Ceramide signaling in mammalian epidermis

Abstract

Ceramide, the backbone structure of all sphingolipids, as well as a minor component of cellular membranes, has a unique role in the skin, by forming the epidermal permeability barrier at the extracellular domains of the outermost layer of skin, the stratum corneum, which is required for terrestrial mammalian survival. In contrast to the role of ceramide in forming the permeability barrier, the signaling roles of ceramide and its metabolites have not yet been recognized. Ceramide and/or its metabolites regulate proliferation, differentiation, and apoptosis in epidermal keratinocytes. Recent studies have further demonstrated that a ceramide metabolite, sphingosine-1-phosphate, modulates innate immune function. Ceramide already has been applied to therapeutic approaches for treatment of eczema associated with attenuated epidermal permeability barrier function. Pharmacological modulation of ceramide and its metabolites signaling can also be applied to cutaneous disease prevention and therapy. The author here describes the signaling roles of ceramide and its metabolites in mammalian cells and tissues, including epidermis.

1. Introduction

Ceramide constitutes the backbone structure of all sphingolipids, as well as being a minor component of cellular membranes. In addition, ceramide has a unique role in the skin, forming the epidermal permeability barrier at the extracellular domains of the outermost layer of skin, the stratum corneum, which is required for terrestrial mammalian survival (Fig. 1) [1,2] (also see J.A. Bouwstra, K. Sandhoff, Y. Uchida et al. chapters elsewhere in this volume). In addition to ceramide’s structural roles in cells and tissues, over the past two decades, ceramide and its metabolites have been recognized as signaling lipids that modulate cellular function (Fig. 1) [3,4]. Concurrent with elucidating the signaling roles of ceramide and its metabolites in cells, pharmacological modulation of cellular ceramide levels has been applied as a therapeutic approach for the treatment and prevention of diseases, such as cancers, cardiovascular disease, immune dysfunction [4–6], and eczema associated with attenuated epidermal permeability barrier function [7]. The author describes here the signaling roles of ceramide and its metabolites in mammalian cells and tissues, including epidermis.

Fig. 1.

Roles of Ceramide and Its Metabolites in Epidermis

Glycosphingolipid lipids, ceramide metabolites, also are signaling lipid (not included in this figure, see 7 and 9.3, below)

2. Ceramide Structures in Epidermis

Ceramides consist of long-chain amino alcohol, sphingoid bases, and amide-linked fatty acid (Fig. 2). Sphingosine (carbon chain lengths 18–20) and non-hydroxy fatty acids (carbon chain length 16–24) are major ceramide constituents in mammalian cells. In addition to these ceramide species, dihydrosphingosine (sphinganine) and 1,3,4-trihydroxydihydrosphingosine (phytosphingosine), which contain ceramide species, are present. Ceramide species containing 6-hydroxysphingosine are contained in epidermis. 2-hydroxy fatty acids also make up ceramide (as amide-linked fatty acids), and are relatively abundant in brain, kidney [8], and differentiated layers of epidermis [9]. Moreover, ultra-long chain non-hydroxy, omega-hydroxy and omega-O-acyl fatty acids (carbon chain lengths 36), essential to forming the epidermal permeability barrier, are contained within ceramide species in differentiated layers of the epidermis [1]. In contrast to glycerolipids, which contain saturated, mono, and polyunsaturated O-esterified fatty acids, saturated or monosaturated fatty acids dominate in amide-linked (N-acylated) fatty acids of ceramides. Recent studies have revealed that different structures of ceramide play distinct signaling roles in cells (see below, 3. Ceramides signal to modulate cellular function).

Fig. 2.

Ceramide (N-octadecanoyl sphingosine) Structure

Ceramides in the stratum corneum show structural variation, including hydroxylation (α, ω, 4, and 6 position). Sphingosine (carbon chain lengths 18–20) and non-hydroxy fatty acids (carbon chain length 16–24) are major ceramide constituents in mammalian cells and these major ceramdie species ubiquitously serve as signaling lipid in mammalian cells, including epidermis.

3. Signaling Ceramide Generation

Three sources of ceramide contribute to the initiation of signals; i.e., 1) Increases in sphingomyelin hydrolysis by activation of sphingomyelinases, as described above. Six isoforms of sphingomyelinase have been identified to date in mammals; i.e., acid sphingomyelinase, four types of neutral sphingomyelinase, and alkaline sphingomyelinase [10]. But the sphingomyelinase(s) that produces ceramide signals are localized in the plasma membrane and endoplasmic reticulum (ER). Alkaline sphingomyelinase catalyzes the hydrolysis of sphingomyelin to lysophosphatidylcholine and platelet activating factor (PAF) to suppress inflammatory responses, in addition to sphingomyelin to ceramide conversion (signaling role of alkaline sphingomyelinase is unknown); 2) increased de novo synthesis of ceramide due to activation of serine palmitoyl transferase and/or ceramide synthase [11,12]. Six isoforms of ceramide synthase have been characterized which showed different substrates as well as tissue specificity; e.g., chain length of fatty acyl-CoA (also see R. Sandhoff chapter elsewhere in this issue). Different chain lengths of the amide-linked fatty acid chain of ceramide, generated by specific isoforms of ceramide synthase, demonstrate different biological activities; i.e., acyl carbon chain length 16 ceramide, synthesized by ceramide synthase 6, protects squamous carcinoma cells from ER stress and apoptosis, while carbon chain length 18 ceramide, synthesized by ceramide synthase 1, inhibits cell growth [13]; and 3) increased sphingosine-1-phosphate hydrolysis by sphingosine-1-phosphatase to produce sphingosine followed by ceramide synthesis by ceramide synthase. In addition to these three pathways, hydrolysis of glucosylceramide, galactosylceramide, and ceramide-1-phosphate by β-glucocerebrosidase, β-galactocerebrosidase and ceramide-1-phosphate phosphatase, respectively, conversely increase cellular ceramide levels and decrease ceramidase activity, albeit these pathways have not yet been defined to generate a subsequent signaling mechanism.

4. Ceramide signaling modulates cellular function

The first report of ceramides modulating cellular function was in 1974 [14]. During studies involving erythropoietic activity of lipid soluble extracts of leukocytes, ceramides were identified as stimulating rabbit erythroblast maturation [14]. This study further characterized the most potent ceramide species that enhanced erythroblast maturation; i.e., ceramides containing amide-linked C24:0 or C24:1 fatty acid and sphingosine, dihydrosphingosine (sphinganine) or 1,3,4, trihydroxy dihydrosphinganine (phytosphingosine); shorter amide-linked fatty acid (<C20) ceramide species were less potent [14]. The signaling roles of glycosphingolipids and their downstream signaling mechanisms began to be explored in the 1980s in several mammalian cell types. Ten years after the first report of ceramide signaling, the signaling roles of ceramide in mammalian cells were rediscovered. Increased cellular ceramide from sphingomyelin hydrolysis by activation of sphingomyelinase, in response to either vitamin D or phorbol ester treatment, induced cell cycle arrest and stimulated differentiation of leukemia cells [15–17]. Moreover, increased cellular ceramide occurred following various stimuli such as oxidative stress [12,18], pathogenic bacterial invasion [19,20], and initiation of an inflammatory cytokine cascade [21]. Shortly thereafter, it was also demonstrated that ceramides induce both apoptosis [22] and autophagy [23,24].

Mimicking activation of endogenous sphingomyelinase, both exogenous bacterial sphingomyelinase and cell-permeable, synthetic short chain amide-linked fatty acid (C2–8) ceramide demonstrated ceramide-dependent regulation of functions in many types of cells, including keratinocytes [25,26]. Because natural ceramides consisting of amide-linked longer chain fatty acid (>C12) have a low solubility in aqueous solutions, short chain ceramides and bacterial sphingomyelinase have been used instead to advance research as ceramide signaling. Yet, since short chain ceramide is absent or only at residual levels in cells, concerns were raised that the effects of short chain ceramides do not represent natural cellular phenomena in response to exogenous stimuli. However, incorporated short chain ceramide cells are hydrolyzed to sphingosine and fatty acid producing sphingosine as a substrate for ceramide synthesis with the increase of endogenous, longer chain length fatty acids (natural ceramide) [27,28]. Therefore, utilization of short chain ceramide can be justified in most studies (although monitoring changes in ceramide levels is important). Alternatively, natural ceramides dissolved in dodecane-ethanol solution become cell permeable [29], but optimization of dodecane-ethanol concentration is necessary to minimize non-specific effects, including cellular toxicity.

It is noted that ceramide (or its metabolites) signaling roles and their mechanisms in carcinoma cells or immortalized cells will not always be identical to those in normal cells. Yet, for example, increased metabolic conversions of ceramide to its metabolites account for a drug-resistant mechanism in some cancer cells. These metabolic conversions serve as a rescue mechanism from ceramide-induced cell death in normal keratinocytes (and possibly in other normal cells), suggesting that the insights gained from carcinoma cells could dictate some signaling roles of ceramide and its metabolites in normal cells. In addition, modulating cellular functions in response to signals by ceramide and its metabolites is dependent upon tissues and cells.

5. Ceramide signaling mechanism

Several downstream signals, initiated by elevation in ceramide, have now been characterized, including activation of ceramide-activating serine/threonine phosphatases (CAPS), i.e., protein phosphatase 1A (PP1A) and protein phosphatase 2A (PP2A) [30], protein kinase C (PKC) ζ [31], catepsin D [32], and kinase suppressor RAS (KSR) [33]. CAPS, PP1A and PP2A inactivate PKCα [34], and AKT (or protein kinase B [PFB]) [35]. These downstream signaling activities mediate diverse responses in a variety of cell responses, which are dependent upon cell and tissue types, and changes in the levels of ceramide (for signal intensities, period, see below, 7.2 Metabolic pathways that rescue keratinocytes from ceramide-induced apoptosis). In addition to these specific targets, ceramide permeabilizes mitochondrial outer membranes releasing molecules <60,000-dalton, leading to mitochondrial-mediated apoptosis [36]. This non-protein-mediated signal of ceramide could occur ubiquitously in mammalian cells.

6. Ceramide-mediated cell death pathways

The mechanisms accounting for ceramide-induced apoptosis have been extensively investigated. Ceramide-mediated cell death, including apoptosis and autophagy, is a mechanism of chemotherapy for cancer [37,38].

A) TNF receptor pathways

The activation of the TNF receptor super family, which includes TNFR1 and the TNF-related apoptosis-inducing ligand (TRAIL) receptors, such as TRAILR1 (DR4), TRAILR2 (DR5) and CD95, increases ceramide production by activation of sphingomyelinase. Both acidic and neutral sphingomyelinases account for these pathways [39–41]. Formation of a platform on plasma membranes and modulating membrane protein localization and/or clustering by increased ceramide have been postulated to initiate specific apoptosis signaling pathways [19,42–47]. Acidic sphingomyelinase localized in the lumen of lysosomes is transferred to plasma membranes in response to TRAIL2 and CD95 activation. In addition to these sphingomyelinase activation mechanisms, internalization of TNF-receptors following ligand binding activates lysosomal acidic sphingomyelinase, followed by increasing ceramide to activating cathepsin D [32,48,49]. Activated cathepsin D then translocates into the cytosol, where it stimulates apoptotic signals [32,48,49].

B) Non-TNF receptor mediated mechanism

Oxidative stress occurs within cells in response to a myriad of stimuli, including irradiation (e.g., ultraviolet, infrared, γ-irradiation), inflammation, bacterial infection, and metal ions. These forms of stress stimulate ceramide production by sphingomyelinase activation, as well as by stimulating de novo ceramide production. Increased ceramide then activates specific proteins (as above in #3. Ceramide signaling mechanism), initiating mitochondrial-mediated-caspase-dependent apoptosis, via cytochrome release [50], activations of SMAC (Second Mitochondria-derived Activator of Caspase) [51], DIABRO (Direct IAP-Binding protein with low PI) [52], and AIF (apoptosis inducing factor) [53]. AIF, HtrA serine protease 2, and endonuclease activation account for caspase-independent apoptosis. In addition to activation of specific apoptosis-inducing proteins, as above, ceramide-mediated mitochondrial outer membrane permeabilization contributes to mitochondrial-dependent apoptosis [36].

DNA breakage occurs in response to γ-irradiation for tumor therapy. Insights from acid sphingomyelinase-deficient mouse studies reveal that increased ceramide by activation of acid sphingomyeliase induces apoptosis in both cancer cells and epithelial cells, where acidic sphingomyelinase is highly expressed; e.g., in the gastrointestinal tract, [54–56]. These studies are not only elucidating roles of acid ceramidase in radiation therapy, but they also could point to more potent therapy for cancer with minimal adverse effects of γ-irradiation to normal cells.

7. Signaling roles of ceramide in epidermis

Signaling roles for ceramide in proliferation, differentiation, and apoptosis also have been demonstrated in epidermis and cultured keratinocytes (Table 1). Exogenous short chain ceramides suppressed cell proliferation of the human squamous cell carcinoma cell line, DJM-1 [25]. DNA synthesis is suppressed for a few hours following exogenous bacterial sphingomyelinase, but then is restored in parallel with increased ceramide to glucosylceramide conversion by glucosylceramide synthase activation in spontaneously immortalized, nontransformed HaCat human keratinocytes [57]. Ceramide also regulates the interferon-gamma-induced intercellular adhesion molecule (ICAM)-1 and human leukocyte antigen (HLA)-DR expression in normal human keratinocytes [26]. Exogenous short chain ceramides activate apoptosis signal-regulating kinase (ASK1), and then p38 MAP kinase, resulting in enhanced differentiation in normal human keratinocytes [58]. Exogenous short chain ceramides also stimulate the production of caspase-14 (a key enzyme involved in epidermal terminal differentiation) [59]. Moreover, ceramides stimulate the transmembrane lipid transporter, ATP binding cassette transporter, family 12 (ABCA12) expression [60]. Ceramide-induced ABCA12 expression is attenuated by silencing proliferator-activated receptor (PPAR) δ expression, suggesting that ceramide activates PPAR δ, leading to stimulation of ABCA12 production [60]. Since PPAR δ stimulates keratinocyte differentiation [61], ceramide-induced keratinocyte differentiation could be via this PPAR δ mechanism. ABCA12 is critical in delivering glucosylceramide into lamellar bodies, a prerequisite for epidermal permeability barrier formation [62]. ABCA12 mutations underlie the pathogenesis of the most severe ichthyosis, Harlequin ichthyosis, as well as a subgroup of autosomal recessive ichthyoses, with lamellar ichthyosis phenotypes [62] (also see M. Akiyama). In keratinocytes, ABCA12 transports glucosylceramides, which are immediate precursors of all ceramide species in the stratum corneum, into epidermal lamellar bodies [62]. Recent studies demonstrate that neonatal demise occurs in glucosylceramide-deficient mice associated with abnormal epidermal lamellar body formation [63,64]. Therefore, both glucosylceramide and ABCA12 are required for epidermal permeability barrier formation [63].

Table 1.

Ceramide and its metabolites signal to modulate keratinocyte function

Treatment	Cell	Effect	Mechanism
Ceramide
Exogenous
short chain ceramide   sphingosine	DJM	↓proliferation, ↑differentiation ↑(modest) proliferation, ↔differentiation	not determined [25]
sphingomyelinase	HaCaT	↓proliferation	not determined [57]
inhibitors of ceramide synthesis   (fumonisin B1, cycloserine)	NHK	↓Interferon gamma-induced ICAM1 and HLA-DR expression	not determined [26]
short chain ceramide	NHK	↑differentiation	ASK1 and p38MAP kinase activation [58]
short chain ceramide	NHK	↑caspase-14 expression	not determined [59]
short chain ceramide and/or   inhibition of ceramide conversion to   its metabolites	NHK	↑ABCA12	PPARδ activation [60]
1α,25-dihydroxyvitamin D3	HaCaT	↑differentiation (1 α,25-dihydroxy- vitamin D3→↑TNFa→↑ceramide)	not determined [65]
ultravioret B irradiation	NHK	irradiation→↑ceramide→↑apoptosis	caspase independent? [12]
ultravioret B irradiation		irradiation→↑ceramide→↑apoptosis	not determined [66]
ultravioret A irradiation	NHK	irradiation→↑ceramide →↑ICAM1 (enzyme independent sphingimyelin→↑ceramide and later ↑Cer→↑serine palmitoyltransferase)	AP2 activation [67,68]
Glucosylceramide
Exogenous
glucosyl ceramides,   including epidermal unique glucosyl	FRSK	↑differentiation	↑intracellular Ca2+
omega-O-acylceramide			and PKC activation [73]
(chemicallysynthesized) glucosyl   omega-O-acylceramide	NHK	↑differentiation	not determined [74]
GM3 ganglioside	A431 and KB	↓proliferation	↓tyrosine phosphorylation of EGF [149]
GM3 ganglioside	NHK	↓proliferation	↓tyrosine phosphorylation of EGF [150]
GT1b ganglioside	NHK	↑differentiation	protein kinase C independent [151]
	NHK	↓cell adhesion	not determined [152]
Sphingosine-1-phosphate
Exogenous
sphingosine-1-phosphate	NHK	↑chemotaxis and induce differentiation	sphingosine-1-phosphate receptor 2/3→↑Ca2+ [153]
sphingosine-1-phosphate	NHK	↑differentiation	↑sphingosine-1-phosphate receptor dependent / independent/[121]
sphingosine-1-phosphate	NHK	protect against TNFα-induced apoptosis	↑ sphingosine-1-phosphate receptor 3→↑eNOS [154]
sphingosine-1-phosphate	NHK	↑laminin 5 synthesis→?↑wound healing	not determined [117]
sphingosine-1-phosphate	NHK	↑migration, ↓cell proliferation	Smad3 activation [113]
sphingosine-1-phosphate	NHK	protect against 1α, 25-dihydroxyvitamin D3-induced apoptosis	not determined [120]
sphingosine-1-phosphate	NHK	restrains insulin-induced cell proliferation	↑sphingosine-1-phosphate receptor 2→↑PKCδ ↓AKT [128]
ER stress→↑ceramide→	NHK	ER stress→↑→↑ceramide→ sphingosine-1-phosphate →↑cathelicidin anti microbial peptide	↑NF-κB→↑c/EBPα [28]

DJM, human squamous cell carcinoma cell; HaCaT, immortalized, nontransformed human keratinocyte; FRSK, fetal rat skin keratinocyte cell; NHK, primary normal human keratinocyte; epidermal cartinoma cell, A431 (human) and KB (mouse) cells

Ceramide signals are responsible in part for TNFα and 1α,25-dihydroxyvitamin D3-mediated keratinocyte differentiation; i.e., 1α,25-dihydroxyvitamin D3 increases TNFα production and TNFα increases ceramide production from sphingomyelin by activating sphingomyelinase [65]. Increased cellular ceramide induces apoptosis in keratinocytes in response to ultraviolet (UV) B irradiation [12,66]. UVA increases ceramide production, without activating ceramide metabolic enzymes, but leads to increased ICAM-1 expression by activation of transcription factor AP2 [67], while increased ceramide following irradiation in turn activates serine palmitoyl transferase (by autocrine pathway), which is a first step and a rate-limiting enzyme of ceramide synthesis (Fig. 3) [68]. Because UV irradiation alters cellular metabolism, including increased hydrolysis of esterified lipids to free fatty acids, an elevated pool of the precursor to ceramide synthesis, free fatty acid, can increase ceramide production without activating enzymes.

Fig. 3.

Ceramide Metabolic Pathway in Epidermis

However, increased sphingomyelin to ceramide conversion occurs in protein-free liposomes following either UVA irradiation or singlet oxygen exposure [67]. Yet, no other studies are available that show non-enzymatic conversion of sphingomyelin to ceramide by oxygen radicals. Finally, ceramides also alter cellular function in other epidermal cells, such as melanocytes. Exogenous, short chain ceramides inhibit melanocyte proliferation via Akt inactivation and induction of melanin production in the spontaneously immortalized mouse melanocyte cell line, Mel-Ab [69]. As noted above, ceramide molecular heterogeneity is unique to epidermis. These heterogeneous ceramides are distributed in the extracellular spaces of the stratum corneum, where they incorporate into stable lamellar membrane structures [2,70]. Therefore, it is unlikely that ceramide in the stratum corneum-derived signals alter cellular function in the nucleated layer of epidermis. Most of the immediate precursors of these barrier ceramides, glucosylceramides, which are not used for cellular membrane constitution, are sequestrated into lamellar bodies [71,72]. These sequestrated lipids are unable to generate signals because of being located in cellular compartments to modulate cellular function; e.g., plasma membranes, mitochondria. Yet, prior studies demonstrated that exogenous glucosyl omega-O-acyl-ultralong chain ceramide induces keratinocyte differentiation [73,74]. If a non-sequestrated pool of epidermal unique glucosylceramide and/or its metabolites is available and/or lamellar bodies diffuse to apical surface of plasma membrane during transition of stratum granulosum to stratum corneum, these lipid species could generate regulatory signals.

Note: Alterations of proliferation, differentiation, cell death, cell-cell attachment, and other cellular functions in epidermis of transgenic mice affect epidermal permeability barrier function. Diminished barrier function also affects cellular proliferation and differentiation in epidermis and results in secondarily changing other epidermal functions. Therefore, it is difficult to distinguish between primary and secondary effects of a targeted enzyme. Hence, utilization of transgenic mice to elucidate a signaling role of ceramide in epidermis should be limited. Pharmacological inhibition or gene silencing of a specific ceramide metabolic enzyme in cultured epidermal keratinocytes should be more precise in representing roles of ceramide and its metabolites in epidermis than use of transgenic animals.

8. Protective mechanisms used against ceramide-induced apoptosis

Ceramide-induced apoptosis plays a beneficial role in cancer treatment, as well as in the elimination of abnormal cells that carry potentially detrimental mutated genes. Yet, increased apoptosis in normal cells allows expansion of cancer cell population. Epidermis is situated uniquely at the environmental interface, so this external organ is continuously at risk from exposure to oxidative stressors such as UV irradiation and xenotoxic compounds. Moreover, during differentiation, keratinocytes produce abundant amounts of ceramides to form the epidermal permeability barrier [71,75,76]. Therefore, normal cells, in particular keratinocytes, need to deploy protective mechanisms against ceramide-induced apoptosis.

8.1 Metabolic conversion of ceramide to non-apoptotic metabolites

The metabolic conversion of ceramides to non-apoptotic metabolites (Figs. 3 and 4) increases in some cancer cells, as well as in drug-resistant cancer cells. For example acidic ceramidase expression increases in head and neck squamous carcinoma cells [77], and in some melanoma [78], prostate [78], and colon cancer [78] cell lines. Sphingosine kinase 1 is overexpressed in head and neck squamous carcinoma cells [79] and prostate cancer [80], suggesting ceramide conversion beyond sphingosine to sphingosine-1-phosphate. Ceramide kinase, which generates ceramide-1-phosphate, is overexpressed in breast cancer cells [81]. Glucosylceramide synthase expression increases in some cell lines (including multidrug resistance) of breast cancer [82], prostate cancer [82], colon cancer [83,84], and leukemia [84,85]. Thus, inhibition of one or more of these ceramide metabolic enzymes could represent a strategy to enhance anti-cancer treatments. Conversely, the metabolic conversions of ceramides to its non-apoptotic metabolites also serve to protect normal cells from ceramide-induced apoptosis.

Fig. 4.

Protective Mechanism Against Ceramide-Induced Apoptosis in Response to Oxidative Stress in Epidermis

Toxic levels of stress overwhelms these protective mechanism and results in increased apoptosis [93]. Since CERT (ceramide-transfer protein) forms stable homotrimer that diminishes ceramide transfer function under oxidative stress, sphingomyelin synthesis does not increase [104].

8.1.1 Conversion of ceramide to sphingosine to sphingosine-1-phosphate

Ceramide is hydrolyzed to sphingoid base and fatty acid by ceramidase (Fig. 3). Five ceramidase isoforms, which show different pH optima and different subcellular distributions, have been characterized in mammals; i.e., 1) acid ceramidase (lysosomal distribution); 2) neutral ceramidase (plasma membrane and mitochondrial membrane distribution) [86,87]; 3) alkaline ceramidase 1 (a differentiated keratinocyte specific isoform expressed in Golgi apparatus and endoplasmic reticulum [ER]) [88]; 4) alkaline ceramidase 2 (Golgi apparatus and ER) [89]; and 5) alkaline ceramidase 3 (or phytoalkaline ceramidase), which hydrolyzes ceramide species containing dihydrosphingosine (sphinganine), 1,3,4-trihydroxydihydrosphingosine (phytosphingosine), and amide-linked unsaturated fatty acids (Golgi apparatus and ER) [90]. Epidermal keratinocytes express all five isoforms of ceramidase, with different expression profiles of each isomer across the different cell layers of epidermis [88]. The sphingosine kinase converts sphingosine to sphingosine-1-phosphate (Fig. 3). Two isoforms of sphingosine kinase, sphingosine kinase 1 [91] and sphingosine kinase 2 have been identified in mammals [92]. The sphingosine kinase isoform, sphingosine kinase 1, is localized in cytosol [91], while sphingosine kinase 2 localizes in ER and nucleus [92]. Both isoforms are expressed in keratinocytes [93]. Sphingosine-1-phosphate can be further metabolized to phosphoethanolamine and hexadecanal by spingosine-1-phosphate lyase [94,95].

8.1.2 Conversion of ceramide to ceramide-1-phosphate

Ceramide kinase phosphorylates ceramide, generating synthesize ceramide-1-phosphate in the trans Golgi network (Fig. 3). Two isoforms of ceramide kinase, ceramide kinase 1 and 2, have been characterized in mammalian tissues [96], including epidermis . Ceramide delivered from ER by ceramide transfer protein (CERT) is utilized for the generation of ceramide-1-phosphate generation [97].

8.1.3 Conversion of ceramide to glucosylceramide

Ceramide is transferred from ER to cis Golgi by vesicle transport and then glucosylated to glucosylceramide at cis Golgi by glucosylceramide synthase (Fig. 3) [98,99]. Glucosylceramide is a dominant glycosphingolipid species (>95%) in epidermis [100]. Other glycosphingolipids’ (such as gangliosides) syntheses also occur in Golgi. Gangliosides also regulate epidermal functions (see Section 9.4).

8.1.4 Conversion of ceramide to sphingomyelin

Differing from ceramide transfer for glucosylceramide synthesis from ER to Golgi, ceramide transferred from ER to trans Golgi by ceramide transfer protein (CERT) [101] precedes ceramide to sphingomyelin conversion, which is synthesized by sphingomyelin synthase 1 (Fig. 3) [102,103]. Sphingomyelin synthesis also occurs at the plasma membrane by sphingomyelin synthase 2 [102,103].

8.2 Metabolic pathways that rescue keratinocytes from ceramide-induced apoptosis

Ceramide-induced apoptosis occurs in keratinocytes following high doses of ultraviolet B (UVB) irradiation [12], while low doses of UVB irradiation only inhibit cell proliferation [93]. Interestingly, both low and high doses of UVB irradiation increase ceramide production to the comparable levels at early time points, but ceramide levels return toward normal ranges following low (but not high) doses of UVB irradiation by metabolic conversion of ceramide to sphingosine, followed by further conversion to sphingosine-1-phosphate in human keratinocytes [93]. This ceramide metabolic pathway does not operate efficiently in cells after high levels of ultraviolet insults [93]. Because the ceramide transfer function, CERT, declines after formation of a stable production of homotrimer following oxidative stress [104], ceramide to sphingomyelin (and likely ceramide to ceramide-1-phosphate) conversion does not upregulate in response to oxidative stress [104].

9. Ceramide metabolites and their downstream signals

9.1 Sphingoid base

Sphingosine inhibits protein kinase C activated by diacylglycerol, Ca²⁺, and phorbol ester [105,106], while sphingosine activates sphingosine dependent kinase, which is produced by proteolysis of protein kinase δ [107,108]. Sphingosine-dependent kinase accounts for apoptosis in astrocytes [107]. Sphingosine induces apoptosis through caspase-dependent pathways [109–111]. In addition, since sphingosine has detergent properties, increased sphingosine could alter membrane fluidity (membrane curvature) and alter cellular functions.

9.2 Sphingosine-1-phosphate

Sphingosine-1-phosphate is generated by sphingosine kinase 1 and has been shown to activate anti-apoptotic activity to protect cells from ceramide-induced apoptosis, to stimulate cell proliferation, to increase cell motility [112,113], and to stimulate wound healing [114–118], while sphingosine kinase 2 localized in nucleus generates sphingosine-1-phosphate, which induces cell cycle arrest [119]. Sphingosine-1-phosphate produced in endoplasmic reticulum by sphingosine kinase 2 is dephosphorylated by sphingosine-1-phosphate phosphatase and is converted to ceramide by ceramidase synthase, resulting in apoptosis [120]. In keratinocytes, sphingosine-1-phosphate induces differentiation, but does not stimulate proliferation [121]. Lipid transporters, ATP binding cassette, i.e., ABCA1, ABCC1 [122], and Spinster 2 [123,124] are involved in sphingosine-1-phosphate efflux from cells. Sphingosine-1-phosphate regulates cellular functions through the activation of plasma membrane localized G-protein coupled sphingosine-1-phosphate receptor. Five isoforms of sphingosine-1-phosphate receptors have been characterized in mammals, and all five receptors are expressed in keratinocytes. Activation of sphingosine-1-phosphate receptors modulates cellular function through a number of downstream signaling pathways, including activations of phospholipase C followed by increased intracellular Ca²⁺ [125,126], and PI3 kinase followed by Akt/Rac activation [127], PKCδ activation accompanied with Akt inactivation [128], and Smad 3 activation [113]. In addition to sphingosine-1-phosphate receptor-dependent signal, sphingosine-1 -phosphate modulates cellular functions through sphingosine-1-phosphate receptor independent pathways; i.e., sphingosine-1-phosphate can directly modulate histone acetylation [129] and is a cofactor in the classical RelA pathway leading to polyubiquitination of receptor interacting protein 1 (RIP1) and NF- κB activation [130].

Moreover, recent studies demonstrated that sphingosine-1-phosphate produced in epithelial cells, including keratinocytes, in response to subtoxic levels of ER stress (which can be initiated by subtoxic levels of external stress, such as UVB irradiation, or epidermal permeability barrier perturbation) stimulate a major innate immune element, cathelicidin antimicrobial peptide via increased ceramide production followed by increasing sphingosine-1-phosphate that activates NF-κB and then c/EBPα (Fig. 5) [28]. In addition, the mechanism of sphingosine-1-phosphate-induced NF-κB activation has been shown through a receptor-independent pathway [28].

Fig. 5.

Ceramide metabolite, sphingosine-1-phosphate signals to stimulate antimicrobial defense.

As above, acid ceramidase expression increases in prostate cancer, suggesting the involvement of tumor growth [131]. It is likely that pro-mitogenic sphingosine-1-phosphate rather than ceramide or sphingosine is responsible for tumor growth. Keratinocytes, acid ceramidase and alkaline ceramidase 1 enhance Ca²⁺-induced cell cycle arrest and differentiation [116]. Overexpression of alkaline ceramidase 2 increases β1 integrine maturation and cell adhesion [132]. Alkaline ceramidase 2 and 3 have been shown to coordinately regulate keratinocyte proliferation and apoptosis [133]; i.e., silencing alkaline ceramidase 3 increases alkaline ceramidase 2 expression and results in increased ceramide, which contains unsaturated fatty acids, that inhibit cell proliferation through upregulation of increased cycline-dependent kinase inhibitor p21C1P/WAF1 expression and suppressed apoptosis [133].

9.3 Ceramide-1-phosphate

Ceramide-1-phosphate stimulates cell proliferation [134] and is implicated in neutrophil phagocytosis [135]. Ceramide-1-phosphate receptor has not been identified. Instead, ceramide-1-phosphate directly interacts with and activates cytosolic phospholipases, and releases arachidonate to increase prostanoid production. Ceramide-1-phosphate-mediated increases in prostaglandin E2 production to stimulate inflammatory responses in cells [136–138]. However, ceramide-1-phosphate inhibits TNFα converting enzyme to suppress TNFα-induced-NF-κB activation [139] and also Toll-like receptor 4 (TLR4)-induced NF-κB activation [140], suggesting ceramide-1-phosphate has an anti-inflammatory effect. Ceramide-1-phosphate stimulates cell proliferation [141,142] and cell migration [143,144], inhibits apoptosis [141,145,146] and increases glucose uptake [147].

9.4 Glycosphingolipids

Prior studies demonstrated the signaling roles of glycosphingolipid in epidermal cellular function, c.f. review articles [148]. In epidermis, glucosylceramide is a dominant glycosphingolipid (>98 %) species, while di- or polyglycosylated sphingolipid species, such as GM3 gangliosides and other gangliosides, are also present as minor components [100]. An earlier study demonstrated that GM3 ganglioside modulates tyrosine phosphorylation of the epidermal growth factor receptor and suppresses cell growth of cell lines of epidermal carcinoma cell, A431 (human) and KB (mouse) cells [149]. In normal human keratinocytes, GM3, GD3, 9-O-acetyl GD3, and GD1b ganglioside also inhibit proliferation, but do not induce differentiation [150], while GT1b ganglioside likely increases keratinocyte differentiation [151] and inhibits cell adhesion [152]. In addition, as above, epidermal unique, glucosyl omega-O-acyl-ultralong chain ceramide induce keratinocyte differentiation [73,74].

10. Conclusion

The role of ceramide in epidermal permeability barrier structure in the stratum corneum is widely-recognized. Ceramide also serves as a signaling lipid to modulate epidermal function. As discussed above, ceramide, including epidermal unique omega-O-acyl-ultralong chain ceramide, which constitute the epidermal barrier in the stratum corneum, becomes a signaling agent. Most of the immediate precursors of these barrier ceramides (glucosylceramides), which are not used for cellular membrane constitution, are sequestrated into lamellar bodies, and are unable to generate signals because of not being located in the cellular compartment (unlike plasma membranes and mitochondria). Yet, it is possible that a non-sequestrated epidermal unique glucosylceramide and/or diffusing lamellar bodies into plasma membrane, these lipid species serve as a signaling lipid. Finally, sphingosine-1-phosphate, a distal metabolite of ceramide, stimulates innate immunity. Modulation of ceramide and its metabolite signals could regulate self defense systems and also modulate inflammatory responses in skin.

Fig. 6.

Signalings of ceramide and its metabolites to alter cellular functions in normal keratinocytes

Highlights.

Ceramide has a unique role in the skin to form the epidermal permeability barrier.

This barrier role of ceramide is not the focus of this review.

Ceramide and its metabolites have a signaling role in regulating cellular function.

Cells deploy protective mechanism against ceramide-induced cell death.