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약학석사학위논문

Genome-scale transcriptional profile analysis of human keratinocytes in

response to hydroquinone

하이드로퀴논에 대한 사람의 각질형성세포에서 유전체 수준의 전사체 반응 연구

2020 년 2 월

서울대학교 약학대학원 약학과 천연물과학전공

표 정 주

Genome-scale transcriptional profile -

analysis of human keratinocytes inresponse to hydroquinone

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This thesis/dissertation is reproduced from the original article with permission from Pyo, J.J., Ahn,

S., Jin, S.H. et al. Arch Toxicol (2019) 93: 2307. https://doi.org/10.1007/s00204-019-02506-6

©Springer-Verlag GmbH Germany, part of Springer Nature 2019 License Number 4715650450814

3

ABSTRACT

Genome-scale transcriptional profile analysis of human keratinocytes in

response to hydroquinone

Jeong Joo Pyo

Department of Pharmacy

The Graduate School of Pharmacy

Seoul National University

Hydroquinone (HQ) is a well-known phenolic agent that can cause

chemical leukoderma, a pigmentary disorder characterized by the loss of functional

melanotic melanocytes in human skin. Despite its leukoderma-inducing potential,

hydroquinone is still widely used as a commercial topical skin whitening agent.

Furthermore, the mechanism of hydroquinone to induce chemical leukoderma is

poorly understood. To elucidate the pathogenesis of hydroquinone-induced chemical

leukoderma, genome-scale transcriptional profile analysis was performed in human

keratinocytes (HKs) treated with sub-cytotoxic hydroquinone concentration.

Differentially expressed genes (DEGs) analysis on transcriptional profile of

hydroquinone treated HKs resulted in the 250 upregulated and downregulated genes,

respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-

4

based functional enrichment analysis of hydroquinone-induced DEGs revealed that

hydroquinone significantly upregulated genes associated with IL-17 signaling

pathway and significantly downregulated genes related to the melanogenesis

pathway in HKs. By comparing the transcriptional profiles of hydroquinone-induced

and cytokines-induced DEGs, 58 commonly upregulated DEGs were found between

hydroquinone - and IL-17A- treated HKs. Interestingly, the expression of IL36G was

significantly upregulated in HKs in response to both hydroquinone and IL-17A.

Further study revealed that cytokine IL-36g directly inhibited melanin biosynthesis

in cultured human melanocytes (HMs) and downregulated key enzymes in the

melanogenesis pathway including TYR, DCT, and TYRP1. Moreover, IL-36g

autocrinally regulated keratinocyte function to produce proinflammatory cytokines

IL-36g, IL-6, and CXCL8/IL-8 in a concentration-dependent manner, suggesting that

IL-36g may initiate the cascade response of cutaneous inflammation. Finally, meta-

analysis of 3 independent transcriptional profile studies from skin of vitiligo patients

revealed the upregulation of IL36G gene expression in the lesioned site of vitiligo,

indicating that IL-36g may be clinically associated with vitiligous phenotypes. In this

regard, hydroquinone-induced IL-36g from human keratinocytes plays a role in the

development of chemical leukoderma by autocrinally or paracrinally regulating the

crosstalk between epidermal keratinocytes and melanocytes.

Keywords: Hydroquinone, Human Keratinocytes, Chemical leukoderma, IL-36g,

Kyoto Encyclopedia of Genes and Genomes (KEGG)

Student Number: 2018-24035

5

CONTENTS

ABSTRACT ............................................................................................ 3

TABLE OF CONTENTS .................................................................... 5

LIST OF FIGURES .............................................................................. 7

LIST OF TABLES ................................................................................ 8

I. Introduction ............................................................................... 9

II. Materials and Methods ........................................................ 12

1. Primary human keratinocyte, melanocyte culture and

cell viability assay ............................................................. 12

2. Total RNA isolation and a genome-scale microarray

experiment .......................................................................... 13

3. KEGG pathway-based functional enrichment analysis

............................................................................................... 13

4. Transcriptional profile comparison analysis ................ 14

5. Quantitative real-time reverse transcriptase chain

reaction and enzyme-linked immunosorbent assay ... 15

6. Western blot analysis ........................................................ 16

7. Melanin content assay ...................................................... 16

6

8. Meta-analysis if transcriptional profile data from skin

of vitiligo patients .............................................................. 17

9. Statistical analysis ............................................................. 17

III. Result ........................................................................................ 18

1. Genome-scale transcriptional profile of HQ-treated

HKs ....................................................................................... 18

2. KEGG pathway-enrichment analysis on HQ-induced

DEGs .................................................................................... 30

3. Transcriptional profile comparison analyses between

HQ-induced DEGs and IL-17A-induced DEGs ......... 33

4. Validation of the HQ-induced DEGs with Q-RT-PCR35

5. IL-36g inhibited melanogenesis in HMs ...................... 38

6. IL-36g stimulated HKs to produce pro-inflammatory

cytokines .............................................................................. 42

7. Meta-analysis: clinical association of IL-36g with

human vitiligo .................................................................... 44

IV. Discussion ................................................................................. 46

V. Reference .................................................................................. 51

요약 (국문초록) .......................................................................... 58

7

LIST OF FIGURES

Figure 1. Effects of hydroquinone (HQ) on the cell viability of

human keratinocyte (HKs) .......................................................... 19

Figure 2. Transcriptional profile comparison analysis between

HQ-induced DEGs and cytokines-induced DEGs in HKs ......... 34

Figure 3. Validation of the upregulated DEGs in the HQ treated

HKs ............................................................................................. 36

Figure 4. Validation of the downregulated DEGs in the HQ treated

HKs ............................................................................................. 37

Figure 5. Melanogenesis inhibitory effects of IL-36g on HMs .. 40

Figure 6. The effects of IL-36g on the regulation of

melanogenesis-associated enzymes in HMs ............................... 41

Figure 7. The effects of IL-36g on HKs to initiate inflammatory

responses ..................................................................................... 43

Figure 8. Meta-analysis of transcriptional profiles of the lesioned

skin from vitiligo patients ........................................................... 45

8

LIST OF TABLES

Table 1. 250 up-regulated DEGs in the HQ-treated HKs ........... 20

Table 2. 250 down-regulated DEGs in the HQ-treated HKs ...... 25

Table 3. Top enriched KEGG pathways in the DEGs of HQ-treated

HKs ............................................................................................. 32

9

Ⅰ. Introduction

Chemical leukoderma, also referred to as occupational vitiligo, is a skin

pigmentary disorder with the phenotype of conspicuous white patches on the skin. It

is believed that the white patches on the skin are due to the loss of melanin producing

melanocytes, either by directly or indirectly affected by certain leukoderma inducing

chemicals (Bonamonte et al. 2016). Monobenzyl ether of hydroquinone (MBEH), an

antioxidant used in the rubber industry decades ago, is known to cause leukoderma

to many rubber industry factory workers (Mosher et al. 1977). Also, the phenolic

compound rhododendrol (RD), once used as a commercial skin whitening agent in

Japan, induced a serious side effect associated with vitiligo in over 16,000 customers

(2% of all users) (Tokura et al. 2015). The reason why certain chemicals induce

chemical leukoderma on human skin is not clearly understood, but the pathogenesis

of chemical leukoderma is thought to be as a multi-etiological process involving

genetic, biochemical, immunological, and diverse environmental factors (Boissy and

Manga 2004; Picardo et al. 2015). Others also suggest that the initiation of chemical

leukoderma is induced by chemical compounds while the progression and

establishment of vitiligo-like phenotypes are maintained by the host auto-

immunologic responses (Boissy and Manga 2004; Speeckaert and van Geel 2017).

Despite the importance of epidermal keratinocytes in diverse dermatological

disease conditions, the pathogenesis studies of leukoderma-inducing chemicals are

mainly focused on using melanocytes or melanocyte-derived cell lines as a biological

model system (Hariharan et al. 2010; Lee et al. 2016a; Toosi et al. 2012; Yang et al.

2015). For instance, tyrosine analogues can be metabolized by melanocytes through

10

tyrosinase, an important enzyme in the biosynthesis of melanin. When tyrosine

analogues are metabolized, reactive metabolites are produced, and these reactive

metabolites can further damage melanocytes and initiate the innate immune

responses (Ito and Wakamatsu 2018; Picardo et al. 2015). In addition, rhododendrol,

cosmetic ingredient that caused serious social vitiligo outbreak in Japan, elicited

leukoderma through selective cytotoxicity against human melanocytes via reactive

oxygen species dependent GADD45 pathway (Kim et al. 2016). Other study showed

that leukoderma inducing MBEH or 4-teriary butyl phenol (4-TBP) can elicit

inflammatory cytokines IL-6 and IL-8 in human melanocytes (HMs) via

endoplasmic reticulum unfolded protein response (ER-UPR), suggesting the

involvement of immunological responses in the pathogenesis of chemical

leukoderma (Toosi et al. 2012). Similar, but different study also showed that MBEH

and 4-TBP exert different toxicological responses in human melanocytes, MBEH

inducing caspase-independent necrotic cell death while 4-TBP is responsible for

caspase-dependent apoptotic cell death (Hariharan et al. 2010; Yang et al. 2000).

Human epidermal skin is mainly constituted of keratinocytes, melanocytes, and

Langerhans cells (Costin and Hearing 2007). Epidermal melanocytes and

keratinocytes together form a functional and structural unit, known as the epidermal-

melanin unit (Costin and Hearing 2007). Each melanocyte form direct contacts with

the surrounding keratinocytes via melanocyte dendrites, and those melanocytes

provide synthesized melanin pigments to the keratinocytes through the complex

melanosome transfer mechanism (Costin and Hearing 2007). Profoundly, epidermal

keratinocytes can also directly affect the differentiation, proliferation, and

melanogenesis of surrounding melanocytes (Imokawa 2004). For example,

11

epidermal keratinocytes can secrete diverse cytokines that can paracrinally modulate

the differentiation or proliferation of melanocytes such as stem cell factor (SCF),

endothelin-1 (EDN1), and basic fibroblast growth factor (bFGF) (Imokawa 2004;

Slominski et al. 2004). Also, under the stressed conditions either by internal or

external stimulants, keratinocytes can secrete IL-1a and IL-6, cytokines that are

known to be able to inhibit melanogenesis of melanocytes (Choi et al. 2012; Morelli

and Norris 1993; Swope et al. 1991). In this regard, when elucidating the potential

mechanisms of chemical leukoderma, the role of keratinocytes in relation to

melanocytes should be investigated altogether in the overall context.

Hydroquinone (HQ) is a well-known phenolic compound that can initiate

chemical leukoderma (Bonamonte et al. 2016; US FDA 2009). HQ was previously

used as a photographic developer in film industries or as a bleaching agent. Due to

the tyrosinase inhibitory effect of HQ, it is also included in a topical cream for skin

whitening (Bonamonte et al. 2016). Many regulatory agencies around the world have

begun to take actions on the use of HQ. Since 2001, the European Union has decided

to ban the use of HQ as a cosmetic whitening agent based on the scientific reports of

side-effects including leukoderma and exogeneous ochronosis (Kooyers and

Westerhof 2005). Recently, the United States Food and Drug Administration

proposed to ban all over-the-counter cosmetic products containing HQ concerning

its potential toxicity. In this study, to elucidate the toxicological role of keratinocytes

in the pathogenesis of chemical leukoderma, genome-scale transcriptional profile

analysis was performed in cultured human keratinocytes (HKs) treated with sub-

cytotoxic HQ concentration.

12

Ⅱ. Materials and Methods

1. Primary human keratinocyte, melanocyte culture and cell

viability assay

HKs originated from neonatal foreskins were purchased from Lonza (Basel,

Switzerland) and cultured in keratinocyte basal medium (KBM) supplied with

hydrocortisone, transferrin, epinephrine, gentamicin/amphotericin B, bovine

pituitary extract, recombinant human epidermal growth factor, and insulin. HKs

were serially passaged at 80% confluency and the third passage of primary HKs was

used in the cytotoxicity assay and other experiments used in this study. HMs from

neonatal foreskin of moderately pigmented donors were purchased from Cascade

Biologics (Portland, OR) and cultured in Medium 254 (Cascade Biologics) supplied

with human melanocyte growth supplement (HMGS/HMGS-2) (Cascade Biologics).

Hydroquinone was purchased from Sigma-Aldrich (St. Louis, MO, USA). A cell

viability assay was performed using Cell Counting Kit-8 (CCK-8, Dojindo,

Kumamoto, Japan) according to the manufacturer’s instruction. HKs were seeded at

a count of 5 ´ 104

cells to each well in 48-well plates and cultured up to 100%

confluence. HQ prepared in KBM/KGM media was treated to HKs for 24 h and

washed with phosphate-buffered saline (PBS) three times. CCK-8 solution was

added to the HQ-treated HKs for 2 h and the absorbance was measured at 450 nm

with a microplate reader (BioTek, Winooski, VT, USA).

13

2. Total RNA isolation and a genome-scale microarray

experiment

Total RNA was extracted using TRIzolTM

reagent (Invitrogen, Carlsbad, CA,

USA). RNA was further purified using a RNeasy Mini Kit (Qiagen, Germantown,

MD, USA). RNA integrity was analyzed with a BioAnalyzer 2100 (Agilent

Technologies, Santa Clara, CA, USA). Complementary DNA (cDNA) library was

prepared from each RNA sample (4 µg) using SuperScriptTM

reverse transcriptase

(Invitrogen) for a microarray experiment. Affymetrix Human Genome U133 Plus 2.0

GeneChip arrays were used for the genome-scale transcriptional analysis

(Affymetrix, Santa Clara, CA, USA). Differentially expressed genes (DEGs) were

selected as previously described (Lee et al. 2016b).

3. KEGG pathway-based functional enrichment analysis

The enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in

the HQ-induced DEGs were determined by analyzing the frequency of pathway map

identifiers defined in the pathway maps of the KEGG database. KEGG pathway map

identifiers for the entire set of genes in the Affymetrix Human U133 2.0 GeneChip

array were obtained from the KEGG webpage (https://www.genome.jp/kegg/). The

KEGG pathway-based functional enrichment analysis was performed using the

KEGG pathway map identifiers available on June 2018. A gene ontology (GO)

enrichment analysis for the common DEGs between the HQ-treated and T helper (Th)

cell cytokine-treated HKs was performed by comparing the frequency of GO

biological process (BP) terms annotated in the common DEGs with that of a total

14

gene set, and the GO annotation files were used from the Gene Ontology Consortium

webpage (the May 2018 version at https://www.geneontology.org). Due to the

redundant probe sets representing the same gene in the Affymetrix Human U133 2.0

GeneChip array, a total of 23,519 genes were used in the KEGG pathway and GO

enrichment analyses. A 2´2 contingency matrices were constructed to compare the

frequency of each KEGG pathway map identifier or GO BP term in DEG sets with

those of the total gene set in the microarray. The 2´2 contingency matrix was

evaluated using the calculation of p values by c2 test (frequency ³ 5) or Fisher’s

exact test (Frequency <5) using the C program.

4. Transcriptional profile comparison analysis

To compare the transcriptional profile between the HQ-induced DEGs and the

cytokine-induced DEGs in HKs, meta-analysis was performed with the genome-

scale transcriptional data on Th-cell cytokine-treated HKs available as GSE53751 at

the National Center for Biotechnology Information (NCBI) Gene Expression

Omnibus (GEO). Briefly, GSE53751 provides the raw data on the genome-scale

transcriptional profile of HKs in response to IFNg, IL-4, IL-6, IL-17A, or IL-22 (Jin

et al. 2014). The microarray raw data (CEL files) from GSE53751 were combined

with those of the HQ-treated HKs data for extracting gene expression values using

the Affymetrix Expression Console software. The number of common DEGs was

counted and illustrated as Venn diagrams.

15

5. Quantitative real-time reverse transcriptase chain reaction

and enzyme-linked immunosorbent assay

Quantitative real-time PCR (Q-RT-PCR) was performed using an AB7500 System

(Applied Biosystems, Foster City, CA, USA) as described (Jin et al. 2014; Lee et al.

2016b). IL-17A was purchased from Sigma-Aldrich (St. Louis, MO, USA). The

TaqMan expression primer sets (Applied Biosystems) used in the Q-RT-PCR were:

matrix metalloprotein 1 (MMP1), Hs00233958_m1; prostaglandin-endoperoxide

synthase 2 (PTGS2), Hs00153133_m1; interleukin 1 beta (IL1B), Hs01555410_m1;

interleukin 36 gamma (IL36G), Hs00219742_m1; S100 calcium binding protein A7

(S100A7), Hs01923188_u1; cystathionine-beta-synthase (CBS), Hs00163925_m1;

cystathionine gamma-lyase (CTH), Hs00542284_m1; cytochrome P450 family 1

subfamily A member 1 (CYP1A1), Hs01054797_g1; peroxisome proliferator

activated receptor delta (PPARD), Hs04187066_g1; frizzled class receptor 2 (FZD2),

Hs00361432_s1; Wnt family member 4 (WNT4), Hs00229142_m1; endothelin 1

(EDN1), Hs00174961_m1; interleukin 33 (IL33), Hs00369211_m1; melanogenesis

associated transcription factor (MITF), Hs01117294_m1; tyrosinase (TYR),

Hs00165976_m1; dopachrome tautomerase (DCT), Hs01098278_m1; tyrosinase

related protein 1 (TYRP1), Hs00167051_m1; interleukin 6 (IL6), Hs00985641_m1;

C-X-C motif chemokine ligand 8 (CXCL8), Hs00174103_m1; C-X-C motif

chemokine ligand 14 (CXCL14), Hs01557413_m1. Human ribosomal protein L13a

(RPL13A), Hs04194366_g1 and human glyceraldehyde-3-phosphate dehydrogenase

(GAPDH), Hs02786624_g1 were used as control genes. A mathematical model

proposed by Pfaffl was adopted for the quantification of the relative mRNA

16

expression level (Pfaffl 2001). Quantitative protein expression for IL-6, CXCL8/IL-

8, and CXCL14 was measured by ELISA assay using DuoSetâ ELISA Development

system (IL-6, CXCL8/IL-8, CXCL14 kits, R&D Systems, Minneapolis, MN, USA).

6. Western blot analysis

Protein levels were determined as described (Lee et al. 2018) with anti-

microphthalmia-associated transcription factor (MITF) antibody (#MS-771-P1,

Thermo Scientific), anti-tyrosinase antibody (#05-647, Sigma-Aldrich), anti-TRP1

antibody (#ab3312, Abcam), anti-TRP2/DCT antibody (#ab74073, Abcam), and

anti-b-actin antibody (#A5441, Sigma-Aldrich).

7. Melanin content assay

The melanin content assay for cultured human melanocytes was determined as

described (Choi et al. 2005, 2013). Briefly, melanocytes were harvested using

trypsin/EDTA solution and centrifuged for 5 min at 1000´g. Cell pellets were lysed

with 1N NaOH in a heat block (80˚C) for 30 min. Cell lysates were transferred into

96-well plates in triplicate and relative melanin content was determined by

measuring the absorbance at 490 nm using a microplate reader (BioTek, Winooski,

VT, USA).

17

8. Meta-analysis of transcriptional profile data from skin of

vitiligo patients

Three independent microarray studies for the skin of vitiligo patient samples were

available in NCBI GEO, accessed with GSE53146, GSE65127, and GSE75819.

Series matrix files for GSE53146 and GSE75819 were obtained and the gene

expression values of IL36G (Probe ID: ILMN_2158713) were normalized by

comparing the level of GAPDH expression across the samples. The raw data CEL

files for the GSE65127 study were available in NCBI GEO and the gene expression

value for IL36G (Probe set ID: 220322_at) was obtained with the Affymetrix

Expression Console software. P-values were calculated using a Wilcoxon signed-

rank test.

9. Statistical analysis

Statistical analysis was performed using SPSSâ for Windows (SPSS Science,

Chicago, IL, USA). For a comparison between a single treated group and the

experimental control, Student’s t test was used. For multiple comparisons, one-way

analysis of variance followed by Tukey’s post hoc tests were used.

18

Ⅲ. Results

1. Genome-scale transcriptional profile of HQ-treated HKs

Before analyzing the transcriptional profile of HQ-treated HKs, we determined

the sub-cytotoxic concentration of HQ in advance, since HQ concentration used in

our daily lives would not cause cell death in epidermal keratinocytes. The maximum

HQ concentration that did not affect the cell viability of HKs was 10 µM and the half

maximal cell viable concentration (CV50 value) was 62.6 µM (Fig. 1). Thus, genome-

scale transcription profile analysis was performed in HKs treated with 10 µM HQ.

The Affymetrix oligonucleotide microarray raw data (CEL files) are publicly

available in NCBI GEO using the accession number GSE126721. Among 23,519

unique genes represented by 54,676 array probes, 250 genes were selected as

upregulated or downregulated DEGs respectively (Table 1 & 2).

19

Figure 1. Effects of hydroquinone (HQ) on the cell viability of human

keratinocytes (HKs)

HKs were cultured in 48-well plate at 100% confluence and treated with HQ for 24

hours. Cell viability of HQ-treated HKs was measured using a Cell Counting Kit-8

assay (CCK-8, Dojindo, Kumamoto, Japan). After washing 3 times with phosphate

buffered saline (PBS), 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-

disulfophenyl)-2H-tetrazolium (WST-8) solution was treated to the HQ-treated HKs.

After 2 hours of incubation, the absorbance was measured at 450nm using a

microplate reader (BioTek, Winooski, VT, USA). Values represent the mean

expression level ± SD (n = 3). *p < 0.05; ** p < 0.01

0

50

100

150

0 1 3 10 30 100

HQ (µM)

**

Cel

l Via

bilit

y (%

)

20

Table 1. 250 up-regulated DEGs in the HQ-treated HKs

Probe Set ID Gene Title Gene Symbol HQFold Change

207302_at sarcoglycan, gamma SGCG 29.93205749_at cytochrome P450, family 1, subfamily A, polypeptide 1 CYP1A1 22.13204818_at hydroxysteroid (17-beta) dehydrogenase 2 HSD17B2 16.91202436_s_at cytochrome P450, family 1, subfamily B, polypeptide 1 CYP1B1 16.89219049_at chondroitin sulfate N-acetylgalactosaminyltransferase 1 CSGALNACT1 14.2741469_at peptidase inhibitor 3, skin-derived PI3 13.86205828_at matrix metallopeptidase 3 MMP3 13.561569583_at epiregulin EREG 13.32237732_at proline rich 9 PRR9 13.07219975_x_at oleoyl-ACP hydrolase OLAH 13.07224009_x_at dehydrogenase/reductase (SDR family) member 9 DHRS9 12.80236039_at LY6/PLAUR domain containing 5 LYPD5 12.31203234_at uridine phosphorylase 1 UPP1 12.221559393_at aldehyde dehydrogenase 1 family, member L2 ALDH1L2 12.15204475_at matrix metallopeptidase 1 MMP1 12.01218963_s_at keratin 23 KRT23 11.52208539_x_at small proline-rich protein 2B SPRR2B 10.53236119_s_at small proline-rich protein 2G SPRR2G 10.02212942_s_at cell migration inducing protein, hyaluronan binding CEMIP 10.01208161_s_at ATP-binding cassette, sub-family C (CFTR/MRP), member 3 ABCC3 9.60227410_at family with sequence similarity 43, member A FAM43A 9.19218000_s_at pleckstrin homology-like domain, family A, member 1 PHLDA1 9.00214088_s_at fucosyltransferase 3 FUT3 8.94209949_at neutrophil cytosolic factor 2 NCF2 8.83224329_s_at cornifelin CNFN 8.781553622_a_at fibrous sheath interacting protein 1 FSIP1 8.42206193_s_at corneodesmosin CDSN 8.18224461_s_at apoptosis-inducing factor, mitochondrion-associated, 2 AIFM2 7.84205899_at cyclin A1 CCNA1 7.66208893_s_at dual specificity phosphatase 6 DUSP6 7.57203946_s_at arginase 2 ARG2 7.56226769_at fin bud initiation factor homolog FIBIN 7.49219563_at long intergenic non-protein coding RNA 341 LINC00341 6.881555788_a_at tribbles pseudokinase 3 TRIB3 6.87219722_s_at glycerophosphodiester phosphodiesterase domain containing 3 GDPD3 6.8438037_at heparin-binding EGF-like growth factor HBEGF 6.66237974_at abhydrolase domain containing 12B ABHD12B 6.63212816_s_at cystathionine-beta-synthase CBS 6.55217127_at cystathionine gamma-lyase CTH 6.42206710_s_at erythrocyte membrane protein band 4.1-like 3 EPB41L3 6.42203304_at BMP and activin membrane-bound inhibitor BAMBI 6.30206662_at glutaredoxin GLRX 6.301554997_a_at prostaglandin-endoperoxide synthase 2 PTGS2 6.12224480_s_at 1-acylglycerol-3-phosphate O-acyltransferase 9 AGPAT9 6.09220620_at cysteine-rich C-terminal 1 CRCT1 6.06205067_at interleukin 1, beta IL1B 6.06208937_s_at inhibitor of DNA binding 1 ID1 6.05220625_s_at E74-like factor 5 ELF5 5.87206604_at ovo-like zinc finger 1 OVOL1 5.82201467_s_at NAD(P)H dehydrogenase, quinone 1 NQO1 5.77

21

Table 1. (Continued)

Probe Set ID Gene Title Gene Symbol HQFold Change

209383_at DNA-damage-inducible transcript 3 DDIT3 5.74209369_at annexin A3 ANXA3 5.69235740_at multiple C2 domains, transmembrane 1 MCTP1 5.63214858_at uncharacterized LOC100130449 PP14571 5.601553454_at repetin RPTN 5.57219181_at lipase, endothelial LIPG 5.51221577_x_at growth differentiation factor 15 GDF15 5.50209616_s_at carboxylesterase 1 CES1 5.481558212_at uncharacterized LOC101930048 LOC101930048 5.42221489_s_at sprouty homolog 4 SPRY4 5.35224328_s_at late cornified envelope 3D LCE3D 5.32206584_at lymphocyte antigen 96 LY96 5.29206628_at solute carrier family 5 (sodium/glucose cotransporter), member 1 SLC5A1 5.28206043_s_at ATPase, Ca++ transporting, type 2C, member 2 ATP2C2 5.26223878_at inositol polyphosphate-4-phosphatase, type II, 105kDa INPP4B 5.21221022_s_at polyamine modulated factor 1 binding protein 1 PMFBP1 5.21221541_at cysteine-rich secretory protein LCCL domain containing 2 CRISPLD2 5.20206932_at cholesterol 25-hydroxylase CH25H 5.20202241_at tribbles pseudokinase 1 TRIB1 5.12212531_at lipocalin 2 LCN2 5.09206115_at early growth response 3 EGR3 5.04235514_at aspartic peptidase, retroviral-like 1 ASPRV1 5.03215209_at SEC24 family member D SEC24D 5.03231930_at ELMO/CED-12 domain containing 1 ELMOD1 5.02202014_at protein phosphatase 1, regulatory subunit 15A PPP1R15A 5.00204733_at kallikrein-related peptidase 6 KLK6 4.981569555_at guanine deaminase GDA 4.93229070_at androgen-dependent TFPI-regulating protein ADTRP 4.92206595_at cystatin E/M CST6 4.92210589_s_at glucosidase, beta, acid pseudogene 1 GBAP1 4.87206177_s_at arginase 1 ARG1 4.82212233_at microtubule-associated protein 1B MAP1B 4.79235737_at thymic stromal lymphopoietin TSLP 4.78203665_at heme oxygenase (decycling) 1 HMOX1 4.78217678_at solute carrier family 7, member 11 SLC7A11 4.76233011_at annexin A1 ANXA1 4.71209093_s_at glucosidase, beta, acid GBA 4.69207710_at late cornified envelope 2B LCE2B 4.65228042_at ADP-ribosylarginine hydrolase ADPRH 4.63219371_s_at Kruppel-like factor 2 KLF2 4.60229740_at small integral membrane protein 5 SMIM5 4.55230574_at uncharacterized LOC100130938 LOC100130938 4.52204351_at S100 calcium binding protein P S100P 4.52207381_at arachidonate 12-lipoxygenase, 12R type ALOX12B 4.49230349_at XK, Kell blood group complex subunit-related, X-linked XKRX 4.46234699_at ribonuclease, RNase A family, 7 RNASE7 4.45228964_at PR domain containing 1, with ZNF domain PRDM1 4.44214636_at calcitonin-related polypeptide beta CALCB 4.43211603_s_at ets variant 4 ETV4 4.39222223_s_at interleukin 36 receptor antagonist IL36RN 4.38

22

Table 1. (Continued)

Probe Set ID Gene Title Gene Symbol HQFold Change

238654_at V-set and immunoglobulin domain containing 10 like VSIG10L 4.32242893_at RP11-3L8.3 RP11-3L8.3 4.32210511_s_at inhibin, beta A INHBA 4.27213256_at E3 ubiquitin protein ligase MARCH3 4.261552532_a_at ATPase, H+ transporting, lysosomal 42kDa, V1 subunit C2 ATP6V1C2 4.24223082_at SH3-domain kinase binding protein 1 SH3KBP1 4.18236140_at glutamate-cysteine ligase, modifier subunit GCLM 4.181553077_at short chain dehydrogenase/reductase family 9C, member 7 SDR9C7 4.18230765_at NACHT and WD repeat domain containing 2 NWD2 4.17206969_at keratin 34 KRT34 4.16203439_s_at stanniocalcin 2 STC2 4.141558044_s_at exosome component 6 EXOSC6 4.09219256_s_at SH3 domain and tetratricopeptide repeats 1 SH3TC1 4.081553081_at WAP four-disulfide core domain 12 WFDC12 4.07209457_at dual specificity phosphatase 5 DUSP5 4.05201631_s_at immediate early response 3 IER3 4.03215977_x_at glycerol kinase GK 4.02220322_at interleukin 36, gamma IL36G 4.01239288_at TRAF2 and NCK interacting kinase TNIK 3.98202847_at phosphoenolpyruvate carboxykinase 2 PCK2 3.97212190_at serpin peptidase inhibitor, member 2 SERPINE2 3.93214599_at involucrin IVL 3.92243222_at alpha-kinase 1 ALPK1 3.92226726_at membrane bound O-acyltransferase domain containing 2 MBOAT2 3.91205047_s_at asparagine synthetase ASNS 3.91242218_at peroxisome proliferator-activated receptor delta PPARD 3.90223195_s_at sestrin 2 SESN2 3.9091826_at EPS8-like 1 EPS8L1 3.90219836_at zinc finger, BED-type containing 2 ZBED2 3.87227123_at RAB3B, member RAS oncogene family RAB3B 3.87210999_s_at growth factor receptor-bound protein 10 GRB10 3.87202510_s_at tumor necrosis factor, alpha-induced protein 2 TNFAIP2 3.83201481_s_at phosphorylase, glycogen; brain PYGB 3.81229764_at tumor protein p63 regulated 1 TPRG1 3.81225799_at long intergenic non-protein coding RNA 152 LINC00152 3.81217966_s_at family with sequence similarity 129, member A FAM129A 3.80242998_at retinol dehydrogenase 12 RDH12 3.79234994_at transmembrane protein 200A TMEM200A 3.79205660_at 2'-5'-oligoadenylate synthetase-like OASL 3.77225822_at transmembrane protein 125 TMEM125 3.76228538_at zinc finger protein 662 ZNF662 3.74232365_at siah E3 ubiquitin protein ligase 1 SIAH1 3.74219270_at ChaC, cation transport regulator homolog 1 CHAC1 3.73205623_at aldehyde dehydrogenase 3 family, member A1 ALDH3A1 3.71226886_at glutamine--fructose-6-phosphate transaminase 1 GFPT1 3.69221050_s_at GTP binding protein 2 GTPBP2 3.69222858_s_at dual adaptor of phosphotyrosine and 3-phosphoinositides DAPP1 3.68207720_at loricrin LOR 3.66201266_at thioredoxin reductase 1 TXNRD1 3.64205428_s_at calbindin 2 CALB2 3.61

23

Table 1. (Continued)

Probe Set ID Gene Title Gene Symbol HQFold Change

1555773_at BPI fold containing family C BPIFC 3.59

1553213_a_at keratin 78 KRT78 3.56

230323_s_at transmembrane protein 45B TMEM45B 3.55

1560848_at uncharacterized LOC101927490 LOC101927490 3.54

202831_at glutathione peroxidase 2 GPX2 3.50

209723_at serpin peptidase inhibitor, clade B (ovalbumin), member 9 SERPINB9 3.49

1569787_at raftlin, lipid raft linker 1 RFTN1 3.49

236429_at zinc finger protein 83 ZNF83 3.47

219998_at lectin, galactoside-binding-like LGALSL 3.41

1569144_a_at cysteine-rich tail protein 1 CYSRT1 3.41

1557389_at SH3PXD2A antisense RNA 1 SH3PXD2A-AS1 3.41

205174_s_at glutaminyl-peptide cyclotransferase QPCT 3.41

1554594_at Rho GTPase activating protein 27 ARHGAP27 3.39

211250_s_at SH3-domain binding protein 2 SH3BP2 3.39

205916_at S100 calcium binding protein A7 S100A7 3.39

218368_s_at tumor necrosis factor receptor superfamily, member 12A TNFRSF12A 3.38

237367_x_at CASP8 and FADD-like apoptosis regulator CFLAR 3.36

221539_at eukaryotic translation initiation factor 4E binding protein 1 EIF4EBP1 3.33

207717_s_at plakophilin 2 PKP2 3.33

202558_s_at heat shock protein 70kDa family, member 13 HSPA13 3.31

210118_s_at interleukin 1, alpha IL1A 3.31

206239_s_at serine peptidase inhibitor, Kazal type 1 SPINK1 3.31

207206_s_at arachidonate 12-lipoxygenase ALOX12 3.30

222290_at olfactory receptor, family 2, subfamily A, member 20 pseudogene OR2A20P 3.30

1558871_at Homo sapiens, clone IMAGE:4105785, mRNA. BC016361 3.29

226834_at CXADR-like membrane protein CLMP 3.26

219095_at JMJD7-PLA2G4B readthrough JMJD7-PLA2G4B 3.26

220771_at long intergenic non-protein coding RNA 328 LINC00328 3.24

208813_at glutamic-oxaloacetic transaminase 1, soluble GOT1 3.23

223484_at chromosome 15 open reading frame 48 C15orf48 3.22

1564333_a_at prosaposin-like 1 PSAPL1 3.22

209291_at inhibitor of DNA binding 4 ID4 3.22

221827_at RanBP-type and C3HC4-type zinc finger containing 1 RBCK1 3.21

236845_at tripartite motif containing 62 TRIM62 3.20

239127_at uncharacterized LOC100288860 LOC100288860 3.20

230729_at RP11-355B11.2 RP11-355B11.2 3.19

222795_s_at phosphatidylinositol-specific phospholipase C, X domain containing 1 PLCXD1 3.18

239739_at sorting nexin 24 SNX24 3.17

217682_at RP11-473I1.9 RP11-473I1.9 3.16

1570490_at RP11-646E18.4 RP11-646E18.4 3.15

203817_at guanylate cyclase 1, soluble, beta 3 GUCY1B3 3.14

222383_s_at arachidonate lipoxygenase 3 ALOXE3 3.12

212912_at ribosomal protein S6 kinase, 90kDa, polypeptide 2 RPS6KA2 3.12

244056_at surfactant associated 2 SFTA2 3.12

241418_at NmrA-like family domain containing 1 pseudogene LOC344887 3.08

205659_at histone deacetylase 9 HDAC9 3.08

238213_at microRNA 5188 MIR5188 3.08

218692_at syntabulin SYBU 3.06

220334_at regulator of G-protein signaling 17 RGS17 3.05

204088_at purinergic receptor P2X, ligand-gated ion channel, 4 P2RX4 3.04

24

Table 1. (Continued)

Probe Set ID Gene Title Gene Symbol HQFold Change

236069_at CTD-2619J13.13 CTD-2619J13.13 3.02

202147_s_at interferon-related developmental regulator 1 IFRD1 3.02

213421_x_at protease, serine, 3 PRSS3 3.02

230944_at chromosome 6 open reading frame 223 C6orf223 3.01

204420_at FOS-like antigen 1 FOSL1 3.01

204979_s_at SH3 domain binding glutamate-rich protein SH3BGR 3.00

227806_at chromosome 16 open reading frame 74 C16orf74 3.00

213524_s_at G0/G1 switch 2 G0S2 3.00

1552685_a_at grainyhead-like 1 GRHL1 3.00

36711_at v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog F MAFF 3.00

206101_at extracellular matrix protein 2, female organ and adipocyte specific ECM2 2.99

214455_at histone cluster 1, H2bc HIST1H2BC 2.99

219554_at Rh family, C glycoprotein RHCG 2.98

240549_at uncharacterized LOC100507459 LOC100507459 2.97

209101_at connective tissue growth factor CTGF 2.93

209193_at Pim-1 proto-oncogene, serine/threonine kinase PIM1 2.93

228442_at nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 NFATC2 2.93

215779_s_at histone cluster 1, H2bg HIST1H2BG 2.92

227747_at myelin protein zero-like 3 MPZL3 2.92

1556321_a_at mesoderm development candidate 1 MESDC1 2.91

235595_at Rho/Rac guanine nucleotide exchange factor (GEF) 2 ARHGEF2 2.91

57163_at ELOVL fatty acid elongase 1 ELOVL1 2.89

221765_at UDP-glucose ceramide glucosyltransferase UGCG 2.88

209309_at alpha-2-glycoprotein 1, zinc-binding AZGP1 2.87

229493_at HOXD cluster antisense RNA 2 HOXD-AS2 2.87

224262_at interleukin 1 family, member 10 (theta) IL1F10 2.87

204802_at Ras-related associated with diabetes RRAD 2.86

219532_at ELOVL fatty acid elongase 4 ELOVL4 2.86

209822_s_at very low density lipoprotein receptor VLDLR 2.85

1552544_at serpin peptidase inhibitor, member 12 SERPINA12 2.85

203889_at secretogranin V SCG5 2.84

222876_s_at ArfGAP with dual PH domains 2 ADAP2 2.84

204072_s_at furry homolog FRY 2.83

223852_s_at serine/threonine kinase 40 STK40 2.82

238482_at Kruppel-like factor 7 KLF7 2.82

232434_at disrupted in renal carcinoma 3 DIRC3 2.80

203921_at carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2 CHST2 2.80

207631_at neighbor of BRCA1 gene 2 NBR2 2.80

239136_at UNC5B antisense RNA 1 UNC5B-AS1 2.79

228205_at transketolase TKT 2.79

220944_at peptidoglycan recognition protein 4 PGLYRP4 2.79

231849_at keratin 80 KRT80 2.79

232082_x_at small proline-rich protein 3 SPRR3 2.79

1562245_a_at zinc finger protein 578 ZNF578 2.78

239648_at DCN1, defective in cullin neddylation 1, domain containing 3 DCUN1D3 2.77

225867_at vasorin VASN 2.77

219529_at chloride intracellular channel 3 CLIC3 2.76

41856_at unc-5 homolog B UNC5B 2.76

231733_at caspase recruitment domain family, member 18 CARD18 2.75

202672_s_at activating transcription factor 3 ATF3 2.75

25

Table 2. 250 down-regulated DEGs in the HQ-treated HKs

Probe Set ID Gene Title Gene Symbol HQFold Change

203708_at phosphodiesterase 4B, cAMP-specific PDE4B 0.07

215039_at long intergenic non-protein coding RNA 1140 LINC01140 0.09

241365_at SATB homeobox 1 SATB1 0.12

203153_at interferon-induced protein with tetratricopeptide repeats 1 IFIT1 0.13

209732_at C-type lectin domain family 2, member B CLEC2B 0.13

209821_at interleukin 33 IL33 0.14

1555145_at centriole, cilia and spindle-associated protein CCSAP 0.14

206149_at calcineurin-like EF-hand protein 2 CHP2 0.14

217203_at glutamate-ammonia ligase (glutamine synthetase) pseudogene 4 GLULP4 0.14

204457_s_at growth arrest-specific 1 GAS1 0.16

204430_s_at solute carrier family 2, member 5 SLC2A5 0.16

214598_at claudin 8 CLDN8 0.16

206758_at endothelin 2 EDN2 0.17

210220_at frizzled class receptor 2 FZD2 0.20

204821_at butyrophilin, subfamily 3, member A3 BTN3A3 0.20

220263_at SMAD5 antisense RNA 1 SMAD5-AS1 0.20

1559126_at ribosomal RNA processing 12 homolog RRP12 0.21

208606_s_at wingless-type MMTV integration site family, member 4 WNT4 0.21

204439_at interferon-induced protein 44-like IFI44L 0.21

213733_at myosin IF MYO1F 0.22

222802_at endothelin 1 EDN1 0.22

230560_at syntaxin binding protein 6 STXBP6 0.23

1552390_a_at glutamate-rich 5 ERICH5 0.23

226702_at cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial CMPK2 0.23

214059_at interferon-induced protein 44 IFI44 0.23

242979_at insulin receptor substrate 1 IRS1 0.24

215491_at v-myc avian myelocytomatosis viral oncogene lung carcinoma derived homolog MYCL 0.24

204519_s_at plasmolipin PLLP 0.25

232060_at receptor tyrosine kinase-like orphan receptor 1 ROR1 0.25

229927_at LEM domain containing 1 LEMD1 0.25

242907_at guanylate binding protein 2, interferon-inducible GBP2 0.25

207826_s_at inhibitor of DNA binding 3, dominant negative helix-loop-helix protein ID3 0.26

222717_at serum deprivation response SDPR 0.26

1553973_a_at serine peptidase inhibitor, Kazal type 6 SPINK6 0.26

204820_s_at butyrophilin, subfamily 3, member A2 BTN3A2 0.27

239750_x_at VAMP (vesicle-associated membrane protein)-associated protein A, 33kDa VAPA 0.27

210095_s_at insulin-like growth factor binding protein 3 IGFBP3 0.27

235752_at AC008746.12 AC008746.12 0.27

239552_at von Willebrand factor D and EGF domains VWDE 0.28

204972_at 2'-5'-oligoadenylate synthetase 2, 69/71kDa OAS2 0.28

204865_at carbonic anhydrase III, muscle specific CA3 0.28

203753_at transcription factor 4 TCF4 0.28

1560011_at Homo sapiens cDNA FLJ35113 fis, clone PLACE6007242. AX747544 0.28

229450_at interferon-induced protein with tetratricopeptide repeats 3 IFIT3 0.28

224348_s_at H19 Opposite Tumor Suppressor HOTS 0.28

209969_s_at signal transducer and activator of transcription 1, 91kDa STAT1 0.29

202086_at myxovirus resistance 1, interferon-inducible protein p78 MX1 0.29

203798_s_at visinin-like 1 VSNL1 0.29

231089_at uncharacterized LOC100505664 LOC100505664 0.30

1559003_a_at coiled-coil domain containing 163, pseudogene CCDC163P 0.30

26

Table 2. (Continued)

Probe Set ID Gene Title Gene Symbol HQFold Change

202037_s_at secreted frizzled-related protein 1 SFRP1 0.30

205483_s_at ISG15 ubiquitin-like modifier ISG15 0.30

200799_at heat shock 70kDa protein 1A HSPA1A 0.30

214298_x_at septin 6 SEPT6 0.30

33767_at neurofilament, heavy polypeptide NEFH 0.30

237083_at RP4-813F11.4 RP4-813F11.4 0.30

216384_x_at prothymosin alpha-like PTMA 0.30

239398_at kelch-like family member 31 KLHL31 0.30

1553111_a_at kelch repeat and BTB (POZ) domain containing 6 KBTBD6 0.31

207761_s_at methyltransferase like 7A METTL7A 0.31

240300_at thymidine kinase 2, mitochondrial TK2 0.31

237247_at ubiquitin specific peptidase 51 USP51 0.31

231292_at EP300 interacting inhibitor of differentiation 3 EID3 0.31

242281_at glutamate-ammonia ligase GLUL 0.31

214022_s_at interferon induced transmembrane protein 1 IFITM1 0.32

204622_x_at nuclear receptor subfamily 4, group A, member 2 NR4A2 0.32

204187_at guanosine monophosphate reductase GMPR 0.32

218986_s_at DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 DDX60 0.32

202963_at regulatory factor X, 5 RFX5 0.32

220577_at GTPase, very large interferon inducible pseudogene 1 GVINP1 0.32

240413_at pyrin and HIN domain family, member 1 PYHIN1 0.32

237245_at BUB3 mitotic checkpoint protein BUB3 0.32

239533_at G protein-coupled receptor 155 GPR155 0.32

236834_at sec1 family domain containing 2 SCFD2 0.32

235355_at cysteine-serine-rich nuclear protein 3 CSRNP3 0.32

223282_at teashirt zinc finger homeobox 1 TSHZ1 0.33

204070_at retinoic acid receptor responder (tazarotene induced) 3 RARRES3 0.33

220026_at chloride channel accessory 4 CLCA4 0.33

244353_s_at solute carrier family 2 (facilitated glucose transporter), member 12 SLC2A12 0.33

233364_s_at Homo sapiens cDNA FLJ11742 fis, clone HEMBA1005508. AK021804 0.33

213167_s_at solute carrier family 5, member 3 SLC5A3 0.33

228884_at leucine rich repeat containing 27 LRRC27 0.33

211329_x_at hemochromatosis HFE 0.33

238428_at potassium inwardly-rectifying channel, subfamily J, member 15 KCNJ15 0.33

1553743_at methyltransferase like 21A METTL21A 0.33

225546_at eukaryotic elongation factor-2 kinase EEF2K 0.33

213348_at cyclin-dependent kinase inhibitor 1C CDKN1C 0.33

1567222_x_at ELOVL fatty acid elongase 5 ELOVL5 0.34

238865_at poly(A) binding protein, cytoplasmic 4-like PABPC4L 0.34

219497_s_at B-cell CLL/lymphoma 11A BCL11A 0.34

205552_s_at 2'-5'-oligoadenylate synthetase 1, 40/46kDa OAS1 0.34

224172_at AC008088.4 AC008088.4 0.34

204415_at interferon, alpha-inducible protein 6 IFI6 0.34

223843_at scavenger receptor class A, member 3 SCARA3 0.34

212148_at pre-B-cell leukemia homeobox 1 PBX1 0.34

229720_at BCL2-associated athanogene BAG1 0.34

203596_s_at interferon-induced protein with tetratricopeptide repeats 5 IFIT5 0.34

202973_x_at family with sequence similarity 13, member A FAM13A 0.34

232563_at zinc finger protein 684 ZNF684 0.35

201416_at SRY (sex determining region Y)-box 4 SOX4 0.35

27

Table 2. (Continued)

Probe Set ID Gene Title Gene Symbol HQFold Change

204557_s_at DAZ interacting zinc finger protein 1 DZIP1 0.35228617_at XIAP associated factor 1 XAF1 0.35209243_s_at paternally expressed 3 PEG3 0.35210538_s_at baculoviral IAP repeat containing 3 BIRC3 0.35229298_at kelch repeat and BTB (POZ) domain containing 7 KBTBD7 0.35238709_at elongator acetyltransferase complex subunit 2 ELP2 0.35229290_at death associated protein-like 1 DAPL1 0.35235621_at fumarylacetoacetate hydrolase domain containing 2A FAHD2A 0.36227735_s_at chromosome 10 open reading frame 99 C10orf99 0.36223496_s_at coiled-coil domain containing 8 CCDC8 0.36228293_at DEP domain containing 7 DEPDC7 0.36219209_at interferon induced with helicase C domain 1 IFIH1 0.36233391_at cadherin 26 CDH26 0.36

214664_at phosphoribosylaminoimidazole carboxylase,phosphoribosylaminoimidazole succinocarboxamide synthetase PAICS 0.36

236068_s_at chromosome 3 open reading frame 14 C3orf14 0.36226625_at transforming growth factor, beta receptor III TGFBR3 0.36201427_s_at selenoprotein P, plasma, 1 SEPP1 0.36209094_at dimethylarginine dimethylaminohydrolase 1 DDAH1 0.36201601_x_at interferon induced transmembrane protein 1 IFITM1 0.36226281_at delta/notch-like EGF repeat containing DNER 0.36230266_at RAB7B, member RAS oncogene family RAB7B 0.37203108_at G protein-coupled receptor, class C, group 5, member A GPRC5A 0.37237075_at uncharacterized LOC101060091 LOC101060091 0.37239576_at microtubule associated tumor suppressor 1 MTUS1 0.37221110_x_at phosphodiesterase 11A PDE11A 0.37236704_at uncharacterized LOC101929787 LOC101929787 0.371557166_at programmed cell death 4 PDCD4 0.37210020_x_at calmodulin-like 3 CALML3 0.37242418_at chromosome 2 open reading frame 27A C2orf27A 0.37208078_s_at salt-inducible kinase 1 SIK1 0.37226974_at neural precursor cell expressed, developmentally down-regulated 4-like NEDD4L 0.371564383_s_at FLJ35934 FLJ35934 0.37236337_at synaptonemal complex protein 2-like SYCP2L 0.37

239846_at methylenetetrahydrofolate dehydrogenase 1,methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase CTD-2555O16.4 0.38

230012_at long intergenic non-protein coding RNA 324 LINC00324 0.38219488_at alpha 1,4-galactosyltransferase A4GALT 0.38243302_at RP11-250B2.6 RP11-250B2.6 0.38223220_s_at poly (ADP-ribose) polymerase family, member 9 PARP9 0.38230113_at muscleblind-like splicing regulator 3 MBNL3 0.38229705_at phosphatidylinositol 3-kinase, catalytic subunit type 3 PIK3C3 0.38212594_at programmed cell death 4 PDCD4 0.381556742_at glucuronidase, beta pseudogene 1 GUSBP1 0.381559034_at signal-regulatory protein beta 2 SIRPB2 0.38221287_at ribonuclease L (2',5'-oligoisoadenylate synthetase-dependent) RNASEL 0.38224646_x_at H19, imprinted maternally expressed transcript H19 0.38210643_at tumor necrosis factor (ligand) superfamily, member 11 TNFSF11 0.38221756_at phosphoinositide-3-kinase interacting protein 1 PIK3IP1 0.38201650_at keratin 19 KRT19 0.38226549_at SH3 domain binding kinase 1 SBK1 0.39219863_at HECT and RLD domain containing E3 ubiquitin protein ligase 5 HERC5 0.39

28

Table 2. (Continued)

Probe Set ID Gene Title Gene Symbol HQFold Change

207757_at ZFP2 zinc finger protein ZFP2 0.39

235768_at SH3 domain containing ring finger 2 SH3RF2 0.39

219528_s_at B-cell CLL/lymphoma 11B BCL11B 0.39

244669_at small nucleolar RNA, C/D box 50A SNORD50A 0.39

228390_at RAB30, member RAS oncogene family RAB30 0.39

228718_at zinc finger protein 44 ZNF44 0.39

207558_s_at paired-like homeodomain 2 PITX2 0.39

221460_at olfactory receptor, family 2, subfamily C, member 1 OR2C1 0.39

207214_at serine peptidase inhibitor, Kazal type 4 SPINK4 0.39

237750_at X-prolyl aminopeptidase (aminopeptidase P) 3, putative XPNPEP3 0.39

228262_at MAP7 domain containing 2 MAP7D2 0.39

207030_s_at cysteine and glycine-rich protein 2 CSRP2 0.39

223129_x_at myosin regulatory light chain interacting protein MYLIP 0.39

217107_at ribosomal protein S4X pseudogene 9 RPS4XP9 0.39

203747_at aquaporin 3 (Gill blood group) AQP3 0.39

227228_s_at coiled-coil domain containing 88C CCDC88C 0.40

219800_s_at threonine synthase-like 1 (S. cerevisiae) THNSL1 0.40

1558859_at uncharacterized LOC101928623 LOC101928623 0.40

212282_at transmembrane protein 97 TMEM97 0.40

205158_at ribonuclease, RNase A family, 4 RNASE4 0.40

1569086_at chromosome 19 open reading frame 83 C19orf83 0.40

206561_s_at aldo-keto reductase family 1, member B10 AKR1B10 0.40

205199_at carbonic anhydrase IX CA9 0.40

229390_at family with sequence similarity 26, member F FAM26F 0.40

215342_s_at RAB GTPase activating protein 1-like RABGAP1L 0.40

228038_at SRY (sex determining region Y)-box 2 SOX2 0.40

1569898_a_at PAXIP1 opposite strand PAXIP1OS 0.40

243880_at golgi SNAP receptor complex member 2 GOSR2 0.40

205225_at estrogen receptor 1 ESR1 0.40

235155_at 3-hydroxybutyrate dehydrogenase, type 2 BDH2 0.40

232187_at palmdelphin PALMD 0.40

206271_at toll-like receptor 3 TLR3 0.40

201641_at bone marrow stromal cell antigen 2 BST2 0.40

208392_x_at SP110 nuclear body protein SP110 0.41

208042_at angiogenic factor with G patch and FHA domains 1 AGGF1 0.41

229848_at zinc finger protein 10 ZNF10 0.41

222121_at Rho guanine nucleotide exchange factor (GEF) 26 ARHGEF26 0.41

1568932_at Homo sapiens cDNA clone IMAGE:4830452 BC034636 0.41

229568_at MOB kinase activator 3B MOB3B 0.41

220267_at keratin 24 KRT24 0.41

217406_at RP1-149A16.17 RP1-149A16.17 0.41

213433_at ADP-ribosylation factor-like 3 ARL3 0.41

231919_at dihydrolipoamide branched chain transacylase E2 DBT 0.41

239483_at uncharacterized LOC399821 FLJ37035 0.41

222663_at RIO kinase 2 RIOK2 0.41

204612_at protein kinase (cAMP-dependent, catalytic) inhibitor alpha PKIA 0.41

1558692_at chromosome 1 open reading frame 85 C1orf85 0.41

229126_at transmembrane protein 19 TMEM19 0.41

1552705_at dual specificity phosphatase 19 DUSP19 0.41

203563_at actin filament associated protein 1 AFAP1 0.41

29

Table 2. (Continued)

Probe Set ID Gene Title Gene Symbol HQFold Change

229497_at ankyrin repeat and death domain containing 1A ANKDD1A 0.42202145_at lymphocyte antigen 6 complex, locus E LY6E 0.42235744_at PTC7 protein phosphatase homolog (S. cerevisiae) PPTC7 0.42230047_at Rho GTPase activating protein 42 ARHGAP42 0.42204380_s_at fibroblast growth factor receptor 3 FGFR3 0.42238179_at spastic paraplegia 7 SPG7 0.42236046_at coiled-coil domain containing 127 CCDC127 0.42215922_at RALBP1 associated Eps domain containing 1 REPS1 0.42202007_at nidogen 1 NID1 0.42244049_at RP11-422P24.11 RP11-422P24.11 0.42221868_at poly(A) binding protein interacting protein 2B PAIP2B 0.42235154_at TAF3 RNA polymerase II TAF3 0.421554067_at chromosome 12 open reading frame 66 C12orf66 0.42219962_at angiotensin I converting enzyme 2 ACE2 0.42221729_at collagen, type V, alpha 2 COL5A2 0.42231195_at killer cell lectin-like receptor subfamily G, member 2 KLRG2 0.42228341_at nudix (nucleoside diphosphate linked moiety X)-type motif 16 NUDT16 0.421553220_at family with sequence similarity 117, member B FAM117B 0.43225415_at deltex 3 like, E3 ubiquitin ligase DTX3L 0.43

230482_atST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 5

ST6GALNAC5 0.43

1568906_at uncharacterized LOC728196 LOC728196 0.43205407_at reversion-inducing-cysteine-rich protein with kazal motifs RECK 0.43200800_s_at heat shock 70kDa protein 1A HSPA1A 0.43230060_at cell division cycle associated 7 CDCA7 0.43230748_at solute carrier family 16, member 6 SLC16A6 0.431553815_a_at transcription elongation factor A (SII) N-terminal and central domain containing TCEANC 0.43218326_s_at leucine-rich repeat containing G protein-coupled receptor 4 LGR4 0.43217019_at ribosomal protein S4X pseudogene 3 RPS4XP3 0.43230774_at prostaglandin reductase 2 PTGR2 0.43211018_at lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase) LSS 0.43235498_at leucine-rich repeats and IQ motif containing 3 LRRIQ3 0.43214579_at NIPA-like domain containing 3 NIPAL3 0.43230127_at RP6-99M1.2 RP6-99M1.2 0.43241863_x_at tetratricopeptide repeat domain 14 TTC14 0.43216490_x_at ribosomal protein, large P2, pseudogene 1 RPLP2P1 0.43225123_at sestrin 3 SESN3 0.43203989_x_at coagulation factor II (thrombin) receptor F2R 0.43227913_at exosome component 3 EXOSC3 0.43204915_s_at SRY (sex determining region Y)-box 11 SOX11 0.43221103_s_at WD repeat domain 52 WDR52 0.43203636_at midline 1 MID1 0.431557331_at polymerase (RNA) I polypeptide B, 128kDa POLR1B 0.43213375_s_at NEDD4 binding protein 2-like 1 N4BP2L1 0.43217353_at heterogeneous nuclear ribonucleoprotein A1 pseudogene 37 HNRNPA1P37 0.44238220_at lysine (K)-specific demethylase 6A KDM6A 0.44207935_s_at keratin 13 KRT13 0.44206752_s_at DNA fragmentation factor, 40kDa, beta polypeptide (caspase-activated DNase) DFFB 0.44226636_at phospholipase D1, phosphatidylcholine-specific PLD1 0.44213273_at teneurin transmembrane protein 4 TENM4 0.44223204_at family with sequence similarity 198, member B FAM198B 0.44

30

2. KEGG pathway-enrichment analysis on HQ-induced DEGs

Transcriptomics analysis such as microarray or RNA-seq is a powerful tool that

can reveal the mRNA transcription status of a cell on whole genome-context.

However, due to the massive amount of data produced by transcriptomics analysis,

the interpretation of its data is very limited. To better understand biological processes

or cellular phenotypes behind the HQ-induced DEGs, a KEGG pathway enrichment

analysis was performed on the HQ-induced upregulated and downregulated DEGs.

Out of total 380 KEGG individual pathways, the HQ-induced upregulated DEGs was

annotated with 81 KEGG pathways while the HQ-induced downregulated DEGs was

annotated with 58 KEGG pathways. To determine significantly associated KEGG

pathways, contingency matrices were generated with 135 annotated KEGG

pathways and Fisher’s exact test or c2 test were performed. Among 81 KEGG

pathways on 250 HQ-induced upregulated DEGs in HKs, IL-17 signaling pathway

(hsa04657) was determined to be the most significantly upregulated KEGG pathway

(Table 3), with a p-value with less than 10-4

. Furthermore, arachidonic acid

metabolism (hsa00590), an important pathway controlling the inflammation

response, was also enriched in the HQ-induced upregulated DEGs. Notably, HQ

induced the upregulation of pathways associated with amino acid metabolism,

including alanine, arginine, aspartate, cysteine, glutamate, and methionine

(hsa00220, hsa00250, hsa00270, and hsa00330) (Table 3). Among 58 KEGG

pathways on 250 HQ-induced downregulated DEGs, signaling pathways regulating

pluripotency of stem cells (hsa04550) was determined to be the most significantly

downregulated KEGG pathway. To our surprise, melanogenesis (hsa04916) was

31

included as one of the major downregulated KEGG pathways in the HQ-induced

downregulated DEGs (Table 3).

32

Table 3. Top enriched KEGG pathways in the DEGs of HQ-treated HKs

‘#’ denotes ‘number’

KEGGpathway Enriched KEGG pathways in DEGs p value

(chi test)

# of DEGs inthe KEGGpathway

# of Genes inthe KEGGpathway

(total 23519)

DEGs(Fold change)

hsa04657 IL-17 signaling pathway 5.34034E-05 8 92 MMP3(13.56),MMP1(12.01),PTGS2(6.12),IL1B(6.06),LCN2(5.09),IL36G(4.01),S100A7(3.39),FOSL1(3.01)

hsa00220 Arginine biosynthesis 0.001183418 3 20 ARG2(7.56),ARG1(4.82),GOT1(3.23)

hsa00270 Cysteine and methionine metabolism 0.001211707 4 44 CBS(6.55),CTH(6.42),GCLM(4.18),GOT1(3.23)

hsa04913 Ovarian Steroidogenesis 0.001318938 4 45 CYP1A1(22.13),HSD17B2(16.91),CYP1B1(16.89),PTGS2(6.12)

hsa00590 Arachidonic acid metabolism 0.002779271 4 55 PTGS2(6.12),ALOX12B(4.49),GPX2(3.5),ALOX12(3.3)

hsa00250 Alanine, aspartate and glutamate metabolism 0.006042993 3 35 ASNS(3.91),GFPT1(3.69),GOT1(3.23)

hsa03320 PPAR signaling pathway 0.006603147 4 70 MMP1(12.01),GK(4.02),PCK2(3.97),PPARD(3.9)

hsa04216 Ferroptosis 0.008179721 3 39 HMOX1(4.78),SLC7A11(4.76),GCLM(4.18)

hsa04350 TGF-beta signaling pathway 0.010050405 4 79 BAMBI(6.3),ID1(6.05),INHBA(4.27),ID4(3.22)

hsa00330 Arginine and proline metabolism 0.010708208 3 43 ARG2(7.56),ARG1(4.82),GOT1(3.23)

hsa04550 Signaling pathways regulatingpluripotency of stem cells

0.01314552 5 131 FZD2(0.2),WNT4(0.21),ID3(0.26),SOX2(0.4),FGFR3(0.42)

hsa04217 Necroptosis 0.018534608 5 143 IL33(0.14),STAT1(0.29),GLUL(0.31),BIRC3(0.35),TLR3(0.4)

hsa04916 Melanogenesis 0.019341988 4 96 FZD2(0.2),WNT4(0.21),EDN1(0.22),CALML3(0.37)

hsa04917 Prolactin signaling pathway 0.037222252 3 69 STAT1(0.29),TNFSF11(0.38),ESR1(0.4)

hsa03018 RNA degradation 0.044330617 3 74 PABPC4L(0.34),NUDT16(0.42),EXOSC3(0.43)

Upregulated DEGs (Total 250)

Downregulated DEGs (Total 250)

33

3. Transcriptional profile comparison analysis between HQ-

induced DEGs and IL-17A-induced DEGs

KEGG pathway enrichment analysis on HQ-induced upregulated DEGs

determined IL-17 signaling pathway (hsa04657) as the most significantly enriched

biological phenotype of HQ-treated HKs (Table 3). Notably, the involvement of IL-

17A in the immunopathology of vitiligo has been previously reported (Bassiouny

and Shaker 2011; Khan et al. 2012). It has been suggested that the infiltration of Th-

17 cells correlated with the severity of disease in vitiligo patients (Kotobuki et al.

2012; Wang et al. 2011). To further identify the relationship between IL-17 signaling

pathway and HQ-induced upregulated DEGs, we compared the transcriptional

profile of HQ-induced upregulated DEGs with cytokines-induced upregulated DEGs

in HKs, publicly available in NCBI GEO using the accession number GSE53751

(Jin et al. 2014) (Fig. 2A). When comparing the transcriptional responses of

GSE126721 and GSE53751, no additional adjustments were needed because the

microarray platform and the cellular model were same. By comparing the HQ-treated

upregulated DEGs with IFNg, IL-4, IL-6, IL-17A, and IL-22 – treated upregulated

DEGs in HKs, 58 DEGs were common in both HQ and IL-17A, statistically

significant compared to other cytokines-treated DEGs (Fig. 2B).

34

Figure 2. Transcriptional profile comparison analysis between HQ-induced

DEGs and cytokines-induced DEGs in HKs

(A) Upregulated transcriptional profiles of HQ and Th-cell associated cytokines

were compared using the data sets from NCBI GEO accession number GSE126721

and GSE53751. (B) 250 upregulated DEGs were selected for both data sets and the

number of commonly upregulated DEGs between IFNg, IL-4, IL-6, IL-17A, and IL-

22 in respect to HQ were determined.

Th1

Th2

Th17

Th22

IFN!

IL-4

IL-17A

IL-22 Keratinocytes

HQ

GSE53751

GSE126721

A

16 13

40

58

12

010203040506070

IFN! IL-

4IL-6

IL-17AIL-22

GSE53751

Num

ber o

f com

mon

ly

upre

gula

ted

DEG

s

B

35

4. Validation of the HQ-induced DEGs with Q-RT-PCR

Oligonucleotide microarray transcriptomics have inherent limitations due to the

premise of statistical assumptions for the normalization of gene expression values

across different gene chips (Chuaqui et al. 2002). Thus, the expression of DEGs

should be validated by alternative measurements such as Q-RT-PCR, ELISA, or

western blotting (Jin et al. 2014; Lee et al. 2018). In Q-RT-PCR analysis, the mRNA

level of the IL-17 signaling pathway associated DEGs MMP1, PTGS2, IL1B, IL36G,

and S100A7 were measured 24 and 48 h after treating HKs with HQ. HQ

significantly increased the transcription of the IL-17 signaling pathway associated

DEGs in a concentration-dependent manner (Figs. 3A-E). The transcription of the

cysteine and methionine metabolism-associated DEGs, CBS and CTH was also

validated in independently prepared HQ-treated HK samples (Figs. 3F, G). The

transcription of CYP1A1 was also significantly upregulated in HKs in response to

HQ (Fig. 3H). In addition, HQ-induced PPARD gene transcription was validated by

Q-RT-PCR (Fig. 3I). In the HQ-induced downregulated DEGs, the downregulation

of the melanogenesis pathway (hsa04916)-associated DEGs, FZD2, WNT4, and

EDN1 was validated by Q-RT-PCR in a dose-dependent manner (Figs. 4A-C).

Interestingly, HQ also downregulated the transcription of IL33, functionally

associated with necroptosis (hsa04217) (Fig. 4D). Therefore, the microarray

expression of the HQ-induced DEGs, especially associated with the IL-17 signaling

pathway and melanogenesis pathway, was longitudinally and dose-dependently

validated in HK samples (Figs 3, 4).

36

Figure 3. Validation of the upregulated DEGs in the HQ treated HKs

Total RNA samples were extracted from confluently cultured HKs treated with HQ

for 24 and 48 h. The mRNA levels of (A) MMP1, (B) PTGS2, (C) IL1B, (D) IL36G,

(E) S100A7, (F) CBS, (G) CTH, (H) CYP1A1, and (I) PPARD were determined by

Q-RT-PCR. Values represent the mean expression level ± SD (n = 3) of the genes of

interest relative to that of human RPL13a. *p<0.05, **p<0.01

S100A7

0

2

4

6

0 10 30 0 10 30

02468

0 10 30 0 10 30

MMP1

Rel

ativ

e m

RN

A

24h 48h

HQ (µM)

** *

**

0

20

40

60

0 10 30 0 10 30

PTGS2

24h 48h

HQ (µM)

****

****

Rel

ativ

e m

RN

A

0

10

20

30

0 10 30 0 10 30

IL1B

24h 48h

HQ (µM)

**

**

**

**

Rel

ativ

e m

RN

A

01234

0 10 30 0 10 30

CBS

24h 48h

HQ (µM)

********

Rel

ativ

e m

RN

A

0

10

20

30

0 10 30 0 10 30

IL36G

24h 48h

HQ (µM)

****

**

**

Rel

ativ

e m

RN

A

0

50

100

0 10 30 0 10 3024h 48h

HQ (µM)

****

**

**R

elat

ive

mR

NA

01234

0 10 30 0 10 30

PPARD

24h 48h

HQ (µM)

**

**

**

**

Rel

ativ

e m

RN

A

0

10

20

30

0 10 30 0 10 30

CYP1A1

24h 48h

HQ (µM)

**

****

**

Rel

ativ

e m

RN

A

CTH

24h 48h

HQ (µM)

***

**

Rel

ativ

e m

RN

A

A B C

E F G

H I J

37

Figure 4. Validation of the downregulated DEGs in the HQ treated HKs

Total RNA samples were extracted from confluently cultured HKs treated with HQ

for 24 and 48 h. The mRNA levels of (A) FZD2, (B) WNT4, (C) EDN1, (D) IL33

were determined by Q-RT-PCR. Values represent the mean expression level ± SD (n

= 3) of the genes of interest relative to that of human RPL13a. *p<0.05, **p<0.01

0

0.5

1

1.5

0 10 30 0 10 30

FZD2R

elat

ive

mR

NA

24h 48hHQ (µM)

0

0.5

1

1.5

0 10 30 0 10 30

WNT4

Rel

ativ

e m

RN

A24h 48h

HQ (µM)

0

0.5

1

1.5

0 10 30 0 10 30

*

** ***

**** ****

EDN1

Rel

ativ

e m

RN

A

24h 48h

HQ (µM)

********

0

0.5

1

1.50 10 30 0 10 30

IL33

Rel

ativ

e m

RN

A

24h 48h

HQ (µM)

**** **

**

A B

C D

38

5. IL-36g inhibited melanogenesis in HMs

Among the 58 commonly upregulated DEGs of HQ and IL-17A, the effects of

keratinocyte derived IL-36g on melanocyte function were further investigated

because its role in melanocyte biology and melanogenesis was not studied. IL36G is

a gene encoding a cytokine IL-36g (IL1F9), one of the newly added interleukin 1

cytokine family member (Johnston et al. 2011). IL-36g is a pro-inflammatory

cytokine predominantly expressed in innate immune cells and epithelial cells,

especially in keratinocytes (Gabay and Towne 2015). Like other IL-1 family

cytokines, the active form of IL-36g requires proteolytic truncation by cathepsin S

or neutrophil elastases to gain its full inflammatory potency (Ainscough et al. 2017;

Henry et al. 2016). The biological functions of IL-36g have been studied in epidermal

keratinocytes and dermal fibroblasts in an autocrine or paracrine manner (Pafaff et

al. 2017). However, the paracrine effect of IL-36g on human epidermal melanocytes

has not been investigated. To study the role of IL-36g on HMs, serine-18 truncated

form of IL-36g was treated to primary HMs. tIL-36g significantly inhibited melanin

biosynthesis in HMs in a dose-dependent manner (Fig. 5). To further validate, the

transcriptional change of melanogenesis-associated genes was investigated in tIL-

36g treated HMs. In Q-RT-PCR analyses, tIL-36g did not significantly change the

transcription of melanogenesis-associated transcription factor (MITF), a gene that is

known to regulate the expression of various enzymes associated with melanin

biosynthesis in HMs (Fig. 6A). However, mRNA levels of the key enzymes involved

in melanogenesis pathway such as tyrosinase (TYR), dopachrome tautomerase (DCT),

and tyrosinase-related protein 1 (TYRP1) were significantly downregulated in HMs

39

treated with tIL-36g (Figs 6B-D). Western blotting analyses were also performed to

determine whether changes in mRNA levels correlate with protein translations (Fig.

6E). As consistent with Q-RT-PCR data, the protein level of MITF was not

significantly changed in tIL-36g treated HMs; whereas the protein expression of

TYR, DCT, and TYRP1 was significantly decreased (Fig. 6F-I). MITF is generally

known as the master regulator for the gene transcription of melanogenic enzymes,

however, MITF-independent anti-melanogenic pathways have also been reported

(Natarajan et al. 2014a, b). For instance, IFNg can also downregulate melanogenic

key enzymes such as TYR, DCT, and TYRP1 through the activation of IRF-1 without

affecting the mRNA or protein expression levels of cellular MITF (Natarajan et al.

2014a, b). These results confirmed that IL-36g inhibits melanogenesis by directly

affecting the tyrosinase-dependent melanin biosynthetic pathway in HMs. Therefore,

it is possible that keratinocyte-derived IL-36g contributes to HQ-induced

leukoderma by inhibiting melanogenesis in HMs in a paracrine manner.

40

Figure 5. Melanogenesis inhibitory effects of IL-36g on HMs

(A) Human melanocytes (HMs) from neonatal foreskin of moderately pigmented

donors were treated with IL-36g twice during the 6 days of culture. Hydroquinone

(HQ) served as a positive control that is known to inhibit melanogenesis in HMs. (B)

on the sixth day of culture, HMs were harvested by trypsinization and washed with

PBS two times. After centrifugation, the cell pellets were photographed. (C) To

determine the relative melanin content, cell pellets were lysed in 1 N NaOH and

lysates were transferred into 96-well plates in triplicate. Melanin content was

determined by measuring the absorbance at 490 nm using a microplate reader

(BioTek, Winooski, VT, USA). Values represent the mean ± SD. *p<0.05, **p<0.01

020406080

100120

01000

2000 HQIL-36! (µg/ml)

Rel

ativ

e m

elan

in c

onte

nt (%

)HQ (µM)

--

1 2 -- - 10

A

Day 0

tIL-36!

Day 2

Day 4

Day 6

tIL-36! harvest

BHMs

Control IL-36!1 µg/ml

IL-36!2 µg/ml

HQ10 µM

C

* ** **

41

Figure 6. The effects of IL-36g on the regulation of melanogenesis-associated

enzymes in HMs

To confirm the anti-melanogenic activity of IL-36g, mRNA levels of melanogenesis

associated genes (A) MITF, (B) TYR, (C) DCT, and (D) TYRP1 were measured by

Q-RT-PCR after treating HMs with tIL-36g for 48 h. (E) For western blotting

analyses of MITF, TYR, DCT, and TYRP1, protein samples were prepared after

treating HMs with tIL-36g for 48 h. Protein levels were quantified for (F) MITF, (G)

TYR, (H) DCT, and (I) TYRP1 relative to the control using the ImageJ software.

Values represent the mean expression level ± SD (n = 3) of the genes of interest

relative to that of human GAPDH. *p<0.05, **p<0.01

0

0.5

1

1.5

01000

2000

MITF

Rel

ativ

e m

RN

A

tIL-36!(ng/ml)

0

0.5

1

1.5

01000

2000

TYR

Rel

ativ

e m

RN

A

tIL-36!(ng/ml)

0

0.5

1

1.5

01000

2000

DCT

Rel

ativ

e m

RN

A

tIL-36!(ng/ml)

0

0.5

1

1.5

01000

2000

TYRP1

Rel

ativ

e m

RN

A

tIL-36!(ng/ml)

Control tIL-36!

TYR

TYRP1

DCT

MITF

"-actin

0

0.5

1

1.5

Control

IL-36!

TYRP1

Rel

ativ

e pr

otei

n

0

0.5

1

1.5

Control

IL-36!

DCT

Rel

ativ

e pr

otei

n0

0.5

1

1.5Co

ntrol

IL-36!

TYR

Rel

ativ

e pr

otei

n

0

0.5

1

1.5

Control

IL-36!

MITF

Rel

ativ

e pr

otei

n

A B C D

EF G H I

* *** * * **

** ** **

42

6. IL-36g stimulated HKs to produce pro-inflammatory

cytokines

HQ-induced secretion of IL-36g from epidermal keratinocytes could also affect

keratinocytes in an autocrine manner. First, the effect of tIL-36g on the gene

expression of major pro-inflammatory cytokines in HKs were evaluated (Fig. 7). In

cultured HKs, mRNA levels of IL36G, IL6, CXCL8/IL8 were significantly

upregulated in 24 and 48 h after tIL-36g treatments (Figs. 7A-C). Notably, it was

reported that CXCL14 is a chemokine constitutively expressed in stromal cells and

its production is significantly downregulated when treated with pro-inflammatory

cytokines such as IFNg, IL-1a, IL-4, IL-6, IL-17A, IL-22, or TNFa (Jin et al. 2014).

Interestingly, tIL-36g also significantly downregulated the gene transcription of

CXCL14 at 24 h after the treatment and the change disappeared at 48 h (Fig. 7D).

When cytokine levels were measured by ELISA, the protein expression of IL-6,

CXCL8, and CXCL14 correlated with those of their gene transcriptional levels (Figs.

7E-G).

43

Figure 7. The effects of IL-36g on HKs to initiate inflammatory responses

To determine the keratinocyte modulatory effects of IL-36g, tIL-36g was treated to

HKs for 24 and 48 h. Q-RT-PCR was performed to measure mRNA levels of (A)

IL36G, (B) IL6, (C) CXCL8, and (D) CXCL14 using human RPL13a as a control

gene. Protein expression levels of (E) IL-6, (F) CXCL8, and (G) CXCL14 were

further determined by ELISA. Values represent the mean expression level ± SD (n =

3) of the genes or proteins of interest. *p<0.05, **p<0.01

A

0

20

40

60

0 50 100

500 0 50 100

500

IL36G

Rel

ativ

e m

RN

A

tIL-36!(ng/ml)

24h 48h

0

20

40

60

0 50 100

500 0 50 100

500

IL6

Rel

ativ

e m

RN

A

tIL-36!(ng/ml)

24h 48h

01020304050

0 50 100

500 0 50 100

500

CXCL8

Rel

ativ

e m

RN

A

tIL-36!(ng/ml)

24h 48h

00.51

1.52

0 50 100

500 0 50 100

500

CXCL14

Rel

ativ

e m

RN

A

tIL-36!(ng/ml)

24h 48h

0100200300400

0 50 100

500 0 50 100

500

IL-6

(pg/ml)

tIL-36!(ng/ml)

24h 48h

0

500

10000 50 100

500 0 50 100

500

CXCL8

(pg/ml)

tIL-36!(ng/ml)

24h 48h

0

1000

2000

3000

0 50 100

500 0 50 100

500

CXCL14

(pg/ml)

tIL-36!(ng/ml)

24h 48h

B C D

E F G

****

**

** ** ****

**** ** **

**

44

7. Meta-analysis: clinical association of IL-36g with human

vitiligo

To further investigate the clinical relevance of IL-36g in chemical leukoderma, a

meta-analysis was performed with the transcriptional profile studies on the lesioned

skin from vitiligo patients. Three transcriptional profile studies, GSE53146,

GSE65127, and GSE75819 were chosen for the meta-analysis of vitiligo lesions in

NCBI GEO (Rashighi et al. 2014; Regazzetti et al. 2015; Singh et al. 2017). In

GSE53146 and GSE75819 data sets, the microarray expression of IL36G was

significantly upregulated in the lesioned skin of vitiligo patients (Figs. 8A, B).

Although the statistical significance was not observed in the GSE65127 study, there

was an increasing tendency for the IL36G gene transcription in the lesioned site of

vitiligo patients (Fig. 8C). The meta-analysis of the lesioned skin from vitiligo

patients supports that the upregulation of keratinocyte-derived IL-36g may be

clinically associated with vitiligous phenotypes and it may play central role in HQ-

induced chemical leukoderma.

45

Figure 8. Meta-analysis of transcriptional profiles of the lesioned skin from

vitiligo patients

The transcriptional profile studies on vitiligo patients were obtained from NCBI

GEO. The gene expression data of IL36G in series matrix files of (A) GSE53146,

(B) GSE75819, and (C) GSE65127 were determined. p values were calculated using

a Wilcoxon signed-rank test.

Normal

Vitiligo

p = 0.00035

IL36

G g

ene

expr

essio

n

GSE75819

B

Normal

Vitiligo

p = 0.032

IL36

G g

ene

expr

essio

n

GSE53146

A

IL36

G g

ene

expr

essio

n

GSE65127

Peri-le

siona

l

Health

yNon

-lesio

nal

Lesion

al

C

46

Ⅳ. Discussion

Vitiligo is the most common form of pigmentary disorder affecting 0.5% - 2% of

the population worldwide (Picardo et al. 2015). Chemical leukoderma is an acquired

form of vitiligo that is triggered by exogeneous chemicals, including HQ, MBEH,

RD, and 4-TBP (Bonamonte et al. 2016; Toosi et al. 2012). Despite the importance

of keratinocytes in diverse dermatological conditions, the pathogenic or

toxicological role of epidermal keratinocytes in chemical leukoderma is poorly

understood. In this study, we aimed to elucidate the keratinocyte-related

toxicological mechanisms associated with chemical leukoderma through a genome-

scale transcriptional analysis of HQ-treated HKs. In the KEGG pathway-based

functional enrichment analysis of the HQ-induced downregulated DEGs, the

melanogenesis pathway (hsa04916) was determined as one of the significantly

suppressed biological phenotypes in the HQ-treated HKs. Among the downregulated

melanogenesis-associated DEGs in HKs, WNT4 and EDN1 are genes that can

promote differentiation and melanogenesis in melanocytes in a paracrine manner (Li

and Hou 2018; Regazzetti et al. 2015; Saldana-Caboverde and Kos 2010). FZD2 is

a gene that codes a receptor for WNT signaling proteins (Schulte and Bryja 2007).

The role of frizzled family receptors has been investigated in mammalian

melanocytes (Yamada et al. 2013). However, the significance of the downregulated

FZD2 in epidermal keratinocytes is not clearly understood. The interactions between

keratinocytes and melanocytes play a key role in the regulation of mammalian

melanogenesis (Choi et al. 2012; Imokawa 2004). It is possible that the signaling

47

pathways associated with WNT and FZD2 may be simultaneously coordinated in

both keratinocytes and melanocytes after the exposure of endogenous or exogeneous

stimuli affecting melanogenesis in human skin. So far, the effects of HQ on skin

have been mainly explained by the direct inhibition of tyrosinase activity in

melanocytes (Kim et al. 2017; Kwon et al. 2018). Downregulation of WNT4 and

EDN1 in keratinocytes suggests that HQ-induced leukoderma is triggered by

affecting not only melanocytes but also keratinocytes leading to decreased melanin

production in the skin. To comprehensively understand the toxicological mechanism

of HQ-induced chemical leukoderma, further studies should be directed to elucidate

the interactive role of melanogenesis-associated signaling pathways in the

pathogenic crosstalk between keratinocytes and melanocytes in response to HQ or

other leukoderma-inducing chemicals.

The upregulation of the IL-17 signaling pathway (hsa04657) was the most

significantly represented biological phenotype in the HQ-treated HKs in the KEGG

pathway enrichment analysis, which was due to the upregulated DEGs MMP1,

PTGS2, IL1B, IL36G, and S100A7. In addition, the transcriptional profile

comparison using GSE53751 showed that both HQ and IL-17A shared their

regulation on epidermal differentiation and early inflammatory responses in HKs.

As mentioned earlier, the pathogenic association of Th-17 cells and their major

cytokine IL-17A with vitiligo has been demonstrated (Bassiouny and Shaker 2011;

Khan et al. 2012; Kotobuki et al. 2012; Wang et al. 2011). In this regard, it is possible

that Th17 cell-mediated immune responses may contribute to the pathogenesis if

chemical leukoderma. In the meta-analysis and experimental validation studies, IL-

36g was identified as a commonly upregulated cytokine in HKs in response to both

48

HQ and IL-17A. In the IL-17 signaling pathway-associated DEGs, it was unclear

whether IL-36g could regulate the biology of melanocytes. To our knowledge, this

is the first study to have demonstrated that IL-36g directly inhibited melanin

biosynthesis in cultured HMs. Our findings revealed that IL-36g suppressed the

transcription of melanin biosynthetic enzymes TYR, DCT, and TYRP1 without

affecting the MITF in HMs.

Interestingly, HQ significantly downregulated the transcription of IL33 in HKs.

IL-33 is an IL-1 family cytokine that induces Th2 cell responses and stimulates the

production of Th2 cell cytokines (Schmitz et al. 2005). Although IL-33 was first

identified as a cytokine, it has a dual function as an intracellular nuclear factor. It has

been recently reported that the intracellular full-length IL-33 inhibits NFk-B

dependent gene transcription and suppress subsequent pro-inflammatory responses

(Ali et al. 2011). The reciprocal regulations between Th-cell subtypes have been well

addressed in T cell immunology (Chitnis et al. 2004; Choy et al. 2015). For instance,

predominant Th2 responses inhibit Th1 or Th17 cell immunity, and vice versa (Ansel

et al. 2006; Gor et al. 2003; Wilson et al. 2009). Therefore, the HQ-induced

downregulation of IL33 may result in a favorable cutaneous microenvironment for

Th17 cell responses by suppressing Th2 cell responses in HKs.

IL-36g also regulate keratinocyte-dependent inflammatory responses in human

skin (Swindell et al. 2018). In this study, we proved that tIL-36g significantly

upregulated the transcription of IL-36g in HKs, suggesting that a positive feedback

mechanism exists in the production of IL-36g. In HKs, tIL-36g increased both

mRNA and protein levels of anti-melanogenic IL-6 and chemokine CXCL8 in a

49

concentration-dependent manner, confirming that IL-36g actively participates in

keratinocyte-mediated cutaneous inflammation. To be in its active form,

extracellular full-length IL-36g should be proteolytically cleaved by neutrophil

elastases or cathepsin S (Henry et al. 2016). The activity of IL-36g can be regulated

by a positive feedback mechanism since CXCL8 recruits neutrophils at the site of

cutaneous inflammation (Griffth et al. 2014). In addition, a three-dimensional skin

equivalent study showed that there was the autocrine feedback regulation of IL-36g

in IL-17A treated keratinocytes (Pfaff et al. 2017). The synergistic interaction

between IL-17A and IL-36g was suggested to initiate an inflammation amplification

cycle, which is essential to the persistence of inflammatory skin phenotypes in

diverse Th17 cell-associated dermatologic conditions (Pfaff et al. 2017). In fact, the

strong correlation of keratinocyte-derived IL-36g to disease severity has been

reported in psoriasis or allergic contact dermatitis (Gabay and Towne 2015).

Furthermore, IL-36g modulates the activity of dendritic cells, resulting in the

stimulation of Th17 responses (Gabay and Towne 2015). The autocrine regulation

of keratinocyte-derived IL-36g and its synergistic interactions with diverse pro-

inflammatory cytokines provide mechanistic insights on pathological phenotypes of

chemical leukoderma. Currently, in vitro toxicological tests are not available to

determine the chemical leukoderma-inducing potential. Additionally, there are no

validated animal models to study or evaluate the leukoderma. Further studies should

be directed to investigate whether IL-36g production of HKs is generally induced by

other leukoderma-inducing chemicals. Furthermore, the interaction network of IL-

36g with keratinocyte-derived cytokines such as IL-6, CXCL8, and IL-33 in response

50

to diverse leukoderma-inducing chemicals should be studied as novel biomarkers to

determine the chemical leukoderma-inducing potential.

In conclusion, the genome-scale transcriptional analysis of HQ-treated HKs

showed that it activates the IL-17A signaling pathway-associated responses in

human keratinocytes. Meta-analysis between GSE126721 and GSE53751 supported

that HQ and IL-17A had the regulation of epidermal differentiation and pro-

inflammatory responses in HKs in common. HQ significantly increased the

production of IL-36g, the IL-17A signaling pathway-associated cytokine.

Importantly, we believe that this is the first study to have demonstrated that IL-36g

directly inhibits the melanin biosynthesis of HMs. In addition, IL-36g regulated

keratinocyte functions in an autocrine manner to produce pro-inflammatory

cytokines, suggesting that IL-36g may stimulate the amplification cycle of cutaneous

inflammation. Notably, the meta-analysis of three independent transcriptional profile

studies available in NCBI GEO showed that the gene transcription of IL36G was

upregulated in the lesioned skin of vitiligo patients, supporting that the upregulation

of IL-36g is associated with the pathogenesis of chemical leukoderma. In this regard,

HQ-induced IL-36g from human keratinocyte plays a pivotal role in the development

of chemical leukoderma by autocrinally or paracrinally modulating the crosstalk

between keratinocytes and melanocytes.

51

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58

요약 (국문초록)

하이드로퀴논에 대한 사람의 각질형성세포에서

유전체 수준의 전사체 반응 연구

표 정 주

서울대학교 약학대학원

약학과 천연물과학전공

하이드로퀴논은 피부의 정상 멜라닌 세포가 손상되어 멜라닌

색소를 만들어 내지 못해 발생하는 화합물 유도 백색증을 일으킨다고

알려져 있는 대표적인 페놀계 화합물이다. 하이드로퀴논이 피부에서

백색증을 일으킬 수 있음에도 불구하고 여전히 시중에서는 피부 미백을

위한 화장품 원료로서 사용되고 있다. 또한, 하이드로퀴논이 어떻게

피부에서 백색증을 유발하는지에 대한 기전 연구도 되어 있지 않다.

따라서 하이드로퀴논 유도 피부 백색증의 병리학적 메커니즘을 밝히기

위해 사람의 정상 각질형성세포에 세포 독성을 보이지 않는 농도의

하이드로퀴논을 처리하여 유전체 수준에서의 전사체 반응을 연구하였다.

하이드로퀴논이 처리된 사람의 각질형성세포의 DEGs (Differentially

Expressed Genes) 분석을 통해 각각 250 개의 과발현된 DEGs 와 발현량이

감소된 DEGs 을 선정하였다. 나아가 선정된 250 개의 차별발현된

유전자들을 대상으로 KEGG (Kyoto Encyclopedia of Genes and Genomes)

pathway enrichment analysis 를 통해 하이드로퀴논이 각질형성세포에서 IL-

17 signaling pathway 와 관련된 유전자들을 과발현시키고, melanogenesis

59

pathway 와 관련된 유전자들의 발현량을 억제한다는 사실을 밝혔다.

하이드로퀴논이 처리된 각질형성세포의 전사체와 사이토카인이 처리된

각질형성세포의 전사체 비교를 통해 58 개의 유전자들이 하이드로퀴논과

IL-17 에서 공통적으로 과발현됬다는 사실을 밝혔으며 특히 IL36G 의

경우 각질형성세포에서 하이드로퀴논과 IL-17A 에 의해 발현량이

유의미하게 증가한다는 사실을 확인하였다. 이후 진행된 실험들에 의해

사이토카인 IL-36g가 사람의 정상 멜라닌 세포에서 멜라닌 생합성을

억제한다는 사실을 검증하였으며 멜라닌 생합 성과 관련된 단백질들인

TYR, DCT, TYRP1 의 발현량을 감소시킨다는 것도 확인하였다. 나아가

각질형성세포에서 발현된 IL-36g는 자가분비 방식으로 다시

각질형성세포에서 염증성 사이토카인 IL-36g, IL-6, 그리고 CXCL8/IL-8 을

농도 의존적으로 유도하였으며 이는 IL-36g가 피부의 염증반응을 촉진

및 매개시킬 수 있다고 가정하였다. 마지막으로 3 개의 독립적인 백반증

환자의 피부 전사체 연구 메타분석을 통하여 IL36G 가 백반증 환자의

흰색 반점 부위에서 발현량이 증가했다는 사실을 확인하였으며 이는 IL-

36g와 피부의 백색증 부위 사이에 임상학적인 상관관계가 있다는 것을

암시한다. 따라서, 사람의 각질형성세포에서 하이드로퀴논에 의해 유도된

IL-36g은 자가분비 또는 주변분비 방식으로 피부의 각질형성세포와

멜라닌 세포 사이의 신호교환을 매개함으로서 화합물 유도 백색증을

유발할 수 있다.

주요어 : 하이드로퀴논, 각질형성세포, 화합물 유도 백색증, IL-36g, Kyoto

Encyclopedia of Genes and Genomes (KEGG)

학번 : 2018-24035