Actives protect epidermal stem cells against stresses

Thus, some models have been developed to extract ESC from epidermis, to culture them and to investigate their specific functions such as colony-forming capacity or their sensitivity to oxidative stress.2 Survivin, an anti-apoptotic protein, has been shown to be specifically expressed in the nucleus of ESC, among an extracted population composed of ESC, transitoryamplifying cells and differentiated keratinocytes.4 ?Np63 is a p53 antiapoptotic homologue which has also been shown to be specifically expressed by ESC.5 It has been described to give UVB resistance to ESC and to decrease in these cells after such a stress.6 The NGF-pathway has also been shown to be implicated in this resistance, inducing an increase in the production of the antiapoptotic Bcl-2 molecule.1,7 Other ESCassociated markers have been described, such as MCSP (Melanoma Chondroitin Sulfate Proteoglycan)8,9 or a high level of ?1-integrin expression.3 The purpose of this study was, first, to develop new in vitro models to investigate ESC response after these different types of stresses and, second, to investigate the preventive effect of active ingredients. We particularly focused on cocoyl alanine (CA), an antioxidant and anti-wrinkle cosmetic product. Indeed, previously obtained results suggested that it was able to protect elderly skin explants from ex vivo culture-induced decrease in survivin expression (nuclear form) within epidermis.10

Methodology

Reagents and materials

Cocoyl Alanine (CA) was manufactured by Seppic. ?-tocopherol was purchased from BASF. Foetal Beef Serum (FBS), human fibroblasts, Modified Eagle’s Medium (MEM), Ham’s F12 medium, M199 medium, L-glutamin, penicillin-streptomycin, gentamycin, trypsin/EDTA and sodium bicarbonate were all purchased from Lonza. Phosphate Buffered Saline (PBS) was purchased from Biomérieux. Dubelcco’s Modified Eagle’s Medium (DMEM), hydrocortisone, transferrin, insulin, EGF, bFGF, mitomycin C, Hoechst reagent, dispase, DMSO, methanol, MTT, collagen IV and H2O2 were all purchased from Sigma- Aldrich. Mouse monoclonal antibodies against human ?1-integrin, against human MCSP and against human ?Np63 were purchased from Tebu. Alexa Fluor568- conjugated secondary goat anti-mouse antibody was purchased from Invitrogen/ Molecular Probes. Mouse monoclonal neutralising antibody against human ?1-integrin was purchased from Beckman Coulter. FragEL DNA fragmentation detection kit and K252a were purchased from Calbiochem. Acetone was purchased from Xilab. Hematoxylin and eosin were purchased from Labonord.

ESC extraction and culture

ESC were isolated from epidermal cell suspensions, themselves extracted from healthy patient abdominal skin explants after standard dispase and trypsin/EDTA procedures. ESC were isolated by selecting the keratinocytes which were able to adhere on type IV collagen-precoated dishes in five minutes, as previously described.11,12 ESC were cultured on postmitotic fibroblasts at 37°C in a humidified incubator under a 5% CO2, 95% air atmosphere. ESC medium was composed of DMEM/F12 (3:1) completed with 0.4 ?g.mL–1 hydrocortisone, 5 ?g.mL–1 transferrin, 5 ?g.mL–1 insulin, 10 ng.mL–1 bFGF, 10 ng.mL–1 EGF, 100 ?g.mL–1 streptomycin, 100 U.mL–1 penicillin and 10% FBS. Medium was replaced every two or three days. ESC were used at passage P2 or P3 and seeded in 48-microwell culture plates. Medium was replaced every two days.

Investigation of ESC phenotype by immunofluorescence

After removal of culture medium, cells were rinsed and fixed with a frozen solution of acetone/methanol (8/2, v/v) then rinsed with a solution of PBS/Montanox 20 0.05% (wash buffer). Non specific sites were saturated with a 10% non-fat dry milk solution. Cells were then incubated with the appropriate primary antibody dilution for one hour at room temperature (anti-MCSP: 1:100, anti-?1-integrin: 1:100). At the end of the incubation, cells were washed and incubated with the Alexa Fluorconjugated secondary antibody dilution for one hour at room temperature in the dark. Cells were then washed again and incubated with the Hoechst dilution (2 ?g.mL–1) for 15 minutes at room temperature in the dark. After washing, microscopical observations were performed with an inverted microscope equipped with UV epifluorescence.

Investigation of ESC resistance to UVB

On day five after seeding, cells were treated or not with K252a (200 nM). On day seven, ESC were irradiated or not with different doses of UVB rays (i.e. 25 to 200 mJ.cm–2). At the end of the irradiation, cells were further cultivated in the ESC medium for 24 hours at 37°C before the investigation of cell viability by a standard MTT procedure.

Investigation of oxidative stress on ESC culture and apoptosis

On day four after seeding, cultures were incubated or not with CA (0.00001%, 0.0001% or 0.001%, cp) or with 10 ?g.mL–1 ?-tocopherol, both diluted in ESC medium containing only 2% FBS, for 24 hours. On day five, cultures were treated with 50 ?M H202 for 18 hours. After this period, medium was replaced by 2% FBS-ESC medium and cells were further cultured for 48 hours. Cultures were then rinsed with PBS and cells were fixed with a frozen solution of acetone/methanol (8/2, v/v). Finally, cells were rinsed either with PBS before performing a standard hematoxylin-eosin (HE) staining or with TBS before detection of DNA fragmentation with the FragEL DNA fragmentation detection kit, according to the instructions provided by the manufacturer.

Investigation of UVB on ESC ?Np63 level and apoptosis

 On day four after seeding, cultures were incubated or not for 72 hours with CA (0.00001%, 0.0001% or 0.001%, cp) or with 20 U.mL–1 IL-1?. After this period, medium was replaced and cells were further cultured for 18 hours. ESC were then irradiated or not with 200 mJ.cm–2 UVB rays. At the end of the irradiation, cells were further cultivated in the ESC medium for 24 or 48 hours. For experiments related to the investigation of the NGF pathway, the inhibitor K252a (200 nM) was added to the 2% FBS-ESC medium. In this case, cells were cultivated 48 hours before and after irradiation with a lower dose of UVB rays (50 mJ.cm–2). In both kinds of experiments, cultures were then rinsed with PBS. Cells were fixed with a frozen solution of acetone/methanol (8/2, v/v). Finally, cells were rinsed either with PBS before performing an anti-?Np63 immunoflorescent staining (24 hours postirradiation) or with TBS before detection of DNA fragmentation.

Investigation of AB1I on ESC anoïkosis

ESC were seeded in 6-microwell culture plates. Medium was replaced every two days. On day four, cultures were incubated or not for 72 hours with CA (0.00001%, 0.0001% or 0.001%, cp). After this period, cells were dissociated by standard trypsin/EDTA procedure then incubated or not during two hours with mouse monoclonal neutralising antibody against human ?1-integrin (1:250). After the end of the incubation, cells were centrifugated and the cellular pellets were resuspended in formaldehyde 4%. After 48 hours at 4°C, fixed cells were deposited on slides and rinsed with TBS before detection of DNA fragmentation.

Image analysis, data analysis and statistical analysis

All experiments were performed in triplicates with at least four samples per experiment. Photographs of each condition were taken with the associated numeric camera. Pictures were analysed with the NIS-Br software. ESC colony size was calculated by the software by measuring the area of colony and expressed as ?m2. Proportions of positive cells for a given marker (or for apoptosis) were calculated by reporting the number of positive cells to that of total ESC nuclei (Hoechst+ cells) and expressed as percentages. For each group, the mean and the standard deviation were calculated, reported to the control group and expressed as variation percentages. Statistical significance was assessed using an ANOVA test followed by a Bonferroni adjustment, with p<0.05 being considered significant (compared with the stress condition). In all cases, a percentage of restoration was calculated. Finally, mean effects were calculated on the basis of results which were significant in each experiment.

Results

Investigation of ESC phenotype

Extracted and cultured cells expressed the ESC-related marker MCSP (Melanoma Chondroitin Sulfate Proteoglycan) and showed a high level of ?1-integrin expression, in comparison with normal human keratinocytes (NHK) (Fig. 1).

Investigation of ESC resistance to UVB

Both NHK and ESC were submitted to a range of UVB ray doses to evaluate their resistance to such a stress. Mortality induced by UVB did not exceed 11% in ESC, even when the cells were exposed to the highest dose of UVB, i.e. 200 mJ.cm–2 (data not shown, DNS). On the contrary, cell viability was highly impaired in NHK, as from the dose of 50 mJ.cm–2 (DNS). was decreased to 70% as from the dose of 25 mJ.cm–2 (DNS), while the differences observed between untreated and K252atreated NHK were not significant (DNS). Thus, two UVB doses were chosen: 200 mJ.cm–2 for the investigation of ESC resistance in basal conditions; and 50 mJ.cm–2 for the investigation of ESC resistance in conditions of NGF pathway inhibition (i.e. treatment with K252a).

Investigation of H2O2 stress on ESC colony size and apoptosis

H2O2 treatment (50 ?M) of cultures induced a significant decrease in the size of ESC colonies, i.e. of 58% (Fig. 2). It also induced a significant increase in the proportion of TUNEL-positive (TUNEL+) cells, i.e. of 284% (Fig. 3). Preliminary results concerning the investigation of the proportion of survivin+ positive cells also showed a decrease after H2O2 treatment (–65%, DNS). The reference molecule ?-tocopherol (10 ?g.mL–1) partially limited such effects, inducing a restoration effect of 94% and 68%, regarding the size of ESC colonies (Fig. 2) and the proportion of TUNEL+ cells (Fig. 3), respectively. It also induced a preventive effect (37%) against the H2O2-induced decrease in the proportion of survivin+ positive cells (DNS). CA tested at the two higher concentrations (0.0001% or 0.001%), also showed a restoration effect regarding both the size of ESC colonies, respectively of 54% and 69% (Fig. 2), and the proportion of TUNEL+ cells, respectively of 32% and 54% (Fig. 3). It also induced a preventive effect (from 37% to 52%) against the H2O2-induced decrease in the proportion of survivin+ positive cells (DNS).

Investigation of UVB stress on ?Np63 level

UVB irradiation (200 mJ.cm-2) of ESC induced a significant decrease in the proportion of ?Np63+ cells, i.e. of 67% (Fig. 4). The reference molecule (IL-1??20 U.mL–1) partially limited such effects, inducing a restoration effect, i.e. of 58%, regarding the proportion of ?Np63+ cells (Fig. 4). CA tested at the two higher concentrations (0.0001% or 0.001%), also showed a restoration effect regarding the proportion of ?Np63+ cells, of 29% and 48% respectively (Fig. 4).

Investigation of the involvement of the NGF pathway in the UVB-induced apoptosis

When ESC were both treated by K252a and UVB, an increase in the proportion of TUNEL+ cells was observed from an UVB dose of 50 mJ.cm–2 to 200 mJ.cm–2 (data not shown). At 50 mJ.cm–2, variations were of +116% (Fig. 5). CA tested at the two higher concentrations (0.0001% or 0.001%) showed a restoration effect regarding the proportion of TUNEL+ cells of respectively 42% and 52% (Fig. 5).

Investigation of the effect of anti- ?1-integrin antibody on ESC anoikis

AB1I treatment (1:250) of cultures induced a significant increase in the proportion of TUNEL+ cells, i.e. of 203% (Fig. 6). CA tested at the highest concentration (0.001%), showed a restoration effect regarding the proportion of TUNEL+ cells, i.e. of 69% (Fig. 6).

Discussion

As expected, extracted and cultured cells expressed the ESC-related marker MCSP and showed a high level of ?1-integrin expression. Furthermore, the extracted and cultured cells showed a particularly high resistance to UVB rays, as described in literature for ESC.1 Thus, taken together and regarding both phenotype and function, these results showed that the extracted cell population was actually mainly composed of ESC. Then, three models have been developed to investigate apoptosis of these cells. In the first model, the protective role of CA was compared with that of ?-tocopherol, a well-known antioxidant molecule which had been previously shown to protect ESC from oxidative stress.2 As expected, and in accordance with its previously described antiradical activity,12 CA did protect ESC against H2O2-induced apoptosis, as illustrated by the restoration effect that it showed regarding both the size of ESCcolonies and the proportion of TUNEL+ cells. In the second model, i.e. UVB model, the protective role of CA was compared with that of interleukin (IL)-1?, which is known to increase the secretion of NGF, protecting thus cells from apoptosis.13 CA exerted a protective effect against UVBinduced decrease in ESC proportion, since it could partially limit the decrease in the proportion of ?Np63+ cells. Such a property could be due, at least partially, to the antiradical property of CA. Whether such protective roles were mediated by the nerve growth factor (NGF)-signalling pathway or not was investigated by decreasing the level of UVB irradiation (50 mJ.cm–2). As expected, in these conditions, inhibition of the NGFsignalling pathway (using a tyrosine kinase inhibitor, i.e. K252a) was necessary to induce an increase in apoptosis in the ESC population (TUNEL+ cells).1,7 The fact that CA could also limit UVB-induced variations suggests that it could partially restore the NGF pathway. Investigation of ?Np63 expression in such conditions will have to be performed in order to confirm these properties and to understand better the mode of action of CA. Finally, in the third model of AB1Iinduced anoïkosis, CA could also exert a preventive effect since it limited the increase in TUNEL+ cells. Thus, these results suggest that CA could act against apoptosis events which would be induced by a loss of extracellular matrix adherence. Taken together, these data suggest that the preventive effect of CA observed in explant cultures, regarding survivin expression within epidermis, is likely to be related to its ability to act against different kinds of stresses on ESC.

Conclusion

In conclusion, after confirmation of ESC phenotype (i.e. MCSP+ ?1-integrinhigh ?Np63+ Surv+) and their functionality (UVB-resistance), three different kinds of models could be validated to evaluate the effect of oxidative stress, UV and loss of adhesion to extracellular matrix on ESC apoptosis. Such models also enable the study of the protective ability of cosmetic active ingredients. More particularly, CA showed a protective effect in the three aforementioned models, which is coherent with its antiradical properties and its clinically proven anti-age activity. In the future, intracellular signalling pathways regulating such effects could be more precisely investigated by studying phosphorylation events and transcription factor activities.

References

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