The concept of sphingolipid rheostat in skin : a driving force for new active ingredients in cosmetic applications

This is anOp Abstract – Skin is a representative model of the complex metabolism that lipids may trigger. It is known that the biosynthesis of these lipids in mammalian cells generally ensures the cell membranes stability and participates to the signaling function. In the inner layers of the skin, the “de-novo” synthesis is the driving force ensuring proliferation, development and intercellular signaling. To promote stratum corneum formation, lipid catabolism leads to the renewal of ceramides, fatty acids and cholesterol that are responsible for the cohesion of the stratum corneum, its permeability, hydration, moisturization and signalling with the outer skin layers, appendages and inner layers secretion (cytokines, neuropeptides). Some actives applied in local treatments (i.e., peptides, n-3 polyunsaturated fatty acids (PUFA), ceramides, urea or an aqueous extract of Gromwell) and in oral treatment (i.e., sphingomyelin, n-3 polyunsaturated fatty acids (PUFA)) promote sphingosine 1-phosphate (S1P) production by the sphingolipid rheostat via triggering the salvage process along with autophagy and detoxification in aged skin. This review gives some basis for using the concept of sphingolipid metabolism rheostat in skin as the driving force for the development of new cosmetic actives ingredients or for repositioning the benefits of other actives for the skin.


Introduction
The term "sphingolipids" was first used by Thudichum in 1884 to describe compounds found in the brain such as phosphosphingolipids, neutral and acidic glycosphingolipids, and sphingomyelin (Thudichum, 1884).Later, a specific accumulation of sphingolipids was reported in several genetic diseases such as Niemann-Pick's disease (sphingomyelin), Gaucher's disease (cerebrosides), characterized by a defective catabolism.
From a functional perspective, they have a biological significance as they are an essential constituent of membranes

Pathways of lipid biosynthesis in mammalian cells
As shown in Figure 1, the de-novo biosynthesis of the long-chain backbone sphingoid base results from the condensation of palmitoyl-CoA and L-serine.The next step is the reduction of the formed 3-keto-sphinganine into sphinganine which is converted into a dihydroceramide (in the endoplasmic reticulum) by a ceramide synthase (Tidhar and Futerman, 2013).Subsequently, dihydroceramide is reduced to ceramide.
These ceramides are transported by ceramide transporter proteins (CERT) (Hanada et al.,, 2003) in the Golgi apparatus where ceramide can have multiple fates.It can be converted into sphingomyelin (SM) by the sphingomyelin synthase (SMS) in the lumen of the Golgi apparatus, into glucosylceramide (GLC) (and to more complex glycosphingolipids, GSL) on the cytosolic surface by the glucosylceramide synthase (GCS), and it can be phosphorylated by a ceramide kinase (CK) to form ceramide-1-phosphate (Futerman and Pagano, 1991).
The complex sphingolipids and phospholipids are transported to the plasma membrane where, in addition to their structural role, they are involved in cell membrane signaling as growth factors, as co-receptors, in cell motility and invasion, and in membrane autophagy recycling.
Endocytosis of complex sphingolipids by the catabolic pathway involves endocytic pathways, hydrolysis and degradation into ceramides and sphingosine that are known as growth inhibitory and pro-apoptotic factors (Merrill et al., 1985).In addition to the de-novo synthesis and the recycling pathways (Fig. 1), the salvage and degradation pathways modulate cellular levels of sphingolipids.By these pathways, ceramide regeneration is obtained from complex sphingolipid reservoirs, such as glycosphingolipids (GSL) and sphingomyelins (SM), through the action of specific hydrolases and phosphodiesterases (Young et al., 2012).

The sphingolipid rheostat defines the cell state
The sphingolipid rheostat (Kolesnick et al., 2000;Spiegel and Milstein, 2000) defines the regulation of sphingolipid cellular metabolism in which ceramide displays a pro- apoptotic role whereas sphingosine 1-phosphate is associated with a mitogenic and anti-apoptotic role.Modulation and regulation of this rheostat thus determines the cell survival versus cell death pathways.
As we will observe in this review, the sphingoid-based rheostat activates or inhibits a remarkable number of cellular events such as growth, division, apoptosis, autophagy, cytoskeleton and junctions formation, or extracellular metabolic events such as intercellular junction formation, extracellular matrix formation, paracrine and autocrine signalling.
Based on the concept of sphingolipid rheostat, several roles have been reported for ceramides such as induction of apoptosis (Fig. 2).Recently, it was found that the expression of CerS1 generating C18-ceramide resulted in the ceramide localization to MAM (Mitochondria-Associated Membranes) (Ardail et al., 2001) that could be involved in mitophagy induction (Babiychuk et al., 2011;Kogot-Levin and Saada, 2014).
It was reported that a specific lipogenic capacity is variable for the cells from different tissues and organs in the body.For example, liver is the major site of biosynthesis of total lipids and fatty acids (up to 37% of the lipid biosynthesis in the whole body), followed by the hypodermis with 24% and the epidermis with 8%, small intestine with 1-2% while the musculature and skeleton contribute for 25% to the lipid synthesis of the body (Gandemer et al., 1983).

Sphingolipid metabolism and skin homeostasis
Skin has a very complex structure and contains four main layers: the innermost subcutaneous fat layer (hypodermis), the overlying dermis, the viable epidermis and the outermost layer of the tissue is a non-viable epidermal layer, the stratum corneum.
The epidermis is itself a complex multiply layered membrane, yet varies in thickness from around 0.06 mm on the eyelids to around 0.8 mm on the load-bearing palms and soles of the feet.
In skin, as in other mammalian cells, sphingolipids are ubiquitous components of cell membranes and are involved in several major biological activities, such as growth regulation (Spiegel and Merrill, 1996), differentiation and apoptosis (Hannun and Obeid, 1997).The modulation of these functions by sphingolipids seems to depend mainly on the constitutive sphingoid bases (Ohta et al., 1994).Sphingolipids regulate receptors for platelet-derived growth factor (PDGF) and epidermal growth factor (EGF).They are also involved in the co-evolution of transmembrane domains (TMD) and the formation of specialized glycosphingolipid-enriched microdomains (Ernst et al., 2014).
During proliferation, differentiation and aging of keratinocytes, sphingolipid metabolism is continuously changing (Ponec, 1991).Whereas the most undifferentiated layers of the epidermis (basal, spinosum and granulosum layers) contain mostly membrane lipids such as phospholipids, the differentiated keratinocytes contain ceramides, cholesterol and fatty acids predominantly.The lamellar bodies, appear in the granulosum layer, contain mostly glucosylceramides that are then hydrolyzed by a glucocerebrosidase into ceramides, the major components of the intercellular matrix of corneocytes layers (Hamanaka et al., 1993).The barrier function of the stratum corneum depends on the high proportion of proteinbound omega hydroxy ceramides along with free ceramides and other lipids (wax esters, cholesterol, fatty acids).Most scientific research underlines the link between the metabolism of several skin lipids and focuses mainly on the ceramides, or more specifically on the protein-bound ceramides, as indicators of homeostasis of the skin barrier, but as shown in a recent article, various lipids species (18 prostanoids, 12 hydroxy fatty acids, 9 endocannabinoids and N-acyl ethanolamides, 21 non-hydroxylated ceramides and sphingoid bases) (Kendall et al., 2015) cooperate with proteins to give the specific conformational ordering and lateral packing required for the maintenance of a good homeostatic barrier (Mojumdar et al., 2014).

Topical Issue
As confirmed also by other laboratories, the barrier function of the stratum corneum depends critically on its unique constituents; 75-80% as proteins, 5-15% as lipids with 5-10% unidentified on a dry weight basis (Wilkes et al., 1973).With regard to stratum corneum permeability, it was also reported that the amount of the 18C sphingosine-containing ceramides and the various acylceramides are key influencing factors (Janu sová et al., 2011).
5 The sphingolipid rheostat in skin and the trends in anti-aging cosmetics As many researchers assumed, skin is a window for intrinsic (chronological) and extrinsic (photoaging) changes.The age-related changes in skin homeostasis are alterations due to the inherent apoptosis and free radical accumulation at a cellular level and translated at skin level to ageing processes and cumulative environmental damage (Fig. 2).
We observe, due to apoptosis and free radical accumulation in the dermis and epidermis, several chronological skin aging signs such as atrophy, laxity, fine wrinkles, loss of elasticity, dryness, contact dermatitis dermatosis, xerosis, fungal infections, seborrheic keratosis (Fig. 2).Photoaging (Krutmann and Gilchrest, 2006) can be associated with changes such as dyspigmentation, freckles, thick skin, deep wrinkles, melasma, citrine skin, senile purpura, pseudostellate scar, acrokeratoelastoidosis marginalis and lentigines ("age-spots").Besides prolonged sun exposure, smoking is also a risk factor for aggravation resulting in sagging, dullness and inflamed skin (Fig. 2).Latterly, the totality of exposures to which an individual is subjected from conception to death is described by the "exposome" (Go and Jones, 2014;Krutmann et al., 2017) and aging is one of them.Otherwise, damage may result from repeated chemical assault of, for example, soaps or cosmetics.
In elder individuals, due to aging processes in their skin, the sphingolipid turnover is slower (Popa and Portoukalian, 2015).A heterogeneous distribution of lipids and the diminution of proteinbound lipids have been observed in aging stratum corneum leading to an impaired skin homeostasis and an impaired integrity (Denda et al., 2002).Dermal ageing was correlated with senescence in the dermis due to increased beta-galactosidase expression and accumulation of ceramides (Mouton and Venable, 2000).
As we can see from the following examples, some actives may induce the salvage way of the sphingolipid rheostat that is reflected by an anti-aging activity in epidermis and dermis, in hydration and water holding in stratum corneum and in barrier integrity, along with a rejuvenated skin presenting resilient, smooth, blemish-free and reduced hyperpigmentation appearance (Fig. 2).
Urea, one of the most widely used cosmetic actives, showed an enhancement of barrier function by inducing epidermal differentiation, increasing lipid content and increasing antimicrobial peptide expression (Grether-Beck et al., 2012) Another specific activity of the PUFA in epidermis is the activation of the TLR2 (co-localized in the lipid rafts of the cellular membrane) receptor pathway that induces the polarization of macrophages into the inflammation-driving M1 phenotype and contributes to immune defense via the synthesis and release of pro-inflammatory cytokines (Hellwing et al., 2018), Alternatively, a B vitamin, niacinamide, has been shown to increase the epidermal production of skin barrier lipids, (ceramides), and proteins (keratin, involucrin, and fillagrin) (Bissett et al., 2005) In other studies, the anti-ageing effect of an aqueous extract of Gromwell (Lithospermum erythrorhizon) was associated (Kim et al., 2012) with the increase in both lipids markers for phopholipids and glucosylceramides in aged fibroblasts.
Otherwise, following oral administration of an active (Cosgrove et al., 2007) such as essential fatty acids n-3 polyunsaturated fatty acids (PUFA), the skin and stratum corneum showed increased production of protein-bound ceramides in atopic subjects (Popa et al., 2011), and in healthy subjects, an increase in free ceramides was shown in the stratum corneum (Popa et al., 2018).
Supplementation with sphingomyelin (SM) containing milk resulted in an significant improvement in facial skin moisture and reduction of wrinkles perception around the eyes (Higurashi et al., 2015) after 12 weeks of treatment.Another study showed a reduction of UV-induced damage by maintaining covalently-bound v-hydroxy ceramides at the physiological levels and down-regulating mRNA levels of acute inflammation-associated genes, including thymic stromal lymphopoietin, interleukin-1 beta, and interleukin-6 (Oba et al., 2015).The bio-distribution of sphingomyelin to the skin represented 20% of the total sphingomyelin metabolized in liver and hydrolyzed to sphingosine and fatty acids.The sphingolipid uptake in skin was correlated also with an increased capacity of water-holding in the hairless mice (Haruta-Ono et al., 2012).
The improvement of the differentiation process in epidermis by vitamin or cannabinoid analogs was shown along with the externalization of cadherin at desmosome localization and the activation of sphingosine-1-phosphate lyase (S1P-lyase) (Celli et al., 2012).This means that the sphingolipid metabolism is representative of the organ integrity state.
As we can see from the afore mentioned work, sphingolipid metabolism modulates and balances cell and organ homeostasis and, based on their intrinsic activity, sphingolipids are proposed to be used alone or as complexes with other molecules as penetration enhancer or molecules (Benson, 2005).
For example, they are used: as complexes with dextrose molecules (a-, b-, g-cyclodextrin) bound in a 1,4-configuration to form rings of various diameters; in vesicles as cosmetic products in which the active ingredients such as humectants like glycerol and urea, sunscreening and tanning agents, enzymes, are encapsulated; in liposomes where, with other colloidal particles, they form concentric biomolecular layers that are capable of encapsulating drugs; in transfersomes composed of phospholipids with 10-25% surfactant (such as sodium cholate) and 3-10% ethanol; in ethosomes containing a high alcohol quantity capable of enhancing penetration to deep tissues and the systemic circulation; in solid lipid nanoparticles (SLN) where they are used as carriers for enhanced skin delivery of sunscreens, vitamins A and E, triptolide and glucocorticoids.
The recovery of barrier homeostasis was also achieved through oral administration of L. reuteri by inducing control over perifollicular inflammation in stressed animals (Arck et al., 2010) or by restoring cutaneous pH and the protease activity in aged skin to the levels seen in young skin.The latter was induced by the fermentation of Lactobacilli and production of free fatty acids (FFAs) and conjugated linoleic acid (CLA) (Yadav et al., 2008).
Similarly, the recovery of the barrier homeostasis was shown in a 2-month pilot clinical study using a 0.2% phytosphingosine solution with antimicrobial activity against P. acnes that reduced by 89% acneiform papules and pustules compared to BPO (benzoyl peroxide) at 3.7% (Pavicic et al., 2007).An anti-microbial efficacy of 90% microbial count reduction on the skin, after application of phytosphingosine at 1% compared to control (triclosan at 0.1%), was also registered in the same work.
In another study, it was shown that the administration of vitamin C enhances the de-novo sphingolipid biosynthesis in epidermis and modulates the ratio sphinganine to sphingosine (Sa/So) in the stratum corneum (Kim et al., 2011).
This Sa/So ratio influences barrier homeostasis because sphinganine induces antimicrobial activity and has a moisturising action in atopic skin lesions in humans, which relies on the enhancement of S1P lyase activity (Park et al., 2013).
In a recent study in mice infected with S. aureus and presenting respiratory cystic fibrosis (CF), inhalation of ceramides and sphingosine resulted in a significant prevention of pulmonary infections (including septic, MRSA exemplification, (Tavakoli et al., 2016).
Due to the microbiota and skin sphingolipids interconnecting and signaling, it was observed that the production of the cathelicidin peptides (CAMP), that are part of antimicrobial peptides (AMP) group, is triggered by the stimulation of sphingosine 1-phosphate content in keratinocytes which activates NFkBèC/EBP a-pathway, (a dependent pathway that enhances CAMP production) (Park et al., 2013).In this study, resveratrol was used as an active to enhance cathelicidin antimicrobial peptide (CAMP) production and promote S. aureus growth inhibition while the sphingosine 1-phosphate content in keratinocyte was increased.
In addition to the above, it was shown that the dietary ingredient genistein stimulates cathelicidin antimicrobial peptide expression through the S1P-dependent rheostat by stimulating ceramide hydrolysis due to the activation of acidic and alkaline ceramidases, along with a corresponding decline in S1P lyase activity.
Similarly to resveratrol, genistein stimulates the pathway of C/EBPa mRNA expression that regulates CAMP gene expression along with the sphingosine 1-phosphate content in keratinocytes.(Park et al., 2014).
In general, sphingoid bases (phytosphingosine, sphingosine) and fatty acids such as sapienic acid show a good skin antibacterial activity on the gram-positive and gram-negative bacteria E. coli, F. nucleatum, S. aureus, S. sanguinis and S. mitis (Benson, 2005).In this respect, the barrier homeostasis could be ensured by the management of a resilient and balanced skin microbiota that is associated with healthy ceramide production in stratum corneum.This leads to a moisturized skin and balanced sebum production (Kober and Bowe, 2015).

Conclusions
It is obvious that the sphingolipid rheostat plays a key role in cell development.As sphingolipids are involved in intracellular signal transduction and cell to cell connexions, they can be modulators of membrane proteins, act as bioactive lipid mediators in human skin (but also in the skin of other mammals such as mice and dogs), act as sensors in complex synapses with integrins and TPS, and co-localize with TLRs (ex: TLR2) within the lipid raft.S1P enhances antimicrobial defense through increased cathelicidin antimicrobial peptide (CAMP) production.
Ceramides, fatty acids, sphingosine, sphingosine 1phosphate are specific lipids modulators in intra-and intercellular triggering and promote skin homeostasis, with anti-aging and anti-inflammatory effects in skin.
Moreover, we notice that GSL (glycosphingolipids) promote modulation of actives, pro-actives, ion-pairs, supersaturated active solutions, eutectic systems, complexation, liposomes and vesicle penetration in skin.

Fig. 1 .
Fig. 1.De-novo and the recycling pathways of the sphingolipid metabolism in cells.Legend: De-Novo biosynthesis pathway of sphingolipid (on light panel and blue writing colors) and the Recycling pathway of sphingolipid in mammalian cells (on blue panel and red writing colours).SM: Sphingomyelin; GSL: Glycosphingolipids; L: Ligand, R: Receptor; GLC-Ceramide: Glucosyl-Ceramide; S1P: Sphingosine 1-Phosphate.