Comprehensive approach to the mechanism of improvement for dry skin

-The effect of moisturizer through time series proteomic analysis-

Shun Sasaoka1, Yu Gabe

2, Masayuki Uchiyama

3, Akira Hachiya

4, Hidehiro Nagasawa

3,

Masahiro Miyaki3, Shun Nakamura

1, Shinichi Tokunaga

1

1Analytical Science Research, Kao Corporation, 2606 Akabane, Ichikai-machi, Haga-gun,

Tochigi 321-3497, Japan, 2Biological Science Research, Kao Corporation, 5-3-28 Kotobuki-cho,

Odawara-shi, Kanagawa 250-0002, Japan, 3Skin Care Products Research, Kao Corporation,

2-1-3 Bunka, Sumida-ku, Tokyo 131-8501, Japan, 4Kao USA Inc. 2535 Spring Grove Avenue

Cincinnati OH 45214, U.S.A.

Key words: time-series proteomic analysis, dry skin, moisturizer, fine fiber

1. Introduction

Dry skin is a condition characterized by flaking, scaling, peeling, and cracking [1] and is a global

concern. In order to develop more effective treatments, it is important to determine how a

moisturizer acts on human skin and why a particular moisturizer is more effective than others,

even though various kinds of moisturizers for dry skin are provided. Due to the lack of an

analytical method to understand epidermal responses post application of moisturizers, the

mechanisms underlying the action of moisturizers in the improvement of dry skin remain unclear.

Proteins in the epidermis play an essential role in a variety of skin functions at the molecular level.

At the outermost layer of the epidermis, stratum corneum (SC), several proteins have been

identified as biomarkers for inflammation, differentiation, and proliferation [2-3]. These protein

markers help us to monitor skin health. Therefore, many researchers have been studying the

proteins in SC, and the epidermal responses post application of various skincare products have

been attracting research interest. However, it is still challenging to analyze sbiological responses.

Therefore, an analytical method to detect not only skin conditions but also skin responses from

non-invasive SC samples, could aid in the development of new skincare technologies for various

disorders, including aging problems.

Recent advances in mass spectrometry (MS) enable the identification and relative quantification

of human skin proteins (Figure 1A). A liquid chromatography-tandem mass spectrometry

(LC-MS/MS)-based proteomic analysis is used to investigate skin diseases, atopic dermatitis [4],

psoriasis [5], and actinic keratosis [6]. In these studies, a comparative protein profiling of skin

samples revealed the differences between patients and healthy controls. On the other hand, to

understand the epidermal response to a skincare product, proteomic analyses are often

performed either at the endpoint or baseline and endpoint. This conventional approach is

referred to as single time point analysis in this study (Figure 1B). The data obtained by this

analysis are only the results of post-application. However, it is challenging to identify biological

responses to skincare treatment based on these data because these responses might be

intricate, inter-related, and time-dependent.

To overcome this limitation, we performed time-series proteomic analysis of SC samples after

dry skin treatment and attempted to acquire information about not only the results but also the

processes of post-application (Figure 1C-F). First, we aimed to clarify the benefits of time-series

analysis compared to the conventional single time point analysis. Second, we sought to unravel

the biochemical mechanism induced by our new skincare technology, fine fiber plus moisturizer.

Figure 1 Comprehensive approach to understanding the biochemical mechanism by

proteomic analysis. (A) Label-free quantitative proteomic analysis using mass spectrometry.

(B) Differential expression of proteins between baseline and endpoint. Single time point analysis

(conventional approach). (C) Time course expression of proteins from baseline through endpoint.

Time-series analysis (our approach). (D) Heatmap of time course protein profiles. (E) Time

course expression of SASPase. (F) Schematic illustration of SASPase function: converts

profilaggrin to filaggrin.

2. Materials and Methods

2.1 Subjects and clinical study design: A moisturizer and a fine fiber plus moisturizer were

respectively applied to the lower leg of 8 healthy American women for two weeks. Informed

consent was obtained from all subjects.

2.2 Materials: Dermalab was obtained from Cortex Technology (Hadsund, Denmark). A film

masking tape was purchased from Teraoka Seisakusho (Tokyo, Japan). A CF15R series

centrifuge was obtained from HITACHI (Tokyo, Japan). Urea and tris hydrochloride were

purchased from Sigma-Aldrich (Deisenhofen, Germany). Thiourea, sodium deoxycholate (SDC),

sodium N-lauroyl sarcosinate (SLS), dithiothreitol, iodoacetamide, ammonium bicarbonate, mass

spectrometry grade Lys-C protease, mass spectrometry grade trypsin protease, ethyl acetate,

and trifluoroacetic acid were purchased from FUJIFILM (Tokyo, Japan). HPLC grade Acetonitrile

was purchased from KANTO (Tokyo, Japan). EZQ protein quantification kit, a SC250EXP and a

SPD111V series evaporator were obtained from Thermo fisher scientific (CA, USA).

2.3 Skin hydration and appearance evaluation: Skin hydration was measured using Dermalab

at 20 °C and 40% relative humidity (RH). Skin condition was evaluated by the observer as

follows: 0; No dryness, 1; Slight dryness, 2; Moderate dryness, 3; Marked dryness, 4; Severe

dryness.

2.4 Sample collection and storage: SC samples were collected with five consecutive tape

strips at Day 0, 7, and 14 (endpoint of treatment) using a film masking tape and then stored at

-80 °C freezer.

2.5 Protein extraction: Tapes were cut into pieces and dipped in 1 mL lysis buffer [7] (7 mol/L

urea and 2 mol/L thiourea, 12 mmol/L sodium deoxycholate, 12 mmol/L sodium N-lauroyl

sarcosinate, 100 mmol/L Tris-HCl). This solution containing tapes was sonicated for 20 minutes.

Dithiothreitol was added to this solution with 0.1 mmol/L final concentration, which was shaken

gently at 37 °C overnight. Iodoacetamide was added with 0.5 mmol/L final concentration at 25 °C

for 30 minutes. Extracted protein concentration was measured by using the EZQ protein

quantification kit according to the manufacturer’s instructions. Furthermore, 2 mL of 50 mM

ammonium bicarbonate was added to the sample solutions.

2.6 Protein digestion: Lys-C protease was added to the sample solutions and incubated for 3

hours at 37 °C. Subsequently, trypsin protease was added and incubated at 37 °C overnight.

The sample solutions were divided equally into three tubes. One milliliter of each ethyl acetate

and trifluoroacetic acid at 0.5% (v/v) final concentration was added to stop protease reaction.

The solutions were shaken for 2 minutes and centrifuged at 20,000 g for 5 minutes. The lower

aqueous layer was collected and dried using an evaporator.

2.7 Peptide purification: The dried residues were dissolved in 0.1% trifluoroacetic acid

containing 5% acetonitrile and then purified by solid phase extraction with styrene divinyl

benzene disks (SUPELCO). The purified peptide solutions were dried using an evaporator and

finally dissolved in 0.1% formic acid containing 2% acetonitrile.

2.8 Nano-LC-MS/MS: Purified peptides were analyzed using nano-liquid chromatography (LC,

Waters nanoAcquity UPLC) coupled mass spectrometry (MS, Thermo Scientific Orbitrap Velos)

and a BEH nanoACQUITY 0.1 mm I.D. × 100 mm column (Waters). In this nanoLC system, a

nanoAcquity binary pump was connected to two mobile phases (A, 0.1% formic acid containing

water; B, 0.1% formic acid containing 80% acetonitrile-water) with a flow rate of 500 nL/min. The

mobile phases were consecutively programmed as follows: an isocratic elution of A 95% (B 5%)

between 0 and 5 min, a linear gradient of A 95-50% (B 5-50%) between 5 and 125 min, an

isocratic elution of A 5% (B 95%) for 25 min, an isocratic elution of A 95% (B 5%) between 150

and 180 min to re-equilibrate the column (a total run time of 180 min). Parameters for mass

spectrometry were as follows: spray voltage, 1,800 V; capillary temperature, 250°C; mass to

charge ratio (m/z) range for full scan, 300 to 1,250; resolution for full scan, 60,000 at m/z 400;

fragmentation method, collision-induced dissociation (CID), data-dependent acquisition, top 15

precursor ions; exclusion time, 180 sec; m/z range for MS2, 100 to 2,000; collision energy, 35.

2.9 Database searching: The raw data were processed using mzR [8], a Bioconductor package.

The processed MS/MS spectra were searched using rTANDEM [9], a Bioconductor package

against the UniProtKB human reference proteome peptide database (release 2014_10).

Parameters for the database search with rTANDEM were as follows: cleavage site, cleavage

C-terminal to every lysine or arginine, except when accompanied by a proline; potential

modification of methionine oxidation; static change of cysteine carbamidomethylation; maximum

miss cleavage is up to one; precursor mass tolerance, 10 ppm; fragment mass tolerance, 0.8 Da;

peptide spectral matches (PSMs) were validated using a target-decoy search [10] at a 1% false

discovery rate (FDR).

2.10 Protein amount index (PAI): To evaluate the protein levels, the protein amount index (PAI)

was defined. The peptide peak intensity (PPI) was defined as the sum of peptide peak intensities

per protein. A weighting factor (wt) was defined as the ratio of i-th PPI to the maximum PPI of the

standard protein. PAI was a log-ratio transformation of PPI to wt. In this study, K1C10 (Keratin,

type I cytoskeletal 10) was used as a standard protein.

2.11 Statistical analysis: Serial PAI changes were evaluated using the paired Student’s t-test. p

< 0.05 was considered significant. Data were expressed as the mean ± SE.

3. Results

3.1 Skin hydration and appearance after application: To investigate the effect of moisturizer

and fine fiber plus moisturizer on severe dry conditions, skin hydration, and appearance were

evaluated. Fine fiber plus moisturizer could significantly elevate skin conductance (Figure 2A)

and lower the skin dryness scale than moisturizer (Figure 2B). After 14 days, skin treated with

fine fiber plus moisturizer appeared healthy and had little or no scales, while the

moisturizer-treated skin showed a few scales.

Figure 2 The effects of fine fiber plus moisturizer on skin hydration and appearance. Serial

changes in skin hydration and appearance after application. (A) Skin conductance values of the

skin hydration indicator. (B) Skin dryness scale values of the skin appearance indicator. Values

are expressed as the mean ± SE (n = 8). The paired Student’s t-test was used for statistical

analysis. **: p < 0.01, *: p < 0.05

3.2 SC protein profile after application: In this study, 172 epidermal proteins were identified

from SC samples by using mass spectrometry-based proteomic analysis. At first, we calculated

the PAI values for each protein and respectively normalized these values for sampling times to

obtain the time course patterns. We found that there were 4 different time course patterns in both

moisturizer and fine fiber plus moisturizer groups: monotonically decreased, decreased with local

maximum, monotonically increased, and increased with a local minimum (Figure 1C). We also

found that some proteins had different patterns according to the application time between

treatment methods. This indicates that a specific protein showed an increasing trend in the fine

fiber plus moisturizer and a decreasing trend in the moisturizer treatment. For this reason, to gain

insights into the differences in the biological mechanisms of these treatments, we next focused

on the skin homeostasis-related proteins.

3.3 Similarities and differences of serial PAI changes between moisturizer and fine fiber

plus moisturizer: We selected 15 skin homeostasis related proteins and categorized these

proteins into seven functional groups (Table 1). We found that fine fiber plus moisturizer not only

elevated the PAI of more proteins than the moisturizer alone (Figure 3) but also exhibited two

characteristics that the moisturizer did not. The first feature is the time lag for significant

differences to occur for the same protein. For example, desmoglein-1 (DSG1) showed a

significant difference only between Day 7 and 14 for moisturizer application and between Day 0

and 14 for fine fiber plus moisturizer application. The second is that protein molecules showed

significant differences. DSG1, kallikrein (KLK), and calpain-1 catalytic subunit (CAPN1) had

substantial differences both in moisturizer and fine fiber plus moisturizer application, whereas

transglutaminase (TGM) was only significantly different for moisturizer application. Some

molecules, e.g., filaggrin (FLG) and saspase (ASPRV1), showed significant differences only for

fine fiber plus moisturizer application. These results indicated that fine fiber plus moisturizer

application had a similar effect on the epidermal biological mechanism as that of conventional

moisturizer application, but it also exhibited other unique features. To clarify these additional

properties, we attempted to quantify time-course changes against application time.

Table 1 Time-series changes in skin homeostasis related proteins according to their

functions.

P values were calculated by the paired Student’s t test.

Figure 3 The effects of fine fiber plus moisturizer on skin protein levels. Serial changes in

PAI values. Values are shown as mean ± SE (n = 8). The paired Student’s t-test was used for

statistical analysis. **: p < 0.01, *: p < 0.05. (A) Moisturizer. white-bar; Day 0, light gray bar; Day

7, gray bar; Day 14. (B) Fine fiber plus moisturizer. White bar; Day 0, light green bar; Day 7, dark

green bar; Day 14.

3.4 Single time point analysis and time-series analysis after moisturizer application:

Conventional single time point proteomic analyses are always performed either at the endpoint

or at baseline and endpoint. This approach can be used to obtain the results of skincare

treatments. We analyzed the time course trend to understand epidermal responses more

precisely. To evaluate the time course responses, we calculated three different mean PAI values

which were represented as follows: type 1) Day 7-Day 0, type 2) Day 14-Day 0, type 3) Day

14-Day 7 (Figure 4). According to single time point analysis with type 2 PAI values, some

molecules increased, and other proteins decreased at Day 14. By using this conventional

analysis, we found that there was no change in the levels of NMF formation related proteins,

except for gamma-glutamylcyclotransferase (GGCT). However, by using whole time course data

with type 1 to type 3 PAI values, we found that arginase-1 (ARG1), histidine ammonia-lyase

(HAL), and GGCT were elevated from Day 7 through Day 14. Additionally, we could detect an

increase in filaggrin after 7 days.

3.5 Biochemical mechanism after fine fiber plus moisturizer application: As shown in

Figure 3, the fine fiber plus moisturizer could change more protein levels than the moisturizer

alone. The two differences between treatments that are significantly different are the timing and

the protein molecules. To investigate these differences, type 1 and 2 PAI values were compared

between moisturizer and fine fiber plus moisturizer (Figure 5). The levels of desmocollin-1

(DSC1), DSG1, ASPRV1 and suprabasin (SBSN) were commonly elevated both at Day 7 and

Day 14 using fine fiber plus moisturizer, while those of bleomycin hydrolase (BLMH) and HAL

were elevated only at Day 7 and ALOX12B and ARG1 were heightened only at Day 14.

Heatmaps of type 1 and 2 PAI are shown in Figure 6. The color patterns of the two heatmaps

were similar to some extent, but there were several differences between treatments. DSC1,

DSG1, CDSN, GGCT, HAL, and ARG1 behaved similarly, whereas fine fiber plus moisturizer

could elevate these levels more. Further, although an increase in ALOX12B and TGM were

observed 14 days after moisturizer application, the same changes were detected 7 days after

fine fiber plus moisturizer application. The same was found in BLMH. Uniquely, SBSN showed a

monotonical increasing trend in the fine fiber plus moisturizer group, and this time-dependent

change was the opposite between the two treatments.

Figure 4 Time-series protein expression post-application of moisturizer.

Serial changes in PAI values. Values are denoted as mean ± SE (n = 8). (A) light gray bar;

Day 7-Day 0, dark gray bar; Day 14-Day 0, dot filled-bar; Day 14-Day 7. (B) PAI values were

represented in the heatmap and colored based on percentile values.

Figure 5 Comparison of protein profiles between moisturizer and fine fiber plus

moisturizer. Mean PAI values normalized against the value on Day 0 after treatment. Values

indicate the mean ± SE (n = 8). The paired Student’s t-test was used for statistical analysis. ***: p

< 0.01, **: p < 0.05, *: p < 0.1. (A) light gray bar; Day 7-Day 0 in Moisturizer, light green bar; Day

7-Day 0 in fine fiber plus moisturizer. (B) dark gray-bar; Day 14-Day 0 in Moisturizer, dark green

bar; Day 14-Day 0 in fine fiber plus moisturizer.

Figure 6 Time-series protein expression post application of fine fiber plus moisturizer.

Serial changes in PAI values. Value, mean PAI normalized against the value on Day 0 after

treatment. PAI values were represented in the heatmap and colored based on percentile. (A)

Moisturizer. (B) Fine fiber plus moisturizer.

4. Discussion

4.1 Time-series proteomic analysis is a powerful tool to understand biological

mechanisms in human skin: Differences and similarities among epidermal responses by

moisturizer estimated using a single time point analysis and time-series analysis are shown in

Table 2. Conventional single time point proteomic analysis revealed that some epidermal

responses were induced by moisturizer treatment as follows: 1) CE maturation was enhanced:

CE is the outermost structure of SC that contributes to the physical strength of human skin and

consists of highly cross-linked proteins, including loricrin, small proline-rich protein (spr),

involucrin, and keratins [11]. TGM catalyzes the cross-linking between protein side chains to

form cornified layers during cell proliferation [12]. Both TGM and ALOX12B catalyze the covalent

binding of lipid to the cornified layer [13]. These proteins were elevated at Day [14], indicating the

enhancement of CE maturation after application. 2) Desquamation and skin cell turnover were

suppressed. KLK catalyzes the degradation of cohesive structures in SC and A2ML1 inhibits

KLK 14. The level of A2ML1 increased, and that of KLK decreased. This indicated that the

enhancer of adhesive component degradation was down-regulated and the inhibitor was

up-regulated, resulting in an increase in DSG1 and CDSN at desmosome junctions to form an

adhesive structure [15]. 3) Filaggrin processing was enhanced. BLMH and CAPN1 catalyze

filaggrin degradation into amino acids [16]. 4) LB secretion was suppressed. SBSN is a marker of

LB secretion, which is involved in epidermal barrier recovery [17].

Time-series proteomic analysis could detect signals that may clarify the responses of NMF

formation and filaggrin supply post-application of moisturizer as follows: 5) NMF formation was

weakly enhanced. GGCT, HAL, and ARG1 convert amino acids into their derivatives, e.g.,

pyrrolidone carboxylic acid (PCA) and urocanic acid (UA) [18]. PCA and UA are the main

components of NMF molecules in SC. There was little difference in the levels of these enzymes

between Day 0 and 14; however, they showed an increasing trend between Day 7 and 14. 6)

Filaggrin supply was enhanced. Although there was no evidence of filaggrin increase between

Day 0 and 14, filaggrin showed a tendency to increase between Day 0 and 7. We, thus, suggest

that filaggrin supply increases till Day 7 and decreases or gets consumed by Day 14. ASPRV1

catalyzes the formation of pro-filaggrin, which is composed of filaggrin monomers [19]. An

increase in ASPRV1 at Day 14 may indicate filaggrin supplementation.

4.2 Fine fiber plus moisturizer activates epidermal responses that are highly effective for

treating dry skin: We developed a novel technique to create a thin fiber network on the skin.

Our results showed that a combination of this network and moisturizer could improve dry skin, for

example, the hydration and appearance of the skin, more effectively than that by using

moisturizer alone. However, it was unclear why a fine fiber plus moisturizer was more effective

than moisturizer alone in dry skin improvement. The characteristics of epidermal responses by

fine fiber plus moisturizer are summarized in Table 3. Using time-series proteomic analysis, we

gained insights into the biological mechanism underlying the effects of fine fiber plus moisturizer,

which contributed to dry skin improvement. We suggest that fine fiber plus moisturizer has three

characteristic effects.

The first is that fine fiber plus moisturizer treatment could rapidly induce epidermal responses;

increases were observed for ALOX12B and TGM on Day 7 in the fine fiber plus moisturizer

groups, but not in the moisturizer group. The same effects were also observed for BLMH. These

results suggest that fine fiber plus moisturizer treatment elicits epidermal responses at a faster

rate than only moisturizer treatment.

The second is that fine fiber plus moisturizer treatment could actively enhance responses.

Substantial differences in protein levels related to skin cell turnover, NMF formation, and filaggrin

supply were observed between fine fiber plus moisturizer and moisturizer groups. These findings

indicate that fine fiber plus moisturizer treatment activates a critical mechanism that regulates

protein dynamics in the epidermis.

The third is that fine fiber plus moisturizer treatment could activate LB secretion, which is

involved in epidermal barrier recovery. Fine fiber plus moisturizer treatment monotonically

up-regulated the SBSN levels, while only moisturizer treatment monotonically down-regulated it.

LBs are lipid particles filled with ceramides and enzymes and play an essential role in the barrier

function of the skin [20]. Ceramides are the predominant lipids in the human SC and play critical

roles in determining the barrier function efficiency and water-holding property of the skin [21],

while enzymes found in LBs are important for normal cornification [22]. For this reason, it is likely

that the enhancement of LB secretion affects epidermal homeostasis.

Fine fiber plus moisturizer treatment could dramatically improve skin hydration and the

appearance of the SC as compared with conventional moisturizer treatment (Figure 2). We

conclude that fine fiber plus moisturizer treatment activates epidermal protein dynamics, which

alleviates dry skin symptoms.

Table 2 Estimated epidermal responses using a single time point and time-series

analysis.

Table 3 Differences and similarities between moisturizer and fine fiber plus moisturizer.

5. Conclusion

We first aimed to clarify the benefits of time-series analysis compared to the conventional single

time point analysis. This approach could detect more responses, indicating that time-series

analysis has the potential to precisely detect epidermal responses that occur post the application

of various cosmetic products. In conclusion, we suggest that time-series proteomic analysis is a

powerful tool for understanding complex and inter-related biological processes in the human skin.

We second aimed to unravel the biochemical mechanism induced by our new skincare

technology, fine fiber plus moisturizer. Time-series proteomic analysis revealed that this could

induce epidermal responses similar to those elicited by moisturizer treatment, but at a faster rate.

We also observed that our treatment uniquely elevated the levels of LB secretion marker

proteins involved in epidermal barrier recovery. These data suggest that our new treatment

activates epidermal responses related to the maintenance of skin homeostasis and results in a

dramatic improvement of skin hydration and appearance compared to conventional moisturizer

treatment (Figure 7). We believe that time-series proteomic analysis is appropriate to investigate

the causal relationship between skincare effects and biological responses in human skin. We

believe that this offers insights into how human skins react to skincare treatments and opens

new avenues for skincare technology development.

Figure 7 Schematic illustration of biochemical responses elicited in the fine fiber plus

moisturizer treatment group. Fine fiber plus moisturizer treatment activates the common

responses (1-4) at a faster rate and shows a unique response (5).

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Supplemental Data

Time-series protein expression post-application of fine fiber plus moisturizer. Serial

changes in PAI values. Values are denoted as mean ± SE (n = 8). (A) light green bar; Day

7-Day 0, dark green bar; Day 14-Day 0, dot filled-bar; Day 14-Day 7. (B) PAI values were

represented in the heatmap and colored based on percentile values.