Restoration of stratum corneum with nacre lipids

Restoration of stratum corneum with nacre lipidsMarthe Rousseau a,⁎,Laurent Bédouet a ,Elian Lati b ,Philippe Gasser b ,Karine Le Ny a ,Evelyne Lopez aaMuséum National d'Histoire Naturelle,Département des Milieux et Peuplements Aquatiques,UMR 5178,CNRS-MNHN «Biologie des Organismes Marins et Ecosystèmes»,7rue Cuvier,F-75231,Paris Cedex 05,FrancebLaboratoire BIO-EC,10Avenue Réaumur,F-92140,Clamart,FranceReceived 20October 2005;received in revised form 30March 2006;accepted 30March 2006Available online 30June 2006AbstractTo discover potential new products for the atopic dermatitis treatment,lipids extracted from nacre from the oyster Pinctada margaritifera were tested on artificially dehydrated skin explants.Expression of filaggrin and transglutaminase 1was investigated after treatment of dehydrated skin with P .margaritifera lipid extracts according to light microscopy after labelling with specific monoclonal antibodies.The lipids were extracted from the nacre with methanol/chloroform mixture at room temperature and the extract composition was determined according to TLC and densitometry measures.Relative to the dry nacre material,a yield of extraction in lipids of 0.54%(w/w)was determined.Fatty acids,triglycerides,cholesterol and ceramides were in low abundance.Then,application of lipid formulations on skin explants previously dehydrated gave after 3h an overexpression of filaggrin and a decrease of transglutaminase expression as shown by light ing immunofluorescence labelling,we showed that lipids extracted from the mother of pearl of P .margaritifera induced a reconstitution of the intercellular cement of the stratum corneum.The signaling properties of the nacre lipids could be used for a development of new active product treatment against the symptoms of the dermatitis.©2006Elsevier Inc.All rights reserved.Keywords:Filaggrin;Lipids;Nacre;Pinctada margaritifera ;Transglutaminase1.IntroductionOne of the most important functions of the skin is to prevent the entry of exogenous substances and the loss of water.Stratum corneum,the major skin barrier,consists of flattened dead cells,embedded in a complex lipid matrix.Stratum corneum lipids are composed of ceramides (50%),cholesterol (25%)and free fatty acids (10%),as well as small amounts of cholesterol esters and cholesterol sulfate (Wertz and van den Bergh,1998).Atopic dermatitis is a common chronic inflammatory skin diseases with a prevalence of 10–20%in children,and a pre-valence of 1–3%in adults (Schultz-Larsen and Hanifin,2002).As a consequence of alterations in barrier function,allergens and irritants can penetrate the skin more easily enhancing theallergic inflammation.Moreover,allergic inflammation would further degrade barrier function to close the vicious circle of atopic dermatitis.Hence,improving the defective barrier may relieve the symptoms,prevent aggravation of the disease and,possibly,decrease the amount of the corticoid needed.Sup-plementation of the deficient complex lipid mixtures is a logical therapeutic approach with minimum side effects.Such barrier recovery was described in several studies,for review see Porksch et al.(2003)and Lodén (2003).Psoriasis and other skin diseases such as ichthyoses and atopic dermatitis are characterized by an epidermic proliferation with an uncompleted and accelerated differentiation accompa-nied with an inflammation of dermis and epidermis.Actually,it is supposed that these diseases are caused or developed by the activation of specific T-cells,autoimmune reactions and an overexpression of factors which could induce a proliferation of keratinocytes (Schon,1999;Prinz,2001).The active phase of the disease is characterized by a lymphocyte proliferation trig-gered by interferon gamma and activation of keratinocytes producing inflammatory cytokines (IL-1,TNF-αand IL-8),andComparative Biochemistry and Physiology,Part B 145(2006)1–9/locate/cbpbAbbreviations:BSA,Bovine Serum Albumin;FITC,Fluorescein Isothio-cyanate;NMF,Natural Moisturing Factor;PBS,Phosphate Buffered Saline;SC,stratum corneum;TGase,Transglutaminase;TLC,Thin Layer Chromatography.⁎Corresponding author.E-mail address:rousseam@ (M.Rousseau).1096-4959/$-see front matter ©2006Elsevier Inc.All rights reserved.doi:10.1016/j.cbpb.2006.06.012the recruitment of endothelial cells,mainly cells of capillary venous network leading to a vasodilatation and an interaction with white blood cells.The cell divisions speed up and an abnormal keratinisation process occurs which leads to an over-proliferation of the epidermic layers and a thickening of the cornified envelope,the stratum corneum(SC),which becomes dry and squamous(Guadially,2002).Filaggrin is largely responsible for the ability of SC of the skin to remain hydrated at low environmental humidity.Filag-grin is a protein located within the more superficial layers of the epidermis and originates from profilaggrin,its precursor is rich in histidin which composes the mayor component of the kera-tohyalin granules of the stratum granulosum(Rawlings et al., 2000).During the final keratinocytes differentiation step,pro-filaggrin is dephosphorylated and processed in intermediate compounds and finally in filaggrin.Filaggrin catalyses the aggregation of the keratin filaments upon formation of disulfide bridges(Rawlings et al.,2002).Filaggrin is programmed to be rapidly degraded and several of its amino acids are metabolized in order to produce the components of the Natural Moisturing Factor(NMF),especially glutamine which is transformed in hydrolidone carboxylic acid and histidin into urocanic acid.The role of these components depends on their hygroscopic power which allows the upper layers of the SC to retain water and to withstand aggression and dehydration.The NMF is localized exclusively in the corneocytes and represents10%of the cel-lular dry weight.Keratin of the SC acquires elastic properties upon interaction with amino acids of the NMF.The conversion of filaggrin into NMF occurs only in a limited variation of the water activity between0.7and0.95when dehydration occurs. Thus a low level in NMF is characteristic of important skin disorders,like the atopic dermatitis(Di Nardo and Wertz,2002).Furthermore,among the6known active transglutaminases in human,3are expressed during the terminal differentiation of keratinocytes.TGase1is a92kDa membrane bound protein, whereas TGase2is an ubiquitous soluble tissue enzyme of 80kDa and the soluble pro-enzyme TGase3of77kDa.Most of TGase activity in the epidermis cells comes from TGase1which is processed in multiple soluble forms(Kim et al.,1995).The TGase catalyzes the isopeptides cross-link between peptide-bound glutamine and lysine in vivo and participates in the for-mation of the cornified envelope.Cross-linking of keratinocytes involucrin to membrane proteins via TGase forms a protective barrier as an insoluble envelope beneath the plasma membrane.To investigate the activity of new products on atopic derma-titis,the lipids of the micronised nacre(mother of pearl)powder of the oyster Pinctada margaritifera were extracted with a metha-nol/chloroform mixture.Next,the biological activity of the total lipid fraction was tested on human skin explants previously de-lipidated and the expression of filaggrin and TGase1after label-ling with specific monoclonal antibodies,was observed.Several studies about bivalve lipids have been conducted on the soft parts(Delaporte et al.,2005;Kraffe et al.,2002,2004, 2005;Soudant et al.,1995)to understand nutritional needs. Indeed bivalves have a very limited or no capability to synthe-tise essential polyunsaturated fatty acids(De Moreno et al., 1976,1977;Waldock and Holland,1984;Chu and Greaves,1991)and require them for their development(Langdon and Waldock,1981;Chu and Webb,1984;Knauer and Southgate, 1997).The lipid and fatty acid composition of the total lipids of the soft parts of the pearl oyster Pinctada fucata martensii,in different seasons and in different areas,were analysed to clarify its lipid physiology and to estimate the possible influence of its prey phytoplankton(Saito,2004).However,in most bivalve lipid studies,only the inner organ lipid composition was dis-cussed.This is the first study on lipids extracted from the nacreous layer of P.margaritifera and on their activity on skin barrier repair based on analysis of the synthesis of fillagrin and TGase expression.2.Materials and methods2.1.Nacre materialThe nacre of the inner shell layer of the giant oyster P. margaritifera from French Polynesia was ground to a fine powder at the“Centre de Transfert de Technologie Céramique”(Limoges,France)in order to obtain particles of50–100μm.2.2.AnimalsSoft tissues were obtained in October2003from pearl oy-sters,P.margaritifera,maintained at a depth of7–12m in French Polynesia.Biological samples were obtained at the breeding site and transported to the laboratory in frozen con-tainers,where they were lyophilized.2.3.Lipid extractionTotal lipids were extracted,according to the method of Folch et al.(1957),modified by Ways and Hanahan(1964),dissolved into chloroform/methanol(2:1)at a concentration of40mg/mL for mollusk body and500mg/mL for nacre powder.2.4.Lipids analysisAnalyses were performed on silica plates with hexane/die-thylether/acetic acid(60,25,15,v/v/v)as mobile phase. Table1Composition of lipids extracted from the nacre and the dehydrated soft tissues of the bivalve P.margaritiferaLipid composition Nacre lipids(%)Lipids in soft tissues(%)Rf Cholesterol sulfate0.50.20.07 Hydroxylated ceramides 2.070.230.21 Nonhydroxylatedceramides1.03 1.590.25Cholesterol 5.9728.090.34 Fatty acids 6.2449.090.51 Triglycerides 4.46 3.180.66 Cholesterol acetate0.70.480.78 Squalene-like lipids79.0317.131 After extraction and solvent evaporation,extracts were analysed by TLC in hexane/diethylether/acetic acid stained with copper sulfate.Quantification was performed after densitometry analysis.2M.Rousseau et al./Comparative Biochemistry and Physiology,Part B145(2006)1–9Samples of 5,10,15and 20μl of the lipid extracts at 20mg/ml in chloroform/methanol (2/1)were loaded and the TLC was developed according to successive ascensions.After drying,lipids were stained with copper sulfate reagent (10%(w/v)copper sulfate,23%ethanol,8%phosphoric acid)and heated in an oven at 105°C for 2h.The amount of each compound was determined using densitometry measures performed with a Chromimage scanner.A standard lipid mixture was run in parallel for the qualitative determination.2.5.Nuclear magnetic resonance analysis1H experiments were conducted on a Bruker Avance 300spectrometer operating at a frequency of 300MHz.Dry samples (5mg)were dissolved in CDCl 3at 10mg/mL concentration.The proton chemical shifts were determined from the 1H NMR experiments using the conventional pulse sequences provided by Bruker.2.6.Tested productsTwo formulations,P1and P2containing respectively 0.5and 1%of total lipids extracted from the nacre and their excipient (demineralized water,glycerin,stearic acid,glyceryl stearate).2.7.Explant preparation and dehydration of skinAn ethylether/acetone mixture (1:1)was applied on a defined zone of a cutaneous plasty for 2min.In this delipidated zone,15explants (10mm diameter)were removed whereas 6others were cut off from a non-delipidated zone.Five groups of ex-plants were determined:delipidated skin (D,n =6),delipidated skin +excipient (DE,n =3),delipidated skin +0.5%nacre lipid (DP1,n =3),delipidated skin +1%nacre lipid (DP2,n =3).The 6control explants were divided into 2lots,normal skin at To (No)and at T +3h (N3).The tested products were applied on explants as soon as their preparation in topic (4mg/explant)forFig.1.TLC analysis of lipids extracted from the nacre of P .margaritifera with hexane/diethylether/acetic acid (60,25,15,v/v/v)as a mobile phase.Samples of 5,10and 20μl of the lipid extracts at 20mg/ml and a standard lipid mixture loaded and stained with copper sulfatereagent.Fig.2.1H NMR analysis of lipids extracted from the nacre of P .margaritifera on a Bruker Avance 300spectrometer.3M.Rousseau et al./Comparative Biochemistry and Physiology,Part B 145(2006)1–93h on the batches DE,DP1and DP2.Batches No.N3and D didnot receive any treatment.Treated and untreated skin explantswere incubated at37°C in a CO2enriched atmosphere.2.8.Histological studiesAt To,immediately after the delipidation,explants frombatches No and Do were removed and a half point was fixed inordinary Bouin solution and the other half was frozen at −80°C.Three hours after the beginning of the treatment,3 explants of each batch(N3,D3,DE,DP1and DP2)wereremoved and treated as described above.Traditional histolog-ical analysis of the general morphology was performed,on5μm sections,after staining according to the Masson's Trich-rom technique.2.9.Immunofluorescence labelling of SC filaggrin and TGase1Filaggrin of the SC was labelled with specific monoclonal antibodies and revealed in immunofluorescence with a biotin/ streptavidin amplification system and the nuclei were counter-stained with propidium iodide.Explants were dried for2h at room temperature before being fixed in acetone at−20°C for 10min.They were air dried and washed in PBS(5min)and incubated in3%goat serum for30min.Excess of serum was wiped off,and explants were incubated for1h with antifil-lagrin monoclonal antibody(BT576,clone OKTB1,Clini-science,France)diluted1/4.000in PBS-BSA0.2%.After3 washes in PBS(15min each),explants were incubated with a biotin labelled goat anti-mouse IgG(final dilution1/400in PBS-BSA0.2%)during1h at room temperature.Following Fig.3.Histological observation of the skin explants morphology.Control at To(A),in dehydrated skin at To(B),in dehydrated skin at T+3h(C),in dehydrated skin at T+3h treated with excipient(D)and in dehydrated skin treated with0.5%(E)and1%(F)of nacre lipids at T+3h.Staining was performed according to the Masson's Trichrom.4M.Rousseau et al./Comparative Biochemistry and Physiology,Part B145(2006)1–9another3washings with PBS(15min),streptavidin–FITC conjugate diluted to1/200in PBS-BSA0.2%was added for 1h.After3washings in PBS(15min),the nucleus were stained with propidium iodide for1min before washing in PBS.The membrane TGase1was labelled with a mouse IgG2a monoclonal antibody(Clone B.C1-Harbor Bio-products, TEBU).Briefly,explants were dried overnight at room tem-perature before being fixed in acetone at−20°C for10min. They were air dried and washed in PBS(5min)and further incubated in3%goat serum containing1%BSA for30min. After wiping off,explants were incubated overnight at4°C with anti-TGase diluted to1/640in PBS-BSA(1%)and after three washes in PBS(15min each),a biotin labelled goat anti-mouse IgG(1/1.250in PBS)was applied.After1h incubation at room temperature,explants were washed in PBS(3×15min),before addition of streptavidin–FITC conjugate diluted1/1.250in PBS for1h at room temperature.After3washings in PBS(15min), nuclei were stained with propidium iodide for1min before washing in PBS.The observation of the labellings was made on a Leica DMLB microscope by using a Leica Filter13with an excitation wavelength of450–490nm.3.Results3.1.Lipid composition of P.margaritifera extractsThe content of lipids in the micronised nacre sample was around0.54%in weight and appeared to be composed ofa Fig.4.Induction of filaggrin expression in the SC shown by immunolabelling.Expression of filaggrin in the control skin at To(A),in dehydrated skin at To(B),in dehydrated skin at T+3h(C),in dehydrated skin at T+3h treated with excipient(D)and in dehydrated skin treated with0.5%(E)and1%(F)of nacre lipids at T+3h. Filaggrin was green labelled with a biotinylated specific monoclonal antibody and visualized after incubation with streptavidin–FITC(white arrow).Observation was performed atλex450–490nm.Cells nuclei were stained with propidium iodide.5 M.Rousseau et al./Comparative Biochemistry and Physiology,Part B145(2006)1–9mixture of different classes of lipids as evidenced after the TLC analysis.The lipid composition of nacre and soft tissue was done only on one pool of animals.The relative proportions of class of lipid were determined,with a low content in cholesterol (6%of the total lipids),in fatty acids (6%)and in triglycerides (4%).Traces of cholesterol derivatives were detected (1%).The most important lipid (79%)was represented by an apolar ma-terial which was identified as consisting of squalene-like mo-lecules and unknown compounds by comparison with the lipid standards (Table 1,Fig.1).On the other hand,lipids contained in the soft tissues of P .margaritifera were also pared to the mother of pearl,the yield of extraction was high,since lipids represented 18%of weight freeze-dried tissue.Cholesterol (28%)and fatty acids (49%)were the main lipids of the mollusk body,whereas the squalene-like lipids and unknown compounds were less abundant,they represented only 17%of the total lipid -pared to the soft parts of P .margaritifera ,nacre was particularly enriched in the squalene-like and apolar unknown compounds.The lipidic nature of our nacre extract is confirmed by 1H NMR spectroscopy (Fig.2).The 1H NMR spectrum shows a large signal between 0.7and 1.5ppm synonymous of the presence of high levels of CH 3and CH 2groups characteristic of squalene lipids and fattyacids.Fig.5.Inhibition of TGase 1expression in the SC shown by immunolabelling.Expression of TGase in control skin at To (A),in dehydrated skin at To (B),in dehydrated skin at T +3h (C),in dehydrated skin at T +3h treated with excipient (D)and in dehydrated skin treated with 0.5%(E)and 1%(F)of nacre lipids at T +3h.TGase was labeled with a biotinylated specific monoclonal antibody.Observation of bound streptavidin –FITC was performed at λex 450–490nm.The immunostaining with anti-TGase 1gave faint green lines (white arrow)in the deeper region of SC.Nuclei all cells were stained with propidium iodide.6M.Rousseau et al./Comparative Biochemistry and Physiology,Part B 145(2006)1–9For the further biological tests on the skin explants,the lipids extracted from the nacre were used,since previous studies had shown that nacre powder implanted between hypodermis and dermis in the rat stimulated the activity of cutaneous fibroblasts (Lopez et al.,2000).3.2.General morphology of the SCTo simulate decreased lipid content in the diseased skin,lipid extraction by organic solvents was used.Immediately after de-hydration of the cultured skin explants,samples treated with either nacre lipid preparations either the excipient or control explants were analysed under light microscope(Fig.3).Micro-scopic examinations of control skin before delipidation(Fig.3A) indicated that the SC was compact and keratinised both in surface and in the basal cellular layers.On the dehydrated skin, SC was more compact and keratinised(Fig.3B),indicating a structure characteristic of dry skin.At T+3h with the delipi-dated skin(Fig.3C),the SC was slightly lamellar and remained compact.Furthermore,on the skin explants treated with P1and P2formulations,the SC was less compact and less keratinised (Fig.3E and F).As the control,the skin treated with excipient for 3h after delipidation showed a less compact and a lamellar appearance,more pronounced than the structure observed after treatment with lipid formulations P1and P2(Fig.3D).3.3.Stimulation of filaggrin synthesis in the presence of the nacre lipid fractionThe expression of filaggrin in the skin explants was in-vestigated according to monoclonal antibody labelling moni-tored with fluorescent microscopy.At To,with normal and delipidated skin,filaggrin was overexpressed and was found in 6to7layers all over the SC(Fig.4A and B).At T+3h,the expression of filaggrin in the control explant was identical to the expression at To(Data not shown).On artificially delipidated skin explants,expression of filaggrin was reduced,the labelling was less regular and filaggrin was found in only several layers (Fig.4C).When the excipient was applied alone on the skin (Fig.4D),expression of filaggrin was slightly higher compared to the control delipided skin explants.On the contrary,addition of the nacre lipid formulations induced an important expression of filaggrin which was found in6to7cellular layers(Fig.4E and F).Compared to the dehydrated skin and the excipient control,the intensity of labelling after the lipid treatment was higher and more homogeneous.The immunolabelling of filag-grin in the cornified layer indicated that the nacre lipids induced an overexpression of filaggrin on the dehydrated skin,higher than the level expressed in the control explants.3.4.Repression of TGase expressionThe expression of TGase in the SC of the skin explants was analysed after labelling with a specific monoclonal antibody.At To,with normal and delipidated skin(Fig.5A and B),a faint expression of TGase was observed in only2 and3cellular layers in the deeper region of the SC.The labelling was regular and higher than the autofluorescence of the SC.Three hours after dehydration,expression of TGase had diminished(Fig.5C),and a discontinuous labelling was observed.The low expression of TGase observed at To is characteristic of a dry skin situation which was amplified after the treatment with the ether/acetone mixture.Upon addition of the excipient(Fig.5D),the expression of TGase was favourable compared to the dehydrated skin control.A different labelling was observed after incubation of the de-hydrated skin explants with the2lipid formulations asso-ciated with the excipient.Three hours after the addition of the formulation P1(0.5%of lipid)on the dehydrated skin, an important reduction of TGase labelling was observed (Fig.5E)compare to treatment with excipient,and no label-ling was obtained when the formulation P2(1%of lipids) was applied(Fig.5F).Only autofluorescence of the surroun-ding cells was visible.It seemed that this immunolabelling, indicated that the expression of TGase1in the dehydrated SC was inhibited3h after the addition of the nacre's P. margaritifera lipids in a dose dependent manner.4.DiscussionFor the last decade,our group has investigated the mor-phogenetic properties of the nacre layer of the shell of the giant oysters P.maxima and P.margaritifera.In vivo studies in vertebrates indicated formation of new bone around the nacre implants(Lamghari et al.,2001;Berland et al.,2005).On the other hand,in vitro studies performed on human fibroblasts MRC-5(Almeida et al.,2000),rat pre-osteoblasts MC3T3-E1 and rat stromal cells indicated that water soluble protein induced a differentiation towards the osteoblast phenotype with an in-hibition of cell proliferation,an increase of alkaline phosphatase expression(Pereira et al.,2001).With preosteoblast cells,nacre proteins induced an overexpression of specific bone collagen (collagen I)and osteocalcin together with an acceleration of the mineralization process(Rousseau et al.,2003).The biochemical studies indicated that proteins are implicated in these phenom-ena,and an active low-molecular weight fraction was recently isolated(Almeida et al.,2000).However,presence of lipids in the nacre layer was until now unexpected.Nacre organic matrix is mainly build up with silk-like proteins rich in alanine and glycine,acidic glycoproteins andβ-chitin which represent approximately1.7%of the nacre weight(Weiner and Traub,1984).Relative to other substances found in the nacre of P.margaritifera,the lipids were abundant around0.5%.In comparison,macromolecules extracted upon acidic demineralization represent0.4%of the nacre weight. Lipids appeared to be a main component of the organic material included in calcium carbonate In the particular case of P.mar-garitifera,we suppose that exportation of lipids from the soft tissues to the nacre may be used to protect the organism from the intense light occurring in the Pacific lagoons(Kohno et al., 1995;Kelly,1999).Another putative function of lipids in nacre could be associated to the shell biomineralization process.Hy-potheses concerning the roles of lipids in calcification were little reported.For example,ethanol pretreatment of glutaraldehyde7M.Rousseau et al./Comparative Biochemistry and Physiology,Part B145(2006)1–9cross-linked porcine aortic valve bioprotheses removed choles-terol and phospholipids,preventing further calcification after implantation(Vyavahare et al.,1997).In vitro,it was shown that the direct deposition of calcium phosphate was accelerated on titanium surface coated with phospholipids in the calcium–phosphate complex compared to the uncoated surface(Satsangi et al.,2003).Such modified surfaces also had a positive effect on osteoblast differentiation and growth.Lipids from the soft parts of P.margaritifera are very similar to those of P.fucata(Saito,2004).A lipid content of 18%was close to values determined for other mollusks bodies like the New Zealand mussel Perna canaliculus(17.9%)and the Tasmanian mussel Mytilus edulis(12.3%)(Murphy et al., 2002).However their lipid compositions were different with a higher concentration in triglycerides(20%)and a low concen-tration in cholesterol(6%).Differences between these organ-isms could be linked to various factors,like the dietary intake and the length of storage where various enzymes are active and could modify the lipid composition(Murphy et al.,2002).In our nacre and tissue extract we did not find any phospholipids. The polar lipids are essential membrane constituents and were described(Saito,2004;Delaporte et al.,2005;Kraffe et al., 2002,2004,2005).Considering the high level of free fatty acids found(49%),this led to suggest of a lipid degradation may be due to the lyophilisation process used for storage or the extraction process used.If the physiological and structural functions of the lipids in the nacre remain elusive,their biological activity on delipi-dated human skin explants is more accurate.Two lipid pre-parations containing respectively0.5and1%of nacre lipids in weight were prepared with an excipient.According to the immunolabelling of filaggrin in skin explants,we showed that after3h of contact,the2lipid formulations induced on the delipidated skin an expression of filaggrin higher to the level found in normal skin.On the other hand,the lipid formula-tions reduced the expression of TGase1in the cornified envelope in a dose dependant manner.Taken together,a better expression of filaggrin and a reduction of TGase expression could be appropriate for the development of nacre lipid-based drugs for the treatment of atopic dermatitis.Indeed the nacre lipids could induce an expression of filaggrin and in parallel could inhibit the expression of the membrane TGase1,the enzyme implicated in the intermolecular protein cross-linking in the corneocytes which is overexpressed in skin diseases. Furthermore,inhibition of TGase expression induced by the nacre lipid fraction may be an alternative to the development of TGase inhibitors which decreased the formation of corni-fied envelope(Kim et al.,1997).It seemed that the lipids extracted from the nacre had brought to the SC the elements necessary to a rapid reconstruction of the intercellular cement,by acting on the expression levels of transglutaminase and filaggrin.The protein components of SC are under the control of several factors which can activate or repress their expression.It was shown in vitro that retinoic acid downregulates the expression at m RNA level of profilaggrin, loricin and TGase1,and represses the proteolytic conversion of profilaggrin into filaggrin(Asselineau et al.,1990).On the other hand,glucocorticoids activate the synthesis of these corneocyte envelope markers(Presland et al.,2001;Komuves et al.,1998).The data obtained in this study on keratinocytes using spe-cific lipids extracted from the nacre of the oyster P.mar-garitifera reinforce the interest represented by this product as a tool for a protection of facial skin from aging,environmental damages and diseases.Thus,action of the nacre from Pinctada maxima on skin fibroblasts was previously observed in the rat (Lopez et al.,2000).Nacre powder implanted in a pocket created between the dermis and the hypodermis for2weeks stimulated the activity of cutaneous fibroblasts.Histological studies showed the formation of an angiogenic network con-verging towards nacre pieces,a recruitment and an activation of fibroblast cells organized around the nacre fragment.These cellular events were accompanied with an intense synthesis of several elements of the extracellular matrix:the decorin proteo-glycan,collagens of type I and III.Such synthesis did not occur in the controls performed with the control CaCO3.It was hy-pothesized that diffusible factors of the organic matrix activated the synthesis of the main proteins of the extracellular matrix that supports the dermis.Otherwise,lipid material of skin could modulate the ex-pression of SC markers,thus expression of TGase m RNA in cultured human keratinocytes was increased after treatment with10μM of sphingosylphosphorylcholine,a lipid epidermis metabolite generated by hydrolysis of sphingomyelin with sphingomyelinase at unusually high levels in patients suffering with atopic dermatitis(Higushi et al.,2001).Sphingosylpho-sphorylcholine is also a powerful mitogen that stimulates the DNA synthesis and cellular proliferation of cultured fibroblasts whereas it inhibits the proliferation of human keratinocytes.Nacre lipids are effective in skin barrier repair after lipid extraction.We suggest that it could supplement the deficient stratum corneum lipids.Moreover it could be beneficial in atopic dermatitis patients,preventing the penetration of aller-gens and irritants and subsequent inflammation.Actually,among the components of the nacre lipid extract, the molecule responsible for the activation of filaggrin and the repression of TGase was not identified as well as the molecular mechanisms.Further fractionations of the whole extract are needed to isolate the active lipid molecule(s).However,nacre of the Pinctada genus represents an attractive source of bioactive molecules(proteins,lipids)having biological activities either on cells of the bone lineage or on skin fibroblasts,and here on the highly differentiated cells of the epidermis,the keratinocytes. AcknowledgementsWe thank Rory Arrowsmith(European Programme Leo-nardo da Vinci,University Keele,UK/MNHN,Paris,France) for English corrections.Thanks are also due to Francine Lallier for her assistance in preparing this manuscript.ReferencesAlmeida,M.J.,Milet,C.,Peduzzi,J.,Pereira,L.,Haigle,J.,Barthélemy,M., Lopez,E.,2000.Effect of WSM fraction extracted from the nacre of8M.Rousseau et al./Comparative Biochemistry and Physiology,Part B145(2006)1–9。