Less Keratinocyte-Derived Factors Related to More Keratinocyte Apoptosis in Depigmented than Normally Pigmented Suction-Blistered Epidermis May Cause Passive Melanocyte Death in Vitiligo

Lee, Ai-Young,Kim, Nan-Hyung,Choi, Won-Ik,Youm, Yun-Hee Blackwell Publishing 2005 The Journal of investigative dermatology Vol.124 No.5

<P>Stem cell factor (SCF) of keratinocyte origin regulates melanocyte growth and survival. Deprivation of survival factors causes the apoptosis of melanocytes. Vitiligo often develops following physical trauma, even if this is minor. The exact mechanism of the Koebner phenomenon in vitiligo is unclear. Apoptosis of keratinocytes, which occurs more in depigmented suction-blistered epidermis than in the normally pigmented counterpart, could reduce levels of keratinocyte-derived factors such as SCF and basic fibroblast growth factor (bFGF). Levels of SCF expression were examined in the depigmented and normally pigmented paired epidermis of 19 patients with vitiligo, and bFGF expression in six patients. The expression of SCF (p<0.001) and bFGF was usually reduced in the depigmented compared with the normally pigmented epidermis. Apoptosis of cultured normal human keratinocytes, which was induced by staurosporine, resulted in a concentration-dependent decrease in levels of SCF mRNA and protein. Normal human melanocytes proliferated more in medium containing SCF or keratinocyte (XB-2) feeder than in medium with neither. Deprivation of SCF or keratinocyte feeder in the culture medium induced a marked decrease in melanocytes as a result of apoptosis. Therefore, lower expression of keratinocyte-derived factors, including SCF, in vitiliginous keratinocytes, which could result from keratinocyte apoptosis, might be responsible for passive melanocyte death and may explain the Koebner phenomenon.</P>

각질세포분비물이 멜라닌세포의 형태 및 멜라닌소체의 이동에 미치는 영향

윤용현(Yong Hyun Yoon),김주영(Joo Young Kim),송인환(In Hwan Song),성언기(Eon Gi Sung) 대한해부학회 2007 Anatomy & Cell Biology Vol.40 No.3

각질세포분비물은 피부색을 결정하는 멜라닌세포의 성장과 분화 혹은 멜라닌의 합성과 이동에 영향을 미친다. 이들의 효과를 알고자 본 실험에서는 인체의 음경껍질에서 분리 배양된 각질세포와 멜라닌세포를 공동배양 하였고, 그 결과를 순수배양과 혼합배양을 시행하여 비교하였다. 멜라닌세포의 활성화 정도를 알고자 가지돌기 수와 세포면적을 계산하였고, 티로시나제의 농도를 측정하여 생성된 멜라닌의 양을 비교하였으며, 투과전자현미경 표본으로 멜라닌소체의 이동을 관찰하였다. 공동배양의 멜라닌세포가 순수배양과 비교하여 세포면적(p⁄0.05)에는 유의한 증가가 있었으나 가지돌기 수에는 유의한 차이가 없었다. 공동배양 2일과 4일째의 티로시나제의 농도는 순수 및 혼합배양과 비교하여 유의하게 높았다(p⁄0.05). 공동배양에서는 배양시간이 경과함에 따라 티로시나제의 농도가 감소하였으나 순수 및 혼합배양에서는 증가하였고, 배양 6일째에 모든 배양에서 유사한 농도를 보였다. 공동배양에서 티로시나제의 농도가 배양액에서는 높았으나 각질세포에서는 검출되지 않았다. 공동배양 및 혼합배양에서 각질세포로 이동된 멜라닌소체를 투과전자현미경 표본에서 관찰할 수 없었다. 결과적으로 각질세포분비물은 멜라닌세포의 형태와 멜라닌의 합성에는 영향을 미쳤으나, 멜라닌소체의 각질세포로의 이동에는 도움을 주지 않았다. Keratinocyte-derived factors are involved in regulating the proliferation, differentiation or melanogenesis of melanocytes. To investigate the effects of keratinocyte-derived factors on the skin pigmentation, human melanocytes and keratinocytes were cultivated in the forms of pure melanocyte or keratinocyte culture system, co-culture system (melanocytes and keratinocytes were cultivated together in a vessel but they were separated with semipermeable membrane) or mixed culture system (melanocytes and keratinocytes were cultivated together in a vessel in mixed form). The authors studied the cellular features (dendritogenesis and area) and the tyrosinase activities and observed the electron microscopic structures for melanosome transfer. Melanocyte in co-culture system increased in the area (p⁄0.05) but not in the dendritogenesis compared with the melanocyte in pure culture system. The tyrosinase activities of co-culture system on the 2nd and 4th day revealed higher compared with the ones of the pure and mixed culture system (p⁄0.05). In the co-culture system, the tyrosinase activities were gradually decreased with the lapse of time and vice versa in the pure and mixed culture system. On the 6th day culture, all of the three melanocyte culture systems showed the same tyrosinase activities. In spite of high tyrosinase activities in the medium, there were no tyrosinase activities in keratinocytes with the co-culture system. In transmission electron microscopic findings, there were scant of melanosomes in keratinocytes with the co-culture and mixed culture systems. In conclusion, keratinocyte-derived factors modulate the activities and melanogenesis of melanocytes but there were no effects on melanosome transfer to keratinocytes.

Keratinocytes negatively regulate the N-cadherin levels of melanoma cells via contact-mediated calcium regulation

Chung, Heesung,Jung, Hyejung,Jho, Eek-hoon,Multhaupt, Hinke A.B.,Couchman, John R.,Oh, Eok-Soo Elsevier 2018 Biochemical and biophysical research communication Vol.503 No.2

<P><B>Abstract</B></P> <P>In human skin, melanocytes and their neighboring keratinocytes have a close functional interrelationship. Keratinocytes, which represent the prevalent cell type of human skin, regulate melanocytes through various mechanisms. Here, we use a keratinocyte and melanoma co-culture system to show for the first time that keratinocytes regulate the cell surface expression of N-cadherin through cell-cell contact. Compared to mono-cultured human melanoma A375 cells, which expressed high levels of N-cadherin, those co-cultured with the HaCaT human keratinocyte cell line showed reduced levels of N-cadherin. This reduction was most evident in areas of A375 cells that underwent cell-cell contact with the HaCaT cells, whereas HaCaT cell-derived extracellular matrix and conditioned medium both failed to reduce N-cadherin levels. The intracellular level of calcium in co-cultured A375 cells was lower than that in mono-cultured A375 cells, and treatment with a cell-permeant calcium chelator (BAPTA) reduced the N-cadherin level of mono-cultured A375 cells. Furthermore, co-culture with HaCaT cells reduced the expression levels of transient receptor potential cation channel (TRPC) 1, −3 and −6 in A375 cells, and siRNA-mediated multi-depletion of TRPC1, -3 and -6 reduced the N-cadherin level in these cells. Taken together, these data suggest that keratinocytes negatively regulate the N-cadherin levels of melanoma cells via cell-to-cell contact-mediated calcium regulation.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Human keratinocyte cells reduce the N-cadherin levels of co-cultured melanoma cells. </LI> <LI> Cell-to-cell contact is necessary for this keratinocyte-mediated N-cadherin reduction. </LI> <LI> Keratinocytes reduce intracellular calcium in co-cultured melanoma cells. </LI> <LI> Keratinocytes reduce the expression of TRPCs in contacting melanoma cells. </LI> </UL> </P>

배양된 정상 인체 각질형성세포에서 자외선 B 조사에 의한 아포프토시스와 p53의 발현

김명남(Myeung Nam Kim),서성준(Seong Jun Seo),홍창권(Chang Kwun Hong),노병인(Byung In Ro),노성욱(Sung Wook Ro),조성인(Sung In Cho) 대한피부과학회 2000 대한피부과학회지 Vol.38 No.4

N/A Cutaneous absorption of ultraviolet B(UVB) in the skin occurs primarily in keratinocyte, causing DNA and protein damage. p53 tumor suppressor gene appeared in the epidermis after UVB irradiation, and the wild type has been known to be responsible for apoptosis and plays an important role in excluding abnormal cells with significant DNA damage. While p53 has been implicated in both DNA repair and apoptosis, it is unclear whether the p53 protein is involved in both of these processes within the same cell. Therefore, UVB-induced apoptosis and changes in p53 expression were studied in cultured normal human keratinocyte to determine that the cellular response to UVB induced DNA damage(DNA repair or apoptosis) correlated with p53 expression. The cultured normal human keratinocytes were irradiated with the doses of UVB(25-150 mJ/cm2) and incubated for various times(3, 6, 12, 24 hour) after radiation. At UVB doses of 100 and 150 mJ/cm2, acridine orange/ethidium bromide(Ao/Eb) staining-positive cells and TUNEL (TdT mediated dUTP-biotin nick end labeling) staining-positive cells increased significantly after 3 hours and 6 hours postirradiation respectively. Twelve hour postirradiation, staining-positive cells increased at each level of UVB-radiation exposure. These results suggest that there were significant influences of UVB doses and time course after irradiation to the number of Ao/Eb and TUNEL staining-positive cells. To determine whether all Ao/Eb and TUNEL-positive cells were actually undergoing apoptosis, cellular DNA was extracted from keratinocytes at 12 hours after UVB irradiation and seperated by electrophoresis on an 2.5% agarose gel to detect the internucleosomal DNA fragmentation(DNA ladder). 'DNA ladder' occurred at every dose of UVB 12 hour after irradiation, but did not appear early after irradiation, suggesting that whether Ao/Eb and TUNEL-positive cells observed early after irradiation were not undergoing apoptosis. Activation of p53 and the response to DNA damage is not observed universally, but is dependent on tissue specificity, species specificity and type of genotoxic damage. To correlate p53 level with UVB-induced apoptosis at the dose of 100mJ/cm2 UVB, p53 levels were determined by western blot analysis. The accumulation of p53 protein was apparent after 6 hours postirradiation, and UVB irradiation caused a dramatic increase in p53 levels at 12 and 24 hours. These results demonstrate that p53 is required for UVB-induced apoptosis in cultured normal human keratinocyte and p53 has a time-dependent effect in the initiation of apoptosis. In this study, the results indicated that a low dose(25mJ/cm2) of UVB irradiation could induce apoptosis in human keratinocyte in vitro and UVB exerts a time-dependent effect on inducing apoptosis. And the results also give support to increasing evidence that p53 may play a role in UVB-induced DNA damage and the induction of apoptosis in cultured normal human keratinocyte and that p53 is involved in the decision process which determines the fate of keratinocyte after UVB -induced DNA damage. (Korean J Dermatol 2000;38(4):481~489)

In vitro evaluation method to monitor pathogenic sensitive skin by co-culture of keratinocytes and neuronal cell line

( Sun Mee Shin ),( Eun Hye Hong ),( Soo Hyun Jeong ),( Eun Joo Park ),( Kwang Joong Kim ),( Kwang Ho Kim ) 대한피부과학회 2020 대한피부과학회 학술발표대회집 Vol.72 No.1

Background: Keratinocytes have recently known to participate in sensory transduction by release of neuroactive molecules which bind to intra-epidermal free nerve endings (FNEs) and modulate nociception. Although reciprocal interactions between keratinocytes and FNEs via soluble mediators are well established, little is known about physical contacts between keratinocytes and sensory neurons. Objectives: We developed for the first time the co-culture of human primary keratinocyte with differentiated SH-SY5Y to reproduce direct interactions in vitro. Methods: We investigated the morphological and functional characteristics of differentiated SH-SY5Y neuronal cell line co-cultured with primary keratinocyte and analyzed the influence of keratinocytes. Results: SH-SY5Y cells survived well in keratinocyte co-culture condition. When SH-SY5Y cells were co-cultured with keratinocytes, they had no significant influence on axonal development. The neuropeptide Substance P (SP) which is released after a cytosolic calcium influx and modulates immediate skin hypersensitivity was also released well after capsaicin treatment in co-cultured condition. Conclusion: This co-culture model in which keratinocytes and neurons may be a useful in vitro alternative for studying and characterizing the close communication between keratinocytes and sensory neurons.

IL-1 Receptor Antagonist Reduced Chemical-Induced Keratinocyte Apoptosis through Antagonism to IL-1α/IL-1β

Lee, Hyejin,Cheong, Kyung Ah,Kim, Ji-Young,Kim, Nan-Hyung,Noh, Minsoo,Lee, Ai-Young The Korean Society of Applied Pharmacology 2018 Biomolecules & Therapeutics(구 응용약물학회지) Vol.26 No.4

Extracellular interleukin 1 alpha (IL-$1{\alpha}$) released from keratinocytes is one of the endpoints for in vitro assessments of skin irritancy. Although cells dying via primary skin irritation undergo apoptosis as well as necrosis, IL-$1{\alpha}$ is not released in apoptotic cells. On the other hand, active secretion has been identified in interleukin-1 receptor antagonist (IL-1ra), which was discovered to be a common, upregulated, differentially-expressed gene in a microarray analysis performed with keratinocytes treated using cytotoxic doses of chemicals. This study examined whether and how IL-1ra, particularly extracellularly released IL-1ra, was involved in chemically-induced keratinocyte cytotoxicity and skin irritation. Primary cultured normal adult skin keratinocytes were treated with cytotoxic doses of chemicals (hydroquinone, retinoic acid, sodium lauryl sulfate, or urshiol) with or without recombinant IL-1ra treatment. Mouse skin was administered irritant concentrations of hydroquinone or retinoic acid. IL-1ra (mRNA and/or intracellular/extracellularly released protein) levels increased in the chemically treated cultured keratinocytes with IL-$1{\alpha}$ and IL-$1{\beta}$ mRNAs and in the chemically exposed epidermis of the mouse skin. Recombinant IL-1ra treatment significantly reduced the chemically-induced apoptotic death and intracellular/extracellularly released IL-$1{\alpha}$ and IL-$1{\beta}$ in keratinocytes. Collectively, extracellular IL-1ra released from keratinocytes could be a compensatory mechanism to reduce the chemically-induced keratinocyte apoptosis by antagonism to IL-$1{\alpha}$ and IL-$1{\beta}$, suggesting potential applications to predict skin irritation.

Role of Keratinocytes in the Development of Vitiligo

( Ai Young Lee ) 대한피부과학회 2012 Annals of Dermatology Vol.24 No.2

Vitiligo is an acquired depigmentary disorder of the skin that results from the loss of functioning epidermal melanocytes. Most studies on vitiligo have concentrated on the abnormality of melanocytes rather than the abnormality of keratinocytes; however, epidermal melanocytes form a functional and structural unit with neighboring keratinocytes. In fact, direct cell-to cell contact stimulates in vitro proliferation of melanocytes, and growth factors produced by adjacent keratinocytes regulate the proliferation and differentiation of melanocytes. The potential role of keratinocyte-derived cytokines has also been presented. We focused on the structural changes in vitiliginous keratinocytes, which may result in loss of melanocytes, to examine the pathomechanism of vitiligo. The results of a comparison between depigmented and normally pigmented epidermis in patients with vitiligo showed that the keratinocytes in the depigmented epidermis were more vulnerable to apoptosis. Impaired Phosphatidylinositol 3-kinase (PI3K)/serine/threonine protein kinase (Akt) activation followed by reduced nuclear factor-κB activation under increased tumor necrosis factor- α levels was demonstrated as a mechanism for keratinocyte apoptosis. The role of aquaporin 3 in keratinocyte apoptosis was addressed based on the relationship between the PI3K/AKT pathway and the E-cadherin-catenin complex. Apoptotic keratinocytes induced a lower expression of keratinocyte-derived factors, including stem cell factor, in depigmented epidermis, resulting in passive melanocyte death. (Ann Dermatol 24(2) 115∼125, 2012)

재조합 Interleukin 1α가 각질형성세포의 증식 및 Superoxide Radical과 Superoxide Dismutase 활성도의 조절에 미치는 영향

서성준,홍창권,노병인 중앙대학교 의과대학 의과학연구소 1993 中央醫大誌 Vol.18 No.1

There are some evidences that an interleukin(IL)1a enhances murine as well as human keratinocyte proliferation in vitro. However, according to its results of experiments using IL-1a by several investigators, it is also conceivable that IL-1a did not stimulate keratinocyte proliferation. To determine whether keratinocyte proliferation by IL-1a occurred, the present study was undertaken. In addition, we measured superoxide radical and superoxide dismutase to identify the intracellular biochemical events associated with effects caused by IL-1a, Primary keratinocyte cultures from neonatal foreskins were grown in complete MCDB 153 medium. Measurements of superoxide radical and superoxide dismutase(SOD)activity were performed according to Babior and Fridovich methods, respectively. The rusults follows: 1. Cell numbers of keratinocytes without treatment of recombinant IL-1a(rIL-1a) were 6.67+-0.91*10^4, 8.23+-0.65*10^4, 1.09+-0.12*10^5 at 4, 6 and 8 days, respectively. Cell numbers of keratinocytes with treatment of 10ng/㎖ rIL-1a were 6.20+-0.92*10^4, 8.73+-1.16*10^4, 1.58+-0.10*10^5 at 4, 6, and 8 days,respectively. Compared with that of untreated case, approximately 1.5fold increase in cell numbers was observed after 8 days culture with treatment of 10ng/㎖ rIL-1a. 2. Release of superoxide radical were measured after 30 minutes exposure to 0.1, 1.0, 10ng/㎖ rIL-1a, the results show 1.61+-0.065nmol/1*10^5, 2.63+-0.59nmol/1*10^5, 3.68+-0.61nmol/1-10^5 respectively, Significant superoxide radical release was detectable at Ing/㎖ and 10ng/㎖ rIL-1a compared with 0.68+-0.19nmol/1*10^5 of untreated case(p<0.05). 3. Total SOD activity without treatment of rIL-1a at 12 hours was 20.50+-8.61, Total SOD activity after 12 hours treatment 0.1, 1.0, 10ng/㎖ rIL-1a were 99.32+-14.84, 115.25+-14.32, 130.85+-10.80 respectively, which suggested that rIL-1a induced increase of total SOD significantly(P<0.05), rIL-1a stimulate total SOD induction in a dose and time dependent manner, although the effects was not outstanding. Mn SOD activity after treatment of 10ng/㎖ rIL-1a at 24 hours was 104.34+-11.61, which was approximately 11 fold of that of untreated case. rIL-1a also induced the Cu, Zn SOD activity, but the effect was not so much as in the case of Mn SOD. In summary, the results indicate that rIL-1a enhances human keratinocyte proliferation and superoxide radical released by rIL-1a may be play a role in human keratinocyte proliferation. Also production of total SOD, especially Mn SOD in response to rIL-1a induced and scavenged overproduced superoxide radical which in turn may result in protection of keratinocytes.

Effect of sandalwood and rose absolute oil on the proliferation and differentiation of epidermal keratinocytes

김진화 ( Jin Hwa Kim ),Dae Kyoung Choi,Chang Deok Kim,Jeung Hoon Lee,Tae Jin Yoon 한국피부장벽학회 2009 한국피부장벽학회지 Vol.11 No.2

The most important role of skin is regarded as a vital barrier against fluid loss, chemical and infectious agents, and UV light. Much of this protective function is dependent on the epidermis, a multi-layered epithelium that is composed of various cell types, including keratinocytes and melanocytes. Keratinocytes are the major cells in the epidermis that make the water-insoluble cornified cell envelope (CE), a key component of skin barrier, through highly sophisticated and tightly regulated process of differentiation. It is important to develop new materials for strengthening the skin texture, thereby providing the additive protective role for maintenance of healthy skin. Recently, a lot of interest has been given to plant-originated oils, because they could be used safely and effectively to protect the skin surface. In this study, we investigated the potential effect of plant oils, which are derived from sandalwood and rose. First, the effect on keratinocyte proliferation was determined by thymidine incorporation assay. Rose absolute oil reduced the cell growth, while sandalwood oil did not affect the growth of cultured keratinocytes. Interestingly, the morphology of rose absolute oil-treated keratinocytes was somewhat similar to that of calcium-treated cells. Thus, we speculated that rose absolute oil has a potential for promoting the keratinocyte differentiation. To test this idea, we next determine the effect of rose absolute oil on keratinocyte differentiation by Western blot analysis. As expected, rose absolute oil induced the expression of involucrin, an important marker for keratinocyte differentiation. Sandalwood oil did not affect the involucrin level, either. We then examine the rose absolute oil effect on the activity of involucrin promoter, by adenoviral transduction of reporter gene. As a result, involucrin promoter activity was increased by rose absolute oil, but sandalwood did not affect the promoter activity. These results suggest that rose absolute oil could be applied for the strengthening of skin texture.

자외선 B를 조사한 각질형성세포의 조건배양액내 Endothelin - 1 이 멜라닌세포의 증식과 기능에 미치는 영향

홍석훈,서성준,홍창권,노병인 ( Seok Hun Hong,Seong Jun Seo,Chang Kwun Haong ) 대한피부과학회 1997 大韓皮膚科學會誌 Vol.35 No.6

Background: The regulatory rnechanisrns of melanoc:ytes underlying ultraviolet(UV) melanogenesis have been an interest tu many investigators. They have shown that several materials produced and secreted frorn norm il human keratinocytes play roles as mitogens of human melanocytes, and demonstrated that UVB exposure stirnulated highly the paracrine linkage of endothelin(ET) be tween keratinocytes and melanocytes. It suggest that ET is one of keratinocytes-derived intrinsic mit.ogens in UVB induced hyperpigmentation. Objective : To evaluate whether ET secreted from keratinocytes under t.he UV irradiation works as paracrine effects such as melanocyte pro)iferation and function, the present, study was under taken. Methpds : Primary kevatinocytes and rnelanocytes cultures from neonatal foreskins were grown in complete MCDB 154 medium. Cultured human keratinocytes were irradiated with UVB 50mJ/ cm2. Twenty fours hour later, conditioned medium of keratinocytes was added into the growth medium of melanocytes of concentration in 10%, 20%, and 30%, respectively. Four days later, in order to detect of melanocytes proliferat.ion and function, number of melanocytes, melanin granules and tyrosinase activity v ere measured. Results : J. The number of me anocytes were higher increase in groups of incubation with conditioned medium of irradiated keratinocytes than that of incubation with conditioned mediurn of irradiated keratinocytes and treatment of anti-ET-1(5ug/ml)(p>0.05). 2. The melanin contents were significant.ly higher increase in groups of incubation with condi t.ioned medium(20%, 30%) of irradiated keratinocyt.es than that of incubation with conditioned medium of irradiated keiatinocytes and treatment of anti-ET-1(5ug/ml)(p<0.05). 3. The tyrosinase acti ity of melanocyte incubated with 30% COllcentration of conditioned medium from cultured keratinocyte irradiated with UVB was significantly higher increase than that of melanocyte incubated with 30% concentration of conditioned mediurn from cultured keratinocytes irradiated with UVB and treated with anti-ET-1(p<0.05). Conclusion : This stud provided an important confirmation of the proposal that ET-1 is intrin sic factor for proliferation and differentiation of human melanocytes. These findings suggest that keratinocyte derived ET-1 make a considerable effect on human melanocyte proliferation and function in UV melanog nesis. (Korean J Dermatol 1997;35(6): 1184-1192)