Issue 36 — April 2004

Issue 36
Category		Titel		Author
Newsletter		What is the most suitable strategy for stress proteins in cosmetics?		Christine Jeanmaire, Vincent Bardey, Louis Danoux, Philippe Moser, Philippe Moussou, and Gilles Pauly

Summary

Under various stress conditions, heat shock protein expression is enhanced in cells. Working on skin pieces, two of the major skin-expressed heat shock proteins, HSP72 and HSP27, were visualized by immunohistochemistry after several stress conditions (UV, pollutants, thermal shocks) and the heat shock protein expression quantified in the epidermis. A strong increase of the epidermal heat shock protein expression, due to these different stresses, was observed. The most suitable cosmetic strategy is discussed: does one need to prevent or to favor heat shock protein induction in cutaneous cells?

A prevention strategy is suitable in the case of acute stress, such as severe UV exposure that provokes cell damage and a strong induction of heat shock proteins. It is demonstrated that, using a photoprotective active ingredient Sunactyl®, suppression of the heat shock protein expression of cells is linked to a protection of the cells against UV induced damages.

An induction strategy is appropriate for chronic daily stress from various sources. Seeing that a small and controlled induction of heat shock proteins in the skin speeds up the response and enhances the cellular resistance to further stress, HSP-Balance® was developed, which boosts the heat shock protein induction in the skin under stress conditions.

Introduction

Heat shock proteins (also called stress proteins) are specific proteins, that are universally expressed by cells and whose synthesis is increased in response to stressful conditions. Heat shock proteins represent a heterogeneous group of proteins between 10 and 110 kDa. According to their molecular weights, heat shock proteins have been grouped into various families: the HSP70, the HSP90, the HSP110, and the small HSP (20 to 28 kDa) families. In humans, the HSP70 family includes several genes coding for either constitutive (HSC70) or inducible proteins (such as the major HSP70 heat-inducible protein HSP72), as well as for the 78-kDa glucose related protein or BiP [1]. Under normal conditions, heat shock proteins are implicated in transport, translocation and folding of new synthesized proteins and are often referred to as molecular chaperones [1, 2]. In stressed cells, heat shock proteins are involved in protecting and repair processes. They bind to unfolded or aggregated stress-damaged polypeptides to restore their active conformation. They are also implicated in the protein-degradation process. Moreover, heat shock proteins prevent the apoptotic pathway during certain stresses [3, 4, 5].

In the skin, both epidermal and dermal cells express heat shock proteins. Under normal conditions, if the constitutive hsc70 gene is expressed by all cutaneous cells, only keratinocytes, throughout the epidermis, produce a moderate level of the heat induced HSP72 [6, 7]. Upon stress, both epidermal cells (such as keratinocytes or melanocytes) and dermal fibroblasts highly express the hsp72 gene [7], resulting in HSP72 expression. In contrast, the stress-inducible HSP27 expression is mainly located in keratinocytes of suprabasal epidermal layers in normal conditions [8, 9].

In this study on human skin pieces, the effect of several stress conditions such as UV irradiation, pollutants, cold shock and heat shock on heat shock protein expression have been investigated. The presence of two of the major skin-expressed heat shock proteins, HSP72 and HSP27, was therefore visualized by immunohistochemistry after inducing stress and their expression quantified in the epidermis of the skin pieces. Based on this study, two different strategies were defined for the action of active ingredients in the protection of the skin from environmental stresses: prevention or boosting heat shock protein expression. Results obtained with two developed active ingredients according to these two strategies are presented.

Experimental

Cell cultures

Human keratinocytes were seeded in a standard medium of cell culture with fetal calf serum. Cells were incubated at 37°C and 5% CO2 until confluence.

Preparation of human organ-cultured skin

Skin cultures consist of human skin pieces cultivated, during a given period, in a humidified incubator at 37°C.This technique was adapted from a technique developed by Boisnic et al. [10]. Briefly, skin breast or abdominal fragments obtained from plastic surgery were cut into 1 cm² and washed three times with an antibiotic solution. Subcutaneous fat and lower dermis were mechanically removed using a surgical scalpel. Skin biopsies were placed with epidermis uppermost at the air/liquid interface on culture inserts (Costar, Polylabo, Fontenay-sous-bois, France) in an incubator at 37°C, 5% CO2 and a relative humidity 95%. Skin biopsies were cultured for several days in a Dulbecco's minimal essential medium (Life technologie, France) containing antibiotics.

Protocols for inducing several types of stress on skin

The effects of several stresses were studied on organotypic cultures of human skin. Heat shock, the first stress inducer of heat shock proteins described in the literature [1,11] was given by incubating the organ-cultured skin pieces at 45°C for 1, 2 or 3 hours. Thereafter, conditions were changed back to normal for 24 hours.

Exposures to exhaust gas or to cigarette smoke were developed in order to mimic daily city stress. For that, skin was incubated, in enclosure, either with car exhaust gas (car motor running) or with cigarette smoke during 16, 24 or 48 hours. These pollution durations correspond to weak (16 hours of incubation) or stronger (24 or 48 hours) pollution conditions.

Mercury (HgCl2 from Sigma) was topically applied for 24 hours at 1% in PBS (phosphate buffered saline from Biomérieux, France). Mercury was selected as a model of topically applied heavy metal pollution.

Cold shocks, another environmental stress that may be encountered during winter, were done by applying either cold anesthetic sprays topically on the skin (freezing stress) or by incubating organotypic cultures overnight at 4°C (cold shock). Thereafter, conditions were changed back to normal for 24 hours.

UV irradiations were delivered either by a solar simulator (Suntest CPS+, Heraeus, Hanau, Germany) or by UVB fluorescent lamps (Duke GL40E - 40 watts, Eurosep, Cergy-St-Christophe, France). Energy delivered was measured by Osram centra probe (Osram, Munich, Germany). After irradiation, conditions were changed back to normal for 24 hours.

For each condition, skin biopsies were done and immediately frozen in liquid nitrogen and stored at -70°C until use. Controls without stress were included in every stress study.

Origin of active ingredients

Sunactyl® LS 9610* is a synergistic complex comprising mainly organic acids, amino acids and their derivatives, peptides, polyphenols, vitamin B6 and Nicotinamide Adenine Dinucleotide, obtained from extracts of Pisum sativum, Yeast and Khaya senegalensis. HSP-Balance® LS 9587* is an aqueous extract of fresh rye sprouts from Natural Agriculture.

Treatment with active ingredients

Skin organotypic cultures were topically treated at a dose of 2 mg/cm² with Sunactyl®, formulated in a cream, with or without sun filters (SPF 6). Thereafter, organotypic cultures were irradiated at three different doses by a solar simulator. Doses of UVA and UVB received by the skins at these different times were: UVA (45.6 J/cm²) + UVB (2.6 J/cm²); UVA (60.8 J/cm²) + UVB (3.5 J/cm²) or UVA (91.2 J/cm²) + UVB (5.2 J/cm²). Controls without irradiation were also done. Biopsies were realized 24 hours after irradiation.
Concerning HSP-Balance®, it was introduced in a culture medium of human keratinocytes after cells reached confluence. Thereafter, the cells were immediately submitted to heat shock at 45°C for 10, 15 or 20 minutes. After this heat shock, the cells were incubated for 2 hours at 37°C and 5% CO2.

Immunocytochemistry and quantification by means of heat shock protein staining

Classical immunocytochemical techniques were used for heat shock protein staining. Briefly, a fixation for 10 minutes in cooled acetone or methanol was applied for skin sections or cultured keratinocytes, respectively. Subsequently, the fixed preparations were incubated for one hour at room temperature using monoclonal anti-HSP72 or anti-HSP27 antibodies, diluted 1:150 and 1:200, respectively (SPA 810 F, SPA 800, TEBU, Le Perray en Yvelines, France). Thereafter, preparations were successively incubated for 45 minutes with biotinylated goat anti-mouse antibody (RPN 1001, Amersham, Saclay, France, diluted 1:50) and with streptavidin-fluorescein complex (RPN 1232, Amersham, Saclay, France, diluted 1:30). Negative controls were produced by omission of the primary antibody. Pictures obtained with a confocal laser scanning microscope (LSM 310, Zeiss, Oberkochen, Germany), were converted into color numeric images and analyzed using a mathematical morphology software (Quantimet Q500IW, Leica, Cambridge, United Kingdom).

For heat shock protein on skin sections, results were expressed as the percentage of the surface occupied by HSP72 staining in the epidermis. For heat shock protein on cultured keratinocytes, results were expressed in percentage of the culture area occupied by heat shock proteins (during the first step of HSP72 induction, the localization is in the cytoplasm) or in number of nuclei (during the second step of HSP72 induction, the localization is nuclear) stained in the observed fields.
For each condition, data correspond to the mean of 6 analyzed pictures standard deviation (SD) and the Mann-Whitney U-test for non-parametric samples was used for statistic evaluation.

Results and discussion

Studies of stress induction on skin organotypic culture

The effects of several stresses such as UV irradiation, pollutants, cold and heat shock have been investigated. HSP72 and HSP27 were visualized by immunohistochemistry and quantified in the epidermis of human skin sections.

In normal skin, without stress, a basal rate of HSP72 is visible inside epidermal cells, while HSP27 is only weakly and patchy expressed in the stratum granulosum (Figure 1, see the first column). For each studied stress, an increase of expression HSP72 and HSP27 was observed in the skin cells. This heat shock protein induction was dose-dependent on the intensity and duration of the stress. After a weak stress only few cells were stained, but after a stronger or longer stress, heat shock protein staining was increased up to occupying a large part of the epidermis area. These results are summarized in Table 1. Only results obtained with exhaust gas and cigarette smoke are illustrated in Figure 1.

In human skin, the expression of HSP27 and HSP72 has been extensively studied after heat shock, UV or solar radiation [8, 9, 11-13] but little data is available concerning the consequence of pollutants on the heat shock protein response. In the present study, it is demonstrated that exposure of skin to exhaust gas or to cigarette smoke leads to an increase of HSP27 and HSP72 expression. These results are consistent with other studies conducted on the effects of deleterious exogenous stimuli present in our city environment. Fenga et al. [14] have demonstrated, in vivo, that chronic exposure of the skin to bitumen compounds leads to HSP27 epidermis up-regulation. Pinot et al. [15] also observed, in vitro, an induction of stress proteins by tobacco smoke in human monocytes. These investigations demonstrate that heat shock proteins are induced in skin cells in response to stresses as different as heat or cold shocks, UV irradiation, cigarette smoke, exhaust gas or mercury. All these stresses correspond to the various environmental aggressions to which skin is daily exposed and that have noticeable negative effects on the skin [14,15, 16].

Increase of heat shock protein expression can be considered both as a marker of cellular suffering and as a protective mechanism set up by cells in response to external injuries. From these first observations, two strategies were developed and two new active ingredients evaluated to ensure the skin protection.

First strategy: preventive approach by Sunactyl®

Solar stress, and essentially acute irradiation during excessive sun exposure, leads to skin photo-aging. Prolonged UV exposures of skin during summer can be very traumatic for cutaneous cells and the use of UV filters is not always sufficient to protect the skin from harmful effects of solar radiation. A photoprotective active, Sunactyl®, was developed to protect the skin from acute UV irradiation. In order to evaluate this photoprotective effect, heat shock protein expression was used as an indicator of acute cellular suffering and as observed above, HSP27 can serve as a sensitive marker of skin irradiation.

Figure 1: Enlarged version Study of HSP72 and HSP27 induction, on an ex vivo model, after exhaust gas or cigarette smoke exposure. HSP are stained in green by immunohistochemistry technique (FITC). After stress, HSP (arrows) expression is increased in cells, in line with the intensity of the stress, up to occupying a large part of the epidermal area (star). E: epidermis, D: Dermis. Scale bar = 25 µm.

Table 1: Enlarged version Summary of the tested stresses and its effects on HSP expression (Np = not performed)

HSP27 was chosen rather than HSP72 to study acute cell stress after solar irradiation, because HSP72 is very sensitive to temperature elevation and, despite the presence of a cooling system (to avoid increase of temperature) during irradiation, a possible crossing effect between UV radiation and heat could arise, so HSP27 seemed to be the better marker for such a study.

Figure 2a: Enlarged version Figure 2b: Enlarged version Evaluation of the cytoprotective effect of Sunactyl® against UV radiation. HSP27 was immunohistochemically studied in an ex vivo model of human skin. The application of our Active Ingredient Sunactyl® has almost completely prevented the expression of the UV-induced HSP27. Scale bar = 25 µm.

Figure 3: Enlarged version Evaluation of the cytophotoprotective properties of Sunactyl® using HSP27 as marker of solar stress.

After solar irradiation, HSP27 expression is highly increased in the epidermis (see Figure 2a and Figure 2b) compared to the control without irradiation. Topical application of a cream with UV filters (SPF 6) has only partially decreased the expression of HSP27, whereas after the application of the cream containing UV filters (SPF 6) and 3% of Sunactyl®, far less expression of stress proteins was observed (see Figures 2 and Figure 3).

Complementary tests not further reported here were performed that verified that the reduced expression of HSP27 after UV radiation following application of Sunactyl® was not due to cytotoxicity or a suppression of cellular defense mechanism against UV irradiation, but due to prevention of stress induced damages.

Figure 4a: Enlarged version Figure 4b: Enlarged version Effect of a heat shock on the expression of HSP72 in human cultured keratinocytes. Scale bar = 50 µm.

Second strategy: heat shock protein induction by HSP-Balance ®

A strategy for cosmetic applications to use heat shock proteins as preconditioning agents of cells was developed. Several data support this approach. First, the skin is submitted every day to not well-defined forms of environmental stress; it is a mixture of mild, but repeated, stress all day long ranging from irradiation, pollution, temperature changes, etc. All these forms of stress increase the synthesis of heat shock proteins that are considered to be part of the protecting mechanism of the cell. Controlling heat shock protein production would therefore allow the control of cellular stress, and help the skin to stay in an optimal physiological state. Second, the heat shock response is attenuated in aged subjects. Aged cells have a reduced capability to express heat shock proteins upon stress exposure [12, 17, 18]. Muramatsu and co-workers [12, 18] have examined the HSP72 expression in skin explants from 30 individuals ranging in age from 17 to 86 years and have observed a reduced expression of HSP72 in aged individuals. Third, the notion of cell's preconditioning. Exposure to mild stress results in an increased expression of heat shock proteins and is followed by a transient state of increased resistance to further stress [11]. Moreover, it has been demonstrated that repeated forms of mild stress, including heat-shock, have beneficial effects on aging in cells and organisms [19, 20]. Using human skin fibroblasts in culture, Rattan showed that exposure to repetitive mild heat shock during their replicative lifespan delays aging in cultured human skin fibroblasts by maintaining their morphology and cytoskeletal organization [19]. Since alterations in cellular morphology are accompanied by accumulation of abnormal proteins, induction of heat shock protein expression could help to restore cellular function.

Figure 5a: Enlarged version Figure 5b: Enlarged version Immunocytochemical study of HSP72 on cultured keratinocytes. Effect of HSP Balance®. Scale bar = 50 µm.

Because "boosters" of heat shock proteins seem to be promising candidates for the prevention and the treatment of cell aging, HSP-Balance® was developed and tested for its heat shock protein-booster effect using a specific test on cultured human keratinocytes. HSP-Balance® was introduced into the culture medium and the culture was submitted to a heat shock lasting different time periods. The heat shock stress applied was used as a stress-model to imitate mild daily environmental stress experienced by keratinocytes. Since HSP72, one of the major members of the HSP70 family, is easily induced by heat shock [12, 18], HSP72 has been chosen as the marker of heat shock protein-induction in this test of evaluation.

The presence of HSP72 was visualised by immunocytochemistry in the cultured keratinocytes. Figure 4a and Figure 4b show the induction of HSP72 protein in human keratinocytes after heat shock at 45°C lasting different time periods. The newly synthesized heat shock proteins appeared first in the cytoplasm, and were detectable in the nuclei or nucleoli of stressed cells at longer stress durations. The intensity of the cell response (i.e., HSP72 expression) is stress dose dependent. Without stress (heat shock) no expression of HSP72 was observed in the cultured keratinocytes (Figure 4 and Figure 5a and Figure 5b).

Indeed after heat shock (45°C - 20 minutes), HSP72 are only expressed in the cytoplasm of untreated culture whereas in presence of HSP-Balance®, HSP72 are expressed already both in cytoplasm and nuclei. It is very important to notice that HSP-Balalnce® did not induce HSP72 in condition "stress-free" (no stressful effect of HSP-Balance®).
Complementary tests not further reported here were performed that verified that the increase of heat shock protein expression was not due to a pro-inflammatory or toxic effect of HSP-Balance®. Therefore, preconditioning skin with a moderate induction of heat shock proteins might have a preventive effect against mild chronic daily forms of stress that accumulate their negative effects day after day.

Conclusion

In conclusion, prevention (for acute stress e.g. during excessive sun exposure) and gentle induction (for chronic and not well-defined forms of daily stress) of heat shock proteins are two possible concepts to develop cosmetic active ingredients in order to protect the skin from environmental forms of stress. But, whatever the circumstances, heat shock protein expression must not be inhibited because heat shock protein production is a protective mechanism set up by cells in response to external injuries, so its suppression would lead to an increase of cellular damages.

* Sunactyl® LS 9610 [INCI name: Mannitol (and) Pisum Sativum (Pea) Extract (and) Histidine HCl (and) Arginine (and) Cyclodextrin (and) Dextrin (and) Yeast Extract (and) Acetyl Tyrosine (and) Pyridoxine HCl (and) Khaya Senegalensis Bark Extract (and) Nicotinamide Adenine Dinucleotide (and) Disodium Succinate (and) Aspartic Acid] and HSP-Balance® LS 9587 [INCI name: Secale Cereale (Rye) Seed Extract (and) dextrin] are registered trademarks of Laboratoires Sérobiologiques-Cognis, France.

Acknowledgments

The authors thank Mrs. Carine Lorio, Mrs. Lydie Martin-Teixeira, Mr. Emmanuel Charrois and Mr. Dominique Gauché for their excellent technical assistance.

References

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Christine Jeanmaire



Christine Jeanmaire, PhD, is in charge of Histological Laboratory at Cognis France, Division Laboratoires Sérobiologiques (R&D Department). Her main activities are the research for new cosmetic concepts and the evaluation of the efficacy of Active Ingredients.

Dr. Christine Jeanmaire
Laboratoires Sérobiologiques,
Division de Cognis France, Pulnoy
5-7 rue de Seichamps
F-54425 Pulnoy
France

E-Mail: christine.jeanmaire@Cognis.com
Internet: http://www.laboratoires-serobiologiques.com
Phone (33) 3 83 29 08 02
Fax (33) 3 83 21 12 15

This article was published as a communication 'What is the most suitable strategy for stress proteins in cosmetics?' at the IFSCC in Edinburgh (September 2002), and in IFSCC Magazine (July/September 2003)