Arachidonate 12-Lipoxygenase Inhibitors Promote S100A3 Citrullination in Cultured SW480 Cells and Isolated Hair Follicles

Abstract

The human hair shaft is covered with multiple scale-like cuticular layers. During the terminal differentiation stage of immature cuticular cells within the hair follicle, cysteine-rich calcium binding S100A3 protein is predominantly translated, and its arginine residues are converted to citrullines by peptidylarginine deiminases (PADI). In this study, we found several naturally occurring compounds (e.g., hinokitiol, escletin, and quercetin) elevate S100A3 citrullination in a human colorectal adenocarcinoma cell line (SW480). Selected compounds similarly promoted cuticular differentiation within isolated human hair follicles. Their promotive activities correlated with the previously reported inhibitory activities of arachidonate 12-lipoxygenase (ALOX12) in vitro. Microarray analysis revealed that ALOX12 inhibitor remarkably up-regulated heparin-binding epidermal growth factor-like growth factor (HBEGF). ALOX12 inhibitor and recombinant HBEGF similarly regulated expression of PADI genes in SW480 cells. In isolated hair follicles, arachidonic acid strongly promoted S100A3 citrullination along with elevation of HBEGF. These results suggest that ALOX12 inhibition efficiently triggers hair cuticle maturation by modulating arachidonate metabolism in concert with HBEGF.

From hair epithelial matrix cells, the cuticle, cortex, and medulla of the hair fiber and the inner root sheathes are formed. This trichogenic epithelial portion, together with the outer root sheath and dermal papilla, comprise an intact hair follicle. Post-translational protein modification within hair epithelial matrix cells is a crucial terminal differentiation process leading to proper hair shaft formation.¹⁾ Hair texture and resilience are largely influenced by physical properties of outermost cuticle layers.²⁾ Furthermore, a number of studies have shown a correlation between various hair shaft disorders and molecular defects.³⁾ S100A3 protein is a cysteine-rich Ca²⁺-binding protein which shares the majority of the soluble proteineous components of hair cuticles.⁴⁾ Peptidylarginine deiminase (PADI) converts several arginine residues of S100A3 protein to citrullines within immature cuticular cells.⁵⁾ Genetic mutations including hair-dominant PADI isotype (PADI3) have recently been shown to cause uncombable hair syndrome.⁶⁾ The extent of S100A3 citrullination has been reported to correlate with the rigidity of individual hair cuticles.⁷⁾ Therefore, promotion of S100A3 citrullination would contribute to conventional maintenance and restoration of defective hair texture.

Although various chemical materials have been adopted in order to physically repair either defective or damaged hair fibers, very little information is available regarding endogenous factors and pharmaceuticals which act on immature trichocytes. Maintenance of the trichogenic potential of hair matrix cells in vitro cultivation still remains to be an unresolved issue,⁸⁾ even though cultured outer root sheathes and dermal papilla cells have already been widely used to screen candidate ingredients in hair restorer development.^9,10) From a public gene expression database, a pathologic cell line SW480, derived from human colorectal adenocarcinoma cells, was found to express the S100A3 gene (http://biogps.org/#goto=genereport&id=6274). We used this cell line to select several plant-originating and its related chemical compounds which enhance citrullination of the S100A3 protein. Selected active compounds were all previously reported inhibitors of platelet-type arachidonate 12-lipoxygenase (ALOX12).¹¹⁾ Their effects on primary cuticular cells within human hair follicles were confirmed using an ex vivo organ culture system.¹²⁾ Here, we would like to report that arachidonate metabolism modulated by ALOX12 inhibitors promotes S100A3 citrullination mediated by heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF).

MATERIALS AND METHODS

Materials

Esculetin (TCI, Tokyo, Japan), quercetin, baicalein, tropolone, PD146176, PD168393 (Sigma-Aldrich, St. Louis, MO, U.S.A.) and Cl-amidine (EMD Millipore, Billerica, MA, U.S.A.) were dissolved in dimethyl sulfoxide. Hinokitiol (Takasago, Tokyo, Japan) and arachidonic acid (Cayman, Ann Arbor, MI, U.S.A.) were used as ethanol solutions. Commercially available 12S-hydroxyeicosatetraenoic acid (12S-HETE) and 12-hydroperoxyeicosatetraenoic acid (12-HPETE) solutions (Cayman) were condensed by evaporating ethanol with a nitrogen gas stream before use. Lyophilized recombinant HBEGF (R&D Systems, Minneapolis, MN, U.S.A.) was reconstituted with distilled water. Serially diluted pharmaceuticals and reagents were used for the following cell and organ cultivation experiments.

Cell Culture

An SW480 cell line derived from human colorectal adenocarcinoma (EC87092801) was cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. When the cell density reached 70% confluence, the supplemented serum concentration reduced to 1%. Subsequently, cells were cultured for 24–48 h in the presence of various concentrations of the test sample.

Organ Culture

Human scalp skin tissue samples were obtained surgically from adult males at the Skin Clinic Group (Japan). Hair follicles were isolated using a scalpel with a tweezer under a stereoscopic microscope, and then cultured in 400 µL of Williams medium E, supplemented with 40 ng/mL of hydrocortisone and antibiotics using 24-well plates.¹³⁾ Test samples were supplemented on the first day. This study was performed in accordance to the principles of the Helsinki Declaration with approval of the Human Research Ethics Committee of Biological Science Laboratories of Kao Corporation.

Gene Expression Analyses

Total RNAs were extracted from SW480 cells and two isolated hair follicles cultured for 24 h under various treatment conditions. cDNA was prepared using SuperScript II reverse transcriptase (Invitrogen, Waltham, MA, U.S.A.). Quantitative real-time PCR analysis was performed using a StepOnePlus system (Applied Biosystems, Waltham, MA, U.S.A.) for the following primer sets: Hs00203458_m1 for PADI1, Hs01042507_m1 for PADI2, Hs00212088.m1 for PADI3, Hs00181813_m1 for HBEGF, Hs00950669_m1 for AREG and, as a standard, Hs99999902_m1 for RPLP0.

Protein Analysis

Proteins were extracted from cultured SW480 cells and isolated hair follicles after various treatments for 48 h, except for arachidonic acid treatment which was for 24 h. Cultured SW480 cells were extracted with 0.1 M Tris–HCl buffer containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 50 mM dithiothreitol (DTT) (pH 7.6). From two cultured hair follicles, soluble proteins under non-reducing conditions were extracted using 0.1 M Tris–HCl containing 0.1% CHAPS, in order to exclude cross-linked S100A3 deposited in mature cuticles. After blocking the extracted proteins with iodoacetamide, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis was carried out using isoelectric point (pI) 3.9-5.1 immobilized pH gradient strips (Bio-Rad, Hercules, CA, U.S.A.). Immunofluorescent signal detection of S100A3 was conducted using a Typhoon 9410 (GE-Healthcare, Chicago, IL, U.S.A.). Citrullination rates of S100A3 derived from a treated sample (CIT_S100A3[TEST]) and a non-treated sample (CIT_S100A3[CONTROL]) were calculated from the relative intensities of isoelectric variants, as previously described.⁵⁾ Elevated citrullination rate of S100A3 (ΔCIT_S100A3) was computed by the following subtraction:   

Microarray Analysis

Total RNA prepared from eight isolated hair follicles and SW480 cells cultured in four wells were pooled and subjected to microarray data collection at Hokkaido System Sciences (Sapporo, Japan) under following conditions. Fifty nanograms of RNA of each treatment condition was used to synthesize double-stranded cDNA and cyanine 3-CTP labelled cRNA using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, CA, U.S.A.). cRNA was hybridized to SuperPrint G3 Human 8×60 K ver. 2.0 (Agilent Technologies) microarray slides. Microarray data were normalized with 75 percentile shift and analyzed fold change for each gene using Gene Spring, version 13.1.1 (Agilent Technologies).

Statistical Analysis

All data were expressed as the mean±standard error along with the number of observations. Statistical analysis was conducted using EXSUS software, version 8.0.0 (CAC Exicare, Tokyo, Japan). Following ANOVA, multiple comparisons between the control and all treated groups were performed by Dunnett’s test. Dose–response relationships were assessed by William’s test.

RESULTS

Citrullinated S100A3 Protein in SW480 Cells

Little information on S100A3 gene expression, far less on the translated protein distribution, in either human tissues or cultured cell lines other than abundance in hair follicles³⁾ has been reported up to now. Using public database information on S100A3 expression in SW480 derived from human colorectal adenocarcinoma, we examined S100A3 protein in these pathologic cell extracts by Western blot analysis using an antibody for S100A3. As shown in Fig. 1A, we could detect an immune-reactive band at a similar position (11-kDa) to the S100A3 bands of recombinant proteins and their natural source extracted from hair follicles. Judging from the intensity of the S100A3 bands, translation level in SW480 was less than 1/20 of the hair follicle extract.

Fig. 1. S100A3 Protein in SW480 Cells and Isolated Hair Follicles

Protein bands and spots were detected by a rabbit specific antibody to S100A3. (A) Western blot analysis of S100A3 Lane 1, recombinant S100A3 protein (0.1 µg); lane 2, hair follicle extract (5 µg); lane 3, SW480 cell extract (100 µg). (B) Two dimensionally migrated S100A3 in the protein extracts derived from two isolated hair follicles and SW480 cells (250 µg). Cl-amidine and esculetin treatments inversely shifted pI of the S100A3 protein.

S100A3 protein derived from hair follicle replaces N-terminal methionine to acetyl group.⁴⁾ S100A3 extracted from either cultured hair follicles or SW480 cells under non-reducing conditions migrated two major spots (pI=4.3, 4.5) on 2D-PAGE gels (Fig. 1B). In the presence of PAD inhibitor Cl-amidine,¹⁴⁾ S100A3 in hair follicles concentrated to single spot corresponding to the non-citrullinated form (pI=4.5). S100A3 in SW480 were separated to a main spot (pI=4.5) and a minor spot (pI=4.3). Because S100A3 separated to triplet spots generating a basic non-modified form (pI=4.7) by Cl-amidine treatment, the major molecular form of S100A3 protein in conventionally cultured SW480 cell appears to be non-acetylated, but a mono-citrullinated form (pI=4.5).

Promotion of S100A3 Citrullination by ALOX12 Inhibitors

In the course of screening naturally occurring compounds using SW480 cells, we first found that almost half of the major spot of S100A3 (pI=4.5) shifted to the acidic side (pI=4.3) after 48 h treatment with hinokitiol and also with esculetin (Fig. 1B). Hinokitiol, also known as β-thujaplicin, is a constituent of cypressaceous plants (Chamaecyparis obtuba, Thuja plicata and etc.).¹⁵⁾ We considered previous reports on the selective inhibitory effect on ALOX12 by tropolone derivatives including hinokitiol¹⁶⁾ and naturally occurring ALOX12 inhibitors,¹¹⁾ such as esculetin,¹⁷⁾ quercetin¹⁸⁾ and baicalein.¹⁹⁾ We could confirm that an elevated intensity of acidic shift of the S100A3 spot (pI=4.3) decreased with Cl-amidine (Fig. 1B).

All ALOX12 inhibitors examined significantly higher citrullination rate of S100A3 in SW480 cells in a concentration-dependent manner (Fig. 2A). There appears to be correlation between the inhibitory activity of ALOX12 enzyme in vitro and the promotive effect of S100A3 citrullination in SW480 cells. For example, the IC₅₀ values of hinokitiol and tropolone were reported to be 0.1 and 2.3 µM, respectively. Consistently, hinokitiol promoted citrullination of S100A3 at a lower concentration compared to tropolone. Reported IC₅₀ values of esculetin, quercetin and baicalein were 0.65, 2.5, and 24.6 µM, respectively.^17–19) Although these values were determined using sonicated platelets originating from different species, ALOX12 inhibitory activities of the three naturally occurring compounds correlated with the promotive effect of S100A3 citrullination measured in SW480 cells. These inhibitors were reported to specifically inhibit ALOX12, but not other lipoxygenases (i.e., ALOX12B, ALOX15). Six different arachidonate lipoxygenases were encoded with six functional ALOX genes on human genome (ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5).²⁰⁾ Among them arachidonate metabolites of ALOX12 and ALOX15 are predominately detected from hair follicles.²¹⁾ A selective ALOX15 inhibitor (PD146176)²²⁾ rarely promoted S100A3 citrullination up to 20 µM: a more than 200-fold higher concentration of the IC₅₀ (data not shown).

Fig. 2. Effects of ALOX12 Inhibitors on S100A3 Citrullination in SW480 Cells (N=4) (A) and Isolated Hair Follicles (N=8) (B)

Statistical differences versus control group using William’s test were indicated by * p<0.025, ** p<0.005, and *** p<0.0005.

Next, we examined the effects of ALOX12 inhibitors on S100A3 citrullination in premature trichocytes. Isolated human hair follicles were cultured in the presence of various concentrations of hinokitiol, esculetin and quercetin for 48 h. 2D-PAGE-Western blot analysis of S100A3 was performed with soluble protein extracted from eight samples of two each hair follicle culture (Fig. 1B). As well as the results obtained using SW480 cells, over 50 µM of hinokitiol, and 200 µM each of esculetin and quercetin, significantly increased the citrullination rate of S100A3 (Fig. 2B).

PAD Isozyme Responsible for S100A3 Citrullination

We previously reported that four arginine residues in S100A3 protein were sequentially citrullinated by PADI isozymes in hair cuticular cells.⁵⁾ PADI3 preferentially converts Arg51 in S100A3, whereas PADI1 and PADI2 primarily convert N-terminal Arg3-harboring peptides²³⁾ and is possible on all four sites of an intact S100A3 protein. Specific conversion of Arg51 in dimeric S100A3 by PADI3 causes a homotetramer to assemble within hair cuticular cells, and following further citrullination of other sites by either PADI1 or PADI2 is predicted to result in enhanced mechanical strength of mature hair cuticles.⁷⁾ We examined how ALOX12 inhibitors modulate expression of PADI genes in SW480 cells and hair follicles.

Quantitative PCR analysis revealed that both hinokitiol and esculetin up-regulated PADI1 significantly and also PADI2 slightly, but remarkably down-regulated PADI3 in SW480 cells (Fig. 3A). The proportion of an acidic shifted spot (pI=4.3), mostly harbors two peptidylcitrullines, increased by either treatment (Fig. 2A). Cl-amidine treatment generated a non-modified form of S100A3 (pI=4.7), and in turn, decreased the acidic form (Fig. 1B). In isolated hair follicles, both hinokitiol and esculetin up-regulated PADI1 and PADI2 mRNA levels, but PADI3 expression level remained constant. Especially, hinokitiol remarkably increased PADI2 mRNA level compared to esculetin (Fig. 3B). Therefore, hinokitiol may be more efficient for hair follicles than esculetin (Fig. 2B). These results suggest that PADI3 is responsible for citrullination of S100A3 in either SW480 cells or hair follicles under conventional conditions, but its dominant role is likely substituted by either PADI1 or PADI2 by supplementation of ALOX12 inhibitor. Broader substrate specificity of PADI1 and PADI2 confers a resulting higher citrullination rate of S100A3 protein.

Fig. 3. Effect of ALOX Inhibitor on PADI Gene Expression

Effect of hinokitiol and esculetin of PADI1, PADI2, and PADI3 gene expression in SW480 cells (A) and in isolated hair follicles (B). Relative mRNA levels were quantified by TaqMan PCR using RPLP0 as standard (N=4). Statistical differences versus control group using Dunnett’s test were indicated by * p<0.05 and *** p<0.001.

Up-Regulation of HBEGF by ALOX12 Inhibitors

The transcriptome profile of isolated hair follicles and SW480 cells after supplementation of ALOX12 inhibitors was analyzed using a microarray custom slide. Hinokitiol and esculetin (100 µM each) treated isolated hair follicles up-regulated 1205 and 448 transcripts (fold change >5) compared to the control, respectively. In order to select transcripts up-regulated within the epithelial portion, we overlaid 313 transcripts up-regulated (fold change >10) in 200 µM esculetin-treated SW480 cells on a Venn diagram (Fig. 4). Six transcripts commonly up-regulated under all three conditions are listed in Table 1.

Fig. 4. Venn Diagram of the Up-Regulated Transcripts by ALOX12 Inhibitors

The number of transcripts in isolated hair follicles and SW480 elevated by hinokitiol or esculetin are shown. Six commonly up-regulated genes refer to Table 1.

Table 1. List of Up-Regulated Genes by ALOX12 Inhibition in Both SW480 Cells and Isolated Hair Follicles

Probe name	Fold change			Gene symbol	Description
	Cultured hair follicle		SW480 cell
	Hinokitiol (100 µM)	Esculetin (200 µM)
A_33_P3295358	18.9	57.0	45.9	ANGPTL4	Homo sapiens angiopoietin-like 4 (ANGPTL4), transcript variant 1, mRNA [NM_139314]
A_21_P0009781	10.2	6.5	10.5	XLOC_012981	Q2VCE5_HUMAN (Q2VCE5) UCA1 protein, partial (98%) [THC2492330]
A_24_P140608	6.7	8.3	37.2	HBEGF	Homo sapiens heparin-binding EGF-like growth factor (HBEGF), mRNA [NM_001945]
A_23_P16469	5.9	6.8	10.0	PLAUR	Homo sapiens plasminogen activator, urokinase receptor (PLAUR), transcript variant 3, mRNA [NM_001005377]
A_33_P3224070	5.5	5.3	12.5	CSRNP1	Homo sapiens cysteine-serine-rich nuclear protein 1 (CSRNP1), mRNA [NM_033027]
A_23_P214079	5.1	5.0	73.9	SPINK1	Homo sapiens serine peptidase inhibitor, Kazal type 1 (SPINK1), mRNA [NM_003122]

This study focused on HBEGF as a candidate mediator of ALOX12 inhibition, possibly leading to S100A3 citrullination. HB EGF treated and overexpressed keratinocytes were previously reported to exhibit abnormally differentiated and poorly proliferative phenotypes.^24,25) We performed quantitative PCR analysis subsequently, and confirmed that the transcription level of HBEGF in isolated hair follicles was up-regulated by either 100 µM hinokitiol (8.2-fold) or 200 µM esculetin (6.8-fold) almost consistent with the array data (Table 1). Among EGF family members, amphiregulin was also significantly up-regulated in esculetin-treated SW480 cells (83-fold) but not in isolated hair follicles (1.6-fold). Therefore, HBEGF appears to be the major EGF family member induced by ALOX12 inhibitors in hair follicles.

Because EGF was previously reported to up-regulate PADI2 gene in mammary epithelial cells²⁶⁾ and cancer cells.²⁷⁾ We examined whether HBEGF modulates expression of PADI genes similarly in SW480 cells. Recombinant HBEGF up-regulated PADI1 and PADI2 as well as ALOX12 inhibitors, and slightly down-regulated PADI3 in SW480 cells (compare Figs. 3A, 5A). Although lesser efficiency of HBEGF, as compared to ALOX12 inhibitors, failed to intrinsically promote S100A3 citrullination (data not shown), an EGF receptor antagonist: PD168393 obviously canceled the S100A3 citrullination increased by esculetin (Fig. 5B).

Fig. 5. HBEGF Mediated S100A3 Citrullination in SW480 Cells

(A) Effect of HBEGF on PADI gene expression in SW480 cells. (B) Cancellation of the esculetin-induced S100A3 citrullination by PD168393, an EGF receptor antagonist. Statistical differences versus control group (N=4) using Williams’ test were indicated by * p<0.025, ** p<0.005, and *** p<0.0005.

S100A3 Citrullination Associated with Arachidonate Metabolism

Arachidonic acid greatly impacts S100A3 citrulliantion compared to its ALOX12 metabolites. Addition of arachidonic acid to cultured prostate cancer and epithelial cells was previously reported to increase its intracellular level.²⁸⁾ Arachidonic acid suppressed both basal and esculetin-induced HBEGF mRNA levels in SW480 cells (Fig. 6A), while 12S-HETE and 12-HPETE (1–10 µM) only partially canceled the elevated citrullination level by esculetin in SW480 cells (data not shown). Consistently, S100A3 citrullination elevated by esucletin in SW480 cells was canceled by arachidonic acid (Fig. 6B), despite its ambivalent effect (100–200 µM) on basal S100A3 citrulliantion. On the other hand, arachidonic acid elevated HBEGF expression (Fig. 6C) and promoted S100A3 citrullination in cultured isolated hair follicles (Fig. 6D).

Fig. 6. Inverse Effect of Arachidonic Acid on SW480 Cells and Hair Follicles

Down-regulation of HBEGF expression (A) and cancellation of esculetin-induced S100A3 citrullination (B) in SW480 cells (N=4). Up-regulation of HBEGF (N=4) (C) and promotion of S100A3 citrullination (N=8) (D) in isolated human hair follicles. Statistical differences versus control group using William’s test were indicated by * p<0.025 and *** p<0.0005.

DISCUSSION

Up to now, little information has been available on how pharmaceuticals directly act on immature trichocytes due to technical difficulty in their cultivation.⁸⁾ In this study, we selected several natural products and their related compounds which could promote S100A3 citrullination conserved within a SW480 cell line. A conventional organ culture system of isolated hair follicles was used to confirm the effect on S100A3 citrullination within cuticular cells. Selected compounds from this combinational experiment were shown to act on the immature cuticles, because distribution of S100A3 was almost all confined to within the hair cuticular cells.

This study has shown that all the selected compounds have been previously reported to inhibit platelet-type lipoxygenase (ALOX12). ALOX12 inhibitors efficiently elevated S100A3 citrullination by substituting its responsible PAD isozymes to broader substrate specificity in either SW480 cells or hair cuticular cells (Fig. 3). The effective concentration for S100A3 modification correlated well with the previously reported IC₅₀ value of the ALOX12 inhibitor in vitro.^11,16–19) ALOX12 is responsible for the conversion of arachidonic acid to 12S-HPETE, as an intermediate reaction product, and 12S-HETE, which was mainly detected from the stratum granulosum and spinosum layers of the epidermis²⁹⁾ and plucked hair root.²¹⁾ 12S-HETE has been reported to stimulate proliferation and to inhibit terminal differentiation of keratinocytes,³⁰⁾ and partially reversed esculetin-induced S100A3 citrullination in SW480 cells. Considering that the predominant isozyme of arachidonate lipoxygenase is reported to be ALOX12 in human skin epidermis,³¹⁾ its inhibition may result in the efficient promotion of S100A3 citrullination within cuticular cells.

Microarray analysis showed that ALOX12 inhibition significantly up-regulated HBEGF in either SW480 cells or isolated hair follicles. ALOX12 inhibitors and HBEGF similarly increased expression of PADI1 and PADI2 genes, which are capable of citrullination of all four peptidylarginines in S100A3 in vitro.⁵⁾ Furthermore, blockage of the EGFR receptor canceled the promotive effect of ALOX12 inhibitor in SW480 cells. Therefore, ALOX12 inhibition appears to be transduced through the autocrine EGFR signaling pathway. Arachidonic acid promoted both HBEGF expression and S100A3 citrullination in hair follicles, while canceling esculetin-induced HBEGF elevation and S100A3 citrullination in SW480 cells (Fig. 6). These different outcomes may be attributed to a specific arachidonate metabolism occurring in superficial epithelium.

From all the results presented here, we propose that action of ALOX12 inhibitors on S100A3 citrullination lead to cuticular maturation. A unique maturation feature of hair cuticular cells is the substitution of a phospholipid membrane to thio-esterified branched fatty acid and formation of A-layer: a very thick, protective structure.²⁾ Squamous epidermal cells, but not SW480 cells, are known to form the similar lipid envelope and the cornified envelope. Although the enzymes responsible for arachidonate metabolism in hair cuticular cells have yet to be defined, the results in this study strongly suggest that ALOX12 plays a crucial role during the terminal differentiation stage. At the hair cuticle maturation stage, arachidonic acid is thought to be abundantly derived from membrane phospholipids by phospholipase A₂, and then oxidized mainly by ALOX12. As well as an elevated arachidonate metabolic turnover, inhibition of ALOX12 enzyme could trigger the terminal differentiation of hair cuticular cells. Considering that ALOX12 enzyme was reported to be localized in stratum spinosum and basal layer but not in stratum corneum and stratum granulosum of normal and psoriatic skin epidermis,³¹⁾ its activity is likely down-regulated at the terminal cuticular maturation stage similarly.

Hinokitiol has been reported to reduce the number of bacterial cells on the skin surface by antimicrobial action,³²⁾ and thereby clinically improves eczematous lesions due to atopic dermatitis.³³⁾ However, little attention has been given to the direct action of hinokitiol on epidermal cells as related to the inhibitory activity of ALOX12 enzyme in vitro.¹⁶⁾ Defective skin barrier function (i.e., increase trans-epidermal water loss) has been reported in ALOX12 deficient mice,³⁴⁾ but its phenotype was less severe compared to lethal ALOX12B and ALOXE3 deficient mice.^35,36) Epithelial type lipoxygenases were previously reported to be pathogenic genes related to non-bullous congenital ichthyosiform erythroderma.³⁷⁾ They are mainly responsible for the formation of corneocyte lipid envelopes rather than arachidonate metabolism.³⁸⁾ Recently, an uncombable hair syndrome causative mutation was reported to exist in PADI3 encoding the hair-dominant PADI isozyme.⁶⁾ Our results clearly showed that ALOX12 inhibition efficiently promotes S100A3 citrullination in concert with HBEGF. Further study is needed to elucidate the pharmacological action of ALOX inhibitors on superficial epithelium maturation of skin and hair follicles.

Acknowledgment

We would like to thank Drs. Y. Ishi (Skin Clinic Group) and H. Taguchi (Kao Corp.) for their kind assistance in human hair follicle cultivation.

Conflict of Interest

The authors are employees of Kao Corporation.

REFERENCES

• 1) Kizawa K, Unno M, Takahara H, Heizmann CW. Importance of citrullination on hair protein: molecular assembly during trichocytic differentiation. Protein Deimination in human health and disease. (Nicholas AP, Bhattacharya SK eds.) Springer, New York, pp. 129–148 (2014).
• 2) Robbins C, Weigmann HD, Ruetsch S, Kamath Y. Failure of intercellular adhesion in hair fibers with regard to hair condition and strain conditions. J. Cosmet. Sci., 55, 351–371 (2004).
• 3) Mirmirani P, Huang KP, Price VH. A practical, algorithmic approach to diagnosing hair shaft disorders. Int. J. Dermatol., 50, 1–12 (2011).
• 4) Kizawa K, Takahara H, Unno M, Heizmann CW. S100 and S100 fused-type protein families in epidermal maturation with special focus on S100A3 in mammalian hair cuticles. Biochimie, 93, 2038–2047 (2011).
• 5) Kizawa K, Takahara H, Troxler H, Kleinert P, Mochida U, Heizmann CW. Specific citrullination causes assembly of a globular S100A3 homotetramer: a putative Ca²⁺ modulator matures human hair cuticle. J. Biol. Chem., 283, 5004–5013 (2008).
• 6) Ü Basmanav FB, Cau L, Tafazzoli A, Méchin MC, Wolf S, Romano MT, Valentin F, Wiegmann H, Huchenq A, Kandil R, Garcia Bartels N, Kilic A, George S, Ralser DJ, Bergner S, Ferguson DJ, Oprisoreanu AM, Wehner M, Thiele H, Altmüller J, Nürnberg P, Swan D, Houniet D, Büchner A, Weibel L, Wagner N, Grimalt R, Bygum A, Serre G, Blume-Peytavi U, Sprecher E, Schoch S, Oji V, Hamm H, Farrant P, Simon M, Betz RC. Mutations in three genes encoding proteins involved in hair shaft formation cause uncombable hair syndrome. Am. J. Hum. Genet., 99, 1292–1304 (2016).
• 7) Takahashi T, Mamada A, Kizawa K, Suzuki R. Age-dependent damage of hair cuticle: contribution of S100A3 protein and its citrullination. J. Cosmet. Dermatol., 15, 211–218 (2016).
• 8) Chan CC, Fan SM, Wang WH, Mu YF, Lin SJ. A two-stepped culture method for efficient production of trichogenic keratinocytes. Tissue Eng. Part C Methods, 21, 1070–1079 (2015).
• 9) Randall VA. The use of dermal papilla cells in studies of normal and abnormal hair follicle biology. Dermatol. Clin., 14, 585–594 (1996).
• 10) Fusenig NE, Limat A, Stark HJ, Breitkreutz D. Modulation of the differentiated phenotype of keratinocytes of the hair follicle and from epidermis. J. Dermatol. Sci., 7 (Suppl.), S142–S151 (1994).
• 11) Schneider I, Bucar F. Lipoxygenase inhibitors from natural plant sources. Part 2: medicinal plants with inhibitory activity on arachidonate 12-lipoxygenase, 15-lipoxygenase and leukotriene receptor antagonists. Phytother. Res., 19, 263–272 (2005).
• 12) Langan EA, Philpott MP, Kloepper JE, Paus R. Human hair follicle organ culture: theory, application and perspectives. Exp. Dermatol., 24, 903–911 (2015).
• 13) Philpott MP, Sanders DA, Kealey T. Effects of insulin and insulin-like growth factors on cultured human hair follicles: IGF-I at physiologic concentrations is an important regulator of hair follicle growth in vitro. J. Invest. Dermatol., 102, 857–861 (1994).
• 14) Knuckley B, Causey CP, Jones JE, Bhatia M, Dreyton CJ, Osborne TC, Takahara H, Thompson PR. Substrate specificity and kinetic studies of PADs 1, 3, and 4 identify potent and selective inhibitors of protein arginine deiminase 3. Biochemistry, 49, 4852–4863 (2010).
• 15) Nozoe T. Uber die Farbstoffe im Holzteile des “Hinoki”-baumers. 1. Hinokitin und Hinokitiol. Ber. Bull. Chem. Soc. Japan, 11, 295–298 (1936).
• 16) Suzuki H, Ueda T, Juránek I, Yamamoto S, Katoh T, Node M, Suzuki T. Hinokitiol, a selective inhibitor of the platelet-type isozyme of arachidonate 12-lipoxygenase. Biochem. Biophys. Res. Commun., 275, 885–889 (2000).
• 17) Sekiya K, Okuda H, Arichi S. Selective inhibition of platelet lipoxygenase by esculetin. Biochim. Biophys. Acta, 713, 68–72 (1982).
• 18) Ferrándiz ML, Nair AG, Alcaraz J. Inhibition of sheep platelet arachonate metabplosm by flavonoids from Spanish and Indian medicinal herbs. Pharmazie, 45, 206–208 (1990).
• 19) Schneider I, Gibbons S, Bucar F. Inhibitory activity of Juniperus communis on 12(S)-HETE production in human platelets. Planta Med., 70, 471–474 (2004).
• 20) Kuhn H, Banthiya S, van Leyen K. Mammalian lipoxygenases and their biological relevance. Biochim. Biophys. Acta, 1851, 308–330 (2015).
• 21) Baer AN, Green FA. Fatty acid oxygenase activity of human hair roots. J. Lipid Res., 34, 1505–1514 (1993).
• 22) Bocan TMA, Rosebury WS, Mueller SB, Kuchera S, Welch K, Daugherty A, Cornicelli JA. A specific 15-lipoxygenase inhibitor limits the progression and monocyte-macrophage enrichment of hypercholesterolemia-induced atherosclerosis in the rabbit. Atherosclerosis, 136, 203–216 (1998).
• 23) Saijo S, Nagai A, Kinjo S, Mashimo R, Akimoto M, Kizawa K, Yabe-Wada T, Shimizu N, Takahara H, Unno M. Monomeric form of peptidylarginine deiminase type I revealed by X-ray crystallography and small-angle X-ray scattering. J. Mol. Biol., 428, 3058–3073 (2016).
• 24) Shirakata Y, Kimura R, Nanba D, Iwamoto R, Tokumaru S, Morimoto C, Yokota K, Nakamura M, Sayama K, Mekada E, Higashiyama S, Hashimoto K. Heparin-binding EGF-like growth factor accelerates keratinocyte migration and skin wound healing. J. Cell Sci., 118, 2363–2370 (2005).
• 25) Stoll SW, Rittié L, Johnson JL, Elder JT. Heparin-binding EGF-like growth factor promotes epithelial-mesenchymal transition in human keratinocytes. J. Invest. Dermatol., 132, 2148–2157 (2012).
• 26) Cherrington BD, Morency E, Struble AM, Coonrod SA, Wakshlag JJ. Potential role for peptidylarginine deiminase 2 (PAD2) in citrullination of canine mammary epithelial cell histones. PLoS ONE, 5, e11768 (2010).
• 27) McElwee JL, Mohanan S, Griffith OL, Breuer HC, Anguish LJ, Cherrington BD, Palmer AM, Howe LR, Subramanian V, Causey CP, Thompson PR, Gray JW, Coonrod SA. Identification of PADI2 as a potential breast cancer biomarker and therapeutic target. BMC Cancer, 12, 500 (2012).
• 28) Meng H, Shen Y, Shen J, Zhou F, Shen S, Das UN. Effect of n-3 and n-6 unsaturated fatty acids on prostate cancer (PC-3) and prostate epithelial (RWPE-1) cells in vitro. Lipids Health Dis., 12, 160 (2013).
• 29) Henneicke-von Zepelin HH, Schröder JM, Smíd P, Reusch MK, Christophers E. Metabolism of arachidonic acid by human epidermal cells depends upon maturational stage. J. Invest. Dermatol., 97, 291–297 (1991).
• 30) Hagerman RA, Fischer SM, Locniskar MF. Effect of 12-O-tetradecanoylphorbol-13-acetate on inhibition of expression of keratin 1 mRNA in mouse keratinocytes mimicked by 12(S)-hydroxyeicosatetraenoic acid. Mol. Carcinog., 19, 157–164 (1997).
• 31) Hussain H, Shornick LP, Shannon VR, Wilson JD, Funk CD, Pentland AP, Holtzman MJ. Epidermis contains platelet-type 12-lipoxygenase that is overexpressed in germinal layer keratinocytes in psoriasis. Am. J. Physiol., 266, C243–C253 (1994).
• 32) Anderson AB, Gripenberg J, Laaksonen T. Antibiotic substances from the heart wood of Thuja plicata D. Don; the constitution of β-thujaplicin. Acta Chem. Scand., 2, 644–650 (1948).
• 33) Arima Y, Nakai Y, Hayakawa R, Nishino T. Antibacterial effect of β-thujaplicin on staphylococci isolated from atopic dermatitis: relationship between changes in the number of viable bacterial cells and clinical improvement in an eczematous lesion of atopic dermatitis. J. Antimicrob. Chemother., 51, 113–122 (2003).
• 34) Johnson EN, Nanney LB, Virmani J, Lawson JA, Funk CD. Basal transepidermal water loss is increased in platelet-type 12-lipoxygenase deficient mice. J. Invest. Dermatol., 112, 861–865 (1999).
• 35) de Juanes S, Epp N, Latzko S, Neumann M, Fürstenberger G, Hausser I, Stark HJ, Krieg P. Development of an ichthyosiform phenotype in Alox12b-deficient mouse skin transplants. J. Invest. Dermatol., 129, 1429–1436 (2009).
• 36) Krieg P, Rosenberger S, de Juanes S, Latzko S, Hou J, Dick A, Kloz U, van der Hoeven F, Hausser I, Esposito I, Rauh M, Schneider H. Aloxe3 knockout mice reveal a function of epidermal lipoxygenase-3 as hepoxilin synthase and its pivotal role in barrier formation. J. Invest. Dermatol., 133, 172–180 (2013).
• 37) Jobard F, Lefèvre C, Karaduman A, Blanchet-Bardon C, Emre S, Weissenbach J, Ozgüc M, Lathrop M, Prud’homme JF, Fischer J. Lipoxygenase-3 (ALOXE3) and 12(R)-lipoxygenase (ALOX12B) are mutated in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17p13.1. Hum. Mol. Genet., 11, 107–113 (2002).
• 38) Zheng Y, Yin H, Boeglin WE, Elias PM, Crumrine D, Beier DR, Brash AR. Lipoxygenases mediate the effect of essential fatty acid in skin barrier formation: a proposed role in releasing omega-hydroxyceramide for construction of the corneocyte lipid envelope. J. Biol. Chem., 286, 24046–24056 (2011).