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Photoprotective effect of a water-soluble extract of Rosmarinus officinalis L. against UV-induced matrix metalloproteinase-1 in human dermal fibroblasts and reconstructed skin

European Journal of Dermatology. Volume 18, Number 2, 128-35, march-april 2008, Investigative report

DOI : 10.1684/ejd.2008.0349

Summary

Author(s) : Richard Martin, Cécile Pierrard, François Lejeune, Pascal Hilaire, Lionel Breton, Françoise Bernerd , L’Oréal Recherche, Clichy and Tours, Centre de Recherche C Zviak, 90 rue du Général Roguet, 92583 Clichy Cedex, France.

Summary : Chronic UV exposure is responsible for long term clinical manifestations such as photoaging and photocancers. One of the major events involved in the development of skin photodamage is up-regulation of matrix metalloproteinase-1 (MMP-1). In this study, the effects of a water-soluble extract of Rosmarinus officinalis (Ro) on the expression of UV-induced MMP-1 were investigated. Using MMP-1 promoter-reporter gene constructs, Ro extract was shown to inhibit UV-induced up-regulation of MMP-1 gene transcription. The anti-MMP-1 effect was confirmed at the protein level in dermal human fibroblasts exposed either to UVB, UVA or Solar Simulated Radiation. Zymographic analysis on casein and gelatin gels revealed that Ro extract more specifically targeted MMP-1 compared to MMP-2. Using a 3D-skin model exposed to Solar Simulated Radiation, anti-MMP-1 activity was confirmed together with a photoprotective effect at the morphological level. Finally the release of cytokines IL1α and IL6 which participate in the up-regulation of MMP-1 induced by UV exposure could be prevented by the Ro extract. All together, from molecular to tissue level, these results illustrate the ability of the Ro water-soluble extract to inhibit UV-induced MMP-1 and its potential benefits in preventing cutaneous photodamage.

Keywords : MMP-1, photodamage, Rosmarinus officinalis, UV light

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ARTICLE

Auteur(s) : Richard Martin, Cécile Pierrard, François Lejeune, Pascal Hilaire, Lionel Breton, Françoise Bernerd

L’Oréal Recherche, Clichy and Tours, Centre de Recherche C Zviak, 90 rue du Général Roguet, 92583 Clichy Cedex, France

accepté le 10 Octobre 2007

Chronic sun exposure is known to be responsible for photoaging and photocancers [1]. Among the solar UV spectrum both UVB (280-320 nm) and UVA (320-400 nm) wavelength ranges have been shown to contribute to detrimental effects through different molecular processes, including direct DNA damage, modulation of gene expression and reactive oxygen species (ROS) generation [2-4].

Photodamaged skin is characterized by major changes in the dermal compartment. Connective tissue structure and organization are altered, leading to visible clinical signs of aging such as sagging, skin shriveling and development of wrinkles [5]. At the biochemical level, photoaging is associated with a progressive decrease in the amount of collagen content [6, 7]. This effect has been shown to result from both a decrease in collagen type I synthesis [8] and its excessive degradation through the action of matrix metalloproteinases (MMP) [9-11]. MMP include a large family of zinc dependent endopeptidases which are able to degrade macromolecules of the extracellular matrix network [12, 13]. MMP play an important role in the tissue remodeling occurring during physiological or pathological processes. MMP-1 degrades fibrillar collagens including collagen type I and is the key enzyme involved in collagen breakdown in the skin. UVB, UVA and solar simulated radiation have been shown to be potent inducers of MMP-1 in vivo and in vitro [14-16]. Over-expression of MMP-1 is also found in aged/photoaged skin in vivo [17]. Molecular mechanisms underlying MMP-1 induction involve UV-induced signal cascades through cell surface cytokines and growth factor receptors [18, 19]. MAP kinase activation results in induction of transcription factor activator protein (AP-1) which regulates the MMP-1 gene transcription [20]. In addition, oxidative stress also plays a role in that process by increasing the ROS level. ROS are involved in the MAP kinase pathway and thereby contribute to the AP-1 induced MMP-1 upregulation [11, 21].

By potentially preventing the imbalance in synthesis/degradation of major fibrillar components of extracellular matrix during the photoaging process, inhibitors of UV-induced MMP-1 are a major approach for maintaining extra cellular matrix homeostasis. Several natural or synthetic MMP inhibitors have been described [22]. However they often inhibit MMP non-specifically, for example through chelating Zn. Since MMP-1 has been clearly identified as an adequate target to prevent the early UV-induced matrix degradation [23], an anti-MMP-1 strategy represents a reasonable approach. Many whole plant extracts contain natural MMP-1 inhibitors such as flavonoids, polyphenols or terpenoids [24-33].

Rosemary (Rosmarinus officinalis L.) is a very common shrub found in various Mediterranean regions. Growing in very arid lands, it shows a very good sun resistance. This plant is well known for its essential oil and its antioxidant extracts containing mainly rosmarinic acid, carnosol and carnosic acid. Surprisingly, rosemary hydrophilic extracts have not really been investigated in the past. Every year, tons of essential oil are produced by steam extraction from the fresh plants. Residual oil-free leaves provide a biomass available to develop oil-free extracts.

Many results show that rosemary gives very different essential oils and methanol extracts with different antimicrobial properties depending on the collection area and with seasonal variations [34]. Fortunately, the biological activity we are interested in is not sensitive to plant origin (data not shown). This is the reason why we decided to work with a normalized “oil free” biomass supplied by an international company, even if the biomass was harvested from different areas.

The aim of the study was to investigate the potential of water soluble Rosmarinus officinalis (Ro) extract in counteracting UV-induced MMP-1. Anti-MMP-1 activity was assessed at the transcriptional level using MMP-1 promoter-reporter constructs. MMP-1 protein synthesis and enzymatic activities were measured in UVB, UVA or SSR exposed dermal fibroblasts. Finally, the Ro extract was tested in a three dimensional skin model which combined more physiological conditions.

Material and methods

Water-soluble Rosmarinus officinalis extract

Plant material: R. officinalis biomass was purchased from Martin Bauer Pharmaceutical Laboratories S.A. (49670 Valanjou, France). This biomass is made of rosemary dry leaves that are said to be “oil free”.

Extract preparation: The leaves were ground using a grinder equipped with a one millimeter sieve. The powder was quickly poured in hot water (1/10 W/V) at 95 °C and stirred with a laboratory propeller for 15 minutes. Then the suspension was filtered as follows: 25 μm sieve then 1.2 μm filter (GFC filter) under vacuum then 1 μm filter (GFB filter) and the last filtration step was made with a 0.44/0.22 μm combined filter (Sartorius, France). Rosemary water extract can be stabilized by two techniques: freeze dried or concentrated under vacuum to obtain a syrup.

Cell and tissue cultures

Normal human epidermal keratinocytes and dermal fibroblasts were isolated from mammary skin obtained after breast plastic surgery after informed patient consent. Keratinocytes were cultured as described [35] on a feeder layer of mouse 3T3 fibroblasts. Fibroblasts were cultured in DMEM 10% calf serum and used at low passages (< 10). HeLa cells (American Tissue Culture Collection, Rockville, MD, USA) were cultured in DMEM 10% fetal calf serum.

Reconstructed skin in vitro (3D-skin model) [36, 37]: Dermal equivalents were prepared as previously described using a 7 ml collagen-fibroblast mixture containing 10⁶ human dermal fibroblasts. After contraction, human normal keratinocytes were seeded on this support. The culture was maintained for 7 days in immersed conditions and raised at the air-liquid interface for another 7 day period to obtain a complete differentiation process.

UV sources

Solar simulated radiation was obtained from a 1000 watt xenon lamp (Oriel corp., CT, USA) equipped with a UG 5/2 mm and a WG 320/1.5 mm Schott filters. The filtered xenon beam provided a simulated solar UV spectrum (290-400 nm). The spectral output delivered was 9% UV-B and 91% UV-A. UVA radiation was obtained using the same xenon lamp equipped with UG5/2 mm and a WG 335/3 mm filters. A Philips TL 20W/12 tube equipped with a Kodacel filter to eliminate radiation wavelengths below 290 nm was used as UV-B source. The wavelength spectra were carefully checked with a Macam SR3010 spectroradiometer (figure 1).

Irradiation protocol and Ro treatment

Just before exposure, the tissue culture medium of sub-confluent (80-90% confluence) dermal fibroblast cultures was replaced by phosphate-buffer (PBS). Following irradiation, PBS was replaced by culture medium without serum. During the post irradiation period, samples were maintained at 37 °C and 5% CO2. Culture medium was harvested at the indicated time after irradiation and frozen at –20 °C until use. The 3-D model was exposed to UV-SSR in the absence of culture medium as described [38]. After irradiation, fresh medium without serum was added. Samples and culture medium were taken 24 h after SSR exposure for histology, MMP-1 detection and cytokine evaluation.

Dermal fibroblasts and reconstructed skins were treated with Ro extract 24 hours before and after UV exposure.

Histology and sunburn cell detection

Reconstructed skin samples were fixed in 10% neutral formalin and embedded in paraffin. 5μm sections were stained with hematoxylin, eosin, sapphron (HES). Sunburn cells were detected by their typical morphology, i.e. an eosinophylic cytoplasm, a condensed picnotic nucleus and a suprabasal localization [38].

Analysis of MMP-1 gene transcription

PCL-CAT3 consists of a 3.8 kb fragment of the 5’-regulatory region of human MMP-1 gene controlling the chloramphenicol acetyl transferase (CAT) reporter gene [39]. PColS consists of a fragment of the 5’-regulatory region of MMP1 gene spanning from position -73 to +63 [40, 41]. Plasmids were stably transfected into HeLa cells using co-transfection with a neomycin-resistant construct (pOG45, Stratagene, France) and selection with geneticin (G418) (Gibco, France). Cells were plated at 16000 cells per cm². Four days later (40% confluency), medium was replaced by fresh medium supplemented with the Ro extract diluted in culture medium for 2 hours at 37 °C 5% CO2. Medium were harvested and cells were rinsed with PBS before being exposed to UV. Immediately after UV exposure, fresh medium supplemented with Ro extract was added and cells were kept at 37 °C, 5% CO2/95% air.

Cellular extracts were obtained 48h after transfection as previously described [42]. Protein content was measured using the Bio-Rad Protein Assay (Bio-Rad) and CAT levels were evaluated using the CAT-ELISA assay (Boehringer Mannheim) on 200 μl of cellular extracts. CAT values were adjusted per μg/mL of protein. These normalized CAT values were expressed in arbritary units (pgCAT/50 μg proteins). Modulations were expressed as fold induction above the basal level corresponding to control sample (no UV exposure, no Ro treatment).

Detection of soluble MMP-1 and TIMP-1 by enzyme-linked immunoassay

Supernatants of monolayered cultures or reconstructed skins were analyzed for the production of human MMP-1 using an ELISA system (Biotrak MMP-1 kit, Amersham Biosciences) according to the manufacturer’s instructions. Human TIMP-1 was measured in supernatants of fibroblasts cultures using ELISA immunoassay (R&D Systems, Quantikine Human TIMP-1).

Zymography analysis

Pre-cast zymogram gels from NOVEX (Invitrogen, France) were used to study metalloproteinases through the detection of proteolytic activity. Samples were analyzed without heating or reduction for SDS-PAGE containing blue casein or gelatine. After gel electrophoresis, gels were washed twice in zymogram renaturating buffer (Biorad, France). Then, gels were incubated for 72 hours at 37 °C in a zymogram developing buffer (Biorad, France). Enzymatic activities were revealed after protein staining with gel code blue (Pierce, France).

Cytokine analysis

Cytokine contents of culture supernatants were measured by ELISA (Biotrak kit RPN2784 for human interleukin 6, kit RPN2140 for human interleukin 1 alpha, Amersham Biotech, France) according to the manufacturer’s instructions.

Results

UVB-induced MMP-1 promoter activity is decreased by Ro extract

Using the MMP-1 promoter-reporter construct containing 3.8 kb of the human MMP-1 promoter sequence, stably transfected into HeLa cells, a UV dose-dependent up-regulation of transcription was observed after UVB exposure (figure 2A). Treatment of cells with Ro extract induced a decrease in UVB-induced up-regulation of promoter activity. The inhibitory effect was linked to the concentration of Ro extract (figure 2A). The concentrations used in the study were not cytotoxic (103.54 ± 2.93% viability at 1 mg (dry powder)/mL Ro, and 97.32 ± 2.24% viability at 2.5 mg/mL Ro).

Since it has been demonstrated that UVB-induced up-regulation of MMP-1 gene transcription is triggered by the AP1 binding site located –72 –65 [20], similar experiments were performed using a shorter construct containing only 73 bp of the 5’ region of human MMP-1 promoter. Similarly to what has been shown for the long construct, up-regulation of transcription was observed after UVB exposure. Ro extract also reduced this up-regulation dose-dependently (figure 2B).

MMP-1 protein produced by dermal fibroblasts is decreased by Ro extract

The level of basal and UV-induced MMP-1 protein was measured on culture supernatant of human primary dermal fibroblasts, using ELISA. Cells were exposed to either UVB, UVA or combined UVB/UVA using Solar Simulated Radiation (SSR). The level of released MMP-1 protein was increased after exposure to the three types of radiation (figure 3). Treatment of dermal fibroblasts with Ro extract decreased the basal as well as the UV-induced level of MMP-1 protein.

Ro extract inhibits MMP-1 activity

The casein gel was used to detect MMP-1 activity in fibroblast culture supernatants. In the absence of Ro extract, the intensity of the band corresponding to MMP-1 was increased by UVB or SSR exposure. In Ro extract treated samples, the intensity of the MMP-1 band was drastically reduced in both unexposed and UV-exposed culture supernatants (figure 4A), confirming ELISA results.

A gelatin zymographic analysis was performed to detect MMP-2 and MMP-9 activity (figure 4B). MMP-2 corresponding bands were intense and not modified after UV exposure. No effect could be noticed in Ro extract-treated samples for MMP-2. The bands corresponding to MMP-9 were thin and of low intensity in the control samples, and became just perceptible after Ro treatment.

Since the natural tissue inhibitor of MMP-1 activity is TIMP-1, its level was evaluated in fibroblast culture supernatants. UVB- or SSR-exposed samples did not reveal any modulation of the level of TIMP-1 protein, neither did treatment with Ro extract (figure 5).

Ro extract could prevent deleterious effects of SSR exposure in reconstructed skin model

Reconstructed skin was exposed to SSR dose (2.2 J/cm² total UV) leading to sunburn cell formation and morphological disorganisation of the tissue at 24 h [43]. Treatment with Ro extract led to an improvement of the morphology of the whole reconstructed skin, and a decrease in sunburn cell formation (figure 6). In the absence of UV exposure, the treatment of reconstructed skin with Ro extract did not alter tissue morphology.

Ro extract prevents UV-induced MMP-1 in reconstructed skin model

In the absence of UV exposure, the treatment with Ro extract slightly decreased the basal level of MMP-1 (figure 7). UV-SSR exposure of reconstructed skin led to increased levels of MMP-1 protein which could be partially counteracted by Ro treatment (by ~ 70% at 1.65 J/cm² and ~60% at 2.2 J/cm²) (figure 6).

RO extract decreased the release of UV-induced cytokines IL1α and IL6

IL1α and IL6 have been shown to be induced by UV exposure and to be involved in MMP-1 up-regulation through autocrine and paracrine pathways [16]. We confirmed that the levels of these two cytokines are increased by UV-SSR exposure in reconstructed skin and that treatment with the Ro extract allowed us to abolish this effect for IL1α, or to decrease it by 56%, for IL6 (figure 8).

Discussion

This study demonstrated that a water-soluble extract of Rosmarinus officinalis could inhibit UV-induced MMP-1. As reflected by the promoter-reporter transfection analysis, this inhibitory effect seemed to be mediated at transcriptional level. It has been previously shown that the AP1 is a key regulator element in the up-regulation of MMP-1 gene transcription by several types of UV radiation [14, 21, 40]. Results obtained with the shorter MMP-1 promoter construct revealed a partial involvement of this AP1 binding site in Ro inhibition of MMP-1 gene transcription. Inhibition at the molecular level was confirmed at the protein level using dermal fibroblasts exposed to UVB, UVA or SSR. UVB radiation primarily acts through induction of direct DNA lesions and modulation of gene expression. Among the UVB-regulated genes, members of fos and jun family are increased, such as c-jun and c-fos [44, 45]. These oncogenes which represent partners in the AP-1 complex are implicated in UVB-induced MMP-1. In UVA exposure conditions, the generation of reactive oxygen species (ROS) has been clearly demonstrated as the major impact of this wavelength range. ROS generation plays a role in MAP kinase mediated signal transduction, which is responsible for UVA-induced MMP-1 via the AP-1 pathway [21]. All together, these results support the assumption that inhibition of the AP1 pathway may participate in the Ro extract inhibitory activity of both UVB and UVA-induced MMP1 upregulation, but also suggest the implication of other responsive elements upstream. The physiological relevance of such a broad UV-spectrum effect is especially important when considering that sun exposure entails the impact on the skin of both UVB and UVA radiation, whose respective dose levels and ratios may vary to a large extent depending on latitude, season, time of the day, weather conditions, light reflection [46]. Our results give clear evidence that the Ro extract can prevent the induction of MMP-1 in a wide range of sun exposure conditions.

The use of several cellular systems with increasing physiological complexity showed that inhibition of MMP-1 transcription by the Ro extract resulted in a decrease in both protein level and enzyme activity. In addition, zymographic studies revealed that the effect of the Ro extract on MMP-1 was considerably higher compared to other major MMPs secreted by cutaneous cells, such as MMP-2 [13]. In addition, the analysis of the natural tissue inhibitor of MMP-1, TIMP-1, did not show any effect of Ro extract. MMP-1 is the key MMP enzyme in the early process of degradation of extracellular matrix during the photoaging process. MMP-1, which belongs to the collagenase subclass, is crucial in physiological or pathological processes since it has the unique property of initiating the cleavage of native fibers of collagens type I and III, which represent more than 90% of the extracellular matrix of the skin. By this process, MMP-1 is involved in the primary events occurring during ECM remodelling and ECM breakdown [18, 23]. Other cutaneous MMPs, such as gelatinases (MMP-2 and MMP-9), only act in a second step by degrading the pre-degraded fibrillar collagen. During the photoaging process, collagen deficiency is the result of extensive and repetitive degradation through UV-induced MMP-1, leading to long term damage according to the theory of the “solar scar formation” [9]. Our results demonstrated that Ro extract treatment could interfere and reduce this process, thus limiting the development of photodamage and the subsequent clinically visible signs.

The use of a 3-D skin model allowed us to demonstrate that the inhibitory effect of the Ro extract observed at the molecular and cellular levels could also be found at the tissue level. A photo-protective effect was observed both on the morphological structure and UV-induced MMP-1. Since we previously showed that, in this model, UV-induced MMP-1 was the combination of direct effects on fibroblasts but also of paracrine effects through the release of epidermal cytokines, [16], the effects of the Ro extract on both MMP-1 and cytokine release were investigated. Indeed, the Ro extract was able to reduce MMP-1 production but also 1Lα and IL6 induction by UV.

Altogether our results demonstrate that the water-soluble Ro extract tested is a powerful anti-MMP-1 agent. Such a biological activity supports this extract as a promising agent for the prevention of skin photodamage.

Acknowledgement

We would like to thank F. Christiaens for expertise and help in monitoring the UV sources. Financial support: the authors are employees of L’Oréal. Conflict of interest: none.

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