• The Korean Journal of Physiology and Pharmacology
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• 제12권4호
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• pp.177-183
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• 2008

The layers of keratinocytes form an acid mantle on the surface of the skin. Herein, we investigated the effects of acidic pH on the membrane current and $[Ca^{2+}]_c$ of human primary keratinocytes from foreskins and human keratinocyte cell line (HaCaT). Acidic extracellular pH ($pH_e{\leq}5.5$) activated outwardly rectifying $Cl^-$ current ($I_{Cl,pH}$) with slow kinetics of voltage-dependent activation. $I_{Cl,pH}$ was potently inhibited by an anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, 73.5% inhibition at 1${\mu}$M). $I_{Cl,pH}$ became more sensitive to $pH_e$ by raising temperature from $24^{circ}C$ to $37^{circ}C$. HaCaT cells also expressed $Ca^{2+}$-activated $Cl^-$ current ($I_{Cl,Ca}$), and the amplitude of $I_{Cl,Ca}$ was increased by relatively weak acidic $pH_e$ (7.0 and 6.8). Interestingly, the acidic $pH_e$ (5.0) also induced a sharp increase in the intracellular [$Ca^{2+}$] (${\triangle}[Ca^{2+}]_{acid}$) of HaCaT cells. The ${\triangle}[Ca^{2+}]_{acid}$ was independent of extracellular $Ca^{2+}$, and was abolished by the pretreatment with PLC inhibitor, U73122. In primary human keratinocytes, 5 out of 28 tested cells showed ${\triangle}[Ca^{2+}]_{acid}$. In summary, we found $I_{Cl,pH}$ and ${\triangle}[Ca^{2+}]_{acid}$ in human keratinocytes, and these ionic signals might have implication in pathophysiological responses and differentiation of epidermal keratinocytes.

• 한국환경성돌연변이발암원학회지
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• 제26권3호
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• pp.69-76
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• 2006

2,3,7,8-Tetrachlorodibenzo-$\rho$-dioxin(TCDD) is a ubiquitous, persistent environmental contaminant and the most powerful carcinogen categorized by IARC. Although the mechanism of carcinogenesis by TCDD is poorly understood, several studies have shown that the skin is one of target organs far TCDD. In this study, we investigated the neoplastic transformation of human keratinocyte-derived cell line, HaCaT, by chemical transformation method using N-methyl-N'-nitro-N-nitrorsoguanidine(MNNG) and TCDD. We found that subsequent exposure to TCDD for 3 weeks after initial exposure to MNNG markedly induced transformed cells. It was suggested that TCDD can act as a potent promoter in HaCaT cells. Furthermore, these transformed cells showed morphological alternations in soft agar and increased telomerase activity. Therefore, the TCDD treatment of HaCaT cells by initiated with MNNG could promote neoplastic transformation without stimulation by exogenous growth factors. As a result, TCDD had a strong potency as a promoter in nontumorigenic immortalized human epidermal keratinocytes.

• BMB Reports
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• 제46권8호
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• pp.410-415
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• 2013

Adhesion molecules such as ICAM-1 are important in the infiltration of leukocytes into the site of inflammation. In this study, we investigated the inhibitory effects of curcumin on ICAM-1 expression and monocyte adhesiveness as well as its underlying action mechanism in the TNF-${\alpha}$-stimulated keratinocytes. Curcumin induced expression of heme oxygenase-1 (HO-1) in the human keratinocyte cell line HaCaT. In addition, curcumin induced Nrf2 activation in dose- and time-dependent manners in the HaCaT cells. Curcumin suppressed TNF-${\alpha}$-induced ICAM-1 expression and subsequent monocyte adhesion, which were reversed by the addition of tin protoporphyrin IX (SnPP), a specific inhibitor of HO-1, or HO-1 knockdown using siRNA. Furthermore, Nrf2 knockdown using siRNA reversed the inhibitory effect of curcumin on the TNF-${\alpha}$-induced ICAM-1 expression and adhesion of monocytes to keratinocytes. These results suggest that curcumin may exert its anti-inflammatory activity by suppressing the TNF-${\alpha}$-induced ICAM-1 expression and subsequent monocyte adhesion via expression of HO-1 in the keratinocytes.

• BMB Reports
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• 제49권1호
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• pp.57-62
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• 2016

Up-regulation of adhesion molecules plays an important role in the infiltration of leukocytes into the skin during the development of various inflammatory skin diseases, such as atopic dermatitis. In this study, we investigated the modulatory effects of 2,3-dimethoxy-2′-hydroxychalcone (DMHC) on tumor necrosis factor (TNF)-α-induced intercellular adhesion molecule-1 (ICAM-1) expression and monocyte adhesiveness, as well as the molecular mechanisms underlying its action in the HaCaT human keratinocyte cell line. Pre-treating HaCaT cells with DMHC significantly suppressed TNF-α-induced ICAM-1 expression and subsequent monocyte adhesiveness. DMHC inhibited TNF-α-induced activation of NF-ᴋB. In addition, DMHC induced HO-1 expression as well as NRF2 activation. Furthermore, HO-1 knockdown using siRNA reversed the inhibitory effect of DMHC on TNF-α-induced ICAM-1 expression and adhesion of monocytes to keratinocytes. These results suggest that DMHC may inhibit TNF-α-induced ICAM-1 expression and adhesion of monocytes to keratinocytes by suppressing the signaling cascades leading to NF-ᴋB activation and inducing HO-1 expression in keratinocytes. [BMB Reports 2016; 49(1): 57-62]

• Applied Biological Chemistry
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• 제46권1호
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• pp.60-65
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• 2003

피부노화는 크게 생리적 노화(chmnological aging)와 광노화(photoaging)로 나누어 진다. 진피세포와 표피세포에서 광노화는 MMP(Matrix metalloproteinase)의 분비량이 증가하면서 MMP와 TIMP(Tissue inhibitors of metalloproteinase)의 균형을 깨뜨려 진피층을 붕괴가 유도된다. 본 연구는 광노화 및 암 전이 과정, 관절염 등 여러 질병에서 주목받고 있는 MMP-1의 생성 억제활성물질을 120여종의 식용식물(edible plants)로부터 활성물질을 분리, 정제하고 정제물질의 일부 물리화학적 성질을 규명하였다. 표피세포(HaCaT)와 진피세포(HS68) 세포주를 이용하여 다양한 강도의 UVB를 조사하여 생성되는 MMP-1의 양과 감수성을 검토한 결과 HS68에서 UVB의 조사량에 비례하여 MMP 분비량이 증가되는 반면, HaCaT세포의 MMP 분비량은 유의적 차이를 보이지 않았다. MMP-1 생성량이 가장 높은 UVB 조사량은 35 $mJ/cm^2$ 내외이었고, UVB조사 후 분비되는 MMP-1의 양은 36-48시간대에 가장 높게 나타났다. MMP-1의 생성 억제활성을 검색한 결과 아가위(Crataegus pinnatifida Bunge)의 냉수분획층(CP)에서 최대활성을 나타내었다. CP를 periodate 산화 처리에서는 변화가 없으나 pronase 처리시 활성이 감소되는것으로 단백질이 활성의 본체임을 확인하였다. 정제는 CP의 ultrafiltration처리, DEAE-Tayopearl 650c, Butyl-Tayopearl 650M의 이온교환크로마토그래프와 Bio-Gel P-30겔 여과 크로마토그래프 통해서 MMP-1 억제 활성 분획층 CP-2Va-2를 정제하였다. CP-2Va-2의 MMP-1억제활성은 88.5%이었으며, 분자량은 HPLC로 확인한 결과 19kDa, SDS-PAGE에서는 20 kDa 확인됨으로써 monomer구조임을 알 수 있었다.

• 대한약침학회지
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• 제15권2호
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• pp.11-14
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• 2012

Objectives: The purpose of this study was to compare the antioxidant and anti-inflammatory effects of Alum (AL) and Burnt Alum (BAL), which are commonly used as external ointments. Methods: Extracts of AL and BAL were classified into three groups: 20, 50, and $100mg/{\mu}{\ell}$. The cytotoxicity was measured by using MTT assays in human keratinocyte cell line (HaCaT). The anti-oxidant effect was measured by using the DPPH (1, 1-diphenyl-2-picryl-hydrazyl-hydrate) radical scavenger. The anti-inflammatory effect was measured by using the inhibitory efficacy for the amount of nitric-oxide (NO) produced in mouse macrophage cell line (RAW 264.7). Results: BAL showed a higher level of cytotoxicity than AL. The AL groups showed a concentration-dependent scavenging effect on DPPH radicals, but no significant relevance was found. The BAL groups showed a concentration-dependent scavenging effect on DPPH radicals. The scavenging effects of the BAL groups were almost insignificant, but the values for the 20, 50, and $100{\mu}g/m{\ell}$ trials were different. The BAL groups showed significant concentration-dependent inhibitory effects on NO production, but the AL groups did not. Conclusions: AL showed an anti-oxidant effect more efficiently than BAL did, which demonstrated a superior anti-inflammatory effect. Therefore, for external usage, AL must be distinguished from BAL.

• 한국해양바이오학회지
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• 제10권2호
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• pp.62-72
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• 2018

An active ingredient with hyaluronidase (HAse) inhibitory effect is one of the anti-aging approaches in cosmeceuticals. Here, red sea cucumbers (RSCs), Stichopus japonicus, from Jeju Island were evaluated to examine their HAse inhibitory and antioxidant activity effect. In this study, RSCs were extracted by six enzymatic hydrolysis (Alcalase; Al, Trypsin; Try, Neutrase; Neu, Pepsin; Pep, Alpha-chymotrypsin; Chy and Protamex; Pro). Alcalase hydrolysate (AlH) showed the highest antioxidant capacities for both of oxygen radical absorbance capacity (ORAC) and trolox equivalent antioxidant capacity (TEAC) methods, compared to those of other hydrolysates, at $66.59{\pm}0.78{\mu}M\;TE/mg$ and $135.78{\pm}3.24{\mu}M\;TE/mg$, respectively. Furthermore, AlH performed the highest capacity of HAse inhibitory with $IC_{50}$ value of 3.21 mg/ml. Thus, RSCs hydrolyzed by Al were chosen to determine the cellular antioxidant activity and hyaluronic acid (HA) production effect on Human immortalized keratinocyte cell line (HaCaT). The results showed that AlH improved the cell viabilities and intracellular reactive oxygen species (ROS) induced by 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) were significantly decreased. In addition, AlH increased HA amount by regulating HYAL2 and HAS2 expressions in the HaCaT cells. Taken together, AlH of RSCs collected from Jeju Island showed HAse inhibitory and antioxidant activities against skin-aging which shows its potentials can be an optional natural bioactive ingredient for novel cosmeceuticals.

• Biomolecules & Therapeutics
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• 제26권3호
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• pp.306-312
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• 2018

In a previous study, we have demonstrated that S-methylmethionine sulfonium (SMMS) confers wound-healing and photoprotective effects on the skin, suggesting that SMMS can be used as a cosmetic raw material. However, it has an unpleasant odor. Therefore, in the present study, we synthesized odor-free SMMS derivatives by eliminating dimethyl sulfide, which is the cause of the unpleasant odor and identified two derivatives that exhibited skin-protective effects: one derivative comprised (2S,4S)- and (2R,4S)-2-phenylthiazolidine-4-carboxylic acid and the other comprised (2S,4R)-, (2S,4S)-, (2R,4R)-, and (2R,4S)-2-phenyl-1,3-thiazinane-4-carboxylic acid. We performed in vitro proliferation assays using human dermal fibroblasts (hDFs) and an immortalized human keratinocyte cell line (HaCaT). The two SMMS derivatives were shown to increase hDF and HaCaT cell proliferation as well as improve their survival by protecting against ultraviolet exposure. Moreover, the derivatives regulated the expression of collagen type I and MMP mRNAs against ultraviolet exposure in hDFs, suggesting that these derivatives can be developed as cosmetic raw materials.

• 대한화장품학회지
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• 제44권1호
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• pp.95-101
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• 2018

표피는 각질형성세포의 분화로부터 재생되어 계층화되는 상피 조직으로서 물리적 장벽을 형성함으로써 다양한 외부 오염원으로부터 개체를 보호한다. 자가포식 작용(autophagy)은 단백질 축적물, 손상된 세포 소기관, 세포내 미생물 등이 리소좀으로 운반되고 분해되도록 매개하는 기작이다. 최근 연구 결과에 의하면 자가포식 작용이 각질형성세포의 대사 기관과 핵을 제거하여 각질층으로 최종 분화하는데 중요한 역할을 하는 것이 보고 되었다. 그러나 자가포식 작용을 촉진함으로써 표피 분화를 유도할 수 있는지는 알려져 있지 않다. 본 연구에서는 천연물 유래 단일 화합물 라이브러리를 스크리닝하여 베타인(betaine)이 인간 각질형성세포주인 HaCaT 세포에서 세포질 내 LC3 punctate 소포체 및 LC3-I에서 LC3-II로의 변환을 증가시켜 자가포식 작용을 촉진함을 규명했다. 자가포식 작용의 억제 신호인 mTOR 경로는 베타인에 의해 영향을 받지 않았으므로, 베타인에 의해 유도된 자가포식 작용은 mTOR에 독립적임을 알 수 있었다. 베타인에 의해 촉진되는 자가포식 작용은 primary keratinocyte 및 skin equivalent에서도 관찰되었다. 또한, 베타인 처리된 인공피부에서 표피층 두께가 증가함을 확인하였다. 이러한 결과들로부터, 자가포식 작용의 새로운 조절소재로서 베타인이 표피의 턴오버를 촉진하여 표피의 장벽기능을 개선하고 피부노화를 방지할 수 있음을 시사한다.

• Biomolecules & Therapeutics
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• 제27권6호
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• pp.553-561
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• 2019

Rab25, a member of the Rab11 small GTPase family, is central to achieving cellular polarity in epithelial tissues. Rab25 is highly expressed in epithelial cells of various tissues including breast, vagina, cervix, the gastrointestinal tract, and skin. Rab25 plays key roles in tumorigenesis, mainly by regulating epithelial differentiation and proliferation. However, its role in skin physiology is relatively unknown. In this study, we demonstrated that Rab25 knock-out (KO) mice show a skin barrier dysfunction with high trans-epidermal water loss and low cutaneous hydration. To examine this observation, we investigated the histology and epidermal differentiation markers of the skin in Rab25 KO mice. Rab25 KO increased cell proliferation at the basal layer of epidermis, whereas the supra-basal layer remained unaffected. Ceramide, which is a critical lipid component for skin barrier function, was not altered by Rab25 KO in its distribution or amount, as determined by immunohistochemistry. Notably, levels of epidermal differentiation markers, including loricrin, involucrin, and keratins (5, 14, 1, and 10) increased prominently in Rab25 KO mice. In line with this, depletion of Rab25 with single hairpin RNA increased the expression of differentiation markers in a human keratinocyte cell line, HaCaT. Transcriptomic analysis of the skin revealed increased expression of genes associated with skin development, epidermal development, and keratinocyte differentiation in Rab25 KO mice. Collectively, these results suggested that Rab25 is involved in the regulation of epidermal differentiation and proliferation.