inflammatory reaction. The aim of this study is to demonstrate the effects of SAA in poly(I:C)-induced inflammatory reaction in skin keratinocytes. Methods: We pre-treated keratinocytes with SAA then stimulated with poly(I:C). Inflammatory reaction of keratinocytes was verified using real-time polymerase chain reaction, enzyme-linked immunosorbent assay and Western blot. Results: When skin keratinocytes were pre-treated with SAA, it significantly inhibited poly (I:C)-induced expression of inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α, and CCL20. SAA inhibited poly(I:C)-induced activation of nuclear factor-κB signaling. And SAA also inhibited inflammasome activation, evidenced by decrease of IL-1β secretion. Finally, SAA markedly inhibited poly(I:C)-induced NLRP3 expression. Conclusion: These results demonstrate that SAA has an inhibitory effect on poly(I:C)-induced inflammatory reaction of keratinocytes, suggesting that SAA can be developed for the treatment of inflammatory skin diseases such as psoriasis. (Ann Dermatol 31(3) 279∼285, 2019)

Ampelopsis japonica Makino extract (AE) inhibits the inflammatory reaction induced by pathogen-associated molecular patterns (PAMPs) in epidermal keratinocytes

( Mi Ra Choi ),( Jin Hyup Lee ),( Dae Kyoung Choi ),( Dong Il Kim ),( Hae Eul Lee ),( Myung Im ),( Young Lee ),( Chang Deok Kim ),( Young Joon Seo ),( Jeung Hoon Lee ) 대한피부과학회 2015 대한피부과학회 학술발표대회집 Vol.67 No.2

Background: Keratinocytes are the major cells inepidermis, providing barrier components such as cornified cells through the sophisticated differentiation process. In addition, keratinocytes exerts their role as the defense cells via activation of innate immunity. It has been knownthat pathogen-associated molecular patterns (PAMPs) including double-strand RNA and nucleotides can provoke inflammatory reaction in keratinocytes. Objectives: The aim of this study is to evaluate the effect of Ampelopsis japonica Makino extract (AE) on PAMPs-induced inflammatory reaction of keratinocytes. Methods: The effects of AE were determined using poly(I:C)-induced inflammation and imiquimod-induced psoriasiform dermatitis models. Results: In cultured keratinocytes, AE significantly inhibited poly(I:C)-induced expression of inflammatory cytokines, such as IL-1モ, IL-6, IL-8 and TNF-メ. AE significantly inhibited poly(I:C)-induced release of caspase-1 active form (p20), and down-regulated NF-リB signaling pathway. In imiquimod-induced psoriasiform dermatitis model, topical application of AE resulted in significant reduction of epidermal hyperplasia. Conclusion: These results suggest that AE may be a potential candidate for the treatment of skin inflammation.