Comprehensive approach to the mechanism of improvement for ... · Key words: time-series proteomic...
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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.